Muscarinic Acetylcholine Receptor Binding Research Articles

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 μM, respectively).

In vivo and in vitro changes in neurochemical parameters related to mercury concentrations from specific brain regions of polar bears (Ursus maritimus).

Mercury (Hg) has been detected in polar bear brain tissue, but its biological effects are not well known. Relationships between Hg concentrations and neurochemical enzyme activities and receptor binding were assessed in the cerebellum, frontal lobes, and occipital lobes of 24 polar bears collected from Nunavik (Northern Quebec), Canada. The concentration-response relationship was further studied with in vitro experiments using pooled brain homogenate of 12 randomly chosen bears. In environmentally exposed brain samples, there was no correlative relationship between Hg concentration and cholinesterase (ChE) activity or muscarinic acetylcholine receptor (mAChR) binding in any of the 3 brain regions. Monoamine oxidase (MAO) activity in the occipital lobe showed a negative correlative relationship with total Hg concentration. In vitro experiments, however, demonstrated that Hg (mercuric chloride and methylmercury chloride) can inhibit ChE and MAO activities and muscarinic mAChR binding. These results show that Hg can alter neurobiochemical parameters but the current environmental Hg exposure level does have an effect on the neurochemistry of polar bears from northern Canada.

Flavonoids with M1 muscarinic acetylcholine receptor binding activity.

Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM), comparable to that of acetylcholine (Ki = 59 µM). Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice.

Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are more severe in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal days 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood.

Phospholipase C-β1 knockout mice exhibit endophenotypes modeling schizophrenia which are rescued by environmental enrichment and clozapine administration

Phospholipase C-beta1 (PLC-beta1) is a rate-limiting enzyme implicated in postnatal-cortical development and neuronal plasticity. PLC-beta1 transduces intracellular signals from specific muscarinic, glutamate and serotonin receptors, all of which have been implicated in the pathogenesis of schizophrenia. Here, we present data to show that PLC-beta1 knockout mice display locomotor hyperactivity, sensorimotor gating deficits as well as cognitive impairment. These changes in behavior are regarded as endophenotypes homologous to schizophrenia-like symptoms in rodents. Importantly, the locomotor hyperactivity and sensorimotor gating deficits in PLC-beta1 knockout mice are subject to beneficial modulation by environmental enrichment. Furthermore, clozapine but not haloperidol (atypical and typical antipsychotics, respectively) rescues the sensorimotor gating deficit in these animals, suggesting selective predictive validity. We also demonstrate a relationship between the beneficial effects of environmental enrichment and levels of M1/M4 muscarinic acetylcholine receptor binding in the neocortex and hippocampus. Thus we have demonstrated a novel mouse model, displaying disruption of multiple postsynaptic signals implicated in the pathogenesis of schizophrenia, a relevant behavioral phenotype and associated gene-environment interactions.

Altered Muscarinic Acetylcholine Receptor Subtype Binding in Neonatal Rat Brain following Exposure to Chlorpyrifos or Methyl Parathion

The neurodevelopmental effects of two organophosphorus (OP) insecticides, chlorpyrifos (CPS) and methyl parathion (MPS), on cholinesterase (ChE) activity and muscarinic acetylcholine receptor (mAChR) binding were investigated in neonatal rat brain. Animals were orally gavaged using an incremental dosing regimen from postnatal day 1 (PND1) until PND8 with a low, medium, and high dosage for both CPS and MPS. On PND4 and PND8, ChE activity was measured in whole brain while the total and subtype densities of mAChRs were measured in three brain sections: area anterior to optic chiasma (anterior forebrain), area from the optic chiasma to the medulla/pons (posterior forebrain); and the medulla/pons excluding the cerebellum. The ligands 3H-pirenzepine, 3H-AF-DX 384, 3H-4-DAMP, and 3H-QNB were used to measure the maximal binding of the M1, M2/M4, and M3 subtypes and total mAChR receptors, respectively. In the anterior and the posterior forebrain, the levels of all mAChRs nearly doubled from PND4 to PND8, while in the medulla/pons, M1- and M3-subtype mAChR densities were low and did not increase and M2/M4 subtype and total mAChR slightly increased from PND4 to PND8. Reduction of ChE activity and mAChR binding by CPS or MPS was more evident in rats at PND8 than at PND4. With respect to mAChR binding, the greatest effects were observed in the medulla/pons and the least effects were observed in the posterior region of the forebrain. These results demonstrate that OPs exert adverse effects on rat central nervous system development through the cholinergic system in an age- and region-dependent manner.

Analysis of Pharmacological Effects of Drugs Used for Treatment of Urinary Disturbance Based on Anticholinergic and Smooth Muscle-Relaxing Effects

Propiverine hydrochloride, oxybutynin hydrochloride and terodiline hydrochloride have both anticholinergic and antispasmodic effects, and are used for the management of urinary frequency and incontinence. The average standard therapeutic doses of these drugs differ greatly. We retrospectively analyzed their pharmacological effects with consideration given to muscarinic acetylcholine receptor binding affinities, anticholinergic activities, and inhibitory effects on KCl-induced contraction. Muscarinic acetylcholine receptor occupancies and the inhibitory ratios of the drugs for both acetylcholine-induced and KCl-induced contraction in a steady state after oral administration of standard doses were calculated based on pharmacokinetics and the receptor occupancy theory. The average muscarinic acetylcholine receptor occupancy and inhibitory ratio of acetylcholine-induced contraction were estimated to be 12.6+/-1.06% and 3.27+/-0.74%, respectively, with no significant differences found between the drugs for those parameters. A significant linear relationship was found between muscarinic acetylcholine receptor occupancy and the maximum ratio of increase in bladder urinary capacity. On the other hand, the inhibitory ratios of KCl-induced contraction varied from 0.01 to 0.48%. The present results suggest that muscarinic acetylcholine receptor occupancy is a principal determinant of the therapeutic effect of a drug used for treatment of urinary disturbance.

Dietary and In Utero Exposure to a Pentabrominated Diphenyl Ether Mixture Did Not Affect Cholinergic Parameters in the Cerebral Cortex of Ranch Mink (Mustela vison)

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are recognized as global environmental contaminants and a potential health risk. They have been shown to elicit neurodevelopmental toxicity through disruption of the cholinergic neurotransmitter system in rodent models, but the effects of environmentally relevant exposures in wildlife species are unknown. The objective of this study was to assess the effects of the commercial pentabrominated diphenyl ether mixture DE-71 on cholinergic parameters in ranch mink (Mustela vison) following dietary exposure of adult females and in utero, lactational, and dietary exposure of their offspring. Adult females were fed diets containing 0, 0.1, 0.5, or 2.5 microg DE-71/g feed from four weeks prior to breeding through weaning of their kits at six weeks of age. A portion of the weaned kits were maintained on their respective diets through 27 weeks of age. Cholinergic parameters, including muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) binding, cholinesterase (ChE) activity, and acetylcholine (ACh) concentration, were assayed in the cerebral cortex, and ChE activity was measured in the plasma. In the cerebral cortex, results indicated a significant exposure-dependent increase in PBDE concentrations, but no significant effects of DE-71 on cholinergic parameters. There was a threefold increase in ChE activity in the plasma of adult females in the 2.5 microg DE-71/g feed group, but was likely due to effects on liver function. This study demonstrated that environmentally relevant exposures to DE-71 did not affect key parameters of the cholinergic neurotransmitter system in the brain of ranch mink.

Sexually dimorphic effects of the Lhx7 null mutation on forebrain cholinergic function

It has been reported recently that mice lacking both alleles of the LIM-homeobox gene Lhx7, display dramatically reduced number of forebrain cholinergic neurons. In the present study, we investigated whether the Lhx7 mutation affects male and female mice differently, given the fact that gender differences are consistently observed in forebrain cholinergic function. Our results show that in adult male as well as female Lhx7 homozygous mutants there is a dramatic loss of choline acetyltransferase immunoreactive forebrain neurons, both projection and interneurons. The reduction of forebrain choline acetyltransferase immunoreactive neurons in Lhx7 homozygous mutants is accompanied by a decrease of acetylcholinesterase histochemical staining in all forebrain cholinergic neuron target areas of both male and female homozygous mutants. Furthermore, there was an increase of M1-, but not M2-, muscarinic acetylcholine receptor binding site density in the somatosensory cortex and basal ganglia of only the female homozygous mutant mice. Such an increase can be regarded as a mechanism acting to compensate for the dramatically reduced cholinergic input, raising the possibility that the forebrain cholinergic system in female mice may be more plastic and responsive to situations of limited neurotransmitter availability. Finally, our study provides additional data for the sexual dimorphism of the forebrain cholinergic system, as female mice appear to have a lower density of M1-muscarinic acetylcholine receptors in the striatal areas of the basal ganglia and a higher density of M2-muscarinic acetylcholine receptors, in a number of cortical areas, as well as the striatal areas of the basal ganglia.

Effects of cochlear ablation on muscarinic acetylcholine receptor binding in the rat cochlear nucleus

Cholinergic synapses in the cochlear nucleus (CN) have been reported to modulate spontaneous activity via muscarinic acetylcholine receptors. In this study, muscarinic receptor binding was measured as specific binding of 1-[N-methyl-(3)H]scopolamine in CN regions of control rats and 7 days, 1 month, and 2 months after unilateral cochlear ablation. In control rats, the strongest binding was found in granular regions, followed in order by fusiform soma, molecular, and deep layers of the dorsal cochlear nucleus (DCN), with much lower binding in the anteroventral CN (AVCN) and posteroventral CN (PVCN). After unilateral cochlear ablation, binding in the AVCN, PVCN, and their associated granular regions on the lesion side became progressively greater than on the control side through 2 months after lesion. A significant asymmetry, with binding higher on the lesion side, was also found in the DCN fusiform soma layer at 7 days, and there and in the DCN deep layer at 1 and 2 months after lesion. There was also evidence of increased binding on the control side in most CN regions. By contrast, binding in the ipsilateral facial nucleus decreased, compared with the control side, by 7 days after the lesion and showed some recovery toward symmetry by 2 months after lesion, and there was no evidence for contralateral changes. These muscarinic receptor binding changes reflect receptor plasticity after loss of auditory nerve innervation. Such plasticity may underlie some of the central auditory functional changes that occur following peripheral lesions, such as tinnitus and hyperacusis.

Biochemical Markers of Neurotoxicity in Wildlife and Human Populations: Considerations for Method Development

Disruption of neurochemical parameters in blood and brain tissues can be used as early biomarkers of neurotoxicity in human and wildlife epidemiological studies. To investigate the feasibility of biomarker measurements in field samples obtained from remote locations, tissue storage limits were determined with human blood and mink cortex tissue using efficient and cost-effective microplate assays. Results show that isolated blood platelets and plasma can be stored at 4°C for 4 wk before measurement of monoamine oxidase (MAO) and cholinesterase (ChE) activities, while human lymphocytes can be stored at 4°C for up to 2 d before muscarinic acetylcholine (mACh) receptor binding analysis. Blood cells stored frozen resulted in decreased MAO activity and mACh receptor function. These data suggest that mink brain tissue obtained from field samples can be stored at various temperatures without affecting dopamine (D2) and mACh receptor densities; however, MAO and ChE activities were most stable in samples stored in a −20°C domestic freezer or at 4°C. Multiple freeze/thaw cycles alter mACh and D2 receptors and MAO activity in mink cortex samples and should therefore be minimized. In conclusion, these neurochemical biomarkers can efficiently be measured in large human and wildlife neurotoxicity studies, provided proper storage conditions are maintained. This study was financially supported by the Collaborative Mercury Research Network (COMERN) and the Natural Science and Engineering Research Council (NSERC) of Canada. C. J. Stamler and N. Basu are both recipients of NSERC postgraduate fellowships. We also thank Mary Gamberg (Gamberg Consulting, Whitehorse, Yukon Territory) and Dr. Steven J. Bursian (Michigan State University, East Lansing) for kind donation of mink tissues. Assistance by Donna Leggee, Leah Tivoli, Kovana M. Loua, and Stephanie Bailey is greatly appreciated.

Functional changes of nicotinic acetylcholine receptor in muscle and lymphocyte of myasthenic rats following acute dimethoate poisoning

The mechanism underlying intermediate myasthenia syndrome (IMS) following acute organophosphate poisoning remains largely unknown. Previous studies indicated that the mechanism of myasthenia in rats and IMS patients is most likely due to a postsynaptic neurotransmission blocking at neuromuscular junctions (NMJ). Nicotinic acetylcholine receptor (nAChR) is a key postsynaptic component at NMJ. Whether functional changes of nAChR are related to the development of myasthenia has not been demonstrated and addressed in vivo so far. In this study, we attempted to investigate temporal and spatial changes of nAChR in the blood lymphocyte, muscle and brain of rats during the course of myasthenia after acute dimethoate poisoning by using radioligand-binding assay. We found that specific nAChR binding activity in the gastrocnemius muscle and blood lymphocytes of myasthenia rats was significantly increased at 48 h after dimethoate poisoning. However, no changes of nAChR binding activity were found in the lymphocytes and muscle of non-myasthenia rats which were sacrificed at 1 h after intoxication. Interestingly, no changes of nAChR and muscarinic acetylcholine receptor (mAChR) binding activity were found in the cerebrum and cerebellum of all rats after dimethoate intoxication either at 1 or 48 h. The change of nAChR specific binding activity in the lymphocytes is parallel with that in the skeletal muscle during the development of myasthenia. This implied that the changes of nAChR receptor binding activity in the skeletal muscle and lymphocytes are highly associated with the development of myasthenia. The functional changes of nAChR at NMJ might play an important role in the paralysis of skeletal muscle following acute organophosphates (OPs) poisoning.

A high n-6 polyunsaturated fatty acid diet reduces muscarinic M2/M4 receptor binding in the rat brain

The aim of this study was to examine the influence of different fat diets on muscarinic acetylcholine receptor binding. Nineteen male Sprague–Dawley rats were divided into four groups and fed a diet of either high saturated fat, n-6 polyunsaturated fatty acid (PUFA), n-3 PUFA or low fat (control) for 8 weeks. Using quantitative autoradiography, [ 3H]pirenzepine binding to muscarinic M1/M4 receptors and [ 3H]AF-DX384 binding to M2/M4 receptors were measured throughout the brain in all four groups. The main findings were that compared to the low fat control group, M2/M4 receptor binding was significantly reduced in the dorsolateral, dorsomedial and ventromedial parts of the caudate putamen (61–64%, p < 0.05), anterior cingulate cortex (59%, p < 0.01), dentate gyrus and CA1–3 fields of the hippocampus (32–43%, p < 0.01) of rats on a high n-6 PUFA diet; however, no differences in M1/M4 receptor binding densities between the four groups were observed. These results suggest that a diet high in n-6 PUFA, but not of n-3 PUFAs or saturated fat, may selectively alter M2/M4 receptor-mediated signal transduction in the rat brain.

An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3H]-quinuclidinyl benzilate ([ 3H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density ( B max) and ligand affinity ( K d). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants ( K i). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

Discrepancy between cell injury and benzodiazepine receptor binding after transient middle cerebral artery occlusion in rats.

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.

Effects of myo-Inositol Versus Fluoxetine and Imipramine Pretreatments on Serotonin 5HT2Aand Muscarinic Acetylcholine Receptors in Human Neuroblastoma Cells

myo-Inositol (mI) is a key metabolic precursor to the phospoinositide (PI) metabolic pathway as a key component of central G-protein coupled receptor signaling systems, including several subtypes of adrenergic, cholinergic, serotonergic and metabotropic glutamatergic receptors. High dose mI has also been shown to be clinically effective in the treatment of obsessive-compulsive disorder, as well as panic and depression, although its mechanism of action remains elusive. The current study aimed to investigate the possible modulatory role of mI versus fluoxetine or imipramine pretreatments on serotonin-2A receptor (5HT2A-R) and muscarinic acetylcholine receptor (mAChR) function and binding in in vitro systems. After pretreating human neuroblastoma cells with different concentrations of mI, fluoxetine, or imipramine, receptor function was measured by second messenger [3H]-IPx accumulation and [35S]-GTPgammaS binding to G alpha(q) protein. Total [3H]-mI uptake into cells was measured, as well as specific receptor binding to determine receptor binding after the pretreatments. Results suggest that mI reduces 5HT2A-R function at the receptor-G protein level. While fluoxetine also reduced 5HT2A-R function, but to a lesser degree, imipramine increased 5HT2A-R function, which may explain why mI seems to be effective exclusively in selective serotonin reuptake inhibitor-sensitive disorders. In addition mI, and at high concentrations fluoxetine and imipramine, also reduces mAChR function. Furthermore the results suggest that the attenuating effect of mI on mAChRs is partially dependent on the PI metabolic pathway. The data provide novel information on understanding the mechanism of action of mI in depression and related anxiety disorders and added to the evidence suggesting a role for the cholinergic system in the pathophysiology of depression.

The effect of chlorpyrifos and chlorpyrifos-oxon on brain cholinesterase, muscarinic receptor binding, and neurotrophin levels in rats following early postnatal exposure.

Chlorpyrifos (CPS) is a widely used diethyl organophosphorus insecticide in agricultural settings. Household and urinary residue analysis has suggested that children in agricultural communities are at risk of exposure to diethyl organophosphorus insecticides. The effects of repeated postnatal exposure to CPS and its metabolite chlorpyrifos-oxon (CPO) on total muscarinic acetylcholine receptor (mAChR) binding, nerve growth factor (NGF) levels, and brain derived neurotrophic factor (BDNF) levels in the forebrain of neonatal rats were investigated. Peak inhibition of brain cholinesterase (ChE) for CPS and CPO was determined after acute exposure to dosages of each compound (a low and a high for each), which produced similar degrees of initial ChE inhibition. Pups were administered CPS (1.5 or 3.0 mg/kg), CPO (0.25 or 0.35 mg/kg), or the corn oil vehicle by daily gavage from postnatal day 1 (PND 1) through PND 6. This exposure paradigm resulted in persistent ChE inhibition by CPS but only transient inhibition by CPO, suggesting that, even though the initial ChE inhibition is similar between compounds, the effects of repeated exposure differ significantly. Forebrain mAChR density, as measured by the binding of 3H-QNB, and NGF levels were significantly reduced on PND 4 and 7 after CPS but not on PND 12. No effects on mAChR density or NGF levels were observed with CPO. No effects on BDNF levels were observed with either compound. The data suggest that the persistent ChE inhibition and decreased mAChR binding may play a role in the decreased NGF levels following CPS exposure.

Sensitivities of benzodiazepine receptor binding and muscarinic acetylcholine receptor binding for the detection of neural cell death caused by sodium nitroprusside microinjection in rat brain.

Sodium nitroprusside (SNP) was microinjected into rat cerebral cortex and changes in muscarinic acetylcholine receptor (mAChR) binding and benzodiazepine receptor (BZR) binding were followed for 24 h after the infusion using [(3)H]-N-methyl-4-piperidyl benzilate ([(3)H]-NMPB) and [(3)H]-flumazenil, respectively, as a radioligand. The microinjection of SNP dose-dependently caused significant neural cell death 3 h after infusion, with the area of cell death becoming extensive 24 h after infusion. Neither SIN-1 nor NOC-18, other types of NO donors, caused neural cell death. Together with the result that deferoxamine, an iron-chelating agent, protected SNP-induced brain injury indicated important roles of iron-related radicals in SNP cytotoxicity in rat brain. In vitro [(3)H]-NMPB binding was significantly reduced in parallel with the time course of neural cell death detected by TTC staining and Nissl staining. In contrast, [(3)H]-flumazenil binding was essentially unaltered during the 24-h period after the SNP infusion. Similar results were observed in in vivo binding experiments. In vivo [(3)H]-NMPB binding was found to be much more sensitive at detecting cell death caused by SNP. On the other hand, [(3)H]-flumazenil binding in vivo was relatively insensitive to SNP-induced cell death. These results indicate that mAChR binding may be superior to BZR binding for detecting cell death in brain tissue, in contrast to what was previously thought.

Effect of rolipram on muscarinic acetylcholine receptor binding in the intact mouse brain.

The effect of rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE(4)) and elevating cyclic AMP (cAMP), on in vivo and in vitro (3)H-N-methylpiperidyl benzilate ((3)H-NMPB) binding to muscarinic acetylcholine receptors in the mouse brain was examined. Rolipram significantly decreased in vivo (3)H-NMPB binding in the cerebral cortex, hippocampus and striatum, whereas in vitro (3)H-NMPB binding in these regions was not altered. Saturation experiments on in vivo binding in conjunction with the kinetic analysis revealed that the apparent association rate constant (k(on)) of (3)H-NMPB binding in vivo was significantly decreased by rolipram. A similar decrease in the apparent association rate constant (k(on)) by rolipram was reported for dopamine D(1) and D(2) receptor binding in vivo. These results indicate that rolipram plays an important role in the global modulation of apparent rates of ligand-receptor interactions in the intact brain.

Changes in histological construction and decrease in 3H-QNB binding in the rat brain after prenatal X-irradiation.

To elucidate the mechanisms involved in deleterious neuronal and behavioral changes after prenatal ionizing irradiation, in vitro muscarinic acetylcholine (mACh) receptor binding and histological construction were investigated in 9-week old rat brains after 1.5 Gy X-ray exposure on embryonic day 15 (E15). A gross anatomical examination with a magnetic-resonance imaging system showed an irregular tissue construction in the hippocampus and cortex of the irradiated rat brain. Histological sections stained with hematoxylin and eosin also indicated that the structures of the hippocampus and cortex were obviously changed. In irradiated rats, the laminar structure of pyramidal cells was selectively deranged in the CA1 region. In vitro 3H-Quinuclidinyl benzilate binding in the hippocampus was significantly decreased (about 10%) in prenatal irradiated rats compared to that in sham-treated rats. On the other hand, no significant change in mACh receptor binding was observed in the cerebral cortex. The present study revealed that prenatal exposure to ionizing radiation may induce dysfunction of the cholinergic neuronal systems, especially in the hippocampus, resulting in deleterious changes in memory and behavior.