Martin Cassell - US grants

Behavioral and physiological studies have implicated the central nucleus of the amygdala (CNA) in the motoric (both somatic and visceral) expression of fear behavior. Anatomically, the CNA is well situated to modulate information concerning the external and internal environment, via limbic cortices, amygdala and hypothalamus, and to affect autonomic and somatc behavior via projections to the basal forebrain, ventral mid-brain and brain stem. The distribution pattern of cortical afferent termination in the CNA is poorly understood though the available evidence indicates that these afferents terminate in specific cytoarchitectonic zones. This may reflect specific interactions with efferent projection neuron populations in the CNA that likewise show highly specific distributions. Moreover, terminals containing met-enkephalin and pro-opiomelanocortin (POMC) derived peptides are distributed over specific parts of the CNA. Preliminary studies suggest that these distribution patterns are congruent with both the distribution of certain cortical afferents and with specific populations of projection neurons. The proposed investigations are designed to test the applicant's hypotheses that (a) cortical afferents that show nonoverlapping distributions in the CNA innervate different populations of projection neurons and (b) that met-enkephalin and POMC peptides interact with cortical afferents contacting similar projection neurons. Results may lead to a better understanding of how the CNA processes cortical information and how endogenous peptides (and drugs that mimic/block these peptides) influence behavior. The distribution of cortical afferents in the CNA will be determined using the anterograde transport of peroxidase conjugated lectins. Data will be correlated with the applicant's previous work on the distributions of projection neurons in the CNA. Evidence of direct cortical innervation of CNA projection neurons and interactions with terminals containing met-enkephalin and POMC peptides will be sought at the ultrastructural level using combined lesion-induced degeneration of cortical afferents, peptide immunocytochemistry and retrograde transport of horseradish peroxidase from focal injections into the medulla and basal forebrain.

DESCRIPTION (provided by applicant): The amygdala is widely recognized as playing a critical role at the interface between emotion and behavior. Much of the interpretation of functional imaging, neuropsychological and pathological studies of the amygdala in anxiety, phobias, depression and drug and alcohol addiction, and the results of a large number of animal experiments, are framed in the context of a linear model of amygdala organization. In this model, inputs from the cortex and thalamus converge on the lateral and basolateral nuclei where learned associations are generated and expressed as autonomic and endocrine changes through the connections of the central, nucleus, the "output" nucleus of the amygdala. However, more and more clinical and experimental findings are becoming difficult to reconcile with this model. The experiments in this proposal are directed at the P.I.'s long term goal of developing a network-based model of amygdala organization with greater interpretative and predictive value. The experiments focus on the central extended amygdala (CEA) not as connecting the rest of the amygdala with autonomic/endocrine areas but as a basal ganglia-like structure involved in re-entrant circuits with the insular cortex, another area increasingly being associated with appetitive drives, emotion and psychiatric disorders. First, combinations of axon tracers will be used to identify the key nodes in re-entrant circuits through the CEA, including a possible "indirect" pathway involving the lateral hypothalamus. Second, immunochemical methods will be used to identify neurochemical compartments in the CEA and determine whether they are organized on the same lines as the nucleus accumbens. Finally, the reconstruction of axons derived from cortical and CEA neurons will be undertaken to examine the likely sequential processing of information by the amygdala network. Preliminary data indicate that the CEA, a so-called "output" structure may receive cortical/thalamic input before the lateral and basolateral nuclei. The experimental results should provide a strong basis for developing a second generation model of amygdala organization offering much greater interpretive value for experimental and clinical studies of psychiatric disorders.

Antibodies; Antigens; blood pressure regulation; Brain; Brain region; Cell Count; Complex; cost; Data; Databases; Dendrites; design; Electron Microscope; Electrons; Elements; Enzymes; Equipment; Equipment and Supplies; experience; Freezing; Genetic; Histocytochemistry; Image; immunocytochemistry; Individual; Injection of therapeutic agent; instrumentation; Light; light microscopy; Link; Methods; Microscopic; Microtomy; Mus; Nerve; Nervous system structure; Neural Pathways; Neuroanatomy; Neurons; Peroxidases; Population; Preparation; programs; Publications; relating to nervous system; Research Personnel; research study; Resolution; Rodent; sample fixation; Services; Signal Transduction; Technical Expertise; Techniques; Tracer; Virus

The purpose of the Neuroanatomy and Neurophysiology core will be to provide expertise, technical assistance and centralized equipment and facilities for all neuroanatomical and sympathetic nerve recording aspects of the program project experiments. The facilities available will allow investigators to access equipment and materials necessary for preparation of the high quality microscopic material and state-of-the-art technique to study sympathetic nervous system activity, all essential for successful completion and interpretation of experiments performed on the brain. As a new addition, a subcontract of the core located at Weill Cornell Medical College will perform ER stress biomarker analysis. This will be achieved in a cost-effective manner by the centralization of facilities and expertise. Specifically, the core will provide: i) Expertise and instrumentation for gene transfer into the mouse brain nuclei in highly specific manner; ii) A full service for the preparation of morphological and immunocytochemical material for analysis with light and electron microscopy. This will include fixation, thin and ultrathin sectioning, and pre-embedding immunocytochemistry, etc. Expertise in the analysis and interpretation of this material will be provided; iii) Quantitative methods for determining cell numbers, etc., and planning, designing and implementing neuroanatomical experiments; iv) Expertise and instrumentation for sympathetic nerve recording, including data analysis and interpretation; v) Training in experimental neuroanatomy and neurophysiology for principal and co-investigators and associated postdoctoral and other students; vi) Measurements of ER stress biomarkers in brain micropunches harvested for Projects 2 and 3 by this Core B at Ul, and harvested at Cornell by Project 1. The core will perform experiments involving a) the precise targeting of brain regions with adenoviral vectors b) the identification of specific neuronal populations in complex brain regions through the use of antibodies and other markers; c) the use of electron microscopic methods to determine the presence of morphological changes associated with ER stress, the interactions of neural elements, and the subcellular localization of antigens not resolvable with light microscopy, d) nerve recordings from sympathetic and other afferent fibers innervating the kidney, muscle and adipose tissue, and e) measures of ER stress biomarkers.