Muscarinic Acetylcholine Receptor Binding Research Articles

Chronic cerebral hypoperfusion induces striatal alterations due to the transient increase of NO production and the depression of glutathione content.

We examined the effects of chronic cerebral hypoperfusion on the endogenous oxidative stress-related indices, nitrite and nitrate (NOx) concentration, glutathione (GSH) content, superoxide dismutase and catalase activities, and thiobarbituric acid-reactive substances level in the rat striatum, to clarify the participation of oxidative stress in the chronic cerebral hypoperfusion-induced alterations. Our present results indicate that chronic cerebral hypoperfusion produces oxidative stress and disturbs intracellular redox regulation in two distinct phases: at 1 day, "acute" and at 6 weeks, "chronic" alterations after the operation. Therefore, striatal neural cell damage may be mainly attributed to the transient increase of NOx production at 1 day after, and the delayed reduction of muscarinic acetylcholine receptor binding in the striatum may be mostly attributed to the continuous depression of GSH content from the 1st to the 6th postoperative week. In particular, the continuous GSH depression may be considered to accompany the pathophysiology of chronic cerebral hypoperfusion.

Changes in mACh, NMDA and GABA(A) receptor binding after lateral fluid-percussion injury: in vitro autoradiography of rat brain frozen sections.

Adult rats were subjected to a moderate lateral fluid percussion injury (FPI), followed by survival periods of 2 and 12 h. Regional NMDA subtype glutamate, muscarinic acetylcholine and GABA(A) receptor binding in various brain regions was analysed by quantitative in vitro autoradiography and short-lived positron emission tomography tracers [11C]cyano-dizocilpine, 4-N-[11C]methylpiperidylbenzilate (4-N-[11C]MPB), and [11C]flumazenil, respectively. The binding potential (BP, Bmax/KD) was calculated. The data with [11C]cyano-dizocilpine showed a significant decrease in BP bilaterally for the frontoparietal cortex and hippocampus at both time points, in comparison with that of the sham-operated controls. At 12 h the decrease was significantly more prominent for the ipsilateral cortex and hippocampus than for the contralateral side. The BP of 4-N-[11C]MPB was significantly decreased after 2 h for the trauma-side hippocampus, and after 12 h it had decreased for the trauma-site cortex and the bilateral hippocampus. The [11C]flumazenil exhibited a significant decrease in BP for the trauma-site cortex and the underlying hippocampus by 2 h after the traumatic brain injury. After 12 h a significantly decreased BP was observed only for the trauma-site cortex. The finding of a decreased BP demonstrates the involvement of these receptor systems in the development of cellular dysfunction, which is widespread and not limited to the site of lateral FPI.

Pharmacology, distribution and development of muscarinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens.

Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5-9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes.These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for excitatory subtypes.

Early treatment with cyclosporin A ameliorates the reduction of muscarinic acetylcholine receptors in gerbil hippocampus after transient forebrain ischemia.

Recent evidence has suggested that cyclosporin A (CsA), an immunosuppressive agent, has neuroprotective properties. However, its mechanisms associated with this activity remain unclear. We have previously shown that post-ischemic administration of CsA daily for 14 days prevented the decrease of muscarinic acetylcholine receptor binding in the hippocampus in the gerbil model of 5-min transient forebrain ischemia. In the present study, CsA (5 mg/kg, subcutaneously) was administered to each animal just after, 2 and 6 h after ischemia so as not to exert its immunosuppressive effect. Initial CsA treatment significantly restored the declined muscarinic acetylcholine receptor binding of the hippocampus 14 days after ischemia similar to the previous report. However, CsA did not alter reactive changes of astrocytes and microglia in the CA1 area of the hippocampus, which had been suppressed by daily administration. These results indicate that CsA could positively modulate the hippocampal acetylcholine neurotransmission system broken down through the ischemia-induced pyramidal cell death and its action mechanism may have no relation to the immunosuppressive properties.

Using single photon emission tomography (SPECT) and positron emission tomography (PET) to trace the distribution of muscarinic acetylcholine receptor (MACHR) binding radioligands

Two [ 18F] labeled ligands for the mAChR were prepared and evaluated in rodents and nonhuman primates. The properties of both compounds, one an agonist and the other an antagonist, were consistent with M 2 subtype specificity.

Nitric oxide modulates muscarinic acetylcholine receptor binding in the cerebral cortex of gerbils.

To determine whether nitric oxide (NO) acts as a modulator of muscarinic acetylcholine receptor (mACh-R) function, we performed a radioligand receptor assay using [3H]quinuclidinyl benzylate ([3H]QNB), the NO radical (NO*) donor 3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC7) and a gerbil brain cortical membrane preparation. NOC7 (at 10 microM, 100 microM or 1 mM concentrations) significantly reduced the [3H]QNB binding Kd values (from 0.196 +/- 0.009 nM in the control, to 0.151 +/- 0.013, 0.144 +/- 0.012 and 0.153 +/- 0.007 nM respectively). NOC7 did not alter the displacement curves of atropine or carbachol. Reduction of SH groups with dithiothreitol, in the presence of the NO donor, significantly increased [3H]QNB binding affinity whereas alkylation by N-ethylmaleimide markedly decreased it. The observed enhancing effect on mACh-R binding affinity for [3H]QNB, may reflect conformational changes in the receptors mediated by the NO generated, and these changes might be explained by NO reactions with such groups through conditions supporting redox reactions intrinsic to the NO molecule, similar to those occurring in redox regulatory sites reported for other neurotransmitter pathways in the CNS.

Chronic cerebral hypoperfusion disrupts discriminative behavior in acquired-learning rats

We describe an `acquired-learning' rat model that was used to investigate the effects of chronic cerebral hypoperfusion on the maintenance of previously acquired discriminative behavior using the discriminating learning task. Male Wistar rats, aged 11 weeks, were trained to discriminate between lamp-on and lamp-off states under an operant-type learning procedure. After 30 sessions, we selected `acquired-learning' rats with an average discrimination ratio higher than 75% recorded during the last three sessions. Chronic hypoperfusion was then induced by permanent ligation of both common carotid arteries under pentobarbital anesthesia. The rats were tested after surgery over a period of 12 weeks and brain tissue was analyzed for muscarinic acetylcholine receptor (mACh-R) binding. Cerebral hypoperfusion resulted in a significant reduction in the discrimination ratio throughout the observation period, compared with sham-operated rats. However, chronic hypoperfusion would not affect on motor function. The maximum number of mACh-R examined 12 weeks after the operation was significantly reduced in the frontal cortex and hippocampus in the hypoperfusion group. Impaired discrimination learning was associated with a reduction in mACh-R. Our findings suggest that chronic cerebral hypoperfusion in acquired-learning rats is a useful model for investigating the pathophysiology of dementia and that cortical and/or hippocampal cholinergic systems contributes to learning impairment, at least, in our learning task.

Repeated administration of tacrine to normal rats: Effects on cholinergic, glutamatergic, and GABAergic receptor subtypes in rat brain using receptor autoradiography

Tacrine, a potent acetylcholinesterae inhibitor, has been reported to improve cognitive function in patients with Alzheimer's disease. The present investigation was conducted to elucidate in vivo any interaction between tacrine-induced cortical cholinergic hyperactivity and glutamatergic and GABAergic neurotransmission, which might influence the therapeutic potential of tacrine. Seven days after a daily dosage of 10 mg/kg tacrine i.p. quantitative receptor autoradiography was performed in coronal sections throughout the brain. Repeated administration of tacrine resulted in decreased binding to high-affinity choline uptake, nicotinic and M 2-muscarinic acetylcholine receptor sites in a number of cortical regions, while reductions in M 1-muscarinic receptor binding were restricted to the cingulate and entorhinal cortex as well as caudate-putamen. Moreover, tacrine injections decreased cortical AMPA receptor binding throughout the brain, while NMDA, kainate, and GABA A receptor binding remained unchanged. Tacrine administration alters cortical AMPA receptor binding in the opposite direction to that observed in patients with Alzheimer's disease, suggesting that tacrine may exert a reversal in up/down-regulation of cortical glutamate receptor subtypes in Alzheimer patients. However, the drug-induced reductions in cortical high-affinity choline uptake sites as well as in nicotinic and in muscarinic acetylcholine receptor binding might partially counteract the cognition-enhancing effects of tacrine produced by acetylcholinesterase inhibition.

Discrepancy between blood flow and muscarinic receptor distribution in rat brain after middle cerebral artery occlusion.

To clarify whether muscarinic acetylcholine receptor (mAChR) binding can be a viable muscarinic neuronal marker which provides therapeutic information different from perfusional information in global brain, we evaluated the discrepancy between the distribution of cerebral blood flow (CBF), mAChR and its five subtypes of messenger ribonucleic acid (mRNA) in the acute (n=9) and chronic (n=8) phases of a middle cerebral artery (MCA) occlusion model and in sham-operated controls (n=6). In the acute phase, regional CBF was markedly reduced in the MCA territory, whereas mAChR was not reduced and the mRNA was reduced only slightly. In the chronic phase, mAChR was reduced markedly in the infarcted lesion and the mRNA was also reduced. The mAChR was slightly reduced in the ipsilateral substantia nigra and pontine nucleus because of remote effects; however, regional CBF in the substantia nigra was slightly increased and did not change in the pontine nucleus. The discrepancy between CBF and mAChR was clarified, and the tendency toward a reduction in mRNA in the acute ischaemic region without a reduction in mAChR suggested the presence of cholinergic neurons which were viable but hypometabolic. It is concluded that mAChR imaging may be of value for the assessment of the viable cholinergic neuron density in vivo.

Long-term effects of chronic ethanol on muscarinic receptor binding in rat brain.

Effects of chronic ethanol treatment (CET) on muscarinic acetylcholine receptor (mAChR) binding properties were investigated via quantitative autoradiography in rats maintained on an ethanol-containing liquid diet for 28 weeks and withdrawn from ethanol for 8 weeks before harvesting of tissues. Controls received an identical diet in which sucrose was substituted isocalorically for ethanol. Maximal binding of the radiolabeled mAChR antagonist quinuclidinyl benzilate ([3H]QNB) was not reduced in hippocampal area CA1, dentate gyrus, neocortex, striatum, or thalamus, suggesting that CET results in no significant mAChR loss in these regions. Binding affinities of the cholinergic agonist carbachol to mAChRs were unaffected by CET in each of these regions, as determined by competitive displacement of [3H]QNB labeling. These results suggest that CET-induced functional deficits in brain cholinergic responses are not due to direct effects of CET on mAChR binding properties.

Co-alteration of dopamine D2 receptor and muscarinic acetylcholine receptor binding in rat striatum after circling training.

CIRCLING training (CT) decreases muscarinic acetyl-choline receptor (mAchR) binding in rat striatum. As cholinergic and dopaminergic systems interact strongly we evaluated the expression of D2-subtype dopamine receptor (DA D2) and mAchR together after CT. Animals trained from 30 to 37 days of age and sacrificed 2 months later showed an enduring drop in Bmax of 40% in DA D2 and 34% in mAchR. Plotting the percentage of binding drop of both receptors for each animal showed that the reduction of one system correlates with the other (r2 = 0.71, p < 0.01; n = 8). Neither mAchR nor DA D2 were affected when training started at 20 or 60 days. We conclude that the presence of a period where CT exerts long term alterations during development involves both cholinergic and dopaminergic systems.

Immunolocalization of muscarinic acetylcholine subtype 2 receptors in rat cochlear nucleus

It has been suggested that cholinergic effects in the rat cochlear nucleus (CN) are mediated by muscarinic acetylcholine receptors. In this study, immunohistochemistry for muscarinic subtype 2 (m2) receptors using a monoclonal subtype-specific antibody (Levey et al. [1995] J. Comp. Neurol. 351:339–356) revealed an m2-like system in the rat CN. A prominent lamina of m2-immunoreactive fibers and puncta was located in a subgranular layer of the caudal anteroventral cochlear nucleus (AVCN) and the posteroventral cochlear nucleus (PVCN). The superficial granular layer of the rostral AVCN and the medial sheet region also contained notable immunoreactivity for m2. Some labeled somata and their processes were found in magnocellular regions of the ventral CN. A network of neurites and puncta was located in the fusiform soma and deep layers of the dorsal CN. The olivocochlear bundle and its branches to the CN were also m2 immunoreactive and possibly contributed m2-labeled fibers and terminals to the CN. Some similarities and some differences were found between this m2 receptor distribution pattern and previous results for choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic acetylcholine receptor immunohistochemistry and binding in the CN. The results suggest that m2 receptors that are located both pre- and postsynaptically mediate many cholinergic effects in the rat CN. © 1996 Wiley-Liss, Inc.

Receptor interaction profile and CNS general pharmacology of metrifonate and its transformation product dichlorvos in rodents

In this study we assessed the possible effects of the putative Alzheimer therapeutic, metrifonate (39, 120, 390 μmol/kg), and its active transformation product, dichlorvos (4.5, 13.6, 45 μmol/kg), on the mammalian central nervous system (CNS). We did this by (1) investigating the receptor interaction profile of the two compounds in a range of in-vitro radioligand binding assays, and (2) by studying the acute compound effects in rats and mice using a battery of behavioral tests after a single oral administration. Metrifonate and dichlorvos failed to displace various radioligands from their respective receptor binding sites on cell membranes at a concentration of 10 μM. In particular, there was no high-affinity interaction with muscarinic or nicotinic acetylcholine receptor binding in-vitro. In the modified Irwin test (rat) both compounds induced transient cholinergic symptoms after oral administration of a single dose of 390 μmol/kg metrifonate and 13.6-45 μmol/kg dichlorvos. The observed symptoms, such as salivation, tremor, and diarrhea, lasted for up to 75 min. In the open field test (rat) metrifonate increased the number of rearings at all doses, whereas dichlorvos had no effect on the parameters tested. Both compounds dose-dependently reduced the pentylenetetrazole threshold dose in mice. In this test, only the highest dose of metrifonate, but all doses of dichlorvos, caused a significant reduction of the convulsion threshold dose. Metrifonate and dichlorvos did not influence traction ability in mice. Metrifonate and dichlorvos did not influence hexobarbital-induced anesthesia in mice. Metrifonate induced hypothermia in rats only at the dose of 390 μmol/kg. Dichlorvos did not affect body temperature. No analgesic potential was observed in the hot-plate test in mice. Furthermore, metrifonate and dichlorvos neither influenced motor coordination nor exhibited any cataleptic potential when administered to rats. Taken together, at cognition-enhancing doses, metrifonate 139-120 μmol/kg) is safe and well tolerated. The adverse symptoms observed at higher doses, together with the apparent lack of high-affinity interaction with neurotransmitter receptors in brain tissue and the similar profile of the active transformation product, dichlorvos, support the assumption that these compounds mediate a highly selective activation of the cholinergic system.

Preventive effects of bifemelane hydrochloride on decreased levels of muscarinic acetylcholine receptor and its mRNA in a rat model of chronic cerebral hypoperfusion

Changes in muscarinic acetylcholine receptor (mACh-R) binding and muscarinic cholinergic m1 receptor (m1-R) mRNA levels were determined in a rat model of cerebral hypoperfusion in which hypoperfusion was induced by permanent bilateral occlusion of the common carotid arteris. After 6 weeks of hypoperfusion, mACh-R binding activity was significantly reduced in the frontal cortex (79.0%, P < 0.01), striatum (74.2%, P < 0.01) and hippocampus (78.6%, P < 0.01), and the m1-R mRNA levels in the frontal cortex (86.6%, P < 0.05) and striatum (89.4%, P < 0.0) compared with sham-operated control. Repeated administration of bifemelane hydrochloide (15 mg/kg/day, p.o., once a day from the day of operation for 6 weeks) prevented the hypoperfusion-induced loss of mACh-R binding and m1-R mRNA levels above described. Since the central cholinergic systems play an important role in learning and memory, these findings suggest that bifemelane hydrochloride is useful to treat and/or prevent vascular dementia which is closely related to cerebral hypoperfusion.

Permanent alteration of muscarinic acetylcholine receptor binding in rat striatum after circling training during development

We evaluated the effect of circling training (CT) in the expression of muscarinic acetylcholine receptor (mAchR) in developing rat striatum. For this, male and female rats were subjected to CT at 20, 30, 40 and 60 days of age during 7 days. Animals trained at 30 days but not at other ages showed an average decreased binding to mAchR of 33% in males and 24% in females, representing a significant difference with respect to control non-trained animals (males P < 0.001, females P < 0.005), and showing also a differential response between sex ( P < 0.01). mAchR drop was found invariably either 2 months or 1 year after training indicating a long term plastic change due to circling training. Scatchard analysis showed that altered binding represents a variation of the total receptor number instead of its binding affinity, with no significant differences found among K d ( P > 0.1). mAchR variation was correlated with the motor performance accomplished in the test. Regarding total distance run, male rats trained for 3 days (300 meters run), for 5 days (600 meters) and for 7 days (900 meters) showed a drop of 19, 28 and 33% respectively ( r 2 = 0.91, P < 0.001), while female changes were of 21, 23 and 24% ( r 2 = 0.78, P < 0.001). Nevertheless, no correlation with running speed was found ( r 2 = 0.13 male, r 2 = 0.02 female; P > 0.1). In summary, these results demonstrate the presence of a limited sensitivity period during striatum development where mAchR expression may be affected by the activity performed during CT, representing a permanent alteration of the receptor levels.

Altered Cholinergic Metabolism in Rat CNS Following Aluminum Exposure: Implications on Learning Performance

The effects of Al on the central cholinergic system have been studied. Al, at a dose of 10 mg/kg of body weight/day for 4 weeks, had a deleterious effect on the activities of biosynthetic (choline acetyltransferase) and hydrolytic (acetylcholinesterase) enzymes of the neurotransmitter acetylcholine. The levels of acetylcholine were also significantly lowered in different brain regions at the end of the dose regimen. There was a significant decrease in high-affinity choline uptake following Al exposure. Muscarinic acetylcholine receptor binding studies revealed a decreased number of binding sites (Bmax), with the maximum effects being manifested in the hippocampus. Exogenous addition of 10 microM desferrioxamine restored the muscarinic receptor binding completely. The impaired cholinergic functioning had severe effects on cognitive functions. Neurobehavioral deficits were manifested in terms of decreased active (52%) and passive (73.30%) avoidance tests. The results suggest that Al exerts its toxic effects by altering cholinergic transmission, which is ultimately reflected in neurobehavioral deficits.

P3-237 - Effects or SDZ ENA-713 on choline acetyltransferase activity and muscarinic acetylcholine receptor binding in the brains of learning impaired rats

P3-237 - Effects or SDZ ENA-713 on choline acetyltransferase activity and muscarinic acetylcholine receptor binding in the brains of learning impaired rats

Activation of inward current associated with M-potassium current inhibition in m1-muscarinic receptor-transformed NG108-15 cells by KST-5452, a novel cognition enhancer

The electrophysiological effects of KST-5452 [3-( m-phenoxybenzylidene)-quinuclidine], an M 1 muscarinic acetylcholine receptor (muscarinic AChR) binding compound, were studied in NG108-15 neuroblastoma × glioma hybrid cells transfected with ml muscarinic AChR cDNA. Application of KST-5452 to m1-transformed NGPM1-27 cells elicited a sustained inward current associated with decreased conductance and reduced M-current relaxations at a holding potential of -20 mV. The KST-5452-induced responses were blocked by pirenzepine, suggesting that KST-5452 acts as a potent excitant via M 1 muscarinic AChRs in brain neurons.

Autoradiographic analysis of second-messenger and neurotransmitter receptor systems in the exo-focal remote areas of postischemic rat brain

We studied the chronological changes of protein kinase C (PKC) and muscarinic acetylcholine receptor binding activities of the rat brain which were determined by using [ 3H]phorbol 12,13-dibutyrate (PDBu) and [ 3H]quinuclidinyl benzilate (QNB) autoradiographic methods, respectively, after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion, followed by different periods of recirculation. After the ischemic insult followed by 3 h of recirculation, [ 3H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [ 3H]QNB binding sites was first detected in those ischemic foci. Moreover, 3 days after ischemic insult, both [ 3H]PDBu and [ 3H]QNB binding sites were concurrently reduced in the ipsilateral thalamus and 1 week after the ischemia, in the substantia nigra, in which both areas had not been directly affected by the original ischemic insult. These alterations of PKC in the postischemic brain areas developed concurrently with 45Ca accumulation, which was detected in our previous study. These results suggest that postischemic alterations of second-messenger (PKC) and neurotransmitter receptor systems were involved not only in the ischemic foci due to ischemia-induced energy failure, but also in the exo-focal remote areas prior to the histologic changes where neuronal damage might be caused by transsynaptic delayed degeneration.

Kinetic analysis of rat parotid gland muscarinic receptors in vivo: comparison with brain and heart

(RR)- and (SS)-quinuclidinyl iodobenzilate enantiomers [(RR)- and (SS)-IQNB, active and inert, respectively] have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor (mAChR) binding. Pharmacokinetic approaches have not been used previously to assess in vivo IQNB binding in nonexcitable tissues. We have applied this method to examine mAChRs in rat parotid gland in comparison to those in brain and heart. Short-term infusion studies in vivo showed that the "instantaneous" reversible binding of (RR)- and (SS)-IQNB was high in the parotid (greater nonspecific binding potential), intermediate in the heart, and lowest in cortex and cerebellum. Long-term bolus injection experiments showed that the parotid gland mAChRs possessed a binding potential for receptor specific sites (380), which was intermediate between that of parietal cortex (930) and cerebellum (10) and greater than that of heart (165). In vitro binding to plasma membranes was generally consistent with the in vivo findings. In aggregate, these studies show that mAChRs can be evaluated in vivo in a nonexcitable tissue with the use of stereospecific ligands and a pharmacokinetic approach. The data suggest that IQNB, a mAChR antagonist, can identify characteristics of specific binding sites, which may reflect tissue differences.