Daniel S. Zahm - US grants

On the basis of the terminal fields of axons projecting from the A9 and A10-A8 mesencephalic dopaminergic cell groups, the striatum in the rat can be parcellated as three subdivisions: the nucleus accumbens and olfactory tubercle are innervated exclusively by A10-A8; the dorsolateral quadrant of the neostriatum is innervated exclusively by A9; a ventromedial part of the caudate-putamen (C-P) extending along its entire rostrocaudal axis is innervated by the A9 and the A10-A8 cell groups. Reported differences in the afferent innervation of the A9 and A10-A8 cell groups and differences in the striatal distribution of synapses from each, both at light and electron microscopic resolutions, magnifies the significance of the striatal region where the projections of A9 and A10-A8 overlap. A series of correlative light and electron microscopic investigations are proposed. In the first, it will be determined if the distribution of tyrosine hydroxylase-immunoreactive (TH-IR) boutons in the zone of overlap resembles the distribution in the nucleus accumbens or neostriatum, or represents a combination of more than one kind of synapse distribution. Complementary studies employing a combination of TH immunocytochemistry and Golgi impregnation will prove the distribution of TH-IR synapses on identified neurons in the ventromedial tier of the C-P. The anterogradely transported lectin, PHA-L, will be used at electron microscopic resolutions to demonstrate the kinds of synapses formed by projections originating in A9 and A10. A combination of tracing methods using the anterogradely transported lectin, PHA-L and wheat germ agglutinin, will be used to demonstrate the convergence of projections originating in A9 and A10 on unidentified neuronal elements in the ventral tier of the C-P. These studies will be supplemented by an investigation in which Golgi-impregnation, transported PHA-L, and anterograde degeneration will be combined to demonstrate the convergence of A9 and A10 projections on identified striatal neurons. Because the dopaminergic innervation of the posteromedial and anterolateral parts of the striatal domain of the olfactory tubercle is a potentially rich source of experimental information about dopaminergic and peptide influences on striatopallidal neurotransmission, these areas will be subjected to a battery of studies similar to those described above using antisera against TH, cholecystokinin, and neurotensin.

This project addresses fundamental neuroantomical issues about parts of the telencephalon, diencephalon and brainstem that are strongly influenced by mesotelencephalic dopamine and subserve spontaneous, purposeful behaviors. The structures involved comprise systems that are adversely affected in disease and drug abuse with resultant physical impairment and abnormal behavioral manifestations of enormous cost to American society. It is proposed to continue to develop a model of cortico- subcortical relationships dependent upon parallel, interconnected "functional-anatomical systems"- differentiated neural networks linking high-order association cortical areas and association cortex-like structures, such as the basal amygdala, with specific telencephalic structures, such as the central division of the extended amygdala and districts in the ventral parts of the basal ganglia associated with the nucleus accumbens core and shell. The patterns of intrinsic and extrinsic connections through which these systems interact with each other and structures in the diencephalon and brainstem will be addressed, as will their relationships with cell groups giving rise to ascending monoaminergic and cholinergic projections reported to control attention, vigilance, reward and locomotor activation. The objectives of the research are: to determine if [1a]cortical afferents of extended amygdala and shell of the accumbens arise from distinct subpopulations of neurons, [1b] outputs from different parts of the extended amygdala give rise to distinct patterns of termination, [2a] projections exist from the nucleus accumbens and extended amygdala to the cholinergic pedunculopontine tegmental nucleus and magnocellular basal forebrain neurons, and [2b] cholinergic neurons in the caudal part of the central extended amygdala are corticopetal. It is proposed also to characterize [3] the pattern of neuronal loss in the ventral tegmentum following neurotoxic lesions, [4] the chemospecificity and connections of basal forebrain neurotensin neurons that project to the ventral tegmental area and substantia nigrapars compacta, and [ 5] the connectional relationships of an until now unaddressed sector of the basal forebrain. The studies will be carried out with contemporary experimental neuroanatomical methods evaluated with the aid of qualitative and quantitative light, epifluorescence and electron microscopy. The experiments represent logical extensions of studies completed in preceding cycles of this grant. Each one addresses a specific question about neural relationships that will either (a) test the "functional-anatomical systems" model as currently configured or (b) extend the scope of the model. Knowledge about the organization and inter-relationships of forebrain functional- anatomical systems acquired through these studies will guide future studies addressing how multiple neural systems cooperate and compete in the synthesis of behavior and how the underlying processes are corrupted individually and collectively by neurodegenerative disease and substance abuse.

DESCRIPTION (provided by applicant): Neurotensin (NT) is a peptide neuromodulator that regulates and is regulated by dopamine (DA). Behavioral effects of psychostimulant drugs, such as cocaine and amphetamine, and injections of NT into the mesencephalic ventral tegmental area (VTA) are similar, and both undergo sensitization - i.e., repetitions produce long-lasting augmentations of neurochemical and behavioral responses to subsequent challenges. Psychostimulant sensitization is blocked by VTA injections of several receptor antagonists, including the NT antagonist, SR-48692, which may attenuate the development of psychostimulant sensitization by blocking an observed stimulatory action of NT on VTA DA neurons. A neuroanatomical study recently carried out in this laboratory revealed that the neurons of origin of a dense NT immunoreactive fiber plexus in the VTA are located largely in the lateral preoptic region and rostral lateral hypothalamus (LPH region). These LPH neurons are not in the striatopallidal circuitry and don?t exhibit the robust regulatory effects that DA exerts on striatal neurons, so it is likely that they are regulated separately. The objectives of the proposed research are 1: to determine if stimulation of the pathway from LPH NT-expressing neurons to the VTA [a] alters extracellular NT levels in the VTA and, if so, [b] the effect is associated with a change in locomotor activation state; 2: to determine if extracellular levels of NT in the VTA are altered by acute or repeated administrations of cocaine or by a cocaine challenge in rats that have developed sensitization to cocaine and whether any changes observed are reversed by lesions of the LPH-VTA pathway; and 3: to determine if responses to cocaine challenge are altered by bilateral lesions of the LPH-VTA NT pathway in a manner that is reversed by delivering NT bilaterally to the VTA. The results of these studies will contribute to a more circuitry-based appreciation of how NT influences normal motivated behavior and behavior modified by psychostimulant drug administration. The data may lead to better, more selective therapeutic approaches to prevention of psychostimulant sensitization, which is thought play a significant role in destructive drug seeking behaviors.

DESCRIPTION (provided by applicant): Corticotropin-releasing factor (CRF) is the major mediator of stress responses in the brain and has been implicated in both normal fear and pathological anxiety. The extended amygdala, a functional-anatomical continuum in the basal forebrain, contains the CRF systems considered most responsible for mediating the behavioral aspects of fear and anxiety produced by stressful situations. Recent progress in the characterization of CRF mechanisms across the brain suggests that two dichotomous anatomical CRF systems, mediated by specific CRF1 versus CRF2a receptor subtypes, modulate unique facets of the stress response. The extended amygdala represents one of few brain regions in which both hypothesized CRF receptor systems are present to a significant degree; yet little detailed knowledge exists regarding CRF receptor systems in this region. The experiments in this proposal are intended to provide basic knowledge both about CRF receptor systems that contribute to fear and anxiety as well as about the organization of the extended amygdala and neighboring caudal accumbens shell. Our combined anatomical and behavioral approach reflects our appreciation of the inter-dependency of form and function, and that a thorough understanding of any neural system requires examination of both. Our specific aims are: 1) To describe the anatomical organization of CRF receptor systems in extended amygdala and caudal accumbens shell. The proposed experiments will describe: a) the distribution and co-localization of CRF receptor subtypes (CRF1 and CRF2a receptors) within extended amygdala and caudal accumbens shell regions (immunohistochemistry combined with in situ hybridization), b) the afferent pathways delivering CRF peptide to extended amygdala and caudal shell (retrograde tracer combined with in situ hybridization), and c) the efferent pathways of extended amygdala and caudal shell cells containing CRF1 versus CRF2a receptors (retrograde tracer combined with in situ hybridization). 2) To determine the roles of CRF receptors in the extended amygdala and caudal accumbens shell in the behavioral expression of fear and anxiety. Proposed experiments will examine how fear and anxiety behavior are modulated within specific regions of extended amygdala and caudal accumbens shell by: a) non-specific CRF ligands, and b) subtype-specific ligands for CRF1 versus CRF2a receptors.

DESCRIPTION (provided by applicant): In life, actions necessary to redress an organism's wants or needs are always accompanied by some level of threat to its well-being, requiring that choice mechanisms involve a capacity to weigh threat against potential gain. One working model of brain systems that might subserve such mechanisms includes the participation of basal forebrain functional-anatomical macrosystems, such as ventral striatopallidum, extended amygdala and septal-preoptic system. Much literature indicates that basal forebrain macrosystems are concerned with the formation of neural associations reflecting potentially rewarding and threatening aspects of internal and external stimuli. While the various macrosystems have their own domains of termination in the basal forebrain, subsets of outputs from all of them converge in the lateral preoptic area (LPO). LPO outputs, in turn, are aimed mainly at structures that directly control dopaminergic neuronal activity, including the lateral habenula (LHb), newly discovered rostromedial tegmental nucleus (RMTg) and the pedunculopontine tegmental nucleus and associated midbrain extrapyramidal area (PPTg/MEA). During the preceding funding cycle we observed that activation of the LPO, but not adjacent basal forebrain sites, such as the ventral pallidum, extended amygdala, sublenticular substantia innominata, or the diagonal band nuclei, stimulates ambulatory locomotion up to 10-fold over baseline. This robust locomotion was attenuated but not abolished by systemic injections of the dopamine receptor antagonist haloperidol, suggesting a significant independence of the response from dopamine neurotransmission. In contrast, locomotion elicited by injection of methamphetamine (1 mg/kg, s.c.) was blocked by an LPO infusion of the GABA A agonist muscimol. Insofar as locomotor activation is viewed as a behavioral expression of a sense of impending consummation from which intended motor acts may be launched, the specificity and sensitivity with which the LPO elicits locomotor activation combined with the convergence there of outputs from reward- and threat-sensitive macrosystems suggests that the LPO is a critical site where macrosystems influence behavior. In order to further pursue these ideas, we propose to provide [1] a detailed experimental chemical-neuroanatomical analysis of the pathways involving the LHb, RMTg and PPTg/ MEA by which the LPO influences the mesolimbic dopamine system and [2] functional analysis of LPO regulation of locomotion, threat response and conditioned place preference in the presence and absence of dopamine neurotransmission. Impulsive, maladaptive decision-making is a major societal concern that accompanies anxiety, depression, panic, compulsion, and attention disorders and underlies chronic joblessness, dysfunctional interpersonal relationships, ruinous child-rearing and addictions. Addiction, if regarded as behavior compulsively repeated in the certain knowledge of its self-destructiveness, is bad decision-making by definition. The proposed studies will reveal non-dopaminergic and dopaminergic mechanisms that modulate LPO-elicited locomotor activation, which we propose contributes to the neural processes that underlie impulsive decision-making.