Peter B. Sargent - US grants

The long term goal of our research is to understand the nature and the consequences of diversity in the expression of neuronal nicotinic acetylcholine receptors (AChRs). Recent molecular biological studies have identified a family of putative AchR genes in the nervous system. However, there is little evidence that AchRs generally serve the same function in the central nervous system that they do in the peripheral nervous system, where they underlie fast, excitatory synaptic transmission. We propose to use both structural and functional approaches to examine AchRs in the chicken ciliary ganglion, where monoclonal antibodies specific for each of the known nicotinic AchRs subunits are available and where AchRs have been found both on the presynaptic terminals and on their target neurons. The target ciliary ganglion neurons make several AchRs classes, some of which may serve novel functions. We will use subunit-specific antibodies to chicken AchRs and laser scanning confocal microscopy to examine the distribution of AchRs subunits on the neuronal surface. At the cellular level, we will examine whether there are differences in AchRs expression between the two neuronal types in the ciliary ganglion (ciliary, choroid). At the subcellular level, we will identify which AchRs subunits are located at synaptic sites, which subunits are located extrasynaptically, and which are located presynaptically. This information will be compared with the results of functional studies that will determine the relative importance of different AchRs classes in underlying rapid excitatory synaptic transmission in the two neuronal populations. At the molecular level, we will use fluorophore-tagged antibodies and fluorescence resonance energy transfer (FRET) to examine the proximity of subunits to each other. This technique should allow us to learn which sudunits are assembled into AchR oligomers. We will pay particular attention to presynaptic nicotinic AchRs, which, although virtually uncharacterized to date, may represent the predominant form of AchR in the central nervous system. We will characterize presynaptic AchRs on the large, calyceal endings in the ciliary ganglion by whole-cell recordings to learn whether these receptors are activated by nerve-released transmitter. In addition, we will characterize presynaptic AchRs by immunofluorescence and confocal microscopy to learn what their subunit composition might be. The studies should enhance our understanding of the structure and function of neuronal nicotinic AchRs.

The long-range objective of my research is to understand the mechanisms that underlie the formation of synapses between neurons during embryogenesis. The specific objectives of this proposal are 1) to complete an analysis of synaptic growth in the cardiac ganglion of postmetamorphic Xenopus laevis and 2) to study the initial formation and the growth of synapses on developing cardiac ganglion cells in larval animals. In mature animals the innervation of individual neurons is dominated by a preganglionic axon which supplies all the synaptic boutons found on the ganglion cell body. During postmetamorphic life the total area of synaptic contact grows by an increase in bouton number, and the increase in bouton number is exactly matched by an increase in ganglion cell size. By studying transmitter release from the principal synaptic input to ganglion cells and by counting boutons on physiologically characterized cells, we shall determine whether boutons represent equivalent amounts of transmitter release and whether they represent the functional unit of synaptic growth. By studying the innervation of both diploid and larger tetraploid ganglion cells in chimeric animals, we hope to determine the basis of the compensatory growth of cell size and bouton number in postmetamorphic animals. The arrival of preganglionic axons in the developing cardiac ganglion will be visualized by filling techniques, and nascent synapses will be studied both anatomically and physiologically. How do the structure and release properties of newly formed boutons compare with those of mature ones? The growth of synapses in larval animals will be analyzed to learn whether boutons are the units of synaptic growth from the very onset of synaptogenesis. Synapse elimination will be investigated by physiological techniques, and we shall ask whether the ganglion cell body is ever innervated by more than one preganglionic axon. Finally, we shall search for transient connections between ganglion cells. Such connections are ordinarily suppressed by preganglionic innervation but may appear early in development. These studies will increase our understanding of how synapses between neurons form and grow during development. They will also identify the stages when the process of synapse formation may go awry.

The objective of this research is to characterize the relationship between synaptic structure and function in the cardiac ganglion of the frog Xenopus laevis. Each parasympathetic neuron in the ganglion is innervated by an axon which deposits several synaptic boutons on its cell body. Extracellular recording techniques will be used to monitor the release of transmitter from individual synaptic boutons. Voltage clamp techniques will be used to measure the quantum content, m, and to calculate the number of quanta available for release, n, which will be compared to the number of active zones. The total number of synaptic boutons on the ganglion cell body is correlated with target cell size in adults. Voltage clamp techniques will be used to learn whether inputs with more synaptic boutons elicit proportionately more synaptic current when stimulated. The functional significance of the observed parallel increase in both cell size and bouton number during postmetamorphic growth will be assessed, again with voltage clamp techniques. If transmitter release from individual boutons elicits a constant amount of synaptic current, regardless of the stage of growth, then synaptic boutons presumably represent functional units of synaptic growth. These studies should increase our understanding of the relationship between the structure, function and growth of interneuronal synapses.

The long-range objective of this research is to understand how innervation regulates the number and distribution of nicotinic acetylcholine receptors (AChRs) on neurons. Our approach has been to use a simple and tractable system, the frog cardiac ganglion, where AChRs on parasympathetic neurons can be analyzed by immunocytochemistry, autoradiography, and biochemistry, and where the influence of innervation upon AChRs can be monitored during denervation and reinnervation in adult frogs (Rana) as well during initial innervation in embryos (Xenopus). In the past award period we found that denervation does not increase the number of AChRs on the surface of cardiac ganglion cells, measured by the binding of 125I-neuronal bungarotoxin, a snake toxin that blocks AChR function. Denervation does increase the sensitivity of cardiac ganglion cells to acetylcholine (ACh) applied via a micropipet; this effect appears to be due to a reduction in the effectiveness of the hydrolytic enzyme acetylcholinesterase (AChE) and not to a change in the number of functional AChRs on the cell surface. We have three specific aims. In the first, we will examine the consequences of denervation and reinnervation upon the distribution of AChRs and AChR clusters in adult Rana pipiens. In the second aim, we will determine whether denervation supersensitivity can be explained by a reduction in the number of AChE molecules in the extracellular space surrounding ganglion cells and/or a change in their kinetic properties. We will also examine whether the reduction in ACh sensitivity that occurs during the first stages of reinnervation is due to a change in AChE, in AChRs, or in both. In the third aim we will examine whether AChRs are expressed on developing cardiac ganglion cells in Xenopus laevis before, during, or after cells are contacted by preganglionic axons. We will determine whether AChRs appear on neurons that have never been innervated by preganglionic axons, and, if so, whether innervation enhances AChR expression. These studies should increase our understanding of how neuronal nicotinic AChRs and ACh sensitivity are regulated by innervation.

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of our research is to understand the nature and the consequences of diversity in the expression of neuronal acetylcholine receptors (nAChRs). Recent molecular biological studies have identified a family of putative nAChR genes in the nervous system. However, there is little evidence that nAChRs generally serve the same function in the central nervous system that they do in the peripheral nervous system, where they underlie fast, excitatory synaptic transmission. We propose to use both structural and functional approaches to examine nAChRs in two preparations from the embryonic chicken, where monoclonal antibodies specific for each of the nicotinic receptor gene products are available: the lateral spiriform nucleus (SpL), located in the mesencephalon, and the ciliary ganglion, located in the orbit. We will use subunit-specific antibodies to chick nAChRs and immunocytochemical techniques to examine the distribution of nAChR subunits among and within neurons. At the cellular level, we will examine whether all cells within the neuronal population (SpL, or ciliary ganglion) express the same subset of subunits. At the subcellular level, we will examine the distribution of receptor subunits at the neuronal surface in order to determine which subunits are located at synaptic sites, which subunits are located extrasynaptically, and which may be transported to axon terminals. Finally, at the molecular level, we will use fluorophore-tagged antibodies and fluorescence resonance energy transfer (FRET) to examine the proximity of subunits to each other. This technique should allow us to learn which subunits are assembled into receptor oligomers. We will pay particular attention to presynaptic nicotinic receptors, which, although virtually uncharacterized to date, may represent the predominant form of nAChR in the central nervous system. We will examine by immunocytochemical techniques whether presynaptic nicotinic receptors are found on the surface of SpL axon terminals that project to the optic tectum and whether they are present on the terminals of preganglionic neurons that project to the ciliary ganglion. Should presynaptic receptors be found in the ciliary ganglion, we will attempt to characterize these receptors functionally by patch clamp recordings. The studies should enhance our understanding of the structure and function of neuronal nicotinic receptors by (1) identifying which subunits associate to form receptor oligomers likely to serve both postsynaptic and presynaptic functions and (2) examining the structure and function of nicotinic presynaptic receptors. It is likely that a fuller understanding of the structure and function of both postsynaptic and presynaptic nicotinic receptors will be essential for understanding nicotine's effects on the nervous system and of the basis of nicotine tolerance and dependence.

DESCRIPTION (provided by applicant): The long term objective of our work is to understand how nicotinic receptors participate in signaling in the nervous system. Nicotinic receptors are found in both the peripheral and central nervous systems, where they are important for a variety of functions, including control of movement, control of excitability, cognition, memory, anesthesia, analgesia, and reward. The specific aim of the current proposal is to explore, using a combination of experimental and modeling approaches, fundamental mechanisms by which nicotinic receptors participate in millisecond time scale signaling. These experiments will be done in a highly accessible model synapse in the embryonic chicken ciliary ganglion. Fast synapses generally operate by the release of quanta of transmitter, via exocytosis, at highly specialized sites on a nerve terminal and their interaction with high-density clusters receptors in the postsynaptic membrane opposite those sites. In the ciliary ganglion, however, there are large numbers of receptors not located opposite specialized sites from which transmitter is known to be released. The rapid time course with which these receptors are activated following release suggests that transmitter must be released directly on them;such release is therefore ectopic, or non-synaptic. A recent modeling study, based on numerical simulations performed on accurate three-dimensional representations of the synaptic volume (MCell), has supported the possibility of ectopic release in this system. In the current proposal we will refine the MCell model, using a variety of measurements on geometry and on receptor distribution, obtained using immuno electron microscopy, and will subject ectopic and conventional models to increasingly demanding tests of their ability to recapitulate actual data sets obtained using whole cell recording methods. We will also test whether transmitter release in this system has the characteristics found for another synapse that has been recently shown to operate by ectopic release. These studies should increase our understanding of the fundamental process by which nicotinic receptors on nerve cells participate in rapid time scale signaling. These studies may also be of use in understanding the basis of pathological states brought about, or exacerbated by, loss or alteration of receptor function (e.g., seizure disorders, neurodegenerative disease).