Toward the identification of peripheral epigenetic biomarkers of schizophrenia.

Schizophrenia (SZ) is a heritable, nonmendelian, neurodevelopmental disorder in which epigenetic dysregulation of the brain genome plays a fundamental role in mediating the clinical manifestations and course of the disease. The authors recently reported that two enzymes that belong to the dynamic DNA methylation/demethylation network-DNMT (DNA methyltransferase) and TET (ten-eleven translocase; 5-hydroxycytosine translocator)-are abnormally increased in corticolimbic structures of SZ postmortem brain, suggesting a causal relationship between clinical manifestations of SZ and changes in DNA methylation and in the expression of SZ candidate genes (e.g., brain-derived neurotrophic factor [BDNF], glucocorticoid receptor [GCR], glutamic acid decarboxylase 67 [GAD67], reelin). Because the clinical manifestations of SZ typically begin with a prodrome followed by a first episode in adolescence with subsequent deterioration, it is obvious that the natural history of this disease cannot be studied only in postmortem brain. Hence, the focus is currently shifting towards the feasibility of studying epigenetic molecular signatures of SZ in blood cells. Initial studies show a significant enrichment of epigenetic changes in lymphocytes in gene networks directly relevant to psychiatric disorders. Furthermore, the expression of DNA-methylating/demethylating enzymes and SZ candidate genes such as BDNF and GCR are altered in the same direction in both brain and blood lymphocytes. The coincidence of these changes in lymphocytes and brain supports the hypothesis that common environmental or genetic risk factors are operative in altering the epigenetic components involved in orchestrating transcription of specific genes in brain and peripheral tissues. The identification of DNA methylation signatures for SZ in peripheral blood cells of subjects with genetic and clinical high risk would clearly have potential for the diagnosis of SZ early in its course and would be invaluable for initiating early intervention and individualized treatment plans.

DNA-methylation gene network dysregulation in peripheral blood lymphocytes of schizophrenia patients.

The epigenetic dysregulation of the brain genome associated with the clinical manifestations of schizophrenia (SZ) includes altered DNA promoter methylation of several candidate genes. We and others have reported that two enzymes that belong to the DNA-methylation/demethylation network pathways-DNMT1 (DNA-methyltransferase) and ten-eleven translocator-1(TET1) methylcytosine deoxygenase are abnormally increased in corticolimbic structures of SZ postmortem brain. The objective of this study was to investigate whether the expression of these components of the DNA-methylation-demethylation pathways known to be altered in the brain of SZ patients are also altered in peripheral blood lymphocytes (PBL). The data show that increases in DNMT1 and TET1 and in glucocorticoid receptor (GCortR) and brain derived neurotrophic factor (BDNF) mRNAs in PBL of SZ patients are comparable to those reported in the brain of SZ patients. The finding that the expressions of DNMT1 and TET1 are increased and SZ candidate genes such as BDNF and GCortR are altered in the same direction in both the brain and PBL together with recent studies showing highly correlated patterns of DNA methylation across the brain and blood, support the hypothesis that a common epigenetic dysregulation may be operative in the brain and peripheral tissues of SZ patients.

Epigenetic GABAergic targets in schizophrenia and bipolar disorder.

It is becoming increasingly clear that a dysfunction of the GABAergic/glutamatergic network in telencephalic brain structures may be the pathogenetic mechanism underlying psychotic symptoms in schizophrenia (SZ) and bipolar (BP) disorder patients. Data obtained in Costa's laboratory (1996-2009) suggest that this dysfunction may be mediated primarily by a downregulation in the expression of GABAergic genes (e.g., glutamic acid decarboxylase67[GAD67] and reelin) associated with DNA methyltransferase (DNMT)-dependent hypermethylation of their promoters. A pharmacological strategy to reduce the hypermethylation of GABAergic promoters is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that induce DNA-demethylation when administered at doses that facilitate chromatin remodeling. The benefits elicited by combining VPA with antipsychotics in the treatment of BP disorder suggest that an investigation of the epigenetic interaction of these drugs is warranted. Our studies in mice suggest that when associated with VPA, clinically relevant doses of clozapine elicit a synergistic potentiation of VPA-induced GABAergic promoter demethylation. Olanzapine and quetiapine (two clozapine congeners) also facilitate chromatin remodeling but at doses higher than used clinically, whereas haloperidol and risperidone are inactive. Hence, the synergistic potentiation of VPA's action on chromatin remodeling by clozapine appears to be a unique property of the dibenzepines and is independent of their action on catecholamine or serotonin receptors. By activating DNA-demethylation, the association of clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be considered a promising treatment strategy for normalizing GABAergic promoter hypermethylation and the GABAergic gene expression downregulation detected in the postmortem brain of SZ and BP disorder patients. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

GABAA receptors and benzodiazepines: a role for dendritic resident subunit mRNAs.

This review is designed to describe the evolution of the seminal observation made simultaneously in 1975 by Dr. W. Haefely's laboratory (Hoffman La Roche, Basel, Switzerland) and in the Laboratory of Preclinical Pharmacology (NIH, St. Elizabeths Hospital, Washington DC), that benzodiazepine action was mediated by a modulation of GABA action at GABA(A) receptors. In fact, our suggestion was that the benzodiazepine receptor was "a receptor on a receptor" and that this receptor was GABA(A). Needless to say, this suggestion created opposition, but we did not abandon the original idea, in fact, as shown in this review, there is now universal agreement with our hypothesis on the mode of action of benzodiazepines. Hence, this review deals with the allosteric modulation of GABA(A) receptors by benzodiazepines, the role of GABA(A) receptors and benzodiazepine structure diversities in this modulation, and describes the results of our attempts to establish a benzodiazepine (imidazenil) devoid of tolerance, withdrawal symptoms, and changes in the expression of GABA(A) receptor subunits during tolerance. It also deals with the idea that the synthesis of GABA(A) receptor subunits triggered by tolerance resides in dendrites and spines where mRNAs and the apparatus for this translation is located. New analytic procedures may foster progress in the understanding of tolerance to and withdrawal from benzodiazepines.

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse.

Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.

Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder: a postmortem brain study.

BACKGROUND: Reelin (RELN) is a glycoprotein secreted preferentially by cortical gamma-aminobutyric acid-ergic (GABAergic) interneurons (layers I and II) that binds to integrin receptors located on dendritic spines of pyramidal neurons or on GABAergic interneurons of layers III through V expressing the disabled-1 gene product (DAB1), a cytosolic adaptor protein that mediates RELN action. To replicate earlier findings that RELN and glutamic acid decarboxylase (GAD)(67), but not DAB1 expression, are down-regulated in schizophrenic brains, and to verify whether other psychiatric disorders express similar deficits, we analyzed, blind, an entirely new cohort of 60 postmortem brains, including equal numbers of patients matched for schizophrenia, unipolar depression, and bipolar disorder with nonpsychiatric subjects. METHODS: Reelin, GAD(65), GAD(67), DAB1, and neuron-specific-enolase messenger RNAs (mRNAs) and respective proteins were measured with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) or Western blot analyses. Reelin-positive neurons were identified by immunohistochemistry using a monoclonal antibody. RESULTS: Prefrontal cortex and cerebellar expression of RELN mRNA, GAD(67) protein and mRNA, and prefrontal cortex RELN-positive cells was significantly decreased by 30% to 50% in patients with schizophrenia or bipolar disorder with psychosis, but not in those with unipolar depression without psychosis when compared with nonpsychiatric subjects. Group differences were absent for DAB1,GAD(65) and neuron-specific-enolase expression implying that RELN and GAD(67) down-regulations were unrelated to neuronal damage. Reelin and GAD(67) were also unrelated to postmortem intervals, dose, duration, or presence of antipsychotic medication. CONCLUSIONS: The selective down-regulation of RELN and GAD(67) in prefrontal cortex of patients with schizophrenia and bipolar disorder who have psychosis is consistent with the hypothesis that these parameters are vulnerability factors in psychosis; this plus the loss of the correlation between these 2 parameters that exists in nonpsychotic subjects support the hypothesis that these changes may be liability factors underlying psychosis.

Expression and function of striatal nAChRs differ in the flinders sensitive (FSL) and resistant (FRL) rat lines.

Rats of Flinders Sensitive (FSL) and Flinders Resistant lines (FRL) differ in their susceptibility to physiological and associated behavioral responses elicited by nicotine. In the present study, we measured dopamine (DA) content in striatal dialysates to investigate the sensitivity of FSL and FRL rats to nicotine delivered locally through a microdialysis probe placed in the striatum. We also measured the expression density of striatal high-affinity nicotinic acetylcholine receptors (nAChRs), and that of mRNAs encoding for alpha3, alpha4, alpha7 and beta2 nAChR subunits in both lines. The DA content of dialysates was measured before and after a 1-min perfusion of nicotine (6, 10 or 20 nmoles/min) and the resulting DA increase was taken as a measure of the alkaloid's intrinsic activity for nAChRs involved in the release of DA. The nicotine-induced increase of striatal DA release was greater in FSL than in FRL rats for all concentrations of nicotine, suggesting that the intrinsic activity of nicotine was greater in the FSL than in the FRL rats. This was further supported by our finding that the density of high-affinity nAChRs in the striatum of FSL rats was 44% greater than in the FRL rats, whereas affinity (K(D)) was virtually the same in the two lines of rats. Also the expression of mRNAs encoding for alpha(4), alpha(7), and beta(2) subunits in the striatum was greater in FSL than in FRL rats (attomol/microg total RNA, alpha(4):98+/-10 vs. 77+/-7; alpha(7):279+/-16 vs. 184+/-16; beta(2):310+/-19 vs. 201+/-12). We hypothesize that the difference in nicotine-induced DA release in the striatum of FSL and FRL rats depends on the difference in nAChR subunit expression in the striatum between the two lines. The Flinders rats could be used as a model for nicotine self-administration studies to evaluate the susceptibilities of FSL and FRL rats to nicotine dependence.

Imidazenil prevention of alprazolam-induced acquisition deficit in patas monkeys is devoid of tolerance.

The partial allosteric modulators (PAMs) of gamma-aminobutyric acid-gated Cl(-) current intensities at gamma-aminobutyric acid type A receptors have high affinity but low intrinsic efficacy on benzodiazepine recognition sites. Unlike the full allosteric modulators (FAM), like alprazolam, triazolam, and diazepam, PAMs are virtually devoid of unwanted side effects, including tolerance. Imidazenil (IMD) is a PAM that elicits potent anxiolytic and anticonvulsant actions in rodents and nonhuman primates and retains its anticonvulsant and anxiolytic effects, even in rodents that are tolerant to FAMs. IMD antagonizes the side effects of FAMs in rodents and nonhuman primates. Using patas monkeys and a multiple schedule with repeated acquisition and performance of chain responses, we report that IMD administration for 17 days antagonized without showing tolerance ALP-induced disruption of acquisition.

Reelin secretion from glutamatergic neurons in culture is independent from neurotransmitter regulation.

Reelin (Reln) is a glycoprotein that in postnatal and adult mammalian brain is believed to be secreted from telencephalic GABAergic interneurons and cerebellar glutamatergic granule neurons into the extracellular matrix. To address the question of whether Reln neurosecretion occurs via a regulated or a constitutive process, we exposed postnatal rat cerebellar granule neurons (CGNs) maintained in culture for 7-9 days to: (i) 100 microM N-methyl-D-aspartate (NMDA) in a Mg(+2)-free medium to stimulate NMDA-selective glutamate receptors and Ca(2+)-dependent neurotransmitter release, (ii) 50 mM KCl to depolarize the cells and elicit Ca(2+)-dependent exocytosis, (iii) 10-100 microM nicotine to activate excocytosis by nicotinic receptors present in these cells, (iv) 10 microM 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide in combination with 10 microM dizocilpine to block alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and NMDA-preferring glutamate receptors activated by endogenously released glutamate, or (v) EGTA (5 mM) to virtually eliminate extracellular Ca(2+) and block Ca(2+)-dependent exocytosis. Although, CGNs express and secrete Reln (measured by quantitative immunoblotting), none of the above-mentioned conditions that control regulated exocytosis alters the stores or the rate of Reln release. In contrast, application of either: (i) a Reln antisense oligonucleotide (5'-GCAATGTGCAGGGAAATG-3') (10 microM) that reduces Reln biosynthesis or (ii) brefeldin A (5 x 10(-5) M), an inhibitor of the traffic of proteins between the endoplasmic reticulum and the Golgi network, sharply curtail the rate of Reln secretion. Because, in subcellular fractionation studies, we have shown that Reln is not contained in synaptic vesicles, these data suggest that Reln secretion from CGNs does not require Ca(2+)-dependent exocytosis, but probably is related to a Reln pool stored in Golgi secretory vesicles mediating a constitutive secretory pathway.

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.

Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues.

The regulation of hippocampal nicotinic acetylcholine receptors (nAChRs) after a protracted treatment with selective or nonselective nAChR agonists.

In rats, 1 mg/kg twice daily for 10 d of nicotine, a nonselective agonist of nicotinic acetylcholine receptors (nAChRs), fails to change alpha4 and beta2 nAChR subunit mRNA but significantly decreased alpha7 nAChR subunit mRNA and protein expression, which is associated with a 35-40% decrease in the number of 125I-alpha-Bgtx binding sites in hippocampus. In addition, this schedule of nicotine treatment produced a 40% increase in the number of high- (K(D) 1 nM), but decreased by 25% the number of low-affinity (K(D) 30 nM) binding sites for 3H-epibatidine in hippocampus. In contrast, repeated treatment with lobeline (2.7 mg/kg twice daily for 10 d), which selectively binds to high-affinity binding nAChRs, fails to change the expression of high- or low-affinity nAChRs. These data suggest that a simultaneous upregulation of high-affinity nAChRs and downregulation of low-affinity nAChRs is elicited by ligands that can bind to both low- and high-affinity nAChRs, but not by selective agonists of high-affinity nAChRs. One might infer that in hippocampus, high- and low-affinity nAChRs may be located in the same cells. When these two receptor types are stimulated simultaneously by nonselective ligands for high- and low-affinity nAChRs, they interact, bringing about an increase in binding site density of the high-affinity nAChRs.

Effects of negative allosteric modulators of gamma-aminobutyric acidA receptors on complex behavioral processes in monkeys.

A multiple schedule of repeated acquisition and performance of conditional discriminations was used to characterize the effects of two negative allosteric modulators of the gamma-aminobutyric acid (GABAA) receptor (ethyl beta-carboline-3-carboxylate [beta-CCE] and N-methyl-beta-carboline-3-carboxamide [FG-7142]), a hallucinogenic beta-carboline derivative (harmine), a benzodiazepine receptor antagonist (flumazenil) and a positive allosteric modulator (alprazolam). In the acquisition component, subjects acquired a different discrimination each session. Acquisition of a discrimination was defined by a decrease in errors as the session progressed. In the performance component, the discrimination was the same each session. Responding in both components was maintained by food presentation under a variable-ratio schedule. Incorrect responses in both components produced a 5-sec timeout. Alprazolam (0.1-18 mg/kg), beta-CCE (0.01-0.32 mg/kg), FG-7142 (0.1-18 mg/kg) and harmine (0.1-1.8 mg/kg) all dose-dependently decreased response rate in both components. However, accuracy of responding-was differentially affected by the drugs. Alprazolam selectively and dose-dependently increased percent errors in acquisition, whereas beta-CCE increased acquisition errors only at the highest doses tested in each subject. In contrast, FG-7142 and harmine had no effects on percent errors at doses that virtually eliminated responding. In all cases, performance accuracy was generally not affected. Flumazenil, at doses that had little or no effect (0.1 and 0.32 mg/kg) or occasionally decreased response rates (1 mg/kg) when administered alone, dose-dependently antagonized the rate-decreasing and error-increasing effects of beta-CCE, FG-7142 and alprazolam. In contrast, flumazenil failed to antagonize the effects of harmine. Thus, the negative allosteric modulators only moderately disrupted acquisition in comparison with the positive allosteric modulator, but the effects of both types of modulator were antagonized by the benzodiazepine antagonist flumazenil.

Imidazenil, a new anxiolytic and anticonvulsant drug, attenuates a benzodiazepine-induced cognition deficit in monkeys.

Imidazenil is a highly potent partial allosteric modulator of gamma-aminobutyric acid action at a great variety of gamma-aminobutyric acid(A) receptors, whereas alprazolam is a full allosteric modulator at these receptors. Data in rats indicate that imidazenil, like alprazolam, has pharmacological effects in animals indicative of anxiolytic, antipanic and anticonvulsant activity in humans, but unlike alprazolam, in animals imidazenil does not produce ataxia, sedation, tolerance or dependence nor does it potentiate the effects of ethanol. To extend the study of the imidazenil pharmacological profile, we administered this drug and alprazolam, alone and in combination, in monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance), the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.01-0.32 mg/kg, p.o.) was administered alone, the overall response rate in both learning and performance decreased and the percent errors in both components increased with increasing doses. Learning, however, was more sensitive than performance; i.e., error-increasing effects were seen in learning at doses that had no effect on performance accuracy.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of full and partial allosteric modulators of GABA(A) receptors on complex behavioral processes in monkeys.

Two baselines involving a repeated acquisition task were used to assess the effects of bretazenil, imidazenil, and triazolam. The first baseline was a multiple schedule of repeated acquisition and performance of conditional discriminations. In the first component, the subject acquired a four-response chain by responding sequentially on three keys in the presence of different combinations of colors and geometric forms displayed on a center key. Acquisition of the discrimination was defined by a decrease in errors as the session progressed. In the performance component, the four-response chain was the same each session. Incorrect responses in either component produced a 5s time out during which responding had no programmed consequence. The second procedure, which has been used to evaluate the effects of drugs on memory, involved the acquisition of a discrimination, followed by a 1h delay and a retest of the same discrimination to assess retention. Triazolam (0.32 and 0.56mg/kg) administered alone, produced dose-related decreases in response rate in each component. In addition, triazolam also produced a dose-related increase in percentage errors in acquisition with no effect in performance. Triazolam (0.32mg/kg) eliminated retention (0 percent savings) in the memory task. Bretazenil (0.1-5.6mg/kg) or imidazenil (0.1-1.8mg/kg) administered alone had little or no effect on either rate of responding or accuracy in either component. Furthermore, bretazenil but not imidazenil disrupted retention at the higher doses tested. The combination of imidazenil or bretazenil with triazolam produced dose-related attenuation of the disruptive effects of triazolam on both behavioral baselines. These data suggest that the disruptive effects of benzodiazepines on learning and memory may be a function of the intrinsic efficacy of these compounds at different GABA(A) receptor subtypes.

Imidazenil, a partial positive allosteric modulator of GABAA receptors, exhibits low tolerance and dependence liabilities in the rat.

Long-term treatment of rats with full (triazolam) or selective (diazepam) allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors rapidly induced tolerance to the protective effect of these drugs against bicuculline-induced convulsion. In contrast, long-term administration of partial allosteric modulators (imidazenil and bretazenil) of GABAA receptors, in doses equipotent to those of diazepam and triazolam that induce anticonvulsant tolerance, failed to elicit such a tolerance. Furthermore, no cross-tolerance was observed between diazepam and imidazenil. Discontinuation of long-term treatment with diazepam or triazolam, but not of long-term treatment with imidazenil or bretazenil, sensitized rats to behavioral inhibition by punishment (electric shock) in a manner that was potentiated by flumazenil. Administration of a single oral dose of [14C]diazepam or [3H] imidazenil to rats treated repeatedly with the corresponding unlabeled drug or vehicle revealed that the brain concentrations of drugs and their metabolites were similar in both groups of animals. This suggests that tolerance to the full or selective allosteric modulators of GABAA receptors may be associated with changes in the efficacy of the allosteric modulation rather than with changes in drug metabolism. Imidazenil has a longer half-life than an equipotent dose of diazepam and protects rats against bicuculline-induced convulsions for a significantly longer time than diazepam or bretazenil.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacology of neurosteroidogenesis.

In adrenal cortex and other steroidogenic tissues including glial cells, the conversion of cholesterol into pregnenolone is catalyzed by the cytochrome P450scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450scc limits the rate of pregnenolone biosynthesis. Participating in this mechanism are DBI (diazepam binding inhibitor), an endogenous peptide that is highly expressed in steroidogenic cells and some of the DBI processing products including DBI 17-50 (TTN). DBI and TTN activate steroidogenesis by binding to a specific receptor located in the outer mitochondrial membrane, termed mitochondrial DBI receptor complex (MDRC). MDRC is a hetero-oligomeric protein: only the subunit that includes the DBI and benzodiazepine (BZD) recognition sites has been cloned. Several 2-aryl-3-indoleacetamide derivatives (FGIN-1-X) with highly selective affinity (nM) for MDRC were synthesized which can stimulate steroidogenesis in mitochondrial preparations. These compounds stimulate adrenal cortex steroidogenesis in hypophysectomized rats but not in intact animals. Moreover, this steroidogenesis is inhibited by the isoquinoline carboxamide derivative PK 11195, a specific high affinity ligand for MDRC with a low intrinsic steroidogenic activity. Some of the FGIN-1-X derivatives stimulate brain pregnenolone accumulation in adrenalectomized-castrated rats. The FGIN-1-X derivatives that increase brain pregnenolone content, elicit antineophobic activity and antagonize punished behavior in the Vogel conflict test in rats. These actions of FGIN-1-X are resistant to inhibition by flumazenil, a specific inhibitor of BZD action in GABAA receptors but are antagonized by PK 11195, a specific blocker of the steroidogenesis activation via MDRC stimulation. It is postulated that the pharmacological action of FGIN-1-X depends on a positive modulation of the GABA action on GABAA receptors mediated by the stimulation of brain neurosteroid production.

Participation of mitochondrial diazepam binding inhibitor receptors in the anticonflict, antineophobic and anticonvulsant action of 2-aryl-3-indoleacetamide and imidazopyridine derivatives.

The 2-hexyl-indoleacetamide derivative, FGIN-1-27 [N,N-di-n-hexyl-2- (4-fluorophenyl)indole-3-acetamide], and the imidazopyridine derivative, alpidem, both bind with high affinity to glial mitochondrial diazepam binding inhibitor receptors (MDR) and increase mitochondrial steroidogenesis. Although FGIN-1-27 is selective for the MDR, alpidem also binds to the allosteric modulatory site of the gamma-aminobutyric acidA receptor where the benzodiazepines bind. FGIN-1-27 and alpidem, like the neurosteroid 3 alpha,21-dehydroxy-5 alpha-pregnane-20-one (THDOC), clonazepam and zolpidem (the direct allosteric modulators of gamma-aminobutyric acidA receptors) delay the onset of isoniazid and metrazol-induced convulsions. The anti-isoniazid convulsant action of FGIN-1-27 and alpidem, but not that of THDOC, is blocked by PK 11195. In contrast, flumazenil blocked completely the anticonvulsant action of clonazepam and zolpidem and partially blocked that of alpidem, but it did not affect the anticonvulsant action of THDOC and FGIN-1-27. Alpidem, like clonazepam, zolpidem and diazepam, but not THDOC or FGIN-1-27, delay the onset of bicuculline-induced convulsions. In two animal models of anxiety, the neophobic behavior in the elevated plus maze test and the conflict-punishment behavior in the Vogel conflict test, THDOC and FGIN-1-27 elicited anxiolytic-like effects in a manner that is flumazenil insensitive, whereas alpidem elicited a similar anxiolytic effect, but is partially blocked by flumazenil. Whereas PK 11195 blocked the effect of FGIN-1-27 and partially blocked alpidem, it did not affect THDOC in both animal models of anxiety.(ABSTRACT TRUNCATED AT 250 WORDS)