Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. This study investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, protein kinases functionally interacting with OAMB were explored by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from the ovary to the uterus (Lee, 2009).

Mating activates highly coordinated physiological processes in the Drosophila female. During copulation, the female receives somatosensory stimulation, sperm and seminal proteins from the male. These mating signals act at multiple sites in the mated female to activate post-mating responses required for successful reproduction. For example, the seminal protein Ovulin stimulates egg-laying for 1 day after mating (Herndon, 1995). Ovulin is not only present in the base of the ovary right after copulation but it also enters the circulatory system, possibly acting at additional sites. Moreover, the seminal sex peptides Acp70A and DUP99B reduce sexual receptivity and stimulate egg-laying. While sex peptides have widespread binding sites in the central nervous system, endocrine glands, and reproductive tissues in the female, it is the sex peptide receptor SPR in the neurons expressing the sex determination factor Fruitless that is indispensable for reduced receptivity as well as increased egg-laying (Lee, 2009).

The downstream targets and mechanisms that Ovulin and SPR activate in the mated female are unknown. Several studies, nonetheless, indicate octopamine as a key neuromessenger for ovulation (Lee, 2003; Monastirioti, 2003; Cole, 2005; Rodriguez-Valentin, 2006; Middleton, 2006), suggesting it being a downstream signal of Ovulin or SPR for egg-laying. Octopamine is a major monoamine in insects and has similar functions to mammalian norepinephrine. Octopamine is synthesized from tyrosine by sequential actions of tyrosine decarboxylase (dTdc) and tyramine beta-hydroxylase (Tβh). The females defective in dTdc2 encoding neuronal dTdc or tβh are sterile due to defective egg-laying (Monastirioti, 2003; Cole, 2005). Octopaminergic neurons innervate numerous brain and thoracico-abdominal ganglion (TAG) areas. In addition, octopaminergic neurons in the TAG project to reproductive tissues such as the ovaries, oviducts, sperm storage organs and uterus. Indeed, the sterility of tβh females is rescued by restored Tβh expression in a subset of neurons including the TAG neurons that innervate the reproductive system. Consistently, octopamine, when applied to the dissected reproductive system, modulates muscle activities in a tissue-specific manner: it enhances muscle contraction in the ovary but inhibits it in the oviduct (Rodriguez-Valentin, 2006; Middleton, 2006). This suggests that distinct octopamine receptors present in the ovary and oviduct mediate the opposite actions of octopamine on muscle activity (Lee, 2009).

Drosophila has four known octopamine receptors: OAMB, Octβ1R, Octβ2R and Octβ3R. The oamb gene encodes two isoforms OAMB-K3 (K3) and OAMB-AS (AS), which are produced by alternative splicing of the last exon, and differ in the third cytoplasmic loop and downstream sequence. Both K3 and AS transcripts are found in the brain, TAG and reproductive system. When assayed in the heterologous cell lines, both isoforms activate an increase in intracellular Ca2+ while K3 also stimulates a cAMP increase. This implies that the two isoforms may activate distinct combinations of signal transduction pathways in vivo. To investigate OAMB's in vivo functions, several oamb mutants defective in both K3 and AS have been generated, and their prominent phenotype is female sterility (Lee, 2003). While oamb mutant females show normal mating, they are impaired in ovulation, causing abnormal retention of mature eggs in the ovary (Lee, 2003). This raises several important questions regarding mechanism of OAMB activity: where (brain, TAG or reproductive system) does OAMB regulate ovulation? Which isoform is critical for this process and what are the downstream signals? This study shows that the critical site for the OAMB's function in ovulation is the oviduct epithelium, in which transgenic expression of either K3 or AS isoform is sufficient to rescue the oamb female's ovulation defect. Moreover, OAMB recruits CaMKII as a key downstream effector for this function (Lee, 2009).

Octopamine, as a major neurotransmitter, neuromodulator and neurohormone, regulates diverse physiological processes in invertebrates that include sensory information processing, egg-laying, fight or flight responses, and complex neural functions such as learning and memory (Roeder, 2005). These astonishingly diverse effects of octopamine are initiated by the binding of octopamine to G-protein-coupled receptors expressed in distinct tissue or cell types; however, very little is known about relevant octopamine receptors and underlying cellular mechanisms that mediate octopamine's physiological functions. This work has shown that OAMB regulates ovulation in the oviduct epithelium and recruits CaMKII for this function. This role of OAMB is physiological, as opposed to developmental, since restored OAMB expression in the oviduct epithelium at the adult stage is sufficient for reinstating ovulation in oamb females. This is consistent with the findings observed in the octopamine-less dTdc2 and tβh females, in which feeding octopamine only at the adult stage rescues the sterility phenotype of both mutants (Cole, 2005; Lee, 2009 and references therein).

Sex peptides transferred to the female during copulation enhance egg-laying upon binding to the receptor SPR expressed in the Fruitless neurons. The mechanism by which the Fruitless neurons stimulate egg-laying is unknown; however, octopaminergic neurons in the TAG likely represent a downstream target since the egg-laying phenotype of tβh females is rescued by restored TβH expression in these neurons (Monastirioti, 2003). The TAG octopaminergic neurons project axons to various areas in the reproductive track including the ovary, lateral and common oviducts, sperm storage organs and the uterus (Rodriguez-Valentin, 2006; Middleton, 2003). Mating induces distinctive changes in vesicle release at the nerve terminals in different areas of the reproductive track, some of which may represent the TAG octopamine neuronal activities. In the dissected reproductive system, octopamine application augments the amplitude of myogenic contractions of the peritoneal sheath in the ovary while it inhibits stimulated muscle contractions of the oviduct (Rodriguez-Valentin, 2006; Middleton, 2003). These opposite effects of octopamine may be crucial for coordinated constriction and relaxation of the ovary and oviduct, respectively, in transferring a mature egg to the uterus. OAMB may serve as a receptor processing the octopamine's input in the oviduct while another octopamine receptor may mediate the constriction signal in the ovarian peritoneal sheath, which lacks OAMB expression (Lee, 2009).

Remarkably, OAMB's activity is required in the epithelium rather than the muscle for normal ovulation. Consistent with this, the histochemical analysis reported here reveals extensive innervation of the TAG octopamine neuronal processes into the oviduct epithelial layer where both OAMB isoforms are enriched, in addition to the muscle. This raises an important question regarding the nature of an OAMB's role in ovulation. While no information is available on the oviduct epithelium in Drosophila or other insects, studies of the mammalian oviduct indicate active roles of the epithelium in fluid secretion and ciliary activity for gamete and embryo transport. Similarly, it is possible that OAMB in the Drosophila oviduct epithelium is involved in regulating fluid secretion to establish proper luminal environment and possibly ciliary action for egg transport. The capacity of either OAMB-K3 or OAMB-AS to reinstate ovulation in oamb females strongly implicates intracellular Ca²⁺ rather than cAMP as a downstream effector. This is corroborated by findings demonstrating CaMKII as a key epithelial component downstream of OAMB. It is uncertain whether individual isoforms or two isoforms together have comparable efficacies in activating CaMKII and ovulation. Future studies employing quantitative manipulation of transgenic OAMB expression may clarify this issue. Taken together, the epithelial OAMB stimulated upon mating likely activates CaMKII via increased intracellular Ca²⁺, which may in turn trigger biochemical changes necessary for fluid secretion. Potential molecules involved in this process may include transporters, ion channels, Na⁺-K⁺-ATPase and the molecules involved in cilia movements. In the absence of OAMB, epithelial cell activities and fluid may be inadequate for egg movement, leading to ovulation failure. Since octopamine induces relaxation in the dissected oviduct, relaxation may involve another octopamine receptor in the muscle, and concerted activities of OAMB and a muscle receptor may be crucial for successful egg transport. This working model is currently under test (Lee, 2009).

Octopamine regulates oviduct activities in other insects as well. In the locust oviduct, octopamine inhibits the basal tonus and neurally evoked muscle contractions, which are mediated by cAMP-dependent mechanisms. These effects of octopamine may be mediated by an OAMB-like receptor with the different intracellular effector cAMP. Alternatively, they may involve another octopamine receptor(s) present in the muscle. Drosophila has three octopamine receptors (OctβR1, 2 and 3) that can also stimulate cAMP increases. Spatial expression patterns of three OctβRs are as yet unknown. It is conceivable that an OctβR or OctβR-like receptor, possibly present in the Drosophila or locust oviduct muscle, respectively, is additionally involved in ovulation by inducing muscle relaxation through a cAMP signaling pathway. At present, molecular components and cellular pathways controlling ovulation are largely unknown and likewise very little is known about the oviduct functions and mechanisms. The current findings uncover the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. Future studies to identify additional downstream effectors of OAMB and their functions should help further understanding of the important reproductive process ovulation and provide novel insights into the development of effective insecticides. Typically, intracellular signals activated by G-protein-coupled receptors are characterized in in vitro cell lines. This study has identified the intracellular signal activated by the G-protein-coupled receptor OAMB in vivo that has functional significance. Similar approaches could be applied to other receptors to investigate rather poorly defined cellular mechanisms that G-protein-coupled receptors activate for their in vivo functions (Lee, 2009 and references therein).

Norepinephrine, a mammalian counterpart of octopamine, also plays profound roles in female reproduction by acting on the reproductive and nervous systems. Sympathetic nerve terminals containing norepinephrine innervate the ovaries, oviducts, and uterus. Moreover, norepinephrine levels in the human fallopian tube vary in a region- and estrous cycle-dependent manner being the highest in the isthmus and the fimbriated end at the time of ovulation. When assayed in vitro, adrenergic receptor agonists not only modulate oviduct muscle activities but they also stimulate fluid secretion possibly via Ca²⁺-dependent mechanisms. Oviduct fluid in mammals is critical for egg transport, maturation and fertilization; however, the cellular process regulating its secretion is largely unknown. Damage in the oviduct epithelium is associated with pelvic inflammatory disorder, leading to hydrosalpinx formation and reduced fertility. Thus, enhanced understanding of physiological and cellular factors and processes controlling oviduct fluid will provide significant insights into healthy reproduction as well as impaired fertility associated with pelvic inflammatory disorder and other related disorders (Lee, 2009).

Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca²⁺-induced Ca²⁺ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca²⁺/calmodulin-dependent kinase II (CamKII). This study shows that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase (SERCA) to deplete presynaptic ER Ca²⁺ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR-CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores (Wong, 2009).

The function of nerve terminals depends on vesicular delivery of proteins synthesized in the soma to synaptic boutons. Transport vesicles are known to contain channels, active zone constituents and neuropeptides. In contrast to synaptic membrane proteins and classical transmitters that are recycled following exocytosis, neuropeptide release is irreversible. Thus, peptidergic transmission depends on replacement of neuropeptide-containing dense core vesicles (DCVs). This is potentially a very slow process because delivery of vesicles synthesized in the soma to nerve terminals by fast axonal transport can take days. However, a cell biological strategy has been discovered that bypasses such delays. Activity-dependent capture of transiting vesicles utilizes a pool of DCVs that rapidly pass through the resting nerve terminal, but that are captured in response to a burst of activity (Shakiryanova, 2006). The onset of this capture, which is evident as decreased DCV efflux and increased neuropeptide content in synaptic boutons, occurs over a period of minutes instead of the hours that would be required for conventional steady state DCV replacement. Essentially, the nerve terminal can tap into the transiting DCV pool to rapidly replenish neuropeptide stores without any direct involvement of the soma. Hence, activity-dependent capture of transiting DCVs eliminates the delay in delivering nascent DCVs, apportions resources based on activity and places control of synaptic neuropeptide storage at sites of release instead of the site of synthesis (i.e., the soma) (Shakiryanova, 2006). A similar recruitment process also occurs with neurotrypsin-containing vesicles, which were concluded to rapidly undergo exocytosis following stimulated capture (Frischknecht, 2008). Likewise, vesicle capture appears to be involved in release of presynaptic Wnt/Wingless protein. Therefore, activity-dependent capture of transiting vesicles supports synaptic neuropeptide, enzyme and developmental peptide release (Wong, 2009).

The signaling required for activity-dependent capture of transiting DCVs is unknown. The long duration of this response in Drosophila motor neurons (i.e., for ~0.5 hour) coupled with the requirement for electrical activity suggests a potential involvement of Ca²⁺-induced phosphorylation. In fact, recent experiments have shown that such signaling increases the mobility of resident DCVs in synaptic boutons. Mobilization, which is triggered by Ca²⁺ influx and persists for ~10 minutes (Shakiryanova, 2005), requires Ca²⁺-induced Ca²⁺ release from presynaptic endoplasmic reticulum (ER) via ryanodine receptors (RyRs) (Shakiryanova, 2007). Subsequently, Ca²⁺/calmodulin-dependent protein kinase II (CamKII) is activated as a necessary step for DCV mobilization (Shakiryanova, 2007). The overlapping onset of the capture and mobilization responses in the first minutes following a brief tetanus stimulated an investigation of whether the RyR-CamKII pathway participates in activity-dependent capture of transiting vesicles (Wong, 2009).

In this study a GFP (Green Fluorescent Protein)-tagged neuropeptide was imaged at the intact Drosophila neuromuscular junction. The rebound in synaptic neuropeptide stores following activity-evoked release, which is caused by capture of transiting vesicles (Shakiryanova, 2006), requires RyR-mediated Ca²⁺ efflux from presynaptic ER and activation of CamKII. Therefore, RyR-CamKII signaling initiates both mobilization of resident DCVs within synaptic boutons and capture of DCVs from the rapidly transiting pool (Wong, 2009).

In vivo imaging has shown that a brief bout of activity elicits prolonged DCV mobilization and capture. These processes are independent because capture requires axonal transport while mobilization does not (Shakiryanova, 2005; Shakiryanova, 2006). Nevertheless, the onsets of mobilization of resident DCVs and capture of transiting DCVs overlap (i.e., both develop over minutes following seconds of activity). This observation stimulated a test of the hypothesis that these two mechanisms are initiated by the same signaling. Previous studies had established that Ca²⁺ influx triggers DCV mobilization by activating RyR-mediated Ca²⁺ release from presynaptic ER that in turn stimulates CamKII (Shakiryanova, 2007). The pharmacological and genetic experiments presented in this study establish that RyR-CamKII signaling is also required for activity-dependent capture of transiting DCVs (Wong, 2009).

This finding raises the issue of how a single signaling pathway produces responses with different durations: after seconds of activity, DCV mobilization lasts ~10 minutes, while the capture response lasts ~40 minutes (Shakiryanova, 2005; Shakiryanova, 2006). One possible consideration is that these kinetic differences could originate in the processes responsible for reversal of mobilization and capture. Specifically, RyR-CamKII signaling could initiate the two processes in parallel, but dephosphorylation of distinct CamKII substrates might occur at different rates, possibly because of the involvement of different phosphatases. This potential explanation suggests that identifying the CamKII substrates that mediate mobilization and capture will be important for understanding the diversity in long-lasting responses initiated by activity-triggered presynaptic RyR-CamKII signaling. Recently, CamKII-dependent phosphorylation of kinesin superfamily protein 17 (KIF17) was found to be essential for unloading NMDA receptor-carrying cargoes from microtubules near the postsynaptic density (Guillaud, 2008). Therefore, CamKII might induce capture by triggering dissociation of transiting DCVs from their molecular motor dynactin complex, which would contain both a kinesin-3 family member UNC-104/Kif1 and a dynein retrograde motor, while mobilization might depend on another CamKII substrate. Alternatively, some process downstream of dephosphorylation might be rate determining for reversal of capture. For example, once captured vesicles are committed to return to the transiting pool, they might need to recruit an unoccupied motor complex to support rapid transiting. If such complexes are rare, then recovery from capture would be very slow. In contrast, recovery from mobilization, which does not require exiting from the bouton, would not be limited in the same way. Regardless of the specific basis for the diverse time courses of mobilization and capture, the use of the same signaling pathway to induce both of these effects is an elegant means to ensure that facilitation of release is coupled to replacement of depleted synaptic neuropeptide stores (Wong, 2009).

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca(2+) mobilization from the endoplasmic reticulum (ER) and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca(2+) release. This study addresses these issues in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. It is reported that neuronal excitation acts with ER Ca(2+) stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca(2+) provided by different presynaptic ER Ca(2+) release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca(2+) in the terminal, while translocation to AZs requires Ca(2+) microdomains generated by IP3Rs (Shakiryanova, 2011).

Although CaMKII alters nerve terminal excitability, neurosecretion, and development, direct studies of CaMKII activation and translocation in living nerve terminals are lacking because of technical limitations. For example, the low level of CaMKII in the nerve terminal compared with the postsynaptic density, as well as the small size of most boutons, hinders light level in immunohistochemical assays of presynaptic CaMKII translocation. Electron microscopy coupled with immunodetection overcomes limitations in spatial resolution, but cannot follow CaMKII dynamics in a living preparation. Thus, the dependence of presynaptic CaMKII enzymatic activity and translocation on stimulation frequency, ER Ca2+ channels, and autophosphorylation was unknown. In this study a FRET indicator that detects both activation and translocation was shown to reproduce known features of presynaptic CaMKII behavior and was then used to determine how CaMKII dynamics is controlled in an intact living nerve terminal (Shakiryanova, 2011).

First, stimulation of presynaptic CaMKII by electrical stimulation, autophosphorylation, and ER Ca2+ channels was quantified in real time. These experiments established that CaMKII activation is surprisingly slow and frequency dependent. This property likely reflects the fact that Ca2+ entry via presynaptic Cav channels alone is not optimal for activating CaMKII. Rather, stimulation of CaMKII also is promoted by presynaptic ER Ca2+ release and autophosphorylation. The similarity of CaMKII kinetics induced by disrupting either of the latter processes suggests autophosphorylation is promoted by the increased activation of CaMKII by ER Ca2+. Furthermore, the participation of RyRs and IP3Rs, the requirement for Cav channels, and the insensitivity to knockdown of BRP, which enhances Cav channel clustering at AZs, imply that presynaptic CaMKII activation is driven by global presynaptic Ca2+ elevation induced by ER and surface Ca2+ channels (Shakiryanova, 2011).

Imaging experiments also revealed that CaMKII translocation toward AZs detected by immunoelectron microscopy in depolarized cultured neurons also occurs with native electrical activity in vivo. Experiments that used BRP immunolocalization or somewhat less efficient transgenic fluorescent protein labeling showed that presynaptic CaMKII clusters usually accumulate at AZs. This does not reflect a simple aggregation of CaMKII already localized near AZs because CaMKII initially localized away from AZs was depleted. Furthermore, once fully formed CaMKII clusters localized near AZs, their motion was limited. Therefore, it seems that initially diffuse presynaptic CaMKII forms clusters that are tethered near AZs (Shakiryanova, 2011).

Strikingly, disrupting autophosphorylation, which reduces activation, does not affect presynaptic CaMKII translocation. This might reflect that activating a subset of the subunits in the CaMKII dodecamer is sufficient for translocation. However, translocation is not a simple consequence of limited activation because, even though CaMKII activation tends to be greater after inhibiting IP3Rs than RyRs, translocation is more markedly disrupted by IP3R inhibition. Furthermore, CaMKII clustering at AZs can long outlast stimulated elevation of presynaptic Ca2+, implying that Ca2+ alone cannot account for persistent translocation. Finally, CaMKII translocates only to a subset of AZs, suggesting heterogeneity between AZs. Differential release between AZs in a single type Ib bouton has recently been demonstrated. Hence, it is possible that a single mechanism induces heterogeneity in AZ-mediated transmitter release and recruitment of CaMKII (Shakiryanova, 2011).

The results suggest that presynaptic CaMKII translocation is preferentially induced by IP3R-generated Ca2+ microdomains located at a subset of AZs. Consistent with this proposal, CaMKII translocation requires the AZ-associated scaffold protein BRP, which is long enough to separately integrate signals from Ca2+ microdomains generated by AZ Cav channel clusters at its C terminus and IP3Rs potentially located at its N terminus. BRP might affect CaMKII translocation also because it enhances Cav channel clustering and it is a potential CaMKII substrate. In contrast, RyRs may be positioned further away from BRP so that their local Ca2+ release cannot mimic the action of IP3Rs. Such a differential distribution of ER Ca2+ channels has not been studied in Drosophila nerve terminals, but is consistent with independent localization and function of RyRs and IP3Rs detected in other cell types and neuronal compartments. According to this view, diffuse CaMKII samples global Ca2+ changes induced by Cav and all ER Ca2+ channels to activate, but local Ca2+ changes generated by IP3Rs are needed for recruitment of CaMKII. With such a model, depletion of the IP3-sensitive Ca2+ pool would account for the observed dispersal of clusters that sometimes occurs during stimulation (Shakiryanova, 2011).

These experiments suggest that translocation is limited to a subset of AZs and is delayed compared with CaMKII activation and synaptic transmission. Thus, it is unlikely that translocation occurs for rapid acute regulation of transmitter release. Indeed, the protocols typically used for quantal analysis (e.g., low-frequency action potentials in the presence of low Ca2+) are unlikely to induce much CaMKII signaling. However, motor neurons fire in bursts that in vivo imaging experiments show are sufficient to translocate presynaptic CaMKII. Thus, future experiments will have to use more physiological stimuli to probe whether CaMKII is recruited to AZs to regulate synaptic transmission. Such effects could be selective, possibly because CaMKII is recruited to AZs that are most active and/or positioned near ER IP3Rs. Interestingly, another consequence of translocation to AZs is that CaMKII is depleted from the presynaptic cytoplasm. Hence, CaMKII activity can be redirected away from substrates that are excluded from AZs (e.g., neuropeptide vesicles and K+ channels) to substrates preferentially localized at AZs. In this way, the role of CaMKII in the nerve terminal changes dynamically depending both on its enzymatic activity and location (Shakiryanova, 2011).

Ligand activation of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K in both the mammalian central nervous system and Drosophila motor nerve terminal. In several subregions of the mammalian brain, mGluR-mediated PI3K activation is essential for a form of synaptic plasticity termed long-term depression (LTD), which is implicated in neurological diseases such as fragile X and autism. In Drosophila larval motor neurons, ligand activation of DmGluRA, the sole Drosophila mGluR, similarly mediates a PI3K-dependent downregulation of neuronal activity. The mechanism by which mGluR activates PI3K remains incompletely understood in either mammals or Drosophila. This study identified CaMKII and the nonreceptor tyrosine kinase DFak as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. Transgene-induced CaMKII inhibition or the DFak^CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. It was also found that CaMKII activation induces other PI3K-dependent effects, such as increased motor axon diameter and increased synapse number at the larval neuromuscular junction. CaMKII, but not PI3K, requires DFak activity for these increases. It is concluded that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak (Lin, 2011).

Metabotropic glutamate receptors (mGluRs), G protein-coupled receptors for which glutamate is ligand, mediate aspects of synaptic plasticity in several systems. In several regions of the mammalian brain, including the hippocampus, the cerebellum, the prefrontal cortex, and others, ligand activation of group I mGluRs induces a long-term depression of synaptic activity, termed mGluR-mediated long-term depression (LTD). Induction of mGluR-mediated LTD both activates and requires the activation of the lipid kinase PI3 kinase (PI3K) and the downstream kinase Tor. Several genetic diseases of the nervous system are predicted to increase sensitivity to activation of mGluR-mediated LTD. For example, increased sensitivity to induction of mGluR-mediated LTD has been observed in the mouse model for fragile X. Furthermore, the genes affected in tuberous sclerosis (Tsc1 and Tsc2) and neurofibromatosis (Nf1) encode proteins that downregulate Tor activity. These observations raise the possibility that hyperactivation of mGluR-mediated LTD plays a causal role in the neurological phenotypes of fragile X, neurofibromatosis and tuberous sclerosis. Because these diseases are each associated with an extremely high incidence of autism spectrum disorders (ASDs), and because several lines of evidence suggest that elevated PI3K activity is associated with ASDs, it has been hypothesized that hyperactivation of this pathway might be responsible for ASDs as well. Thus it would be of interest to identify additional molecular components by which mGluR activation activates PI3K, and yet despite recent advances, this mechanism remains incompletely understood (Lin, 2011).

In Drosophila larval motor neurons, glutamate activation of the single mGluR, called DmGluRA, downregulates neuronal excitability; glutamate both activates PI3K and requires PI3K activity for this downregulation). Because glutamate is the excitatory neurotransmitter at the Drosophila neuromuscular junction (NMJ), it was hypothesized that this DmGluRA-mediated downregulation of neuronal excitability carried out a negative feedback on activity: glutamate released from motor nerve terminals would activate DmGluRA autoreceptors, which would then depress excitability (Lin, 2011).

This study identified additional molecular components that mediate the activation of PI3K by DmGluRA in Drosophila larval motor nerve terminals. It was found that activity of the calcium/calmodulin-dependent kinase II (CaMKII) is necessary for glutamate application to activate PI3K, and expression of the constitutively active CaMKII^T287D is sufficient both to activate PI3K even in the absence of glutamate and to confer several other neuronal phenotypes consistent with PI3K hyperactivation. It was also found that CaMKII^T287D requires the nonreceptor tyrosine kinase DFak for this PI3K activation: the DFak^CG1 null mutation blocks the ability of glutamate application to activate PI3K and prevents CaMKII^T287D from hyperactivating PI3K. Finally, CaMKII^T287D expression completely suppresses the hyperexcitability conferred by the DmGluRA null mutation DmGluRA^112b. It is concluded that ligand activation of DmGluRA activates PI3K via CaMKII and DFak (Lin, 2011).

In both mammalian central synapses and Drosophila larval motor neurons, activation by glutamate of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K, but the mechanism by which this activation occurs has not been elucidated. This study identified CaMKII as a critical intermediate in the ability of the single Drosophila mGluR (DmGluRA) to activate PI3K and shows that the ability of both activated DmGluRA and CaMKII to activate PI3K requires the nonreceptor tyrosine kinase, DFak (see A proposed mechanism for the DmGluRA-dependent activation of PI3K via CaMKII and DFak). These results provide novel insights into the mechanism by which DmGluRA activation triggers the observed downregulation of subsequent neuronal activity in Drosophila motor neurons. These results might also be relevant to the mechanism by which mGluR activates PI3K in mammalian central synapses, a process implicated in fragile X, ASDs, neurofibromatosis, and tuberous sclerosis (Lin, 2011).

How might CaMKII lead to the DFak-dependent activation of PI3K? Although the ability of CaMKII to activate PI3K has only recently been reported, it has been well established in mammals that CaMKII phosphorylates both Fak and Pyk2 on multiple serines on the C terminus. These phosphorylation events can activate Pyk2 by enabling subsequent tyrosine phosphorylations (particularly at Tyr402) via mechanisms that are incompletely understood. It has also been well established that Fak and Pyk2, when activated by tyrosine phosphorylation, are each able to activate PI3K: tyrosine-phosphorylated Fak binds p85, the PI3K regulatory subunit, via both the SH3 and SH2 domains. In addition, both tyrosine-phosphorylated Fak and Pyk2 are capable of activating Ras via the conserved Grb2-SoS pathway, which could in principle lead to the Ras-dependent, p85-independent activation of PI3K. These observations raise the possibility that Drosophila CaMKII might similarly activate PI3K by directly phosphorylating and activating DFak. Alternatively, DFak might function in a more indirect fashion, perhaps as a scaffold linking CaMKII and PI3K in a signaling complex. This alternative possibility would suggest that additional intermediates linking CaMKII and PI3K activation exist but are currently unidentified (Lin, 2011).

The observation that DmGluRA-mediated activation of PI3K requires CaMKII implies that DmGluRA activation increases intracellular Ca²⁺ levels in Drosophila motor nerve terminals as a necessary step in PI3K activation. The source of Ca²⁺ for this activation is not known. However in mammals, activation of group I mGluRs, which are responsible for mGluR-mediated LTD in the hippocampus and cerebellum, induce phospholipase C and IP3-mediated Ca²⁺ transients, which are essential intermediates in cerebellar mGluR-mediated LTD. Although the Drosophila DmGluRA is most similar to mammalian group II mGluRs, which are not known to activate Ca²⁺ transients, given that DmGluRA is the sole mGluR in Drosophila, it seems possible that DmGluRA might carry out many of the functions carried out by each of the three groups of mGluRs in mammals, as suggested previously. Alternatively, it is possible that DmGluRA activation might increase intracellular Ca²⁺ via the ryanodine receptor, which was previously shown to be an essential activator of CaMKII in Drosophila larval motor nerve terminals (Lin, 2011).

The ability of CaMKII to activate PI3K requires the nonreceptor tyrosine kinase DFak; the DFak^CG1 null mutation completely blocks the ability of glutamate applied to motor nerve terminals to activate PI3K, completely suppresses the increase in basal p-Akt levels conferred by CaMKII^T287D, and blocks the ability of CaMKII^T287D to confer two additional PI3K-dependent phenotypes: increased synapse number at the NMJ and increased motor axon diameter. These results identify DFak as an essential intermediate in PI3K activation by DmGluRA and CaMKII. However, DFak^CG1 mutants fail to exhibit other phenotypes conferred by decreased PI3K activity: in an otherwise wild-type background, DFak^CG1 larvae exhibit only minor effects on NMJ synapse number or motor axon diameters, which are each significantly decreased by decreased PI3K. These results raise the possibility that, whereas PI3K activation by DmGluRA and CaMKII is blocked in DFak^CG1, total PI3K activity is not strongly decreased because other significant routes to PI3K activation are DFak independent. Alternatively, DFak might participate in signaling pathways distinct from the CaMKII-DFak-PI3K pathway identified in this study that would oppose the effects of PI3K on synapse number and axon diameter. In this view, CaMKII would preferentially promote the ability of DFak to activate PI3K, rather than other DFak-dependent pathways (Lin, 2011).

In several subregions of the mammalian brain, ligand activation of group I mGluRs induces LTD, a type of synaptic plasticity. This induction both activates and requires the activity of PI3K as well as the PI3K-activated kinase Tor. Several lines of evidence have led to the proposal that increased sensitivity to mGluR-mediated LTD induction might underlie specific neurogenetic disorders. In particular, mice null for the gene affected in fragile X, which is associated with an extremely high incidence of autism as well as other cognitive deficits, exhibit increased sensitivity to mGluR-mediated LTD induction in both the hippocampus. Furthermore, the genes identified in two additional diseases associated with a high incidence of autism, neurofibromatosis (Nf1) and tuberous sclerosis (Tsc1 and Tsc2), each encode negative regulators of the PI3K pathway: Nf1 encodes a Ras GTPase activator, which inhibits the PI3K activator Ras, whereas the Tsc proteins are Tor inhibitors that are in turn inhibited by PI3K activity. Thus loss of Nf1 or Tsc might also increase sensitivity to mGluR-mediated LTD. Finally, several lines of direct evidence suggest that PI3K hyperactivation plays a causal role in autism. For example, DNA copy number variants observed in individuals with autism but not unaffected individuals identify at high frequency PI3K subunits or regulators, and each genetic change is predicted to elevate PI3K activity. In addition, a translocation that increases expression of the translation factor eIF-4E, which is known to be activated by the PI3K pathway, plays a direct, causal role in autism. The potential involvement of mGluR-mediated LTD in these neurogenetic disorders increases interest in identifying the molecular intermediates that participate in this pathway, but these intermediates are for the most part unidentified. Thus, the possibility that CaMKII and Fak might participate in mGluR-mediated PI3K activation in mammals as well as Drosophila might have significant medical interest (Lin, 2011).

Development of neural circuitry relies on precise matching between correct synaptic partners and appropriate synaptic strength tuning. Adaptive developmental adjustments may emerge from activity and calcium-dependent mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been associated with developmental synaptic plasticity, but its varied roles in different synapses and developmental stages make mechanistic generalizations difficult. In contrast, this study focused on synaptic development roles of CaMKII in a defined sensory-motor circuit. Thus, different forms of CaMKII were expressed with UAS-Gal4 in distinct components of the giant fiber system, the escape circuit of Drosophila, consisting of photoreceptors, interneurons, motoneurons, and muscles. The results demonstrate that the constitutively active CaMKII-T287D impairs development of cholinergic synapses in giant fiber dendrites and thoracic motoneurons, preventing light-induced escape behavior. The locus of the defects is postsynaptic as demonstrated by selective expression of transgenes in distinct components of the circuit. Furthermore, defects among these cholinergic synapses varied in severity, while the glutamatergic neuromuscular junctions appeared unaffected, demonstrating differential effects of CaMKII misregulation on distinct synapses of the same circuit. Limiting transgene expression to adult circuits had no effects, supporting the role of misregulated kinase activity in the development of the system rather than in acutely mediating escape responses. Overexpression of wild-type transgenes did not affect circuit development and function, suggesting but not proving that the CaMKII-T287D effects are not due to ectopic expression. Therefore, regulated CaMKII autophosphorylation appears essential in central synapse development, and particular cholinergic synapses are affected differentially, although they operate via the same nicotinic receptor (Kadas, 2012).

A schematic diagram of the GFS is depicted in GFS schematic. Visual input evokes the sequential activation of most likely cholinergic neurons in lamina, medulla, and lobula. A bundle of columnar lobula neurons, the ColA interneurons, output to the bilaterally symmetrical pair of giant fiber (GF) interneurons. The cell bodies of these interneurons are in the protocerebrum and possess large diameter (~8 microm) axons that descend to the mesothoracic neuromere to output to follower neurons. GF follower neurons are the tergotrochanteral motoneurons (TTMns) that innervates the tergotrochanteral muscle (TTM, jump muscle) and the peripherally synapsing interneuron (PSI), which outputs to five dorsolongitudinal muscle motoneurons (DLMns). DLMns supply the large indirect dorsal longitudinal muscles (DLMs: Kadas, 2012).

This study provides evidence that postsynaptic accumulation of transgenically derived, Ca²⁺-independent, constitutively active CaMKII in developing Drosophila giant fibers and motoneurons permanently impairs, but with different severity, the function of central cholinergic synapses in giant fiber (GF) and motoneuron dendrites, while the neuromuscular junctions seem unaffected. Although not supported by the morphological data, the possibility cannot be excluded that the physiological phenotypes describe arise from undetectable wiring changes (e.g., additional, or different interneuron(s) incorporated into the circuit) and not from altered synaptic functions in an otherwise normal circuit (Kadas, 2012).

Forced expression data do not necessarily emulate the native CaMKII distribution or activity state in these neurons. In addition, the manipulations and perturbations of the system may induce homeostatic regulation, especially during development when plasticity is prominent. This suggests that some of the observed effects may be the consequence of CaMKII accumulation in non-GFS cells. However, the latter explanation is not favored based on previous work on giant neurons in culture that respond to CaMKII antagonists, and on results with the constitutively active CaMKII-T287D suggest that these neurons and relevant synapses of the GFS contain the molecular machinery to respond to the elevated kinase activity. This, along with the abundant neuronal expression of the kinase, suggest that it is normally present in the circuit, although its unequivocal demonstration is not trivial and is the subject of ongoing work (Kadas, 2012).

The data indicate that the electrophysiological phenotypes result from sustained CaMKII activity during development of cholinergic synapses. In contrast, expression of the CaMKII-R3 and T287A transgenes did not alter cholinergic transmission. It is suggested that CaMKII elevation per se did not appear to result in altered neurotransmission, but rather it resulted from sustained Ca²⁺/CaM-independent activity during a critical developmental period when low or no activity is required. This is consistent with the lack of effects due to CaMKII-R3 expression that, although it elevated wild-type protein in the circuit, was nevertheless dependent on Ca²⁺/CaM and consequent autophosphorylation to exert its effects. Similarly, the Ca²⁺-sensitive mutant CaMKII-T287A, which cannot sustain the activated state and mimic CaMKII-T287D, did not precipitate defects. Moreover, if the deficits are a consequence of constitutive CaMKII activity in developmental periods when it should either be inactive or at least Ca²⁺/CaM-dependent, then reducing the endogenous kinase with RNAi or its activity with a competitive inhibitior (ala peptide) even with the highest efficiency would likely have no adverse consequences. However, in contrast to the current findings, synaptic transmission and growth in larval NMJs and K⁺ currents and membrane excitability in GF neurons in culture are altered by CaMKII antagonists or/and ala peptide expression. It is formally possible that this reflects differences in functional properties between larval abdominal NMJs and adult DLM, TTM NMJs. In addition, differences in the membrane properties between the cultured GF and adult GF in situ, may explain the differential response in CaMKII abrogation (Kadas, 2012).

Characteristics of synaptic improvement were detected upon expression of CaMKII-T287D and CaMKII-R3 with chaGal4 in GFS neurons afferent to the GF. This improvement in GFS synaptic function is either of developmental origin or reflects an activity-dependent mechanism since the chaGal4 driver is expressed by the end of synapse formation and then strongly in the adult. Unfortunately, the locus of the phenotype cannot be defined due to the lack of specific drivers for particular groups of neurons of the sensory part of the GFS (Kadas, 2012).

The data indicate that presynaptic forced expression of CaMKII-T287D and the other forms of the kinase in GFs and motoneurons does not impair synaptic transmission in GF/TTMn synapses, probably in PSI/DLMn synapses and the NMJs. In contrast, presynaptic expression of CaMKII-T287D in cholinergic chordotonal (sensory) neurons eliminates habituation of a reflex controlling the leg position, while at the larval glutamatergic NMJs, presynaptic expression in the motoneurons increases the number of boutons without affecting neurotransmitter release and alters potassium conductance in motoneuron axons. Thus, both GFS central cholinergic and peripheral glutamatergic NMJs in the adult appear to be nonresponsive to kinase alterations. Consistent with this, a recent study provides evidence that in mammalian cultured cortical neurons, expression of constitutively active CaMKII? has no effect on presynaptic release (Kadas, 2012).

Expression of CaMKII-T287D in GF neurons during metamorphosis could affect each or a combination of axon pathfinding (larval stages, 24 h APF), target recognition and initial synapse formation (24-55 h APF), or synapse formation, stabilization, and maintenance (55-100 h APF). This study reports that the constitutively active CaMKII exerts its effects during a 60 h window (24-84 h APF) in the pupa. The end of this time window seems to coincide with completion of synapse formation and stabilization. In congruence, GFS synapses have been reported susceptible to permanent disruptions if endocytosis is temporarily blocked, but only before 85 h APF. Interestingly, even if CaMKII-T287D activity is continuously present in the circuit when the TARGET system was not used, the effect was still specific to the ColA/GF synapse. This suggests at least temporal specificity in the effects of Ca²⁺-independent CaMKII. Nevertheless, in affected neurons the dendrites appear normal, and their axons reach the mesothoracic neuromere and show their characteristic bends and functional electrochemical contacts with PSI and TTMn neurons. Therefore, the initial phase of axon pathfinding (larval stages, 24 h APF) can be excluded from the effect of Ca²⁺-independent CaMKII on GF development. Thus, the effects of CaMKII-T287D seem specific to synapse formation and stabilization of central cholinergic neurons rather than on dendritic and axonal growth. This may be distinct from similar processes in central glutamatergic neurons, and this will be explored in the future (Kadas, 2012).

How does constitutive CaMKII activation affect cholinergic synapses? The cholinergic GFS central synapses operate, mainly if not exclusively, through the Dα7 nicotinic acetylcholine receptor, and GFS-mediated escape response is abolished in nAChR receptor mutant animals. Because the locus of the CaMKII-T287D defect is postsynaptic and CaMKII participates in the suppression of nAChR gene expression in mammalian muscles, one likely explanation for the observed GFS phenotypes is a decreased number of nAChRs. In the class of interneurons known as Kenyon cells, activation of nAChRs causes rapid and reversible intracellular calcium increase that depends on indirect calcium influx through voltage-gated calcium channels directly via nAChRs (Campusano, 2007). In accord with these studies, it is proposed that CaMKII may act as a negative feedback element regulating nAChR density during normal development (Kadas, 2012).

An alternative explanation could be the abnormal function or localization of the nAChRs in the postsynaptic area due to defective formation and/or stabilization of the synapses. The constitutively active CaMKII-T287D is known to cause structural abnormalities in the larval NMJ by phosphorylating Discs Large (DLG) and increasing its extrasynaptic localization. Dephosphorylated DLG associates with the synaptic complex and clusters the cell adhesion molecule Fasciclin II (Fas II) and the Shaker K⁺ channels. Importantly, FasII is essential for synapse maintenance and plasticity. Thus, either the homolog of PSD93 in mammalian central cholinergic synapses, DLG, or another scaffolding protein regulated by CaMKII could participate in a similar mechanism for the correct localization of the nAChRs in the postsynaptic area of GFS neurons (Kadas, 2012).

Finally, a third possible explanation for the CaMKII-T287D phenotypes could be impaired potassium conductance in GFS neurons. Indeed, Shaker K⁺ channels mutations (Sh) cause longer RP and lower FF₅₀ values in the GFS of the mutant animals, and shaker knock down also strongly affects MN5 motoneuron firing responses. Furthermore, Eag (Ether-a-go-go) potassium channels are known to interact with CaMKII, and DLMn5 motoneuron firing behavior is altered upon expression of an Eag dominant negative isoform (Kadas, 2012).

GF/TTMn, PSI/DLMn and ColA/GF cholinergic synapses of the GFS exhibit similar malfunctions, but postsynaptic, constitutively active CaMKII is differentially detrimental for the ColA/GF synapses. Distinctions in the severity of the synaptic phenotypes may reflect functional differences between the stereotypic GF/TTMn, PSI/DLMn, and the weak-plastic ColA/GF synapses. Synapses in GFS motoneuron dendrites (GF/TTMn) and axons (PSI/DLMn) receive strong synaptic input and must generate a single action potential only per GF activation. These synaptic sites can operate at activation frequencies above 150 Hz and are not designed to summate postsynaptic potentials. Thus, synaptic transmission is unlikely to be affected by subtle postsynaptic alterations. In contrast, synapses in GF dendrites integrate sensory input and operate at a maximum of 30 Hz, and small postsynaptic defects uncovered by CaMKII activation likely result in failure of action potential generation and light-induced escape response. If the constitutively active kinase downregulates the cholinergic receptor or compromises synaptic stabilization and/or neurotransmitter release, then the lower number of active synapses may be sufficient for GFS motoneurons, but not for the GF neurons to reach threshold for action potential generation (Kadas, 2012).

In Drosophila, short-term (STH) and long-term habituation (LTH) of olfactory avoidance behavior are believed to arise from the selective potentiation of GABAergic synapses between multiglomerular local circuit interneurons (LNs) and projection neurons in the antennal lobe. However, the underlying mechanisms remain poorly understood. This study shows that synapsin (syn) function is necessary for STH and that syn⁹⁷-null mutant defects in STH can be rescued by syn⁺ cDNA expression solely in the LN1 subset of GABAergic local interneurons. As synapsin is a synaptic vesicle-clustering phosphoprotein, these observations identify a presynaptic mechanism for STH as well as the inhibitory interneurons in which this mechanism is deployed. Serine residues 6 and/or 533, potential kinase target sites of synapsin, are necessary for synapsin function suggesting that synapsin phosphorylation is essential for STH. Consistently, biochemical analyses using a phospho-synapsin-specific antiserum show that synapsin is a target of Ca²⁺ calmodulin-dependent kinase II (CaMKII) phosphorylation in vivo. Additional behavioral and genetic observations demonstrate that CaMKII function is necessary in LNs for STH. Together, these data support a model in which CaMKII-mediated synapsin phosphorylation in LNs induces synaptic vesicle mobilization and thereby presynaptic facilitation of GABA release that underlies olfactory STH. Finally, the striking observation that LTH occurs normally in syn⁹⁷ mutants indicates that signaling pathways for STH and LTH diverge upstream of synapsin function in GABAergic interneurons (Sadanandappa, 2013).

Mechanisms for synaptic plasticity have been intensively analyzed in reduced preparations, e.g., in hippocampal or cortical-slice preparations and in neuromuscular synapses, e.g., of Aplysia, crayfish, frog, lamprey, and Drosophila. In contrast, synaptic mechanisms underlying specific forms of behavioral learning are less well understood in vivo, in terms of the signaling pathways engaged by experience as well as the cell types in which these mechanisms operate. The analysis of such in vivo mechanisms of plasticity requires an accessible neural circuit, whose properties are measurably altered by experience (Sadanandappa, 2013).

The olfactory response in Drosophila is initiated by the activation of odorant receptors expressed in the olfactory sensory neurons (OSNs), which project into the antennal lobe (AL) and form synapses with glomerulus-specific projection neurons (PNs) that wire to both, the mushroom bodies (MB) and the lateral protocerebrum. OSNs and PNs also activate multiglomerular excitatory and inhibitory local interneurons (LNs), which mediate lateral and intraglomerular inhibition in the AL. Strong evidence has been proved for a model in which Drosophila olfactory habituation, i.e., reduced olfactory avoidance caused by previous exposure to an odorant arises through potentiation of inhibitory transmission between GABAergic LNs and PNs of the AL (Das, 2011). This study analyzes the molecular basis of this potentiation and propose a mechanism that could lead to increased presynaptic GABA release after odorant exposure (Sadanandappa, 2013).

Synapsins are a conserved family of synaptic vesicle-associated proteins. They are predominantly associated with the reserve pool of synaptic vesicles and their phosphorylation by kinases such as calcium-dependent protein kinases (CaMKs), protein kinase A (PKA), and MAPK/Erk, result in the mobilization of these vesicles and thereby induces presynaptic facilitation. While synapsin may play key roles in behavioral plasticity in mammals, its functions in learning and memory remain mysterious in part because mammals have three synapsins (I, II, and III) encoded by three different genes (Sadanandappa, 2013).

Recent studies of the single synapsin gene in Drosophila show that it is required for adult anesthesia-sensitive memory of odor-shock association (Knapek, 2010). Larval associative memory requires synapsin with intact phosphorylation target sites (Ser6 and Ser533) likely operating in a subset of MB neuron (Sadanandappa, 2013).

This study asked whether, where, and how synapsin functions in olfactory habituation. The observations indicate that short-term habituation (STH) requires synapsin function as well as its CaMKII-dependent phosphorylation in the LN1 subset of inhibitory interneurons in the AL. These point to presynaptic facilitation of GABA release as being a crucial mechanism for STH. The observation that long-term habituation (LTH) forms normally in syn⁹⁷ mutants indicates that this form of long-term memory can be encoded without transition through a short-term memory stage (Sadanandappa, 2013).

Previous studies have established the important roles for vertebrate and invertebrate synapsins in short-term synaptic plasticity. The conclusion that increased GABA release underlies olfactory STH is built on previous studies that have established the role of synapsin in synaptic vesicle mobilization and presynaptic facilitation. Several in vivo and in vitro studies have provided evidence that synapsin phosphorylation and dephosphorylation regulate the effective size of different vesicle pools and thereby control neurotransmitter release. In the mollusks Aplysia californica and Helix pomatia/aspersa, the phosphorylation and redistribution of synapsin induced by the PKA or MAPK/Erk-pathways mediates short-term facilitation of transmitter release (Byrne, 1996; Angers, 2002; Giachello, 2010). In Drosophila larval neuromuscular junctions, post-tetanic potentiation of transmitter release requires synapsin function and is accompanied by mobilization of a reserve pool of synaptic vesicles. And in the mouse CNS (Cui, 2008), enhanced ERK signaling in the inhibitory neurons results in increased levels of synapsin-1 phosphorylation and enhanced GABA release from hippocampal interneurons (Sadanandappa, 2013).

In context of these prior studies of synapsin, the current observation that synapsin and its phosphorylation are necessary in GABAergic local interneurons for STH indicates that the facilitation of GABA release from LNs attenuates excitatory signals in the AL and thereby results in the reduced behavioral response, characteristic of habituation. The molecular reversibility of phosphorylation could potentially account for the spontaneous recovery of the olfactory response after STH. These in vivo observations on synapsin function constitute a substantial advance as they show that synapsin-mediated plasticity, usually observed in response to experimentally enforced electrophysiological stimulations, can underlie behavioral learning and memory induced by sensory experience. In addition, by placing these changes in an identified subpopulation of local inhibitory interneurons in the AL, they implicate presynaptic plasticity of LNs as a mechanism necessary for habituation and thereby substantially clarify a neural circuit mechanism for this form of nonassociative memory (Sadanandappa, 2013).

A significant question is how synapsin phosphorylation is regulated in vivo for synaptic plasticity and how this contributes to altered circuit function that underlies behavioral learning. This has been difficult to address for two reasons: first, due to the complexity and potential promiscuity of kinase signaling pathways, and second, this requires not only the identification of neurons that show synapsin-dependent plasticity, but also concurrent understanding of the circuit functions of these neurons and their postsynaptic target(s) in vivo. The latter has been a particular challenge in neural networks for behavioral memory. For instance, although synapsin- and S6/S533-dependent odor-reward memory trace localized to the MBs (Michels, 2011), the downstream targets of the MB to mediate learned behavior are only beginning to be unraveled. This makes it difficult to comprehensively interpret the complex role of synapsin in associative memory illustrated by the finding that 3 min memory and anesthesia-sensitive 2 h memory require synapsin, whereas 5 h memory and anesthesia-resistant 2 h memory do not (Knapek, 2010). In the Drosophila neural circuit that underlies olfactory habituation, this study presents evidence that is most parsimoniously explained by a model in which CaMKII regulates synapsin phosphorylation in GABAergic LNs of the AL, neurons that are known to inhibit projection neurons that transmit olfactory input to higher brain centers (Sadanandappa, 2013).

in vivo CaMKII studies not only show the requirement of its function in olfactory habituation, but also demonstrate that the predominant form of synapsin, which arises from pre-mRNA editing, is a potent substrate for CaMKII. Thus, expression of an inhibitory CaMKII peptide in neurons not only reduces (yet does not abolish) the S6-phosphorylated form of synapsin detected using S6 phospho-specific antibody, it also blocks olfactory habituation, thereby providing experimental evidence for presynaptic function of CaMKII. These findings provide in vivo support for a model that proposed the primacy of CaMKII regulation of synapsin function; however, the structural basis for this regulation may be slightly different as invertebrate synapsins lack the D domain found on mammalian homologs that appear to be the major targets of CaMKII-dependent synapsin phosphorylation (Sadanandappa, 2013).

These conclusions on the role of CaMKII must be qualified in two ways. First, though tight correlations were shown between CaMKII phosphorylation of synapsin and the protein's function in STH, the data fall short of establishing causality. Second, it was not possible to directly demonstrate that odorant exposure that induces STH results in CaMKII-dependent synapsin phosphorylation in LNs. Attempts to address the latter issue failed because of technical difficulties in using phospho-specific antibodies for in vivo immunohistochemistry. Finally, although it is proposed that CaMKII phosphorylation is necessary for synapsin function during STH, it remains possible that other additional kinases, e.g., PKA and CaMKI, also contribute to synapsin regulation in vivo required for STH (Sadanandappa, 2013).

Several studies have discriminated between mechanisms of short-term and long-term memory (STM and LTM) formation. Most have focused on the distinctive requirement for protein synthesis in LTM and have not established whether STM is a necessary step in the formation of LTM or whether STM and LTM arise via distinctive, if partially overlapping molecular pathways. However, a few reports describe experimental perturbations that greatly reduce STM, without altering LTM (Sadanandappa, 2013).

In cultured sensorimotor synapses, which provide a surrogate model for behavioral sensitization in Aplysia, it has been shown that synaptic application of a selective 5-HT receptor antagonist blocked short-term facilitation while leaving long-term facilitation unaffected. In contrast, somatic application of the same antagonist selectively blocked long-term facilitation. This showed that long-term synaptic plasticity, though triggered by similar inputs (5-HT in this case), can progress through a pathway that does not require short-term plasticity (Sadanandappa, 2013).

In mammalian systems, it has been shown that a variety of pharmacological infusions into the hippocampal CA1 region or in entorhinal/ parietal cortex inhibited STM without affecting LTM of a shock avoidance memory task. In Drosophila, different isoforms of A-kinase anchor protein (AKAP) interacts with cAMP-PKA and play a distinct role in the formation of STM and LTM (Lu, 2007; Zhao, 2013). While these studies indicate that LTM is not built on STM, they do not rule out the possibility that they share an early common synaptic mechanism, but differ in subsequent mechanisms of consolidation, which occur in anatomically distinct brain regions (Sadanandappa, 2013).

The current observations on the absolute requirement for synapsin in STH but not LTH extend a model in which LTM can be encoded without transition through STM, particularly because STH and LTH involve different timescales of plasticity in the very same olfactory neurons. In the simple learning circuit for STH and LTH, the current observations are interpreted in a biochemical model. While both STH and LTH occur through potentiation of iLN-PN synapses, it is proposed that the signaling mechanisms for short- and long-term synaptic plasticity diverge before the stage of synapsin phosphorylation in GABAergic local interneurons. Thus, odorant exposure results in the activation of key signaling molecules such as PKA and CaMKII in LNs required for both forms of olfactory habituation. However, the kinases affect STH through synapsin phosphorylation, which results in a rapid but transient increase in evoked GABA release. Meanwhile, the same signaling molecules also participate in the activation of translational and/or transcriptional control machinery, which act, on a slower timescale, to cause the formation of additional GABAergic synapses that persist stably for much longer periods of time (Sadanandappa, 2013).

The unusual behavioral state, in which an animal is unable to form an STM but capable of longer term memory may have some clinical significance. Recently, a comprehensive mouse model for Down syndrome, which is trisomic for ~92% of the human chromosome 21, was shown to have defective STM of novel object recognition, while still showing normal LTM in this task, which is conceptually similar to behavioral habituation. Thus, it is suggested that further studies may eventually identify several other molecules which, like synapsin, are selectively necessary for short-term but not for long-term memory (Sadanandappa, 2013).

Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by Protein kinase C (PKC). The Drosophila adducin, Hu-li tai shao (Hts), has been shown to play a role in larval neuromuscular junction (NMJ) growth. This study finds that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), this study shows that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. Evidence is provided that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. It was also shown that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014: PubMed ID).

The Drosophila neuromuscular junction (NMJ) is the site of contact between motor neuron and muscle, and is stably maintained but remodelled during the growth and development of the fly. To permit these differing functions, the NMJ uses an actin- and spectrin-based cytoskeleton both pre- and post-synaptically, where a number of synaptic proteins modify the cytoskeleton dynamically. One such protein involved in the dynamic responses of the synapse to stimuli in vertebrates is the actin- and spectrin-binding protein adducin, a heteromeric protein composed of α, β and γ subunits that is widely expressed in many cell types including neurons and myocytes. The adducins are composed of a globular N-terminal head domain, a neck domain and a C-terminal myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain containing an RTPS-serine residue which is a major phosphorylation site for protein kinase C (PKC), as well as cAMP-dependent protein kinase (PKA). Phosphorylation of adducin in the MARCKS-homology domain inhibits adducin-mediated promotion of actin-spectrin interactions, resulting in cytoskeletal reorganization (Wang, 2014).

Multiple studies have demonstrated that the mammalian MARCKS protein, or more specifically its MARCKS effector domain, can bind to and sequester the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP₂), in artificial lipid vesicles. This interaction has been linked to the regulation of the actin cytoskeleton during the growth and branching of dendrites in rat brains, as well as the directed migration of bovine aortic endothelial cells in wound healing assays. Notably, it has been proposed that aberrant MARCKS regulation of PIP₂ signaling may be implicated in the formation of amyloid plaques in Alzheimer's disease. A recent study has also provided evidence that reduced hippocampal levels of MARCKS, and thus PIP₂, in mice contributes to age-related cognitive loss (Wang, 2014).

MARCKS binds to PIP₂ as the MARCKS effector domain carries basic residue clusters that can interact with acidic lipids in the inner leaflet of the cell membrane. By analogy to other MARCKS-homology domain-containing proteins, it is hypothesized that phosphorylation of adducin at the RTPS-serine may alter the electrostatic interaction between adducin and phosphoinositides, thus reversing the binding between them and causing translocation of adducin from the membrane to the cytosol. In this way, adducin might act as a molecular switch in its regulation of synaptic plasticity, with its localization at the synapse controlled by phosphorylation (Wang, 2014).

In Drosophila, orthologs of adducin are encoded by the hu-li tai shao (hts) locus, and the Hts protein is present at both the pre- and post-synaptic sides of the larval NMJ where it regulates synaptic development. Previous studies have shown that Hts interacts with the scaffolding protein Discs large (Dlg), and regulates Dlg localization at the postsynaptic membrane by promoting its phosphorylation through Partitioning-defective 1 (PAR-1) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), two known regulators of Dlg postsynaptic targeting. Dlg is an important regulator of synaptic plasticity, and likely constitutes a major route by which Hts controls NMJ development. This study found that the main isoforms of Hts at the NMJ are the MARCKS-homology domain-containing isoforms, Add1 and/or Add2. There, the adducin-like isoforms form complexes with Dlg and PIP₂, interactions that were identified through Proximity Ligation Assay (PLA). Evidence is provided that Hts promotes the phosphorylation, and thus delocalization, of Dlg at the postsynaptic membrane by regulating the re-distribution of par-1 and camkII transcripts from the muscle nucleus to the cytoplasm. It was also shown that these Hts interactions with Dlg and PIP₂ are impeded through phosphorylation of the MARCKS-homology domain, further establishing that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014).

Through the use of PLA, this study has shown that Hts forms complexes with Dlg and PIP₂ at the postsynaptic region of the larval NMJ, with its ability to associate with these proteins being negatively regulated through phosphorylation of the MARCKS-homology domain. Studies on mammalian adducin have demonstrated that phosphorylation of the MARCKS-homology domain impedes its actin-binding and spectrin-recruiting functions, reduces its affinity for these cytoskeletal components and the membrane, and targets it for proteolysis. It is proposed that phosphorylation of the MARCKS-homology domain in the Add1/Add2 isoforms of Hts in response to upstream signaling events at the synapse reduces their affinity for spectrin-actin junctions and Dlg at the NMJ, but may also hinder their interactions with PIP₂ and other phosphoinositides in line with the electrostatic switch model for phosphoinositide binding by the MARCKS-homology domain (Wang, 2014).

It was proposed previously that Hts regulates Dlg localization at the NMJ by controlling the protein levels of PAR-1 and CaMKII, which phosphorylate Dlg and disrupt its postsynaptic targeting. This study now shows that regulation of these kinases appears to occur at the level of transcript processing, with Hts promoting the accumulation of par-1 and camkII transcripts in the muscle cytoplasm. Cytoplasmic accumulation of the transcripts would then presumably lead to higher levels of PAR-1 and CaMKII protein. How is Hts achieving this mode of regulation when it is residing with Dlg at the postsynaptic membrane? One possibility is that Hts at the NMJ is activating a signaling pathway that promotes the transcription and/or stability of par-1 and camkII transcripts, as well as their transport out of the nucleus. Another possibility is that Hts itself, which contains predicted NLS and NES sequences, translocates to the nucleus in response to events at the NMJ, similar to the way that mammalian α-adducin translocates to the nucleus upon loss of cell-cell adhesion in epithelia. This study was unable to detect endogenous Hts in muscle nuclei, however, nuclear Hts levels might be tightly restricted and undetectable under wild-type conditions. Over-expressed wild-type Hts, on the other hand, is readily observable in the nucleus, though not its phosphorylated form - a result also seen with α-adducin. Whatever the mechanism may be, the presence of Hts in a complex with Dlg may allow it to evaluate the status of Dlg and the synapse, and execute a response in the form of regulating Dlg localization through PAR-1 and CaMKII mediated phosphorylation (Wang, 2014).

A recent study has uncovered a novel nuclear envelope budding mechanism that can export select transcripts from muscle nuclei during larval NMJ development, and involves Lamin C (LamC) and the Wnt receptor, DFrizzled2 (DFzz2) (Speese, 2012). Interestingly, camkII, but not dlg, transcripts are regulated by this process, which is consistent with the findings that CaMKII, but not Dlg, expression is regulated by Hts. Future work will determine whether Hts is involved in this LamC/DFzz2-dependent mechanism (Wang, 2014).

Two papers have underscored the importance of phosphoinositides in synaptic development at the Drosophila NMJ (Forrest, 2013; Khuong, 2010). Binding of Hts to PIP₂ and probably other phosphoinositides at the NMJ, as seen with other MARCKS-homology domain-containing proteins, may affect the availability of these lipids for processes such as signal transduction, thus affecting synaptic development. Conversely, the localization of Hts at the NMJ may be regulated by the distribution of phosphoinositides. In line with this, postsynaptic knockdown of the phosphoinositide phosphatase Sac1 via transgenic RNAi expression disrupts Hts localization at the NMJ (Wang, 2014).

The observations reported in this study may have important implications for understanding diseases that affect synaptic function in humans and other mammals. Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a disorder characterized by the progressive loss of motor neurons, have been assumed until recently to be a consequence of neuronal death within the central nervous system. However, there is substantial recent evidence indicating that neuron pathology in ALS and other neurodegenerative diseases is due to a degenerative process that begins in the presynaptic terminal, NMJ or distal axon. This may also be the case in normal aging (Wang, 2014).

Initial interested in adducin arose when elevated levels of phospho-adducin protein was found in the spinal cord tissue of patients who died with ALS, compared to individuals who died without neurological disease. Similar observations were also made in mSOD-expressing mice, a transgenic animal model of ALS. Multiple studies have shown that adducin plays important roles in synaptic plasticity, and that mice mutant for β-adducin display defects in memory, learning and motor coordination. It is clear that modulation of Hts expression and phosphorylation can affect synaptic development. This study provides evidence here that phosphorylation of Hts impedes its function at the larval NMJ, a result that is consistent with the mammalian adducins. In addition, overexpression of phospho-mimetic Hts has dominant negative effects over endogenous Hts. Thus, loss of adducin function through aberrant phosphorylation of the MARCKS-homology domain may be a contributing factor for human neurodegenerative diseases (Wang, 2014).

Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. This was addressed with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. Two motor input types, Ib and Is, were found to provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing 'wakes up' silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function (Newman, 2017).

The transfer of information between neurons throughout the nervous system relies on communication across inherently unreliable chemical synapses. Synaptic communication is further complicated by the fact that individual neurons can receive inputs from many functionally diverse neurons. Even synapses formed by one presynaptic neuron and one or more postsynaptic target neurons can vary greatly in neurotransmitter release, postsynaptic sensitivity, and plasticity. Additionally, postsynaptic cells are not passive receivers of information but can produce retrograde signals, including homeostatic signals that modulate synaptic release. The mechanisms that regulate diversity in transmission at individual synapses are not well understood, nor are their relationships to the plasticity and homeostatic mechanisms that adjust synaptic strength. In particular, it is unclear whether retrograde signaling is input or synapse specific and able to maintain input context among diverse convergent synapses (Newman, 2017).

The Drosophila larval neuromuscular junction (NMJ) is a model system for studying glutamatergic transmission, with pre- and postsynaptic molecular machinery similar to that of central excitatory synapses in vertebrates, while also possessing activity-dependent adjustments in synaptic strength, including short- and long-term plasticity, as well as homeostatic plasticity. Two morphologically distinct glutamatergic motor neurons, larger type Ib and smaller type Is, converge onto most of the larval body wall muscles used for locomotion. There is evidence that the transmission properties of these inputs differ (Kurdyak, 1994; Lnenicka, 2000; Lu, 2016), thus providing a powerful system for investigating the role of input and synapse specificity in the regulation of basal synaptic strength, plasticity, and homeostasis. In addition, there is great heterogeneity, in that both the basal release probability (Pr) of evoked release and the frequency of spontaneous release differ greatly between synapses of the same Ib axon (Peled, 2011; Peled, 2014). This study set out to understand how plasticity and homeostasis function in a diverse pool of synapses and to determine whether synaptic homeostasis operates globally or has input specificity (Newman, 2017).

To address these questions requires high-resolution, high- speed analysis of function at many synapses simultaneously. Quantal resolution measurements of excitatory transmission through Ca2+-permeant glutamate receptors (GluRs) has been achieved by imaging chemical or genetically encoded Ca2+ indicators (GECIs) in the postsynaptic cell (Cho, 2015; Guerrero, 2005; Lin, 2016; Melom, 2013; Muhammad, 2015; Peled, 2011; Peled, 2014; Reese, 2015, Reese, 2016; Siegel, 2013). This study has generated a vastly improved postsynaptically targeted GECI based on GCaMP6f. When expressed in Drosophila larval muscle, 'SynapGCaMP6f' enables quantal imaging without voltage clamping. SynapGCaMP6f was combined with an optical platform that immobilizes larvae without anesthetics to measure synaptic transmission simultaneously at hundreds of Ib and Is synapses in the intact, behaving animal (Newman, 2017).

The quantal imaging makes it possible to connect the elementary properties of transmission at single synapses, to the synaptic drive that is generated by convergent synaptic inputs, to the operation of muscle groups in large parts of the animal, and finally to behavior. These observations show that basal synaptic strength, short-term plasticity, and, most strikingly, synaptic homeostasis are input specific, diversifying excitatory transmission and behavioral output (Newman, 2017).

The Ib and Is inputs to the Drosophila larval muscle respectively resemble tonic (low release probability, facilitating) and phasic (high release probability, depressing) inputs seen at neuro-muscular and neuro-neuronal connections in other organisms. Given these differences, the Is input has been proposed to initiate strong, phasic muscle contractions (Schaefer, 2010). However, this study found that the primary excitatory drive and main cause of muscle contraction in larval Drosophila is actually the Ib input. This is despite the fact that Is synapses have a higher basal release probability (Pr) and larger quantal sizes. These functional differences likely reflect the relatively short duration of the Is bursts of activity and the relatively small number of active zones of the Is terminal. Whereas elimination of transmission from Is inputs had little or no effect on muscle contraction during restrained locomotion, just as it had earlier been shown to have negligible effect on general behavior, this study found that the Is input is needed for normal intersegmental coordination of contraction waves. This is consistent with the broad, multi-muscle Is innervation pattern (Newman, 2017).

Ib synaptic activity begins before and ends after Is activity, consistent with their common presynaptic inputs and different intrinsic excitabilities. Recent work has shown that excitatory and inhibitory interneurons regulate the differential recruitment of MNs between hemi- segments and within hemisegments, respectively (Heckscher, 2015; Zwart, 2016). While Ib inputs ramp up their synaptic drive during locomotor bursts, Is inputs abruptly reach a maximum, which they often sustain for the duration of the shorter burst. Given their substantial depression during high-frequency stimulation, the ability of the Is input to sustain a fixed output level during locomotion suggests that it increases the firing frequency during the bursts. The common interneuron drive within the VNC suggests that the progressive increase in synaptic drive during the Ib burst may also be partly due to an increase in frequency during the burst, in addition to the recruitment of silent synapses and increase in Pr of active synapses, which is observed during facilitating high-frequency trains. Thus, a combination of circuit-level and cell-autonomous differences combine to generate the complex behavioral output of the larva from a limited number MNs and muscles (Newman, 2017).

Spontaneous glutamate release has been shown to play a role in synapse development (Choi, 2014). However, this study found that spontaneous release in vivo represents only ~1% of total release, suggesting that evoked release would drown out the influence of spontaneous release. One possible explanation is that in late third-instar larvae, spontaneous release may represent a larger fraction of total synaptic transmission than earlier in development when the developmental influences are exerted. An intriguing alternative explanation derives from the finding that, at both inputs, synapses preferentially participate in either evoked or spontaneous release. This suggests that developmental signals could occur at a subset synapses that are dominated by spontaneous release (Newman, 2017).

Synapses of both inputs were found to differ in key properties, with Is synapses having larger quantal sizes, less total spontaneous release, higher Pr, and short-term depression (as opposed to facilitation in Ib synapses) during high-frequency trains. The difference in short-term plasticity between the inputs is consistent with electrophysiological analysis of responses to separate stimulation of Is or Ib axons (Lnenicka, 2000; Lu, 2016), as well as modeling. These resemble input-specific differences seen in the mammalian brain, such as between parallel and climbing fibers converging on Purkinje cells (Dittman, 2000; Mapelli, 2015) or interneurons in the cortex. The tendency to facilitate or depress is shaped by basal Pr, where high Pr results in greater depletion of immediately releasable vesicles leading to depression. Although overall this agrees with the observation that Is synapses have higher Pr and depress, it was found that despite overlapping distributions of single-synapse Pr, overall release tended to depress in Is and facilitate in Ib, suggesting additional differences in regulation. This greater complexity comes into stark relief when the behavior of hundreds of individual synapses is examined during high-frequency trains of presynaptic firing. This study found that the heterogeneity of short-term plasticity within an input is much greater than was previously appreciated. Local plasticity dynamics were catagorized by how transmission changed during a stimulus train, and it was found that the inputs differ only in two categories, Is inputs having a larger number of active sites that depress and Ib having a larger number of silent sites that are recruited to a releasing state. These observations reveal a previously unknown level of specialization in basal release and plasticity between neighboring synapses of a common input (Newman, 2017).

The Drosophila larval NMJ has proven to be a powerful system for studying synaptic homeostasis. The mechanism of this homeostasis is that reduction in quantal size (because of reduced GluR conductance that results from either mutation of a GluR subunit or partial pharmacological block) triggers a retrograde signal that leads to increased transmitter release, restoring the normal the normal level of excitatory drive (Davis, 2015). The current results reveal a new aspect to homeostatic plasticity by showing that it is specific to input and acts primarily at synapses with certain properties of basal transmission, short-term plasticity and physiological function. Only glutamate release from the Ib input is boosted during homeostatic compensation. This is attributed to the fact that despite the smaller quantal size and unitary Ca2+ influx at Ib synapses, Ib inputs have longer bouts of activity, resulting in much larger aggregate Ca2+ elevation in the Ib postsynapse during locomotion. The larger Ca2+ influx drives a higher activation of CaMKII in the Ib postsynapse. Critically, mutation of the GluR subunit to reduce quantal size at both Ib and Is synapses exclusively reduces activated CaMKII at the Ib postsynapse. Why there is no reduction in activated CaMKII at the Is postsynapse is not clear, though nonlinearity in the relationship between the Ca2+ concentration profile and CaMKII activation (Stratton, 2013) and differences in GluR subunit composition that influence Ca2+ influx may contribute (Newman, 2017).

A second mechanistic difference ensures exclusivity for homeostatic signaling to the Ib input: even when CaMKII activity is inhibited throughout the muscle, only Ib presynapses are boosted in Pr. This could mean that Is presynapses are not responsive to the homeostatic signal. Alternatively, it could mean that Is postsynapses are incapable of generating the homeostatic signal, an idea that would be consistent with the finding that elimination of release from Is axons does not induce homeostatic compensation at Ib synapses. However, this would also require that the retrograde homeostatic signal act very locally so that it could not travel even a few microns from signaling-capable Ib postsynapses to Is boutons that are often located very close by. Another possibility is that basal Pr is high at Is synapses partly due to lower levels of activity in vivo resulting in greater homeostatic enhancement in presynaptic release, which in turn could occlude further increases in synaptic reliability following changes in GluR composition or global postsynaptic CaMKII inhibition (Newman, 2017).

Evidence for signaling compartmentalization can be seen in the structural differences between Ib and Is NMJs, particularly on the postsynaptic side. Ib axons are surrounded by a significantly thicker and more elaborate SSR. There are also substantial differences in the localization of key organizing postsynaptic proteins such as Dlg which is present at higher levels in Ib postsynapses. CaMKII activity has also been shown to affect SSR structure and Dlg localization. Thus, the different activity patterns during native behaviors may also directly regulate postsynaptic properties other than presynaptic release to maintain functional diversity (Newman, 2017).

The presynaptic changes that mediate homeostatic compensation in Pr are only partly known. Homeostasis has been shown to be accompanied by an increase in presynaptic AP-evoked Ca2+ elevation in Ib boutons. Although a number of molecules have been demonstrated to play a role in mediating presynaptic homeostatic compensation (Davis 2015), it remains to be determined how these mechanisms interact with input-specific differences in strength and plasticity (Newman, 2017).

Although homeostasis has been generally thought to be a global mechanism to regulate synaptic strength, evidence has emerged that homeostasis can be regulated with variable degrees of specificity. This study has now demonstrated that homeostatic changes can also propagate retrogradely to the presynaptic neuron in an input-specific and synapse-autonomous manner. Given the divergent innervation of multiple muscles by Is inputs and unique innervation by Ib inputs, a Ib-specific homeostatic mechanism can provide greater functional flexibility (Newman, 2017).

By combining in vivo, physiological measurements of glutamate release with high-resolution quantal analysis at single synapses in the semi-dissected preparation, this study has clarified the relative properties of convergent glutamatergic inputs in the larva during native behaviors. Importantly, this study has demonstrated that the regulation of basal synaptic strength, short-term plasticity, and homeostasis are shaped in a precise input-specific manner. The Ib input has higher levels of in vivo activity, lower Pr synapses, and a propensity for facilitation by recruiting silent synapses during spike trains. Furthermore, the Ib input is the primary determinant for contraction dynamics in the behaving animal. Consistent with its dominant role, when postsynaptic sensitivity to glutamate is altered at both inputs, there is a selective homeostatic adjustment in the amount of neurotransmitter released from Ib input. CaMKII, localized to the postsynaptic density, is ideally placed to detect local changes in postsynaptic activity at both inputs, and postsynaptic inhibition of CaMKII activity is sufficient to enhance release at the Ib input, demonstrating a high degree of signaling compartmentalization within a single muscle cell. Together, these results demonstrate how synaptic activity at the Drosophila larval NMJ is precisely regulated to ensure both functional diversity and stability (Newman, 2017).

Retrograde signaling systems are fundamental modes of communication synapses utilize to dynamically and adaptively modulate activity. However, the inductive mechanisms that gate retrograde communication in the postsynaptic compartment remain enigmatic. This study investigated retrograde signaling at the Drosophila neuromuscular junction, where three seemingly disparate perturbations to the postsynaptic cell trigger a similar enhancement in presynaptic neurotransmitter release. This study shows that the same presynaptic genetic machinery and enhancements in active zone structure are utilized by each inductive pathway. However, all three induction mechanisms differ in temporal, translational, and CamKII activity requirements to initiate retrograde signaling in the postsynaptic cell. Intriguingly, pharmacological blockade of postsynaptic glutamate receptors, and not calcium influx through these receptors, is necessary and sufficient to induce rapid retrograde homeostatic signaling through CamKII. Thus, three distinct induction mechanisms converge on the same retrograde signaling system to drive the homeostatic strengthening of presynaptic neurotransmitter release (Goel, 2017).

The Drosophila neuromuscular junction (NMJ) is an established system to study retrograde synaptic signaling. At this model glutamatergic synapse, genetic or pharmacological perturbations to postsynaptic receptor functionality trigger retrograde signaling that instructs the neuron to precisely increase presynaptic neurotransmitter release, maintaining stable levels of synaptic strength. This process is termed presynaptic homeostatic potentiation (PHP) and can be induced through two distinct disruptions to postsynaptic glutamate receptor functionality. First, acute pharmacological blockade of receptors by application of philanthotoxin-433 (PhTx) reduces miniature excitatory postsynaptic potential (mEPSP) amplitude, initiating rapid expression of PHP (increase in quantal content) within 10 min. Second, genetic loss of the postsynaptic glutamate receptor subunit GluRIIA leads to a similar reduction in mEPSP amplitudes over chronic timescales (days) and a similar expression of PHP. Although these perturbations each disrupt receptors and lead to adaptive increases in presynaptic neurotransmitter release, PhTx- and GluRIIA-mediated PHP signaling exhibit important differences. First, some genes have been identified that are only necessary for GluRIIA-dependent PHP expression, whereas PHP is robustly expressed following acute PhTx application in larvae with mutations in these genes (Frank, 2009, Kauwe, 2016, Penney, 2016, Spring, 2016, Tsurudome, 2010). In addition, PhTx-induced PHP expression is translation-independent (Frank, 2006), whereas GluRIIA-induced PHP is blocked by inhibitions to postsynaptic translation through loss of the translational regulator target of rapamycin (Tor) (Kauwe, 2016, Penney, 2012). Although several genes and mechanisms necessary for the expression of PHP in the presynaptic neuron have been identified, far less is known about the mechanistic differences in postsynaptic transduction between PhTx- and GluRIIA-induced PHP signaling (Goel, 2017).

Recently, a novel manipulation to the postsynaptic muscle that does not affect glutamate receptors was demonstrated to induce retrograde PHP signaling at the Drosophila NMJ. This was accomplished by postsynaptic overexpression of the non-specific translational regulator Tor (Tor-OE) (Penney, 2012), which leads to a chronic and global increase in muscle protein synthesis (Chen, 2017). Although Tor-OE does not functionally affect glutamate receptors, somehow the increased muscle protein synthesis is converted into an instructive retrograde signal that appears to induce an enhancement in presynaptic glutamate release of a magnitude comparable with that observed in PhTx- and GluRIIA-mediated PHP (Penney, 2012). Although PhTx application, loss of GluRIIA, and Tor-OE each induce a similar enhancement in presynaptic release, to what extent they utilize separate or shared postsynaptic induction pathways, retrograde signaling systems, and modulations to presynaptic function is not known (Goel, 2017).

This study has characterized PHP signaling and expression when induced through PhTx application, loss of GluRIIA, and Tor-OE. This analysis has revealed that a common retrograde signaling system drives similar homeostatic adaptations in the presynaptic terminal but that separate inductive pathways differentially respond to glutamate receptor perturbation, Ca2+/calmodulin-dependent protein kinase II (CamKII) activity, and protein synthesis (Goel, 2017).

There appears to be a core set of genes necessary for both acute and chronic PHP expression, including ones involved in the homeostatic modulation of synaptic vesicle trafficking, presynaptic excitability, calcium channel activity, and active zone remodeling. However, other genes appear to be dispensable for this core program and may rather be involved in secondary functions, such as maintaining PHP expression over chronic timescales or supporting other aspects of homeostatic adaptation. Interestingly, the existence of multiple retrograde signaling pathways may be one reason for the failure of forward genetic approaches to identify any individual genes required in the muscle for the core process of PHP induction, suggesting some level of redundancy. This convergence of diverse induction mechanisms in the postsynaptic cell enables multiple pathways to detect and respond to homeostatic challenges by feeding into a unitary retrograde signaling system that potentiates presynaptic neurotransmitter release to stabilize synaptic strength (Goel, 2017).

CamKII activity plays a crucial role in gating diverse forms of synaptic plasticity. At the Drosophila NMJ, transgenic manipulations that affect postsynaptic CamKII activity have been reported to modulate the expression of PHP in GluRIIA mutants (Haghighi, 2003, Newman, 2017). The current results indicate that pCamKII levels are reduced to similar levels in GluRIIA mutants or following acute pharmacological receptor blockade, consistent with CamKII activity being capable of modulation in seconds at postsynaptic compartments. An attractive model would be that a reduction in calcium influx, either over 10 min or during chronic timescales, triggers diminished pCamKII levels and activates PHP signaling. However, this study found that pharmacological blockade of receptors is necessary and sufficient to reduce pCamKII levels at postsynaptic densities, independent of extracellular calcium, and that incubation in calcium-free saline alone is not sufficient to acutely induce PHP expression. Although there are several indications that reduced calcium influx in the postsynaptic muscle over chronic timescales likely contributes to PHP signaling, perhaps necessitating translation-dependent pathways, a calcium-independent system drives the acute expression of PHP following PhTx application, implying a distinct mechanism (Goel, 2017).

Two possibilities are considered to explain how PhTx application to glutamate receptors is transduced into PHP retrograde signaling without requiring calcium signaling through extracellular sources. First, PhTx binding to receptors may induce a conformational perturbation, distinct from ion influx through the receptor, to initiate PHP signaling. Such a mechanism could operate through a metabotropic mechanism, which would be unanticipated but not unprecedented. For example, at mammalian central synapses, the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long term depression (LTD) does not require calcium influx through NMDA, but, rather, pharmacological perturbation to the receptor is sufficient. A metabotropic pathway has been proposed. Further, mammalian kainate receptors, to which the Drosophila glutamate receptors are homologous, are also capable of signaling through metabotropic mechanisms. Thus, pharmacological perturbation to GluRIIA-containing receptors could, in principle, initiate PHP signaling through an undefined metabotropic mechanism. However, at present, there is no evidence for such a mechanism in Drosophila (Goel, 2017).

Alternatively, pharmacological disruption of glutamate receptors may lead to local signaling at the NMJ through interactions with scaffolds such as Discs large (DLG)/PSD-95 and Dalcium/calmodulin dependent serine protein kinase (CASK). These scaffolds are known to be in complexes with CamKII and capable of modulating CamKII activity and phosphorylation at the subsynaptic reticulum (SSR). Intriguingly, defects in the elaboration of the SSR have recently been reported to disrupt retrograde homeostatic plasticity (Koles, 2015). CamKII signaling during PHP appears to be restricted to postsynaptic densities of type 1b boutons (Newman, 2017), suggesting that compartmentalized signaling at the SSR orchestrates local PHP signal transduction. In contrast, Tor-OE is capable of initiating PHP signaling independent of pCamKII reduction, where it promotes translation throughout the cell. This implies that protein synthesis modulates retrograde signaling downstream of or in parallel to CamKII signal transduction but ultimately feeds back into local post-translational signaling pathways. Future experiments probing the interactions between glutamate receptors, postsynaptic scaffolds, translation, and CamKII activity will clarify the signaling at this compartmentalized synapse (Goel, 2017).

The finding that PHP can be acutely induced by pharmacological perturbation of glutamate receptors and not through reductions in calcium influx over rapid timescales may help to explain perplexing observations regarding the phenomenology of PhTx-mediated PHP. For example, it was noted that PHP can be induced and expressed by a 10-min incubation of PhTx with only mEPSP events occurring. Although a reduction in calcium during these mEPSP events was discussed as a possible induction mechanism, estimates are that, at most, six mEPSP events occur per active zone during this induction time, a very low level and frequency of activity to reliably and robustly produce PHP expression. Indeed, a recent study demonstrated that mEPSP events account for a very small fraction (<1%) of the total postsynaptic calcium signal at individual NMJs (Newman, 2017), making a reduction in calcium even more implausible to explain acute PHP induction. Hence, pharmacological perturbation of postsynaptic glutamate receptors, rather than a reduction in calcium through these receptors, is an attractive mechanism to explain the characteristics of the acute induction of PHP by PhTx and raises interesting questions for future studies about how pharmacological receptor perturbation is transduced into PHP induction (Goel, 2017).

Why might a single retrograde signaling system exist to homeostatically stabilize synaptic strength at the Drosophila NMJ? In central neurons, diverse forms of synaptic plasticity, including Hebbian and homeostatic, dynamically operate over multiple timescales to bi-directionally adjust synaptic strength. Further, translation-dependent and independent processes also contribute to retrograde homeostatic signaling in the hippocampus following AMPA receptor blockade. In contrast, the NMJ is built for stable excitation and is acutely sensitive to reductions in receptor function. However, when neurotransmitter sensitivity in muscle is enhanced by increased receptor expression, no retrograde signaling system exists to homeostatically downregulate presynaptic efficacy. Thus, the muscle is endowed with multiple signaling systems to respond to perturbations but appears limited to signal retrograde increases in neurotransmitter release. Hence, a single retrograde signaling system might provide an efficient means to ensure non-additive potentiation in synaptic strength and prevent hyper-excitation when conflicting signals and multiple inductive mechanisms are simultaneously activated (Goel, 2017).

The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian alpha, beta, gamma, and delta isozymes show the greatest divergence from one another. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologs of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences are observed. First, the R3A isoform is found to have a significantly higher K of activation for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted is that the R6 isoform has a significantly lower K(m) for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity (GuptaRoy, 1996a).

Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins: B1K-B4K and B1Q-B4Q (Maune, 1992). These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produce activation defects, the most severe being binding site 4 and B1Q mutants. Activation differs substantially between isoforms. R4, R5, and R6 are the least sensitive to mutations in calmodulin, while R1, R3, and R3A are the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms show almost identical binding patterns with the mutants. High binding affinity does not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II (GuptaRoy, 1996b).

Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate multiple isoforms that vary only in a region between the calmodulin-binding domain and the association domain. This variation has been shown to modulate activation of the enzyme by calmodulin. In this study the ability is examined of seven of the Drosophila isoforms to phosphorylate purified protein substrates and to be inhibited by a substrate analog, and the response of six of the isoforms to a mutant form of calmodulin (V91G) that was isolated in a genetic screen. Significant variation in Kms for Eag, a potassium channel, and Adf-1, a transcription factor, were found. In the case of a peptide inhibitor, AC3I, there are significant variations in Ki between isoforms. Kact for V91G calmodulin is increased for all of the isoforms. In addition, one isoform, RI, exhibits a lower Vmax when assayed with this mutant CaM. These results indicate that the variable domain of calcium/calmodulin-dependent protein kinase II is capable of altering the substrate specificity of the catalytic domain and the activation response to calmodulin (GuptaRoy, 2000).

Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells (the visual signal transduction pathway). Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. The identity of the protein kinase responsible for the phosphorylation of phosrestin I was sought, to link the transmembrane signaling with the light-adaptive inactivation response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. Partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II; the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin-dependent kinase II, such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with the hypothesis that Ca2+/calmodulin-dependent kinase II phosphorylates phosrestin I. It is concluded that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation (Kahn, 1997).

Phototransduction in vertebrates and invertebrates is a complex signal transduction cascade, based on rhodopsin-G-protein coupling interactions. The phototransduction process has the capacity to amplify single photon events into large electrical signals and to regulate the photoresponse output in a broad dynamic range. Major advances have been made in characterizing the molecular components of phototransduction and the mechanisms of light adaptation and response termination. However, the modulation of these latter processes is not yet fully understood. The powerful combination of molecular genetics and electrophysiology makes Drosophila photoreceptors an exquisite preparation for studying these processes. In Drosophila, light activation of rhodopsin activates phospholipase C via G-proteins; phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). This process leads within a few tens of milliseconds to the opening of cation-selective channels encoded by the trp and trp-like genes. The feedback control of the activation process involves calcium-calmodulin (Ca2+/calmodulin), which tightly regulates the adaptation and termination of the light response (Peretz, 1998 and references).

The photoreceptor potential has a complex waveform that arises from the opening of light-activated channels as well as from voltage-dependent conductances. Interestingly, Drosophila photoreceptors are endowed with high densities of voltage-gated K+ channels. In neurons, K+ channels were recognized to regulate action potential duration, firing patterns, and resting membrane potential. A great diversity of K+ channel subtypes appears to underlie these pleiotropic functions. Analysis of Drosophila mutants enables the initial molecular characterization of several classes of K+ channels. Four different voltage-sensitive K+ channel genes are initially identified in Drosophila: Shaker and Shal, encoding A-type K+ currents (I_A), and Shab and Shaw, encoding delayed-rectifier K+ currents (I_K). Subsequently, other classes of K+ channels were characterized molecularly in Drosophila (Peretz, 1998 and references).

Drosophila photoreceptors express both I_A and I_K currents, with the former mediated by subunits encoded by the Shaker locus. However, the genes encoding the delayed-rectifier channel subunits have not yet been identified, and very little is known about I_K modulation. Although the functional significance of I_A and I_K remains to be clarified in Drosophila phototransduction, in Limulus and in the blowfly Calliphora vicina I_A and I_K may regulate the gain and frequency response during light and dark adaptation. In Drosophila photoreceptors, the sustained depolarization generated by light activation of transient receptor potential (TRP) and transient receptor potential-like (TRPL) cationic channels is expected to open voltage-gated K+ channels. One can predict that the subsequent hyperpolarizing K+ currents will oppose the light-induced depolarizing currents to shape the photoreceptor potential (Peretz, 1998).

Drosophila photoreceptors I_A andI_K are regulated by calcium-calmodulin (Ca2+/calmodulin). Photoreceptors I_K and I_A are specifically inhibited by different Ca2+/calmodulin antagonists such as W7 or TFP, with I_K being far more sensitive than I_A. This regulation occurs via a Ca2+/calmodulin-dependent protein kinase (CaM kinase), with I_K once again being far more sensitive than I_A. Inhibition of Ca2+/calmodulin by N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide or trifluoperazine markedly reduces the K+ current amplitudes. Likewise, inhibition of CaM kinases by KN-93 potently depresses I_K and accelerates its C-type inactivation kinetics. Thus the modulation by the Ca2+/calmodulin antagonists is mimicked by two selective CaM kinase antagonists, KN-62 and KN-93. The effect of KN-93 is specific because its structurally related (but functionally inactive) analog KN-92 is totally ineffective. In Drosophila photoreceptor mutant ShKS133, which allows isolation of I_K, it is demonstrated by current-clamp recording that inhibition of I_K by quinidine or tetraethylammonium increases the amplitude of the photoreceptor potential, depresses light adaptation, and slows down the termination of the light response. Similar results are obtained when CaM kinases are blocked by KN-93. The mechanisms whereby I_K and I_A are depressed after exposure to CaM kinase inhibitors are not elucidated yet, but may involve either a direct channel phosphorylation or an indirect modulation. With respect to indirect regulation, it is possible that the CaM kinase mediates its effect via the Eag channel subunit. Shab 1 and Shab 2 isoforms as well as Shaw transcripts are expressed in Drosophila retina, indicating that Shab and Shaw gene products could possibly be direct or indirect substrates of CaM kinase regulation. In this regard, it is worth noting that the Shab channel subunit contains four consensus sites for phosphorylation by CaM kinase [XRXXS] at its intracellular amino and C termini. These findings place photoreceptor K+ channels as an additional target for Ca2+/calmodulin and suggest that I_K is well suited to act in concert with other components of the signaling machinery to sharpen light response termination and fine tune photoreceptor sensitivity during light adaptation (Peretz, 1998).

Slob has been identified as a novel protein that binds to the carboxy-terminal domain of Slowpoke. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation of these residues. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity (Zhou, 1999).

What are the molecular details of the profound downregulation of dSlo channel activity by 14-3-3? Members of the family of K_Ca channels are subject to modulation by a variety of molecular mechanisms, ranging from protein phosphorylation to oxidation/reduction reactions. It is conceivable that simply the binding of 14-3-3 to dSlo via Slob is sufficient to alter the gating of the channel, as appears to be the case for ß subunit interactions with Slowpoke and other potassium channels. Alternatively, 14-3-3 may act as another scaffolding component, to bring one of the protein kinases that it is known to bind into the proximity of the channel. Indeed, because 14-3-3 dimerizes, it might bridge the interactions of several different signaling proteins with the channel. It will be interesting to determine whether the Raf protein kinase, one of the kinases that binds 14-3-3, can phosphorylate and modulate dSlo, because Raf is a key player in the mitogen-activated protein (MAP) kinase pathway that conveys signals from the plasma membrane to the cell nucleus. Activation of this pathway can influence ion channel expression and activity, and potassium channel activity in turn can modulate tyrosine kinase signaling in cells. Thus, the present findings raise the intriguing possibility that a potassium channel regulatory complex is involved in MAP kinase signaling and the regulation of many fundamental cell processes (Zhou, 1999 and references).

The finding that the interaction of Slob with 14-3-3 requires Slob phosphorylation is consistent with studies of other 14-3-3 binding proteins. It is especially intriguing that the binding can be regulated in vivo by changes in the activity of CaMKII; these results suggest that there may be dynamic physiological regulation of dSlo channel activity by 14-3-3 that depends on the phosphorylation state of Slob. In view of the presynaptic colocalization of the three proteins described here, it is interesting that it is the CaMKII phosphorylation of Slob that regulates 14-3-3 binding. CaMKII is also present at a high concentration presynaptically in Drosophila, and thus the same calcium rise that evokes transmitter release might promote phosphorylation of Slob, binding of 14-3-3, and downregulation of dSlo (Zhou, 1999 and references).

Discs large (DLG) mediates the clustering of synaptic molecules. Synaptic localization of Dlg itself is regulated by CaMKII. Dlg and CaMKII colocalize at synapses and exist in the same protein complex. Constitutively activated CaMKII phenocopies structural abnormalities of dlg mutant synapses and dramatically increases extrajunctional Dlg. Decreased CaMKII activity causes opposite alterations. This was most clearly demonstrated by examining the size of the postsynaptic junctional membrane -- the SSR. Constitutive CaMKII activation results in poorly developed SSR, while CaMKII inhibition results in an overdeveloped SSR. These alterations phenocopy previously described effects of changing Dlg levels at the synaptic membrane. Mutations in dlg result in a poorly developed SSR, while overexpressing wild-type Dlg results in an overdeveloped SSR. The similarity between the phenotypes elicited by constitutive activation of CaMKII and those observed in dlg mutants is not solely restricted to the morphology of the SSR. An increase in the number of active zones, an enlargement of the boutons, and an abnormal Fas II clustering around these boutons is observed. Thus, these results strongly suggest that the changes in synaptic structure and composition elicited by changing CaMKII activity are the result of changes in DLG distribution. In vitro, CaMKII phosphorylates a Dlg fragment with a stoichiometry close to one. Moreover, expression of site-directed dlg mutants that block or mimic phosphorylation has effects similar to those observed upon inhibiting or constitutively activating CaMKII. It is proposed that CaMKII-dependent Dlg phosphorylation regulates the association of Dlg with the synaptic complex during development and plasticity, thus providing a link between synaptic activity and structure (Koh, 1999).

Antibodies against Drosophila CaMKII were used to determine its distribution at the larval NMJ. CaMKII is localized at wild-type NMJs around type I synaptic boutons in a pattern similar to Dlg. Double labeling with anti-Dlg reveals that Dlg and CaMKII are colocalized at bouton borders. In addition, transgenic CaMKII is targeted to synaptic boutons, as visualized by increased immunoreactivity levels at NMJs, upon expression of a CaMKII transgene in both motor neurons and muscles, by using muscle specific and motor neuron specific GAL4 activators. CaMKII and DLG colocalization is supported by immunoprecipitation experiments. Both proteins exist in the same complex in body wall muscles. When anti-CaMKII is used for immunoprecipitations of body wall muscle extracts, Dlg coimmunoprecipitates from wild-type, CaMKII+, or CaMKII-T287D overexpressing extracts. Moreover, transgenic Dlg expressed in dlg^X1-2 mutants is also coimmunoprecipitated by CaMKII. Thus, Dlg and CaMKII are closely associated at type I NMJs (Koh, 1999).

It is proposed that the anchoring of Dlg at the synapse is optimal when Dlg is in the dephosphorylated state. Upon phosphorylation, Dlg is less restricted to the synaptic complex. Because Dlg is essential for proper synaptic localization of Fas II and Shaker, these binding partners are similarly free to move away from the synaptic complex upon CaMKII-dependent phosphorylation. An alternative possibility however, and one that is not mutually exclusive, is that CaMKII-dependent phosphorylation of Dlg affects its targeting to the synapse during development. However, CaMKII and DLG colocalize at synapses. Therefore, it is likely that CaMKII-dependent phosphorylation of Dlg occurs at the synapse. It is known that synaptic Fas II is downregulated by increased cAMP levels, suggesting that PKA is also involved in coupling synaptic activity to structural plasticity at NMJs. Both PKA and CaMKII can be activated by various synaptic stimuli. Whether both signal transduction pathways act together or in parallel to regulate Dlg-dependent localization of Fas II remains to be determined (Koh, 1999 and references).

Drosophila Uba2 and Ubc9 (Lesswright) SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. At least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 (see SUMO) in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, a characterization of the system was undertaken. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function (Long, 2000).

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (Dlg), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (Fas2) and the position specific (PS) integrin receptors, including ßPS (Myospheroid) and its alpha partners (alphaPS1, alphaPS2, PS3/alpha Volado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that Dlg and Fas2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. ßPS integrin physically associates with the synaptic complex anchored by the Dlg scaffolding protein, which contains CaMKII and Fas2. This study demonstrates an alteration of the Fas2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory ßPS integrin mutations increase the expression and synaptic localization of Fas2. Synaptic structural defects in ßPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of Fas2 (distal in pathway). These studies demonstrate that ßPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology (Beumer, 2002).

ßPS integrin is a transmembrane receptor present in both pre- and post-synaptic membranes at the larval NMJ. Dlg is a synaptic scaffolding protein associated with both pre-and post-synaptic membranes and is also involved in the regulation of synaptic morphology, through the localization of diverse transmembrane proteins (including Fas2). Studies were undertaken to determine whether ßPS integrin associates with the DLG complex to tie together these disparate receptor components in a common molecular machine. In confocal analyses, ßPS integrin and Dlg co-localize at the larval NMJ. Dlg clearly has less extensive expression, more tightly localized at NMJ boutons, whereas ßPS is more extensive through the subsynaptic reticulum (SSR) and also localized at extrasynaptic sites in the muscle, including the muscle sarcomere and attachment sites. However, both proteins are most intensively expressed at the NMJ presynaptic/postsynaptic interface, where they co-localize. Therefore, tests were performed to determine if ßPS integrins form part of the synaptic complex linked by Dlg. Protein immunoprecipitation assays were performed using rabbit anti-DLG to probe Oregon R head extracts. Dlg antibodies clearly co-immunoprecipitate Dlg and ßPS integrin protein, consistent with co-localization observed in confocal analysis. Inspection of the ratio of both bound proteins and proteins not bound to beads (immunoprecipitation versus flow through lanes) indicates that a large portion of the ßPS integrin protein associates with a complex containing Dlg. The fact that ßPS was found to co-immunoprecipitate with the complex mediated by Dlg provides support for integrins existing in a synaptic complex with Fas2 and CaMKII at the synapse (Beumer, 2002).

To assay the mys requirement in synaptic development comprehensively, two regulatory alleles of mys with opposing structural phenotypes were compared and contrasted: mys^b9, which causes NMJ undergrowth, and mys^ts1, which causes NMJ overgrowth. In both mutants, a correlation was observed between the morphological alterations and synaptic function, in contrast to the homeostasis seen in Fas2 mutant synapses but consistent with the parallel alterations seen in CaMKII-inhibited synapses. However, the correlation between synaptic structural and functional alterations in mys mutants is not striking, and it is clear that integrins primarily mediate architectural regulation. Therefore, the focus in this study was exclusively on the mechanism(s) by which integrins modulate NMJ structural development. This article presents molecular and genetic evidence that strongly support the hypothesis that synaptic integrin receptors containing ßPS modulate CaMKII activation on both sides of the synaptic cleft and, through CaMKII, control both the expression and the synaptic localization of Fas2 at the synapse. The primary experimental results supporting these conclusions are detailed below (Beumer, 2002).

(1) Transgenic expression of CaMKII is sufficient to completely rescue all synaptic structure defects in mys mutants. Genetically increasing CaMKII in postsynaptic muscle, but not presynaptic neuron, completely rescues the structural undergrowth of the mys^b9 integrin mutant, whereas it is necessary to elevate CaMKII both pre- and post-synaptically to rescue structural overgrowth in the mys^ts1 mutant. These results are consistent with the postsynaptic mislocalization of ßPS integrin in the mys^b9 mutant, as opposed to the global loss of synaptic integrin in the mys^ts1 mutant. These data indicate that coordinate regulation of CaMKII in the muscle and motoneuron is necessary for proper development of synaptic architecture (Beumer, 2002).

(2) At the distal end of the cascade, both the expression and synaptic localization of Fas2 are increased in mys mutants, although the extent of Fas2 misregulation is significantly different between the two regulatory mutants. Importantly, both the NMJ overgrowth (mys^ts1) and undergrowth (mys^b9) phenotypes are rescued towards wild-type structure by genetically reducing the amount of Fas2 available at the synapse to near normal levels. In mys^ts1 mutants, correcting for Fas2 level suppresses the synaptic overgrowth, while in mys^b9 mutants, correcting the Fas2 level suppresses the synaptic undergrowth. These results support the conclusion that Fas2 is centrally involved in mediating synaptic growth, but suggest that the Fas2-mediated mechanism is more complex than previously thought (Beumer, 2002).

The results demonstrate that integrins regulate morphological growth at the postembryonic NMJ through activation of CaMKII in both pre- and post-synaptic compartments. One important target of CaMKII is Fas2 and it is clear that regulation of Fas2 expression and localization is an important component of integrin regulation. However, the modulation of Fas2 levels alone is unlikely to account fully for the alterations in synaptic architecture in integrin mutants. In particular, both mys^ts1 and mys^b9 upregulate synaptic Fas2 levels, albeit to different degrees, yet show opposite alterations in synaptic growth. Moreover, reducing Fas2 levels rescues both under- and overgrowth defects, but the rescue is not perfect (Beumer, 2002).

Precise control of Fas2 levels finely tunes morphological development at the NMJ. Different degrees of reduced Fas2 expression can either facilitate or inhibit the growth/maintenance of the NMJ, and reduced Fas2 expression has been demonstrated in other overgrowth mutants. However, overexpression of Fas2 in specific muscles drives increased NMJ elaboration/bouton differentiation and selective preference for a high-expressing muscle over a low-expressing muscle. How can this complexity be interpreted? One likely explanation is interaction between Fas2 and other developmental pathways regulated by CaMKII. To date, the only known Fas2-independent regulation of synaptic morphology involves a deubiquitinating protease encoded by fat facets (faf), and a putative ubiquitin ligase encoded by highwire (hiw), which have been shown to work together to modulate the degree of NMJ growth. Loss-of-function hiw mutants display NMJ structural overgrowth, importantly without a concomitant decrease in Fas2. Indeed, overexpression of Fas2 cannot suppress the overgrowth seen in hiw mutants. These observations are consistent with the overgrowth combined with overexpression of Fas2 observed in mys^ts1 mutants in this study. However, any putative interaction between Fas2-dependent and -independent mechanisms of morphological growth regulation at the Drosophila NMJ synapse remain to be elucidated. It is expected that further study will reveal an additional structural control mechanism regulated by CaMKII, acting in parallel to and interacting with Fas2. The ubiquination mechanism discussed here is one candidate mechanism, but as CaMKII is known to have many synaptic targets, it is clearly not the only candidate (Beumer, 2002).

In summary, it is concluded that architectural developmental defects observed in NMJ synapses mutant for ßPS integrin are due to the loss of the ability to regulate synaptic CaMKII properly. One function of CaMKII is to phosphorylate the scaffolding protein Dlg, and thus regulate the proteins synaptically localized by this scaffold. Fas2 is the central known output of this regulatory cascade. Loss of this regulation is central to the mys mutant defects in the postembryonic elaboration of NMJ structure. It is clear from this study, however, that regulation of Fas2 localization via CaMKII is only one facet of how integrins function at the synapse (Beumer, 2002).

CaMKII is critical for structural and functional plasticity. Camguk (Cmg, also known as Caki), the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca2+/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca2+/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca2+/CaM, can provide a localized source of active kinase. When Ca2+/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses (Lu, 2003).

Cmg, mammalian CASK, and C. elegans Lin-2 form a MAGUK subfamily that is defined by an N-terminal CaMKII-like domain in addition to the SH3, PDZ, and guanylate kinase domains typical of the MAGUK family. In mammalian neurons, CASK has been shown to be localized to synapses both pre- and postsynaptically where it associates with cell surface molecules like neurexins, calcium channels, and syndecans, the cytoskeletal protein 4.1, PSD95, and other adaptor molecules. CASK may also participate in signaling to the nucleus by directly interacting with a transcription factor (Lu, 2003 and references therein).

Cmg null animals have impaired locomotion, but their ability to learn had not been tested. Modification of courtship behavior in Drosophila has been used to test both associative and nonassociative memory formation; CaMKII is required for both. Exposure of a male to a mated female leads to decreased courtship of a subsequently presented virgin. This suppression reflects formation of an associative memory that requires mushroom body and central complex circuits. Courtship suppression also occurs during the training period via a non-mushroom body central circuit. Habituation to a courtship stimulating cue can be demonstrated after training with immature males. In these behavioral assays, the locomotor defects of the Cmg null flies could only cause an underestimate of behavioral defects since 'failure' in the assay is signified by an increase in courtship activity. Wild-type, Cmg null, and cmg deficiency heterozygotes were tested for mated female conditioning. All genotypes were normal for associative memory formation when compared to males that had been sham conditioned in an empty chamber. Behavior during the training period, however, was abnormal; Cmg null males fail to decrease courtship during training. Deficiency heterozygotes are intermediate. To determine if the Cmg null has a habituation defect, the response to immature males, who emit courtship promoting pheromones distinct from the female type, was examined. Cmg null animals fail to habituate. Courting Cmg null males have normal initial courtship levels and the ability to discriminate between virgin and mated females, indicating that peripheral sensory pathways are intact (Lu, 2003).

Association of Cmg with CaMKII is stimulated by ATP bound to the CaMKII catalytic domain. At physiological levels of ATP, Cmg and CaMKII are likely to be associated. ATP hydrolysis in the presence of Ca2+/CaM leads to autophosphorylation of CaMKII on T287 and production of a Cmg-localized constitutively active kinase. Cmg can also promote ATP hydrolysis by CaMKII in the absence of Ca2+/CaM. Under these conditions and without prior T287 autophosphorylation, CaMKII preferentially autophosphorylates its own CaM binding domain. This reaction leads to the fast release of the CaMKII in a form that cannot be activated by Ca2+/CaM. The ability of genetic manipulation of Cmg, PP2A, and synaptic activity levels in vivo to alter the amount of postsynaptic CaMKII that is in the inactivated state argues strongly that these reactions are physiologically relevant and define a new mechanism for the regulation of CaMKII activity (Lu, 2003).

What is the function of this interaction in vivo? One possible role is to provide a basis for maintaining differences between active and inactive synapses. The effect of cmg on courtship conditioning, a behavior known to require CaMKII, is suggestive of a role for this interaction in behavioral plasticity. Differences in synaptic strength can fine tune networks and are crucial to most memory models. Local CaMKII inactivation by Cmg could serve to decrease the gain on CaMKII-mediated calcium signaling at synapses that have been inactive. At low Ca2+, T306 autophosphorylation would occur, and inactive kinase would be released from Cmg. This provides a 'use it or lose it' mechanism for preserving synapse-specific strength differentials, since CaMKII tethered to Cmg at synapses that see higher Ca2+ levels would be protected from inactivating autophosphorylation by bound CaM. This complexed kinase can undergo T287 autophosphorylation, resulting in constitutive kinase activity and a slowed CaM dissociation rate that would further prolong the lifetime of the complex. Whether the Cmg-mediated inactivation of CaMKII operates locally or globally remains to be determined and likely depends on the ability of the inactivated kinase to escape from complexes with its other binding partners in the postsynaptic density (Lu, 2003).

Establishment of a pool of autophosphorylated CaMKII that cannot be directly stimulated by Ca2+/CaM also provides a mechanism for phosphatase activity to regulate CaMKII. In vitro, both PP1 and PP2A are able to dephosphorylate the CaM binding domain of CaMKII. In Drosophila, PP2A is the relevant in vivo phosphatase for postsynaptic pT306. The opposing actions of Cmg and PP2A provide machinery for dynamic regulation of the maximum level of calcium-activatable CaMKII at synapses (Lu, 2003).

In mouse, knockin of a CaMKII T305D mutation blocks LTP and learning and decreases postsynaptic localization of the kinase, suggesting that this mutant is unable to interact with one or more of its normal synaptic binding partners. Inability to undergo inactivating autophosphorylation (T305VT306A knockin) is associated with a lower threshold for LTP and loss of behavioral fine tuning. These data argue that, in mammals, autophosphorylation of the CaM binding domain of CaMKII is also an important regulatory mechanism for both function and localization of CaMKII. Whether in mammals CASK (the Cmg homolog) is the critical synaptic binding partner and regulator of synaptic T305 autophosphorylation is unknown (Lu, 2003).

This study also demonstrates a novel binding mechanism for CaMKII on a MAGUK scaffolding protein. It is proposed that this interaction consists of two discrete steps. First, binding of ATP to the catalytic domain of CaMKII reveals an interaction surface on the kinase that forms an initial bond to the Cmg protein. This initial interaction can occur either in the presence or absence of Ca2+/CaM, implying that it does not involve the C-terminal CaM binding sequences of the autoinhibitory domain. The ability of a peptide corresponding to the N terminus of the autoinhibitory domain to partially block complex formation suggests that this region, which is known to be involved in regulation of ATP binding or the catalytic domain surface that the N terminus of the autoinhibitory domain binds to, is the site of this initial interaction (Lu, 2003).

Once an initial interaction is established, bound ATP is no longer required. This could be due to a stabilization of the ATP-bound conformation by Cmg binding. Stabilization of the open form of the kinase autoregulatory domain is postulated to occur after Ca2+/CaM-dependent NR2B binding, since this interaction is insensitive to CaM stripping. Another possibility for the loss of ATP requirement is that a secondary interaction occurs that abrogates the need for the initial interaction. Such a secondary interaction does occur, but this does not rule out a concurrent stabilization of the ATP-bound conformation (Lu, 2003).

The secondary interaction that forms after ATP-dependent binding of Cmg involves the C-terminal region of the autoinhibitory domain, including the CaM binding sequences. Three lines of evidence suggest that this secondary interaction provides the most stable form of the complex. (1) N-terminal peptides only partially block complex formation, while peptides that contain the CaM binding domain completely block complex formation. (2) CaMKIIs with mutations of T306/7 do not bind to Cmg at all. These mutants are fully capable of binding ATP, but the exposure of the initial binding domain is insufficient to maintain a salt-stable interaction in the absence of a normal CaM binding domain. The failure of the relatively subtle threonine to serine mutant to bind suggests a direct interaction of Cmg with T306/7. (3) The natural release mechanism of this complex is via autophosphorylation of the CaM binding domain. Protection of this region by Ca2+/CaM can prevent both the autophosphorylation and dissociation of CaMKII (Lu, 2003).

The two-step association mechanism and the autophosphorylation-regulated dissociation of CaMKII from Cmg provide a new way in which CaMKII can be localized and regulated using its autoinhibitory domain. As defined for catalytic activation, the regulatory region of CaMKII is a small but complicated protein interaction domain. It is becoming clear that this region can support multiple intra- and intermolecular interactions that have profound effects on local CaMKII activity and synaptic plasticity (Lu, 2003).

The ability of CaMKII to act as a molecular switch, becoming Ca²⁺ independent after activation and autophosphorylation at T287, is critical for experience-dependent synaptic plasticity. This study shows that Caki, the Drosophila homolog of CASK, also known as Camguk, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's Calmodulin (CaM) binding domain. Phosphorylation of T306 blocks Ca²⁺/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch (Hodge, 2006).

Autophosphorylation of CaMKII at a site in the N terminus of its autoregulatory domain (T287 in Drosophila and T286 in mammalian αCaMKII) confers Ca²⁺-independent activity on the enzyme. This switch-like property of the kinase is crucial to its role in long-term potentiation and memory formation in mammals and flies, and generation of the autonomous form of the kinase can be stimulated by a number of different behaviors and activity paradigms. Interestingly, in mice, both mutations that prevent T286 phosphorylation, and transgenes that increase constitutive activity have been shown to block plasticity. This implies that the level of constitutive activity needs to be tightly controlled to remain in a range optimal for learning. This balance has been believed to be exerted by the positive effects of Ca²⁺/CaM and the negative effects of phosphatases. To date, no proteins other than calmodulin have been shown to influence the development of autonomous activity via T287 autophosphorylation (Hodge, 2006).

The function of autophosphorylation in the C terminus of the regulatory domain within the CaM binding region (T306 in Drosophila and T305 in mammalian αCaMKII) has been more mysterious, but also has consequences for plasticity. In the test tube, with purified kinase, this phosphorylation occurs only after T287 phosphorylation and removal of CaM with EGTA because the site is protected from phosphorylation by bound CaM. With purified kinase, this means that pT306 is only found in doubly phosphorylated pT287, pT306 enzyme. Recently, a novel mechanism for phosphorylation of T306 in the absence of pT287 has been described (Lu, 2003). dCASK interacts with the regulatory domain of the kinase and, when the CaM binding site of the kinase is unoccupied (e.g., when synaptic calcium is low), stimulates the kinase to autophosphorylate at T306. This reaction releases CaMKII from the dCASK complex in a form that is incapable of binding Ca²⁺/CaM and has been suggested to provide a mechanism for downregulation of the activatable kinase pool at quiescent synapses. The effects of dCASK on T287 phosphorylation have not been addressed, and modulation of this site would have important consequences for plasticity (Hodge, 2006).

This study presents evidence that dCASK can also act as an activity-dependent modulator of the levels of constitutively active CaMKII in vivo. CaMKII pT287 autophosphorylation occurs within the dodecameric holoenzyme and requires Ca²⁺/CaM to be bound to both the subunit doing the catalysis and the subunit that contains the substrate threonine. This cooperativity implies that modifications that alter the ability of Ca²⁺/CaM to bind would affect the ability of the kinase to become Ca²⁺ independent. Because it promotes phosphorylation of T306, interaction with dCASK regulates T287 phosphorylation by altering the occupancy of CaM binding sites in the holoenzyme. In vivo this postsynaptic interaction provides a synapse-specific mechanism to alter the probability of generating autonomous activity that is controlled by the activity history of the synapse. This makes dCASK an important gain controller for the CaMKII molecular switch (Hodge, 2006).

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. This study found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and this study show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and evidence is presented that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation (Wang, 2011).

Dlg is a Drosophila member of a family of MAGUK scaffolding proteins that has four mammalian members, SAP97/hDlg, PSD-93/Chapsyn-110, PSD-95/SAP90 and SAP102/NE-Dlg, and which has important developmental and regulatory roles in the nervous system and in epithelia. Phosphorylation is emerging as a mechanism for the regulation of the localization and function of the Dlg family. The post-synaptic targeting of Dlg to the Drosophila NMJ is inhibited by phosphorylation of the first PDZ domain by CaMKII and of the GUK domain by PAR-1. In mammalian neurons, CaMKII phosphorylation of PDZ1 of PSD-95 terminates long term potentiation-induced spine growth by inducing translocation of PSD-95 out of the active spine. Furthermore, phosphorylation of PDZ1 of either PSD-95 or SAP97/hDlg by CaMKII regulates their interaction with NMDA subunits, and additional cell culture studies in epithelial cells demonstrate effects of SAP97/hDlg phosphorylation by various kinases (Wang, 2011).

An important function of Dlg at the NMJ is involvement in synaptic plasticity during muscle growth, at least in part by controlling the localization at the pre-synaptic and post-synaptic membranes of Fas2, a homophilic cell adhesion molecule of the immunoglobulin superfamily that binds Dlg. The breaking and restoration of Dlg/Fas2-mediated adhesion between the pre- and post-synaptic membranes is likely critical to synaptic growth during development. This study has shown that Hts exists in a complex with Dlg, probably at both the post-synaptic membrane of the NMJ and at the lateral membrane in epithelia, where the two proteins are likely brought into close proximity by a shared association with the spectrin-actin junction. Hts is therefore positioned to locally regulate Dlg and participate in synaptic plasticity at sites of association with the membrane cytoskeleton, and it positively contributes to phosphorylation of Dlg on its GUK domain. This site of phosphorylation is a target for PAR-1, and consistent with this it was found that Hts can regulate the levels of PAR-1. Phosphorylation of Dlg impedes its targeting to the post-synaptic membrane and it is proposed that Hts regulates this targeting at the synapse by controlling the levels of PAR-1 at the post-synaptic membrane. This could occur through Hts acting as a scaffold participating in localized stabilization or translation of PAR-1 at the post-synaptic membrane and, consistent with the latter mechanism, localized post-synaptic translation of NMJ proteins has been reported in Drosophila. Over-expression of Hts permits synaptic overgrowth, which may in part be due to effects on Dlg targeting to the post-synaptic membrane, but unpublished results indicating additional routes of action. Furthermore, if Hts were acting mainly by elevating PAR-1 levels, synaptic undergrowth would be expected rather than overgrowth with Hts over-expression, as the post-synaptic over-expression of PAR-1 leads to decreased bouton number and an oversimplified synapse. CaMKII also regulates synaptic growth and Dlg targeting at the NMJ through phosphorylation. There is currently no antibody available to examine phosphorylation of Dlg by CaMKII in vivo, but it was determined that Hts controls the levels of this kinase similarly to PAR-1, suggesting that Hts may also be regulating Dlg targeting via CaMKII-dependent phosphorylation (Wang, 2011).

Two other studies address Hts function in neurons, one describing a role in photoreceptor axon guidance and the other characterizing pre-synaptic Hts function at the NMJ (Ohler, 2011; Pielage, 2011). The latter study reported a phenotype of synaptic retraction at the hts mutant NMJ, revealed by the absence of the pre-synaptic marker Bruchpilot from extensive stretches of the NMJ, which is consistent with the observation of small synapses in hts mutants stained for the synaptic vesicle marker CSP-2 (Pielage, 2011). However, in addition to retraction, this group observed overgrowth of the hts mutant NMJ, visible with anti-HRP staining. Although no quantification was performed using anti-HRP, qualitative examination of hts mutant muscle 6/7 NMJs in the current study did not indicate an overgrowth phenotype. A possible explanation for this discrepancy is the choice of the muscle 6/7 NMJ for analysis in contrast to Pielage, who focused on muscle 4. Unlike most other larval muscles, muscles 6 and 7 are not innervated by type II boutons. A component of the synaptic overgrowth reported by Pielage is the extension of thin, actin-rich extensions somewhat similar in appearance to type II and type III processes and, accordingly, growth of these processes is impaired with pre-synaptic over-expression of Hts (Pielage, 2011). Pielage proposes that pre-synaptic Hts restricts synaptic growth through its function as an actin-capping protein. At the muscle 6/7 NMJ this role may not be so prevalent and the post-synaptic growth-promoting function of Hts prevails. By examining NMJs other than muscle 6/7 in hts mutant larvae it was possible to confirm the overgrowth phenotype observed by Pielage (Wang, 2011).

Synaptic plasticity at the Drosophila NMJ has parallels with the structural changes seen at synapses in cellular models of learning such as long-term facilitation (LTF) in Aplysia and long-term potentiation in the mammalian hippocampus, and several studies indicate that the current results with hts at the NMJ may be relevant to these other systems. Phosphorylation of the conserved serine residue in the MARCKS domain of adducin is increased during LTF in Aplysia, and mice lacking β-adducin exhibit impaired synaptic plasticity and learning (Bednarek, 2011; Gruenbaum, 2003; Porro, 2010; Rabenstein, 2005; Ruediger, 2011; Wang, 2011 and references therein).

Similar to the plasticity exhibited by the NMJ during development, the morphogenesis of epithelia involves what has been referred to as 'epithelial plasticity' in which cell-cell adhesions are disassembled and cells become motile, for example in epithelial-mesenchymal transitions. The acquisition of motility by epithelial cells is also involved in tumor cell invasion and metastasis. The Drosophila follicular epithelium is a model for studying both developmental and pathological epithelial plasticity, and recent observations indicate that Dlg and Fas2 collaborate to prevent inappropriate invasion of follicle cells between neighboring germ cells. Furthermore, border cell migration, an invasion of a subset of follicle cells between germ cells occurring during normal oogenesis, requires downregulation of Fas2 expression in the border cells. PAR-1 is required for detachment of border cells from the follicular epithelium and it is interesting to speculate that this might involve regulation of Dlg localization. These various results indicate that regulation of the Dlg/Fas2 complex is important for the epithelial plasticity exhibited by follicle cells and this mechanism likely applies to epithelia in the developing embryo. As in muscle, Hts over-expression causes elevated levels of PAR-1 and CaMKII in epithelial cells. While Hts over-expression does not cause discernible Dlg delocalization in the epidermis, it disrupts the membrane localization of Dlg in amnioserosa cells. Dlg in the amnioserosa might be particularly susceptible to Hts function, as it not incorporated into septate junctions in this tissue, unlike in the epidermis. Furthermore, if the delocalization of Dlg by Hts is CaMKII-dependent, it would be more pronounced in the amnioserosa than the epidermis as Hts can only weakly promote CaMKII accumulation in the epidermis. In addition to showing that Hts can regulate Dlg localization in the amnioserosa, this study has determined that proper cortical localization of Hts in amnioserosa cells in the early stages of dorsal closure is dependent on Dlg. This result suggests that where Dlg and Hts are together in a complex, Dlg stabilizes the membrane localization of Hts. This stabilization may occur in cells other than the amnioserosa but might not be readily detectable. Hts localizes all around the epithelial membrane, with much of it not co-localizing with Dlg. In the very flat, squamous cells of the amnioserosa early in dorsal closure, the proportion of lateral membrane Hts dependent on Dlg for stabilization may be greater than in more columnar epithelial cells such as those in the epidermis (Wang, 2011).

Consistent with the effects that they have on each other's localization, the frequency of cuticle defects seen in dlg hts zygotic double mutant embryos suggests that Hts and Dlg co-operate in epithelial development, and it is of interest that mammalian adducin has recently been implicated in the stabilization and remodeling of epithelial junctions (Naydenov, 2010). Embryos deficient in Hts likely have a diminished ability to delocalize Dlg, effectively reducing the pool of Dlg available for de novo junction formation, and this situation would be worsened by reducing Dlg levels with a dlg allele. Conversely, reducing Dlg in an embryo deficient in Hts may further compromise Hts function through effects on Hts membrane localization. One of the phenotypes seen in hts mutants, the frequency of which is increased by additionally reducing Dlg, is that of embryos which secrete only small pieces of cuticle. This phenotype is characteristic of maternal zygotic dlg mutant embryos (Wang, 2011).

The interaction of Hts with Dlg suggests that adducin could be a regulator of the septate junction and it will be of interest to examine adducin effects on septate junction morphology in the nervous system. In the Drosophila nervous system septate junctions are formed between glial cells, whereas in mammals they are found between glial and neuronal membranes at the paranodal junction. Interestingly, β-adducin was recently identified as a paranodal junction protein localized to the neuronal membrane where it co-localizes with NCP1, the vertebrate homolog of the Drosophila septate junction protein Neurexin IV (Ogawa and Rasband, 2009) (Wang, 2011).

Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. Enhanced green fluorescent protein-tagged arrestin 2 (Arr2) was used to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. It was demonstrated that during degeneration of norpA^P24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA^P24 is the failure to activate CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca²⁺ entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. How reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration was investigated. To uncover the consequence underlying a lack of CaMKII activation, ala¹ flies were characterized in which CaMKII was suppressed by an inhibitory peptide, and it was shown that morphology of rhabdomeres was not affected. In contrast, it was found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA^P24 photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, it is concluded that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration (Kristaponyte, 2012).

Fragile X mental retardation protein (FMRP) and Ataxin-2 (Atx2) are triplet expansion disease- and stress granule-associated proteins implicated in neuronal translational control and microRNA function. This study shows that Drosophila FMRP (dFMR1) is required for long-term olfactory habituation (LTH), a phenomenon dependent on Atx2-dependent potentiation of inhibitory transmission from local interneurons (LNs) to projection neurons (PNs) in the antennal lobe. dFMR1 is also required for LTH-associated depression of odor-evoked calcium transients in PNs. Strong transdominant genetic interactions among dFMR1, atx2, the deadbox helicase me31B, and argonaute1 (ago1) mutants, as well as coimmunoprecitation of dFMR1 with Atx2, indicate that dFMR1 and Atx2 function together in a microRNA-dependent process necessary for LTH. Consistently, PN or LN knockdown of dFMR1, Atx2, Me31B, or the miRNA-pathway protein GW182 increases expression of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) translational reporter. Moreover, brain immunoprecipitates of dFMR1 and Atx2 proteins include CaMKII mRNA, indicating respective physical interactions with this mRNA. Because CaMKII is necessary for LTH, these data indicate that fragile X mental retardation protein and Atx2 act via at least one common target RNA for memory-associated long-term synaptic plasticity. The observed requirement in LNs and PNs supports an emerging view that both presynaptic and postsynaptic translation are necessary for long-term synaptic plasticity. However, whereas Atx2 is necessary for the integrity of dendritic and somatic Me31B-containing particles, dFmr1 is not. Together, these data indicate that dFmr1 and Atx2 function in long-term but not short-term memory, regulating translation of at least some common presynaptic and postsynaptic target mRNAs in the same cells (Sudhakaran, 2013).

Observations presented in this study lead to three significant insights into the endogenous functions of dFmr1 and Atx2 in the nervous system and their contribution to long-term synaptic plasticity. First, the data strongly indicate that both proteins function in the same pathway, namely translational control, to mediate the form of long-term memory analyzed in this study. Second, the remarkably similar effects of knocking down these proteins in LNs and PNs provide in vivo support for an emerging idea that translational control of mRNAs in both presynaptic and postsynaptic compartments of participating synapses is necessary for long-term synaptic plasticity. Finally, although both dFmr1 and Atx2 have isoforms containing prion-like, Q/N domains, the different effects of loss of Atx2 and dFmr1 on neuronal Me31B aggregates indicate important differences in the mechanisms by which the two proteins function in translational control (Sudhakaran, 2013).

The different molecular and clinical consequence of pathogenic mutations in FMRP and Atx2 encoding genes has led to largely different perspectives on their functions. Fragile X causative mutations cause reduced levels of the encoding mRNA and lower levels of FMRP, leading to increased protein synthesis and a range of pathologies evident in children and young adults. These pathologies importantly do not include the formation of inclusion bodies. In contrast, SCA-2 and amyotrophic laterosclerosis causative mutations in Atx2 result in the dominant formation of inclusion body pathologies and age-dependent degeneration of the affected neuronal types. Observations made in this article indicate that the distinctive pathologies of the two diseases have obscured common molecular functions for the two proteins in vivo (Sudhakaran, 2013).

The genetic, behavioral, and biochemical observations show (1) shared roles of the two proteins in olfactory neurons for long-term but not short-term habituation, and (2) striking transdominant genetic interactions of dfrm1 and atx2 mutations with each other as well as with miRNA pathway proteins, which is not only consistent with prior genetic and behavioral studies of the two respective proteins but also strongly indicative of a common role for the two proteins in translational repression of neuronal mRNAs. This conclusion is supported at a mechanistic level by (3) the finding that both proteins are required for efficient repression mediated by the 3' UTR of CaMKII, a 3' UTR that this study shows to be repressed by the miRNA pathway, and (4) strong evidence for in vivo biochemical interaction among dFmr1 and Atx2 and for binding of these regulatory proteins with the UTR of the CaMKII transcript that they jointly regulate. Thus, dFMR1 and Atx2 function with miRNA pathway proteins for the regulation of a dendritically localized mRNA in identified olfactory neurons (Sudhakaran, 2013).

An unexpected observation was that dFMR1 and Atx2 seemed to be necessary for LTH as well as for CaMKII reporter regulation in both inhibitory LNs and excitatory PNs of the antennal lobe (Sudhakaran, 2013).

Until recently mammalian FMRP was regarded as a postsynaptic protein, consistent with the view that translational control of mRNAs essential for long-term plasticity occurs exclusively in postsynaptic dendrites. In contrast, work in Aplysia indicated that translational control of mRNAs is required in presynaptic terminals for long-term synaptic plasticity. This conflict between vertebrate and invertebrate perspectives is beginning to be resolved by findings that (1) mammalian FMRP is present in axons and presynaptic terminals; and that (2) translational control of both presynaptic and postsynaptic mRNAs is essential for long-term plasticity of cultured Aplysia sensorimotor synapses (Sudhakaran, 2013 and references therein).

Prior studies at the Drosophila neuromuscular junction have strongly indicated presynaptic functions for dFmr1 and translational control but have also pointed to their significant postsynaptic involvement in neuromuscular junction maturation, growth, and plasticity. More direct studies of experience-induced long-term plasticity have been performed in the context of Drosophila olfactory associative memory, wherein a specific dFmr1 isoform in particular and translational control in general are necessary for long-term forms of memory. However, the incomplete understanding of the underlying circuit mechanism has made it difficult to conclude presynaptic, postsynaptic, or dual locations for dFmr1 function in long-term memory. In contrast, recent work showing an essential role for Atx2 and Me31B in PNs for LTH more strongly indicate a postsynaptic requirement for translational control mediated by these proteins; however, this did not address a potential additional presynaptic function (Sudhakaran, 2013).

The finding that dFmr1 and Atx2 are necessary in both LNs and PNs for LTH, a process driven by changes in the strength of LN–PN synapses, provides powerful in vivo support for a consensus model in which translational control on both sides of the synapse is necessary for long-term plasticity. A formal caveat is that the anatomy of LN–PN synapses in Drosophila antennal lobes remains to be clarified at the EM level. If it emerges that these are reciprocal, dendrodendritic synapses, similar to those between granule and mitral cells in the mammalian olfactory bulb, then a clear assignment of the terms 'presynaptic' and 'postsynaptic' to the deduced activities of dFmr1 and Atx2 in this context may require further experiments (Sudhakaran, 2013).

Previous studies in Drosophila have indicated a broader role for Atx2 than dFmr1 in miRNA function in nonneuronal cells. Although Atx2 is necessary for optimal repression of four miRNA sensors examined in wing imaginal disk cells, dFmr1 is not necessary for repression of any of these sensors. The resulting conclusion that dFmr1 is required only for a subset of miRNAs to function in context of specific UTRs is consistent with the observation that only a subset of neuronal miRNAs associate with mammalian FMRP and that the protein shows poor colocalization with miRNA pathway and P-body components in mammalian cells. Parallel studies have shown that Atx2 in cells from yeast to man is required for the formation of mRNP aggregates termed stress granules, which in mammalian cells also contain Me31B/RCK and FMRP. In addition, biochemical interactions between these proteins and their mammalian homologs with each other as well as with other components of the miRNA pathway have been reported. However, neither the mechanisms of Atx2-driven mRNP assembly, nor the potential role for FMRP in such assembly, have been tested in molecular detail (Sudhakaran, 2013).

The demonstration that loss of Atx2 in neurons results in a substantial depletion of Me31B-positive foci in PN cell bodies and in dendrites is consistent with Atx2 being required for the assembly of these two different (somatic and synaptic) in vivo mRNP assemblies. Thus, the mechanisms that govern their assembly, particularly of synaptic mRNPs in vivo, overlap with mechanisms used in P-body and stress granule assembly in nonneuronal cells (Sudhakaran, 2013).

The finding that loss of dFmr1 has no visible effect on these Me31B-positive foci can be explained using either of two models. A simple model is that dFmr1 is not required for mRNP assembly, a function mediated exclusively by Atx2. This would suggest that Atx2 contains one or more functional domains missing in dFmr1 that allow the multivalent interactions necessary for mRNP assembly. This is most consistent with the observation that that although dFMR1 is a component of stress granules in Drosophila nonneuronal cells, it is not required for their assembly. An alternative model would allow both dFmr1 and Atx2 to mediate mRNP assembly but posit that dFmr1 is only present on a small subset of mRNPs, in contrast to Atx2, which is present on the majority. In such a scenario, loss of dFmr1 would only affect a very small number of mRNPs, too low to detect using the microscopic methods used in this study. In the context of these models, it is interesting that both dFmr1 and Atx2 contain prion-like Q/N domains, potentially capable of mediating mRNP assembly. It is to be noted here that the dFmr1 Q/N domain, although lacking prion-forming properties, is capable of serving as a protein interaction domain enabling the assembly of dFmr1 into RNP complexes. This observation would support the view that dFmr1 may be involved in the formation of only a subset of cellular mRNP complexes. Future studies that probe the potential distinctive properties of these assembly domains may help discriminate between these models. In addition, potential interaction of Atx2 with other proteins that are involved in mRNP formation across species, like Staufen, could help to understand the mechanisms behind Atx2-dependent function in mRNP assembly (Sudhakaran, 2013).

However, the observations presented in this study clearly show that despite the remarkable similarities in the roles of dFmr1 and Atx2 for repression of CaMKII expression at synapses and the control of synaptic plasticity that underlies long-term olfactory habituation, both proteins also have distinctive molecular functions in vivo (Sudhakaran, 2013).

Mutations that affect neuronal translational control are frequently associated with neurological disease, particularly with autism and neurodegeneration. Although these clinical conditions differ substantially in their presentation, a broadly common element is the reduced ability to adapt dynamically to changing environments, a process that may require activity-regulated translational control at synapses. Taken together with others, the observations of this study suggest that there may be two routes to defective activity-regulated translation. First, as in dFmr1 mutants, the key mRNAs are no longer sequestered and repressed, leading to a reduced ability to induce a necessary activity-induced increase in their translation. Second, it is suggested that increased aggregation of neuronal mRNPs (indicated by the frequent occurrence of TDP-43 and Atx2-positive mRNP aggregates in neurodegenerative disease) may result in a pathologically hyperrepressed state from which key mRNAs cannot be recruited for activity-induced translation. Thus, altered activity-regulated translation may provide a partial explanation not only for defects in memory consolidation associated with early-stage neurodegenerative disease but also for defects in adaptive ability seen in autism spectrum disorders (Sudhakaran, 2013).

DEVELOPMENTAL BIOLOGY

Embryonic

A trace amount of transcript is detected in cleavage stage embryos. After blastoderm formation, basal levels of zygotic expression are observed throughout the embryo. During germ band elongation high levels of expression become apparent in the anterior and posterior midgut. Expression in the midgut becomes more prominent during germ band shortening. At the same time, expression is observed in the neuroblasts of the central nervous system. Transcripts of CaM kinase II are expressed in great quantities in the central and peripheral nervous systems in the late embryonic stage of development (Ohsako, 1993).

In situ hybridization shows CaM kinase mRNA is present in both neuronal and nonneuronal tissues in adult Drosophila. No differential tissue distribution of isoforms was observed (Griffith, 1993b).

Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest (Ca2+-responsive transactivator, a syntaxin-related nuclear protein that interacts with CREB-binding protein and is expressed in the developing brain) or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. Genetic manipulation, imaging and quantitative dendritic architecture analysis were combined in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dalpha7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter it was demonstrated that AP-1 transcriptional activity is downstream of Dalpha7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. These results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies (Vonhoff, 2013).

By combining genetic and neuroanatomical tools with imaging in a single-cell model, the adult MN5 in Drosophila, this study demonstrates that: (1) AP-1 is transcriptionally active during all stages of postembryonic motoneuron dendritic growth, (2) AP-1 and excitatory cholinergic inputs are required for normal dendrite growth in MN5, (3) AP-1 transcriptional activity is enhanced via a CaMKII-dependent mechanism by increased neural activity during pupal development but not in the adult, and (4) both activity and AP-1 can promote or inhibit dendritic branching, depending on the developmental stage. AP-1 is required for normal MN5 dendrite growth downstream of activity and CaMKII (Vonhoff, 2013).

Although AP-1 has been thought to regulate dendrite development in an activity-dependent manner via global changes in gene expression, probably in a calcium-dependent manner as described for CREB or Crest, direct evidence for this hypothesis was sparse (Vonhoff, 2013).

This study demonstrated that excitatory cholinergic input to MN5 and AP-1 transcriptional activity were required for normal dendrite growth of MN5 during pupal life. MN5 total dendritic length and branch numbers were significantly reduced (~50%) by inhibition of AP-1 [by Jbz (a dominant-negative form of Jun) expression] and in Dα nAChR mutants. Conversely, overexpression of AP-1 or increased MN5 excitability as induced by potassium channel knockdown (by EKI) increased dendritic branching (Duch, 2008). Clearly, AP-1 acted downstream of activity as inhibition of AP-1 by Jbz completely attenuated EKI (electrical knock-in) mediated dendritic growth and branching (Vonhoff, 2013).

A new AP-1 reporter was employed to measure activity-induced AP-1 transcriptional activity by imaging, and to gain insight into the pathway that might connect MN5 activity to AP-1-dependent transcription. Although the detection threshold of this reporter might be too low to detect small changes in AP-1 activity, sensitivity was sufficient to reliably report increased AP-1 activity following overexpression of fos and jun, inhibition of AP-1 transcriptional activity by Jbz expression, and changes in AP-1 activity as induced by various manipulations of cellular signaling. Therefore, the reporter was deemed suitable for testing changes in AP-1 transcriptional activity in MN5 (Vonhoff, 2013).

Targeted expression of TrpA1 channels in MN5 allowed the induction of firing in vivo by temperature shifts during selected developmental periods. Activation of MN5 during pupal life for 36 hours (P9 to adult) or longer (P5 to adult) caused significant increases in AP-1-induced nuclear GFP fluorescence. By contrast, in adults neither similar nor longer durations of TrpA1 activation resulted in any detectable increase in AP-1 reporter-mediated nuclear GFP fluorescence in MN5. Similarly, live imaging in semi-intact adult preparations did not reveal any detectable AP-1 activity upon acute TrpA1 activation for various durations. This indicated that activity-dependent AP-1 activation was restricted to pupal life. However, whether AP-1 activation in the adult MN5 occurred upon patterned activity was not tested. Spaced stimuli that reflect endogenous activity patterns are required for insect motoneuron axonal and dendritic development and can regulate mammalian neuron dendritic morphology. However, during flight, MN5 fires tonically at frequencies between 5 and 20 Hz, a pattern that is well reflected by temperature-controlled TrpA1 channel activation. Therefore, adult flight behavior is unlikely to induce AP-1 activity, which is involved in dendrite and synapse development (Freeman, 2010). This is consistent with the assumption that dendritic structure is fairly stable in the adult (Vonhoff, 2013).

cAMP and Jun N-terminal kinase (Jnk) have been implicated as potential links between activity and AP-1 activation. Cell culture studies on Drosophila larval motoneurons and giant neurons demonstrate a role of calcium. This study showed that Dα7 nAChRs, which are highly permeable to calcium, were required for normal MN5 dendritic growth. Combining genetic manipulation of Dα7 nAChRs, AP-1 and CaMKII with imaging of AP-1 reporter activity revealed that CaMKII was required downstream of Dα7 nAChRs to cause AP-1-dependent transcription. These data show that activity-dependent calcium influx through nAChRs might activate AP-1 during pupal life via a CaMKII-dependent mechanism in vivo. Activity and AP-1 can promote or inhibit dendritic growth during pupal life, depending on timing (Vonhoff, 2013).

In larval motoneurons, AP-1 is required for dendritic overgrowth as induced by artificially increased activity (Hartwig, 2008). In MN5, AP-1 is required downstream of nAChRs and CaMKII for normal dendritic growth. By contrast, premature expression of AP-1 in MN5 inhibited dendritic growth. These data were consistent with the hypothesis that timing is the crucial factor. First, P103.3 and D42 both caused similar overgrowth but exhibited fairly different expression patterns. Second, C380-GAL4 and Dα7 nAChR-GAL4 both inhibited MN5 dendrite growth but expressed in largely different sets of neurons. Therefore, the common factor of C380 and Dα7 nAChR on the one hand and D42 and P103.3 on the other hand was timing. Third, shifting the timing of C380-GAL4-driven AP-1 expression to later stages prevented dendritic defects. Fourth, imposed activity prior to P5 by TrpA1 activation also inhibited dendritic branching. Dendritic defects as induced by imposed premature activity were rescued by inhibition of AP-1 via Jbz expression in MN5 (Vonhoff, 2013).

MN5 early dendritic growth starts at early pupal stage 5 (P5), and expression of Dα7 nAChRs begins 2.5 hours later, at mid stage P5. Similarly, Xenopus optic tectal and turtle cortical neurons receive glutamatergic and GABAergic inputs as soon as the first dendrites are formed. In vertebrates, early synaptic inputs and neurotransmitters play essential roles in dendrite development. The current data are consistent with the hypothesis that the endogenous expression of nAChRs caused increased activity throughout the developing motor networks, which, in turn, upregulated AP-1-dependent transcription and dendritic growth via a CaMKII-dependent mechanism. During zebrafish spinal cord development, activity is required for strengthening functional central pattern generator (CPG) connectivity. As dendrites are the seats of input synapses to motoneurons, an activity-dependent component in motoneuron dendritic growth that follows early synaptogenesis might function to refine dendrite shape during the integration into the developing CPG (Vonhoff, 2013).

Adult

Transcripts are more abundant in the head than in the body of the adult fly (Ohsako, 1993).

Expression is seen in ovaries in the somatic follicle cells and germ line oocytes. Adult expression is also observed in gut epithelium (Griffith, 1993b).

Courtship and courtship conditioning are behaviors that are regulated by multiple sensory inputs, including chemosensation and vision. Globally inhibiting CaMKII activity in Drosophila disrupts courtship plasticity while leaving visual and chemosensory perception intact. Light has been shown to modulate CaMKII-dependent memory formation in this paradigm and the circuitry for the nonvisual version of this behavior has been investigated. In this paradigm, volatile and tactile pheromones provide the primary driving force for courtship, and memory formation is dependent upon intact mushroom bodies and parts of the central complex. In the present study, the GAL4/UAS binary expression system has been used to define areas of the brain that require CaMKII for modulation of courtship conditioning in the presence of visual, as well as chemosensory, information. Visual input suppresses the ability of mushroom body- and central complex-specific CaMKII inhibition to disrupt memory formation, indicating that the cellular circuitry underlying this behavior can be remodeled by changing the driving sensory modality. These findings suggest that the potential for plasticity in courtship behavior is distributed among multiple biochemically and anatomically distinct cellular circuits (Joiner, 2000).

To analyze the distribution of Drosophila calcium/calmodulin-dependent protein kinase II in the adult brain, monoclonal antibodies were generated against the bacterially expressed 490-amino acid form of CaMKII. One of those, named #18 antibody, was used for this study. Western blot analysis of the adult head extracts showed that the antibody specifically detects multiple bands between 55 and 60 kDa corresponding to the molecular weights of the splicing isoforms of CaMKII. Epitope mapping revealed that it was in the region between amino acids 199 and 283 of CaMKII. Preferential CaMKII immunoreactivity in the embryonic nervous system, adult thoracic ganglion and gut, and larval neuro-muscular junction (NMJ) was consistent with previous observations by in situ hybridization and immunostaining with a polyclonal antibody at the NMJ, indicating that the antibody is applicable to immunohistochemistry. Although CaMKII immunoreactive signal was low in the retina, it was found at regular intervals in the outer margin of the compound eye. These signals were most likely to be interommatidial bristle mechanosensory neurons. CaMKII immunoreactivity in the brain was observed in almost all regions and relatively higher staining was found in the neuropilar region than in the cortex. Higher CaMKII immunoreactivity in the mushroom body (MB) was found in the entire gamma lobe including the heel, and dorsal tips of the alpha and alpha' lobes, while cores of alpha and beta lobes were stained lightly. Finding abundant CaMKII accumulation in the gamma lobe suggests that this lobe might especially require high levels of CaMKII expression to function properly among MB lobes (Takamatsu, 2003).

A model for the role of sarah in egg activation

The Drosophila modulatory calcineurin-interacting protein (MCIP) sarah (sra) is essential for meiotic progression in oocytes. Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract; it is unknown whether calcium signaling is involved. MCIPs [also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)] are highly conserved regulators of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 1 and 2. Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity, their in vivo functions remain unclear. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrestphenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. This is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process (Takeo, 2006; Horner, 2006).

In vertebrates and marine invertebrates, fertilization triggers a transient rise in free Ca²⁺, and this rise is responsible for subsequent activation events such as modification of the eggshell, prevention of polyspermy, and cell-cycle resumption. This study shows that a protein involved in a calcium signal-transduction pathway is necessary for several egg-activation events in Drosophila. The sarah gene product is a calcipressin; the human calcipressin DSCR1 can directly bind to and inhibit calcineurin, the only known phosphatase that is dependent on both calcium and calmodulin. The accompanying paper by Takeo (2006) verifies that the fly Sarah protein shares these biochemical properties (Horner, 2006).

The present understanding of egg activation in Xenopus and the activities of calcipressin make strong predictions for the role of sra in meiotic reactivation. Upon fertilization of frog eggs, Ca²⁺ activates calmodulin-dependent protein kinase II (CaMKII), which in turn directs the inactivation of Anaphase Promoting Complex (APC) inhibitors such as Erp1/Emi2. The APC in turn inactivates MPF through the destruction of cyclin, relieving the meiotic block and initiating other egg-activation events. The current results suggest that essentially the same pathway operates during the activation of Drosophila eggs. It is hypothesized that Sarah acts early in the pathway by mediating the antagonistic relationship between calcineurin and CaMKII; that is, CaMKII activity upon egg activation requires the inhibition of calcineurin by Sarah (Horner, 2006).

Consistent with this model is the similarity of the sarah phenotype to that associated with mutations in another Drosophila gene called cortex. The Cortex protein is a member of the Cdc20 protein family, whose members serve as specificity factors and activators for the APC. Meiosis arrests normally at metaphase I in cortex oocytes and resumes when the eggs are laid but then soon arrests again at metaphase II. The variation in the phase of the meiotic arrest (metaphase II versus anaphase I) being an exception, other aspects of egg activation are similarly affected by mutations in cortex and sra (Horner, 2006).

The meiotic arrest in both sra and cortex eggs is most simply explained by the failure of the APC to target certain molecules for degradation upon egg laying. In cortex eggs, cyclin A fails to be degraded. The cyclin B component of MPF remains undegraded in sra eggs. These results are consistent with the mitotic-arrest phenotypes seen in early Drosophila embryos expressing nondegradable cyclin A (metaphase arrest) or nondegradable cyclin B (early anaphase arrest). Different APC targets could remain undegraded in sra and cortex eggs because there are at least two Cdc20-like proteins (Cortex and Fzy) in Drosophila eggs. APC^Cortex and APC^Fzy may have different substrate specificities and may be differently regulated by APC inhibitors downstream of CaMKII (Horner, 2006).

The failure of the eggs laid by sra mutant mothers to translate the maternal bcd mRNA can also be understood in the same theoretical framework. The translation of bcd requires the polyadenylation of its mRNA; the enzyme poly(A) polymerase that catalyzes polyadenylation is phosphorylated and thus negatively regulated by MPF. Failure of APC activation in sra mutant eggs would therefore prevent both MPF inactivation and bcd mRNA translation (Horner, 2006).

Although meiosis and bcd translation are both disrupted in sra eggs, vitelline membrane (VM) cross-linking is apparently normal. VM cross-linking also occurs in other mutants that block female meiotic progression; such mutants include cortex, grauzone, prage, and wispy. In fact, VM reorganization can occur independently of every other known egg-activation event, including bcd mRNA translation, the degradation of other maternal mRNAs, and microtubule depolymerization. If there is only a single trigger for egg activation, it must therefore be able to activate at least two autonomous downstream pathways (one for VM cross-linking and a second for other events). Because in vitro activated eggs are defective in several aspects of embryonic development, it is difficult to interpret the finding of delayed VM modification in sra eggs upon in vitro activation. Although sra function is not formally required for VM organization, sra-dependent processes might nonetheless impinge on its efficiency (Horner, 2006).

In summary, results indicate that despite its independence from a sperm trigger, egg activation in Drosophila involves calcium-mediated pathways that are likely to be analogous to those in other animals. It is intriguing that among these downstream events is the acquisition of the egg's competence to remodel the sperm nucleus into the male pronucleus (Horner, 2006).

The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014; PubMed).

Effects of Mutation or Deletion

CaM kinase II is capable of directly regulating its own activity by autophosphorylation. To assess the involvement of CaM kinase in experience-dependent behavior in an intact animal, a specific peptide inhibitor of CaM kinase was expressed in transgenic Drosophila under control of an inducible promoter. These flies fail to learn normally in two behavioral plasticity paradigms: acoustic priming, a nonassociative measure of sensitization, and courtship conditioning, a measure of associative learning. The magnitude of the learning defect in the associative paradigm appears to be proportional to the level of expression of the peptide gene in the two transgenic lines and can be increased by heat shock induction of gene expression. These results suggest that CaM kinase activity is required for plastic behaviors in an intact animal (Griffith, 1993a).

CaM kinase II has been implicated in neural plasticity that underlies learning and memory processes. Transformant strains of Drosophila, ala1 and ala2, expressing a specific inhibitor of CaM kinase have altered short-term plasticity in synaptic transmission along with abnormal nerve terminal sprouting and directionality of outgrowth. These results represent an interesting parallel with the activity-dependent regulation of synaptic physiology and morphology by the cAMP cascade in Aplysia and Drosophila (See cAMP dependent protein kinase 1). In contrast to the learning mutants dunce and rutabaga, which are defective in the cAMP cascade, inhibition of CaM kinase in ala transformants causes increased sprouting at larval neuromuscular junctions near the nerve entry point, rather than altering the higher order branch segments. In addition, synaptic facilitation and potentiation are altered in a manner different from that observed in the cAMP mutants. Furthermore, synaptic currents in ala transformants are characterized by greater variability, suggesting an important role of CaM kinase in the stability of transmission (Wang, 1994).

Similar defects in both synaptic transmission and associative learning are produced in Drosophila melanogaster by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene ether à go-go (eag). These behavioral and synaptic defects are not simply additive in animals carrying both an eag mutation and a transgene for a protein kinase inhibitor. This raises the possibility that the phenotypes share a common pathway. At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. These similarities in behavior and synaptic physiology, the genetic interaction, and the in vitro biochemical interaction of the two molecules suggest that an important component of neural and behavioral plasticity may be mediated by modulation of Eag function by calcium/calmodulin-dependent protein kinase II (Griffith, 1994).

Transgenic Drosophila strains expressing an inhibitory peptide of CaM Kinase II, or a constitutive activated CaM kinase show altered neuronal process morphology compared to wild type in scanning electron microscopy of cultured mature neurons from embryonic neuroblasts. Significantly enhanced neural process growth is observed in cells with inhibited enzyme, and reduced process growth is observed with activated enzyme, suggesting that active CaM kinase is involved in the inhibition of neurite growth during development. The subcellular distribution of CaM kinase was examined in wild type neuronal cultures. Prior to neuronal contact there is little CaM kinase in neuronal processes, but after connections have been made the processes exhibit a high level of CaM kinase. These results suggest that the transport of CaM kinase to terminals does not occur until after or during the formation of neuronal connections when a functional synapse might be formed. These results also suggest a target-dependent transport of the enzyme along processes and an inhibitory role for CaM kinase on neurite branching. One of the roles of CaM kinase in the mature synapse is the phosphorylation of synapsin I (see Drosophila Synapsin), which is involve in the Ca2+ dependent release of neurotransmitter from neurosecretory vesicles. Synapsin, like CaM kinase translocates to the terminus following contact with the postsynaptic target. The down-regulatory role of CaM kinase contrasts with the apparent up-regulatory role of protein kinase C in neurite elongation (Broughton, 1996).

Flexing the femorotibial joint elicits a resistance response from the tibial extensor motor neurons, and this reflex response decays on repetition. The frequency of excitatory junction potentials (EJPs) was monitored throughout a repetitive series of sinusoidal flexion-extension movements, and the data were collected in 50 msec bins. The peak response corresponds to the flexion phase of the movement. With 2 Hz of stimulation the peak response decays smoothly, with a time constant of one second in these examples to a plateau of ~40% of the initial response. This response was readily fit with a single exponential decay function, as is the case for many other examples of habituation. This response decrement conforms to other features of habituation as well. When the 2 Hz stimulus is terminated, the initial response recovers spontaneously to ~75% of the initial amplitude in 2 sec and is fully recovered within 60 sec. As is the case for most other examples of habituation, the rate of decrement in this reflex is dependent on the frequency and amplitude of the stimulus. For example, decreasing the rate of flexion from 2 to 0.5 Hz increases the time constant of decay from 1 to 22 sec. The time constant also is correlated with stimulus strength. Finally, the response can be dishabituated by tactile stimuli to the abdomen. When an animal was stimulated with 15 successive flexion movements and a tactile stimulus is provided during the ninth and tenth stimulus, the response recovers to ~60% of the initial response (Jin, 1998).

To study the presynaptic function of CaMKII in this nonassociative form of learning, a P[Gal4] insertion line was used to target the expression of mutant forms of CaMKII to the sensory neurons controlling the reflex. Targeted expression of a calcium-independent CaMKII construct (T287D) in the sensory neurons eliminates habituation. Targeted expression of a mutant CaMKII incapable of achieving calcium independence (T287A) reduces the initial reflex response, but a strong facilitation then occurs, and this eliminates most of the habituation. Finally, when a CaMKII inhibitory peptide (ala) is expressed in sensory neurons, the initial response is reduced, followed by facilitation. Thus, increasing the amount of calcium-independent kinase activity with the T287D construct eliminates habituation, and the response level remains at its maximum throughout the stimulus trial. This implies that the additional kinase activity is able to mobilize transmitter release and relieve the presynaptic depression that is normally responsible for habituation. Even with reduced stimulus strength that enhances depression in most habituation paradigms, only a weak depression is seen. An increased amount of calcium-dependent activity with the T287A construct has a similar effect, slowing the rate of depression and supporting the idea that additional CaMKII activity can relieve habituation. These results suggest that basal CaMKII levels in the presynaptic neurons set the response level and dynamics of the entire neural circuit (Jin, 1998).

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, it is shown that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, presynaptic BMP receptor Wishful thinking is required for the retrograde signal to function. These results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Reducing the function of postsynaptic glutamate receptors at the neuromuscular junction (NMJ) of Drosophila triggers a retrograde signal from the postsynaptic muscle to the presynaptic motor neuron, leading to an increase in the amount of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of CaMKII. Reducing postsynaptic CaMKII activity by expressing a CaMKII inhibitory peptide in somatic muscles increases quantal content, mimicking the effect of reducing postsynaptic glutamate receptor activity. Furthermore, in glutamate receptor GluRIIA^-/- mutants, constitutive activation of CaMKII in muscles inhibits the retrograde signal and decreases quantal content. These changes in retrograde signaling and neurotransmitter release are not accompanied by any significant changes in the number of synaptic boutons per muscle surface area or any gross structural or ultrastructural alterations. However, upon inhibition of CaMKII postsynaptically the number of T bars per active zone in presynaptic boutons is significantly increased. Similarly, the number of T bars per active zone is doubled in GluRIIA^-/- mutant larvae. This increase is suppressed by constitutive activation of CaMKII in postsynaptic muscles in GluRIIA^-/- mutants. These results point to CaMKII as a key regulator of the retrograde signal controlling homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Postsynaptic inhibition of CaMKII activity is sufficient to increase presynaptic neurotransmitter release in a retrograde fashion. This increase in quantal content can be potentiated by expressing additional doses of the inhibitory transgene and suppressed by expressing a constitutively active CaMKII transgene simultaneously. These results suggest a direct involvement of CaMKII in controlling the retrograde signal that maintains the homeostasis of neurotransmitter release at the Drosophila NMJ (Haghighi, 2003).

While increasing the postsynaptic activity of CaMKII in wild-type larvae has no effect on neurotransmitter release, once the retrograde signal is induced (i.e., in GluRIIA^-/- mutants), activation of CaMKII can inhibit the signal. This is consistent with the observation that while removal of GluRIIA causes a decrease in quantal size and an increase in quantal content, overexpression of GluRIIA, which leads to an increase in quantal size, does not change quantal content. These results suggest that quantal content may be increased only when CaMKII activity is reduced to a critical threshold. As long as CaMKII activity remains above this critical threshold, quantal content is unchanged. This could be a mechanism through which the synapse can compensate for any reduction in muscle activity and ultimately maintain homeostasis (Haghighi, 2003).

In a recent study, Kazama (2003) has provided evidence for the involvement of postsynaptic CaMKII in the retrograde control of neurotransmission at the Drosophila NMJ. Changes in the activity of postsynaptic CaMKII have been shown to affect both neurotransmitter release and synaptic structure in early first instar larvae. Kazama also reports an apparent change in localization of postsynaptic glutamate receptors in response to postsynaptic activation of CaMKII. This is in contrast to the current observations; no changes were found in the Highaghi (2003) study in overall synaptic structure or localization of glutamate receptors in response to either inhibition or activation of postsynaptic CaMKII. The differences in these findings could be partially due to differences in the level and pattern of expression of transgenes (using different Gal4 lines) or due to the fact that the NMJ was examined at very different developmental stages. Haghighi (2003) examined late third instar larvae, while Kazama (2003) examined early first instar larvae. Interestingly, the results are in agreement in that both studies observed no changes in the amplitude or kinetics of spontaneous potentials, indicating that CaMKII does not directly modulate glutamate receptors at the Drosophila NMJ; this is not the case with vertebrates (Haghighi, 2003).

When the activity of postsynaptic glutamate receptors at the Drosophila NMJ is reduced, a retrograde signal from the muscle to the motor neuron is triggered that causes an increase in quantal content. It has been suggested that this retrograde signal could be triggered in response to changes in muscle depolarization or in response to Ca²⁺ conducted by glutamate receptors. The data indicate that postsynaptic activity of CaMKII plays an important role in controlling the signal. Both muscle depolarization and Ca²⁺ flux through glutamate receptors could be involved in changing the levels of intracellular Ca²⁺ and thus that of CaMKII. The idea is favored that calcium influx through glutamate receptors is at least in part responsible for activating CaMKII and triggering the retrograde signal. There are several lines of evidence that support this hypothesis (Haghighi, 2003).

One line of evidence is based on the glutamate receptor ion channel properties and how they are changed in GluRIIA^-/- mutants. Compared to wild-type receptors, glutamate receptors in these mutants have a greatly reduced single-channel mean open time; this also affects the kinetics of EPSPs. Therefore, evoked currents that give rise to similar EPSP peak amplitudes in wild-type and GluRIIA^-/- mutants will lead to less ion influx in the mutants (ion flux is a product of time and current). Considering the high Ca²⁺ permeability of glutamate receptors (P_Ca/P_Na = 9.55), due to this reduced ion influx, Ca²⁺ influx will also be reduced in GluRIIA^-/- mutants both during spontaneous and evoked activities. Therefore, it is conceivable that this change in Ca²⁺ influx, monitored by CaMKII, could act as a trigger for the retrograde signal (Haghighi, 2003).

This hypothesis is further supported by data demonstrating that the retrograde increase in quantal content in GluRIIA^-/- mutants can be counteracted by overexpressing multiple copies of GluRIIA, independent of the size of EPSPs. The conclusion from these results is that the retrograde control of presynaptic release for these genotypes is not directly related to muscle depolarization. In addition, it is argued that if muscle depolarization were the sole trigger for the homeostatic retrograde signaling, then quantal content in highwire (hiw) mutants should have been compensated for. hiw mutants have 60%-70% less quantal release, while retrograde control of neurotransmission is still intact in these mutants. Therefore, it is proposed that postsynaptic CaMKII regulates presynaptic release by responding to calcium influx through glutamate receptors during evoked and spontaneous neurotransmitter release (Haghighi, 2003).

The role of postsynaptic membrane depolarization in homeostatic control of presynaptic release at the Drosophila NMJ has been investigated by Paradis (2001). This study demonstrates that the expression of an inward-rectifying potassium channel, Kir2.1, in postsynaptic muscles leads to an increase in quantal content. Kir2.1-expressing muscles show severe defects in muscle properties, including input resistance and membrane potential. More importantly, muscle excitability is affected to the point that mEPSP amplitude is reduced to less than half of wild-type levels (Paradis, 2001). The authors further show that mEPSCs are still wild-type under voltage-clamp conditions, suggesting no change in glutamate receptor function. However, considering the kinetics of membrane depolarization, it is conceivable that, under physiological conditions, GluRIIA function could be compromised during an evoked response that is severely reduced in duration. In other words, while glutamate receptor function is not affected directly, these results suggest that ion influx through glutamate receptors could be affected due to membrane defects. Therefore, the moderate increase in quantal content could be partially due to this apparent reduction in glutamate receptor activity (Haghighi, 2003).

How does the motor neuron respond to the retrograde signal? Based on the results, inhibition of postsynaptic CaMKII mimics the reduction in postsynaptic activity in glutamate receptor mutants and triggers the retrograde signal, leading to an increase in neurotransmitter release at the NMJ. This increase in neurotransmitter release does not appear to induce the NMJ to grow more synaptic boutons, since the numbers of synaptic boutons remained unchanged. Similarly, the overall ultrastructure of boutons remained indistinguishable from wild-type. In contrast, Koh (1999) has reported an overdevelopment of the subsynaptic reticulum in larvae expressing a CaMKII inhibitory peptide (Ala) (Haghighi, 2003).

In that study, Ala or CaMKII^T287D were expressed in both muscles and the nervous system simultaneously, whereas Haghighi (2003) manipulated CaMKII only in neurons or muscles exclusively. It is conceivable that the level and the pattern of expression of these transgenes could have led to differences between experiments. Furthermore, Koh analyzed boutons at the midline section only, while the Haghighi study looked at complete serial sections of boutons. These differences in the levels or pattern of Ala expression as well as differences in analyses could underlie this discrepancy (Haghighi, 2003).

The Haghighi study found a 60% increase in the number of T bars per active zone in response to inhibition of CaMKII in muscles. Often present at active zones at the Drosophila NMJ, T bars are electron-dense structures associated with clusters of synaptic vesicles. Higher numbers of active zones and T bars appear to correlate with an increase in the strength of synaptic transmission. For example, hyperexcitable eag shaker mutants contain a higher number of T bars than wild-type at NMJ synapses. The Haghighi study further demonstrates that induction of retrograde signaling in GluRIIA^-/- mutants leads to a doubling of the number of T bars per active zone, similar to the effect of postsynaptic inhibition of CaMKII. Reiff (2002) has recently reported an increase in T bars in another allelic combination of glutamate receptor mutants. Finally, the Haghighi study shows that the increase in T bars could be surpressed by postsynaptic activation of CaMKII. Since postsynaptic activation of CaMKII in glutamate receptor mutants also suppresses quantal content, these results further support a direct correlation between presynaptic T bars and neurotransmitter release at the Drosophila NMJ. These findings suggest that postsynaptic reduction of CaMKII activity may boost presynaptic neurotransmitter release by upregulating T bars at active zones in presynaptic boutons, a potential mechanism for the control of synaptic transmission induced by the retrograde signal. The number of T bars per active zone could therefore be used as an index for the presence of the homeostatic retrograde signal, independent of quantal content measurements (Haghighi, 2003).

It has been demonstrated that a BMP type II receptor, wishful thinking (wit), is required for both growth and function of the NMJ in Drosophila. To further explore the mechanism by which motor neurons respond to the retrograde signal, whether the retrograde enhancement of quantal content can occur in wit mutants was examined. The results indicate that the retrograde signal cannot increase neurotransmitter release in the absence of Wit. Activation of the retrograde signal by either postsynaptic expression of GluRIIA^M/R or postsynaptic inhibition of CaMKII did not lead to any increase in quantal content. These results indicate a requirement for wit presynaptically for the functioning of the retrograde mechanism that controls the homeostasis of neurotransmitter release at the NMJ of Drosophila and that postsynaptic inhibition of CaMKII requires the function of presynaptic BMP signaling to enhance quantal release (Haghighi, 2003).

Glass bottom boat (Gbb), a BMP ortholog, functions as a retrograde ligand for Wit at the Drosophila NMJ. Mutations in gbb lead to NMJ defects similar to those observed in wit mutants, and postsynaptic transgenic expression of Gbb can rescue many of these defects. In light of these findings, it is possible that there is a link between postsynaptic activity of CaMKII and the level and function of Gbb at the NMJ of Drosophila (Haghighi, 2003).

Another candidate protein for interacting with CaMKII in controlling retrograde signaling is Discs large (DLG). DLG has been shown to be phosphorylated by CaMKII (Koh, 1999) and to be involved in synaptic transmission at the NMJ of Drosophila. However, the defects in synaptic transmission in dlg mutants are rescued by presynaptic rather than postsynaptic expression of DLG. This suggests that the role of DLG in neurotransmission is primarily presynaptic. Furthermore, in the rho-type guanine nucleotide exchange factor dpix mutants quantal release is not greatly affected, while DLG levels are reduced by 80%. Therefore, it seems unlikely that the effects observed are due to changes in DLG phosphorylation levels. Additional experiments are needed to further elucidate the mechanism through which CaMKII activity controls the homeostasis of neurotransmitter release and to identify target proteins that CaMKII may interact with in the postsynaptic cell (Haghighi, 2003).

Calcium-independent calcium/calmodulin-dependent protein kinase II in the adult Drosophila CNS enhances the training of pheromonal cues

Calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the CNS and is crucial for cellular and behavioral plasticity. It is thought that the ability of CaMKII to autophosphorylate and become Ca2+ independent allows it to act as a molecular memory switch. Inhibition of Drosophila CaMKII leads to impaired performance in the courtship conditioning associative memory assay, but it was unknown whether the constitutive form of the kinase had a special role in learning. In this study, a tripartite transgenic system combining GAL4/UAS with the tetracycline-off system was used to spatially and temporally manipulate levels of Ca2+-independent CaMKII activity in Drosophila. An enhancement of information processing during the training period was found with Ca2+-independent, but not Ca2+-dependent, CaMKII. During training, control animals have a lag before active suppression of courtship begins. Animals expressing Ca2+-independent CaMKII have no lag, implying that there is a threshold level of Ca2+-independent activity that must be present to suppress courtship. This is the first demonstration, in any organism, of enhanced behavioral plasticity with overexpression of constitutively active CaMKII. Anatomical studies indicate that transgene expression in antennal lobes and extrinsic mushroom body neurons drives this behavioral enhancement. Interestingly, immediate memory was unaffected by expression of T287D CaMKII in mushroom bodies, although previous studies have shown that CaMKII activity is required in this brain region for memory formation. These results suggest that the biochemical mechanisms of CaMKII-dependent memory formation are threshold based in only a subset of neurons (Mehren, 2004; full text or article).

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