Charles Wilson - US grants

The function of the basal ganglia in the initiation and elaboration of movement depends upon the generation of properly times episodes of action potential generation by neurons in the neostriatum. The goal of these studies is to determine the anatomical and neurophysiological mechanisms that determine whether or not such an episode will occur in an individual neostriatal neuron, and the timing of these episodes when they do occur. Anatomical experiments will employ intracellular staining with biocytin and light and electron microscopy- to examine the neostriatal innervation from single axons from the largest and most important of the inputs to the neostriatum, the cerebral cortex. The experiments will directly determine the number of synapses made by a single cortical axon which enters the neostriatum, and the volume of neostriatum innervated by one axon. Estimates of the number of synapses made onto one neostriatal neuron by a single cortical axon and the number of axons that contacts each neuron will be made from these data. These estimates will be made separately for three functionally and anatomically different types of corticostriatal cells and for the patch (striosomal) and matrix compartments of the neostriatum. Electron microscopy will be used to identify the identity of postsynaptic neurons, the location of synaptic contacts on the postsynaptic cells, and the morphological features of the synapses formed by each corticostriatal cell type in the patch and matrix compartments. This information is essential to understand how many cortical neurons must be active simultaneously in order to activate a neostriatal neuron. Physiological experiments will use intracellular recording in vivo to determine the firing patterns of corticostriatal neurons that give rise to episodes of activity in neostriatal neurons. This information is analogous to that of the anatomical experiments because it helps to determine the strength of the influence that one cortical neuron may be expected to assert on one neostriatal cell. These experiments will examine each of several types of corticostriatal neurons separately. These experiments provide information required for the interpretation of firing patterns observed during voluntary and goal-directed movements and for interpretation of changes in firing that may accompany disorders of the neostriatum, including Parkinson's disease or Huntington's disease.

Neuronal populations in motor areas of cerebral cortex that act as input to the basal ganglia will be identified by retrograde transport of tritiated acetyl wheat germ agglutinin or wheat germ agglutinin conjegated to horseradish peroxidase. Paired injections in neostriatum and each of several other major targets of motor and premotor cortex will also be performed. The neurons projecting to neostriatum will be compared to those giving rise to other important motor cortex output pathways, and the extent to which nerurons in cerebral cortex have branched axons projecting to neostriatum and another structure will be assessed. Corticostriatal enruons from each of the populations defined in this way will also be identified by antidromic activation in intracellular recording experiments. These cells will be stained by intracellular injection of horseradish peroxidase to examine their dendritic branching patterns (as an indication of what inputs they might receive), their patterns of intracortical synaptic connections (inferred from the distribution of their intracortical axonal arborizations) and the size and shape of the area in neostriatum that receives an input from a single axon. In addition, major inputs to these cortical areas will be stimulated and the responses of intracellularly recorded corticostriatal neurons analysed to determine the extent to which different populations of corticostriatal neurons recieve synaptic input from ventral thalamic nuclei, contralateral motor and premotor cortex, and axon collaterals of pyramidal tract neurons. These experiments are designed to help to define the nature of the motor cortex input to the basal ganglia by comparing the neurons that convey it to those carrying cortical efferent activity in pathways whose behavioral importance is more clearly understood. They will also contribute to the process of defining the operations performed by the basal ganglia on its cortical input.

electron microscopy; biomedical equipment purchase; image processing;

The goal of this project is to investigate the electrophysiological and neurochemical correlates of hippocampal neuronal pathology found in temporal lobe epilepsy. Hypotheses on neuronal excitability, neuronal inhibition and disinhibition, and synchronization of neurons will be tested during: l) the interictal period, 2) the transition to ictus, 3) the propagation of ictal activity and 4) the termination of ictal activity. Bilateral neurophysiological measures of these epileptic phenomena include I) the pattern of single neuron firing, 2) degree and extent of neuronal synchronization, 3) frequency and extent of epileptiform spikes and sharp waves, and 4) level of paired pulse excitability. Simultaneously, bilateral in vivo microdialysis measures of neurotransmitters will be sampled from the extracellular space immediately adjacent to the microelectrodes to look for differences between the hippocampal area of seizure onset and the contralateral homotopic area. Microdialysis measures will include the excitatory amino acid neurotransmitters glutamate and aspartate, the inhibitory neurotransmitter gamma-aminobutyric acid, and the opioid peptide neuromodulators, met- /leu-enkephalin, and dynorphin. The four hypotheses to be tested are: 1) During the interictal state, the epileptogenic hippocampus in human mesial temporal lobe epilepsy will display significantly enhanced inhibition and significantly enhanced excitation, as compared to homotopic hippocampal areas of the contralateral temporal lobe. 2) Two distinct mechanisms of transition to ictus occur, one disinhibitory due to decreased inhibition and increased excitation (Type A), the other hypersynchronous, requiring both increased inhibition and increased excitation (Type B). 3) Transition from a Type B ictal pattern to a Type A ictal pattern requires propagation to adjacent structures with different anatomical and physiological properties, prior to subsequent modifications in the electrophysiological patterns of activity of the structures initiating the seizure. 4) Cessation of Type A ictal events are associated with release of opioid peptides, which suppress unit discharges, while Type B ictal events do not release opioid peptides and seizures terminate as the involved neuronal aggregates become desynchronized. Results of these tests will be correlated with molecular pathophysiology measures of reorganization of excitatory and inhibitory hippocampal circuitry from Subproject #1, with Ca2+ signalling properties of glial cells in Subproject #2, and with in vitro measures of dentate gyrus extracellular negativity in Subproject #3. Knowledge of the physiology of the neuronal circuitry underlying interictal excitability, ictal onset and termination and propagation of ictal discharges in vivo in the intact human temporal lobe provides unique information unobtainable from histopathological, in vitro slice, cell culture, or animal seizure models. Such information may have important implications for effective pharmacological and surgical control of seizures.

Grafts to embryonic rat striatal tissue placed into the striatum of adult rats will be studied using anatomical and neurophysiological methods. The goal of these studies is to determine the extent to which the grafted neurons posses the characteristic phenotypic features of striatal neurons, and whether they resemble mature or immature striatal cells. Neurons in the graft will be identified as located in one of two major tissue compartments that are known to form there, called P and NP. Dopamine-innervated neurons of the P region will be identified by their projections to globus pallidus and/or cytochemical identification of the dopaminergic fibers. Morphological studies will employ intracellular staining of P and NP cells to determine whether these neurons show the characteristics morphological features of adult or immature striatal neurons, whether they have dendritic or axonal projects into the other tissue compartments, and whether these are related to the presence of axonal projections to the globus pallidus. Neurophysiological studies will concentrate on the characteristic set of ion conductances of striatal neurons. Developmentally-regulated ion channels, including a group responsible for transient potassium conductances (A-channels), inwardly-rectifying potassium conductances, and both high and low- threshold calcium conductances will have the highest priority. These conductances, as well as helping to specify the functional properties of the graft neurons, can also be used to access the maturational state of the cells in the grafts. Ionic conductances will be assessed using whole-cell voltage clamp recordings using acutely dissociated striatal graft neurons. The function of these channels will be confirmed using conventional intracellular recording in slices from striatal grafts. Neurons identified as P or NP cells based upon their projects to the globus pallidus will also be studied using single-cell mRNA amplification methods to determine the presence of specific ion channel mRNAs related to the neurophysiological experiments and to allow comparison of the constellation of cell-type specific mRNAs expressed in normal adult, immature, and grafted striatal projection neurons.

DESCRIPTION (Adapted from applicant's abstract) : The function of the basal ganglia in the initiation and elaboration of movement depends upon the generation of properly timed episodes of action potential generation by the projection neurons of the neostriatum. The goal of these studies is to discover the anatomical and neurophysiological mechanisms that determine whether or not such an episode will occur in an individual neostriatial neuron, and the timing of these episodes when they do occur. Anatomical experiments will employ in vivo intracellular biocytin staining of the cortical neurons that project to the neostriatum, to visualize single axons from this largest and most important of the inputs to the basal ganglia. The experiments will allow specific identification of neurons in the cerebral cortex that project to the strisomes, matrisomes, and extended matrix of the neostriatum. The boundaries between striosome and matrix will be visualized using immunocytochemistry for calbindin-D-28k, which specifically labels the matix. Quantitative studies of their axonal arborization will reveal the degree to which single axonal avbors may innervate individual neostriatal spiny neurons, the degree to which nearby neostiatal neurons receive similar inputs, and the spatial organization of converging cortical inputs to neostriatal projection cells. Electron microscopy will be used to insure that the light microscopic analyses of axonal varicosities are accurate estimates of the distribution of synaptic contacts. Physiological experiments will employ in vivo intracellular recording of striatal spiny neurons during selective stimulation of corticostriatal inputs to the stiosmal, matrisomal, and extended matrix stained compartment. The spiny cell studied will be stained intracellularly and their axons traced to determine whether they participate in the direct or indirect pathway. These experiments will allow a test of the idea that neurons of the indirect pathways receive separated and specific corticostriatal inputs.

technology /technique development; magnetic resonance imaging; mother /infant health care; gas; eye; biomedical resource; bioengineering /biomedical engineering; biological products; Mammalia;

DESCRIPTION (provided by applicant): Altered function of the neostriatal cholinergic interneurons has been implicated in the pathology of Parkinson's disease, Huntington's disease, and a variety of other disorders. The observation that cholinergic antagonists are clinically effective in treating Parkinson's disease has led many investigators to suggest that within the striatum there is a balance of opposing actions of dopamine and acetylcholine. Despite the explosion of information on the pharmacology of acetylcholine in the neostriatum, physiological information has been difficult to obtain due to the rarity of cholinergic interneurons compared to the other cells in the striatum. Using infrared differential interference contrast microscopy, we have recorded from identified cholinergic neurons in slices, and have shown that they are intrinsic pacemakers that exhibit three distinctly different spontaneous filing patterns, even in the absence of fast synaptic input (but with neuromodulators intact). One of the firing patterns resembles that seen in experimental Parkinsonism. This finding provides a window on several otherwise inexplicable observations, including the rhythmic synchronous activity of these neurons in monkeys rendered Parkinsonian by experimental treatment with MPTP. In the proposed experiments, we will employ whole ceil recording of identified cholinergic interneurons and calcium imaging in single cells to determine (1) The ionic mechanisms of the rhythmic bursting firing mode, which most resembles that seen in Parkinsonism, which we already know is related to modulation of calcium and calcium dependent ion channels (2) The basis for synchronization of cholinergic interneurons when they are firing in the bursting mode, including the synaptic connectivity among cholinergic cells and (3) The influence of D1 and D2 dopaminergic agonists and antagonists on the firing patterns of cholinergic interneurons. The effects of dopamine on firing pattern will be directly related to other studies on dopaminergic modulation of specific ion channels to provide an integrated understanding of the actions of dopamine on cholinergic interneurons and the neostriatal circuitry.

Several articles reporting data obtained at the Facility by Dr. Bruce Berkowitz have been published and are included in this report. A new faculty member, Dr. Nadir Alikacem, has been recruited and has begun work on this project. (Service 15) REPORT PERIOD: (09/01/97-08/31/98)

In the past few years, evidence from both experimental and clinical studies suggests a key role for the subthalamic nucleus in the pathogenesis of Parkinson's disease. As the principal excitatory pathway interconnecting basal ganglia nuclei, it is one of the most likely suspects as the origin of the synchronous rhythmic firing patterns in the globus pallidus that are associated with the motor symptoms of that disease. In slices of the subthalamic nucleus, the neurons spontaneously fire rhythmically at a rate approximating that seen in dopamine-denervated animals and in Parkinsonism in humans. The proposed experiments will determine the ionic mechanisms responsible for the rhythmic firing of subthalamic neurons seen in slices, and determine the degree to which rhythmic activity may become synchronized by recurrent excitatory interactions between subthalamic neurons. The experiments will employ infrared-visualized whole cell recording, imaging of calcium entry during spontaneous and evoke rhythmic firing, and intracellular staining of recurrent axonal connections. A possible role of extrinsic excitatory inputs in desynchronizing the cells of the nucleus, and in disrupting their rhythmic activity will be explored by micro-stimulation and cortico- subthalamic fibers in slices preserving a portion of that pathway. A knowledge of the mechanism underlying rhythmic and possibly synchronous activity in vitro may offer new pharmacological approaches to treatment of the motor symptoms of Parkinson's disease, and provide a more mechanistic explanation of stereotaxic surgical treatments currently in use.

In humans with Parkinson's disease, and in monkeys treated with the neurotoxin MPTP, neurons in the subthalamic nucleus (STN) and globus pallidus external segment (GPe) exhibit correlated periodic firing not normally seen in these structures. This correlated rhythmic firing is strongly implicated in the pathogenesis of Parkinson's disease. A variety of experimental evidence and theoretical considerations suggest that the STN and GPe may generate rhythmic but not correlated firing patterns intrinsically when not subject to suppressing inputs from the cerebral cortex and striatum. The rhythmic activity arises largely from the intrinsic pacemaking of the neurons of the STN, and the correlation of inputs arise from synaptic connections between the STN and GPe, which interact with the pacemaker mechanism in the STN neurons. Although the cellular properties of STN and GPE neurons are entirely due to the interactions among ion channels which have been and continue to be studied in isolation, they do not allow an immediate prediction of the physiological properties of the cells under natural conditions, or their peculiar responses to synaptic input. We propose to make the connection between biophysical/molecular properties and cellular activity using mathematical modeling and computer simulation. Similarly the rhythmic activity seen in the intact network do not resemble those of isolated neurons, and this again offers an opportunity for a theoretical approach. This project proposes to use mathematical modeling and computer simulations to bridge the gap between biophysical information on ion channels (including their modulation after dopaminergic denervation) and cellular properties playing a key role in STN-GPe rhythmogenesis in Parkinsonism. Biophysical and cellular data from other projects in the program will be the principal source of new information on ion channel properties and modulation. Some aspects of the models will be tested directly within the project using whole cell recording and calcium imaging to reveal voltage and calcium dynamics in single neurons of the STN or GPe in slices. The results will reveal the dynamic properties of single neurons in the two structures that are critical in generating network rhythmic correlated firing in vivo.

transmission electron microscopy; image enhancement; minority institution research support; biomedical facility;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There has been no change in the support of key personnel since the last reporting period. There will not be any significant change in the level of effort for key personnel in the next budget period. The estimated unobligated balance will not be greater than 25% of the current year's budget.

DESCRIPTION (provided by applicant): We propose a SNRP at the University of Texas at San Antonio. UTSA is a young institution with a commitment to rapid growth in graduate training and research in Neuroscience, and an emphasis on quantitative and computational research. In the past 3 years the Department of Biology at UTSA has hired 6 promising young tenure-track neurobiology faculty from among the best labs in the country to represent a cross-section of neuroscience disciplines and advance Neuroscience within our institution. Most of these new scientists are assistant professors, who have never previously held faculty positions. In the upcoming 5 years we are committed to hiring 5 additional tenure-track Neuroscientists with research interests in topics fundamental to the understanding of nervous system function and disorders, including neural control of movement, central pattern generation, structural plasticity in the adult nervous system, ion channel structure and function, and behavioral genetics. During this time of rapid growth of our Neuroscience group, we envision the SNRP functioning as the center of the Neuroscience community at UTSA. The program will develop a community of UTSA researchers and their collaborators at other institutions. It will support a shared research infrastructure tailored to Neurobiology research by providing resources and expertise in the design, collection and quantitative analysis of numerical and image data. We will offer training and leadership to foster the development of our promising new faculty. We will provide critical evaluation, advice and support for their collaborative pilot research, by providing guidance and direct help in the administrative and management issues that arise in establishing their new laboratories. The SNRP leadership will also act as an advocate for their interests at the college and university levels as they establish their independent research programs and develop into productive managers, scientists and scholars. A second focus of the SNRP will be to provide training opportunities that supplement the Neurobiology Ph.D program, including seminars, symposia, and year-long competitive training fellowships for students interested in pursuing advanced mathematical or statistical studies while in our program. REVIEW OF INDIVIDUAL PROJECTS AND CORES: Project 1: Origin and regulation of motor neuron identity in hindbrain. Dr. Gary Gaufo and Dr. Anne Moon DESCRIPTION (provided by applicant): The vertebrate hindbrain is essential for controlling an array of behaviors, from voluntary movements of the craniofacial musculature to autonomic functions of the cardiovascular and gastrointestinal systems. These behaviors rely on the precise registration of motor neurons with their peripheral targets along the head and body's anterior-posterior (AP) axis. This highly ordered relationship originates from a simple embryonic body plan in which motor neurons develop within individual rhombomeres and their prospective targets in adjacent branchial arch tissues. A major cellular contribution to this motor neuron-peripheral target relationship;omes from the neural crest cell, a restricted stem cell population that arises from the dorsal rhombomere and migrates into the surrounding branchial arch tissue. The positional information imposed upon the varied cell types constituting this motor neuron circuit is largely provided by the AP-restricted expression of the Hox genes. However, the mechanism that maintains the AP-restricted expression of the Hox genes and their ability to control the differentiation of the neurons derived from the hindbrain and the neural crest cell remain to be defined. In the first aim, we will use a genetic fate map of the rhombomeres to identify the neuronal lineages that arise from neural crest cells and their possible regulation by the Hox genes. In the second aim, we will address the role of Hox genes in neuronal differentiation through the use of a conditional mutagenesis system to disrupt Hox gene function among progenitors and postmitotic motor neurons in the ventral neural tube. In the third aim, we will explore a mechanism by which Fgf signaling regulates motor neuron-subtype identity by repressing the activity of the Hox genes in the hindbrain. The latter aim may reveal a mechanism that establishes the different motor neuron identities along the entire AP axis of the central nervous system. An understanding of the molecular and cellular determinants contributing to the formation of the motor neuron-peripheral target circuit may provide therapeutic insight into damaged nervous tissue and diseases associated with motor neurons and nerve conduction.

DESCRIPTION (provided by applicant): Recent advances in the study of human Parkinson's disease and experimental animal models of the disease have directed attention to oscillatory electrical activity in the basal ganglia. Low frequency oscillations in the beta range (13-30 Hz) have been shown to be exaggerated in Parkinson's disease, and to occur normally when movements are inhibited. The oscillations are measured as field potentials using gross electrodes, so their cellular origins are not known, but they are in part generated in the striatum. Because field potentials are a population measure, they must reflect synchronous activity in groups of neurons. Studies of experimental parkinsonism in monkeys have shown that neurons firing in rhythm with the low frequency oscillation are all tonically active striatal interneurons. These cells maintain their background firing, even when animals are not moving. In contrast, the principal cells of the striatum, the spiny neurons, and the best-studied interneurons (the fast-spiking interneurons) fire episodically in relation to movement and are mostly silent otherwise. Thus, the striatal generator for the low frequency oscillations associated with bradykinesia is probably tonically firing interneurons. It has previously been thought that all tonically active interneurons in the striatum are cholinergic interneurons. Recently, it has become clear that there are two kinds of tonically active interneurons in the striatum (i.e. active in the absence of excitation from elsewhere). They are the cholinergic interneuron and the LTS (low-threshold spike) bursting interneuron. Together, these two neuron types comprise a spontaneously active network in the striatum that generates continuous oscillatory activity, even in the absence of input. The spontaneously active network receives sparser synaptic input from striatal afferents, and interacts with the phasic striatal cells primarily by way of neuromodulatory control of excitability and synaptic plasticity via acetylcholine, nitric oxide, somatostatin, and neuropeptide Y. The experiments proposed here will determine the connectivity and dynamic properties of the network of spontaneously-active interneurons. They will determine whether the intrinsic resonant properties of the network consisting of tonically active striatal interneurons are appropriate for generation of oscillations in the beta frequency band. We will determine the mechanism of spontaneous oscillations in LTS cells, and whether synaptic connections between them act to promote or oppose synchronous activity. We will also examine the changes in the intrinsic oscillations and synchronization that follow chronic dopamine depletion with 6- hydroxydopamine. The two autonomously active cell types can be readily identified in slices, and targeted for study. These experiments will reveal mechanisms promoting synchronization that may be points of action of dopaminergic depletion and possible targets for future anti-parkinsonian therapies.

Deep brain stimulation (DES) of the subthalamic nucleus is a useful treatment for Parkinson's disease (PD), but its therapeutic mechanism is unknown. Effective DES requires high frequency stimulation, well above the average firing rate of basal ganglia output neurons. Periodicity of DES is also essential; random stimulation patterns at the same mean frequency are ineffective. Three mechanisms for its effect on basal ganglia output neurons have been proposed. DES may correct a pathological change in: (1) firing rate of basal ganglia output cells and their targets in the thalamus, (2) bursting or oscillations of those cells, or (3) the degree to which the firing of the cells are correlated. Neither the rate nor the bursting model for the action of DES adequately explains either the frequency or periodicity requirements. We have shown that a periodically-driven oscillator model of basal ganglia output cells exhibits a sequence of synchronizing entrainment and then failure of entrainment and desynchrony as the frequency of an excitatory stimulus is increased. In this model, the range of stimulus frequency, intensity and periodicity required for chaotic desynchronization matches that of the therapeutic effectiveness of DES. This application will test our desynchronization hypothesis by measuring the degree of correlation among pairs of simultaneously recorded neurons in slices of the substantia nigra pars reticulata (SNR) during application of DES-like natural and artificial synaptic conductances. Aim 1 will test the fundamental mechanisms at work in the model using purely excitatory input. Aim 2 will add an inhibitory component to the synaptic input, and test our method for designing the optimal stimulus. Aim 3 will determine the influence of inhibitory coupling between output neurons on normal firing patterns and during DES. Aim 4 will determine whether the cellular dynamics or synaptic connections underlying DES are altered after chronic dopamine depletion. Our model offers a mechanistic explanation of DES and its properties, and a mathematical model that can be used to predict the effects of future DES-like stimulation therapies. At this point, the model has not been validated, and this proposal will provide a test of the proposed mechanism. If it survives experimental test, our idea may be useful for explaining DES and for designing future stimulation therapies.

Models of basal ganglia function and dysfunction have long been based on a circuit diagram sketching the flow of electrical activity through the nuclei. These models have guided the development of treatments for neurodegenerative diseases of the basal ganglia, and continue to be useful for design and refinement of new treatments. The validity of the functional models and their predictive value for design of new treatments is tightly linked to accurate information on the circuitry in each of the nuclei. Our knowledge of the anatomical connectivity among neurons in basal ganglia nuclei is good and improving rapidly, but our understanding of circuit effect on the flow of electrical activity is in its infancy. In this proposal, the functional characteristics of circuits in the main input nucleus of the basal ganglia (the striatum) and that in the main output nucleus (the substantia nigra) are analyzed. The experiments use sinusoidal inputs and combinations of sinusoids over a range of frequencies to characterize the effects of local circuits on neuronal responses. These inputs engage the known resonance properties of the neurons and synapses, and they will reveal other resonances that arise from circuit interactions. In some experiments, the input is intracellularly or optogenetically delivered to some or all cell-types in the circuit, whereas in others the periodic input signal to the circuit is delivered synaptically by continuously controlling firing of upstream neurons. These experiments will reveal the circuit mechanisms that amplify or suppress rhythmic activity in the basal ganglia, including both the beta oscillations that are a principal pathophysiological component of Parkinson's disease, and the therapeutic periodic signal generated during DBS.