Regulation of cerebral microvessels by glutamatergic mechanisms

We studied changes in glutamate receptors, expression of immediate early genes, and AP-1 DNA binding activity in the brains of phenobarbital (PB)-dependent and -withdrawn rats to investigate the possible involvement of activation of glutamate receptors in PB withdrawal syndrome. PB-dependent rats were prepared by feeding drug-admixed food for 5 weeks. Autoradiographic analysis showed that binding of [3H(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin e (MK-801), an antagonist of N-methyl-D-aspartic acid (NMDA) receptors, increased significantly in the cerebral cortices of PB-dependent and 24-h-withdrawn rats. However, [3H]MK-801 binding in the hippocampus and [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainic acid binding in the hippocampus and cerebral cortex were essentially unchanged in both groups. PB withdrawal seizures were followed by increased expression of c-fos mRNA in the hippocampus and cerebral cortex and of c-jun mRNA in the cerebral cortex. The induction of c-fos and c-jun mRNA was suppressed by administration of MK-801. Furthermore, PB withdrawal enhanced AP-1 DNA binding activity in the brain. The present findings suggest functional enhancement of glutamatergic neurotransmission during the development of PB withdrawal syndrome.     

Differential effects of aging on binding sites of the activated NMDA receptor complex in mice

The NMDA receptor site has been shown to be vulnerable to the effects of aging. Decreases in binding to the receptor site of up to 50% have been reported in aged animals. The present study was designed to quantitate and compare the effects of aging on multiple binding sites of the NMDA receptor complex in various brain regions. Autoradiography with [3H]glutamate, [3H]CPP, [3H]glycine, [3H]MK801 and [3H]TCP was performed on brain sections from 3, 10 and 28-30 month old C57B1/6 mice. The percent declines between 3 and 28-30 months of age in [3H]-glutamate (15-35% declines) and [3H]CPP (20-42% declines) binding were similar within most cortical regions and the caudate nucleus but [3H]glutamate binding showed less change (0-11% declines) than [3H]CPP (13-27% declines) in the occipital/temporal cortex and hippocampal regions. [3H]MK801 and [3H]TCP binding, stimulated by 10 microM glutamate, exhibited intermediate aging changes between the glycine and NMDA sites, both in percent decline (3-28% and 0-26%, respectively) and in the number of brain regions involved. [3H]Glycine binding, stimulated by 10 microM glutamate, showed no significant overall effect of age (declines ranged from 0-34%). [3H]CPP binding was significantly more affected than [3H]glycine binding in many regions. These results suggest that aging has heterogeneous effects on different sites on the NMDA receptor complex throughout the brain and on NMDA receptor agonist versus antagonist binding in selected brain regions.     

Up-regulation of brain N-methyl-D-aspartate receptors following multiple intracerebroventricular injections of [D-Pen2, D-Pen5] enkephalin and [D-Ala2, Glu4]deltorphin II in mice

The effects of chronic administration of [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II, the selective agonists of the delta 1- and delta 2-opioid receptors, on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2, D-Pen5]enkephalin or [D-Ala2, Glu4]deltorphin II (20 micrograms/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [3H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM-concentration, the binding of [3H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [D-Pen2, D-Pen5]enkephalin treated mice. In [D-Ala2, Glu4]deltorphin II treated mice, the binding of [3H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the Bmax value of [3H]MK-801. It is concluded that tolerance to delta 1- and delta 2-opioid receptor agonists is associated with up-regulation of brain N-methyl-D-aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl-D-aspartate receptors antagonists block tolerance to the analgesic action of delta 1- and delta 2-opioid receptor agonists.     

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.     

Research on factors affecting neonatal tetanus and its prevention through immunization

This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.     

Long-term adrenalectomy decreases NMDA receptors in rat hippocampus

The effect of long-term adrenalectomy on NMDA receptors in the rat hippocampus was studied. Hippocampal sections of control and adrenalectomized rats were incubated with [3H]MK-801, a radiolabeled non-competitive inhibitor of the NMDA receptor. Analysis by in vitro autoradiography showed a significant decrease in [3H]MK-801 binding in the dentate gyrus, CA1 and CA4 areas, as well as the temporal cortex. Results of this study suggest that glucocorticoids are vital for the regulation of the NMDA receptors.     

Stress test in the elderly. Clinical, hemodynamic, metabolic and electrocardiographic variables]

Pentylenetetrazol is a convulsive drug acting on gamma-aminobutyric acid-A (GABA[A]) gated-chloride receptors. In this study we used a subconvulsive dose (30 mg/kg) of pentylenetetrazol to induce a fully kindled state in rats. Glutamate receptors were evaluated using [3H]-[1(2-thienylcyclohexyl)]-piperidin (TCP) and [3H]kainate receptor autoradiography and [3H]muscimol autoradiography was used to study GABA(A) receptors. In fully kindled rats decreased N-methyl-D-aspartate receptor binding was found in parietal cortex, area CA2 of hippocampus and piriform cortex. Decreased kainate receptor binding was observed in all areas of the hippocampus, the medial amygdala and in the piriform cortex in the kindled rats. In contrast, GABA(A) receptor binding increased in the dentate gyrus. It is concluded that modulatory neuronal plasticity events are induced in fully pentylenetetrazol kindled rats, which appears to lead to decreased glutamatergic excitation and increased GABAergic inhibition in brain regions implicated in the development of seizure activity.     

Differences in NMDA receptor antagonist-induced locomotor activity and [3H]MK-801 binding sites in short-sleep and long-sleep mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl D-aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater locomotor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [3H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [3H]MK-801 and [3H]CPP and brain levels of [3H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [3H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

Effects of subacute lead exposure on [3H]MK-801 binding in hippocampus and cerebral cortex in the adult rat

We used the NMDA receptor non-competitive antagonist, [3H]MK-801, as a ligand for an autoradiographic study to determine the effects of lead on NMDA receptor in the rat brain. Adult male rats were administered lead acetate, 100 mg/kg, or sodium acetate, 36 mg/kg (control), by i.p. for 7 days. High lead levels were detected in blood (41.1 microg/dl) and in brain (16.7-29.4 microg/g). Concentrations of lead in brain regions were not significantly different. The [3H]MK-801 binding was heterogeneously distributed throughout the rat brain with the following order of binding densities: hippocampal formation > cortex > caudate-putamen > thalamus > brainstem. Lead exposure produced a significant decrease in [3H]MK-801 binding to the NMDA receptor in the hippocampal formation including CA2 stratum radiatum, CA3 stratum radiatum, hilus dentate gyrus and presubiculum, and in the cerebral cortex including agranular insular, cingulate, entorhinal, orbital, parietal and perirhinal areas. The hippocampal formation is known as a critical neural structure for learning and memory processes, whereas, cortical and subcortical regions have been demonstrated to be involved in the modulation of complex behavioral processes. The NMDA receptor has been demonstrated to play a key role in synaptic plasticity underlying learning and memory. Lead-induced alterations of NMDA receptors in the hippocampal formation and cortical areas may play a role in lead-induced neurotoxicity.     

Effects of antisense oligodeoxynucleotides to NR1 on suppression of seizures and protection of cortical neurons from excitotoxicity in vivo and in vitro

OBJECTIVE: To investigate the effects of NR1 subunit on the initiation and development of seizures and protection of cortical neurons from excitotoxicity by using antisense oligodeoxynucleotides (ODN) to NR1 in vivo and in vitro. METHODS: Intracerebroventricular injection, temporal cortex slices discharge, cerebral cortical neuronal culture, induction of neurotoxicity and [3H]MK-801 binding were used in this study. RESULTS: After an antisense ODN for NR1 was administered intracerebroventricularly (i.c.v. 100 micrograms in 10 microliters) once daily, for three days in genetically epilepsy-prone rats (GEPR, P77PMC), the animals did not develop any clonic and tonic convulsions and their seizure scores were significantly lower compared to the control groups. The frequency and amplitude of early seizure-like events (SLEs) and late recurrent discharges (LRD), induced by lowering Mg2+, were reduced in entorhinal cortex (EC) of the temporal slice treated by antisense ODNs. Pretreatment with antisense ODN (2 microM) protected more than 52% of glutamate-sensitive neurons and reduced the [3H]MK-801 binding to 50% in cultured cerebral cortical neurons. CONCLUSIONS: N-methy-D-aspartate-receptors (NMDAR), specifically the NR1 subunit, may participate and play important roles in the initiation and propagation of epilepsy in the P77PMC rat.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Pharmacological characterization of N-methyl-D-aspartate receptors in spinal cord of rats with a chronic peripheral mononeuropathy

N-Methyl-D-aspartate (NMDA) receptor antagonists, acting in the spinal cord, are analgesic. However, the clinical utility of these antagonists is diminished by their adverse effects on cognition and behavior. To facilitate the development of spinal cord-selective NMDA receptor antagonists, we characterized ligand interactions at NMDA receptors in spinal cord of normal rats and rats with a chronic peripheral neuropathy. NMDA receptors in spinal cord were distinguished from those in cerebral cortex on the basis of differences in the potencies of competitive and noncompetitive antagonists and on the basis of differences in their response to spermidine. D(-)-2-Amino-5-phosphonopentanoic acid (AP-5) and (+)-(1-hydroxy-3-aminopyrrolidine-2-one) (HA-966) were more potent in inhibiting NMDA-dependent [3H]TCP binding in spinal cord while, conversely, MK-801 was more potent in inhibiting [3H]TCP binding to NMDA receptors in cerebral cortex. Spermidine increased [3H]TCP binding to NMDA receptors in cerebral cortex (39+/-8%) but not spinal cord (2+/-1%). Based on these properties, NMDA receptors in spinal cord more closely resembled those in cerebellum than those in cerebral cortex. Generation of a chronic neuropathy had no effect on the density of NMDA receptors in lumbar spinal cord. There were also no major changes in the potencies of competitive antagonists or channel blocking ligands, although there was a trend for kynurenic acid and D-CPP to be more potent in the spinal cords of neuropathic animals. These findings indicate that, in both normal and neuropathic pain states, NMDA receptors in spinal cord can be distinguished pharmacologically from those in cerebral cortex. These findings underscore the feasibility of developing spinal cord-selective NMDA receptor antagonists as novel analgesics.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.     

Flow threshold for reduction of cyclic AMP binding in the hippocampus CA1 and other brain regions during stroke development in gerbils

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.     

Differential profiles of binding of a radiolabeled agonist and antagonist at a glycine recognition domain on the N-methyl-D-aspartate receptor ionophore complex in rat brain

Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [3H]5,7-dichlorokynurenic acid ([3H]-DCKA) a Gly recognition domain on the N-methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [3H]DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidine ethanol, with [3H]Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [3H]DCKA binding virtually without affecting those of four different agonists. Phospholipases A2 and C and p-chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [3H]DCKA binding with [3H]Gly binding being unaltered. Moreover, the densities of [3H]DCKA binding were not significantly different from those of [3H]-Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [3H]Gly binding than of [3H]DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Preliminary evaluation in humans of Tc-99m-Q3, a new tracer for myocardial perfusion imaging

We studied the alterations in binding of cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase binding activity following transient cerebral ischemia in Mongolian gerbils and examined the effects of vinconate and pentobarbital against alterations in the binding. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Cyclic AMP binding was significantly reduced in the hippocampus 5 h after ischemia, whereas the striatum showed no significant change in the binding. Seven days after ischemia, a severe reduction of [3H]cyclic AMP binding was noted in the dorsolateral striatum, hippocampal CA1 and CA3 sectors, and dentate gyrus. Intraperitoneal administration of vinconate (100 or 300 mg/kg) showed a significant elevation of [3H]cyclic AMP binding in the striatum, stratum pyramidale of hippocampal CA1 and CA3 sectors, and dentate gyrus 5 h after ischemia. By contrast, the intraperitoneal administration of pentobarbital (40 mg/kg) showed no significant alteration of [3H]cyclic AMP binding in most of these regions. However, vinconate and pentobarbital prevented a significant reduction of [3H]cyclic AMP binding in the dorsolateral striatum and stratum pyramidale of hippocampal CA3 sector 7 days after ischemia, although both drugs failed to prevent damage to the hippocampal CA1 sector. These results suggest that alteration in cyclic AMP binding may not be a major factor in causing ischemic neuronal damage.     

Neuroprotective properties of lifarizine compared with those of other agents in a mouse model of focal cerebral ischaemia

1. Changes in the peripheral type benzodiazepine binding site density following middle cerebral artery occlusion in the mouse, have been used as a marker of neuronal damage. These sites can be identified using the selective ligand [3H]-PK 11195 located on non neuronal cells, macrophages and astroglia, within the CNS. Glial cell proliferation and macrophage invasion is an unvoidable sequelae to cerebral ischaemic injury, secondary to neuronal loss. Following occlusion of the left middle cerebral artery (left MCA) a reproducible lesion was found in the parietal cortex within 7 days which gave rise to a significant increase in [3H]-PK 11195 binding. 2. Treatment of animals with the sodium channel blocker, lifarizine, significantly reduced the ischaemia-induced increase in [3H]-PK 11195 binding when given either 30 min pre-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.01) or delayed until 15 min post-ischaemia and three times daily for 7 days at 0.5 mg kg-1, i.p. (P < 0.001). Lifarizine was an effective neuroprotective agent in this model of focal ischaemia in the mouse. 3. Lifarizine also showed a dose-related protection against the ischaemia-induced increase in [3H]-PK 11195 binding with significant protection at doses of 0.1 mg kg-1, i.p. (P < 0.05), 0.25 mg kg-1, i.p. (P < 0.01) or 0.5 mg kg-1, i.p. (P < 0.01) 15 min post-ischaemia and b.i.d. for 7 days. No significant change is seen in the Kd for [3H]-PK 11195. The first dose could be delayed for up to 4 h after cerebralartery cauterization and protection was maintained.4. Phenytoin (28 mg kg-1, i.v. 15 min and 24 h post-ischaemia) was also neuroprotective in this model(P<0.01). This agent is thought to interact with voltage-dependent sodium channels to effect its anticonvulsantactions and this mechanism may also underlie its neuroprotective actions in focal cerebralischaemia.5. Agents with other mechanisms of action were also shown to have significant neuroprotection in this model. The non-competitive NMDA antagonist, MK 801, showed significant neuroprotection in the model when given at 0.5 mg kg-1, i.p. 30 min pre-ischaemia with t.i.d. dosing for 7 days (P< 0.001). The dihydropyridine calcium antagonist, nimodipine was not protective when given using the same dosing protocol as MK 801, 0.5 mg kg-1 30 min pre-occlusion and three times daily for 7 days but showed significant protection when given at 0.05 mg kg-1 15 min post-ischaemia and three times daily for 7days. The lipid peroxidation inhibitor, tirilazad (single dose 1 mg kg-1, i.v.) showed significant neuroprotection when given 5 min post-ischaemia but not when the first dose was delayed for 4 h.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Reconstitution of membrane fusion between pancreatic islet secretory granules and plasma membranes: catalysis by a protein constituent recognized by monoclonal antibodies directed against glyceraldehyde-3-phosphate dehydrogenase

This study was conducted to assess the involvement of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptor systems, located in specific limbic brain regions. in the discriminative stimulus effects of ethanol. Male Long-Evans rats were trained to discriminate between intraperitoneal (i.p.) injections of ethanol (1 g/kg) and saline on a two-lever drug discrimination task. The rats were then implanted with bilateral injector guides aimed at the nucleus accumbens core (AcbC), prelimbic cortex (PrLC), hippocampus area CA1 (CA1), or extended amygdala (i.e., at the border of the central and basolateral nuclei). Infusions of the non-competitive NMDA antagonist MK 801 in the AcbC or CA1 resulted in dose-dependent full substitution for i.p. ethanol. MK 801 infusion in the PrLC or amygdala failed to substitute for ethanol. Injection of the competitive NMDA antagonist CPP in the AcbC also failed to substitute for ethanol. Co-infusion of MK 801 in the hippocampus potentiated the effects of MK 801 in the AcbC, whereas NMDA infusion in the hippocampus attenuated the ability of MK 801 in the AcbC to substitute for ethanol. The direct GABA(A) agonist muscimol resulted in dose-dependent full substitution for i.p. ethanol when it was injected into the AcbC or amygdala, but failed to substitute when administered in the PrLC. Co-infusion of MK 801, but not CPP, potentiated the effects of muscimol in the AcbC. These results demonstrate that ethanol's discriminative stimulus function is mediated centrally by NMDA and GABA(A) receptors located in specific limbic brain regions. The data also suggest that the discriminative stimulus effects of ethanol are mediated by interactions between ionotropic GABA(A) and NMDA receptors in the nucleus accumbens, and by interactions among brain regions.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.