PROJECTS ON LEARNING/MEMORY SYNAPTIC MECHANISMS:
Scientific Background: Learning and Memory Mutants in Drosophila:
The most powerful approach to biological questions is systematic genetic analysis. This becomes more obvious with the increasing complexity of the question or system studied. The most complex biological system known to us is the brain and among the most complex questions concern the mechanisms of learning and memory. This proposal is to apply systematic genetic analysis to understanding the mechanisms of learning and memory. Since we believe that learning/memory mechanisms occur at synaptic connections (Bailey and Kandel, 1993; Jessel and Kandel, 1993), the site of information transfer between neurons, this approach requires a system which is both genetically malleable and permits detailed assays of synaptic function and architecture. The Drosophila system is the most attractive candidate. Drosophila is one of the most classic genetic systems and is the only advanced genetic system which permits detailed synaptic electrophysiology (Broadie and Bate, 1995; Keshishian et al., 1996; Zhong and Pena, 1995). Most importantly, a host of learning/memory mutants are already known in Drosophila which have defined defects in learning and specific stages of memory storage (Davis, 1996; Dezazzo and Tully, 1995). I propose to investigate the cellular and molecular basis of these learning/memory defects.
Drosophila is a classic genetic system for the investigation of the mechanisms underlying learning and memory (Davis, 1996; Dezazzo and Tully, 1995). The basis of this work is Pavlovian associative training paradigms. Associative conditioning is accomplished in tests using either reward or aversive unconditioned stimuli including tests of "classical" and "operant" conditioning (Tully and Quinn, 1985). Drosophila adults are placed in a training machine and given a choice between two stimuli, for example two odors (A and B). Odor A is always paired with a mild electrical shock and odor B either unpaired or given positive reinforcement with a food reward. Under these conditions, wild-type animals will quickly learn to avoid odor A after a series of training trials (10 is the usual number). If several training trials are spaced over time, the animals will remember the lesson for long-periods of time (Tully and Quinn, 1985). Different components of the learning to memory pathway can be defined based on experimental dissection; for example, memory retention following an electrical or temperature shock and dependence of memory retention on protein synthesis. Drosophila shows all the memory components found in other animal systems: the immediate learning phase, rapid short-term memory, amnesia-resistant intermediate-term memory and long-term memory lasting for most of the animal's lifetime (Dezazzo and Tully, 1995).
Genetic mutations can be generated in Drosophila which specifically block the ability to learn and memorize. These mutants are isolated based on an inability to learn in the associative training trials outlined above (Tully and Quinn, 1985). Briefly, such mutants are isolated as follows. First, the Drosophila genome is mutagenized with a chemical mutagen, irradiation or a transposable DNA element. Second, mutations on a particular chromosome are isolated and saved in a stable mutant stock. Third, each stock is then trained in the olfactory paradigm and assayed for the ability to learn and remember the training trials. Mutants impaired in this ability are isolated and characterized in detail. Genetic screens of this kind have uncovered dozens of genetic mutants with altered learning and memorizing abilities (Davis, 1996; Dezazzo and Tully, 1995). Many of these mutants show a specific change of only one component of the learning to memory pathway; for example, loss of only short-term, amnesia-resistant or long-term memory. These studies have shown that the process of learning and memory formation is genetically dissectable and have produced high expectations that the genetic and molecular mechanisms of learning and memory can be uncovered rapidly through this genetic approach.
The learning mutants isolated in Drosophila define a number of distinct phenotypic classes. Perhaps the most expected class is genetic mutations that result in gross abnormalities in the neural architecture, particularly in the learning centers of the brain. In Drosophila, the primary olfactory learning centers are the brain mushroom bodies (Davis, 1993; Heisenberg et al., 1985). A large number of learning mutants affect the structure of these organs. For example, mushroom body deranged (mbd) mutants show deranged mushroom bodies including a loss of axonal projections and aberrant neuronal cell body locations, and olfactory learning is lost or greatly reduced (de Belle and Heisenberg, 1995). In mushroom body miniature (mbm) mutants, the mushroom bodies are reduced in size and the animals show a corresponding decrease in learning and memorizing abilities (de Belle and Heisenberg, 1995). In no bridge (nob) mutants, the cerebral bridge between the left and right sides of the adult brain is absent and adult learning is extremely reduced (Bouhouche and Vaysse, 1991). In many of these cases, the molecular nature of the genetic defect remains unknown. However, in a few cases, the molecular characterization has already been completed. For example, the linotte (lio) gene is known to encode a protein-tyrosine kinase (Bolwig et al., 1995) and the minibrain (mnb) gene encodes a distinct protein kinase (Tejedor et al., 1995). Both genes mutate to cause abnormal brain development and are thought to be part of the signal transduction cascade required for the development of learning centers in the adult brain (Miyamoto et al., 1995).
A large number of the learning/memory mutants in Drosophila have normal brain morphology yet show severe learning and memorizing defects. One interesting class of mutations occur in genes encoding central neurotransmitters. For example, the amnesiac (amn) gene encodes a small neuropeptide (Feany and Quinn, 1995). Mutations in amnesiac show normal associative learning, but their ability to remember training decays four times faster than normal. This memory decay is biphasic; rapid for the first hour and much slower thereafter. Thus, it appears that short-term memory is defective with long-term memory being normal (Feany and Quinn, 1995; Quinn et al., 1979). A similar situation occurs in inactive (iav) mutants which show a selective decrease in octopamine levels in the adult brain (O'Dell et al., 1987). Finally, a recently defined gene, called K13sc, encodes a dopamine receptor (Han et al., 1996). K13sc mutants show a significant depression in learning/memory abilities suggesting that the dopamine receptor is involved in signal transduction processes leading to memory formation.
Many Drosophila learning/memory genes encode products involved in signal transduction pathways downstream of neurotransmission. The best studied examples are elements of the cAMP second messenger cascade. For example, the rutabaga (rut) gene encodes an adenylate cyclase with strong expression in the mushroom bodies of the adult brain (Duerr and Quinn, 1982; Livingstone et al., 1984). Mutants in the rut gene show subnormal learning immediately following training in negatively reinforced olfactory paradigms and rapid memory decay thereafter. Likewise, the dunce (dnc) gene, which encodes a cAMP-specific phosphodiesterase with strong expression in the mushroom bodies (Byers et al., 1981), plays a distinctive role in learning and memory mechanisms. Mutants in the dnc gene have increased levels of cAMP, apparently learn normally, but have abnormally short memory retention. Modifications of the original shock-odor testing system reveal that dnc is defective in short-term memory, with long-term memory similar to wild-type [Dezazzo, 1995 #19; Davis, 1996 #196]. These results indicate that the cAMP second messenger cascade plays an important role in short-term memory formation, reinforcing complementary results from experimental systems ranging from Aplysia to mice (Jessel and Kandel, 1993; Schacher et al., 1993). Moreover, genetic analysis in Drosophila has revealed other signal transduction cascades involved in learning and memory mechanisms. For example, the turnip (tur) gene encodes a GTP-binding protein of the rho-subfamily (Choi et al., 1991). Mutations in turnip result in blocked or impaired learning, very rapid short-term memory loss and slower long-term memory loss. Finally, disruption of the calcium/calmodulin dependent protein kinase II (CamKII) gene function leading to impaired learning performance in animals ranging from Drosophila to mice (Bach et al., 1995). Thus, genetic analyses has revealed that the cAMP, GTP and the Cam Kinase pathways all play roles in learning and memory mechanisms.
Many additional genetic mutants that effect learning and memory abilities have been isolated in Drosophila which have unknown functions and so cannot yet be categorized. These mutants include classes with both increased and decreased learning and memorizing abilities. For example, consolidated memory is lacking in radish (rad) mutants. Mutants in radish learn normally but display abnormally rapid memory decay following training (Folkers et al., 1993). Anesthesia resistant memory is cycloheximide-insensitive (does not require protein synthesis) and is disrupted in rad mutants. Long-term memory is cycloheximide-sensitive, but is not disrupted in rad mutants (Folkers et al., 1993). In contrast, the agnostic (agn) gene can be mutated so that certain mutant alleles show increased learning abilities whereas other mutant alleles show decreased learning (Savvateeva et al., 1993; Savvateeva et al., 1991). Thus, the agn gene seems to be pivotal in determining the extent of learning capabilities. Finally, many other genes fall into the class of disrupting learning/memory without molecular characterization or obvious anatomical defects. These mutations include tetanic (tta) (Orgad et al., 1989) and cabbage (cab) (Duerr and Quinn, 1982), both of which show blocked or impaired learning abilities.
In summary, a large class of learning and memory mutants have been isolated and behaviorally characterized in Drosophila. These mutants have allowed us to begin to genetically dissect the process of learning and memory formation (Davis, 1996; Dezazzo and Tully, 1995). Mutants have been identified which specifically block learning and/or short-term, amnesia-resistant and long-term memory. Many of these mutants fall into defined phenotypic and/or mechanistic classes including anatomical brain development, neurotransmission and signal cascade events within central neurons. This body of mutants represents an important resource which should lead directly to the cellular and molecular mechanisms underlying the ability to learn and remember.
Scientific Background: The NMJ as a Model Synapse for Learning and Memory Mechanisms in Drosophila
The Drosophila NMJ shows all the plastic features identified in central synapses. First, the NMJ shows use-dependent synaptic morphological alterations. Synaptic growth, branching and formation of presynaptic boutons (sites of neurotransmitter release) are all positively regulated by neuronal activity (Keshishian et al., 1996). Mutations in ion channel genes which artificially alter the amount of neuronal activity, lead to striking alteration in synaptic size and architecture (Budnik et al., 1990). Second, the NMJ shows use-dependent physiological plasticity. These features include transmission facilitation and post-tetanic potentiation (PTP) (Zhong et al., 1992; Zhong and Wu, 1991). This latter property is an experimentally-induced condition similar to the long-term potentiation recorded in the mammalian brain. Thus, the Drosophila NMJ appears to be a good model system for synaptic plasticity at both morphological and physiological levels.
All the Drosophila learning/memory genes which have been studied in detail are expressed at the NMJ and have roles there consistent with their proposed functions in central synapses of the brain (Davis, 1996). For example, dunce and rutabaga mutants are impaired in learning (Byers et al., 1981; Livingstone et al., 1984) and influence growth and disrupt synaptic facilitation and potentiation at the NMJ (Zhong et al., 1992; Zhong and Wu, 1991). Both mutants are defective in a step of the cAMP cascade and show abnormal responses to direct application of dibutyryl cAMP at the NMJ. These results suggest that the cAMP cascade plays a role in synaptic facilitation and potentiation and provide a direct genetic link between synaptic plasticity and learning/memory mechanisms. Likewise, inhibition of calmodulin kinase II (CamKII) affects learning (Bach et al., 1995) and CamKII is strongly expressed at the Drosophila NMJ where it influences sprouting and elaboration of synaptic terminals (Wang et al., 1994). These results suggest that CamKII regulates morphological synaptic plasticity involved in learning/memory mechanisms. Finally, the amnesiac gene encodes a PACAP-like neuropeptide involved in learning mechanisms (Feany and Quinn, 1995) and PACAP peptide modulates synaptic and K+ currents at the Drosophila NMJ (Zhong and Pena, 1995). PACAP modulation of the K+ current is mediated by coactivation of the Ras/Raf and rut cyclase pathways (Zhong, 1995). These results suggest that the amnesiac-encoded neuropeptide is playing similar modulatory roles centrally to regulate learning and memory mechanisms. The simplest hypothesis based on all of these studies is that the products of genes involved in learning and memory play conserved roles at the synapse, whether it is the peripheral NMJ or synapses in the mushroom bodies of the brain. In summary, both central and peripheral synapses appear to function using conserved cellular, genetic and molecular pathways. A large body of work suggests that detailed anatomical and functional assays at the NMJ will give direct insight into the synaptic mechanisms underlying learning and memory in the brain. However, despite the exceptional promise of this approach, very little work has yet been done. Of the 25+ defined Drosophila learning/memory mutants, only four (dunce, rutabaga and, indirectly, CamKII and amnesiac; see above) have been examined for synaptic defects. Moreover, a number of new mutants have recently been identified (see below) which also must be pursued with detailed analyses. Therefore, assays of synaptic structure, function and adaptive plasticity in Drosophila learning/memory mutants is a key priority in our investigation of learning/memory mechanisms.
Scientific Background: Our Earlier Work on the Synapse
The first, crucial stage in any genetic investigation is to gain a thorough understanding of the system before generating mutants. I spent several years charting the wild-type development of the Drosophila neuromuscular synapse (Broadie et al., 1993; Broadie, 1994; Broadie and Bate, 1993a). These studies included examining the morphological and functional development of the body muscles. Among other properties, I assayed the complement of voltage-gated ion channels in the muscle membrane and charted the development of muscle electrical properties during embryogenesis using a combination of current-clamp and voltage-clamp techniques (Broadie and Bate, 1993b). I examined the morphological and functional development of the NMJ. This work included intracellular dye-injection, antibody staining, immuno-SEM/EM and ultrastructural analyses (Broadie et al., 1995; Broadie and Bate, 1993a). It also included functional analyses of endogenous synaptic transmission during synaptogenesis, examination of the maturation of synaptic transmission parameters during synaptogenesis and definition of the transmission properties of the mature NMJ at the end of development (Broadie and Bate, 1993a; Broadie and Bate, 1993e). The combination of all of these studies has provided the techniques and knowledge required for the genetic approach outlined in this proposal and a framework for subsequent experimentation. It has also shown the similarity between Drosophila and higher mammalian synaptic development (Broadie and Bate, 1995; Broadie, 1994) and suggested that Drosophila would be a good model system for the genetic investigation of synaptic mechanisms.
The second stage in defining this genetic system was to use known genetic mutants to dissect the cellular interactions underlying synaptic and neuronal development. I used a neural fate mutant, prospero, to alter the developmental fate of motor neurons and genetically separate them from the developing muscles. I used this genetic tool to assay the innervation-dependence of muscle properties and postsynaptic development in the NMJ (Broadie and Bate, 1993c; Broadie and Bate, 1993d). Similarly, in collaboration with others, I have used a series of muscle fate mutants (twist, mef-2, myoblast city), to block muscle development at different defined points of maturation so as to assay the influence of the muscle on peripheral nerve formation and the presynaptic development of the NMJ (Prokop et al., 1996). Finally, I have used ion channel mutants to either increase (K+ channel mutants: Shaker, ether-a-gogo, etc.) of decrease (Na+ channel mutants: paralytic, no action potential, etc.) electrical excitability in the peripheral nerves during development. These genetic tools have allowed me to assay the activity-dependence synapse formation (Broadie and Bate, 1993e). A combination of these and similar experiments have allowed me to chart the cellular interactions occurring between the motor neurons and the muscles during the functional wiring of the neuromusculature. The important result from this body of work is that the developmental and functional interactions underlying synaptogenesis have been strikingly conserved between Drosophila and higher vertebrate systems.
Most recently, I have begun to characterize single gene mutations in this defined neural system. These genetic analyses have been of two types: 1) Forward genetics on novel genes discovered in Drosophila to have essential roles in neuronal/synaptic function, and 2) reverse genetics on the Drosophila homologues of genes already believed to function in the synapse based on work in other experimental systems. The forward genetic approaches have involved work on glial function (e.g. gliotactin (Auld et al., 1995) ), adhesion/intercellular communication molecules (e.g. connectin (Meadows et al., 1994) ) and synaptic function (e.g. rop, an n-sec1 homologue (Harrison et al., 1994) ). The reverse genetic work has largely been on synaptic function genes. So far, I have characterized the null mutant phenotypes of synaptotagmin, synaptobrevin, neurexin and syntaxin, among others (Broadie et al., 1994; Broadie et al., 1995; Broadie, 1995; Schulze et al., 1995; Sweeney et al., 1995). This work has conclusively shown that single gene mutations in neuronal function genes can be isolated in Drosophila. It has also shown that these mutants can be characterized in as great (or greater) detail as in the more historically established synaptic experimental systems (Broadie, 1995). Finally, it has established, without doubt, the strong conservation in genetic and molecular pathways in the neuronal function between Drosophila and higher mammalian systems (Bate and Broadie, 1995; Broadie and Bate, 1995).
A combination of all these studies has laid the essential groundwork for the present research proposal, which would not have been feasible prior to this work. First, this work has defined the synaptic system in detail at a cellular, morphological and functional level. Second, this work has provided the tools and knowledge for the detailed assessment of genetic mutants affecting neuronal and synaptic function. Third, this work has shown that single essential gene mutations can be isolated and meaningfully assayed in the Drosophila nervous system. Finally, this work has shown that neuronal and synaptic development and function has been conserved on cellular, molecular and genetic levels, proving that Drosophila is a good model system for the forward genetic investigation of the synaptic mechanisms.
Specific Projects
1. The role of integrins in synaptic mechanisms
Recent work in Drosophila has identified a novel alpha-integrin subunit called Volado which plays a dominant role in short-term memory formation. Weak hypomorphs in the Volado gene show specific learning defects in the adult fly. Stronger mutations in the gene cause severe incoordination which results in larval lethality. The Volado integrin protein is expressed in an intrigueing, dynamic pattern in both cntral synapses and the NMJ. Moreover, the three Drosophila position-specific (PS) integrins are also expressed at the NMJ synapse. All four integrin subunits are expressed at the synapse only during post-embryonic staages. These data are consistent with a role for integrins in synaptic plasticity mechanisms. What is the role of the integrins in synaptic plasticity? This investigation will require a combination of molecular genetics, electrophysiology and ultrastructural anatomy.
2. The role of the 14-3-3 proteins in synaptic mechanisms
Recent work in Drosophila has identified a homologue of the 14-3-3 protein family encoded by the Leonardo gene. Weak hypomorphs in Leonardo show specific defects in learning in the adult fly. Stonger mutations in the gene result in embryonic lethality. The Leonardo protein is strongly expressed at both central synapses and the NMJ from an early stage of synaptogenesis. Work in other systems has shown that the 14-3-3 proteins interact with RAF in the MAP kinase pathway. The proteins have also been implicated in the regulation of the actin cytoskeleton and are found associated with synaptic vesicles.Therefore, these proteins are prime candidates for regulators of synaptic plasticity mechanisms underlying learning. What is the role of 14-3-3 proteins in synaptic plasticity? This investigation will require the analysis of genetic interactions, molecular analyses, electrophysiology and ultrastructural anatomy.
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