Joseph T. Coyle - US grants

Twelve years ago, we first demonstrated that intrastriatal injection of the potent excitatory analogue of glutamate, kainic acid (KA), reproduced many of the synaptic neurochemical and histopathologic features of Huntington's Disease (HD) in the rat. Subsequent studies indicated that agonists at all three glutamate receptor subtypes - quisqualic acid (QA), N-methyl-D-aspartic acid (NMDA) and KA - caused specific patterns of selective neuronal degeneration upon intracerebral injection, leading to the proposal that endogenous excitotoxic mechanisms might account for neuronal death in HD and other neurodegenerative disorders. During the last period of support, we have focused on the endogenous processes - both presynaptic and postreceptor - that may contribute to the excitotoxic mechanisms. We have identified an excitatory dipeptide, N-acetyl aspartyl glutamate (NAAG) which has been localized by immunocytochemical staining, to subsets of putative glutamatergic pathways. Using the neuronal-like cell line, N18-RE-105, we have demonstrated that oxidative stress is the proximate cause of Ca++-dependent cell death due to glutamate or QA exposure in this in vitro model for excitotoxicity. In the proposed studies, we will characterize the enzymes that synthesize and catabolize NAAG and determine whether NAAG is subject to Ca++ -dependent depolarization induced release in vivo, an important criterion for a neurotransmitter candidate. We will examine the metabolic events that underlie the oxidative stress in glutamate/QA induced degenemtion of the N18-RE-105 cells. Using primary cultures of the hippocarnpus, we will determine the contribution of oxidative stress to KA, QA and NMDA induced delayed neuronal degeneration. Finally, we will exploit a mammalian cell replica plating method to isolate clones expressing QA, NMDA or KA receptors, in order to further characterize post-receptor mechanisms responsible for excitotoxin-induced neuronal degeneration.

Clinical and neurochemical studies have provided compelling evidence that cortical cholinergic pathways are consistently and severely affected in Alzheimer's type dementia (AD). We have demonstrated in the rat that the primary source of cortical cholinergic innervation is the nucleus basalis (nBM) and that these neurons atrophy and/or degenerate in patients dying with AD. In studies supported by this grant, we have characterized the organization of the cortical/hippocampal cholinergic projections in the rat and have demonstrated that they regulate cortical muscarinic receptors. We have also demonstrated that rats with lesions either in the nBM, medical septal area or both sites exhibit selective impairments in working memory but not reference memory, analogous to the cognitive defects observed early in AD. However, we have found that the lesioned rats exhibit behavioral and neurochemical recovery unlike AD. In the proposed studies, we will focus on the issues of selective vulnerability of cholinergic neurons and mechanisms involved in behavioral and neurochemical recovery after lesions of the basal forebrain cholinergic complex. 1. We will use a combination of neurochemical and neuroanatomic techniques to determine the source of recovering cholinergic markers in the prefrontal cortex following nBM lesions. 2. We will characterize the mechanism of cytotoxicity of ethylcholine aziridinium ion against cholinergic neurons. We will use this drug to characterize the choline carrier. 3. We will determine the factors that account for behavioral recovery following lesions of the basal forebrain cholinergic complex and we will characterize the differential behavioral impairments associated with either the nBM or medial septal lesions that result in a similar defect in working memory. These basic studies in the rat may clarify the role of the nBM cholinergic deficit in the pathophysiology of AD.

? DESCRIPTION (provided by applicant): Schizophrenia is the 7th most costly illness to Society because of the lack of drugs to effectively treat the persistent negative symptoms and cognitive deficits. These two symptom domains correlate with the degree of cortical atrophy, which results from dendritic atrophy and loss of synapses. We proposed 20 years ago that this pathology resulted from hypofunction of NMDA receptors (R). Recent GWAS and CNV results in schizophrenia have substantiated our hypothesis by demonstrating that risk genes are markedly enriched in ones encoding proteins localized to glutamatergic synapses. During the previous 4 years of support, we have shown that genetic silencing in mice of serine racemase (SR-/-), an established risk gene for schizophrenia and the source of D- serine, the co-agonist at cortical NMDARs, replicates many of the neuropathologic, neurochemical and behavioral features of schizophrenia. Treating the SR-/- mice for 3 weeks with doses of D-serine that restored cortical levels to normal reversed these deficits. We seek three years of additional support to build upon these findings to advance our understanding of the role of SR in psychiatric disease and to determine whether the deficits due to the NMDAR hypofunction endophenotype of schizophrenia can be pharmacologically reversed. Aim 1. What is D-serine doing in cortical GABAergic interneurons? We were surprised to discover that astrocytes do not express SR or D-serine but that over half of the cortical cells containing D-serine are GABAergic neurons. Having successfully crossed SRfl/fl mice with mice with Cre driven by a GAD65 promoter, we will determine how the suppression of SR expression only in GABAergic neurons affects their electrophysiology, D-serine distribution and behavior. Aim 2. Can D-serine deficiency be overcome by heteroreceptor pharmacologic interventions? We will examine the efficacy of a positive allosteric modulator for the mGluR5 receptor, which enhances NMDAR function, and a positive allosteric modulator for the alpha-7 nicotinic receptors, which are concentrated on PV+GABAergic neurons and promote glutamate release, in reversing neurochemical, behavioral, electrophysiologic and dendritic deficits in SR-/-. Aim 3. Does SR induced in reactive astrocytes contribute to symptoms? Inflammation is an important feature of the pathology of schizophrenia, correlating with the deficit state. Recently, we found that reactive astrocytes express SR and D- serine. We hypothesize that D-serine released by reactive astrocytes activates extra-synaptic NMDARs, contributing to cognitive deficits and negative (depressive-like) symptoms. We will characterize the expression of SR and D-serine and the transporter, Asc-1, in tissue culture and in a model of reactive astrocytosis: systemic LPS treatment. We will determine whether the behavioral phenotype is reduced in astrocyteSR-/- mice after LPS treatment and whether LPS exacerbates the phenotype in neuronalSR-/- mice.

This project will test the hypothesis that schizophrenia is associated with NMDA hypofunction by examining in-vivo brain data acquired through the combined use of proton magnetic resonance spectroscopy (MRS) and BOLD functional magnetic resonance imaging (fMRI). We propose to measure proton metabolite concentrations, including N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG), in temporal and prefrontal cortical regions in schizophrenic patients and healthy controls. MRS data will be acquired on a 4T scanner with newly developed methods that provide improved resolution of peaks and signal components within the proton spectrum, thus improving interpretability of NAA measurements, as well as extending MRS capabilities to include quantification of NAAG. The quantification of these metabolites is relevant for the main hypothesis of the center grant as work from our group has reported reduced NAA in temporal cortex bilaterally. Further, NAAG has been implicated in NMDA hypofunction. At present in-vivo measurements of these metabolites, have not been concurrently characterized in schizophrenia. Moreover, it has been shown that NMDA receptors in the hippocampus are essential for spatial learning and that pharmacological blockade of NMDA receptors impairs spatial navigation. Therefore, as an exploratory endpoint, we propose to acquire fMRI data on a 3Tscanner during the completion of a virtual analogue of a spatial navigation task, the water maze, to assess spatial memory performance. We will also acquire fMRI data during a transitive inference task to assess relational memory, and during a facial recall challenge, tasks previously shown to be mediated by the hippocampus. Finally, in collaboration with Dr. Don Goff (Clinical Trials Section), we will enroll 60 schizophrenic patients into a placebo-controlled trial of D-serine, an agonist at the glycine site on the NMDA receptor. Patients will be imaged with the MRS/fMRI protocols described above to examine the effects of D-serine treatment on spatial and relational memory performance, BOLD activation and NAA and NAAG concentrations.

The Biostatistical Research Core (BRC) will continue to serve the six projects through applications of classical, modern, and newly-emerging quantitative methods for experimental design and analysis of empirical findings. In addition, the BRC will work with the Computational and Animal Behavioral Cores to develop new computational and biostatistical technologies for the projects. The BRC will also serve to integrate the efforts of all three cores. The major continuing challenge of the BRC is to push biostatistical interactions with the projects beyond mere confirmation of their expectations towards the discovery of previously undetected signals hidden in noisy data. With the Benes project and the Animal Behavioral Core, the BRC will apply mixed-effects Poisson analyses of variance models to test the hypotheses that GABA blockade and reduced NMDA receptor function in hippocampal CA sectors can result in aspects of cognitive dysfunction found in schizophrenia by assessing the significance of Treatment X Environment interactions in the novelty detection study. Computer-intense Bayesian Markov Chain Monte Carlo (MCMC) methods will also be employed. In a combined analysis, the DISH experiments will employ similar yet weighted Poisson models with silver grain NR2A densities as weights for the counts of GAD67-positive hippocampal interneurons. We will also apply our existing semi-automatic algorithms for detection of Fos bodies. With the Lisman project and the Computational Core, we will apply MCMC technology to revise prior connectivitymorphology probability mappings based on existing literature by new data likelihoods obtained from studies in the Benes project to yield updated posterior probability mappings. The Greene project and the Animal Behavioral Core will benefit from our longitudinal mixed-effects logistic regression models applied to their odor familiarity and recognition study. The BRC will serve as consultant to the Coyle, Yurgelun-Todd and Goff projects on an as needed basis. Further biostatistical applications include tests to distinguish between local spatial heterogeneity (different neuronal densities in different regions) and spatial dependence (correlations between neuronal locations) and Poisson random field methods to detect features of 3D brain cell assemblies at multiple levels of spatial resolution.

DESCRIPTION (provided by applicant): Substance abuse (SA) affects up to 20% of the population at some point in their lives and exhibits a heritability rate between 40 and 60%. SA is likely due to complex genetics, i.e., multiple risk alleles of modest effect interacting with environmental factors to produce the addiction phenotype. Schizophrenia, which affects ~1% of the population and exhibits substantial heritability (~80%), is a disorder with a very high prevalence of SA. Nearly 90% of individuals with schizophrenia smoke cigarettes heavily, ~50% have ethanol dependence and high rates of stimulant abuse. Pharmacologic, post-mortem and recent genetic research have implicated NMDA receptors (NMDAR) in the pathophysiology of schizophrenia. NMDARs have also been implicated in the acquisition and extinction of SA in animal models. We hypothesize that the high prevalence of SA in schizophrenia is due to shared risk genes that disrupt NMDAR function. Specifically, 3 risk genes for schizophrenia affect the availability D-serine, a co-agonist at the NMDAR in cortico-limbic regions of the brain. We have developed mice, in which serine racemase, the enzyme that synthesizes D-serine has been genetically inactivated (SR-/- ). The SR-/- mice exhibit structural, neurochemical and behavioral homologies to schizophrenia. They also present abnormalities in the acquisition and extinction of conditioned hyperactivity to amphetamine, consistent with an increased vulnerability to SA. We will use SR-/- mice and, as positive controls, GlyT1+/- mice, which have increased NMDAR function, to assess the role of NMDAR function in two animal models of SA: the cocaine self-administration paradigm, which measures the reinforcing effects of cocaine and intracranial self-stimulation, which measures the propensity of the mouse to self-administer a rewarding brain stimulus, in essence the hedonic status of the subject. Alterations in the performance of SR-/- and GlyT1+/- mice on cocaine self- administration will be correlated with neuronal activity as monitored by cFos and DFosB expression in brain regions relevant to SA. Finally, we will determine whether behavioral abnormalities in the SR-/- mice can be reversed by treatments that replace the deficient D-serine.