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PubMed1991

Age-related decreases in corticotropin-releasing factor (CRF) receptors in rat brain and anterior pituitary gland.

Corticotropin-releasing factor (CRF) receptors were measured in discrete areas of brain and in anterior pituitary of 4-, 12-, 18-, and 24-month-old male Fischer rats. No significant age-related alterations in [125I]ovine CRF binding were observed in the olfactory bulb, cerebral cortex, hippocampus, brainstem, and cerebellum; there was a trend for CRF binding to decrease in the striatum as a consequence of aging. Significant age-related decreases were observed in 125I-ovine CRF binding in the anterior pituitary and hypothalamus with maximal reductions of 60 and 27%, respectively. Saturation analysis in the anterior pituitary indicated an age-related reduction in the density of CRF receptors (i.e. Bmax) without an alteration in the affinity (i.e. Kd) of CRF for its binding site. Northern analysis of proopiomelanocortin (POMC) mRNA in the anterior pituitary indicated no significant differences in the levels of POMC mRNA between 4- and 24-month-old rats. These and other data suggest that the age-related decrease in anterior pituitary CRF receptors may be due to hypersecretion of hypothalamic CRF rather than a loss of corticotropes in the anterior pituitary.

Aging↗
PubMed1993

Intracellular filling in fixed brain slices using Miniruby, a fluorescent biocytin compound.

Biocytin is useful for intracellular filling in living slices because it is soluble, has high electrophoretic mobility and a high affinity for avidin. In fixed slices, however, membrane potential cannot be used to signify that a cell is impaled. Thus, it is necessary to inject a fluorescent molecule so that impalement and filling can be visually monitored. As biocytin does not fluoresce, it cannot be used by itself in fixed slices. Here, we report that a biocytin-dextran (MW 10 kDa and 40 kDa) compound, Miniruby (MR), is a useful intracellular marker for injecting neurons in fixed slices. Fixed slices (200-400 microns) of adult rat olfactory bulb, piriform cortex, midbrain periaqueductal gray and locus coeruleus were used. Slices were stained by 0.001% ethidium bromide so that cell bodies could be visualized. The slices were imaged and filled using a specially designed hinged, epi-fluorescent microscope. A cell was impaled with a pipette containing 1-5% MR; positive pulsed constant current (1-5 nA; 300-400 ms on and 600-700 ms off; approximately 10 min) was applied until the fine dendrites were brightly fluorescent. Slices were post-fixed for 6-12 h, then reacted by a conventional ABC-DAB protocol. Miniruby has several advantages: (1) it is easy to visualize the electrode in relation to the cell bodies; (2) the staining procedure is very sensitive, does not require immunohistochemistry, and the reaction product is light stable; (3) injected neurons, dendrites and initial part of axons are well visualized by bright-field microscopy. It should be possible to analyze MR filled cells at the EM level.

Animals↗
PubMed1993

Characterization of metabotropic glutamate receptors negatively linked to adenylyl cyclase in brain slices.

We have characterized the pharmacological profile of activation of metabotropic glutamate receptors negatively linked to adenylyl cyclase (mGluR decreases cAMP) in brain slices. Among the putative mGluR agonists, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), were the most potent inhibitors of forskolin-stimulated cAMP formation in hippocampal slices, followed by ibotenate, L-2-amino-3-phosphonopropionate (AP3), quisqualate, L-glutamate and beta-N-methylamino-L-alanine (BMAA). Inhibition of forskolin-stimulated cAMP formation by DL-2-amino-4-phosphonobutanoate (AP4) was biphasic, suggesting that the drug interacts with more than one mGluR decreases cAMP subtype. Both L-AP4 and L-serine-O-phosphate (a restricted analogue of AP4) were much more effective in inhibiting forskolin-stimulated cAMP formation than their D-isomers, indicating that interaction of these drugs with the mGluR decreases cAMP is stereoselective. Despite the fact that DCG-IV and ibotenate behave as NMDA receptor agonists, their effect was insensitive to MK-801. The regional pattern of expression of mGluR decreases cAMPS, as estimated by using 1S,3R-ACPD as an agonist, did not correlate with the steady-state levels of mGluR2 mRNA. Thus, 1S,3R-ACPD inhibited forskolin-stimulated cAMP in slices from hippocampus, cerebral cortex, corpus striatum, olfactory tubercle or hypothalamus, but not in slices from olfactory bulb or cerebellum; in contrast, mGluR2 mRNA levels were high in the olfactory bulb and very low in the corpus striatum. 1S,3R-ACPD also inhibited forskolin-stimulated cAMP formation in cortical membranes, excluding the involvement of trans-synaptic mechanisms in the activity of mGluR decreases cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1986

In vitro autoradiographic localization of guanine nucleotide binding sites in sections of rat brain labeled with [3H]guanylyl-5'-imidodiphosphate.

In vitro labeling of guanine nucleotide binding proteins has been accomplished for the microscopic localization and quantitation of these sites in individual brain regions. Cryostat sections of rat brain were labeled with [3H]guanylyl-5'-imidodiphosphate [( 3H]Gpp(NH)p) using parameters which resulted in a high specific to non-specific (signal to noise) binding ratio. Gpp(NH)p binding sites were identified to some extent in all brain regions examined. The highest densities existed in the olfactory bulb, cerebral cortex, caudate-putamen, interpeduncular nucleus, substantia nigra and cerebellum. Thus, GTP binding proteins can be labeled and localized using a novel application of an autoradiographic technique developed for microscopic detection of receptors.

Animals↗
PubMed1995

Systemic administration of kainate induces marked increases of endogenous kynurenic acid in various brain regions and plasma of rats.

The endogenous neuroinhibitory and neuroprotective excitatory amino acid receptor antagonist kynurenic acid has been hypothetically linked to the pathogenesis of epilepsy and several other brain disorders. In the present study, alterations in kynurenic acid levels were examined in the kainate model of temporal lobe epilepsy. Kainate was systemically injected in rats at a dose (10 mg/kg s.c.) which induces a characteristic behavioural syndrome with stereotypies and focal (limbic) and generalized seizures, eventually progressing into severe status epilepticus. Kynurenic acid was determined 3 h after kainate injection in various brain regions (olfactory bulb, frontal cortex, piriform cortex, amygdala, hippocampus, nucleus accumbens, caudate/putamen, thalamus, superior and inferior colliculus, pons and medulla, and cerebellar cortex) and in plasma, using a sensitive high-performance liquid chromatographic method. When data were analysed irrespective of individual seizure severity, significant increases in kynurenic acid were determined in all brain regions examined except the hippocampus, nucleus accumbens and pons/medulla. The most marked (200-500%) increases above controls were seen in the piriform cortex, amygdala, and cerebellar cortex. Furthermore, a significant kynurenic acid increase of about 200% above control was determined in plasma. When kynurenic acid levels were determined in subgroups of rats with different behavioural alterations in response to kainate, the most marked kynurenic acid increases were seen in subgroups with status epilepticus. Rats which only developed mild (focal) seizures or stereotyped behaviours (wet dog shakes) also exhibited significantly increased kynurenic acid levels, thus indicating that the increase in kynurenic acid in response to kainate was not solely due to sustained convulsive seizure activity. Whereas it was previously proposed that kynurenic acid is involved only in later stages of seizure disorders, the present data demonstrate that marked increases in central and peripheral kynurenic acid levels occur early after the onset of neuroexcitation, at least in the kainate model.

Animals↗
PubMed1996

Regulation of glutamic acid decarboxylase mRNA expression in rat brain after sertraline treatment.

We now investigated the effect of chronic treatment with sertraline on glutamic acid decarboxylase mRNA expression in different rat brain areas by means of in situ hybridization. We found a reduced glutamic acid decarboxylase mRNA expression in the prefrontal cortex, accumbens nucleus, olfactory tubercle and reticular nucleus of the thalamus. The involvement of presynaptic modulation of gamma-amino-butyric acid transmission in the anxiolytic effect of sertraline is discussed.

1-Naphthylamine↗
PubMed1996

Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney.

We have isolated and characterized a rat brain cDNA clone which encodes a new protein of 474 amino acids in length which contains two C2 domains structurally homologous to those present in synaptotagmins. The overall amino acid identity in C2 domains between this protein and the synaptotagmins is 36-44%. This protein also contains 3 putative consensus sequences for phosphorylation by cAMP-dependent protein kinase. RNA blot hybridization revealed a 3.0 kb transcript abundantly expressed only in the rat brain and the kidney. Thus, we called this brain/kidney protein (B/K). In situ hybridization and Northern blot analyses showed that the B/K transcript was found in forebrain including the olfactory bulb, cerebral cortex, hippocampus, and hypothalamus. In the kidney, high levels of B/K transcript were expressed in the papillary region of the inner medulla, the inner stripe of the outer medulla and the cortex. The selective expression in forebrain and kidney suggests that B/K may be involved in similar cAMP-dependent processes at these very different sites.

Amino Acid Sequence↗
PubMed1988

Metaphit prevents locomotor activation induced by various psychostimulants and interferes with the dopaminergic system in mice.

Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine.

Amphetamine↗
PubMed1989

Changes of mouse brain gangliosides during aging from young adult until senescence.

The brain gangliosides from young adult to senescent mice (BDF1 and C57BL/6) were studied. The total ganglioside concentrations of the whole brains were almost constant from young adulthood until the beginning of senescence, but decreased constant from young adulthood until the beginning of senescence, but decreased during senescence to about 80% of the constant level observed at the period before the beginning of senescence. In spite of the constancy of the ganglioside concentrations at the period before the beginning of senescence, the composition gradually changed, with an increase of GM1 and decreases of GD1b, GT1b and GQ1b. During the senescence, all of the gangliosides decreased in their concentrations, but GD1a, GT1b and GQ1b decreased to a markedly greater extent (GT1b greater than GD1a greater than GQ1b). The regional gangliosides in olfactory bulb, cerebrum cortex, cerebrum white matter, hippocampus, hypothalamus, cerebellum and medulla oblongata were compared between 3- and 30-month-old mice of both strains. Significant changes in ganglioside concentrations were observed in both strains in the cerebrum and the hippocampus. In the cerebrum white matter, cerebellum and medulla oblongata, GM1 and GM4 contents significantly increased in the senescent mice.

Aging↗
PubMed1988

Characterization of [3H] CCK4 binding sites in mouse and rat brain.

We have investigated the possible occurrence of distinct CCK8 and CCK4 binding sites in the brain by comparing the binding characteristics of [3H] CCK4 to those of the CCK8 analogue, [3H] Boc (Nle28,31]CCK27-33 (BDNL-CCK7). [3H] CCK4 and [3H] BNDL-CCK7 were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 +/- 2.1 fmol/mg prot (KD = 3.78 +/- 0.47 nM) and 38.9 +/- 2.2 fmol/mg prot (KD = 0.26 +/- 0.02 nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of [3H] CCK4 binding sites in rat forebrain was the same as that of [3H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK4 labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for [3H] CCK4 in mouse and rat brain, with characteristics similar to those already reported with CCK33, CCK8 and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.

Animals↗
PubMed1992

Transfer of [3H]leucine across the blood-brain barrier at high blood-side oxytocin concentrations in normal and morphine-dependent rats.

The effects of circulating oxytocin on permeability of the blood-brain barrier (BBB) to L-[3H]leucine were studied in anaesthetized rats using the intracarotid, single pass, bolus injection technique. After bolus intracarotid oxytocin injection (10(-9) M), there were no differences in [3H]leucine uptake, compared with controls, in any of eight brain regions with a 'tight' BBB (olfactory bulb, frontal cortex, visual cortex, corpus striatum, hippocampus, thalamus, hypothalamus and colliculi) or in BBB-free, 'leaky' structures (pineal gland, choroid plexus, neuro-intermediate pituitary, anterior pituitary). [3H]leucine uptake by the 'leaky' structures was 2.4x and 2.6x uptake by 'tight' regions in the oxytocin and control groups respectively. In morphine-dependent rats, naloxone increased oxytocin secretion 28-fold within 5 min, but did not affect [3H]leucine uptake for any BBB-protected brain region or BBB-free 'leaky' structure. Accumulation of [3H]leucine was 8.3x and 7.0x greater in the 'leaky' structures than in the 'tight' regions in the naloxone and control groups respectively; [14C]inulin accumulation by each 'tight' region (measured simultaneously with [3H]leucine to determine the vascular space) was not affected by naloxone. It is concluded that even very high blood plasma concentrations of oxytocin do not affect BBB permeability for leucine. It is unlikely that altered BBB permeability, at least for amino acids, contributes to CNS changes during naloxone-provoked morphine withdrawal.

Animals↗
PubMed1979

Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [3H]propylbenzilylcholine mustard and its distribution in the forebrain.

[3H]Propylbenzilylcholine mustard ([3H]PrBCM) is a synthetic, potent muscarinic antagonist, which binds specifically and irreversibly by means of a covalent linkage to muscarinic receptors. Ten micrometer coronal cryostat sections taken through unfixed rat brain at the level of the maximum extent of the caudate nucleus were mounted on glass slides and incubated with 2.4 nM [3H]PrBCM at 30 degrees C for 25 min. They showed a total binding of 3250 pmol/g protein, of which 2130 pmol/g protein was sensitive to pretreatment with 10-6 M atropine. The specific (atropine-sensitive) binding was saturable. Saturation was reached at 15 min, with a rate constant of 1.3 x 106 M-1 sec-1. Binding was unaffected by drugs acting at nicotinic receptors (D-tubocurarine, hexamethonium), or by physostigmine, but was inhibited by muscarinic drugs (pilocarpine, oxotremorine, 3-quinuclidinylbenzilate). Postfixation for 15 min in Carnoy's fixative reduced the specific binding by 10% and the non-specific by 50%. Prefixation (i.e. before incubation with [3H]PrBCM) with any fixatives containing formaldehyde largely prevented specific binding, but a range of concentrations of glutaraldehyde (2% to 0.05%) caused only small reductions in specific binding (e.g. 0.1% glutaraldehyde caused only a 6% reduction). Clear, regionally specific patterns of localization of specific label in light microscope autoradiographs could be obtained from cryostat sections prefixed with 0.1% glutaraldehyde, incubated with 2.4 nM [3H]PrBCM for 15 min at 30 degrees C, and postfixed for 15 min in Carnoy's solution. Of the 105 forebrain areas studied 12 had grain counts between 6 and 9 times the non-specific level and a further 30 had counts 4 to 6 times non-specific. The higher grain counts were in the external plexiform layer of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, pyriform cortex, stratum radiatum of the hippocampus, stratum moleculare of the dentate gyrus, lateral amygdaloid nucleus, cortico-amygdaloid transition zone, anteroventral thalamic nucleus, hypothalamic supraoptic nucleus, caudate-putamen, nucleus accumbens, and in laminae 3 and 6 of the neocortex (parietal region). There were high grain densities over the choroid plexus the lateral but not the third or fourth ventricles.

Animals↗
PubMed1996

Ontogeny of NMDA R1 subunit protein expression in five regions of rat brain.

A polyclonal antiserum to a fusion protein corresponding to a region of the NMDA R1 (NR1) subunit (amino acids 656-811) was produced and affinity purified. A quantitative immunoblotting technique was developed using the fusion protein as a standard. By employing this method, ontogenic studies (day 2-42) of the density of NR1 protein were carried out in several regions of rat brain. The results showed that in all five of the brain regions examined [olfactory bulb (Ob), cortex (Cx), hippocampus (Hp), midbrain (Mb) and cerebellum (Cb)], levels of NR1 protein are low at birth and increase with similar patterns having a sharp rise within the first 3 weeks after birth. Levels increased 2.0 to 4.5-fold from postnatal day 2 to postnatal day 42. Although the general patterns of developmental expression are similar, large differences in the absolute amounts of NR1 protein among the five brain regions were observed. The maximal levels (pmol of fusion protein equivalent/mg +/- S.E.) of NR1 subunit attained during development in the five regions are: Hp 2.0 +/- 0.37 > Cx 1.4 +/- 0.11 > Ob 1.3 +/- 0.2 > Mb 1.0 +/- 0.10 > Cb 0.57 +/- 0.13. The temporal patterns of expression of NR1 protein are similar to results from studies examining the expression of NR1 mRNA. Furthermore, the absolute numbers obtained from our studies are close to those found using [(3)H]MK-801 binding suggesting that many of the NR1 subunits expressed in the brain exist in an active form.

Aging↗
PubMed1992

Distribution and cellular localization of preproenkephalin mRNA in the ovine brain and pituitary.

In this study in situ hybridization histochemistry was used to determine the regional and cellular localization of preproenkephalin (PPE) mRNA in the sheep brain and pituitary. Coronal brain sections were hybridized with an 35S-labelled synthetic 45-mer deoxyribonucleotide probe complementary to a portion of the bovine PPE gene. The specificity of the probe was confirmed by Northern blot analysis. The highest density of labelled cell bodies was found in the nucleus accumbens, caudate-putamen, olfactory tubercle, the central nucleus of the amygdala, the paraventricular nucleus of the hypothalamus, the suprachiasmatic nucleus and in the gigantocellular division of the medullary reticular formation. Labelled cells were also found in the olfactory bulb, prefrontal cortex, piriform cortex and cerebral cortex and in the vicinity of the locus coeruleus, parabrachial nucleus and the nucleus of the solitary tract. In the pituitary a dense PPE mRNA signal was observed in the intermediate lobe; cells in the anterior or neural lobe did not express PPE mRNA. The widespread distribution of cells containing PPE mRNA transcripts within the ovine brain agrees with a similar distribution in the rat. The data suggest that PPE neurons may be involved in diverse physiological functions including the processing of sensory and nociceptive information and in the regulation of endocrine and motor responses.

Animals↗
PubMed1993

Distribution of GAP-43 (B50/F1) mRNA in the adult rat brain by in situ hybridization using an alkaline phosphatase labeled probe.

GAP-43 (B-50,F1, pp46) is a calmodulin binding protein which is specific to the nervous system and also a substrate for the protein kinase C. Furthermore an enrichment of this protein in the growth cone and developmental brain indicate that this protein is related to nerve development, regeneration, and outgrowth. While its level dramatically decreases after the completion of synaptogenesis, the protein is still to some extent continuously expressed in certain regions of the mature brain. In order to clarify GAP-43 localization in mature normal rats, we investigated the distribution of GAP-43 mRNA in the rat central nervous system by using a non-radioisotopic in situ hybridization histochemistry. This method demonstrated GAP-43 mRNA expressing cells with high resolution. GAP-43 mRNA was more abundant in the forebrain than in the lower brainstem. Intense hybridization signal was observed in the mitral cells of olfactory bulb, cerebral cortex, CA3 region of hippocampus, diagonal band, substantia nigra, raphe nuclei, locus coeruleus, and dorsal motor nucleus of vagus. Weak to moderate hybridization signals were also widely expressed in thalamus, hypothalamus, and midbrain. Moreover, most noradrenergic, adrenergic, serotonergic, histaminergic, and caudal part of dopaminergic cells exhibited an intense GAP-43 mRNA signal. Thus, GAP-43 mRNA is abundantly expressed under normal conditions in the brain and may play an important physiological role particularly in the forebrain and in monoaminergic neurons supporting the findings that GAP-43 could be implicated in plasticity and monoamine release.

Alkaline Phosphatase↗
PubMed1995

Molecular cloning of cDNA encoding a bovine selenoprotein P-like protein containing 12 selenocysteines and a (His-Pro) rich domain insertion, and its regional expression.

When cDNA containing proteins enriched in the bovine cerebellar cortex were cloned, a clone which seemed to encode a selenoprotein P-like protein was isolated. The coding nucleotide sequence of its cDNA insert displayed high homology to rat and human selenoprotein P cDNA but contained 12 rather than 10 TGAs (12 rather than 10 selenocysteines in deduced amino acids), a tandem repeat of one CACTCC (His-Ser) and seven CATCCCs (His-Pro), and a 3' untranslated region approximately 890 bases shorter than that of rat liver selenoprotein P. RT-PCR using a set of primers flanking to the repeat displayed the existence of mRNA without the repeat. The tandem repeat and its adjacent region consisted of a similar motif of CAC/TCC/AC/T. Thus, these proteins included a (His-Pro) rich domain with a slightly negative free energy change irrespective of having the tandem repeat or not. Such His-Pro repeats reportedly exist in the segmentation gene paired or homeobox protein Om(1D) of Drosophila. Moreover, both this selenoprotein P-like protein mRNA and selenoprotein P mRNA were expressed in all the areas of the brain but most prominently in the cerebellar cortex, hippocampus, and olfactory bulb. These findings suggest the possibility that these selenoproteins are major selenium carriers in the brain and play a role in the morphological response of nerve or glial cells.

Amino Acid Sequence↗
PubMed1994

Localization and age-dependent expression of hexokinase mRNA in the rat brain.

Localization of mRNA encoding for the enzyme hexokinase and its regulation in aged animals was carried out in rat brain using the in situ hybridization technique. The highest levels of the hybridization signal were observed in the olfactory bulb, piriform cortex, tenia tecta, hippocampus and granular cells of the cerebellum. Other brain areas and nuclei including cerebral cortex, thalamus, hypothalamus, substantia nigra, subiculum, choroid plexus and superior colliculus displayed moderate to low density of transcripts. Correlation between relative hexokinase content and levels of its mRNA was found only for some brain regions such as caudate-putamen, geniculate nucleus, ventral and lateral thalamic nuclei, superior colliculus and granular cells of the cerebellum. In the cerebral cortex and hippocampus of old animals the expression of hexokinase was significantly increased at 18 and 24 months of age. From the present data we conclude that although hexokinase is an ubiquitous enzyme, sites of synthesis display a discrete and uneven localization in rat CNS and expression, in the aging brain, might be regulated to compensate for reduced oxidative phosphorylation in the brain tissue.

Aging↗
PubMed1995

A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), is expressed in rat brain.

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

3-Hydroxysteroid Dehydrogenases↗