Differential effects of M1 and M2 muscarinic drugs on septohippocampal, hippocampal and cortical neurons in the rat

The effects of muscarinic agonists (McN-A-343, pilocarpine, oxotremorine-M, carbachol) and antagonists (pirenzepine, gallamine) applied by iontophoresis were studied on several neuronal populations in the central nervous system of rats anesthetized with urethane. Septohippocampal neurons and neurons from hippocampus, subiculum and somatic sensory cortex were studied. Oxotremorine-M and carbachol had (almost exclusively) potent excitatory effects whereas pilocarpine had some and McN-A-343 had almost exclusively inhibitory effects on the 4 populations of neurons studied. Pirezepine blocked more easily the effects of pilocarpine and McN-A-343 than those of oxotremorine-M or carbachol. These results suggest (i) that many central neurons may bear different functional muscarinic receptors and (ii) that the various agonists studied might act through (at least partially) different mechanisms.     

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

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

Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats

The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5–25 μg: 5 μl) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 μg) increased MAP and HR by 32 ± 3 mmHg and 45 ± 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 μg; i.c.v.) blocked the effects of physostigmine (25 μg; i.c.v.). Also, the pre-administration of the M₂ muscarinic antagonist, methoctramine (25 μg; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M₁ muscarinic antagonist, pirenzepine (100 μg; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M₃ muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 μg; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25–30 mg/kg), physostigmine (25 μg; i.c.v.) evoked a decrease in MAP and HR of 13 ± 6 mmHg and 15 ± 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats arepossibly mediated through stimulation of central M₂ muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.     

Astrocytes have both M1 and M2 muscarinic receptor subtypes

In astrocytes, carbachol evoked the turnover of membrane inositol phospholipids prelabelled with [3H]inositol, as revealed by [3H]inositol phosphate accumulation in the presence of 5 mM lithium. This effect was blocked by atropine and by pirenzepine (IC50 2.2 nM and 56 nM, respectively). Carbachol partially attenuated the isoproterenol-stimulated cyclic adenosine 3',5'-monophosphate production in astrocytes by a direct effect on adenylate cyclase, an effect blocked by atropine and pirenzepine. These results suggest that astrocytes express muscarinic receptor subtypes.     

Biphasic effect of tacrine on acetylcholine release in rat brain via M1 and M2 receptors

Rat cortical synaptosomes preloaded with [³H]choline were superfused and stimulated with K⁺ in order to investigate the effect of the cholinesterase inhibitor tacrine on the in vitro release of acetylcholine (ACh). Tacrine was found to biphasically both increase (10⁻⁶ and 5 × 10⁻⁶ M) and decrease (10⁻⁵−10-⁻⁴ M) the release of ACh in a concentration-dependent manner. The facilitatory effect of tacrine was prevented by atropine and the M₁ antagonist pirenzepine, whereas the inhibitory effect induced by tacrine was blocked by atropine and the M₂ antagonist AF-DX 116. These results indicate that tacrine causes a biphasic effect on K⁺ stimulated ACh release in the brain via M₁ and M₂ muscarinic receptors. The tacrine induced enhancement of the ACh release occurs at clinical relevant tacrine concentrations and might therefore be of importance for the treatment outcome of Alzheimer's disease.     

Localization of M1 Muscarinic Receptors in Rat Brain Using Selective Muscarinic Toxin-1

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M₁ receptors in homogenates of rat cerebral cortex. Iodination of the toxin, ¹²⁵I-muscarinic toxin-1 (¹²⁵I-MT-1), renders the toxin selective for M₁ muscarinic receptors. Quantitative measurement of ¹²⁵I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of ¹²⁵I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.     

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M₁- and M₂-receptor subtypes through semi-quantitative in vitro autoradiography. [³Quinuclidinyl benzilate] (QNB) and [³H]pirenzepine (PZ) were used for labeling total mAChR and M₁-receptors, respectively. In the cerebral cortex of adult rats, [³H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M₁-receptors were diffusely observed in all the layers. This means that M₂-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M₁- and M₂-receptor first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M₁-type in the adult rat brain, ontogenic patterns of M₁-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M₂-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M₁-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M₁- and M₂-receptors show distinct developmental behaviors in the rat brain.     

Buspirone impairs performance of a three-choice working memory water escape task in rats

Two studies were conducted to assess the effects of buspirone and alprazolam on a three-choice working memory water escape task. Both studies involved giving rats three daily trials, each trial consisting of an information run during which guillotine doors forced the rats to swim into the correct escape alley and a test run during which the rats could enter any of the three alleys but escape only on entering the same alley to which they had been forced on the information run. In the first experiment, rats were trained to a 70% correct choice criterion with 5-min interrun intervals and then tested for performance with 5, 20, 20 with distraction, and 60-min interrun intervals. In the second experiment, rats in each of the drug groups were tested after receiving one of three different doses of their respective drug. Results suggest that 1.0, 3.0, and 10.0 mg/kg buspirone but not 0.5, 1.0, and 2.0 mg/kg alprazolam impaired performance of rats on a three-choice working memory water escape task.     

[H]Pirenzepine identifies putative M1 muscarinic receptors in human stellate ganglia

The specific binding of [³H]pirenzepine ([³H]PZ) and [³H](−) quinuclidinyl benzilate ([³H](−)QNB) was investigated in homogenates of human stellate ganglia. [³H]PZ saturation isotherms yielded aK_d of 14 nM and B_max of 16.7 fmol/mg protein, while [³H](−)QNB binding curves yielded aK_d of 59 pM and B_max of 33.0 fmol/mg protein. This represents the greatest proportion of high affinity [³H]PZ labeling, relative to [³H](−)QNB, seen in any peripheral tissue examined thus far.     

Bidirectional modulation of long-term potentiation by carbachol via M1 and M2 muscarinic receptors in guinea pig hippocampal mossy fiber-CA3 synapses

Participation of muscarinic M₁ and M₂ receptors in the modulation of long-term potentiation (LTP) was studied in the mossy fiber-CA3 synapse of guinea pig hippocampal slices. The magnitude of tetanus-induced LTP was attenuated in the presence of 0.01–0.1 μM carbachol, at which concentration the pre-tetanus amplitude of field excitatory postsynaptic potential (fEPSP) was not affected. The attenuation of LTP by the low concentration of carbachol was reversed by an M₂ muscarinic antagonist, AF-DX 116, but not by an M₁ antagonist, pirenzepine. On the contrary, a high concentration (10 μM) of carbachol decreased the pre-tetanic amplitude of fEPSP, however, the magnitude of LTP was significantly larger than that in control slices in which pre-tetanic amplitude of fEPSP was reduced to the level of carbachol-treated slices by reducing the intensity of stimulation or extracellular Ca²⁺ concentration. The augmentation of LTP by 10 μM carbachol was blocked by pirenzepine but not by AF-DX 116. These results suggest that the synaptic plasticity in the guinea pig hippocampal mossy fiber-CA3 synapse is inhibited and facilitated by muscarinic agonist through muscarinic M₂ and M₁ receptors to inhibit and facilitate the LTP, respectively.     

Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1–5 min) and the second phase (7–30 min) after the injection. The muscarinic antagonist atropine (0.1–10 ng, i.t.) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.1–20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20–100 ng, i.t.) did not cause inhibition. The M₁ muscarinic receptor antagonist pirenzepine (10–100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M₂ muscarinic receptor antagonist 11-{(2-[(diethylamino)methyl]-1-piperidinyl}acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AF-DX116; 10–1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 μg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M₃ muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.     

Involvement of M2 muscarinic receptors in relaxant response of circular muscle of mouse gastric antrum

Our previous study showed that atropine significantly inhibited the sustained relaxation induced by electrical field stimulation (EFS) in the circular muscle strips prepared from the mouse antrum, and that pituitary adenylate cyclase activating peptide (PACAP) partially mediated the sustained relaxation. The muscarinic receptor subtype associated with the sustained relaxation was examined in the present study by using each muscarinic receptor subtype of knockout (KO) mice. EFS-induced sustained relaxation in the antrum prepared from M(2) receptor KO mice was significantly less than that of wild-type mice. Atropine failed to inhibit this relaxation. On the other hand, similar sustained relaxation and inhibitory effects of atropine to those of wild-type mice were observed in M(1), M(3) and M(4) receptor KO mice. Exogenously added PACAP-27 relaxed the antral strips of wild-type and M(2) receptor KO mice to a similar extent. Immunohistochemical study revealed that M(2) receptor immunoreactivity was localized with PACAP-immunoreactivity in enteric neurons within the antrum of wild-type mice. In contrast, atropine did not affect the EFS-induced sustained relaxation in the gastric fundus. These results suggest that M(2) receptors modulate the sustained relaxation, probably through the regulation of PACAP release, in the mouse antrum.     

Working memory deficits following muscarinic blockade combined with depletion of brain somatostatin in rats

In a working memory task with three-panel runway paradigm, cysteamine, a depletor of somatostatin, at 100 or 200 mg/kg i.p. given 24 h before testing, had no effect on the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). Cysteamine at 100 mg/kg caused a significant reduction in somatostatin-like immunoreactivity in the rat brain, including the hippocampus and cerebral cortex. Working memory errors were significantly increased by scopolamine, a muscarinic receptor antagonist, at 0.32 mg/kg i.p. given 20 min before testing, whereas errors were not affected by the 0.1 mg/kg dose. Combined administration of 100 mg/kg cysteamine and 0.1 mg/kg scopolamine significantly increased the number of working memory errors. However, cysteamine at 100 mg/kg and scopolamine at 0.1 mg/kg had no effect on reference memory errors, whether they were administered alone or in combination. These results suggest that depletion of brain somatostatin aggravates working memory deficits induced by blockade of muscarinic receptors.     

Differential effects of 5-HT3 receptor antagonism on working memory failure due to deficiency of hippocampal cholinergic and glutamatergic transmission in rats

The muscarinic acetylcholine receptor antagonist scopolamine significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task with a three-panel runway setup, when injected bilaterally at 3.2 μg/side into the dorsal hippocampus. Concurrent infusion of the selective and potent 5-hydroxytryptamine₃ (5-HT₃) receptor antagonist Y-25130 (0.32 and 1.0 μg/side) significantly attenuated the increase in working memory errors induced by intrahippocampal 3.2 μg/side scopolamine. Intrahippocampal Y-25130 (1.0 μg/side) by itself did not affect working memory errors. On the other hand, intrahippocampal administration of the competitive NMDA receptor antagonist (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) at 32 ng/side caused a significant increase in the number of working memory errors. However, Y-25130 at 1.0 μg/side did not affect the increase in working memory errors when infused intrahippocampally together with 32 ng/side CPP. These results suggest that antagonism of hippocampal 5-HT₃ receptors is ineffective against working memory failure resulting from blockade of NMDA receptor-mediated neurotransmission, but that it can compensate deficiency of septohippocampal cholinergic activity involved in working memory function of rats.     

Antagonism of muscarinic M1 receptors by dicyclomine inhibits the consolidation of morphine-associated contextual memory

M1 muscarinic receptor has been shown to be involved in cognitive functions of the brain. Conditioned place preference (CPP) paradigm involves memory for the association between environmental stimuli and the rewarding properties produced by a treatment. Using a balanced CPP design, we studied the possible involvement of M1 muscarinic receptors on the acquisition, expression and consolidation of morphine place conditioning in male mice. Subcutaneous administration of morphine sulphate-induced CPP in a dose-dependent manner. Using a 6-day schedule of conditioning, it was found that dicyclomine, an M1 muscarinic antagonist, significantly reduced the time spent by mice in the morphine compartment when given immediately, but not 6 h, after each conditioning session (consolidation). It had no effect when administered 30 min before each conditioning session during CPP training period (acquisition) or 30 min before testing for place preference in the absence of morphine (expression). It is concluded that M1 muscarinic receptors may play a time-dependent role in the consolidation of reward-related memory of morphine.     

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [³H]pirenzepine or [³H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [³H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [³H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [³H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.     

M4 muscarinic receptors are involved in modulation of neurotransmission at synapses of Schaffer collaterals on CA1 hippocampal neurons in rats

All five subtypes of muscarinic acetylcholine receptors (mAChR; M(1)-M(5)) are expressed in the hippocampus, where they are involved both in cognitive functions and in synaptic plasticity, such as long-term potentiation (LTP). Muscarinic toxins (MTs) are small proteins from mamba snake venoms that display exquisite discrimination between mAChRs. MT1 acts as an agonist at M(1) and an antagonist at M(4) receptors, with similar affinities for both. MT3, the most selective antagonist available for M(4) receptors, infused into the CA1 region immediately after training caused amnesia in the rat, indicating the participation of M(4) receptors in memory consolidation. Our goal was to investigate the participation of M(4) receptor in neurotransmission at the hippocampal Schaffer collaterals-CA1 synapses. Two different preparations were used: 1) field potential recordings in freshly prepared rat hippocampal slices with high-frequency stimulation to induce potentiation and 2) whole-cell voltage clamp in cultured hippocampal organotypic slices with paired stimuli. In preparation 1, a dose of MT3 that was previously shown to cause amnesia blocked LTP; the nonselective antagonist scopolamine blocked LTP without affecting basal transmission, although it was depressed with higher concentration. In preparation 2, basal transmission was decreased and LTP induction was prevented by an MT3 concentration that would bind mainly to M(4) receptors. Although M(1) receptors appeared to modulate transmission positively at these excitatory synapses, M(1) activation concomitant with M(4) blockade (by MT1) only allowed a brief, short-term potentiation. Accordingly, M(4) blockade by MT3 strongly supports a permissive role of M(4) receptors and suggests their necessary participation in synaptic plasticity at these synapses.     

Sustained unilateral blockade of rat striatal M1 muscarinic receptors with m1-toxin1 in vivo

m1-Toxin1 is a trace component of the venom of the green mamba that antagonizes M₁ muscarinic receptors specifically and irreversibly in vitro. It was injected into the right caudatoputamen of Wistar rats to occlude M₁ receptors specifically for several days (‘M₁ knockdown’), and to begin to establish the unknown effects of striatal M₁-neurotransmission on movement. The extent and duration of M₁-blockade were evaluated with receptor binding assays and light microscopic autoradiography, using 1–2 nM [³H]pirenzepine to assess unoccupied M₁ receptors. An injection of 27 pmol m1-toxin1 blocked almost all of the M₁ receptors in one caudatoputamen (4 pmol), followed by their slow recovery to supranormal levels (115%) during a week. During maximum M₁-blockade, the binding of 1 nM [³H]N-methylscopolamine to striatal membranes was reduced by only half, indicating no blockade of M₄ receptors, which comprise 51% of striatal muscarinic receptors. It is concluded that m1-toxin1 can produce acute, focal, selective, unilateral and long-lasting M₁-blockade for investigations of the effects of M₁-neurotransmission in vivo. Rats with extensive unilateral striatal M₁-blockade appeared normal, and did not show unilateral deficits in spontaneous forearm use, in contrast to rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. Since M₁-blockade should decrease the activity of both striatonigral and striatopallidal projection neurons, and the relative activity of these neurons is believed to control movement, the results suggest that a balanced decrease in the activity of these neurons on one side of the brain does not produce a right/left imbalance in spontaneous movement.     

Facilitation of amphetamine-induced rotation by muscarinic antagonists is correlated with M2 receptor affinity

This study examined the relationship between the affinity of cholinergic drugs for muscarinic receptor subtypes and their potency in potentiating or inhibiting amphetamine-induced rotation. The ascending nigrostriatal dopaminergic pathway was unilaterally lesioned in male Wistar rats using 6-hydroxydopamine. In these rats, ipsiversive rotation induced by amphetamine sulphate (1 mg/kg, s.c.) was dose-dependently inhibited by the cholinergic agonists oxotremorine, RS86 and pilocarpine and by the acetylcholinesterase inhibitor physostigmine. In contrast the cholinergic antagonists scopolamine, secoverine and dicyclomine facilitated amphetamine-induced rotation. Agonist and antagonist potencies were then compared with M1 and M2 binding site affinities estimated by displacing [3H]pirenzepine from forebrain and [3H]QNB from brainstem homogenates. The data suggest a relationship between antagonist potency and M2 binding site affinity.