CHRONIC CENTRAL ADMINISTRATION OF VALPROIC ACID: INCREASED PRO-SURVIVAL PHOSPHO-PROTEINS AND GROWTH CONE ASSOCIATED PROTEINS WITH NO BEHAVIORAL PATHOLOGY

2013·1.1 MB·English

by  Bates, Ryan Christopher

CHRONIC CENTRAL ADMINISTRATION OF VALPROIC ACID: INCREASED
PRO-SURVIVAL PHOSPHO-PROTEINS AND GROWTH CONE
ASSOCIATED PROTEINS WITHb yN O BEHAVIORAL PATHOLOGY
RYAN CHRISTOPHER BATES
B.S., Metropolitan State University of Denver, 2006

A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Integrative Biology
2013
This thesis for the Master of Science degree by
Ryan Christopher Bates
has been approved for the
Integrative Biology Program
by

Bradley J. Stith, Chair
Jefferson Knight
Amanda Charlesworth

July 9, 2013

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Bates, Ryan, Christopher. (M.S., Integrative Biology)
Chronic Central Administration of Valproic Acid: Increased Pro-Survival Phospho-
Proteins and Growth Cone Associated Proteins with no Behavioral Pathology

Thesis Directed by Professor Bradley J. Stith

ABSTRACT
Valproic acid (VPA) is the most widely prescribed antiepileptic drug due to its ability to
treat a broad spectrum of seizure types. However, potential complications of this drug
include anticonvulsant polytherapy metabolism, organ toxicity and teratogenicity which
limit its use in a variety of epilepsy patients. Direct delivery of VPA
intracerebroventricularly (ICV) could circumvent the toxic effects normally seen with the
oral route of administration. An additional potential benefit would be significantly
reduced dosing while achieving high brain concentrations. Epileptogenic tissue from
patients with intractable seizures has shown significant cell death which may be mitigated
by maximizing cerebral VPA exposure. Here we show ICV administration of VPA
localized to the periventricular zone increased pro-survival phospho-proteins (pAktSer473,
pAktThr308, pGSK3βSer9, pErk1/2Thr202/Tyr204) and growth cone associated proteins (2G13p,
GAP43) in a whole animal system. No significant changes in DCX, NeuN,
synaptotagmin, and synaptophysin were detected. Assessment of possible behavioral
alterations in rats receiving chronic central infusions of VPA was performed with the
open field and elevated plus mazes. Neither paradigm revealed any detrimental effects of
the drug infusion process.

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The form and content of this abstract are approved. I recommend its publication.

Approved: Bradley J. Stith

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DEDICATION

I dedicate this thesis to my loving parents, Dale & Karen Bates, and to my “little”
brother, Steven.

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ACKNOWLEDGEMENT

I would like to thank Dr. Brad Stith and Dr. Doug Petcoff for their countless hours spent
advising me about the complex world of bioscience and the games a scientist must play
in order to win.

The contents of this thesis were published on August 31st 2012: Bates RC, Stith BJ,
Stevens KE. Chronic central administration of valproic acid: increased pro-survival
phospho-proteins and growth cone associated proteins with no behavioral pathology.
Pharmacology, Biochemistry and Behavior 2012;103:237-244.
DOI Link: http://dx.doi.org/10.1016/j.pbb.2012.08.023

Paper featured on Global Medical Discovery with additional data figure (Fig. 4.) on
March 19th 2013: http://globalmedicaldiscovery.com/key-scientific-articles/chronic-
central-administration-of-valproic-acid-increased-pro-survival-phospho-proteins-and-
growth-cone-associated-proteins-with-no-behavioral-pathology/
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TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION ............................................................................................... 1
II. MATERIALS AND METHODS ....................................................................... 3
Animals ............................................................................................................... 3
Implant Surgery .................................................................................................. 3
In Vivo Behavioral Studies ................................................................................. 4
Tissue Preparations ............................................................................................. 5
Immunohistochemistry ....................................................................................... 5
Sample Lysate Preparation ................................................................................. 6
Western Immunoblotting .................................................................................... 7
Statistics .............................................................................................................. 8
III. RESULTS ......................................................................................................... 9
Behavior of ICV Implanted Rats in Open Field and Elevated Plus Maze……22
VPA, Phospho-Proteins, and Growth Cone Associated Protein Localization in
Ventricle………………………………………………………………………28
Phospho-Protein and Total Protein Levels in Regional Brain Tissue Lysates..28
IV. DISCUSSION ................................................................................................. 18
V. CONCLUSION ................................................................................................ 24
REFERENCES ..................................................................................................... 25

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LIST OF FIGURES
FIGURE
1 Behavioral effects of ICV VPA administration ................................................. 11
2 Immunochemical results of ICV VPA administration ....................................... 14
3 Growth cone-associated proteins after ICV VPA administration ...................... 16
4 Drug penetrance and cases of hydrocephaly ...................................................... 17

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LIST OF TABLES
TABLE
1 ANOVA results for Open Field and Elevated Plus Paradigms .......................... 10
2 ANOVA and Scheffe a posteriori results for Immunohistochemistry and Western blot
studies ................................................................................................................... 12

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LIST OF ABBREVIATIONS
Erk, extracellular signal-regulated kinase
GSK3β, glycogen synthase kinase 3 beta
GAP43, growth associated protein 43
ICV, intracerebroventricle
VPA, valproic acid
CSF, cerebrospinal fluid
BBB, blood brain barrier
EP, elevated plus maze
OF, open field maze
SUDEP, sudden unexpected death in epilepsy
DCX, doublecortin
NeuN, neuronal nuclei
CC, corpus callosum
LV, lateral ventricle
TV, third ventricle
H, hippocampus

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