Autoradiographic distribution of M1, M2, M3, and M4 muscarinic receptor subtypes in Alzheimer's disease

We studied the autoradiographic densities of all pharmacologically characterised muscarinic receptors (MR) in frontal, temporal, and visual cortex, hippocampal formation, and striatum in autopsied brains from 19 histopathologically verified patients of Alzheimer's disease (AD) and in matched controls. Almost all (16 of 19) of the AD cases were severe. In AD brains, total MR, M1, and M3 MR subtypes were found to be significantly decreased in entorhinal cortex and in most hippocampal strata. Total MR and M1 receptors were also significantly reduced in visual area and in frontal cortex of AD brains, respectively. M2 receptors were significantly reduced over hippocampal formation but increased significantly in striatum of AD brains as compared with controls. M3 receptors in AD were in the range of controls in neocortex and striatum, whereas the M4 receptor subtype was also preserved in all brain regions in AD brains when compared with controls. This is the first autoradiographic study analysing the distribution of all MR subtypes in AD brains. These changes in MR densities concur with the general pattern of neuronal degeneration occurring in AD brains and partly explain the poor response of AD cognitive decline to present cholinergic supplementation therapies. Although M3 and M4 MR were labelled with nonselective approaches, the preservation of M4 and to a lesser degree M3 MR subtypes in AD brains could open an alternative way for the symptomatic therapy of AD dementia. Synapse 26:341–350, 1997. © 1997 Wiley-Liss Inc.     

M1 muscarinic receptor signaling in mouse hippocampus and cortex

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.     

Role of specific muscarinic receptor subtypes in cholinergic parasympathomimetic responses,in vivo phosphoinositide hydrolysis,and pilocarpine-induced seizure activity

Muscarinic agonist-induced parasympathomimetic effects, in vivo phosphoinositide hydrolysis and seizures were evaluated in wild-type and muscarinic M1-M5 receptor knockout mice. The muscarinic agonist oxotremorine induced marked hypothermia in all the knockout mice, but the hypothermia was reduced in M2 and to a lesser extent in M3 knockout mice. Oxotremorine-induced tremor was abolished only in the M2 knockout mice. Muscarinic agonist-induced salivation was reduced to the greatest extent in M3 knockout mice, to a lesser degree in M1 and M4 knockout mice, and was not altered in M2 and M5 knockout mice. Pupil diameter under basal conditions was increased only in the M3 knockout mice. Pilocarpine-induced increases in in vivo phosphoinositide hydrolysis were completely absent in hippocampus and cortex of M1 knockout mice, but in vivo phosphoinositide hydrolysis was unaltered in the M2-M5 knockout mice. A high dose of pilocarpine (300 mg/kg) caused seizures and lethality in wild-type and M2-M5 knockout mice, but produced neither effect in the M1 knockout mice. These data demonstrate a major role for M2 and M3 muscarinic receptor subtypes in mediating parasympathomimetic effects. Muscarinic M1 receptors activate phosphoinositide hydrolysis in cortex and hippocampus of mice, consistent with the role of M1 receptors in cognition. Muscarinic M1 receptors appear to be the only muscarinic receptor subtype mediating seizures.     

Muscarinic blockade weakens interaction of gamma with theta rhythms in mouse hippocampus

theta (4-12 Hz) and gamma (40-90) oscillations are prominent rhythms in the mammalian brain. A striking feature of these rhythms, possibly vital to memory encoding, is their specific coordination in a manner that has been termed 'nesting', i.e. the preferred occurrence of bouts of gamma activity during specific phases of theta. Both rhythms are shaped by the neuromodulator acetylcholine, but it is unknown to what degree their coordination is influenced by cholinergic neuromodulation. Here, we investigated the effects of a blockade of muscarinic acetylcholine receptors by atropine on theta and gamma oscillations, and their interaction, in mouse hippocampus in vivo. Multi-site recordings from area CA1 of freely moving mice showed that under control conditions gamma activity was amplitude-modulated at theta frequencies. This coordination of theta and gamma oscillations, as assessed by cross-correlation of theta with the gamma envelope, was prominent in basal and apical dendritic laminae but not in intermediate laminae. It was stronger during active exploration than during awake immobility. Atropine (50 mg/kg intraperitoneal) altered several aspects of the individual and nested rhythms. It rendered theta activity irregular, decreased theta oscillation frequency and reduced gamma power. Atropine also reduced the amplitude-modulation of gamma oscillations at theta frequencies, in part by perturbing the coordination of the rhythms on a short time scale. Thus, our findings demonstrate that phase locking of the amplitude of gamma oscillations to theta in hippocampal area CA1 is partially governed by neuronal elements harbouring muscarinic receptors.     

Sphingomyelinase selectively reduces M1 muscarinic receptors in rat hippocampal membranes

Although there is evidence that nicotinic acetylcholine (Ach) receptors are influenced by ceramides, we do not currently know whether or not these sphingolipids can also regulate the muscarinic subtypes of Ach receptors. Using the whole-cell patch technique, we demonstrated that the effectiveness of the muscarinic receptor agonist pilocarpine, in enhancing spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, was completely abolished in hippocampal slices pre-exposed to the ceramide-generating enzyme sphingomyelinase (SMase). Western blot experiments, performed with biotinylated hippocampal membranes, showed that this electrophysiological defect possibly relies on the loss of M1 muscarinic Ach receptors at the cell surface. However, the effect appears to be relatively specific as the cell-surface expression of M4 muscarinic receptors was not found to be impacted by SMase treatment. Interestingly, we observed that G protein-coupled receptor kinases 2 and β-arrestin1/2 interactions with M1-immunoprecipitated proteins were substantially augmented in SMase-treated slices and that the reduction of cell-surface M1 muscarinic receptor expression generated was completely suppressed by the muscarinic antagonist atropine. Collectively, our data suggest that selective internalization of M1 muscarinic receptors can be accentuated in neurons subjected to high ceramide levels. The potential physiopathological implications of this finding are presented.     

Ontogeny of muscarinic receptors in rat brain

The ontogeny of muscarinic acetylcholine receptors in the rat brain has been examined using the radioligards, [³H]N-methylscopolamine, [³H]propylbenzilylcholine and [³H]oxotremorine-M. In the 3 regions of the brain selected for study, the cerebral cortex, the diencephalon and the medulla-pons, the receptors develop at different rates. The most rapid development takes place in the medulla with considerably slower maturation in the diencephalon and cerebral cortex. In the cortex, the agonist binding properties of the muscarinic receptors vary during development. There appears to be a 6–7 day lag in the appearance of high affinity sites following formation of low affinity sites.     

The effect of entorhinal cortical ablation on the distribution of muscarinic cholinergic receptors in the rat hippocampus

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.     

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.     

Rat Schwann cells express M1-M4 muscarinic receptor subtypes

The expression of different muscarinic receptor subtypes was analyzed in immature Schwann cells obtained from sciatic nerve of 2-day neonatal rats. By using RT-PCR analysis, we demonstrated the presence of M1, M2, M3, and M4 receptor subtypes in cultured Schwann cells, with M2 displaying the highest expression levels. Muscarinic subtypes were also quantified by immunoprecipitation and [3H]QNB binding. With this approach, we found the levels of receptor expression to be M2 > M3 > M1. M4 is expressed at very low levels, and M5 receptor was not detectable. Moreover, we also demonstrated that stimulation of the receptors by muscarinic agonists activates previously described signal transduction pathways, leading to a decrease of cAMP and an increase of IP3 levels not associated with an efficient intracellular Ca2+ release. The presence and activity of particular muscarinic receptors in immature Schwann cells suggest that ACh may play an important role in Schwann cell development.     

Distinct directional couplings between slow and fast gamma power to the phase of theta oscillations in the rat hippocampus

It is well‐established that theta (~4–10 Hz) and gamma (~25–100 Hz) oscillations interact in the rat hippocampus. This cross‐frequency coupling might facilitate neuronal coordination both within and between brain areas. However, it remains unclear whether the phase of theta oscillations controls the power of slow and fast gamma activity or vice versa. We here applied spectral Granger causality, phase slope index and a newly developed cross‐frequency directionality (CFD) measure to investigate directional interactions between local field potentials recorded within and across hippocampal subregions of CA1 and CA3 of freely exploring rats. Given the well‐known CA3 to CA1 anatomical connection, we hypothesized that interregional directional interactions were constrained by anatomical connection, and within‐frequency and cross‐frequency directional interactions were always from CA3 to CA1. As expected, we found that CA3 drove CA1 in the theta band, and theta phase‐to‐gamma power coupling was prominent both within and between CA3 and CA1 regions. The CFD measure further demonstrated that distinct directional couplings with respect to theta phase was different between slow and fast gamma activity. Importantly, CA3 slow gamma power phase‐adjusted CA1 theta oscillations, suggesting that slow gamma activity in CA3 entrains theta oscillations in CA1. In contrast, CA3 theta phase controls CA1 fast gamma activity, indicating that communication at CA1 fast gamma is coordinated by CA3 theta phase. Overall, these findings demonstrate dynamic directional interactions between theta and slow/fast gamma oscillations in the hippocampal network, suggesting that anatomical connections constrain the directional interactions.     

Corticosterone and corticotropin‐releasing factor acutely facilitate gamma oscillations in the hippocampus in vitro

Stressful experiences do not only cause peripheral changes in stress hormone levels, but also affect central structures such as the hippocampus, implicated in spatial orientation, stress evaluation, and learning and memory. It has been suggested that formation of memory traces is dependent on hippocampal gamma oscillations observed during alert behaviour and rapid eye movement sleep. Furthermore, during quiescent behaviour, sharp wave‐ripple (SW‐R) activity emerges. These events provide a temporal window during which reactivation of memory ensembles occur. We hypothesized that stress‐responsive modulators, such as corticosterone (CORT), corticotropin‐releasing factor (CRF) and the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one (THDOC) are able to modulate gamma oscillations and SW‐Rs. Using in vitro hippocampal slices, we studied acute and subacute (2 h) impact of these agents on gamma oscillations in area cornu ammonis 3 of the ventral hippocampus induced by acetylcholine (10 μm ) combined with physostigmine (2 μm ). CORT increased the gamma oscillations in a dose‐dependent fashion. This effect was mediated by glucocorticoid receptors. Likewise, CRF augmented gamma oscillations via CRF type 1 receptor. Lastly, THDOC was found to diminish cholinergic gamma oscillations in a dose‐dependent manner. Neither CORT, CRF nor THDOC modulated gamma power when pre‐applied for 1 h, 2 h before the induction of gamma oscillations. Interestingly, stress‐related neuromodulators had rather mild effects on spontaneous SW‐R compared with their effects on gamma oscillations. These data suggest that the alteration of hippocampal gamma oscillation strength in vitro by stress‐related agents is an acute process, permitting fast adaptation to new attention‐requiring situations in vivo.     

Autoradiographic localization of muscarinic cholinergic receptors in the hippocampus of patients with senile dementia

Muscarinic cholinergic receptors were localized by autoradiography in the hippocampi from 4 patients with senile dementia (S.D.) and 4 neurologically normal age-matched controls. A large number of senile plaques were observed in the hippocampi from S.D. patients, whereas they were not observed in control brains. The distribution and density of muscarinic receptors was similar in control and S.D. patients. In the hippocampus, areas rich in receptors were subiculum, strata oriens, pyramidalis and radiatum and the molecular layer of the dentate gyrus. The density of autoradiographic grains over the senile plaques was comparable to that over the surrounding neuropil, indicating that senile plaques have muscarinic receptors.     

Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: Relation to cholinergic nuclei and projections

The autoradiographic distributions of receptors with high affinity for [3H]oxotremorine-M (the M2 receptor) and [3H]pirenzepine (the M1 receptor) were studied in the rat brain. M1 receptors were seen in highest density only in telencephalic structures: cerebral cortex (layers I-II), hippocampus, dentate gyrus, medial and basolateral amygdala, nucleus accumbens and caudate/putamen. M2 receptors were detected throughout the brain, with highest levels observed in cerebral cortical layers III and V, forebrain cholinergic nuclei, caudate/putamen, various thalamic areas, inferior and superior colliculus, interpeduncular and pontine nuclei, brainstem cholinergic nuclei and cervical spinal cord regions. M2 receptors were found to be good markers for cholinergic cell groups and the majority of cholinergic projection areas, whereas M1 receptors were only found in a large sub-group of telencephalic cholinergic projection areas, and the pattern of distribution of receptors in these areas differed from that of M2 receptors. Scatchard analysis of [3H]oxotremorine-M binding to inferior collicular slices revealed one site with a dissociation constant (Kd) of 1.9 nM and a receptor density (Bmax) of 1.4 pmol/mg protein. Our data support the hypothesis that M1 and M2 receptors are physically distinct sub-types of the muscarinic acetylcholine receptor.     

Gamma oscillations during episodic memory processing provide evidence for functional specialization in the longitudinal axis of the human hippocampus

The question of whether the anterior and posterior hippocampus serve different or complementary functional roles during episodic memory processing has been motivated by noteworthy findings in rodent experiments and from noninvasive studies in humans. Researchers have synthesized these data to postulate several models of functional specialization, However, the issue has not been explored in detail using direct brain recordings. We recently published evidence that theta power increases during episodic memory encoding occur in the posterior hippocampus in humans. In our current investigation we analyzed an expanded data set of 32 epilepsy patients undergoing stereo EEG seizure mapping surgery with electrodes precisely targeted to the anterior and posterior hippocampus simultaneously who performed an episodic memory task. Using a repeated measures design, we looked for an interaction between encoding versus retrieval differences in gamma oscillatory power and anterior versus posterior hippocampal location. Our findings are consistent with a recently articulated model (the HERNET model) favoring posterior hippocampal activation during retrieval related processing. We also tested for encoding versus retrieval differences in the preferred gamma frequency band (high versus low gamma oscillations) motivated by published rodent data.     

Effects of antipsychotic medication on muscarinic M1 receptor mRNA expression in the rat brain

Alterations in muscarinic M1 receptor protein and mRNA expression have been revealed in post-mortem brains of schizophrenia patients. Most patients had been treated with antipsychotics, so medication effects cannot be excluded as a possible explanation for these results. With in situ hybridization, this study investigated M1 receptor mRNA expression in rats treated with the typical antipsychotic haloperidol (0.3 mg/kg/day) and the atypical antipsychotics olanzapine (1.5 mg/kg/day) and aripiprazole (2.25 mg/kg/day) for 1 or 12 weeks. Compared with the control group, haloperidol significantly increased (approximately 13-21%, P < 0.05) M1 mRNA expression in the CA1, CA2, and CA3 regions of the hippocampus after both 1 and 12 weeks of treatment, and it also increased (approximately 17%, P < 0.01) M1 mRNA expression in the substantia nigra compacta after 1 week of treatment. Olanzapine significantly increased (14-22%, P < 0.05) M1 mRNA expression in the hippocampus (CA1, CA2, and CA3) and substantia nigra compacta after 12 weeks of treatment, but not after 1 week. Aripiprazole significantly increased (17%, P < 0.01) M1 mRNA expression in the hippocampus (CA1) after both 1 and 12 week treatments and increased (12%, P < 0.05) M1 mRNA expression in the nucleus accumbens after 1 week of treatment. Despite their different affinities for muscarinic M1 receptors, all three antipsychotic medications induced a similar trend of change in M1 mRNA expression in selected brain regions. These data suggest that the decreased M1 receptor protein and mRNA expression observed in schizophrenia patients is unlikely to be a consequence of drug treatments and implicates muscarinic M1 receptors in the pharmacotherapy of the disease.     

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.     

Evidence for an agonist independent down regulation of hippocampal muscarinic receptors in kindling

Kindling induces a decline of hippocampal muscarinic cholinergic receptors. To test the hypothesis that the decline was mediated by the agonist, acetylcholine, adult male Sprague-Dawley rats were lesioned in the medial septum prior to kindling. Despite the marked destruction of presynaptic cholinergic terminals in the hippocampus, amygdala kindling proceeded normally and the hippocampal muscarinic receptor decline was not blocked. A small but significant decline in choline acetyltransferase activity was demonstrated in non-lesioned kindled rats. It is proposed that the kindling induced decline of hippocampal muscarinic receptors is mediated by repeated neuronal depolarization.     

Recognition memory and theta–gamma interactions in the hippocampus

Neuronal oscillations and cross‐frequency interactions in the rat hippocampus relate in important ways to memory processes and serve as a model for studying oscillatory activity in cognition more broadly. We report here that hippocampal synchrony (CA3–CA1 coherence) increased markedly in the low gamma range as rats were exploring novel objects, particularly those for which the rat subsequently showed good memory. The gamma synchrony varied across phases of the theta rhythm such that coherence was highest at the falling slope and trough of the theta wave. Further, the shape of the theta wave was more asymmetric and elongated at the falling slope during exploration of objects for which the rat subsequently showed good memory as compared with objects for which the rat subsequently showed poor memory. The results showed a strong association between event‐related gamma synchrony in rat hippocampus and memory encoding for novel objects. In addition, a novel potential mechanism of cross‐frequency interactions was observed whereby dynamic alterations in the shape of theta wave related to memory in correspondence with the strength of gamma synchrony. These findings add to our understanding of how theta and gamma oscillations interact in the hippocampus in the service of memory. © 2013 Wiley Periodicals, Inc.     

Computational model of carbachol-induced delta, theta, and gamma oscillations in the hippocampus.

Field potential recordings from the rat hippocampus in vivo contain distinct frequency bands of activity, including delta (0.5-2 Hz), theta (4-12 Hz), and gamma (30-80 Hz), that are correlated with the behavioral state of the animal. The cholinergic agonist carbachol (CCH) induces oscillations in the delta (CCH-delta), theta (CCH-theta), and gamma (CCH-gamma) frequency ranges in the hippocampal slice preparation, eliciting asynchronous CCH-theta, synchronous CCH-delta, and synchronous CCH-theta with increasing CCH concentration (Fellous and Seinowski, Hippocampus 2000;1 0:187-197). In a network model of area CA3, the time scale for CCH-delta corresponded to the decay constant of the gating variable of the calcium-dependent potassium (K-AHP) current, that of CCH-theta to an intrinsic subthreshold membrane potential oscillation of the pyramidal cells, and that of CCH-gamma to the decay constant of GABAergic inhibitory synaptic potentials onto the pyramidal cells. In model simulations, the known physiological effects of carbachol on the muscarinic and K-AHP currents, and on the strengths of excitatory postsynaptic potentials, reproduced transitions from asynchronous CCH-theta to CCH-delta and from CCH-delta to synchronous CCH-theta. The simulations also exhibited the interspersed CCH-gamma/CCH-delta and CCH-gamma/CCH-theta that were observed in experiments. The model, in addition, predicted an oscillatory state with all three frequency bands present, which has not yet been observed experimentally.