Sequential changes in muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia.

We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinuclidinyl benzilate (QNB). [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-min ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation. Following 6-min ischemia and three 2-min ischemic insults. [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.     

Mapping second messenger systems in the rat hippocampus after transient forebrain ischemia: in vitro [H]forskolin and [H]inositol 1,4,5-triphosphate binding

Autoradiographic imaging demonstrated predominant and reciprocal localization of forskolin and inositol 1,4,5-trisphosphate (IP₃) binding sites in synaptic areas in the hippocampus. We produced selective damage to the CA₁ pyramidal cells in the rat hippocampus by means of transient forebrain ischemia and analyzed the alteration of the intracellular signal transduction using quantitative autoradiography of these second messenger systems. The dendritic fields (stritum oriens, radiutum and lacunosummoleculare) in the CA₁ showed 20% decrease in [³H]IP₃ binding activity 3 h after ischemia, when no morphological abnormalities contrast, the stratum pyramidale of the CA₁ showed no significant change until 2 days after recirculation. Seven days after ischemia, when CA₁ pyramidal cells were depleted, all layers in the CA₁ subfield lost 85% of [³H]IP₃ binding sites. In the CA₃ subfield, only a small and transient alteration in the [³H]IP₃ binding was noticed during recirculation. Postischemic reduction of [³H]forskolin binding sites was obvious in the CA₁ only 1 h after ischemia followed by loss of 50% of binding activity 7 days after recirculation. These results suggest that forskolin and IP₃ binding sites are predominantly distributed on the pyramidal cells in the CA₁ subfield and that marked alteration of intracellular signal transduction precedes the delayed CA₁ pyramidal cell death.     

Postischemic alteration of muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in selectively vulnerable areas: an autoradiographic study of gerbil brain.

We performed receptor autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of a voltage dependent L-type calcium channel blocker 1 h-1 month after transient cerebral ischemia in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Transient ischemia was induced for 10 min. [3H]QNB and [3H]CHA binding showed no significant alteration in selectively vulnerable areas at an early stage (1-24 h) of recirculation. However, the dentate molecular layer which was resistant to ischemia revealed a significant decrease in the [3H]CHA binding sites 24 h after ischemia. Thereafter, the [3H]QNB and [3H]CHA binding showed significant reduction in most of selectively vulnerable areas. Marked reduction was especially found in the dorsolateral part of striatum and the hippocampal CA1 sector which was the most vulnerable to ischemia. In contrast, [3H]PN200-110 binding showed a transient elevation in the hippocampal CA1 sector, the dentate molecular layer and the thalamus 1 h of recirculation. However, the striatum and neocortex revealed no alteration in the [3H]PN200-110 binding. Thereafter, the reduction in the [3H]PN200-110 binding was seen only in the dorsolateral part of the striatum and the hippocampal CA1 sector. The results suggest that transient cerebral ischemia can cause the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of L-type calcium channel blocker in most of selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium antagonist, adenosine A1, and muscarinic bindings in rat hippocampus after transient ischemia

The protective roles of Ca2+ channel blockers against ischemic hippocampal damage are still debated. We used autoradiography to study postischemic L-type Ca2+ channels (1,4-dihydropyridine Ca2+ channel blocker binding), adenosine A1 receptors, and muscarinic cholinergic receptors in the rat hippocampus using [3H]PN200-110 (PN), [3H]cyclohexyladenosine (CHA), and [3H]quinuclidinyl benzilate (QNB), respectively, in 49 rats subjected to 20 minutes of forebrain ischemia. The rats were decapitated after 1 (n = 7), 3 (n = 7), 6 (n = 8), 12 (n = 7), 24 (n = 6), 48 (n = 6), or 168 (n = 8) hours of recirculation; eight control rats were sham-operated but experienced no cerebral ischemia. Reduced receptor binding preceding the delayed death of CA1 pyramidal cells was first observed in the stratum oriens of the CA1 subfield. Significant reductions in [3H]PN, [3H]CHA, and [3H]QNB bindings of this stratum compared with control were noticed after 3 (35%, p less than 0.01), 12 (31%, p less than 0.01), and 1 (10%, p less than 0.05) hours of recirculation, respectively. By 168 hours after ischemia (when the populations of CA1 pyramidal cells were depleted) all strata in the CA1 subfield had lost most of their receptor sites, and [3H]PN, [3H]CHA, and [3H]QNB bindings in the stratum oriens were decreased to 23%, 30%, and 63% of control (p less than 0.01). Although [3H]PN binding in the CA3 subfield did not change significantly during 168 hours after ischemia, the histologically intact dentate gyrus exhibited a 31% loss of binding sites compared with control (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]diphenyl-acetoxy-N-methyl-piperidine ([³H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [³H]QNB and [³H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M₁, M₃ subtypes selective antagonist) > atropine (general) > pirenzepine (M₁). Methoctramine (M₂) didn’t antagonise the [³H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca²⁺ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M₁ and M₃ muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Sequential changes of cholinergic and dopaminergic receptors in brains after 6-hydroxydopamine lesions of the medial forebrain bundle in rats

Summary. We studied sequential changes in muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D₂ receptors in the brain after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [³H]Quinuclidinylbenzilate (QNB), [³H]hemicholinum-3 (HC-3) and [³H]raclopride were used to label muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D₂ receptors, respectively. The degeneration of nigrostriatal pathway produced a transient decrease in [³H]QNB binding in the parietal cortex of both ipislateral and contralateral sides at 2 and 8 weeks postlesion. [³H] QNB binding also showed a mild but insignificant decrease in the ipsilateral striatum throughout the postlesion periods. No significant change was observed in the substantia nigra (SN) of both ipsilateral and contralateral sides throughout the postlesion periods. In contrast, [³H]HC-3 binding showed no significant change in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. However, [³H]HC-3 binding was upregulated in the ipsilateral dorsolateral striatum throughout the postlesion periods. The ventromedial striatum also showed a significant increase in [³H]HC-3 binding at 1 week and 2 weeks postlesion. On the other hand, no significant change in [³H]raclopride binding was found in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. [³H]Raclopride binding showed a conspicuous increase in the ipsilateral striatum (35–52% of the sham-operated values in the lateral part and 39–54% in the medial part) throughout the postlesion periods. In the contralateral side, a mild increase in [³H]raclopride binding was also found in the striatum (10–15% of the sham-operated values in the lateral part and 22% in the medial part) after lesioning. However, a significant decline in [³H]raclopride binding was observed in the ipsilateral SN and ventral tegmental area during the postlesion. The present study indicates that 6-hydroxydopamine injection of medial forebrain bundle in rats can cause functional changes in high-affinity choline uptake site in the striatum, as compared with muscarinic cholinergic receptors. Furthermore, our studies demonstrate an upregulation in dopamine D₂ receptors in the striatum and a decrease in the receptors in the SN and ventral tegmental area after the 6-hydroxydopamine injection. Thus, these findings provide further support for neurodegeneration of the nigrostriatal pathway that occurs in Parkinson's disease. Received April 26, 1999; accepted November 12, 1999     

A1 and A2A adenosine receptors and A1 mRNA in mouse brain: effect of long-term caffeine treatment

The effect of oral treatment with caffeine, in doses that are known to produce marked adaptive effects, was investigated on A₁ and A_2A receptors in the mouse brain. Caffeine (0.1, 0.3 or 1 g/l) was added to the drinking water and the animals were sacrificed after a 14-day treatment period. Ligand binding to A₁ receptors was studied, using quantitative autoradiography, with the agonist [³H]cyclohexyladenosine (CHA) and the antagonist [³H]1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Caffeine did not remain in the sections during the autoradiography experiments. Caffeine treatment (1 g/l, but not 0.1 or 0.3 g/l) tended to increase [³H]CHA binding to the CA3 subfield of the hippocampus, but in no other region studied. There was no change in the number of A₁ receptors since [³H]DPCPX binding to the CA3, cerebral and cerebellar cortex was not influenced by caffeine treatment. There was similarly no change in the ability of CHA to displace [³H]DPCPX binding, suggesting that there are no major changes in the proportion of A₁ receptors that are coupled to G-proteins. mRNA for the A₁ receptor, measured by in situ hybridization, did not differ significantly between caffeine-treated and control mice in the structures examined. Thus, higher doses of caffeine can cause an increase in A₁ agonist binding without a corresponding change in A₁ mRNA or in A₁ antagonist binding, suggesting that the adaptive changes seen upon prolonged caffeine treatment may be in sites different from A₁ receptors. Caffeine (1 g/l) increased A_2A receptors in the striatum measured as binding of the agonist [³H]CGS 21680 suggesting that up-regulation of A_2A receptors may be an adaptive effect of caffeine intake.     

Effects of naftidrofuryl oxalate, a 5HT2 antagonist, on neurotransmission and transduction systems in the gerbil hippocampus

We investigated the effects of age and naftidrofuryl oxalate (Naftidrofuryl), a 5-HT₂ antagonist, on neurotransmission and transduction systems in the gerbil hippocampus using quantitative autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]cyclohexyl-adenosine (CHA), [³H]MK-801, and [³H]muscimol were used to label muscarinic acetylcholine, adenosine A1, N-methyl-d-aspartate (NMDA), and γ-aminobutyric acid-A (GABA_A) receptors, respectively. [³H]PN200-110 labeled L-type Ca²⁺ channels. [³H]Forskolin, [³H]cyclic adenosine monophosphate (cAMP), [³H]phorbol 12,13-dibutyrate (PDBu), and [³H]inositol 1,4,5-triphosphate (IP₃) were used to label adenylate cyclase, cAMP-dependent protein kinase, protein kinase C (PKC), and IP₃ receptors, respectively. Approximately 20% reductions in [³H]QNB, [³H]forskolin, and [³H]PDBu binding were observed in the hippocampus of 9-month-old gerbils in comparison with 5-week-old gerbils. Treatment with Naftidrofuryl (10 mg/kg, i.p., once a day for 7 days) ameliorated these reductions. No changes were found in [³H]CHA, [³H]MK-801, [³H]muscimol, [³H]PN200-110, [³H]cAMP, and [³H]IP₃ binding. The results suggest that Naftidrofuryl may have beneficial effects on the age-related alterations in signal transmission and transduction systems in the brain. Because the acetylcholine system, adenylate cyclase, and PKC are considered to be involved in learning and memory processes, the result may have clinical implications.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.     

Autoradiographic analysis of regional alterations in brain receptors following chronic administration and withdrawal of typical and atypical neuroleptics in rats

Summary Rats were administered haloperidol, clozapine, raclopride, or no drug for 28 days or 8 months. Following a 3 week withdrawal period, in vitro autoradiography was utilized to examine receptor binding for dopamine D2 ([³H]spiperone and [³H]raclopride), dopamine D1 ([³H]SCH23390), GABA_A ([³H]muscimol), benzodiazepine ([³H]RO15-1788), and muscarinic ACh receptors ([³H]QNB). [³H]spiperone was elevated in striatal subregions only in haloperidol-treated rats, with the largest increases seen in the 8 month duration animals. Striatal [³H]raclopride binding was increased after both short- and long-term treatment in both haloperidol and raclopride, but not clozapinetreated animals. Clozapine-treated rats showed significant increases in [³H]SCH23390 in the nucleus accumbens after 28-day administration; otherwise no changes were seen for this ligand in any other groups. Increases in [³H]muscimol binding in the substantia nigra reticulata were seen in haloperidol-treated rats after 8 month treatment. Binding of [³H]QNB and [³H]RO15-1788 were not significantly different from control for any of the drug-treated groups. These data suggest that persisting alterations in receptor binding are primarily seen in dopamine D2 and GABA receptors after withdrawal from chronic administration of haloperidol but not the atypical neuroleptics, clozapine and raclopride.     

Regional changes in [H]inositol 1,4,5-triphosphate binding in the gerbil brain following repeated sublethal ischemia

We investigated the regional changes in [³H]inositol 1,4,5-triphosphate (IP₃) binding in the brain following ischemia using in vitro autoradiography. Three 2-min ischemic insults at 1-hr intervals and a 6-min period of ischemia were induced in gerbils and they were killed after 1, 4, and 28 days. Normal animals had high [³H]IP₃ binding in the CA1 subfield of the hippocampus and the striatum. The binding in the CA1 decreased strikingly after both 6-min ischemia and three 2-min ischemic insults. The [³H]IP₃ binding also decreased in the lateral striatum after three 2-min ischemic insults but not after 6 min of ischemia. Histological observations confirmed neuronal damage to these areas of reduced binding. By contrast, we found a marked increase in [³H]IP₃ binding in the ventral thalamus 28 days after three 2-min ischemic insults. Histological observations with Nissl staining revealed an accumulation of fine granular deposits there. Thus, repeated ischemic insults produced more extensive neuronal damage and changes in [³H]IP₃ binding than a single equivalent period of ischemia. The increased [³H]IP₃ binding in the thalamus coincidentally with an accumulation of Nissl-positive granules at the chronic stage after repeated ischemia is of considerable interest.     

Effect of NG-nitro-L-arginine methyl ester a nitric oxide synthase inhibitor on neurotransmitter receptor systems in aged rats

In order to examine the effect of age and nitric oxide synthase inhibitor N^G-nitro-L-arginine methl ester (l -NAME) we studied the changes on major neurotransmitter receptor systems in 6 (adult and 24-month-old (aged) Fischer male rats using receptor autoradiography. l -name was administrated intraperitoneally in aged rats once a day for 4 weeks. [³H]QNB (quinuclidinyl benzilate) ³HC (hemicholinium-3) [³H] muscimol ³H] SCH 23390 ([N-methyl-³H] N-methyl-³H]R[+]-8-chloro-2 3 4 5-tetrahydro-3-methyl-5-phenyl-7-il-benzazepine) ³H] mazindol were used as markers of muscarinic acetylcholine receptors high-high-affinity choline uptake sites GABA_A (γ-aminobutyric acid (SP2)_A) receptors dopamine D₁ receptors dopamine D₂ receptors and dopamine uptake sites respectively. The age-related change in ³H muscimol binding in the brain was more pronounced than that in [³H] QNB ³H]HC ³H]SCH 23390 ³H] nemonapride and ³H] nemonapride and ³H] mazindol binding.Chronic treatment (4 weeks) with l -NAME caused no significant changes in [sp1)³H] muscimol ³H SCH 23390 and [³H] nemonapride binding in most areas of aged rat brain as compared with vehicle-treated aged animals. However chronic treatment with l -NAME caused a significant reduction in ³H] HC and ³H] mazindol binding in any brain regions of aged rats in comparison with the vehicle-treated aged animals. These results demonstrate that the GABAergic system is more susceptible to aging processes than cholinergic and dopaminergic systems in teh brain. Furthermore our findings suggest that nitric oxide may play some role in the regulation of choline uptake and dopamine uptake systems during aging processes.     

Antagonism of muscarinic receptors in the rabbit iris-ciliary body by 8-OH-DPAT and other 5-HT1A receptor agonists

Summary Physiological studies have shown that serotonin and 5-HT_1A agonists can influence muscarinic function in the rabbit iris-ciliary body (ICB). The purpose of this study was to examine whether a direct interaction exists between muscarinic and 5-HT_1A receptors in the ICB. At high concentrations, the 5-HT_1A agonist 8-OH-DPAT attenuated the carbachol-induced stimulation of inositol phosphates (InsPs) production, but this was not blocked by the presence of 5-HT_1A antagonists. In contrast, serotonin failed to influence carbachol-induced InsPs formation. Moreover, 8-OH-DPAT but not serotonin displayed affinity for [³H]QNB binding sites in the ICB. The combined data suggest that activation of 5-HT_1A receptors in the ICB does not cause a modulation of muscarinic receptor-stimulated phosphoinositide turnover. The data instead suggest that, at high concentrations, 8-OH-DPAT acts as an antagonist at muscarinic receptors and in this way influences muscarinic receptor function. The mechanism of 5-HT-induced modulation of muscarinic function in the ICB therefore remains to be elucidated.     

GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography

Quantitative receptor autoradiography was used to measure the binding of gamma-aminobutyric acid (GABA) and benzodiazepine receptors after ischemia by means of transient occlusion of bilateral common carotid arteries in the gerbil. [3H]Muscimol was used to label the GABAA receptors and [3H]flunitrazepam to label central type benzodiazepine receptors. In the superolateral convexities of the frontal cortices, [3H]muscimol binding was increased in 60% of the animals killed 3 days after ischemia, and decreased in 67% of the animals killed 27 days after ischemia. Twenty-seven days after ischemia, [3H]flunitrazepam binding in the substantia nigra pars reticulata increased to 252% of the control, though the increase in [3H]muscimol binding was not significant. In the dorsolateral region of the caudate putamen, marked neuronal necrosis and depletion of both [3H]muscimol and [3H]flunitrazepam binding sites were observed 27 days after ischemia, the ventromedial region being left intact. In spite of the depletion of pyramidal cells in the CA1 region of the hippocampus, both [3H]muscimol and [3H]flunitrazepam binding sites were preserved 27 days after ischemia. Since our previous study revealed that adenosine A1 binding sites were depleted in the CA1 subfield of the hippocampus after ischemia correlating with neuronal damage, GABAA and benzodiazepine receptors may not be distributed predominantly on the pyramidal cells in the CA1 region.     

KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

Autoradiographic analysis of second messenger and neurotransmitter system receptors in the gerbil hippocampus following transient forebrain ischemia.

Changes in second messenger and neurotransmitter system receptor ligand binding induced by transient forebrain ischemia were studied in the gerbil hippocampus. The animals were allowed variable periods of recovery ranging from 2 h to 7 days after 5-min bilateral carotid artery occlusion. The binding of second messenger systems ([3H]inositol 1,4,5-trisphosphate ([3H]IP3)to inositol 1,4,5-triphosphate, [3H]forskolin to adenylate cyclase and [3H]phorbol 12,13-dibutylate to protein kinase C) and neurotransmitter receptor systems ([3H]PN200-110 to L-type calcium channels. [3H]N6-cyclohexyl-adenosine to adenosine A1 and [3H]quinuclidinyl benzilate to muscarinic cholinergic receptor) were assayed using quantitative autoradiography. In the CA1 subfield, 2 h after ischemia, [3H]IP3, [3H]forskolin, and [3H]quinuclidinyl benzilate binding activities significantly decreased by 25, 17 and 13%, respectively, though no morphological abnormalities were obvious. Six hours after ischemia, the [3H]phorbol 12,13-dibutylate binding activity in the stratum oriens of the CA1 subfield increased by 15%. One day after ischemia, [3H]PN200-110 binding activity in this subfield decreased by 26%, and 7 days after ischemia, [3H]phorbol 12,13-dibutylate and [3H]N6-cyclohexyl-adenosine receptor binding activities decreased in this subfield. In particular, at 7 days after ischemia, [3H]IP3 binding activity in the CA1 subfield showed a complete decline. In the CA3 subfield, [3H]PN200-110 binding activity decreased 2 days after ischemia, and [3H]IP3 and [3H]N6-cyclohexyl-adenosine binding activities decreased 7 days after ischemia. In the dentate gyrus, the structure of which remained histologically intact after ischemic insult, [3H]IP3 and [3H]forskolin binding activities decreased 7 days after ischemia. In contrast, the [3H]phorbol 12,13-dibutylate binding activity increased in the molecular layer of the dentate gyrus 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield and that the histologically intact CA3 and dentate gyrus also shows modulated neuronal transmission after ischemia.     

Differential effects of the adenosine A1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine, [3H]muscimol, and [3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting down-regulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.     

Amnesic effects of the anticholinergic drugs, trihexyphenidyl and biperiden: differences in binding properties to the brain muscarinic receptor

An amnesic effect of anticholinergic drugs was previously described from several behavioral studies. We examined this effect induced by trihexyphenidyl and biperiden, clinically used in the parkinsonism and schizophrenic patients, by using passive avoidance tasks. Both of these drugs (0.1–10 mg/kg, s.c.) showed dose-dependent amnesic effects in the acquisition and retrieval phases. However, the effect induced by trihexyphenidyl was transient, whereas that of biperiden was long-lasting. To clarify the reason for the different duration of the amnesic activity, binding to the muscarinic receptor was examined. In the Scatchard analysis, trihexyphenidyl competed with [³H]quinuclidinyl benzilate ([³H]QNB) on the muscarinic receptor (showed increased K_d and unchanged B_max value), while biperiden decreased [³H]QNB binding (B_max value) significantly. Furthermore, in an exchange assay for receptor inactivation, trihexyphenidyl binding to muscarinic receptors was exchanged by [³H]QNB completely, but biperiden decreased the exchangeable binding of [³H]QNB in a dose dependent manner (0.1–100 nM). These results suggested that the binding of trihexyphenidyl and biperiden to muscarinic receptor might be completely reversible and partially irreversible, respectively, whereas the K_i values of these two drugs were similar. In conclusion, this difference in binding property may explain the difference in the time-course of the amnesic effect induced by trihexyphenidyl and biperiden.     

Regional distribution of cholinergic muscarinic receptors in spinal cord

Quinuclidinylbenzilate ([³H]QNB) binding sites are present in the rat spinal cord. The binding sites are muscarinic in character based on displacement of [³H]QNB by cholinoceptive drugs. They are distributed rather uniformly along the cord, although the receptor density is greater in gray matters than in white matter. Binding to white matter may be associated with glial cells. Within the gray matter, the receptor density is higher in the ventral born than in the dorsal horn. In the thoracic region receptor density is about equal in the intermediate zone and ventral horn. Midthoracic transection of the cord does not change the receptor density or the dissociation constant of [³H]QNB in the lumbar cord. In contrast, treatment with the neurotoxin, 6-aminonicotinamide, which produces lesions of the cord, loss of motor control and paralysis, reduces the receptor density and affinity of [³H]QNB for lumbar gray matter but not white matter. The presence of [³H]QNB binding sites throughout the spinal cord as well as the documented presence of acetylcholine-containing neurons, suggest that muscarinic receptors play a role in all phases of spinal cord physiology.