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A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control. In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes. Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*. A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P. The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times. The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ã-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP. Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).

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Dramatic growth in substituted amphetamines (‘Ecstasy’) use since the 1980’s has correlated with increased incidence of acute toxicity and residual neuropsychological deficits. This thesis aimed to characterise the acute neurochemical mechanisms and residual neurochemical alterations produced by p-methoxyamphetamine (PMA), which is usually sold as ‘ecstasy’ and is associated with greater acute toxicity than 3,4-methylenedioxymethamphetamine (MDMA). While both PMA and MDMA primarily modulate dopaminergic and serotonergic neurotransmission, little is known of the differences in the neurochemical effects of PMA within the central nervous system, in vivo. This thesis used in vivo chronoamperometry to elucidate the acute neurochemical alterations in monoaminergic pharmacology in vivo after local application of PMA or MDMA within discrete brain nuclei in anaesthetised rats. Measurement of evoked release of monoamines including serotonin (5-HT), and inhibition of neurotransmitter uptake via membrane transporters were assessed. Initial studies compared pharmacodynamic responses of PMA and MDMA, showing PMA to have greater efficacy and potency for alteration of core body temperature in rats, a primary cause of acute toxicity, within minimal alteration in locomotion. Dose-response studies indicated local PMA application within striatum resulted in significantly greater 5-HT evoked release than MDMA, yet lesser dopaminergic release, as predicted by the pharmacodynamic data. Only PMA-evoked release could be partially blocked by pre-treatment with a 5-HT reuptake inhibitor (SERT). Differences in both the qualitative and quantitative nature of striatal evoked-release of 5HT and dopamine were noted for both drugs, which had not been previously seen. Both PMA and MDMA inhibited 5-HT clearance, but only MDMA inhibited dopamine clearance in striatum. Doseresponse studies in the CA3 region of hippocampus indicated PMA was also more efficacious than MDMA in the inhibition of 5-HT clearance in vivo. While the question of whether long term MDMA use induces selective neurodegeneration (reductions in serotonergic in vitro biomarkers) is still unclear, it was not known for PMA prior to this work. Repeated PMA administration was shown to result in reductions in cortical SERT (indicative of potential loss of 5-HT terminal axons), cortical 5-HT content was unaltered. A subsequent comprehensive study followed, comparing the residual effects of PMA or MDMA administration on in vitro serotonergic biomarkers (markers of selective neurodegeneration) and SERT function in vivo. PMA administration resulted in reductions in hippocampal SERT binding and [3H]-5HT synaptosomal uptake, correlating with in vitro biomarkers previously used. SERT function in vivo using chronoamperometric techniques was reduced, as would be predicted. However, hippocampal 5-HT content was again not reduced, indicating that selective neurodegeneration of 5-HT fibres may not in fact be occurring. MDMA administration reduced all measured in vitro serotonergic biomarkers, however SERT function in vivo was completely unaltered. These data indicate that reductions of in vitro biomarkers of 5-HT axonal degeneration do not necessarily predict the potential compensatory mechanisms that maintain SERT function in vivo. Compensatory mechanisms appear to exist in vivo to maintain clearance of extracellular 5- HT that may be disrupted or eliminated during tissue preparation for in vitro assays. In summary, while PMA produced significantly greater alterations, compared to MDMA, in processes intrinsic to 5-HT neurotransmission in both striatum and hippocampus, the magnitude of these responses did not explain the significantly higher risk of acute toxicity seen clinically with PMA use. The second component of the thesis extended beyond prior work, investigating the potential neurodegenerative effects of PMA and MDMA through the assessment of changes in key functional processes in 5-HT neurotransmisson. It is hoped this will contribute to the subsequent characterisation of the mechanism(s) of functional compensation in 5-HT neurotransmission which may lead to more targeted treatments to modulate potential psychological/psychiatric deficits that occur in regular ‘ecstasy’ users. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346193 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

Here, we investigated the effects of modulation of the GABAergic system using a targeted neuropharmacological, 1H/13C NMR spectroscopy and metabolomic approach in Guinea pig cortical brain slices. The effects of exogenous GABA, agonists, antagonists and allosteric modulators at GABAA receptors were described and classified on the basis of metabolic activity; this corresponded to receptor location rather than pharmacology. The effects of agonists and antagonists at the GABAB receptor were described and classified into inhibitory and excitatory components, consistent with context dependent outcomes of receptor activity. Metabolic evidence for GABAC mediated activity in the cerebral cortex was identified for the first time indicating a strong role for this receptor in the control of neuronal activity. Inhibition of GABA uptake was examined using inhibitors of these transporters. The major effect of individual transporter subtype blockade was increased synaptic inhibition. The paradoxical activity of the GABA-transaminase inhibitor vigabatrin was resolved, with a direct demonstration of a single inhibitory mechanism mediated via this drug, via a mechanism also induced by antagonists at the GABAC receptor. These data were then integrated using multivariate statistics to identify 5 subclasses of activity which corresponded to receptor location (e.g. synaptic or extrasynaptic) rather than receptor pharmacology. This represents a novel and powerful new approach to the study of brain metabolism and the GABAergic system.

Here, we investigated the effects of modulation of the GABAergic system using a targeted neuropharmacological, 1H/13C NMR spectroscopy and metabolomic approach in Guinea pig cortical brain slices. The effects of exogenous GABA, agonists, antagonists and allosteric modulators at GABAA receptors were described and classified on the basis of metabolic activity; this corresponded to receptor location rather than pharmacology. The effects of agonists and antagonists at the GABAB receptor were described and classified into inhibitory and excitatory components, consistent with context dependent outcomes of receptor activity. Metabolic evidence for GABAC mediated activity in the cerebral cortex was identified for the first time indicating a strong role for this receptor in the control of neuronal activity. Inhibition of GABA uptake was examined using inhibitors of these transporters. The major effect of individual transporter subtype blockade was increased synaptic inhibition. The paradoxical activity of the GABA-transaminase inhibitor vigabatrin was resolved, with a direct demonstration of a single inhibitory mechanism mediated via this drug, via a mechanism also induced by antagonists at the GABAC receptor. These data were then integrated using multivariate statistics to identify 5 subclasses of activity which corresponded to receptor location (e.g. synaptic or extrasynaptic) rather than receptor pharmacology. This represents a novel and powerful new approach to the study of brain metabolism and the GABAergic system.