The Distal-less (Dlx) homeobox genes of mammals are expressed in many tissues of the developing organism including the limbs, craniofacial skeleton and the forebrain. In the forebrain, Dlx1, Dlx2, Dlx5 and Dlx6 play a critical role in driving tangential migration of GABAergic progenitors from the ventral telencephalon to their final destinations, notably the neocortex and the striatum. These Dlx genes are organised into convergently transcribed clusters with short intergenic regions that contain notable cis regulators elements (CREs) that drive Dlx expression in unique subdomains of the developing ventral telencephalon. Previous studies have characterised Dlx regulation including but not limited to the direct activation of these CREs by effector proteins. However, to date very little work has been done to examine how the forebrain Dlx genes may be regulated at the level of the chromatin. To explore this, I used in silico and in vivo methods to examine some key histone modifications of the Dlx1/2 and Dlx5/6 bigene clusters in the developing forebrain; namely H3K27Ac, H3K4me3, H3K4me1 and H3K27me3. I found that within the Dlx expressing ganglionic eminences (GE), at midgestation, the Dlx loci are marked by bivalent chromatin which is enriched in both permissive H3K4me3 and repressive H3K27me3 marks. By performing ChIP-qPCR on the GE tissue of embryonic mice with targeted deletions of enhancer CREs, I found that these CREs play unique roles in shaping the chromatin. Removal of one of these CREs has widespread effects on the chromatin at both loci. Since these changes in chromatin signatures do not accompany significant changes in expression of histone modifying genes, we believe these CREs play yet-to-be determined roles in recruiting the modifying proteins to the loci, thereby establishing bivalent chromatin to fine-tune Dlx expression.

Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains vi, 86 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 75-85).

Synaptic plasticity, which is thought to represent the neuronal substrate for learning and memory is influenced by the degree of GABAergic inhibitory tone. In particular, γ-aminobutyric acid subtype A receptors (GABAARs), which mediate the majority of inhibitory neurotransmission in the mammalian brain regulate learning and plasticity. In these studies I examined a subpopulation of α5 subunit-containing GABAA receptors (α5GABAARs), which are preferentially expressed in the hippocampus, to determine whether they have a specific role in memory processes. I hypothesized that α5GABAAR-activity constrains hippocampus-dependent learning and CA1 synaptic plasticity. The main research objective of this thesis was to investigate the electrophysiological changes within the hippocampus that accompany genetic and pharmacological targeting of α5GABAARs and how these changes impact behaviour. I found that the general anesthetic etomidate enhanced a tonic inhibitory conductance generated by α5GABAARs, and this action correlated with an impairment of long-term potentiation (LTP) and hippocampus-dependent memory performance for fear-associated memory and spatial navigation. Mice with a genetic deletion of the α5 subunit gene (Gabra5–/–) were resistant to the LTP- and memory-impairing effects of etomidate. Additionally, the LTP- and memory-impairing effects of etomidate were rescued by pharmacologically inhibiting α5GABAARs. Genetic and pharmacological inhibition of α5GABAARs enhanced associative learning in trace fear but not contextual fear conditioning tasks. Interestingly, genetic deletion and pharmacological inhibition of α5GABAARs did not result in the common adverse side-effects associated with non-selective inhibition of GABAARs such as anxiogenesis or seizures. Further, I found that blocking the tonic inhibition generated by α5GABAARs lowered the threshold for LTP, such that lower stimulation frequencies enhanced LTP. Synaptic changes within this frequency band were modified independently of phasic GABAAR inhibition. Inhibiting the α5GABAAR-dependent membrane conductance was associated with an increase in the depolarizing envelope during 10 Hz stimulation. These experiments provide new insights into the in vitro and in vivo physiology of α5GABAARs and suggest that a tonic inhibition generated by α5GABAARs constrains learning and glutamate plasticity through regulation of the membrane’s electrical properties.

The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABA(A) receptor because of sensitivity to GABA(A) receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity.

The role of inhibitory compounds such as adenosine and GABA in modulating neuronal activity in invertebrate species is not well described. Here I investigate their role in modulating excitability of cluster F neurons in the pedal ganglia of Lymnaea stagnalis. Receptor-specific agonists and antagonists were used to determine that the inhibitory effects of adenosine were mediated through the adenosine A1 receptor, and that action potential frequency varied linearly with intracellular calcium concentrations. These effects had a seasonal dependence, as neurons were resistant to adenosinergic modulation during the summer months. GABAergic modulation of neuronal activity was also seasonal as demonstrated by ionic plasticity in GABAergic transmission. GABA application led to inhibition or excitation of electrical activity in neurons obtained during the fall and winter months, respectively. These effects were mediated through the GABA(A) receptor because of sensitivity to GABA(A) receptor antagonist bicuculline and were likely due to differential cation-chloride cotransporter activity.

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in the brains extracellular potassium (K+) and pH homeostasis. Pathological brain states have been shown to cause astrocyte swelling. However, these volume changes have never before been verified to occur in response to physiological activity. In the present thesis, two-photon laser scanning microscopy was used to visualize real-time astrocyte volume changes in the stratum radiatum of the CA1 region of the hippocampus. Astrocyte somas and primary processes were observed to swell by 19.0±0.9% in response to a physiological (3 mM) increase in the concentration of extracellular K+. Astrocyte swelling was partially mediated by K+ influx through inwardly rectifying K+ channels (Kir), as their inhibition resulted in a significant decrease of the increased K+ induced astrocyte swelling (13.9±0.9%). In addition, the bicarbonate ion (HCO3-) was found to play a significant role in the increased K+ induced astrocyte swelling. The astrocyte swelling was significantly decreased when the influx of HCO3- was decreased in 1) a HCO3- free extracellular solution (5.4±0.7%), 2) in the presence of an extracellular carbonic anhydrase inhibitor (11.4±0.6% ), and 3) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked (8.3±0.7%) . Conversely, astrocytes were found to shrink by 7.7±0.5% in response to ã-Amino-butyric Acid (GABA) receptor activation. GABAA receptor mediated astrocyte shrinkage was significantly decreased to 5.0±0.6% when HCO3- efflux was reduced. Furthermore, in this thesis it was shown for the first time that astrocytes swell in response to neuronal stimulation (4.0±0.4%). This activity induced astrocyte swelling was significantly decreased to 1.5±0.2% in a HCO3- free extracellular solution. These astrocyte volume changes may have important implications for the regulation of brain activity under both physiological and pathological brain states.

Synaptic plasticity, which is thought to represent the neuronal substrate for learning and memory is influenced by the degree of GABAergic inhibitory tone. In particular, γ-aminobutyric acid subtype A receptors (GABAARs), which mediate the majority of inhibitory neurotransmission in the mammalian brain regulate learning and plasticity. In these studies I examined a subpopulation of α5 subunit-containing GABAA receptors (α5GABAARs), which are preferentially expressed in the hippocampus, to determine whether they have a specific role in memory processes. I hypothesized that α5GABAAR-activity constrains hippocampus-dependent learning and CA1 synaptic plasticity. The main research objective of this thesis was to investigate the electrophysiological changes within the hippocampus that accompany genetic and pharmacological targeting of α5GABAARs and how these changes impact behaviour. I found that the general anesthetic etomidate enhanced a tonic inhibitory conductance generated by α5GABAARs, and this action correlated with an impairment of long-term potentiation (LTP) and hippocampus-dependent memory performance for fear-associated memory and spatial navigation. Mice with a genetic deletion of the α5 subunit gene (Gabra5–/–) were resistant to the LTP- and memory-impairing effects of etomidate. Additionally, the LTP- and memory-impairing effects of etomidate were rescued by pharmacologically inhibiting α5GABAARs. Genetic and pharmacological inhibition of α5GABAARs enhanced associative learning in trace fear but not contextual fear conditioning tasks. Interestingly, genetic deletion and pharmacological inhibition of α5GABAARs did not result in the common adverse side-effects associated with non-selective inhibition of GABAARs such as anxiogenesis or seizures. Further, I found that blocking the tonic inhibition generated by α5GABAARs lowered the threshold for LTP, such that lower stimulation frequencies enhanced LTP. Synaptic changes within this frequency band were modified independently of phasic GABAAR inhibition. Inhibiting the α5GABAAR-dependent membrane conductance was associated with an increase in the depolarizing envelope during 10 Hz stimulation. These experiments provide new insights into the in vitro and in vivo physiology of α5GABAARs and suggest that a tonic inhibition generated by α5GABAARs constrains learning and glutamate plasticity through regulation of the membrane’s electrical properties.

Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in the brains extracellular potassium (K+) and pH homeostasis. Pathological brain states have been shown to cause astrocyte swelling. However, these volume changes have never before been verified to occur in response to physiological activity. In the present thesis, two-photon laser scanning microscopy was used to visualize real-time astrocyte volume changes in the stratum radiatum of the CA1 region of the hippocampus. Astrocyte somas and primary processes were observed to swell by 19.0±0.9% in response to a physiological (3 mM) increase in the concentration of extracellular K+. Astrocyte swelling was partially mediated by K+ influx through inwardly rectifying K+ channels (Kir), as their inhibition resulted in a significant decrease of the increased K+ induced astrocyte swelling (13.9±0.9%). In addition, the bicarbonate ion (HCO3-) was found to play a significant role in the increased K+ induced astrocyte swelling. The astrocyte swelling was significantly decreased when the influx of HCO3- was decreased in 1) a HCO3- free extracellular solution (5.4±0.7%), 2) in the presence of an extracellular carbonic anhydrase inhibitor (11.4±0.6% ), and 3) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked (8.3±0.7%) . Conversely, astrocytes were found to shrink by 7.7±0.5% in response to ã-Amino-butyric Acid (GABA) receptor activation. GABAA receptor mediated astrocyte shrinkage was significantly decreased to 5.0±0.6% when HCO3- efflux was reduced. Furthermore, in this thesis it was shown for the first time that astrocytes swell in response to neuronal stimulation (4.0±0.4%). This activity induced astrocyte swelling was significantly decreased to 1.5±0.2% in a HCO3- free extracellular solution. These astrocyte volume changes may have important implications for the regulation of brain activity under both physiological and pathological brain states.

Dissertation (Ph.D.)--University of Toledo, 2009. / "Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Table of contents (p. iv) gives incorrect starting page numbers for "Bibliography" and "Abstract". "Bibliography" starts on p. 120 (not p. 119); "Abstract" starts on p. 150. Bibliography: p. 64-70, 97-100, 120-149.

Ph.d.