Ph D.

Fast postsynaptic inhibition in the brain is mediated by ionotropic GABA_A receptors (GABA_ARs), which are activated by the release of the neurotransmitter GABA from presynaptic interneurons. The GABA_AR is primarily permeable to chloride ions (Cl-) and therefore the transmembrane gradient for Cl- sets the reversal potential of the receptor (E_GABA-A). When intracellular Cl^- concentrations are relatively low, E_GABA-A is more negative than the membrane potential and GABA_AR responses will have a hyperpolarising and inhibitory effect upon the postsynaptic cell. In contrast, when intracellular Cl^- concentrations are relatively high, E_GABA-A will be more positive and GABA_AR activation will have a depolarising effect. How a neuron controls its intracellular Cl^- concentrations is a fundamental question that has direct relevance to hyperexcitability conditions such as epilepsy. Recently, it has become clear that Cl^- homeostasis is altered in epileptic tissue such that postsynaptic inhibition via the GABA_AR is reduced and, under some conditions, GABA_AR signalling may even be excitatory. In my thesis I explore some of the mechanisms and factors that are responsible for regulating postsynaptic GABA_AR signalling in the context of epileptic seizure activity in the rat hippocampus. In the first series of experiments I combined pharmacological approaches with electrophysiological recordings from pyramidal neurons in the CA3 region of the hippocampus to trigger seizure activity. My results show that intense neuronal activity during a seizure leads to a transient accumulation of intracellular Cl^-, which generates a pronounced depolarising shift in E_GABA-A. Under these conditions, GABAergic synapses become excitatory and contribute to ongoing neuronal activity rather than exerting their normal inhibitory role. I found that the same seizure activity also induces the release of a neuromodulator called adenosine, which serves to limit the deleterious effects of excitatory GABA_AR responses. Adenosine exerts these effects by activating downstream potassium channels, which increase the postsynaptic cell’s membrane conductance and, in doing so, ‘shunt’ incoming GABA_AR responses. In the second series of experiments I examined Cl^- homeostasis and E_GABA-A in the context of neonatal seizures. One of the main mechanisms by which neurons maintain their intracellular Cl^- levels is through the activity of ion transporter proteins that reside in the membrane and move Cl^- either into, or out of, the cell. I discovered that the intracellular trafficking of an important Cl^- transporter protein, NKCC1, correlates with changes in Cl^- homeostasis. Using a combination of biochemical and molecular techniques, I then identified a novel molecular association between NKCC1 and a motor protein, Myosin Va, which has been implicated in the intracellular trafficking of membrane proteins. Using electrophysiological recordings I found that Myosin Va is required for NKCC1’s contribution to Cl^- homeostasis, which may be important for E_GABA-A changes in epilepsy. In the final series of experiments I developed methods to study the temporal dynamics in E_GABA-A during a single seizure. These revealed a Cl^- unloading mechanism that emerges at the end of a seizure and which depends upon hyperpolarisation of the postsynaptic membrane potential. This mechanism aids E_GABA-A recovery after the seizure and moves E_GABA-A to more hyperpolarised values. This mechanism could boost postsynaptic inhibition after a seizure and thereby help to protect against further seizure episodes. In conclusion, this work extends our understanding of postsynaptic GABAergic transmission in the context of epileptic seizure activity and suggests new mechanisms that could be relevant for the development of rational anti-epileptic treatments.

Dissertation (Ph.D.)--The University of North Carolina at Greensboro, 2009. / Directed by Keith Erikson; submitted to the Dept. of Nutrition. Title from PDF t.p. (viewed Apr. 29, 2010). Includes bibliographical references (p. 154-191).

Whole-cell patch recordings were made from immature (six- to 12-day- old) rat rostral ventrolateral medulla neurons in brainstem slices. GABA or the specific GABA(B) receptor agonist (-)baclofen (10-50 μM) by superfusion or by pressure ejection induced an outward current or a hyperpolarization, which persisted in a tetrodotoxin (0.3 μM)-containing Krebs' solution in nearly every cell tested. The GABA(B) receptor antagonists 2-hydroxy saclofen (50-200 μM) and CGP 35348 (50-200 μM) dose-dependently suppressed baclofen- currents. Baclofen-currents were suppressed by barium (1 mM) but not by tetraethylammonium (20 mM), low Ca2+ (0.24 mM) solution or in a solution containing the Ca2+ chelator BAPTA-AM (10 μM). The outward current had an estimated reversal potential of -98, -77 and -52 mV in 3.1, 7 and 15 mM [K+](o). Pre-incubation of slices with pertussis toxin (500 μg/ml for 5-7 h) or intracellular dialysis with GDP-β-S (500 μM) markedly reduced baclofen-currents. Baclofen in low concentrations (1-3 μM) that caused slight or no change of holding currents and of inward or outward currents induced by exogenously applied glutamate or glycine/GABA, decreased excitatory and inhibitory postsynaptic currents by an average of 86.5 ± 4.3% and 78.4 ± 2.7%. The GABA(B) antagonist CGP 35348 (100 μM) increased the excitatory postsynaptic currents by an average of 64%, without causing a significant change in holding currents in 10/18 cells tested. Our results indicate the presence of post- and presynaptic GABA(B) receptors in the rostral ventrolateral medulla neurons. Activation of postsynaptic GABA(B) receptors induces an outward K+ current which is barium-sensitive, Ca2+- independent and may be coupled to a pertussis-sensitive G-protein. Activation of presynaptic GABA(B) receptors attenuates excitatory or inhibitory synaptic transmission. More importantly, the observation that CGP 35348 enhanced excitatory synaptic currents implies a removal of tonic activation of presynaptic GABA(B) receptors by endogenously released GABA (disinhibition), supporting the hypothesis that these receptors may have a physiological role in regulating the input and output ratio in a subset of rostral ventrolateral medulla neurons in vivo.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Plusieurs variables biologiques, psychologiques, ainsi que des différences culturelles, ont été mises en cause afin d'expliquer la différence de perception de la douleur existante entre les hommes et les femmes. Il est connu que les hormones gonadiques influencent la réponse nociceptive chez l’animal et chez l’humain. Le cerveau a aussi la capacité de synthétiser ses propres "hormones sexuelles", les neurostéroïdes. L'objectif de cette thèse était: 1) évaluer les facteurs physiologiques et psychologiques influençant de perception de la douleur chez les hommes et les femmes, 2) évaluer l'implication des androgènes et du cortisol sur les symptômes cliniques et la perception de la douleur chez des sujets atteints de fibromyalgie et sains, et 3) évaluer l'implication des hormones gonadiques et de leurs métabolites 3α5α-réduits dans la transmission et la modulation de la douleur chez animaux les mâles et les femelles par l'utilisation de modèles de douleur comportementaux. / Several biological, psychological, and cultural differences can explain the difference in pain perception between men and women. It is known that gonadal hormones influence the nociceptive response in animals and humans. The brain also has the ability to synthesize its own "sex hormones", also named neurosteroids. The aims of this thesis were: 1) to assess the physiological and psychological factors influencing the difference in pain perception between men and women, 2) to relate the levels of androgens and cortisol with clinical symptoms and pain perception in healthy volunteers and patients with fibromyalgia, and 3) to evaluate the involvement of gonadal hormones and of their 3α5α-reduced metabolites in the transmission of pain and the effectiveness of descending pain modulation systems (DPMS) in males and females using behavioral pain model in rats and mice.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.