Several studies suggest profound effects on mood and cognition by neuroactive steroids. Estrogen alone or in combination with antidepressant drugs affecting the serotonin system has been used to treat mood disorders. On the other hand, progesterone is related to negative effects on mood and memory. A major part of the progesterone effects on the brain can be mediated by its metabolite allopregnanolone, which is also de novo synthesized in the brain, and affects the GABAA receptors. It would be of great importance to find a substance that antagonize allopregnanolone adverse effects. To investigate how long term supplementation of estradiol and progesterone, resembling postmenopausal hormone replacement therapy, affects serotonin receptors in different brain areas important for mood and memory functions, we used ovariectomized female rats. After 2 weeks of supplementation with 17β-estradiol alone or in combination with progesterone, or placebo pellets, estradiol alone decreases but estradiol supplemented together with progesterone increases 5HT1A mRNA expression in the hippocampus. Estradiol decreases the 5HT2C receptor gene expression, while estradiol in combination with progesterone increases the 5HT2A mRNA expression in the ventral hippocampus. Thus, estradiol alone has opposite effects compared to the estradiol/progesterone combination. To detect if acute tolerance develops to allopregnanolone, an EEG method was used where male rats by continuous allopregnanolone infusion were kept on anesthesia level of the silent second (SS). After different time intervals (first SS, 30 min or 90 min of anesthesia) several GABAA receptor subunit mRNAs were measured for detecting if changed expression of any GABAA receptor subunits is involved in development of acute tolerance. There is development of acute tolerance to allopregnanolone and brain regions of importance are hippocampus, thalamus and hypothalamus. The GABAA receptor alpha4 subunit in thalamus and alpha2 subunit in the dorsal hippocampus are related to development of acute tolerance. For assessing allopregnanolone behavioral effects, we studied how this neurosteroid affects spatial learning in the Morris water maze task Allopregnanolone inhibits spatial learning short after the injection and shows a specific behavioral pattern with swimming close to the pool wall. The steroid UC1011 can inhibit the increase in chloride ion uptake induced by allopregnanolone. UC1011 decreases allopregnanoloneinduced impairment of spatial learning in the water maze, as well as the specific behavioral swim pattern. In conclusion, the present work demonstrates that neuroactive steroids affect the 5HT and GABA systems in a brain region specific way. GABAA receptor subunit changes in hippocampus and thalamus are related to acute allopregnanolone tolerance. Allopregnanolone induces cognitive deficits, like spatial learning impairment and UC1011 can inhibit allopregnanolone-induced effects in vitro and in vivo. Key words: Estradiol, progesterone, HRT, allopregnanolone, UC1011, serotonin receptor, GABAA receptor, mRNA, Morris water maze, silent second, tolerance.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

Background: Premenstrual dysphoric disorder (PMDD) affects 3–8% of women in fertile ages. Combined oral contraceptives (OCs) are widely used and some users experience adverse mood effects. The cyclicity of PMDD symptoms coincides with increased endogenous levels of allopregnanolone after ovulation. Allopregnanolone enhances the effect of γ-aminobutyric acid (GABA) on the GABAA receptor, the principal inhibitory transmitter system in the brain. The sensitivity to other GABAA receptor agonists than allopregnanolone (i.e. benzodiazepines, alcohol and the 5 β epimer to allopregnanolone, pregnanolone) has been reported to depend on menstrual cycle phase and/or PMDD diagnosis. Isoallopregnanolone, the 3 β epimer to allopregnanolone, has previously been used to verify specific allopregnanolone GABAA receptor effects. Saccadic eye velocity (SEV) is a sensitive and objective measurement of GABAA receptor function. Aims: To study the pharmacological effects, and any effect on gonadotropin release, of intravenous allopregnanolone in healthy women. A second aim was to explore whether allopregnanolone sensitivity differs over the menstrual cycle or during OC use in healthy women, and thirdly in PMDD patients. Methods: Ten women were challenged with a cumulative dose of intravenous allopregnanolone in the follicular phase of the menstrual cycle. The effect on FSH and LH was compared to women exposed to isoallopregnanolone. A single dose of allopregnanolone was administered once in the follicular phase and once in the luteal phase in another ten healthy women and in ten PMDD patients, and additionally in ten women using OCs. Repeated measurements of SEV, subjectively rated sedation and serum concentrations after allopregnanolone injections were performed in all studies. Results: Allopregnanolone dose-dependently reduced SEV and increased subjectively rated sedation. Healthy women had a decreased SEV response in the luteal phase compared to the follicular phase. By contrast, PMDD patients had a decreased SEV response and subjectively rated sedation response to allopregnanolone in the follicular phase compared to the luteal phase. There was no difference in the SEV response to allopregnanolone between women using oral contraceptives and healthy naturally cycling women. Allopregnanolone decreased serum levels of FSH and LH whereas isoallopregnanolone did not affect FSH and LH levels. Conclusion: Intravenous allopregnanolone was safely given and produced a sedative response in terms of SEV and subjectively rated sedation in women. The sensitivity to allopregnanolone was associated with menstrual cycle phase, but in the opposite direction in healthy women compared to PMDD patients. The results suggest mechanisms of physiological tolerance to allopregnanolone across the menstrual cycle in healthy women and support that PMDD patients have a disturbed GABAA receptor function. In addition, one of our studies suggests that allopregnanolone might be involved in the mechanism behind hypothalamic amenorrhea.

La douleur neuropathique est un syndrome secondaire à une maladie ou à une lésion affectant le système nerveux somatosensoriel. Les causes biologiques de ces comorbidités ne sont pas clairement établies. En utilisant un modèle murin de douleur neuropathique, nous avons cherché à amplifier l'inhibition nerveuse médiée par les récepteurs GABAA afin de tenter de réduire les symptômes douloureux neuropathiques et les troubles émotionnels associés. Pour cela nous avons utilisé l’étifoxine, un anxiolytique non benzodiazépinique qui possède une action originale sur les récepteurs GABAA mais, surtout, semble avoir des effets secondaires limités comparativement à d'autres molécules comme les benzodiazépines par exemple. Son effet passe par la potentialisation directe du récepteur GABAA (site modulateur situé sur les sous-unités β2 et β3) mais aussi par une action indirecte : la stimulation de la production de neurostéroïdes 3α-réduits capables de potentialiser la fonction des récepteurs GABAA. Quelques rares études ont montré que les neurostéroïdes endogènes, surtout ceux réduits en position 3α comme l'allopregnanolone (THP, allotétrahydroprogestérone) ou le THDOC (tétrahydrodéoxycorticostérone), pouvaient réduire les symptômes douloureux. Les effets obtenus dans ces études sont similaires à ceux issus d’injections exogènes des mêmes composés. / Neuropathic pain is associated with significant co-morbidity, including anxiety and depression, which impact considerably on the overall patient experience. Several lines of evidence suggest that anxiolytics may be of interest to alleviate pain symptoms and the associated negative emotions in chronic pain states. Among them, the non-benzodiazepine anxiolytic etifoxine (EFX: stresam™) has an interesting pharmacological profile. In patients, it has a weak sedative action, with limited functional tolerance and dependence, and without cognitive declines. In this study, we aim at analyzing the preclinical therapeutic potential of etifoxine on the anxiodepressive consequences of neuropathic pain.

La sclérose en plaques (SEP) a longtemps été considérée comme une affection inflammatoire démyélinisante de la substance blanche. (SB) Hors, de nombreuses études ont démontré l’implication extensive de la substance grise (SG). La souffrance neuronale joue un rôle majeur dans la détérioration physique et cognitive des patients atteints de SEP. Le développement de nouvelles techniques d’imagerie capables de quantifier cette atteinte neuronale est devenu crucial. Grace à la tomographie par émission de positons (TEP) et au radiotraceur [11C]flumazenil ([11C]FMZ), antagoniste du récepteur central aux benzodiazépines, nous avons quantifié de façon non-invasive la souffrance neuronale de la SG chez des patients atteints de SEP à différents stades de la maladie. Une cohorte de 18 patients atteints de SEP a été comparée à 8 sujets sains. Les participants ont bénéficié d’une évaluation clinique, cognitive, et radiologique par TEP au [11C]FMZ et IRM. Les données TEP ont été évaluées par région d’intérêt et vertex-à- vertex. Des réductions significatives de l’activité TEP au [11C]FMZ ont été mises en évidence au sein de la SG corticale et sous-corticale des patients comparés aux contrôles. Ces changements étaient présents dès le stade rémittent de la maladie et corrélaient modérément avec la charge lésionnelle de la SB. L’activité TEP corticale était aussi associée à la performance cognitive des patients. Cette étude pilote est la première à quantifier in vivo la souffrance neuronale chez des patients atteints de SEP. Nos résultats permettent de proposer la TEP au [11C]FMZ comme marqueur spécifique et discriminant de l’atteinte neuronale de la SG dans la SEP. / Multiple sclerosis (MS) has long been regarded as an inflammatory demyelinating disorder of the white matter. But post-mortem studies have recently shed light on the extensive involvement of the grey matter (GM). Neuronal damage, characterized by synaptic and dendritic loss as well as neuronal apoptosis, is thought to be a major substrate of physical and cognitive deterioration in MS patients. There is a crucial need for new imaging techniques able to specifically assess neuronal damage in MS. Using positron emission tomography (PET) with [11C]flumazenil ([11C]FMZ), an antagonist of the central benzodiazepine site located within the GABAA receptor, and a non-invasive quantification method, we measured and mapped neurodegenerative changes in the GM of patients with MS at distinct disease stages. A cohort of 18 MS patients was compared to 8 healthy controls and underwent neurological and cognitive evaluations, high-resolution dynamic [11C]FMZ PET imaging and brain MRI. PET data were evaluated using a region of interest and a surface-based approach. [11C]FMZ binding was significantly decreased in the cortical and subcortical GM of MS patients compared to controls. These changes were significant in both progressive and relapsing-remitting forms of the disease and correlated moderately with white matter lesion load. [11C]FMZ cortical binding was also associated with cognitive performance. This pilot study is the first to quantify in vivo the neurodegenerative changes occurring in MS. Our results show that PET with [11C]FMZ could be a promising and sensitive quantitative marker to assess and map the neuronal substrate of GM pathology in MS.

Gamma-Amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system (CNS) and inhibits the neurotransmission by targeting the ionotropic transmembrane GABAA receptor. Modulators of the GABAA receptor targets the allosteric binding sites and modifies the GABA effect and these sites acts as superior drug targets within psychopharmacology.   Gabather AB has developed the novel compound GT-002 that is known to target the receptor and cause a behavioral effect in rodents. This project characterized the binding of the lead compound GT-002 to GABAA receptor in mammalian brain tissue by development and validation of a radioligand binding assay. In the assay a comparative evaluation was performed using the benzodiazepine (BZ) antagonist Flumazenil (FLU).   All experiments were performed using GABAA receptors originating from porcine and mouse brain tissue membrane, where no significant difference between the mammals was displayed. GT-002 binds with higher affinity and associates faster than FLU to the receptor and implies a two-binding site model. GT-002 displaced FLU and no tested competitive analytes targeting various modulatory sites of the receptor displaced GT-002, implying independent binding of GT-002 and allosterically impacts the BZ binding site.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

<p>The formation and retention of distinct membrane domains in the fluidic membrane bilayer is the key process in establishing spatial organization for mediating physiological functions in metazoans. The spectrin-ankyrin network organizes diverse membrane domains including T-tubule and intercalated disc of cardiomyocytes, basolateral membrane of epithelial cells, costameres of striatal muscle, and axon initial segments and nodes of Ranvier in nervous system. This thesis identifies a novel function of 480 kDa ankyrin-G, an alternatively spliced isoform of the ankyrin family, in promoting somatodendritic GABAA receptor synaptogenesis both in vitro and in vivo. In the nervous system, an insertion of a neuronal specific exon (exon 37) occurs in ankyrin-G polypeptide which results in a 480 kDa isoform. 480 kDa ankyrin-G (giant ankyrin-G) has been shown to coordinate formation and maintenance of the axon initial segment (AIS) and nodes of Ranvier. This thesis research began with the discovery that giant ankyrin-G, previously thought to be confined to the axon initial segment, forms developmentally-regulated and cell-type specific somatodendritic "outposts" on the plasma membrane of pyramidal neurons. This somatodendritic 480 kDa ankyrin-G outpost forms micron-scale membrane domains where it associates with canonical AIS binding partners including voltage-gated sodium channel and neurofascin. This thesis further discovered that the giant insert of 480 kDa ankyrin-G interacts with GABARAP, a GABAA receptor-associated protein. Both the interaction with GABARAP and the membrane association through palmitoylation of giant ankyrin-G are required for the formation of somatodendritic GABAergic synapses. This work further found that ankyrin-G associates with extrasynaptic GABAA receptors and stabilizes receptors on the extrasynaptic membrane through opposing endocytosis. This story demonstrates for the first time the existence of giant ankyrin-G somatodendritic outpost as well as its function in directing the formation of GABAergic synapses that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and neurodevelopmental disorders.</p><p>Additional work presented in the Appendix characterized novel ankyrin-G full length transcripts in the heart and kidney with unique domain compositions though alternative splicing. The preliminary work further identified biochemical properties and potential role of an insert C in the C-terminus of ankyrin-G in mediating cytokinesis and cellular migration in mouse fibroblasts. Together, this thesis work expands the knowledge of giant ankyrin-G functions in the nervous system and offers insights into the diversified roles of distinct ankyrin-G peptides acquired from alternative splicing in organizing specific membrane domains and interacting with defined intracellular pathways in different tissues.</p> / Dissertation

Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.

No description available.