Muscarinic cholinergic receptor subtypes in normal human brain and Alzheimer's presenile dementia

Brain muscarinic M1 and M2 binding sites, as defined by their affinity for pirenzepine, were studied in 24 regions of 3 normal control brains. For each region, the binding of [3H]QNB, a non-subtype selective antagonist, was saturable with a mean KD of 0.117 +/- 0.066 nM. Computer-assisted analysis of the pirenzepine competition binding curves yielded the amount of high affinity (M1) and low affinity sites (M2) as well as their respective Ki values for this ligand. The neocortex contained a mixed population of 67% M1 and 33% M2 sites, without locoregional heterogeneity. The muscarinic receptors of the caudate nucleus, putamen, pallidum, hippocampus and nucleus amygdalis were predominantly of the M1 type as well. The cerebellum and the brain-stem are examples of regions containing over 90% M2 sites. White matter structures like the centrum ovale appeared to contain low concentrations of [3H]QNB binding sites, predominantly of the M1 subtype. The data for the frontal cortex were compared with data obtained in 3 patients who died from Alzheimer's disease with very early onset: the muscarinic receptor concentrations were somewhat lower but their M1/M2 ratio remained unchanged. GTP caused an appreciable rightward shift and steepening of the carbachol competition binding curve in M2 predominant regions such as the pons, whereas only a slight shift was observed in the cortex. In the presence of GTP, the alkylating reagent N-ethylmaleimide caused a 35-fold increase of the affinity for carbachol in M2 predominant regions. In contrast, regions where the receptors are predominantly of the M1 type, N-ethylmaleimide caused only a 5-fold increase in agonist affinity. These findings confirm our previously formulated hypothesis that the ability of N-ethylmaleimide to modulate the agonist affinity is an additional criterion for the characterization of M1 and M2-type receptors.     

Characterization of muscarinic receptor subtypes in Alzheimer and control brain cortices by selective muscarinic antagonists.

Subtypes of muscarinic receptors were characterized in the frontal cortices of control and Alzheimer brains, with labelled quinuclidinyl benzilate [3H]QNB and the unlabelled muscarinic antagonists pirenzepine, AF-DX 116, hexahydro-sila-diphenidol (HHSiD), para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and himbacine. High and low affinity sites were observed for both pirenzepine and AF-DX 116 in human control frontal cortices. The majority (76%) of the pirenzepine binding sites showed high affinity to the muscarinic receptors (M1), while the rest of the binding sites had an affinity that was 40 times less. AF-DX 116 displayed two sets of binding sites where the high affinity AF-DX 116 (M2) sites constituted 27%, while the low affinity AF-DX 116 (non-M2 site) was 73%. A single class of binding sites was observed for HHSiD, p-F-HHSiD and himbacine in human frontal cortices. HHSiD showed an affinity in the frontal cortices that was comparable to that of the pirenzepine high affinity binding (M1) sites. The affinity of p-F-HHSiD was three times lower than that of HHSiD but similar to himbacine. A significant increase in the affinity (+ 40%) as well as in the Bmax (+ 99%) value was observed for the pirenzepine high affinity binding sites (M1) in the frontal cortices of Alzheimer brains compared to controls. Similarly, a significant increase was observed in the Bmax value (+ 60%) for the AF-DX 116 low affinity binding sites (non-M2), while no change was found for the high affinity binding sites (M2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.     

Presynaptic M2-muscarinic receptors on noradrenergic nerve endings and endothelium-derived M3 receptors in cat cerebral arteries.

The muscarinic (M) receptors involved in the vasodilation elicited by acetylcholine (ACh) and in the carbachol inhibition in electrically induced [3H]noradrenaline (NA) release in cat cerebral arteries was investigated. For this, atropine, pirenzepine, AF-DX 116, 4-DAMP, non-specific, M1, M2 and M3 receptor antagonists, respectively, were used. ACh elicited concentration-dependent relaxations up to 10(-6) M which were attenuated by these antagonists; the order of potency (pA2 values) to inhibit the ACh-induced relaxation was: atropine (10.1) 4-DAMP (8.9) greater than pirenzepine (7.6) greater than AF-DX 116 (5.9). The electrical stimulation (200 mA, 0.3 ms, 2 Hz, during 1 min) of these arteries preincubated with [3H]NA caused tritium release which was inhibited by carbachol (10(-6) M). The 4 antagonists attenuated the action of the M agonist; the order of potency (pIC50 values) was: atropine (8.7) greater than 4-DAMP (8.1) greater than AF-DX 116 (7.9) greater than pirenzepine (5.8). The action of McN-A-343, a putative M1 agonist, was also investigated. This agent produced small vasodilator responses and elevated concentrations (5 x 10(-5) M) inhibited the stimulated NA release, which was partially antagonized by atropine (10(-7) M) and pirenzepine (10(-8) and 10(-7) M). These results suggest the existence of M3 and M2 receptors mediating the relaxation induced by ACh and the NA release inhibition evoked by carbachol, respectively.     

In vitro electro-pharmacological and autoradiographic analyses of muscarinic receptor subtypes in rat hypothalamic ventromedial nucleus: implications for cholinergic regulation of lordosis

Muscarinic agonists can act through the hypothalamic ventromedial nucleus (VMN) to facilitate lordosis. To elucidate the neuronal mechanism(s) underlying this muscarinic facilitation, effects of muscarinic agents on the single-unit activity of VMN neurons recorded in brain tissue slices of estrogen-primed female rats were analyzed. All the agonists tested, including acetylcholine (ACh), oxotremorine-M (OM), carbachol (CCh) and McN-A-343 (McN), evoked primarily excitation (80–100%), some inhibition (0–20%) and occasional biphasic responses (0–8%). By comparing the response magnitude and the effectiveness in evoking a response, the rank order for evoking excitation, the primary response, was found to be: OM > CCh > ACh McN, which is consistent with that (OM > CCh McN) for facilitating lordosis reported by others. This consistency and the frequency of its occurrence suggest that the excitatory electric action of the muscarinic agonists is related to their facilitatory behavioral effect. Experiments with antagonists selective for M1 (pirenzepine), M2 (AF-DX 116) and M3 (4-DAMP and p-F-HHSiD) indicate that muscarinic excitations are mediated by M1 and/or M3, but not M2. Since M1 receptors have been shown to be neither sufficient nor necessary to mediate the muscarinic facilitation, M3 receptor may be crucially involved in this behavioral effect. Autoradiographic assays of binding to [³H]4-DAMP with or without pirenzepine and AF-DX 116, also indicate the presence of M3 receptors in the VMN. Quantitative analyses show that the M3 binding was not affected by the in vivo estrogen priming required to permit muscarinic agonists to facilitate lordosis. Thus, while the excitation mediated by M3 is likely to be involved in muscarinic facilitation of lordosis, the regulation of M3 receptor density does not seem to be involved in the permissive action of estrogen.     

The effect of subtype-selective muscarinic receptor antagonists on the cholinergic current in motoneurons of the lobster cardiac ganglion

Muscarinic agonists evoke a voltage-dependent inward current in motoneurons of the lobster cardiac ganglion. In this study, a number of drugs, known to show muscarinic receptor subtype selectivity in mammals, were used to determine the pharmacological profile of the muscarinic receptor on lobster motoneurons. The neurons were held under voltage-clamp, and various concentrations of the antagonists were applied in the presence of 1 mM methacholine. From competition curves plotting agonist-induced current against antagonist concentration, the inhibitor affinity constant and the slope factor were determined. The rank order of potencies of antagonists having an effect was: atropine greater than pirenzepine greater than 4-DAMP greater than methoctramine greater than HHSiD = (R)-HHD greater than (S)-HHD. Neither AF-DX 116 nor gallamine were effective at concentrations as high as 10 mM. The M1-selective agonist McN-A-343 had no effect. Although this crustacean muscarinic receptor resembles the mammalian M1 muscarinic receptor because of its relatively high affinity for pirenzepine, the rank order of other subtype-specific antagonists does not otherwise resemble that of any of the pharmacologically defined muscarinic receptors in mammals. It may be preferable, therefore, to use a term such as 'pirenzepine-sensitive' muscarinic receptor rather than M1 or 'M1-like' for invertebrate muscarinic receptors with pharmacological characteristics like those reported here.     

Muscarinic receptors and second-messenger responses of neurons in primary culture

The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were maintained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [3H]N-methylscopolamine (NMS) bound specifically and with high affinity (Kd = 70 pM) to a homogeneous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [3H]NMS by pirenzepine indicated the presence of heterogeneous sites (81% high affinity sites, Kh = 51 nM, K1 = 1.5 microM); AF-DX 116 showed the opposite selectivity (15% high affinity sites, Kh = 56 nM, K1 = 1.3 microM). The dopamine agonist SKF-38393 (1 microM) enhanced the accumulation of cyclic adenosine monophosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC50, 1.7 microM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC50, 61 microM). Both responses were antagonized by pirenzepine (apparent Ki of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent Ki 540 nM and 160 nM, respectively). In binding studies on brainstem cultures, AF-DX 116 indicated the presence of two sites of approximately equal abundance (Kh = 170 nM, K1 = 2.9 microM); data for pirenzepine were adequately fit by a one-site model (Kd = 630 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography

The muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M1 (pirenzepine), M2 (AF-DX 116), and M3 (4-DAMP) and the classical nonselective antagonist atropine. pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.50, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M3.     

Muscarinic1 and 2 receptor mRNA in the human caudate-putamen: no change in m1 mRNA in schizophrenia

Studies using tissue obtained at autopsy suggest that changes in cholinergic neurons could be important in the pathology of schizophrenia.1-4 We have previously reported a decrease in [3H]pirenzepine binding5 and [3H]AF-DX 384 binding6 to caudate-putamen (CP) from subjects who had schizophrenia. Under the conditions chosen, [3H]pirenzepine would predominately bind to muscarinic1 (M1) and muscarinic4 (M4) receptors,7whereas [3H]AF-DX 384 would mainly bind to muscarinic2 (M2) and M4 receptors.8 Given the relative concentrations of M1, M2 and M4 receptors in the human CP and the magnitude of the decreases in radioligand binding in schizophrenia, our results most likely reflected a change in the density of M1 and M2 receptors in the CP from the schizophrenic subjects. In situ hybridisation has now been used to determine levels of m1 and m2 mRNA in CP from 14 schizophrenic and 16 control subjects previously used for radioligand binding. m2 mRNA in the CP from the schizophrenic and control subjects was below the sensitivity of in situhybridisation. There was no difference in the levels of m1 mRNA in CP from schizophrenic and control subjects (mean +/- SEM: 103 +/- 16 vs106 +/- 17 fmol [35S]oligonucleotide probe g-1estimated tissue equivalents, P = 0.91). In conclusion, data from our radioligand binding studies show decreases in [3H]pirenzepine binding that are likely to reflect a decrease in the density of M1 receptors in CP from schizophrenic subjects. Our data in this study show the absence of a concomitant change in mRNA coding for that receptor.     

Selectivity of pirenzepine in the central nervous system. I. Direct autoradiographic comparison of the regional distribution of pirenzepine and carbamylcholine binding sites

The binding capacities of the novel antagonist pirenzepine and the agonist carbamylcholine were examined autoradiographically to compare their abilities to reduce the binding of 1-[3H]quinuclidinyl benzilate ([3H]-1-QNB). This technique, which is applicable to any muscarinic ligand, permits a direct comparison between the binding of carbamylcholine and pirenzepine in the same assay. Analysis of the binding curves generated by standard scintillation counting of whole-brain slices indicated that the ligands bound heterogeneously to muscarinic receptors in the brain. Following apposition of slides to tritium-sensitive film, the binding profile for each ligand was examined visually and by microdensitometry. Regional analyses indicated that the agonist carbamylcholine displayed highest potency for thalamic nuclei, lower potency for cortical regions, and the lowest affinity for layers of the hippocampus. The M1-selective ligand pirenzepine displayed the highest potency for the dentate gyrus of the hippocampus, with lower inhibition levels in the cortex, and the lowest levels of inhibition found in the thalamus. The distribution of high affinity agonist sites was found to be distinct from the distribution of high-affinity antagonist binding sites. In a separate assay, the regional inhibition of pirenzepine and scopolamine was compared for the hippocampus and the forebrain. While scopolamine did not distinguish between muscarinic receptor sites in the hippocampus and cortex, pirenzepine inhibited [3H]-1-QNB labeling in the hippocampus significantly greater than in the cerebral cortex, providing additional evidence for the hypothesis that pirenzepine is a selective antagonist.     

Characterization of [3H]AF-DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic-M2 receptor sites

This study shows that [³H]AF-DX 116 binds specifically, saturably, and with high affinity to putative muscarinic-M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [³H]AF-DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (K_d < 4.0 nM) and one class of lower affinity sites (K_d > 50 nM, except in the cerebellum). The apparent maximal capacities (B_max) of [³H]AF-DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [³H]AF-DX 116 binding sites was 10.4 ± 0.4 (K_d = 1.9 ± 0.2 nM) and 39.1 ± 2.6 (K_d = 26 ± 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the K_d for the higher and lower affinity [³H]AF-DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF-DX 116 is more potent than (–)QNB > atropine > methoctramine > 4-DAMP > gallamine > NMS > carbamylcholine > oxotremorine > pirenzepine > > nicotine in competing for the higher affinity [³H]AF-DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites. For example, (–)QNB was more potent than AF-DX 116 and pirenzepine was more potent than either oxotremorine or 4-DAMP at the lower affinity [³H]AF-DX 116 sites. In addition, pirenzepine was modestly more potent at the lower compared to the higher affinity sites. Neither the higher nor the lower affinity [³H]AF-DX 116 sites were sensitive to the effects of Gpp(NH)p or N-ethylmaleimide. In addition, the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding to the higher and the lower affinity sites was altered by Gpp(NH)p and NEM, to a similar extent. However, Gpp(NH)p decreased the affinity of carbamylcholine (i.e., increased the IC₅₀), whereas N-ethylmaleimide had the opposite effect. Furthermore, N-ethylmaleimide also appeared to steepen the curve for the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding, as evidenced by the increased n^H. Thus it appears that [³H]AF-DX 116 binds to two subsets of muscarinic-M2 receptors in the rat brain, which can be differentiated by their affinity for certain agonists and antagonists.     

Modulation by adenosine of both muscarinic M1-facilitation and M2-inhibition of [3H]-acetylcholine release from the rat motor nerve terminals

The crosstalk between adenosine and muscarinic autoreceptors regulating evoked [3H]-acetylcholine ([3H]-ACh) release was investigated on rat phrenic nerve-hemidiaphragm preparations. Motor nerve terminals possess facilitatory M1 and inhibitory M2 autoreceptors that can be activated by McN-A-343 (1-30 microm) and oxotremorine (0.3-100 microm), respectively. The muscarinic receptor antagonist, dicyclomine (3 nm-10 microm), caused a biphasic (inhibitory/facilitatory) effect, indicating that M1-facilitation prevails during 5 Hz stimulation trains. Concomitant activation of AF-DX 116-sensitive M2 receptors was partially attenuated, as pretreatment with M1 antagonists, muscarinic toxin 7 (MT-7, 0.1 nm) and pirenzepine (1 nm), significantly enhanced inhibition by oxotremorine. Activation of A2A-adenosine receptors with CGS 21680C (2 nm) (i) potentiated oxotremorine inhibition, and (ii) shifted McN-A-343-induced facilitation into a small inhibitory effect. Conversely, the A1-receptor agonist, R-N6-phenylisopropyl adenosine (R-PIA, 100 nm), attenuated the inhibitory effect of oxotremorine, without changing facilitation by McN-A-343. Synergism between A2A and M2 receptors is regulated by a reciprocal interaction with facilitatory M1 receptors, which may be prevented by pirenzepine (1 nm). During 50 Hz-bursts, facilitation (M1) of [3H]-ACh release by McN-A-343 disappeared, while the inhibitory (M2) effect of oxotremorine became predominant. This muscarinic shift results from the interplay with A2A receptors, as it was precluded by the selective A2A receptor antagonist, ZM 241385 (10 nm). In conclusion, when the muscarinic M1 positive feedback loop is fully operative, negative regulation of ACh release is mediated by adenosine A1 receptors. During high frequency bursts, tonic activation of A2A receptors promotes M2 autoinhibition by braking the M1 receptor operated counteraction.     

Himbacine recognizes a high affinity subtype of M2 muscarinic cholinergic receptors in the rat cerebral cortex

The in vitro receptor binding properties of a muscarinic antagonist himbacine have been studied in rat cerebral cortical, cardiac and ileal membranes. Himbacine displayed high affinity (KH = 2.94 nM) for 19%, and low affinity (KL = 71.2 nM) for the remaining muscarinic receptors in rat cerebral cortex. This high affinity of himbacine agrees with its affinity for the 62% of cerebral cortical [3H]AF-DX 116 binding sites (KH = 2.30 nM). The affinity of himbacine for cardiac receptors (Ki = 9.06 nM) and ileal receptors (Ki = 12.4 nM) was the same. Therefore, himbacine appears to be a high-affinity M2-selective ligand which recognizes a subtype of M2 receptors in the cerebral cortex.     

Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.

It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits.     

Altered hippocampal muscarinic M4, but not M1, receptor expression from subjects with schizophrenia.

BACKGROUND: Having shown a decrease in [3H]pirenzepine binding in the hippocampus from subjects with schizophrenia, we wished to determine whether such a change in radioligand binding was associated with changes in hippocampal mRNA for the muscarinic1 (M1) and muscarinic4 (M4) receptors in tissue from different cohorts of subjects. METHOD: The [3H]pirenzepine binding using autoradiography and in situ hybridization with oligonucleotides specific for muscarinic M1 and M4 receptors were completed using hippocampal tissue obtained postmortem from 20 control subjects and 20 subjects with schizophrenia. RESULTS: The [3H]pirenzepine binding was decreased in the dentate gyrus (p < .05), CA3 (p < .01), CA2 (p < .05), and CA1 (p < .01) regions of the hippocampus from subjects with schizophrenia. Levels of M4 mRNA varied with the diagnosis of schizophrenia (p = .01), but significant region-specific changes were not apparent. Changes in levels of mRNA for the muscarinic M1 receptor were not detected with diagnosis. CONCLUSIONS: This study suggests that decreases in hippocampal [3H]pirenzepine binding in subjects with schizophrenia are most likely associated with widespread changes in expression levels of the M4 receptor. These data further implicate the hippocampal formation in the pathology of schizophrenia.     

Role of muscarinic receptors, G-proteins, and intracellular messengers in muscarinic modulation of NMDA receptor-mediated synaptic transmission.

Previously, we reported that activation of muscarinic receptors modulates N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in auditory neocortex [Aramakis et al. (1997a) Exp Brain Res 113:484-496]. Here, we describe the muscarinic subtypes responsible for these modulatory effects, and a role for G-proteins and intracellular messengers. The muscarinic agonist oxotremorine-M (oxo-M), at 25-100 microM, produced a long-lasting enhancement of NMDA-induced membrane depolarizations. We examined the postsynaptic G-protein dependence of the modulatory effects of oxo-M with the use of the G-protein activator GTP gamma S and the nonhydrolyzable GDP analog GDP beta S. Intracellular infusion of GTP gamma S mimicked the facilitating actions of oxo-M. After obtaining the whole-cell recording configuration, there was a gradual, time-dependent increase of the NMDA receptor-mediated slow-EPSP, and of iontophoretic NMDA-induced membrane depolarizations. In contrast, intracellular infusion of either GDP beta S or the IP3 receptor antagonist heparin prevented oxo-M mediated enhancement of NMDA depolarizations. The muscarinic receptor involved in enhancement of NMDA iontophoretic responses is likely the M1 receptor, because the increase was prevented by pirenzepine, but not the M2 antagonists methoctramine or AF-DX 116. Oxo-M also reduced the amplitude of the pharmacologically isolated slow-EPSP, and this effect was blocked by M2 antagonists. Thus, muscarinic-mediated enhancement of NMDA responses involves activation of M1 receptors, leading to the engagement of a postsynaptic G-protein and subsequent IP3 receptor activity.     

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.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Distribution and function of cholinergic receptors in the sheep detrusor muscle

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.