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

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.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xi, 130 p.; also includes graphics (some col.). Includes bibliographical references (p. 109-130). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 131-154).

No description available.

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

The pedunculopontine nucleus (PPN) is a brainstem structure involved in motor control, sleep and arousal. The boundaries of the PPN are defined by its cholinergic neurons, but it also contains GABAergic, glutamatergic and calcium-binding protein- positive neurons. To further understand the physiological roles of the PPN it is necessary to understand which neuronal subtypes are present in the PPN and how they are connected with other regions of the brain in normal and pathological conditions. In order to address these issues, the total numbers, distributions and neurochemical phenotypes of neurons, positive for the calcium-binding proteins calbindin and calretinin, were studied in the rat PPN. Sagittal, perfuse-fixed rat brain sections were double or triple-immunolabelled to reveal the cholinergic marker choline acetyltransferase (ChAT) with calbindin and/or calretinin. A stereological approach revealed that calbindin- and calretinin-positive neurons account for a large proportion of PPN neurons, but they rarely eo-express ChAT. A combination of immunolabelling for calbindin or calretinin with in situ hybridisation for GAD65/67 or VGluT2 mRNAs revealed that about one third of the calbindin- and calretinin-expressing neurons are GABAergic and preferentially located in the rostral PPN, whereas approximately two thirds are glutamatergic and principally located in the caudal PPN. Additionally, retrograde tracer injections in the subthalamic nucleus (STN) and the gigantocellular nucleus (GiN) showed that the majority of PPN neurons, projecting to one or both of these nuclei, were not cholinergic (70-90%). Less than 10% of STN-projecting neurons expressed calbindin or calretinin and 5% of the GiN-projecting neurons contained calretinin but none contained calbindin. Finally, the expression of the immediate early gene, Egrl, a marker of neuronal activation, was evaluated in STN- and GiN-projecting neurons of the PPN in control and 6-0HDA lesioned animals. No statistically significant differences, in the number of Egr l-positive neurons, were observed between control and 6- OHDA lesioned animals. These findings show that calbindin- and calretinin-positive neurons are abundant in the PPN, heterogeneously distributed and display a GABAergic or glutamatergic phenotype. Additionally, calbindin- and calretinin-positive neurons represent only a minority of the PPN neurons projecting to either the STN, GiN or both nuclei. Results also suggest that the hyperactivity seen in the PPN in the 6-0HDA model of Parkinson's disease may not necessarily be due to the neurons projecting to the STN and/or GiN. Overall, this thesis supports the notion that the PPN is composed of a rich diversity of neuronal cell-types, which are heterogeneously distributed along its rostro-caudal axis. The heterogeneous neurochemistry, connectivity and physiology of these neurons allow the PPN to influence a wide range of brain regions through a variety of pathways presumably underlying its various functional roles.

A common organizational feature of any nervous system is the existence of groups of neurons that share a set of common traits but that can be further divided into individual neuron types and subtypes. Understanding the mechanistic basis of neuron type and subtype diversification processes will constitute a major step toward understanding brain development and evolution. In this dissertation, I have explored the mechanistic basis for the specification of motor neuron classes in the nematode C. elegans which serves as a paradigm for neuron diversification processes. Cholinergic motor neurons in the C. elegans ventral nerve cord share common traits, but are also comprised of many distinct classes, each characterized by unique patterns of effector gene expression (e.g. motor neuron class-specific ion channels, signaling molecules, and neurotransmitter receptors). Both the common as well as class-specific traits are directly activated by the terminal selector of cholinergic motor neuron identity, the EBF/COE-like transcription factor UNC-3. Via forward genetic screens to identify mutants that are defective in class specification, I have discovered that the diversification of UNC-3/EBF-dependent cholinergic motor neurons is controlled by distinct sets of phylogenetically conserved, motor neuron class-specific transcriptional repressors. One such repressor is in fact a novel gene previously uncharacterized in C. elegans or any nervous systems and is now named bnc-1. By molecularly dissecting the cis-regulatory region of effector genes, I found that the repressor proteins prevent UNC-3/EBF from activating class-specific effector genes in specific motor neuron subsets via discrete binding sites that are adjacent to those of UNC-3/EBF. And by using CRISPR/Cas9-mediated genome engineering to tag repressor proteins with inducible degrons, I demonstrate that these repressors share the important feature of being continuously required throughout the life of the animal to counteract, in a class-specific manner, the function of the UNC-3/EBF terminal selector that is active in all motor neuron classes. I propose that the strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a neuron subtype-specific manner may constitute a general principle of neuron subtype diversification.