The Pennsylvania State University the Graduate School College of Medicine L-TYPE CALCIUM CHANNEL REGULATION in the P/Q-TYPE CALC
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The Pennsylvania State University The Graduate School College of Medicine L-TYPE CALCIUM CHANNEL REGULATION IN THE P/Q-TYPE CALCIUM CHANNEL MUTANT MOUSE, TOTTERING, A MODEL FOR EPISODIC NEUROLOGICAL DISORDERS A Thesis in Neuroscience by Brandy Ellen Fureman 2001 Brandy Ellen Fureman Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2001 We approve the thesis of Brandy Ellen Fureman. Date of Signature __________________________________ ________________ Ellen J. Hess Associate Professor of Neuroscience and Anatomy Thesis Advisor Co-Chair of Committee __________________________________ ________________ Robert J. Milner Professor of Neuroscience and Anatomy Director, Graduate Program in Neuroscience Co-Chair of Committee __________________________________ ________________ Melvin L. Billingsley Professor of Pharmacology __________________________________ ________________ Robert Levenson Professor of Pharmacology __________________________________ ________________ Theresa L. Wood Associate Professor of Neuroscience and Anatomy iii ABSTRACT Mutations in calcium channels cause episodic neurological disorders in humans and paroxysmal phenotypes in mice. Tottering mice display stress-induced attacks of a movement disorder due to a P/Q-type calcium channel mutation and cerebellar L-type calcium channel upregulation.L-type calcium channels were assessed in studies of calcium uptake, radioligand binding and in situ hybridization in tottering mice. These studies confirmed the increase in tottering cerebellar L-type calcium channels. Mutant mice also exhibited unique responses to repeated exposure to an L-type calcium channel antagonist (nimodipine) and agonist (BAY K8644). These studies provide further evidence of L-type calcium channel misregulation in the tottering mouse cerebellum. In developing tottering mice, restraint stress did not produce motor attacks until twenty- two days of age; attack frequencies at p22 were similar to adults. A second phenotype, aberrant cerebellar tyrosine hydroxylase mRNA expression, was not detected until after the onset of stress-induced motor attacks. These studies more precisely define the temporal relationship between these phenotypes, suggesting that aberrant calcium-responsive gene expression is a consequence of the intense cerebellar activation associated with motor attacks. In adult tottering mice, attacks were reliably precipitated by stressful environmental disturbances, caffeine and alcohol administration; these agents are widely known triggers in human episodic disorders. Thus, tottering mice provide an excellent model to study common pathophysiological mechanisms underlying trigger phenomena in ion channelopathies. As iv activation of hormonal systems is a common feature of tottering mouse triggers, the influence of stress hormones on tottering mouse attacks was assessed. Glucocorticoids were neither necessary nor sufficient for the expression of this behavior; pharmacological blockade of the corticotropin- releasing hormone receptor type 1 (CRF-1) did not prevent stress-induced motor attacks. Potential therapeutics including ethosuximide, phenytoin and carbamazepine failed to prevent motor attacks. However, L-type calcium channel antagonist nimodipine prevented caffeine- and alcohol-induced attacks, and the glutamatergic antagonist MK-801 was effective in preventing attacks induced by restraint stress. The results of these studies suggest that abnormal ionic signaling triggered through calcium-dependent mechanisms in cerebellar networks produces periods of hyperexcitability in the tottering mouse brain. The unique neurocircuitry of the cerebellum may amplify these abnormal signals once they are produced. v TABLE OF CONTENTS List of Figures.......................................................................................................................... vii List of Tables ............................................................................................................................ ix List of Abbreviations...................................................................................................................x Acknowledgments................................................................................................................... xiii Chapter 1: Introduction and Literature Review............................................................................1 Chapter Summary....................................................................................................................1 Voltage Dependent Calcium Channels.....................................................................................3 Human Episodic Disorders......................................................................................................9 The tottering Mouse..............................................................................................................13 Conclusions and Experimental Questions ..............................................................................24 Chapter 2: Tottering mouse development ..................................................................................26 Chapter Summary..................................................................................................................26 Rationale...............................................................................................................................28 Materials and Methods ..........................................................................................................32 Results ..................................................................................................................................38 Discussion.............................................................................................................................44 Chapter 3: L-type calcium channels in tottering mouse phenotypes ...........................................50 Chapter Summary..................................................................................................................50 Rationale...............................................................................................................................52 Materials and Methods ..........................................................................................................54 Results ..................................................................................................................................58 Discussion.............................................................................................................................67 Chapter 4: Chronic L-type calcium channel activation in tottering mice ....................................73 Chapter Summary..................................................................................................................73 Rationale...............................................................................................................................75 Materials and Methods ..........................................................................................................77 Results ..................................................................................................................................80 Discussion.............................................................................................................................84 Chapter 5: Triggers and potential therapeutics in tottering mouse attacks ..................................89 Chapter Summary..................................................................................................................89 Rationale...............................................................................................................................91 Materials and Methods ..........................................................................................................94 Results ..................................................................................................................................98 Discussion...........................................................................................................................105 vi Chapter 6: Conclusions ...........................................................................................................111 Development of the tottering mouse phenotypes..................................................................113 Perturbed L-type calcium channel regulation in tottering mice ............................................115 Cerebellar TH mRNA expression in tottering mice..............................................................118 Attack triggers and potential therapeutics ............................................................................120 General model of hyperexcitability in the tottering mouse cerebellum.................................122 References ..............................................................................................................................126 vii LIST OF FIGURES Figure Title Page α 1.1 Predicted protein topology of voltage dependent calcium channel 1 subunits 4 1.2 Model diagram of predicted voltage dependent calcium channel subunit 5 interactions 2.1 Developmental onset of restraint-induced attacks in tottering mice 38 2.2 TH mRNA expression in the developing mouse brain 40 2.3 Calcium uptake in cerebellar synaptosomes from developing mice 41 2.4 Calcium uptake in forebrain synaptosomes from developing mice 42 3.1 Calcium