Synaptic transmission - the process whereby electrical signals are conveyed between a neuron and its target cell - is the fundamental activity which allows animals to possess an integrated nervous system. Behavior is largely determined by the pattern of synaptic connectivity between cells and by information processing that occurs at the synapse. Movement is controlled by the output of central neural circuits, the information mediated to the muscles through a specialized synapse, the neuromuscular junction (NMJ). Plastic changes in synaptic connectivity and transmission strength allow the nervous system to adapt to environmental challenges. Such plastic synaptic processes are believed to underlie higher brain function and form the cellular basis of learning and memory. Thus, understanding the molecular mechanism regulating the construction of synapses is of central importance to the field of neurobiology.

Essentially all of our knowledge of synaptic development and function comes from the vertebrate NMJ. A variety of new techniques - brain slices, culturing systems and optical monitoring assays - have increased our ability to analyze central synapses but, nevertheless, the difficulty of studying identified synapses in the CNS remains daunting. By comparison, NMJs are simple, large and easily accessible to experimental manipulation. Moreover, the NMJ can be assayed as it develops both in vivo and in a variety of culture systems. The formation of the NMJ entails six primary steps: 1) recognition of the muscle target by the growth cone, 2) cessation of growth, 3) growth cone conversion to a presynaptic terminal, 4) signaling from the terminal to the postsynaptic cell, 5) postsynaptic specialization, and 6) retrograde signaling from the postsynaptic cell to maintain the synapse. Of these steps, we only have detailed molecular understanding of steps 4 and 5, the formation of postsynaptic specializations.

Signals from the presynaptic terminal take two forms: a receptor clustering signal and a signal to increase transcription of appropriate postsynaptic genes. Biochemical methods, mostly utilizing the abundant specialized NMJs in the electric organ of the marine ray (Torpedo), have been instrumental in identifying these signals and their receptors. For example, the presynaptic cell induces the differentiation of the postsynaptic acetylcholine receptor (AChR) field through four complementary signaling pathways: 1) Agrin induced clustering, 2) trophic factor induced synaptic AChR transcription, 3) activity-dependent suppression of extrasynaptic AChR transcription and 4) modification of AChR channel properties (see Figure, below). First, the Agrin protein is secreted from the presynaptic terminal to induce the aggregation of receptors in the muscle membrane. Agrin imbedded in the synaptic basal lamina binds to a Dystrophin-associated glycoprotein complex in the muscle membrane (a-dystroglycan, b-dystroglycan, Dystrophin, Utrophin), to stimulate tyrosine phosphorylation (TyrP) in the cytoplasmic domain of an AChR subunit and anchor the AChR to the underlying actin cytoskeleton. The Rapsyn protein is strongly implicated in this clustering/anchoring mechanism and Rapsyn mutant mice fail to show the postsynaptic specialization. Second, secreted neural trophic factors up-regulate AChR synthesis. Two possible signals are AChR-inducing activity (ARIA) and Calcitonin gene-related peptide (CGRP). ARIA is homologous to the mammalian gene Heregulin which acts via tyrosine phosphorylation of target proteins.CGRP stimulates AChR synthesis and insertion via a G protein induced elevation in cAMP. Third, activity-dependent suppression of extrasynaptic AChR transcription, an effect apparently overcome in the synaptic nuclei by the up-regulating trophic factor(s). The activity-induced increase in intracellular calcium is believed to increase protein kinase C (PKC) activity which, in turn, may regulate the level of transcription factors like MyoD. Fourth, the postnatal alteration of the AChR channel properties. At least two changes occur: 1) synaptic AChRs are metabolically stabilized and 2) the subunit composition of the AChR changes with the substitution of an adult (epsilon) subunit for an embryonic (gamma) subunit. The AChR stabilization occurs through calcium-dependent protein phosphorylation and the AChR subunit switch occurs at a transcriptional level and appears to be induced by both trophic factors (e.g. ARIA) and activity-dependent mechanisms. In comparison to this molecular description, considerably less is known about the mechanisms of presynaptic synaptogenesis. However, again inductive interactions between the synaptic partners appears to be a driving force, since contact with the target muscle triggers the differentiation of synaptic characteristics in the motor terminal, pointing to the existence of retrograde signals. Many retrograde signaling pathways have been proposed, including gap junctions, secreted growth factors and selective adhesion. Three possible retrograde signaling pathways are particularly interesting: 1) a calcium signal, 2) arachidonic acid (AA), and 3) S-laminin. First, a presynaptic calcium transient is induced by muscle-membrane associated molecules. This signal enhances local calcium influx via a cAMP-dependent protein kinase. These data suggest that the muscle target retrogradely regulates calcium accumulation in the presynaptic nerve terminal by inducing the local activity of a presynaptic cAMP-dependent protein kinase cascade and that this pathway, in turn, regulates differentiation of neuronal signaling. Second, AA is believed to be secreted from the muscle to act as a retrograde signal acting through a G-protein signaling cascade in the presynaptic terminal. Third, S-laminin protein is synthesized by the muscle and inserted into the synaptic basal lamina to form an ECM attachment site for the presynaptic terminal. Mice with a mutation in the S-laminin gene show severe NMJ abnormalities; they have relatively unbranched terminals, form few active zones, have dispersed synaptic vesicle concentrations and a reduction in the machinery of transmitter release. Thus, S-laminin plays a key role in presynaptic differentiation, though the mechanisms of this action remain unclear.

In summary, the vertebrate NMJ model of synaptogenesis has revealed a hierarchy of inductive signals, both anterograde and retrograde, and a complex pathway of regulation involved in the cooperative differentiation of the signaling and receptor fields in the two synaptic partners. The best studied aspect of synaptogenesis, the development of a postsynaptic receptor field, occurs through the directed aggregation of AChRs and a complex degree of transcriptional regulation of AChR subunit mRNA, involving both activity-dependent suppression and local activation mediated by neural trophic factors. Thus, the NMJ model has provided a framework in which to decipher the mechanisms of synaptogenesis. However, considerably less is known about presynaptic synaptogenesis and , in general, we are a very long way from understanding the genetic and molecular mechanisms of synapse formation. It is our ultimate goal to understand the in vivo function of the constituent molecules in the developmental cascade underlying synaptogenesis. Ideally, we would now systematically remove or alter the basic elements of this cascade in order to assess their exact role in synaptic development. Unfortunately, the genetic and molecular techniques required to accomplish this analysis are inherently difficult in the vertebrate system. I would like to suggest that the Drosophila NMJ provides a new experimental model with which we can proceed.

Abstract of Research

Nervous system formation depends upon the construction of synapses, the communication links which transfers information between cells and therefore allows the establishment of an integrated network. We are undertaking a systematic molecular genetic investigation into the mechanisms of synapse development and adaptive plasticity. This approach includes both forward and reverse genetics in the classic system of the fruit fly, Drosophila melanogaster. Analysis of synaptic mutations takes place at the neuromuscular junction (NMJ), a classic synaptic system valued for its simplicity and accessibility. This research project has three related components. First, a systematic forward genetic screen of the Drosophila genome for essential synaptic genes. This will be the first systematic study of the molecular and genetic mechanisms of the synapse. Second, a reverse genetic approach to synapse formation mechanisms. This approach involves mutating Drosophila homologues of genes already implicated in synapse formation from work in other systems. The function of such genes can be quickly determined through mutant analyses. Third, a genetic investigation of the mechanisms of mainted synaptic developmental potential (plasticity), particularly in relation to higher brain function such as learning and memory. In Drosophila, mutants in learning and memorizing abilities can be directly screened for and the synaptic causes of these defects assayed (see section on plasticity mechanisms). Our system is the only one available in which systematic genetic analyses can be married to detailed assays of synapse function and structure. Since synaptic mechanisms have been highly conserved through evolution, the information gained in Drosophila will be directly applicable to higher vertebrate neural development. This research has important impacts in areas ranging from neural circuit formation to neurotransmission to higher brain functions such as learning and memory.

Background on the Drosophila NMJ system

Drosophila is one of the classic genetic systems and has been used to address a number of fundamental biological questions. In neuroscience, this system has been used widely to look at early neural development, mature neural function and behavior. However, the fundamental areas of synaptogenesis and neural circuit formation had not been examined in Drosophila due to the lack of a working system and experimental techniques. My thesis work was aimed at developing a system for the genetic analysis of synaptogenesis. I choose the neuromuscular junction in the embryo and developed all the physiological and morphological techniques with which to chart normal synaptogenesis in this system. First, I used these techniques to monitor normal synapse formation; both at a morphological (LM and EM) and physiological level in the pre- and postsynaptic cells. Second, I used a number of mutants to dissect developmental interactions during synaptogenesis. For example, I used ion channel mutants to modify electrical activity and define activity-dependent steps in synapse formation. Likewise, I used neural fate mutants to interrupt or delay innervation to define innervation-dependent steps in synaptogenesis. Finally, I demonstrated that single gene mutations show distinct, quantifiable defects in synaptic transmission. My Ph.D. work laid the foundation for the systematic investigation of synapse formation and function which I am now pursuing.

As a post-doctoral fellow, I focused on the genetic dissection of synapse function. Several labs in Europe and the US had been using reverse genetics techniques to identify and mutate Drosophila homologues of important synaptic genes involved in neurotransmission. I collaborated with these groups to define the role of such genes in synaptic transmission using the embryonic NMJ system I had developed as a Ph.D. student. My aim was to use electrophysiological and morphological techniques to characterize mutations in essential synaptic genes, all of which mutate to cause early lethality. I characterized mutants in synaptotagmin and showed that it is not the predicted sole Ca++-sensor but plays an important role in excitation-secretion coupling. I characterized, rop (the n-sec1 homologue) and showed that it is essential to synaptic transmission. I characterized gliotactin and showed that it is required in peripheral glia to insulate the motor axons from ion imbalances in the blood. I characterized syntaxin and showed that it is not the predicted t-SNARE in vesicle docking but rather plays an essential role in vesicle fusion during synaptic transmission. Finally, I characterized transgenic lines in which tetanus toxin was expressed to eliminate synaptobrevin at the synapse. I showed that synaptobrevin is not the predicted v-SNARE in vesicle docking but rather plays an essential role in neurotransmission between docking and Ca++-dependent fusion. My post-doctoral work was the first description of synaptic mutants described in any system. The work on mouse and C.elegans that came out soon after (on synaptotagmin mutants) confirmed my phenotype descriptions and provided proof that synaptic mechanisms have been highly conserved through evolution. I am presently continuing the genetic analysis of synapse function using a combination of forward and reverse genetics.

Rationale of Research Project

We take a genetic approach to understanding synaptic mechanisms. In particular, we use two general approaches; reverse and forward genetics. In the reverse approach, genetic mutations are targeted to a gene already implicated in synaptic processes from earlier biochemical or pharmacological purification. In the forward approach, the genome is mutated at random and new synaptic genes are uncovered based purely on their mutant phenotypes.

We have several major reverse and forward genetic investigation currently in progress. First, a major thrust of our effort is directed to forward genetic screens designed to uncover new genes involved in synapse formation. Our intention is to systematically screen the genome to uncover all the genes essential for the construction of a synapse. This work involves designing and executing genetic screens, characterizing mutants with electrophysiological and anatomical approaches, and using molecular biology techniques to isolate and characterize the gene and gene product thus identified. Second, another major effort is to use reverse genetic approaches to characterize the role of genes involved in synapse formation. In the past, we have collaborated with other laboratories who have isolated mutants in important synapse function genes and I have used these mutations to characterize the roles of particular gene products in neurotransmission. This work is continuing and expanding and compliments the analyses of novel synapse function genes uncovered in our forward genetic screens. Third, we are interested in synapse plasticity, particularly as it relates to learning and memory. A new major effort in our research is to characterize the synaptic role of genes implicated in learning and memory mechanisms.

We are currently engaged on a saturation mutagenesis of the Drosophila genome for novel genes essential to synapse formation. The initial screen is focussing on the 3rd chromosome (40% of the genome). The mutagenesis is being conducted with a non-selective chemical mutagen (EMS) and continue until every isolated locus has sustained 2 or more independent hits (estimated at 7-10,000 mutant lines). A secondary screen is being conducted using P-elements. This screen will not be saturating but should provide convenient molecular handles on genes of interest. Both screens are conducted in a parent line marked with muscle and nerve reporter gene constructs to eliminate mutants with severe morphological abnormalities. Isolation of mutants will involve a 5-nested screening protocol involving: 1) a screen for pre-adult lethality (using adult visible markers), 2) a screen for embryonic/early postembryonic lethality (hatching assays), 3) a screen for gross morphology (muscle/neural reporter gene constructs, antibody stains), 4) a screen for movement/coordinated locomotion and 5) functional screens using physiological techniques. Selected lines will be embryonic/early postembryonic lethals with normal morphology but movement/locomotion defects attributable to dysfunction in the peripheral nerves, muscles or NMJ. Fine analyses will be required to limit this pool of mutants to those affecting only the synapse. In earlier work, we have developed whole-cell patch clamp techniques for recording from embryonic lethal mutant synapses [2]. we have also developed techniques to stimulate and assay synapse function. To complement these functional studies, we have developed a range of LM, SEM and TEM techniques to assay the morphology and ultrastructure of the synapse [3,5]. A combination of these techniques will allow us to isolate novel synaptic mutants and pinpoint the pre- or post-synaptic location of the genetic defect. Novel synaptic mutants will be complementation tested and mapped to a chromosomal location on the giant polytene salivary chromosomes using standard Drosophila methods. Novel mutations with interesting functional defects will be cloned and molecularly characterized using well established Drosophila techniques.

We plan to use reverse genetics to complement our forward screens. Synapse formation has been highly conserved through evolution [6]. Individual synaptic genes show 70+% identity between Drosophila and higher vertebrates and their functional roles appears highly similar [6]. We will continue with the reverse genetic approach of isolating Drosophila homologues of important synaptic genes and then mutating them to examine their functional role in synaptic transmission. In the past, we have focussed on genes playing roles in neurotransmission (see Projects on Synapse Function). We propose to expand our studies to include genes implicated in synapse formation mechanisms (see above). As previously, we will focus our energy on the phenotypic description of mutant defects using functional and anatomical studies at the embryonic NMJ.

Our effort will be divided into two components; 1) the detailed analysis of previously isolated putative synapse formation mutants and 2) the characterization of newly generated mutants. For the first effort, we will focus on phenotypic characterization of mutants within the known synaptogenesis pathways. For the second effort, we will need to decide which genes to target and mutate those genes using established Drosophila techniques. This research will require geneticists, molecular biologists and, latterly, cell biologist interested in the characterization of the mutant phenotypes.

Scientific References