Many basal forebrain and mesopontine tegmental cholinergic projection systems tend to overlap in their origins. This raises the possibility that these projection systems are collateralized to innervate divergent areas. In experiment one, the degree to which basal forebrain and mesopontine tegmental neurons that innervate the reticular thalamic nucleus have axons that collateralize to innervate the cortex as well was examined with a retrograde fluorescence labeling method combined with immunohistochemistry. A significant portion of the labeled neurons in the region of the nucleus basalis magnocellularis and pedunculopontine tegmental nucleus projecting to the reticular thalamic nucleus were observed to be also labeled (double-labeled) following intracortical tracer injections. Many of these double-labeled neurons displayed choline acetyltransferase choline acetyltransferase immunoreactivity. It was also shown that numerous basal forebrain neurons that innervated the reticular thalamic nucleus contained the calcium-binding protein, parvalbumin. These neurons tended to be located more rostrally than the ChAT immunoreactive neurons; primarily in the region of the ventral pallidum. There was some indication that parvalbumin-containing neurons in the basal forebrain that innervate the reticular thalamic nucleus also have axons that branch to innervate the cortex. Finally, none of the basal forebrain neurons innervating the reticular thalamic nucleus was found to contain somatostatin. In experiment two, the degree to which basal forebrain neurons have axons that collateralize to innervate the interpeduncular nucleus and hippocampus was examined with retrograde fluorescence labeling methods. Labeled neurons projecting to both of these limbic structures were observed only occasionally. Comparison of the distribution of single labeled neurons innervating each of these structures revealed that within the region of origin, in the horizontal limb of the diagonal band, neurons innervating the interpeduncular nucleus tended to be located dorsally to those innervating the hippocampus. The results of these experiments are discussed in relation to their anatomical and functional implications toward a greater understanding of the basal forebrain and mesopontine cholinergic and non-cholinergic projection systems. / Medicine, Faculty of / Graduate

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Acetylcholine modulates neuronal activity in the brain with different responses in activity depending on the region of the brain. Our study was focused on the cholinergic modulation of excitatory synaptic transmission in the monkey anterior cingulate cortex (ACC) and lateral prefrontal cortex (LPFC), with specific focus on the effects of carbachol, a cholinergic agonist, on spontaneous excitatory postsynaptic currents (sEPSCs) and on the expression the muscarinic cholinergic type II (M2) receptor in these regions. We used electrophysiology to analyze the effects of carbachol on sEPSC of layer 3 (LIII) pyramidal neurons from each area. We used confocal microscopy to study the M2 colocalization with axon terminals labeled with vesicular glutamate transporter 1 (VGLUT1) in the ACC and LPFC, and the colocalization of M2 with specific axon terminals from the amygdala labeled with tracer and terminating in the ACC. Results from the electrophysiological experiments showed that both the ACC and LPFC L3 neurons responded to carbachol by decreasing the frequency of sEPSCs. Cells from the LPFC showed a decrease in sEPSC frequency after 4 minutes in carbachol, an earlier timepoint than ACC neurons, which showed a decrease in sEPSCs frequency after 6 minutes in carbachol. In the confocal studies, M2 expression and colocalization with VGLUT1 terminals in the ACC and LPFC were observed. However, we observed a greater total area of M2 expression in the ACC versus the LPFC in layer 1. We found minimal colocalization of the M2 receptor with axon terminals from the amygdala in the ACC. Together, our data show that acetylcholine has distinct interactions with neurons and pathways in ACC and LPFC, which may be related to the distinct function of the two areas in cognition, learning and memory.

Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references.

With medical advancements extending the life span, age-related cognitive decline is a growing problem for the United States. A rat model of cognitive aging was used to investigate the GABAergic neurotransmitter system in relation to changes in learning and memory functions. Confocal stereology was used to determine the number of GABAergic and cholinergic projection neurons in the rostral basal forebrain of spatially characterized young and aged male F344 rats. The GABAergic system was then assessed as a potential target for improving age-related cognitive decline using an odor discrimination task sensitive to decline in aging. Performance of aged rats was impaired compared to young rats on the spatial version of the Morris water maze. Notably, a high degree of variability in individual abilities was observed among aged rats such that some aged rats performed on par with young (aged-unimpaired) and others performed outside the range of young, demonstrating impairment (aged-impaired). The number of basal forebrain neurons expressing multiple immunomarkers for GABAergic septohippocampal projection cells was selectively increased in aged-impaired rats in comparison to both young and aged-unimpaired rats. Indeed, among aged rats, worse performance in the water maze was reliably associated with higher GABAergic cell number. The number of cholinergic neurons, quantified in adjacent sections did not differ as a function of chronological age or cognitive status. These data suggest that aging can dysregulate GABAergic systems in circuitry important for learning and memory and such alterations may contribute to age-related cognitive decline. To test whether the GABAergic system may be a viable target for treating age-related cognitive decline, a second cohort of young and aged rats was characterized in an odor discrimination task. Similar to aged rat water maze performance, some aged rats performed odor learning discrimination problems on par with the young cohort (i.e. aged-unimpaired) and some aged rats were impaired compared to young (i.e. aged-impaired). Using a within-subjects design, the GABA(B) antagonist, CGP 55845 completely ameliorated odor discrimination learning deficits in aged-impaired rats in a dose-dependent manner. These data support the hypothesis that the GABAergic system should be a novel target for therapies aimed at treating age-related cognitive decline.

This thesis investigates the involvement of the non-neuronal cholinergic system in endothelium-dependent relaxations and the impact of hypertension on the function of this system. In Study1 the contribution of nicotinic receptors (nAChRs) to endothelium-dependent relaxations evoked by acetylcholine was examined. Both muscarinic (mAChRs) and nAChR were expressed in the aortic endothelium of spontaneously hypertensive (SHR)and Wistar-Kyoto rats (WKY). However, isometric tension measurements showed that, the muscarinic antagonist atropine abolished the relaxations to acetylcholine in WKY aortae, but only partially inhibited those in SHR aortae. While the nicotinic antagonist mecamylamine inhibited the remaining response in SHR aortae, it did not significantly affect the response solely in either SHR or WKY preparations. Hence, nAChRs mediate endothelium-dependent relaxations to the acetylcholine only in the SHR aorta and only when mAChRs are inhibited. Nicotine, the prototypical nicotinic agonist, also induced endothelium-dependent relaxations in both SHR and WKY aortae which were due to activation of α7-nAChRsbut not by mecamylamine-sensitive α3-nAChR. The acetylcholine-induced, atropine-insensitive relaxations and that to nicotine both involve the PI3K pathway. Thus, activation of nAChRscan contribute to acetylcholine-induced endothelium-dependent relaxations via PI3K signaling pathway in aortae of hypertensive animals. Study 2 examined the involvement of non-neuronal cholinergic system in endothelium-dependent relaxations. Isometric tension measurements showed that mild hypothermia (37℃–31℃) induced endothelium-dependent relaxations, which were reduced by atropine, tubocurarine, acetylcholinesterase (enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake) and vesamicol (inhibitor of acetylcholine release) in SHR but not in WKY aortae, indicating that the non-neuronal cholinergic system is involved in mild hypothermia-induced endothelium-dependent relaxations. Compared with WKY, SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but lesser vesicular acetylcholine transporter, located mainly in the endothelium. A choline/acetylcholine assay showed that, mild hypothermia increased the uptake of choline by the endothelium of SHR,but not WKY, aortae from extracellular environment for acetylcholine production. To define possible different mechanisms employed by SHR and WKY endothelial cells, the involvement of transient receptor potential (TRP)channels in mild hypothermia-induced response were examined using selective pharmacological inhibitors of different subtypes of TRP channels, namelyAMTB (TRPM8 antagonist),HC-030031 (TRPA1 antagonist)and HC-067047 (TRPV4 antagonist).The results suggest that both TRPM8 and TRPA1 play a role in the response to mild hypothermia in the WKY aorta; however, in the SHR aortaTRPV4,but not TRPA1, channels are activated by mild hypothermia. Moreover, the observation that the mild hypothermia-induced increases in cyclic guanosine monophosphate (cyclic GMP)and choline uptake were inhibited by HC-030031 in WKY but by HC-067047 in SHR aortae further indicate that in the hypertensive strain compensatory TRPV4 activation can make up for the loss of TRPA1-mediated NO production, and that the endothelial cells of the hypertensive animal utilize TRPV4 channels to activate the production of endogenous acetylcholine in response to mild hypothermia. Taken in conjunction, the results reported in this thesis together suggest that hypertension alters the function of the non-neuronal cholinergic system (e.g. n-AChR sensitivity or acetylcholine production) to modulate endothelium-dependent relaxations. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Alcoholism is a complex disease that exists as a specific set of behaviors, such as the preoccupation with obtaining alcohol and compulsive alcohol drinking. Currently, more than 18 million adults in the United States suffer from alcohol abuse or alcoholism. This disease poses serious medical and economic consequences for society. Identifying the neurobiological mechanisms that underlie alcohol drinking, specifically the transition from initiation to binge drinking is critical for improved treatments for alcoholics and the vulnerability for relapse in those recovering. Many studies have identified brain regions and molecular mechanisms that underlie various stages of alcohol abuse; however few have investigated the role of specific cell types within these areas. The overarching hypothesis of the studies in this dissertation is that cholinergic interneurons of the nucleus accumbens (NAc) are key neural substrates that underlie alcohol drinking, and as drinking continues; neuroadaptations within these cells then facilitate such behaviors as compulsive alcohol drinking. More specifically, these studies tested whether 1) cholinergic cell ablation in the NAc causes a decrease in alcohol drinking in C57BL/6J mice, 2) neuroadaptive changes in dopamine (DA) D2 receptor and cyclin dependent kinase 5 (Cdk5) occur within these cells following initiation alcohol drinking, and to a greater extent following binge alcohol drinking in C57BL/6J mice, and 3) neuroadaptive changes in DA D2 receptor and Cdk5 also occur in brain regions that have been implicated in the rewarding and reinforcing effects of alcohol in inbred alcohol-preferring (iP) rats. The present findings report a causal role for accumbal cholinergic neurons in binge alcohol drinking and identify DA D2 receptor and Cdk5 neuroadaptations following initiation and binge alcohol drinking. These studies identify the involvement of cholinergic interneurons in binge drinking and reveal alcohol-induced region- and cell-specific receptor and molecular changes that occur with continued drinking. These findings contribute to the understanding of the neurobiological mechanisms that underlie alcohol drinking, and provide the basis for cholinergic targeted treatments designed to attenuate binge drinking. These data also provide the groundwork for future studies aimed to examine receptor and intracellular molecular changes that occur with compulsive alcohol drinking, craving, and relapse.