InteractiveFly: GeneBrief

radish : Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Long-term memory in Drosophila is separable into two components: consolidated, anesthesia-resistant memory and long-lasting, protein-synthesis-dependent memory. The Drosophila memory mutant radish is specifically deficient in anesthesia-resistant memory and so represents the only molecular avenue to understanding this memory component. The radish gene was identified by positional cloning and comparative sequencing, a mutant stop codon was found in in gene CG15720 from the Drosophila Genome Project. Induction of a wild-type CG15720 transgene in adult flies acutely rescues the mutant's memory defect. The phospholipase A2 gene, previously identified as radish [Chiang, 2004), maps 95 kb outside the behaviorally determined deletion interval and is unlikely to be radish. The Radish protein is highly expressed in the mushroom bodies, centers of olfactory memory. It encodes a protein with 23 predicted cyclic-AMP-dependent protein kinase (PKA) phosphorylation sequences. The Radish protein has recently been reported to bind to Rac1 (Formstecher, 2005), a small GTPase that regulates cytoskeletal rearrangement and influences neuronal and synaptic morphology (Folkers, 2006).

The specific defect of the radish mutant, diminished ARM, normal long-lasting protein-synthesis-dependent memory, provides a key to obtaining mechanistic information about ARM. The radish mutant was isolated in a screen for learning-defective flies after chemical mutagenesis. Genetic recombination and deletion mapping of the memory defect placed the gene in a 140-kb stretch in region 11D of the X chromosome. a transcript analysis and information from the Drosophila genome project indicated 17 genes in this interval. 105 kb of the genomic 11D region DNA was PCR-amplified from radish flies and from the parental wild-type Canton-S (C-S) flies, the products were sequenced, and the sequenceswere compared. The sequences analyzed included the ORFs of all identified genes in or near the interval. In this comparison, only a 1-nt difference was detected between mutant and parent strains, a C-to-T transition in the ORF of the CG15720 predicted gene. This mutation converts a glutamine codon in the wild-type ORF into an amber stop codon in the mutant radish DNA (Folkers, 2006).

Previous mapping of the memory phenotype revealed that the radish mutation is uncovered by the Df (1)105 X-chromosome deficiency (Folkers, 1993). However, the CG15720 ORF lies proximal of the Df (1)105 breakpoint, but close enough so that the deletion could plausibly eliminate its transcription. To verify that this was the case, RT-PCR was used to isolate the CG15720 transcript from C-S/Df (1)105, radish/Df (1)105, and radish/C-S flies. The region of the mutation was further amplified and the products were sequenced. The results were all consistent with Df (1)105 cis-acting elimination of radish gene transcription, C-S/Df (1)105 gave only wild-type sequence; radish/Df (1)105 gave only mutant sequence; radish/C-S gave 50:50 wild-type and mutant sequence (Folkers, 2006).

The CG15720 gene is expressed in the fly head. Northern blot analysis revealed a single 6-kb transcript. This transcript was amplified by RT-PCR, its ORF was sequenced, and it was found to span five exons. The radish mutation is at the 3' end of the fourth exon, which should truncate the protein in radish mutant flies. The inferred mutant protein lacks the C-terminal 64 aa (Folkers, 2006).

To ascertain whether CG15720 is, in fact, the radish gene, wild-type CG15720 gene expression was reestablished in radish mutant flies and ARM was assayed. The wild-type CG15720 ORF DNA was subcloned into the germ-line transformation vector hs-CaSpeR under the control of the inducible promoter for heat-shock protein 70. The hs-CG15720 construct was injected into mutant radish embryos and two resulting transformant flies were bred into homozygous populations (hs-rsh-1, hs-rsh-2). The hs-CG15720 transgene was induced with a 25-min heat shock (37°C), transgene expression required a wait of 1h, and the flies were trained in a odor-discrimination paradigm. Two hours later, the flies were anesthetized by cooling on ice for 2 min, they were allowed to recover, and memory was tested 1 h thereafter (Folkers, 2006).

Expression of the hs-CG15720 transgene in mutant radish flies restored normal ARM. Without heat-shock induction of the hs-CG15720 transgene, the flies remembered as poorly as mutant radish flies. Western blot analysis indicated that the transgene was expressed after heat shock and not in its absence. (Transgenic rescue of memory was also obtained without anesthesia). These results strongly indicate that the CG15720 gene is radish (Folkers, 2006).

Induction of this transgene in radish⁺ flies elicited no additional ARM, indicating that this transgene specifically rescues the radish memory defect. When ARM was assayed after heat shock, rsh⁺ scores were 0.15 ± 0.05; scores for transformed w, rsh⁺[hs-rsh-1] flies were 0.15 ± 0.03. Without heat-shock treatment, scores for w, rsh⁺ were 0.18 ± 0.04; scores for transformed w, rsh⁺[hs-rsh-1] flies were 0.19 ± 0.04 (Folkers, 2006).

Chiang (2004) have reported that the radish gene encodes a phospholipase A2 gene (PLA2) with the designation CG4346. However, this gene was found to be far outside the deletion, Df(1)N105, that defines the proximal end of the radish interval. PCR-amplification of Df(1)N105 DNA indicates that the proximal breakpoint of this deletion is 95 kb distal of the PLA2 gene. Recently Dubnau and colleagues have done additional experiments, including repeating their complementation crosses with reciprocal parental genotypes. Their recent results indicate that PLA2 and radish are different genes (J. Dubnau, personal communication to Folkers, 2006) (Folkers, 2006).

It is concluded that the Radish protein functions acutely in the adult fly to engender ARM, because its expression 1 h before training is sufficient to rescue memory in mutant radish flies. PKA-dependent phosphorylation of the Radish protein, as suggested by numerous PKA target sites, would logically link ARM to the cAMP pathway that functions in short-term memory in Drosophila. The idea is inconsistent with the findings that rutabaga flies have substantial ARM, although the reduction in cAMP levels caused by the mutation is partial (Folkers, 2006).

The high Arg/Ser content of the inferred amino acid sequence and the observed homology to Arg/Ser-rich splicing factors both suggest a role in RNA processing for the Radish protein. This notion appears to be contradicted by the finding that the translation inhibitor cycloheximide does not affect ARM. However, protein synthesis in these experiments was reduced by only 50% (Folkers, 2006).

Finding the Radish protein in the MBs is consistent with the known importance of the MBs in olfactory memory, the expression of many other memory-relevant genes (including cAMP cascade components) there, and the observation that blocking MB output abolishes ARM. Staining in the MB calyx and lobes indicates the presence of the Radish protein in neurites. Nuclear localization is not apparent, in current immunostains, but it may be present under other conditions, e.g., on activation of PKA (Folkers, 2006).

Long-term memory in Aplysia and mice is correlated with changes in synaptic morphology. One last property of the Radish protein argues for its role in synaptic morphology. Formstecher (2005) carried out an extensive high-throughput yeast two-hybrid screen for interacting Drosophila gene products using 102 proteins as bait. Intriguingly, this analysis identified an interaction between CG15720 (Radish) and Rac1. Rac1 is a small GTPase of the Rho family that regulates cytoskeletal assembly to influence neuronal and synaptic morphology in Drosophila and mammals. Furthermore, Rac1 lies in the same cell-signaling pathways as Pak1 and fragile-X protein (FMRP), both of which have documented effects on synaptic and behavioral plasticity. Strikingly, amino acids 538–579 of the Radish protein are sufficient for its binding to Rac1, and these residues are all deleted in the mutant Radish protein. Therefore, it is plausible that the critical function of Radish in ARM is to change synaptic morphology through Rac1 interaction (Folkers, 2006).

Additive expression of consolidated memory through Drosophila mushroom body subsets

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α'β', and γ neurons. It has been previously demonstrated that two parallel pathways mediate ARM consolidation: the serotonergic dorsal paired medial (DPM)-αβ neurons and the octopaminergic anterior paired lateral (APL)-α'β' neurons. This study shows that blocking the output of αβ neurons and that of α'β' neurons each impairs ARM retrieval, and blocking both simultaneously has an additive effect. Knockdown of radish and octβ2R in αβ and α'β' neurons, respectively, impairs ARM. A combinatorial assay of radish mutant background rsh¹ and neurotransmission blockade confirms that ARM retrieved from α'β' neuron output is independent of radish. The MB output neurons MBON-β2β'2a and MBON-β'2mp were identified as the MB output neurons downstream of αβ and α'β' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, α'β' neurons can be functionally subdivided into α'β'm neurons required for ARM retrieval, and α'β'ap neurons required for ARM consolidation. These data demonstrate that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval (Yang, 2016).

The key finding in this study is the identification of two parallel neural pathways that additively express 3-h aversive ARM through Drosophila MB αβ and α'β' neurons. After training, Radish in MB αβ neurons and octopamine signaling in α'β' neurons independently consolidate ARM, which is additively retrieved by αβ-MBON-β2β'2a and α'β'm-MBON-β'2mp circuits for memory expression. Five lines of evidence support this scenario. First, the output from αβ or α'β' neurons is required for ARM retrieval, and the effect of blocking αβ output and that of blocking α'β' output during retrieval are additive. Second, knockdown of radish in αβ neurons, but not in α'β' neurons, impaired ARM, while knockdown of octβ2R in α'β' neurons further impaired the residual ARM in rsh¹ mutant flies. Third, blocking output from α'β' neurons, but not from αβ neurons, during retrieval further impaired the residual ARM in rsh¹ mutant flies. Forth, glutamatergic output from neurons downstream of the αβ or α'β' neurons, i.e., MBON-β2β'2a or MBON-β'2mp neurons, is required for ARM retrieval, and the effects of knockdown of VGlut are additive. Finally, output from α'β'm neurons, but not α'β'ap neurons, is required for ARM retrieval, consistent with the dendritic distribution of MBON-β'2mp neurons (Yang, 2016).

The parallel pathways for 3-h ARM expression were spatially defined by the requirements of neurotransmission from two sets of circuits during retrieval, the αβ-MBON-β2β'2a neurons and the α'β'm-MBON-β'2mp neurons. In addition, blocking neurotransmission from αβ or α'β' neurons during retrieval reduced ARM expression by about 50% whereas simultaneous blockade produced an additive effect that completely abolished ARM expression. Similar additive effects were repeatedly observed in experiments that utilize manipulations in both pathways: an rsh¹ mutant background plus octβ2R RNAi knockdown or plus retrieval blockade in α'β' neurons and knockdown of VGlut in MBON-β2β'2a plus MBON-β'2mp neurons. Thus, total four lines of evidence support the additive expression of 3-h ARM (Yang, 2016).

The parallel pathways for 3-h ARM expression shown in this study differ from the degenerate parallel pathways for the stomatogastric ganglion of the crab or CO₂ avoidance in the fly, as the latter enable mechanisms by which the network output can be switched between states. In the current study, the two parallel neural pathways additively contribute to the expression of 3-h ARM. The nature of the ARM parallel pathways may be similar to that for cold avoidance behavior in the fly, where parallel pathways in the β' and β circuits additively contribute but only the β circuit allows age-dependent alterations for potential benefits against aging (Shih, 2015). Considering the robustness of ARM through the course of senescence, it's unlikely to be age-dependent alterations in ARM system (Yang, 2016).

In studies of Drosophila neurobiology, C305a-GAL4 is a common GAL4 line for α'β' neurons. In this study, by examining three different zoom-in sections of the MB lobes and counting the cells, the following GAL4 lines expressing in α'β' neurons were extensively characterized: VT30604-GAL4 and VT57244-GAL4, which cover most α'β'ap and α'β'm neurons; VT37861-GAL4 and VT50658-GAL4, which cover α'β'ap neurons; and R42D07-GAL4 and R26E01-GAL4, which cover most α'β'm neurons. In contrast, C305a-GAL4 sporadically expresses in about half as many MB neurons as VT30604-GAL4 or VT57244-GAL4 does. Although covering both subsets of α'β' neurons, the expression pattern of C305a-GAL4 in α'β'm neurons is too few and/or weak to lead to a perturbation of synaptic transmission. This is shown by the data that retrieval of 3-h ARM was disrupted by shibire manipulation using all-α'β' neurons driver or α'β'm-specific driver, but neither α'β'ap-specific driver nor C305a-GAL4 for 3-h memory. Note that the GFP signals were acquired from flies carrying two copies of 5XUAS-mCD8::GFP reporter and without any immunostaining-mediated amplification. With the assistance of immunostaining and/or advanced reporter such as increasing copy number of UAS or incorporating a small intron to boost expression, some studies have shown appreciable GFP signal in most α'β' neurons. Given that shibire-mediated neurotransmission blockade and RNAi-mediated knockdown require high enough expression level, the imaging method adopted in this study can faithfully reflect the regions that were effectively manipulated in these behavioral assays. Regarding the pervasive use of C305a-GAL4 for shibire or RNAi manipulation, some functional studies of α'β' neurons might need to be carefully revisited. This study showed, by close examination and cell counting, that VT30604-GAL4, VT37861-GAL4, and R42D07-GAL4 are useful GAL4 lines to study α'β', α'β'ap, and α'β'm neurons, respectively, especially when split-GAL4 lines that span the second and third chromosomes are not genetically feasible (Shih, 2015).

ARM was thought to be diminished in radish mutant flies, in which a truncated RADISH is expressed. It's noteworthy that radish mutants still show a residual 3-h ARM with a PI of roughly 10, which is equal to the 3-h ARM score in wild-type flies fed with an inhibitor of serotonin synthesis to hinder the serotonergic DPM neurotransmission. Interestingly, feeding radish mutant flies with the drug didn't make the 3-h memory score worse, which has already implied that RADISH mediates the consolidation of ARM in the serotonergic DPM-αβ neurons circuit. Indeed, in this study advantage was taken of RNAi-mediated knockdown to identify αβ neurons with RADISH-mediated ARM consolidation. However, only the output from αβs neurons among three subsets of αβ neurons is required for aversive memory retrieval. Whether the αβs neurons are the only aversive ARM substrate of RADISH remains to be identified (Yang, 2016).

APL and DPM neurons are two pairs of modulatory neurons broadly innervating the ipsilateral MB, although the DPM neuron's fiber is lacking in the posterior part of pedunculus and the calyx. Broad, extensive fiber and non-spiking feature allow these two pairs of neurons to have multiple functional roles through different types of neurotransmission. The APL neuron has been shown to receive odor information from the MB neurons and provide GABAergic feedback inhibition as the Drosophila equivalent of a group of the honeybee GABAergic feedback neurons. This feedback inhibition has been proposed to maintain sparse, decorrelated odor coding by suppressing the neuronal activity of MB neurons, which can be somewhat linked to the mutual suppression relation with conditioned odor and the facilitation of reversal learning. Interestingly, Pitman (2011) proposed that the feedback inhibition from APL neurons sustains the labile appetitive ASM based on shibire manipulation. Since shibire manipulation can impact small vesicle release, and APL neurons have been demonstrated to co-release at least GABA and octopamine, it might worth conducting GABA-specific manipulation in APL neurons to confirm the role in appetitive ASM. For aversive olfactory memory, acute RNAi-mediated knockdown of Glutamic acid decarboxylase in APL neurons had no effect on 3-h memory. Instead, the octopamine synthesis enzyme mutant, Tβh^nM18, knockdown of Tβh in APL neurons, the octopamine receptor mutant, PBac{WH}octβ2R^f05679, and knockdown of octβ2R in α'β' neurons all phenocopied the 3-h ARM impairment caused by shibire-mediated neurotransmission blockade in APL neurons. Together with the serotonergic DPM-αβ neurons circuit , a model that is favored that two sets of triple-layered parallel circuits, octopaminergic APL-α'β'-MBON-β'2mp and serotonergic DPM-αβ-MBON-β2β'2a, additively contribute to 3-h aversive ARM (Yang, 2016).

Although the data showed that 3-h ARM consolidation requires recurrent output from α'β'ap neurons but not from α'β'm neurons, RNAi-mediated knockdown of octβ2R in α'β'ap or α'β'm neurons impaired ARM, suggesting that Octβ2R functions for normal ARM expression in the entire population of α'β' neurons. On the other hand, neuronal activity during memory consolidation is naturally more quiescent than that during memory retrieval, and the shibire-mediated neurotransmission blockade requires an exhaustion of already-docked vesicles. Together with the unfavorable performance for experiments blocking the output from α'β'm neurons during consolidation, the possibility cannot be excluded that output from α'β'm neurons is also required for ARM during consolidation. Alternatively, octopamine signaling may also be involved in ARM retrieval (Yang, 2016).

Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca²⁺ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn⁹⁷ mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn⁹⁷ mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn⁹⁷ mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut¹, rut²⁰⁸⁰ and dnc¹ mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

DEVELOPMENTAL BIOLOGY

Adult

To localize the Radish protein in the fly brain a polyclonal rabbit antibody was raised to a synthetic peptide was used corresponding to amino acids 522–542 of the inferred Radish protein. (This region is deleted in the Radish mutant protein.) This antibody was used to immunostain wild-type and radish mutant brains. Radish immunoreactivity was evident throughout the neuropil of wild-type brains and was particularly strong in the calyx, peduncle, and lobes of the mushroom bodies (MBs) and ellipsoid body of the central complex. Critically, this staining was absent in brains of radish mutant flies (Folkers, 2006).

EFFECTS OF MUTATION

The Drosophila mutant radish has been characterized. Initial learning of radish flies in two olfactory discrimination tests is high, but subsequent memory decays rapidly at both early and late times after training. Anesthesia-resistant memory (consolidated memory) is undetectable in radish flies 3 hr after training. The mutant shows normal locomotor activity and normal sensitivity to the odor cues and electric-shock reinforcement used in the learning tests. The radish gene maps within a 180-kb interval in the 11D-E region of the X chromosome (Folkers, 1993; full text of article).

Behavioral and pharmacological experiments in many animal species have suggested that memory is consolidated from an initial, disruptable form into a long-lasting, stable form within a few hours after training. These traditional approaches have been combined with genetic analyses in Drosophila to show that consolidated memory of conditioned (learned) odor avoidance 1 day after extended training consists of two genetically distinct, functionally independent memory components: anesthesia-resistant memory (ARM) and long-term memory (LTM). ARM decays within 4 days, is resistant to hypothermic disruption, is insensitive to the protein synthesis inhibitor cycloheximide (CXM), and is disrupted by the radish single-gene mutation. LTM showed no appreciable decay over 7 days, was sensitive to CXM, and was not disrupted by the radish mutation (Tully, 1994).

Learning and memory in the Drosophila mutants dunce, amnesiac and radish, that were isolated originally from the classical olfactory learning paradigm, were analyzed in an operant visual learning paradigm. Dunce appears to show normal ability to learn during training, but its memory is significantly affected. Though the learning index during the first minute after training is normal, its short-term memory (STM), anesthesia-resistant memory (ARM) and long-term memory (LTM) are all significantly damaged. Amnesiac displays disrupted middle-term memory (MTM), while its STM and LTM remain unchanged. Learning and memory in radish mutants seem to be unaffected. These results lend support to the argument that there are certain common molecular mechanisms underlying learning and memory through different tasks and the previous multi-phase model of visual memory is modified in a genetic way (Gong, 1998).

Memory enhancement and formation by atypical PKM activity in Drosophila

Synaptic stimulation activates signal transduction pathways, producing persistently active protein kinases. PKMzeta is a truncated, persistently active isoform of atypical protein kinase C-zeta (aPKCzeta), which lacks the N-terminal pseudosubstrate regulatory domain. Using a Pavlovian olfactory learning task in Drosophila, it was found that induction of the mouse aPKMzeta (MaPKMzeta) transgene enhances memory. The enhancement requires persistent kinase activity and is temporally specific, with optimal induction at 30 minutes after training. Induction also enhances memory after massed training and corrects the memory defect of radish mutants, but does not improve memory produced by spaced training. The 'M' isoform of the Drosophila homolog of MaPKCzeta (DaPKM) is present and active in fly heads. Chelerythrine, an inhibitor of PKMzeta, and the induction of a dominant-negative MaPKMzeta transgene inhibits memory without affecting learning. Finally, induction of DaPKM after training also enhances memory. These results show that atypical PKM is sufficient to enhance memory in Drosophila and suggest that it is necessary for normal memory maintenance (Drier, 2002).

The study of PKC in memory formation has a long history. However, most previous work was done before the current appreciation of the complexity of the PKC gene family. The PKC family can be divided into three classes based on their cofactor requirements. Whereas all PKC proteins require phosphatidylserine for activation, the 'conventional' (cPKC) isotypes require diacylglycerol (DAG) and Ca²⁺ for full activity; 'novel' (nPKC) isotypes are Ca²⁺ independent but still require DAG, and the 'atypical' (aPKC) isotypes are both DAG and Ca²⁺ independent. Structurally, these kinases can be divided into an N-terminal regulatory domain, which contains a pseudosubstrate region as well as the binding sites for the required cofactors, and the C-terminal catalytic domain. Removal of the N-terminal regulatory domain produces a persistently active kinase, referred to as PKM. Persistently active kinases have received attention as components of memory mechanisms (Drier, 2002).

The roles of PKC in hippocampal models of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) have been studied extensively. PKC/M activities may have several roles in the mechanisms that initiate and sustain LTP. However, Western blot analyses with antibodies specific for each of the rat PKC isoforms demonstrate that the only one whose levels specifically increase and remain elevated during the maintenance phase of LTP is PKMzeta, which is the truncated form of the atypical isozyme PKCzeta. Expression analyses also show that the maintenance of LTD is associated with decreasing levels of PKMzeta. Most interestingly, LTP maintenance is abolished by sustained application of low concentrations of the PKC inhibitor chelerythrine, whereas perfusion of PKMzeta into CA1 pyramidal cells produces an increase in AMPA receptor-mediated synaptic transmission (Drier, 2002 and references therein).

Experiments in honeybees also indicate a role for PKC in memory formation. Biochemical analyses of extracts made from the antennal lobes of associatively trained bees show a sustained increase in cytosolic, Ca²⁺-independent PKC activity. This persistent increase correlates with long-lasting bee memory in four ways: it requires multiple training trials; it persists for up to three days; it is insensitive to a drug that blocks cPKC activity, and it is blocked by protein-synthesis inhibitors. Together with the LTP data, these studies point to an important role for a nonconventional PKC activity in the maintenance of memory (Drier, 2002).

In Drosophila, the best characterized assay for associative learning and memory is an odor-avoidance behavioral task. This classical (Pavlovian) conditioning involves exposing the flies to two odors (the conditioned stimuli, or CS), one at a time, in succession. During one of these odor exposures (the CS⁺), the flies are simultaneously subjected to electric shock (the unconditioned stimulus, or US), whereas exposure to the other odor (the CS^-) lacks this negative reinforcement. After training, the flies are placed at a 'choice point', where the odors come from opposite directions, and they decide which odor to avoid. By convention, learning is defined as the fly's performance when testing occurs immediately after training. A single training trial produces strong learning: a typical response is that >90% of the flies avoid the CS⁺. Performance of wild-type flies from this single-cycle training decays over a roughly 24-hour period until flies once again distribute evenly between the two odors. Flies can also form long-lasting associative olfactory memories, but normally this requires repetitive training regimens (Drier, 2002).

This task was used in Drosophila to examine the role of atypical PKM in memory formation. Induction of the mouse aPKMzeta (MaPKMzeta) transgene enhances memory, and corrects the memory defect of radish mutants. There is a single atypical PKC in Drosophila, and the truncated 'M' isoform, DaPKM, is preferentially expressed and active in fly heads. Both pharmacological and dominant-negative genetic intervention of DaPKC/M activity disrupts normal memory. Finally, induction of the predicted DaPKM also enhances memory, further suggesting a general role of aPKM in memory processes (Drier, 2002).

To investigate the role of PKC in learning and memory in Drosophila, transgenic lines of flies were made bearing heat shock-inducible, murine atypical PKC (MaPKC) isoforms. Considering that LTP experiments indicate that MaPKMzeta levels increase after the presentation of the stimuli required for long-lasting potentiation, whether inducing MaPKMzeta after training affected olfactory memory was tesed. Induction by mild heat shock (32°C) after training strongly enhances 24-hour memory. This enhancement is not due to transgene-independent heat-shock effects, because the wild-type flies show no enhanced memory when exposed to heat shock. The transgenic flies were made in this wild-type strain, so the enhancement is not due to differences in genetic background. Finally, the memory enhancement does not result from an insertional mutation caused by the transgene, because two independent lines (MaPKMzeta-14 and MaPKMzeta-43) have similar effects (Drier, 2002).

Whether 24-hour memory could be enhanced after single-cycle training was tested by inducing MaPKMzeta with a strong heat shock (37°C) 3 hours before training, but this regimen has no effect. Because transgene induction after behavioral training enhances memory, whereas induction before training does not, the temporal specificity of this MaPKMzeta-dependent effect was examined. Optimal enhancement occurs when heat-shock induction begins 30 minutes after training ends, and the effect is absent if heat shock occurs before, or is delayed until 2 hours after training (Drier, 2002).

The memory enhancement is not observed when a kinase-inactive (KI) mutant of MaPKMzeta is induced either before or after training. The enhancement is also not observed when full-length (FL)-MaPKCzeta is induced before or after training. The failure of either the KI-MaPKMzeta or the FL-MaPKCzeta transgene to enhance memory is not due to lack of expression, because both are expressed at levels comparable to the MaPKMzeta protein. Together, these results indicate that the memory enhancement requires a persistently active aPKM isoform (Drier, 2002).

Inducible increases in MaPKMzeta protein levels and kinase activity have been detected in extracts made from Drosophila heads. Western blot analyses shows that both the mild and strong heat-shock regimens induce the MaPKMzeta and MaPKCzeta isoforms, and that these proteins persisted for ~18 hours after heat shock. The induced MaPKMzeta protein is active, since enhancement of Ca²⁺/DAG-independent PKC activity is observed in fly head extracts from induced but not from uninduced transgenic flies (Drier, 2002).

Drosophila can form associative olfactory memories lasting 24 hours and longer, but this normally requires repetitive training. Multiple-trial training regimens have been established that produce both anesthesia-resistant memory (ARM) and long-term memory (LTM). ARM can be produced by 10 cycles of 'massed' training with no rest intervals between the individual training trials, and lasts 2-3 days. LTM results from repetitive training that contains rest intervals (15 min each), and 10 cycles of this 'spaced' training generates LTM that lasts at least 7 days. To test whether MaPKMzeta can enhance ARM or LTM, flies were subjected to massed or spaced training regimens; the transgene was induced for 30 minutes after training, and then 4-day memory was measured (Drier, 2002).

MaPKMzeta induction substantially increases 4-day memory after massed training but does not improve 4-day memory after spaced training. These data indicate that MaPKMzeta induction enhances massed training-induced, but not spaced training-induced memory (Drier, 2002).

Previous work indicates that consolidated memory in Drosophila consists of two biochemically separable components: ARM and LTM. ARM is produced by either massed or spaced training, and it is insensitive to cycloheximide treatment. LTM is produced by spaced training and is blocked by cycloheximide treatment; thus it is considered to require acute protein synthesis. A previously identified Drosophila memory mutant, radish, is deficient in ARM, since this mutation blocks memory produced by massed training. Spaced training of radish mutants does produce memory, but this memory can be completely blocked by treating the mutants with cycloheximide. These results led to a two-pathway model of consolidated memory, one dependent on the Radish gene product (ARM) and the other dependent on activity-induced, acute protein synthesis (LTM) (Drier, 2002).

Because MaPKMzeta induction enhances memory after massed but not after spaced training, the dependence of this effect on radish was tested. The radish gene is on the X chromosome in Drosophila, and homozygous radish mutant females were crossed to males homozygous for an autosomal copy of the heat shock-inducible MaPKMzeta transgene. The radish mutant is recessive, thus the heterozygous female progeny of this mating will have normal memory after massed training, whereas the hemizygous males will display the radish memory deficit in the absence of induction. The progeny were subjected to massed training, followed by the standard MaPKMzeta induction after training, and then tested at 24 hours. Males and females were trained and tested en masse, and then separated and counted. The radish mutation did not block the memory effect of MaPKMzeta induction. The memory defect of radish males was apparent in the absence of heat-shock induction (HS-), but memory was clearly present in induced males (HS+). A lesser, but significant induction-dependent memory enhancement of the heterozygous radish females by MaPKMzeta was also observed (Drier, 2002).

There is a single atypical PKC (DaPKC) gene in the Drosophila genome, and it is highly homologous to the MaPKCzeta gene that was used. (The kinase domain shows 76% identity and 87% similarity). A Western blot of extracts made from wild-type fly heads and bodies shows that the antiserum used to detect MaPKMzeta and MaPKCzeta recognizes two bands in fly extracts, the smaller of which is enriched in head extracts. This antiserum is directed against the C-terminal 16 amino acids of MaPKC/Mzeta, which shares substantial homology with DaPKC. Antiserum from mice immunized with peptides derived from DaPKC recognizes these same bands. The molecular weights of these two bands indicate that they are probably the DaPKC (~73 kDa) and DaPKM (~55 kDa) isoforms (Drier, 2002).

The N-terminal sequence of the lower molecular weight band has not been established; however, it likely represents an endogenous DaPKM isoform. The immunoreactivity is competitively reduced by a peptide from the corresponding region of DaPKC, but not one outside of this epitope. In agreement with the Western blot data, fly heads contains more Ca²⁺ and DAG-independent PKC activity than does bodies. The presence of the putative DaPKM correlates strongly with this enriched activity, suggesting that most, if not all, of the endogenous atypical kinase activity measured in head extracts is due to this DaPKM isoform. These data indicate that flies possess both 'C' and 'M' forms of an atypical PKC that is highly homologous to MaPKC/M, and that the DaPKM is enriched in heads (Drier, 2002).

A P-element insertional mutant in DaPKC has been described; however, it is an embryonic lethal and thus is not suitable for examining a possible role in adult learning and memory formation. To assess whether this gene's product is necessary for memory formation, two approaches were taken. First, the effects on memory of feeding flies the PKC inhibitor chelerythrine were monitored. This drug is reported to selectively inhibit PKMzeta at low concentrations; however, its specificity is controversial, and it inhibits other PKC isotypes at higher concentrations. Memory effects produced by inducing the kinase-inactive KI-MaPKMzeta protein were tested: this form of the protein displays 'dominant-negative' activity that is likely to be specific to the atypical PKCs, leaving cPKC and nPKC responses intact (Drier, 2002).

Feeding flies chelerythrine inhibits 24-hour memory formation in a dose-dependent manner, and induction of the KI-MaPKMzeta inhibits 24-hour memory after massed training. The inhibitory effects of both chelerythrine and the KI-MaPKMzeta are not likely due to effects on olfactory acuity or shock reactivity because learning is unaffected by either treatment (Drier, 2002).

The memory enhancement produced by MaPKMzeta could have been due to properties unique to this mammalian protein. The expression data showing that DaPKM is expressed and active in Drosophila heads, when combined with the chelerythrine and dominant-negative data, suggests that DaPKM is involved in normal memory processes in Drosophila. The extensive structural homology between MaPKMzeta and DaPKM also argues against functional uniqueness. The hypothesis of functional homology makes a strong prediction: induction of DaPKM after training should also enhance memory (Drier, 2002).

Based on the approximate molecular weight of the DaPKM, the DaPKC gene was truncated within the hinge region separating the regulatory from the catalytic domains such that the putative DaPKM gene begins at methionine 223. Induction of the DaPKM transgene after training enhances 24-hour memory after single-cycle training. One of these lines was then used to show that 4-day memory after massed training is also enhanced. As with the MaPKMzeta transgenes, the DaPKM lines shows rapid heat-shock induction. These results confirm those obtained with MaPKMzeta, and thus indicate that aPKM is fundamental in the mechanisms underlying memory across species (Drier, 2002).

These results provide strong evidence that atypical PKM activity is sufficient to enhance memory in Drosophila. Ideally, necessity should have been tested by assessing potential memory deficits of flies bearing null mutations in the DaPKC/M gene. However, the lethality of such mutants precluded these analyses, and no special alleles exist that might have preserved the gene's vital function while disrupting its role in memory. In an attempt to circumvent these problems, both pharmacological and dominant-negative interventions were used. Chelerythrine inhibits normal memory in a dose-dependent manner, and induction of a predicted dominant-negative atypical PKM produces the same memory deficit (Drier, 2002).

It was found that heat-shock induction of MaPKMzeta does not enhance long-term memory, because it does not improve memory after spaced training. One explanation for this is that spaced training induces endogenous maintenance mechanisms, and thus occludes the effect of inducing the MaPKMzeta transgene. Thus, memory after single-cycle or massed training may be prolonged by transgene induction because these training regimens do not normally induce prolonged atypical PKM activity. Work in honeybees shows that single-cycle training produces neither persistent PKC activity nor long-lasting memory, but multiple-cycle training produces both. The memory enhancement observed when inducing MaPKMzeta may simply bypass the endogenous requirements (normally provided by spaced training) for prolonged activation of aPKM (Drier, 2002).

The MaPKMzeta-induced enhancement of massed, but not spaced training prompted an examination of the involvement of the radish gene product in this process. If radish were required for the enhancement, the radish mutation would have blocked the MaPKMzeta-induced effect, and this was clearly not the case. Although MaPKMzeta induction phenotypically rescues the memory defect of radish, it does not do so because radish encodes for the Drosophila aPKM. DaPKM is on the second chromosome and radish is on the X, and no Drosophila PKC gene maps to the genetically defined radish locus. There are two principal possibilities explaining how MaPKMzeta-induced memory enhancement bypasses the defect of radish mutants: (1) MaPKMzeta is downstream of radish or (2) MaPKMzeta activates a pathway that is parallel to and independent of radish. The first interpretation is favored because memory after massed training can be either enhanced or disrupted and the radish phenotype can be partially rescued (Drier, 2002).

The temporal specificity of the MaPKMzeta-dependent memory enhancement implies restrictions on its biochemical mechanism(s); enhancement requires that prior activity-dependent mechanisms be in place, and MaPKMzeta has a narrow post-training interval in which to act. If these kinetic restrictions do exist, the rapid induction achievable with the heat-shock promoter is essential for the detection of memory enhancement in these experiments (Drier, 2002).

There are two general interpretations of these data: PKMzeta acts to increase either (1) the magnitude or (2) the duration of the synaptic potentiation that underlies the behavior. In the first model, PKMzeta enhances the synaptic machinery induced by training, making a 'stronger' synaptic connection that decays more slowly. In the second model, PKMzeta acts solely to maintain the synapses previously modified by experience, with no effect on the induction of the potentiation. If one considers the behavioral measurements of learning (testing done immediately after training) and memory (testing done after a longer time) with induction and maintenance, respectively, the chelerythrine and dominant-negative data argue for a role in maintenance. Neither of these treatments affect learning, but each inhibits memory. No enhancement of learning was detected by prior induction of PKMzeta, nor was there an improvement of 3-hour memory if PKMzeta was induced 30 minutes after training. Although the magnitude and duration models may be artificially exclusive, taken together these data are most consistent with a role of PKMzeta in the maintenance of experience-dependent synaptic plasticity (Drier, 2002).

The stability of a synapse varies in response to different regimens of stimuli. Long-lasting changes normally require multiple stimuli and depend on new protein synthesis. Recent experiments support the existence of a synaptic marking system that enables neurons to tag recently active synapses, thus maintaining synaptic specificity during the cell-wide process of protein synthesis-dependent long-term memory formation. A synapse that would normally be stable for only a short period of time can be potentiated for a much longer period of time. However, to do so it must be activated within 2-4 hours of stimulation that produces long-term changes at a second and separate synapse within the same neuron. Although there is no direct evidence for a role of PKMzeta in this process, the similarity between the temporal windows for the proposed synaptic tag and the memory enhancement observed suggests a mechanistic relationship between them (Drier, 2002).

DaPKC is part of a multiprotein complex important for both cell polarity and the asymmetrical cell divisions of early Drosophila neurogenesis. These processes show strong structural and functional parallels with the first asymmetrical cell division of Caenorhabditis elegans embryogenesis. The Drosophila homologs of C. elegans proteins important for this process, Par-3 (Bazooka) and Par-6 (DmPar-6), interact with each other and with DaPKC to direct a specific and interdependent subcellular localization of the complex. During early Drosophila embryogenesis, Bazooka, DmPar-6, and DaPKC are localized to the zonula adherens, a cell junction structure. Mutation in any one of these genes disrupts the ability of the remaining two proteins to localize to this structure properly, and this disrupts cell polarity. This mutual dependence for localization is also apparent during neurogenesis, and causes the inappropriate segregation of cell determinants. This multiprotein complex is critical in mammalian cell polarity and in organizing junctions between epithelial cells. The mouse homologs of Bazooka and Par-6 are expressed in various regions of the CNS, and their subcellular localization within CA1 hippocampal neurons is consistent with a role in synaptic plasticity. Bazooka and DmPar-6 are expressed in Drosophila heads, as are DaPKC and DaPKM. It remains unclear how DaPKM activity is regulated during memory mechanisms; however, the subcellular localization affected by the Bazooka-DmPar-6-DaPKC complex provides hypotheses with attractive physical properties (Drier, 2002).

Atypical PKM is sufficient to enhance memory in Drosophila, and the chelerythrine and dominant-negative data suggest that it is also necessary for normal memory. Strikingly corroborative results have also been obtained for the role of PKMzeta in the maintenance phase of LTP. Injection of MaPKMzeta into CA1 pyramidal cells is sufficient to potentiate evoked excitatory postsynaptic currents. The potentiation occludes LTP and is reversed by chelerythrine. The introduction of the KI-MaPKMzeta into a CA1 cell abolishes its ability to support LTP. The non-NMDA receptor antagonist CNQX blocks this potentiation, indicating that it occurs via AMPA receptors. When these physiological results, obtained in rat hippocampal slice preparations, are combined with the Drosophila behavioral data, they point to a central role of atypical PKM in the mechanism of memory maintenance. Understanding the regulation of atypical PKMzeta, as well as what it in turn regulates, may be critical to unraveling this process (Drier, 2002 and references therein).

Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila

This study distinguishes the memory response of flies to appetitive vs. aversive long-term memory. In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, this study shows that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body α'β' neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Finally, this study describes experiments in which feeding and/or starving flies after training reveal a critical motivational drive that enables appetitive LTM retrieval (Krashes, 2008).

A single 2 min training session pairing odor with sucrose forms appetitive memory that lasts for days. The term 'session' rather than 'trial' is used cautiously because, although the conditioned odor stimulus is continuously presented for 2 min, it is not known how often the flies sample the sugar unconditioned stimulus. One session of the established aversive training paradigm presents 12 shocks at 5 s intervals overlapping with 1-min-long odor exposure, and therefore neither protocol is strictly 'single-trial' learning. Nevertheless, the results present a profound difference between the training protocol requirements to form aversive and appetitive LTM in flies. Formation of aversive LTM requires 5-10 training sessions with rest intervals, whereas a single 2 min session is sufficient to form robust protein synthesis-dependent appetitive LTM. Appetitive LTM is disrupted by cycloheximide (CXM) feeding, inhibition of CREB-dependent transcription, and the crammer (Comas, 2004) and tequila (Didelot, 2006) genes, which suggests that it is bona fide LTM. Furthermore, these data indicate some mechanistic parallel between aversive and appetitive LTM. Appetitive conditioning forms more distributed memory traces in the brain and more efficiently forms LTM than aversive conditioning. It is speculated that these properties of appetitive memory result from the ethological relevance of feeding and the salience of sucrose reinforcement. Furthermore, the salience is likely to be enhanced in hungry flies because they are motivated to seek food. There are a few other reports of single-trial training forming LTM. With the notable exception of fear conditioning in rodents, most involve feeding behavior and the gustatory pathway. In conditioned taste aversion experiments, rodents develop a long-lasting avoidance of a novel tastant after a single exposure of the tastant and delayed drug-induced malaise. Similarly, pond snails develop long-lasting conditioned taste aversion if carrot juice is paired with salt exposure, and 1-d-old chicks develop LTM to avoid pecking a colored bead if that bead was tainted with a bitter tasting compound when first presented. There are also examples in which single-trial conditioning forms appetitive LTM. Rats deficient in thiamine can be trained to prefer non-nutritious saccharin-flavored water by pairing it with delayed an intramuscular thiamine injection. Pond snails form appetitive LTM for the odorant/tastant amylacetate after a single trial of appetitive conditioning pairing it with sucrose. Last, a single trial of appetitive conditioning in honeybees forms robust day-long memory that, surprisingly, does not require new protein synthesis after training. Therefore, it is possible that the innate importance of food-seeking behavior and memory makes it particularly prone to fast consolidation to LTM (Krashes, 2008).

The single training session appetitive LTM assay provides a unique advantage for the study of memory consolidation because one can manipulate the brain immediately after training during the initial period of memory formation. In contrast, 10 cycles of aversive spaced training takes 150 min to complete, and therefore one cannot perturb neural processing during this period without also interfering with acquisition. Using cold-shock anesthesia, it was found that appetitive memory is quickly, and perhaps entirely, consolidated to anesthesia-resistant forms within 2 h after training (Krashes, 2008).

Previous work in flies suggests that cold shock-resistant memory can be broken into two independent components, ARM that depends on the radish (rsh) gene and is resistant to CXM and LTM that is unaffected by rsh and is sensitive to CXM. Feeding flies CXM disrupted appetitive LTM and produced a statistically significant defect 6 h after training, suggesting that protein synthesis-dependent LTM guides behavior at that time. Although the effect of CXM feeding is estimated to inhibit only 50% of global protein synthesis and has to be partial, these data are consistent with the notion that consolidated memory before 6 h might be ARM. However, whereas aversive LTM requires protein synthesis and is not affected by rsh, appetitive LTM requires new protein synthesis and rsh, suggesting appetitive LTM and rsh-dependent appetitive memory do not represent separable memory phases. This result highlights a potentially major mechanistic difference between aversive and appetitive LTM, and that the relationship between ARM and LTM is worth revisiting. Unfortunately, the cloning of rsh does not provide any mechanistic insight because its primary sequence does not contain any known functional domains (Krashes, 2008).

These data reveal a slight discrepancy in the notion that rsh, dCreb-dependent transcription and new protein synthesis are all necessary components of appetitive LTM. Cold-shock anesthesia indicates that appetitive memory consolidation is nearly complete 2 h after training and rsh mutant flies display defective performance 3 h after training, but neither dCreb2-b repressor transgene nor CXM feeding produced a significant difference in memory performance 3 h after training. It is speculated that expression of early forms of appetitive LTM (E-LTM) depend on rsh and that because Radish protein immunolocalizes to neuropil, Radish might function in a synaptic tagging process that marks the relevant synapses for capture of dCreb2-dependent transcripts. This idea provides a plausible reason why radish is required both for E-LTM and for later appetitive LTM (L-LTM), whereas dCreb2-b only interferes with L-LTM. Similarly, it is posited that CXM feeding blocks the translation of mRNAs that are direct and indirect targets of CREB and that are necessary for L-LTM. Similar models have been proposed based on work in rodents and Aplysia (Krashes, 2008).

Previous work has determined that stable olfactory memory (MTM) observed 3 h after aversive and appetitive training requires the sequential involvement of different MB neuron subsets. MB α' β' neurons are required during and after training to acquire and stabilize memory (Krashes, 2007), whereas MB αβ neuron output is required only to retrieve the memory. Stable aversive and appetitive MTM also requires the action of MB-innervating dorsal paired medial (DPM) neurons during the first hour after training. Similarly timed manipulation of these distinct neural circuit elements strongly impairs appetitive LTM, suggesting a tight mechanistic link between appetitive MTM and LTM (Krashes, 2008).

Finding that consolidation of appetitive memory to a protein synthesis-dependent form requires the DPM-MB neural circuitry and that retrieval requires MB αβ neuron output is consistent with the idea that consolidated memory is represented in MB αβ neurons themselves. Several studies have now reported that MB neuron output is required to retrieve olfactory memory, and a few have indicated that MB αβ neurons are particularly important to retrieve aversive and appetitive MTM. A recent live-imaging study provided additional evidence that consolidated aversive LTM is represented in MB αβ neurons (Yu, 2006). Flies that had been space trained with odor and shock exhibited enhanced odor-evoked Ca²⁺ signals in the vertical α branch of MB αβ neurons 9-24 h after conditioning. The development of this memory 'trace' was disrupted by CXM administration, by mutations in the amnesiac gene, and by expressing a transgenic dCreb2-b in MB αβ neurons. Furthermore, expression of the dCreb2-b transgene in MB αβ neurons also impaired aversive LTM behavior. These data are highly consistent with the current findings for appetitive LTM after a single training session and therefore indicate that there are common mechanistic components to aversive and appetitive LTM. It is also worth noting that radish is strongly expressed in MB αβ neurons. Therefore, this collection of findings provides strong evidence that consolidated aversive and appetitive LTM involves MB αβ neurons (Krashes, 2008).

These results do not support the recently proposed idea that LTM consolidation involves transfer from MB to EB (Wu, 2007). Although an appetitive memory assay was used, it was found that Feb170;uas-shi^ts1 flies have a pronounced locomotor defect and therefore these flies are not suitable for memory analysis. Furthermore, Ruslan GAL4 (Wu, 2007) and c305a (Krashes, 2007) express in EB ring neurons, but blocking these neurons does not affect appetitive LTM retrieval. These data are instead consistent with the notion that the transfer of the MB lobe requirement within the first few hours after training may be the fly equivalent of systems consolidation (Krashes, 2008).

These data clearly demonstrate that flies have to be hungry to effectively retrieve appetitive memory. Feeding them ad libitum after training suppressed memory performance, but restarving them restored memory performance. It is proposed that this apparent context dependence of appetitive memory retrieval reflects a motivational state to seek food and therefore it is predicted to be regulated by neuromodulatory systems that signal hunger (Krashes, 2008).

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).

An octopamine-mushroom body circuit modulates the formation of anesthesia-resistant memory in Drosophila

Drosophila olfactory aversive conditioning produces two components of intermediate-term memory: anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Recently, the anterior paired lateral (APL) neuron innervating the whole mushroom body (MB) has been shown to modulate ASM via gap-junctional communication in olfactory conditioning. Octopamine (OA), an invertebrate analog of norepinephrine, is involved in appetitive conditioning, but its role in aversive memory remains uncertain. This study shows that chemical neurotransmission from the anterior paired lateral (APL) neuron, after conditioning but before testing, is necessary for aversive ARM formation. The APL neurons are tyramine, Tβh, and OA immunopositive. An adult-stage-specific RNAi knockdown of Tβh in the APL neurons or Octβ2R OA receptors in the MB α'β' Kenyon cells (KCs) impaired ARM. Importantly, an additive ARM deficit occurred when Tβh knockdown in the APL neurons was in the radish mutant flies or in the wild-type flies with inhibited serotonin synthesis. It is concluded that OA released from the APL neurons acts on α'β' KCs via Octβ2R receptor to modulate Drosophila ARM formation. Additive effects suggest that two parallel ARM pathways, serotoninergic DPM-αβ KCs and octopaminergic APL-α'β' KCs, exist in the MB (Wu, 2013).

The key finding of this study is that OA from the single APL neuron innervating the entire MB is required specifically for ARM formation in aversive olfactory conditioning in Drosophila. This conclusion is supported by five independent lines of evidence. First, blocking neurotransmission from APL neurons after training, but before testing, impaired ARM. Second, the APL neurons are tyramine, Tβh, and OA antibody immunopositive. Third, adult-stage-specific reduction of Tβh levels in the APL neurons, but not in dTdc2-GAL4 neurons that do not include the APL neurons, specifically abolishes ARM without affecting learning or ASM. Fourth, Octβ2R is expressed preferentially in the α'β' lobes, and adult-stage-specific reduction of Octβ2R expression in the α'β' KCs impaired ARM. Fifth, the additive memory impairments demonstrated in flies subjected to Tβh plus inx7 knockdowns and Tβh knockdown plus cold shock, but not inx7 knockdown plus cold shock, confirm that a single APL neuron modulates both ASM and ARM through gap-junctional communication and OA neurotransmission, respectively. Although it has been shown that the APL neurons are also GABAergic, the current results showed that OA is the primary neurotransmitter from the APL neurons involved in ARM formation because reduced GABA levels induced by Gad1^RNAi inhibition in the APL neurons did not affect 3 hr memory (Wu, 2013).

In Drosophila olfactory memories, OA and dopamine have been shown to act as appetitive and aversive US reinforcements, respectively. It is important to point out that the original claim that Tβh plays no role in aversive learning only examined 3 min memory, not 3 hr memory or ARM. It is not surprising to find that OA modulates ARM in aversive memory because dopamine has also been attributed to diverse memory roles, including a motivation switch for appetitive ITM and appetitive reinforcement. Intriguingly, dopamine negatively inhibits ITM formation, but OA positively modulates ARM formation (Wu, 2013).

Food deprivation in Drosophila larvae induces behavioral plasticity and the growth of octopaminergic arbors via Octβ2R-mediated cyclic AMP (cAMP) elevation in an autocrine fashion. This study showed that the APL neurons release OA acting on the Octβ2R-expressing α' β' KCs for ARM, instead of inducing autocrine regulation. Applying OA directly onto the adult brain results in an elevation of cAMP levels in the whole MB, and OA has been shown to upregulate protein kinase A (PKA) activity in the MBs. Intriguingly, ARM is enhanced by a decreased PKA activity and requires DUNCE-sensitive cAMP signals. It is speculated that APL-mediated activation of Octβ2R may lead to an intricate regulation of cAMP in the α' β' KCs for ARM formation. (Wu, 2013).

Although it has generally been assumed that, in a particular neuron, the same neurotransmitter is used at all synapses, exceptions continue to accumulate in both vertebrates and invertebrates. Scattered evidence suggests that co-release may be regulated at presynaptic vesicle filling and postsynaptic activation of receptors, but the physiologic significance remains poorly understood. This study reports that the APL neurons co-release GABA and OA. In the APL neurons, a reduced GABA level affects learning, but not ITM, whereas a reduced OA level has no effect on learning, but impairs ITM, suggesting that the two neurotransmitters are regulated in different ways in the same cell (Wu, 2013).

It has been proposed that the APL neurons might be the Drosophila equivalent of the honeybee GABAergic feedback neurons, receiving odor information from the MB lobes and releasing GABA inhibition to the MB calyx. This negative feedback loop for olfactory sparse coding has been supported by electrophysiological recording of the giant GABAergic neuron in locusts. However, the function of Drosophila APL neurons is complicated by the existence of functioning presynaptic processes in the MB lobes, mixed axon-dendrite distribution throughout the whole MB, and GABA/OA cotransmission (Wu, 2013).

Normal performance of ARM behavior requires serotonin from the DPM neurons acting on ab KCs via d5HT1A serotonin receptors and function of RADISH and BRUCHPILOT in the ab KCs. Surprisingly,the current results show that ARM formation also requires OA from the APL neurons acting on the α' β' KCs via Octβ2R OA receptors, suggesting the existence of two distinct anatomical circuits involved in ARM formation. However, it remains uncertain whether two branches of ARM occur in parallel because combination of various molecular disruptions (i.e., TβhRNAi and pCPA feeding/rsh¹ mutant) did not completely abolish ARM and partial disruption of one anatomical circuit will allow additive effects of another treatment even if they act on the same ARM. The hypothesis of the existence of two distinct forms of ARM is favored based on the following observations. First, neither d5HT1A^RNAi knockdown in α' β' KCs nor Octβ2R^RNAi knockdown in ab KCs affects ARM, suggesting that the two signaling pathways act separately in different KCs and do not affect each other in the same KCs. Second, each of the three ways of molecular disruption (i.e., Tβh^RNAi, pCPA feeding, and rsh¹ mutant) results in a similar degree of ARM impairment, but additive effect did not occur in rsh¹ mutant flies fed with pCPA and was evident when Tβh^RNAi treatment combines with either pCPA feeding or rsh¹ mutant. It's noteworthy that ARM is also affected by dopamine modulation because calcium oscillation within dopaminergic MB-MP1 and MB-MV1 neurons controls ARM and gates long-term memory, albeit a different view has been brought up. The target KCs of these dopaminergic neurons on ARM remain to be addressed (Wu, 2013).

Both the APL and DPM neurons are responsive to electric shock and multiple odorants, suggesting that they likely acquire olfactory associative information during learning for subsequent ARM formation. However, the DPM neurons may receive ARM information independently because their fibers are limited within MB lobes and gap-junctional communications between the APL and DPM neurons are specifically required for the formation of ASM, but not ARM. Given that all dopamine reinforcement comes in via the γ KCs, it is possible that the DPM neurons obtain ARM information from γ KCs. Together, these data suggest that two parallel neural pathways, serotoninergic DPM-αβ KCs and octopaminergic APL-α'β' KCs, modulate 3 hr ARM formation in the MB (Wu, 2013).

Aging accelerates memory extinction and impairs memory restoration in Drosophila

Age-related memory impairment (AMI) is a phenomenon observed from invertebrates to human. Memory extinction is proposed to be an active inhibitory modification of memory, however, whether extinction is affected in aging animals remains to be elucidated. Employing a modified paradigm for studying memory extinction in fruit flies, this study found that only the stable, but not the labile memory component was suppressed by extinction, thus effectively resulting in higher memory loss in aging flies. Strikingly, young flies were able to fully restore the stable memory component 3 h post extinction, while aging flies failed to do so. In conclusion, these findings reveal that both accelerated extinction and impaired restoration contribute to memory impairment in aging animals (Chen, 2015).

Simultaneously exposing the flies with one odor (conditioned odor) and electric shock, then another odor (unconditioned odor) without electric shock sequentially make them learn to avoid the conditioned odor. Cycles of extinction procedures, which are performed as the presentation of conditioned odor without electric shock, impair aversive olfactory memory. In a previous report, memory was reduced about 10% following 10 cycles of odor presentation. To improve extinction efficiency, this study modified the original paradigm by performing the extinction procedures between the presentation of the conditioned odor and unconditioned odor, and named this treatment PCOP (Chen, 2015).

The performance index was found to decrease gradually with the increase of extinction cycle numbers. Furthermore, when equal or more than four cycles of PCOP was performed, the ratio of memory reduction was more than 30%, which was a more significant decrease than previous paradigm. These findings suggested that the presenting time of PCOP and the unconditioned odor affected extinction efficiency. By adjusting the sequence of 4 cycles of PCOP and the unconditioned odor, it was found that the earlier presentation of PCOP, the more significant memory extinction induced. Therefore, four cycles of PCOP before the unconditioned odor were used in all subsequent experiments (Chen, 2015).

To investigate the effect of aging on extinction, the memory index upon extinction procedures in flies at 2, 10, 20, 30 and 50 days of age was measured. It was found that the aversive olfactory memory was reduced significantly by PCOP among these flies. Strikingly, the memory reduction ratio in flies at 20, 30 and 50 days of age was statistically higher than the younger flies. These results indicate that memory extinction in aging flies is more severe than in younger flies, in accordance with the faster extinction performance in aging rats (Chen, 2015).

Several earlier reports showed that extinguished memory can be restored, in the presence of an unconditioned stimulus. To test whether the extinction effect changed over time in flies, the memory 3 h post conditioning was evaluated. It was found that PCOP-induced memory reduction was spontaneously recovered within 3 h in flies at 2 days or 10 days of age. Strikingly, this memory restoration was not observed in flies at 20, 30 and 50 days of age, suggesting more severe memory deficiencies in aging flies (Chen, 2015).

It was reported that aging specifically impaired anesthesia-sensitive memory (ASM) while leaving anesthesia-resistant memory (ARM) intact. Given these findings that aging flies exhibited higher ratios of memory extinction, whether extinction affected ARM and ASM differently, using amnX8 and rsh1 mutant flies was examined. It was found that amnX8 mutant flies exhibited significant memory extinction, with a reduction comparable to that in wild-type flies. Unexpectedly, little memory extinction was observed in rsh1 mutant flies. These results suggested that PCOP specifically suppressed ARM, whereas ASM was unaffected (Chen, 2015).

Radish was reported to be strongly expressed in both the mushroom body (MB) and ellipsoid body in the adult fly brain. It was found that expression of radish with c739-Gal4 in the MB α/β lobes rescued the ARM formation, and re-established PCOP-induced memory extinction. In contrast, expressing Radish in the MB α′/β′ lobes and ellipsoid body with c305a-Gal4 failed to do so. Together, these findings suggested that Radish expression in MB α/β lobes was required for memory extinction (Chen, 2015).

Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, has been reported to be specifically required in the MB for ARM formation. Similar to rsh1 mutant flies, MB-specific brp-knocking down flies exhibited no significant memory extinction in the PCOP assay. Taken together, these results suggest that prolonged odor presentation specifically impairs ARM, but not ASM (Chen, 2015).

To test if the impaired ARM was restored, or whether ASM was elevated after 3 h, a 2-min cold shock was introduced 2 h after the conditioning step to examine ARM. In young wild-type flies, the PCOP group showed comparable ARM to that in the air control group, indicating that the extinction-induced impairment of ARM was restored. In contrast, amnX8 mutant flies still exhibited significant PCOP-induced reduction of ARM, and showed almost no detectable ARM in the PCOP group. Since amnX8 mutant flies are deficient in ASM, the study proposes that recovery of the suppressed ARM requires the presence of ASM. Overall, these findings reveal that upon aging, memory extinction is becoming more and more severe, and once in place, this reduction cannot be restored (Chen, 2015).

Place memory retention in Drosophila

Some memories last longer than others, with some lasting a lifetime. Using several approaches memory phases have been identified. How are these different phases encoded, and do these different phases have similar temporal properties across learning situations? Place memory in Drosophila using the heat-box provides an excellent opportunity to examine the commonalities of genetically-defined memory phases across learning contexts. This study determines optimal conditions to test place memories that last up to three hours. An aversive temperature of 41°C was identified as critical for establishing a long-lasting place memory. Interestingly, adding an intermittent-training protocol only slightly increased place memory when intermediate aversive temperatures were used, and slightly extended the stability of a memory. Genetic analysis of this memory identified four genes as critical for place memory within minutes of training. The role of the rutabaga type I adenylyl cyclase was confirmed, and the latheo Orc3 origin of recognition complex component, the novel gene encoded by pastrel, and the small GTPase rac were all identified as essential for normal place memory. Examination of the dopamine and ecdysone receptor (DopEcR) did not reveal a function for this gene in place memory. When compared to the role of these genes in other memory types, these results suggest that there are genes that have both common and specific roles in memory formation across learning contexts. Importantly, contrasting the timing for the function of these four genes, plus a previously described role of the radish gene, in place memory with the temporal requirement of these genes in classical olfactory conditioning reveals variability in the timing of genetically-defined memory phases depending on the type of learning (Ostrowski, 2014).

Temperature as an aversive reinforcer interacts with training conditions to induce place memories of different stabilities. Previous work showed that intermittent training for Drosophila in space and place memory increases memory performance up to two hours after training. Shown in this study is that temperatures at or above 41°C are needed for induction of this longer lasting memory. That is, 37°C and below can act as an aversive reinforcer and condition flies to avoid a part of the training chamber, but continued avoidance decays within minutes of training. It is only with a temperature of 41°C that an hours-long memory is induced with massed and intermittent training. This abrupt difference in the length of the memory after training with the higher temperature may reflect a threshold of some sort, the steepness of which is currently unknown. This could arise from a differential input to the reinforcing circuit from separate sensory systems, like the Trp family of receptors, or from altered output from one of these sensory systems. Future studies on different temperature responsive proteins may differentiate between these possibilities (Ostrowski, 2014).

Genetic analysis challenges the use of time as a critical factor in determining a memory phase. Memory phases in the fly were initially examined after classical olfactory conditioning where an odorant is typically paired with an aversive electric shock or a rewarding sugar. Four different memory phases have been classified based roughly on time after training and genetic/pharmacological manipulations. Short-term memory after olfactory learning is measured within minutes of training; long-term memory and anesthesia resistant memory start to be active within hours and are increasingly important for memories at the 24 h range and longer. An intermediate memory is thought to be important in the interval between short-term and long-term memories. That time alone is a critical factor in determining these phases loses support when comparing flies with different mutations in aversive and rewarded olfactory memory. For example, the long-known mutant radish was originally shown to be important in the hours-long range after aversive olfactory training and genetically classified the anesthesia-resistant memory. Interestingly, this gene is important within minutes of training in rewarded olfactory memory (Ostrowski, 2014).

Several genes that are important for early to late phases of classical olfactory conditioning are critical on a finer time scale in place memory. Mutation of both the rut and lat genes leads to reduced aversive olfactory memory tested immediately after training, as well as longer time points. Although it is currently unclear when during the life-cycle these genes are important for place memory, mutation of rut and lat reduces memory directly after training. Furthermore, both the rut and lat products have been implicated in synaptic plasticity at the neuromuscular junction (NMJ), which suggests a role for these genes in early stages of learning and memory. It is pretty straight-forward that the rut-encoded type I adenylyl cyclase is also acting early on in associative processes in place learning. The lat gene encoding a subunit of the origin of replication (orc3) is also localized to the pre-synaptic specializations at the NMJs). The lat-orc3 also acts early-on in associative processes for place learning. How the lat-orc3 product is related to regulation of cAMP levels is, however, not as clear. The rut and lat results add to our understanding of an apparently common set of short-term changes in memory between olfactory and place memory, which include a common function of the S6 kinase II, an atypical tribbles kinase, and the arouser EPS8L3. And, the recently identified role of the foxp transcription factor specifically in operant learning, as tested in a flight simulator, suggests another set of genes that could be important for operant place memory in the minutes range (Ostrowski, 2014).

Late memory phases in classical olfactory conditioning depend on a set of genes that are important for place memory within minutes. The first challenge to a common timing of a memory phase came from the radish gene. In contrast to a role in the hours range after olfactory learning, radish mutant flies have a deficit in operant place memory within minutes of training. Furthermore, the pst gene (CG8588), encoding a novel product, has been previously shown to have a specific defect in aversive olfactory memory 24 h after spaced training. That is, the pst mutant flies have a normal short-term olfactory memory but a defective memory 1 day later. Interestingly, in the heat-box pst mutant flies already show a significant decrement in place memory immediately after training. This place memory defect seems to get worse within the first hour after training, reduced to ~50% of normal after 60 min. Thus, this 'long-term memory gene' is also involved in a memory within minutes of training in a second learning situation (Ostrowski, 2014).

Using the classical aversive olfactory learning paradigm the rac small GTPase has been identified as a key regulator in memory retention. Inhibition of Rac activity slows early olfactory memory decay, leading to elevated memory levels one hour after training, but becoming increasingly important 2 h after training. There does not appear to be an effect of Rac inhibition in olfactory memory in the minutes range after training. Transgenic flies with inhibited Rac function also have an increase in memory retention after place memory training. However, the first evidence of an increase in memory performance is within 10 min. Impressively, significant place memory was still evident up to 5 h after training, far beyond the range that can be typically measured in wild-type flies. Thus, while rac has a more general role in stabilizing memories, the timing of this function depends again on the type of memory trace that is formed (Ostrowski, 2014).

Not all memory genes first identified in other contexts, however, play a significant role in place memory. The DopEcR gene has been implicated in several behaviors, including a 30 min memory after courtship conditioning. This G-protein linked receptor is responsive to both dopamine and the steroid hormone ecdysone. Remarkably, DopEcR has been shown to interact with the cAMP cascade through double mutant and pharmacological tests. Using conditions that induce a robust and lasting place memory, the DopEcR mutant flies do not show a defect in memory directly after training or at 1 h post-training. This is despite the fact that the rut and cAMP-phosphodiesterase genes (dunce) are critical for place memory. It may be that DopEcR is not required for this type of learning and would be consistent with the independence of place memory from dopamine signaling. Alternatively, other redundant pathways may compensate for the reduction in DopEcR function caused by the DopEcRPB1 allele. One might further speculate that other types of behavioral plasticity, such as reversal learning or memory enhancement after unpredicted high temperature exposures in the heat-box might be more sensitive to DopEcR changes. Future experiments will determine if this is the case (Ostrowski, 2014).

Memory stability across learning contexts in Drosophila has some common genetic mechanisms, but the timing for gene action depends on the type of learning. That this study has added several genes here, including lat, pst, and rac as regulators of memory stability in operant place memory suggests that there are at least some common molecular processes in memory stability across different learning types. However, the timing of these genetically-defined phases depends on what is learnt. It is speculated that an ideal system to regulate memory stability would be one that activates its own decline. That is, a given memory type should activate the process of decreasing memory expression. This might work with the recruitment of a reinforcing pathway, like the dopaminergic signal that is important for both the acquisition of an associative olfactory memory and the active process of forgetting that association. In this case an odor associated with shock gives rise to a memory trace in mushroom body neurons that depends on a set of dopamine neurons that is important for both memory acquisition and decline. Whether this type of aminergic-based system applies to other forms of memory is not yet known. However, if an aminergic-based signal is common in memory decline, as appears to be the case with the Rac-based mechanism, differences in the types of aminergic neurons or innervation targets could give rise to the altered stabilities of behaviorally expressed memories (Ostrowski, 2014).

REFERENCES

Search PubMed for articles about Drosophila radish

Chen, N., Guo, A. and Li, Y. (2015). Aging accelerates memory extinction and impairs memory restoration in Drosophila. Biochem Biophys Res Commun 460: 944-948. PubMed

Chiang, A. S., Blum, A., Barditch, J., Chen, Y. H., Chiu, S. L., Regulski, M., Armstrong, J. D., Tully, T. and Dubnau, J. (2004). radish encodes a phospholipase-A2 and defines a neural circuit involved in anesthesia-resistant memory. Curr. Biol. 14(4): 263-72. 14972677: see Chiang, 2007 for a retraction of this article.

Comas, D., Petit, F., Preat, T. (2004). Drosophila long-term memory formation involves regulation of cathepsin activity. Nature 430: 460-463. PubMed Citation: 15269770

Didelot, G., et al. (2006). Tequila, a neurotrypsin ortholog, regulates long-term memory formation in Drosophila. Science 313: 851-853. PubMed Citation: 16902143

Drier, E. A., et al. (2002). Memory enhancement and formation by atypical PKM activity in Drosophila melanogaster. Nature Neurosci. 5: 316-324. 11914720

Folkers, E., Drain, P. and Quinn, W. G. (1993). Radish, a Drosophila mutant deficient in consolidated memory. Proc. Natl. Acad. Sci. 90(17): 8123-7. 8367473

Folkers, E., Waddell, S. and Quinn, W. G. (2006). The Drosophila radish gene encodes a protein required for anesthesia-resistant memory. Proc. Natl. Acad. Sci. 103(46): 17496-500. Medline abstract: 17088531

Formstecher, E., et al. (2005). Protein interaction mapping: a Drosophila case study. Genome Res. 15(3): 376-84. Medline abstract: 15710747

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Khurana S, Abu Baker MB, Siddiqi O. (2009). Avoidance learning in the larva of Drosophila melanogaster. Journal of Biosciences. 34(4): 621-31. PubMed ID: 19920347

Krashes, M. J., Keene, A. C., Leung, B., Armstrong, J. D. and Waddell, S. (2007). Sequential use of mushroom body neuron subsets during Drosophila odor memory processing. Neuron 53: 103-115. PubMed Citation: 17196534

Krashes, M. J. and Waddell, S. (2008). Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila. J. Neurosci. 28(12): 3103-13. PubMed Citation: 18354013

Ostrowski, D., Kahsai, L., Kramer, E. F., Knutson, P. and Zars, T. (2015). Place memory retention in Drosophila. Neurobiol Learn Mem 123: 217-224. PubMed ID: 26143995

Pitman, J. L., Huetteroth, W., Burke, C. J., Krashes, M. J., Lai, S. L., Lee, T. and Waddell, S. (2011). A pair of inhibitory neurons are required to sustain labile memory in the Drosophila mushroom body. Curr Biol 21: 855-861. PubMed ID: 21530258

Shih, H. W., Wu, C. L., Chang, S. W., Liu, T. H., Lai, J. S., Fu, T. F., Fu, C. C. and Chiang, A. S. (2015). Parallel circuits control temperature preference in Drosophila during ageing. Nat Commun 6: 7775. PubMed ID: 26178754

Shuai, Y., Lu, B., Hu, Y., Wang, L., Sun, K. and Zhong, Y. (2010). Forgetting is regulated through Rac activity in Drosophila. Cell 140: 579-589. PubMed ID: 20178749

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date revised: 15 August 2017

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