GABAA receptors (GABAARs) mediate the majority of fast inhibition in the CNS and as such are crucial to neuronal function. Two distinct modes of GABAAR mediated inhibition exist: “phasic” involves the transient activation of postsynaptic GABAARs following presynaptic vesicular GABA release and “tonic” whereby high-affinity extrasynaptic GABAARs are persistently activated by ambient GABA. GABAARs exhibit a rich pharmacology and are the target for a number of clinically useful compounds including benzodiazepines, barbiturates and certain general anaesthetics. In addition, several naturally occurring steroids typified by the progesterone metabolite 5a-pregnan-3a-ol-20-one (5a3a) are potent positive allosteric modulators of GABAAR function - a property that endows these steroids with anxiolytic, sedative and anti-convulsant actions. Importantly, in addition to importing steroids from the periphery, the brain also harbours a steroidogenic capacity and can manufacture GABA-modulatory steroids, termed “neurosteroids”, from cholesterol. Although several studies have demonstrated neurosteroids to be implicated in a variety of physiological processes including neurodevelopment, important questions remain. In particular, are neurosteroids neurone selective? Do they discriminate between particular neuronal GABAARs subtypes? Under what conditions do endogenous neurosteroids influence neuronal function? And importantly, which cells synthesise neurosteroids in the central nervous system? Therefore, the principal aims of this study were: 1) to characterise the properties of GABAAR mediated inhibition in postnatal day (P) 17-24 neurones of the mouse ventrobasal (VB) nucleus of the thalamus – neurones which have a well defined GABAAR expression profile and which exhibit both synaptic and tonic GABAAR mediated inhibition. 2) To investigate the effects of 5a3a on synaptic and tonic GABAAR mediated inhibition in VB neurones. 3) To investigate whether there is a role for endogenous neurosteroids in regulating synaptic GABAAR function during postnatal development (P7-P20) in VB neurones and 4) cortical layer 2/3 neurones – a neuronal population with a more heterogenous, and less well defined GABAAR expression profile. To address these aims, whole-cell voltage clamp recordings were performed on acute brain slices derived from wild type and transgenic GABAAR subunit “knock-out” mice in conjunction with pharmacological approaches. Synaptic inhibition in WT VB neurones, as inferred by recording miniature inhibitory postsynaptic currents (mIPSCs) was characterised by relatively large amplitude and fast decaying mIPSCs. A large tonic conductance in WT VB neurones was inhibited following application of the competitive GABAAR antagonist bicuculline (30 µM). Deletion of the a4 subunit (a40/0) revealed alteration to both synaptic and tonic inhibition. Most notably, a40/0 VB neurones displayed a greatly diminished tonic conductance. These results are in agreement that the majority of extrasynaptic GABAARs in VB neurones contain the a4 subunit. Exogenous application of the neurosteroid 5a3a (100 nM) to WT VB neurones only modestly enhanced the tonic conductance and also gave rise to mIPSCs with prolonged decay kinetics. It is concluded 5a3a has a relatively low potency at the extrasynaptic GABAAR population in VB neurones and furthermore that this concentration does not discriminate between synaptic and tonic inhibition. Between P7 and P20, mIPSCs recorded from VB and L2/3 neurones became progressively faster decaying, a feature that has been associated with an increase in a1-GABAAR subunit expression during development. However, the developmental decrease in mIPSC decay time was still observed in recordings from a10/0 L2/3 pyramidal neurones indicating the contribution of additional factors. Here I show that blocking neurosteroid synthesis using the 5a-reductase inhibitor, finasteride, or treating the brain slice with the steroid-scavenger molecule, ?-cyclodextrin (?-CD), results in significantly faster mIPSC decay times between P7 and P10 in VB and L2/3 neurones. These results provide the first indication that an endogenous neurosteroid tone may influence the mIPSC decay kinetics of VB and L2/3 neurones during development. Moreover, compared to ?-CD pre-incubation, application of ?-CD to the intracellular compartment via the patch pipette was found to be equally effective thus suggesting a possible autocrine mechanism of neurosteroid action. For VB neurones at P20-24 (an age range at which the mIPSCs are insensitive to ?-CD treatment), incubation with the GABAAR-inactive neurosteroid precursor 5a-DHP resulted in a robust prolongation of the mIPSC decay time thus revealing the presence of functional neurosteroidogenic enzymes in the brain slice tissue. In summary, these results reveal for the first time that during the first ~2 weeks of life the developmental decrease in the duration of the mIPSCs recorded from VB and L2/3 neurones is largely due to a reduction in local neurosteroid synthesis. Moreover, P20-24 VB neurones are capable of reinitiating neurosteroid production thus giving rise to a mechanism that may, under certain conditions, locally influence neuronal inhibition. These results may have important physiological consequences as the developmental timing of the neurosteroid tone observed here coincides with a myriad of crucial neurodevelopmental processes including the transition of GABA from a depolarising to a hyperpolarising action.

Η Νόσος του Πάρκινσον (ΝΠ) χαρακτηρίζεται κυρίως από εκλεκτική και βαθμιαία εκφύλιση των ντοπαμινεργικών νευρώνων της μέλαινας ουσίας (SNPc) του μεσεγκεφάλου. Ο "weaver" μυς αποτελεί ένα ιδανικό μοντέλο για τη μελέτη της Νόσου του Πάρκινσον δεδομένου ότι η εκφύλιση του μοντέλου "weaver" είναι προοδευτική, ξεκινάει την 7η μέρα μετά τη γέννηση, είναι δραματική μέχρι την 21η μέρα όπου το 50% των ντοπαμινεργικών νευρώνων έχει ήδη πεθάνει και στην ηλικία των 2 μηνών φτάνει στο 65%. Στη νόσο του Πάρκινσον, η εκφύλιση της μελαινοραβδωτής οδού, έχει ως αποτέλεσμα να χάνεται ο έλεγχος της εύρυθμης λειτουργίας των δύο νευρικών οδών εξόδου της πληροφορίας από τα βασικά γάγγλια, της "άμεσης" και της "έμμεσης" οδού. Το αποτέλεσμα είναι υπέρμετρη δραστηριότητα της "έμμεσης" οδού που οδηγεί στις κινητικές δυσλειτουργίες της νόσου (βραδυκινησία, τρόμος σε ηρεμία, ακαμψία, αστάθεια θέσης). Πρώτος στόχος της παρούσας εργασίας ήταν να διερευνηθεί κάτω από ντοπαμινεργική απονεύρωση στο μυ "weaver" η μεταγωγή σήματος των Α2A υποδοχέων αδενοσίνης (A2ARs), οι οποίοι αλληλεπιδρούν ανταγωνιστικά με τους D2 υποδοχείς ντοπαμίνης στους νευρώνες της "έμμεσης" οδού. Για το λόγο αυτό μελετήσαμε μετά από διέργεση των Α2A υποδοχέων, την "in vivo" φωσφορυλίωση της DΑRΡΡ-32 στη θέση Τh34, κεντρικό μόριο στο μονοπάτι μεταγωγής σήματος των Α2A υποδοχέων. Επιπλέον μελετήθηκε εάν κάτω από τη ντοπαμινεργική απονεύρωση οι Α2Α υποδοχείς αλλάζουν μονοπάτι μεταβίβασης σήματος. Οπότε μελετήσαμε την εμπλοκή της οδού των MAP-κινασών με την ανίχνευση της φωσφορυλίωσης των ERK1/2 κινασών. Τα αποτελέσματά μας δείχνουν, αυξημένα βασικά επίπεδα των φωσφορυλιωμένων DARPP-32 στη θέση Thr34 και ERK1/2 πιθανώς, ως ένα φαινόμενο προσαρμογής στο ντοπαμινεργικό νευροεκφυλισμό. Η διέγερση των Α2Α υποδοχέων οδηγεί σε περαιτέρω αύξηση των επίπεδων φωσφορυλίωσης της DARPP-32 αυξάνοντας περαιτέρω τη δραστηριότητα της "έμμεσης" οδού και επιδεινώνοντας τα συμπτώματα της νόσου. Η διέγερση των Α2Α υποδοχέων ρυθμίζει αρνητικά την αυξημένη φωσφορυλίωση των ERK1/2 κινασών ενώ οι ανταγωνιστές των Α2Α υποδοχέων έχουν ακριβώς αντίθετη δράση. Αυτό το αποτέλεσμα ενδεχομένως να έχει κλινική σημασία δεδομένου ότι, ο αποκλεισμός των υποδοχέων Α2Α με χρήση ειδικών ανταγωνιστών χρησιμοποιείται ήδη σε κλινικές δοκιμές για τη μείωση των συμπτωμάτων της νόσου. Σύμφωνα με τα δικά μας αποτελέσματα όμως η χρήση ανταγωνιστών των Α2Α υποδοχέων αυξάνει τη φωσφορυλίωση των ERK1/2 κινασών, η ενεργοποίηση των οποίων έχει συνδεθεί με τις δυσκινησίες που προκαλούνται από τη χρόνια χορήγηση της L-Dopa. Για το λόγο αυτό η ρύθμιση της ενεργοποίησης των ERK1/2 κινασών μέσω των Α2Α υποδοχέων κάτω από τη ντοπαμινεργική απονεύρωση πρέπει να διερευνηθεί περαιτέρω. Δεδομένου ότι δεν υπάρχει θεραπευτική αγωγή για τη ΝΠ, δεύτερος στόχος της παρούσας εργασίας ήταν η διερεύνηση της νευροπροστατευτικής δράσης βασικά των "Νευροστεροειδών" αλλά και άλλων ήπιων παραγόντων, μεμονωμένα και σε συνδυασμό, στο μοντέλο "weaver". Η χορήγηση των φαρμακευτικων παραγόντων εφαρμόστηκε πριν την έναρξη της εκφύλισης, από την 1η μετεμβρυϊκή ημέρα (Ρ1) και καθ’ όλη τη διάρκεια που αυτή εξελίσσεται με καταιγιστικό ρυθμό, μέχρι την 21η μέρα της γέννησης, (Ρ21) που η εκφύλιση αγγίζει το 50%. Για την εκτίμηση της νευροπροστατευτικής δράσης μετρήθηκε με ανοσοϊστοχημεία ο αριθμός των ντοπαμινεργικών κυττάρων της μέλαινας ουσίας που επιβίωσαν. Μελετήθηκαν ακόμα, η έκφραση της τυροσίνης της υδροξυλάσης (TH) και η έκφραση του μεταφορέα της ντοπαμίνης (DAT) των τελικών απολήξεων των ντοπαμιμνεργικών ινών στο ραβδωτό σώμα. Η "in vivo" χρόνια χορήγηση (Ρ1-Ρ21) των "Νευροστεροειδών" DHEA, BNN-50 και του συνδυασμού BNN-50 και NAC οδήγησε σε σημαντική επιβίωση των κυττάρων της μέλαινας ουσίας στο "weaver" μυ. Επιπλέον η χορήγηση του BNN-50 καθώς και του συνδυασμού BNN-50/NAC παρέχουν προστασία και στους τελικούς ντοπαμινεργικούς νευράξονες στο ραβδωτό σώμα, υποδεικνύοντας ότι διέσωσαν τις λειτουργικές ντοπαμινεργικές απολήξεις. Τέλος, τρίτος στόχος ήταν να διερευνηθεί ο μηχανισμός μέσω του οποίου πραγματοποιείται η "in vivo" νευροπροστατευτική δράση των "Νευροστεροειδών". Διερευνήσαμε λοιπόν την έκφραση των TrkA υποδοχέων στα κύτταρα της μέλαινας ουσίας των φυσιολογικών και "weaver" μυών. Ο αντιαποπτωτικός μηχανισμός δεν φαίνεται να πραγματοποιείται μέσω του TrkA υποδοχέα όπως έχει δειχθεί σε "in vitro" πειράματα σε PC12 κυτταρικές σειρές. Ο TrkA υποδοχέας δεν ανιχνεύεται στους ντοπαμινεργικούς νευρώνες της μέλαινας ουσίας ούτε των φυσιολογικών, ούτε των "weaver" μυών, ούτε υπάρχει επαγωγή του μετά τη χρόνια χορήγηση των φαρμάκων. Οπότε ο μοριακός μηχανισμός μέσω του οποίου ασκείται η νευροπροστατευτική δράση των DHEA-S και BNN-50 στους ντοπαμινεργικούς νευρώνες "in vivo" πρέπει να διερευνηθεί περαιτέρω. Με βάση το γεγονός ότι η χορήγηση του BNN-50 και του συνδυασμού BNN-50 και NAC παρέχει πλήρη νευροπροστασία, ο παρόν συνδυασμός θα μπορούσε μελλοντικά να προταθεί ως προληπτικό νευροπροστατευτικό σχήμα για τη ΝΠ. / Parkinson's disease (PD) is mainly characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SNPc) of the midbrain. The "weaver" mouse is an ideal model for the study of Parkinson's disease since the degeneration of dopaminergic neurons is progressive, starting at day 7 after the birth, continuing until day 21 where 50% of dopaminergic neurons has already died and reaching 65% at the age of 2 months. In Parkinson disease, degeneration of the nigrostriatal pathway, leads to an impairement of the operational control of the two neuronal output pathways from the basal ganglia, the "direct" and "indirect". The result is excessive activity of the "indirect" pathway leading to motor dissabilities like (bradykinesia, tremor at rest, rigidity and postural instability). The first objective of this study was to investigate, under dopaminergic denervation in the "weaver" mice, the signal transduction of A2A adenosine receptor (A2ARs), which are colocalized and interact competitively with D2 dopamine receptors on the neurons of the "indirect" pathway. For this reason, we studied after stimulation of A2A receptors, the "in vivo" phosphorylation of DARRR-32 at Th34, a central molecule in the signaling pathway of A2A receptors. Moreover, we studied whether under dopamine denervation, A2A receptors switch on an other transduction pathway. Thus, we examined the involvement of MAP-kinases pathway by detecting the phosphorylation state of ERK1/2 kinases. Our results show elevated basal levels of phosphorylated DARPP-32 at position Thr34 and ERK1/2, probably as an adjustment mechanism on the dopaminergic neurodegeneration. Stimulation of A2A receptors leads to a robust increase of DARPP-32 phosphorylation levels, which would further increase the activity of "indirect" pathway worsening thus the PD symptoms. Stimulation of A2A receptors negatively regulates the increased phosphorylation levels of ERK1/2 kinases, while A2A receptor antagonists have the opposite effect. This effect is likely to have clinical importance since A2A receptor blockade is already used in clinical trials, to ameliorate the symptoms of the disease. However, according to our results, the use of A2A receptors’ antagonists increase the phosphorylation state of ERK1/2 kinases, the activation of which is connected with dyskinesias induced by chronic administration of L-Dopa. For this reason, the regulation of ERK1/2 kinases activity through A2A receptors under dopamine denervation, must be further investigated. Since there is no curative treatment for PD, the second objective of this study was to investigate the neuroprotective effect basically of "Neurosteroids" and other mild pharmaceutical agents, individually and in combination, on the "weaver" model. The pharmaceutical substrates were applied prior to the degeneration, on the first postnatal day until day 21 of birth when the degeneration reaches 50%. In order to evaluate the neuroprotective effect, we measured by immunohistochemistry, the number of dopaminergic neurons in the substantia nigra that survived. We also studied the expression of tyrosine hydroxylase (TH) and the expression of the dopamine transporter (DAT) on the terminals of dopaminergic fibers in the striatum. Chronic "in vivo" administration (P1-P21) of the "neurosteroids" DHEA, BNN-50 and the combination BNN-50, and NAC resulted to a significant survival of the dopaminergic cells in the substantia nigra of the "weaver" mice. The administration of BNN-50 and the combination BNN-50/NAC provided also protection to the final dopaminergic axons in the striatum, suggesting that they rescued functional dopaminergic terminals. Finally, the third objective was to investigate the mechanism by which the "in vivo" neuroprotective action of "neurosteroids" takes place. Therefore we investigated the expression of TrkA receptor on the cells of the substantia nigra of normal and "weaver" mice. The antiapoptotic mechanism does not seem to be mediated through the TrkA receptor as it has been shown "in vitro" experiments using PC12 cells. The TrkA receptor was not detected on the dopaminergic neurons of the substantia nigra nor of the normal or of the "weaver" mice and its expression was not induced after chronic administration of the neurosteroids. Thus, the molecular mechanism through which the dopaminergic neuroprotective effect of DHEA-S and BNN-50 is mediated "in vivo", must be further investigated Based on the fact that administration of BNN-50 and the combination BNN-50, and NAC provides complete neuroprotection, the present combination could be proposed in future as a neuroprotective agent for PD..

The classical model of sexual differentiation states that genes influence gonadal differentiation, and gonadal hormones then drive sexual differentiation throughout development. This model has been called into question by research, especially in songbirds, providing evidence for alternative mechanisms like direct effect of genes and local production of steroids via de novo synthesis or local metabolism of steroid precursors like DHEA, which can be metabolized to testosterone and E₂. In order to assess the role of local steroid production on sexual differentiation in songbirds, levels of DHEA and E₂ were measured in brachial and jugular plasma, as well as brain and peripheral tissues in zebra finches at critical ages during development and in adulthood. DHEA levels in brachial and jugular plasma peaked at P30 and higher DHEA levels in jugular plasma were found in males relative to females at P30. Also, at P30, higher DHEA levels were found in rostral telencephalon in females relative to males. The findings of this study indicate that DHEA may play a role in sexual differentiation of songbirds. Surprisingly, E₂ was non-detectable in many plasma and tissue samples. Higher E₂ was found in the diencephalon in females relative to males at P3/P4 and higher E₂ was found in gonads in adult females relative to males. There was little evidence to suggest that E₂ is synthesized de novo in the brain, although perhaps E₂ is being rapidly metabolized into another estrogen or E₂ synthesis is more localized in the synapse. The findings of this study support the role of alternative mechanisms like de novo steroid synthesis and local metabolism of steroid precursors and challenge the role of classical mechanisms of sexual differentiation in songbirds. Also, these findings may have important implications for sex differences, which develop independently of gonadal hormones, in other animal species.

The classical model of sexual differentiation states that genes influence gonadal differentiation, and gonadal hormones then drive sexual differentiation throughout development. This model has been called into question by research, especially in songbirds, providing evidence for alternative mechanisms like direct effect of genes and local production of steroids via de novo synthesis or local metabolism of steroid precursors like DHEA, which can be metabolized to testosterone and E₂. In order to assess the role of local steroid production on sexual differentiation in songbirds, levels of DHEA and E₂ were measured in brachial and jugular plasma, as well as brain and peripheral tissues in zebra finches at critical ages during development and in adulthood. DHEA levels in brachial and jugular plasma peaked at P30 and higher DHEA levels in jugular plasma were found in males relative to females at P30. Also, at P30, higher DHEA levels were found in rostral telencephalon in females relative to males. The findings of this study indicate that DHEA may play a role in sexual differentiation of songbirds. Surprisingly, E₂ was non-detectable in many plasma and tissue samples. Higher E₂ was found in the diencephalon in females relative to males at P3/P4 and higher E₂ was found in gonads in adult females relative to males. There was little evidence to suggest that E₂ is synthesized de novo in the brain, although perhaps E₂ is being rapidly metabolized into another estrogen or E₂ synthesis is more localized in the synapse. The findings of this study support the role of alternative mechanisms like de novo steroid synthesis and local metabolism of steroid precursors and challenge the role of classical mechanisms of sexual differentiation in songbirds. Also, these findings may have important implications for sex differences, which develop independently of gonadal hormones, in other animal species.

The classical model of sexual differentiation states that genes influence gonadal differentiation, and gonadal hormones then drive sexual differentiation throughout development. This model has been called into question by research, especially in songbirds, providing evidence for alternative mechanisms like direct effect of genes and local production of steroids via de novo synthesis or local metabolism of steroid precursors like DHEA, which can be metabolized to testosterone and E₂. In order to assess the role of local steroid production on sexual differentiation in songbirds, levels of DHEA and E₂ were measured in brachial and jugular plasma, as well as brain and peripheral tissues in zebra finches at critical ages during development and in adulthood. DHEA levels in brachial and jugular plasma peaked at P30 and higher DHEA levels in jugular plasma were found in males relative to females at P30. Also, at P30, higher DHEA levels were found in rostral telencephalon in females relative to males. The findings of this study indicate that DHEA may play a role in sexual differentiation of songbirds. Surprisingly, E₂ was non-detectable in many plasma and tissue samples. Higher E₂ was found in the diencephalon in females relative to males at P3/P4 and higher E₂ was found in gonads in adult females relative to males. There was little evidence to suggest that E₂ is synthesized de novo in the brain, although perhaps E₂ is being rapidly metabolized into another estrogen or E₂ synthesis is more localized in the synapse. The findings of this study support the role of alternative mechanisms like de novo steroid synthesis and local metabolism of steroid precursors and challenge the role of classical mechanisms of sexual differentiation in songbirds. Also, these findings may have important implications for sex differences, which develop independently of gonadal hormones, in other animal species. / Medicine, Faculty of / Graduate

Peripheral sensitisation and central sensitisation are implicated in the development of neuropathic pain with neuroplasticity occurring at multiple levels of the pain pathway. Hypersensitivity of the spinothalamic tract has been described in neuropathic animal models of diabetes. Spinal dorsal horn neurones of diabetic rats exhibit abnormally high spontaneous firing, suggesting an imbalance between excitatory and inhibitory signals converging within this structure. GABAergic neurones within the spinal cord and thalamus are crucial for the transmission of painful stimuli to higher centres of the brain that are involved in pain perception. GABAA receptors (GABAARs) are an important target for many clinical drugs, and certain endogenous neurosteroids act as potent allosteric modulators of these receptors. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurones and this may be related in part to fluctuations in endogenous neurosteroid tone. The objective of this study was to investigate changes to inhibitory neurotransmission with development in three levels of the pain pathway and to explore potential mechanisms underlying diabetic neuropathy. The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in lamina II of the spinal cord, the nucleus reticularis (nRT) of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Neurosteroids and their precursors were employed along with compounds that prevented their activity at the GABAAR such as ?-cyclodextrin, which is a barrel-shaped cyclic oligosaccharide with a lipophilic interior that sequesters neurosteroids. Behavioural experiments were also performed using von Frey filaments and the tail flick test to examine mechanical and thermal nociception. Recordings from the spinal cord, the thalamus and the cerebral cortex revealed that the decay time of miniature inhibitory postsynaptic currents are significantly reduced with development. The neurosteroids allopregnanolone and ganaxolone were significantly more effective in neurones from the older mice. In contrast, ?-cyclodextrin had significantly less effect in neurones from the older mice. In mature diabetic mice (ob/ob mice), the endogenous neurosteroid tone is reduced compared to control mice, but certain neurosteroid compounds have a greater effect on the GABAARs of these diabetic mice. In addition, the diabetic mice exhibit mechanical allodynia and hyperalgesia, which is responsive to exogenously applied neurosteroids. These results are consistent with the hypothesis that a dramatic reduction in endogenous neurosteroid tone occurs as development progresses and that this impacts on the exponential decay time of GABAergic mIPSCs within neurones of the pain pathway. The higher neurosteroid tone in the youngest mice may confer a degree of neural protection over the nervous system as it develops. The reduction of endogenous neurosteroid tone in diabetic mice may be associated with their hypersensitivity. It is possible that pregnane-derived neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone that is observed in immature animals.

Peripheral estradiol treatment enhances hippocampus-dependent memory and morphology in ovariectomized rats. Although these enhancements are traditionally thought to be due to circulating estradiol, recent data suggest these changes are brought on by hippocampus-derived estradiol, the synthesis of which depends on GnRH activity. The goal of the current work is to test the hypothesis that peripheral estradiol affects cognition and the hippocampus through brain-derived estradiol via hippocampal GnRH receptor activity. In Experiment 1, we investigated if peripheral estradiol exerts its effects through regulation of brain-derived estradiol. Intracerebroventricular infusion of letrozole, which prevents the synthesis of estradiol, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task and blocked or attenuated estradiol-induced increases in hippocampal synaptic proteins. In Experiment 2, we investigated if peripheral estradiol affects cognition and the hippocampus through hippocampal GnRH receptors. Hippocampal infusion of antide, a long-lasting GnRH receptor antagonist, blocked the ability of peripheral estradiol administration in ovariectomized rats to enhance hippocampus-dependent memory in a radial-maze task and blocked estradiol-induced increases in hippocampal synaptic proteins and aromatase, the final enzyme involved in estradiol synthesis. In Experiment 3A, we investigated whether hippocampal GnRH infusion was sufficient to enhance hippocampus-dependent memory and affect the hippocampus in ovariectomized rats and whether this was dependent on neuroestradiol synthesis. Hippocampal infusion of GnRH enhanced hippocampus-dependent memory in a radial maze task, the effects of which were blocked by letrozole infusion. Unexpectedly, GnRH treatment decreased levels or had no effect on a hippocampal postsynaptic proteins or aromatase. Due to the unexpected molecular results of Experiment 3A, Experiment 3B investigated if shorter treatment of GnRH affects hippocampal proteins differently. As opposed to the 10-12 days of GnRH treatment in Experiment 3A, five days of GnRH treatment increased levels of synaptic proteins via neuroestradiol synthesis as coadministration of letrozole blocked these effects. Results indicate that peripheral estradiol-induced enhancement of cognition and hippocampal morphology is mediated by brain-derived estradiol via hippocampal GnRH receptor activity. / acase@tulane.edu

(AJ) Tinnitus is not seen as a separate disease, but in terms of symptoms accompanying various diseases. The emergence of tinnitus is involved in a variety of risk factors. Relationship between tinnitus and blood flow of the ear is described in a relation to impaired microcirculation, which plays a key role in the proper function of the inner ear and therefore we evaluated the relationship prothrombogenic factors for tinnitus. From the original group of 853 patients we excluded patients with organic cause problems. We excluded patients with hearing impairment, cardiovascular and other comorbidities and also patients taking ototoxic drugs or patients with laboratory abnormalities. Then we have a homogenous group of 40 patients without the evidence of an organic cause of tinnitus and without associated diseases and the effect of ototoxic medications. On this basis there has been created a control group matched by the age and sex. In our group as the main marker of protrombogenic state was used a level of 11- dehydrotromboxan B2. Patients with tinnitus have significantly higher values of 11- dehydrotromboxane-B2. The average concentration in tinnitus patients was 2.02±1.81 ng/ml compared to 1.32±1.33 ng/ml in the control group. At the same time we evaluated other coagulation parameters. We checked...

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Les troubles dépressifs constituent un problème majeur de santé publique puisqu’ils affectent plus de 350 millions de personnes dans le monde. La plupart des médicaments antidépresseurs prescrits actuellement ciblent les systèmes monaminergiques centraux. Cependant, ces molécules ont un délai d’action de plusieurs semaines et produisent des effets secondaires substantiels, souvent responsable de l’arrêt du traitement. De plus, un pourcentage non négligeable de patients ne répond que partiellement ou négativement à ces traitements. C’est pourquoi le développement de nouvelles molécules antidépressives constitue un axe majeur de recherche en psychopharmacologie. Le 3β-methoxyprégnénolone (MAP4343), un neurostéroide de synthèse, constitue une nouvelle molécule dont l’efficacité antidépressive a été récemment établie dans un modèle d’isolement social chez le rat. Le mode d’action du MAP4343 serait différent de celui des antidépresseurs conventionnels, puisqu’il a été montré in vitro qu’il pouvait se lier à la protéine associée aux microtubules de type 2 (MAP2) pour ainsi modifier la fonction microtubulaire, ce qui, in fine, favoriserait la plasticité cérébrale.L’objectif de cette thèse est de consolider les preuves de l’efficacité antidépressive du MAP4343 et d’élucider les mécanismes d’action cellulaires associés à son effet pharmacologique. Pour cela, nous avons utilisé un modèle animal, de dépression à savoir le Toupaye de Belanger (toupaia belangeri) soumis au stress psychosocial chronique qui développe des troubles comportementaux, fonctionnels et hormonaux similaires à ceux observés chez les patients dépressifs.Chez le Toupaye, l’administration chronique de MAP4343 (50 mg/kg/jour ; per os) pendant quatre semaines permet d’inhiber la diminution de l’activité locomotrice et le comportement d’évitement induit par le stress. De plus, les altérations physiologiques comme la diminution du poids corporel, l’hyperthermie ou encore les troubles du sommeil sont enrayés par le traitement au MAP4343. Enfin, la molécule agit en inhibant partiellement l’hyperactivation de l’axe adreno-corticotrope. Les mesures dans l’hippocampe de l’expression des isoformes de l’α-tubuline, élément constitutif des microtubules, montrent une diminution de l’α-tubuline tyrosinée, une isoforme plus abondante dans des microtubules néoformés. Ce phénomène n’est pas renversé par le MAP4343. Cependant, le stress chronique induit une réduction de l’expression de la forme acétylée de l’α-tubuline, un phénomène qui serait plus tardif lors d’une altération de la fonction microtubulaire. De façon intéressante, le MAP4343 permet de prévenir cette baisse de l’acétylation. Ce travail de thèse démontre un effet antidépresseur robuste du MAP4343, confirmant ainsi une première étude réalisée auparavant chez le rat. Son efficacité peut se maintenir lors d’un traitement prolongé chez le Toupaye. Cette molécule constituerait ainsi une nouvelle classe d’antidépresseurs ayant comme cible potentielle le système microtubulaires. Des expériences complémentaires seront nécessaires afin de préciser le mode d’action du MAP4343 sur les fonctions neuronales. / Depressive disorders affect more than 350 million people worlwide generating major public health. Most of the antidepressant drugs currently used target monaminergic systems. However, the onset of action of these drugs is delayed for several weeks and they produce important side effects responsible for the discontinuation of treatment. Furthermore, a large rate of patients responds poorly or not at all to this kind of treatment. Accordingly, the development of new antidepressant drugs constitutes a major axis of psychopharmacology research. 3ß-methoxypregnenolone (MAP4343) is a new synthetic neurosteroid whose antidepressant efficacy was recently established in a rat model of social isolation. The mechanism of action of MAP4343 is very likely different from that of conventional antidepressants, as it was shown in vitro that this compound can bind the type 2 microtubule associated protein (MAP2) and may promote in fine the neuronal plasticity. The objective of this thesis is to further demonstrate the antidepressant efficacy of MAP4343 and to elucidate the cellular mechanisms associated to its pharmacological effect. For this purpose, we used one animal models of depression: tree shrews (toupaia belangeri) subjected to a chronic psychosocial stress. Stressed tree shrews develop behavioral, functional and hormonal alterations similar to those observed in the depressive patients. Chronic administration (four weeks) of MAP4343 (50 mg / kg / day) inhibits both the decrease of locomotor activity and the avoidance behavior induced by chronic stress. Furthermore, physiological changes such as hyperthermia or sleep disorders are also reversed by MAP4343 treatment. Finally, the molecule acts by partially inhibiting the stress-induced hyper-activation of hypothalamo-pituitary-adrenal axis. The quantitative study of post-translational modifications of α-tubulin (a major component of microtubules) in the hippocampus shows a decrease of tyrosinated α-tubulin, a dynamic microtubules marker. This phenomenon is not reversed by MAP4343 treatment. However, chronic stress exposure decreases the expression of acetylated α-tubulin, a later phenomenon occurring during microtubular alteration. Interestingly, MAP4343 prevents this decrease. To conclude, this thesis demonstrates a strong antidepressant effect of MAP4343, reproducible in one relevant and translational animal model of depression, thus confirming a previous study realized in an isolated rat model. The efficacy of this compound is observed from the first day of treatment in the rat and persists during a prolonged treatment. This molecule belongs to a new class of antidepressants potentially targeting the microtubular system. Additional experiments will be necessary to well understand the mechanism of action of the MAP4343 on neuronal functions.