Alteration of benzodiazepine receptors in mouse cerebellum following methylazoxymethanol treatment during development

The specific binding of [3H]flunitrazepam was studied to biochemically specify the morphological alterations induced in mouse cerebellum by a single injection of an antimitotic agent, methylazoxymethanol (MAM) performed at the beginning of the postnatal life. The MAM injection causes a general reduction of the benzodiazepine receptors in the adult mice which is particularly severe in mice having been injected the 1st day of postnatal life (so-called MAM0 mice) as compared to animals injected the 5th day (MAM5 mice): in MAM0 mice the benzodiazepine receptor is reduced to half of the control value. The affinity of the benzodiazepine towards its receptor was not affected and the topographic and biochemical action of MAM in the central nervous system was ascertained. Correlations could be made between the biochemical modifications and the morphological alterations otherwise described.     

Differential regulation of type I and type II interleukin-1 receptors in focal brain inflammation

Most pathologies of the brain have an inflammatory component, associated with the release of cytokines such as interleukin-1beta (IL-1beta) from resident and infiltrating cells. The IL-1 type I receptor (IL-1RI) initiates a signalling cascade but the type II receptor (IL-1RII) acts as a decoy receptor. Here we have investigated the expression of IL-1beta, IL-1RI and IL-1RII in distinct inflammatory lesions in the rat brain. IL-1beta was injected into the brain to generate an inflammatory lesion in the absence of neuronal cell death whereas neuronal death was specifically induced by the microinjection of N-methyl-D-aspartate (NMDA). Using TaqMan RT-PCR and ELISA, we observed elevated de novo IL-1beta synthesis 2 h after the intracerebral microinjection of IL-1beta; this de novo IL-1beta remained elevated 24 h later. There was a concomitant increase in IL-1RI mRNA but a much greater increase in IL-1RII mRNA. Immunostaining revealed that IL-1RII was expressed on brain endothelial cells and on infiltrating neutrophils. In contrast, although IL-1beta and IL-1RI were elevated to similar levels in response to NMDA challenge, the response was delayed and IL-1RII mRNA expression was unchanged. The lesion-specific expression of IL-1 receptors suggests that the receptors are differentially regulated in a manner not directly related to the endogenous level of IL-1 in the CNS.     

Kainic acid differentiates GABA receptors from benzodiazepine receptors in the rat cerebellum

In a combined psychophysical-electrophysiological study on 29 patients with multiple sclerosis (MS), a compromised ability to make interaural time discriminations was nearly always found to be associated with ‘abnormal’ brain stem potentials evoked by clicks to at least one ear. However, no obvious relationships were found between evoked brain stem potentials and several other auditory behavioral measures such as interaural intensity discrimination, pure-tone thresholds and speech discrimination.     

Localization of type I benzodiazepine receptors to postsynaptic densities in bovine brain

The subcellular localization of central-type benzodiazepine receptors in bovine cerebral cortex, cerebellum, hippocampus, and corpus striatum has been studied. In all regions except for the corpus striatum, benzodiazepine receptors are most highly enriched in purified postsynaptic densities (PSDs) prepared by Triton X-100/hypotonic lysis of purified synaptosomal plasma membranes. Benzodiazepine receptor enrichment in PSDs varies regionally, following the order cerebellum (approximately 8.5-fold enriched relative to crude P2 membranes) greater than cerebral cortex greater than hippocampus greater than striatum (no significant enrichment); the percentage of putative type I benzodiazepine receptors in each of these brain regions follows the same rank order. In cerebral cortex, analysis of displacement of the benzodiazepine antagonist [3H]Ro-15-1788 by the type I-selective drug CL-218,872 reveals that PSDs contain type I benzodiazepine receptors exclusively; other subcellular fractions contain mixtures of type I and type II benzodiazepine receptors. Benzodiazepine receptors in PSDs resist further extraction with detergent but can be solubilized with detergent containing greater than or equal to 0.2 M NaCl. The enrichment of detergent-resistant/detergent-plus-salt extractable type I benzodiazepine receptors in PSDs might account in part for the differential solubilization of type I and type II benzodiazepine receptors from crude brain membranes previously reported. The benzodiazepine-binding protein in cerebral cortical PSDs was identified by photoaffinity labeling with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The PSD benzodiazepine-binding protein is identical in molecular weight to the binding protein from whole brain; partial tryptic and alpha-chymotryptic fingerprints are also very similar in PSDs and whole brain.     

Further chemical differentiation of type I and type II adrenocorticosteroid receptors in mouse brain cytosol: evidence for a new class of glucocorticoid receptors

There are at least two classes of intracellular receptors for adrenocorticosteroid hormones in brain. Type I receptors have a high affinity for the naturally occurring gluco- and mineralocorticoids, corticosterone (CORT) and aldosterone (ALDO), respectively, and a very low affinity for synthetic glucocorticoids such as dexamethasone (DEX). type II receptors have a high affinity for the synthetic glucocorticoids, a lower affinity for CORT and a very low affinity for ALDO. In recent studies with mouse brain cytosol we have found a number of other biochemical differences between these two receptor types. In the present study, brain cytosol from adrenalectomized mice was prepared in HEPES buffer and subjected to various potentially inactivating treatments prior to assessment of Type I and Type II receptor specific binding capacity by incubation for 24 h at 0 degrees C with [3H]ALDO +/- [1H]RU 26988 (to prevent or permit the cross-binding of [3H]ALDO to Type II receptors) or [3H]DEX +/- [1H]Prorenone (to prevent or permit the cross-binding of [3H]DEX to Type I receptors), respectively. These studies revealed that 10-20% of the high-affinity (Kd = 3 nM) [3H]DEX specific binding capacity remained even after extensive, high concentration and repeated pretreatments with dextran-coated charcoal (DDC. to remove endogenous sulfhydryl-reducing reagents and other biochemicals). These procedures had little effect on Type I receptors. Further analyses revealed that DCC-resistant [3H]DEX binders were not Type I receptors since they were not saturated by [1H]Prorenone. These binders were also not inactivated by aging steroid-free cytosol at 0 degree C or by treating it with buffers containing 0.3 M KCl. Since these     

Ontogeny of type I and type II corticosteroid receptors in the rat hippocampus

The ontogeny of the corticoid receptors in the rat hippocampus was examined by in vitro [3H]corticosterone (CORT) binding to soluble molecules in the cytosol, using the selective Type II glucocorticoid agonist, RU 28362, to discriminate between Type I and Type II receptor sites. Type I receptors were undetectable until 8 days after birth. From this age on, the receptor showed adult characteristics for both the binding capacity (Bmax) and affinity (Kd). The Type II receptor concentration increased gradually over the observed period; however, at 3 weeks of age concentrations were still only about 65% those found in adults. The binding affinity of Type II to CORT was high during the first week of life but decreased thereafter towards adult value. These data thus suggest clear distinctions in the developmental patterns of Type I and Type II receptors for corticosteroids in the rat.     

Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum.

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

Development of beta-adrenergic receptors in normal and mutant mouse cerebellum

[3H]-dihydroalprenolol was used to examine the development of beta-adrenoceptors in the cerebellum of weaver, reeler, staggerer, and jimpy neurologically mutant mice and their normal counterparts. In normal animals the greatest increase in [3H] binding occurred during the postnatal interval of 10-15 days, and maximum values were obtained at about 3 weeks. Binding was saturable with an apparent dissociation constant of 0.26 nM, and the affinity of [3]-dihydroalprenolol for its receptor did not change significantly during development. [3H]-dihydroalprenolol binding was significantly reduced in weaver, reeler, and staggerer (14-22% of control values) at 20 days, but not in the jimpy mutant. These results are discussed in relation to the ontogeny of beta-adrenoreceptors with the known noradrenergic innervation of the cerebellum, and to the paucity of both granule neurons and myelination which occurs in the neurological mutants.     

Ontogeny of high-affinity GABA and benzodiazepine receptors in the rat cerebellum: an autoradiographic study

High-affinity GABA and benzodiazepine receptors were localized by light microscopic autoradiography in the developing rat cerebellum. [3H]muscimol was used for the labeling of GABA receptors and [3H]flunitrazepam for benzodiazepine receptors. Very low densities of GABA sites were found during the first postnatal week. GABA receptors start increasing linearly at the end of the second week up to adult levels around the fourth postnatal week. The increase in receptor density is concentrated in the developing granule cell layer. Benzodiazepine receptors are present at birth and increases in the density of receptors were observed already during the first postnatal week. Receptor concentrations reached adult values around the third to fourth weeks postnatally. The increase in benzodiazepine receptors in concentrated in the growing molecular layer with little change in the granule cell layer. The immature cell of the external granule layer were characterized by the absence of receptor sites. At least partial association of high-affinity GABA receptors with granule cells and benzodiazepine receptor with Purkinje cell dendrites is suggested by these developmental profiles.     

Differential induction of chemokines in human microglia by type I and II interferons

Chemokines are secreted proteins that function as chemoattractants, mediating the recruitment of specific subsets of leukocytes to sites of tissue damage and immunological reactions. Chemokines may also function as antiviral agents, since viruses such as human immunodeficiency virus type 1 (HIV-1) use chemokine receptors as co-receptors for viral entry. This study examines whether virus-induced interferon, IFNbeta, or immune-related interferon, IFNgamma, affects the production of beta-chemokines by CNS microglia and peripheral monocytes. When IFNbeta was used as the stimulus, induction of MIP-1alpha, MIP-1beta, MCP-1, and RANTES mRNA and protein was observed within 12 h of stimulation in microglia. By contrast, when IFNgamma was used as the stimulus, only MCP-1 was induced. IFNbeta stimulation of blood monocytes resulted in upregulation of MIP-1alpha, MIP-1beta, and MCP-1. Thus, type I and II interferons differentially regulate beta-chemokines in human fetal microglia and peripheral blood monocytes. These observations may have relevance for the therapeutic activity of IFNbeta in multiple sclerosis and for the antiviral effects of IFNbeta for HIV-1 infection of monocytes and microglia.     

Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum

Four lines of mutant mice were used to investigate (1) the neuronal localization of cannabinoid receptors in the cerebellar molecular layer and (2) the anatomical association of these receptors with elements of the two second messenger systems in the brain. Two of the mutant lines--Purkinje cell degeneration and nervous--are selectively deficient in Purkinje cells; the other two--weaver and reeler--are deficient in granule cells. In the heterozygous mice, [3H]CP 55,940 binding to cannabinoid receptors was discretely and densely localized to the molecular layer, as was [3H]forskolin binding to adenylate cyclase and [3H]phorbol 12,13-dibutyrate binding to protein kinase C, a component of the phosphoinositide cycle. [3H]CP 55,940 and [3H]forskolin binding was selectively reduced in weaver and reeler homozygous mice but unchanged in Purkinje cell deficient and nervous homozygotes. No decreases in [3H]phorbol 12,13-dibutyrate binding were found in any of the homozygous mutants relative to the heterozygous littermates. The results suggest that cannabinoid receptors and adenylate cyclase are localized to granule cell axons in the molecular layer, whereas protein kinase C is equally distributed in parallel fibers and Purkinje cell dendrites.     

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.     

Expression of corticotropin releasing hormone receptors type I and type II mRNA in suicide victims and controls

Corticotropin-releasing hormone (CRH) is a key neuroendocrine factor implementing endocrine, immune and behavioral responses to stress. CRH exerts its action through two major receptors, CRH-R1 and CRH-R2. Recently novel non-peptidic antagonists directed against CRH-R1 or CRH-R2 have been proposed as promising agents in the treatment of depression, anxiety and eating disorder. However, so far the CRH-receptor system has not been widely studied in humans. Therefore, we employed quantitative TaqMan PCR to analyze the expression and distribution of both CRH-R1 and CRH-R2 in human brain tissue and peripheral organs. Furthermore the expression of CRH receptors was analyzed for the first time in pituitaries of suicide victims by in situ hybridization and quantitative PCR. Our data demonstrated a different expression pattern in humans as compared to rodents. Both CRH-R1 and CRH-R2 were expressed in high amounts in the brain with the strongest expression in the pituitary. As described in rodents, however the CRH-R1 in human was the predominant receptor in the brain (82.7 +/- 11.0%), whilst CRH-R2 was the predominant receptor in peripheral organs (77.0 +/- 15.8%). There was a shift in the ratio of CRH-R1/R2 in the pituitaries of suicide victims. In conclusion, both CRH-R1 and CRH-R2 are widely expressed in human tissues with a distribution substantially different from rodents. Strong expression of both CRH-R1 and CRH-R2 in human pituitaries suggests that particularly under stress, activation of the HPA axis can be maintained through both receptors.     

Sex differences in the binding of type I and type II corticosteroid receptors in rat hippocampus.

Binding parameters of soluble Type I and Type II receptors were assessed in hippocampus of adult, adrenalectomized, male and female rats. No sex differences in the number of either Type I or Type II receptors could be demonstrated between gonadally intact animals. When females treated with 17 beta-estradiol benzoate (10 micrograms/day) were compared with males, a statistically significant reduction in Type II receptors was observed in the females; progesterone produced no further decrease in receptor numbers. The amount of tissue-associated corticosteroid-binding globulin in gonadally intact animals (perfused with dextran-saline) was twice as great in females as males. Sex-dependent differences in these gonadally intact rats were found in the affinity, measured as the dissociation constant (Kd), of both the Type I and Type II receptors. For both receptors, affinity in cytosols from females was reduced. The difference for the Type II receptor was slight, but the Kd value of the Type I receptor was several-fold higher in females. The difference in affinity was evident with both natural and synthetic steroid ligands. There appears to be little, if any, difference in affinity between the hippocampal Type I and the Type II receptors in females. This suggests that the occupancy of Type I receptors in females is substantially less than that of males at low circulating concentrations of corticosteroids.     

Comparison of the molecular structure of GABA/benzodiazepine receptors purified from rat and human cerebellum

The use of improved affinity chromatographic techniques has allowed for the substantial copurification of both the benzodiazepine and the GABA receptor from brain. These preparations have been used to begin characterization of the benzodiazepine receptor at a molecular level. We have recently purified benzodiazepine receptor from human and rat cerebellum, and SDS-PAGE has revealed that both preparations consist of a major protein of 50 kDa and a minor protein of 55 kDa. These proteins are recognized by a series of monoclonal antibodies prepared against the rat benzodiazepine receptor suggesting the rat and human receptors share several common antigenic domains. Several other approaches have been employed to further investigate possible homology between the rat and human receptors. Proteolytic degradation studies have shown that the major limiting photolabeled peptide fragment generated in rat and human is similar as determined by HPLC analysis. Isoelectric focusing and SDS (two-dimensional) electrophoresis have revealed that the immunoreactive, photolabeled 50 kDa protein, and the purified receptor have identical PI values. The receptor from both human and rat are glycoproteins as determined by lectin binding studies. However, exposure of these proteins to neuraminidase fails to alter the pharmacology of the receptors indicating possible similarities in their posttranslational glycosylation. Thus, it appears that some degree of structural homology exists between the rat and human benzodiazepine receptors.     

Hippocampal type I and type II corticosteroid receptors are modulated by central noradrenergic systems.

The effects of corticosteroids on various brain functions, including the negative feedback control of hypothalamo-pituitary-adrenal (HPA) axis activity, are mediated by two types of receptors (type I, or mineralocorticoid, and type II, or glucocorticoid) in the central nervous system. Although receptor numbers are thought to be regulated by circulating levels of corticosterone, there may be a direct neural control of corticosteroid receptors. In the present experiments, we demonstrate that 6-OHDA lesioning of noradrenergic (NA) ascending pathways in the pedunculus cerebellaris superior (PCS) reduces corticosterone secretion in response to novelty and increases the number of hippocampal type I corticosteroid receptors in rats 24 hr after adrenalectomy. The same lesion in adrenalectomized animals in which corticosterone levels were maintained within normal limits by corticosterone replacement implants also led to an increase in the number of type I corticosterone receptors and a decrease in the apparent affinity (Kd) of type II receptors in the hippocampus. These results suggest that the NA system may regulate HPA axis activity via a direct control of the number of type I receptors and the apparent affinity of type II receptors in the hippocampus. The possibility that there is a neural control of corticosteroid receptors may throw light on mechanisms controlling HPA axis activity and may suggest other approaches to the treatment of dysregulation of the HPA axis observed during stress and in certain psychopathological conditions.     

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.     

In vivo and in vitro modulation of central type benzodiazepine receptors by phosphatidylserine

The in vivo and in vitro modulation of central benzodiazepine binding sites (BDZ-R) by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was studied. Five days i.p. administration of 15 mg/kg/day of BC-PS liposomes increased the maximal number of binding sites (Bmax) for [3H]flunitrazepam in cerebral cortical membranes. In contrast, the density of hippocampal benzodiazepine recognition binding sites decreased. In cerebellar membranes, BC-PS treatment did not alter the characteristics of [3H]flunitrazepam binding. Similar experiments using phosphatidylcholine extracted from bovine brain (BC-PC) resulted in no changes in the [3H]flunitrazepam binding in the 3 neural structures studied. Confirming previous results, rats submitted to an acute swimming stress showed a decrease in the density of cerebral cortex BDZ-R. Animals treated with BC-PS liposomes before stress showed cortical [3H]flunitrazepam binding significantly below treated, unstressed animals but not below controls. The effects of BC-PS liposomes appeared to be selective for the central type of BDZ-R since no changes were observed in [3H]RO 5-4864 binding, a radioligand specific for the peripheral type BDZ-R. Preincubation of cerebral cortical and cerebellar synaptosomal membranes with BC-PS liposomes (1-300 micrograms per assay) significantly increased in a concentration-dependent manner (up to 100 micrograms) the [3H]flunitrazepam binding. Scatchard analysis revealed changes in the apparent affinity without alterations in the Bmax. Very similar results were obtained using a purified PS from spinal cord. BC-PC, phosphatidylinositol, phosphatidic acid and the lyso derivatives of PS and PC (lysoPS and lysoPC) were found to be ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)