Localization of Cav3.1 T-Type calcium channels in the thalamus of normal and parkinsonian monkeys: light and electron microscopic immunocytochemistry using subtype-specificantibodies Open Access

Abstract

The motor dysfunction of Parkinson's disease results from degeneration of the nigrostriatal dopaminergic system, and the consequent functional changes of basal ganglia-thalamocortical circuits. Changes in thalamic activity, including an increase in burst discharges of thalamic neurons, are associated with the development of parkinsonism. In part, the abnormal bursting activity in PD may involve de-inactivation of T-type calcium channels (Ca v 3) following neuronal hyperpolarization. It is unclear whether this abnormal thalamic burst activity is the result of altered hyperpolarization from basal ganglia inputs, or changes in T-type calcium channel localization and function. To address the involvement of T-type calcium channels in abnormal thalamic activity, we studied the cellular, subcellular, and subsynaptic localization of the Ca v 3.1 channel in the ventrolateral (VL) and centromedian/parafascicular (CM/Pf) thalamic nuclei, the main thalamic targets of basal ganglia outflow, in normal and parkinsonian monkeys. At the light microscopic level, strong Ca v 3.1 neuropil immunoreactivity was found throughout the thalamus. The intensity of immunolabeling in CM/Pf was lower than in VL. There was no significant difference in the overall pattern and intensity of immunostaining between normal and parkinsonian monkeys. At the electron microscopic level, most Ca v 3.1 immunoreactivity was found in dendritic shafts of various sizes, with 40-60% and 30-40% dendritic profiles displaying Ca v 3.1 immunoreactivity in the VL and CM/Pf, respectively. At the subcellular level, aggregates of Ca v 3.1 immunoperoxidase and immunogold labeling were commonly found in the post-synaptic densities of putative asymmetric glutamatergic synapses and putative symmetric GABAergic synapses, suggesting potential roles in both excitatory and inhibitory neurotransmission. The pattern of subcellular and subsynaptic localization of Ca v 3.1 between normal and parkinsonian monkeys was not significantly different. Thus, any involvement of T-type calcium channels in increased thalamic bursting firing activities in the parkinsonian state is likely mediated by altered hyperpolarization of thalamic neurons rather than changes in channel expression.

Table of Contents

I. Introduction...1

A. Background...1

B. Pathophysiology of parkinsonism...1

C. Current treatments, challenges, and research motivations...2

D. Basal ganglia-thalamocortical dysrhythmia in parkinsonism...4

E. T-type calcium channels and parkinsonism...4

F. Current research and study rationale...5

II. Materials and Methods...7

A. Animals...7

B. Perfusion of animals and sectioning of tissue...8

C. Immunohistochemistry...9

i. Antibodies used...9

ii. Specificity test of Cav3.1 antibody...9

iii. Selection of tissues...10

iv. Light microscopic pre-embedding immunoperoxidase...10

v. Electron microscopic pre-embedding immunoperoxidase...11

vi. Electron microscopic pre-embedding immunogold...12

D. Data analysis

i. Densitometric analysis of light microscopy material...12

ii. Analysis of electron microscopy material...13

a. Immunoperoxidase...13

b. Immunogold...14

III. Results...15

A. Western blot for Cav3.1 expression in thalamic and striatal tissue...15

B. Tyrosine hydroxylase stain reveals dopamine depletion in the basal ganglia following treatment with MPTP...15

C. Light microscopic immunohistochemical staining for Cav3.1...15

D. Immunoperoxidase localization of Cav3.1 in the VL and CM/Pf...16

E. Immunogold localization of Cav3.1 in the VL and CM/Pf...18

IV. Discussion...19

V. Tables and Figures...26

A. F1: Impact of Parkinson's disease on neural circuitry...26

B. F2: Burst firing in thalamic neurons...27

C. T1: Commercial sources and characteristics of primary antibodies used...28

D. F3: Western blot analysis to show the specificity of the Cav3.1 antibody...28

E. T2: Summary of tissue areas used for densitometric analysis...29

F. T3: Summary of tissue areas used for electron microscopic analysis...29

G. F4: Tyrosine hydroxylase immunoreactivity in the basal ganglia...29

H. F5: Light micrographs of Cav3.1 immunoreactivity in the thalamus...30

I. F6: EM immunoperoxidase localization of Cav3.1 in the thalamus...31

J. F7: EM immunoperoxidase localization of Cav3.1 in thalamic dendrites...32

K. F8: EM immunogold localization of Cav3.1 in thalamic dendrites...33

L. F9: EM immunogold synaptic localization of Cav3.1...34

VI. References...36

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Physiology Biology, Neuroscience Biology, Animal Physiology
Keyword	Parkinson's Disease Thalamus T-type Calcium Channel Basal Ganglia
Committee Chair / Thesis Advisor	Smith, Yoland, Emory University
Committee Members	Jaeger, Dieter, Emory University Sober, Samuel J, Emory University

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