Density Of Muscarinic Cholinergic Receptors Research Articles

Effects of galanin subchronic treatment on memory and muscarinic receptors

The cholinergic pathways, which originate in the basal forebrain and are responsible for the control of different cognitive processes including learning and memory, are also regulated by some neuropeptides. One of these neuropeptides, galanin (GAL), is involved in both neurotrophic and neuroprotective actions. The present study has evaluated in rats the effects on cognition induced by a subchronic treatment with GAL by analyzing the passive avoidance response, and the modulation of muscarinic cholinergic receptor densities and activities. [3H]-N-methyl-scopolamine, [3H]-oxotremorine, and [3H]-pirenzepine were used to quantify the density of muscarinic receptors (MRs) and the stimulation of the binding of guanosine 5′-(γ-[35S]thio)triphosphate by the muscarinic agonist, carbachol, to determine their functionality.Some cognitive deficits that were induced by the administration of artificial cerebrospinal fluid (aCSF) (i.c.v. aCSF 2μl/min, once a day for 6days) were not observed in the animals also treated with GAL (i.c.v. 1.5mmol in aCSF, 2μl/min, once a day for 6days). GAL modulates the changes in M1 and M2 MR densities observed in the rats treated with aCSF, and also increased their activity mediated by Gi/o proteins in specific areas of the dorsal and ventral hippocampus. The subchronic administration of the vehicle was also accompanied by an increased number of positive fibers and cells for GAL around the cortical tract of the cannula used, but that was not the case in GAL-treated rats. In addition, the increase of GAL receptor density in the ventral hippocampus and entorhinal cortex in the aCSF group was avoided when GAL was administered. The number of acetylcholinesterase (AChE)-positive neurons was decreased in the nucleus basalis of Meynert of both GAL- and aCSF-treated animals. In summary, GAL improves memory-related abilities probably through the modulation of MR density and/or efficacy in hippocampal areas.

Differences in neonatal neurotoxicity of brominated flame retardants, PBDE 99 and TBBPA, in mice

Flame retardants such as polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A are used as flame retardants and detected in the environmental, wildlife species and human tissues. Exposure to PBDEs during the neonatal development of the brain has been shown to affect behavior and learning and memory in adult mice, while neonatal exposure to TBBPA (another brominated flame retardant) did not affect behavioral variables in the adult. In this study, we hypothesized that the effects of these compounds could be reflected by changes in biochemical substrates and cholinergic receptors and have examined the levels of four proteins involved in maturation of the brain, neuronal growth and synaptogenesis and the densities of both muscarinic and nicotinic cholinergic receptors. We measured the levels of radioactivity in the brain after administration of 14C-labelled TBBPA at different time points and saw that levels of TBBA peaked earlier and decreased faster than the earlier reported levels of PBDE 99. The protein analysis in the neonatal brain showed changes in the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), growth associated protein-43 (GAP-43) and synaptophysin following neonatal exposure to PBDE 99 (21μmol/kg body weight), but not following exposure TBBPA. Furthermore, neonatal exposure to PBDE 99 and TBBPA caused a decrease in binding sites of the nicotinic ligand cytisine in frontal cortex. These results confirm earlier reported data that PBDE 99 can act as a developmental neurotoxicant, possibly due to its different uptake and retention in the brain compared to TBBPA. In addition, the changes in protein levels are interesting leads in the search for mechanisms behind the developmental neonatal neurotoxicity of PBDEs in general and PBDE 99 in particular, since also other compounds inducing similar adult behavioral disturbances as PBDE 99, affect these proteins during the period of rapid brain development.

Methylmercury Impairs Components of the Cholinergic System in Captive Mink (Mustela vison)

The effects of methylmercury (MeHg) on components of the cholinergic system were evaluated in captive mink (Mustela vison). Cholinergic parameters were measured in brain regions (occipital cortex, cerebellum, brain stem, basal ganglia) and blood (whole blood, plasma, serum) following an 89-day exposure to MeHg at dietary concentrations of 0, 0.1, 0.5, 1, and 2 ppm (n = 12 animals per treatment). There were no effects of MeHg on brain choline acetyltransferase, acetylcholine, and choline transporter. However, significantly higher densities of muscarinic cholinergic receptors, as assessed by 3H-quinuclidinyl benzilate binding, were measured in the occipital cortex (30.2 and 39.0% higher in the 1 and 2 ppm groups, respectively), basal ganglia (67.5 and 69.1% higher in the 0.5 and 1 ppm groups, respectively), and brain stem (64.4% higher in the 0.5 ppm group), compared to nonexposed controls. The calculated positive relationship between MeHg exposure and muscarinic cholinergic receptor levels in this dosing study were consistent with observations in wild mink. There were no MeHg-related effects on blood cholinesterase (ChE) activity, but ChE activity was significantly higher in the occipital cortex (17.0% in the 1 ppm group) and basal ganglia (34.1% in the 0.5 ppm group), compared to nonexposed controls. The parallel increases in muscarinic cholinergic receptor levels and ChE activity following MeHg exposure highlight the autoregulatory nature of cholinergic neurotransmission. In conclusion, these laboratory data support findings from wild mink and demonstrate that ecologically relevant exposures to MeHg (i.e., 0.5 ppm in diet) have the potential to alter the cholinergic system in specific brain regions.

Deranged spontaneous behaviour and decrease in cholinergic muscarinic receptors in hippocampus in the adult rat, after neonatal exposure to the brominated flame-retardant, 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE 99)

Polybrominated diphenyl ethers (PBDEs), which are used as flame-retardants, have recently been shown to be increasing in the environment and in human milk. We have recently reported that neonatal exposure to PBDEs, including 2,2′,4,4′,5-pentaBDE (PBDE 99), can induce persistent aberrations in spontaneous behaviour and also affect learning and memory functions and nicotinic cholinergic receptors in the adult mice. The present study indicates that spontaneous behaviour, along with the cholinergic system during its developing stage, can be targets for PBDEs in the rat as well. Neonatal oral exposure of male Sprague–Dawley rats, on postnatal day 10, to 0.8, 8.0, and 16 mg PBDE 99/kg body weight, was shown to disrupt normal spontaneous behaviour in a dose–response related manner at 2 months of age. Also, the animals of the present study showed a decrease in density of muscarinic cholinergic receptors in hippocampus, at an adult age. These findings show similarities to observations made from neonatal exposure of mice to PBDE 99, 2,2′,4,4′,5,5′-hexaBDE (PBDE 153) and certain PCBs, compounds shown to affect both spontaneous behaviour and the cholinergic system.

Effect of training on beta1 beta2 beta3 adrenergic and M2 muscarinic receptors in rat heart.

Physical training is known to alter several cardiovascular parameters. These adaptations are for a great part linked to an alteration of the myocardial responses to its autonomic nervous regulation. To further explain the parasympathetic and catecholamine effects, we hypothesized that endurance training could modify rat myocardial beta1, beta2, beta3 adrenoreceptors (AR) and M2 muscarinic cholinergic receptor (AchR) densities. Two groups of adults female Wistar rats were studied: controls (C) (N = 7) and trained (T) (N = 9). An 8-wk treadmill training protocol was performed, 5 d x wk and of 1 h x d. At the end of the training session, left ventricle and atria muscle were isolated and weighed. Then, quantification of beta1, beta2, beta3 AR and M2 AchR was performed using Western blot analysis. M2 AchR densities were not modified in left ventricle or in atria by training (respectively, 100 +/- 22%, C vs 101 +/- 14%, T and 100 +/- 23%, C vs 119 +/- 30%, T). Concerning the left ventricle beta AR isoforms, beta1AR density was decreased in T (80 +/- 10% T vs 100 +/- 14% C, P = 0.01), beta2AR was unaltered (102 +/- 12%, T vs 100 +/- 17%, C), and beta3 AR density was increased in T (139 +/- 38% T vs 100 +/- 15% C; P < 0.05). Our results show for the first time that in female rats an 8-wk treadmill training protocol alters specifically the left ventricle beta AR isoforms densities but not the M2 AchR one. These results could explain some of the beneficial cardiovascular adaptations of the physically trained heart.

Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na +,K +-ATPase in rats

This study demonstrated that chronic aspartame consumption in rats can lead to altered T-maze performance and increased muscarinic cholinergic receptor densities in certain brain regions. Control and treated rats were trained in a T-maze to a particular side and then periodically tested to see how well they retained the learned response. Rats that had received aspartame (250 mg/kg/day) in the drinking water for 3 or 4 months showed a significant increase in time to reach the reward in the T-maze, suggesting a possible effect on memory due to the artificial sweetener. Using [ 3H]quinuclidinyl benzilate (QNB) (1 nM) to label muscarinic cholinergic receptors and atropine (10 −6 M) to determine nonspecific binding in whole-brain preparations, aspartame-treated rats showed a 31% increase in receptor numbers when compared to controls. In aspartame-treated rats, there was a significant increase in muscarinic receptor densities in the frontal cortex, midcortex, posterior cortex, hippocampus, hypothalamus and cerebellum of 80%, 60%, 61%, 65%, 66% and 60%, respectively. The midbrain was the only area where preparations from aspartame-treated rats showed a significant increase in Na +,K +-ATPase activity. It can be concluded from these data that long-term consumption of aspartame can affect T-maze performance in rats and alter receptor densities or enzymes in brain.

Abnormal kainic acid receptor density and reduced seizure susceptibility in dystrophin-deficient MDX mice

Duchenne muscular dystrophy is characterized by a defect in dystrophin, which often causes mental retardation in addition to progressive muscular weakness. As dystrophin is localized in synaptic regions of the CNS, cognitive abnormalities associated with Duchenne muscular dystrophy are attributable to synaptic dysfunction. We report that dystrophin-deficient mdx mice were more resistant to kainic acid-induced seizures but not to GABA antagonist-induced seizures compared with the control mice. The kainic-acid receptor density in the brain was significantly lower in the mdx than in the control, although the density of muscarinic cholinergic receptors, another important neurotransmitter receptor for cognitive function, was normal. Moreover, mdx had significantly lower Timm staining intensity in the mossy fibers, which originate from the dentate granule cells and terminate on the pyramidal cells in the CA3 of the hippocampus. These results suggest that an instability of neurotransmitter receptors, such as kainate-type glutamate receptors, on synaptic membranes due to the disruption of dystrophin complex induces inefficient neurotransmission in Duchenne muscular dystrophy patients.

Systemic effects of dietary n-3 PUFA supplementation accompany changes of CNS parameters in cerebral hypoperfusion.

Dietary supplementation with long-chain polyunsaturated fatty acids (PUFAs) has become an attractive possibility to alleviate or prevent cerebrovascular pathophysiology. To characterize the potentially beneficial cerebrovascular action of n-3 PUFAs that predominantly occur in fish oil, we set up an experimental paradigm where rats with chronic cerebral hypoperfusion were supplied with n-3 PUFA-enriched diets. Cerebral hypoperfusion was created by a permanent, bilateral occlusion of the common carotid arteries (2VO) of rats at the age of 4 months, with a survival of 3 months. Simultaneously, the rats were provided with experimental diets from the time of weaning until the termination of the experiments. The control diet was comparable to standard rat chow, while diet 1 contained additional n-3 PUFAs and diet 2 was further enriched with structural phospholipids and neurotransmitter precursors. In summary, the data show that diet 2 improved spatial learning of 2VO rats in the Morris water maze. Both diet 1 and diet 2 augmented blood-brain barrier parameters and increased the density of the M1-type muscarinic cholinergic receptors in the hippocampus independent of the rate of cerebral perfusion. In addition to an overview of these results, changes that were supportive or accompanying those described in the CNS are also presented. Briefly, plasma corticosterone concentration was elevated most explicitly by 2VO, while the relative weight of the liver and spleen increased due to the diets. The data draw attention to changes not only in the CNS, but also in the periphery as a consequence of chronic supplementation with n-3 PUFA-enriched diets.

Piracetam improves cognitive performance by restoring neurochemical deficits of the aged rat brain.

In order to test the hypothesis that piracetam improves cognitive functions by restoring biochemical deficits of the aging brain, we investigated the effects of piracetam treatment (300 mg/kg daily for 6 weeks) on the active avoidance performance of young and aged rats. After testing, the rats were killed and membrane fluidity and NMDA as well muscarinic cholinergic receptor densities were determined in the frontal cortex, the hippocampus, the striatum, as well as the cerebellum. Piracetam treatment improved active avoidance learning in the aged rats only and elevated membrane fluidity in all brain regions except the cerebellum in the aged animals. Moreover, we observed a positive effect of piracetam treatment on NMDA receptor density in the hippocampus and on muscarinic cholinergic receptor densities in the frontal cortex and the striatum and to a lesser extent in the hippocampus. Again, these effects were only observed in aged animals. Discrimination analysis indicated that piracetam effects on membrane fluidity in the frontal cortex, the hippocampus, and the striatum and its effects on NMDA densities in the hippocampus might be involved in its positive effects on cognitive performance.

Differential expression of muscarinic subtype mRNAs after exposure to neurotoxic pesticides

We have recently reported an increase in the density of muscarinic cholinergic receptors in mice neonatally exposed to a persistent environmental agent, dichlorodiphenyltrichloroethane (DDT), and a subsequent exposure as adults to nonpersistent toxicants, such as bioallethrin or paraoxon. Here we have examined the effects of an exposure like this on muscarinic receptor mRNA expression. Ten-day-old Naval Medical Research Institute mice received a single oral dose of DDT (0.5 mg/kg body weight). When aged 5 months, they received bioallethrin (0.7 mg/kg body weight per day for 7 days) or paraoxon (1.4 mg/kg body weight every second day for 7 days). mRNA expression of subtypes m1, m3, and m4 was studied in 7-month-old animals. Changes could only be discovered in the DDT-bioallethrin treated mice, where expression of subtype m4 was elevated in cortex and caudate putamen. Moreover, the expression pattern of the subtypes m1, m3, and m4 in mouse brains was found to be very similar to that seen in rats, except for slight differences in the pyramidal cell layer of the hippocampus, where the outermost part of the CA3 region did not show any m4 hybridization. The present study indicates that the earlier observed increase in muscarinic receptor density in mice exposed as neonates to DDT and as adults to bioallethrin can be attributed to changes in the expression of m4.

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1. Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [3H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2. [3H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (Kd) values of [3H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (Bmax) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3. The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4. This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.

Down-regulation of phospholipase C-β1 following chronic muscarinic receptor activation

To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation.

Effect of partial volume correction on muscarinic cholinergic receptor imaging with single-photon emission tomography in patients with temporal lobe epilepsy.

Animal experiments and preliminary results in humans have indicated alterations of hippocampal muscarinic acetylcholine receptors (mAChR) in temporal lobe epilepsy. Patients with temporal lobe epilepsy often present with a reduction in hippocampal volume. The aim of this study was to investigate the influence of hippocampal atrophy on the quantification of mAChR with single photon emission tomography (SPET) in patients with temporal lobe epilepsy. Cerebral uptake of the muscarinic cholinergic antagonist [123I]4-iododexetimide (IDex) was investigated by SPET in patients suffering from temporal lobe epilepsy of unilateral (n=6) or predominantly unilateral (n=1) onset. Regions of interest were drawn on co-registered magnetic resonance images. Hippocampal volume was determined in these regions and was used to correct the SPET results for partial volume effects. A ratio of hippocampal IDex binding on the affected side to that on the unaffected side was used to detect changes in muscarinic cholinergic receptor density. Before partial volume correction a decrease in hippocampal IDex binding on the focus side was found in each patient. After partial volume no convincing differences remained. Our results indicate that the reduction in hippocampal IDex binding in patients with epilepsy is due to a decrease in hippocampal volume rather than to a decrease in receptor concentration.

Low-dose effects of paraoxon in adult mice exposed neonatally to DDT: changes in behavioural and cholinergic receptor variables

This study revealed increased susceptibility in adult mice, exposed neonatally to a low dose of DDT (1,1,1-trichloro-2,2-bis( p-chlorofenyl)ethane), to develop changes in behaviour and cholinergic muscarinic receptors when exposed as adults to the organophosphorus insecticide paraoxon. 10-day-old NMRI male mice were given a single oral dose of DDT (0.5 mg/kg body weight). At the age of 5 months, paraoxon was administered by gavage as a single dose (0.7 or 1.4 mg/kg body weight) every 2nd day for 1 week. These doses caused approximately 15% and 45% inhibition of acetylcholinesterase respectively, 48 h after the last exposure. 24 h after the last paraoxon administration, a spontaneous motor activity test revealed no differences between any of the adult paraoxon-treated mice and their corresponding controls, though when the test was performed again 2 months later, mice exposed neonatally to DDT and given paraoxon as adults had developed changes in spontaneous behaviour. The density of muscarinic cholinergic receptors was significantly increased in this group. No significant changes were seen in either behaviour or muscarinic receptors in mice exposed neonatally to the vehicle and receiving paraoxon as adults and there were no significant differences in the muscarinic or nicotinic subpopulations investigated, between any of the treatment groups. These results show that a dose of paraoxon not having any effect in vehicle-treated animals can cause effects in animals neonatally exposed to DDT.

Beta-adrenergic and muscarinic cholinergic receptor densities in the human sinoatrial node: identification of a high beta 2-adrenergic receptor density.

The objective of this study was to measure autonomic receptor densities in the human sinoatrial node and adjacent atrial myocardium to gain further insights into autonomic regulation of sinoatrial node function in the human heart. Sinoatrial nodes (n = 9) were acquired from human donors. Quantitative light microscopic autoradiography of radioligand binding sites in tissue sections was used to compare beta-adrenergic and muscarinic cholinergic receptor densities within specific tissue compartments of the sinoatrial node and adjacent myocardium. Total beta-adrenergic receptors were measured with the nonsubtype selective radioligand [125I]iodocyanopindolol. beta 2-Adrenergic receptors were determined by measuring the amount of radioactivity bound to sections incubated with radioligand in the presence of the highly beta 1-selective antagonist CGP-20712A. Specific autoradiographic grain densities were normalized to myocyte area/unit tissue area. Myocytes in the sinoatrial node occupied 47.7% +/- 0.1% of the total tissue area compared with 92.8% +/- 0.1% in myocardium (P < 0.001). Total specific beta-adrenergic receptor density per unit myocyte area was 3.5 +/- 0.9 times greater in the sinoatrial node than in myocardium (P < 0.001). The relative densities of beta 1-(4.2, P < 0.002), beta 2-(2.6, P < 0.002), and muscarinic (3.3, P < 0.001) receptors were significantly greater in the sinoatrial node than in the atrium. Thus, total beta-adrenergic and muscarinic cholinergic receptor densities are > 3-fold higher in the sinoatrial node than adjacent atrial myocardium, reflecting their specialized roles in regulating cardiac rate and rhythm. The beta 1-subtype is predominant in both regions. The beta 2-subtype, however, is > 2.5-fold more abundant in the sinoatrial node than in atrial myocardium. The relatively high beta 2-receptor density in the human sinoatrial node is consistent with physiologic studies that implicate this receptor in regulating cardiac chronotropism.

Evaluation of 1-azabicyclo[2.2.2]oct-3-yl α-fluoroalkyl-α-hydroxy-α-phenylacetates as potential ligands for the study of muscarinic receptor density by positron emission tomography

Both 1-azabicyclo[2.2.2]oct-3-yl α-(1-fluoroeth-2-yl)-α-hydroxy-α-phenylacetate (FQNE, 5) and 1-azabicyclo[2.2.2]oct-3-yl α-(1-fluoropent-5-yl)-α-hydroxy-α-phenylacetate (FQNPe, 6) were prepared and evaluated as potential candidates for the determination of muscarinic cholinergic receptor (mAChR) density by positron emission tomography (PET). The results of in vitro binding assays demonstrated that although both 5 and 6 had high binding affinities for m 1 and m 2 mAChR subtypes, 6 displayed a higher affinity (nM, m 1; K D, 0.45, m 2; K D, 3.53) as compared to 5 (nM, m 1 K D, 12.5, m 2; K D, 62.8). It was observed that pretreatment of female Fisher rats with either 5 or 6 prior to the i.v. administration of Z-(−)(−)-[ 131I]-IQNP, a high-affinity muscarinic ligand, significantly blocked the uptake of radioactivity in the brain and heart measured 3 h postinjection of the radiolabeled ligand. These new fluoro QNB analogues represent important target ligands for evaluation as potential receptor imaging agents in conjunction with PET.

Relative Densities of Muscarinic Cholinergic and β-Adrenergic Receptors in the Canine Sinoatrial Node and Their Relation to Sites of Pacemaker Activity

The site of earliest extracellular electrical activation in the sinoatrial node (SAN) is known to shift in response to autonomic stimuli, but the mechanisms underlying this phenomenon and the determinants of the location of dominant pacemaker activity have not been elucidated. The present study was designed to characterize the spatial distribution of muscarinic cholinergic and beta-adrenergic receptors in the canine SAN and to determine whether a consistent relationship exists between autonomic receptor densities and the site of dominant pacemaker activity. We used quantitative light-microscopic autoradiography of radioligand binding sites to characterize the spatial distribution of muscarinic cholinergic and beta-adrenergic receptor subtypes in tissue sections containing the SAN and adjacent right atrial muscle from 18 canine hearts. Muscarinic receptor density was 5.4 times greater in SAN cells than in atrial myocytes (P < .01). Total beta-adrenergic receptor density was more than 3 times greater in SAN cells than in atrial myocytes (P < .0001), due entirely to the significantly greater number of beta 1-adrenergic receptors in the SAN. The region of dominant pacemaker activity, localized in 4 hearts with in vitro mapping, consistently exhibited greater densities of muscarinic and beta 1-adrenergic receptors than other SAN regions. Muscarinic receptor density in the dominant pacemaker region was 18 +/- 2% and 29 +/- 7% higher than in adjacent superior and inferior regions, respectively. beta 1-Receptor density in the dominant site was 53 +/- 5% and 26 +/- 4% higher than in adjacent superior and inferior SAN regions, respectively. Thus, the SAN is richly endowed with both muscarinic cholinergic and beta 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure to an organophosphate (DFP) during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice

The organophosphate Diisopropylfluorophosphate (DFP) is a well-known inhibitor of cholinesterases. We have recently observed that neonatal exposure to a single subsymptomal dose of DFP induces permanent alterations in muscarinic cholinergic receptors (MAChRs) and in spontaneous behaviour, in the mice as adults. In order to determine if there is a critical period for these effects, neonatal mice were given a single oral dose of 1.5 mg/kg DFP b.wt. on postnatal day 3, 10 or 19, causing equal inhibition of AChE. At the adult age of 4 months the mice were tested for spontaneous motor behaviour, and were subsequently sacrificed for measurement of density of MAChRs and subpopulations of MAChRs in the cerebral cortex by using the antagonist quinuclidinyl benzilate ([ 3H]QNB), and agonist carbachol, respectively. At adult age, mice exposed to DFP on postnatal day (PND) 3 or 10 showed significant ( P ≤ 0.01) alterations in spontaneous motor behaviour and a significant ( P ≤ 0.01) decrease in muscarinic receptor density. There were no alterations mice exposed on PND 19. The proportions and affinity-constants of high- and low-affinity MAChR binding sites were not affected in mice showing altered MAChR density. The lack of effect on mice exposed on PND 19 was not due to differences in AChE activity.

Diabetes-Induced Alterations in the Properties of Muscarinic Cholinergic Receptors in Rat Vas Deferens

Muscarinic cholinergic receptors were identified and characterized by radioligand receptor binding assay using [3H]quinuclidinyl benzilate (QNB) in rat vas deferens membrane particulates of three experimental groups: 1) 8-week diabetic, 2) 8-week diabetic insulin-treated and 3) age-matched control. Diabetes was induced by the intravenous injection of 65mg./kg. streptozotocin (STZ). The density of muscarinic receptors (Bmax values), as determined by saturation experiments with [3H]QNB, was demonstrated to be higher in the vas deferens of diabetic rats than in the vas deferens of control and diabetic insulin-treated rats. The equilibrium dissociation constants (KD values), however, were similar in all three groups. Muscarinic cholinergic antagonists competed with [3H]QNB binding sites in the vas deferens membrane particulates with the following rank order of Ki values: atropine < methoctramine ≤ 4-DAMP < AF-DX 116 < HHSiD < pirenzepine = pfHHSiD. The pharmacological profile of muscarinic receptors was similar in all three groups. Additional pharmacological studies showed a similar rank order of Ki values for vas deferens, bladder dome and heart, but this rank order was significantly different in cerebral cortex and prostate. This is consistent with the predominance of the M2 muscarinic cholinergic receptor subtype in the rat vas deferens. It is concluded that STZ-induced diabetes causes an upregulation of muscarinic cholinergic receptor density in the rat vas deferens that can be prevented by the administration of insulin.

Intracerebroventricular administration of nerve growth factor affects muscarinic cholinergic receptors in the cerebral cortex of neonatal rats.

The repeated intracerebroventricular administration of nerve growth factor (5 micrograms/2.5 microliters) to neonatal rats induced the activation of choline acetyltransferase in forebrain cholinergic neurons that was paralleled by a concomitant change in the density of muscarinic cholinergic receptors in the cerebral cortex. The administration of nerve growth factor altered muscarinic binding sites in a biphasic fashion during postnatal development. A significant stimulation of the developmental increase in the density of muscarinic binding sites occurred in nerve growth factor-treated animals at days 2 and 3 after birth. Conversely, nerve growth factor induced a significant decrease in the receptor number at postnatal days 8 and 14. Muscarinic receptor number returned to control values after treatment, suggesting that nerve growth factor-induced changes to muscarinic cholinergic receptors are reversible. Nerve growth factor administration did not affect muscarinic cholinergic receptor density in striatal membranes and did not alter the relative content of cortical messenger RNAs encoding m1 and m3 muscarinic cholinergic receptor subtypes at postnatal day 14, as determined by reverse transcriptase-polymerase chain reaction. The up- and down-regulation of muscarinic cholinergic receptors induced by nerve growth factor during postnatal development may be temporally related events associated with concomitant changes in the activity of choline acetyltransferase.