Sukumar Vijayaraghavan - US grants

DESCRIPTION (Applicant's Abstract): Nicotine is a habit forming drug that is widely used in our society. The widespread distribution of the nicotinic acetylcholine receptors (AChRs) in the brain suggests that the dynamics of nicotine use in humans is likely to be complex. Understanding mechanisms underlying nicotines action in the CNS must begin with the elucidation of the functions of various CNS-AChR subtypes. The findings that many AChRs are highly permeable to calcium makes them candidates for mediating many essential calcium-dependent events in neurons by altering intracellular calcium concentrations ([Ca]i). The main aim of this proposal is to examine the calcium signalling properties of a subtype of hippocampal AChRs that bind the snake venom toxin alpha-bungarotoxin (a-Bgt-AChRs). Calcium permeability of the a-Bgt-AChR channel will be determined from cultured hippocampal neurons using direct calcium fluororescence measurements using electrodes filled with the fluo-3 pentapotassium salt coupled with whole cell voltage clamp determinations. The ability of aBgt-AChRs to alter [Ca]i in response to nicotine application in intact neurons will be examined by calcium imaging from cells loaded with fluo-3 AM. The temporal patterns of calcium signals generated by the various cholinergic receptors on these neurons and their interactions with other sources contributing to [Ca]i will be examined in detail. This will elucidate the role aBgt-AChRs play in maintaining calcium homeostasis in hippocampal neurons. The ability of aBgt-AChRs to regulate calcium dependent events like activation of phospholipase A2, CaM kinases and calmodulin sensitive adenylate cyclases will be studied using established biochemical procedures and spatial requirements of calcium increases for their activation determined. These studies will establish aBgt-AChRs as candidates for mediating calcium dependent events in the hippocampus. Further the ability of calcium to directly modulate aBgt-AChR function and thus maintain a rapid and tight control on cellular calcium levels will be examined using whole cell voltage clamp measurements. The structural determinants of calcium dependent regulation of aBgt-AChR function will be examined by site-directed mutagenesis using the alpha7 gene and the Xenopus oocyte expression system. The effects of altering a putative EF-hand domain on the function of the gene product will be examined by 2-electrode voltage clamp techniques. These studies will be the first detailed study of CNS aBgt-AChRs at the molecular and cellular level. Results obtained in this project will suggect roles for aBgt-AChRs in regulating hippocampal functions.

DESCRIPTION (provided by applicant): The role of central cholinergic pathways in a variety of cognitive behaviors and its role in neurodegenerative diseases has been well documented. It is also recognized nicotine's actions on the brain involves disruption of endogenous cholinergic signaling. In spite of the importance of this signaling pathway, almost nothing is known about the properties of cholinergic transmission at a cellular level. This proposal will establish a model system for the examination of cholinergic transmission and elucidade some fundamental properties of nicotinic transmission in the brain. Transgenic mice expressing GFP or GFP-tau protein under the control of the choline acetyltransferase promoter (ChAT) will be generated. The selective targetting of the transgene to cholinergic neurons in the brain will be determined by immunocytochemical techniques using antibodies against ChAT and GFP. Using the ChAT GFP-tau mice, evoked nAChR responses on interneurons at the stratum radiatum of the hippocampus will be examined by stimulating cholinergic fibers in septo-hippocampal slices using a patch electrode. The effects of acetylcholine esterases on nAChR responses will be examined in order to characterize transmitter diffusion and its ability to act on non-synaptic receptors. Quantal content, probability of release, and number of sites will be determined. Using whole cell voltage-clamp recordings from CA3 pyramidal cells of the hippocampus, the modulation of glutamate release from mossy fiber terminals by synaptically released acetylcholine will be examined. Direct monitoring of ACh release from presynaptic boutons will be monitored using styryl dyes and live cell imaging from acute slices as well as from septal neurons in culture. These studies will be the first detailed examination of cholinergic transmission in the central nervous system and will also provide an invaluable tool to researchers for studying cholinergic processes. The proposal will also begin examining the role of central cholinergic system in mediating nicotine addiction.

DESCRIPTION (provided by applicant): Olfactory dysfunction is one of the earliest symptoms of neurodegenerative disorders like Alzheimer's disease and Parkinsonism. A dominant hypothesis is that these diseases have a primary cholinergic etiology. Understanding how this system modulates olfactory processing, therefore, sets the stage for early detection and intervention. Acetylcholine modulates olfactory perceptual learning and working memory. This modulation of olfaction results from the incoming cholinergic innervation from the basal forebrain and subsequent activation of two classes of cholinergic receptors - the muscarinic acetylcholine receptors and nicotinic acetylcholine receptors (nAChRs). In this proposal, we examine the role of nAChRs in modulating the excitability of mitral cells in the mouse main olfactory bulb. Using a synergistic approach incorporating olfactory slice electrophysiology and awake behaving recording we ask how interactions between various nAChR subtypes in the olfactory bulb result in the modulation of the glomerular output to incoming odor signals thereby affecting behavior. The proposal examines this question at multiple levels. In olfactory bulb slices we examine the nAChR modulation of the glomerular microcircuit using gene knockout mice, pharmacology, electrophysiology and calcium imaging. We test the model that nAChR modulation of glomerular output is due to efficient feedback inhibition from periglomerular (PG) cells. We also test a novel idea that effective inhibition of glomerular output is driven by amplification of GABA release due to PG-PG interactions. Using optogenetic approaches, we will examine how the glomerular circuit is modulated by ACh released from cholinergic fibers. Timing of transmitter release, relative to incoming odor input, will be examined both in slices, as well as with recordings from awake behaving animals. The research proposed here will greatly enhance our understanding of the principles governing cholinergic modulation in the brain to lay the foundation for rational drug design to treat neurodegenerative disorders.

DESCRIPTION (provided by applicant): Nicotine addiction is a major health problem in our society and accounts for millions in health care costs. A fundamental prerequisite for understanding the actions of nicotine is to elucidate the physiological role for nicotinic acetylcholine receptors (nAChRs) in the brain. Recently, it has been shown that nAChRs are expressed in astrocytes. This proposal will examine the role of astrocytic nAChRs on synaptic signaling in the CA3 region of the mouse hippocampus and how these receptors contribute to glia-neuron interactions in the brain. The proposal will address two issues. First, we will characterize nicotinic signaling in astrocytes using acute hippocampal slices from mice expressing GFP linked to the glial fibrillary acidic protein (GFAP). Electrophysiological analyses will be carried out examining nicotinic currents on astrocytes. Calcium imaging will be used to elucidate the details of calcium signaling in these cells, including the contributions from various sources of calcium within astrocytes. nAChR-induced intra- and inter-cellular calcium waves will be examined. We will use transgenic mice that express tau-GFP driven by the cholineacetyl transferase (ChAT) promoter in order to label cholinergic axons. The ability of local stimulation of cholinergic fibers to induce nAChR currents and calcium signals in astrocytes will be examined. These studies will establish the existence of functional nAChRs in astrocytes and their ability to transduce signals in these cells in response to both nicotine and endogenous acetylcholine. The second part of the proposal will address a unique signaling resulting in an action potential- independent form of transmission at the mossy fiber-CA3 synapse in the hippocampus. We will examine the role of astrocytes in mediating or modulating this form of short-term plasticity by its action on the presynaptic mossy fiber terminals. A combination of electrophysiology and calcium imaging will be used in these studies. It is increasingly becoming apparent that addiction is a problem of hedonic homeostasis that involves multiple regions of the brain, not just the mesolimbic dopaminergic system. Understanding the totality of nAChR actions in the brain, in order to arrive at a rational drug design to combat smoking must include the understanding of these receptors on glia as well as neurons. This proposal makes a start in that direction. PUBLIC HEALTH RELEVANCE: Methods that have been developed to combat smoking have been inadequate mainly because of our lack of knowledge on how this drug affects brain functions. It is now being recognized that drugs of abuse like nicotine have far reaching consequences for the brain. They affect not just functions of nerve cells in the brain but also support cells known as glia. These support cells have been shown to play a very active role in determining how the nerve cells function. Here we study the effects of nicotine on astrocytes, a type of glial cells, and determine how nicotine, acting via these cells can affect the functioning of, and signaling by neurons. This study will open a new direction in understanding the consequences of smoking for the brain and aid in the development of better pharmacological interventions to assist people in breaking nicotine addiction.

DESCRIPTION (provided by applicant): Nicotine is a drug that is widely abused in this society costing millions of dollars in health care. It is clear that the starting point of any pharmacological approach to target nicotine addiction must begin with the understanding of the mechanistic details of the action of this drug. It is known that the actions of nicotine are mediated by its ability to activate nicotinic acetylcholine receptors (nAChRs). In this proposal we ask what physiological functions of nAChRs in the brain are. One effect of activation of nAChRs is that they increase neurotransmitter release in the brain, possibly facilitating synaptic plasticity. This effect is commonly mediated by a subset of nAChRs, one containing the alpha7 subunit (a7-nAChRs), a subset that is very effective at raising intracellular calcium levels and mediating calcium-dependent signal transduction in the brain. In this proposal we examine the mechanisms underlying increased glutamate release mediated by these receptors at the mossy fiber-CA3 synapses in the hippocampus. In the previous funding period we show that nAChR activation results in a dramatic increase in frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) dependent on ER store calcium. In this proposal we investigate whether there are ER calcium stores at the mossy fiber terminals and what their properties might be using fluorescence microscopy. We then ask whether the mechanisms underlying nAChR modulation of release are specific to mEPSCs or whether they can also influence evoked release at these synapses. We will also investigate the nature and population of vesicles that are mobilized by nAChRs using styryl dyes and photoconversion followed by electron microscopy. These studies will arrive at the mechanistic details of a novel form of plasticity discovered in our lab. Lastly, we investigate the biological significance of the effect using relevant doses of nicotine and a newly developed transgenic mouse model where cholinergic fibers are labeled with GFP-tau. These studies will set the stage for the development of pharmacological tools and therapeutic strategies to combat nicotine addiction, ones that are based on realistic models of receptor physiology. PUBLIC HEALTH RELEVANCE: The abuse of nicotine costs our society millions in health care costs to combat the resultant heart disease, cancer, and other debilitating illnesses. In this project we examine how nicotine acts as a powerful modulator of brain functions, by examining its ability to usurp important signaling pathways in the brain. Our studies will form the basis for developing effective drugs to combat nicotine addiction, one that will take into account the entirety of nicotine's effects on the brain, an approach sorely lacking at present.

Project Summary Loss of cholinergic innervation is an early pathological sign in many neurodegenerative disease like Alzheimer's disease (AD) and Parkinson's disease (PD) and it is thought to underlie cognitive deficits in these diseases. Another early symptom, one that precedes the canonical symptoms of these diseases by many years is deficits in olfaction. It stands to reason that if we can provide a mechanistic explanation linking these early deficits to AD and PD, we will have an early biomarker for these diseases and allow for the design of strategies for early intervention and treatment. In this proposal we test the idea that olfactory insults can cause the degeneration of central neurons that express genes that confer susceptibility to degeneration. To test this idea, we will use mouse models that over-express these genes only in cholinergic neurons. The two genes being tested are the tau, implicated in AD (and now in PD) and the A53T mutant ? synuclein (A53T ?syn) along with a flurophore (GFP or Td-Tomato). The mice will be subject to either chemically-induced anosmia or treatments that specifically target dopaminergic neurons in the olfactory bulb (intranasal MPTP or stereotactically injected 6-OHDA). We will then examine the cholinergic projections in these mice. For this we will use the CLARITY technique for rendering the brain transparent coupled with light sheet microscopy that allows us to image deep into tissues. Using this technique we will reconstruct and render in 3-D the cholinergic innervation from the basal forebrain to the olfactory bulb. This will allow us to compare changes in cholinergic innervation under conditions of olfactory insults. This project is the first attempt at providing a link between olfactory dysfunction and the cognitive symptoms seen in AD and PD. It will also be the first to provide a detailed 3-D rendering of the basal forebrain cholinergic system. Results from this study will provide the basis for using olfactory dysfunction as an early biomarker for neurodegenerative diseases.

? DESCRIPTION (provided by applicant): This is a competitive renewal of a Jointly Sponsored Training Grant that currently funds 6 slots for pre-thesis Ph.D. students in the Neuroscience Training Program (NSP) at the University of Colorado, School of Medicine. The NSP is an interdisciplinary Ph.D. granting degree started in 1986 that has been funded by the Jointly Sponsored Training Grant since 2001. The NSP has 60 faculty members of whom 56 are on this application. The faculty have an outstanding training record. Our graduates have a strong record of achievements as academicians and scientists. The average number of manuscripts published by our graduate students during their tenure was 3 manuscripts. The focus of the NSP is on training outstanding neuroscientists and academicians who will make significant contributions to neurobiology, become leaders in the field and impart these qualities to future generations of neuroscientists. In addition, we aim to foster development of students who approach research in a responsible, professional manner. In the last funding period, the NSP had its external review and acted quickly to put the reviewers' recommendations into practice. The Curriculum Committee, working in close collaboration with the Director, refined the curriculum designed to attain these goals. The emphasis of NSP is on fostering increasing independence, responsible conduct and critical thinking through courses and laboratory rotations in the first year of instruction so that, in the second year and beyond, we have students who think independently and develop, troubleshoot and communicate effectively the results of their own hypothesis-driven projects.