In primary cultures of cerebellar granule cells the activation of N-methyl-D-aspartate-sensitive glutamate receptors induces c-fos mRNA expression.

L-Glutamate, the natural agonist of quisqualate- and N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors, elicits a rapid, transient, dose-dependent increase of the steady state level of c-fos mRNA followed by an accumulation of c-fos protein immunostaining in cell nuclei. This induction is prevented by 2-amino-5-phosphonovalerate, an isosteric glutamate receptor antagonist, and by Mg2+ ion and phencyclidine, two noncompetitive allosteric antagonists of NMDA-sensitive glutamate receptors. Kainate and quisqualate (up to 150 microM) failed to alter the basal expression of c-fos mRNA.

Hippocampal membranes contain a neurotrophic activity that stimulates cholinergic properties of fetal rat septal neurons cultured under serum-free conditions.

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, approximately 98% of the septal cells are neurons, and approximately 6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons.

Modulation of glutamate receptors by phencyclidine and glycine in the rat cerebellum: cGMP increase in vivo.

In rats receiving N-methyl-D-aspartate (NMDA) intraventricularly or intracisternally the cerebellar cyclic guanosine monophosphate (cGMP) content increases in a dose-related manner. This response was used to study phencyclidine (PCP) and glycine interactions with the glutamate receptor subtype stimulated by NMDA. The increase of cGMP elicited by NMDA was inhibited by PCP and potentiated by glycine.

Kynurenate and 2-amino-5-phosphonovalerate interact with multiple binding sites of the N-methyl-D-aspartate-sensitive glutamate receptor domain.

By studying the binding of [3H]glycine and [3H]glutamate to rat synaptic membranes in the presence of 2-amino-5-phosphonovalerate (APV) and kynurenate (KYN) we have demonstrated that KYN is more potent than APV in displacing [3H]glycine, while an opposite order of potency was seen in displacing [3H]glutamate. Moreover, 2-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) inhibited only [3H]glutamate binding. The [3H]MK-801 specific binding was inhibited by all of the above antagonists; this action was abolished by glutamate, while glycine partially reversed only the action of KYN.

Down-regulation of proopiomelanocortin synthesis and beta-endorphin utilization in hypothalamus of morphine-tolerant rats.

We investigated regulation of the dynamic state of enkephalin and endorphin brain stores during morphine tolerance and dependence using cDNA hybridization and radioimmunoassay of the biologically active peptide(s) and their respective peptide precursors. Rats were made tolerant to morphine with the subcutaneous implantation of three morphine pellets (75 mg each) for a period of five days. Hypothalamic proopiomelanocortin (POMC) mRNA, POMC, and corticotropin-like intermediate lobe peptide content were decreased by 50% in morphine-dependent rats.

Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites.

The photoaffinity ligand [3H]PK 14105 was utilized to modify covalently peripheral-type benzodiazepine binding sites in rat adrenal mitochondrial preparations. The photolabeled membrane preparations were then solubilized in 1% digitonin and the detergent-soluble extracts subjected to fractionation by ion-exchange chromatography and reversed-phase high performance liquid chromatography. This scheme resulted in the purification of the putative binding site protein for PK 14105 which we have entitled PKBS.

Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes.

In previously frozen and extensively washed brain membranes [3H]glutamate binds to a single population of sites characteristic of the NMDA-sensitive glutamate receptor subtype. This binding cannot be displaced by glycine and D-serine, but actually is enhanced by these amino acids in a dose-dependent manner. Glycine and D-serine increase the affinity of glutamate binding without changing the density of binding sites.

Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex.

Using a sensitive histofluorescence staining method that allows for a quantitation of neuronal death, we compared the protective effects of gangliosides (a group of naturally occurring glycosphingolipids), phencyclidine (PCP), and MK-801 (dibenzocyclohepteneimine) on glutamate- and kainate-induced neuronal death in primary cultures of cortical and cerebellar neurons prepared from neonatal rats. PCP and MK-801 block neurotoxicity induced by glutamate doses 50 times higher than the LD50 (LD50 in Mg2+-free medium, 10 microM) but only partially block the kainate neurotoxicity (LD50 in presence of Mg2+, 100 microM). In contrast, pretreatment with gangliosides (GT1b greater than GD1b greater than GM1) results in complete and insurmountable protection against the neurotoxicity elicited by glutamate or kainate.

Diazepam binding inhibitor gene expression: location in brain and peripheral tissues of rat.

Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a 35S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle.

Chromosomal localization of the human diazepam binding inhibitor gene.

We have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the gamma-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q).

Molecular characterization and mitochondrial density of a recognition site for peripheral-type benzodiazepine ligands.

In a previous report, mitochondria were proposed as a subcellular structure where recognition sites for peripheral benzodiazepine ligands are located in adrenal glands. The present study examines the subcellular distribution of specific binding sites for PK 11195 in eight tissues and compares the relative densities of these binding sites in mitochondrial-enriched fractions with the relative activities of two mitochondrial marker enzymes. In all eight tissues examined, PK 11195 binding sites were found to subfractionate in a manner nearly identical to that of the mitochondrial enzyme succinate dehydrogenase.

Beta adrenergic and prostaglandin receptor activation increases nerve growth factor mRNA content in C6-2B rat astrocytoma cells.

C6-2B rat astrocytoma cells were used to test whether the increase of cellular nerve growth factor (NGF) content and secretion induced by isoproterenol treatment are associated with an increase in the content of mRNA that encodes NGF (NGF mRNA). Incubation of cells with isoproterenol (10 microM) for different time periods produced an increase of NGF mRNA content (3- to 5-fold over control) reaching maximum levels at 3 hr and lasting up to 24 hr. The isoproterenol effect on NGF mRNA was antagonized by the beta adrenergic receptor antagonist I-propranolol (10 microM) but not by phentolamine (10 microM), an alpha adrenergic receptor antagonist.

Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-activated cationic channels in rat cortical neurons in culture.

Single-channel currents were recorded by means of the patch clamp method in outside-out patches excised from rat cortical neurons in primary culture. The excitatory amino acid N-methyl-D-aspartic acid activated mainly 40-50 pS conductance channels. Channel opening durations were characterized by a series of rapid openings and closures induced by the presence of Mg2+ ions.

Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells.

In cultured granule cells prelabeled with [3H]arachidonate the activation of excitatory amino acid receptors by various agonists results in a dose-dependent stimulation of [3H]arachidonic acid release. Glutamate and aspartate were the most potent agonists, whereas N-methyl-D-aspartate, kainate and quisqualate were less potent. Other neurotransmitter receptor agonists--GABA, baclofen and norepinephrine--were inactive, while carbachol induced only a slight effect.

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.

Pertussis toxin inhibits signal transduction at a specific metabolotropic glutamate receptor in primary cultures of cerebellar granule cells.

In primary cultures of cerebellar granule cells, glutamate receptors have been classified into metabolotropic (GP1 and GP2) and ionotropic (GC1 and GC2). The GP1 and GC1 receptors are negatively modulated by magnesium and noncompetitively inhibited by phencyclidine; GP2 and GC2 receptors are insensitive to inhibition by magnesium and phencyclidine (Costa, Fadda, Kozikowski, Nicoletti and Wroblewski, 1988). Exposure of cultured cerebellar granule cells to pertussis toxin (PTX, 1 microgram/ml for 14-16 hr) reduced the stimulation of the hydrolysis of inositol phospholipids (PI) by the GP2 receptor agonists, glutamate and quisqualate in the presence of magnesium, but did not inhibit the stimulation of the hydrolysis of PI by GP1 receptor agonists.

Negative feedback regulation of the content of proenkephalin mRNA in chromaffin cell cultures.

The content of proenkephalin messenger RNA (PEmRNA) in cultured bovine adrenal chromaffin cells was reduced in the presence of reserpine (1 nM to 0.1 microM) with a return to basal levels 3 days after removal of the drug. In these cells, the basal release of Met5-enkephalin-Arg6-Phe7 immunoreactivity (MERF-IR) into the medium was significantly decreased when the cultures were pretreated with 0.

Subsets of GABAergic neurons in dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides.

The GABAergic properties of dissociated neurons from cerebral cortex of neonatal rats were studied in primary culture using electrophysiological, biochemical and immunohistochemical methods. Cultured neurons had a resting potential of -50 to -60 mV and exhibited spontaneous excitatory and inhibitory synaptic currents. Non-spontaneous (elicited) ionic currents were produced by direct application of GABA and glutamate.

Heterogeneity of gamma-aminobutyric acid/benzodiazepine/beta-carboline receptor complex in rat spinal cord.

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites.

Dihydropyridine binding sites regulate calcium influx through specific voltage-sensitive calcium channels in cerebellar granule cells.

In primary cultures of cerebellar granule cells, [3H]nitrendipine binds with high affinity to a single site (KD 1 nM and Bmax 20 fmol/mg protein). The 1,4-dihydropyridine (DHP) class of compounds such as nitrendipine, nifedipine, and BAY K 8644 displace [3H]nitrendipine binding at nanomolar concentrations. Verapamil partially inhibits whereas diltiazem slightly increases the [3H]nitrendipine binding.

Phencyclidine and glycine modulate NMDA-activated high conductance cationic channels by acting at different sites.

Glutamate activates high (40-50 pS) and low (5-15 pS) conductance cationic channels in outside-out patches excised from cultured cortical and cerebellar granule neurons of neonatal rats. In these neurons, the excitatory amino acid N-methyl-D-aspartic acid (NMDA) activates mainly high conductance channels. Phencyclidine (PCP) at 2 microM selectively reduces the number of NMDA-activated channel openings, at 20 microM it reduces the channel open-time.