Stephen L. Foote - US grants

The studies proposed here further test the hypothesis that corticotropin-releasing factor (CRF), the peptide responsible for initiating neuroendocrine stress responses, also functions as a neurotransmitter in the nucleus locus coeruleus (LC) during stress. It is proposed that stressors release CRF from axons innerving the LC, thereby activating these noradrenergic neurons which in turn mediate autonomic and/or behavioral aspects of stress responses. Previous experiments have demonstrated: 1) CRF-immunoreactive (CRF-ir) fibers in LC; 2) LC activation by intracerebroventricular (ICV) or locally applied CRF; and 3) LC activation by a hemodynamic stressor is prevented by microinfusion of a CRF antagonist into LC. The proposed Specific Aims will further test the hypothesis, identify neuronal circuits involved in stress-induced LC activation, and determine the function of LC activation in stress. These aims are: 1) To further characterize the morphology of CRF-ir fibers in LC and to determine the source(s) of these fibers. Laser confocal microscopy, CRG immunohistochemistry, and retrograde tracing will be used; 2) To identify stressors which activate LC, determine whether this activation is mediated by neurotransmitter CRF, and characterize the neuronal circuits involved in this activation. The CRF antagonist, alpha-helical CRF 9- 41 (AHCRF), and dexamethasone will be used to determine whether stress-induced LC activation is due to extrahypophyseal (i.e., neurotransmitter) CRF release. Injections of AHCRF into LC and of GABA into nuclei that are potential sources of CRF innervation will elucidate circuits mediating LC activation; 3) To determine the effects of stress-induced LC activation on autonomic measures, electroencephalographic (EEG) activity, norepinephrine release, and stress-related behaviors. LC discharge, EEG activity, and blood pressure will be recorded in unanesthetized rats. Local LC injection of AHCRF, clonidine, or GABA will be used to assess the role of CRF in the LC and/or of LC activation in arousal or autonomic endpoints of stress. A behavioral index, a proconflict effect, will be similarly evaluated; 4) To determine whether CRF effects on LC discharge, previously demonstrated in rat, extend to monkey. The effects of CRF and hemodynamic stress on LC spontaneous and sensory-evoked discharge will be characterized in monkey. Thus, integrated anatomic, physiologic, and behavioral approaches will reveal some of the mechanisms by which neurotransmitter CRF mediates non-endocrine aspects of stress responses.

The studies proposed here are designed to elucidate the functions of noradrenergic (NA) and serotonergic (S) afferents in primate neocortex. These projections originate in brainstem nuclei (the locus ceruleus (LC) and raphe nuclei (R), respectively) and innervate the entire neocortical mantle. Our immunohistochemical studies indicate that both systems innervate monkey neocortex more densely than previously thought, with each exhibiting a unique pattern of specialization in terms of region-specific densities and laminar distributions of fibers. This suggests differing functions for the two transmitters in terms of the cytoarchitectonic regions and the classes of neurons which are their principal targets. We have described enhanced acoustic signal processing by monkey auditory cortex neurons during microiontophoresis of NA. These and other observations suggest an important role for NA input in modulating spontaneous and stimulus-elicited neocortical neuronal activity. Other data suggest that the physiologic functions of S innervation differ from those of NA innervation. To demonstrate specific functions for these systems in neocortex, multiple coordinated experiments are proposed. Microiontophoresis, manipulation of source-cell activity, and transmitter-specific lesions will be combined with single-cell recording from neocortical sensory regions to determine the impact of each system on cortical neuronal responses in unanesthetized monkeys. Histological reconstruction of recording sites will include immunohistochemical visualization of NA and S fibers to permit direct comparison of the distribution of their effects on cortical neuronal activity with the regional and laminar distribution of their axons. Our previous data describing the activity of monkey LC neurons and the conduction properties of their axons will be used to make experiments on the LC-NA system as physiologically relevant as possible. Experiments to determine the discharge characteristics and axonal conduction properties of primate R neurons are also proposed. These studies will yield: 1) detailed maps of NA and S innervation of specific cortical fields, 2) quantified measurements of the effects of iontophoretically applied S, NA and other putative neurotransmitters on functionally characterized neocortical neuronal activity, 3) comparable data on LC and R activation and lesion effects, 4) correlation between LC or R and neocortical neuronal activity, 5) hypotheses of the roles of these systems in neocortical information processing.

Biochemical data suggest that noradrenergic (NA) neurons in the locus ceruleus (LC) are activated by stress-inducing conditions. Recent biochemical, immunohistochemical and electrophysiological data suggest that this activation may be mediated by neurotransmitter corticotropin-releasing hormone (CRH) secreted from axons terminating in LC. The proposed experiments will extend these observations and test three hypotheses: a) LC is activated during stress, producing certain components of stress responses, b) neurotransmitter CRH is released during stress, also producing components of stress responses, c) stress-induced LC activation is mediated by neurotransmitter CRH. The proposed aims are: 1) To further characterize, at the light microscopic level, the CRH innervation of LC and other brain regions in both rat and monkey. Immunohistochemical and transport methods will determine the source of CRH innervation of LC, map CRH-containing neurons and processes in monkey neocortex, and verify that CRH is contained in the primate olivocerebellar pathway. 2) To determine whether particular "stressors" electrophysiologically activate rat LC neurons and whether this activation can be antagonized by pharmacologic means. Microelectrode recordings in LC of anesthetized and unanesthetized rats will determine whether hemorrhage, temperature fluctuations and audiogenic stress activate LC neurons and whether this activation can be blocked by CRH antagonists, CRH antisera, or steroid treatment. 3) To determine, utilizing in vitro intracellular recording, the membrane events which mediate CRH effects on LC activity. A slice preparation will be used. 4) To characterize the involvement of LC neurons in generating behavioral components of stress responses utilizing an audiogenic stress paradigm. Verifiable, reversible manipulations of LC activity by discrete drug infusions will test whether LC activation and/or CRH infusion produce stress-induced behaviors, and whether these effects can be blocked by LC inactivation and/or CRH antagonist infusion. 5) To test the hypothesis that CRH innervation of a subpopulation of dopamine-containing neurons functions to activate these neurons in response to stressful stimuli. Anatomic observations suggest a dense CRH innervation of a subpopulation of DA neurons. Methods similar to those in Aims 1-4 will be used to study possible CRH-DA interactions. The proposed studies will furnish cellular anatomic and physiologic information about NA, DA, and CRH functions and interactions in stress.

The widespread abuse of cocaine and other stimulant drugs constitutes a major public health concern. In experimental situations, repeated stimulant administration produces progressive changes in behavioral responses, and these changes are believed to reflect processes that are involved in addiction and relapse in humans, as well as in the induction of stimulant psychosis with higher-dose patterns of stimulant intake. Extensive studies in rodents have greatly contributed to current perspectives regarding the relationships between neurochemical and behavioral actions of stimulants. These studies indicate that brain monoamines, particularly dopamine, are involved in mediating the behavioral effects of these drugs. However, little is known regarding the neurochemical effects of these drugs in humans or other primates. This lack of knowledge about the extent to which the neurochemical- behavioral relationships observed in rodents extend to primates is a serious deficiency in understanding the neurobiology of stimulant effects. It is proposed here to use in vivo microdialysis in awake monkeys to: 1) characterize the effects of acute cocaine administration on dopamine, norepinephrine, and serotonin dynamics in basal ganglia and neocortex and on concomitant behavioral and physiological measures; 2) characterize changes in transmitter, physiological, and behavioral response profiles with the repeated administration of cocaine; 3) assay the same measures during withdrawal and subsequent cocaine administration. MRI scans will be used to accurately implant guide cannulae for subsequent microdialysis sampling from motor cortex, caudate nucleus and nucleus accumbens. Animals will be chaired daily for 33 days, and behavioral, EEG and autonomic data will be collected each day. There will be a 5-day baseline period followed by 14 days of cocaine administration, 13 days of withdrawal, and a final day of cocaine challenge. On all days, animals will be treated similarly, except for receiving either i.v. cocaine (1.5 mg/kg) or saline. Dialysis samples will be collected on the first and last days of cocaine administration and on the challenge day, through dialysis probes inserted on the preceding day. The collection of neurochemical, behavioral, EEG, and autonomic measures in a controlled regimen will provide novel information concerning the effects of acute and chronic cocaine, as well as withdrawal and subsequent drug challenge, on these variables and their interrelationships.