David G. Amaral, Ph.D - US grants

The studies proposed in this application are directed at the analysis of certain aspects of the morphological development of the dentate gyrus of the hippocampal formation in the rat. The long term goal of this research in an understanding of the mechanisms by which the principal neuronal cell type (the dentate granule cell) of this simple, mammalian cortical structure, develops a mature dendritic configuration and, in turn, receives an appropriate complement of inputs from a variety of afferent systems. We have chosen the dentate gyrus as a model system for the analysis of cortical development because of its relatively simple cytoarchitectonic organization and the rather strict laminar arrangement of its major extrinsic afferents. But we would hope that the principles of organization operating in this simple cortical structure would have widespread applicability to more complex, neocrotical regions. The particular projects we propose are: 1) to carry out a quantitative analysis of the development of the dendritic tree of the dentate granule cell, (2) to study the development of the major afferents of the dentate gyrus, and (3) to survey the development and structure of glial cells in the dentate gyrus. The development of granule cell dendrites will be studied in preparations stained by several variants of the Golgi method and also by direct intracellular injection of horseradish peroxidase into granule cells in the hippocampal slice preparation. In both cases, analysis will be aided by a computer-linked microscopic reconstruction system. The development of the afferents of the dentate gyrus will be studied with the retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase, and with 3H-amino acid autoradiography for anterograde studies. The survey of glial development will be carried out both in Golgi preparations and with the immunohistochemical localization of astrocytes with antibodies directed against glial fibrillary acidic protein. The hippocampal formation has long been implicated in processes dealing with memory consolidation and, in pathological cases, with epilepsy. This region appears to be particularly sensitive to birth traumas such as anoxia and is often damaged as a result of neonatal febrile convulsion. By gaining an understanding of the normal development of neuronal form and connections in the hippocampal formation we may get some insight not only into the mechanisms underlying its normal function, but also the means by which genetic and environmental perturbations cause developmental defects.

The proposed work is both a continuation and an extension of the studies that we have been engaged in for some years. The longterm objectives of this work are to provide as complete an analysis of the organization and development of the hippocampal formation as possible, and, by taking advantage of the unusual simplicity of these cortical areas, to provide a model for understanding the development of cortical areas in general. In the coming grant period we plan to focus our attention on several specific problems associated with the development of the dentate gyrus, the structural organization of which is sufficiently well known (in large part from our own earlier studies) to enable us to address a number of fundamental questions concerning cortical development. These include: (1) Analyzing in detail three-dimensional morphology of individual dentate granule cells to establish on a quantitative basis the degree of variability in such cells; (2) Examining, again quantitatively, the development of these neurons both in vivo and in vitro; (3) To determine, using a variety of immunocytochemical procedures, the unusual pattern of migration of the dentate granule cells; (4) To reexamine the origin of the glial cells in the dentate gyrus; (5) To examine the role played by the so-called nerve cell adhesion molecule (nCAM) in the assembly of the dentate gyrus and in the initial formation of its connections; and (6) To use the dentate gyrus to screen for the expression of a variety of cellular oncogenes during normal development, and to determine if some of these genes are reactivated during the reactive synaptogenesis that occurs in the dentate when it is deprived of certain of its afferent inputs.

The amygdaloid complex has long been implicated in the organization of visceral, endocrine and autonomic aspects of species-specific behavior, and such affective responses as aggression and defense, sexual arousal, and appetitive behavior. More recently, however, neuropsychological studies of the primate amygdala have clearly shown that it also has a role in cognitive functions such as associative memory. These newly uncovered roles for the primate amygdala are consistent with its widespread connections with the cerebral cortex. While there is little evidence of direct projections between the amygdala and the neocortex in non-primates, in the monkey brain, it has recently been found to project to much of the frontal, temporal, insular and occipital cortices including primary sensory, unimodal associational, and polymodal associational areas. Most of these amygdalocortical projections have only just been identified and the analysis of their anatomical organization is at a very early stage. The goals of the proposed studies are: first, to determine the topographic organization of the amygdalocortical projections with special reference to regional and cross modality divergence; and second, to determine candidate neurotransmitters for these projections. Specifically, the topography of the amygdalocortical projections will be studied by injecting fluorescent dyes with distinct emission spectra into different cortical regions and analyzing sections through the amygdaloid complex for single and/or double retrogradely labeled cells. Neurotransmitter candidates will be examined by two different strategies. In the first, the possibility that these connections are mediated by an excitatory amino acid will be studied by injecting D-3H-aspartate into cortical terminal fields and examining the amygdaloid complex for retrogradely labeled cells. In the second strategy, the distribution of neurons containing other putative neurotransmitters will be studied immunohistochemically using antisera directed against a variety of peptides, such as somatostatin, and specific enzymatic markers, such as glutamic acid decarboxylase. Lesions of the human amygdaloid complex have been found to result in a variety of behavioral and cognitive disturbances. For example, such damage has been linked to the development of temporal lobe epilepsy. It is also known that the amygdala is one of the first structures to demonstrate histologic changes in Alzheimer's disease. The anatomical organization of the amygdala also appears to be abnormal in childhood autism. Moreover, the fact that the amygdala has one of the highest brain levels of benzodiazepine receptors is suggestive that it may mediate the antianxiety effects of these drugs. Knowledge of the amygdalocortical interconnections will advance our understanding of the normal and pathological functioning of this prominent component of the limbic system.

The results of recent clinicopathological studies of human amnesic patients strongly support the long held view that the hippocampal formation plays an essential role in normal memory processing. Individuals with damage limited to the hippocampal formation demonstrate a profound inability to recreate a record of day-to- day events though most post memories remain intact. The hippocampal formation thus appears to play a role in consolidating new memories but is clearly not the long term repository of memory. Research carried out in the post several years of this program has dealt with a wide spectrum of issues including the neurogenesis and morphogenesis of the hippocampal formation, the maturation of its cells in tissue culture, the organization of its intrinsic and extrinsic connections in the developing and mature brain, and morphological plasticity following partial experimental denervation. In the present proposal we will narrow the scope of the work and focus on elucidating the organization of the mature neuronal circuitry which might underlie the role of the hippocampal formation in memory processing. We will continue, and substantially increase, our efforts in studying the connections of the primate hippocampal formation. Using both anterograde and retrograde tracers we will: study the topographic organization of the perforant path projection from the entorhinal cortex to the dentate gyrus and hippocampus; determine the full complement of neocortical interconnections with the subicular complex; precisely define the topographic organization of cortical inputs to the entorhinal cortex and subicular complex; and investigate the cortical and subcortical efferent projections of the entorhinal cortex. The overall objective of these studies is to determine the route by which sensory information enters the hippocampal formation and to uncover the areas to which the hippocampal formation relays its processed information. In the rat, we will carry out studies designed to provide a comprehensive and, where possible, a quantitative summary of the intrinsic circuitry of the hippocampal formation. We will analyze the axonal and dendritic organization of single hippocampal pyramidal cells which have been injected with horseradish peroxidase in the in vitro slice preparation. The processes of these cells will be reconstructed and quantitatively analyzed with a computer-linked three dimensional digitizing system. A second series of experiments will employ the recently developed lectin, anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) to study the topographic organization of the major intrinsic connections of the dentate gyrus and hippocampus. These studies are intended to provide the anatomical information necessary for realistic computational modeling and computer stimulation of hippocampal activity in memory function.

biomedical equipment purchase;

Damage to the human hippocampal formation following surgical ablation, cerebral ischemia or neurodegenerative conditions such as Alzheimer's disease results in a profound impairment of memory characterized by an enduring inability to recreate a record of day-to-day events. Experimental studies in the monkey indicate that the hippocampal formation is but one component of a distributed memory system. Recent neuroanatomical and behavioral studies conducted under this research program have determined that other temporal lobe structures, such as the perirhinal and parahippocampal cortices, also make important contributions to normal memory. Moreover, it appears that the perirhinal cortex may carry out its memory function independently of the hippocampal formation. We propose to investigate the efferent connections of the perirhinal cortex in order to determine whether it interacts with other brain regions, such as the mediodorsal nucleus of the thalamus or the frontal cortex, that are nonhippocampal components of the memory system. Since modern attempts at developing a unified neurobiology of memory rely on rat, monkey and human models, it is problematic that there is relatively little neuroanatomical information about the rat perirhinal region. We propose to fill this gap in essential information by carrying out a series of cytoarchitectonic and connectional studies in the rat perirhinal region that parallels our ongoing work in the monkey. Neuroanatomical and clinical evidence suggest that the retrosplenial area of the cingulate cortex may be yet another important memory-related region. It may contribute to memory by acting as an interface between working memory regions of the frontal lobe and the long term memory mechanisms of the medial temporal lobe. We have previously discovered strong projections from the monkey retrosplenial cortex to the hippocampal formation and to the parahippocampal cortex. We propose here to conduct additional neuroanatomical studies and initiate a new program of behavioral studies to investigate the functional organization of the monkey retrosplenial cortex. While it is important to investigate new components of the brain's memory system, there remain substantial gaps in our understanding of the functional circuitry of the primate hippocampal formation. We propose to use the sensitive anterograde tracer PHA-L to study the patterns of intrinsic connections of the monkey hippocampal formation. The results of these studies will be compared with similar studies already completed in the rat. We will also study the intralaminar and inter-areal organization of local circuits in the monkey entorhinal cortex. These data will provide insight into the level of sensory integration and input/output relations that take place in this pivotal cortical region. Taken together, these studies will provide important new information concerning the functional organization of memory systems in the rat and monkey brains.

mental disorders; cognition; nervous system; Primates; Mammalia;

transplantation; nervous system; Primates; Mammalia;

Significance Evidence from animal studies and from brain damaged human subjects indicates that certain brain regions such as the amygdala, the temporal polar cortex and the orbitofrontal cortex play essential roles in the orchestration of social behavior. Understanding these neural systems will have implications for understanding normal social behavior, for understanding, treating and ultimately preventing neurodevelopmental social disorders such as autism and societal concerns such as psychopathology and criminality. Objectives Studies under way are investigating the role of the amygdala in dyadic and tetradic social interactions between macaque monkeys. Highly specific lesions are produced in the amygdala and alterations of social function are observed and quantified by using computer assisted behavioral analysis. Results We have recently completed a 2 1/2 year intensive study of six monkeys with bilateral amygdala damage and 6 age, sex and dominance matched controls. Not only do animals with amygdala lesions demonstrate abnormal interactions with inanimate objects (they lack a normal reluctance to engage a novel potentially threatening object) but their social interactions are altered as well. A novel finding is that the normal animal rapidly detects a difference in the amygdala lesioned animals and demonstrates more affiliative behaviors towards them. Future Directions A second cohort of animals for total amygdala lesions is currently under selection. These animals will be used for refined analysis of social interaction. Preparations are currently under way to initiate funded studies of the effects of neonatal amygdala disconnection on the emergence of social behavior. Finally, the effects of amygdala lesions will be investigated on the ability of macaque monkeys to discriminate facial expressions. KEY WORDS amygdala, social behavior FUNDING NIH Grant MH/HD57502

animal tissue; cognition; nervous system; Primates;

This is a revised proposal that seeks funding to support multidisciplinary predoctoral and postdoctoral training in Systems Neuroscience--the analysis of neural systems that underlie behavior--by creating the Training Program in Systems Neuroscience. The Program is designed to produce new Ph.D.'s and postdoctoral scientists who are capable of establishing independent research programs in neuroscience. It will operate under the auspices of the Graduate Program in Neuroscience, a recently established multidisciplinary program that encompasses faculty from several departments at the University of California, Davis. This application is prompted by the establishment of the Center for Neuroscience and the rapid expansion of the neuroscience community at UC Davis. In the past five years, UC Davis has recruited 10 new faculty members in the area of systems neuroscience, and the strong institutional commitment to the continued development of neuroscience has made UC Davis an ideal environment for the multidisciplinary training of neuroscientists. Funds are requested for five years to support four predoctoral and four postdoctoral students. The Trainers for this program include 26 extramurally funded UC Davis neuroscientists whose research interests range from the molecular biology of calcium channels, to neurophysiological analyses of primate sensory cortices, to neuropsychological analyses of patients with focal brain damage. Many of the faculty emphasize research in mental-health related areas. Graduate trainees will participate in course work, including a comprehensive Core Course in Neuroscience, laboratory rotations designed to aid them in selecting an appropriate research sponsor, mentored research projects, journal clubs, and lecture series that equip them to conduct research responsibly, professionally, and successfully. While postdoctoral students will also participate in that lecture series, their training will emphasize intensive, mentored programs of research. Particular emphasis will be placed on training postdoctoral students to achieve independent research funding. The Training Program will sponsor an annual retreat, during which trainees and other neuroscience graduate students will present their research. Trainees will also participate in the organization of a biweekly seminar series in neuroscience. In order to provide additional instructional resources to trainees and members of the graduate and postgraduate neuroscience community, a Neurosciences Resource Center will be developed that will contain computer-aided instructional materials in neuroscience and facilities for independent research in computer simulation of neural systems.

DESCRIPTION (adapted from applicant's abstract): The primate amygdaloid complex has long been identified as an important component of the brain system involved in mediating appropriate species-specific behaviors such as threat and defense. Large lesions of the inferior temporal lobe, which include the amygdala, produce the so called "Kluver-Bucy Syndrome" which has been characterized as an inability to attribute emotional significance to perceived stimuli. Critical reading of the literature, however, indicates that the experimental basis for this characterization remains tenuous. Previous studies have often suffered either from the lack of discrete lesions, comprehensive histological analysis or ethologically appropriate and sophisticated behavioral assessment. Moreover, recent studies in the human indicate that congenital impairment of amygdaloid function may be more debilitating to normal emotional and social interactions than lesions produced in the mature individual. The program of studies outlined in this revised application uses sophisticated neurobiological and behavioral methods to reassess the role of the primate amygdala in normal social interaction. The studies are carried out in the context of a long-standing program of neuroanatomical studies which demonstrate that the amygdala receives sensory information from widespread regions of the neocortex. The overarching hypothesis guiding this program is that the amygdala functions as a high level perceptual filter that interprets ongoing sensory information in order to orchestrate an appropriate species-specific response. The proposed studies will employ MRI-guided, axon-sparing stereotaxic ibotenic acid lesions of the amygdala along with the assessment of behavioral and physiological responses during dyadic and tetradic social interactions and videotape presentations. Behavioral data will demonstrate whether complete or partial amygdala lesions in the mature primate produce deficits in affiliative and/or agonistic behaviors. In a second phase of these studies, complete amygdala lesions will be produced in neonatal macaque monkeys. The effects of these lesions on mother-infant and juvenile-juvenile interactions will be evaluated. Future studies (when the neonates have matured) will analyze dyadic and tetradic social interactions and thus allow comparisons of the severity of effects of neonatal or mature amygdala lesions on social behavior. During these experiments, the pituitary-adrenal activation of lesioned and control monkeys in response to social and restraint stressors will also be analyzed. These studies will provide important insights into the neurobiology of normal social behavior and may contribute to an understanding of pathologies of social communication in disorders such as autism.

psychology; nervous system; behavioral /social science research tag; Mammalia;

DESCRIPTION (Provided by applicant): The UC Davis M.I.N.D. (Medical Investigation of Neurodevelopmental Disorders) Institute was founded in 1998 as an interdisciplinary organization to study autism and other neurodevelopmental disorders. This unique collaboration among parents, community leaders, and researchers and clinicians at UC Davis takes a three-pronged approach to solving neurodevelopmental disorders; it brings together resources at UC Davis, from the Sacramento community and from the broader UC system to focus on basic and clinical research, patient treatment, and the education of patients, families and providers. Current activities include: an aggressive recruitment campaign to develop a critical mass of researchers, clinicians and educators; the construction of a campus of five buildings comprising approximately 142,000 sq. ft. of space that will include a comprehensive pediatric clinic, two research buildings, a community center and a laboratory school; and the expansion of an active intramural and extramural research program. The vision of the M.I.N.D. Institute is entirely consistent with the establishment of a translational Center of Excellence in Autism Research as outlined in the Children's Health Act of 2000. However, with the intense activity currently underway at the Institute and our need to incorporate many new Institute faculty into the preparation of a Center grant, we believe that it is premature to submit an application for the December deadline. The Developmental Grants for Autism Centers of Excellence provide a useful mechanism for enabling comprehensive discussion, consultation and decision-making in preparation for submission of such an application in 2002. During the planning year we will focus on defining those areas of translational research and community outreach that the M.I.N.D. Institute is best suited to pursue as an Autism Center of Excellence. A broad-based, twenty member, leadership team will participate in weekly brainstorming sessions to shape the aims of our expected application. Intensive two-day Focus Group sessions, during which consultants will not only present their latest data, but also advise the Institute on such promising areas as Biomarkers Research, Clinical Drug Trials, and Innovative Treatments for Autism, will inform the deliberations of this group. We will pursue collaborative relationships with California State University, Sacramento, and several Sacramento County school districts to establish pilot educational programs for children with autism. Evaluation, selection and implementation of a comprehensive database management system will be a priority for ensuring the seamless flow of information between the Institute's clinical and research efforts. Finally, we will explore the feasibility of using telemedicine technology for clinical care and education in autism.

Autism is a neurodevelopmental syndrome defined by deficits in social reciprocity and communication and by unusual repetitive behaviors. While there is clearly an underlying genetic predisposition, the etiology(ies) of autism is/are currently unknown. Development of animal models of autism have been hobbled by the lack of knowledge concerning its etiology(ies) and by the paucity of data on the characteristic neuropathology of autism, i.e., it is not clear what a successful model of autism would look like. If one focuses on the root deficit in autism, i.e., the impairment of social interaction, however, successful mouse and nonhuman primate models are achievable. The overarching goal of this project is to establish batteries of behavioral tasks that will provide sensitive assessments of normal mouse and rhesus monkey social behavior. With the establishment of the animal models, two hypotheses will be tested: 1) that prenatal and/or postnatal exposure to xenobiotics will decrease normal conspecific social behavior; and 2) that changes in social behavior will be associated with alterations of brain regions, such as the amygdala, that have been implicated in social behavior. Perinatal mice will be parametrically exposed to thimerosal, methyl mercury, and to a mixture of PCB congeners (PCB 153, 180, 118, 138, and 170) to determine whether these xenobiotics alter normal social behavior. Based, in part, on the mouse studies and on information concerning expected environmental exposure in autistic children, neonatal monkeys will also be exposed to thimerosal, methyl mercury and PCBs. The mouse battery of social and cognitive testing will include: Response to maternal separation and relocation; response to maternal separation and relocation; response to novel objects; response to a human intruder; response to social videotapes. A specially designed ethogram will also be used to evaluate maternal < - >infant interactions and to study the emergence and quality of social behaviors through daily dyadic social interactions with "stimulus" animals. At the termination of behavioral testing, morphological changes will be evaluated in brain regions, such as the amygdala, known to be involved in normal social behavior. Additional tissue will be distributed to Core I for analysis of xenobiotic distribution, to Core II for analysis of cytokines and autoantibody production and Core III for altered brain gene expression.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The primate amygdala is a complex brain region comprised of 13 nuclei and cortical regions in the rostral portion of the medial temporal lobe. This grant has funded studies with the overarching goal of defining the cytoarchitectonic organization and intrinsic and extrinsic connections of the macaque monkey amygdaloid complex. We have also been investigating neuropathology in the autistic amygdala. We reported (Schumann et al, 2004) that the amygdala in typically developing boys undergoes a 40% increase in volume between 7 and 18 years of age. This expansion occurs at a time when the cerebral volume decreases by about 10%. In boys with autism, the amygdala reaches its adult size by 7 years and does not increase thereafter. Given the association of the amygdala with a variety of psychiatric disorders including anxiety, depression, autism and schizophrenia, many of which are first manifest during the peripubertal period, it would be valuable to determine the morphological features of the amygdala's postnatal development. It is not feasible, however, to carry out this type of analysis in postmortem human brains.

Funding provided by this grant has led to the establishment of a sophisticated infrastructure for evaluating the role of putative brain regions in nonhuman primate social behavior. Highly selective lesions of the amygdaloid complex and of the hippocampal formationhave been made in adult rhesus monkeys and in two week old infants.A unique aspect of this program is that all neonatal subjects are raised by their mothers following neurosurgery and experience daily periods of normal socialization. Both the social behavior and emotional responsiveness of adult and infant animal subjects have been extensively analyzed using quantitative behavioral analytic techniques. During the current funding period, we have also carried out two microPET studies of brain metabolism that opens the door to future noninvasive imaging studies of monkey brain function. We have also developed a new fear-potentiated startle apparatus for the rhesus monkey and have found, contrary to results in the rodent, that the amygdala is essential for learning a fear response but not for expressing a fear memory. The combined protocols for brain imaging, neurosurgery and behavioral analyses that have been developed are now being applied to other brain regions. Currently, a new group of animals with lesions of the orbitofrontal cortex are being studied. Finally, we have further developed the use of the allatostatin transient inactivation technique in rats and are now ready to begin studies with this technology in the rhesus monkey. For the proposed extension period, we have planned seven specific aims. These include: 1) To conclude longitudinal analyses of rhesus monkeys that received bilateral lesions of the amygdala or hippocampal formation at two weeks of age;2) To continue analyses of social and emotional behavior in a group of adult rhesus monkeys that were prepared with bilateral lesions of the orbitofrontal cortex; 3) To prepare a new cohort of adult animals with lesions of the anterior cingulate cortex; 4) To transfect the amygdala of a new cohort of adult rhesus monkeys with the receptor for allatostatin 5) To prepare a new cohort of infant animals either with permanent lesions of the orbitofrontal cortex and/or the anterior cingulate cortex; 6) To carry out in vivo and postmortem morphological analyses of animals that received lesions of the amygdala and hippocampus as neonates; and 7) To carry out microPET studies with social stimuli to explore the organization of regions involved in the social brain.

[unreadable] DESCRIPTION (provided by applicant): Autism is a neurodevelopmental disorder characterized by deficits in social behavior and communication and the presence of repetitive or stereotyped behaviors. The etiology(ies) of autism are currently unknown. However, evidence suggests that maternal autoantibodies directed against fetal brain tissue are a putative cause for a subset of autism cases. In support of this, we have recently identified a characteristic pattern of autoantibody production to human fetal brain tissue (and to rhesus monkey brain tissue) in 20% of mothers of multiple children with autism. Our preliminary studies indicate that rhesus monkeys, prenatally exposed to IgG class antibodies from these mothers, produce more whole body motor stereotypies (a defining symptom of autism) compared to control monkeys. These preliminary findings, while striking, are based on a small number of treated subjects, a restricted window of exposure to the purified IgG and a relatively limited period of behavioral observations. It is possible that extending the duration of IgG exposure may result in other behavioral abnormalities more indicative of the complete autistic syndrome. We propose to replicate and extend our research on this promising immunological model of autism by increasing the number of experimental subjects and increasing the duration of IgG exposure into the second trimester for a subset of the experimental subjects. We will enhance and extend the behavioral observations of the treated animals and carry out a structural neuroimaging study. We propose first to conduct an extensive behavioral assessment of the subjects during the first two years of development. We will quantitatively analyze the emergence of species typical behaviors in a variety of social contexts and in experiments designed to probe attachment, social dominance, social motivation and fear reactions. These studies will also evaluate the quality of transactional interactions and detect abnormal behaviors such as stereotypies. We will also carry out a detailed longitudinal magnetic resonance imaging (MRI) study to evaluate differences in the time course of brain development. We will focus the MRI analyses on brain regions most commonly implicated in the neuropathology of autism, including the frontal lobes, amygdala and cerebellum and on structures associated with stereotypies such as the basal ganglia. This work represents a promising animal model of autism and may have direct implications for diagnosis, prevention and treatment of this disorder. PUBLIC HEALTH RELEVANCE: Autism spectrum disorders affect 1:150 children in the United State though the cause(s) are unknown. This project is designed to provide additional support for the hypothesis that abnormal maternal autoantibodies may be one cause of autism. If maternal autoantibodies are identified as a risk factor for autism, this finding would quickly lead both to the development of a diagnostic assay for maternal autoantibodies and to the development of therapeutic interventions to diminish this autism risk factor. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal responds to NOT-OD-09-058, "Recovery Act Funds for Competitive Revision Applications." This revision application will support new research aims that are outside the scope of the currently funded parent grant, "Infants at Risk of Autism: A longitudinal Study" (2 R01 MH068398-06;1/1/09 - 12/31/13;PI Ozonoff). The parent study examines the earliest possible behavioral indices of autism, but the original aims did not include potential biological markers. Responding to three NIMH areas of interest outlined in the RFA ("Biological Measures for the Study of Mental Disorders, "Understanding Postnatal Brain Development," and "Supporting Longitudinal Neuroimaging Studies in Mental Disorders"), we propose to increase the scope of the parent grant by evaluating brain structure and developmental trajectory as possible early biological markers of autism. We propose to carry out a longitudinal neuroimaging study to investigate brain growth during a critical period of development in autism. High resolution structural MRI will be combined with diffusion-weighted imaging and resting state functional connectivity MRI to evaluate multiple aspects of brain organization in infants at risk for autism. Through the parent study, two groups will be recruited: a high risk group of 40 male infant siblings of children with autism and a low risk group of 20 male infant siblings of children with typical development. They will be imaged beginning at 6-9 months of age and followed at six-month intervals for a total of three time points. At the end of the two year grant period, we will have acquired a rich dataset of 180 scans of infants ranging from 6-21 months of age. Through the parent grant, outcomes of Autism/ASD, Other Delays, or Typical Development are determined at 24 and 36 months of age. We will then compare brain maturation trajectories for the different outcome groups. The parent grant provides a unique opportunity for prospectively evaluating the very early abnormal brain development that has been reported in children with autism. The combined parent and revision projects have the potential to identify both behavioral and biological markers for very early autism and ultimately contribute towards the early identification and treatment of autism. PUBLIC HEALTH RELEVANCE: Autism is a neurodevelopmental disorder that occurs in as many as 1 in 150 children, but is rarely diagnosed before the age of 3. Earlier detection could lead to earlier interventions and reduction of lifelong disability. The aim of the revision study is to identify changes in brain structure and connectivity in infancy that may be present before the clinical diagnosis of autism is made and indicate increased risk for development of the disorder.

DESCRIPTION (provided by applicant): This application, entitled, Interdisciplinary Investigation of Biological Signatures of Autism Subtypes is in response to RFA-MH-09-170 titled, Recovery Act Limited Competition: Research to Address the Heterogeneity of Autism Spectrum Disorders (R01). There is near universal agreement that there are multiple biological subtypes of autism and each of these types may have a quite distinct etiology. A major roadblock to understanding the causes of the different types of autism, and ultimately to identifying preventions or best treatments for each type, is that there is currently no way to distinguish and parse one type of autism from another at diagnosis. What is the best strategy to deal with the heterogeneity of ASD? This application proposes to study a very large cohort of children with autism spectrum disorders (ASD) using a multidisciplinary analytic approach in order to identify biological signatures that define distinct phenotypes of autism. The proposal is an extension of a pilot project initiated at the M.I.N.D Institute called the Autism Phenome Project (APP). One hundred sixty preschoolers with ASD and with typical development (TD) have already received full medical evaluation, medical record review, environmental exposure assessment;blood based genetic and immune profiling, structural magnetic resonance imaging, EEG based studies of auditory processing and comprehensive developmental and neuropsychological evaluations. We have established, therefore, that the intensive bio-behavioral studies proposed in this project are entirely feasible. Moreover, preliminary data from the APP have already highlighted at least three biological signatures of autism phenotypes. These include: (1) the presence of auto antibodies to GABAergic neurons which are observed in 21% of ASD children and 0% of TD (maternal antibodies and cytokine profiles are additional putative immune biosignatures);(2) abnormal auditory evoked potential responses (4 distinct response profiles have been observed) and (3) abnormal brain size (evidence is accumulating for both enlarged and small brains and frontal lobes). Having accomplished the pilot phase of this project and obtaining assurance that all procedures are feasible and well tolerated, we are ready to complete Phase 1 of this project. This will complete ongoing recruitment of 250 children with autism and 150 age-matched controls. This is a sufficiently large number to realistically expect to identify several subtypes of autism. All children will undergo the comprehensive multidisciplinary analyses described previously. Blood samples, DNA and RNA will be banked for future studies. We will establish a new data management and biostatistician group to organize and analyze data and transfer it to NDAR. Not only will this research program accomplish a unique, in depth analysis of a very large cohort of young children with autism and age-matched controls, but it will also provide the substrate for future longitudinal studies aimed at determining how early biological signatures of autism predict subsequent behavioral, biological and medical outcomes. It will also foster effective prevention and treatment research. PUBLIC HEALTH RELEVANCE: Autism is a neurodevelopment disorder that occurs in as many as 1 in 150 children and a major roadblock to preventing and more adequately treating autism is that there are many causes and many types. It is currently impossible to determine what type of autism a child has at diagnosis so in order to solve this problem;we will study the biology of a large number of children with autism to identify biological signatures of different types of autism. We already have strong evidence for at least an autoimmune type of autism and this research will lead to more effective efforts at preventing future cases of autism and treating existing children with autism.

DESCRIPTION (provided by applicant): In this application, we propose a series of experiments that, if successful, will change forever our approaches to evaluating the nonhuman primate social brain, and could have a profound influence on behavioral neuroscience more broadly. Electrophysiological recordings, brain lesions and functional neuroimaging have been the most common methods used to identify and dissociate the function of brain structures in humans and animals. However, invasive neural recordings or lesions can result in unintended damage and compensatory functional reorganization, both of which complicate the interpretation of behavioral results. Functional neuro- imaging, while having the advantage of being noninvasive and amenable to repeated studies, can indicate that a brain region is active during a particular behavior, but cannot determine that it is essential for the behavior. Complementary studies that temporarily manipulate brain function in animal models are also needed. All options currently available for transient brain activation or inactivation in nonhuman primates, however, require repeated injections into the brain and/or permanent cranial implants, both of which cause physical trauma and preclude long-term study of awake, behaving animals. A new technique, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), offers a minimally-invasive means to control brain function during long-term studies. Viral vectors transfect neurons in specific areas with a DREADD gene. These novel receptors are triggered by intravenous or oral administration of a nontoxic molecule called clozapine-N-oxide (CNO). Activation or inactivation of neural activity occurs within 15 minutes after CNO administration and lasts for up to 9 hours. This technique has been successfully used to study complex behavioral patterns in rodents. Our overall objective is to implement DREADD-based transient inactivation in nonhuman primates, which is the animal model of choice for studying the social brain. In Specific Aim 1, we will compare DREADD-based inactivation of one social brain component, the amygdala, with behavioral and metabolic deficits already characterized with permanent amygdala lesions. In Specific Aim 1A, we will use high-resolution positron emission tomography to measure how CNO infusion affects metabolism in the amygdala and other brain areas that are heavily interconnected with it. We will also examine how DREADD- based amygdala inactivation affects fear learning (Specific Aim 1B) and social interactions (Specific Aim 1C). Once we have verified that the DREADD method can reliably inhibit amygdala function, Specific Aim 2 will measure how amygdala inactivation modulates eye gaze patterns as animals view pictures or videos of species-typical social signals. Beyond providing a powerful new tool for minimally-invasive transient inactivation studies with nonhuman primates, the proposed research will also advance our understanding of how the amygdala contributes to social information processing, and how amygdala dysfunction may contribute to the profound social deficits that characterize many human psychiatric disorders.

DESCRIPTION (provided by applicant): The primary, long-term goal of this training program is to prepare a diverse pool of highly trained scientists to conduct interdisciplinary biological, behavioral, and clinical research to understand, treat, and prevent autism spectrum and related neurodevelopmental disorders. Rationale: This interdisciplinary training program addresses the current scientific and public focus on the lifelong effects of autism spectrum disorders (ASD) on social relations, educational and vocational success, community participation and contribution, and lifelong supports. According to Dr. Thomas Insel, Director of NIMH, .. the nation, recognizes the urgent needs of the autism community, and presses on toward the goal of transformative scientific discoveries and enhanced services and supports that will make a difference in the lives of individuals and families living with ASD. This training program, now in its 10th year, prepares scientists to make transformative discoveries in the biology and psychology of persons with autism, discoveries that will improve services and supports, prevent or ameliorate disability, and enhance the lives of persons with ASD and related disorders and the lives of their families. Program objectives involve: 1. Provision of 24 months of postdoctoral interdisciplinary research training to 8 NIH-funded MD and PhD trainees from diverse scientific fields and from cultural backgrounds under-represented in science. 2. Development and delivery of an interdisciplinary curriculum that imparts to trainees the current research questions, methods, and findings on autism spectrum and related neurodevelopmental disorders in diverse fields of study, including genetics/genomics; animal models; epidemiology; immunology; cultural competency and health disparities; neuroanatomy; neuroimaging; metabolomics/proteomics; neurochemistry/psychopharmacology; neurophysiology; and research design and analysis. 3. Development and delivery of training content that grounds trainees in the ethics of research involving children, persons with disabilities, family members, and research access by underrepresented groups; 4. Delivery of training that provides experiential and conceptual understanding of the clinical and psycho- social effects of autism and related disabilities on persons with the disorders and on their families. Design of the trainin program Four main aspects of the training program provide interdisciplinary and disciplinary research expertise: (1) a weekly, interdisciplinary two year curriculum that focuses on the biology and psychology of ASD and related disorders, and on the ethics of research; (2) ongoing disciplinary mentorship and research training from a faculty member with autism research expertise; (3) interdisciplinary research experience with a mentor and team from a different discipline; and (4) experiences of the clinical and everyday effects of ASD on persons and their families.

SUMMARY: PROJECT 3 Common psychiatric disorders such as schizophrenia (SZ), in which symptoms manifest in late adolescence or early adulthood, are increasingly considered neurodevelopmental disorders. There is substantial evidence, for example, that maternal viral disease with resulting immune activation (maternal immune activation, or MIA) significantly increases the risk of having a child who is ultimately diagnosed with SZ. Yet, very little is known about the consequences of MIA on the developmental trajectory of the brain. This knowledge might offer enhanced opportunities for prevention or early treatment. Over the last 8 years, the Center team has developed a nonhuman primate (NHP) model of MIA: a modified form of a viral mimic adapted for use in primates (poly IC/LC) is recognized as foreign by the primate immune system and induces a transient innate inflammatory response. Offspring from this NHP MIA model display abnormal behaviors from early postnatal development that increase in severity up to adulthood. This is the first use of poly IC/LC-induced MIA in the rhesus monkey, which directly parallels the much larger literature on the rodent poly IC model. In addition to behavioral disturbances, members of this Center recently discovered several other phenotypes that are expected in a model for SZ. At 3-4 years of age, offspring exhibit enhanced striatal dopamine uptake, a hallmark of psychosis, as well as neural inflammation. The working hypothesis is that there will be MIA-induced prodromal modifications of the trajectory of brain development that presage the emergence of behavioral abnormalities. Project 3 will carry out longitudinal structural and resting state functional MRI studies of a cohort of 24 rhesus monkeys, half of which will be exposed to MIA; the entire cohort will be behaviorally tested by the NHP Core. The objectives are to establish MIA-induced abnormal brain organization signature in the NHP and compare it to that of MIA-exposed mice and human patients undergoing their first episode of SZ. To test this working hypothesis, the project will pursue the following aims: 1) determine normal and abnormal trajectories of structural brain development for NHP controls and individuals exposed to MIA during gestation; 2) Identify atypical functional connectivity in NHP controls and individuals exposed to MIA during gestation; 3) Compare the MIA-induced abnormal brain organization signature established in NHP to brain abnormalities observed in MIA-exposed mice and human patients undergoing their first episode of SZ. These studies will reveal hitherto unknown information on the alterations in brain development caused by MIA that may precede psychosis. Moreover, establishing a common developmental neuropathology across mice, monkeys and humans will lead to clinically useful biomarkers for detecting individuals at high risk for SZ and will promote earlier prevention and more effective treatment.

DESCRIPTION (provided by applicant): Despite increasing evidence that circulating maternal antibodies can produce disease in the fetus and that anti- brain antibodies are associated with an ever-expanding number of neurological and psychiatric disorders, there is little information concerning when and how antibodies enter the brain, particularly of the fetus during pregnancy. We have previously proposed that gestational exposure to maternal antibodies is associated with the development of autism spectrum disorder (ASD) in human offspring. To support this contention, we exposed pregnant rhesus monkeys to purified IgG obtained from mothers who harbor anti-fetal brain antibodies and have given birth to children with ASD. The rhesus offspring demonstrated behavioral and brain growth alterations that are consistent with ASD. Nonetheless, substantial skepticism remains with the notion that unusual maternal antibodies are pathogenic for ASD. This is due, in part, to the fact that there is so little evidence concernig the entry of antibodies into the fetal brain and the mechanisms by which they might alter brain development leading to ASD. In fact, while papers by Coe and colleagues indicate that maternal antibodies cross the placenta during the latter half of pregnancy in the rhesus monkey, we have not been able to find a single publication that determines whether and when these antibodies enter the fetal monkey brain. To investigate this issue, we have recruited a team of investigators with expertise in neuroscience, immunology, fetal development of the nonhuman primate, and positron emission tomography (PET) to carry out studies with the overarching goal of providing evidence for transplacental transfer of maternal antibodies and entry into the fetal brain. While there are several potential strategies for carrying out these studies, we have focused on PET imaging with the hope that this will establish expertise and a protocol for noninvasively evaluating antibody entry into the central nervous system of the nonhuman primate at various ages. Specific Aim 1 will evaluate the occurrence of transplacental transport of 124I-labeled IgG into the fetal rhesus monkey brain at three developmental time points in the second and third trimesters. Entry of IgG into the brain will be assessed both by PET and by fetal tissue harvests followed by brain dissection and evaluation for radioactivity using a gamma counter. We will use a monoclonal antibody to GPM6A which is a tetraspan membrane protein abundant on neuronal surfaces in the central nervous system. Evaluation of gene and protein databases indicate that this protein is abundant throughout fetal life both in the nonhuman primate (http://www.blueprintnhpatlas.org) and in the human (http://www.brainspan.org/), and is a component of the human surfaceome. Specific Aim 2 will carry out longitudinal PET scans to identify quantitative changes of IgG entry into the fetal brain from midgestation to near term. The studies described here are a first step towards developing a nonhuman primate model of antibody-induced psychiatric illness and establishing the mechanisms through which anti-brain antibodies might influence brain development, brain function and behavior.

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) has a common set of diagnostic features that nonetheless vary substantially in severity in different individuals. There are also many co-morbid features ranging from developmental delay to epilepsy to gastrointestinal disturbances that further complicate the phenotypes of ASD. To discover and integrate multilevel phenotypic information that would allow definition of biological subtypes and potentially predict and facilitate optimal outcomes, the MIND Institute initiated the Autism Phenome Project (APP) in 2006. To date, behavioral, medical, immunological and magnetic resonance imaging (MRI) data have been acquired on 279 children (189 ASD, 90 typically developing controls - TD) at 2-3.5 years of age. Of these, 210 (134 ASD, 76 TD) have received a second MRI scan one year later and thus far 142 (83 ASD, 59 TD) have received a third scan at 5-6.5 years of age. This program of research has identified a number of neurophenotypes of ASD related to abnormal amygdala growth and abnormal brain enlargement. But, do these brain differences really matter? An overarching goal of the proposed research is to determine whether identified neural phenotypes persist into middle childhood and are associated with the quality and severity of core and co-morbid behavioral impairments. A unifying hypothesis is that different morphometric patterns will be associated with clinical features and with the quality of behavioral outcome. We are particularly interested in whether there are different patterns of brain organization that may predict optimal behavioral outcomes in ASD. Using recently developed behavioral modification procedures that yield high quality MRI images from children at all severity levels of ASD, we propose to obtain an additional MRI time point and conduct extensive behavioral assessment of children enrolled in the APP when they reach 9-11 years of age. Based on previous return rates, we estimate that 195 children will participate. We will also recruit 100 new subjects into the APP program. This research would allow an unprecedented exploration of the relationship between brain development, behavioral abnormalities, and cognitive and functional outcome in children transitioning from early to middle childhood. We propose: 1. To evaluate brain and behavioral consequences of three patterns of early amygdala growth; 2. To evaluate brain and behavioral consequences of abnormal brain enlargement in early childhood; and 3. To identify a pattern of brain organization that is associated with optimal behavioral outcome. These projects are consistent with Objectives 1 and 2 of the NIMH Strategic plan and address the 2009 IACC Strategic Plan crosscutting themes of Heterogeneity and Lifespan Perspective. They also contribute to the still unmet research opportunity for Multi-disciplinary, longitudinal, biobehavioral studies of children, youths, and adults beginning during infancy that characterize neurodevelopmental and medical developmental trajectories across the multiple axes of ASD phenotype.... An important goal is to identify children who will need additional or specialized help to achieve the highest quality of life.

PROJECT SUMMARY ? OVERALL If there is one issue that unites the diverse and vocal community of families affected by autism spectrum disorder (ASD), it is the frustration that despite the hundreds of millions of dollars that have been spent on autism research, there are so few treatment options available to decrease the disabilities of their loved ones. The overarching goal of our proposed Center for the Development of Phenotype-Based Treatments of Autism Spectrum Disorder is to discover targets for effective treatments in groups of children with ASD with rigorously defined phenotypic characteristics. It has become abundantly clear that there are many causes and trajectories of ASD. Moreover, some of the most debilitating aspects of ASD are due to the serious co-morbid conditions such as anxiety, seizures and intellectual disability. Considering the broad range of clinical and behavioral features of ASD, it is unlikely that a single treatment will correct all of these problems. This proposal is based on the premise that identifying clinically meaningful subtypes of ASD will facilitate the analysis of etiologies and the development of more effective therapeutics. The Specific Aims for the Center include: Aim #1: To use enhanced clinical evaluations of children with ASD, particularly those with intellectual disability, to better characterize the sub-group that exhibits clinically significant anxiety. Proper diagnosis and effective treatment holds the promise of a much-improved quality of life for these children. Aim #2: To conduct a 16-week randomized comparative treatment trial of Behavioral Intervention for Anxiety in Children with Autism (BIACA), sertraline, and pill placebo in youth with ASD. Aim #3: To use fMRI to investigate neural predictors of treatment efficacy, markers of treatment-induced change, and signatures of anxiety sub-types defined in Aim 1. Aim #4: To carry out behavioral, neuroimaging and electrophysiological analyses of a newly recruited group of children (2-3 1/2-years-old) with ASD and brains that are disproportionately enlarged relative to body size. The major goal of this aim is to increase our understanding of the cognitive functions and brain systems that are so impacted as to lead to a poorer prognosis for these children. This would inform the design of more targeted behavioral interventions. While there is no evidence that these children have less access to standard behavioral therapies, it has become clear that they constitute an ASD phenotype that benefits less from standard interventions. How to treat these children is not yet clear and the Center endeavors to fill this gap. Aim #5: To generate an iPSC patient resource from a subset of the children that are investigated in Aim #4. Lines of iPSCs for each subject will be differentiated into neural progenitor cells, oligodendrocytes and microglial cells to identify gray and white matter contributions to the development of enlarged brains. The cell lines will also be studied by RNA-sequencing to identify gene networks and signaling mechanisms that are altered. These studies may provide additional targets for pharmacological treatment of this form of ASD.

PROJECT SUMMARY ? CORE A: ADMINISTRATIVE CORE The Administrative Core of the UC Davis MIND Institute Center for the Development of Phenotype-Based Treatments of Autism Spectrum Disorder will provide general oversight and management for Center operations. The Core will coordinate development of budgets and insure that expenditures are in line with allocations to projects and cores. It will supervise and assess the research plans and provide intellectual leadership to achieve maximal integration of results across projects and cores. It will also oversee compliance, data sharing, technology transfer, and all aspects of privacy. The Administrative Core will set goals and objectives for the Center and review and update these as work progresses. The Administrative Core, with advice from its advisory committees, will evaluate proposals for new Center faculty and new Center projects. The specific objectives of the Administrative Core are to: ? Provide administrative and budgetary support and oversight to all projects and cores. ? Establish Internal and External Scientific Advisory Committees and a Community Advisory Committee. ? Develop a multi-component program to enhance communication. This will include: a. Establish monthly meetings of the Steering Committee and Center staff. b. Coordinate semiannual meetings of the Internal Advisory and Community Advisory Committees. c. Coordinate an Annual Retreat and annual meetings of the External Advisory Committee. d. Establish an ACE web site for dissemination of Center findings. e. Use the web site and other forms of communication to promote new collaborations that capitalize on the rich dataset from the large cohort of individuals analyzed in this Center. f. Coordinate media presentations of Center findings. ? Safeguard human subjects; oversee establishment and renewal of Center IRB documents. ? Integrate the activities of the Center with those of the MIND Institute Intellectual and Developmental Disabilities Research Center (IDDRC, U54 HD079125). ? Integrate the Center with other Centers and Projects at the MIND Institute and UC Davis. ? Foster interdisciplinary training and include young trainees in Center research. ? With the advice of the Internal and External Advisory Committees, develop mechanisms to launch new Center projects, encourage the participation of new faculty members in autism research, and apply for and acquire additional funding for new projects resulting from Center research. ? Forge links with other ACE Centers and Networks. ? Coordinate and oversee the data and resource sharing plan. Insure data transfer to the National Database of Autism Research (NDAR).

Autism Spectrum Disorder (ASD) affects 1-2% of children in the United States. The etiology(ies) and neurobiological underpinnings of autism remains unclear and hence targets for effective medical treatments are rare. While myriad genetic mouse models have been created, and many demonstrate some phenotypic features of autism, there is growing concern that rodent models may not be the best approach for creating phenocopies of childhood disorders, such as autism, that have cognitive and social disabilities as their core features. Over the last 5 years, through the use of techniques such as CRISPR/Cas9, there has been a revolution in genetically modifying organisms that can be applied to large species such as nonhuman primates (NHP). A first goal of this application is to develop a nonhuman primate model of ASD through loss of function modifications to the CHD8 gene. The gene encoding the chromatin remodeler CHD8 is among the most frequently mutated genes in individuals with ASD. The CHD8 form of autism is unique in being both highly penetrant and having a behavioral and neurobiological phenotype. Individuals with loss of function of this gene not only have autism but typically demonstrate macrocephaly/megalencephaly. We have selected this gene as a starting point because UC Davis Co-investigators on this application have been developing mouse models with Chd8 mutations and analysis of megalencephaly is a major focus of a recently funded Autism Center of Excellence at the MIND Institute. A second goal of the application is to build capacity and expertise in generating genetically modified nonhuman primate models of neurodevelopmental disorders. We argue that UC Davis, with its California National Primate Research Center that houses over 4000 rhesus monkeys, the Mouse Biology Program that has expertise in genetic manipulations leading to hundreds of clinically significant mouse models, and the MIND Institute which houses expertise on all facets of neurodevelopmental disorders research from genetics to clinical trials, is extraordinarily well-positioned to generate and comprehensively evaluate these animal models. We will establish a Leadership Group that will guide this program to successful development of valuable nonhuman primate models of neurodevelopmental disorders. For this initial phase of studies we propose 1) to implement strategies for gene editing of the nonhuman primate, validation of gene editing and efficient production of embryos for later implantation 2) to produce up to ten live rhesus monkeys with Chd8 loss of function mutations 3) to determine normal and abnormal trajectories of structural and functional brain development for rhesus monkeys with Chd8 loss of function mutations and 4) to carry out behavioral analyses of the genetically modified offspring. While the proximal goal of this application is to develop a valuable NHP model to facilitate understanding of the neurobiological underpinnings of autism, a long-term goal is to establish infrastructure to enable the generation of genetically modified monkeys for translational biomedical research more globally - which we believe to be in the national interest.