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).
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).
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).
Reference names in red indicate recommended papers.
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.
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
Gong, Z., Xia, S., Liu, L., Feng, C. and Guo A. (1998). Operant visual learning and memory in Drosophila mutants dunce, amnesiac and radish. J. Insect Physiol. 44(12): 1149-1158. 12770314
Tully, T., Preat, T., Boynton, S. C., Del Vecchio, M. (1994). Genetic dissection of consolidated memory in Drosophila. Cell 79(1): 35-47. 7923375
date revised: 1 November 2007
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