dunce

DEVELOPMENTAL BIOLOGY

Larval

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs have been performed. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (Fas II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that Fas II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, ectopic expression of Fas II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain (Kurusu, 2002).

Studies of MB development with mosaic clones have shown that MB neurons in the adult brain are classified into three groups that project dorsally to the alpha and alpha' lobes and medially to the ß, ß' and gamma lobes. Based on this classification, all the Kenyon cells born before the mid-third larval instar belong to the gamma group. Only in the late third instar, the second group of neurons projecting into the alpha' and ß' lobes is produced. In this study, using various MB markers, it has been demonstrated that the larval Kenyon cells can be further subdivided into distoproximal concentric groups surrounding each of the neuroblasts. Furthermore, the axonal projections of the Kenyon cells are also organized into concentric layers in the peduncle and lobes. Axons of newly born Kenyon cells project into the core that is constituted of densely packed thin fibers rich in actin filaments (Kurusu, 2002).

Distoproximal expression patterns of nuclear regulatory genes in the larval MB cell clusters have been described. In particular, whereas ey is expressed in all the MB cells, including the neuroblasts and ganglion mother cells (GMCs), dac is expressed in differentiated Kenyon cells but not in the centrally located proliferating cells. GAL4 MB markers, such as 201Y and c739, are expressed in an outer group of the differentiated Kenyon cells that is located several cell diameters away from the proliferating neuroblasts (Kurusu, 2002).

While the four MB neuroblasts continue dividing up to the late pupal stage supplying increasing numbers of Kenyon cells, the newly formed larval MB axons follow the medial and the dorsal lobe projections that were pioneered at the embryonic stage with a concomitant increase in the sizes of the lobes. By contrast, a set of genes is turned on in the Kenyon cells after the hatching of the first instar larvae in slightly different patterns in both the cell bodies and their projections. As development proceeds, these differential gene expression patterns became more evident in the second instar larval stage. While the Dac protein is expressed in most of the Kenyon cells, dnc-lacZ is expressed in a small subset of cells peripherally positioned in each of the Kenyon cell clusters originated by the four MB neuroblasts. Expression of 201Y is detected in another subset of cells located more centrally in each of the Kenyon cell clusters, whereas c739 is widely expressed in most of the Kenyon cells (Kurusu, 2002).

Remarkably, these differential expression patterns observed in the Kenyon cells were topologically reflected in their axonal projections in the peduncle and lobes: dnc-lacZ is detected in the outermost surface layer of the peduncle and lobes; 201Y is detected in both the surface and middle layers; and c739 is detected in most axons, a pattern similar to that of FAS II (Kurusu, 2002).

As development proceeds further, further subdivisions emerge in the third instar larval stage with increasing numbers of Kenyon cells and their axons. Moreover the expression patterns of the 201Y and c739 markers change in both cell bodies and their projections; 201Y is then detected in many of the Kenyon cells and their projections, obscuring the 201Y peripheral pattern in the previous larval instar; c739 is then detected in a group of cells located near each of the neuroblasts. The axons of the c739-expressing cells project into an inner layers of the peduncle and lobes. By contrast, dnc-lacZ is maintained in the peripheral subdivisions both in the Kenyon cells and their projections. Double staining with anti-Fas II antibody confirms discrete internal organization of the peduncle and lobes, which are concentrically subdivided into at least three layers surrounding a core that is not labeled with the MB markers, including Fas II (Kurusu, 2002).

Interestingly, the reporter molecule for dnc-lacZ exhibits a characteristic patchy appearance in the calyx, peduncle and lobes, suggesting uneven distribution of the dnc-lacZ fibers. Indeed, higher magnification of the calyces double labeled with anti-ß-gal and anti-synaptotagmin antibodies reveals extensive arborization of the dnc-lacZ expressing neurons around the synaptic terminals, which are likely to represent the afferent terminals of axonal collaterals of the antennocerebral neurons (Kurusu, 2002).

Based on these expression profiles of nuclear regulatory genes and GAL4 markers in the cell bodies, it is suggested that the Kenyon cells that are labeled with both Dac and 201Y project their axons into the concentric layers that also are labeled with Fas II. However, the proximally located Kenyon cells that are labeled with DAC but not 201Y may correspond to the newly differentiated MB neurons that project thin fibers into the core of the peduncle and lobes. Recently described (using a DsRed variant) has been a similar concentric generation of Kenyon cell fibers in the surrounding layers of the peduncle and lobes, in which younger axons extend into the inner layer to shift older fibers into the outer layers. Clonal studies on the larval projection patterns support this temporal order of layer generation and further show that axons of the newly produced Kenyon cells first project into the core as actin-rich thin fibers to shift to the surrounding layers as they undergo further differentiation (Kurusu, 2002).

Adult

Antibodies to the dnc PDE show that the most intensely stained regions in the adult brain are the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that DNC RNA is enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains are also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development (Nighorn, 1991).

Effects of Mutation or Deletion

Several chromosomal deletions and inversions that remove increasingly larger portions of the dnc gene from its 5' end and progressively more of the five known transcription start sites (tss) were used to assess the functions of the various transcriptional units. Surprisingly, the dnc PDE activity, female fertility, mushroom body expression, learning, and memory are unaffected by the removal of tss1 and tss2. tss3 is required for elevated mushroom body expression but not for female fertility nor initial learning. tss4 contributes to learning and the female fertility function, whereas tss5 contributes to female fertility. The results indicate that the structural complexity of the gene is of biological significance, with individual transcriptional units serving different biological functions (Qui, 1993).

The numbers of terminal varicosities and branches are increased in dnc mutants, suggesting a role for dunce in axonogenesis. Such increase is suppressed in dunce-rutabaga double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether a go-go (eag) and Shaker (Sh) alleles, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. The expanded projections in dnc are further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggests a role of cAMP cascade in mediating activity-dependent synaptic plasticity (Zhong, 1992).

Increase in synaptic growth in eag, Shaker and dunce mutants is accompanied by approximately 50% reduction in synaptic levels of Fasciclin2. This decrease in Fas2 is both necessary and sufficient for presynaptic sprouting; Fas2 mutants that decrease Fas2 levels by 50% lead to sprouting similar to eag, Shaker and dunce mutants, while Fas2 transgenetic animals that maintain synaptic Fas 2 levels suppress sprouting in eag, Shaker and dunce mutants. However, Fas2 mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons. Thus these results show a requirement for the presynaptic downreguation of Fas2 in activity and cAMP-induced synaptic sprouting. It is speculated that activity or cAMP may trigger the down-regulation of synaptic Fas2 by actively removing it from the presynaptic terminal (Schuster, 1996).

Since Fas2 mutants lead to an increase in number of boutons without affecting synaptic strength, and increased cAMP in dnc mutants increases both synaptic structure and quantal content, there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB, the cyclic AMP response element-binding protein, mediates the transcriptional requirement of cAMP-dependent long-term synaptic change. Thus CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with FAS2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, dCREB2-b (an isoform of CREB), in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, dCREB2-a, increases synaptic strength, by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of dCREB2-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release and expression of dCREB2-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity. Thus cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, G. W. 1996).

Mutants of the Drosophila dunce and rutabaga genes, which encode a cAMP-specific phosphodiesterase and a calcium/calmodulin-responsive adenylyl cyclase, respectively, are deficient in short-term memory. Altered synaptic plasticity has been demonstrated at neuromuscular junctions in these mutants, but little is known about how their central neurons are affected. This problem was examined by using the "giant" neuron culture, which offers a unique opportunity to analyze mutational effects on neuronal activity and the underlying ionic currents in Drosophila. On the basis of instantaneous frequency and first latency of spikes evoked by current steps, four categories of firing patterns (tonic, adaptive, delayed, and interrupted) were identified in wild-type neurons, revealing interesting parallels to those commonly observed in vertebrate CNS neurons. The distinct firing patterns are correlated with expression of different ratios of 4-aminopyridine- and tetraethylammonium-sensitive K+ currents. Subsets of dnc and rut neurons display abnormal spontaneous spikes and altered firing patterns. Altered frequency coding in mutant neurons was demonstrated further by using stimulation protocols involving conditioning with previous activity. Abnormal spike activity and reduced K+ current remain in double-mutant neurons, suggesting that the opposite effects on cAMP metabolism by dnc and rut do not counterbalance the mutual functional defects. The aberrant spontaneous activity and altered frequency coding in different stimulus paradigms may present problems in the stability and reliability of neural circuits for information processing during certain behavioral tasks, raising the possibility of modulation in neuronal excitability as a cellular mechanism underlying learning and memory (Zhao, 1997).

In response to suprathreshold step current injections, wild-type neurons of different categories follow a defined temporal pattern in firing frequency, and each operates within a restricted frequency range. In contrast, erratic firing patterns in subsets of dnc and rut mutant neurons deviate from a clear scheme of frequency coding for each cell category. Some details of the abnormalities are noteworthy. (1) The periodic bursting activity of single mutant neurons reaches an instantaneous spike frequency as high as 120 Hz, whereas the maximum instantaneous frequency seldom approaches 30 Hz in wild-type controls. Such bursting activities apparently occur more frequently in tonic and delayed neurons than in adaptive neurons. (2) Unlike wild-type neurons that return to quiescence at the termination of stimulation, some mutant neurons frequently generate prolonged firing activities outlasting current steps for seconds. These long-lasting potentials seem to be more frequent in neurons of rut than those of dnc. (3) Extreme cases of abnormal patterns of regenerative potentials were found in subpopulations of mutant neurons that do not fall into the four categories in response to step current injections. Additional subtleties of mutational effects on neuronal excitability have been revealed with stimulation paradigms involving preconditioning, such as a progressive increment of stimulation strength in the ramp or long-duration depolarization in the twin-pulse protocol. In general, mutant neurons displayed in these two paradigms show considerably greater variability than wild-type controls. Moreover, the overall trend found in each category of wild-type controls with a twin-pulse paradigm becomes blurred in dnc and rut mutant neurons. So far, these paradigms have examined only short-term plasticity in neuronal excitability. The long-term effects of conditioning by prolonged previous activity on firing patterns in Drosophila neurons must await further investigation (Zhao, 1997 and references).

Synchronous activities and oscillations at characteristic firing frequencies in neuronal populations are thought to be important for the proper functioning of isolated neuronal networks of the rat hippocampus and neocortex. Recently, theoretical analysis and computational modeling have proposed that multiple short-term memory events could be represented by oscillatory activities in a network, with each memory event stored at a different high-frequency subcycle imbedded in a low-frequency oscillation. Progress made in insects reveals that the frequency of field potential oscillations in the mushroom bodies of the locust is odor-dependent, with the processing of different features of olfactory information distributed among neural subassemblies. The observed aberrant spontaneous activity, disrupted frequency coding, and abnormal modulation by previous conditioning in dnc and rut neurons of Drosophila might present problems in the stability of neural circuits and the reliability of information processing, causing poor performance in certain learning tasks in mutants. These results thus lend strong support for the notion that in addition to the well established synaptic mechanisms, modulation of neuronal excitability represents a potentially important cellular mechanism for learning and memory processes (Zhao, 1997 and references).

One target of cAMP modulation of synaptic function is ion channels. In Drosophila, cAMP has been shown to modulate K+ conductances, and a hyperpolarized resting potential has bee reported in mutant dunce muscle. In dunce mutants, synaptic plasticity in motor end plates can be restored by K+ channel blockers, suggesting that the effects of cAMP are mediated by K+ channel conductances. In support of this notion, abnormal spontaneous spikes and altered firing patterns in correlation with altered K+ conductances are observed in embryonic neuroblasts ('giant neurons') from dnc and rut mutants (Zhao, 1997). Despite this, the fundamental question remains open: whether mushroom body (mrb) neurons from Drosophila mutants deficient in the cAMP cascade exhibit altered K+ conductances. Outward current modulation by cAMP was investigated in wild type (wt) and dunce (dnc) Drosophila larval neurons. dnc is deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mrb. In Drosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons are classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs is reversible and is found only in type I and type IV neurons. In the general wt neuron population, approximately half of the neurons exhibit cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. Because mrb neurons are expected to contribute only 1% to 5% of total neurons, it is concluded that downregulation by cAMP of K+ conductances occurs in many more neurons than are expected to be contributed by the mrb. However, a significantly larger fraction of mrb neurons in wt (70%) is endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the dnc neurons display outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents is most severe in neurons derived from the mrb of dnc individuals. Only 4% of the mrb neurons of dnc are cAMP-modulated. The dnc defect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with the dnc defect. Moreover, this study demonstrates that in dnc mutants such a deficiency affects most severely neurons in brain centers of acquisition and retention. A suitable candidate to account for the maintained, 4-AP-sensitive outward currents downmodulated by cAMP is the K+ channel encoded for by Shaw. Downmodulation of K+ currents by cyclic nucleotide may operate indirectly through protein kinase A (Delgado, 1998).

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. Pacemaker resetting and free-running activity rhythms have been examined in flies mutant for dunce and for DC0 [DCO is the catalytic subunit of cAMP-dependent protein kinase (PKA). dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Circadian rhythms in cAMP content in head tissues demonstrate that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity (Levine, 1994).

Genetic studies using Drosophila have advanced an understanding of the molecular mechanisms upon which different forms of learning are based, including habituation, but the relevant neural components of the learning pathways have not been as fully explored. A well defined neural circuit that underlies an escape response can be habituated, providing excellent opportunities for studying the physiological parameters of learning in a functional circuit in the fly. Compared with other forms of conditioning, relatively little is known of the physiological mechanisms responsible for habituation (Engel, 1996).

The giant nerve fiber pathway mediates a jump-and-flight escape response to visual stimuli. In the tethered fly, the jump may also be triggered electrically at multiple sites. The jump-and-flight response exhibits various parameters of habituation, including frequency-dependent decline in responsiveness, spontaneous recovery, and dishabituation by a novel stimulus (attributable to plasticity in the brain).

Mutations of rutabaga that diminish cAMP synthesis reduce the rate of habituation, whereas dunce mutations that increase cAMP levels lead to a detectable but moderate increase in habituation rates. Surprisingly, habituation is extremely rapid in dunce/rutabaga double mutants. This corresponds to the extreme defects shown with other learning tasks in double mutants, and demonstrates that defects of the rutabaga and dunce products interact synergistically in ways that could not have been predicted on the basis of simple counterbalancing biochemical effects (Engel, 1996).

Although habituation is localized to afferent neurons that innervate the giant fiber, cAMP mutations also affect performance in thoracic portions of the pathway on a millisecond time scale not otherwise accounted for by behavioral plasticity. More significantly, spontaneous recovery and dishabituation are not as clearly affected as is habituation in mutants; this indicates that these processes may not overlap entirely in terms of cAMP-regulating mechanisms. The analysis of the habituation of the giant fiber response in available learning and memory mutants could be a crucial step toward realizing the promise of memory mutations to elucidate mechanisms in neural circuits that underlie behavioral plasticity (Engel, 1996).

dunce mutations are semi-dominant for initial learning and genetic variants carrying the enzymatically hypomorphic dnc2 mutation produce learning scores lower than those of the amorphic dncM11. There are no discernable effects of the different dunce mutations on memory formation 30 to 180 min after training. These results are consistent with a model of memory formation, in which dunce is hypothesized to disrupt acquisition and/or short-term memory (Tully, 1993).

Synaptic transmission was examined in Drosophila memory mutants. In both dunce and rutabaga larvae, voltage-clamp analysis of neuromuscular transmission reveals impaired synaptic facilitation and post-tetanic potentiation as well as abnormal responses to direct application of dibutyryl cAMP. In addition, the calcium dependence of transmitter release is shifted in dunce. The results suggest that the cAMP cascade plays a role in synaptic facilitation and potentiation and indicate that synaptic plasticity is altered in Drosophila memory mutants (Zhong, 1991).

There is a muscle potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening (Delgado, 1991).

Various K+ currents in Drosophila muscles are affected by altered cAMP cascades in dunce and rutabaga mutants. Four distinct K+ currents have been identified in Drosophila larval muscle fibers, i.e. the voltage-activated transient IA and delayed IK and the Ca(2+)-activated fast ICF and slow ICS. Results from voltage-clamp studies indicated that both IA and IK are increased in dnc alleles. Normal muscle fibers treated with dibutyryl-cAMP showed a similar increase of IA, but no significant effect on IK. In contrast to the dnc alleles, the rut mutations appeared to enhance ICS greatly while leaving the amplitude of other currents largely unchanged. In addition, the dibutyryl-cAMP-induced increase in IA is not observed in rut fibers. Caffeine and W7, which are known to interfere with several second messenger pathways, also modulate K+ currents in larval muscle fibers. The currents in dnc and rut fibers show strikingly altered responses to caffeine and W7 (Zhong, 1993)

The opposing biochemical defects of rutabaga and dunce allow rut mutants to partially suppress the female sterility caused by elevated cyclic AMP levels in dunce flies. Selection of mutations that suppress dunce sterility has led to the isolation of two rutabaga alleles. The alleles (rut2 and rut3) decrease basal adenylate cyclase activity but, unlike the original rutabaga mutation, leave the calcium/calmodulin-stimulated activity intact. Behaviorally, the two alleles also differ from rut1. One of the mutations partially rescues the dunce learning defect, and flies bearing both alleles learn (Feany, 1990).

Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, although some do develop to adults. These adult progeny exhibit morphological defects confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells (Bellen, 1987).

Conditional expression of dunce transgenes in dnc adults shortly before training significantly improves learning over nontransgenic controls. Remarkably, behavioral rescue is also observed after induction of a transgene carrying a rat counterpart of dunce. Induction of the transgenes in adult dnc females confers partial rescue of the female sterility phenotype. These data are consistent with a major physiological requirement for the gene's activity in the learning process and show that a rat counterpart can substitute functionally for the Drosophila gene (Dauwalder, 1995).

The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mutations at two loci, dunce and shibire, act synergistically with certain stoned temperature sensitive mutations to cause lethality, but fail to interact with stnC (Petrovich, 1993).

Upon exposure to ethanol, adult Drosophila display behaviors that are similar to acute ethanol intoxication in rodents and humans. Within minutes of exposure to ethanol vapor, flies first become hyperactive and disoriented and then uncoordinated and sedated. After approximately 20 min of exposure they become immobile, but nevertheless recover 5-10 min after ethanol is withdrawn. cheapdate, a mutant with enhanced sensitivity to ethanol, has been identified as a contributory factor, using an inebriometer to measure ethanol-induced loss of postural control. An inebriometer is a device that allows a quantitative assessment of ethanol-induced loss of postural control. The inebriometer is an approximately 4 ft long glass column containing multiple oblique mesh baffles through which ethanol vapor is circulated. To begin a "run," about 100 flies are introduced into the top of the inebriometer. With time, flies lose their ability to stand on the baffles and gradually tumble downward. As they fall out of the bottom of the inebriometer, a fraction collector is used to gather them at 3 min intervals, at which point they are counted. The elution profile of wild-type control flies follows a normal distribution; the mean elution time (MET), approximately 20 min at a standard ethanol vapor concentration, is inversely proportional to their sensitivity to ethanol. A genetic screen was carried out to isolate P element-induced mutants with altered sensitivity to ethanol intoxication using the inebriometer as the behavioral assay. One X-linked mutation isolated in this screen was named cheapdate (chpd) to reflect the increased ethanol sensitivity displayed by hemizygous mutant male flies. chpd males elute from the inebriometer with a MET of 15 min compared with 20 min for the wild-type controls. This increased ethanol sensitivity of chpd males was observed at all ethanol vapor concentrations tested. Genetic and molecular analyses reveals that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, it is found that the enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol (Moore, 1998).

Flies carrying mutations in three molecules involved in cAMP signaling were tested for response to ethanol: (1) rutabaga (rut), encoding the Ca²⁺-calmodulin-sensitive AC; (2) dunce (dnc), encoding the major cAMP-phosphodiesterase (PDE), and (3) DCO, encoding the major catalytic subunit of cAMP-dependent protein kinase (PKA-C1). Males hemizygous for rut mutations display an ethanol-sensitive phenotype similar to that of amn mutants. Flies heterozygous for the loss-of-function DCO alleles, which show reduced cAMP-stimulated PKA activity, also display increased ethanol sensitivity (homozygotes cannot be tested because they die as embryos). Ethanol sensitivity of males hemizygous for dnc mutations, however, are nearly normal. These data show that flies unable to increase cAMP levels normally (such as rut and possibly amn) or to respond properly to increased cAMP levels (such as DCO/+) are more sensitive to ethanol-induced loss of postural control. The converse, however, is not observed; dnc flies, whose cAMP levels are several times higher than wild type, display nearly normal ethanol sensitivity, a phenotype that is also observed in males doubly mutant for dnc and amn. Unexpectedly, whereas both rut and amn are ethanol sensitive, males doubly mutant for rut and amn are not significantly different from control (Moore, 1998).

In mammalian cells and tissues, ethanol potentiates receptor-mediated cAMP signal transduction; the mechanisms underlying this effect, however, remain poorly understood. While a direct link between cAMP signaling and ethanol-induced behaviors has not been established in mammals, the responses to acute ethanol are thought to be mediated by alterations in the function of various ligand-gated ion channels. Certain subtypes of GABA_A and NMDA receptors are potentiated and inhibited by ethanol, respectively, and both these channels can be phosphorylated by PKA in cells, tissues, or heterologous expression systems. It is tempting to speculate that PKA phosphorylation of neurotransmitter receptors is altered by ethanol and that this contributes to the behavior of the inebriated animal (Moore, 1998 and references).

Relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. An examination was made of the role of the cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current is increased in dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current is decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered, thereby decreasing the cAMP concentration. The dunce effect is mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect is rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduces the current and inhibits the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations (Bhattacharya, 1999).

The Drosophila mutants amnesiac, dunce, and rutabaga were isolated after associative conditioning tests, during which animals were trained to associate the presence of an odor with that of electric shocks (ES). In the absence of conditioning, the odor avoidance (OA) of these mutants is normal, indicating that their poor associative conditioning performance is attributable to specific learning or memory deficits. However, the OA of the mutants is greatly decreased after their exposure to ES. This effect can last for hours. These results strongly suggest that part of the defect displayed by these mutants in associative conditioning tests does not correspond to a learning or memory deficit but might arise from abnormal sensitivity to stressful stimuli. OA after ES of two previously characterized dnc mutants was examined. Df(1)N79f specifically decreases Dnc expression in the mushroom bodies, leading to a normal level of learning but decreased memory. Df(1)N79f mutants displays a normal OA after ES. Df(1)N64j15 affects the entire brain expression of Dnc, leading to decreased learning and memory. Df(1)N64j15 animals show a strong decrease of their OA after ES. Thus, the lack of Dnc 'general' expression is most likely responsible for the OA defect, which would be responsible for the apparent learning defect after conditioning. In contrast, the Dnc phosphodiesterase accumulated in the mushroom bodies is thought to be involved specifically in memory formation (Preat, 1998).

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission were examined in cultured embryonic Drosophila neurons. Chronic elevation in neuronal cAMP [in dunce neurons or wild-type neurons grown in dibutyryl-cAMP (db-cAMP)] results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation (Lee, 2000).

Thus neuronal cAMP levels can regulate functional plasticity, independent of differentiation, at cholinergic synapses between cultured Drosophila neurons. Although numerous studies have demonstrated that the cAMP signaling cascade plays a role in modulating plasticity at central glutamatergic (hippocampus) synapses and presumed glutamatergic (Aplysia) synapses, this is the first direct evidence that cAMP regulates plasticity at central cholinergic synapses by mediating fast excitatory transmission. Since acetylcholine appears to be a primary excitatory neurotransmitter in the fly brain, these data are consistent with the hypothesis that cAMP-dependent plasticity at cholinergic synpases mediates changes in neuronal communication in the Drosophila CNS. Although the majority of fast excitatory synaptic transmission in the mammalian brain is mediated by glutamate, recent reports indicate the presence of fast synaptic signaling via nAChRs in both the rodent hippocampus and visual cortex. In light of these findings in Drosophila, it seems likely that cAMP may also be important in modulating fast cholinergic synaptic transmission in the mammalian CNS (Lee, 2000).

The data demonstrating a significant increase in mEPSC frequency in three different dunce alleles, isolated in two independent screens, strongly support the hypothesis that mutations in dunce, a gene encoding a cAMP phosphodiesterase, result in alterations in cholinergic synaptic transmission. The phosphodiesterase activity is higher in the dnc¹ versus dnc² mutant. However, the cAMP levels in these two dunce alleles are similar and significantly higher than wild-type. The similarity in the mEPSC frequency in dnc¹ and dnc² and the observation that chronic exposure to db-cAMP induces a concentration-dependent increase in mEPSC frequency in wild-type neurons, are consistent with the suggestion that elevated levels of cAMP in the mutant regulate mEPSC frequency. The smaller increase in mEPSC frequency in neurons within the dnc^M11 cultures (as compared to dnc¹ and dnc² cultures) is not unexpected. The dnc^M11 cultures are genetically heterogenous with only 25% of the neurons homozygous for the mutant allele. In contrast, in the dnc¹ and dnc² cultures, all of the neurons are genetically homozygous for the mutations in the dnc locus. Assessment of mEPSC frequency after experimental manipulations resulting in a reduction in cAMP levels in dnc mutant neurons will be important in further examining the role of cAMP in regulation of synaptic transmission in the mutant neurons (Lee, 2000).

In this study, brief exposures of cultured wild-type neurons to db-cAMP demonstrate that the cAMP-signaling cascade is involved in both short-term and long-lasting modulation of activity at cholinergic synapses in Drosophila. The short-term change is characterized by a rapid onset increase in cholinergic sEPSC frequency in differentiated wild-type neurons. Because there are no immediate changes in action potential (AP) independent synaptic transmission, and the increase in sEPSC frequency is blocked in the presence of a protein kinase inhibitor (staurosporine), it suggests that the alterations involve posttranslational modifications of existing proteins that do not affect the synaptic machinery involved in mediating constitutive release. The increase in AP-dependent release is transient in that the majority of the elevation in synaptic current frequency observed 24 hr after cAMP exposure could be accounted for by AP-independent synaptic currents. In Aplysia, it has been shown that cAMP, through protein kinase A (PKA), mediates a phosphorylation-induced reduction in conductance of existing potassium channels resulting in a rapid, transient increase in neuronal excitability contributing to short-term facilitation. Phosphorylation induces a rapid and reversible decrease in outward potassium currents in Drosophila neurons. This suggests that an increase in AP duration and/or excitability, similar to that seen in Aplysia sensory neurons, may contribute to the rapid cAMP-induced increase in sEPSC frequency at cholinergic synapses in Drosophila neurons (Lee, 2000).

The long-lasting change induced by brief db-cAMP exposure in cultured Drosophila neurons is characterized by a delayed onset increase in mEPSC frequency that peaks 24 hr after exposure and is blocked by cycloheximide. Time course, requirement for de novo protein synthesis, and the indication that the changes are presynaptic (absence of changes in the biophysical properties of each mEPSC), are all consistent with cAMP-inducing synaptic growth resulting in an increase in the number of functionally identical cholinergic release sites. A similar mechanism has been proposed to underlie cAMP-induced long-term facilitation in Aplysia where studies in cell culture have revealed that enhancement of synaptic strength between identified sensory and motor neurons, observed at 24 hr after the stimulus, requires protein and RNA synthesis and the growth of new synaptic connections between the neurons. However, further analysis will be necessary to determine if cAMP regulates evoked transmitter release at cholinergic synapses in Drosophila and if so whether the mechanism involves regulation of the number of synaptic sites or affects other processes such as efficacy of transmission at each bouton (Lee, 2000).

In Aplysia cell culture, while a single pulse (1x) of serotonin can induce cAMP-dependent short-term facilitation, repetitively spaced (5x) application of serotonin is necessary to induce cAMP-dependent long-term facilitation. Consolidation of changes induced by spaced stimuli involve cAMP response element-binding protein (CREB)-mediated gene transcription. A role for cAMP-CREB-mediated transcription has also been demonstrated in long-term potentiation at glutamatergic synapses in the rodent hippocampus. Furthermore, studies in Drosophila indicate that cAMP-initiated changes in CREB activity play a role in long-term synaptic enhancement at peripheral glutamatergic synapses. The observation that repetitive spaced treatments with db-cAMP induces a more persistent change than a single db-cAMP treatment, suggests that plasticity at cholinergic synapses induced by spaced exposure to db-cAMP in Drosophila involves activation of CREB-mediated gene transcription. It will be possible to test this hypothesis by examining cAMP-dependent modulation of cholinergic transmission in neurons from transgenic flies carrying CREB activators and CREB inhibitors (Lee, 2000).

The data clearly demonstrate that cAMP plays an important role in short-term modulation of transmission, as well as initiating events that contribute to long-lasting synaptic changes requiring new protein synthesis, at interneuronal cholinergic synapses in Drosophila. Several lines of evidence support the hypothesis that the cAMP-dependent regulation of cholinergic plasticity reported in the cultured neurons is likely to represent a mechanism involved in modulation of functional transmission important for behavior in the adult fly. (1) It was found that repetitive spaced exposure to db-cAMP induces a more persistent increase in mEPSC frequency than an equivalent length single exposure in wild-type Drosophila neurons. These results represent a remarkable parallel to those of behavioral studies in Drosophila, demonstrating that repetitive spaced trails, where an olfactory stimulus is paired with a foot shock, induces more persistent memory than an equivalent number of training trials presented in the absence of a rest interval between trials in wild-type flies. (2) The data from dnc mutant neurons reveals that chronic disruption of cAMP signaling in neurons, previously shown to result in associative learning deficits in the fly, alters the basal levels of AP-independent transmission at cholinergic synapses. Even more significant is the finding that a persistent increase in mEPSC frequency could not be induced by spaced exposure to db-cAMP in the dnc mutant neurons. The inability of dnc neurons to respond to transient, activity-induced changes in cAMP levels that are likely to occur during olfactory training episodes in the adult fly, could contribute to the reduced performance index when compare with wild-type. Finally, although there are no studies directly demonstrating cholinergic synaptic transmission in the adult Drosophila CNS, cholinergic transmission takes place at synapses in the mushroom body of the adult honeybee, an anatomical structure critical for learning and memory in Drosophila as well as the honeybee. Taken together these findings support the hypothesis that cholinergic synapses in Drosophila, similar to central glutamatergic synapses in other species, are sites of cAMP-dependent synaptic plasticity important for learning and memory. Insights gained from functional studies in this Drosophila culture system, well suited to molecular genetic and biochemical manipulations, will be useful in delineating the molecular mechanisms underlying modulation of central synaptic transmission, a process thought to contribute to learning and memory in all animals (Lee, 2000).

At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). An extracellularly applied fluorescent dye, FM1-43, is incorporated into synaptic vesicles in nerve terminals during endocytosis and subsequently released by exocytosis. Using this dye, the two synaptic vesicle pools, ECP and RP have been identified in the larval NMJ. Vesicles in ECP can be loaded with FM1-43 by high K+ stimulation and are located at the periphery of individual boutons. Loaded dye is completely released by a second challenge of high K+ saline. Both pools are loaded with FM1-43 by enhancing endocytosis with cyclosporin A or by incubating at room temperature after complete depletion of vesicles in shibire at a nonpermissive temperature. The dye in ECP can be unloaded by a second challenge of high K+ saline, while vesicles in RP still maintain the dye. The RP appears to be more broadly distributed toward the center of individual boutons. In this report, this distribution is referred to as being in the center of the bouton because of its appearance in fluorescence microscopy without intending any implication as to its composition or exact boundary. In the animals whose RP is disconnected from the cycling pathway by treatment with cytochalasin D, high-frequency stimulation causes an accelerated decline of synaptic potentials, while low-frequency stimulation does not, suggesting that RP is required for sustaining high rate release of transmitter (Kuromi, 2000 and references therein).

During high-frequency nerve stimulation, vesicles in RP are recruited for release, and endocytosed vesicles are incorporated into both pools, whereas with low-frequency stimulation, vesicles are incorporated into and released from ECP. Release of vesicles from RP can be detected electrophysiologically after emptying vesicles in the ECP of transmitter by a H+ pump inhibitor. Recruitment from RP is depressed by inhibitors of steps in the cAMP/PKA cascade and enhanced by their activators. In rutabaga (rut) mutants, which have low cAMP levels, mobilization of vesicles from RP during tetanic stimulation is depressed, while it is enhanced in dunce (dnc) mutants, which have high cAMP levels (Kuromi, 2000).

The present electrophysiological studies have shown that, in wild type larvae, recruitment of synaptic vesicles from RP induced by 10 Hz stimulation for 10 s continues for some period, even after tetanic stimulation, but such recruitment is not observed in rut and does not continue after tetanic stimulation in dnc. Reduced recruitment of vesicles from RP in rut mutants may be caused by a failure in Ca2+/calmodulin-dependent cAMP production. However, even in rut, repeated tetanic stimulation does recruit vesicles from RP, and enhanced synaptic potentials continue after tetanic stimulation. A similar phenomenon is observed in rut after treatment with db-cAMP. Thus, it is likely even in rut that cAMP production can occur through another pathway during tetanic stimulation. FM1-43 loading experiments show that during high-frequency stimulation, some recycling vesicles are incorporated into RP in wild type, whereas recycling vesicles are minimally stored in RP in dnc mutants. Although RP may be refilled slowly with vesicles recycled or transported by axonal flow, the high level of cAMP produced by tetanic stimulation causes a marked translocation of vesicles from RP to ECP, resulting in no net accumulation of recycling vesicles into RP in dnc. Together, these findings suggest that recruitment of vesicles from RP to ECP is one of the mechanisms that control synaptic efficacy and plasticity (Kuromi, 2000).

The dunce and rutabaga mutations of Drosophila affect a cAMP-dependent phosphodiesterase and a Ca²⁺/CaM-regulated adenylyl cyclase, respectively. These mutations cause deficiencies in several learning paradigms and alter synaptic transmission, growth cone motility, and action potential generation. The cellular phenotypes either are Ca²⁺ dependent (neurotransmission and motility) or mediate a Ca²⁺ rise (action potential generation). However, interrelations among these defects have not been addressed. Conditions have been established for fura-2 imaging of Ca²⁺ dynamics in the 'giant' neuron culture system of Drosophila. Using high K+ depolarization of isolated neurons, a larger, faster, and more dynamic response was observed from the growth cone than the cell body. This Ca²⁺ increase depends on an influx through Ca²⁺ channels and is suppressed by the Na+ channel blocker TTX. Altered cAMP metabolism by the dnc and rut mutations reduces response amplitude in the growth cone while prolonging the response within the soma. The enhanced spatial resolution of these larger cells allow an analysis of Ca²⁺ regulation within distinct domains of mutant growth cones. Modulation by a previous conditioning stimulus was altered in terms of response amplitude and waveform complexity. Furthermore, rut disrupts the distinction in Ca²⁺ responses observed between the periphery and central domain of growth cones with motile filopodia (Berke, 2002).

This study describes the first use of fura-2 imaging for intracellular Ca²⁺ in a dissociated culture system of Drosophila. Unlike in vivo preparations such as the neuromuscular junction, optical imaging in culture can relate subcellular Ca²⁺ dynamics throughout different regions of single neurons. The spatial resolution offered by optical imaging complements electrophysiological studies of Drosophila giant neurons in culture and may indicate the functional significance of membrane excitability differences in subneuronal regions. The two approaches in combination will greatly enhance the neurogenetic study of Ca²⁺-dependent processes involved in neuronal development and physiology (Berke, 2002).

The initial characterization of regional differences in high K⁺-induced Ca²⁺ regulation in the soma and growth cone indicates that both Ca²⁺ and Na⁺ channels are involved. Drosophila central neurons in culture contain two types of Ca²⁺ currents (L and T type) distinguished by their activation voltage, decay kinetics, voltage dependence, and underlying single-channel activities. In other species, electrical recordings from the growth cone indicate that similar L- and T-type Ca²⁺ channels are expressed throughout the neuron, but that channel density may be higher and the channels more clustered in the growth cone. Such variation may relate to findings that the growth cone and soma differ in sensitivity and response kinetics during depolarization. In future investigations, it will be interesting to use identified mutations in Na⁺ and Ca²⁺ channel genes to dissect their involvement in Ca²⁺ regulation throughout the neuron (Berke, 2002).

Local Ca²⁺ levels regulate filopodial formation and play an important role in directing growth cone turning in cultured neurons from other species. The data from Drosophila show that the magnitude of the high K⁺ response is larger in the periphery of motile, as opposed to nonmotile, growth cones. Distinct regions within the growth cone exhibit differences in the localization of cytoskeletal elements and cytoplasmic organelles. It may be questioned whether the cytokinesis inhibition technique used to generate giant neurons affects the distribution of Ca²⁺ channels in the soma and growth cone because of actin cytoskeletal disruption. However, previous electrophysiological studies did not detect differences in action potential and ion current properties with and without the removal of cytochalasin B. This result is consistent with a preliminary study indicating that differences in response characteristics between the soma and growth cone are still evident in embryonic cultures made in the absence of CCB. In future studies, untreated larval neurons in culture can be used to examine Ca²⁺ signaling in the same dnc and rut growth cones that display retarded motility (Berke, 2002).

The analysis of these well studied mutants by fura-2 imaging has revealed previously unknown mutant phenotypes and suggests the usefulness of Ca²⁺ imaging for a wide range of other mutations. dnc and rut decrease sensitivity to high K⁺ stimulation for a cytosolic Ca²⁺ increase while affecting both the activity dependence and spatial distribution of [Ca²⁺] within motile and nonmotile growth cones. Chronic changes in cAMP metabolism imposed by the dnc and rut mutations decrease sensitivity most strongly in the growth cone while prolonging the Ca²⁺ increase only in the soma (Berke, 2002).

It is known that dnc and rut alter the modulation of K⁺ currents gated by the Sh and eag channel subunits. Moreover, enhanced spike activity has been detected with patch-clamp recordings from the soma of dnc and rut giant neurons. Such altered excitability may explain the prolonged soma response observed and should be further examined during patch-clamp experiments with high K⁺ depolarization. Electrophysiological studies of other currents in dnc and rut neurons have been lacking, but a Ba²⁺ current flowing through wild-type Ca²⁺ channels increases during the application of cAMP analogs. Further support for Ca²⁺ channel defects stems from findings that dnc and rut increase and decrease an L-type (dihydropyridine-sensitive) Ca²⁺ current in larval muscle, phenotypes that are mimicked by short-term pharmacological manipulations on wild-type muscles. However, some dnc and rut physiological phenotypes at the larval neuromuscular junction cannot be mimicked by acute pharmacological treatments and are attributed to long-term, developmental effects of the mutations. Different aspects of the Ca²⁺ signaling phenotypes may be caused by either acute or chronic effects. Therefore, the combination of genetic and pharmacological analyses will offer a more comprehensive picture of how the cAMP pathway regulates neuronal Ca²⁺ (Berke, 2002).

In Drosophila, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, the involvement of a candidate gene, cAMP response-element binding protein (CREB), was investigated. The duration of rest is inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies does not affect the clock, but increases rest rebound. CREB mutants also have a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increases after rest deprivation and remains elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila (Hendricks, 2001).

The daily rest of flies carrying mutations and/or transgenes that alter cAMP signaling was examined at several points in the pathway. dunce flies have a mutation in the phosphodiesterase enzyme and therefore have increased cAMP. The null mutant (dnc^ML) rests significantly less than the background yw strain. Similarly, increasing PKA activity in flies with a heat-shock-inducible transgene of the catalytic subunit of PKA significantly decreases daily rest durations compared to pre-heat-shock rest levels. Decreased adenylyl cyclase enzyme activity and thus decreased cAMP characterize rutabaga (rut) mutants, which rest more than the Canton S background strain. Similarly, S162 flies that carry a mutation that abolishes dCREB2 activity rest more than their comparison group (siblings without the mutation). The mutation is a stop codon just upstream of the basic leucine-zipper motif of the dCREB2 gene (Hendricks, 2001).

Dunce role in oogenesis

Because of the potential role of the G protein-coupled receptor kinase 2 gene in G protein signaling it was hypothesized that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger. To examine this hypothesis, tests were performed for genetic and biochemical interactions between dunce and Gprk2 mutants; dunce is able to suppress the sterility defect of a gprk2 mutant. Similarly, gprk2 mutation is able to rescue the viability and sterility defects of dunce mutants. Results of the biochemical analysis of cAMP levels in the dunce;gprk2 mutant combinations further strengthen the genetic findings. These results suggest that Gprk2 is involved in a receptor-mediated signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

The genetic interaction between dunce and Gprk2 was examined through the effects of these two genes on oogenesis. A mutant allele of Gprk2, called gprk2⁶⁹³⁶, has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2⁶⁹³⁶. Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2⁶⁹³⁶ background suppresses all of these defects to a significant degree. Suppression is also observed when a single copy of gprk2⁶⁹³⁶ is introduced into a dunce background. Like rutabaga mutants (rutabaga encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2⁶⁹³⁶ has reduced levels of cAMP. Ovaries from gprk2⁶⁹³⁶ females contain about one third the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels (Lannutti, 2001).

Because of the possibility that dunce may be acting in a common pathway with Gprk2, the dunce ovarian phenotype was reexamined. Females homozygous for hypomorphic (dnc²) and null (dnc^M14 ) alleles have been shown to lay few or no eggs. This has been attributed to defects in the chorion and vitelline membrane. In addition to these defects, nurse cell dumping is incomplete in egg chambers from dnc²homozygous females. This incomplete cytoplasmic transfer may cause or contribute to the formation of misshapen dorsal appendages. Incomplete cytoplasmic dumping is also apparent in dnc^M14 homozygotes and dnc²/dnc^M14 transheterozygotes. In both of these genotypes, egg chambers have severely truncated dorsal appendages or the dorsal appendages fail to form altogether. The percentage of egg chambers that fail to complete cytoplasmic dumping does not increase significantly in the stronger allelic combinations, probably because many egg chambers fail to develop to that stage (Lannutti, 2001).

In addition to the dumping and dorsal appendage defects, ovaries from dunce mutant mothers contain degenerating egg chambers. In dnc² homozygotes, 70% of stages 10B and 11 egg chambers are degenerating, as determined by the presence of condensed, DAPI-bright nuclei. In dnc²/dnc^M14 and dnc^M14 homozygotes, egg chamber degeneration is detected in earlier stages (beginning at stage 6) and occurs more frequently. All three aspects of the dunce ovary phenotype, the incomplete cytoplasmic dumping, the malformed dorsal appendages, and the degeneration of egg chambers, resemble those of the gprk2⁶⁹³⁶ mutant (Lannutti, 2001).

There is a marked increase in fertility in allelic combinations of dunce and gprk2. This increase suggests that there should be a corresponding improvement in the ovary morphology of combinations of both mutants. To test this, a quantitative analysis of the ovaries from the different mutant combinations was performed. The feature of the dunce phenotype that is most readily quantified is egg chamber degeneration; this defect can be assayed by the presence of DAPI-bright nuclei. In dnc² homozygotes, DAPI-bright, nurse cell nuclei were observed in 39% of stages 9 and 10 egg chambers. This defect is more severe in dnc²/dnc^M14 transheterozygotes and dnc^M14/dnc^M14 homozygotes. In these genotypes 52% (dnc²/dnc^M14) and 58% (dnc^M14/dnc^M14) of stages 9 and 10 egg chambers display a degenerating phenotype. These numbers are an underestimate of the level of degeneration in dnc²/dnc^M14 and dnc^M14/dnc^M14 allelic combinations because egg chambers often degenerate earlier in oogenesis. As a result, ovaries from dnc²/dnc^M14 and dnc^M14/dnc^M14 females produce few egg chambers past stage 11. One copy of gprk2⁶⁹³⁶, which suppresses the sterility of the two dunce alleles, also suppresses degeneration in the dnc²/dnc², dnc²/dnc^M14, and dnc^M14/dnc^M14 mutants. Although degeneration is dramatically reduced in all three genotypes, rescue is not complete. These results support the suppression of sterility that is observed in dunce;gprk2⁶⁹³⁶ allelic combinations (Lannutti, 2001).

Mutations in the dunce gene cause an increase in embryonic and adult cAMP levels (two- to fivefold, depending on the allele) and most dunce alleles are sterile. In contrast, mutations in rutabaga cause a slight decrease in cAMP levels but are completely fertile. Double mutants of rutabaga and dunce have intermediate levels of cAMP and fertility is partially restored. Similarly, one copy of gprk2⁶⁹³⁶ partially restores fertility in dunce mutants. By analogy to the dunce-rutabaga interaction, the simplest explanation for the mutual suppression of gprk2⁶⁹³⁶ and dunce is that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are lower than those in wild type flies. In agreement with this suggestion, it was found that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are almost threefold lower than those in wild type females. Furthermore, the suppression of the gprk2⁶⁹³⁶ phenotype by dunce (and vice versa) is reflected in the cAMP levels of the dunce, gprk2⁶⁹³⁶ allelic combinations. Introducing a single copy of the dnc² or dnc^M14 mutant into gprk2⁶⁹³⁶ homozygotes resulted in a wild-type cAMP content, threefold higher than that in gprk2⁶⁹³⁶ homozygotes. The gprk2⁶⁹³⁶ mutant also suppresses the elevated levels of cAMP seen in mutant dunce alleles. In short, in every mutant combination in which an increase in fertility is observed, the cAMP content in the ovaries changes in the expected direction. Taken together, these results strongly support the hypothesis that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

It is interesting that gprk2⁶⁹³⁶ and dunce mutants have similar ovary phenotypes, even though they have opposite effects on cAMP levels in the ovaries. It appears that cAMP levels must be maintained at an optimum level; both an increase and a decrease cause defects in dorsal appendage formation, cytoplasmic dumping, and probably other maternal and zygotic functions. This effect has also been documented in assays of learning in the fly. Both rutabaga and dunce mutants cause defects in learning and memory, although they cause opposite changes in cAMP levels. Apparently, the level of cAMP must be tightly regulated for proper functioning of cAMP-dependent pathways (Lannutti, 2001 and references therein).

Another parallel with the dunce, rutabaga studies is the observation that the degree of suppression does not always correlate with the level of cAMP in the ovaries. In earlier studies it has been shown that a dunce, rutabaga double mutant (homozygous for both alleles) is still defective in learning, although cAMP levels are rescued. Similarly, rescue of fertility is not complete, even in combinations that produced cAMP levels very close to those of wild type. For example, the ovaries from dnc²/+;gprk2⁶⁹³⁶ /gprk2⁶⁹³⁶ females have cAMP levels that are not statistically different from wild type levels. However, the fertility of these females is still statistically less than that in wild type. There are several possible explanations for this effect. (1) It has been suggested that the kinetics of cAMP metabolism are as important as the level of cAMP per se. Similarly, the subcellular distribution of cAMP may play an equally important role in fertility. (2) The Gprk2 and dunce genes may have functions that are not dependent on one another. For example, because there are many heptahelical receptors in Drosophila and only two known GRKs, it is likely that Gprk2 protein phosphorylates many different receptor types. If some of these receptors act through second messengers other than cAMP, then they would not be rescued by a decrease in the dosage of dunce. Similarly, Dunce is the major source of phosphodiesterase activity in Drosophila. Therefore, a decrease in phosphodiesterase activity could disrupt signaling from Gprk2-independent receptors. (3) Gprk2 and Dunce could carry out functions that are not directly related to production or metabolism of second messengers. For example, mammalian GRKs have been shown to interact with cytoskeletal proteins, suggesting that they have a potential scaffolding or docking function (Lannutti, 2001 and references therein).

In germline clones of dnc^M14, eggs are laid but fail to hatch, suggesting that dunce activity is required in the somatic cells for egg laying and in the germline for hatching. This appears to contradict the data suggesting a germline requirement for dunce in egg laying. However, there are several possible explanations for this apparent discrepancy. (1) The analysis of egg laying from females with dnc^M14germline clones could not be quantitative because the ovarioles do not all contain clones. Therefore, while the data demonstrate that somatic expression of dunce is necessary for egg laying, it does not exclude a role in the germline as well. (2) By the same argument, a role for Gprk2 activity in the somatic cells has not been ruled out. Although Gprk2 expression in the follicle cells cannot be detected, it is possible that a low level of somatic expression plays a role in egg laying. (3) Although the Gprk2 gene plays a role in regulating the level of cAMP in the ovaries, it has not been directly shown that Gprk2 and dunce are acting in the same molecular pathway. Perhaps dunce and Gprk2 act in different signaling pathways that are active in somatic and germline cells, respectively, and the interaction of the two pathways is necessary for normal levels of egg laying (Lannutti, 2001).

In summary, analysis of the interaction between gprk2⁶⁹³⁶ and dunce mutants demonstrates that the regulation of cAMP synthesis and metabolism is critical for development. The gprk2⁶⁹³⁶ mutant has not only reduced levels of cAMP but also decreased egg laying and hatching. These defects were all suppressed by weak and null alleles of dunce. Similarly, the increased levels of cAMP and sterility of dunce are suppressed by the gprk2⁶⁹³⁶ mutant. Further analysis of the developmental functions of gprk2⁶⁹³⁶ in vivo will continue to complement the biochemical characterization of this protein (Lannutti, 2001).

A screen was performed for female sterile mutations on the X chromosome of Drosophila and new loci were identified that are required for developmental events in oogenesis: new alleles of previously described genes were identified as well. The screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. New roles are described for the genes dunce, brainiac and fs(1)Yb, and new alleles of Sex lethal, ovarian tumor, sans filles, fs(1)K10, singed, and defective chorion-1 have been identified (Swan, 2001).

Waking experience affects sleep need in Drosophila: Experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes

Sleep is a vital, evolutionarily conserved phenomenon, whose function is unclear. Although mounting evidence supports a role for sleep in the consolidation of memories, until now, a molecular connection between sleep, plasticity, and memory formation has been difficult to demonstrate. Drosophila as a model to investigate this relation; the intensity and/or complexity of prior social experience stably modifies sleep need and architecture. Furthermore, this experience-dependent plasticity in sleep need is subserved by the dopaminergic and adenosine 3',5'-monophosphate signaling pathways and a particular subset of 17 long-term memory genes (Ganguly-Fitzgerald, 2006).

Sleep is critical for survival, as observed in the human, mouse, and fruit fly, and yet, its function remains unclear. Although studies suggest that sleep may play a role in the processing of information acquired while awake, a direct molecular link between waking experience, plasticity, and sleep has not been demonstrated. Advantage was taken of Drosophila genetics and the behavioral and physiological similarities between fruit fly and mammalian sleep to investigate the molecular connection between experience, sleep, and memory (Ganguly-Fitzgerald, 2006).

Drosophila is uniquely suited for exploring the relation between sleep and plasticity for at least two reasons. (1) Fruit flies sleep. This is evidenced by consolidated periods of quiescence associated with reduced responsiveness to external stimuli and homeostatic regulation -- the increased need for sleep that follows sleep deprivation. (2) Drosophila has been successfully used to elucidate conserved mechanisms of plasticity. For example, exposure to enriched environments, including the social environment, affects the number of synapses and the size of regions involved in information processing in vertebrates and Drosophila. In the fruit fly, these structural changes occur in response to experiential information received within a week of emergence from pupal cases. Although brain plasticity is not limited to this period, the first week of emergence does coincide with the development of complex behaviors in Drosophila, including sleep. Hence, daytime sleep, which accounts for about 40% of total sleep in adults, is highest immediately after eclosion and stabilizes to adult levels 4 days after emergence (Ganguly-Fitzgerald, 2006).

To assess the impact of waking experience during this period of brain and behavioral development, individuals from the wild-type C-S strain were exposed to either social enrichment or impoverishment immediately at eclosion and were tested individually for sleep 5 days later. Socially enriched individuals (E), exposed to a group of 30 or more males and females (1:1 sex ratio) before being tested, slept significantly more than their socially impoverished (I) siblings, who were housed individually. This difference in sleep [DeltaSleep (E)] was restricted to daytime sleep. Socially enriched individuals consolidated their daytime sleep into longer bouts of ~60 min compared with their isolated siblings, who slept in 15-min bouts. In contrast, nighttime sleep was unaffected by prior social experience, corresponding with observations that daytime sleep is more sensitive to sex, age, genotype, and environment, when compared with nighttime sleep. This effect of social experience on sleep persisted over a period of days. Moreover, it was a stable phenotype: When socially enriched, longer-sleeping individuals and socially impoverished, shorter-sleeping siblings were sleep-deprived for 24 hours, they defended their respective predeprivation baseline sleep quotas by returning to these levels after a normal homeostatic response (Ganguly-Fitzgerald, 2006).

Experience-dependent modifications in sleep have long been observed in humans, rats, mice, and cats. But what is the nature of the experiential information that modifies sleep need in genetically identical Drosophila? Differences in sleep need in socially enriched and socially impoverished individuals were not a function of the space to which they were exposed -- flies reared in 2-cc tubes slept the same as those reared in 40-cc vials. Neither did it arise out of differences in reproductive state or sexual activity between the two groups: Socially impoverished mated and virgin individuals slept the same, as did socially enriched individuals from mixed-sex or single-sex groups. Further, differences in sleep were not a reflection of differences in overall activity (measured as infrared beam breaks) between the two groups. Although social context can reset biological rhythms, mutations in clock (Clk^jerk), timeless (tim⁰¹), and cycle (cyc⁰¹) disrupt circadian rhythms but had no effect on experience-dependent responses in sleep need (Ganguly-Fitzgerald, 2006).

Because social interaction requires sensory input, fly strains that were selectively impaired in vision, olfaction, and hearing were evaluated . Blind norpA homozygotes failed to display a response in sleep to waking experience: Sleep need in norpA mutants did not increase after exposure to social enrichment. In contrast, norpA/+ heterozygotes with restored visual acuity slept more when previously socially enriched. Attenuating visual signals by rearing wild-type (C-S) flies in darkness also abolished the effect of waking experience on sleep. Compromising the sense of smell while retaining visual acuity also blocked experience-dependent changes in sleep need: Socially enriched smellblind¹ mutants slept the same as their impoverished siblings. As confirmation, neurons carrying olfactory input to the brain were specifically silenced [Or83b-Gal4/UAS-TNT, and it was observed that sleep in these flies was also not affected by prior waking experience. Auditory cues, however, did not affect the relation between experience and sleep. Finally, sleep need in individual Drosophila increased with the size of the social group to which they were previously exposed. Socially isolated flies slept the least, whereas those exposed to social groups of 4, 10, 20, 60, and 100 (1:1 sex ratio) showed proportionately increased daytime sleep need. When rendered blind, however, flies did not display this relation between sleep need and the intensity of prior social interactions (Ganguly-Fitzgerald, 2006).

If sensory stimulation received during a critical period of juvenile development directs the maturation of the adult sleep homeostat, then subsequent environmental exposure should not affect adult sleep time and consolidation. Alternatively, if experience-dependent modifications in sleep are a reflection of ongoing plastic processes, this phenomenon would persist in the adult. It was observed that sleep in flies was modified by their most recent social experience regardless of juvenile experience. Shorter sleeping socially impoverished adults became longer sleepers when exposed to social enrichment before being assayed. Conversely, longer sleeping socially enriched flies became shorter sleepers after exposure to a period of social isolation. Moreover, repeated switching of exposure between the two social environments consistently modified sleep, reflecting an individual's most recent experience (Ganguly-Fitzgerald, 2006).

An estimation of neurotransmitter levels in whole brains revealed that short-sleeping, socially impoverished individuals contained one-third as much dopamine as their longer-sleeping, socially stimulated isogenic siblings. Silencing or ablating the dopaminergic circuit in the brain [TH-Gal4/UAS-TNT and TH-Gal4/UAS-Rpr specifically abolished response to social impoverishment in individuals that were reared in social enrichment. Similar results were obtained when endogenous dopamine levels were aberrantly increased, by disrupting the monoamine catabolic enzyme, arylalkylamine N-acetyltransferase, in Dat^lo mutants. Hence, abnormal up- or down-regulation of the dopaminergic system prevented behavioral plasticity in longer sleeping, socially enriched individuals when switched to social impoverishment (Ganguly-Fitzgerald, 2006).

The observation that dopaminergic transmission affects experience-dependent plasticity in sleep need is particularly compelling, given its role as a modulator of memory. Mutations in 49 genes implicated in various stages of learning and memory were screened to assess their impact on experience-dependent changes in sleep need. Of these, only mutations in short- and long-term memory genes affected experience-dependent plasticity in sleep need. Mutations in dunce (dnc¹) and rutabaga (rut²⁰⁸⁰) have opposite effects on intracellular levels of adenosine 3',5'-monophosphate (cAMP), but are both correlated with short-term memory loss. In dnc¹ mutants, waking experience had no impact on subsequent sleep need. This effect was partially rescued in dnc¹/+ heterozygotes, but complete rescue was only achieved when a fully functional dunce transgene was introduced into the null mutant background. rut²⁰⁸⁰, however, selectively abolished the ability of socially enriched adults to demonstrate decreases in sleep after exposure to social impoverishment, which was reminiscent of aberrant dopaminergic modulation. Similarly, of the long-term memory genes screened, 17 (~40%) specifically disrupted the change in sleep need in socially enriched adults after exposure to social impoverishment. For example, overexpression of the Drosophila CREB gene repressor, dCREB-b, resulted in socially enriched flies that continued to be longer sleepers even after exposure to social impoverishment. As a control, overexpression of the dCREB-a activator yielded wild-type phenotypic read out. It is noteworthy that not all long-term memory mutants had a disrupted relation between experience and sleep. Instead, the particular subset of genes identified, only half of which are expressed in the mushroom bodies, may specifically contribute to pathways that underlie sleep-dependent consolidation of memories (Ganguly-Fitzgerald, 2006).

Finally, to assess the correlation between sleep and memory, male flies trained for a courtship conditioning task that generated long-term memories were measured for sleep after training. Males whose courtship attempts are thwarted by nonreceptive, recently mated females or by males expressing aphrodisiac pheromones form long-term associative memories as evidenced by subsequently reduced courtship of a receptive virgin female. Trained males that formed long-term memories slept significantly more than their untrained siblings and wake controls (ones that were sleep-deprived while the experimental flies were being trained). Exposure to a virgin female did not alter sleep need. As before, this increase in sleep was associated with longer daytime sleep bouts in trained individuals compared with controls. Further, sleep deprivation for 4 hours immediately after training abolished training-induced changes in sleep-bout duration, as well as courtship memory. Although these results are intriguing, invertebrate memory is particularly sensitive to extinction by mechanical perturbations. However, gentle handling that ensured wakefulness, but not mechanical stimulation, immediately following training, also abolished subsequent courtship memory. Furthermore, sleep deprivation per se did not affect the formation of long-term memory: Trained flies that were allowed to sleep unperturbed for 24 hours and then subjected to 4 hours of sleep deprivation retained courtship memory (Ganguly-Fitzgerald, 2006).

In summary, this study has demonstrate a rapid and dynamic relation between prior social experience and sleep need in Drosophila. In particular, experience-dependent changes in sleep need require dopaminergic modulation, cAMP signaling, and a particular subset of long-term memory genes, supporting the hypothesis that sleep and neuronal activity may be inexorably intertwined. These observations are compelling given two recent studies have demonstrating a central role of the mushroom bodies in sleep regulation and emphasize the importance of establishing Drosophila as a model system to investigate the molecular pathways underlying sleep and plasticity (Ganguly-Fitzgerald, 2006).

Analysis of a spatial orientation memory in Drosophila

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Fruit flies possess a similar spatial memory during locomotion. Using a new detour setup, flies are shown to be able to remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, it was found that the GABAergic (stainable with antibodies against GABA) ring neurons (Hanesch, 1998) of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. The protein kinase S6KII (ignorant; Putz, 2004) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies (Neuser, 2008).

Previous studies have shown that walking flies heading for an object maintain their direction even when the target disappears. This persistence of orientation can last for several seconds, indicating that flies store the position of, or the path towards, the hidden object for further targeting. It is therefore proposed that flies form a spatial memory for objects that is similar to the working memory in vertebrates. To investigate this putative memory in Drosophila a detour paradigm for walking flies was established. Single flies were put into a cylindrical virtual-reality arena, in which two dark vertical stripes were presented at opposite sides. Normally, flies patrol between the two visual objects for a considerable length of time. In the new paradigm, the stripes disappeared when the fly crossed the invisible midline of the circular walking platform, and a new target appeared laterally at a 90° angle to the fly. In most cases wild-type flies turned towards this new target if it was presented for more than 500 ms. After the fly had oriented itself towards the new object (deviation of the fly's longitudinal body axis from the ideal course to the stripe below +/-15°), this target also disappeared within 1 s and no objects were visible to the fly. It was then determined whether the fly turned back to continue its approach to its initial, but still invisible, target. The walking traces reveal a direct course towards the former location of the first target. The flies therefore retained positional information on the former object, although it was no longer present in the environment (Neuser, 2008).

Wild-type (Canton-S) flies recall the old target and integrate it into a guided behaviour with a median frequency of 80% as measured in ten consecutive trials for each fly. Longer presentation of the distracter stripe did not significantly change the percentage of positive choices. These data strongly suggest that flies stored the relative position of the first target in a spatial orientation memory for at least 4 s. To exclude the possibility that flies used chemical traces of former runs for their orientation the absolute positions of the stripes were randomly changed after each trial. As a result of this randomization, flies had to update their memory continuously. Moreover, no training effect could be observed, because the frequency of positive turns did not change during the ten consecutive trials. Similar performances were observed when two opposing distracters were presented to the fly. This orientation memory for vanished objects is considered to be to be idiothetic. Because no visible landmarks were presented to the fly after the distracter disappeared, the fly could not use a stored reference picture of the environment for its guidance. It is therefore suggested that the fly uses online stored information of its own angle towards the former target, a strategy known as path integration. Path integration has been shown to be used by other insects, such as ants and bees, to navigate through a familiar landscape (Neuser, 2008).

In an attempt to localize this type of memory to discrete parts of the insect brain, several mutant lines with structural central-complex defects of Drosophila were analysed. The central complex is composed of four different neuropils and has been implicated in supervising motor output during locomotion. First tests showed that the persistence of orientation towards a removed target is reduced or lost whenever the ellipsoid body of the central complex was defective. Therefore the ellipsoid body open mutant (ebo^KS263) was tested in the detour paradigm; these flies did not show a preference for the first target after the detour, suggesting that an intact ellipsoid body is required for establishing a spatial orientation memory. In contrast, the use of hydroxyurea to ablate the mushroom bodies, which are important in olfactory memory, did not disturb the orientation memory (Neuser, 2008).

One prominent type of neuronal cells of the ellipsoid body is the group of GABAergic ring neurons. The fibres of these neurons run in a prominent tract, the RF tract (ring-neuron and tangential fan-shaped-body neuron tract), and form bushy thin endings in the ipsilateral lateral triangle and bleb-like endings in the ellipsoid body. Four different kinds of ring neuron (R1-R4) can be distinguished by their arborization pattern around the ellipsoid body canal. R1-R3 neurons project outwards from the ellipsoid body canal, whereas the arborization of R1 is restricted to the inner zone, that of R2 to the outer zone, and that of R3 to both zones. R4 neurons project from the periphery inwards and arborize in the outermost zone. It was next proposed that the ring neurons might be necessary for the orientation memory. The GAL4/UAS system was used to silence distinct subsets of ring neurons through the expression of tetanus toxin (TNT) by using the GAL4 driver lines c232, c481 and c105. For temporal control, TNT was induced conditionally by using the temperature-sensitive GAL4 repressor GAL80^ts under the control of the ubiquitous Tubulin promoter (Tub-GAL80^ts). Experimental and control flies were raised at 18°C, tested within the detour paradigm, and retested after the induction of TNT. Pairwise comparison revealed that the preference for the original target was lost whenever the toxin was expressed in ring neurons of the ellipsoid body. This finding confirms the hypothesis that the ellipsoid-body ring neurons are necessary components of the orientation memory (Neuser, 2008).

To investigate which molecular pathways are involved in this kind of memory, focus was placed on the cyclic-AMP signalling pathway. Variable levels of cAMP have been shown to have a crucial function in memory formation during associative learning in Drosophila. cAMP levels are modulated by the opposing actions of adenylyl cyclases and cAMP phosphodiesterases. Mutants for the adenylyl cyclase gene rutabaga (rut¹ and rut²⁰⁸⁰) were unable to target visual objects and could not be tested in the paradigm. Therefore mutants of the dunce gene (dnc), which encodes a cAMP phosphodiesterase, were tested in the detour paradigm. The dnc¹ mutant is a hypomorph and displays about half of the enzyme activity in the wild type. dnc¹ mutant flies show deficits in several paradigms of associative classical learning and operant conditioning. In contrast, dnc¹ mutants showed no defects in the detour paradigm, indicating that a tight modulation of cAMP levels might not be critically required for spatial orientation memory (Neuser, 2008).

Another molecule involved in memory formation in Drosophila is a member of the ribosomal serine kinase family. ignorant (ign) encodes the S6 kinase II (S6KII), which interacts with mitogen-activated protein (MAP) kinase signalling in Drosophila. S6KII does not seem to be involved in cAMP signalling pathways. The null allele ign^58/1 has been shown to be defective in classical aversive conditioning and operant learning (Putz, 2004). Therefore ign^58/1 flies were tested in the detour paradigm. Although the mutants readily targeted visible objects, they showed no directional preference for the position of the original target after it disappeared, suggesting that they had lost their memory. In contrast, walking speed, walking activity and orientation towards visual objects were similar to those of the wild type. Next whether ign is required in the ring neurons targeted by c232-GAL4 was tested with the use of a UAS-ign RNA-mediated interference (RNAi) effector line. RNAi silencing in these ring neurons decreased the performance by half. This decrease in memory constitutes only a partial phenocopy of the null mutant, because the performance was not significantly different from that of the wild type or ign^58/1. Nevertheless, this result is interpreted to suggest that ign is required in the ring neurons for spatial orientation memory (Neuser, 2008).

To address the question of whether restoring S6KII levels is sufficient for regaining memory, neuron-specific rescue experiments were performed in the ign^58/1 mutant background. S6KII was expressed pan-neuronally with Appl-GAL4 and elav-GAL4, and also specifically in the R3 and R4 ring neurons with c232-GAL4. In all three cases a complete rescue was observed. Next, whether ign function in the R3 and R4 ring neurons is acutely required for orientation memory was examined. Therefore, again use was made of the GAL80^ts transgene to rescue the ign phenotype only in the adult. Conditional expression of S6KII only in the R3 and R4 ring neurons resulted in a perfect rescue of the ign mutant. This result -- that acute S6KII expression in the R3 and R4 ring neurons accomplished a complete rescue -- reveals that this very narrow subset of cells is sufficient for regaining a functional orientation memory. It has been reported that Drosophila S6KII negatively regulates extracellular signal-regulated kinases (ERKs) by acting as a cytoplasmic anchor of the MAP kinase. Further studies will determine whether the MAP kinase signalling pathway is required for this kind of memory task (Neuser, 2008).

It is concluded that the relevant ring neurons use the inhibitory neurotransmitter GABA. Their circuitry and interconnections within the ellipsoid body are not yet known. Expression of the dDA1 dopamine receptor in the ellipsoid body has recently been shown. It is therefore possible that the same neurotransmitter systems as those used for visual-spatial memory in the monkey prefrontal cortex are used to establish orientation memory in the central complex of flies (Neuser, 2008).

Attention-like processes in Drosophila require short-term memory genes

Although there is much behavioral evidence for complex brain functions in insects, it is not known whether insects have selective attention. In humans, selective attention is a dynamic process restricting perception to a succession of salient stimuli, while less relevant competing stimuli are suppressed. Local field potential recordings in the brains of flies responding to visual novelty revealed attention-like processes with stereotypical temporal properties. These processes were modulated by genes involved in short-term memory formation, namely dunce and rutabaga. Attention defects in these mutants were associated with distinct optomotor effects in behavioral assays (van Swinderen, 2007).

Studies of visual discrimination in flies have revealed sophisticated perceptual effects that are relevant to selective attention, such as associative learning, context generalization, cross-modal binding, and position invariance. Visual choice behavior in Drosophila is correlated with local field potential (LFP) activity in the brain, centered around 20 to 30 Hz (van Swinderen, 2003). This activity is transiently increased in amplitude by classical conditioning, is suppressed during sleep or light anesthesia (van Swinderen, 2003), and is modulated by dopamine. Electrophysiological and behavioral measures of visual attention in flies were developed to test whether these short-term processes depend on the effect of genes involved in memory formation and plasticity (van Swinderen, 2007).

LFP responses to two distinct visual objects (a cross or a box, 180° apart, each moving around the fly once every 3 s) were investigated. When the objects were presented individually to wild-type flies, they evoked brain responses that were maximal when the single object swept directly in front of the flies. In contrast, dunce mutants (dnc¹), which are defective in short-term memory, displayed attenuated and delayed brain responses to each visual object, as compared to wild-type flies (van Swinderen, 2007).

To test for visual selection between these objects, they were presented together after having increased the salience for one object specifically in a recurrent-novelty paradigm. To measure visual selection, the 20- to 30-Hz brain response (mapped onto the 3-s sequence) was averaged for 10 s (about three rotations) after each transition to novelty, and this was compared to the response for the 10 s before novelty transitions. When wild-type flies were trained with two identical boxes for 100 s before one of the boxes changed to a cross, the response mapped selectively to the sectors of the rotation sequence associated with the (novel) cross, and the response for the competing box was significantly suppressed. Converse experiments attaching novelty salience to the alternate image (the box) after 100 s of cross training mapped 20- to 30-Hz responses to the novel box, showing that novelty selection was plastic. Novelty selection was also found to be position-invariant in a subset of trials, suggesting a cognitive effect rather than habituation (van Swinderen, 2007).

By decreasing the time between transitions in otherwise identical experiments, this paradigm provided a way to estimate the minimum exposure required for selection of recurrent novelty. When the training time was decreased to 50 s (~16 rotations), significant selection of the novel object and corresponding suppression of the competing object were still seen in wild-type flies. However, when the training time was further decreased to 25 s (about eight rotations), these novelty effects were lost (van Swinderen, 2007).

To control for the effect of change alone without novelty, transitions from a cross and a box back to two boxes were tested. In this case, an object changed to one that was already present during training. Such changes did not produce any selective 20- to 30-Hz responses for any training time in wild-type flies. The response is therefore unlikely to emanate from a startle reflex or an electrical artifact (van Swinderen, 2007).

Salience is a transient phenomenon. To investigate the extinction of novelty, the temporal sequence of selective brain responses were analyzed for successive rotations of a novel panorama after a transition. In wild-type flies, 20- to 30-Hz activity was strongly selective for the novel object (the cross) for 9 s (three successive panorama rotations) on average, and this was matched at a lower level for 10- to 20-Hz activity. Responses to training for the alternate object (making the box novel) revealed similar temporal dynamics: Wild-type flies in the 100-s paradigm stereotypically 'attended' to novelty for about 9 s or three sweeps of the panorama (van Swinderen, 2007).

The robust 100-s training effect was used for subsequent experiments in short-term memory mutants. There, dnc¹ flies failed to show any selective 20- to 30-Hz response to the novel visual stimulus after a transition. Instead, they revealed some selective responsiveness in the 10- to 20-Hz range. Further analysis of dnc¹ flies showed that brain responses in this mutant were greatest in the lower-frequency (10 to 20 Hz) bracket, as compared to greater responses at 20 to 30 Hz in the wild type (van Swinderen, 2007).

Mutations in rutabaga (rut) affect the same signaling network as mutations in dnc¹ [by producing opposite effects on adenosine 3', 5'-monophosphate (cAMP) levels], and flies display similar behavioral phenotypes in olfactory memory assays. Electrophysiology uncovered differences between rut and dnc mutants. Unlike dnc¹, rut²⁰⁸⁰ showed some responsiveness in the 20- to 30-Hz range, but without the sustained 9-s selection characteristic of wild-type flies. Similar to dnc¹, rut²⁰⁸⁰ responded strongly in the 10- to 20-Hz range (van Swinderen, 2007).

To investigate visual behavior in these strains, an optomotor paradigm was used that provides an efficient alternative to flight paradigms, because many mutant Drosophilae do not fly well [notably, dnc¹. The defective responsiveness to visual novelty seen in dnc¹ brain recordings (described above) may have predicted poor behavioral responsiveness to visual stimuli, but the opposite was the case: dnc¹ flies displayed the strongest optomotor response of ~100 different strains tested. The optomotor performance was analyzed of seven olfactory learning and memory mutants; these spanned a broad range of optomotor phenotypes. Like dnc¹, rut mutants also showed unusually strong optomotor responsiveness (van Swinderen, 2007).

To better describe optomotor performance, individual choices were filmed and quantified as flies progressed through a maze. Wild-type flies showed a preference for turning into the direction of perceived motion (a positive optomotor response) throughout most successive choice points . Another characteristic of wild-type optomotor behavior is some decreased responsiveness at choice points in the middle of the maze. In contrast, dnc¹ flies proceeded through the first two choice points without displaying any optomotor response but then responded strongly at the remaining six choice points in the maze (van Swinderen, 2007).

The delayed optomotor response in dnc¹ flies reveals a defect in processing a novel visual stimulus (a moving grating) as flies enter the maze. Attention-like behavior in dnc¹ was addressed more directly by adding a competing visual object to the optomotor paradigm. In wild-type flies, a static bar placed to one side of the transparent maze abolishes responsiveness to the moving grating, presumably by acting as a visual distractor. The effect of competing visual stimuli on optomotor responsiveness has also been previously observed in tethered flight experiments, where it has been described as evidence of limited perceptual resources (i.e., attention) partitioned among visual stimuli. In the walking analog of this paradigm, it was found that dnc¹ animals were not distracted by the competing visual stimulus (unlike wild-type flies), even though dnc¹ flies clearly perceived the distractor alone. The rut²⁰⁸⁰ brain-response defects were also matched by behavioral anomalies: The rut mutant was unresponsive to the distractor and responded more strongly than did the wild type, throughout the maze, to the grating presented alone without an initial delay. Subtle differences between rut and dnc mutants [also observed in habituation and socialization) suggest that common performance defects in these memory mutants may conceal differences at the level of short-term behavioral and brain processes (van Swinderen, 2007).

Finally, whether the conditionally expressed dnc gene product (cAMP phosphodiesterase) could modulate the corresponding electrophysiological and behavioral phenotypes described here, was investigated by expressing wild-type Dunce protein in a dnc¹ mutant background by using RU486-induced gene activation of a functional dnc transgene. When wild-type Dunce protein was expressed throughout the brain (via ElavGAL4 GeneSwitch) in adult mutant animals (by feeding adult flies RU486 for 24 hours), optomotor responsiveness remained high and brain responsiveness to novelty remained correspondingly insignificant, resembling dnc¹ flies. When the same construct was activated throughout development (by growing transgenic flies on RU486-laced food), optomotor responsiveness decreased to wild-type levels, and brain responsiveness to novelty was correspondingly increased to wild-type levels. A temporal examination of 20- to 30-Hz responses in the brain revealed that extinction dynamics were rescued as well, with the strong selective response persisting for at least 9 s in RU486-grown flies. The constitutive requirement of Dunce suggests that short-term plasticity for visual responsiveness in Drosophila adults is dependent on cAMP effects in the brain during its growth and development (van Swinderen, 2007).

Light-induced structural and functional plasticity in Drosophila larval visual system

How to build and maintain a reliable yet flexible circuit is a fundamental question in neurobiology. The nervous system has the capacity for undergoing modifications to adapt to the changing environment while maintaining its stability through compensatory mechanisms, such as synaptic homeostasis. This study describes findings in the Drosophila larval visual system, where the variation of sensory inputs induces substantial structural plasticity in dendritic arbors of the postsynaptic neuron and concomitant changes to its physiological output. Furthermore, a genetic analysis has identified the cyclic adenosine monophosphate (cAMP) pathway and a previously uncharacterized cell surface molecule as critical components in regulating experience-dependent modification of the postsynaptic dendrite morphology in Drosophila (Yuan, 2011).

Proper functions of neuronal circuits rely on their fidelity, as well as plasticity, in responding to experience or changing environment, including the Hebbian form of plasticity, such as long-term potentiation, and the homeostatic plasticity important for stabilizing the circuit. Activity-dependent modification of neuronal circuits helps to establish and refine the nervous system and provides the cellular correlate for cognitive functions, such as learning and memory. Multiple studies have examined synaptic strength regulation by neuronal activity, whereas to what extent and how the dendritic morphology may be modified by neuronal activity remain open questions (Yuan, 2011).

The model organism Drosophila melanogaster has facilitated genetic studies of nervous system development and remodeling. Notwithstanding the relatively stereotyped circuitry, flies exhibit experience-induced alterations in neuronal structures and behaviors such as learning and memory). In a study of experience-dependent modifications of the Drosophila larval CNS, it has been found that different light exposures dramatically altered dendritic arbors of ventral lateral neurons [LN(v)s], which are postsynaptic to the photoreceptors. Unlike the visual activity-induced dendrite growth in Xenopus optic tectum, extending the light exposure of Drosophila larvae reduced the LN(v)s' dendrite length and functional output, a homeostatic plasticity for compensatory adaptation to alterations in sensory inputs. It was further shown that the cyclic adenosine monophosphate (cAMP) pathway and an immunoglobulin domain-containing cell surface protein, CG3624, are critical for this sensory experience-induced structural plasticity in Drosophila CNS (Yuan, 2011).

In Drosophila larvae, Bolwig's organ (BO) senses light, and its likely postsynaptic targets are LN(v)s. As the major circadian pacemaker, LN(v)s are important for the entrainment to environmental light-dark cycles and larval light avoidance behavior. In the larval brain, Bolwig's nerve (BN), the axonal projection from BO, terminates in an area overlapping the dendritic field of LN(v)s. Using the FRT-FLP system [in which DNA sequences flanked by flippase recognition targets (FRT) are snipped out by flippase (FLP)] along with three-dimensional (3D) tracing, the dendritic arbor of individual LN(v) neurons were labeled and analyzed. Then potential synaptic connections were demonstrated between BN and LN(v)s using the GRASP [green fluorescent protein (GFP) reconstitution across synaptic partners] technique to drive expression of one-half of the split GFP in the BN by means of Gal4/UAS and expression of the other half of the split GFP in LN(v)s via LexA/LexAop. The proximity of putative synaptic connections between BN and LN(v)s' dendrites reconstituted GFP fluorescence for photoreceptors expressing either rhodopsin 5 (Rh5) or rhodopsin 6 (Rh6) in BO, which suggested that both groups of photoreceptors may have synaptic connections with LN(v)s (Yuan, 2011).

To test whether LN(v)s can be activated by BN inputs through light stimulation, calcium imaging was performed using GCaMP3 transgenic flies with the larval brain-eye preparation, which included BO, BN, developing eye disks, the larval brain, and ventral nerve cord. Because BO senses blue and green light, the confocal laser at 488 nm (blue) and 543 nm (green) were used to stimulate these larval photoreceptors. LN(v)s' axon terminals displayed a relatively stable baseline of GCaMP3 fluorescence and, upon light stimulation, yielded large calcium responses, which increased with the greater intensity and longer duration of the light pulses (Yuan, 2011).

Recent studies suggest that Cryptochrome (CRY) in adult large LN(v)s senses light and elicits neuronal firing. In larvae, however, severing BN abolished light-induced calcium responses in LN(v)s. The loss-of-function mutation of NorpA (no-receptor-potential A), encoding a phospholipase C crucial for phototransduction, also eliminated these calcium responses, which indicated that light-elicited responses in LN(v)s are generated via phototransduction in larval photoreceptors rather than as a direct response to light by LN(v)s (Yuan, 2011).

In animals with BO genetically ablated, the dendritic field of LN(v) is absent. To test whether BO is required for LN(v)s' dendrite maintenance, the expression of cell death genes rpr and hid was induced in BO after synapse formation, and the LN(v) dendrite length was also found to be greatly reduced. Whereas physical contacts with BN or growth-promoting factors released from presynaptic axons could be important for LN(v)s' dendrite maintenance, it is also possible that synaptic activity from BN promotes LN(v) dendrite growth, as suggested by previous studies. To explore the latter scenario, newly hatched larvae were provided with different visual experiences through various light regimes—including the standard 12 hours of light and 12 hours of dark daily cycle (LD); constant darkness (DD) for sensory deprivation; constant light (LL) for enhanced light input; 16-hour light and 8-hour dark cycle, mimicking a long day; and 8-hour light and 16-hour dark cycle, mimicking a short day. The dendrite morphology of LN(v)s of late third instar larvae was examined. Whereas different light exposure had no detectable effects on larval developmental timing, increasing light exposure reduced the total dendrite length of individual LN(v) neurons, with the longest dendrite in constant darkness and the shortest dendrite length in constant light condition. Thus, not only is the LN(v) dendrite dependent on the presence of presynaptic nerve fibers, its length is modified by the sensory experience in a compensatory fashion, whereby an increase in sensory inputs causes a reduction in the dendrite length and vice versa (Yuan, 2011).

Whereas adult LN(v)s alter their axon terminal structures in a circadian cycle-controlled fashion, no difference was found in dendrite morphology of LN(v)s from larvae collected at four different time points around the clock, which indicated that circadian regulation is not involved in the light-induced modification of LN(v) dendrites. Under regular light-dark conditions, LN(v) dendrites expanded as the larval brain size increased from the second to the third instar stage. However, the dendrite length of the LL group increased at a significantly slower rate than the DD group. It thus appears that light exposure retards the growth of LN(v) dendrites throughout the larval development (Yuan, 2011).

To test the contribution of different light-sensing pathways, loss-of-function mutations of Cry (cry⁰¹) or NorpA (norpA³⁶) and of double mutants lacking both Rh5 and Rh6 (rh5²;rh6¹) were examined. Although wild-type and cry⁰¹ larvae displayed differences in their dendrite length when exposed to constant darkness versus constant light, such light-induced changes were absent in the rh5²;rh6¹ double mutant and the norpA³⁶ mutant. Thus, similar to the calcium response to light, light-induced modification of LN(v) dendritic structure requires visual transduction mediated by rhodopsin and NorpA in BO but not Cry function in LN(v)s (Yuan, 2011).

To manipulate the level of synaptic activity, the BO excitability was weither increased by expressing the heat-activated Drosophila transient-receptor-potential A1 (dTrpA) channel, or transmitter release from BN was reduced through a temperature-sensitive form of the dominant-negative dynamin, Shibire^ts (Shi^ts). These manipulations eliminated light-induced modification of LN(v) dendrites at 29°C. Reducing BO activity by means of Shi^ts caused dendrite expansion, as if the animal detected no light, whereas increasing BO activity by means of the dTrpA channel resulted in reduction of LN(v) dendrites, a process reminiscent of constant light exposure (Yuan, 2011).

Whether intrinsic LN(v) neuronal activity drives modification of its dendrite morphology was further tested by expression of either the sodium channel NaChBac to increase excitability or the potassium channel Kir2.1 to reduce excitability. LN(v)s expressing Kir2.1 showed reduced or no calcium responses upon light stimulation. In contrast, LN(v)s expressing NaChBac displayed numerous peaks in GCaMP3 signals in the presence or absence of light stimulation, indicative of elevated spontaneous activities. Upon examining LN(v) dendrites, it was found that neuronal excitability of the LN(v) was inversely proportional to its dendrite length (Yuan, 2011).

These results obtained using genetic approaches agreed with findings in experiments with different environmental light conditions. They suggested that LN(v)'s dendritic structures are modified according to its neuronal activity, which varies with light-induced synaptic inputs (Yuan, 2011).

To test whether synaptic contacts of BN on LN(v)s are modified by light, synapses formed by BN with EGFP (enhanced green fluorescent protein)-tagged Cacophony (Cac-EGFP) were marked, because Cacophony is a calcium channel localized at presynaptic terminals and its distribution correlates with the number of synapses. Close association was found of Cac-EGFP-expressing structures with LN(v)s' dendritic arbors. Compared with regular light-dark conditions, constant darkness increased, whereas constant light reduced, the total intensity of Cac-EGFP, which suggested that light modified not only dendritic arbors of LN(v)s but also the number of synaptic contacts impinging on LN(v) dendrites (Yuan, 2011).

Next, using calcium imaging, whether there are light-induced functional modifications of LN(v)s was examined. Increased light exposure caused LN(v)s to be less responsive. Conversely, sensory deprivation in constant darkness increased LN(v)s' sensitivity to light. Thus, in contrast to stable synaptic responses observed in synaptic homeostasis, light-induced responses of central neurons postsynaptic to photoreceptors in the Drosophila larval visual circuit have a dynamic range, modifiable by sensory experiences and positively correlated to the dendrite length (Yuan, 2011).

In dunce¹, a loss-of-function mutant of the fly homolog of 3'5'-cyclic nucleotide phosphodiesterase, the LN(v)s' dendrite length was comparable among LD, LL, and DD groups. Reducing dunce gene expression specifically in LN(v)s through RNA interference (dncIR) resulted in a similar indifference of LN(v)s' dendrite size to the light exposure, which implicated a cell-autonomous action of dunce in LN(v) neurons (Yuan, 2011).

To explore the possibility that the elevated cAMP level caused by the dunce mutation interfered with dendrite plasticity, tests were performed for the involvement of downstream components of the cAMP pathway, including the catalytic subunit of protein kinase A (PKAmc), which up-regulates cAMP signaling, and a dominant-negative form of the cAMP response element-binding protein (CREBdn), which inhibits cAMP-induced transcription activation. Expression of either transgene specifically in LN(v)s obliterated their ability to adjust dendrite length under different light-dark conditions. Calcium imaging further revealed that the expression of PKAmc or CREBdn eliminated changes of LN(v)s' light responses produced by different light-dark conditions. Thus, the cAMP pathway regulates both structural and functional plasticity of LN(v)s (Yuan, 2011).

The screen for mutants with defective LN(v) dendritic plasticity also identified babos-1, a mutant with a P-element insertion near the transcriptional start site of CG3624, a previously uncharacterized immunoglobulin domain-containing cell surface protein. The LN(v) dendrite length of babos-1 mutant larvae was comparable to controls in LD and LL but has no compensatory increase in DD. Similar phenotypes were found in larvae expressing an RNAi transgene targeting CG3624 in LN(v)s. Moreover, flies carrying a hypomorphic allele of CG3624, CG3624^[KG05061], also showed defective light-induced dendritic plasticity, which was fully rescued by expressing the UAS-CG3624 transgene specifically in LN(v)s. Thus, the function of this immunoglobulin domain-containing protein in LN(v)s is important for the dendrite expansion in constant darkness (Yuan, 2011).

Bioinformatic analyses suggest that CG3624 is a cell surface protein containing an N-terminal signal peptide, extracellular immunoglobulin domains followed by a transmembrane helix, and a short C-terminal cytoplasmic tail. CG3624 is widely expressed in the nervous system throughout development. Its specific requirement for the adjustment of LN(v)s' dendrite length in constant darkness suggests that elevation or reduction of sensory inputs likely invokes separate mechanisms for compensatory modifications of central neuronal dendrites (Yuan, 2011).

A functioning nervous system must have the capacity for adaptive modifications while maintaining circuit stability. This study of the Drosophila larval visual circuit reveals large-scale, bidirectional structural adaptations in dendritic arbors invoked by different sensory exposure. Whereas the circuit remains functional with modified outputs, this type of homeostatic compensation may modify larval light sensitivity according to its exposure during development and could facilitate adaption of fly larvae toward altered light conditions, such as seasonal changes. The observations also suggest shared molecular machinery between homeostasis and the Hebbian plasticity with respect to the cAMP pathway and indicate the feasibility of genetic studies of experience-dependent neuronal plasticity in Drosophila (Yuan, 2011).

Altered gene regulation and synaptic morphology in Drosophila learning and memory mutants

Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in

radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. This study demonstrates that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, synaptic connectivity was assayed and an expression analysis was performed to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed approximately 375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila (Guan, 2011).

Drosophila has proven to be a powerful model for identifying gene products involved in learning and memory based on olfactory, visual, and courtship behavioral assays. How proteins identified in these studies regulate neuronal function or physiology to specifically alter behavioral plasticity is an ongoing area of investigation. Using the well-characterized 3rd instar larval NMJ as a model glutamatergic synapse, the effects on synaptic connectivity were compared of several learning mutants that alter cAMP signaling (dnc¹, rut¹, amn¹) with the poorly characterized ARM mutant rsh¹. Each mutant altered synaptic connectivity at NMJs in a specific manner, suggesting that changes in neuronal connectivity in the CNS might contribute to the behavioral defects found in these strains. The observations in dnc¹ and rut¹ are similar to previous studies of synaptic morphology in these mutants. Gene expression was assayed in the mutants using microarray analysis, which revealed many neuronal transcripts that were transcriptionally altered. A long-term goal is to link transcriptional changes in specific loci to the behavioral and morphological defects found in learning and memory mutants (Guan, 2011).

Experimental approaches to define the biochemical transition from short-term plasticity to long-term memory storage have suggested a key role for cAMP signaling. At the molecular level, one of the best-characterized pathways for STM has been described for gill withdrawal reflex facilitation in Aplysia. In this system, conditioned stimuli act through a serotonergic G protein-coupled receptor pathway to activate adenylyl cyclase in the presynaptic sensory neuron, resulting in the synthesis of cAMP. cAMP activates PKA, which phosphorylates a presynaptic potassium channe, leading to prolonged calcium influx and enhanced neurotransmitter release from the sensory neuron. Insights into the LLTM pathway in Aplysia have implicated CREB function. Robust training or stimulation with serotonin induces translocation of the catalytic subunit of PKA into the nucleus, where it activates the transcription factor CREB-1 and inhibits the transcriptional suppressor CREB-2. CREB-1 acts on additional transcription factors to produce specific mRNAs that are transported to dendrites and captured by activated synapses. Local synthesis of new proteins and subsequent growth of synaptic connections is predicted to underlie long-term memory in the system. It is likely that similar molecular pathways exist in other species. Transgenic Drosophila with inducible inhibition of PKA show memory impairment. PKA is also activated during hippocampal LTP induction in mammals, and transgenic mice that express an inhibitor of PKA have defective LTP and hippocampal-dependent memory, suggesting a general role for cAMP/PKA in the transition from learning to memory storage (Guan, 2011).

In addition to CREB-dependent LLTM, which requires transcription and translation for its formation, the Radish-dependent ARM pathway represents a distinct long-term memory storage mechanism. These various memory pathways partially overlap in time. Three hours after training ~50% of memory is stored as STM, with the rest present as ARM, which is formed immediately after training in flies and can last for days depending on training intensity. ARM is not blocked by agents that disrupt electrical activity in the brain, suggesting that a biochemical pathway for ARM is likely initiated by learning stimuli, but does not require continued neuronal excitation for its expression. ARM is also not as sensitive to translation inhibition, as a 50% reduction of protein synthesis by cycloheximide does not affect ARM, but blocks LLTM (Guan, 2011).

Similar to the role of CREB in LLTM, Radish appears to be a key regulator of the ARM phase of memory. In contrast to the molecular pathways underlying STM (cAMP/PKA cascade) and LLTM (PKA/CREB), the signaling mechanisms mediating ARM are unknown. Unfortunately, the amino acid sequence of the radish locus gives little insight into its function, as it lacks known structural motifs or domains. Radish contains a serine/arginine-rich sequence with very limited homology to splicing factors, hinting that it may be involved in RNA processing. The Radish protein also contains PKA phosphorylation sites and multiple NLS sites within its sequence. Consistent with these sequence features, This study found that Radish is phosphorylated by PKA in vitro, linking ARM to the cAMP/PKA pathway. By generating a GFP-tagged Radish transgenic animal, it was possible to characterize Radish localization. Radish was prominently localized to cell bodies of neurons in the CNS, but was enriched in the nucleus in other cell types such as salivary gland and muscle cells. Given the overlap between several of the NLS and PKA sites in Radish, it will be interesting to explore whether the phosphorylation state of Radish regulates its subcellular distribution. An attractive hypothesis is that activated PKA phosphorylates Radish at synapses, resulting in transport to the nucleus with accompanying effects on transcription or RNA processing that would modify long-term synaptic function. Given that ARM can last for days, a change in nuclear function is an attractive biological underpinning, even though ARM has been suggested to be a translation-independent form of memory. Given that general protein synthesis was reduced by only 50% in the previous studies, it is quite possible that ARM and LLTM have different thresholds for translational inhibition (Guan, 2011).

In terms of synaptic modifications in rsh¹ mutants, this study found that larval NMJ synapses were altered compared with controls. Specifically, rsh¹ mutants had shorter axonal projections onto target muscles and displayed more synaptic boutons within the innervated region. These alterations gave rise to a more compact innervation pattern than observed in controls. Overgrowth of synapses at larval NMJs was also observed in dnc¹ mutants, whereas reduced innervation length was found in rut¹ mutants. As such, rsh¹ mutant NMJs display a unique phenotype compared with mutants that increase or decrease cAMP levels. The molecular mechanisms by which Radish regulates synaptic growth are unclear. Radish could directly interface with growth regulators at the synapse in a PKA-dependent fashion. Indeed, an interaction between Radish and Rac1 was found in a high-throughput yeast two-hybrid screen for interacting Drosophila proteins. Rac1 is a Rho family GTPase that regulates neuronal and synaptic morphology via reorganization of the cytoskeleton. Rac1 function has also been linked to PAK1 and the Fragile-X Mental Retardation protein (FMRP), which alter synaptic and behavioral plasticity in mammals. Recently, Rac activity has been linked to memory decay in Drosophila (Shuai. 2010), indicating that a Radish-Rac link might control memory processing via alterations in cytoskeletal modulation of synaptic function or stability. Although it is possible that Radish regulates synaptic properties through a Rac1 interaction, no robust Rac1-Radish interaction was observed in either yeast-two hybrid or GST pull-down experiments. No Radish-GFP enrichment was observed at larval synapses where the synaptic growth defect was quantified, although the protein was present in larval axons. As such, it may be that NMJ defects in rsh¹ arise through downstream effects secondary to the loss of Radish function in a neuronal compartment besides the synapse (Guan, 2011).

To further explore this possibility and examine links between rsh and the STM pathway, genome-wide microarray studies were performed on several learning and memory mutants. Although there were some shared transcriptional changes between rsh¹ and the other mutants (dnc¹, rut¹, amn¹), most of the changes in rsh¹ were unique. Although linking these changes to a direct effect on the underlying biology will require more work, several interesting loci were identified that could contribute to synaptic plasticity defects. The Drosophila NFAT homolog, a transcription factor that binds to the activity-regulated AP-1 (Fos/Jun) dimer, was robustly up-regulated by sevenfold in rsh¹ mutants. The RNA-binding protein smooth (sm) was also up-regulated in rsh¹ mutants. Mutations in sm have been shown to alter axonal pathfinding. Other genes that were transcriptionally altered in rsh¹ mutants and that would be predicted to influence synaptic connectivity were the Sh potassium channel, the adapter protein Disabled, and the Lapsyn cell adhesion protein. The potential role of Lapsyn was intriguing, as LRR-containing proteins have been implicated in the regulation of neurite outgrowth and synapse formation. In particular, netrin-G ligand and synaptic-like adhesion molecule (SALM) are known LRR proteins that regulate neuronal connectivity and synapse formation. In Drosophila, LRR repeat proteins have been implicated in motor neuron target selection. Given the roles of other LRR-containing proteins in the regulation of neuronal connectivity, this study explored whether Lapsyn might also function in this pathway. Lapsyn was up-regulated by neuronal activity in addition to being up-regulated in rsh¹, making it an interesting transcriptional target to assay for a role in synaptic modification (Guan, 2011).

Lapsyn mRNA expression was broadly up-regulated in the brain by neuronal activity, suggesting a potential widespread effect on neuronal function. Lapsyn-GFP transgenic protein targeted to the presynaptic terminal, partially overlapping with the periactive zone, a region of the nerve terminal enriched in proteins that regulate synaptic vesicle endocytosis and synaptic connectivity. Animals lacking Lapsyn died at the end of embryogenesis, although the early stages of nervous system formation appeared normal. It was possible to partially rescue Lapsyn mutants with neuronal expression of a Lapsyn transgene, indicating an essential function for the protein in the nervous system. Rescue to adulthood required expression outside the nervous system, suggesting Lapysn is likely to have functions in other tissue types as well. Manipulations of Lapsyn expression in the nervous system resulted in distinct defects in synaptic connectivity at the NMJ. Heterozygotes expressing only a single copy of the Lapsyn gene displayed supernumerary satellite bouton formation, a phenotype commonly associated with mutants that disrupt synaptic endocytosis or that alter the transmission or trafficking of synaptic growth factors through the endosomal system. This increase in satellite boutons in Lapsyn heterozygotes suggests that the protein plays a role in the regulation of synaptic growth signaling. Overexpression of Lapsyn, as induced by activity or observed in rsh¹ mutants, also elicited a change in synaptic growth, resulting in an increase in overall bouton number at larval NMJs. Thus, regulation of Lapsyn levels modulate synaptic growth mechanisms at NMJs. Lapsyn mutant heterozygotes also display defects in larval associative learning, although this phenotype could not be rescued with pan-neuronal overexpression. The lack of a specific rescue makes it unclear whether the learning defects are linked to a non-Lapsyn function, or if a more specific spatial and temporal expression of Lapsyn is required for functional rescue (Guan, 2011).

How Lapsyn participates in synaptic signaling is currently unclear. The closest mammalian homologs of Lapsyn are the NGL family of synaptic adhesion molecules. Three isoforms are found in mammals, NGL-1, NGL-2, and NGL-3, which interact with netrin-G1, netrin-G2, and the receptor tyrosine phosphatase LAR, respectively. NGL-1 promotes axonal outgrowth, whereas NGL-2 is capable of triggering synapse formation. The interaction of NGL-3 with LAR is intriguing, as the Drosophila LAR homolog has been shown to bind the heparan sulfate proteoglycans Syndecan and Dallylike to regulate synaptic growth at the NMJ. The homology between Lapsyn and the mammalian NLG family is restricted to the extracellular LRR domain, with no homology observed in the intracellular C terminus. The three mammalian NLGs also lack homology to each other at the C terminus, except for the presence of a PDZ-binding domain at the end of the intracellular domain. It will be important to identify binding partners for Lapsyn at the synapse to define how it may regulate synaptic adhesion or signaling between the pre- and postsynaptic compartments to regulate synaptic growth. Likewise, additional studies into the Radish-dependent ARM phase of memory may reveal how rsh-dependent changes in Lapsyn levels contribute to the synaptic and behavioral defects of this memory mutant (Guan, 2011).

Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations

Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. This study asked how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. This study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes were reviewed. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. Factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue are discussed (Ueda, 2012).

It has been demonstrated that cAMP levels are decreased in rut. The results clearly contrast the differential effects of disruptions in synthesis and degradation of cAMP on synaptic function and nerve terminal morphology. Mutations in dnc, including dnc¹, dnc^M11, and dnc^M14, can lead to severe defects in nerve terminal branching and bouton morphology. Aside from this study, previous reports have documented in identified larval muscles that total bouton numbers and motor terminal branching pattern are severely affected by dnc, but these defects were not detected in rut. A similar situation has been reported in the adult CNS: axon terminal growth in the mushroom body is enhanced in dnc but is not affected in rut. In contrast, rut and dnc mutations both have clear effects on synaptic transmission but in distinct manners. Increased cAMP levels in dnc could enhance transmitter release (as indicated by increased ejp sizes with a minimal disturbance in the temporal precision of the release process. In comparison, rut mutations more severely disrupt temporal control of release, regardless of the rearing temperature. In addition, the rearing temperature affects the amplitude of synaptic transmission in rut, with strongly depressed transmission at high temperature. This likely reflects a decrease in vesicle release because the miniature ejp size was unaltered at different temperatures (data not shown) (Ueda, 2012).

A number of mutant alleles of the rut gene have been described in the literature of developmental studies, but the alleles frequently used in neurogenetic experiments are limited to rut¹, rut², rut³, rut¹⁰⁸⁴, and rut²⁰⁸⁰. Furthermore, only three mutant alleles have been biochemically characterized in Drosophila: rut¹, rut², and rut³. It should be mentioned that these rut mutations can cause significant decrease in total cAMP synthesis despite the fact that there are at least four adenylyl cyclase (AC) homologous genes that have been identified molecularly and biochemically in Drosophila. This raises the possibility that rut may represent a major AC gene but all AC genes may play differential roles in regulating cAMP levels, depending on their subcellular localization and conditions to activate their actions. As demonstrated in this study as well as in earlier reports, a general pattern of relative severity among several rut mutant alleles is observed across different phenotypes, as represented in the following sequence: rut¹ = rut¹⁰⁸⁴ = rut² = rut³ = WT (Ueda, 2012).

Compared to the AC genes, there appears to be fewer PDE homologous genes and only two genes are known for their cAMP degradation action besides dnc. However, dnc gene products are represented by more than 10 splicing variants as opposed to 2 rut splicing variants. There are a large number of dnc mutant alleles reported in literature but only a small number of them are frequently used in neurogenetic studies, i.e., dnc¹, dnc², dnc^M11, and dnc^M14. Interestingly, a consistent pattern of phenotypic severity can be observed across different phenotypes among these four alleles: dnc^M11 = dnc^M14 = dnc¹ = dnc² (Ueda, 2012).

A comparison of their effects on a variety of phenotypes includes PDE enzyme activity disruption, defective growth cone motility of cultured neurons, enhanced growth of larval NMJ, enhanced K ⁺ and Ca^{2 +} currents in larval muscles, decrease in the larval motor neuron firing frequency upon depolarization, increase in whole-cell ejps or ejcs, and decrease in activity-dependent facilitation of synaptic transmission at larval NMJ, decrease in the habituation rate of olfactory jump response and odor-electric shock association in adult flies, and female sterility. In a different approach, overexpression of a UAS-dnc ⁺ transgene in motor neurons results in reduced NMJ growth and decreased ejp size even in larvae reared at room temperature. These phenotypes demonstrated the effects of increased cAMP degradation in contrast to those caused by dnc mutations (Ueda, 2012).

When considering their mechanisms of action, several reported phenotypic effects of dnc alleles may be complicated by the implications of contributions from the genetic background. Notably, the dnc^M11 mutant line has been reported to affect protein kinase C (PKC) activity in addition to PDE. In addition, the severity of dnc¹ may in fact be more extreme than reported, since dnc¹ has been shown to be female sterile once a second-site mutation near the dnc locus is removed from the original fertile line. It is possible that many dnc¹ lines used in neurogenetic investigations contain this mutation in the background (Ueda, 2012).

A number of experimental paradigms have been used to characterize behavioral and physiological phenotypes of dnc and rut mutants with defined quantitative parameters. For a majority of phenotypes examined, dnc and rut mutations do not lead to opposite effects on these quantitative indices, even though they alter the cAMP levels in opposite directions. Only for certain phenotypes, the dnc and rut mutations affect the parameters in opposite directions. For example, in larval neuromuscular synaptic boutons, mobilization of synaptic vesicles from the reserve pool to exo/endo cycling pool is suppressed in rut and enhanced in dnc. Similarly, the number of docked vesicles at synapses is decreased in rut and increased in dnc. Ca^{2 +} current measured in larval muscles is decreased in rut and increased in dnc. Hyperexcitability-induced overgrowth of larval NMJ can be suppressed by rut but enhanced by dnc. Similarly, dnc and rut mutations exert opposite effects on Kenyon cell axon counts in the mushroom body of developing adult flies. Finally, habituation rate of the giant fiber escape circuit is decreased by rut and increased by dnc (Ueda, 2012).

In contrast, for some other phenotypes, rut alleles have no apparent effects while dnc mutants display clear alterations. For instance, the larval NMJ terminal projection pattern and adult mushroom body axonal terminal growth were altered in dnc but not in rut. Moreover, identified K ⁺ currents in larval muscles are increased in dnc but unaltered in rut. In these cases, increased cAMP levels can produce abnormalities but underlying mechanisms may be tolerant to depleted cAMP levels (Ueda, 2012).

For another group of phenotypes, dnc and rut mutations can affect separate parameters and sometimes produce superficially similar effects by altering a parameter in the same direction. Such cases include decreased growth cone motility, irregular action potential firing pattern, and modified intracellular Ca^{2 +} dynamics in cultured neurons. In larval neuromuscular junctions, both dnc and rut decrease synchronicity of synaptic transmitter release and presynaptic facilitation of neuromuscular transmission. During post-eclosion development of adult flies, both dnc and rut mutations enhance the axon terminal growth of mechanosensory cells and decrease the structural and functional adaptation of the olfactory system to odor exposure. Neither dnc nor rut mutants respond to environmental or social deprivation in modifying Kenyon cell axon counts of young adults. Mutations of either dnc or rut decreases habituation rate of the proboscis extension reflex and olfactory avoidance and jump response, and the performance indices of both classical and operant conditioning. Studies on alcohol response have demonstrated increased sensitivity in rut alleles but no apparent change in dnc alleles. Although it is reassuring to observe opposite effects of dnc and rut mutations on some of the quantitative parameters, it should be noted that it is not straightforward in associating most of the indicators with the defective mechanisms directly regulated by cAMP signaling. Dysfunction in AC and PDE may exert opposite effects on some cell biological mechanisms or neural circuit components but can still lead to apparently similar deficiencies of a cellular function or behavioral task (Ueda, 2012).

Some insights may be gained through examining the genetic interactions between dnc and rut in double mutants about how rut AC and dnc PDE are involved in particular aspects of physiological or behavioral plasticity. At the present time, only a limited number of reports document the resultant phenotypes in dnc rut double mutants. Significantly, the majority of the single-mutant phenotypes of dnc or rut mutations do not become less severe in dnc rut double mutants, even though the overall cAMP levels are largely restored. The phenotypes that are not rescued in double mutants include increased bouton size in dnc and impaired synchronicity of transmitter release in larval NMJs, irregular firing of cultured neurons, and habituation and olfactory associative learning of adult flies (Ueda, 2012).

However, a few cases of successful rescue in double mutants have been described. Decreased growth cone motility in dnc and rut neurons in culture can be restored by combining two mutations and the overgrowth and altered projection patterns of dnc larval motor terminals is suppressed in dnc rut. Interestingly, none of the above cases of successful rescue involve opposite effects of dnc and rut single-mutant phenotypes. Notably, both cases of restoration involve a particular allele, rut¹. The allele rut¹ is different from other alleles with characterized AC enzyme activity (rut² and rut³) in that the Ca^{2 +} /CaM-dependent activation of AC is eliminated in rut¹ flies, but retained in rut² and rut³. Unlike rut¹, the allele rut² is not able to rescue the dnc mutational effects of enhanced larval NMJ growth and irregular firing in cultured neurons. In the present study of NMJ focal recording, it was clears that rut² did not affect the precision in release timing (ejc peak time) and ejc amplitudes, although rut¹ decreased the temporal precision of release (increased variability in ejc peak time) and the ejc amplitude significantly. It will be helpful if further experiments are performed on additional allele combinations of dnc and rut to delineate the role of Ca^{2 +} -dependent regulation of AC in specific phenotypes of interest (Ueda, 2012).

In addition to peculiarities of enzymatic properties in mutant alleles, e.g., rut¹ AC devoid of Ca^{2 +} /calmodulin (CaM) sensitivity, other factors influencing interactions between dnc and rut must be considered. As summarized above, counterbalancing rescue of dnc and rut phenotypes in double mutants is likely to be exceptions rather than a general rule. Therefore, it would be desirable to identify the conditions and factors that could facilitate their counterbalancing interactions, which may provide insights into the orchestration of dnc PDE and rut AC underlying the phenotype of interest (Ueda, 2012).

First, it is important to consider the temporal and spatial characteristics of expression and operation of these enzymes. In the temporal domain, their effects on a variety of phenotypes are mediated through integration among different biochemical pathways and cellular processes, some of which may function with rapid kinetics, whereas others may represents slow accumulation of products through a number of steps. Some of the resultant phenotypes may require continuous adjustment in response to internal or environmental conditions while others may appear relatively permanent and irreversible, possibly associated with developmental events (Ueda, 2012).

The spatial factors to be considered include the cellular expression and subcellular localization of the enzymes. To the best of our knowledge, there is little information about whether dnc PDE and rut AC are colocalized in molecular assemblies or aggregates within certain functional domains in specific neuronal cell types. Close proximity of AC and PDE localization facilitates local regulation of cAMP levels within a short time. Certain cellular processes with slower kinetic steps also facilitate integration of dnc and rut interactions, extending their balancing acts to a broader spatial range (Ueda, 2012).

For the few examples of successful counterbalancing rescue, growth cone motility seems to be a continuous adjustment by cAMP on a time scale of tens of seconds to minutes. This relatively slow kinetics makes it possible to readily manipulate the cAMP signaling pathway, e.g., bath application of db-cAMP increases rut growth cones motility, mimicking dnc counter balancing effects in double-mutant growth cones. Some developmental or maintenance processes, such as axonal path finding, branch formation, target interaction, and synaptogenesis, are also slow adjustment processes (in the order of hours to days). In these cases, restoration of cAMP levels through long-range interactions of AC and PDE may be sufficient to rescue the single-mutant phenotype. For example, dnc defects in larval motor terminal growth are suppressed by rut¹ (Ueda, 2012).

In contrast, defects in some physiological properties (K ⁺ currents, neuronal firing, and transmitter release timing) and behavioral conditioning (habituation and classical conditioning) cannot be rescued by combining dnc and rut, which sometimes leads to even more extreme deficiencies, e.g., the extremely rapid habituation in dnc rut. One possibility is the requirement of dynamic cAMP regulation within a short time period (millisecond to second range) during which a counterbalancing act is difficult to achieve. Another possible explanation is the requirement of unimpaired cellular machinery laid down during development (e.g., proper channel and receptor localization) and functional connectivity among synaptic partners (inhibitory and excitatory elements in the circuit) underlying behavioral phenotypes under consideration. Deviation from coordinated actions of such subcellular machinery or circuit components will make it difficult to obtain compensatory rescue (Ueda, 2012).

It should be noted that well-defined abnormalities in central fiber projection have been reported in dnc and rut single mutants that reflect the alterations in peripheral motor terminals in larval NMJs. Furthermore, dnc PDE and rut AC are preferentially expressed in mushroom bodies, which are important in odor-associated learning. Therefore, it is reasonable to speculate that defects in higher functions, including classical associative learning and habituation, may involve anatomical defects in the CNS, such as altered dendritic arbors and synaptic connections detectable in certain defined circuits, in addition to potential changes in synaptic physiology (Ueda, 2012).

Cell-specific expression and subcellular localization of AC and PDE isoforms may affect dnc and rut single-mutant, as well as double-mutant phenotypes. These include splicing variants of the dnc and rut gene as well as the products of their homologous genes. Such complexity needs to be considered in the interpretation of dnc and rut interactions in order to appreciate contributions of individual splicing variants and to delineate influence from their homologous genes (Ueda, 2012).

Finally, cross-talk between the cAMP and other signaling pathways can also modify dnc and rut phenotypes. For example, variety of signalling pathways are known to converge onto the CREB transcription factor. It is also established that not only the cAMP cascade but also other signaling pathways, including PKG and CaMKII, can modify larval NMJ physiology and morphology as well as adult habituation, courtship conditioning and classical conditioning. It will be of particular interest to establish the consequences of such genetic interactions across signaling pathways. Double mutant analysis in conjunction with transgenic and genomic approaches remains a powerful and profitable direction for revealing the genetic network underlying neural and behavioral plasticity (Ueda, 2012).

AKAPs act in a two-step mechanism of memory acquisition

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. This study explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) this study identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. It is proposed that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition (Scheunemann, 2013).

Conditioned odor avoidance is subject to a general dichotomy since multiple memory components are engaged in control of behavior. This is usually analyzed at two time points, i.e., 3 min and 3 h after training. At 3 min, basal and dynamic STM are separable by genetic means as revealed by opposing phenotypes of rut¹ and dnc¹ mutants. Moreover, those components are also separable due to characteristic differences in their acquisition dynamics as revealed by different effects of shock number. A similar dichotomy applied to 3 h memory when ASM and ARM were separable by means of amnestic treatment. It was striking that basal STM and consolidated ARM were instantaneously acquired, resulting in a front line of protection by eliciting conditioned avoidance after a singular experience of a CS/US pairing. Interestingly, consolidated ARM (as defined by means of resisting amnestic treatment) was installed quickly after training. However, it remains to be addressed at the genetic and molecular level, whether 3 h ARM linearly results from the functionally similar basal SMT component (Scheunemann, 2013).

In contrast, the two components of dynamic STM and labile ASM acquire in a dynamic fashion but are clearly dissociated from each other by characteristic chronologies of CS/US pairings required for their acquisition. However, either component contributes to behavioral performance in addition to the appropriate instantaneous component, and hence, increases avoidance probability during the test situation. Considering a potential benefit from avoiding aversive situations this overall dichotomy of behavioral control seems plausible and is also reflected at the genetic level since rut-dependent cAMP signals are limited to support of dynamic but not instantaneous memory components. Rut-dependent STM and ASM, however, are dissociated by means of shock impact and discontinuous formation of ASM is limited to situations where animals repeatedly experience high-impact CS/US pairings within a predefined time window. Experience that does not meet this criterion, however, is not discounted but adds to continuously acquired dynamic STM. By functional means these two components are thus clearly separated but commonly dependent on rut-derived cAMP signals within the KC layer, forming ties between genetically and functionally defined memory components (Scheunemann, 2013).

Genetic dissection of memory has a long-standing history in Drosophila and provided a powerful means to define molecular, cellular, and neuronal networks involved in regulation of conditioned odor avoidance. Among others, the cAMP-signaling cascade has been identified as one central tenet of aversive odor memory foremost by means of two single-gene mutants affecting either a Ca²⁺-sensitive type 1 adenylyl cyclase (AC1) and/or a cAMP-specific type 4 phosphodiesterase (PDE4) affected in the Drosophila learning mutants rutabaga (rut-AC1) and dunce (dnc-PDE4). Although originally isolated due to poor performance in the aversive odor learning paradigm, a general dichotomy has been recently revealed that separates memory components by their dependency on either rut-AC1 or dnc-PDE4 function, and the view was established that two different types of cAMP signals are engaged during the single-cycle training procedure (Scheunemann, 2012). A similar dichotomy is observed at level of acquisition dynamics and suggests that rut-dependent cAMP signals are limited to formation of dynamically acquired memory components, i.e., dynamic STM and ASM. Interestingly, rut-dependent cAMP is also required for long-term memory (LTM), which acquires after spaced and repeated training sessions. Downstream the signaling cascade, however, appropriate cAMP signals are differently channeled to either support LTM in a CREB-dependent manner, ASM via tomosyn-dependent plasticity, or basal STM via synapsin-dependent regulation of synaptic efficacy. It appears that the chronology of CS/US pairings is an important determinant of which downstream effect is triggered and hence molecular mechanisms must be installed that are sensitive to the temporal order of training (Scheunemann, 2013).

At the level of molecular scaffolds, literature suggests that AKAPs serve the integration of cAMP with other signaling processes and are crucially involved in the control of a plethora of cellular functions in any organ. For example, AKAP79 coordinates cAMP and Ca²⁺ signaling in neurons to control ion channel activities. The recognized design principle of AKAPs to serve as molecular switch is well in line with the recognized two-step register mechanism involved in ASM formation. An increasing body of evidence shows that AKAPs are involved in memory processing across phyla and accordantly those studies revealed a contribution for support of matured, but not immediate memories. Communality among all those AKAP-dependent memories is the need for repeated and temporally organized training sessions, i.e., only spaced training sessions are effective to induce protein synthesis-dependent LTM in flies and mammals. Similarly, ASM requires the precise timing of two training sessions and mechanistically acts via an 'activated' state generated by the initial CS/US pairing that persists within the brain for ~5 min. Such temporal integration might well take place at level of AKAPs within the KC layer to operate rut-AC1-dependent cAMP signals finally onto phosphorylation of tomosyn. Identification of the particular AKAPs involved in two-step ASM formation will require further analysis of appropriate mutants. To date, only four Drosophila AKAPs are characterized, i.e., rugose, a 550 kDa protein that impacts on STM performance probably via molecular domains other than its AKAP function; yu/spoonbill that supports LTM; and Nervy and AKAP²⁰⁰ have not been tested for their impact on aversive odor memory (Scheunemann, 2013).

Together, the benchmarking of Drosophila aversive odor memory performance by means of acquisition dynamics that were demonstrated in this study will provide a valuable tool since dynamic aspects of acquisition are obviously informative and add to the steady-state condition determined by the single-cycle training procedure (Scheunemann, 2013).

Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).

Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).

Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain

DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca²⁺/calmodulin-responsive adenylate cyclase) mutants is rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants is counteracted when a loss-of-function DopEcR mutation is introduced. A memory defect caused by suppressing dopamine synthesis is also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB) - a brain region central to learning and memory in Drosophila - is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. The multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids (Ishimoto, 2013).

Steroid hormones are essential modulators of a broad range of biological processes in a diversity of organisms across phyla. In the adult nervous system, the functions of steroids such as estrogens and glucocorticoids are of particular interest because they have significant effects on the resilience and adaptability of the brain, playing essential roles in endocrine regulation of behavior. Reflecting their importance in neural functions, steroid hormones are implicated in the etiology and pathophysiology of various neurological and psychiatric disorders, and are thus often targeted in therapies. The biological actions of steroids are mediated mainly by nuclear hormone receptors - a unique class of transcription factors that activate or repress target genes in a steroid-dependent manner. Substantial evidence suggests, however, that steroid hormones can also exert biological effects quickly and independently of transcriptional regulation, by modulating intracellular signaling pathways. Such 'non-genomic' effects might be induced by direct allosteric regulation of ion channels, including receptors for GABA and NMDA. Alternatively, in certain contexts, non-genomic steroid signaling could be mediated by classical nuclear hormone receptors acting as effector molecules in the cytosol (Ishimoto, 2013).

G-protein coupled receptors (GPCRs) that directly interact with steroids have the potential to play an important role in non-genomic steroid signaling. So far, however, only few GPCRs have been identified as bona fide steroid receptors in vertebrates. The G-protein coupled estrogen receptor 1 (GPER, formally known as GPR30) is the best studied GPCR that is responsive to steroids. Pharmacological and gene knockout approaches suggest that this protein has widespread roles in the reproductive, nervous, endocrine, immune and cardiovascular systems (Prossnitz, 2011). Although other G-protein coupled receptors were predicted to be responsive to steroids (e.g., the Gq-coupled membrane estrogen receptor and estrogen receptor-X), their molecular identity is not known (Qiu, 2006; ToranAllerand, 2002). Overall, the physiological roles of the GPCR-mediated actions of steroids and the underlying molecular mechanisms remain poorly understood, and sometimes controversial, in spite of their importance. In particular, it is unknown how this non-canonical steroid mechanism influences neural functions and complex behaviors (Ishimoto, 2013).

Drosophila genetics has been extensively used to study the roles and mechanisms of action of steroid hormones in vivo. The major steroid hormone in Drosophila is the molting hormone 20-hydroxy-ecdysone (20E), which orchestrates a wide array of developmental events, including embryogenesis, larval molting and metamorphosis. Recent studies revealed that 20E also plays important roles in adult flies, regulating: the innate immune response, stress resistance, longevity, the formation of long-term courtship memory and the active/resting state. In general, the functions of 20E during development and adulthood are thought to be executed by ecdysone receptors (EcRs), members of the evolutionarily conserved nuclear hormone receptor family (Ishimoto, 2013).

In addition to canonical ecdysone signaling via EcRs, Srivastava (2005) identified a novel GPCR called DopEcR, and showed that it propagates non-genomic ecdysone signaling in vitro. DopEcR shares a high level of amino-acid sequence similarity with vertebrate β-adrenergic receptors. In situ hybridization and microarray data revealed that DopEcR transcripts are preferentially expressed in the nervous system. In heterologous cell culture systems, DopEcR is localized to the plasma membrane and responds to dopamine as well as ecdysteroids (ecdysone and 20E), modulating multiple, intracellular signaling cascades (Srivastava, 2005). Furthermore, Inagaki (2012) recently detected DopEcR expression in the sugar-sensing gustatory neurons of adult flies, and showed that DopEcR-mediated dopaminergic signaling enhances the proboscis extension reflex during starvation. Nonetheless, little is known about whether DopEcR functions as a steroid receptor in vivo, and about how it drives responses in the central nervous system (CNS) to modulate complex behaviors. This study reports that DopEcR mediates non-genomic ecdysone signaling in the adult brain, and that it is critical for memory processing. It was also shown that, during memory processing, DopEcR transmits information via novel steroid signals that interact with the cAMP pathway, a signaling cascade that is universally important for neuronal and behavioral plasticity. This genetic study thus uncovers underappreciated GPCR-mediated functions and mechanisms of action that employ non-canonical steroid signaling to regulate the adult nervous system and, thereby, behavior (Ishimoto, 2013).

This study used genetic, pharmacological, and behavioral approaches in Drosophila to demonstrate that the steroid hormone 20E rapidly regulates behavioral plasticity via a non-genomic mechanism that is mediated by the GPCR-family protein DopEcR. This non-canonical steroid signaling pathway was found to have strong functional interactions with the classical 'memory genes' rut and dnc, which encode the central components of the cAMP pathway. The identification of 20E as an important modulator of cAMP signaling in the adult Drosophila brain reveals an unprecedented opportunity - that of taking advantage of fly genetics to dissect the molecular and cellular mechanisms responsible for the non-genomic steroid signaling that underlies neuronal and behavioral plasticity (Ishimoto, 2013).

Electrophysiological analyses revealed that the adult giant-fiber (GF) pathway of DopEcR mutant flies is more resistant to habituation than that of control flies. Direct excitation of GF or its downstream elements would lead to a short-latency response of the dorsal longitudinal flight muscle (DLM), which could follow high-frequency stimuli up to several hundred Hz. In contrast, the afferent input to the GF leads to a long-latency response that is labile and fails to follow repetitive stimulation well below 100 Hz and displays habituation even at 2-5 Hz. Although there is the possibility that DopEcR-positive thoracic neurons may modulate thoracic motor outputs and contribute to certain parameters of the habituation process not characterized in this study, the more effective modulation would occur in the more labile element afferent to the GF circuit rather than the robust GF-PSI-DLMn downstream pathway (PSI referring to peripherally synapsing interneuron), which is responsible for the reliability of the escape reflex. Thus, the mutant phenotype in habituation indicates that DopEcR positively controls activity-dependent suppression of neuronal circuits afferent to the GF neurons in the brain (Ishimoto, 2013).

Moreover, the finding that DopEcR and rut mutants have a similar GF habituation phenotype raises the possibility that DopEcR positively regulates cAMP levels in the relevant neurons following repetitive brain stimulation. Besides GF habituation, Drosophila displays olfactory habituation, which is mediated by the neural circuit in the antennal lobe. Interestingly, Das (2011) found that olfactory habituation is induced by enhancement of inhibitory GABAergic transmission, and that rut function is required for this neuronal modulation. Similar modulation of GABAergic transmission may also be responsible for habituation of the GF pathway. It will be interesting to examine whether and how DopEcR contributes to the regulation of rut and enhanced GABAergic transmission in GF habituation (Ishimoto, 2013).

Several studies already suggested that 20E has rapid, EcR-independent effects in Drosophila and other invertebrate species. For example, 20E was shown to reduce the amplitude of excitatory junction potentials at the dissected Drosophila larval neuromuscular junction (NMJ), and to do so within minutes of direct application (Ruffner, 1999). Whereas treatment with 20E did not change the size and shape of the synaptic currents generated by spontaneous release, it led to a reduction in the number of synaptic vesicles released by the motor nerve terminals following electrical stimulation. A similar effect of 20E was observed in crayfish, and it was suggested that the suppression of synaptic transmission by 20E may account for the quiescent behavior of molting insects and crustaceans. These observations suggested that 20E suppresses synaptic efficacy under certain conditions by modulating presynaptic physiology through a non-genomic mechanism. It is possible that such actions of 20E are mediated by DopEcR. To detail the mechanisms underlying DopEcR-dependent neural plasticity, it will be worthwhile to determine if and how DopEcR contributes to 20E-induced, rapid synaptic suppression at the physiologically accessible larval NMJ, and to determine the extent to which non-genomic mechanisms of steroid actions are shared between the larval NMJ and the adult brain (Ishimoto, 2013).

One surprising finding made in this study is that ecdysone signaling can modify the phenotypes associated with mutations in the classic 'memory genes', namely rut and dnc, through the actions of DopEcR. rut and dnc encode central components of the cAMP pathway, which is required for memory processing in vertebrates as well as invertebrates. The demonstration that genetically and/or pharmacologically enhancing DopEcR-mediated ecdysone signaling restores the courtship memory phenotype of loss-of-function rut mutants suggests that 20E-mediated DopEcR activation triggers an outcome similar to rut activation, i.e., increased cAMP levels. This assumption is supported by the finding that loss-of-function dnc mutants restore courtship memory when DopEcR activity is suppressed. A similar restoration of the dnc memory phenotype also occurs in a dnc and rut double mutant, again supporting the idea that DopEcR positively regulates cAMP production (Ishimoto, 2013).

The results of rescue and phenocopy experiments indicate that the MB is critical for the DopEcR-dependent processing of courtship memory. Although the endogenous expression pattern of DopEcR is not known, DopEcR is thus likely to modulate cAMP levels in the MB in response to 20E during courtship conditioning. A new Gal4 line has been generated in which a portion of the first coding exon of DopEcR is replaced with a DNA element that contains the Gal4 cDNA whose translation initiation codon is positioned exactly at the DopEcR translation start site. When this line was used to drive UAS-GFP, the reporter gene was widely expressed in the adult brain with prominent signals in the MB. This preliminary result strongly indicates the endogenous expression of DopEcR in the MB. It has also been directly shown that cAMP levels in the MB increase rapidly in flies fed 20E, and that this increase does not occur when DopEcR expression is down-regulated specifically in the MB. Taken together, these findings suggest that DopEcR expressed in the MB responds to 20E and acts upstream of cAMP signaling in a cell-autonomous manner (Ishimoto, 2013).

Surprisingly, enhancement of DopEcR-mediated ecdysone signaling restored courtship memory in flies harboring a strong hypomorphic allele of rut (rut¹⁰⁸⁴). A similar result was obtained even in mutants harboring a presumptive rut null allele rut¹. These results suggest that, upon stimulation by 20E, DopEcR may be able to signal via another adenylyl cyclase that can compensate for the lack of Rut. This interesting possibility requires further investigation (Ishimoto, 2013).

This study has focused on the roles and mechanisms of action of DopEcR-mediated, non-genomic ecdysone signaling. Since it has been found that 20E levels rise in the head during courtship conditioning (Ishimoto, 2009), the data presented in this study suggest that DopEcR is activated by 20E during conditioning, triggers a rise in cAMP levels and induces physiological changes that subsequently suppress courtship behavior. This interpretation assumes that 20E directly activates DopEcR to increase cAMP levels. Previous cell-culture studies suggested that DopEcR also responds to dopamine to modulate intracellular signaling (Srivastava, 2005). Furthermore, Inagaki (2012) has demonstrated that flies respond to starvation by sensitizing gustatory receptor neurons to sugar via dopamine/DopEcR signaling. It is therefore necessary to consider whether dopamine is directly involved in the processing of courtship memory through DopEcR. There is a possibility that 20E initially stimulates the production and/or release of dopamine, and that it in turn activates DopEcR and elevates cAMP levels to induce courtship memory. This possibility is thought unlikely because even when courtship memory is disrupted by pharmacological suppression of dopamine synthesis, 20E feeding can compensate for decreased dopamine and allow restoration of memory. Although dopamine plays a significant role in courtship memory, the results suggest that DopEcR does not act as the major dopamine receptor in this particular learning paradigm. The possibility is thus favored that dopamine contributes to courtship memory in parallel with, or upstream of, DopEcR-mediated ecdysone signaling. Consistent with this view, Keleman (2012) reported that the formation of courtship memory depends on the MB γ neurons, which express DopR1 dopamine receptors, receiving dopaminergic inputs. Notably, the current results indicate that the processing of courtship memory requires DopEcR expression in the αβ, but not γ, neurons of the MB, which makes it unlikely that DopEcR is directly influenced by the dopaminergic neurons innervating γ neurons (Ishimoto, 2013).

Ecdysone signaling through nuclear EcRs is necessary for forming long-term courtship memory that lasts at least 5 days, but appears not to have a significant effect on short-term courtship memory (Ishimoto, 2009). In contrast, we found that DopEcR-mediated ecdysone signaling is critical for habituation and 30-minute courtship memory. These findings suggest that DopEcR and EcRs control distinct physiological responses to courtship conditioning, and that the former regulates short-term memory, while the latter regulates long-term memory. Although non-genomic actions of steroid hormones have been implicated in vertebrate learning and memory, such actions have been attributed mainly to the classical nuclear hormone receptors that function outside of the nucleus and exert roles distinct from those of steroid-activated transcription factors. Although recent evidence has shown that membrane-bound receptors independent of the classical estrogen receptors are involved in estradiol-induced consolidation of hippocampal memory, the molecular identities of these proteins have not been established. The current findings in this study provide a novel framework for dissecting GPCR-mediated steroid signaling at the molecular and cellular levels. Furthermore, future analysis of the functional interplay between genomic and non-genomic steroid signaling pathways is expected to reveal novel mechanisms through which steroid hormones regulate plasticity of the nervous system and other biological phenomena (Ishimoto, 2013).

Metabolic learning and memory formation by the brain influence systemic metabolic homeostasis

Metabolic homeostasis is regulated by the brain, but whether this regulation involves learning and memory of metabolic information remains unexplored. This study use a calorie-based, taste-independent learning/memory paradigm to show that Drosophila form metabolic memories that help in balancing food choice with caloric intake; however, this metabolic learning or memory is lost under chronic high-calorie feeding. Loss of individual learning/memory-regulating genes causes a metabolic learning defect, leading to elevated trehalose and lipid levels. Importantly, this function of metabolic learning requires not only the mushroom body but also the hypothalamus-like pars intercerebralis, while NF-κB activation in the pars intercerebralis mimics chronic overnutrition in that it causes metabolic learning impairment and disorders. Finally, this study evaluated this concept of metabolic learning/memory in mice, suggesting that the hypothalamus is involved in a form of nutritional learning and memory, which is critical for determining resistance or susceptibility to obesity. In conclusion, these data indicate that the brain, and potentially the hypothalamus, direct metabolic learning and the formation of memories, which contribute to the control of systemic metabolic homeostasis (Zhang, 2015).

This work consists of a series of studies in Drosophila: these animals were found to temporarily develop metabolic learning to balance food choice with caloric intake. In Drosophila research, sugar has often been used for studying the appetitive reward value of food taste. Of interest, recent research has suggested that fruit flies can distinguish caloric values from the taste property of food. Using tasteless sorbitol as a carbohydrate source to generate an environmental condition that contained NC versus HC food, this study revealed that Drosophila can develop a form of metabolic learning and memory independently of taste, by which flies are guided to have a preference for normal caloric environment rather than high-caloric environment. However, this form of metabolic memory does not seem robust, as it is vulnerably diminished under genetic or environmental influences. It is postulated that this vulnerability to overnutrition is particularly prominent for mammals (such as C57BL/6J mice), and overnutritional reward-induced excess in caloric intake can quickly become dominant. This effect can be consistently induced in Drosophila when learning/memory-regulating genes are inhibited in the brain or the hypothalamus-like PI region. It was observed that each of these genetic disruptions led to impaired metabolic learning, resulting in increased caloric intake and, on a chronic basis, the development of lipid excess and diabetes-like phenotype. Indeed, it has been documented that chronic high-sugar feeding is sufficient to cause insulin resistance, obesity and diabetes in Drosophila. It is yet unclear whether this metabolic learning can induce an appetitive memory of normal caloric environment or an aversive memory of high-caloric environment. Regardless, the findings in this work have provoked a stimulating question, that is, whether this form of metabolic learning and memory is present in the mammals and, if so, whether this mechanism can be consolidated to improve the control of metabolic physiology and prevent against diseases. These mouse studies may provide an initial support to this concept and strategy, but clearly, in-depth future research is much needed (Zhang, 2015).

In light of the underlying neural basis for this metabolic learning, this study indicates that multiple brain regions are required, including the hypothalamus-like PI region in addition to the MB (equivalent to the hippocampus in mammals), which is classically needed for learning and memory formation. Anatomically, the PI region is located in the unpaired anteromedial domain of the protocerebral cortex, which is near the calyces of the MB and the dorsal part of the central complex (another brain region for regulating learning and memory). Functionally, the PI region has been demonstrated to coordinate with the MB in regulating various physiological activities in Drosophila. Thus, it is very possible that some PI neurons present nutritional information to the MB and thus induce metabolic learning and memory formation. However, the underlying detailed mechanism is still unknown, especially if this process involves a role of dilps, which represent the prototypical neuropeptides produced by the PI neurons. Considering that the PI region in flies is equivalent to the mammalian hypothalamus, this study was extended to mouse models by comparatively analysing A/J versus C57BL/6J mice—which are known to have different diet preference as well as different susceptibilities to obesity development. While A/J mice showed a learning process of distinguishing NC versus HC food, C57BL/6J mice failed to do so. It is particularly notable that this difference of learning and memory between these two strains is associated with differential expression profiles of learning/memory genes in the hypothalamus rather than the hippocampus. This finding, in conjunction with the Drosophila study, highlights a potential that the hypothalamus has a unique role in mediating metabolic learning and memory formation. Although the mouse experiments cannot exclude the impacts from the taste/smell properties of the studied food, the results demonstrated that there is a form of nutritional memory, which seems dissociable from the memory of overnutritional reward. These initial observations in mice lend an agreement with findings in Drosophila, suggesting that the brain and potentially the hypothalamus can link nutritional environment to a form of metabolic learning and memory homeostasis (Zhang, 2015).

From a disease perspective, metabolic learning in Drosophila is impaired under chronic overnutrition, and the mouse study was in line with this understanding. This response to overnutrition is useful when famine is outstanding; however, it is a dilemma when metabolic disease is of concern, much like the scenario pertaining to leptin resistance under chronic overnutrition, whereas an increase in leptin sensitivity is demanded to reduce obesity. Recently, it was established that NF-κB-dependent hypothalamic inflammation links chronic overnutrition to the central dysregulation of metabolic balance. This study showed that activation of the NF-κB pathway in the PI region weakened the function of metabolic learning and, conversely, NF-κB inhibition in this region provided a protective effect against chronic overnutrition-impaired metabolic learning. These findings are in alignment with the literature, for example, pan-neuronal NF-κB inhibition was shown to improve activity-dependent synaptic signalling and cognitive function including learning and memory formation, and persistent NF-κB activation inhibits neuronal survival and the function of learning and memory formation. Hence, overnutrition-induced neural NF-κB activation has a negative impact on metabolic learning and memory formation in regulating metabolic homeostasis homeostasis (Zhang, 2015).

To summarize the findings in this work, a series of behavioural studies was performed revealing that Drosophila have a form of metabolic learning and memory, through which the flies are directed to balance food choice with caloric intake in relevant environments. Several learning/memory-regulating genes including rut, dnc and tequila are involved in this function, and brain regions including the PI in addition to the MB are required to induce this mechanism. On the other hand, metabolic learning is impaired under chronic overnutrition through NF-κB activation, leading to excess exposure to calorie-enriched environment, which causes metabolic disorders. Overall, metabolic learning and memory formation by the brain and potentially the hypothalamus play a role in controlling metabolic homeostasis homeostasis (Zhang, 2015).

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