Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.     

Modulation of NMDA Receptor Subunit mRNA in Butorphanol-Tolerant and —Withdrawing Rats

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/l/h) resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR 1 and NR2) as well as NMDA receptor binding ([³H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.     

Carbamazepine inhibits electroconvulsive shock-induced inositol trisphosphate (IP3) accumulation in rat cerebral cortex and hippocampus

Carbamazepine is used to treat manic-depressive disorder, and is also an anticonvulsant. Rats were injected with this drug 90 min prior to this experiment, when mild inhibition of convulsions took place. Intraventricular injections of 14 muCi [3H]myoinositol were made 20-24 hrs prior to the experiment. Ninety min after intraperitoneal injection of carbamazepine or vehicle, rats were given electroconvulsive shock or sham procedure and sacrificed 30 sec later. Incorporation of radiolabel into inositol lipids and inositol phosphates was analyzed in cerebral cortex and hippocampus. Carbamazepine's effects on the brain inositol lipid cycle, studied here for the first time, showed 1) enhanced labeling in the polyphosphoinositides (carbamazepine-ECS groups showed increases of about 40% in PIP2); 2) decreased [H]IP1 levels; and 3) inhibition of ECS-induced [3H]-IP3 accumulation.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Rapid Down-Regulation of GABAA Receptors in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABA_A/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down-regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5-min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABA_A receptor antagonist [³H]SR-95531 and the hydrophobic benzodiazepine agonist [³H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [³H]SR-95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the B _max as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in K _D). As expected, there were no changes in the binding of [³H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABA_A receptors to high concentrations of synaptic GABA in vivo causes receptor down-regulation, perhaps via receptor internalization.     

Autoradiographic Imaging of [3H]Phorbol 12,13-Dibutyrate Binding to Protein Kinase C in Alzheimer''s Disease

Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being greater than 60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CA1 of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex; e.g., layers I-II control, 491 +/- 46 versus AD, 537 +/- 39 pmol/g of tissue; middle temporal cortex, e.g., layers I-II control, 565 +/- 68 versus AD, 465 +/- 72 pmol/g of tissue; and hippocampal formation, e.g., CA1 control, 511 +/- 28 versus AD, 498 +/- 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well-defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical correlates of AD.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

Cannabinoid receptor and WIN-55,212-2-stimulated [S]GTPγS binding and cannabinoid receptor mRNA levels in several brain structures of adult male rats chronically exposed to R-methanandamide

We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [³⁵S]-GTPγS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon’s horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon’s horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [³⁵S]-GTPγS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon’s horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.     

Alteration of ryanodine receptor in the hippocampus CA1 after hemispheric cerebral ischemia

Alterations in ryanodine binding and local cerebral blood flow (LCBF) were examined at 30 minutes and 2 hours post-ischemia in the gerbil brain in order to evaluate the influence of cerebral ischemia on the intracellular channels of Ca²⁺-induced Ca²⁺ release (CICR). Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. LCBF was measured at the end of the experiment using [¹⁴C]iodoantipyrine method, and the ryanodine binding was evaluated in vitro using [³H]ryanodine as a specific ligand for CICR channels. An autoradiographic method developed in our laboratory enabled us to determine both parameters within the same brain. A group of gerbils who underwent a sham procedure served as controls. LCBF was found to be significantly reduced in most of the cerebral regions on the occluded side at both 30 minutes as well as 2 hours post-ischemia. In contrast, a significant reduction in ryanodine binding was noted only in the hippocampus CA1 on the occluded side at 30 minutes and 2 hours after the occlusion. These findings suggest that regionally specific changes of CICR may be the cause of decreased ryanodine binding in the hippocampus CA1, and that these changes may be related to the pathophysiological mechanisms that cause this region to be particularly vulnerable to ischemia.     

CNS Adenosine A1 Receptors Are Altered After the Administration of Convulsant 3-Mercaptopropionic Acid and Cyclopentyladenosine: An Autoradiographic Study

Rat CNS adenosine A₁ receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N⁶-[cyclopentyl-2,3,4,5-³H adenosine]-([³H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [³H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [³H]CCPA binding in CA1 and CA2 areas (17–18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [³H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A₁ receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Decreased density of [3H]TCP binding following antipsychotic drug withdrawal in rats

Antipsychotic drugs have been reported to increase the expression of subunits of the NMDA receptor at the level of mRNA but it is not clear whether such effects are apparent at the level of the radioligand binding or receptor protein. Therefore, we examined the effect of treatment of, and withdrawal from, haloperidol, chlorpromazine, olanzapine or clozapine on the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP ) to the open ion channel of the NMDA receptor in rat caudate-putamen, hippocampus and frontal cortex. [3H]TCP binding was not significantly different in the caudate-putamen, hippocampus and cortex after three months of treatment with any antipsychotic drug. There were significant decreases in [3H]TCP binding in rat caudate-putamen and cortex, but not hippocampus, one month after ceasing treatment. Decreases in the caudate-putamen were detected in rats previously treated with chlorpromazine (0.1 mg/kg/day) and clozapine (0.1 and 1.0 mg/kg/day). In the cortex, decreases in [3H]TCP binding were also detected in rats previously treated with olanzapine (0.1 mg/kg/day) for three months. These data suggest that changes in the NMDA receptor associated ion channels occur following antipsychotic drug withdrawal.     

Electroconvulsive Shock (ECS) and the Adenosine Neuromodulatory System: Effect of Single and Repeated ECS on the Adenosine A1 and A2 Receptors, Adenylate Cyclase, and the Adenosine Uptake Site

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.     

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor mRNA levels in several brain structures of adult male rats chronically exposed to R-methanandamide.

We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [³⁵S]-GTPγS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon’s horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon’s horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [³⁵S]-GTPγS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon’s horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.     

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: Results of a quantitative autoradiographic study

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [³H]glutamate (displaced by NMDA), [³H]-kainate, and [³H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [³H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [³H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.     

Histamine Stimulation of Inositol 1-Phosphate Accumulation in Lithium-Treated Slices from Regions of Guinea Pig Brain

Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of 10 mM LiCl in [3H]inositol-loaded tissue slices from several regions of guinea pig brain. The level of [3H]inositol 1-phosphate increased approximately linearly, after an initial lag period, up to a time of 120 min. In the absence of lithium ions the accumulation of the 1-phosphate stimulated by histamine in cerebral cortical and hippocampal slices was markedly reduced. Lithium ions had much less effect on the response to histamine in cerebellar slices. The characteristics of the response to histamine were consistent with mediation by H1 receptors, and the affinity constants derived for mepyramine (2.3 X 10(9) M-1) and methapyrilene (1.8 X 10(8) M-1) were similar to those reported from measurements on other H1 responses in the guinea pig. The EC50 for histamine was similar in cerebellum, cerebral cortex, hippocampus, and hypothalamus. The position of the dose-response curve for histamine in cerebral cortical slices was similar to that of the curve for the receptor binding of histamine deduced from histamine inhibition of [3H]mepyramine binding.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes

A high affinity [3H]ryanodine receptor has been solubilized from rabbit brain membranes and biochemically characterized. [3H]Ryanodine binding to rabbit brain membranes is specific and saturable, with a Kd of 1.3 nM. [3H]Ryanodine binding is enriched in membranes from the hippocampus but is significantly lower in membranes from the brain stem and spinal cord. Approximately 60% of [3H]ryanodine-labeled receptor is solubilized from brain membranes using 2.5% CHAPS and 10 mg/ml phosphatidylcholine containing 1 M NaCl. The solubilized brain [3H]ryanodine receptor sediments through sucrose gradients like the skeletal receptor as a large (approximately 30 S) complex. Solubilized receptor is specifically immunoprecipitated by sheep polyclonal antibodies against purified skeletal muscle ryanodine receptor coupled to protein A-Sepharose. [3H]Ryanodine-labeled receptor binds to heparin-agarose, and a protein of approximately 400,000 Da, which is cross-reactive with two polyclonal antibodies raised against the skeletal muscle ryanodine receptor, elutes from the column and is enriched in peak [3H]ryanodine binding fractions. These results suggest that the approximately 400,000-Da protein is the brain form of the high affinity ryanodine receptor and that it shares several properties with the skeletal ryanodine receptor including a large oligomeric structure composed of approximately 400,000-Da subunits.