Brent Vogt - US grants

Anterior cingulate cortex has been implicated in affective responses to noxious stimuli contains opioid synthesizing neurons and has high densities of mu and delta opioid receptors. It is not known which neuronal processes bind opioid receptor ligands. The present experiments will employ experimental localization techniques developed by the P.I. for localizing axonal and dendritic muscarinic acetylcholine receptor subtypes. For whole section analysis one of three types of lesions will be made in rat brain followed by assay of mu and delta-selective ligand binding in cryomicrotome sections, coverslip autoradiography and single grain counting in anterior cingulate cortex: 1) full depth or laminar ibotenic acid, 2) selective destruction of cortical projection neurons following injections of the immunotoxin OX7-ss- saporin into the caudate or periaqueductal gray nuclei or into posterior cingulate cortex, 3) deafferentation undercut lesions. A second localization approach will involve prelabeling of caudate, periaqueductal gray or cortical projection neurons with a stable, retrogradely transported fluorescent dye. Cortical neurons will then be enzymatically and mechanically dissociated and assayed for mu-selective binding of beta-funaltrexamine to prelabeled pyramidal neurons and unlabeled pyramidal and multipolar neurons. Another series of experiments will evaluate mu and delta sites in cortical sublaminae and thalamic subnuclei in cryomicrotome sections of rabbit and human brains. As a number of thalamic and cortical connections are likely involved in sensorimotor aspects of responses to noxious stimuli, multiple label connection studies are also proposed to analyze thalamocortical and corticocortical connections of rabbit cingulate cortex. Studies in thalamus will emphasize the lateral magnocellular nucleus because its connections have not been described and it is not known to exist in any other mammal. Studies at the cortical level will emphasize topographic relations among cingulate areas and among cingulate and visual areas. The main goals of these studies are to elucidate a direct means by which opiate and opioid compounds influence cortical neurons which project to autonomic and somatic motor centers, characterize the distribution of opioid receptors on the dendrites and somata of pyramidal projection neurons and multipolar interneurons and describe the connections of a virtually unknown nucleus in rabbit thalamus. Dissociated adult cortical neurons can be used in future studies to evaluate mechanisms of up and down regulation of opioid receptors as well as gating of ionic channels by opioid compounds.

Anterior cingulate cortex has been implicated in affective responses to noxious stimuli contains opioid synthesizing neurons and has high densities of mu and delta opioid receptors. It is not known which neuronal processes bind opioid receptor ligands. The present experiments will employ experimental localization techniques developed by the P.I. for localizing axonal and dendritic muscarinic acetylcholine receptor subtypes. For whole section analysis one of three types of lesions will be made in rat brain followed by assay of mu and delta-selective ligand binding in cryomicrotome sections, coverslip autoradiography and single grain counting in anterior cingulate cortex: 1) full depth or laminar ibotenic acid, 2) selective destruction of cortical projection neurons following injections of the immunotoxin OX7-ss- saporin into the caudate or periaqueductal gray nuclei or into posterior cingulate cortex, 3) deafferentation undercut lesions. A second localization approach will involve prelabeling of caudate, periaqueductal gray or cortical projection neurons with a stable, retrogradely transported fluorescent dye. Cortical neurons will then be enzymatically and mechanically dissociated and assayed for mu-selective binding of beta-funaltrexamine to prelabeled pyramidal neurons and unlabeled pyramidal and multipolar neurons. Another series of experiments will evaluate mu and delta sites in cortical sublaminae and thalamic subnuclei in cryomicrotome sections of rabbit and human brains. As a number of thalamic and cortical connections are likely involved in sensorimotor aspects of responses to noxious stimuli, multiple label connection studies are also proposed to analyze thalamocortical and corticocortical connections of rabbit cingulate cortex. Studies in thalamus will emphasize the lateral magnocellular nucleus because its connections have not been described and it is not known to exist in any other mammal. Studies at the cortical level will emphasize topographic relations among cingulate areas and among cingulate and visual areas. The main goals of these studies are to elucidate a direct means by which opiate and opioid compounds influence cortical neurons which project to autonomic and somatic motor centers, characterize the distribution of opioid receptors on the dendrites and somata of pyramidal projection neurons and multipolar interneurons and describe the connections of a virtually unknown nucleus in rabbit thalamus. Dissociated adult cortical neurons can be used in future studies to evaluate mechanisms of up and down regulation of opioid receptors as well as gating of ionic channels by opioid compounds.

Neurons in the hippocampal formation and entorhinal cortex are consistently destroyed in end-stage cases of dementia of the Alzheimer type (DAT). In contrast, there is wide variation in the extent of neuron degeneration in posterior cingulate cortex and in the deposition of neurofibrillary tangles (NFT) and neuritic plaques (NP). Cases can be classified into five groups based on the layer(s) which express the most severe neuron degeneration. The laminar pattern of neuron losses, onset and length of the disease and deposition of NFT and NP are similar for each class. Experiments are proposed in postmortem tissue that will explore these hypotheses: a) laminar specificities in cingulate neuron degeneration reflect independent classes of DAT and b) alterations in muscarinic receptor binding may be associated with disruption of cholinergic and thalamocortical connections in a class-dependent manner. The four experiments are as follows. First, the sample of cases for neuron, NFT and NP densities in cingulate cortex will be expanded to 75 to enhance the reliability of this data, including determination of the age at disease onset for each class, and to determine if there is a unique group of cases with neuron degeneration mainly in layer II. Second, it has been shown that the anterior thalamic nuclei degenerate in DAT. These nuclei project to cingulate cortex and have presynaptic muscarinic receptors that can be identified in cortex with oxotremorine-M or AF-DX 116 binding. Thus, each division of this, the lateral nucleus and the medial pulvinar will be analyzed for neuron degeneration and NFT and NP to determine if there are differences within each class of the disease. Third, pilot studies show that a case from class 1 has reduced 3H-oxotremorine-M mainly in layers III-V, while in a case from class 3 binding is massively increased in layers IlIc-VI. These phenomena suggest that there may be different dynamics at work in reorganization of cingulate cortex in different classes of DAT. Thus binding of oxotremorine-M will be analyzed in at least 4 classes of this disease as will that for AF-DX 116 binding which, in the presence of unlabeled pirenzepine, is a selective protocol for presynaptic muscarinic receptors. Fourth, the laminar distribution of hemicholinium-3 binding will be analyzed to determine if there is a compensatory alteration in binding to high affinity choline uptake sites to cholinergic terminals in the layers of cases which have altered ligand binding to muscarinic receptors. The data from these studies will provide new perspectives on neuropathological subtypes of DAT and characterize new features of receptor reorganization in limbic neocortical areas.

Neurons in the hippocampal formation and entorhinal cortex are consistently destroyed in end-stage cases of dementia of the Alzheimer type (DAT). In contrast, there is wide variation in the extent of neuron degeneration in posterior cingulate cortex and in the deposition of neurofibrillary tangles (NFT) and neuritic plaques (NP). Cases can be classified into five groups based on the layer(s) which express the most severe neuron degeneration. The laminar pattern of neuron losses, onset and length of the disease and deposition of NFT and NP are similar for each class. Experiments are proposed in postmortem tissue that will explore these hypotheses: a) laminar specificities in cingulate neuron degeneration reflect independent classes of DAT and b) alterations in muscarinic receptor binding may be associated with disruption of cholinergic and thalamocortical connections in a class-dependent manner. The four experiments are as follows. First, the sample of cases for neuron, NFT and NP densities in cingulate cortex will be expanded to 75 to enhance the reliability of this data, including determination of the age at disease onset for each class, and to determine if there is a unique group of cases with neuron degeneration mainly in layer II. Second, it has been shown that the anterior thalamic nuclei degenerate in DAT. These nuclei project to cingulate cortex and have presynaptic muscarinic receptors that can be identified in cortex with oxotremorine-M or AF-DX 116 binding. Thus, each division of this, the lateral nucleus and the medial pulvinar will be analyzed for neuron degeneration and NFT and NP to determine if there are differences within each class of the disease. Third, pilot studies show that a case from class 1 has reduced 3H-oxotremorine-M mainly in layers III-V, while in a case from class 3 binding is massively increased in layers IlIc-VI. These phenomena suggest that there may be different dynamics at work in reorganization of cingulate cortex in different classes of DAT. Thus binding of oxotremorine-M will be analyzed in at least 4 classes of this disease as will that for AF-DX 116 binding which, in the presence of unlabeled pirenzepine, is a selective protocol for presynaptic muscarinic receptors. Fourth, the laminar distribution of hemicholinium-3 binding will be analyzed to determine if there is a compensatory alteration in binding to high affinity choline uptake sites to cholinergic terminals in the layers of cases which have altered ligand binding to muscarinic receptors. The data from these studies will provide new perspectives on neuropathological subtypes of DAT and characterize new features of receptor reorganization in limbic neocortical areas.

DESCRIPTION (provided by applicant): Anterior cingulate cortex is the most frequently activated cortex in human pain studies and is comprised of perigenual and midcingulate regions. Nociceptive information passes through the midline and intralaminar thalamic nuclei, however, links between the spinothalamic tract and spinoreticulothalamic system to the midline nuclei has never been made directly to cingulate cortex. A case of irritable bowel syndrome with severe visceral pain shows alleviation of symptoms with psychotherapy and reduced midcingulate and increased anterior insular activity. Two hypotheses guide these studies: Functions of perigenual areas 32, 25, 24a-c and midcingulate areas 32' and 24a'-d are driven by different midline thalamic inputs and these nuclei conduct parallel circuits for nociception and stress. Each animal will receive horseradish peroxidase either in the spinal cord or dorsocaudal medullary reticular nucleus, and three fluorescent dextran amine retrograde tracers in contralateral cingulate and/or insular areas followed by reactions for 4 antibodies. Specific Aim #1 will evaluate the immunoarchitecture of the midline and dorsocaudal medullary nuclei with a neuron-specific antibody and antibodies for neurofilaments and calbindin. #2 will determine the projections of the midline nuclei to perigenual, midcingulate, and insular cortices with multiple fluorescent tracers that have the same uptake, transport, and signal properties. #3 will evaluate the topography of inputs to the midline nuclei with anterograde horseradish peroxidase following cervical spinal cord injections. #4 will assess the topography of inputs to the midline nuclei with horseradish peroxidase in the dorsocaudal medullary nucleus. #5 will evaluate overlap of dopamine-13 hydroxylase in each of the midline nuclei with inputs from spinothalamic and medullary nuclei to determine the overlap of nociceptive and stress circuits in the thalamus. These studies will provide the first evidence for differential projections of the midline thalamus to limbic cortex and the extent of spinothalamic, medullary, and locus coeruleus overlap in the medial thalamus and modulatation of sensory, affective, and motor domains of pain processing in anterior cingulate and insular cortices. The mechanisms of medial pain system processing will be identified and this will help interpret human imaging studies and assist in developing objective cdteria and therapies for treating the psychiatric consequences of chronic pain.

The NEUROANATOMY CORE is and integral part of every Main Project in the DEARC. It collaborates with DEARC members on a variety of scientific issues and methods. The CORE provides specific expertise on the organization of neuropathological methods for analyzing neuronal plasticity associated with the prenatal and adolescent exposure to ethanol. The Specific Aims are as follows: (1) CORE personnel work with DEARC members to understand the structure/function of cell, layer, and nuclear architecture, and connections in the developing (e.g., fetal and adolescent) rodent brain; (2) in conjunction with the key personnel for a particular project, the CORE is responsible for tissue preparation. Methods to explore brain structure include histological methods and immunohistochemistry. Metabolic studies rely on 2-deoxy-Dglucose autoradiography and cFos immunohistochemistry which are paired with immunohistochemical methods to focus on specific neuronal phenotypes; (3) light and confocal microscopy and digital imaging will be provided for quantification of findings with microdensitometry, stereology, and grain counting methods; and (4) digital image processing will be provided with a live microscopic, video-conferencing system for group evaluation of methods and findings and another system for producing illustrations for publication. Thus, the NEUROANATOMY CORE provides invaluable expertise in the basic science and methods necessary to conduct all of the Main Projects and may of the Pilot Projects.

The NEUROANATOMY CORE is and integral part of every Main Project in the DEARC. It collaborates with DEARC members on a variety of scientific issues and methods. The CORE provides specific expertise on the organization of neuropathological methods for analyzing neuronal plasticity associated with the prenatal and adolescent exposure to ethanol. The Specific Aims are as follows: (1) CORE personnel work with DEARC members to understand the structure/function of cell, layer, and nuclear architecture, and connections in the developing (e.g., fetal and adolescent) rodent brain;(2) in conjunction with the key personnel for a particular project, the CORE is responsible for tissue preparation. Methods to explore brain structure include histological methods and immunohistochemistry. Metabolic studies rely on 2-deoxy-Dglucose autoradiography and cFos immunohistochemistry which are paired with immunohistochemical methods to focus on specific neuronal phenotypes;(3) light and confocal microscopy and digital imaging will be provided for quantification of findings with microdensitometry, stereology, and grain counting methods; and (4) digital image processing will be provided with a live microscopic, video-conferencing system for group evaluation of methods and findings and another system for producing illustrations for publication. Thus, the NEUROANATOMY CORE provides invaluable expertise in the basic science and methods necessary to conduct all of the Main Projects and may of the Pilot Projects. PERFORMANCE SITE(S) (organization, city, state) Department of