Erminio Costa

Neurosteroids act on recombinant human GABAA receptors

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Coupling of Inositol Phospholipid Metabolism with Excitatory Amino Acid Recognition Sites in Rat Hippocampus

Abstract: Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, l‐glutamate, and l‐aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N‐methyl‐d‐aspartate, kainate, quinolinate, and N‐acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above‐mentioned excitatory amino acids is potently and selectively antagonized by dl‐2‐amino‐4‐phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.

An epigenetic mouse model for molecular and behavioral neuropathologies related to schizophrenia vulnerability

Reelin and glutamic acid decarboxylase (GAD)67 expressed by cortical γ-aminobutyric acid-ergic interneurons are down-regulated in schizophrenia. Because epidemiological studies of schizophrenia fail to support candidate gene haploinsufficiency of Mendelian origin, we hypothesize that epigenetic mechanisms (i.e., cytosine hypermethylation of CpG islands present in the promoter of these genes) may be responsible for this down-regulation. Protracted l-methionine (6.6 mmol/kg for 15 days, twice a day) treatment in mice elicited in brain an increase of S-adenosyl-homocysteine, the processing product of the methyl donor S-adenosyl-methionine, and a marked decrease of reelin and GAD67 mRNAs in both WT and heterozygous reeler mice. This effect of l-methionine was associated with an increase in the number of methylated cytosines in the CpG island of the reelin promoter region. This effect was not observed for GAD65 or neuronal-specific enolase and was not replicated by glycine doses 2-fold greater than those of l-methionine. Prepulse inhibition of startle declined at a faster rate as the prepulse/startle interval increased in mice receiving l-methionine. Valproic acid (2 mmol/kg for 15 days, twice a day) reverted l-methionine-induced down-regulation of reelin and GAD67 in both WT and heterozygous reeler mice, suggesting an epigenetic action through the inhibition of histone deacetylases. The same dose of valproate increased acetylation of histone H3 in mouse brain nearly 4-fold. This epigenetic mouse model may be useful in evaluating drug efficacy on schizophrenia vulnerability. Hence the inhibition of histone deacetylases could represent a pharmacological intervention mitigating epigenetically induced vulnerability to schizophrenia in individuals at risk.

Regional distribution of leu and met enkephalin in rat brain

Abstract The regional distribution of leu- and met-enkephalin in the brain of rats killed with a microwave beam focused on the skull was studied using a radioimmunoassay. The enkephalin content varied significantly in the seven regions of rat brain studied: the highest concentration was found in striatum and the lowest in cerebellum and hippocampus. In every brain region studied, the content of met- was higher than that of leuenkephalin. In the brain of rats killed with focused microwave, the content of enkephalins is higher than in brains of rats killed by decapitation. It seems unlikely that this difference is due to an artifact caused by microwave radiation.

Diazepam binding inhibitor (DBI): A peptide with multiple biological actions

Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.

DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains

A down-regulation of reelin and glutamic acid decarboxylase (GAD) 67 mRNAs was detected in γ-aminobutyric acid (GABA)ergic cortical interneurons of schizophrenia (SZ) postmortem brains (10), suggesting that the availability of GABA and reelin may be decreased in SZ cortex. In situ hybridization of the mRNA encoding for DNA-methyltransferase 1, which catalyzes the methylation of promoter CpG islands, shows that the expression of this mRNA is increased in cortical GABAergic interneurons but not in pyramidal neurons of SZ brains. Counts of reelin mRNA-positive neurons in Brodmanns area 10 of either nonpsychiatric subjects or SZ patients show that the expression of reelin mRNA is decreased in layer-I, -II, and -IV GABAergic interneurons of SZ patients. These findings are consistent with the hypothesis that the increase of DNA-methyltransferase 1 expression in telencephalic GABAergic interneurons of SZ patients causes a promoter hypermethylation of reelin and GAD67 and perhaps of other genes expressed in these interneurons. It is difficult to decide whether this dysfunction of GABAergic neurons detected in SZ is responsible for this disease or is a consequence of this disorder. Although at present we cannot differentiate between these two alternatives, it is important to consider that so far a molecular pathology of cortical GABAergic neurons appears to be the most consistent finding associated with SZ morbidity.

Projections of substance P containing neurons from neostriatum to substantia nigra.

Substance P has been found to be most concentrated in the substantia nigra in hurnanlo~lz and rat brai+. Subcellular distribution studies have revealed that most of the substance P present in the hypothalamus and substantia nigra can be recovered in the nerve ending fractionss. However, precise information on the location of substance P cell bodies which project to substantia nigra is still lacking. We have attempted to obtain such information by making various types of brain lesion and determining the substance P content in substantia nigra and other brain regions. Sprague-Dawley rats weighing 175-225 g were used (from Allison Park, Pa.). Hemitransections of the brain with procedures similar to those described by McGeer et al.9 were performed at A 4380 pm, A 6860 pm, and A 7600 ym, according to Kanig and Klippel atlas7. Electrolytic lesions of globus pallidus were made stereotaxically by placing a temperature sensing probe at A 6.3; L 2.8; V -0.6 (according to Kiinig and Klippel atlas7) and raising the tissue temperature to 50 “C for 1 min by a radiofrequency lesion generator as described previously 5. Brain tissue was dissected and substance P concentration was determined by radioimmunoassay as reported befores.

Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis

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: 5α-reductase (5α-R) type I and 3α-hydroxysteroid dehydrogenase (3α-HSD). This study evaluates 5α-R type I and 3α-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100β immunohistochemistry. We demonstrate that 5α-R type I and 3α-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5α-R type I nor 3α-HSD mRNAs are expressed in S100β- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5α-R type I and 3α-HSD in cortical and hippocampal GABAergic interneurons. However, 5α-R type I and 3α-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.

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

Abstract: The glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA) stimulated a rapid, extracellular Ca2+‐dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor‐mediated activation of nitric oxide (NO) synthase. The NMDA‐induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled l‐arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose‐dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK‐801 and by the inhibitors of NO synthase, NG‐monomethyl‐L‐arginine (MeArg) and NG‐nitro‐L‐arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 μM) stimulated 45Ca2+ influx to the same extent as 100 μM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.

Learning impairment in rats by N-methyl-D-aspartate receptor antagonists

2-Amino-5-phosphonovalerate (APV, icv) phencyclidine (PCP, ip) and scopolamine (sc) dose-dependently disrupted short term working memory in radial maze. These drugs injected before, but not after training attenuated retention of long term memory in passive avoidance task. A relation of PCP action to its antagonism at NMDA receptors may be suggested.