Lynn D. Selemon, Ph.D. - US grants

The developmental hypothesis of schizophrenia suggests that prenatal perturbation of early brain development results in a latent defect expressed in post-adolescent life in the form of profound mental illness. In the Section of Neurobiology at Yale, a primate animal model of schizophrenia has been developed over the past decade which produces behavior deficits in working memory capacity, perhaps the most prominent cognitive deficit observed in schizophrenic patients, and mimics several prominent morphologic correlates of the disease. Irradiation of the fetal rhesus monkey brain during a critical period in early gestation results in deletion of developing thalamic neurons and, ultimately in the adult monkey, in behavioral deficits on tasks that are mediated by the prefrontal cortex, features that have been reported in schizophrenic patients. The goal of the study is (1) to determine the extent to which anatomical abnormalities generated in the non-human primate brain by fetal exposure to x-irradiation replicate anomalies previously described in postmortem studies of the human schizophrenic brain and (2) to utilize this animal model to explore morphological disruption of the cortex that is not feasible in the disease human brain. These goals will be achieved by a comprehensive anatomical investigation of the fetally x-irradiated primate model with particular focus on the dorsolateral prefrontal cortex, including application of the latest stereologic methodology to assess neuronal and glial density, cell size, as well as cortical and ventricular volume, morphometric analysis of neuronal dendritic structure, and quantitation of chemically identified cell populations. In addition, ultra-structural analyses, which is not feasible in postmortem human brains will be used to directly examine the neuropil compartment of the prefrontal cortex. These finding should provide insights into neurodevelopmental mechanisms leading to cognitive dysfunction and cortical pathology in schizophrenia.

developmental disease /disorder; brain morphology; schizophrenia; mental health epidemiology; brain mapping; thalamus; developmental neurobiology; psychopathology; neurogenesis; brain injury; frontal lobe /cortex; hippocampus; disease /disorder model; radiation; early experience; longitudinal animal study; magnetic resonance imaging; histology; Macaca; postmortem;

The dopamine hypothesis remains a prominent influence on research into the pathophysiology of schizophrenia (SCHIZ), yet the presence of consistent SCHIZ-linked abnormalities either in the presynaptic components of the dopamine system or in dopamine receptors (DRs) still remains a matter of debate. The present proposal focuses on a recently recognized group of DR-interacting proteins (DRIPs) as possible novel sites of dysfunction in SCHIZ. In pursuing this hypothesis, we have already demonstrated that the dorsolateral prefrontal cortex from SCHIZ cases of the Stanley Foundation Neuropathology Consortium express significantly increased levels of the D 1 DRIP, calcyon, and the D2 DRIP, NCS-1. These findings support our proposition that SCHIZ is associated with altered levels of specific DRIPs. It is also interesting that these two proteins strongly associate with Ca2+ signaling. Therefore, they have joined the growing list of molecules involved in Ca2+ signaling and whose abnormalities have been reported in SCHIZ. Consequently, our data uphold the central hypothesis of our Center that SCHIZ, while may be induced by multifactorial causes, is, at its core, a disease of Ca2+ signaling. The main goal of this project is to confirm and expand our examinations of DRIPs in SCHIZ, which is essential for proving both the specific hypothesis of this project and the general hypothesis of the entire Center. This will be achieved in the following specific aims: 1. We will determine whether in SCHIZ patients levels of calcyon and NCS-1 are affected in areas other than the previously-examined dorsolateral prefrontal cortex. We will also examine whether the changes in these DRIPs could be consistently observed in samples from several brain collections. 2. We will determine whether detected changes in levels of calcyon and NCS-1 proteins are associated with increased transcription of these DRIPs by regional cells. 3. We will analyze possible SCHIZ-linked changes in the proportion of calcyon- and NCS-1-containing neurons and glia among the cells of specific brain regions and their structural subdivisions. 4. We will conduct a detailed examination of the cellular and subcellular distributions of calcyon and NCS-1 in specific regions of the primate brain. 5. We will continue to evaluate additional novel DRIPs.

The hypothesis that the thalamus is a primary site of pathology in schizophrenia and that thalamic pathology will be tested, as well as consequent pathology in other brain regions, could arise from a prenatal insult. Toward this aim, fetal monkeys have been exposed to x-irradiation in order to substantially reduce thalamic volume and neuron number. During the current grant cycle, the high dimensional brain mapping (HDBM) method developed by and colleagues has been used to assess thalamic volume in fetally irradiated monkeys (FIMs) and has established that fetal irradiation during the time of thalamogenesis produces thalamic pathology in the fully mature macaque that can be detected with neuroimaging techniques. In this grant period, HDBM will be used to examine how other brain regions, specifically the frontal cortex and hippocampus, are affected by early gestational depletion of thalamic neurons and whether the pathology in these regions is comparable to that observed in schizophrenia. The volume and thickness of individual cortical areas, e.g., dorsolateral prefrontal area 46, will be assessed through a novel combination of postmortem histologic and magnetic resonance imaging methodologies. In addition, postmortem analyses of the cytoarchitecture of the hippocampus will probe the cellular basis of alterations in volume and shape of the hippocampus that have been detected via in vivo imaging in FIMs; these analyses may provide insight into the structural underpinnings of similar conformational alterations in schizophrenia patients. Finally, application of the HDBM methodology to a new cohort of FIM and control monkeys at selected developmental intervals will provide a longitudinal picture of the normal development of brain morphology and the interaction of a prenatal lesion on the normal maturational process.

The projects in this Center explore the molecular and morphologic basis of aging in pyramidal cells in the prefrontal cortex, neurons that play a critical role in working memory function. The mission of the Histology and Morphometry Core is to process brain tissue in a manner that is suitable for quantitative analysis of dendritic arborization and spine density, i.e., Golgi impregnation and Lucifer Yellow (LY) intracellular filling. The Core is also responsible for application of quantitative neuroanatomic methods, specifically neuronal tracing with the Neurolucida system and photoimaging of dendrites on the confocal microscope. For Golgi impregnation, we have chosen to use the FD Rapid GolgiStainTM kit as this method represents a synthesis of the Golgi-Cox and rapid Golgi techniques, exploiting the advantages of both methods to optimize the quality of impregnated somata and dendritic spines. Golgi-impregnated pyramidal neurons in layer III will be traced with the aid of a computer-integrated microscopy system (Microbrightfield, Williston, VT) featuring Neurolucida software. Measurements of linear spine density, dendritic length and dendritic complexity (Sholl analysis) will be derived from tracings for between-group comparisons, e.g., aged vs. young rats. Slice injection and intracellular filling of pyramidal neurons with LY will confirm the findings from Golgi analyses and provide a complete 3D reconstruction of dendritic spines on selected dendritic segments via confocal microscopy. Dr. Patrick Hof, Mount Sinai School of Medicine, will lend his expertise as a consultant for the LY injections and analyses. Brain tissue from rats and monkeys studied in Projects 2-4 will be processed and analyzed by the Core. Our goals are to provide histologic processing of uniformly high quality and to collect data in a systematic and consistent manner across the experimental designs of individual projects. In so doing, the Core will facilitate integration of data among projects and further the overall goal of the Center, i.e., to understand the structural basis of age-related cognitive dysfunction. LAY SUMMARY: Declining memory and cognitive function are generally regarded as inevitable and intractable consequences of the normal aging process in human populations. Understanding the underlying structural and molecular basis of the age-related deficits in mental capacity and exploring the potential reversibility of dendritic atrophy may lead to prophylactic treatments that preserve mental functioning in elderly subjects.

PROJECT SUMMARY Because of its close phylogenetic relationship to the human, the non-human primate (NHP) brain holds enormous potential for understanding structure, function, development and pathology of the human brain. However, many obstacles impede studies of NHPs, among these, significant logistic, ethical, and financial burdens. The Department of Neuroscience at Yale houses a unique collection of brain tissue from NHP macaques, ranging in age from embryonic to adult, which was generated over four decades, in the laboratories of Dr. Pasko Rakic and the late Dr. Patricia Goldman-Rakic. The collection includes four Case Sets of slides: (1) [3H]thymidine-labeled serially cut brains for cell birth dating, (2) brains labeled with tritiated amino acids for tract tracing, (3) brains from monkeys with enucleations and selected cortical lesions, (4) and brains from prenatally X-irradiated and ultrasound-exposed monkeys sacrificed postnatally. A 5th Case Set is comprised of EM blocks from multiple brain regions of fetal and postnatal monkeys. The objective of this grant is to make this unique collection an international research resource (MacBrainResource) that is readily available to the neuroscience community at-large. To accomplish this, we propose (1) to inventory and catalogue the collection, (2) to restore the slides to optimal condition by re-coverslipping and in some instances re-staining them, (3) to publicize the resource via mailings, presentations at the Society for Neuroscience meetings and our website (MacBrainResource.org), and (4) to facilitate use of MacBrainResource by outside investigators both on site and remotely by transmission of high resolution images via the internet. The value of the MacBrainResource collection is immeasurable. Primate brain research not only requires animal sacrifice but is very expensive. Making existing NHP brain tissue available will avoid costly duplication of experiments and unnecessary sacrifice of animals. With increasing pressure to find alternatives to primate research, establishment of the MacBrainResource would allow researchers worldwide to conduct primate studies without the need to sacrifice a single new animal. Proof that this resource represents a viable and valuable alternative for de novo primate research comes from the publication of several recent papers that utilized this material (e.g. Duque at al., 2011; 2015; 2016; Ratnanather et al. 2013; Selemon et al., 2013) as well as demand for material from investigators all over that world. Dedicated personnel, proper cataloguing, and adequate equipment would ensure that these slides and EM blocks are preserved for future generations of neuroscientists to exploit, perhaps using methods not yet developed.