InteractiveFly: GeneBrief

NMDA receptor 1 and NMDA receptor 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

N-methyl-D-aspartate (NMDA) receptors are one of three pharmacologically distinct subtypes of ionotropic receptors that mediate a majority of excitatory neurotransmission in the brain via the endogenous amino acid, L-glutamate. NMDARs form heteromeric complexes usually comprised of the essential NR1 subunit and various NR2 subunits. Molecular and electrophysiological properties of NMDARs suggest that they may be the Hebbian 'coincidence detectors' hypothesized to underlie associative learning. Because of the nonspecificity of drugs that modulate NMDAR function or the relatively chronic genetic manipulations of various NMDAR subunits from mammalian studies, conclusive evidence for such an acute role for NMDARs in adult behavioral plasticity, however, is lacking. Moreover, a role for NMDARs in memory consolidation remains controversial (Xia, 2005; full text of article).

The Drosophila genome encodes two NMDAR homologs, dNR1 and dNR2. When coexpressed in Xenopus oocytes or Drosophila S2 cells, dNR1 and dNR2 form functional NMDARs with several of the distinguishing molecular properties observed for vertebrate NMDARs, including voltage/Mg²⁺-dependent activation by glutamate. Both proteins are weakly expressed throughout the entire brain but show preferential expression in several neurons surrounding the dendritic region of the mushroom bodies. Hypomorphic mutations of the essential dNR1 gene disrupt olfactory learning, and this learning defect is rescued with wild-type transgenes. Importantly, Pavlovian learning is disrupted in adults within 15 hr after transient induction of a dNR1 antisense RNA transgene. Extended training is sufficient to overcome this initial learning defect, but long-term memory (LTM) specifically is abolished under these training conditions. In conclusion, this study uses a combination of molecular-genetic tools to (1) generate genomic mutations of the dNR1 gene, (2) rescue the accompanying learning deficit with a dNR1⁺ transgene, and (3) rapidly and transiently knockdown dNR1⁺ expression in adults, thereby demonstrating an evolutionarily conserved role for the acute involvement of NMDARs in associative learning and memory (Xia, 2005).

The NMDAR channel is highly permeable to Ca²⁺ and Na⁺, and its opening requires simultaneous binding of glutamate and postsynaptic membrane depolarization. Once activated, the NMDAR channel allows calcium influx into the postsynaptic cell where calcium triggers a cascade of biochemical events resulting in synaptic changes (Xia, 2005).

Cellular studies have suggested that NMDAR is involved in several forms of synaptic plasticity, including long-term potentiation and long-term depression. The NMDAR possesses an interesting molecular property, namely, a voltage-dependent blockade of glutamate-induced calcium flux. This suggests NMDAR's role as the above mentioned 'Hebbian coincidence detector' underlying associative learning. Additional, non-Hebbian cellular mechanisms appear necessary, however, to model associative learning adequately. To that end, behavioral studies attempting to demonstrate an acute role for mammalian NMDARs in associative learning and/or memory have been limited by (1) the nonspecificity of drugs that modulate NMDAR function or (2) the relatively chronic genetic manipulations of various NMDAR subunits. Whether NMDARs also are involved with memory consolidation is even more controversial (Xia, 2005).

In invertebrates, pharmacological manipulations have suggested that NMDA-like receptors mediate associative learning in Aplysia (Roberts, 2003) and memory recall in honeybee (Si, 2004), and the function of an NR1 homolog, NMR-1, has been characterized in C. elegans (Brockie, 2001). These studies did not determine which potential NMDAR homologs form functional NMDARs, (Dingledine, 1999), however, direct demonstrations of roles for specific NMDAR genes in behavioral plasticity still are lacking in these model systems. Therefore molecular, genetic, electrophysiological, and behavioral experiments were persued on the Drosophila NMDAR subunit genes, dNR1 (Ultsch, 1993) and dNR2, which together establish an acute role for NMDAR in associative learning and in long-term memory consolidation (Xia, 2005).

Homology searches of the Drosophila genome database and cloning suggest dNR1 is the only gene bearing high amino acid sequence similarity to the mammalian NMDA receptor subunit NR1. Compared with its vertebrate counterpart, dNR1 shows high homology with respect to its entire size, domain structures, and active physiological sites. dNR2 appears to be the sole gene encoding the Drosophila homolog of mammalian NR2, although there are four NR2 family members in vertebrates (Yamakura, 1999). dNR2 undergoes alternative splicing, however, to generate eight different transcripts and three protein variants. The domain structures of dNR2 show high homology to vertebrate NR2, but its entire size, active physiological sites, and molecular function are only moderately conserved from its mammalian counterparts (Xia, 2005).

The dNR1 transcript is highly regulated during development and is expressed at high levels in late embryos when the larval nervous system is formed, in late pupae when the adult central nervous system develops, and in adult head. Western blots confirmed that both proteins are expressed at a high level in adult head but not in the body. Immunostaining also indicates that they may be expressed throughout the whole brain and at especially high levels in several neurons surrounding the calyx of the MBs. The interpretation of generally weak expression of dNR1 and dNR2 is further supported by Western blots showing a detectable band from single-head preparations. Thus, dNR1 and dNR2 likely function together in most places, which is in agreement with functional analyses. In contrast, dNR1 appears to have a broader pattern of preferential expression than dNR2 in adult brain, suggesting alternative associations with other endogenous glutamate receptors. Alternatively, dNR1 alone may form functional NMDAR channels in vivo, given its weak but significant NMDA-selective response in Xenopus oocytes. It might be noted, however, that functional NMDA receptors can be formed by expression of NR1 alone in Xenopus oocytes but not in mammalian cell lines. Finally, dNR1 has an RSS (Retention Signal Sequence) motif at its C terminus, similar to its mammalian homolog, suggesting that dNR1, when not associated with dNR2 or other glutamate receptors, may be retained in the ER rather than inserted in the cell membrane (Xia, 2005).

Coexpression of dNR1 and dNR2-2 in Xenopus oocytes generated NMDA-selective responses. Similarly, functional homomeric receptors can be formed within the AMPA and kainate subunit families but probably not for NMDA receptors in vertebrates, and highly active NMDAR channels are only formed when the NR1 subunit is expressed in combination with one of the four NR2 subunits (Dingledine, 1999; Mori, 1995). Pharmacological, anatomical, biochemical, and immunological studies also have established heteromeric, but not homomeric, assembly of NMDAR channel subunits in vivo (Mori, 1995). The physiological features that distinguish NMDAR from other ionotropic glutamate receptors are (1) high permeability to Ca²⁺, (2) selective activation by NMDA and L-asparate, (3) modulation by glycine as the coagonist for glutamate, and (4) voltage-dependent blockade by Mg²⁺ (Dingledine, 1999). The electrophysiological profile of dNR1 and dNR2 coexpressed in Xenopus oocytes or Drosophila S2 cells reveals that the functional NMDARs produce most of these distinguishing characteristics including selective activation by NMDA and L-asparate, modulation by glycine as the coagonist for glutamate, and voltage- and Mg²⁺-dependent conductance. Thus, Drosophila likely has functional NMDARs consisting of two subunits, dNR1 and dNR2 (Xia, 2005).

The NMDA-selective conductance was sensitive to Mg²⁺ blockade only in Drosophila S2 cells but not in Xenopus oocytes up to 10 mM, which is highly reminiscent of NMDA receptors in C. elegans (Brockie, 2001). Proper external ionic conditions for oocytes and insect cells are remarkably different. The endogenous Mg²⁺ concentration for fly muscle, for instance, is about ten times higher than that for oocytes, suggesting that invertebrate NMDA receptors have evolved to be less sensitive to Mg²⁺. Molecular evidence exists in support of this conclusion. Replacement of the asparagine residue in the pore-forming TM2 domain reduces but does not abolish Mg²⁺ block for mammalian NR receptors (Dingledine, 1999). This crucial asparagine residue in dNR2 subunits is replaced by glutamine. In addition, TM1, TM4, and the short linker between TM2 and TM3 domains also are critical determinants for Mg²⁺ block (Kuner, 1996). Although the linker appears conserved in dNR2, TM1 and TM4 are not (Xia, 2005).

Fly NMDA receptors have been shown to regulate the larval locomotor rhythm (Cattaert, 2001). This effect can be blocked completely by MK801, requiring binding to the same asparagine residue to execute its antagonist effect (Ferrer-Montiel, 1995). MK801 also suppresses NMDAR-mediated juvenile hormone biosynethesis in cockroach (Chiang, 2002) (Xia, 2005).

This study provided the first demonstration that NMDARs are required acutely for associative learning in Drosophila. Pavlovian task is a form of fear conditioning which uses well-defined odors as conditioned stimuli (CSs) and footshock as an unconditioned stimulus (US). When tested immediately after Pavlovian conditioning (one training session), flies homozygous for either of two different hypomorphic mutations performed poorly in this task, although they seem to grow normally, do not show any obvious behavioral abnormalities, and most importantly, show normal sensorimotor responses to the stimuli used for this task. The learning deficit in dNR1 mutants can be rescued fully in transgenic flies carrying either of two different genomic constructs containing the dNR transcription unit, which constitutes definitive proof that this transcription unit is responsible for the phenotypic defect observed in these mutants (Xia, 2005).

dNR1 is acutely required for associative learning. Disruption of dNR1, with an hs-GAL4 driver to induce expression of a dNR1 antisense message, yielded a learning deficit specifically and transiently. These results rule out any potential developmental explanation for the adult learning defect. The data extend to insects similar findings from pharmacological and genetic studies in mammals and provide the strongest argument to date that adult learning and memory depend on proper NMDA receptor function (Xia, 2005).

Acute disruption of dNR1 also disrupts 1-day memory after spaced training, without affecting 1-day memory after massed training. The specific abolition of LTM, without affecting 1-day memory after massed training, is similar to that produced by induced expression of a CREB-repressor transgene and indicates a specific disruption of cycloheximde-sensitive LTM with no effect on cycloheximide-insensitive ARM. Hence, CREB-dependent LTM formation appears to depend on normal NMDA receptor function. The cAMP/PKA/CREB signaling pathway has been shown to be involved in diverse processes ranging from hippocampal LTP and barrel formation to learning and memory in mammals. In most of these experimental contexts, activation of NMDARs is required for LTM formation (Riedel, 2003). Recent experiments in mammals also have revealed NMDAR-dependent activation of CREB during LTP and LTM in both amygdala and hippocampus (Schulz, 1999; Cammarota, 2000). Interestingly, two functionally distinct NMDA receptor signaling complexes have been identified: synaptic and extrasynaptic (Hardingham, 2002). Synaptic NMDARs can cause sustained CREB phosphorylation and CRE-mediated gene expression, whereas extrasynaptic NMDARs actively suppress CREB activity via an as yet unknown mechanism. Hence, it seems likely that synaptic NMDAR complexes regulate memory formation by controlling nuclear signaling to CREB (Xia, 2005).

This characterization of a role for NMDA receptors in behavioral plasticity of Drosophila again reinforces the notion that the functional homologies among various model systems is appreciable. Many intracellular signaling proteins are known to be physically associated with vertebrate NMDA receptors (Husi, 2000). The newly identified NMDAR complex consist of more than 80 different proteins, organized into receptor, adaptor, signaling, cytoskeletal, cell adhesion, and novel proteins (Husi, 2000). Genetic and pharmacological disruptions of several components of the NMDAR complex produce learning impairments in rodents. Obvious Drosophila homologs can be identified for a majority of these 80 proteins. Among of them are NR1, PKA subunits, PKC isoforms, and NF1. Disruptions of these genes yield associative learning deficits in flies (Xia, 2005).

The conservation of NMDA-dependent behavioral plasticity in invertebrates further demonstrates that a unified mechanism underlies associative learning and memory. Because behavioral plasticity is tightly associated with synaptic plasticity, it is speculated that similar cellular mechanisms of NMDAR-mediated long-term changes, such as LTP and LTD, may also exist in the adult insect brain. Drosophila genetics now can be applied to discover additional genes and signaling pathways important for NMDAR-dependent plasticity (Xia, 2005).

In conclusion, this study has established that Drosophila likely has functional NMDARs consisting of two subunits, dNR1 and dNR2. Combined expression of both dNR1 and dNR2 generated NMDA-selective responses, whereas expression of either of them individually no significant NMDA-dependent responses in oocytes. The eletrophysiological profile of dNR1 and dNR2 coexpressed in Xenopus oocytes or Drosophila S2 cells reveals that the functional NMDARs produce most of these distinguishing properties specific to mammalian counterparts including selective activation by NMDA and L-asparate, modulation by glycine as the coagonist for glutamate, and voltage- and Mg²⁺-dependent conductance (Xia, 2005).

This study also demonstrates that NMDARs not only are involved acutely for associative learning but also are required for LTM consolidation. Genomic mutations of the essential dNR1 gene yield defects in a Pavlovian olfactory learning task, and these learning defects are fully rescued by two different genomic transgenes containing the dNR1⁺ coding sequence. Importantly, it was shown that Pavlovian learning is disrupted within 15 hr via transient induction in adults of a dNR1 antisense RNA transgene. Finally, the transient knockdown of dNR1 also specifically abolishes the consolidation of protein synthesis- and CREB-dependent LTM (Xia, 2005).

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ~20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) Rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. These observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation (Larkin, 2010).

Experience-induced plasticity of synapses is believed to be a fundamental mechanism of learning and memory. However, central synaptic changes that underlie memory have not been clearly defined, even for relatively simple nonassociative learning processes such as habituation (Larkin, 2010).

During habituation, unreinforced exposure to a repeated or prolonged stimulus results in a reversible decrease in response to that stimulus. Habituation probably serves as an important building block for more complex cognitive function. By allowing unchanging or irrelevant stimuli to be ignored, it allows cognitive resources to be focused on more salient stimuli (Larkin, 2010 and references therein).

The neural basis of short-term habituation (STH) is best studied in the marine snail, Aplysia californica. Here STH (lasting ~30 min) of the defensive gill-withdrawal reflex in response to tactile stimulation of the siphon is thought to arise from presynaptic depression of transmitter release at sensorimotor synapses. However, even here, presynaptic plasticity may not be cell-autonomous, potentially requiring, for instance, activity of yet-to-be-identified interneurons (Larkin, 2010).

Several forems of habituation have been described in Drosophila and are often shown to require the function of genes that regulate cAMP-dependent forms of associative memory. For instance, habituation of proboscis extension reflex as well as odor-evoked startle reflex in adult Drosophila requires rutabaga (rut)-encoded Ca2+/calmodulin-sensitive adenylyl cyclase. In addition, habituation of the ethanol-induced startle response requires the shaggy/GSK-3 signaling pathway. Despite such pioneering observations, the mechanisms of these various forms of habituation, even whether the primary neuronal changes are purely sensory or involve plasticity of central synapses (involving centrally located interneurons that may integrate various different kinds of modulatory, inhibitory, and excitatory inputs), remain poorly understood (Larkin, 2010).

Recent advances in understanding the circuitry that underlies Drosophila olfactory behavior, as well as the development of new tools to perturb identified neurons in vivo, has opened the opportunity for understanding mechanisms of olfactory habituation at the level of the underlying neural circuitry (Larkin, 2010).

In the larval olfactory system, 21 olfactory sensory neurons (OSNs), each expressing a single odorant receptor (together with the broadly expressed Or83b co-receptor), synapse, respectively, onto 21 cognate projection neurons (PNs) within 21 glomeruli in the larval antennal lobe (AL). Local, predominantly GABAergic interneurons (LNs) synapse widely within the antennal lobe, interlinking different glomeruli. Various neuromodulatory synapses also form on the larval antennal lobe and mushroom body. Thus, odorant-stimulated signals in sensory neurons are processed in the antennal lobe, modulated by motivational or emotional states, and relayed through projection neurons to higher brain centers (Larkin, 2010).

Previous work has shown that in Drosophila larvae, olfactory chemotaxis decreases after odorant pre-exposure. This study shows that this behavioral habituation, alternatively referred to as 'adaptation' by some previous investigators, arises from mechanisms of synaptic plasticity. This study demonstrates that odorant receptor activation is not necessary for olfactory habituation; however, local interneuron activity and projection neuron signaling is necessary. These observations suggest a model in which habituation occurs by a pathway in which NMDA receptors in projection neurons signal depression of OSN-PN synapses and/or facilitation of LN-PN synapses (Larkin, 2010).

Previous studies have not clearly discriminated between peripheral and central mechanisms. Indeed, the term 'adaptation,' better applied to sensory neuron changes such as receptor desensitization, has often been used interchangeably with the term 'habituation', which is usually restricted to behavioral changes arising from central synaptic mechanisms (Larkin, 2010).

The form of larval olfactory STH characterized in this study displays at least some of the defining behavioral characteristics of habituation. First, there is a behavioral decrement in response to repeated or sustained application of a particular stimulus. Second, STH shows spontaneous recovery with time in the absence of the habituating stimulus. And third, STH is susceptible to dishabituation when habituated larvae are presented with of a strong or noxious stimulus. The property of dishabituation is particularly significant, as an important way of distinguishing between habituation and either fatigue or sensory adaptation. Dishabituation shows that the habituated animal retains the capability to respond and suggests that the attenuated behavioral response arises from some form of active suppression. Thus, the behavioral data suggest (1) that the term 'habituation' may be better used in place of 'adaptation,' while referring to the behavioral phenomenon that was studied; and (2) that STH probably arises from central synaptic mechanisms, rather than sensory neuron adaptation (Larkin, 2010).

Three main lines of data support the conclusion that STH arises from a central synaptic mechanism that resides in the antennal lobe, rather than from adaptation of olfactory receptor signaling in the OSN. First, behavioral decrements similar to STH can be induced by direct depolarization of OSNs, indicating that STH may potentially be induced by processes stimulated by activation action-potential firing in OSNs, independently of olfactory receptor activation. Second, and more striking, STH requires synaptic-vesicle exocytosis from local interneurons during the process of odorant exposure, when STH is being established. This requirement is incompatible with an exclusively sensory mechanism. Third, STH requires the function of NMDA receptors on postsynaptic projection neurons. This last observation also provides a particularly strong argument for a synaptic mechanism, indicating a need for plasticity of OSN and/or LN synapses made onto dendrites of projection neurons in the antennal lobe. Given that OSNs are excitatory and LNs are primarily inhibitory, it appears most likely that NMDAR functions in PNs to depress excitatory OSN-PN synapses and/or to potentiate inhibition by strengthening the LN-PN synapse. It is suggestd that the LN-PN mechanism may be involved because (1) LN transmission seems necessary for both induction and expression of habituation; and (2) the process of dishabituation could be attractively explained as arising from the inhibition of local inhibitory synapses through descending neuromodulation. A requirement for facilitation of the LN-PN synapse would be consistent with previous studies (Sachse, 2007) showing that adult-long-term olfactory habituation is associated with an increase in odor-evoked calcium fluxes in GABAergic processes within the Drosophila antennal lobe (Larkin, 2010).

Based both on experimental and theoretical arguments, a simple model is suggested for short-term olfactory habituation. Since this is a model, no claim is being made to to having ruled out additional major contributing mechanisms, It is suggested that during initial odorant pre-exposure, dendritic NMDA receptors on projection neurons detect and respond to membrane depolarization occurs coincident with transmitter release from LNs. Calcium entry through dendritic NMDA receptors may trigger a local retrograde signal required for facilitation of transmitter release from the LNs. Although existing data do not rule out functions for rutabaga in higher larval brain centers, it is suggested that either the generation of a retrograde signal in PN dendrites or the presynaptic response of LNs to this signal could be dependent on the rut adenylate cyclase. In habituated animals, facilitation of GABA release would reduce odor-evoked projection neuron outputs to higher brain centers, thereby reducing olfactory behavior. As NMDAR signaling would only occur at active glomeruli, this mechanism can account not only for the observed odor selectivity of habituation, but also the instances of cross-habituation (Larkin, 2010).

Such a model also naturally suggests a hypothesis for the mechanism of dishabituation: namely, that dishabituating stimuli cause release of neuromodulators that act to reduce GABA release from local inhibitory synapses (Larkin, 2010).

Given the remarkable similarities in the anatomical organization of insect and mammalian olfactory systems, a significant conservation of olfactory mechanisms would be expected. In rodents, at least two forms of habituation have been described, lasting 2-3 and 30-60 min, respectively: the latter equivalent in timescale to larval STH described in this study. Consistent with a similar underlying mechanism, the more persistent form of olfactory habituation can be blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist in the olfactory bulb, a structure homologous to the insect antennal lobe. Thus, larval STH described in this study has some similarities to a previously characterized form of mammalian olfactory habituation. Analysis of the underlying mechanisms is therefore likely to provide directly transferable insights in mammalian olfaction. The data make the prediction that the activity of mammalian olfactory interneurons, either periglomerular or granule cells, is critical for the establishment and display of at least one timescale of olfactory habituation (Larkin, 2010).

In addition to providing some insight into mechanisms of olfactory habituation in mammals, it possible that circuit mechanisms of larval olfactory habituation are relevant to other forms of behavioral habituation. In at least three previous instances, increased inhibition has been associated with attenuated behavior. For example, habituation of an escape reflex mediated by the lateral giant fibers in the crayfish has been associated with enhanced GABAergic transmission onto giant fibers. Similarly, LTP of inhibitory synapses controlling excitability of the Mauthner cell has been associated with reduced escape behavior in goldfish. Furthermore, ethanol, a potentiator of GABA synapses, has been shown to enhance habituation of a motor pathway in the frog spinal cord. Could these different instances of habituation all involve circuit mechanisms similar to those used in Drosophila larval olfactory behavior (Larkin, 2010)?

In all brain regions, principal/projection neurons are subject to inhibitory feedback modulation and a pathway that has been appreciated as potentially essential for neuronal homeostasis. Potentiation of inhibitory feedback triggered by the pattern of principle cell activation would be predicted to preferentially dampen this particular output pattern. Thus, the circuit mechanism suggest in this study is theoretically generalizable to other and more complex forms of habituation. Further experiments will be required to determine the validity of this very testable hypothesis (Larkin, 2010).

The importance of habituation has been underlined by the fact that deficits in sensory gating and pre-pulse inhibition (PPI), processes with similarities to habituation, have been linked with various neurological problems, including autism and schizophrenia. Indeed, a circuit model for understanding schizophrenia has specifically proposed that altered negative feedback in the hippocampus may underlie both positive and negative symptoms of schizophrenia (Larkin, 2010).

In addition, defects in habituation or habituation-like processes have been described in Fragile X syndrome and migraines. It has also been shown to have important effects relating to learning disabilities, age-related changes in learning, and substance abuse. If mechanisms of olfactory habituation prove to be general, then studies of olfactory plasticity may prove relevant for other forms of cognition as well as for human neurological disease (Larkin, 2010).

Network-specific synchronization of electrical slow-wave oscillations regulates sleep drive in Drosophila

Slow-wave rhythms characteristic of deep sleep oscillate in the delta band (0.5-Hz) and can be found across various brain regions in vertebrates. Across phyla, however, an understanding of the mechanisms underlying oscillations and how these link to behavior remains limited. This study discovered compound delta oscillations in the sleep-regulating R network of Drosophila. The power of these slow-wave oscillations increases with sleep need and is subject to diurnal variation. Optical multi-unit voltage recordings reveal that single R neurons get synchronized by activating circadian input pathways. This synchronization depends on NMDA receptor (NMDAR) coincidence detector function, and an interplay of cholinergic and glutamatergic inputs regulates oscillatory frequency. Genetically targeting the coincidence detector function of NMDARs in R5, and thus the uncovered mechanism underlying synchronization, abolished network-specific compound slow-wave oscillations. It also disrupted sleep and facilitated light-induced wakening, establishing a role for slow-wave oscillations in regulating sleep and sensory gating. It is therefore proposed that the synchronization-based increase in oscillatory power likely represents an evolutionarily conserved, potentially 'optimal,' strategy for constructing sleep-regulating sensory gates (Raccuglia, 2019).

In vertebrates, oscillatory electrical compound patterns are associated with fundamental brain functions and specific behaviors. Characteristic of vertebrate deep sleep are compound slow-wave oscillations in the delta band (0.5- Hz), which are thought to derive from the synchronization of neuronal activity. However, how specific neural networks contribute to generating compound oscillations and whether these oscillations represent a functional unit for sleep regulation largely remains unclear. This is partially due to methodological constraints, as readouts either focus on single-cell/unit or local compound potential recordings (electroencephalograms and local field potentials) that, confined by poor spatial resolution, do not permit for dissecting interactions of genetically identified multi-units (Raccuglia, 2019).

Like vertebrates, invertebrates' sleep and behavior selection is sensitive to an animal's sleep need. However, in invertebrates, it is unknown whether electrical oscillations can gate specific behaviors and whether an electrophysiological sleep correlate, such as slow-wave oscillations, exists or is involved in sleep regulation. Local field potential (LFP) measurements in the Drosophila brain indicate that the frequency of large-scale compound neuronal activity is reduced during sleep, opening up the possibility that, comparable to vertebrates, slow oscillatory activity could be involved in mediating sleep. Yet such oscillations have not been identified, and it remains unknown which and how neural networks would generate slow-wave oscillations that could be crucial for sleep regulation (Raccuglia, 2019).

This study made use of recent technological advancements in Drosophila melanogaster and combined targeted expression of a genetically encoded voltage indicator (GEVI) with that of optogenetic actuators. This all-optical electrophysiological approach bypasses common methodological constraints, allowing monitoring of multi-unit electrical patterns within a specific network and gaining of mechanistic insight into how sleep-relevant neural activity might be generated (Raccuglia, 2019).

This study has discovered sleep-regulating, network-specific delta oscillations within the R network of the Drosophila ellipsoid body, which is situated at a crossroad involved in sleep regulation and sensory processing. Compound delta oscillations are not detectable in the morning (Zeitgeber time [ZT] 0-3) but become apparent, and increase in power, over the day. Activating circadian input pathways leads to multi-unit synchronization that depends on NMDA receptor (NMDAR)-mediated coincidence detection. Disrupting this synchronization and thus the emergence of compound delta oscillations affects the flies' sleep patterns and alters sensory gating during sleep. This study thus identified delta oscillations as an electrophysiological correlate of sleep pressure regulation in invertebrates and ties these oscillatory patterns to behavioral readouts. This work suggests that slow-wave oscillations and sleep could be fundamentally interconnected across phyla. Slow-wave oscillations may therefore potentially represent an evolutionarily conserved strategy for network mechanisms regulating internal states and sleep (Raccuglia, 2019).

The data suggest that compound delta oscillations specific to the sleep-regulating R network are generated by circadian drive transduced via TuBu neurons. Optogenetic activation of TuBu neurons increases single-unit power and synchronizes R neurons, which should result in an increase of compound delta power and thus internal sleep drive. This is consistent with thermogenetic activation of TuBu neurons increasing the total amount of sleep in flies. High levels of TuBu neuron output could be generated by altering activity in sleep-modulating DN circadian clock neurons, which form direct connections with the TuBu neurons. Of note, following R synchronization mediated via activation of TuBu neurons, a prolonged hyperpolarization becomes apparent that typically can be observed following highly synchronized states (Raccuglia, 2019).

The R network also receives excitatory input from Helicon cells, a potential source of cholinergic input. This study provides evidence that nAChRs act as prime candidates to provide concurrent depolarization required for NMDAR coincidence detection in R neurons. Analogous to the relationship between AMPARs and NMDARs in mammals, the interplay of nAChRs and NMDARs could provide a substrate for coincidence detection in Drosophila. By reducing the temporal variability of mixed synaptic inputs, NMDARs could promote the temporal summation of excitatory postsynaptic potentials, thus increasing the synchrony between R neurons. Indeed, rhythmic application of acetylcholine to R dendrites is sufficient to induce oscillations but only in the presence of functional NMDARs. Helicon cells also receive visual information and are part of a recurrent circuit mediating homeostatic sleep pressure regulation. Thus, R oscillatory activity is likely regulated via a complex interplay of sensory input, circadian rhythms, and homeostatic sleep pressure regulation (Raccuglia, 2019).

These experiments further indicate that additional inhibitory input via glutamate-gated chloride channels could antagonize excitatory drives to retain a frequency within the delta range, potentially defining the time window for recurrent input. It is therefore proposed that the interplay between glutamatergic and cholinergic signals sets a frequency within the delta range (0.5-1. Hz), which permits temporal synchronization of single units to define the power and frequency of compound oscillatory activity and thus the strength of sleep drive (Raccuglia, 2019).

Expression levels of NMDARs in R neurons have previously been associated with the regulation of sleep drive. However, the physiological contribution of NMDARs remained open. The current data suggest that NMDAR coincidence detection gates neuronal synchronization of delta-wave activity within the R network to increase the power of sleep-relevant compound oscillations. Indeed, at the single-cell level, expressing Mg2+ block-deficient NMDARs in R neurons led to irregular activity patterns, which could be at the basis of impaired synchronization and disrupted compound oscillations. It is therefore hypothesized that NMDAR-coincidence detection can lead to a state-dependent transition of activity patterns permitting synchronization. Disrupting synchronization by expressing Mg2+-block-deficient NMDARs interfered with multi-unit synchronization, directly altering the animals' sleep drive, sleep quality, and stimulus-induced wakening. This tightly links the mechanisms underlying network-specific delta-band oscillations to shaping animal behavior (Raccuglia, 2019).

This study has identified slow-wave oscillations as an electrophysiological correlate of sleep pressure regulation involved in sensory gating in Drosophila. Interestingly, oscillatory activity in Drosophila R neurons is reminiscent of up- and down-states occurring at the level of mammalian cortical networks during deep sleep. It is thus hypothesized that the oscillations observed in this study are comparable to sleep-regulating thalamocortical oscillations as well as network-specific oscillations observed during sleep deprivation in vertebrates (local sleep). Thus, the R network could be functionally analogous to the thalamus, as network-specific synchronization of slow-wave activity within the thalamus plays a crucial role in maintaining sleep and sensory gating. Comparable to the potential function of network-specific oscillations during 'local sleep' in vertebrates, slow-wave oscillations within the flies' R network may well be involved in the homeostatic regulation of synaptic strength. It is thus suggested that oscillatory network synchronization may represent an evolutionarily selected optimal strategy for signaling sleep pressure as well as for the internal representation of sleepiness. One prediction from this analogy would be that slow-wave oscillations would also be detected in other Drosophila brain regions. Indeed, extracellular local field potential recordings suggest that overall activity in the fly brain does change during sleep. Future work should address whether network oscillations in the delta band can also be detected in other brain regions, for instance, the sleep-inducing fan-shaped bodies or the mushroom bodies. Additionally, whether cell-autonomous conductances contribute to sustained rhythmic activities of single R neuron remains an open question. This framework should pave the road for identifying evolutionarily conserved fundamental principles that link slow-wave oscillations as electrophysiological hallmarks of sleep to the neuronal processes underlying memory consolidation (Raccuglia, 2019).

A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female (Ishimoto, 2020).

Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling. This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics (Ishimoto, 2020).

The pre-mating rejection should continue if the encounter does not match the female's criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances. cVA is a male-specific pheromonal cue that elicits female sexual arousal via the olfactory sensory system. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete-that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel. Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless or dsx, and no morphological sexual dimorphism has been detected. Thus, a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior is proposed (Ishimoto, 2020).

Genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females, but either suppression of R2/R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time. In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship; however, the walking speed of the mated females increases with the progression of the male courtship. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB (Ishimoto, 2020).

This study found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females. Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL). I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z. Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with the current findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior (Ishimoto, 2020).

Genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization, the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed (Ishimoto, 2020).

The distribution pattern of neurotransmitters in EB neurons has been documented. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release. These intriguing analogies between vertebrate findings and the current results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, others have proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster). The current genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom (Ishimoto, 2020).

The NMDA receptor regulates competition of epithelial cells in the Drosophila wing

Cell competition is an emerging principle that eliminates suboptimal or potentially dangerous cells. For 'unfit' cells to be detected, their competitive status needs to be compared to the collective fitness of cells within a tissue. This study reports that the NMDA receptor controls cell competition of epithelial cells and Myc supercompetitors in the Drosophila wing disc. While clonal depletion of the NMDA receptor subunit NR2 results in their rapid elimination via the TNF/Eiger>JNK signalling pathway, local over-expression of NR2 causes NR2 cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue through clonal expansion that causes, but also relies on, the killing of surrounding cells. Consistently, NR2 is utilised by Myc clones to provide them with supercompetitor status. Mechanistically, this study found that the JNK>PDK signalling axis in 'loser' cells reprograms their metabolism, driving them to produce and transfer lactate to winners. Preventing lactate transfer from losers to winners abrogates NMDAR-mediated cell competition. These findings demonstrate a functional repurposing of NMDAR in the surveillance of tissue fitness (Banreti, 2020).

Cell competition is an evolutionary conserved quality control process, which ensures that suboptimal, but otherwise viable, cells do not accumulate during development and aging. How relative fitness disparities are measured across groups of cells, and how the decision is taken whether a particular cell will persist in the tissue ('winner cell') or is killed ('loser cell') is not completely understood. This is an important issue as competitive behaviour can be exploited by cancer cells (Banreti, 2020).

Various types of cell competition exist. While structural cell competition is triggered upon loss of cellular adhesion or changes in epithelial apico-basal polarity, metabolic cell competition occurs in response to alterations in cellular metabolic states. Growth signalling pathways involved in metabolic cell competition seem to funnel through Myc, which functions as an essential signalling hub in many types of cancers. Myc regulates expression of components that control proliferation, cell death, differentiation, and central metabolic pathways. Particularly, acute changes in cellular metabolism appear to be critical for the winner phenotype during Myc supercompetition in Drosophila, where robustly growing Myc-expressing cells are able to not only outgrow but also actively trigger the elimination of nearby wild-type cells from the tissue (Banreti, 2020).

Recent in vivo data demonstrate that some tumours can uptake lactate and preferentially utilize it over glucose to fuel tricarboxylic acid (TCA) cycle and sustain tumour metabolism. Moreover, the growth-promoting effect of stromal cells is impaired by glycolytic inhibition, suggesting that the stroma provides nutritional support to malignant cells by transferring lactate from cancer-associated fibroblasts (CAFs) to cancer cells. Such energy transfer from glycolytic stromal cells to epithelial cancer cells closely resembles the physiological processes of metabolic cooperativity, such as in 'neuron-astrocyte metabolic coupling' in the brain, and the 'lactate shuttle' in the skeletal muscle. Activation of glycolysis in astrocytes and MCT-mediated transfer of lactate to neurons supports neuron mitochondrial oxidative phosphorylation and energy demand. These observations raise the intriguing possibility that lactate serves as fuel to complement glucose metabolism during cell competition (Banreti, 2020).

This study reports that the NMDA receptor controls the competitiveness of epithelial cells in the Drosophila wing discs. While tissue-wide depletion of NR2 has no effect on cell viability and growth, clonal depletion of NR2 results in their rapid elimination via the TNF>JNK signalling pathway. Conversely, local over-expression of NR2 causes NR2-overexpressing cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue. These data indicate that relative levels of NR2 underpins cell competitive behaviour in the wing epithelia. Moreover, this study finds that Myc-induced supercompetition also depends on upregulation of NMDAR. Genetic depletion of NR2 abrogates Myc-induced supercompetition. Mechanistically, this study finds that the JNK>PDK signalling axis in 'loser' cells (lower NMDAR) results in phosphorylation and inactivation of PDH, the enzyme that converts pyruvate to Acetyl-CoA to fuel the TCA in the mitochondria. In such loser cells, phospho-dependent inactivation of PDH causes mitochondrial shutdown and metabolic reprogramming, thus loser cells produce and secrete lactate to winners. Preventing lactate transfer from losers to winners removes fitness disparities and abrogates NMDAR-mediated cell competition. Together these data are consistent with the notion that NMDAR underpins cell competition and that targeting NMDAR converts Myc supercompetitor clones into superlosers (Banreti, 2020).

The elimination of unfit cells via competitive interactions plays an important role for the maintenance of tissue health during development and adulthood. The data indicate that the NMDA receptor NR2 influences the competitive behaviour of epithelia cells and Myc supercompetitors in the Drosophila wing disc. Genetic depletion of NR2 reprograms metabolism via TNF-dependent and JNK-mediated activation of PDK, which in turn phosphorylates and inactivates PDH. This causes a shutdown of pyruvate catalysis and results in a switch to aerobic glycolysis. Upon phospho-dependent inhibition of PDH, pyruvate is reduced to lactate via LDH, and secreted. While lactate exits cells to avoid acidification, it can be recaptured and used as carbon source by other cells, leading to metabolic compartmentalisation between adjacent cells. In normal physiology as well as in murine and human tumours, lactate is an important energy source that fuels mitochondrial metabolism. For example, lactate produced and secreted by astrocytes is transported to neighbouring neurons where it is used as source of energy to support neuronal function. This is akin of the 'reverse Warburg effect', also named 'two-compartment metabolic coupling' model, where cancer-associated fibroblasts (CAFs) undergo aerobic glycolysis and production of high energy metabolites, especially lactate, which is then transported to adjacent cancer cells to sustain their anabolic need (Banreti, 2020).

These data suggest that the epithelial NMDA receptor is responsible for fitness surveillance and to provide Myc clones with supercompetitor status. Cells with decreased epithelial NMDA receptor are metabolically reprogrammed to transfer their carbon fuel to their neighbours. According to the current model, differential NMDAR signalling in adjacent cells triggers lactate-mediated metabolic coupling, and underpins cell competition in epithelia. Consistently, preventing loser cells from 'transferring' lactate to their neighbours, via inhibition of MCT1, Impl3 or PDK, removes the fitness disparity and nullifies cell competition. Likewise, exposure to elevated levels of systemic lactate, blocks elimination of NR2 loser clones. This suggests that cell competition may be based on NMDAR-mediated metabolic coupling between winners and losers. Importantly, this metabolic coupling only occurs under competitive conditions. Consistently, NR2 losers are only eliminated if they are surrounded by cells with functional NMDAR. This is evident as tissue-wide inhibition of NMDAR by AP5, a selective inhibitor of NR2, blocks elimination of NR2 loser clones in a heterotypic genetic setting (Banreti, 2020).

NR2 is upregulated in Myc expressing clones and Myc cells co-opt epithelial NR2 to promote cell competition, subduing their neighbouring wild-type cells that become re-classified as 'unfit'. Interestingly, Myc clones lose their supercompetitor status upon tissue-wide depletion of NR2. Under this condition, WT cells are no longer eliminated and survive among Myc supercompetitors. This indicates that NR2 underpins Myc-induced supercompetition. Given that Myc is a major driver of cancer cell growth, and is a hallmark of the disease in nearly seven out of ten cases, blocking Myc's function would be a powerful approach to treat many types of cancer. However, the properties of the Myc protein itself make it difficult to design a drug against it. Since the NMDAR signalling circuit is hijacked in many types of human cancers, and its expression level is associated with poor patient survival, it is attractive to speculate that targeting NMDAR may be a promising strategy to improve patient care (Banreti, 2020).

REGULATION

Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila larvae

The noncompetitive antagonists of the vertebrate NMDA receptor dizocilpine (MK 801) and phencyclidine (PCP), delivered in food, were found to induce a marked and reversible inhibition of locomotor activity in Drosophila larvae. To determine the site of action of these antagonists, an in vitro preparation of the Drosophila third-instar larva was used, preserving the central nervous system and segmental nerves with their connections to muscle fibers of the body wall. Intracellular recordings were made from ventral muscle fibers 6 and 7 in the abdominal segments. In most larvae, long-lasting (>1 h) spontaneous rhythmic motor activities were recorded in the absence of pharmacological activation. After sectioning of the connections between the brain and abdominal ganglia, the rhythm disappeared, but it could be partially restored by perfusing the muscarinic agonist oxotremorine, indicating that the activity was generated in the ventral nerve cord. MK 801 and PCP rapidly and efficiently inhibited the locomotor rhythm in a dose-dependent manner, the rhythm being totally blocked in 2 min with doses over 0.1 mg/mL. In contrast, more hydrophilic competitive NMDA antagonists had no effect on the motor rhythm in this preparation. MK 801 did not affect neuromuscular glutamatergic transmission at similar doses, as demonstrated by monitoring the responses elicited by electrical stimulation of the motor nerve or pressure applied glutamate. The presence of oxotremorine did not prevent the blocking effect of MK 801. These results show that MK 801 and PCP specifically inhibit centrally generated rhythmic activity in Drosophila, and suggest a possible role for NMDA-like receptors in locomotor rhythm control in the insect CNS (Cattaert, 2001)

Mg²⁺ block of Drosophila NMDA receptors is required for long-term memory formation and CREB-dependent gene expression

NMDA receptor (NMDAR) channels allow Ca²⁺ influx only during correlated activation of both pre- and postsynaptic cells; a Mg²⁺ block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg²⁺ block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg²⁺ block, it was found that Mg²⁺ block mutants are defective for LTM formation but not associative learning. It was demonstrated that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg²⁺ block defective NMDARs. Furthermore, it was shown that genetic and pharmacological reduction of Mg²⁺ block significantly increases expression of a CREB repressor isoform. These results suggest that Mg²⁺ block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction (Miyashia, 2012).

Although the mechanism through which Mg²⁺ block restricts NMDAR activity is well known, the cellular and behavioral functions of Mg²⁺ block have not been extensively studied. In this study, transgenic flies expressing dNR1^N63IQ to show that Mg²⁺ block is important for formation of LTM. Previous studies of hypomorphic mutants have shown that NMDARs are required for both learning and LTM. In contrast, our Mg²⁺ block mutants do not have learning defects. This suggests that although Ca²⁺ influx through NMDARs is important for learning, inhibition of influx during uncorrelated activity is not. Notably, elav/dNR1^N63IQ flies have slightly enhanced learning. Consistent with this result, NMDAR-dependent induction of hippocampal LTP is enhanced in the absence of external Mg²⁺. In the current studies, Mg²⁺-block-defective dNR1 was overexpressed in an otherwise wildtype background, so it cannot be definitively concluded that Mg²⁺ block is dispensable for learning. However, electrophysiology experiments indicate that Mg²⁺ block is abolished in the flies at physiological potentials. Furthermore, it was demonstrated that expression of Mg²⁺-block-defective dNR1 rescues learning defects in dNR1 hypomorphs, consistent with a model in which Mg²⁺ block is not required for learning. Interestingly, the dNR1^N63IQ transgene does not rescue the semilethality of dNR1 hypomorphs, suggesting that Mg²⁺ block has an essential biological function unrelated to learning (Miyashia, 2012).

The results suggest that Mg²⁺-block-dependent suppression of NMDAR activity and Ca²⁺ influx at the resting state is critical for LTM formation. Supporting this idea, chronic reduction of NMDAR-mediated Ca²⁺ influx at the resting state has been shown to enhance long-term synaptic plasticity. Extending these results, it was found that Mg²⁺ block is required for CREB-dependent gene expression during LTM formation. A CREB-dependent increase in staufen expression upon spaced training is essential for LTM formation, and this study shows that Mg²⁺ block is required for this increase. Two other genes, activin and homer, were identified that are expressed upon LTM induction in a CREB-dependent manner. It is proposed that all three genes are maintained in an LTM-inducible state by Mg²⁺-block-dependent inhibition of CREB repressor, and it was shown that the amount of increase in expression of dCREB2-b in Mg²⁺ block mutants correlates with the ability of dCREB2-b to suppress LTM. The 4-fold increase in dCREB2-b protein in Mg²⁺ block mutant flies is comparable to the increase in dCREB2-b in heat-shocked hs-dCREB2-b flies showing equivalent defects in LTM (Miyashia, 2012).

Next the homer gene was further characterized and it was determined to be required specifically for LTM but not for learning or ARM. It was determined that spaced training increases HOMER expression in several brain regions, including the antennal lobes, lateral protocerebrum, protocerebral bridge, and calyx of the MBs. This increase does not occur in the absence of Mg²⁺ block. Significantly, when Mg²⁺ block is abolished by dNR1^N63IQ expression, specifically in the MBs, increased Homer expression is suppressed in the MBs but not in other regions, including the protocerebral bridge, indicating that Mg²⁺ block regulates CREB repressor and LTM-associated gene expressions in a cell autonomous manner (Miyashia, 2012).

Electrophyisiological experiments demonstrate that 20 mM Mg²⁺ is sufficient to block Drosophila NMDAR currents at the resting potential (-80 mV). Although this concentration is higher than the concentrations needed to block mammalian NMDARs, the Mg²⁺ concentration in Drosophila hemolymph has been shown by various groups to be between 20 and 33 mM, which is correspondingly higher than the Mg²⁺ concentration reported in mammalian plasma. In mammals, Mg²⁺ concentration is higher in cerebrospinal fluid than in plasma, further suggesting that the 20 mM Mg²⁺ concentration used in this study is likely to be within the physiologically relevant range (Miyashia, 2012).

An N/Q substitution at the Mg²⁺ block site of mammalian NR1 disrupts Mg²⁺ block and reduces Ca²⁺ permeability, while a W/L substitution in the TM2 domain of NR2B disrupts Mg²⁺ block and increases Mg²⁺ permeability. This raises the possibility that Mg²⁺-block-independent changes in channel kinetics and Mg²⁺ permeability may be responsible for the effects observed in the dNR1^N63IQ-expressing flies. While this possiblity cannot be completely ruled out, increases were observed in dCREB-2b protein in wild-type neurons in Mg²⁺-free conditions, indicating that disruption of Mg²⁺ block, rather than changes in other channel properties, causes increased CREB repressor expression and decreased expression of LTM-associated genes (Miyashia, 2012).

A chronic elevation in extracellular Mg²⁺ enhances Mg²⁺ block of NMDARs, leading to upregulation of NMDAR activity and potentiation of NMDA-induced responses at positive membrane potentials (during correlated activity) (Slutsky, 2010). This raised the possibility that the Mg²⁺ block mutations may cause a downregulation of NMDAR-dependent signaling and decreased NMDA-induced responses at positive membrane potentials. Since this study recorded NMDA-induced responses from various sizes of cells, it was not possible to directly compare amplitudes of NMDA-induced responses between elav/dNR1^wt cells and elav/dNR1^N63IQ cells. However, training-dependent increases in ERK activity, required for CREB activation, occurred normally in both elav/dNR1^wt cells and elav/dNR1^N63IQ cells, while it was significantly suppressed in dNR1 hypomorphs. These results suggest that the Mg²⁺ block mutations do not alter NMDA-induced responses at positive membrane potentials (Miyashia, 2012).

Similar to dNR1 Mg²⁺ block mutants, dNR1 hypomorphic mutants also have defects in CREB-dependent gene expression upon LTM formation. However, dNR1 hypomorphs and Mg²⁺ block mutants are likely to have opposing effects on Ca²⁺ influx. While hypomorphic dNR1 mutants should have decreased Ca²⁺ influx during spaced training because of a reduction in the number of dNMDARs, elav/dNR1^N63IQ flies are unlikely to have this effect. Conversely, while elav/ dNR1^N63IQ flies should have increased Ca²⁺ influx during the resting state when uncorrelated activity is likely to occur, dNR1 hypomorphs should not. Supporting a model in which dNR1 hypomorphs and Mg²⁺ block mutants inhibit LTM-dependent gene expression through different mechanisms, it was shown that Mg²⁺ block mutants increase basal expression of dCREB2-b repressor while NMDAR hypomorphs do not. Conversely, the data indicating that NMDAR hypomorphs are defective for training dependent increases in ERK activity, while elav/dNR1^N63IQ flies are not. These data fit a model in which there may be two equally important requirements for NMDARs in regulating LTM-dependent transcription. First, during correlated, LTM-inducing stimulation, a large Ca²⁺ influx through channels, including NMDARs, may be required to activate kinases, including ERK, necessary to activate CREB. dNR1 hypomorphs are defective for this process. However, a second and equally important requirement for NMDARs may be to inhibit low amounts of Ca²⁺ influx during uncorrelated activity to maintain the intracellular environment in a state conducive to CREB-dependent transcription. Mg²⁺ block is required for this process (Miyashia, 2012).

Although it is unclear what types of uncorrelated activity are suppressed by Mg²⁺ block, one type may be spontaneous, action potential (AP)-independent, single vesicle release events (referred to as 'minis'). Supporting this idea, an increase in dCREB2-b was observed in cultured wild-type brains in Mg²⁺-free medium in the presence of TTX, which suppresses AP-dependent vesicle releases but does not affect minis. In addition, a significant increase was observed in cytosolic Ca²⁺, [Ca²⁺]i, in response to 1 mM NMDA in the presence of extracellular Mg²⁺ in neurons from elav/dNR1^N63IQ pupae. In neurons from transgenic control and wild-type pupae, which have an intact Mg²⁺ block mechanism, 1 mM NMDA does not cause Ca²⁺ influx and membrane depolarization. The concentration of glutamate released by minis is on the order of 1 mM at the synaptic cleft, suggesting that an increase in frequency of mini-induced Ca²⁺ influx due to decreased Mg²⁺ block may contribute to the increase in dCREB2-b in elav/ dNR1^N63IQ flies (Miyashia, 2012).

Correlated, AP-mediated NMDAR activity has been proposed to facilitate dCREB2-dependent gene expression by increasing activity of a dCREB2 activator. The present study suggests that, conversely, Mg²⁺ block functions to inhibit uncorrelated activity, including mini-dependent Ca²⁺ influx through NMDARs, which would otherwise cause increased dCREB2-b expression and decreased LTM. Other studies have also suggested opposing roles of AP-mediated transmitter release and minis. For activity-dependent dendritic protein synthesis, local protein synthesis is stimulated by AP-mediated activity and inhibited by mini activity. In the case of NMDARs, the opposing role of low Ca²⁺ influx in inhibiting CREB activity must be suppressed by Mg²⁺ block for proper LTM formation (Miyashia, 2012).

Protein Interactions

Functional Expression of Drosophila NMDARs in Xenopus Oocytes or Drosophila S2 Cells

To determine whether the cloned dNR1 and dNR2 subunits associate to form functional ionotropic receptor channels, they were coexpressed in Xenopus oocytes and the resulting electrophysiological properties were examined. Coexpression of dNR1 and dNR2-2 induced robust NMDA-selective responses, whereas dNR2-1 in combination with dNR1 induced no NMDA-dependent responses in oocytes, suggestive of some functional difference between the two dNR2 isoforms. Coexpression of dNR1 and dNR2-3 has not been tested yet. The oocytes, expressing both dNR1 and dNR2-2, exhibited significant inward currents upon application of NMDA but not AMPA, and the NMDA-activated responses were concentration dependent. This suggests that dNR1 and dNR2 can form a functional ion channel in oocytes, which selectively responds to NMDA. Mammalian NMDA receptors are modulated by glycine (Kleckner, 1998). This also is the case for fly NMDA receptors, although application of glutamate in the presence of glycine appears much less effective than NMDA alone, which may reflect the facts that the relevant structural domains for glycine and glutamate binding are not completely conserved in dNR1 and dNR2 or that residual glycine may alter the response in this heterologous system. Mammalian NMDA receptors are activated by L-aspartate as well as glutamate (Patneau, 1990). Consistent with this observation, fly NMDA receptors are activated by various concentrations of aspartate. When expressed in oocytes, however, conductance through fly NMDA receptors is not voltage dependent. Consequently, dNR1 and dNR2 was also coexpressed in Drosophila S2 cells, thereby revealing a voltage-dependent conductance that is blocked by external Mg²⁺. Thus, this eletrophysiological profile of coexpressed dNR1 and dNR2 reveals most of the distinguishing characteristics of vertebrate NMDARs (Xia, 2005).

DlgS97/SAP97, a neuronal isoform of discs large, regulates ethanol tolerance

From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, intolerant (intol), a novel allele of discs large 1 (dlg1) was identified. Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly's brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol's sedative/hypnotic effects. It is proposed that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity (Maiya, 2012).

DEVELOPMENTAL BIOLOGY

Adult

To examine expression of the dNR1 protein, a rabbit anti-dNR1 polyclonal antibody was generated. The antibody recognized a single protein of the appropriate size on Western blot. dNR1 seems to be weakly expressed throughout the entire brain (see dNR1 and dNR2 protein expression in the adult brain). Higher expression levels were observed in some scattered cell bodies and part of their fibers, including those from several pairs of DPM (dorsal-posterior-medial) neurons surrounding the calyx, DAL (dorsal-anterior-lateral) and DPL (dorsal-posterior-lateral) neurons in the lateral protocerebrum (LP), VAL (ventral-anterior-lateral) neurons in the anterior protocerebrum, and two pairs of VP (ventral-posterior) neurons in the posterior protocerebrum. Many cell bodies in the optic lobes also were labeled preferentially. Notably, punctuate staining was detected in many brain regions including the superior medial protocerebrum, suggesting synaptic localization of dNR1 (Xia, 2005).

The anti-dNR1 antibody does not preferentially label MB neurons. This is notable because MBs are critically required for olfactory learning. Instead, preferential dNR1 expression was detected in 12 pairs of cell bodies surrounding the MB calyx. Interestingly, a pair of DPM2 (dorsal-paired-medial 2) neurons are located just next to the previously identified DPM neurons in which no dNR1 expression is detectable. The DPM neurons innervate all the MB lobes and appear involved in early memory. Three additional pairs of DPM3 neurons with cell bodies smaller than DPM2 also showed strong immunolabeling. The spatial distributions of these neurons are highly symmetrical. Four other DPM4 neurons are located medially to the MB calyx and send descending fibers along a common tract. DPM4 neurons are clustered together in some flies but scattered in others. Another two pairs of neurons, DPM5 and DPL (dorsal-posterior-lateral), are located above the MB calyx. They appear to project descending fibers together with DPM4 neurons . The cell bodies of the VP (ventral-posterior) neurons are located beneath the MB calyx. DAL (dorsal-anterior-lateral) neurons are located in the LP region. LP receives extensive olfactory projections through the antennalglomerular tract of the antennal lobe, which itself receives olfactory input from antennae. The function of LP in olfaction and olfactory learning is largely unknown. dNR1 appears only weakly expressed in antennal lobes and central complex (Xia, 2005).

One of the mouse monoclonal anti-dNR2 antibodies allowed evaluation of the distribution of dNR2 proteins in adult brain. This antibody labels two bands with molecular weights close to the deduced sizes of dNR2 proteins. Similarly to dNR1, weak expression of dNR2 was detected in most, if not all, brain neurons. Again, preferential expression was found in several pairs of large neurons. Notably, dNR1 and dNR2 colocalized in four cell bodies of DPM4 neurons. Both proteins also colocalized in many synapse-like punctuate structures including those along the fibers of DPM4 neurons. Nevertheless, not all dNR1-positive neurons appear to express dNR2 at equivalent levels or verse visa. dNR2 is strongly expressed in a pair of DAL2 neurons and two pairs of VAL2 neurons, for instance, whereas dNR1 is strongly expressed in DAL and VAL neurons. These observations suggest that NR1 and NR2 may be regulated differentially during development or by experience or that these subunits may partner in vivo with other unknown subunits to form functional NMDARs (Xia, 2005).

The 3D staining patterns of dNR1 and dNR2 were superimposed into a volume model of adult fly brain to analyze NR-positive fibers in more detail. VAL appears to be the only neurons sending dNR1-positive projections to the front of contralateral MB calyx. Remarkably, all other NR-positive neurons do not appear to send projections to MBs. DPL and DPM5 are descending neurons and project in parallel with DPM4 neurons to the ventral-posterior ipsilateral protecerebrum and then extend anteriorly. The NR-positive fibers from other neurons surrounding the MB calyx do not enter the calyx or lobes of MBs. This, however, does not exclude the possibility that they may contact MBs through presynaptic fibers where no dNR proteins are expressed. DAL projects ascending fibers toward the superior medial protocerebrum with dNR1 protein distributed at the cell bodies and synapse-like puncta along its fibers. Thus, at least in DAL neurons, dNR1 appears to localize both pre- and postsynaptically (Xia, 2005).

Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila

The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes (see Mushroom body is a quadruple structure). In young adult Drosophila, the last-born KCs extend their processes in the alpha/beta lobes as a thin core (alpha/beta cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the alpha/beta cores (alpha/betac) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. To address the role of Glu signaling in the development and maturation of the MB, the time course of Glu immunoreactivity was compared with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. It was observed that last-born alpha/betac KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the alpha/betac neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the alpha/beta lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the alpha/betac neurons. In contrast, evidence is provided that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB alpha/betac immediately and for a short time after eclosion. The distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the alpha/betac KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life (Sinakevitch, 2010).

In Drosophila and other arthropods, Glu is well characterized as the excitatory neurotransmitter of the neuromuscular junction. However, this amino acid has important signaling functions in the Drosophila brain as well. The Drosophila genome was predicted to encode 30 iGluR subtypes, including 3 AMPA- and 15 kainate-like, 2 NMDA-like, 4 δ-like and 6 divergent receptors. For now, the best characterized of these are the postsynaptic iGluRs expressed at the neuromuscular junction. Drosophila NMDA-like receptors are expressed in the central nervous system and have been implicated in learning and memory and locomotor control. The Drosophila genome encodes a single functional mGluR, DmGluRA, an ortholog of vertebrate group II mGluRs (Parmentier, 1996). This mGluR is presynaptic and expressed at the periphery of the active zones at the glutamatergic neuromuscular junctions, where it modulates both synapse excitability and fine structure (Bogdanik, 2004). DmGluRA is also expressed in the brain, in particular in lateral clock neurons, where it regulates circadian locomotor behavior (Hamasaka, 2007; Sinakevitch, 2010 and references therein).

The mushroom bodies (MBs) are paired centers located in the protocerebrum of Drosophila and other dicondylic insects that play essential roles in olfactory learning and memory and other brain functions, such as the control of locomotor activity, courtship behavior, courtship conditioning, visual context generalization, and sleep. The intrinsic structure of the MB is provided by the Kenyon cells (KCs), which have their cell bodies in the brain cortex and their dendrites in the MB calyx, where they receive input from the antennal lobe projection neurons. Axon-like processes of KCs project anteriorly and ventrally in the peduncle to form the vertical and medial lobes, which are subdivided into discrete parallel entities, the vertical α, α' and the medial β, β' and γ lobes. In addition to the KCs, there are other MB intrinsic neurons and several classes of MB extrinsic neurons that connect the MB to other areas of the brain neuropil. Emerging evidence suggests that different subtypes of MB KCs may be involved in distinct mechanisms of memory formation due to their connections to different MB extrinsic neurons (Sinakevitch, 2010).

Developmental studies have shown that the KCs are produced in each hemisphere of the brain by the division of four neuroblasts born early during the embryonic stage. The division of these neuroblasts sequentially produces the three morphologically and spatially distinct subtypes of KCs: γ, α'/β' and α/β. The γ neurons are generated up to the mid-third instar larval stage; they form the larval dorsal and medial lobe. The next KC subtype to be generated is the α'/β' neuron, which continues to be produced until puparium formation. Lastly, the α/β neurons are generated from the time of puparium formation until adult eclosion. In the α/β lobes, the KCs are organized in concentric layers. The youngest axon-like processes situated in the inner layer of the lobes are successively displaced outwards as they differentiate and newer α/β processes are added to the structure from the most recently born KCs (Kurusu, 2002). This volume of the α/β lobes into which grow the last-born axons contains densely packed and extremely thin fibers that are rich in actin filaments. This subset of processes has been named the α/β core (α/βc) (Sinakevitch, 2010).

An increased response to mGluR activation may play a prominent role in the fragile X syndrome (FXS), the most common form of inherited mental retardation and the predominant cause of autism. Mutations in dFmr1, the Drosophila homologue of the gene implicated in FXS, lead both to learning deficits and altered development of the MB, of which the most common feature is a failure of β lobes to stop at the brain midline. These behavioral and developmental phenotypes can be successfully rescued in Drosophila by treatment with mGluR antagonists (McBride, 2005), implicating Glu in the pathology, as is the case in mammalian models. Recent studies showed that dFmr1 interacts with DmGluRA in the regulation of synaptic architecture and excitability at glutamatergic synapses (Gatto, 2008; Repicky, 2009). However, until now the precise role of Glu and mGluRs in FXS and MB development has remained obscure (Sinakevitch, 2010).

This study presents evidence that Glu and its receptor DmGluRA are directly involved in construction of the MB neural circuits. Previous studies suggested that the Drosophila last-born α/βc KCs are immunoreactive to anti-Glu antibodies. The present study shows that these neurons express a high level of Glu-like immunoreactivity in newly eclosed adult flies. Interestingly, newborn KCs in late larval and pupal stages also appear to express as a rule a high level of Glu. To understand further the role and fate of Glu during KC maturation, the dynamics of Glu, DmGluRA and other Glu signaling-associated proteins were analyzed in the MB of young adult Drosophila from the time of their eclosion until 10 days post-eclosion. The results indicate that a transient Glu release likely regulates functional maturation of newborn KCs by a paracrine action during Drosophila post-embryonic development and the first hours after adult eclosion (Sinakevitch, 2010).

One intriguing question in neuroscience is how newborn neurons establish a functional network during their period of growth and maturation. This work describes a study of the late maturation of a subset of the α/β intrinsic MB KCs, the α/βc neurons, during a short period after adult eclosion. Glu-like immunoreactivity has been observed in the ingrowth lamina of the cockroach MB, which contains axons of the youngest KCs. Similarly in Drosophila, Glu accumulates in the α/βc, which contains newly generated neurons, whereas taurine-expressing neurons were found in the outer α/βc and aspartate-expressing neurons in the rest of the α/β lobes. It has been shown in vertebrates that Glu can have a strong influence on cone motility and induce rapid filopodia protrusion from hippocampal neurites or cultured astrocytes. In the present study an extensive analysis was performed of the distribution of various glutamatergic markers in the MBs of young adult Drosophila. The results suggest that the α/βc neurons are not simply glutamatergic. Rather, the evidence provided in this study indicates that these newborn KCs may transiently use Glu as a paracrine agent to favor interactions with glial cell processes and become mature neurons forming functional circuits (Sinakevitch, 2010).

Although the last-born α/βc KCs show a high level of Glu immunoreactivity a few hours prior and after adult eclosion, Glu immunostaining is dramatically reduced in these cells 24 hours after eclosion and is entirely absent a few days later. Disappearance of this signal could result from the release or intracellular metabolism of this amino acid. Similarly, it was observed in cockroach MBs that newborn KCs loose Glu immunoreactivity when they become mature and establish contacts with extrinsic neurons. This study also presents the first evidence that Glu transiently accumulates at a high level in developing newborn KCs of Drosophila in late larva and during pupal stages. Therefore, transient Glu expression could correlate with KC growth and maturation not only in the α/βc around eclosion time but also in other lobes during earlier stages of MB development (Sinakevitch, 2010).

Three subtypes of vesicular Glu transporters (VGluTs) have been identified in the mammalian nervous system with similar Glu transport functionality. Two of these (VGluT1 and VGluT2) present complementary distribution in central glutamatergic neurons. The third isoform, VGluT3, appears to be primarily expressed in neurons that release another transmitter (serotonin, dopamine, acetylcholine or GABA), where it may be required for efficient synaptic transmission. In the present study, neither the α/βc neurons nor any other intrinsic MB KCs were found to express the Drosophila vesicular transporter DVGluT. This may indicate that the Glu that is accumulated in the inner α/βc neurons is not stored in synaptic vesicles. However, the possibility cannot be excluded that these cells express another vesicular Glu transporter not yet identified in Drosophila. DVGluT immunoreactivity was observed in the MBs, particularly in the γ lobe and spur region and in the α lobes, but the punctuate labeling and localization suggest that this distribution corresponds to glutamatergic synapses belonging to extrinsic neurons (Sinakevitch, 2010).

Can the Glu transiently stored in the newborn MB neurons be released into the extracellular space? In the absence of DVGluT or another similar transporter, this could involve a non-vesicular release of Glu. Non-conventional release of Glu from immature neurons has been demonstrated in the developing rat hippocampus where Glu release exerts a paracrine action that seems to particularly affect the migration of neighboring maturing neurons. To address this question indirectly, the presence in the MB of other proteins known to be involved in the recycling and degradation of Glu at glutamergic synapses was sought (Sinakevitch, 2010).

An important role of glial cells is to capture Glu released from the synapse with specific transporters and then convert Glu to glutamine with GS. The only Drosophila high-affinity Glu transporter, dEAAT1, is expressed in subtypes of glial cells and is associated with Glu-release sites. GS2 is similarly expressed in glial cells in the Drosophila nervous system. This study shows that glial cells expressing dEAAT1 and GS surround the Drosophila MB lobe neuropiles, closely enwrapping the α/β lobes, thus isolating them from other lobes, and sending a mesh-like system of extensions inside these lobes. Enwrapping and invading of the MB β lobes by glia was also observed to occur in cockroach MBs, where glial cells are implicated in the removal of degenerating transient KC processes that occur during their establishment of mature connections with extrinsic cell dendrites. The high levels of glial dEAAT1 and GS within the Drosophila MB lobes suggest that this neuropil is tightly cordoned off from other parts of the brain and regulates the extracellular Glu level between the axons (Sinakevitch, 2010).

These data show that GS expression is highly dynamic in the MB during the first day of adult life, suggesting that glial cells play a role in establishing the MB's functional network. During the first hour after eclosion, the meshwork of glial processes expressing Glu signaling-associated molecules (GS and dEAAT1) is not present in the inner α/βc region, but within 24 hours this area becomes covered by glial extensions. These glial elements are possibly guided towards the α/βc area by the gradient of Glu released by the last born KCs. Glia could be involved in reducing Glu concentration in this area and play a role in axonal guidance and final maturation of KCs. Evidence that Glu transporters are required for coordinated brain development has been previously reported for mice: the absence of two glial Glu transporters resulted in excess of extracellular Glu and abnormal formation of the neocortex (Sinakevitch, 2010).

Assuming Glu is released by the newborn MB neurons, it has to interact with specific receptors. Therefore, the expression was sought of Glu receptors in MB neuropiles of young adult Drosophila, particularly those receptors that are likely to regulate neuronal growth and maturation through second-messenger pathways. Once activated by simultaneous Glu binding and membrane depolarization, the NMDAR channel allows calcium influx into the postsynaptic cell, where this ion triggers a cascade of biochemical events resulting in synaptic maturation and plasticity. Available antibodies against the constitutive dNR1 subunit of the Drosophila NMDAR were used. Immediately after eclosion, many processes in the MB neuropil were found to be dNR1-positive, with the exception of the α/βc neurons. The Glu released from either these α/βc neurons, or the surrounding glial cells, or extrinsic MB glutamatergic neurons may activate these NMDAR receptors. Thus, a widespread localization of NMDAR characterizes the MB immediately after eclosion, at the beginning of adult life when the MB is expected to receive the least inputs from sensory interneurons. Subsequently, with increasing sensory data being received and relayed to projection neurons, there is a dramatic and concomitant restructuring of NMDAR signaling: the majority of MB neurons no longer express these receptors. It is only those neurons that receive constant glutamatergic signaling that still address the dNR1 subunit in the vicinity of glutamatergic synapses expressing DVGluT. This occurs in particular within the spur region of the MB and the lateral horn. Such developmentally related regulation of NMDAR expression in the MBs of young adult flies may relate to adaptations of synaptic activity in response to sensory experience (Sinakevitch, 2010).

mGluRs are neuromodulatory G-protein-coupled receptors that are involved in many aspects of brain physiology, including neuronal development, synaptic plasticity, and neurological diseases. Whereas eight distinct mGluRs are present in the mammalian genome, a single functional mGluR is expressed in Drosophila, DmGluRA. The fly mGluR is structurally and pharmacologically closer to the mammalian group II mGluRs, which are mainly presynaptic receptors negatively coupled to adenylate cyclase. Attempts to locate DmGluRA with the commercially available monoclonal antibody 7G11 were not successful because the antibody produced by the hybridoma clone recently lost its binding specificity. To monitor DmGluRA distribution, a new GAL4 line was used that carries an enhancer trap insertion close to the mGluR start site of transcription, keeping in mind that expression of this GAL4 reporter may, in part, differ from the mGluR pattern. Strikingly, the DmGluRA-GAL4 line was found to express GFP selectively in the Glu-accumulating α/βc KCs of newly eclosed adult flies. This is in contrast to commonly used MB GAL4 driver lines (17d-, c739- and 201Y-GAL4) that do not express GFP in these neurons immediately after eclosion. Ten days later, the GFP staining in the DmGluRA-GAL4 line appeared strongly reduced in the α/βc; in contrast, the MB drivers now expressed GFP in these neurons (Sinakevitch, 2010).

Because the GAL4 reporter method reveals whole neurons, it could not be determined where the receptor is addressed restrictively in cell bodies, dendrites or axons. A previous study performed with an active lot of 7G11 antibody indicated that DmGluRA is present in nearly all neuropiles of the mature adult fly brain, including the MB calyces, but not in the MB lobes (Devaud, 2008). However, thas study did not report on the localization of DmGluRA in newly eclosed Drosophila. Further work is required to precisely locate the subcellular localization of DmGluRA in the newborn α/βc neurons, either with a new antibody or a DmGluRA-GFP fusion gene. The source of Glu binding to this mGluR receptor may be the neighboring glial cells or newborn KCs themselves, or both. Through activation of these receptors, Glu is likely to have a transient paracrine action on the α/βc neurons during the first day after eclosion that could be required for dendrite growth or synaptic maturation (Sinakevitch, 2010).

Although the α/βc KCs represent a minor part of the α/β lobe neurons, the maturation of these cells appears to be essential for proper MB functioning. Selective expression of the rutabaga (rut)-encoded adenylate cyclase in the α/βc neurons with 17d-GAL4 was shown to partially restore olfactory learning and memory in 2- to 5-day-old rut mutant flies. In contrast, no rescue of the rut defect was observed with c739-GAL4, which expresses in more peripheral α/β neurons at this stage. Therefore, the network involved in olfactory learning and memory apparently requires the α/βc neurons and is already functional in 2- to 5-day-old flies. Furthermore, treatment with mGluR antagonists restored courtship behavior, memory deficits and MB structural defects in DFmr1 mutants, a Drosophila model of FXS. These positive effects are even stronger when the pharmacological treatment is applied both during larval development and after eclosion. This suggests that these behavioral defects relate to an abnormally high level or prolonged duration of DmGluRA expression in the α/βc neurons of DFmr1 mutants. Further study should determine the distribution of Glu and DmGluRA during MB development in Drosophila FXS models (Sinakevitch, 2010).

The ubiquitin-proteasome system is one of the major conserved cellular pathways controlling protein turnover in eukaryotic cells. Substrate protein ubiquitination plays important roles in neuronal differentiation, axonal targeting, synapse formation and plasticity. In addition to strong Glu immunolabeling in the inner α/βc KCs, a high level of anti-ubiquitin immunoreactivity was also observed in these neurons immediately after eclosion. Such a high staining level was no longer detected in 10-day-old flies. In contrast, the spur region of the MB showed a constant high ubiquitin immunoreactivity that did not change with the age of the animal. This could suggest that synaptic plasticity is particularly active in this MB area (Sinakevitch, 2010).

Similarly, labeling of the cockroach MB β lobe with anti-ubiquitin showed, at specific stages in each developmental instar, as well as at an early adult stage, consistent staining of newly generated KC axons. Anti-ubiquitin also labeled the extending transiently Glu-immunoreactive collateral processes from developing KCs in the ingrowth zone, the hemimetabolous homologue of Drosophila's core KCs. This study showed that ubiquitin expression precedes degeneration of these collaterals and their subsequent removal by scavenging glial cells. Glu receptors can be endocytosed by an ubiquitin-dependent mechanism. The down-regulation of Glu and its receptor protein, possibly mediated by ubiquitin, thus appear to be important steps in the maturation and differentiation of the α/βc KCs (Sinakevitch, 2010).

In conclusion the present study suggests that the Glu accumulated in the α/βc KCs of young adult Drosophila is used for cell growth and maturation rather than for neurotransmission. The distribution and dynamics of glutamatergic markers indicates that Glu released from newborn KCs can bind to intrinsic mGluRs in the α/β cores and to NMDARs in the rest of the MB neuropil before being captured and metabolized by surrounding glial cells. As an autocrine or paracrine agent, Glu is likely to play a role in pathway finding within the lobe, namely, interactions between maturing KCs and extrinsic neuron dendrites, guidance of glial cell outgrowth and glial process targets into and around the relevant lobes, and maturation of synaptic networks required for a functional MB. Further study of the paracrine function of Glu in wild-type flies and in the Drosophila FXS model may shed light on similar actions of this neurotransmitter in the developing human brain in normal and pathological conditions (Sinakevitch, 2010).

EFFECTS OF MUTATION

Mutations of dNR1 disrupt learning

The dNR1 gene consists of 15 exons scanning more than 24 kb of genomic DNA. The 5′ end overlaps with Itp-r83A, the fly homolog of an inositol 1,4,5-tris-phosphate receptor. Flies homozygous for an F-element insertion in the third intron of dNR1 are subviable and female-sterile. Two independent EP element insertions also lie in dNR1 or nearby. EP3511 inserts in the first intron of the dNR1 gene, 718 bp upstream of the start codon in exon 2. EP331 is inserted 425 bp downstream of the 3′ end of the dNR1 transcription unit. Expression levels of dNR1 protein are reduced but not eliminated in homozygous EP3511/EP3511 or EP331/EP331 flies, indicating that both EP insertions represent hypomorphic mutations of dNR1. EP3511/EP3511 or EP331/EP331 homozygotes are viable, which allowed evaluation olfactory learning. Compared to wild-type flies, learning was reduced in both homozygotes (Xia, 2005).

The learning defects of EP3511 or EP331 mutants were rescued by cosmids containing genomic DNA from the dNR1 region. Cosmid-A contains the full-length Itp-r83A coding sequence and upstream elements that include only partial coding sequence of dNR1. Conversely, Cosmid-B and Cosmid-C contain all of the dNR1 transcription unit and only part of Itp-r83A. Cosmid-A, but not Cosmid-B or Cosmid-C, rescues the lethality associated with two different mutations of Itp-r83A, whereas Cosmid-B and Cosmid-C, but not Cosmid-A, rescued the learning defect of the EP3511 and EP331 mutants. These results establish that the learning defects of the EP mutants are due to disruption of the dNR1 gene not the Itp-r83A gene (Xia, 2005).

Acute disruption of dNR1 via an anti-dNR1 mRNA produces a learning defect

EP331 also allowed the use the EP-element to control the expression of dNR1 conditionally. The EP element in EP331 flies is inserted downstream of, and in an opposite orientation to, the transcription start site of dNR1. When combined with a GAL4 driver, this EP element yields an antisense transcript of dNR1. In transheterozygous EP331/+, hs-GAL4/+ flies, an anti-dNR1 message was induced by heat shock and was still detected 15 hr later, leading to a significant reduction in dNR1 protein. This antisense message was also detected before heat shock in EP331/+, hs-GAL4/+ flies but absent in heterozygous EP331/+ flies, suggesting some leaky expression of hs-GAL4 was driving low-level expression of anti-dNR1. This leaky expression did not produce any measurable effect on NR1 protein levels from Western blot analysis (Xia, 2005).

The disruption of dNR1 in EP331/+, hs-GAL4/+ flies was further confirmed with immunohistochemistry. Anti-dNR1 immunostaining was diminished throughout the entire brain after heat shock as compared with no heat shock. This reduction in dNR1 was quantified in a pair of dorsal-anterior-lateral (DAL) and a pair of ventral-anterior-lateral (VAL) neurons, where the protein is expressed at high levels. In both DAL and VAL neurons, the immunofluorescence intensity was reduced significantly 15 hr after heat shock (Xia, 2005).

Accordingly, learning was severely disrupted 15 hr after heat shock. In contrast, learning was disrupted only mildly in EP331/+, hs-GAL4/+ flies in the absence of heat shock. This mild disruptive effect is consistent with the observation that hs-GAL4 yields some leaky expression of anti-dNR1 message through development, though a concommitant reduction in NR1 protein was not detected. Alternatively, this transgenic line might harbor slight, nonspecific differences in genetic background (Xia, 2005).

Acute disruption of dNR1 abolishes long-term memory

Whether dNR1 was required for long-lasting memory produced by extended training was evaluated. EP331/+, hs-GAL4/+ flies were subjected to spaced or massed training 15 hr after heat shock and then tested for 1-day memory. In the absence of heat shock, 1-day memory after both spaced and massed training was normal. When trained 15 hr after heat shock, 1-day memory after massed training was normal, whereas that after spaced training was significantly reduced. Typically, 1-day memory after spaced training is composed of 50% LTM and 50% ARM (Anesthesia-Resistant Memory), and LTM specifically is disrupted in transgenic flies inducibly overexpressing CREB repressor. 1-day memory after massed training, in contrast, is composed only of ARM. Accordingly, these results suggest that ARM is normal and LTM is completely abolished in EP331/+, hs-GAL4/+ flies after acute disruption of dNR1. The observation that 1-day memory after massed training was normal also suggested that extended training might overcome the learning defect (after one training session) observed for EP331/+, hs-GAL4/+ flies subjected to heat shock. Indeed, this was the case for both spaced and massed training (Xia, 2005).

A modified massed training protocol was used, in which flies sat in the training chamber for 150 min before training began. With this protocol, massed training ends at the same time as spaced training, but 1-day memory after massed training is slightly higher than that after the standard protocol, which does not include pretraining exposure to the training chamber. Hence, the above experiments were repeated with the original massed training protocol with only heat-shocked wild-type and EP331/+, hs-GAL4/+ flies. Here again, 1-day memory after massed training was normal, whereas that after spaced training was disrupted (Xia, 2005).

Disruption of dNR1 does not affect sensorimotor responses to odors or shock

Although dNR1 was expressed throughout the adult brain and especially also at the lateral protocerebrum (LP), sensorimotor responses to the odors and footshock stimuli were not affected in transheterozygous EP331/+, hs-GAL4/+ flies before or after heat shock. Homozygous EP3511/EP3511 and EP331/EP331 mutants also performed normally to these sensory stimuli (Xia, 2005).

Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body

In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. This study demonstrates an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. The results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila (Wu, 2007).

The predominant view of olfactory memory is that it is processed in the mushroom body. The data demonstrate a role for R4m neurons in the ellipsoid body during LTM consolidation, which now suggests a much broader and more complex neuronal circuitry sub-serving olfactory memory consolidation in Drosophila. Because genetic modulations of NMDARs in the ellipsoid body produce effects that are specific to LTM, components of this complex neural circuitry appear to subserve at least one specific temporal phase (LTM) of memory processing. Consistent and complementary to this notion, consolidation of LTM remains normal when NMDAR function is disrupted in the mushroom body. Thus, the transference of memory from one anatomical location to another as consolidation progresses to LTM appears to occur in Drosophila, as in various other species (Wu, 2007).

Specific disruption of functional NMDARs by dsRNA-mediated knockdown of either dNR2 or dNR1 in the R4m neurons of the ellipsoid body specifically abolishes protein synthesis-dependent LTM, suggesting that the ellipsoid body is important during memory consolidation. NMDAR function is physiological rather than developmental, as induction of dsRNA transgenes in adults was sufficient to abolish LTM. This role for NMDARs is also specific for memory phase (LTM) and brain region (ellipsoid body). Initial learning and early memories were not affected when NMDARs were knocked down in the ellipsoid body, and LTM was not affected when NMDARs were knocked down in the mushroom body. These results, together with recent observations that LTM formation may require neuronal activity from the vertical lobes of the mushroom body and correlates with the appearance of a Fas II-immunoreactive asymmetrical body near the central complex, support a broader neuroanatomical circuitry involving both the mushroom body and ellipsoid body that subserves olfactory memory consolidation (Wu, 2007).

It was also postulated that NMDARs in the ellipsoid body are involved with LTM consolidation and storage, but not retrieval. Disruption of NMDAR function during training, but not during testing, severely diminished LTM, excluding the possibility that memory retrieval was affected. Reversal memory was stronger when NMDARs were disrupted during an initial spaced training session compared with disruption during reversal training, directly demonstrating these flies' ability to retrieve memory. That retrieval is normal and that consolidation and storage have failed is consistent with an initial study and with various studies of mammals (Wu, 2007).

Blocking synaptic output from the mushroom body, but not from the ellipsoid body, during training and in the first 6 h after training abolished consolidation of LTM, suggesting that consolidation of LTM occurs downstream of the mushroom body (or possibly in the mushroom body if the efferents from the ellipsoid body were to project back to the mushroom body). In contrast, blocking synaptic output from the ellipsoid body or mushroom body disrupted retrieval, but not acquisition and consolidation. This result is functionally analogous to STM, which is formed in or upstream of the mushroom body, probably without the involvement of extrinsic neurons or structures, and synaptic output from the mushroom body is required specifically for its retrieval, but not its acquisition. Taken together, these observations support a model where memory is first acquired in the mushroom body and is then transferred to the ellipsoid body for storage during memory consolidation, in agreement with various observations from other species where memory transfer from one brain region to another may occur. Combined with other recent studies, it is proposed (1) that acquisition involves an initial association of an odor and shock in and/or upstream of the mushroom body, (2) that STM resides in the mushroom body, (3) that middle-term memory (MTM) is acquired in mushroom body α/β neurons and persists there via recurrent activity involving the mushroom body α′/β′ DPM (dorsal-paired medial) neurons and the mushroom body α/β neurons themselves, (4) that LTM consolidation involves the transference of memory to the ellipsoid body and (5) that LTM retrieval requires neural activity output from the ellipsoid body and from mushroom body neurons. Therefore, the results support a broader neuroanatomical circuitry involving both the mushroom body and ellipsoid body that subserves memory processing and retrieval (Wu, 2007).

Consolidation is the progressive stabilization of memory from a short, labile form to a long-lasting, stable form. Memory consolidation commonly refers to two types of processes, early and late consolidation. Early (or cellular or local) consolidation is accomplished within the first few minutes to hours after learning and occurs in all of the species studied to date. This relatively fast type of consolidation takes place in local nodes in the neuronal circuit(s) and depends on cross talk among synapses, somata and nuclei. Late consolidation, which so far has only been demonstrated in humans and mammals, takes much longer and involves multiple brain systems. Late consolidation is initiated in parallel to, or as a consequence of, early consolidation, and is characterized by much slower temporal kinetics. Recent molecular and cellular studies in rodents have shown that a memory initially depends on the hippocampus, but eventually becomes independent of hippocampal function and may be consolidated into neocortical circuits. The existence of multiple memory systems has not clearly been demonstrated in any invertebrate model system. This demonstration that NMDARs are specifically required in the ellipsoid body rather than in the mushroom body during LTM consolidation shows that memory consolidation is a systems-level phenomenon in Drosophila (Wu, 2007).

It has recently been shown that LTM requires branch-specific neural activity and CREB in α/β mushroom body neurons. Notably, the only GAL4 driver used in that study, C739, is not specific to the mushroom body; it also labels some ellipsoid body neurons that appear to be different from the NMDAR-positive R4m neurons. Consistent with this observation, C739-targeted knockdown of functional NMDARs left LTM intact. When neural activity was silenced in UAS-shi^ts1/+; C739/+ flies, however, memory retrieval was disrupted. Considering that the cyclic AMP–dependent neuronal activity in the mushroom body is required for retrieval of LTM, these results raise the interesting possibility that neural activity in the mushroom body α lobes may be correlated with memory retrieval rather than memory consolidation (Wu, 2007).

An intriguing difference exists in systems-level memory processing between Drosophila and rodents. Though LTM may eventually recruit the ellipsoid body, the mushroom body appears to be crucial for both memory consolidation and retrieval in Drosophila. In contrast, the hippocampus is required for consolidation, but not for retrieval, of long-lasting memories transferred to cortical systems. This difference may highlight an unusual aspect of olfactory memory in Drosophila. It was recently have found that the encoding of odor identity and odor intensity is experience-dependent and mushroom body–dependent. Thus, the mushroom body appears to be central to olfactory discrimination as a perceptual task, providing a possible explanation as to why the mushroom body has to be involved with retrieval of LTM (Wu, 2007).

The mushroom body is clearly involved in olfactory learning, memory consolidation and retrieval. Nevertheless, consolidation of LTM occurred normally even when the function of NMDARs in the mushroom body was disrupted. MTM depends on NMDAR function in the mushroom body and on amnesiac (amn)-encoded neuropeptides expressed in dorsal-paired medial (DPM) neurons. DPM neurons are extrinsic to the mushroom body, but nevertheless send extensive arborizations to all the mushroom body lobes. Synaptic output from DPM neurons is required for the persistence of MTM, but does not appear to be involved in its acquisition and retrieval. Consistently, MTM is proposed to be formed in the mushroom body α/β neurons, and blocking mushroom body output diminishes the retrieval of MTM. Taken together, these observations suggest that the involvement of the mushroom body in LTM consolidation may be through an NMDAR-independent pathway, and that the mushroom body and ellipsoid body may participate independently in distinct temporal stages of memory consolidation, a neurobehavioral phenomena that has been reported in other species (Wu, 2007).

Understanding how the NMDAR-containing protein complexes are independently involved, at the cellular level, with different memory phases in distinct brain regions, and how these distinct anatomical sites communicate with each other to yield adaptive behavior from prior experience will be of particular importance in the future (Wu, 2007).

Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling

The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).

This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).

Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of K_ir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).

Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca²⁺, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).

This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).

In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).

The NMDA receptor promotes sleep in the fruit fly, Drosophila melanogaster

A short sleeper mutant of Drosophila, fumin (fmn),was previously identified as mutation in the Dopamine transporter gene. In aversive olfactory learning tasks, fmn mutants demonstrate defective memory retention, which suggests an association between sleep and memory. In an attempt to discover additional sleep related genes in Drosophila, this study carried out a microarray analysis comparing mRNA expression inheads of fmn and control flies and found that 563 genes were differentially expressed. Next, using the pan-neuronal Gal4driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, a functional screen was performed. It was found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1)(also known as dNR1) reduced sleep. The NMDA receptor (NMDAR) plays an important role in learning and memory both in Drosophilaand mammals. The application of the NMDAR antagonist, MK-801, reduced sleep in control flies, but not in fmn. These results suggest that NMDAR promotes sleep regulation in Drosophila (Tomita, 2015).

EVOLUTIONARY HOMOLOGS

NMDA receptors in C. elegans

The NMDA subtype of glutamate receptor is important for synaptic plasticity and nervous system development and function. Genetic and electrophysiological methods were used to demonstrate that NMR-1, a Caenorhabditis elegans NMDA-type ionotropic glutamate receptor subunit, plays a role in the control of movement and foraging behavior. nmr-1 mutants show a lower probability of switching from forward to backward movement and a reduced ability to navigate a complex environment. Electrical recordings from the interneuron AVA show that NMDA-dependent currents are selectively disrupted in nmr-1 mutants. A slowly desensitizing variant of a non-NMDA receptor can rescue the nmr-1 mutant phenotype. It is proposed that NMDA receptors in C. elegans provide long-lived currents that modulate the frequency of movement reversals during foraging behavior (Brockie, 2001).

The C. elegans polymodal ASH sensory neurons detect mechanical, osmotic, and chemical stimuli and release glutamate to signal avoidance responses. To investigate the mechanisms of this polymodal signaling, the role of postsynaptic glutamate receptors in mediating the response to these distinct stimuli was characterized. By studying the behavioral and electrophysiological properties of worms defective for non-NMDA (GLR-1 and GLR-2) and NMDA (NMR-1) receptor subunits, it has been shown that while the osmotic avoidance response requires both NMDA and non-NMDA receptors, the response to mechanical stimuli only requires non-NMDA receptors. Furthermore, analysis of the EGL-3 proprotein convertase provides additional evidence that polymodal signaling in C. elegans occurs via the differential activation of postsynaptic glutamate receptor subtypes (Mellem, 2002; full text of article).

Nitrous oxide (N₂O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N₂O and VAs may act by distinct mechanisms; N₂O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in C. elegans, whether the action of N₂O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs was tested. Distinct from the action of VAs, N₂O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N₂O, whereas a non-NMDA receptor-null mutant is normally sensitive. The N₂O-resistant nmr-1(null) mutant is not resistant to VAs. Likewise, VA-resistant mutants have wild-type sensitivity to N₂O. Thus, the behavioral effects of N₂O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N₂O is the NMDA receptor (Nagele, 2004; full text of article).

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in C. elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. These results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex (Corrigan, 2005).

Learning and memory are essential processes of both vertebrate and invertebrate nervous systems that allow animals to survive and reproduce. The neurotransmitter glutamate signals via ionotropic glutamate receptors (iGluRs) that have been linked to learning and memory formation; however, the signaling pathways that contribute to these behaviors are still not well understood. A genetic and electrophysiological analysis of learning and memory was undertaken in the nematode Caenorhabditis elegans. This study shows that two genes, nmr-1 and nmr-2, are predicted to encode the subunits of an NMDA-type (NMDAR) iGluR that is necessary for memory retention in C. elegans. nmr-2 was cloned, a deletion mutation was generated in the gene, and it was shown that like nmr-1, nmr-2 is required for in vivo NMDA-gated currents. Using an associative-learning paradigm that pairs starvation with the attractant NaCl, it was also shown that the memory of a learned avoidance response is dependent on NMR-1 and NMR-2 and that expression of NMDARs in a single pair of interneurons is sufficient for normal memory. These results provide new insights into the molecular and cellular mechanisms underlying the memory of a learned event (Kano, 2008).

NMDA receptors in insects

In vertebrates, the NMDA receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. Immunohistochemically glutamatergic nerve terminals have been demonstrated in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibit elevated cytosolic (Ca²⁺), but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg²⁺. Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 microM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg²⁺. Having obtained substantive evidence of functioning NMDAR in insect corpora allata, RTPCR was used to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, the same primers for DNMDAR1 were used to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. These results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution (Chiang, 2002).

In contrast to vertebrates the involvement of glutamate and N-methyl-D-aspartate (NMDA) receptors in brain functions in insects is both poorly understood and somewhat controversial. This study examined the behavioural effects of two noncompetitive NMDA receptor antagonists, memantine (low affinity) and MK-801 (high affinity), on learning and memory in honeybees (Apis mellifera) using the olfactory conditioning of the proboscis extension reflex (PER). Memory deficit was induced by injecting harnessed individuals with a glutamate transporter inhibitor, L-trans-2,4-pyrrolidine dicarboxylate, that impairs long-term (24 h), but not short-term (1 h), memory in honeybees. L-trans-2,4-PDC-induced amnesia is 'rescued' by memantine injected either before training, or before testing, suggesting that memantine restores memory recall rather than memory formation or storage. When injected alone memantine has a mild facilitating effect on memory. The effects of MK-801 are similar to those of L-trans-2,4-PDC. Both pretraining and pretesting injections lead to an impairment of long-term (24 h) memory, but have no effect on short-term (1 h) memory of an olfactory task (Si, 2004).

Structure of NMDA receptors

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn2+ ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. This study identified the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs (Gielen, 2008)

The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production. This study report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. This study also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine. Unexpectedly, it was also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. These observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism (Neame, 2019).

D-Serine Signaling and NMDAR-Mediated Synaptic Plasticity Are Regulated by System A-Type of Glutamine/D-Serine Dual Transporters

D-serine is a physiologic coagonist of NMDA receptors (NMDARs; see Drosophila Nmdar1 and Nmdar2) required for synaptic plasticity, but mechanisms that terminate D-serine signaling are unclear. In particular, the identity of unidirectional plasma membrane transporters that mediate D-serine reuptake has remained elusive. This study reports that D-serine and glutamine share the same neuronal transport system, consisting of the classic system A transporters Slc38a1 and Slc38a2 (see Drosophila CG32081). This study shows that these transporters are not saturated with glutamine in vivo and regulate the extracellular levels of D-serine and NMDAR activity. Glutamine increased the NMDAR-dependent long-term potentiation and the isolated NMDAR potentials at the Schaffer collateral-CA1 synapses, but without affecting basal neurotransmission in male mice. Glutamine did not increase the NMDAR potentials in slices from serine racemase knock-out mice, which are devoid of D-serine, indicating that the effect of glutamine is caused by outcompeting D-serine for a dual glutamine-D-serine transport system. Inhibition of the system A reduced the uptake of D-serine in synaptosomes and neuronal cultures of mice of either sex, while increasing the extracellular D-serine concentration in slices and in vivo by microdialysis. When compared with Slc38a2, the Slc38a1 transporter displayed more favorable kinetics toward the D-enantiomer. Biochemical experiments with synaptosomes from Slc38a1 knock-down mice of either sex further support its role as a D-serine reuptake system. This study identifies the first concentrative and electrogenic transporters mediating D-serine reuptake in vivo. In addition to their classical role in the glutamine-glutamate cycle, system A transporters regulate the synaptic turnover of D-serine and its effects on NMDAR synaptic plasticity (Bodner, 2020).

Alternative Splicing of Presynaptic Neurexins Differentially Controls Postsynaptic NMDA and AMPA Receptor Responses

AMPA- and NMDA-type glutamate receptors mediate distinct postsynaptic signals that differ characteristically among synapses. How postsynaptic AMPA- and NMDA-receptor levels are regulated, however, remains unclear. Using newly generated conditional knockin mice that enable genetic control of neurexin alternative splicing (see Drosophila Neurexin), this study shows that in hippocampal synapses, alternative splicing of presynaptic neurexin-1 at splice site 4 (SS4) dramatically enhanced postsynaptic NMDA-receptor-mediated, but not AMPA-receptor-mediated, synaptic responses without altering synapse density. In contrast, alternative splicing of neurexin-3 at SS4 suppressed AMPA-receptor-mediated, but not NMDA-receptor-mediated, synaptic responses, while alternative splicing of neurexin-2 at SS4 had no effect on NMDA- or AMPA-receptor-mediated responses. Presynaptic overexpression of the neurexin-1beta and neurexin-3beta SS4+ splice variants, but not of their SS4- splice variants, replicated the respective SS4+ knockin phenotypes. Thus, different neurexins perform distinct nonoverlapping functions at hippocampal synapses that are independently regulated by alternative splicing. These functions transsynaptically control NMDA and AMPA receptors, thereby mediating presynaptic control of postsynaptic responses (Dai, 2019).

Presynaptic PTPsigma regulates postsynaptic NMDA receptor function through direct adhesion-independent mechanisms

Synaptic adhesion molecules regulate synapse development and function. However, whether and how presynaptic adhesion molecules regulate postsynaptic NMDAR function (see Drosophila Nmdar1 and Nmdar2) remains largely unclear. Presynaptic LAR family receptor tyrosine phosphatases (LAR-RPTPs; see Drosophila Lar) regulate synapse development through mechanisms that include trans-synaptic adhesion; however, whether they regulate postsynaptic receptor functions remains unknown. This study reports that presynaptic PTPsigma, a LAR-RPTP, enhances postsynaptic NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity in the hippocampus. This regulation does not involve trans-synaptic adhesions of PTPsigma, suggesting that the cytoplasmic domains of PTPsigma, known to have tyrosine phosphatase activity and mediate protein-protein interactions, are important. In line with this, phosphotyrosine levels of presynaptic proteins, including neurexin-1 (see Drosophila Neurexin), are strongly increased in PTPsigma-mutant mice. Behaviorally, PTPsigma-dependent NMDAR regulation is important for social and reward-related novelty recognition. These results suggest that presynaptic PTPsigma regulates postsynaptic NMDAR function through trans-synaptic and direct adhesion-independent mechanisms and novelty recognition in social and reward contexts (Kim 2020).

NMDA receptor interaction with scaffolding proteins

Chapsyn-110, a member of the membrane-associated putative guanylate kinase (MAGUK) family and related to Drosophila DLG, binds directly to the N-methyl-D-aspartate (NMDA) receptor and Shaker K+ channel subunits. In rat brain, chapsyn-100 protein shows a somatodendritic expression pattern that overlaps partly with PSD-95 but that contrasts with the axonal distribution of SAP97, other MAGUK proteins. Chapsyn-110 associates tightly with the postsynaptic density in brain, and mediates the clustering of both NMDA receptors and K+ channels in heterologous cells. Chapsyn-110 andd PSD-95 can heteromultimerize with each other and are recruited into the same NMDA receptor and K+ channel clusters. Thus, chapsyn-110 and PSD-95 may interact at postsynaptic sites to form a multimeric scaffold for the clustering of receptors, ion channels, and associated signalling proteins (Kim, 1996)

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 binds to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins (Irie, 1997).

Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-D-aspartate (NMDA) receptor function. Analysis of the physical and functional interaction between Fyn and NMDA receptors was carried out to see how Fyn participates in synaptic plasticity. Tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, is reduced in fyn-mutant mice. NR2A is tyrosine-phosphorylated in 293T cells when coexpressed with Fyn. Therefore, NR2A would be a substrate for Fyn in vivo. Results also show that PSD-95, which directly binds to and coclusters with NMDA receptors, promotes Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associate with NR2A and Fyn, respectively; therefore, PSD-95 could mediate complex formation of Fyn with NR2A. PSD-95 also associates with other Src-family PTKs: Src, Yes, and Lyn. These results suggest that PSD-95 is critical for regulation of NMDA receptor activity by Fyn and other Src-family PTKs, serving as a molecular scaffold for anchoring these PTKs to NR2A (Tezuka, 1999).

The postsynaptic density (PSD) can be visualized as an ultrastructural thickening of the postsynaptic membrane that is characteristic of excitatory synapses. Among the glutamate receptor complexes, the NMDA receptor/PSD-95 complex is the one most tightly associated with the PSD. In biochemical preparations of the PSD, NMDA receptors and PSD-95 are highly enriched and resistant to extraction by Triton X-100 and sarkosyl detergents, while AMPA receptors/GRIP and mGluRs/Homer (see Drosophila Homer) are relatively soluble. It is possible that the components of the NMDA receptor/PSD-95 complex comprise the major constituents of the core PSD, which remains after extraction with strong detergents. Because they are likely to play critical roles in the structural organization of the synapse and in the transduction of NMDA receptor signals, these core PSD proteins are important to define and study. A family of proteins (termed GKAP, SAPAP, or DAP) has been characterized that is highly concentrated in the PSD and that binds to the guanylate kinase (GK) domain of PSD-95. GKAP appears to be tightly associated with PSD-95; it can be immunoprecipitated from the brain in a complex with PSD-95 family proteins, and it is consistently colocalized with PSD-95 in neurons, even in the absence of associated NMDA receptors. The GKAP family of proteins contains at least four members and undergoes complex alternative splicing, but the physiological roles of these variants are unknown. To gain insight into GKAP function, a screen was carried out for binding partners of GKAP, hoping to extend the network of protein interactions emanating from NMDA receptors into the PSD (Naisbitt, 1999 and references).

A novel family of postsynaptic density (PSD) proteins, termed Shank, is described that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton (Naisbitt, 1999).

Originally identified as a substrate of Src tyrosine kinase, cortactin is an F-actin-binding protein enriched in cell-matrix contact sites, membrane ruffles and lammelipodia of cultured cells, and in growth cones of neurons. The translocation of cortactin to the cell periphery is stimulated by the small GTPase Rac1, and its F-actin cross-linking activity is inhibited by Src tyrosine phosphorylation. Thus, a large body of evidence implicates cortactin in regulation of the actin cytoskeleton in dynamic regions of the cell periphery. This study suggests that cortactin may also play a role in neuronal synapses, based on the following findings: biochemically, cortactin is loosely associated with the PSD, and immunocytochemically, it colocalizes with Shank in a subset of synapses. Most interestingly, a significant redistribution of cortactin to synaptic sites in response to glutamate stimulation has been demonstrated. The glutamate-induced synaptic localization of cortactin is reminiscent of cortactin recruitment to the cortical cytoskeleton by growth factor stimulation of nonneural cells. Their coexistence in growth cones supports the suggestion that Shank and cortactin may function at sites of active cytoskeletal remodeling in neurons. In mature synapses, it is speculated that a regulated Shank-cortactin interaction may be a mechanism for linking NMDA receptor activation to the control of the postsynaptic actin cytoskeleton. Shank is highly related to CortBP1, a protein isolated by yeast two-hybrid screening with the SH3 domain of cortactin. CortBP1 has been shown to colocalize with cortactin in membrane ruffles of cultured cells and in growth cones of cultured neurons, analogous to the colocalization of Shank and cortactin in growth cones and synapses. Based on their similarity in primary structure and cell biological properties, it seems reasonable to consider CortBP1 and Shank as members of the same family of proteins (Naisbitt, 1999 and references).

The efficiency with which N-methyl-D-aspartate receptors (NMDARs) trigger intracellular signaling pathways governs neuronal plasticity, development, senescence, and disease. Excessive Ca influx triggers excitotoxicity, damaging neurons in diverse neurological disorders. Rapid Ca2+-dependent neurotoxicity is triggered most efficiently when Ca2+ influx occurs through NMDARs, and cannot be reproduced by loading neurons with equivalent quantities of Ca2+ through non-NMDARs or voltage-sensitive Ca2+ channels (VSCCs). This suggests that Ca2+ influx through NMDAR channels is functionally coupled to neurotoxic signaling pathways. In cultured cortical neurons, suppressing the expression of the NMDAR scaffolding protein PSD-95 (postsynaptic density-95) selectively attenuates excitotoxicity triggered via NMDARs, but not by other glutamate or calcium ion (Ca2+) channels. NMDAR function is unaffected, because receptor expression, NMDA currents, and 45Ca2+ loading are unchanged. Suppressing PSD-95 blocks Ca2+-activated nitric oxide production by NMDARs selectively, without affecting neuronal nitric oxide synthase expression or function. Thus, PSD-95 is required for efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to excitotoxic Ca2+ signaling. This raises the possibility that the preferential activation of neurotoxic Ca2+ signals by NMDARs is determined by the distinctiveness of NMDAR-bound MAGUKs, or of the intracellular proteins that they bind (Sattler, 1999).

Enduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. This study demonstrates that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. β-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, β-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of ß-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy (Tai, 2007).

mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. mPins (mammalian homologue of Drosophila Partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR - MAGUK (N-methyl-D-aspartate receptor - membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs (Sans, 2005).

mPins is a ubiquitously expressed protein that is critical for the regulation of mitotic spindle organization in dividing cells. mPins interacts with several functionally distinct proteins, including NuMA, Ras, LKB1 and Galphai. The finding that mPins interacts with the PSD-95 family adds another group of important proteins to those whose trafficking depends on mPins. Drosophila Pins is required for asymmetric division of sensory organ precursor cells (pI) and dividing neuroblasts. Whereas the roles of Pins in cell division are relatively well-characterized, the function of mPins in the mature mammalian central nervous system remains enigmatic. The related protein, AGS3, may affect cocaine-induced plasticity by regulating G-protein signalling in the prefrontal cortex. The data show that mPins and AGS3 are both expressed in the developing hippocampus but have different subcellular localizations, perhaps because mPins, but not AGS3, interacts with SAP102. Moreover, AGS3 is down-regulated in adult hippocampus and seems to be absent from the PSD, whereas mPins is expressed throughout development and is enriched in synaptic membranes. mPins and AGS3 are found in different domains throughout the cell body and dendrites in primary cultures of hippocampal neurons. mPins, but not AGS3, redistributes into punctate structures after ionomycin or NMDA treatment, suggesting that calcium signalling functions in trafficking of mPins complexes. These findings strongly suggest that these two orthologues of Drosophila Pins have different functions in neurons (Sans, 2005).

The MAGUKs do not compete with the other known interacting proteins of mPins suggesting that the association of these other interacting proteins may indirectly influence the trafficking of the MAGUK and its associated proteins, such as NMDARs. Both Ras and Galphai are particularly interesting in this context. Ras has been implicated in the trafficking of GluRs. Characterized as molecular switches that alternate between GTP-bound ('on') and GDP-bound ('off') forms, these proteins are involved in the reorganization of synaptic structure. G-proteins, such as Galphai, influence NMDAR trafficking through metabotropic GluRs. In this study, it is shown that Galphai proteins function in NMDAR trafficking through a direct interaction with the mPins-SAP102 complex. mPins mediates G-protein signalling through binding to Galphai1-3^GDP, thereby inhibiting binding of Galphai to Gßγ (and consequently enhancing Gßγ signalling in the absence of a G-protein-coupled receptor). mPins shifts between a closed state, when the N- and C-terminal halves of the protein bind to one another, and an open state when NuMA binds to mPins to switch it open, allowing the binding of Galphai. SAP102, similarly to SAP97, may exist in the cytoplasm as a folded molecule in which the GK domain is folded onto the SH3 domain. The data suggest that SAP102 binds to mPins in its closed state, as the two proteins localized in ring-like structures in COS cells. Therefore, mPins could be required upstream of, or in parallel to, the NR2B-SAP102 interaction. It is also shown that SAP102-mPins complexes have a different fate from that of NR2B-SAP102-mPins complexes, since the three proteins form clusters in COS cells and synaptic clusters in spines. These data suggest that NMDARs can open the SAP102-mPins complexes. Interestingly, cotransfection of the linker region of mPins with NR2B and SAP102 results in the formation of ternary complexes that are rapidly degraded, suggesting that interaction of Galphai with GoLoco domains (or an unidentified protein with TPR domains) is important for stabilization of the complex. mPins can bind four Galphai molecules, and it is unclear at present whether all of the sites need to be occupied for proper folding and targeting of mPins. As a modulator of G-protein signalling, the possibility cannot be excluded that Galphai binds to the NMDAR-MAGUK-mPins complex at synapses after activation of a G-protein-coupled receptor. Studies have suggested that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) can exhibit some of their effects through interactions with heterotrimeric G-proteins in addition to their ionic channel function. For instance, it has been shown that AMPA can induce dissociation of Galphai1 from the Galphai1/ß heterotrimeric complex and its association with GluR1 through an adaptor protein. In light of the current data, the possibility exists that Galphai signalling proteins may also be recruited to certain MAGUK-mPins complexes through simultaneous dissociation from AMPARs (Sans, 2005).

The results suggest that the NMDAR associates indirectly through SAP102 with two molecular complexes -- the exocyst and mPins-Galphai complexes -- and that these associations are necessary for proper trafficking of receptors in neurons. The results also suggest that this complex is formed in the ER in heterologous cells and early in the secretory pathway in neurons. Although this has not been demonstrated directly for native proteins, an association of MAGUK with AMPARs in the ER (or cis-Golgi) has been shown for native AMPARs in brain by using the endo-H sensitivity of immature AMPARs, so such an association is not unprecedented. These results suggest that NMDARs are trafficked as part of a large complex from their site of synthesis in the cell body to the postsynaptic membrane, presumably in a transport vesicle. The identification of other components of the SAP102 cargo complex (containing NMDARs, the exocyst and mPins-Galphai complexes) will undoubtedly help to clarify the steps involved in trafficking of NMDARs from assembly and ER exit to transport in dendrites and spines in normal and disease states (Sans, 2005).

Citron binds to PSD-95 at glutamatergic synapses on inhibitory neurons in the hippocampus: Signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons

Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho, preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95 and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons. SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).

The results presented here support the notion that differential expression of PSD-95-binding proteins in different neurons helps to determine the composition of signal transduction complexes formed by association with PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define the nature of local biochemical signaling associated with activation of NMDA receptors. The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made onto glutamatergic neurons in the hippocampus. CaM kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses will differ dramatically between excitatory and inhibitory neurons. High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus. In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often found together in excitatory neurons. Thus, the composition of signal transduction machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in other areas of the brain (Zhang, 1999).

Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines

NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. The Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with either RNAi or dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines (Tolias, 2005).

Casein kinase-II regulates NMDA channel function

Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. CKII is identified as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings is enhanced by CKII or spermine and is decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduce synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function (Lieberman, 1999).

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depends on the NMDA receptor. Complexes of NMDA receptors with the multivalent adaptor protein PSD-95 are found in the dorsal horn of spinal cord; PSD-95 plays a key role in neuropathic reflex sensitization. Mutant mice expressing a truncated form of the PSD-95 molecule fail to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but develop normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes; elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr286 CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice (Garry, 2003).

Modulation of NMDA receptors by PACAP

A novel mechanism has been identified for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. This study identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. Activation of the cAMP/protein kinase A pathway in hippocampal slices induces the release of RACK1 from NR2B and Fyn. This results in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. The neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)) was identified as a ligand that induces phosphorylation of NR2B and enhances NMDA receptor potentials. Finally, it was found that activation of the cAMP/protein kinase A pathway induces the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn regulates the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together these results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1 (Yaka, 2003).

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gα_s-coupled receptors and protein kinase A. However, the major hippocampal PACAP1 receptor (PAC₁R) also signals via Gα_q subunits and protein kinase C (PKC). In CA1 neurons, PACAP38 enhances synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC₁R, Gα_q, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gα_q pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca²⁺, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons (Macdonald, 2005).

LTP inhibits LTD in the hippocampus via regulation of GSK3β

Glycogen synthase kinase-3 (GSK3) has been implicated in major neurological disorders, but its role in normal neuronal function is largely unknown. GSK3β mediates an interaction between two major forms of synaptic plasticity in the brain, NMDA receptor-dependent long-term potentiation (LTP) and NMDA receptor-dependent long-term depression (LTD). In rat hippocampal slices, GSK3β inhibitors block the induction of LTD. Furthermore, the activity of GSK3β is enhanced during LTD via activation of PP1. Conversely, following the induction of LTP, there is inhibition of GSK3β activity. This regulation of GSK3β during LTP involves activation of NMDA receptors and the PI3K-Akt pathway and disrupts the ability of synapses to undergo LTD for up to 1 hr. It is concluded that the regulation of GSK3β activity provides a powerful mechanism to preserve information encoded during LTP from erasure by subsequent LTD, perhaps thereby permitting the initial consolidation of learnt information (Peineau, 2007).

NMDA receptor-dependent LTD is due to the internalization of AMPA receptors and involves protein interactions directly associated with the AMPA receptor subunits, particularly GluR2. It was reasoned that GSK3β might form a complex with AMPA receptors, and thus attempts were made to investigate this by probing for an association of native GSK3β with AMPA receptors in the CA1 area of hippocampal slices. A specific antibody against GSK3β was able to coimmunoprecipitate the GluR1 and GluR2 AMPA receptor subunits, and conversely, immunoprecipitation of AMPA receptors produced coimmunoprecipitation of GSK3β. To determine the functional status of AMPA receptor-associated GSK3β, AMPA receptors were immunoprecipitated, using antibodies against either GluR1 or GluR2, and then assayed for kinase activity. GSK3β activity was readily detected in both GluR1 and GluR2 immunoprecipitates relative to the background IgG control, demonstrating that endogenous GSK3β associates with native AMPA receptors in the brain, and that the bound GSK3β is functionally active. This association of GSK3β with AMPA receptors suggests a compartmentalization of this enzyme for the efficient regulation of AMPA receptors during LTD (Peineau, 2007).

It was asked whether the GSK3β activity that is associated with AMPA receptors could be regulated. Previous work has shown that transient exposure of cultured neurons to a solution containing sucrose plus glycine leads to an NMDA receptor-dependent insertion of AMPA receptors into the plasma membrane. Interestingly, this effect is associated with an increase in AMPA receptor-associated PI3K activity. Since PI3K is an upstream regulator of GSK3β, whether this treatment also affected the AMPA receptor-associated GSK3β enzyme activity was investigated. Neurons were treated with sucrose (200 mM) plus glycine (100 µM) for 2 min, and this led to the insertion of AMPA receptors into the plasma membrane as determined approximately 15 min later using surface biotinylation assays. This chemically induced AMPA receptor insertion was associated with a decrease in AMPA receptor-associated GSK3β activity (Peineau, 2007).

This study identified a form of regulation of synaptic plasticity in which the transient synaptic activation of NMDA receptors, as occurs during LTP, leads to inhibition of LTD. This regulation is very powerful since LTD is fully inhibited immediately following the conditioning stimulus and the effect lasts for approximately 1 hr. Also some of the signaling pathways responsible for this potent regulation of synaptic plasticity have been identified. GSK3β activity is an absolute requirement for the induction of LTD and the conditioning stimulus inhibits its activity via activation of the PI3K-Akt pathway. Finally, there is a correlation between the phosphorylation state of GSK3β ser9 and whether NMDA receptor activation leads to the induction or inhibition of LTD (Peineau, 2007).

GSK3β is an unusual kinase that has been implicated in many diseases. However, very little is known about its normal function in the nervous system. It is important during early development and it has been shown to play a key role in cell polarity and in the growth of neuromuscular junctions. Recently, it has been shown that GSK3β is important for determining neuronal polarity during the development of hippocampal neurons. However, though GSK3β is also highly expressed in the mature brain, its function in the nervous system has, hitherto, been largely unexplored. In the nucleus of hippocampal neurons, GSK3β is involved in the regulation of gene transcription by promoting the nuclear export of the transcription factor NF-ATc4. In addition, it has been shown that overexpression of GSK3β impairs spatial learning, though the mechanism underlying this effect is unknown. This study shows that in 2-week-old rats, an age at which the expression of GSK3β is near its peak, GSK3β activity is essential for NMDA receptor-dependent LTD in the hippocampus. This form of LTD is widespread throughout the brain and has been strongly implicated in development and learning and memory. Therefore, this novel GSK3β-dependent mechanism may be of general significance in regulating the interaction between LTP and LTD throughout the brain (Peineau, 2007).

GSK3β, unlike most enzymes, possesses high basal level constitutive activity and can be bidirectionally regulated to either further increase or decrease its activity. During LTD there is additional activation of GSK3β, probably via dephosphorylation of ser9. This effect is prevented by an inhibitor of PP1/PP2A. This suggests that the activation of PP1, which is known to occur during LTD, is responsible for the activation of GSK3β, via its dephosphorylation of ser9. LTD is associated with inhibition of Akt, probably also via the activation of PP1. These data suggest that GSK3β activity is increased during LTD because the phosphatase concomitantly inhibits Akt and directly dephosphorylates ser9 of GSK3β (Peineau, 2007).

Interestingly, the alteration in the phosphorylation status of GSK3β persists beyond the delivery of low-frequency stimulation (LFS), and lithium completely blocks LTD when applied after the delivery of LFS. These data suggest that GSK3β is required for the LTD process beyond the initial induction phase. Further studies are required to determine the full time course of the involvement of GSK3β in LTD (Peineau, 2007).

GSK3β has several upstream regulators and numerous downstream targets. In the present study, two of its upstream regulators have been identified. During LTD, GSK3β is activated via an okadaic acid-sensitive protein phosphatase, which is probably PP1. During LTP, GSK3β is inhibited via the PI3K-Akt pathway. Since GSK3β is such a ubiquitous kinase, it needs mechanisms to localize its access to its substrates. This is achieved in part via direct interactions with other proteins to form complexes. For example, in the canonical Wnt pathway, GSK3β binding proteins control access of β-catenin. It seems likely that the association between GSK3β and AMPA receptors serves to localize the kinase close to substrates that are involved in the trafficking of these receptors during synaptic plasticity. Further studies are required to establish the mechanism of this interaction as well as the downstream pathways mediated by GSK3β in the regulation of LTD (Peineau, 2007).

The finding that the synaptic activation of NMDA receptors during LTP inhibits NMDA receptor-dependent LTD raises an intriguing issue: what determines whether the synaptic activation of NMDA receptors leads to the induction or inhibition of LTD? Evidence is presented that the phosphorylation state of ser9 of GSK3β is a critical determinant. Thus, during LTP, activation of the PI3K-Akt pathway results in phosphorylation of GSK3β, and hence inhibition of its activity. In contrast, during LTD, activation of PP1 results in inhibition of Akt and the dephosphorylation of GSK3β at ser9, and this leads to an increase in the enzyme's activity. The activation of PI3K-Akt and inhibition of PP1 during LTP, but inhibition of Akt during LTD as well as the selective activation of PP1 during LTD, can be explained by the differences in the magnitude and spatiotemporal properties of the Ca2+ rise associated with the synaptic activation of NMDA receptors during these two forms of synaptic plasticity (Peineau, 2007).

Previous work has described other ways in which synaptic plasticity can be powerfully influenced by the prior history of synaptic activity. However, the mechanisms involved in these forms of metaplasticity are not known. Why synapses need such regulatory mechanisms is a matter of conjecture. One intriguing possible role for the regulation described in this study is to stabilize a synaptic modification over the short term by protecting synapses from the effects of additional NMDA receptor-dependent plasticity until the information can be either consolidated or erased by NMDA receptor-independent mechanisms (Peineau, 2007).

The regulation of synaptic plasticity is further complicated by the involvement of mGluRs, which are involved in depotentiation, LTD of baseline transmission, heterosynaptic LTD, and metaplasticity. So that focus could be placed on interactions between the NMDA receptor-dependent forms of synaptic plasticity, the additional complication of mGluR-dependent synaptic plasticity were eliminated by using the broad spectrum mGluR antagonist LY341495 and by employing stimulus protocols optimized for NMDA receptor-dependent synaptic plasticity. However, given that PI3K has been implicated in a chemically induced form of mGluR-dependent LTD and heterosynaptic LTD, it will be interesting to determine whether GSK3β is also involved in these forms of synaptic plasticity. One possibility is that the PI3K-Akt-GSK3β pathway serves to inhibit NMDA receptor-dependent LTD both homosynaptically following the induction of LTP and heterosynaptically following the induction of LTD (Peineau, 2007).

The finding that in the normal brain activation of GSK3β is essential for NMDA receptor-dependent LTD, and that its activity can be regulated by LTP, may offer clues to the pathological role of this enzyme in neurological disorders. For example, the primary therapeutic action of lithium in bipolar disorders may be via inhibition of GSK3β. Indeed, specific inhibition of GSK3β has recently been shown to produce antidepressive-like activity in vivo. Overactivity of GSK3β may, therefore, lead to this mood disorder by affecting the balance and interplay between NMDA receptor-dependent LTP and LTD (Peineau, 2007).

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. This study shows that in addition to this 'classical' LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network (Kwon, 2008).

Adrenergic modulation of NMDA receptors in prefrontal cortex is differentially regulated by RGS proteins and spinophilin

The noradrenergic system in the prefrontal cortex (PFC) is involved in many physiological and psychological processes, including working memory and mood control. To understand the functions of the noradrenergic system, regulation of NMDA receptors , key players in cognition and emotion, by alpha1- and alpha2-adrenergic receptors (alpha1-ARs, alpha2-ARs) was studied in PFC pyramidal neurons. Applying norepinephrine or a norepinephrine transporter inhibitor reduced the amplitude but not paired-pulse ratio of NMDAR-mediated excitatory postsynaptic currents (EPSC) in PFC slices. Specific alpha1-AR or alpha2-AR agonists also decreased NMDAR-EPSC amplitude and whole-cell NMDAR current amplitude in dissociated PFC neurons. The alpha1-AR effect depended on the phospholipase C-inositol 1,4,5-trisphosphate-Ca(2+) pathway, whereas the alpha2-AR effect depended on protein kinase A and the microtubule-based transport of NMDARs that is regulated by ERK signaling. Furthermore, two members of the RGS family, RGS2 and RGS4, were found to down-regulate the effect of alpha1-AR on NMDAR currents, whereas only RGS4 was involved in inhibiting alpha2-AR regulation of NMDAR currents. The regulating effects of RGS2/4 on alpha1-AR signaling were lost in mutant mice lacking spinophilin, which binds several RGS members and G protein-coupled receptors, whereas the effect of RGS4 on alpha2-AR signaling was not altered in spinophilin-knockout mice. This work suggests that activation of alpha1-ARs or alpha2-ARs suppresses NMDAR currents in PFC neurons by distinct mechanisms. The effect of alpha1-ARs is modified by RGS2/4 that are recruited to the receptor complex by spinophilin, whereas the effect of alpha2-ARs is modified by RGS4 independent of spinophilin (Liu, 2006).

The Neuregulin-1 receptor ErbB4 controls glutamatergic synapse maturation and plasticity

Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. This study shows that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. These results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction (Li, 2007).

Splice variants of NMDA receptors and their interaction

The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. The contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons was investigated. An ER retention signal (RRR) was identified in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels. Surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron (Standley, 2000).

NMDA receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. This study used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, the data indicated that the conformation of NR1 subunit homodimers is affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function (Qiu, 2005).

Excitatory neurotransmission mediated by NMDA receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. This study reports crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation (Furukawa, 2005).

Subunits of the NMDA receptor (NMDAR) associate with many postsynaptic proteins that substantially broaden its signaling capacity. Although much work has been focused on the signaling of NR2 subunits, little is known about the role of the NR1 subunit. This study set out to elucidate the role of the C terminus of the NR1 subunit in NMDAR signaling. By introducing a C-terminal deletion mutant of the NR1 subunit into cultured neurons from NR1(-/-) mice, it was found that the C terminus was essential for NMDAR inactivation, downstream signaling, and gene expression, but not for global increases in intracellular Ca2+. Therefore, whereas NMDARs can increase Ca2+ throughout the neuron, NMDAR-dependent signaling, both local and long range, requires coupling through the NR1 C terminus. Two major NR1 splice variants differ by the presence or absence of a C-terminal domain, C1, which is determined by alternative splicing of exon 21. Analysis of these two variants showed that removal of this domain significantly reduced the efficacy of NMDAR-induced gene expression without affecting receptor inactivation. Thus, the NR1 C terminus couples to multiple downstream signaling pathways that can be modulated selectively by RNA splicing (Bradley, 2006).

NMDA receptor activity is important for many physiological functions, including synapse formation and alterations in synaptic strength. NMDA receptors are composed most commonly of NR1 and NR2 subunits. There are four NR2 subunits (NR2A-NR2D). NR2 subunit expression varies across both brain regions and developmental stages. The identity of the NR2 subunit within a functional NMDA receptor helps to determine many pharmacological and biophysical receptor properties, including strength of block by external Mg2+ (Mg(o)2+). Mg(o)2+ block confers strong voltage dependence to NMDA receptor-mediated responses and is critically important for many of the functions that the NMDA receptor plays within the CNS. This study describes the NR2 subunit dependence of the kinetics of Mg(o)2+ unblock after rapid depolarizations. Mg(o)2+ unblocks from NR1/2A and NR1/2B receptors with a prominent slow component similar to that previously described in native hippocampal and cortical NMDA receptors. Strikingly, this slow component of Mg(o)2+ unblock is completely absent from NR1/2C and NR1/2D receptors. Thus currents from NR1/2C and NR1/2D receptors respond more rapidly to fast depolarizations than currents from NR1/2A and NR1/2B receptors. In addition, the slow component of Mg(o)2+ unblock from NR1/2B receptors is consistently slower than from NR1/2A receptors. This makes rapid depolarizations, such as action potential waveforms, more efficacious at stimulating Mg(o)2+ unblock from NR1/2A than from NR1/2B receptors. These NR2 subunit differences in the kinetics of Mg(o)2+ unblock are likely to help determine the contribution of each NMDA receptor subtype to current flow during synaptic activity (Clarke, 2006).

The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C-terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, it was found that the three amino-acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino-acids following the TM4 region (NR22) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR22 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR22 subunits were cotransfected with either the NR1-4a or NR1-1aAAA mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, it was found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2B5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence specificity-dependent. Taken together, these results show that the C-terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C-terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface (Yang, 2007).

Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation

Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice is attributed to reduced NR2B degradation, which causes elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitates the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning (Hawasli, 2007).

NMDA receptors regulate AMPA receptor function

To monitor changes in AMPA receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) is functional and is transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP is intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induces a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events require synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediated transmission observed during long-term potentiation and activity-dependent synaptic maturation (Shi, 1999).

These results build on a number of studies suggesting that the delivery of AMPA receptors to synapses contributes to activity-dependent plasticity. Inhibition of membrane fusion processes in the postsynaptic cell blocks the action of LTP. Furthermore, the COOH-termini of AMPA receptor subunits GluR2 and GluR4c bind N-ethylmaleimide-sensitive fusion protein, a protein involved in membrane fusion processes. Vesicular organelles, possibly undergoing exocytosis and endocytosis, have been detected with electron microcopy in spines. And last, dendrites can display a calcium-evoked exocytosis of trans-Golgi-derived organelles that is mediated by the calcium/calmodulin-dependent protein kinase II, an enzyme thought to mediate LTP. Other postsynaptic mechanisms, such as an increase in conductance of AMPA receptors, may also occur in parallel. These results also do not rule out a contribution by presynaptic modifications. In addition to the spine delivery of GluR1-GFP, tetanic stimulation induces the formation of clusters of the tagged receptor within dendrites. These structures may be related to the spine apparatus, membranous structures at the base of spines that appear to contain AMPA receptors. The entry of calcium through synaptic NMDA receptors may cause nucleation of AMPA receptor-containing membranes close to active synapses. Once formed, such sites may serve several functions. These sites may replenish those receptors delivered to spines during plasticity. Additionally, they may serve as a 'synaptic tag', providing a docking site for AMPA receptors synthesized at distant sites. Last, they could provide a site for local AMPA receptor synthesis. In these capacities, such clusters could represent a structural modification serving as a long-lasting memory mechanism (Shi, 1999 and references).

The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).

The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. A Ca2+-sensitive protein complex is described involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of (1) MUPPI, a multi-PDZ domain-containing protein, (2) SynGAP, a synaptic GTPase-activating protein, and (3) the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+, entering the cell via the NMDAR, drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirms these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking (Krapivinsky, 2004).

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission (Kim, 2005).

The ERK1/2 signaling pathway is activated by calcium influx through NMDARs and plays an important role in synaptic plasticity and cell survival. NMDAR-dependent ERK activation involves the small GTPase Ras, which is stimulated by specific guanine nucleotide exchange factors (GEFs) and inhibited by GTPase activating proteins (GAPs). The RasGEF RasGRF1 is reported to bind directly to the NR2B subunit of NMDARs. SynGAP, a RasGAP highly enriched in the postsynaptic density (PSD), can associate with NMDARs through binding to PSD-95 family proteins. The exact function of these Ras regulatory proteins in synaptic plasticity has not been established, and how they are functionally coupled to NMDARs remains unclear (Kim, 2005).

Altered AMPAR trafficking has emerged as a major postsynaptic mechanism for the expression of synaptic plasticity. A prevailing model is that NMDAR-dependent LTP is mediated by the surface insertion and synaptic delivery of GluR1, that is driven by CaM kinase II and the Ras-ERK pathway. In contrast, LTD is supposed to result, at least in part, from the removal of synaptic AMPARs by the increased endocytosis and/or reduced recycling of GluR2/3 subunits (Kim, 2005).

This study investigates the links between NMDAR subtypes, Ras-ERK signaling, and AMPAR trafficking. NR2A and NR2B are found to have antagonistic actions on Ras-ERK activation and AMPAR distribution in mature neurons. NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1 -- primarily by regulating GluR1 surface insertion. Potentially accounting for this difference is that NR2B is coupled to the inhibition rather than the activation of the Ras-ERK pathway. This functional coupling is correlated with the specific biochemical association of SynGAP with NR2B-NMDARs (Kim, 2005).

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. Evidence is presented that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibits NMDA-induced AMPA receptor internalization and blocks the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295 (Kim, 2007).

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA-type (but not NMDA-type) glutamate receptors at excitatory synapses on their dendritic shafts, in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. The NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further shows that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization is preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons (Mi, 2004).

Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB

p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine-nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons (Tian, 2004).

cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an A-kinase anchoring protein scaffold protein

NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, it has been demonstrated that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP-PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150-PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, these observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components (Smith, 2006).

NCAM promotes assembly and activity-dependent remodeling of the postsynaptic signaling complex

The neural cell adhesion molecule (NCAM) regulates synapse formation and synaptic strength via mechanisms that have remained unknown. This study shows that NCAM associates with the postsynaptic spectrin-based scaffold, cross-linking NCAM with the NMDA receptor and Ca²⁺/calmodulin-dependent protein kinase II α (CaMKIIα) in a manner not firmly or directly linked to PSD95 and α-actinin. Clustering of NCAM promotes formation of detergent-insoluble complexes enriched in postsynaptic proteins and resembling postsynaptic densities. Disruption of the NCAM-spectrin complex decreases the size of postsynaptic densities and reduces synaptic targeting of NCAM-spectrin-associated postsynaptic proteins, including spectrin, NMDA receptors, and CaMKIIα. Degeneration of the spectrin scaffold in NCAM-deficient neurons results in an inability to recruit CaMKIIα to synapses after NMDA receptor activation, which is a critical process in NMDA receptor-dependent long-term potentiation. The combined observations indicate that NCAM promotes assembly of the spectrin-based postsynaptic signaling complex, which is required for activity-associated, long-lasting changes in synaptic strength. Its abnormal function may contribute to the etiology of neuropsychiatric disorders associated with mutations in or abnormal expression of NCAM (Sytayk, 2007).

Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. In cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits (Chubykin, 2007).

Calmodulin functions downstream of NMDA receptors

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, mutant CaMs were designed that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the 'closed' Ca2+-free conformation. Their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation, were measured. CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. These results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways (Shifman, 2006).

BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation

The NMDA receptor, brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and phosphatidylinositol 3-kinase (PI3K) have all been implicated in long-term potentiation. This study shows that these molecules are involved in a single pathway for synaptic potentiation. In visual cortical neurons in young rodents, the neurotrophin receptor TrkB is associated with PSD-95. When BDNF is applied to cultured visual cortical neurons, PSD-95-labeled synaptic puncta enlarge, and fluorescent recovery after photobleaching (FRAP) reveals increased delivery of green fluorescent protein-tagged PSD-95 to the dendrites. The recovery of fluorescence requires TrkB, signaling through PI3K and the serine-threonine kinase Akt, and an intact Golgi apparatus. Stimulation of NMDARs mimics the PSD-95 trafficking that is induced by BDNF but requires active BDNF and PI3K. Furthermore, local dendritic contact with a BDNF-coated microsphere induces PSD-95 FRAP throughout the dendrites of the stimulated neuron, suggesting that this mechanism induces rapid neuron-wide synaptic increases in PSD-95 and refinement whenever a few robust inputs activate the NMDAR-BDNF-PI3K pathway (Yoshii, 2007).

NMDA-receptor activation induces Calpain-mediated β-catenin cleavages for triggering gene expression

The canonical Wnt-β-catenin signaling pathway is important for a variety of developmental phenomena as well as for carcinogenesis. In hippocampal neurons, NMDA-receptor-dependent activation of calpain induces the cleavage of β-catenin at the N terminus, generating stable, truncated forms. These β-catenin fragments accumulate in the nucleus and induced Tcf/Lef-dependent gene transcription. Fosl1, one of the immediate-early genes, was identified as a target of this signaling pathway. In addition, exploratory behavior by mice resulted in a similar cleavage of β-catenin, as well as activation of the Tcf signaling pathway, in hippocampal neurons. Both β-catenin cleavage and Tcf-dependent gene transcription are suppressed by calpain inhibitors. These findings reveal another pathway for β-catenin-dependent signaling, in addition to the canonical Wnt-β-catenin pathway, and suggest that this other pathway could play an important role in activity-dependent gene expression (Abe, 2007).

Requirement of JIP scaffold proteins for NMDA-mediated signal transduction

JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, the phenotype was examined of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in NMDA receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca++, and gene expression. The decreased NMDA receptor activity in JIP-deficient neurons is associated with reduced tyrosine phosphorylation of NR2 subunits of the NMDA receptor. JIP complexes interact with the SH2 domain of cFyn and may therefore promote tyrosine phosphorylation and activity of the NMDA receptor. It is concluded that JIP scaffold proteins are critically required for normal NMDA receptor function (Kennedy, 2007).

JIP scaffold proteins are important for the normal activity of NMDA receptors. It is established that JIP proteins are localized at post-synaptic densities in neurons, but the mechanism that accounts for the functional interaction of JIP proteins with NMDA receptors is unclear. One possibility is that the PTB domain of JIP1 and JIP2 may contribute to this regulatory process. Indeed, several ligands for this PTB domain have been described, including the low-density lipoprotein receptor-related protein LRP8, the Rac exchange factor Tiam1, and the Ras exchange factor Ras-Grf. Interestingly, all three of these proteins (LRP8, Tiam1, and Ras-Grf) are known to bind NMDA receptors. These proteins may therefore recruit JIP/cFyn complexes to the NMDA receptor to regulate NR2 subunit tyrosine phosphorylation (Kennedy, 2007).

The interaction of LRP8 with JIP1/2 is particularly intriguing because it is established that the site of interaction of the JIP1/2 PTB domain with the cytoplasmic domain of LRP8 is encoded by exon 19 (Stockinger, 2000). This is an alternative spliced exon that is selectively included in Lrp8 mRNA in response to neuronal activity. Thus, only exon 19-positive LRP8 can bind to the JIP1/2 PTB domain. Gene targeting studies have demonstrated that ablation of Lrp8 exon 19 results in mice that exhibit defects in NMDA receptor signaling associated with markedly decreased NR2 subunit tyrosine phosphorylation (Beffert, 2005). Exon 19-positive LRP8 may therefore regulate NMDA receptor activity. Interestingly, the defect in NMDA receptor signaling and NR2 subunit phosphorylation caused by loss of Lrp8 exon 19 (Beffert, 2005) is similar to that caused by compound mutation of JIP1/2. Together, these data suggest that the physical interaction between the JIP1/2 PTB domain and the segment of the LRP8 cytoplasmic tail encoded by Lrp8 exon 19 is functionally significant. Indeed, it is possible that JIP/cFyn complexes may mediate the effects of LRP8 on NMDA receptors. Engagement of cell surface LRP8 by its ligand Reelin may trigger this regulatory pathway to control NMDA receptor function (Kennedy, 2007).

The postnatal role of LRP8 to regulate NMDA receptor activity requires Lrp8 exon 19. However, LRP8 also plays an important developmental role in determining the positioning of neurons in the brain. Mice lacking the ligand Reelin (reeler mice) or mice with targeted ablation of the Lrp8 gene exhibit marked defects in neuronal positioning. However, neither targeted ablation of the alternatively spliced Lrp8 exon 19 nor compound deficiency of JIP1/2 causes a reeler-like neuronal positioning defect during development. Thus, the JIP1/2 PTB domain interaction with the cytoplasmic tail of LRP8 does not play a role in the early developmental function of LRP8 to regulate neuronal positioning (Kennedy, 2007).

A critical role for cFyn in NMDA receptor regulation is established by the finding that cFyn^-/- mice exhibit severely reduced tyrosine phosphorylation of NR2A and NR2B and display major defects in long-term potentiation and spatial learning. Nevertheless, other members of the SRC family of tyrosine kinases (including Lck, Lyn, Src, and Yes) have also been implicated in the regulation of NMDA receptor function. cFyn may therefore represent only one member of a larger group of SRC family kinases that are recruited by JIP scaffold proteins. This function of JIP proteins may be coordinated with the actions of other scaffold proteins that can bind SRC family kinases and are present within post-synaptic densities (e.g., PSD-95 or RACK1). Since Jip1^-/- and Jip2^-/- neurons exhibit different defects in NMDA receptor function, it is possible that these JIP scaffold proteins may cooperate by influencing different steps in the regulatory process. It is also possible that the structurally unrelated scaffold proteins JIP3 and JIP4 may also contribute to NMDA receptor regulation (Kennedy, 2007).

In conclusion, this study has demonstrated that JIP1/2 scaffold proteins represent novel components of the NMDA receptor regulatory network required for normal NMDA-mediated signal transduction (Kennedy, 2007).

Rapid bidirectional switching of synaptic NMDA receptors

Synaptic NMDA-type glutamate receptors (NMDARs) play important roles in synaptic plasticity, brain development, and pathology. In the last few years, the view of NMDARs as relatively fixed components of the postsynaptic density has changed. A number of studies have now shown that both the number of receptors and their subunit compositions can be altered. During development, the synaptic NMDARs subunit composition changes, switching from predominance of NR2B-containing to NR2A-containing receptors, but little is known about the mechanisms involved in this developmental process. This study reports that, depending on the pattern of NMDAR activation, the subunit composition of synaptic NMDARs is under extremely rapid, bidirectional control at neonatal synapses. This switching, which is at least as rapid as that seen with AMPARs, will have immediate and dramatic consequences on the integrative capacity of the synapse (Bellone, 2007).

At early ages synaptic NMDARs are at least as dynamic as AMPARs. LTP-inducing stimuli causes a switch in subunit composition within seconds and the change lasts for at least an hour. However, depotentiating stimuli rapidly reverses the change. These data indicate that the activity-dependent loss of NR2B receptors is coincident with the simultaneous recruitment of NR2A receptors. How this swapping of receptors is coordinated is unclear, but is likely to be dependent on the differences in the C terminus of the two receptor subunits and the partners with which they interact as well as to differences in phosphorylation (Bellone, 2007).

Specific roles of NMDA and AMPA receptors in direction-selective and spatial phase-selective responses in visual cortex

Cells in the superficial layers of primary visual cortex (area 17) are distinguished by feedforward input from thalamic-recipient layers and by massive recurrent excitatory connections between neighboring cells. The connections use glutamate as transmitter, and the postsynaptic cells contain both NMDA and AMPA receptors. The possible role of these receptor types in generating emergent responses of neurons in the superficial cortical layers is unknown. NMDA and AMPA receptors are both involved in the generation of direction-selective responses in layer 2/3 cells of area 17 in cats. NMDA receptors contribute prominently to responses in the preferred direction, and their contribution to responses in the nonpreferred direction is reduced significantly by GABAergic inhibition. AMPA receptors decrease spatial phase-selective simple cell responses and generate phase-invariant complex cell responses (Rivadulla, 2001).

By combining extracellular recording and iontophoresis of receptor blockers, the following results have been demonstrated: (1) Blocking AMPA receptors removes a proportionately larger component from nonpreferred compared with preferred responses and increases direction selectivity. The remaining responses are mediated by NMDA receptors and are overwhelmingly in the preferred direction. (2) Blocking NMDA receptors removes proportional components from preferred and nonpreferred responses and preserves directional selectivity. Because the remaining responses are mediated by AMPA receptors, these receptors are sufficient for direction selectivity. (3) Blocking inhibition preferentially enhances the contribution of NMDA receptors to nonpreferred responses and reduces direction selectivity. Thus, inhibition contributes to direction selectivity by reducing NMDA responses in the nonpreferred direction. (4) Blocking AMPA receptors increases the modulation of complex cell responses by a drifting grating stimulus. Thus, AMPA receptors decrease the selectivity of complex cells for spatial phase or the spatial location of visual stimuli. (5) Blocking NMDA receptors or inhibition has little effect on the temporal modulation of simple or complex cell responses. Together, these results allow for the proposal specific roles for NMDA and AMPA receptors in direction selectivity in the superficial layers of area 17 and in the generation of phase selectivity by simple and complex cells in these layers (Rivadulla, 2001).

Direction selectivity first appears in simple cells of layer 4 in area 17, where NMDA receptors are not present in significant proportions and contribute little to visual responses. The mechanism(s) by which direction selectivity is generated and whether the mechanism is similar in various cortical layers remain unresolved. One hypothesis is that inhibition reduces the response in the nonpreferred direction. The hypothesis is supported by pharmacological studies in cat and monkey, demonstrating that blockade of inhibition in cortical cells induces a loss of selectivity to the direction of stimulus motion. An alternative hypothesis is that there is enhancement of excitation in the preferred direction. It has been shown that simple cells in area 17 have asymmetries in the time course of the response evoked from different positions of the receptive field. Linear summation of these asymmetries allows one to predict the direction preference of the cell but also leads to an overestimation of the response in the nonpreferred direction. Recurrent excitation has been proposed as a nonlinear mechanism by which responses can be increased in the preferred direction. Recently, it has been postulated that inhibition can sculpt the spatiotemporal profile of the receptive field, accentuating spatiotemporal asymmetry and increasing direction selectivity, particularly in layer 4 (Rivadulla, 2001 and references therein).

A fundamental difference between layers 2/3 and 4 is the presence in supragranular layers of NMDA receptors, where they have been shown to participate in transmission in vivo and in vitro. AMPA and NMDA receptors both contribute to direction selectivity in supragranular layers. AMPA receptors are sufficient for generating direction selectivity, either because inputs to the superficial layers conveyed by AMPA receptors are already biased for direction or because feedforward and recurrent connections mediated by AMPA receptors generate direction selectivity within these layers. NMDA receptors by themselves can generate highly direction-selective responses, by summing and/or amplifying responses to the preferred stimulus while contributing less to nonpreferred responses because of close GABAergic control. One possibility is that NMDA receptor activation is possible only with enough excitation in the preferred direction. However, a comparison of the nonpreferred and spontaneous responses that remain after application of AMPA receptor inhibitor CNQX (spontaneous activity in this population of cells is reduced on average by only 28% under CNQX, whereas nonpreferred responses are reduced by 88%) suggests that the reduced contribution of NMDA receptors to nonpreferred responses is likely mediated by active inhibition rather than simply being a function of overall response magnitude: spontaneous activity occurs under less inhibition than nonpreferred responses and remains significantly greater after AMPA receptor blockade (Rivadulla, 2001).

Two lines of evidence indicate that GABAergic inhibition regulates the reduced contribution of NMDA receptors to nonpreferred responses. (1) Blocking inhibition by application of bicuculline decreases the direction selectivity of cells, but this effect is reversed by simultaneous application of APV, indicating that release of inhibition facilitates NMDA responses in the nonpreferred direction. (2) Blockade of inhibition concurrent with AMPA receptor blockade by CNQX reduces direction selectivity. Bicuculline preferentially increases nonpreferred responses, leaving a higher contribution of NMDA responses in the nonpreferred direction (Rivadulla, 2001).

These data confirm and extend the findings that NMDA inhibitor APV causes a proportional reduction in responses of area 17 cells to optimally oriented moving bars as stimulus contrast is increased, whereas application of NMDA increases responses by a proportional amount and quisqualate increases responses by an absolute amount at all contrasts. Importantly, responses in different directions (as also simple and complex cell responses) were studied with APV and CNQX and the modulation of NMDA and AMPA responses by inhibition. NMDA-mediated responses in the nonpreferred direction are reduced nonlinearly by inhibition. Furthermore, the contribution of AMPA receptors to complex cell responses is much more than addition of a constant response component; rather, there is a nonlinear change in the temporal modulation of the response. These observations argue for specific circuits that engage inhibition for generating direction-selective responses and AMPA receptors for generating complex cell responses (Rivadulla, 2001).

The possibility that inhibition regulates NMDA-mediated activity is consistent with other lines of evidence in area 17. The relationship between GABAergic inhibition and NMDA function is probably related to the voltage dependence of NMDA receptors; the binding of extracellular Mg2+ to the channel pore is highly dependent on membrane potential, and changes in the latter could significantly modulate NMDA receptor-mediated activity. The likely source of inhibition is GABAergic interneurons located within layer 2/3 itself (Rivadulla, 2001).

One caveat is that the iontophoresis technique does not allow definitive conclusions about whether all receptors on a cell are affected or whether other cells (either excitatory or inhibitory) in the vicinity could be modifying the responses of the recorded cell. However, the temporal effects of drug application were studied in the first and the second half of the iontophoresis period in several cells and found to be similar. Furthermore, the effect of iontophoresis does not change with ejection time, indicating that most of the affected receptors are in the volume covered by the antagonist since the start of iontophoresis (Rivadulla, 2001).

In addition to examining the role of AMPA and NMDA receptors in direction selectivity, an examination was made of their role in generating simple and complex responses in the supragranular layers by analyzing the temporal pattern of response of cells when they were stimulated with drifting gratings. CNQX caused a dramatic change in complex cell responses, causing them to increase their temporal modulation and respond in a manner similar to that of simple cells. APV does not affect the response modulation of complex cells, and the modulation of simple cell responses is unaffected by CNQX or APV (Rivadulla, 2001).

Recently, it has been proposed that complex cell responses arise as a consequence of decreasing the phase selectivity of simple cell responses by recurrent intracortical connections. The model predicts that a decrease in intracortical excitation should cause complex cells to respond like simple cells. If AMPA receptors primarily mediate short-range intracortical excitation, the results presented here agree strongly with this prediction, demonstrating that during blockade of AMPA receptors complex cells behave as simple cells when stimulated with drifting gratings (Rivadulla, 2001).

It is proposed that AMPA and NMDA receptors in layer 2/3 have different spatial distributions on cells, with both present on the same cell but in different proportions at different inputs. Both receptors mediate feedforward connections, and these afferents provide the necessary input for direction selectivity in layer 2/3. The data are consistent with spatiotemporal asymmetry and enhancement of excitation in feedforward pathways as crucial for direction selectivity in layer 2/3, with a prominent role for NMDA receptors in generating the preferred response and a role for GABAergic inhibition in reducing the nonpreferred response. In contrast to feedforward connections, local recurrent connections are mainly mediated by AMPA receptors (with a possible small contribution from NMDA receptors), and they are responsible for smearing the phase selectivity of simple cells to create phase-invariant complex cell responses (Rivadulla, 2001).

The suggestion that short-range excitation between cortical cells is mediated primarily by AMPA receptors is consistent with the fact that fast EPSCs that are evoked in supragranular layer cells in area 17 by adjacent intralaminar stimulation are not APV sensitive. In somatosensory cortex, intracellular recording of unitary EPSCs in layer 4 and the supragranular cortex indicates that both thalamocortical and intracortical EPSCs are mediated by AMPA receptors and have similar characteristics. In slices of area 17, white matter stimulation evokes EPSPs in the supragranular layers that have NMDA- and AMPA-mediated components. Because of feedforward and local recurrent connections, short-latency responses are primarily AMPA mediated, whereas long-latency responses, because of horizontal connections, have significant NMDA components. Furthermore, long-range horizontal inputs to layer 2/3 cells in area 17 can sum nonlinearly with feedforward or short-range inputs, indicative of NMDA receptor involvement in the long-range connections. Thus, it is likely that there is even finer spatial segregation of glutamate receptors associated with specific inputs on layer 2/3 cells. Together with the modulation of responses (particularly those mediated by NMDA receptors) by inhibition, the specific relationship between receptor types and anatomical connections provides a rich substrate for dynamic control of emergent responses in the cortex (Rivadulla, 2001).

Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network

Forming distinct representations of multiple contexts, places, and episodes is a crucial function of the hippocampus. The dentate gyrus subregion has been suggested to fulfill this role. This hypothesis was tested by generating and analyzing a mouse strain that lacks the gene encoding the essential subunit of the N-methyl-D-aspartate (NMDA) receptor NR1, specifically in dentate gyrus granule cells. The mutant mice performed normally in contextual fear conditioning, but are impaired in the ability to distinguish two similar contexts. A significant reduction in the context-specific modulation of firing rate was observed in the CA3 pyramidal cells when the mutant mice were transferred from one context to another. These results provide evidence that NMDA receptors in the granule cells of the dentate gyrus play a crucial role in the process of pattern separation (McHugh, 2007).

Using conditional genetic-engineering techniques, it has been shown that NRs in the CA3 play a crucial role in rapid learning and pattern completion-mediated recall, whereas CA1 NRs are required for the formation of both spatial and nonspatial memory. The DG-NR1 KO mice described here allowed extension of the study to the roles of DG NRs and NR-dependent plasticity. The data support the notion that DG GC NRs play an important role in rapidly forming a unique memory of a context and discriminating it from similar contexts previously encountered (pattern separation), although they are dispensable for the acquisition of contextual memory per se (McHugh, 2007).

DG-NR1 KO mice exhibited impaired context discrimination early during training in the incremental fear-conditioning paradigm and impaired context-modulated place cell activity in CA3 on the initial day of recording. Both deficits were overcome with training or experience. Together, these data suggest that NR function at perforant path (PP)-GC synapses is important for the animals' ability to discriminate similar contexts rapidly with limited experience, but not for slower acquisition of this ability over more trials. It is suggested that common mechanisms underlie the DG-NR1 KO deficits observed at both the behavioral and physiological levels, despite the different timelines of recovery to control levels. These differences may reflect differences between the cues used to define the contexts, the use of conditioning footshocks in the behavioral experiment, the contribution of non-hippocampal structures in fear conditioning, or the greater sensitivity of the readout in the place cell recordings relative to the behavioral task. The eventual acquisition of the discriminating power by DG-NR1 KO mice may be due to the gradual development of synaptic plasticity at sites downstream of the PP-GC synapses. For example, the recurrent collateral-CA3 synapses may provide a complementary site at which small differences in PP input can be amplified; this is supported by a recent study reporting a contribution of CA3 NRs to pattern separation. Although the large number of cells and sparse connectivity of the DG would provide the ultimate substrate for the pattern separation, synaptic plasticity may be the tool that allows rapid and efficient separation of representations (McHugh, 2007).

CA3 receives excitatory input from two external sources, the DG and the EC. Input from the DG is most likely to contribute to the orthogonalization of CA3 representations by virtue of the high GC number and the sparse GC-CA3 connectivity. Loss of NRs in the GCs may decrease drive from the DG to CA3. This would increase the relative proportion of EC drive to CA3, thus reducing the CA3 ensemble's ability to detect, amplify, and reflect small differences in EC activity generated in similar contexts. Indeed, rate remapping in CA3 (induced by changes in recording chamber shape or color) can occur in the absence of detectable changes in medial EC firing rates or locations. However, despite unvarying input from the EC, DG GCs did respond to contextual changes robustly and rapidly under these conditions. These data suggest that NR-mediated activity or plasticity in the GCs may underlie these changes, subsequently shaping CA3 encoding (McHugh, 2007).

It is puzzling that rate remapping in CA3 did not always affect spatial or rate coding downstream in CA1. It is possible that under different conditions, such as in the behavioral discrimination task, small differences in context-specific coding parameters, including firing rates, could be amplified by contextual salience (such as footshock) and may be manifested in CA1. It remains to be seen whether the context specificity of CA3 coding will be transferred to CA1 under the conditions of behavioral discrimination (McHugh, 2007).

NMDA receptors and photoperiod response

Many physiological and behavioral phenomena are controlled by an internal, self-sustaining oscillator with a periodicity of approximately 24 hr. In mammals, the principal oscillator resides in the suprachiasmatic nucleus (SCN). A light pulse during the subjective night causes a phase shift of the circadian rhythm via direct glutamatergic retinal afferents to the SCN. Along with the accepted theoretical models of the clock, it is suggested that behavioral resetting of mammals is completed within 2 hr; however, the molecular mechanism has not been elucidated. The real-time image of the transcription of the circadian-clock gene mPer1 is shown in the cultured SCN by using the transgenic mice that carry a luciferase reporter gene under the control of the mPer1 promoter. The real-time image demonstrates that the mPer1 promoter activity oscillates robustly in a circadian manner and that this promoter activity is reset rapidly (within 2-3 hr) when a phase shift occurs (Asai, 2001).

Insufficient sleep reversibly alters bidirectional synaptic plasticity and NMDA receptor function

Insufficient sleep impairs cognitive functions in humans and animals. However, whether long-term synaptic plasticity, a cellular substrate of learning and memory, is compromised by sleep loss per se remains unclear because of confounding factors related to sleep deprivation (SD) procedures in rodents. Using an ex vivo approach in C57BL/6J mice, it has been shown that sleep loss rapidly and reversibly alters bidirectional synaptic plasticity in the CA1 area of the hippocampus. A brief (approximately 4 h) total SD, respecting the temporal parameters of sleep regulation and maintaining unaltered low corticosterone levels, shifted the modification threshold for long-term depression/long-term potentiation (LTP) along the stimulation frequency axis (1-100 Hz) toward the right. Reducing exposure to sensory stimuli by whisker trimming did not affect the SD-induced changes in synaptic plasticity. Recovery sleep reversed the effects induced by SD. When SD was combined with moderate stress, LTP induction was not only impaired but also occluded. Both electrophysiological analysis and immunoblotting of purified synaptosomes revealed that an alteration in the molecular composition of synaptically activated NMDA receptors toward a greater NR2A/NR2B ratio accompanied the effects of SD. This change was reversed after recovery sleep. By using an unparalleled, particularly mild form of SD, this study describes a novel approach toward dissociating the consequences of insufficient sleep on synaptic plasticity from nonspecific effects accompanying SD in rodents. A framework was established for cellular models of cognitive impairment related to sleep loss, a major problem in modern society (Kopp, 2006).

Glia-derived D-serine controls NMDA receptor activity and synaptic memory

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. Since D-serine is released exclusively by astrocytes, the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity was studied. To this end, advantage was taken of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. Direct evidence is provided that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling (Panatier, 2006).

Growth factor-dependent trafficking of cerebellar NMDA receptors via protein kinase B/Akt phosphorylation of NR2C

NMDA receptor subunit composition varies throughout the brain, providing molecular diversity in NMDA receptor function. The NR2 subunits (NR2A-D) in large part dictate the distinct functional properties of NMDA receptors and differentially regulate receptor trafficking. Although the NR2C subunit is highly enriched in cerebellar granule cells and plays a unique role in cerebellar function, little is known about NR2C-specific regulation of NMDA receptors. This study demonstrates that PKB/Akt directly phosphorylates NR2C on serine 1096 (S1096). In addition, 14-3-3epsilon was identified as an NR2C interactor, whose binding is dependent on S1096 phosphorylation. Both growth factor stimulation and NMDA receptor activity lead to a robust increase in both phosphorylation of NR2C on S1096 and surface expression of cerebellar NMDA receptors. Finally, NR2C expression, unlike NR2A and NR2B, supports neuronal survival. Thus, these data provide a direct mechanistic link between growth factor stimulation and regulation of cerebellar NMDA receptors (Chen, 2009).

Growth factors play a critical role in regulating neuronal survival during development. Cerebellar granule cells, in particular, are known to undergo a period of extensive apoptosis during development, which is precisely regulated by growth factors. IGF-1 has been demonstrated to promote cerebellar granule cell survival both in vitro and in vivo by blocking apoptosis. IGF-1 increases PI3 kinase activity leading to increased PKB activity and stimulation of downstream kinase/signaling cascades. PKB phosphorylates a variety of substrates, many of which regulate mitochondrial function and apoptosis. Mice lacking various isoforms of PKB have an overall reduction in cell number in many tissues, and specifically in the brain when the PKBγ isoform is absent. The growth factor/PKB signaling pathway has been specifically implicated in regulating cerebellar granule cell survival. Furthermore, there is evidence that PKB can potentiate NMDA receptor responses in cerebellar granule cells, suggesting a link between growth factor signaling and receptor activation in these neurons. However, until now, there was no mechanism described linking growth factor signaling, PKB activity, and NMDA receptor expression (Chen, 2009).

This study found that growth factor signaling and PKB phosphorylation increase surface expression of NR2C-containing NMDA receptors. Importantly, overexpression of NR2C was shown to protect neurons from NMDA-induced toxicity. Consistent with a role for phosphorylation of S1096 in receptor trafficking, NR2C S1096A was less effective at neuronal protection than NR2C WT, supporting a role for the PI3K/PKB pathway in neuronal survival. Overexpression of NR2C S1096A also has a protective effect compared to NR2A or NR2B, which is probably due to the increased level of NR2C surface expression due to exogenous overexpression. For example, the S1096A mutation does not completely abolish the surface expression of NR2C when overexpressed in cerebellar granule cells. These results suggest that surface-expressed NR2C S1096A, even if decreased compared to that of NR2C WT, is sufficient to support neuronal survival. How does NR2C contribute to neuronal survival? The answer probably lies in the fact that NR2C-containing NMDA receptors have unique channel properties and that NR2C may mediate specific signaling pathways to activate downstream survival molecules. In conclusion, these findings that PKB directly phosphorylates cerebellar NMDA receptors and increases their surface expression reveals a novel molecular mechanism by which growth factor signaling can directly affect NMDA receptor activity (Chen, 2009).

Downregulation of NR3A-containing NMDARs is required for synapse maturation and memory consolidation

NR3A is the only NMDA receptor (NMDAR) subunit that downregulates sharply prior to the onset of sensitive periods for plasticity, yet the functional importance of this transient expression remains unknown. To investigate whether removal/replacement of juvenile NR3A-containing NMDARs is involved in experience-driven synapse maturation, a reversible transgenic system was used that prolonged NR3A expression in the forebrain. Removal of NR3A is required to develop strong NMDAR currents, full expression of long-term synaptic plasticity, a mature synaptic organization characterized by more synapses and larger postsynaptic densities, and the ability to form long-term memories. Deficits associated with prolonged NR3A were reversible, as late-onset suppression of transgene expression rescued both synaptic and memory impairments. These results suggest that NR3A behaves as a molecular brake to prevent the premature strengthening and stabilization of excitatory synapses and that NR3A removal might thereby initiate critical stages of synapse maturation during early postnatal neural development (Roberts, 2009).

NMDARs are heteromers containing NR1 subunits and combinations of four NR2 (A-D) and two NR3 (A-B) subunits. Most research to date has focused on the role of the developmental switch between predominantly NR2B- to NR2A-containing NMDARs because it occurs in many brain regions and can be driven by neural activity and experience. In contrast, little is known about the importance of the removal/replacement of NR3A-containing NMDARs despite growing evidence that nonconventional NR3A subunits are well positioned to be potent regulators of NMDAR function. First, inclusion of NR3A results in the formation of heteromeric receptors (NR1/NR2/NR3A) with reduced Ca2+ permeability and low sensitivity to Mg2+ blockade, thus modifying two major properties of classical NMDARs (NR1/NR2 heteromers). Second, NR3A is prominently expressed during a narrow temporal window of postnatal development that correlates with periods of intense synaptogenesis and pruning and later becomes downregulated, just prior to the onset of critical period plasticity. Third, genetic deletion of NR3A increases spine density, indicating that NR3A might participate in synapse formation or elimination. Finally, removal of NR3A-containing NMDARs can be regulated by activity via selective recruitment of the endocytic adaptor PACSIN/syndapin 1 (see Drosophila Syndapin), suggesting that regulated removal might occur at the level of individual synapses rather than nonselectively along entire dendritic branches or trees. Yet, if and how NR3A subunits modulate synaptic plasticity, synapse maturation, or learning is unknown, leaving an important gap in understanding of NMDAR-mediated development. Assessing the role of NR3A may also be important for understanding human disease. For example, genetic variations in NR3A alter human brain function, and abnormal NR3A expression is associated with schizophrenia and bipolar disorder (Roberts, 2009 and references therein).

To address the role of NR3A subunits in synapse development and plasticity, transgenic mice were generated in which NR3A expression could be prolonged beyond its natural time window in postnatal forebrain neurons. Prolonging NR3A expression results in synaptic NMDAR hypofunction, deficits in long-term potentiation (LTP), reduced postsynaptic maturation at Schaffer collateral-CA1 synapses of the hippocampus, and impairs behavioral flexibility and memory consolidation. Conversely, genetic deletion of endogenous NR3A yielded a premature concentration of NMDARs at postsynaptic sites, enhancing synaptic NMDAR currents and promoting an earlier developmental onset of LTP. Both the synaptic and cognitive deficits resulting from persistent NR3A expression could be reversed by suppressing transgene expression at later stages. These findings indicate that the presence of NR3A maintains synapses in an anatomically and functionally juvenile state that is refractory to the induction of LTP and structural plasticity and incapable of supporting long-term memory storage (Roberts, 2009).

NMDA receptor and learning and memory

Brief bath application of N-methyl-D-aspartate (NMDA) to hippocampal slices produces long-term synaptic depression (LTD) in CA1 that is (1) sensitive to postnatal age, (2) saturable, (3) induced postsynaptically, (4) reversible, and (5) not associated with a change in paired pulse facilitation. Chemically induced LTD (chem-LTD) and homosynaptic LTD are mutually occluding, suggesting a common expression mechanism. Using phosphorylation site-specific antibodies, induction of chem-LTD is found to produce a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at serine 845, a cAMP-dependent protein kinase (PKA) substrate, but not at serine 831, a substrate of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). These results suggest that dephosphorylation of AMPA receptors is an expression mechanism for LTD and indicate an unexpected role for PKA in the postsynaptic modulation of excitatory synaptic transmission (Lee, 1998).

The classically conditioned vertebrate eye-blink response is a model in which to study neuronal mechanisms of learning and memory. In this paradigm, an eye-blink reflex in response to a tone can be evoked when the tone is repeatedly paired with an air puff to the cornea that normally elicits the blink response. A neural correlate of this response recorded in the abducens nerve can be conditioned entirely in vitro using an isolated brainstem-cerebellum preparation from the turtle by pairing trigeminal and auditory nerve stimulation. Conditioning requires that the paired stimuli occur within a narrow temporal window of <100 msec. Conditioning is blocked by a NMDA receptor antagonist. Moreover, there is a significant positive correlation between the levels of conditioning and greater immunoreactivity with the glutamate receptor 4 (GluR4) AMPA receptor subunit in the abducens motor nuclei, but not with NMDAR1 or GluR1. It is concluded that in vitro classical conditioning of an abducens nerve eye-blink response is generated by NMDA receptor-mediated mechanisms that may act to modify the AMPA receptor by increasing GluR4 subunits in auditory nerve synapses (Keifer, 2001).

The mechanisms behind the induction of cellular correlates of memory by sensory input and their contribution to meaningful behavioral changes are largely unknown. A graded memory in the form of sensorimotor adaptation has been reported in the electromotor output of electric fish. This study shows that the mechanism for this adaptation is a synaptically induced long-lasting shift in intrinsic neuronal excitability. This mechanism rapidly integrates hundreds of spikes in a second, or gradually integrates the same number of spikes delivered over tens of minutes. Thus, this mechanism appears immune to frequency-dependent fluctuations in input and operates as a simple pulse counter over a wide range of time scales, enabling it to transduce graded sensory information into a graded memory and a corresponding change in the behavioral output. This adaptation is based on an NMDA receptor-mediated change in intrinsic excitability of the postsynaptic neurons involving the Ca2+-dependent activation of TRP channels (Oestreich, 2006).

Memories are dynamic and can change when recalled. The process that returns memories to a labile state during remembering is unclear. The presence of NMDA, but not AMPA, receptor antagonists in the amygdala prior to recall prevented the consolidated fear memory from returning to a labile state. These findings suggest that NMDA receptors in the amygdala are critical for transforming a memory from a fixed to a labile state (Ben Mamou, 2006).

The hippocampus is considered to play a role in allocentric but not in egocentric spatial learning. How does this view fit with the emerging evidence that the hippocampus and possibly related cortical areas are necessary for episodic-like memory, i.e., in all situations in which events need to be spatially or sequentially organized? Are NMDA receptor-dependent mechanisms crucial for the acquisition of spatiotemporal relationships? To address this issue, knock-out (KO) mice lacking hippocampal CA1 NMDA receptors and presenting a reduction of these receptors in the deep cortical layers (NR1-KO mice) were used. A new task (the starmaze) was designed, allowing the distinguishing of allocentric and sequential-egocentric memories. NR1-KO mice were impaired in acquiring both types of memory. These findings suggest that memories composed of multiple spatiotemporal events require intact NMDA receptors-dependent mechanisms in CA1 and possibly in the deep cortical layers (Rondi-Reig, 2006).

Emotions generally improve memory, and the basolateral amygdala (BLA) is believed to mediate this effect. After emotional arousal, BLA neurons increase their firing rate, facilitating memory consolidation in BLA targets. The enhancing effects of BLA activity extend to various types of memories, including motor learning, which is thought to involve activity-dependent plasticity at corticostriatal synapses. However, the underlying mechanisms are unknown. The NMDA-to-AMPA ratio is nearly twice as high at BLA as compared with cortical synapses onto principal striatal neurons; activation of BLA inputs greatly facilitates long-term potentiation induction at corticostriatal synapses. This facilitation is NMDA-dependent, but it occurs even when BLA and cortical stimuli are 0.5 s apart during long-term potentiation induction. Overall, these results suggest that BLA activity opens long time windows during which the induction of corticostriatal plasticity is facilitated (Popescu, 2007).

Animals recognize a taste cue as aversive when it has been associated with post-ingestive malaise; this associative learning is known as conditioned taste aversion (CTA). When an animal consumes a new taste and no negative consequences follow, it becomes recognized as a safe signal, leading to an increase in its consumption in subsequent presentations (attenuation of neophobia, AN). It has been shown that the nucleus accumbens (NAcc) has an important role in taste learning. To elucidate the involvement of NMDA and muscarinic receptors in the NAcc during safe and aversive taste memory formation, bilateral infusions of DL-2-amino-5-phosphonopentanoic acid (APV) or scopolamine were administrated in the NAcc shell or core respectively. The results showed that pre-training injections of APV in the NAcc core and shell disrupted aversive but not safe taste memory formation, whereas pre-training injections of scopolamine in the NAcc shell, but not core, disrupted both CTA and AN. These results suggest that muscarinic receptors seem to be necessary for processing taste stimuli for either safe or aversive taste memory, whereas NMDA receptors are only involved in the aversive taste memory trace formation (Ramirez-Lugo, 2007).

Extinction of conditioned fear is an active learning process requiring NMDA receptors, but the timing, location, and neural mechanisms of NMDAR-mediated processing in extinction are a matter of debate. This study shows that infusion of the NMDAR antagonist CPP into the ventromedial prefrontal cortex (vmPFC) prior to, or immediately after, extinction training impairs 24 hr recall of extinction. These findings indicate that consolidation of extinction requires posttraining activation of NMDARs within the vmPFC. Using multichannel unit recording, it is observed that CPP selectively reduces burst firing in vmPFC neurons, suggesting that bursting in vmPFC is necessary for consolidation of extinction. In support of this, it was found that the degree of bursting in infralimbic vmPFC neurons shortly after extinction predicts subsequent recall of extinction. It is suggested that NMDAR-dependent bursting in the infralimbic vmPFC initiates calcium-dependent molecular cascades that stabilize extinction memory, thereby allowing for successful recall of extinction (Burgos-Robles, 2007).

NMDA-receptor-mediated, cell-specific integration of new neurons in adult dentate gyrus

New neurons are continuously integrated into existing neural circuits in adult dentate gyrus of the mammalian brain. Accumulating evidence indicates that these new neurons are involved in learning and memory. A substantial fraction of newly born neurons die before they mature and the survival of new neurons is regulated in an experience-dependent manner, raising the possibility that the selective survival or death of new neurons has a direct role in a process of learning and memory--such as information storage--through the information-specific construction of new circuits. However, a critical assumption of this hypothesis is that the survival or death decision of new neurons is information-specific. Because neurons receive their information primarily through their input synaptic activity, whether the survival of new neurons is regulated by input activity in a cell-specific manner was investigated. A retrovirus-mediated, single-cell gene knockout technique was developed in mice and it was shown that the survival of new neurons is competitively regulated by their own NMDA-type glutamate receptor during a short, critical period soon after neuronal birth. This finding indicates that the survival of new neurons and the resulting formation of new circuits are regulated in an input-dependent, cell-specific manner. Therefore, the circuits formed by new neurons may represent information associated with input activity within a short time window in the critical period. This information-specific addition of new circuits through selective survival or death of new neurons may be a unique attribute of new neurons that enables them to play a critical role in learning and memory (Tashiro, 2006).

Adenylyl cyclase 1 is a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. This study reports that genetic deletion of AC1 significantly attenuates neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion does not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, neuronal excitotoxicity induced by an intracortical injection of NMDA was studied. Cortical lesions induced by NMDA are significantly reduced in AC1 but not in AC8 knock-out mice. These findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases (Wang, 2007).

Decoding NMDA receptor signaling: Identification of genomic programs specifying neuronal survival and death

NMDA receptors promote neuronal survival but also cause cell degeneration and neuron loss. The mechanisms underlying these opposite effects on neuronal fate are unknown. Whole-genome expression profiling revealed that NMDA receptor signaling is decoded at the genomic level through activation of two distinct, largely nonoverlapping gene-expression programs. The location of the NMDA receptor activated specifies the transcriptional response: synaptic NMDA receptors induce a coordinate upregulation of newly identified pro-survival genes and downregulation of pro-death genes. Extrasynaptic NMDA receptors fail to activate this neuroprotective program, but instead induce expression of Clca1, a putative calcium-activated chloride channel that kills neurons. These results help explain the opposing roles of synaptic and extrasynaptic NMDA receptors on neuronal fate. They also demonstrate that the survival function is implemented in neurons through a multicomponent system of functionally related genes, whose coordinate expression is controlled by specific calcium signal initiation sites (Zhang, 2007).

Role of NMDA receptors in dopamine neurons for plasticity and addictive behaviors

A single exposure to drugs of abuse produces an NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA receptor (AMPAR) currents in DA neurons; however, the importance of LTP for various aspects of drug addiction is unclear. To test the role of NMDAR-dependent plasticity in addictive behavior, functional NMDAR signaling was genetically inactivated exclusively in DA neurons (KO mice). Inactivation of NMDARs results in increased AMPAR-mediated transmission that is indistinguishable from the increases associated with a single cocaine exposure, yet locomotor responses to multiple drugs of abuse were unaltered in the KO mice. The initial phase of locomotor sensitization to cocaine is intact; however, the delayed sensitization that occurs with prolonged cocaine withdrawal did not occur. Conditioned behavioral responses for cocaine-testing environment were also absent in the KO mice. These findings provide evidence for a role of NMDAR signaling in DA neurons for specific behavioral modifications associated with drug seeking behaviors (Zweifel, 2008).

Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B

N-methyl-D-aspartate receptors (NMDARs) play important functions in neural development. NR2B is the predominant NR2 subunit of NMDAR in the developing brain. This study used mosaic analysis with double markers (MADM) to knock out NR2B in isolated single cells and analyze its cell-autonomous function in dendrite development. NR2B mutant dentate gyrus granule cells (dGCs) and barrel cortex layer 4 spiny stellate cells (bSCs) have similar dendritic growth rates, total length, and branch number as control cells. However, mutant dGCs maintain supernumerary primary dendrites resulting from a pruning defect. Furthermore, while control bSCs restrict dendritic growth to a single barrel, mutant bSCs maintain dendritic growth in multiple barrels. Thus, NR2B functions cell autonomously to regulate dendrite patterning to ensure that sensory information is properly represented in the cortex. This study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning can be separated from those that control general dendrite growth and branching (Espinosa, 2009).

The dendrites of CNS neurons play a critical role in integrating synaptic inputs from a multitude of presynaptic partners and, subsequently, in determining the extent to which a neuron transmits this information to its postsynaptic partners. Characteristic dendritic arborization patterns allow neurons to perform signal processing and computation appropriate for their functions. For example, in the adult somatosensory cortex of rodents, most layer 4 stellate neurons orient their dendrites toward a single barrel center to maximize contacts with thalamocortical afferents representing a single whisker. The development of dendritic trees characteristic to specific neuronal types is believed to result from the interplay between intrinsic genetic programs, extracellular signals, and electrical activity. Despite an increasing understanding of dendrite development, it is unclear if mechanisms that sculpt specific dendrite patterns are an integral part of those that control dendrite growth and branching, or if independent mechanisms can regulate these two aspects of dendrite development(Espinosa, 2009).

N-methyl-D-aspartate-type glutamate receptors (NMDARs) play a central role in activity-dependent regulation of dendrite development. NMDARs function mainly as heterotetramers of two obligate NR1 subunits and a combination of two NR2 subunits (A-D). Each NMDAR subunit combination confers distinct functional properties, including the regulation of unitary conductance, binding affinity, and gating and desensitization kinetics. For example, compared to NR2A-containing receptors, NR2B-containing NMDARs have a 3- to 4-fold slower decay time course of NMDAR-mediated excitatory postsynaptic currents, resulting in a larger Ca2+ influx. In the mammalian brain, the NR1 mRNA is found ubiquitously, whereas the NR2 subunits are differentially expressed, both temporally and spatially. At embryonic stages, the NR2B subunit is expressed in the entire brain, while the NR2D subunit is expressed selectively in the diencephalon, the mesencephalon, and the spinal cord. From the time of birth to adulthood, expression of NR2B becomes restricted to the forebrain and NR2D expression peaks 1 week after birth but is then strongly reduced. During this time, the expression levels increase for NR2A in the forebrain and for NR2C in the cerebellum. The expression pattern of NR2B suggests that it plays a more important role during development than do other NR2 subunits. Indeed, NR2B knockout mice die shortly after birth, similar to NR1 knockout mice, but knockout mice for NR2A, 2C, or 2D are fully viable (Espinosa, 2009).

Pharmacological agents that block NMDARs have been used to study their function in dendrite development. In the Xenopus retinotectal system, NMDAR antagonists inhibit dendritic arborization of tectal neurons during development (Rajan, 1998) or in response to visual stimulation (Sin, 2002). NMDAR function in dendrite development has also been examined in knockout mice. In the cortex-specific NR1 knockout, individual layer 4 stellate cells lose oriented arborization and grow exuberant dendrites and spines (Datwani, 2002). NMDARs are also necessary for dendritic spine formation induced by sensory activity and long-term potentiation (Engert, 1999; Maletic-Savati, 1999). Cortex-specific NR1 knockout results in reduced spine densities (Ultanir, 2007). Although these studies have revealed important functions for NMDARs in multiple aspects of dendrite development, it is unclear to what extent the observed defects are caused by the cell-autonomous perturbation of NMDAR function. These experiments cannot exclude secondary consequences of perturbing the NMDAR in other neurons in the circuit. In the Xenopus retinotectal system, NMDAR blockade also affects the arborization of the retinal ganglion cell axon termini (Cline, 1990; Ruthazer, 2003), which may indirectly perturb tectal cell dendrite development. In cortex-specific NR1 knockout mice, although thalamocortical axons are genetically unperturbed, their terminal arborization patterns are grossly altered in response to NR1 knockout in cortical cells (Lee, 2005), and barrels do not form properly (Datwani, 2002). It is therefore difficult to determine if the unoriented dendrites of layer 4 stellate neurons reflect the cell-autonomous requirement for NMDAR or if they are a secondary consequence of the general pattern formation defects in the barrel cortex. Recently, genetic perturbations of NR2A and NR2B subunits have been reported using overexpression and morpholino-mediated knockdown in single Xenopus tectal cells. Compared to overexpression, knockdown of NR2B has minor consequences on dendrite development (Espinosa, 2009).

This study used the mosaic analysis with double markers (MADM) system to knock out NR2B in isolated single neurons to assess the cell-autonomous function of NR2B in dendrite development. MADM permits simultaneous gene inactivation and distinct labeling of homozygous mutant cells and their wild-type siblings in the same animal through Cre/LoxP-mediated interchromosomal mitotic recombination events. Moreover, infrequent recombination generates isolated single knockout cells, allowing unambiguous assessment of cell-autonomous function of genes. This study found that in two types of neurons analyzed, dGCs and bSCs, NR2B is dispensable for general dendrite growth and branching but is required for dendrite patterning critical for information processing. This study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning are separable from those that control general dendrite growth and branching (Espinosa, 2009).

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date revised: 5 August 2023

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