Formation of normal olfactory memory requires the expression of the wild-type amnesiac gene in the dorsal paired medial (DPM) neurons. Imaging the activity in the processes of DPM neurons revealed that the neurons respond when the fly is stimulated with electric shock or with any odor that was tested. Pairing odor and electric-shock stimulation increases odor-evoked calcium signals and synaptic release from DPM neurons. These memory traces form in only one of the two branches of the DPM neuron process. Moreover, trace formation requires the expression of the wild-type amnesiac gene in the DPM neurons. The cellular memory traces first appear at 30 min after conditioning and persist for at least 1 hr, a time window during which DPM neuron synaptic transmission is required for normal memory. DPM neurons are therefore 'odor generalists' and form a delayed, branch-specific, and amnesiac-dependent memory trace that may guide behavior after acquisition (Yu, 2005).
Memory traces together represent the memory engram that directs behavior of the organism after learning or conditioning events. Classical conditioning is one form of learning whereby a conditioned stimulus (CS) becomes predictive of an unconditioned stimulus (US) when the two stimuli are paired in an appropriate way. The prototypic example of classical conditioning stems from studies on dogs conducted by Ivan Pavlov in which tone cues (CS) paired with a food reward (US) became predictive of the food reward, shown by the dog's salivation upon hearing the tone cue after conditioning. In Drosophila, olfactory classical conditioning is a robust and well-studied type of learning in which olfactory cues (CS) are usually paired with electric shock (US), such that conditioning leads to learned avoidance behavior of the CS. Learning to associate two forms of sensory information likely involves specific neurons that respond to both sensory cues and can integrate the information to produce learning. Thus, memory traces for olfactory classical conditioning in Drosophila are expected to form in neurons positioned at the intersections of the olfactory nervous system, the pathway that conveys and processes the CS (CS pathway), and the pathways that convey and process the US (US pathway) (Yu, 2005 and references therein).
The insect olfactory nervous system begins with olfactory receptor neurons (ORNs) distributed between the antennae and maxillary palps. The ORNs project axons to the antennal lobe, where they terminate in morphologically discrete and synapse-dense areas known as glomeruli. There, the ORNs are thought to form excitatory synapses with at least two classes of neurons, one of these being the projection neurons. The projection neurons then convey the olfactory information along their axons in the antennal cerebral tract to at least two higher brain centers, the mushroom bodies and the lateral horn. A large body of evidence has accumulated indicating the importance of the mushroom bodies for olfactory learning. Thus, odors are represented first in the olfactory nervous system by the activation of overlapping sets of ORNs; second by the activation of overlapping sets of projection neurons, and third by the activation of mushroom body and lateral horn neurons (Yu, 2005 and references therein).
An olfactory memory trace forms in the projection neurons after olfactory classical conditioning. Synaptic release of neurotransmitter from presynaptic specializations of projection neurons in the antennal lobe was monitored optically using the transgenically supplied indicator of synaptic transmission, synapto-pHluorin (spH). The memory trace was detected in this case by a rapid but short-lived recruitment of new synaptic activity into the representation of the learned odor. More specifically, distinct odors stimulate distinct sets of projection neurons in naive animals. Within 3 min after conditioning, additional sets of projection neurons become activated by the learned odor. This recruitment is odor specific; different odors recruit different sets of projection neurons into the representation of the learned odor. The recruitment of new sets of projection neuron synapses into the representation of the learned odor, however, is short lived, lasting only 5 min before the synaptic release from the recruited sets of projection neurons decays to the undetectable levels observed prior to conditioning. The short-lived memory trace of projection neurons, although potentially important for guiding behavior for a few minutes after conditioning, cannot account for the time course for behavioral memory, which can last for days. Thus, memory traces in other areas of the nervous system must provide for the persistence of behavioral memory (Yu, 2005).
The dorsal paired medial (DPM) neurons are large neurons that express neuropeptides encoded by the amnesiac (amn) gene and are critical for normal memory. The encoded neuropeptides are related to pituitary adenylyl cyclase-activating peptide (PACAP). The DPM neurons have been widely hypothesized to be part of the US pathway through the release of the expressed modulatory neuropeptides, in part because their processes invade the mushroom body neuropil and are thought to intersect the olfactory nervous system. There is also evidence that these neurons release acetylcholine as a co-neurotransmitter along with neuropeptides (Yu, 2005).
The hypothesis that DPM neurons are solely part of a US pathway predicts that the US but not the CS should activate them and that their response properties should not change after olfactory classical conditioning. A change in their response pattern after conditioning would indicate the presence of a memory trace. A surprising observation is reported that not only do DPM neurons respond to the US of electric shock -- predicted by the hypothesis that they are part of the US pathway -- but they are odor generalists, responding to all odors that were tested. Moreover, they form odor-specific memory traces as registered by increased odor-evoked calcium influx and synaptic transmission. In contrast to the memory trace that forms immediately after conditioning in projection neurons, the memory trace that forms in the DPM neurons is delayed, appearing at 30 min after olfactory classical conditioning. Temporally distinct memory traces that form within projection neurons and DPM neurons after classical conditioning may be partly responsible for guiding behavior during different time windows after learning (Yu, 2005).
There are two DPM neurons, each with a large cell body residing in the dorsal aspect of each brain hemisphere. They have no obvious dendritic field and extend a single neurite in an anterior direction toward the neuropil regions (lobes) that contain the axons of the mushroom body neurons. The neurite from each DPM neuron splits, and one branch broadly innervates the vertical mushroom body lobes while the other innervates the horizontal mushroom body lobes. Careful examination of 12 different confocal stacks highlighting the DPM neurons with the DPM neuron driver, c316-GAL4, revealed that all of the fluorescence in the vertical and horizontal lobes of the mushroom bodies in c316-GAL4/UAS-mCD8GFP flies can be traced to the DPM neuron cell bodies rather than other c316-GAL4-expressing neurons in the brain (Yu, 2005).
It was first asked whether DPM neurons respond to electric-shock pulses delivered to the abdomen of living flies. The processes of DPM neurons in the mushroom body lobes of flies carrying c316-GAL4 and the synaptic transmission reporter UAS-synapto-pHluorin (UAS-spH) or the calcium reporter UAS-G-CaMP were visualized before and during the application of electric-shock pulses delivered to the abdomen. Electric-shock pulses were used of the same intensity, duration, and frequency as those used for behavioral conditioning. The calcium influx in the DPM neuron processes innervating the vertical mushroom body lobes that occurs with electric shock was examined. There was a dramatic response in the processes at the distal tip of the vertical lobes as well as at the vertical-lobe stalk. The change in fluorescence (ΔF/Fo) was examined that occurs with 12 shock pulses delivered at a rate of 1 shock pulse every 5 s. There was an increase in ΔF/Fo coincident with each shock pulse in the vertical lobes and the horizontal lobes with both G-CaMP and spH. These data indicate therefore that electric-shock pulses to the abdomen produce both calcium influx into the DPM neuron processes and synaptic release from their terminals, observations consistent with the possibility that DPM neurons provide US input to the mushroom body neurons (Yu, 2005).
The hypothesis that DPM neurons provide US input into the mushroom body neurons for olfactory memory formation predicts that these neurons should not be activated by the CS of an olfactory stimulus. To test this prediction, DPM neuron calcium influx and synaptic transmission was examined in flies presented with odor stimuli to their antennae and maxillary palps. Stimulation with pure odors like 3-octanol (OCT), 4-methylcyclohexanol (MCH), and benzaldehyde (BEN) elicited robust calcium influx into the DPM neuron processes innervating the vertical mushroom body lobes. The magnitude of the response was dependent on the odor concentration; no responses were observed using air blown over mineral oil, which was used as an odorant diluent. Moreover, these pure odors elicited synaptic activity of the DPM neuron as well as increased calcium influx. Odor responses were also observed in the DPM neuron processes innervating the horizontal mushroom body lobes. Finally, the generality of the DPM odor-evoked response was tested using a battery of 17 different odorants, ranging from pure odors to complex odors such as apple, banana, and grape. In all cases, the DPM neurons responded with increased calcium influx into their processes. Therefore, the DPM neurons are odor generalists in the sense that they apparently respond to all odors administered to the fly. However, the circuitry that provides odorant information to the DPM neurons is unknown (Yu, 2005).
Since the DPM neurons responded to both an odor conditioned stimulus and an electric-shock unconditioned stimulus, the possibility was considered that the neurons might form a memory trace and exhibit a changed response to the CS after olfactory classical conditioning. Either calcium influx into the DPM neuron processes or synaptic release after olfactory classical conditioning were measured. For all experiments, each animal was used for only one measurement in order to avoid potential complications produced by odor habituation, adaptation, or generalization that could occur with multiple exposures (Yu, 2005).
A within-animal experimental design was employed in which the response of the DPM neuron processes within each animal was first evaluated with a single, 3 s presentation of odor. This was followed by forward conditioning, in which a 60 s odor stimulus was presented simultaneously with 12 electric-shock pulses or by backward conditioning in which the 60 s odor stimulus was presented after the onset of the electric-shock stimuli. Forward conditioning leads to robust behavioral conditioning, whereas backward conditioning does not. The response of the DPM neuron processes was then tested at various times after conditioning, again within each animal, and the postconditioning response was compared to the preconditioning response (Yu, 2005).
The postconditioning responses of the DPM neurons to any of three conditioned odors, OCT, MCH, or BEN, did not differ from the preconditioning responses when assayed at 3 min after forward conditioning. This is in marked contrast to the odor-specific memory trace responses that occur in the projection neurons of the antennal lobe within 3 min after conditioning. However, amn mutant flies are only slightly impaired in 3 min memory and have a more pronounced impairment at later times beginning 10-60 min after training. Furthermore, synaptic transmission from DPM neurons is not required for 3 min memory and is dispensable during acquisition and retrieval for robust 3 hr behavioral memory. DPM synaptic transmission is instead required during the interval between training and testing. These observations led to a consideration of the possibility that the DPM neurons might form a memory trace with a delayed onset (Yu, 2005).
The postconditioning responses at 30 min after forward conditioning to three different odors compared to the preconditioning responses revealed that a delayed memory trace registered by increased calcium influx was detectable at this time. The increase in calcium influx was not observed at 30 min after backward conditioning, indicating that the memory trace is dependent on the order in which the CS and US are presented, like behavioral conditioning. Furthermore, the increased calcium influx after forward conditioning was also detectable at 60 min after conditioning but not at 15 or 120 min after forward conditioning. However, the variability of the postconditioning responses at 120 min was larger than at other time points, probably because the physiological state of the flies becomes compromised and more variable from the prolonged immobilization. Thus, the DPM neuron memory trace, detectable first at 30 min postconditioning, extends to at least 1 hr and perhaps 2 hr after conditioning. Moreover, the delayed memory trace registered by increased calcium influx into the DPM neuron processes at 30 min after conditioning was also registered as increased synaptic transmission using spH as a reporter. Therefore, odor evokes both increased calcium influx and increased synaptic transmission from DPM neurons 30 min after forward conditioning (Yu, 2005).
Two general models were considered for the role of the amn gene and the DPM neurons in the process of olfactory memory formation in Drosophila. The first model, the possibility that the amn gene and the DPM neurons provide solely US information for the process of acquisition, is unlikely for several different reasons. (1) amn mutants have normal levels of memory acquisition, shown by memory growth curves with multiple training trials relative to control flies. Impairment in the processing of the US information would likely cause the mutant flies to exhibit performance scores that reach asymptote at levels lower than controls. (2) The parallel nature of the memory growth curves also suggests that the processing of CS information is unimpaired since CS impairment should slow the memory growth rate relative to control flies. (3) These data suggest that the association process itself, or acquisition, is unimpaired since a defect in the association of the CS with the US would also alter the memory growth curve. (4) The discovery of a delayed olfactory memory trace within the DPM neurons themselves, unless fortuitous, is inconsistent with a role specific to US processing. Rather, the data are strongly consistent with a second model alternative envisioning amn and DPM neuron involvement in the formation of intermediate-term memory. The amn mutants exhibit no obvious deficit in acquisition but are impaired in memory. Synaptic transmission is required from the DPM neurons during the interval between training and testing but not at the time of training or testing. The latter observation indicates either that DPM neurons are chronically active or that acquisition itself leads to sustained DPM neuron activity since blocking synaptic activity after acquisition produces a memory impairment at 3 hr. The delayed memory trace formed in DPM neurons that is coincident in time with their requirement for normal memory formation argues for their involvement in an intermediate stage of memory (Yu, 2005).
The delayed olfactory memory trace that forms in the DPM neurons is different from previously observed projection neuron memory trace in several interesting ways. (1) The memory trace formed by antennal-lobe projection neurons occurs by the recruitment of new synaptic activity into the representation of the learned odor. In other words, there is a qualitative change in the brain's representation of the learned odor as represented by projection neuron activity. The memory trace formed by DPM neurons, in contrast, is a quantitative one, being manifest as an increase in calcium influx and synaptic release with CS stimulation after acquisition. Despite this, the trace formed in the DPM neurons is odor specific. (2) The memory trace formed by projection neurons is detectable very early (as little as 3 min) after training, whereas the memory trace formed by DPM neurons is delayed, forming between 15 and 30 min after training. (3) The memory trace formed by projection neurons is very short lived, existing for about 5 min after training. The memory trace established in DPM neurons persists for at least 2 hr after training. The existence of multiple memory traces in distinct areas of the olfactory nervous system with different times of formation and duration leads to the interesting hypothesis that memory of a singular event over time is due to multiple and distinct memory traces that guide behavior during different windows of time after learning, a conclusion also reached from studies with the honeybee (Menzel, 2001; Yu, 2005).
These observations show that the delayed olfactory memory trace is established in the DPM neuron branch that innervates the vertical mushroom body lobes and not in the branch that innervates the horizontal mushroom body lobes. Thus, there exists an intriguing branch specificity to the formation of the delayed olfactory memory trace. The significance of this observation is not yet clear. However, other studies have pointed to the possibility that mushroom body neurons have branch-specific information processing. Some flies mutant for the α lobes absent (ala) gene lack the vertical branch or the horizontal branch of the mushroom body neurons. Intriguingly, mutant animals missing only the vertical branch of the mushroom body neurons have been reported to exhibit normal short-term memory but no long-term memory. Thus, long-term memory may form only in the vertical branch of the mushroom body neurons or be retrieved specifically from this branch. The formation of a delayed olfactory memory trace in the DPM neuron branch that innervates the vertical mushroom body lobes is consistent with the possibility that branch-specific long-term memory processes occurring in the vertical branch of the mushroom body lobes are dependent on the delayed memory trace that forms in this DPM neuron branch (Yu, 2005).
The delayed memory trace that forms in the DPM neurons is dependent on the normal function of the amn gene product since the trace fails to form in amn mutants but can be rescued by expression of the wild-type amn gene in the DPM neurons. This observation raises at least three possibilities for the role of the amn-encoded neuropeptides in the formation of the delayed olfactory memory trace. (1) It is possible that the released neuropeptides exert their effects in an autocrine fashion, interacting with neuropeptide receptors on the DPM neurons themselves in order to initiate the formation of the memory trace. (2) It is also possible that the released neuropeptides interact with receptors on postsynaptic neurons, such as mushroom body neurons, and that this stimulates a retrograde signal that leads to the formation of the DPM neuron memory trace. (3) It is possible that the amn-encoded neuropeptides are not employed for physiological changes in the adult brain but are required in a developmental capacity for DPM neurons to be competent to form the memory trace (Yu, 2005).
There exist at least two broad explanations for the role of the DPM neurons and the amn-encoded neuropeptides in olfactory learning. One possibility is that the DPM neurons integrate CS and US information independently of integration events that occur elsewhere in the nervous system. In this scenario, the CS information may be transmitted to the DPM neurons via unknown interneurons from the antennal lobe or lateral horn, or, alternatively, the DPM neurons might receive CS information from the mushroom body axons. In other words, DPM neurons may be postsynaptic to the mushroom body neurons. This could explain why the DPM neurons are odor generalists since their broad innervation of the mushroom body lobes would allow them to sample the odorant-stimulated activity of many or all mushroom body neurons. This possibility predicts that the DPM neurons should exhibit postsynaptic specializations on some of their processes -- perhaps those that innervate the horizontal lobes, as one possibility. The strengthening of specific mushroom body-DPM neuron synapses after olfactory learning could explain how the DPM neurons form odor-specific memory traces despite being odor generalists. Other DPM neuron processes may be presynaptic to the mushroom bodies such that the CS/US integration events that occur within the DPM neurons might be passed on to the mushroom bodies to reinforce their output. The presynaptic interactions may be through synapses onto the mushroom body fibers in the vertical lobes, reinforcing mushroom body output over the intermediate term and perhaps establishing the permissive signaling events for long-term memories to form in the vertical lobes. The DPM neurons may also receive US information indirectly from the mushroom body neurons or from other neurons. The contributions of the two putative DPM neuron neurotransmitters -- acetylcholine and neuropeptides -- to these processes remain to be clarified. Both acetylcholine and amn neuropeptides are required for behavioral memory (from experiments with Shibire and amn mutants, respectively). The amn neuropeptides are also required autonomously for the formation of the DPM neuron memory trace (Yu, 2005).
The second broad explanation envisions the DPM neurons as maintaining already integrated information through a networked association with the mushroom bodies. The complete integration of CS and US information may occur in the projection neurons and mushroom body neurons. DPM neurons, in a postsynaptic role to the mushroom bodies, would receive integrated information leading to increased excitability. The transfer of the CS/US-integrated information from the mushroom bodies to the DPM neurons may occur immediately after learning, initiating a process intrinsic to the DPM neurons that produces a delayed increase in odor-evoked transmission 30 min later, or the transfer of the integrated information itself from the mushroom bodies to the DPM neurons may occur through a delayed process after learning. In either case, the increased excitability of the DPM neurons would feed back onto and strengthen the output of the mushroom body neurons, leading to robust intermediate-term memory (Yu, 2005).
The memory mutant amnesiac was induced by mutagenesis. Testing was carried out in an apparatus desiged to distinguish flies who successfully made an association of a specific odor with an electric shock, from those who who were unable to do so. (Quinn, 1979).
In Drosophila, adult or larval rearing conditions influence brain structure. In particular, larval density affects the number of fibers forming the mushroom bodies, a neuropil structure involved in olfactory learning. The mushroom bodies receive chemosensory inputs from the antennal lobes at the level of the calyx. Larval density affects calyx volume measured shortly after eclosion from the pupal case. In the memory mutant amnesiac this form of experience-dependent structural plasticity is missing, whereas it is not affected in the learning mutant rutabaga and in the memory mutant radish. Independent of the plasticity effect, the calyces are on average slightly bigger than wild type in amnesiac and smaller in rutabaga flies (Hitier, 1998).
The Drosophila memory gene amnesiac has been proposed to encode a neuropeptide protein, which includes regions homologous to vertebrate pituitary adenylyl cyclase-activating peptide (PACAP). Definitive experiments to link this gene to memory formation, however, have not yet been accomplished. The experiments described here demonstrate that the putative amn transcript is involved in adult memory formation. With the use of a UAS-amn(+) transgene, complete rescue of memory defects has been shown in amn(28A), a mutant allele caused by the insertion of a GAL4 enhancer trap transposon. Study of the amn(28A) reporter reveals widespread expression in the adult brain but also enriched expression in the embryonic and larval nervous systems. To begin addressing the temporal requirement of amn in memory, it was asked whether the memory defects could be rescued by restricting transgenic expression to the adult stage. A heat-shock regimen shown previously to rescue fully the amn ethanol sensitivity defect failed to rescue the memory defect. These results, coupled with previous genetic and anatomical studies, suggest that adult memory formation and ethanol sensitivity have different temporal and spatial requirements for amn (DeZazzo, 1999).
Mutations in the amnesiac gene in Drosophila affect both memory retention and ethanol sensitivity. The predicted amnesiac gene product is an apparent preproneuropeptide, and previous studies suggest that it stimulates cAMP synthesis. Unlike other learning-related Drosophila proteins, Amnesiac is not preferentially expressed in mushroom bodies. Instead, it is strongly expressed in two large neurons that project over all the lobes of the mushroom bodies, a finding that suggests a modulatory role for Amn in memory formation. Genetically engineered blockade of vesicle recycling in these cells abbreviates memory as in the amnesiac mutant. Moreover, restoration of amn gene expression to these cells reestablishes normal olfactory memory in an amn deletion background. These results indicate that Amn neuropeptide release onto the mushroom bodies is critical for normal olfactory memory (Waddell, 2000).
The AMN mRNA transcript is extremely rare, detectable only by nested RT-PCR techniques (Moore, 1998). This rarity has precluded localization of the sites of amnesiac expression in the brain by in situ hybridization to mRNA. To circumvent this problem polyclonal antibodies raised against synthetic peptides were affinity-purified with sequences corresponding to the predicted amn open reading frame (Feany, 1995; Moore, 1998). Purified antiserum was used for immunohistochemical analysis of the adult Drosophila brain (Waddell, 2000).
The previously cloned learning mutant genes, rutabaga, dunce, and DCO, that encode components of the cAMP cascade show dramatically enhanced expression in mushroom bodies. In contrast, Amnesiac immunoreactivity is notably absent there as is reporter gene expression in amn mutants. The Kenyon cell bodies are situated above the MB calyces. The Amnesiac protein is expressed in neurons that are near, but not intrinsic to, the mushroom bodies. The most prominent of these is a pair of large cell bodies near the protocerebral bridge of the central complex. These cells, which have been termed dorsal paired medial (DPM) cells, stain with Amnesiac-specific antisera and are labeled in enhancer-trap patterns of all three relevant amnesiac lines (Waddell, 2000).
The expression patterns of amnc651 [a P{GAL4} enhancer-trap line] and amnchpd (a P-element based enhancer trap line) suggest that amn is expressed preferentially in DPM cells, but also at low levels throughout the brain neuropil. However, the expression pattern of these amn lines differs from the pattern of the amn28A line. The analysis of amn28A adult brains reveals enhancer-expression in many places that are not labeled in amnc651 and amnchpd brains, including some Kenyon cells. Therefore, it is suspected that additional regulatory sequences are driving ectopic expression in amn28A and that the pattern seen in amnc651 and amnchpd is the real one (Waddell, 2000).
It is possible that separately cleaved and processed Amnesiac peptides are present in different brain cells. Antibody was raised against a synthetic polypeptide corresponding to the putative C-terminal cleavage product. It is predicted that the antibody might recognize unprocessed Amnesiac, because it appears to immunostain only the DPM cell bodies and not their processes. If the antibody does in fact react to unprocessed Amnesiac, most of the cells expressing the amn gene should be detected, irrespective of variations in Amnesiac peptide processing (Waddell, 2000).
A P{GAL4} enhancer-trap line, c316, has been isolated that almost exclusively stains the DPM cells. This line was used to examine the DPM cells at higher resolution. The cell bodies of the DPM neurons are large (~12 µm), and they send a single large-diameter neurite toward the MB lobes. This neurite splits into two main branches. One branch leads to the vertically projecting alpha and alpha' lobes, while the other projects to the horizontally arranged beta, beta', and gamma lobe complexes. The processes form a network of fibers and synaptic boutons throughout all of the lobes, and into the spur and anterior region of the pedunculus. There are no detectable projections from the DPM cells to any other brain neuropil region (Waddell, 2000).
To ablate the two DPM cells from the adult fly, the Drosophila cell death genes, either hid or rpr, were expressed with the amnc651 or c316 drivers. This deletion approach, however, leads to lethality. To circumvent this lethality, a new approach was used that allows for the temporary inactivation of DPM cell function in adult flies. The shibire gene encodes a mictrotubule-associated GTPase, dynamin, that is involved in endocytosis and is essential for synaptic vesicle recycling. The temperature-sensitive allele, shits1, is defective in vesicle recycling at restrictive temperature (>29°C) resulting in a rapid blockade of synaptic transmission. In wild-type flies, restricted expression of the shits1 allele (using tissue specific GAL4 expression and a uas-shits1 transgene) in photoreceptor cells or in cholinergic neurons leads to temperature-dependent blindness or to paralysis, respectively. shits1 probably acts dominantly by forming nonfunctional multimers. In this study the c316{GAL4} driver line was used to restrict expression of uas-shits1 to DPM cells in otherwise wild-type flies. Olfactory learning and memory was assayed at both the permissive temperature of 25°C and at the restrictive temperature of 34°C. Memory was tested in an olfactory learning paradigm in which the original amn1 mutant has a 1 hr memory score of zero (Waddell, 2000).
Wild-type flies learn and remember at 25°C and 34°C. Double transgenic c316; uas-shits1 flies learn and remember indistinguishably from wild-type flies at 25°C. However, at 34°C the c316; uas-shits1 flies have undetectable memory after 1 hr although their initial learning scores are the same as wild-type flies. Neither the c316 insertion alone, nor the uas-shits1 transgene alone produces this memory loss. Flies heterozygous for either the c316 driver or for the uas-shits1 transgene remember as well as wild-type flies at 34°C (Waddell, 2000).
Elevated temperature (34°C) has a significant detrimental effect on memory of all strains tested. However at 34°C memory scores of wild-type and the singly transgenic flies are statistically indistinguishable. This contrasts with the complete abolition of memory in the c316; uas-shits1 combination at the restrictive temperature. In addition, the restrictive temperature does not have a significant effect on the olfactory acuity or shock reactivity of wild-type or c316; uas-shits1 flies. Therefore, functional perturbation of DPM cell function (the predominant site of amn expression) with the uas-shits1 transgene mimics the amn1 phenotype. These data support a role for the DPM cells as a crucial locus of Amnesiac in olfactory memory (Waddell, 2000).
The target neuropil of the secreted amn gene product is almost certainly the mushroom bodies because of the striking neuroanatomy of DPM cells. The MBs are thought to integrate multimodal sensory information, and the output of such processed information travels down the Kenyon cell neurites to the MB lobes. The location of the Amnesiac neuropeptide strongly suggests that it modulates information processing in the MBs. Consistent with this, a genetically induced blockade of Amnesiac neurosecretion from a limited number of cells, of which the DPM cells are by far the most prominent, abbreviates memory as does the original amn1 mutation. Furthermore, selective reestablishment of amn expression in DPM cells, in flies otherwise deleted for the amn gene, restores normal memory. DPM cells are a critical site of amn gene expression: expression of Amnesiac product in these cells is necessary and sufficient for normal olfactory memory. The finding that DPM cells lie outside the MBs, but that they ramify over all the MB lobes, lends credence to the critical role of MBs in olfactory learning and memory (Waddell, 2000).
Evidence for related mechanisms has been found in other organisms, specifically in bees, in Aplysia, and in mammals. In honeybees, the VUMmx neuron appears functionally related to the Drosophila DPM cells. The single VUMmx1 neuron is demonstrably crucial for reward learning. Honeybees can associate specific odors (CS) with a sucrose reward (US), resulting in an enhanced probability of odor-evoked proboscis-extension response (PER). Stimulating the VUMmx1 neuron substitutes for the gustatory US in paired training. This neuron, which synthesizes octopamine, innervates three neuropil areas of the bee brain: antennal lobes, mushroom body calyces, and lateral protocerebra. Local injection of octopamine into either the antennal lobe or the MB calyx can substitute for US presentation or VUMmx1 stimulation in PER conditioning (see Tyramine β hydroxylase). Evidently the VUMmx1 (and its secreted octopamine) mediates a positive reinforcement in honeybee PER learning, just as fly DPMs, secreting Amnesiac peptides, mediate persisting negative reinforcement in Drosophila olfactory learning. A second invertebrate system has similarity to Drosophila. In Aplysia the gill and siphon withdrawal reflex can be sensitized and associatively conditioned. In this creature, the reinforcing behavioral stimulus appears to be mediated by a monoamine (serotonin) released in parallel with several peptide neurotransmitters, such as the small cardiac peptides (Waddell, 2000).
There are also evident molecular parallels in mammals. In rats and mice, monoaminergic neurons have demonstrable reinforcing effects in learning and memory; and dopamine regulates late-phase hippocampal long-term potentiation. As for peptides, a mammalian homolog of Amnesiac, PACAP, may be involved. Genes for its receptor are expressed in the hippocampus (Shioda, 1997; Otto, 1999). Nevertheless, in mammals, the involvement of neuropeptides in behavioral reinforcement is currently not understood, perhaps because there are so many peptides, perhaps because the anatomy is so complex (Waddell, 2000).
Studies in mammals and particularly in Drosophila strongly suggest that these molecular substrates of behavioral reinforcement are related to drug addiction. Across animal phyla, addictive drugs engage the same molecular pathways as does associative learning -- release of monamine and peptides and production of cAMP. The addictive drugs cocaine and methamphetamine both potentiate monoamine transmitter action by inhibiting reuptake. In Drosophila, octopamine mediates acute responses to cocaine, nicotine, and ethanol. More strikingly, Drosophila learning mutants that have altered components of the cAMP cascade (amn, dnc, rut, DCO, and PKA-RII) also display dramatically altered responses to ethanol and cocaine. Recent work shows that expression of the rut gene in Kenyon cells is sufficient for olfactory learning, and indicates that the MBs are a critical locus for the signal-integrating properties of the RUT Ca2+/calmodulin-stimulated adenylyl cyclase. The current study markedly refines this anatomical picture of fly learning. Amnesiac is strongly expressed in two DPM cells that innervate all the lobes of the two mushroom bodies (but not the MB calyces or the pedunculi). The rut gene is expressed in the Kenyon cells that give rise to the mushroom bodies; and the Rut cyclase is much more abundant in the lobes than in other parts of the MBs. This expression pattern of Rut suggests that the cyclase functions principally in the MB lobes, where it is directly accessible to secreted Amnesiac peptide(s). Most plausibly, Amnesiac acts directly on the MB lobes, to lengthen and potentiate behavioral associations that have been made in these lobes by persistent activation of the Rut adenylyl cyclase (Waddell, 2000 and references therein).
The mushroom bodies are key features of the brain circuitry involved in insect associative learning, especially when evoked by olfactory cues. Mushroom bodies are also notable for the close-packed parallel architecture of their many intrinsic neuronal elements, known as Kenyon cells. Kenyon cells of adult Drosophila exhibit synchronous oscillation of intracellular calcium concentration, with a mean period of approximately 4 min. Robust oscillation within a dissected brain persists for hours in insect saline and is strongly modulated in amplitude by the product(s) of the memory consolidation gene, amnesiac. It is also sensitive to pharmacological agents specific for several classes of ion channel and for acetylcholine and GABA receptors. A role in memory consolidation involving transcriptionally mediated synaptic strengthening is proposed (Rasay, 2001).
Mutations of the amnesiac (amn) locus, which putatively encodes one or more neuropeptides, perturb olfactory memory consolidation in Drosophila. amnX8 is a deletion of the locus caused by imprecise excision of a P element insertion. When tested for an effect on oscillation, all three alleles cause a very significant increase in oscillation amplitude. Female compound heterozygotes for amnc651 and amnX8 display the same phenotype. A less severe amn allele, amn1, shows no clear effect. Preliminary experiments with the olfactory learning mutations dunce, rutabaga, and radish did not reveal a major effect on either oscillation amplitude, period, or waveform (Rasay, 2001).
amnX8 flies are highly sensitive to ethanol, an effect that on the basis of chemical ablation experiments appears not to involve the MBs. Ethanol appears to have an inhibitory effect in amnX8 and controls. In all configurations, the amplitude ratio of amnX8 to control is greater than 4:1. The ratio of amplitude for amnX8/wild-type after removing the ethanol is 4:1 and ratio of amplitude for amnX8/wild-type after subsequent addition of ethanol to 0.25% is 4:1. The amn product has been shown to be expressed in two large neurons that project all over the lobes of the MBs and through which it likely performs a modulatory role in memory formation. The finding of increased oscillation amplitude in several strong amn mutant backgrounds suggests that the modulatory effect may involve calcium oscillation (Rasay, 2001).
The prominent closely packed parallel organization of KC axons, reciprocally connected by chemical synapses, and perhaps also by nonsynaptic transmission mechanisms, or by gap junctions, may partially explain how the activities of individual KCs are synchronously coordinated. Do KCs oscillate 'spontaneously' or as part of a larger circuit? If the later, then the specific anatomical focus of any applied pharmacoactive compound may be presynaptic to the KCs (Rasay, 2001).
The involvement of voltage-gated sodium channels, as demonstrated by the effects of TTX and veratridine application, strongly suggests concomitant oscillations of electrical activity within a neural pathway that involves the MBs. A clear role has been demonstrated for both ACh-mediated and GABA-mediated synaptic mechanisms. Taken as a whole, the data suggest the following model. Cholinergic input, most probably via the antennal glomerular tract pathway, generates excitatory postsynaptic potentials within KC dendrites, and in certain KCs will summate to cause sodium spike activity. Activation of some KCs triggers the recruitment of others as a consequence of the network properties discussed above. Thus, during the rising phase of each calcium oscillation, a large number of KCs become activated. Spike activity in KCs in turn causes activation of voltage-gated calcium channels and, thus, the influx of external calcium. Hence, the peak levels of cytosolic calcium observed. Relatively little contribution is made by internal calcium stores (Rasay, 2001).
Two phenomena may account for the cessation of calcium accumulation. in one case, activation of potassium channels, either voltage-gated or calcium activated, causes hyperpolarization of KCs, inhibiting further spike activity. In the other, KC-mediated excitation of GABAergic recurrent neurons causes inhibition, both directly at KC dendrites, and at the AGT terminals immediately presynaptic to them. In the longer term, the strength of recurrent inhibition determines the refractory period by preventing cholinergic excitation reaching the threshold for spike initiation. Only when inhibition decays is the oscillation cycle reinitiated, such that the strength of inhibition determines the oscillation period. In support of this model, potassium channel blockers and GABA receptor agonists increase and decrease oscillation frequency, respectively. In such a model, there is no specific requirement for matched periodicity of input (from the antennal glomerular tract or elsewhere) (Rasay, 2001).
What role might the oscillations play in the biology of Drosophila? Although the physiological properties of KCs are still rather obscure, there is a large body of evidence to suggest that KCs play an important role in associative learning and memory; in an olfactory context in Drosophila, and with respect to additional modalities in several other insects. Consistent with the above, KCs are also a major site of expression for several Drosophila 'learning' genes. Drosophila that are selectively ablated for ~90% of their KCs have otherwise fairly normal brain anatomy. Behavioral consequences so far described appear restricted to severely defective olfactory learning abilities, some minor olfactory defects and defects with context generalization in visual learning. While it is tempting to contemplate a role for oscillations in learning and memory, to what aspects of these processes is the minute range relevant? Memory consolidation in the honeybee appears to be particularly sensitive to interference within a 3-5 min window after a conditioning trial. Moreover, induction of long-term olfactory memory in Drosophila requires a spaced training regime in which conditioned and unconditioned stimuli are repeatedly paired at intervals of up to 15 min. The product of the memory consolidation gene amn is expressed in two neurons that are critical for memory formation, and that ramify widely throughout the MB lobes. It is thus of considerable interest that amn alleles cause altered oscillation amplitude. A deletion of the gene, two independent P element insertions, and a compound heterozygote all have nearly identical oscillation characteristics, making it unlikely that any extraneous genetic factors are responsible for the effect. The data with the amn mutants are supportive of a role for oscillations in memory consolidation (Rasay, 2001).
Long-term memory formation in Drosophila requires transcription, as illustrated by experiments involving ectopic expression of activators and blockers of CREB, the cAMP response element binding factor. Non-cAMP-mediated pathways are also anticipated, likely to involve additional transcriptional mechanisms. It is therefore of considerable interest that experimentally induced calcium oscillations, in a similar frequency range as those described here, can affect both the efficiency and specificity of nuclear gene expression. It is suggested that calcium oscillations in the Drosophila MBs have a similar role, affecting the expression of genes involved in the induction of synaptic modification relevant to memory consolidation (Rasay, 2001).
Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. The Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. Growth of the perineurial glia is controlled by interactions among five genes: inebriated (ine), which encodes a member of the Na+/Cl--dependent neurotransmitter transporter family; ether a go-go (eag), which encodes a potassium channel; pushover (push), which encodes a large, Zn2+-finger-containing protein; amnesiac, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide, and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. These results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis (Yager, 2001).
Mutations in two genes that affect neuronal excitability also affect the structure of the peripheral nerve: double mutants defective in ine, and push exhibit an extremely thickened nerve, which is a phenotype that is clearly visible with the dissecting microscope. To understand the cellular basis for this phenotype, transmission electron microscopy was performed on cross-sections of peripheral nerves. This analysis demonstrated that the push1 and ine1;push1 double mutants exhibit a normal axon and peripheral glial layer, but a thickened perineurial glial layer. This increased perineurial thickness is expressed only moderately in push1 but very strongly in the ine1;push1 double mutant. This increase in thickness is accompanied by an increase in the number of mitochondria within perineurial glial thin sections, suggesting that an increase in cell material accompanies this increased thickness. The ine1;push1 phenotype is significantly rescued in transgenic larvae expressing the 943-aa Ine isoform, called Ine-P1, under the transcriptional control of the heat-shock promoter. In particular, perineurial glial thickness in ine1 push1; hs-ine-P1 larvae, even in the absence of heat shock, was reduced to 2.0 ± 0.2 µm from 3.1 ± 0.3 in ine1;push1. The observed synergistic interaction between ine and push mutations suggests that each gene controls perineurial glial growth through partially redundant pathways (Yager, 2001).
In certain respects, mutations in ine confer phenotypes similar to mutations in the K+ channel structural gene eag. In particular, both eag and ine mutations interact synergistically with mutations in the K+ channel encoded by Shaker to cause a characteristic 'indented thorax and down-turned wings' phenotype, which is not exhibited by any of the single mutants. Because of this phenotypic similarity, the possibility that eag mutations might also affect perineurial glial thickness was tested. eag1 resembles ine1 in the control of perineurial glial growth: eag1;push1 double mutants, but not the eag1 single mutant, exhibit strongly potentiated perineurial glial growth. This increased growth is similar to, but less extreme than, what is observed in ine1;push1. Double mutants for eag1; push2 also exhibit a thickened perineurial glial layer. In contrast, eag and ine mutations fail to display a comparable synergistic interaction (Yager, 2001).
Mutations in push were identified independently on the basis of defective segregation of nonrecombinant chromosomes in the female meiosis. push was implicated in this process as an intermediate in a signaling pathway mediated by the PACAP-like neuropeptide encoded by amn (S. Hawley, personal communication to Yager, 2001). This observation raised the possibility that push likewise affects perineurial glial growth by acting as an intermediate from an Amn signal. Consistent with this hypothesis, the amnX8 deletion mutation increases perineurial glial thickness, and this increase is significantly rescued in transgenic flies expressing amn+ (Yager, 2001).
A second signaling pathway mediated by a PACAP-like neuropeptide has been identified in Drosophila. In this pathway, the larval muscle responds to application of PACAP by activating a voltage-gated potassium channel. This activation requires NF1, the ortholog of the human gene responsible for type 1 neurofibromatosis. The possibility was tested that NF1 might affect perineurial glial growth. The NF1P2-null mutant exhibits strong potentiation of perineurial glial thickness in combination with ine1. This thickness is much greater than the thickness observed in ine1 mutants carrying K33, the NF1+ parent chromosome of NF1P2. The increased glial thickness of ine1; NF1P2 is fully rescued by heat-shock-induced expression of the NF1+ transgene. However, unlike push, the phenotype of NF1P2 is potentiated only moderately by the eag1 mutation. In contrast, perineurial glial thickness in the push1; NF1P2 double mutant was 2.1 ± 0.15 µm, which is significantly thicker than either push1 or NF1P2, but not significantly different from amnX8. These results are consistent with the possibility that push and NF1 mediate the amn signal through parallel partially redundant pathways (Yager, 2001).
These results are consistent with a model in which two neurotransmitter-mediated signaling pathways exert opposing effects on perineurial glial growth. One pathway, mediated by the Amn neuropeptide, inhibits perineurial glial growth. This pathway requires NF1 and Push activity. The second pathway, mediated by the substrate neurotransmitter of Ine (which will be called NT here), promotes perineurial glial growth. In this pathway, mutations in ine or eag each increase signaling by NT: ine mutations increase NT signaling by eliminating the NT reuptake transporter thus increasing NT persistence, whereas eag mutations increase NT signaling by increasing NT release as a consequence of increased excitability. These pathways interact such that the most extreme effects on perineurial glial growth are observed when the NT pathway is overstimulated and the Amn pathway is disrupted simultaneously. The genetic interactions that form the basis for this interpretation require that the mutations under investigation be null. Although the eag1 mutation tested has not been characterized molecularly, the mutations in each of the other four genes analyzed are known to be or are strongly suspected to be null. Direct neuron-perineurial glia signaling is unlikely because the peripheral glia, which form the blood-brain barrier, are expected to be an impervious barrier to intercellular traffic. Two alternative mechanisms could underlie this signaling. In the first mechanism (direct peripheral glia-perineurial glia signaling), the peripheral glia release each neurotransmitter, and the perineurial glia respond. In the second mechanism (indirect signaling), each neurotransmitter is released by neurons, and the peripheral glia respond by regulating the release of a trophic factor that acts on perineurial glia (Yager, 2001).
Although direct signaling seems to be the simplest possibility, indirect signaling is most consistent with previous studies. As described above, both invertebrate and mammalian motor neurons can release small molecule and peptide neurotransmitters that affect properties of Schwann cells. A similar motor nerve terminal-peripheral glia communication could occur in Drosophila, because first boutons at the larval neuromuscular junction are covered by peripheral glia. This observation raises the possibility that Drosophila peripheral glia might respond to Amn and NT released from motor nerve terminals, and propagate these signals along the length of the nerve via gap junctions. However, the alternative possibility of NT release from along the length of axons, as has been suggested in other systems, cannot be ruled out. In addition, mammalian Schwann cells release trophic factors such as Desert hedgehog (Dhh) to induce growth of the surrounding perineurium, and astrocytes can respond to glutamate application by releasing a substance that affects blood vessels. This model predicts that peripheral glia release a trophic factor that behaves similarly to Dhh. The prediction that Drosophila NF1 acts within peripheral glia is consistent with the likelihood that mammalian NF1 acts within Schwann cells as well (Yager, 2001).
The possible effects of the thickened perineurial glia on motor neuron function are unclear. Mutations in four of the genes that affect perineurial glial thickness (eag, NF1, ine, and push) were each shown in previous studies to increase either neuronal or muscle membrane excitability, which raises the possibility of a correlation between excitability and perineurial glial growth. However, no increases in neuronal excitability have been detected in the amn mutant or the ine; NF1 double mutant (greater than that conferred by the ine mutation alone), despite the presence of greatly thickened perineurial glia in these genotypes. It is possible that the effects on neuronal excitability of these genotypes might be subtler than the assays can detect, or that the participation of these genes in both perineurial glial growth and excitability is coincidental (Yager, 2001).
These results are consistent with the previous observations that push and NF1 act downstream of the Amn/PACAP receptor. However, the precise nature of the interactions among these proteins is unknown. Thus, it is possible that the interactions are direct, and that Push, the NF1-encoded protein Neurofibromin, and the Amn receptor bind to each other in a macromolecular complex. Alternatively, it is possible that Push and Neurofibromin mediate the effects of Amn only indirectly. In either case, the observation that the push1; NF1P2 double mutant exhibits a perineurial glial thickness much greater than push1 or NF1P2 alone is consistent with the possibility that Push and Neurofibromin mediate the Amn signal through parallel partially redundant pathways (Yager, 2001).
The indirect signaling model could explain the partial cell-nonautonomy of NF1 in neurofibroma formation. Neurofibromas most likely initiate in individuals heterozygous for NF1 mutations by loss of the NF1+ allele in Schwann cells. However, neurofibromas contain, in addition to Schwann cells, cells derived from fibroblasts, perineurial cells, and neurons, which are thought to remain phenotypically NF1+. It is suggested that NF1 mutant Schwann cells cause the overproliferation of their wild-type neighbors by oversecreting trophic factors, and that this oversecretion might ultimately occur as a consequence of defective receipt of a neurotransmitter signal from neurons (Yager, 2001).
Age-related memory impairment (AMI) is observed in many species. However, it is uncertain whether AMI results from a specific or a nonspecific decay in memory processing. In Drosophila, memory acquired after a single olfactory conditioning paradigm has three distinct phases: short-term memory (STM), middle-term memory (MTM), and longer-lasting anesthesia-resistant memory (ARM). This study demonstrates that age-related defects in olfactory memory are identical to those of the MTM mutant amnesiac (amn). Furthermore, amn flies do not exhibit an age-dependent decrease in memory, in contrast to other memory mutants. The absence of AMI in amn flies is restored by expression of an amn transgene predominantly in dorsal paired medial (DPM) cells. Thus, it is proposed that AMI in flies results from a specific decrease in amn-dependent MTM (Tamura, 2003; full text of article).
Although Drosophila is known to be an excellent model for genetic studies, it has not been well studied for AMI. A significant age-related decay in courtship learning has observed in mutants of the kynurenine pathway (tryptophan metabolism) upon aging. However, this was not observed in wild-type flies. In the current study, AMI in wild-type flies was observed for Pavlovian olfactory memory. Initially, a performance deficit appears immediately after training in 10-day-old flies. This effect is slight, however, and does not increase upon further aging, suggesting a minor contribution to AMI. In contrast, the disruption of 1 hr memory in flies 20 days old and older is much more severe. In temporal dynamics and magnitude, this type of disruption is similar to that observed in amn mutants, suggesting a linkage between AMI and amn-dependent memory (Tamura, 2003).
Since 1-day-old amn flies already resemble aged flies, it is possible that amn flies age prematurely. However, no shortening of average lifespan is observed, but rather an extension of lifespan. Given the behavioral similarities between amn and aged flies and the absence of AMI in amn flies, it is likely that AMI is neither a general nor nonspecific disruption of memory processing upon aging, but rather a disruption of a specific phase of memory formation or its underlying neuroanatomy. This amn-dependent memory component has been characterized as MTM (Tamura, 2003).
An alternative interpretation for AMI is that aging simply affects the ability to acquire information, due to a less-attentive state during training or difficulties with sensory perception. In fact, 20-day-old flies show a reduction in odor avoidance as previously reported. However, flies 20 days and older showed 0 hr memory comparable to that in 10-day-old flies, which have normal odor avoidance and shock reactivity. Aging may also affect motor activity. However, shock avoidance was normal in flies up to 50 days of age. These observations strongly suggest that flies 20 days and older retain sufficient attentive state, sensory perception, and motor activity to perform this Pavlovian task (Tamura, 2003).
Since the amn gene product is preferentially expressed in DPM cells, it was possible that DPM cells degenerate upon aging, resulting in AMI. However, significant growth was found of DPM terminals rather than degeneration. Interestingly, despite the growth of DPM terminals during aging, no concomitant increase in amn expression was observed. If the amounts of amn gene products per synapse are reduced during aging due to the increase in numbers of release sites, one might expect that overexpression of the amn transgene would ameliorate AMI. However, AMI could not be reversed by overexpressing the amn transgene either in DPM cells (driven by c316-GAL4) or in panneuronal cells (driven by elav-GAL4) in a wild-type background (data not shown). Therefore, it is unlikely that changes in expression of amn per se are responsible for AMI, but rather an attenuation of cAMP signaling downstream of amn (Tamura, 2003).
Although it could not be concluded whether AMI is absent in mutants of rut-adenylyl cyclase (AC), the present results suggest the importance of cAMP signaling in AMI, since the amn gene encodes a putative peptide with sequence homology to PACAP, which exerts its effects via AC. Supporting this possibility, AMI is ameliorated by the drugs that facilitate cAMP signaling in aged rodents. The expression of rut-AC is required exclusively in the MBs for normal olfactory memory, and synaptic output from MBs (MB lobes) is required for retrieval of olfactory memory for up to 3 hr. Therefore, one possible explanation is that a PACAP-like peptide released from DPM cell terminals may prolong rut-AC activation in the MB lobes to process olfactory memory. Notably, expression of the amn+ transgene predominantly in DPM cells is sufficient to restore AMI to similar levels to wild-type. Taken together, it is proposed that amn-dependent processing of MTM, probably involving signaling between DPM cells and MB lobes via cAMP, is important in young flies and decays at 20 days of age, leading to AMI (Tamura, 2003).
Similar to the situation in Drosophila, PACAP, the putative homolog to the amn gene product in mammals, has been shown to be critical for memory retention in rodents. Mice lacking the PACAP receptor show normal learning (0 hr memory) for one-trial contextual fear conditioning, but memory decay thereafter is abnormally rapid. In addition, administration of PACAP-38 immediately after acquisition of a passive avoidance task improves memory retention in rats. In the rodent model, severe memory decay appears around 10 to 12 months after birth. Given the average lifespan in rodents, 10 to 12 months of age is roughly equivalent to 20 days in flies. Hence, these findings may be conserved in mammalian systems, and it will be of great interest to examine whether mouse mutants lacking the PACAP receptor show the absence of AMI (Tamura, 2003).
γ-hydroxybutyric acid (GHB) can be synthesized in the brain but is also a known drug of abuse. Although putative GHB receptors have been cloned, it has been proposed that, similar to the behavior-impairing effects of ethanol, the in vivo effects of pharmacological GHB may involve metabotropic γ-aminobutyric acid (GABA) GABAB receptors. A fruitfly model has been developed to investigate the role of these receptors in the behavioral effects of exogenous GHB. Injecting GHB into male flies produces a dose-dependent motor impairment (measured with a computer-assisted automated system), that is greater in ethanol-sensitive cheapdate/amnesiac mutants than in wild-type flies. These effects of pharmacological concentrations of GHB require the presence and activation of GABAB receptors. The evidence for this was obtained by pharmacological antagonism of GABAB receptors with CGP54626 and by RNA interference (RNAi)-induced knockdown of the GABAB(1) receptor subtype. Both procedures inhibit the behavioral effects of GHB. GHB pretreatment diminishes the behavioral response to subsequent GHB injections; i.e., it triggers GHB tolerance, but does not produce ethanol tolerance. In contrast, ethanol pretreatment produces both ethanol and GHB tolerance. It appears that in spite of many similarities between ethanol and GHB, the primary sites of their action may differ and that recently cloned putative GHB receptors may participate in actions of GHB that are not mediated by GABAB receptors. These receptors do not have a Drosophila orthologue. Whether Drosophila express a different GHB receptor should be explored (Dimitrijevic, 2005).
γ-hydroxybutyric acid (GHB) is a naturally occurring metabolite of γ-aminobutyric acid (GABA), found in mammalian tissues including the brain. Pharmacologically, GHB (sodium oxybate) is considered in the treatment of narcolepsy and occasionally as an anesthetic. The pharmacological profile of GHB is similar to the profile of ethanol. Abuse of GHB, which shares its behavioral effects with a number of classical sedative/hypnotics, is an increasing problem. Clinically, there are reports of severe GHB withdrawal symptoms, and in a rat model, repeated administration of GHB produces both behavioral tolerance and withdrawal (Dimitrijevic, 2005).
It has been proposed that metabotropic GABA receptors, GABAB receptors, and GHB receptors may mediate the actions of GHB. Although a direct binding of GHB to GABAB receptors has not been conclusively demonstrated, it appears that to produce its behavioral effects, GHB requires these receptors. In GABAB(1) receptor knockout mice, which lack functional GABAB receptors, GHB application failed to produce either the behavioral or the biochemical responses seen in wild-type mice. In contrast, the binding of a putative GHB antagonist, NCS-382, to the specific [3H]GHB-binding sites is unchanged in GABAB(1) receptor knockout mice, suggesting that the behavioral and biochemical effects of GHB are GABAB receptor-dependent whereas the nature and signaling properties of the specific [3H]GHB-binding sites remain elusive (Dimitrijevic, 2005).
Although GABAB receptor knockout mice are useful for behavioral studies, they are developmentally altered and their use is complicated by inherited pathologies such as seizures. To circumvent these drawbacks, a Drosophila model for the adult GABAB(1) receptor knockdown has been developed via the injectable RNA interference (RNAi) method. Recent studies have demonstrated that Drosophila can be successfully used in neuropharmacological research. Fruit flies possess a physiologically active endogenous GABA system (Leal, 2004), express GABAB receptors (Mezler, 2001), and when treated with GABAB receptor ligands display distinct behavioral responses (Dzitoyeva, 2003 and Dimitrijevic, 2004) and developmental abnormalities. Previously, GHB was administered to Drosophila either via food or by injection and in both conditions GHB impairs their locomotor activity. Similar to mammals, Drosophila possess the machinery for GHB synthesis and are capable of metabolizing 1,4-butanediol into GHB in vivo (Dimitrijevic, 2005).
In flies (Dzitoyeva, 2003), similar to mice, the cAMP-linked GABAB receptors participate in the behavior-impairing effects of ethanol. Experiments with mutant flies provided evidence that the cAMP signaling system plays a crucial role in the acute response of fruitflies to ethanol vapor (Moore, 1998). This study found that lack of the amnesiac gene, which is thought to encode a peptide that increases levels of cAMP, or a mutation in this gene called cheapdate increase sensitivity to ethanol. The current study examined whether the cheapdate mutation influences behavioral effects of GHB. In this work, it was hypothesized that GABAB receptors participate in the behavioral actions of GHB in flies, and that flies can be used as an in vivo model to investigate the behavioral interactions of GHB and ethanol (Dimitrijevic, 2005).
Pharmacological concentrations of GHB produce behavioral effects in adult Drosophila that require the presence and activation of GABAB receptors. The evidence for this was obtained by pharmacological antagonism of GABAB receptors and by RNAi-induced knockdown of GABAB(1) receptor subtypes. Both procedures are capable of inhibiting the behavioral effects of GHB. These findings with in vivo experiments in Drosophila are consistent with observations from in vivo experiments with GABAB(1) receptor knockout mice (Kaupmann, 2003), and are somewhat at odds with the recent notion that GHB does not bind GABAB receptors in vitro in GABAB receptor-expressing HEK 293 cells. However, similar experiments in COS cells found that GHB is a weak agonist of recombinant GABAB receptors. It is possible that the cell type-specific environment could contribute to recombinant metabotropic receptor functionality. Furthermore, the specific GHB binding in the brain of GABAB(1) receptor knockout mice is significantly lower than in wild-type mice, suggesting that a component of in vivo GHB binding, and possibly its pharmacological actions may involve GABAB receptors. In contrast, the functional role of putative GHB receptors, particularly with respect to behavior, is unclear. This study found that a GHB receptor antagonist, NCS-382, has no effect on the behavioral GHB actions in Drosophila. Others reported that NCS-382 does not block the behavioral actions of GHB in rats whereas these actions in rats are inhibited by the GABAB receptor antagonist CGP 35348 (Dimitrijevic, 2005).
Adult RNAi via dsRNA injection into adult insects is a useful tool for investigating the loss-of-function phenotypes that circumvent developmental alterations. An inhibition of the behavioral effects of GHB was found with injectable GABAB(1) RNAi. These results, along with the data obtained with CGP 54626 inhibition of GHB effects, suggest that functional GABAB receptors are needed to produce behavioral GHB activity in Drosophila (Dimitrijevic, 2005).
GABAB receptors, which mediate some of ethanol’s behavioral effects in mice and Drosophila, are linked to complex transduction mechanisms and involve negative coupling to the cAMP pathway. Regulation of the cAMP pathway is critical for modifying the sensitivity of Drosophila to ethanol. Thus, cheapdate mutants, which are characterized by defective cAMP signaling, are sensitive to ethanol (Moore, 1998). Using ethanol injections, the increased sensitivity of cheapdate compared to wild-type flies was confirmed in a dose range from 50–200 nmol ethanol/fly. The highest ethanol dose used (400 nmol/fly) was not toxic and prolonged immobility in a manner that was no longer sensitive to cheapdate-dependent mechanisms. Interestingly, it was found that cheapdate mutants are also more sensitive to the behavior-impairing actions of GHB. The increased sensitivity to GHB does not appear to be caused by alterations in GHB metabolism. Both cheapdate and wild-type flies eliminate the injected GHB equally well. Although the exact mechanism for the increased ethanol sensitivity of cheapdate is not clear, Moore (1998) found that this enhanced ethanol sensitivity can be reversed by treatment with agents that increase cAMP levels or protein kinase A (PKA) activity. Conversely, genetic or pharmacological reductions in PKA activity result in increased sensitivity to ethanol, providing functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to impairing levels of ethanol. These data extend this notion to the behavior-impairing effects of GHB. Considering the involvement of GABAB receptors in the behavioral effects of GHB, whether the content of GABAB(1) mRNA is altered in cheapdate was investigated and no difference was found between these and wild-type flies. It is possible that the cAMP-linked functioning of GABAB receptors is altered by the cheapdate mutation (Dimitrijevic, 2005).
Rapid tolerance to repeated ethanol exposures was observed under various regimens of ethanol delivery to a number of different species including Drosophila. Repeated administration of GHB to rats leads to diminished GHB intoxication; i.e., tolerance. It has been proposed that the development of tolerance to ethanol and GHB may involve GABAB receptors. Although the data in Drosophila indicate that GABAB receptors participate in the acute behavioral effects of GHB and ethanol, it does not appear that a single mechanism; e.g., a direct action of GHB on these receptors, is responsible for GHB-induced GHB tolerance and ethanol crosstolerance. Namely, although GHB pretreatment produced GHB tolerance it did not produce ethanol tolerance. In contrast, ethanol pretreatment is able to produce both ethanol and GHB tolerance. Thus, it appears that in spite of many similarities between ethanol and GHB, the primary sites of their action may differ (Dimitrijevic, 2005).
It is possible to speculate about the mechanisms regulated by GABAB receptors that modify the behavioral responses of Drosophila to ethanol (Dzitoyeva, 2003) and GHB. The behavior-impairing effects of ethanol in flies can be modified by genetic manipulations that impair the function of insulin-producing cells or of the insulin-receptor signaling pathway (Corl, 2005). Interestingly, GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in rat pancreatic beta cells (Brice, 2002). It should be investigated whether an interaction of GABAB receptors with Drosophila insulin signaling plays any role in modifying the behavioral effects of ethanol and/or GHB (Dimitrijevic, 2005).
In conclusion, the Drosophila model, similar to recent experiments with GABAB(1) receptor knockout mice, indicates that GABAB receptors rather than NCS-382-sensitive GHB receptors mediate the acute locomotor-impairing effects of GHB. Although GABAB receptors also participate in the behavioral actions of ethanol, they do not appear to be involved in all of the behavioral interactions between GHB and ethanol, for example, in crosstolerance. A recently cloned GHB receptor does not have a Drosophila orthologue. Whether Drosophila express a different GHB receptor should be explored (Dimitrijevic, 2005).
Memories are formed, stabilized in a time-dependent manner, and stored in neural networks. In Drosophila, retrieval of punitive and rewarded odor memories depends on output from mushroom body (MB) neurons, consistent with the idea that both types of memory are represented there. Dorsal Paired Medial (DPM) neurons innervate the mushroom bodies, and DPM neuron output is required for the stability of punished odor memory. Stable reward-odor memory is also DPM neuron dependent. DPM neuron expression of amnesiac (amn) in amn mutant flies restores wild-type memory. In addition, disrupting DPM neurotransmission between training and testing abolishes reward-odor memory, just as it does with punished memory. DPM-MB connectivity was examined by overexpressing a DScam variant that reduces DPM neuron projections to the MB α, β, and γ lobes. DPM neurons that primarily project to MB α′ and β′ lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, these results suggest that the fly employs the local DPM-MB circuit to stabilize punitive- and reward-odor memories and that stable aspects of both forms of memory may reside in mushroom body α′ and β′ lobe neurons (Keene, 2006).
It is widely believed that memory is encoded as changes in synaptic efficacy between neurons in a network. This concept of synaptic plasticity predicts that it will be possible to localize memory to discrete synapses in neural networks in the brain. The relatively small brains of insects are well suited to this endeavor, and genetic manipulation in the fruit fly Drosophila has greatly aided neural circuit mapping of odor memory. Flies can be taught to associate an odor conditioned stimulus (CS) with either a punitive electric shock or a rewarding sugar unconditioned stimulus (US). Strikingly, learning and memory with these opposing unconditioned stimuli requires differential transmitter involvement: sugar-rewarded odor memory is dependent on intact octopamine signaling, while shock-punished (punitive) odor memory is dependent on dopamine signaling. However, despite the differential requirement for these monoamine transmitters, blocking MB output during retrieval impairs both punitive- and reward-odor memories, implying that these memories rely on overlapping brain regions. Stability of reward-odor memory is reliant on the same MB extrinsic neurons that are required for stability of punitive-odor memory (Keene, 2006).
amnesiac mutant flies can associate odors with a punitive or a rewarding US, but they quickly forget this information, which suggests that amn might be generally involved in memory. The amn gene is expressed throughout the brain and strongly in Dorsal Paired Medial neurons—two large modulatory neurons that appear to ramify throughout the approximately 5000 neurons of the MBs. Prolonged DPM neuron output is required for the stability of punitive-odor memories. Since DPM neurons heavily ramify in the MBs, these data support the importance of the MB as a crucial locus for memory and also suggested that the neural network involving MB and DPM neurons could be critical for all MB-dependent memory. Therefore whether the circuitry involving DPM neurons was involved in the stability of rewarded olfactory memory was tested (Keene, 2006).
It was first confirmed that amn mutant flies have a memory defect when conditioned with odors and sugar reward. A modified protocol was used that more closely resembles the odor-shock conditioning protocol and that produces robust memory that lasts for more than 6 hr. In brief, approximately 100 starved flies were exposed to an odor for 2 min in the absence of sugar, followed by a clean air stream for 30 s and a second odor with sugar reward for 2 min. Olfactory memory was tested 3, 60, 180, and 360 min after training. Flies homozygous for the strong amn alleles—amn1 or amnX8—learn to associate the appropriate odor with sugar reward (they have a small but significant initial performance defect), but they forget this association within 60 min of training. These data are consistent with the earlier report that amn1 flies have defective reward-odor memory (Keene, 2006).
Since amn mutant flies forget quickly when trained with either a punitive or a rewarding US, it was of interest to see whether similar neural circuitry was involved in both types of memory. Although the amn gene is expressed throughout the brain, expressing the amn gene in DPM neurons restores punitive odor memory performance to amn mutant flies. Therefore whether restoring amn expression in DPM neurons of amn mutant flies would rescue the reward-odor memory defect was tested. The c316 {GAL4} line was used to transgenically express the amn gene in DPM neurons of amn mutant flies. 3 hr memory of amnX8/amn1;c316/uas-amn and amn1;c316/uas-amn flies was similar to wild-type flies and was statistically different from the memory of amnX8 and amn1; uas-amn mutant flies. These data demonstrate that amn expression in DPM neurons is sufficient to restore reward-odor memory to amn mutant flies and suggest that DPM neurons are generally critical for olfactory memories (Keene, 2006).
Next an acute role of DPM neurons in reward-odor memory was directly tested by temporally blocking their output during the course of the experiment. The dominant temperature-sensitive shibirets1 transgene was tested in DPM neurons by using the c316{GAL4} and Mz717{GAL4} drivers and a sugar reward conditioning experiment was performed at either the permissive (25°C) or the restrictive (31°C) temperature. At the restrictive temperature, shibirets1 blocks vesicle recycling and thereby blocks synaptic vesicle release. At 25°C, reward-odor memory of c316; uas-shits1 and Mz717; uas-shits1 flies was comparable to memory of wild-type and uas-shits1 flies. However, at 31°C, memory of c316; uas-shits1 and Mz717; uas-shits1 flies was statistically different from wild-type and uas-shits1 flies. Therefore, DPM synaptic release is necessary for stable reward-odor memory as it is with punitive-odor memory (Keene, 2006).
Stable punitive-odor memory requires prolonged DPM output between acquisition and retrieval, and DPM output is dispensable during training and testing. Therefore whether DPM neurons were similarly required for reward-odor memory was tested. Again DPM output was blocked by expressing uas-shits1 with c316{GAL4}, but this time the inactivation was restricted to either the training, testing, or storage period. Blocking DPM neurons during acquisition did not produce memory loss; memory of c316; uas-shits1 flies was comparable to wild-type and uas-shits1 flies. Similarly, DPM neuron output was not required during memory retrieval; memory of c316; uas-shits1 flies was comparable to wild-type and uas-shits1 flies. However, blocking DPM output for 30 min after training significantly reduced reward-odor memory; memory of c316; uas-shits1 flies is statistically different from wild-type and uas-shits1 flies. These data parallel results with punitive-odor memory and suggest that there is a similar requirement for DPM neuron output to stabilize both punitive- and reward-odor memory. DPM block from 30 to 60 min after training decreased punitive-odor memory similar to a 0–30 min block. However, disrupting DPM neuron output from 30 to 60 min had an insignificant effect on reward-odor memory. These data imply that the role of DPM neurons is diminished at 30–60 min for reward-odor memory. The Mz717 driver was used to increase the confidence that the temporal uas-shits1 disruptive effect can be ascribed to blocking DPM neurons. Blocking DPM output for 60 min after training with Mz717 significantly reduced reward-odor memory. Memory of Mz717; uas-shits1 flies is statistically different from wild-type and uas-shits1 flies (Keene, 2006).
DPM neurons innervate all the lobes of the MBs, and previous imaging studies suggest that the DPM projections there may be both transmissive and receptive. In addition, expression of n-syb::GFP in DPM neurons has been reported to label DPM projections to the MB lobes. In an attempt to gain further insight into DPM neuron organization, a collection of pre- and postsynaptic compartment markers was overexpressed in DPM neurons. However, no clear evidence was seen for asymmetry within DPM neurons or between projections to individual MB lobes. Therefore, understanding DPM polarity and organization will require further work (Keene, 2006).
During this analysis, it was found that expression of the DScam17-2::GFP fusion protein (which has been described to label the presynaptic compartment when overexpressed in certain neurons, in DPM neurons, with c316{GAL4}, affected DPM neuron development and resulted in DPM neurons that predominantly project to the MB α′ and β′ lobe subsets. Coexpressing uas-DScam17-2::GFP and uas-CD2 or uas-lacZ in DPM neurons reveals that DScam17-2::GFP labels the remaining projections rather than a subset of existing projections. To identify projections to MB α/β neurons versus α′/β′ neurons, brains were costained with anti-FASII, which labels α/β and γ neurons , and anti-TRIO, which labels α′/β′ and γ neurons (Keene, 2006).
A functional role for the MB α′ and β′ lobes in memory has not been reported. Therefore, uas-DScam17-2::GFP; c316 flies were used to assess the role of DPM neuron projections to the MB α′ and β′ lobe subset in punitive- and reward-odor memory. Heterozygous uas-DScam17-2::GFP flies were included as a control as well as wild-type and amnX8 flies for comparison. The presence of the uas-DScam17-2::GFP transgene did not significantly affect punitive-odor memory. Remarkably, DPM neurons that primarily project to the α′ and β′ lobe subsets retain punitive-memory function. Memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amnX8 flies. Therefore, DPM neuron projections to the α′ and β′ lobes of the MB are apparently sufficient for punitive-odor memory. Next the function of uas-DScam17-2::GFP; c316 flies in reward-odor memory was tested. Again, memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amnX8 flies. These data indicate that the DPM neuron projections to the α′ and β′ MB lobe subsets are also apparently sufficient for reward-odor memory and imply that the circuit requirements for the stability of rewarded and punished odor memory are very similar. Although redundancy of DPM projections or retention of a few critical projections to the α, β, and γ lobes cannot currently be ruled out, these data are consistent with the notion that DPM projections to the α′ and β′ MB lobes are sufficient for stabilizing memory. The data also suggest that DScam may play a role in wiring the DPM-MB circuit (Keene, 2006).
In Drosophila, there is a striking dissociation of monoamine transmitters for reward and punishment. Dopamine is required for aversive-odor memory formation, whereas octopamine is necessary for appetitive-odor memory. Octopaminergic and dopaminergic neurons project throughout the brain including to the MBs. Although it is not known whether the MB arborization of these monoaminergic neurons is required for odor memories, blocking MB output is required to retrieve both aversive- and appetitive-odor memory (Keene, 2006).
DPM neurons ramify throughout the MB lobes and provide a general stabilizing mechanism for both punitive- and reward-odor memory. This DPM neuron analysis enhances resolution of memory processing and provides further weight to the idea that components of both punitive- and reward-odor memory reside at synapses within MB neurons. Imaging studies suggest that DPM neurons are both receptive and transmissive to MB neurons, and a model is favored where DPM neurons represent recurrent feedback neurons that consolidate conditioned changes in synaptic weight in MB neurons. However, if MB neurons provide drive to DPM neurons, one would expect MB neuron output and DPM neuron output to have similar temporal requirements. Current published data conclude that MB neuron output is not required during memory storage (but is exclusively required for retrieval) when DPM neuron output is required. However, the work described here suggests a role for MB α′ and β′ lobe neurons in memory stability, and it is noteworthy that MB studies did not employ GAL4 drivers with extensive expression in MB α′/β′ neurons. Further detailed analysis of the role of MB α′/β′ neurons in memory should resolve this conundrum (Keene, 2006).
Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca(2+) channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. Data is presented suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6-38, an antagonist at type-I PACAP receptor. 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2',5'-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca(2+) channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila (Bhattacharya, 2004; full text of article).
The study of age-related memory impairment (AMI) has been hindered by a lack of AMI-specific mutants. In a screen for such mutants in Drosophila, it was found that heterozygous mutations of DCO (DCO/+), which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), delay AMI more than twofold without affecting lifespan or memory at early ages. AMI is restored when a DCO transgene is expressed in mushroom bodies, structures important for olfactory memory formation. Furthermore, increasing cAMP and PKA activity in mushroom bodies causes premature AMI, whereas reducing activity suppresses AMI. In Drosophila AMI consists of a specific reduction in memory dependent on the amnesiac (amn) gene. amn encodes putative neuropeptides that have been proposed to regulate cAMP levels in mushroom bodies. Notably, both the memory and AMI defects of amn mutants are restored in amn;DCO/+ double mutants, suggesting that AMI is caused by an age-related disruption of amn-dependent memory via PKA activity in mushroom bodies (Yamazaki, 2007).
The molecular mechanisms affecting both AMI and aging are likely to be tightly linked. In general, mechanisms and mutations that extend lifespan also delay the onset of AMI. For example, calorie restriction both increases longevity and improves memory in aged animals. Ames dwarf mice, which have reduced growth hormone, thyroid-stimulating hormone and prolactin, live longer than their wild-type siblings and show delayed age-related declines in performance of an inhibitory avoidance task. Furthermore, antioxidant activity has been associated with both extension of lifespan and delayed AMI (Yamazaki, 2007).
There is also evidence that aging and AMI can be separated, however. The optimal level of calorie restriction required for maximal extension of lifespan is not necessarily optimal for delaying AMI, and signaling by growth hormone and insulin-like growth factor activity, which promotes aging, ameliorates AMI. Thus, although it is likely that aging and AMI share pathways in common, at some point these pathways diverge (Yamazaki, 2007).
In Drosophila, genetic studies of memory after a single cycle of training in a Pavlovian olfactory association task have revealed four temporal memory phases: initial learning (LRN), short-term memory (STM), middle-term memory (MTM) and anesthesia-resistant memory (ARM). These phases have been shown to be distinct based on the existence of mutants and pharmacological sensitivities that are specific to particular memory phases. To study how aging affects these phases, AMI has been characterized in Drosophila, and it has been determined that aging specifically impairs MTM, which is dependent on the function of the amn gene (Tamura, 2003). Memory defects in aged flies become identical to those of amn mutants and amn mutants do not show any memory decay upon aging (Yamazaki, 2007).
However, amn expression does not decrease upon aging and overexpression of an amn transgene does not ameliorate AMI. The amn gene products are proposed to be putative neuropeptides secreted from two dorsal-paired medial (DPM) neurons that innervate the mushroom bodies, neural centers for learning and memory. Therefore, it was theorized that an age-related disruption of signaling downstream of amn leads to memory impairment. This suggests that AMI may arise in the mushroom bodies, and led to a screen for genes regulating AMI in the mushroom bodies (Yamazaki, 2007).
In the present study identified heterozygous mutations in DCO (also called Pka-C1), the gene encoding the catalytic subunit of PKA, as strong AMI suppressors. Notably, heterozygous mutations in DCO affect neither memory at early ages nor lifespan, suggesting that DCO hypomorphs are AMI-specific mutants. PKA activity in the mushroom bodies mediates an age-related decline in amn-dependent MTM (Yamazaki, 2007).
From a screen of 54 fly lines with mutations in genes expressed predominantly in mushroom bodies, two lines were identified with altered AMI. Among these, heterozygous mutations in DCO function as strong AMI suppressors. Although it has been shown that mutations and conditions that extend lifespan tend also to delay
AMI, the DCO mutations seem to function by a different mechanism. In contrast to low temperature and calorie restriction, which seem to extend lifespan and ameliorate AMI proportionately, DCO/+ mutants have no effect on lifespan but delay AMI onset and severity to much greater extents than manipulations that alter lifespan. Thus, it is envisioned that these mutants lie downstream of aging pathways and specifically suppress the negative effects that aging has on memory (Yamazaki, 2007).
Decreasing cAMP and PKA activity by using four distinct heterozygous DCO mutations or by expressing PKI significantly delays AMI. Increasing cAMP and PKA activity either by using a heterozygous dnc1 mutant or by overexpressing DCO results in disruption of memory reminiscent of premature AMI. In addition, AMI is restored in DCOB3/+ flies when DCO is expressed in the mushroom bodies but not when it is expressed in the fan-shaped body or ellipsoid body. From these data, it is concluded that cAMP and PKA signaling activity in the mushroom bodies is a cause of AMI (Yamazaki, 2007).
Several PKA substrates have been associated with AMI. For example, calcium dysregulation caused by increased activity of the L-type voltage-gated calcium channel (LVGCC) Cav1.2 has been proposed to cause AMI. Notably, LVGCC expression is highly upregulated in aging and channel activity is strongly enhanced by PKA phosphorylation, suggesting that LVGCCs may be a candidate substrate that can cause AMI during aging. In support of this hypothesis, LVGCC phosphorylation has been shown to be increased in the hippocampus of aged rats. Phosphorylated tau protein is a major component of neurofibrillary tangles (NFT), and accumulation of NFTs shows strong correlation with age-related memory loss. PKA is one of several kinases that phosphorylate tau protein and facilitate NFT formation (Yamazaki, 2007).
AMI results from a specific reduction of amn-dependent MTM, and amn mutants show memory and AMI defects reminiscent of the phenotype of flies overexpressing DCO+. Both these memory and AMI defects are suppressed in amn;DCO/+ double mutants, strongly suggesting that amn and DCO function in a common pathway and DCO function is downstream of amn function. The amn gene encodes at least three peptides, two of which have homologies to mammalian pituitary adenylyl cyclase-activating peptide (PACAP) and growth hormone-releasing hormone (GHRH). In mammalian systems, receptors of PACAP and GHRH function to increase adenylyl cyclase activity. At the Drosophila neuromuscular junction, amn mutations cause decreases in Ca2+ currents through LVGCCs as a result of decreased PKA activity. These results support the idea that Amn peptides stimulate adenylyl cyclase activity. It has also been reported, however, that Ca2+ currents through LVGCCs in the mushroom bodies are greatly enhanced by amn mutations. Thus amn mutants seem to have opposite phenotypes at the neuromuscular junction and in the mushroom bodies. Although it remains possible that amn functions differently at these two locations, it is hypothesized that, instead, indirect effects may occur in the mushroom bodies of amn mutants that increase intracellular Ca2+ concentrations. A drastic increase in Ca2+ influx may enhance activity of the Ca2+- and calmodulin-dependent adenylyl cyclase rutabaga, leading to aberrant increases in activity-dependent PKA activity that can be suppressed by DCO/+ mutations (Yamazaki, 2007).
It has been reported that inhibition of cAMP and PKA activity in the prefrontal cortex (PFC) improves working memory in aged, cognitively impaired rats but not in young rats, whereas activation of cAMP and PKA impairs memory in aged rats at lower concentrations than in young rats. Basal levels of PKA, and of several adenylyl cyclase and phosphodiesterase isoforms, do not show age-related changes in the aged rat PFC, a finding similar to results obtained from flies. CREB phosphorylation, which is likely to be downstream of cAMP/PKA signaling, is increased in the aged rat PFC. These results suggest that several aspects of AMI are conserved between Drosophila and mammals and support a model in which increasing PKA-dependent phosphorylation at a post-translational step may be responsible for AMI (Yamazaki, 2007).
As PKA activity is essential for memory formation, it seems counterintuitive that PKA is also responsible for an age-dependent memory reduction. Indeed, in mammalian systems, it has been widely reported that increasing cAMP levels can enhance both the protein synthesis-dependent phase of hippocampal long-term potentiation (LTP) and hippocampus-dependent long-term memory in aged mice. These results have been used to suggest that an age-dependent reduction in cAMP and PKA activity may cause AMI. However, increasing cAMP improves memory in young as well as old mice, indicating that this is a general rather than an age-specific effect (Yamazaki, 2007).
If activation of the cAMP/PKA pathway improves hippocampal memory and LTP, why isn't the activity of this pathway higher in the wild-type organism? Although speculative, it seems likely that cAMP/PKA activity must have adverse effects, preventing high PKA expression. The current data are consistent with a model in which acute PKA activity is required for memory but long-term effects of PKA promote AMI. Thus, the levels of PKA observed naturally may be the result of a balance between two antagonistic pleiotropic effects of PKA (Yamazaki, 2007).
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).
Sleep has many important biological functions, but how sleep is regulated remains poorly understood. In humans, social isolation and other stressors early in life can disrupt adult sleep. In fruit flies housed at different population densities during early adulthood, social enrichment was shown to increase subsequent sleep, but it is unknown if population density during early development can also influence adult sleep. To answer this question, Drosophila larvae were maintained at a range of population densities throughout larval development, kept them isolated during early adulthood, and then tested their sleep patterns. The findings reveal that flies that had been isolated as larvae had more fragmented sleep than those that had been raised at higher population densities. This effect was more prominent in females than in males. Larval population density did not affect sleep in female flies that were mutant for amnesiac, which has been shown to be required for normal memory consolidation, adult sleep regulation, and brain development. In contrast, larval population density effects on sleep persisted in female flies lacking the olfactory receptor or83b, suggesting that olfactory signals are not required for the effects of larval population density on adult sleep. These findings show that population density during early development can alter sleep behavior in adulthood, suggesting that genetic and/or structural changes are induced by this developmental manipulation that persist through metamorphosis (Chi, 2014).
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).
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date revised: 23 August 2017
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