All ETDs from UAB

Advisory Committee Chair

Joh J Hablitz

Advisory Committee Members

David Sweatt

Lori L McMahon

Robin Lorenz

Michelle Olsen

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Learning and memory rely on long-lasting, experience-dependent adaptations in synaptic and non-synaptic forms of neuronal plasticity. Previous evidence implicates transcriptional and epigenetic mechanisms, including DNA cytosine methylation, as critical regulators of site-specific, Hebbian alterations in synaptic efficacy such as long-term potentiation (LTP) and long-term depression (LTD). However, whether DNA methylation modulates cell-wide, non-Hebbian homeostatic adaptations like synaptic scaling and intrinsic plasticity (IP) is unclear. Whereas synaptic scaling involves bidirectional changes in postsynaptic receptor density in response to chronic alterations in neuronal activity, IP involves the activity-dependent attunement of passive and/or active membrane properties that govern action potential (AP) firing. This dissertation utilized dissociated primary cultures of rat cortical neurons in combination with pharmacological, genetic, and electrophysiological approaches to directly investigate whether DNA cytosine methylation acts a tonic regulator of synaptic and intrinsic homeostatic adaptations. Chronic activity blockade with the Na+ channel blocker tetrodotoxin (TTX) not only recapitulated previously described electrophysiological synaptic upscaling but was further associated with decreased cytosine methylation and increased expression of genes encoding glutamate receptors and glutamate receptor trafficking proteins. Remarkably, prolonged inhibition of DNA methyltransferase (DNMT) activity, either by small-molecule antagonisms via RG108 or by antisense oligonucleotide (ASO)-mediated knockdown of Dnmt1 and Dnmt3a, resulted in glutamatergic synaptic upscaling, evidenced by a multiplicative increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Furthermore, in response to somatic current injections, prolonged DNMT inhibition enhanced intrinsic membrane excitability (IME) of cortical pyramidal neurons. Importantly, both DNMT inhibition-mediated upscaling and enhanced excitability were blocked by ASO-mediated knockdown of the TET1 dioxygenase, as well as RNA polymerase inhibition with actinomycin D, suggesting a likely role for secondary cytosine demethylation and de novo transcription in the induction of these non-Hebbian adaptations. Mechanistic studies revealed transcriptional repression of small conductance Ca2+-activated K+ (SK) channels and subsequent reduction in the medium afterhyperpolarization (mAHP) as a probable mechanism underlying DNMT inhibition-enhanced IME. Collectively, the data presented in this dissertation suggests that genomic cytosine dynamics control transcription-dependent cell-wide synaptic and intrinsic membrane plasticities. Hence, in addition to regulating Hebbian synaptic plasticity, covalent DNA modifications may contribute to long-term behavioral adaptation by impinging on cell-wide, non-Hebbian forms of plasticity.

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