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Neuroactive steroids and rat CNS
Umeå University, Faculty of Medicine, Clinical Sciences, Obstetrics and Gynaecology. Umeå University, Faculty of Medicine, Public Health and Clinical Medicine, Medicine.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
2004. , 91 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 898
Keyword [en]
Estradiol, progesterone, HRT, allopregnanolone, UC1011, serotonin receptor, GABAA receptor, mRNA, Morris water maze, silent second, tolerance
Research subject
Clinical Neurophysiology
Identifiers
URN: urn:nbn:se:umu:diva-296ISBN: 91-7305-668-5 (print)OAI: oai:DiVA.org:umu-296DiVA: diva2:142981
Public defence
2004-09-16, E04, 6E, 09:00 (English)
Opponent
Available from: 2004-08-11 Created: 2004-08-11 Last updated: 2009-12-18Bibliographically approved
List of papers
1. Serotonin 5-HT(1A) receptor mRNA expression in dorsal hippocampus and raphe nuclei after gonadal hormone manipulation in female rats.
Open this publication in new window or tab >>Serotonin 5-HT(1A) receptor mRNA expression in dorsal hippocampus and raphe nuclei after gonadal hormone manipulation in female rats.
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2001 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 74, no 2, 135-142 p.Article in journal (Refereed) Published
Abstract [en]

Female ovarian steroids influence mood and cognition, an effect presumably mediated by the serotonergic system. A key receptor in this interplay may be the 5-HT(1A) receptor subtype. We gave adult ovariectomized female rats subcutaneous pellets containing different dosages of 17 beta-estradiol alone or in combination with progesterone, or placebo pellets, for 2 weeks. 5-HT(1A) receptor mRNA levels were analyzed by in situ hybridization in the dorsal hippocampus, dorsal and median raphe nuclei, and entorhinal cortex. Estradiol treatment alone reduced 5-HT(1A) gene expression in the dentate gyrus and the CA2 region (17 and 19% decrease, respectively). Estradiol combined with progesterone supplementation increased 5-HT(1A) gene expression versus placebo in the CA1 and CA2 subregions of the dorsal hippocampus (16 and 30% increase, respectively). Concomitantly, 5-HT(1A) mRNA expression was decreased by 13% in the ventrolateral part of the dorsal raphe nuclei, while no changes were found in the median raphe nucleus and entorhinal cortex. Chronic effects of ovarian hormones on 5-HT(1A) receptor mRNA expression appear tissue-specific and involve hippocampal subregions and the raphe nuclei. Modulation of 5-HT(1A) receptor gene expression may be of importance for gonadal steroid effects on mood and cognition. Copyright 2001 S. Karger AG, Basel

Identifiers
urn:nbn:se:umu:diva-4028 (URN)10.1159/000054679 (DOI)11474221 (PubMedID)
Available from: 2004-08-11 Created: 2004-08-11 Last updated: 2017-12-14Bibliographically approved
2. Ovarian hormone effects on 5-hydroxytryptamine (2A) and 5-hydroxytryptamine (2C) receptor mRNA expression in the ventral hippocampus and frontal cortex of female rats.
Open this publication in new window or tab >>Ovarian hormone effects on 5-hydroxytryptamine (2A) and 5-hydroxytryptamine (2C) receptor mRNA expression in the ventral hippocampus and frontal cortex of female rats.
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2002 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 319, no 3, 157-161 p.Article in journal (Refereed) Published
Abstract [en]

Alterations in female gonadal hormones are associated with anxiety and mood changes. The aim of the present study was to determine influences of chronic gonadal hormone supplementation on 5-HT(2A) and 5-HT(2C) receptor mRNA levels in the ventral hippocampus and the frontal cerebral cortex. Ovariectomized adult female Sprague-Dawley rats (n=37) received implantation of subcutaneous pellets containing different dosages of 17beta-estradiol alone or in combination with progesterone, or placebo pellets, for 2 weeks. Serotonin receptor mRNA levels were analyzed by in situ hybridization in the ventral hippocampus and 5-HT(2A) receptor mRNA also in the frontal cortex. Estradiol treatment in combination with low-dose progesterone increased 5-HT(2A) receptor mRNA by 43% in the CA2 region of the ventral hippocampus, while estradiol combined with high-dose progesterone increased the expression of this gene by 84% in ventral CA1. 5-HT(2A) mRNA expression in the frontal cortex was not influenced by hormone manipulation. 5-HT(2C) receptor gene expression was in the ventral hippocampus decreased in the CA2, ventral CA1 and the subiculum subregions by high-dose estradiol treatment (8-20% decreases). Effects on mood by gonadal hormones can be mediated, at least partly, through influences on 5-HT(2A) and 5-HT(2C) receptor expression.

Identifiers
urn:nbn:se:umu:diva-4029 (URN)10.1016/S0304-3940(01)02570-8 (DOI)11834317 (PubMedID)
Available from: 2004-08-11 Created: 2004-08-11 Last updated: 2017-12-14Bibliographically approved
3. GABA(A) receptor changes in acute allopregnanolone tolerance
Open this publication in new window or tab >>GABA(A) receptor changes in acute allopregnanolone tolerance
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2006 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 535, no 1-3, 125-134 p.Article in journal (Refereed) Published
Abstract [en]

To study acute tolerance, rats were anesthetized with interrupted i.v. allopregnanolone infusions where the "silent second" in the electroencephalogram (EEG) was the target. Animals were killed either directly at the first silent second or at the silent second level after 30 or 90 min of anaesthesia. Acute tolerance was demonstrated at 90 min of anaesthesia as earlier shown. In situ hybridization showed a decreased expression of the gamma-aminobutyric acid(A) (GABA(A)) receptor subunit alpha4mRNA amount in the thalamus ventral-posteriomedial nucleus of the tolerant rats. A parallel change in the abundance of the alpha4 subunit was detected with immunohistochemistry. The increase in maintenance dose rate (MDR) was significantly negatively correlated with the alpha4mRNA in the thalamus ventral-posteriomedial nucleus, and positively correlated with alpha2mRNA in different hippocampal subregions. There was also a positive relationship between the alpha1mRNA amounts in the different hippocampal subregions, with significant differences between groups. These changes in GABA(A) receptor subunits mRNA expression and protein (alpha4) might be of importance for the development of acute tolerance to allopregnanolone.

Keyword
Anesthetics/administration & dosage/blood/pharmacokinetics, Animals, Brain/*drug effects/metabolism, Brain Chemistry/drug effects, Dentate Gyrus/chemistry/drug effects/metabolism, Dose-Response Relationship; Drug, Drug Tolerance, Gene Expression/drug effects, Immunohistochemistry, In Situ Hybridization, Infusions; Intravenous, Male, Pregnanolone/*administration & dosage/blood/pharmacokinetics, Protein Subunits/analysis/genetics, RNA; Messenger/genetics/metabolism, Rats, Rats; Sprague-Dawley, Receptors; GABA-A/analysis/*genetics, Receptors; GABA-B/analysis/genetics, Ventral Thalamic Nuclei/chemistry/drug effects/metabolism
Identifiers
urn:nbn:se:umu:diva-16927 (URN)10.1016/j.ejphar.2006.01.059 (DOI)16513107 (PubMedID)
Available from: 2007-12-02 Created: 2007-12-02 Last updated: 2017-12-14Bibliographically approved
4. Allopregnanolone inhibits learning in the Morris water maze
Open this publication in new window or tab >>Allopregnanolone inhibits learning in the Morris water maze
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2002 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 934, no 2, 125-131 p.Article in journal (Refereed) Published
Abstract [en]

The progesterone metabolite allopregnanolone (3alpha-OH-5alpha-pregnane-20-one) inhibits neural functions, enhancing the GABA induced GABA(A) receptor activation. This effect is benzodiazepine like and benzodiazepines are known to impair memory. Acute effects of allopregnanolone on the hippocampus dependent spatial learning in the Morris water maze have not been studied. Adult male Wistar rats where injected (i.v.) with allopregnanolone (2 mg/kg), or vehicle, daily for 11 days. At 8 or 20 min after each injection, studies of place navigation were performed in the Morris water maze. Allopregnanolone concentrations in plasma and in nine different brain areas where analyzed by radioimmunoassay. The latency to find the platform was increased 8 min after the allopregnanolone injection, while normal learning was seen after 20 min. Swim speed did not differ between groups. A higher number of rats were swimming close to the pool wall (thigmotaxis) in the 8 min allopregnanolone group compared to the other groups. Allopregnanolone concentrations in the brain tissue at 8 min were 1.5 to 2.5 times higher then at 20 min after the allopregnanolone injections. After vehicle injections the brain concentrations of allopregnanolone were at control levels. Plasma concentrations of allopregnanolone followed the same pattern as in the brain, with the exception of an increase 8 min after vehicle injections. The natural progesterone metabolite allopregnanolone can inhibit learning in the Morris water maze, an effect not caused by motor impairment. The learning impairment might be due to a combination of changed swimming behavior and difficulties in navigation.

Identifiers
urn:nbn:se:umu:diva-4031 (URN)10.1016/S0006-8993(02)02414-9 (DOI)11955475 (PubMedID)
Available from: 2004-08-11 Created: 2004-08-11 Last updated: 2017-12-14Bibliographically approved
5. 3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the learning impairment induced in rats by allopregnanolone.
Open this publication in new window or tab >>3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the learning impairment induced in rats by allopregnanolone.
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2004 (English)In: European Journal of Neuroscience, ISSN 0953-816X, E-ISSN 1460-9568, Vol. 20, no 6, 1604-1612 p.Article in journal (Refereed) Published
Abstract [en]

Allopregnanolone is a progesterone metabolite and GABA-A receptor modulator with benzodiazepine like effects, including decreased learning and memory. In vitro 3beta-hydroxypregnane steroids antagonize allopregnanolone-induced effects, but no antagonism has been shown in vivo. Our purpose was to evaluate 3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) as a blocker of allopregnanolone-induced effects in vivo and in vitro in rats. We tested adult male Wistar rats in the Morris water maze 8 min after daily injections (i.v.) of allopregnanolone 2 mg/kg (n = 21); allopregnanolone : UC1011 2 : 6 (n = 7), 2 : 8 (n = 7), 2 : 20 (n = 14) mg/kg; UC1011 20 mg/kg (n = 14); or vehicle (10% 2-hydroxypropyl-beta-cyclodextrin, n = 4). Studies of chloride ion uptake into cortical and hippocampal membrane preparations were performed. The latency to find the hidden platform was still high in the allopregnanolone-injected group on day 6. Day 3-6 rats injected with allopregnanolone and UC1011 (2 : 20 mg/kg) had lower latency (P < 0.05), compared to the allopregnanolone-injected group. The group that only received UC1011 learned the location of the platform as fast as the controls. There was no significant difference in swim speed between groups. The time spent swimming close to the pool wall was in the allopregnanolone : UC1011 group (2 : 20 mg/kg) significantly decreased (P < 0.05, day 3-6), compared to the allopregnanolone-injected group. The increased chloride ion uptake induced by increasing dosage of allopregnanolone in the presence of 10 micro m GABA was significantly decreased with UC1011 (P < 0.01), in both cortical and hippocampal homogenates. In conclusion, UC1011 can via antagonism at the GABA-A receptor reduce the negative allopregnanolone effect on learning in the water maze.

Keyword
Analysis of Variance, Animals, Behavior; Animal, Cerebral Cortex/drug effects/metabolism, Chlorides/metabolism, Chromatography; High Pressure Liquid/methods, Dose-Response Relationship; Drug, Drug Synergism, GABA Antagonists/*therapeutic use, GABA Modulators/blood/*toxicity, Hippocampus/drug effects/metabolism, Learning Disorders/chemically induced/*drug therapy, Male, Maze Learning/drug effects, Pregnanediol/pharmacology/*therapeutic use, Pregnanolone/blood/pharmacology/therapeutic use/*toxicity, Radioimmunoassay/methods, Rats, Rats; Wistar, Reaction Time/drug effects, Statistics; Nonparametric, Time Factors, gamma-Aminobutyric Acid/physiology
Identifiers
urn:nbn:se:umu:diva-16941 (URN)10.1111/j.1460-9568.2004.03610.x (DOI)15355327 (PubMedID)
Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2017-12-14Bibliographically approved

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