Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5␣-reductase (5␣-R) type I and 3␣-hydroxysteroid dehydrogenase (3␣-HSD). This study evaluates 5␣-R type I and 3␣-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67͞65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100␤ immunohistochemistry. We demonstrate that 5␣-R type I and 3␣-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5␣-R type I nor 3␣-HSD mRNAs are expressed in S100␤-or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67͞65 antibodies to mark GABAergic neurons, we failed to detect 5␣-R type I and 3␣-HSD in cortical and hippocampal GABAergic interneurons. However, 5␣-R type I and 3␣-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABAA receptors, either with an autocrine or a paracrine mechanism or by reaching GABAA receptor intracellular sites through lateral membrane diffusion.3␣-hydroxysteroid dehydrogenase ͉ 5␣-reductase (type I) ͉ GABAergic neurons ͉ glutamatergic neurons T he neurosteroids 3␣-hydroxy-5␣-pregnan-20-one [allopregnanolone (ALLO)] and 3␣,21-dihydroxy-5␣-pregnan-20-one [tetrahydrodeoxycorticosterone (THDOC)] are potent positive allosteric modulators of GABA action at GABA A receptors (1-6). These neurosteroids can be synthesized in the brain from progesterone (7) or deoxycorticosterone (8, 9), respectively, by the sequential action of two enzymes, 5␣-reductase (5␣-R) type I and 3␣-hydroxysteroid dehydrogenase (3␣-HSD) (10).Two types (I and II) of 5␣-Rs, which convert progesterone into 5␣-dihydroprogesterone (5␣-DHP) or convert deoxycorticosterone into 5␣-dihydrodeoxycorticosterone (5␣-DHDOC), have been identified in tissues of rodents and humans (11). Whereas 5␣-R type I and II are abundantly expressed in several peripheral tissues, 5␣-R type I is the most abundant 5␣-R molecular form detected in the adult brains of rats, mice, and humans (11-17). The human brain expresses four types of 3␣-HSD, which, under different optimal conditions, either catalyze the reduction of 5␣-DHP into ALLO or reverse this reaction (18). So far, only one 3␣-HSD isoform has been identified in the rat or mouse brain (19)(20)(21)(22). The mRNA sequences of 5␣-R type I (Ϸ88%) and 3␣-HSD (Ϸ89%) are highly homologous in mouse (5␣-R type I GeneBank access...

Cortical DNA-methyltransferase 1 (DNMT1) is preferentially expressed in interneurons secreting GABA where it very likely contributes to promoter CpG island hypermethylation, thus causing a down-regulation of promoter functions. To consolidate and expand on previous findings that, in the cortex of schizophrenia (SZ) brains, glutamic acid decarboxylase 67 (GAD67) expression is downregulated whereas that of DNMT1 is up-regulated, we studied both parameters in Brodmann's area (BA) 9 from the McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center, Belmont, MA). In BA9 of SZ and bipolar disorder patients with psychosis, DNMT1 mRNA and protein expression preferentially increases in layer I, II, and IV interneurons, and this increase is paralleled by a decreased number of GAD67 mRNA-positive neurons. The increase in DNMT1 and the decrease in GAD67-expressing neurons were unrelated to postmortem interval, pH, RNA quality, or to the presence, dose, or duration of antipsychotic (APS) medication, with the exception of a subgroup of SZ patients treated with a combination of valproate and APS in which the expression of DNMT1 failed to change. The DNMT1 increase and the GAD67 decrease in BA9 interneurons are significant features of SZ and bipolar disorder with psychosis. Interestingly, the DNMT1 increase failed to occur when patients with psychosis received a combination of valproate and APS treatment but not APS monotherapy. bipolar disorder ͉ glutamic acid decarboxylase ͉ schizophrenia ͉ valproate ͉ antipsychotics A lthough monozygotic twins carry identical genomic sequences, they can exhibit a number of differences. In fact, in the absence of nucleotide sequence mutations, DNA can be modified by methylation of the cytosine ring. Such structural DNA modifications can be termed ''epigenetic,'' to indicate a modification that occurred after the operation of conventional genetic mechanisms (1).In neurons, epigenetic hypermethylation in the 5Ј position of the cytosine ring expressed in promoter CpG islands of various genes including that of glutamic acid decarboxylase 67 (GAD 67 ) and reelin is catalyzed by DNA-methyltransferases (2, 3). One of them, the DNA-methyltransferase 1 (DNMT1), is preferentially expressed in cortical GABA-secreting neurons of Brodmann's area (BA) 10, and BA17, and in the medium spiny neurons of the caudate nucleus (4).GABAergic interneurons of schizophrenia (SZ) patient (SZP) brains (BA10) exhibit a decrease of reelin and GAD 67 expression (4-6) and an increased expression of DNMT 1 (4).In a recent study (7), it was found that the selective expression of the human reelin gene in GABAergic neurons is regulated by an epigenetic cytosine hypermethylation of promoter CpG islands. In fact, when DNMT1 function is inhibited by the addition of aza-2Ј-deoxycytidine or down-regulated by the induction of demethylation elicited by histone deacetylase inhibitors, the in vitro expression of reelin increases severalfold (7). Moreover, in the hippocampus and frontal cortex of mice receiving methionine (6.6 m...

The association of the histone deacetylase (HDAC) inhibitor valproate (VPA) with atypical antipsychotics has become a frequent treatment strategy for schizophrenia and bipolar disorder. Because the VPA doses administered are elevated, one cannot assume that the benefits of the VPA plus antipsychotic treatment are exclusively related to the covalent modifications of nucleosomal histone tails. We compared the actions of N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide derivative (MS-275), which is a potent HDAC inhibitor in vitro, with the actions of VPA for their ability to (i) increase the acetylated status of brain nucleosomal histone tail domains and (ii) to regulate brain histone-RELN and histone-GAD67 promoter interactions. MS-275 increases the content of acetylhistone 3 (Ac-H3) in the frontal cortex. Whereas this response peaks after a s.c. injection of 15 mol͞kg, the increase in Ac-H3 content in the hippocampus becomes significant only after an injection of 60 mol͞kg, suggesting that MS-275 is 30-to 100-fold more potent than VPA in increasing Ac-H3 in these brain regions. In contrast to VPA, MS-275, in doses up to 120 mol͞kg, fails to increase Ac-H3 content in the striatum. Chromatin immunoprecipitation shows that MS-275 increases Ac-H3-RELN and Ac-H3-GAD67 promoter interaction in the frontal cortex. These results suggest that MS-275 is a potent brain region-selective HDAC inhibitor. It is likely that, in addition to MS-275, other benzamide derivatives, such as sulpiride, are brain-region selective inhibitors of HDACs. Hence, some benzamide derivatives may express a greater efficacy than VPA as an adjunctive to antipsychotics in the treatment of epigentically induced psychiatric disorders. bipolar disorder ͉ reelin ͉ schizophrenia ͉ histone code ͉ chromatin remodeling A prefrontal cortex GABAergic neuron dysfunction, which is characterized by a reduction of the 67-kDa form of glutamic acid decarboxylase (GAD 67 ) and reelin expression, is one of the most consistent neuropathological findings in postmortem brain studies of schizophrenia (SZ) and bipolar (BP) disorder (1-9). These expression deficits cannot be explained by reelin or GAD 67 gene haploinsufficiency (10, 11). Converging epidemiological (12), histological, and biochemical (10, 13-17) evidence suggests that the pathogenesis of this dysfunction may be related to a disruption of epigenetic signaling, resulting in the selective hypermethylation of several GABAergic gene promoters that characterize SZ as a selective defect of gene transcription in GABAergic cortical neurons (13,14). Such hypermethylation is very likely mediated by the overexpression of DNA methyltransferase 1 (DNMT1) (15, 18), which has been found to be operative in cortical and subcortical GABAergic interneurons of SZ and BP patients.The long-term objective of this line of research is to identify drugs that selectively correct a basic defect of SZ, which is an epigenetic GABAergic neuron dysfunction, by directly or indirectly reducing the RELN and GAD 67 pr...

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD 67 expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 mol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD 67 expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD67 promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 mol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD67 expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.antagonists ͉ epigenetic mechanisms ͉ nicotinic acetylcholine receptor agonists ͉ schizophrenia T obacco smoking is frequently abused by schizophrenia patients (SZP) (for reviews see refs. 1 and 2). Because nicotine is a potent cholinergic receptor agonist that is inhaled with tobacco smoking and both the expression and function of nicotinic acetylcholine receptors (nAChRs) are down-regulated in the brain of SZP, one may conclude that the high level of tobacco smoking in these patients represents an attempt to self-medicate; i.e., correction of some disease-associated abnormalities of cholinergic (nicotinic) neurotransmission (3, 4), possibly related to the decrease of GABAergic function occurring in the brain of SZP (5-9).Typically, plasma nicotine levels in heavy smokers (Ϸ20-30 cigarettes a day) oscillate between 0.3 and 0.6 M. Because in humans nicotine half-life is Ϸ2 h, the nicotine plasma levels in heavy smokers progressively increase during the day but fluctuate in a ''peak and trough'' fashion after each cigarette (10, 11). These submicromolar concentrations of nicotine, which act at heteroolig...

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