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.

In rats, the subnuclei of the inferior olive (IO) and thalamus exist topographic spatial representation to sinusoidal horizontal linear translations along either the antero-posterior or interaural direction. To examine the effect of GABAergic neurotransmission within the vestibular nucleus on the establishment of gravity-related topographic spatial representation in relay station of the central vestibular pathway, GABAA receptor antagonist bicuculline was used to chronically perturb GABA transmission within the vestibular nucleus of postnatal rats. Implantation of bicuculline-loaded or saline-loaded Elvax slice onto the dorsal surface of vestibular nucleus was performed in P1 rats which were allowed to recover into adulthood. Fos protein expression was used as an indicator to identify central neurons responsive to horizontal linear accelerations. In stationary or labyrinthectomized rats, Fos-immunoreactive (ir) neurons were either absent or sporadically scattered throughout the IO and thalamic subnuclei, indicating that the Fos expression in these neural area was otolithic in origin. In the saline control group, Fos expression induced by horizontal antero-posterior linear acceleration was observed in both the IO and thalamus. Responsive IO subnuclei include β subnucleus of IO and dorsomedial cell column while those in the thalamus include central medial nucleus, paracentral nucleus, mediodorsal nucleus, central lateral nucleus, zona incerta and subparafascicular nucleus of thalamus. For-ir neurons responsive to horizontal interaural linear acceleration were found in those IO subnuclei and thalamic subnuclei. When compared with the saline-treated group, the number of Fos-ir IO neurons responsive to horizontal linear acceleration was significantly lower in adult rats perturbed with bicuculline at P1. Besides, the pattern of Fos expression in both the IO and thalamus was altered in adult rats pretreated with bicuculline. In the utricle-related thalamic subnuclei, the postnatal time when Fos-ir neurons were found triggered by otolithic stimulation was delayed and the number of these Fos-ir neurons was fewer in the bicuculline-treated group than those in the saline-treated group. To investigate whether there exists a critical period for postnatal establishment of topographic spatial representation in the IO and thalamus, implantation of bicuculline-loaded Elvax slice onto the vestibular nucleus was carried out in P14 rats. The topographic spatial representation in IO and thalamus of those rats were unchanged as compared with adult rats pretreated with saline at P14. These results indicate that the GABAergic neuronal circuit in the vestibular nucleus plays an important role in postnatal establishment of topographic spatial representation in the central vestibular system. Most importantly, we documented the occurrence of a postnatal critical period (between P1 and P14) during which GABAergic transmission regulated the formation of a gravity-related spatial framework in the brain. / published_or_final_version / Physiology / Master / Master of Philosophy

Inhibitory synaptic transmission within the vestibular circuits plays an essential regulatory role in coordinating vestibular functions. The maturation profile of γ- aminobutyric acid (GABA) synapses in the vestibular system remains unknown. To address this, we first used double immunohistochemistry to document the postnatal expression profile of GABAA receptors in canal-related and saccule-related vestibular nuclear neurons of rats. The proportion of Fos / GABAA receptors α1 subunit doublelabeled neurons progressively increased with age. Whole-cell patch-clamp experiments on brainstem slice preparations were also employed to characterize the developmental properties of these synapses within the medial vestibular nucleus. The frequency of GABAA receptor-mediated miniature inhibitory postsynaptic currents (IPSC) progressively increased during the first two postnatal weeks and reached a plateau thereafter. This is in agreement with an increase in sensitivity to GABAA receptor α1 subunit agonist zolpidem during the same period. The rise time and decay time however decreased by 2-fold. These results suggest that change in the composition of GABAA receptor occurs during the functional maturation of medial vestibular neurons. To further investigate whether GABA receptors contribute to synaptic plasticity in the developing vestibular nucleus, two stimulus protocols were used. Repetitive depolarizing pulses induced long-lasting decrease in the frequency of GABAA receptormediated spontaneous IPSCs between P3 and P7. The probability of inducing such frequency decline of sIPSCs decreased after the first postnatal week. High frequency stimulation on the other hand, induced long-term depression (LTD) of GABAA receptormediated evoked IPSCs between P3 and P5. The probability of inducing LTD decreased after P14. These results indicate that LTD at GABAergic synapses could be easily induced in developing medial vestibular neurons before maturation of GABAergic synaptic transmission. To examine if GABAergic transmission within the vestibular nucleus is crucial for establishment of gravity-related spatial organization, an intervention approach was adopted to perturb GABAergic transmission within the postnatal vestibular nucleus. A slice of Elvax loaded with either GABAA receptor agonist muscimol or antagonist bicuculline was inserted into the fourth ventricle and covered the bilateral vestibular nuclei at different ages. Expression of Fos protein in functionally activated neurons was used to demarcate the topographic spatial map in the inferior olive. The spatial map in subnuclei IOβ and DMCC was disturbed in each adult rat that was implanted with bicuculline- or muscimol-loaded Elvax at P1. However, no change was observed in adult rats that were pretreated with bicuculline or muscimol at P14 or P21. Vestibularrelated behavior tests were also performed. The acquisition of negative geotaxis, an otolith-related orientation reflex, was delayed in postnatal rats pretreated with bicuculline but was advanced in those rats pretreated with muscimol. Furthermore, the acquisition of motor learning, evaluated by rotarod test, was impaired in adult rats treated with bicuculline or muscimol. Taken together, our results indicated that maturation of GABAergic transmission within the vestibular nucleus play important roles in development of spatial recognition and vestibular-related behavior. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy