Higher brain functions of PACAP and a homologous Drosophila memory gene amnesiac: insights from knockouts and mutants

Neuropeptides usually exert a long-lived modulatory effect on the small-molecule neurotransmitters with which they colocalize via regulation of the response times of second messenger systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuromodulator and neurotransmitter and regulates a variety of physiological processes. PACAP is structurally highly conserved during evolution, implying its vital importance. In Drosophila, loss-of-function mutations in a PACAP-like neuropeptide gene, amnesiac (amn), affect both memory retention and ethanol sensitivity. The amnesiac gene is expressed in neurons innervating the mushroom body lobes, the olfactory associative learning center. Conditional genetic ablation of neurotransmitter release from these neurons mimics the amnesiac memory phenotypes, suggesting an acute role for amnesiac in memory. However, genetic rescue experiments also suggest developmental defects in amnesiac mutants, implying a role in neuronal development. There is a parallel between memory formation in Drosophila and mammals. PACAP-specific (PAC(1)) receptor-deficient mice show a deficit in hippocampus-dependent associative learning and mossy fiber long-term potentiation (LTP). Meanwhile, PACAP-deficient mice display a high early mortality rate and additional CNS phenotypes including behavioral and psychological phenotypes (e.g., hyperlocomotion, intense novelty-seeking behavior, and explosive jumping). A functional comparison between PACAP and amnesiac underlines phylogenetically conserved functions across phyla and may provide insights into the possible mechanisms of action and evolution of this neuropeptidergic system.     

Aging specifically impairs amnesiac-dependent memory in Drosophila

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). Here, we demonstrate 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 DPM cells. Thus, we propose that AMI in flies results from a specific decrease in amn-dependent MTM.     

Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation

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. Here we show that 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. We further examined DPM-MB connectivity by overexpressing a DScam variant that reduces DPM neuron projections to the MB alpha, beta, and gamma lobes. DPM neurons that primarily project to MB alpha' and beta' lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, our 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 alpha' and beta' lobe neurons.     

Functional anatomy: from molecule to memory

The Drosophila memory gene amnesiac is expressed in neurons that project to mushroom body axons. Blockade of synaptic transmission in the amnesiac-expressing cells disrupts memory, but not learning, suggesting presynaptic and postsynaptic sites for memory formation.     

Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output

Amnesiac mutant flies have an olfactory memory defect. The amn gene encodes a homolog of vertebrate pituitary adenylate cyclase-activating peptide (PACAP), and it is strongly expressed in dorsal paired medial (DPM) neurons. DPM neurons ramify throughout the mushroom bodies in the adult fly brain, and they are required for stable memory. Here, we show that DPM neuron output is only required during the consolidation phase for middle-term odor memory and is dispensable during acquisition and recall. However, we found that DPM neuron output is required during acquisition of a benzaldehyde odor memory. We show that flies sense benzaldehyde by the classical olfactory and a noncanonical route. These results suggest that DPM neurons are required to consolidate memory and are differently involved in memory of a volatile that requires multisensory integration.     

Mushroom bodies, Ca(2+) oscillations, and the memory gene amnesiac.

The memory of odors in Drosophila is mediated by mushroom body neurons. Memory is formed, in part, by a modulation of the physiology of these neurons brought about by neuropeptides that are encoded by the amnesiac gene and released from peptidergic neurons that innervate mushroom body neurons. Slow and spontaneous oscillations of calcium levels are elevated in the mushroom body neurons of amnesiac mutants and may contribute to memory consolidation.     

Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects.

Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.     

Bridging behavior and physiology: ion-channel perspective on mushroom body-dependent olfactory learning and memory in Drosophila

An important body of evidence documents the differential expression of ion channels in brains, suggesting they are essential to endow particular brain structures with specific physiological properties. Because of their role in correlating inputs and outputs in neurons, modulation of voltage-dependent ion channels (VDICs) can profoundly change neuronal network dynamics and performance, and may represent a fundamental mechanism for behavioral plasticity, one that has received less attention in learning and memory studies. Revisiting three paradigmatic mutations altering olfactory learning and memory in Drosophila (dunce, leonardo, amnesiac) a link was established between each mutation and the operation of VDICs in Kenyon cells, the intrinsic neurons of the mushroom bodies (MBs). In Drosophila, MBs are essential to the emergence of olfactory associative learning and retention. Abnormal ion channel operation might underlie failures in neuronal physiology, and be crucial to understand the abnormal associative learning and retention phenotypes the mutants display. We also discuss the only case in which a mutation in an ion channel gene (shaker) has been directly linked to olfactory learning deficits. We analyze such evidence in light of recent discoveries indicating an unusual ion current profile in shaker mutant MB intrinsic neurons. We anticipate that further studies of acquisition and retention mutants will further confirm a link between such mutations and malfunction of specific ion channel mechanisms in brain structures implicated in learning and memory.     

The cyclic AMP phosphodiesterase encoded by the Drosophila dunce gene is concentrated in the mushroom body neuropil.

Drosophila dunce (dnc) flies are defective in learning and memory as a result of lesions in the gene that codes for a cAMP-specific phosphodiesterase (PDE). Antibodies to the dnc PDE showed that the most intensely stained regions in the adult brain were the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that dnc RNA was enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains were also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development. The role of the dnc PDE in mushroom body physiology is discussed, and a circuit model describing a possible role of the mushroom bodies in mediating olfactory learning and memory is presented.     

Behavioral functions of the insect mushroom bodies

New methods of intervention in Drosophila and other insect species reveal that the mushroom bodies are involved in a diverse set of behavioral functions. The intrinsic Kenyon cells (those neurons with projections within the mushroom bodies) house part of the short-term memory trace for odors and are required for courtship conditioning memory. A pair of extrinsic mushroom body neurons (neurons with projections both inside and outside the mushroom bodies) provides a neuropeptide important for 1-hour olfactory memory. In addition, the mushroom bodies are necessary for context generalization in visual learning and for regulating the transition from walking to rest.     

Altered outward K(+) currents in Drosophila larval neurons of memory mutants rutabaga and amnesiac.

K(+) currents in cultured Drosophila larval neurons have been classified into four categories according to their inactivation time constants, relative amplitude, and response to K(+) channel blockers 4-AP and tetraethylammonium. The percentage (65%) of neurons displaying K(+) currents which were reduced to 30% in amplitude by 5 mM cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP in both Drosophila memory mutants rutabaga (rut) and amnesiac (amn) was significantly larger than that (50%) in wild type. This initial characterization provides evidence for altered K(+) currents in both rut and amn mutants. Arachidonic acid, a specifical inhibitor of Kv4 family (shal) K(+) channels, was found to inhibit K(+) currents in cultured Drosophila neurons, suggesting the presence of shal channels in these neurons.     

GABAA receptor RDL inhibits Drosophila olfactory associative learning

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABAA receptor, resistance to dieldrin (Rdl), is highly expressed in the Drosophila mushroom bodies (MBs), a group of neurons playing essential roles in insect olfactory learning. Flies with increased or decreased expression of Rdl in the MBs were generated. Olfactory associative learning tests showed that Rdl overexpression impaired memory acquisition but not memory stability. This learning defect was due to disrupting the physiological state of the adult MB neurons rather than causing developmental abnormalities. Remarkably, Rdl knockdown enhanced memory acquisition but not memory stability. Functional cellular imaging experiments showed that Rdl overexpression abolished the normal calcium responses of the MBs to odors while Rdl knockdown increased these responses. Together, these data suggest that RDL negatively modulates olfactory associative learning, possibly by gating the input of olfactory information into the MBs.     

Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid

Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.     

Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway

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. We now present data 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.     

Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila

A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process.     

Diverse mechanisms to remember various odors

Two dorsal paired medial (DPM) neurons express the Amnesiac neuropeptide and project onto mushroom bodies, the Drosophila olfactory memory center. In this issue of Neuron, Keene et al. show that higher-level brain circuits process various olfactory memories differently. DPM neurons are required during acquisition of some odors and during memory consolidation of others. These findings reveal a surprising level of complexity for the formation of olfactory memories in Drosophila.     

Metamorphosis and adult development of the mushroom bodies of the red flour beetle, Tribolium castaneum

The insect mushroom bodies play important roles in a number of higher processing functions such as sensory integration, higher level olfactory processing, and spatial and associative learning and memory. These functions have been established through studies in a handful of tractable model systems, of which only the fruit fly Drosophila melanogaster has been readily amenable to genetic manipulations. The red flour beetle Tribolium castaneum has a sequenced genome and has been subject to the development of molecular tools for the ready manipulation of gene expression; however, little is known about the development and organization of the mushroom bodies of this insect. The present account bridges this gap by demonstrating that the organization of the Tribolium mushroom bodies is strikingly like that of the fruit fly, with the significant exception that the timeline of neurogenesis is shifted so that the last population of Kenyon cells is born entirely after adult eclosion. Tribolium Kenyon cells are generated by two large neuroblasts per hemisphere and segregate into an early-born delta lobe subpopulation followed by clear homologs of the Drosophila gamma, alpha'/beta' and alpha/beta lobe subpopulations, with the larval-born cohorts undergoing dendritic reorganization during metamorphosis. BrdU labeling and immunohistochemical staining also reveal that a proportion of individual Tribolium have variable numbers of mushroom body neuroblasts. If heritable, this variation represents a unique opportunity for further studies of the genetic control of brain region size through the control of neuroblast number and cell cycle dynamics.     

A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a neurotransmitter system

Vesicular transporters are required for the storage of?all classical and amino acid neurotransmitters in synaptic vesicles. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male's position during copulation that is rescued by expression in KCs. Because prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning.