Abstract
Neuronal communication is dependent on the generation of action
potentials. The normal functioning of voltage-gated sodium channels
is required for the generation of an action potential. Scn8a
mutations in mice, mutations in a channel coding for voltage-gated
sodium channel six (Nav1.6), are associated with the generation of
spike-wave discharges (SWDs) and absence epilepsy (Papale et al.,
2009). Absence epilepsy is a non-convulsive form of genetic
generalized epilepsy showing characteristic SWDs on EEG recordings
of patients afflicted with this condition. Several studies have
established that SWDs arise from disturbances in the
thalamocortical circuitry (Lacey et al., 2012 ; Seidenbecher et
al., 1998). Although it is known that certain genetic factors may
contribute to absence epilepsy, a full understanding of the
genetic, molecular and cellular mechanisms of absence epilepsy
remains elusive. The purpose of this study was to survey the
expression of Scn8a in the cerebral cortex, reticular
nucleus, hippocampus, and thalamus of the mouse in order to further
elucidate the mechanisms which underlie absence seizure generation.
Using immunohistochemistry and fluorescence imaging, we observed
broad Scn8a expression and confirmed that Scn8a is
expressed in GABAergic neurons in various regions of the mouse
brain. We found that the expression of Scn8a varies between
different brain structures and layers of the cortex. Based on these
results, we developed a model which may explain how SWDs arise from
altered Scn8a function.
Table of Contents
Contents
Introduction 1
Materials and Methods 5
Animals 5
Perfusion of Animals 5
Sectioning of Tissue 6
Immunohistochemistry 6
Free-Floating 6
Pre-mounted Protocol 7
Fluorescent Microscopy and Image Acquisition 8
Cell Counting 8
Statistical Analysis 9
Results 10
Optimization of Nav1.6 Antibody 10
Optimization of Ankyrin-G, Glutamate Decarboxylase (GAD-67) and
GABA Antibodies 10
Tomato ROSA Reporter Mice 12
Counts of Dlx-expressing cells co-localized with Scn8a 12
Scn8a is differentially expressed in Dlx+ cells in various cortical
layers 13
Counts of ankyrin G+ cells expressing Scn8a 13
Scn8a is differentially expressed in ankyrin-G+ cells of the cortex
and thalamus 14
Discussion 15
Effect of fixative concentration in tissue on anti-Nav1.6 labeling
15
Looking at isotype in the triple label reaction: GAD-67, ankyrin-G
and Nav1.6 stain 16
Using endogenously expressed fluorescent protein as a marker for
cell type 17
Implications of differential expression of Scn8a 18
Conclusion and Future Directions 22
Figures and Tables 23
Table 1. Cell counts of Dlc+ neurons expressing Scn8a and counts of
Scn8a+ neurons expressing Dlx. 23
Table 2. Observed counts vs expected counts of Dlx+ cells
expressing Scn8a. 24
Table 3. Cell counts of ankyrin-G+ neurons expressing Scn8a and
counts of Scn8a+ neurons expressing ankyrin-G. 24
Table 4. Observed versus expected counts of ankyrin-G+ cells
expressing Scn8a. 25
Table 5. Shows the confidence interval for the difference of
proportions in co-localization. 25
Figure 1.Anti-Nav1.6 staining in 1% PFA vs 4% PFA 26
Figure 2. General integrity of 1% and 4% PFA tissue. 26
Figure 3. pAb GABA failed stain in hippocampal section 27
Figure 4. Anti-GAD-67 stain of the nRT in 1% PFA tissue 27
Figure 5. Anti-ankyrin-G immunolabeling of 1% PFA tissue 28
Figure 6. Image of endogenously fluorescent DLX+ cells. 28
Figure 7. Characteristic co-localization of Scn8a and Dlx 29
Figure 8. Anti-ankyrin-G staining of the cortex. 29
Figure 9. Co-localization between ankyrin-G and Scn8a in
hippocampal slice. 30
Figure 10. Failed triple stain. 30
Figure 11. Model circuitry for absence seizure generation. 32
References 33
About this Honors Thesis
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