M1 Receptor Antagonist Pirenzepine Research Articles

Reactivation-dependent transfer of fear memory between contexts requires M1 muscarinic receptor stimulation in dorsal hippocampus of male rats.

Memory updating is essential for integrating new information into existing representations. However, this process could become maladaptive in conditions like post-traumatic stress disorder (PTSD), when fear memories generalize to neutral contexts. Previously, we have shown that contextual fear memory malleability in rats requires activation of M1 muscarinic acetylcholine receptors in the dorsal hippocampus. Here, we investigated the involvement of this mechanism in the transfer of contextual fear memories to other contexts using a novel fear memory updating paradigm. Following brief reexposure to a previously fear conditioned context, male rats (n = 8-10/group) were placed into a neutral context to evaluate the transfer of fear memory. We also infused the selective M1 receptor antagonist pirenzepine into the dorsal hippocampus before memory reactivation to try to block this effect. Results support the hypothesis that fear memory can be updated with novel contextual information, but only if rats are reexposed to the originally trained context relatively recently before the neutral context; evidence for transfer was not seen if the fear memory reactivation was omitted or if it occurred 6 h before neutral context exposure. The transferred fear persisted for 4 weeks, and the effect was blocked by M1 antagonism. These findings strongly suggest that fear transfer requires reactivation and destabilization of the original fear memory. The novel preclinical model introduced here, and its implication of muscarinic receptors in this process, could therefore inform therapeutic strategies for PTSD and similar conditions.

Abstract #1401109: Quetiapine-induced Hypoglycemia in an Elderly Non-diabetic Patient with Dementia

Quetiapine is a second-generation antipsychotic agent (SGA) widely prescribed for the treatment of psychotic and mood disorders. In elderly patients, quetiapine is often used to treat psychotic symptoms associated with dementia. A metabolic syndrome phenotype of weight gain, hyperglycemia, and hyperlipidemia is a potential adverse effect of SGA’s, though hypoglycemia caused by SGA’s is documented. We report a case of quetiapine-induced hypoglycemia in a non-diabetic patient. A 79-year-old male with Lewy body dementia was admitted to hospital for management of a myocardial infarction. The patient had no history of diabetes mellitus, renal failure, or hepatic insufficiency, and nobody living with the patient was treated for diabetes. Plasma glucose on admission was 84 mg/dL. Quetiapine was started to control delirium, but within 24 h the patient had worsening confusion and a capillary blood glucose (CBG) measurement of 52 mg/dL. Symptoms improved with administration of dextrose, and the patient ultimately required a 10% dextrose infusion to maintain CBG’s >80 mg/dL. An ACTH-stimulation test (30 min stimulated cortisol 24.8 μg/dL, expected >18) and hypoglycemic medications panel (sulfonylureas and meglitinides) were unremarkable. Quetiapine was stopped, and hypoglycemia resolved. No hypoglycemia occurred during a subsequent 48-h diagnostic fast followed by a mixed meal tolerance test. Due to the patient’s advanced age and comorbidities, it was decided not re-challenge with quetiapine and observe for recurrence of hypoglycemia. Hypoglycemia is a rare complication of SGA’s, and diagnosis may be delayed or missed in patients with baseline confusion or agitation. In addition to quetiapine, there are case reports of hypoglycemia occurring during treatment with risperidone, olanzapine, aripiprazole, and paliperidone. Clozapine and olanzapine increase insulin release from isolated pancreatic islets in vitro, though quetiapine did not demonstrate this effect. However, endogenous hyperinsulinemia has been documented in case reports of quetiapine-induced hypoglycemia. The physiologic pathway by which SGA’s increase insulin release is unknown; some authors have focused on the antagonism of M1 muscarinic receptors, but the muscarinic M1 receptor antagonist pirenzepine has been demonstrated to reduce insulin secretion induced by the muscarinic receptor agonist carbachol from isolated rat pancreatic islets in vitro. Though the mechanism by which hypoglycemia occurs is unclear, this case demonstrates that hypoglycemia should be considered in patients with worsening mental status during treatment with SGA’s.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Muscarinic Depolarization of Layer II Neurons of the Parasubiculum

The parasubiculum (PaS) is a component of the hippocampal formation that sends its major output to layer II of the entorhinal cortex. The PaS receives strong cholinergic innervation from the basal forebrain that is likely to modulate neuronal excitability and contribute to theta-frequency network activity. The present study used whole cell current- and voltage-clamp recordings to determine the effects of cholinergic receptor activation on layer II PaS neurons. Bath application of carbachol (CCh; 10–50 µM) resulted in a dose-dependent depolarization of morphologically-identified layer II stellate and pyramidal cells that was not prevented by blockade of excitatory and inhibitory synaptic inputs. Bath application of the M1 receptor antagonist pirenzepine (1 µM), but not the M2-preferring antagonist methoctramine (1 µM), blocked the depolarization, suggesting that it is dependent on M1 receptors. Voltage-clamp experiments using ramped voltage commands showed that CCh resulted in the gradual development of an inward current that was partially blocked by concurrent application of the selective Kv7.2/3 channel antagonist XE-991, which inhibits the muscarine-dependent K+ current I M. The remaining inward current also reversed near EK and was inhibited by the K+ channel blocker Ba2+, suggesting that M1 receptor activation attenuates both I M as well as an additional K+ current. The additional K+ current showed rectification at depolarized voltages, similar to K+ conductances mediated by Kir 2.3 channels. The cholinergic depolarization of layer II PaS neurons therefore appears to occur through M1-mediated effects on I M as well as an additional K+ conductance.

Additive interaction of intrathecal ginsenosides and neostigmine in the rat formalin test

BackgroundThe authors evaluated the effect of intrathecal mixture of ginsenosides with neostigmine on formalin-induced nociception and made further clear the role of the spinal muscarinic (M) receptors on the activity of ginsenosides.MethodsA catheter was located in the intrathecal space of male Sprague-Dawley rats. Pain was evoked by injection of formalin solution (5%, 50 µl) to the hindpaw. Isobolographic analysis was done to characterize drug interaction between ginsenosides and neostigmine. The antagonism of ginsenosides-mediated antinociception was determined with M1 receptor antagonist (pirenzepine), M2 receptor antagonist (methoctramine), M3 receptor antagonist (4-DAMP), M4 receptor antagonist (tropicamide). The expression of muscarinic receptor subtypes was examined with RT-PCR.ResultsIntrathecal ginsenosides and neostigmine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed an additive interaction between ginsenosides and neostigmine in both phases. Intrathecal pirenzepine, methoctramine, 4-DAMP, and tropicamide reversed the antinociception of ginsenosides in both phases. M1-M4 receptors mRNA detected in spinal cord of naïve rats and the injection of formalin decreased the expression of M1 receptor mRNA, but it had no effect on the expression of other three muscarinic receptors mRNA. Intrathecal ginsenosides little affected the expression of all of muscarinic receptors mRNA in formalin-injected rats.ConclusionsIntrathecal ginsenosides additively interacted with neostigmine in the formalin test. Furthermore, M1-M4 receptors exist in the spinal cord, all of which contribute to the antinocieption of intrathecal ginsenosides.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

Cholinergic Modulation Differs between Basal and Apical Dendritic Excitation of Hippocampal CA1 Pyramidal Cells

We hypothesize that endogenous cholinergic modulation of dendritic processing of hippocampal CA1 is layer specific, and it specifically enhances spike output resulting from basal as compared with the apical dendritic excitation. Laminar profiles of evoked field potentials were recorded in the CA1 area of urethane-anesthetized rats using multichannel silicon probes and analyzed as current source density. High-frequency stimulation of the pontis oralis (PnO) attenuated the midapical more than the basal or distal apical dendritic excitatory sink. Population spike (PS) and excitatory sink-PS potentiation resulting from basal dendritic excitation were facilitated, while the PS evoked by apical dendritic stimulation was attenuated by PnO stimulation. Perfusion of cholinergic agonist carbachol onto hippocampal slices in vitro also attenuated the apical more than the basal dendritic excitatory postsynaptic potentials. Excitatory sink attenuation and PS changes after PnO stimulation were blocked by systemic or local scopolamine and by intracerebroventricular (icv) M1 receptor antagonist pirenzepine but not by icv M2 receptor antagonist AFDX-116 or nicotinic antagonists. However, a hippocampal theta rhythm activated by PnO stimulation was blocked by systemic but not by local scopolamine. We conclude that endogenous acetylcholine mediates a stronger presynaptic inhibition of the midapical than basal and distal apical excitation mainly through M1 receptors.

Cholinergic modulation of hippocampal CA1 basal-dendritic long-term potentiation

Extensive research suggests that long-term potentiation (LTP) may serve as a cellular mechanism for memory and that alterations in synaptic plasticity may underlie the gross memory impairments observed in patients with Alzheimer’s disease. Cholinergic facilitation of hippocampal LTP in the behaving rat is a useful model for the study of the effects of anticholinesterase or other drugs on synaptic plasticity. Field excitatory postsynaptic potentials were recorded from the hippocampal CA1 region following excitation of the basal dendrites in behaving male Long-Evans rats. LTP was induced by a high-frequency train (200 Hz for 0.5 s duration) following injection of the acetylcholinesterase inhibitor eserine sulfate (0.5 mg/kg, i.p.), specific muscarinic M1 receptor antagonist pirenzepine (21.2 μg/μl, i.c.v.), or saline (i.p. or i.c.v.). Pirenzepine was found to block basal-dendritic LTP when LTP was induced during walking, but not when LTP was induced during immobility. Eserine facilitated LTP when induction occurred during either immobility or walking. The present study demonstrates that an anticholinesterase enhances LTP in CA1 of the behaving rat, and that facilitation of basal-dendritic LTP during walking is mediated by muscarinic M1 receptors.

Excitatory muscarinic M1 receptors mediate in the regulation of guinea-pig terminal ileum motility

In vitro experiments were performed on smooth muscle strips cut out longitudinally or circularly from the terminal ileum about 5 cm from the ileo-coecal sphincter. The aim of this study was to determine the functional role of M1 muscarinic receptor subtype in the regulation of cholinergic contractions of the guinea-pig terminal ileum. Electrical field stimulation (EFS; 16 Hz) produced in vitro contractile responses of the guinea-pig terminal ileum muscle strips (longitudinal and circular muscle layer). Those contractions were inhibited by 1 μM tetrodotoxin (2±2% of the control response) and 1 μM atropine (1±1% of the control response), thus indicating the activation of intrinsic cholinergic nerves. Exogenous ACh (1 μM) induced contractions that were inhibited by 1 μM atropine, but not by 1 μM tetrodotoxin, indicating a direct effect on the smooth muscle. MCN-343 specific agonist of M1 receptor subtype (5 μM) induced contractions that were inhibited by 1 μM atropine and by 1 μM tetrodotoxin. M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh - induced contractions but reduced EFS - induced contractions by 11±3% and MCN-A-343 induced contractions by 17±4%. In conclusion, specific M1 receptors modulate terminal ileum contractions by regulating the release of ACh from cholinergic nerves. M1 receptors, present on cholinergic nerves, function as prejunctional facilitatory autoreceptors.

Effects of anticholinergic drugs selective for muscarinic receptor subtypes on prepulse inhibition in mice

The effects of anticholinergic drugs selective for muscarinic receptor subtypes on prepulse inhibition of acoustic startle response were determined in mice. The prepulse inhibition is associated with sensorimotor information processing in the brain. The anticholinergic agent scopolamine (0.3 mg/kg, s.c.) significantly attenuated prepulse inhibition, while the drug (1–10 mg/kg, s.c.) had no effects on startle amplitude as an indicator of startle response. The muscarinic M1 receptor antagonist pirenzepine (0.1–10 μg/mouse, i.c.v.) and the muscarinic M2 receptor antagonist AF-DX116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (0.1–10 μg/mouse, i.c.v.) had no effects on prepulse inhibition or startle amplitude. The muscarinic M3 receptor antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxy-piperidinium iodide) (30 μg/mouse, i.c.v.) and the muscarinic M4 receptor antagonist tropicamide (0.1 μg/mouse, i.c.v.) significantly attenuated prepulse inhibition, while tropicamide (0.01 μg/mouse, i.c.v.) but not 4-DAMP (10 and 30 μg/mouse, i.c.v.) produced a significant increase in startle amplitude. These results suggest that the blockade of muscarinic M3 and M4 receptors leads to the disruption of prepulse inhibition.

Differential modulation by carbachol of four separate excitatory afferent systems to the rat subiculum in vitro.

The subiculum is a limbic cortical region that receives inputs from hippocampus and other parahippocampal regions. We used horizontal brain slices to study the modulatory effects of muscarinic receptor activation on excitatory afferent systems of the subiculum. Multiple inputs are preserved in these slices. Carbachol (CCh, applied to the bath) induced a decrease in the field responses (40-50% at 50 microM; 60% at 100 microM) to CA1, presubicular (PreS), and medial entorhinal (MEC) stimulation. Subicular responses to lateral entorhinal (LEC) stimuli were not depressed. The M1 receptor antagonist pirenzepine at 1 microM was sufficient to reverse most of the CCh-induced depression of afferent excitation, but 10 microM concentrations were required to eliminate the CCh-induced firing in the isolated subiculum. A partial reversal of the CCh-induced depression of afferent excitation was achieved by the M2 receptor antagonist methoctramine (1 or 10 microM), but these concentrations did not prevent CCh-induced firing. When CA1 afferents were repetitively activated with submaximal stimuli in the presence of CCh, population excitatory postsynaptic potentials (EPSPs) showed modest summation, but every response was smaller than a corresponding events in normal media. Population spikes, particularly late spikes in a train, showed pronounced facilitation during CCh exposure. The NMDA receptor antagonist CPP (10 microM) prevented facilitation of responses to repetitive stimulation in the presence of carbachol. We conclude that CA1, PreS, and MEC afferents to the subiculum exhibit CCh sensitivity similar to that established for area CA3 afferents to CA1, and LEC afferents to subiculum exhibit CCh resistance. Our data suggest that much of the hippocampal formation circuitry is modulated by CCh and the properties of this modulation can explain some specific firing characteristics of hippocampal formation neurons in "cholinergic" versus "noncholinergic" brain states.

Role of cholinergic and GABAergic systems in the feedback inhibition of dorsal raphe 5-HT neurons.

Several observations indicate that 5-HT1A receptors found on a long neuronal feedback loop, originating from the medial prefrontal cortex, regulate 5-HT neuronal firing. In the present study, the muscarinic (M) receptor antagonists atropine and scopolamine as well as the M2 receptor antagonist AF-DX 116, but not the preferential M1 receptor antagonist pirenzepine, reduced the suppressant effect of the 5-HT1A receptor agonist 8-OH-DPAT on the spontaneous firing activity of rat dorsal raphe 5-HT neurons. Moreover, AF-64A-induced lesions of cholinergic neurons directly in the medial prefrontal cortex and after its i.c.v. injection attenuated the effect of 8-OH-DPAT. Finally, the NMDA receptor antagonist (+)MK-801 and the GABA(B) receptor antagonist SCH-50911, but not the GABA(A) receptor antagonist (-)bicuculline, dampened the latter response. The present study unveiled a key role for the cholinergic and GABAergic systems in the feedback inhibition of dorsal raphe 5-HT neurons.

Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro

Whole-cell patch-clamp recordings were made from subthalamic nucleus (STN) neurones in brain slices from rats. Stimulation with bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA (IPSCs) receptors. Dopamine reversibly reduced the amplitude of GABAA IPSCs by up to 48 % with an IC50 value of 3.4 +/- 0.8 microM. The dopamine D2 receptor agonist quinpirole, but not the D1 receptor agonist SKF 82958, also inhibited GABAA IPSCs. This effect was completely reversed by the D2 receptor antagonist sulpiride but not by SCH 23390, a D1 antagonist. Muscarine reversibly reduced the amplitude of GABAA IPSCs by up to 70 % with an IC50 value of 0.6 +/- 0.1 microM. Inhibition of IPSCs by muscarine was completely blocked by scopolamine (10 microM), a muscarinic receptor antagonist. The M3 muscarinic receptor antagonist 4-DAMP effectively reversed muscarine-induced inhibition of IPSCs with an IC50 of 0.11 +/- 0.03 microM. Although the M1 receptor antagonist pirenzepine also reversed the inhibition of IPSCs by muscarine, this effect was only observed at relatively high concentrations (IC50 = 21.7 +/- 9.4 microM). Dopamine and muscarine both increased the paired-pulse ratio of GABAA IPSCs. Neither agent produced sustained changes in postsynaptic holding current. Glutamate EPSCs were also inhibited reversibly by dopamine (by up to 29%; IC50 = 16 +/- 3 microM) and muscarine (by up to 41%; IC50 = 1.0 +/- 0.4 microM). However, both agents were more potent and efficacious for reducing GABA IPSCs compared with glutamate EPSCs. These results suggest that the most significant effect of dopamine and muscarine in the STN is to reduce inhibitory synaptic input by acting at presynaptic dopamine D2 and muscarinic M3 receptors, respectively.

Paeoniflorin, a Major Constituent of Peony Root, Reverses Muscarinic Mi-Receptor Antagonist-Induced Suppression of Long-Term Potentiation in the Rat Hippocampal Slice

We previously reported that paeoniflorin but not albiflorin, components of peony root, produced ameliorative effects on scopolamine-induced spatial cognitive impairment in rats. In this study, we examined the effects of paeoniflorin and muscarinic receptor antagonists on long-term potentiation (LTP) of population spike recorded from the CA1 region of rat hippocampal slices. Bath applications of an M1 and M2-receptor antagonist scopolamine and a selective M1-receptor antagonist pirenzepine, at a concentration of 10 μM, significantly suppressed LTP, whereas AF-DX116, a selective M2-receptor antagonist, failed to affect it. Paeoniflorin (0.1-1 μM), which alone was ineffective on LTP induction, significantly reversed the suppressive effects of scopolamine and pirenzepine (10 μM). In contrast, albiflorin (0.1-Ι μM) had no effect on the scopolamine-induced LTP suppression. These results suggest that paeoniflorin reversal of the muscarinic M1-receptor-mediated inhibition of LTP may be implicated in the ameliorative effect of paeoniflorin on spatial cognitive impairment caused by cholinergic dysfunction.

Paeoniflorin, a major constituent of peony root, reverses muscarinic M1-receptor antagonist-induced suppression of long-term potentiation in the rat hippocampal slice.

We previously reported that paeoniflorin but not albiflorin, components of peony root, produced ameliorative effects on scopolamine-induced spatial cognitive impairment in rats. In this study, we examined the effects of paeoniflorin and muscarinic receptor antagonists on long-term potentiation (LTP) of population spike recorded from the CA1 region of rat hippocampal slices. Bath applications of an M1- and M2-receptor antagonist scopolamine and a selective M1-receptor antagonist pirenzepine, at a concentration of 10 microM, significantly suppressed LTP, whereas AF-DX116, a selective M2-receptor antagonist, failed to affect it. Paeoniflorin (0.1-1 microM), which alone was ineffective on LTP induction, significantly reversed the suppressive effects of scopolamine and pirenzepine (10 microM). In contrast, albiflorin (0.1- 1 microM) had no effect on the scopolamine-induced LTP suppression. These results suggest that paeoniflorin reversal of the muscarinic M1-receptor-mediated inhibition of LTP may be implicated in the ameliorative effect of paeoniflorin on spatial cognitive impairment caused by cholinergic dysfunction.

Spontaneously hypertensive rats cholinergic hyper-responsiveness: central and peripheral pharmacological mechanisms.

1. The mechanisms and the subtypes of muscarinic receptors implicated in the cardiovascular effects of physostigmine were investigated in conscious normotensive and spontaneously hypertensive rats. 2. Intravenous (i.v.) physostigmine (50 microg kg-1) induced in both strains a long pressor response, accompanied by a bradycardia. This pressor response was larger in spontaneously hypertensive (+41+/-6 mmHg) than in Wistar-Kyoto (+25+/-2 mmHg) rats (P<0.05). 3. Pretreatment with atropine sulphate (0.4 mg kg-1 i.v.), completely abolished the physostigmine-induced pressor response in both normotensive and hypertensive rats. In both strains, the physostigmine pressor response was significantly reduced by the systemic administration of either an alpha1-adrenoceptor antagonist (prazosin, 1 mg kg-1) or a V1A-vasopressin receptor antagonist (AVPX, 20 microg kg-1). This physostigmine pressor effect was completely abolished in both strains when both antagonists were administered concomitantly. 4. In WKY rats, the pressor response to physostigmine (50 microg kg-1 i.v.) was inhibited in a dose-dependent manner by i. c.v. administration of atropine (ID50=3.70 nmoles), the M1 receptor antagonist pirenzepine (ID50=10.71 nmoles), the M2 receptor antagonist methoctramine (ID50=4.31 nmoles), the M3 receptor antagonist p-F-HHSiD (ID50=60.52 nmoles) and the M4 receptor antagonist tropicamide (ID50=214.20 nmoles). In the hypertensive strain, the ID50 were found to be significantly higher for atropine (7.34 nmoles), pirenzepine (21.60 nmoles) and p-F-HHSiD (139.50 nmoles) (P<0.05). 5. The present results indicate that physostigmine acts in normotensive and spontaneously hypertensive rats, through stimulation of both central M2 and M1 cholinoceptors to induce a rise in blood pressure mediated by an increase in plasma vasopressin and sympathetic outflow. Moreover, our results suggest that some modifications of the M1 receptor subtypes in terms of expression or affinity could be responsible for the hyper-responsiveness of the hypertensive strain to cholinomimetic agents.

Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions.

The aim of this study was to determine the functional role of muscarinic receptor subtypes regulating gallbladder cholinergic contractions. Electrical field stimulation (EFS; 16 Hz) produced contractile responses of guinea pig gallbladder muscle strips in vitro that were inhibited by 1 microM tetrodotoxin (2 +/- 2% of control) and 1 microM atropine (1 +/- 1% of control), indicating activation of intrinsic cholinergic nerves. Exogenous ACh (5 microM)-induced contractions were inhibited by atropine (1 +/- 1% of control) but not tetrodotoxin (102 +/- 1% of control), indicating a direct effect on smooth muscle. The M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh-induced contractions but inhibited EFS-induced contractions by 11 +/- 3%. The M2 antagonist methoctramine (10 nM) had no effect on ACh-induced contractions but augmented EFS-induced contractions by 5 +/- 2%. The M3 antagonist 4-DAMP (10 nM) inhibited ACh-induced contractions by 14 +/- 4% and EFS-induced contractions by 22 +/- 5%. In conclusion, specific M1, M2, and M3 receptors modulate gallbladder muscle contractions by regulating ACh release from cholinergic nerves and mediating the contraction. Cholinergic contractions are mediated by M3 receptors directly on the smooth muscle. M2 receptors are on cholinergic nerves and function as prejunctional inhibitory autoreceptors. M1 receptors are on cholinergic nerves and function as prejunctional facilitatory autoreceptors.

Presynaptic muscarinic M1 and M2 receptor modulation of auriculotemporal nerve transmission in the rat

Parotid secretory and vascular responses to electrical stimulation of the parasympathetic innervation were measured in anaesthetized rats. Stimulation was performed at 1, 10 and 40 Hz. Atropine (1.5 μmol/kg I.V.) almost abolished the secretion to stimulation of peptide depleted nerves at 40 Hz, thus confirming the existence of a pure cholinergic response. Atropine also reduced secretion by 74% during stimulation of non-depleted nerves at the same frequency. Selective blockade by the muscarinic M1 receptor antagonist pirenzepine and by the muscarinic M2 receptor antagonist methoctramine was found to occur at doses (50 nmol/kg I.V. and of 300 nmol/kg I.V., respectively) that did not inhibit the responses to exogenous acetylcholine. In the presence of methoctramine, the nerve-evoked fluid responses were increased by 200% at 1 Hz independently of the total number of impulses (10–300), suggesting that M2 receptor activation normally has an inhibitory effect on transmitter release. The magnitude of the increase was inversely related to frequency of stimulation, and changes in the secretory responses occurred at 40 Hz only when non-depleted nerves were stimulated over the longest period employed. The fluid response then increased by 35% and protein concentration by 200%. The vasodilator responses increased at 1 and 10 Hz, but not at 40 Hz. Pirenzepine reduced the secretory and vascular responses at 10 and 40 Hz but only during stimulation over short periods of time. This suggests that M1 receptor activation normally has a facilitatory effect on neurotransmitter release. During stimulation of non-depleted nerves at 10 Hz for 10 impulses, the fluid response was reduced by 29% and the protein concentration by 26%. When the peptide depleted nerves were stimulated at 10 Hz, pirenzepine also reduced the fluid response (by 43%), but not the protein concentration. It is concluded that the release of transmitter from postganglionic nerve fibres in the rat auriculotemporal nerve is modulated by presynaptic muscarinic receptors. Muscarinic M1 receptors normally facilitate cholinergic and peptidergic transmission during short, intense stimulation. On the other hand, muscarinic M2 receptors normally inhibit cholinergic transmission at low frequencies; at higher frequencies, peptidergic transmission is also inhibited, but only after some delay.

Postsynaptic Integration of Cholinergic and Dopaminergic Signals on Medium-Sized GABAergic Projection Neurons in the Neostriatum

The effects of cholinergic drugs and the interaction between cholinergic and dopaminergic compounds were studied on electrically evoked [ 3H]γ-aminobutyric acid (GABA) overflow in slices of the rat neostriatum. Slices were prepared and loaded with [ 3H]GABA in the presence of β-alanine and then superfused with Krebs-bicarbonate buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA uptake and metabolism, respectively. The nonselective muscarinic agonist oxotremorine (0.1–10 μM) increased the release of [ 3H]GABA and the selective M1 receptor agonist McN-A-343 (0.1–10 μM) exerted similar effect. The stimulatory effect of oxotremorine (10 μM) on [ 3H][GABA overflow was antagonized by the nonselective muscarinic antagonist atropine (1 μM) and the selective M1 receptor antagonist pirenzepine (0.1–1.0 μM). The M2 receptor antagonist methoctramine (1.0 μM) did not alter the stimulatory effect of oxotremorine. Of the muscarinic receptor antagonists atropine, pirenzepine, and methoctramine (1.0 μM) failed to affect [ 3H]GABA overflow. The M3 receptor antagonist p-F-HHSiD (1 μM) increased [ 3H]GABA overflow and p-F-HHSiD and oxotremorine were found to be additive in increasing this effect. The D2 dopamine receptor antagonist sulpiride (10 μM) increased the electrical stimulation-induced [ 3H]GABA overflow, and this stimulation was counteracted by concomitant administration of atropine (1 μM). McN-A-343 and sulpiride also increased the KCl-induced [ 3H]GABA overflow from superfused neostriatal slices and tetrodotoxin (1 μM) did not affect these stimulations. These data indicate that the release of GABA in the neostriatum is under the control of M1 stimulatory and M3 inhibitory muscarinic receptors. Dopamine, which exerts inhibition on GABA release via D2 receptors, may counteract the M1 facilitation, and M1 and D2 receptors involved in the cholinergic-dopaminergic interaction may be located postsynaptically on medium-sized spiny GABAergic projection neurons.

Cocaine-induced convulsions: pharmacological antagonism at serotonergic, muscarinic and sigma receptors.

The concurrent influence of multiple neurotransmitter systems in mediating cocaine-induced convulsions is predicted by the results of previous receptor binding studies. The present results demonstrate that pharmacological manipulations of these predicted neurotransmitter systems alters the occurrence of cocaine-induced convulsions. The 5-HT reuptake inhibitor fluoxetine enhanced the occurrence and severity of convulsions produced by 100 mg/kg (-) cocaine, while the 5-HT2 receptor antagonists cinanserin, ketanserin and pirenperone antagonized cocaine-induced convulsions in a dose-dependent manner. Further, the M1 receptor antagonist pirenzepine antagonized cocaine-induced convulsions, but atropine did not. In addition, both the (+) and (-) stereoisomers of the sigma ligand SKF 10047 significantly attenuated cocaine-induced convulsions. (+)SKF 10047 was more potent than (-)SKF 10047 in this effect, suggesting a stereoselective effect at sigma receptor sites. In constrast, DA and NE neurotransmission do not appear to modulate the proconvulsant effects of cocaine in a specific, dose-dependent manner. Thus, of the CNS binding sites with which cocaine is known to interact, the results are consistent with the conclusion that 5-HT transporters and 5-HT2 receptor sites appear to be direct and primary sites related to cocaine-induced convulsions, while M1 and sigma binding sites appear to play important but secondary and modulatory roles in this response.