Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: a light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1-M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1>M2>M4>M3>M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3-M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.     

Muscarinic desensitization after septal lesions in rat hippocampus: evidence for the involvement of G-proteins.

Three days after bilateral septal lesions, regional and laminar densities of the muscarinic acetylcholine receptors of the dorsal rat hippocampus were studied. The concentration of [3H]N-methylscopolamine binding sites and muscarinic M1 and M2 receptor subtypes, as well as the affinity of muscarinic receptors and their sensitivity to modulation by 5-guanylylimidodiphosphate were analysed by quantitative receptor autoradiography. The measurement of these parameters was performed with a computerized image-processing system allowing a spatial resolution down to the level of single hippocampal layers. No postlesional changes of the density of M1 receptors were detected. M2 receptors showed a remarkable decrease in concentration (less than 21%) in some hippocampal layers after septal lesions. In competition experiments the affinity of muscarinic receptors for the cholinergic agonist carbamylcholine chloride decreased significantly in all hippocampal subregions and layers of the lesioned animals. In contrast to controls, the sensitivity of muscarinic receptors of the lesioned animals could not be modulated by 5-guanylylimidodiphosphate. These findings demonstrate a desensitization of muscarinic receptors in the rat hippocampus three days after septal lesions, which is caused by changes of the coupling of guanine nucleotide-binding proteins to muscarinic receptors. Therefore, the lesion-induced alteration of the muscarinic receptor-effector complex is a major aspect of the hippocampal plasticity after cholinergic denervation.     

Cholinergic drug effects and brain muscarinic receptor binding in aged rats

Muscarinic systems are significantly altered in the brains of laboratory animals and man as a result of normal aging. Cholinergic neurotransmission in cerebral cortex and hippocampus is also severely impaired in a major age-related neurological disorder. Alzheimer's disease. The objective of these studies was to assess specific ³H-quinuclidinyl benzilate (³H-QNB) binding to brain muscarinic receptors in young, adult and senescent Fischer 344 rats, and to relate receptor changes to differences in the pharmacologic actions of cholinergic drugs. Muscarinic receptor density declined with advanced age in the frontal cortex, corpus striatum and hypothalamus, but no age-related changes in receptor affinity were observed. Specific binding of ³H-QNB in hippocampus was not significantly altered. In contrast, the in vivo effects of oxotremorine (hypothermia and antinociception) were markedly enhanced in aged rats, whereas scopolamine-induced locomotor activity was reduced. Hence, senescent rats were more sensitive to the pharmacologic actions of a cholinergic agonist, but less responsive than young rats to a muscarinic antagonist. These seemingly contradictory results of binding experiments and pharmacological studies could be due, in part, to changes in subtypes of brain muscarinic receptors with advanced age. Alternatively, the age-related differences in cholinergic drug effects may reflect a decreased ability of the senescent animal to adapt to changes in its environment.     

Age-related changes of dopamine receptors in the rat hippocampus: a light microscope autoradiography study

Hippocampus is a brain region involved in learning and memory and is particularly sensitive to ageing. It is supplied with a dopaminergic innervation arising from the midbrain, which is part of the mesolimbic dopaminergic pathway. Dysfunction of the dopaminergic mesolimbic system is probably involved in the pathophysiology of psychosis and behavioural disturbances occurring in the elderly. The present study was designed to assess the density and localisation of dopamine D1- and D2-like receptor subtypes in the hippocampus of male Sprague-Dawley rats aged 3 months (young), 12 months (adult) and 24 months (old). Dopamine D1-like receptors, labelled by [3H]-SCH 23390, in young rats displayed a dentate gyrus-CA1 subfield gradient. The expression was increased in the cell body of dentate gyrus, CA4 and CA3 subfield of old rats compared to younger cohorts, as well as in the neuropil of dentate gyrus. A decreased density of dopamine D1-like receptors was found in the stratum oriens of CA1 and CA3 subfields. Dopamine D2-like receptors, labelled using [3H]-spiperone as radioligand, were expressed rather homogeneously throughout different subfields of the hippocampus. In old rats, the density of dopamine D2-like receptors was decreased in the dentate gyrus, unchanged in the CA4 and CA1 subfields and increased in the CA3 subfield. The above results indicate the occurrence of inhomogeneous changes in the density of dopamine D1- and D2-like receptors in specific portions of hippocampus of old rats. These findings support the hypothesis of an involvement of dopaminergic system in behavioural abnormalities or psychosis occurring in ageing.     

Effects of aging on signal transmission and transduction systems in the gerbil brain: morphological and autoradiographic study.

The Mongolian gerbil was used as a model of aging because of its relatively short lifespan, genetic homogeneity and the fact that data had been collected previously. Furthermore, gerbils have been widely used in biomedical investigations of stroke and epilepsy. Age-related differences in signal transmission and transduction systems were investigated in brains of three-, 11- and 21-month-old gerbils by morphological and in vitro receptor autoradiographic studies. Morphometric analysis revealed a decreased number of neurons in layer III of the occipital cortex and also a decrease in cerebellar Purkinje cells in 21-month-old animals. However, no statistical differences were observed in the hippocampal formation, the dorsolateral striatum and layer III of the frontal cortex. Autoradiography was used to map muscarinic cholinergic (labeled with [3H]quinuclidinyl benzilate), serotonin2 ([3H]spiperone), dopamine D2 ([3H]spiperone), adenosine A1 ([3H]cyclohexyladenosine), GABAA ([3H]muscimol), naloxone ([3H]naloxone), protein kinase C ([3H]phorbol 12,13-dibutyrate), adenylate cyclase ([3H]forskolin), cyclic AMP ([3H]cyclic AMP) and L-type Ca2+ channels ([3H]PN200-110). Muscarinic cholinergic receptor and protein kinase C, cyclic AMP and L-type Ca2+ channels were significantly decreased in the cerebral cortex and/or in the CA1 subfield of the hippocampus in the 21-month-old group. Muscarinic cholinergic receptor and L-type Ca2+ channel binding sites were significantly reduced in the dentate gyrus. In contrast, protein kinase C was increased in this area in the 21-month-old group. Also, naloxone binding sites were increased in the CA3 subfield, hilus, dentate gyrus and molecular layer of the cerebellum in the 11- and 21-month-old groups. Muscarinic cholinergic, serotonin2 and dopamine D2 receptors and adenylate cyclase were significantly decreased in the striatum. On the other hand, adenosine A1 and GABAA receptors remained unchanged in the 21-month-old group. Although age-related histopathological abnormalities were only observed in the occipital cortex and in the cerebellum, alterations of signal transmission and transduction systems were noticed in all areas examined (e.g. cerebral cortex, CA1 subfield, dentate gyrus and striatum). These data indicate that changes in these receptors and binding sites may be related to dysfunction of learning and memory and to the loss of motor function. The aged gerbil model is a good system for studying aging and is of value for simulating aging after epilepsy and stroke.     

Quantitative light microscopic autoradiographic localization of cholinergic muscarinic receptors in the human brain: forebrain

The distribution of muscarinic cholinergic receptors in the human forebrain and cerebellum was studied in detail by quantitative autoradiography using N-[3H]methylscopolamine as a ligand. Only postmortem tissue from patients free of neurological diseases was used in this study. The highest densities of muscarinic cholinergic receptors were found in the striatum, olfactory tubercle and tuberal nuclei of the hypothalamus. Intermediate to high densities were observed in the amygdala, hippocampal formation and cerebral cortex. In the thalamus muscarinic cholinergic receptors were heterogeneously distributed, with densities ranging from very low to intermediate or high. N-[3H]Methylscopolamine binding was low in the hypothalamus, globus pallidus and basal forebrain nuclei, and very low in the cerebellum and white matter tracts. The localization of the putative muscarinic cholinergic receptors subtypes M1 and M2 was analysed in parallel using carbachol and pirenzepine at a single concentration to partially inhibit N-[3H]methylscopolamine binding. Mixed populations of both subtypes were found in all regions. M1 sites were largely predominant in the basal ganglia, amygdala and hippocampus, and constituted the majority of muscarinic cholinergic receptors in the cerebral cortex. M2 sites were preferentially localized in the diencephalon, basal forebrain and cerebellum. In some areas such as the striatum and substantia innominata there was a tendency to lower densities of muscarinic cholinergic receptors with increasing age. In general, we observed a slight decrease in M2 sites in elderly cases. Muscarinic cholinergic receptor concentrations seemed to be reduced following longer postmortem periods. The distribution of acetylcholinesterase was also studied using histochemical methods, and compared with the localization of muscarinic cholinergic receptors and other cholinergic markers. The correlation between the presence of muscarinic cholinergic receptors and the involvement of cholinergic mechanisms in the function of specific brain areas is discussed. Their implication in neurological diseases is also reviewed.     

Layer-specific processing of excitatory signals in CA1 interneurons depends on postsynaptic M₂ muscarinic receptors

The hippocampus receives a diffuse cholinergic innervation which acts on pre- and postsynaptic sites to modulate neurotransmission and excitability of pyramidal cells and interneurons in an intricate fashion. As one missing piece in this puzzle, we explored how muscarinic receptor activation modulates the somatodendritic processing of glutamatergic input in CA1 interneurons. We performed whole-cell recordings from visually identified interneurons of stratum radiatum (SR) and stratum oriens (SO) and examined the effects of the cholinergic agonist carbachol (CCh) on EPSP-like waveforms evoked by brief glutamate pulses onto their proximal dendrites. In SO interneurons, CCh consistently reduced glutamate-induced postsynaptic potentials (GPSPs) in control rat and mice, but not in M₂ muscarinic receptor knockout mice. By contrast, the overwhelming majority of interneurons recorded in SR of control and M₂ receptor-deficient hippocampi exhibited muscarinic enhancement of GPSPs. Interestingly, the non-responding interneurons were strictly confined to the SR subfield closest to the subiculum. Our data suggest that postsynaptic modulation by acetylcholine of excitatory input onto CA1 interneurons occurs in a stratum-specific fashion, which is determined by the absence or presence of M₂ receptors in their (somato-)dendritic compartments. Thus cholinergic projections might be capable of recalibrating synaptic weights in different inhibitory circuits of the CA1 region.     

M2 muscarinic acetylcholine receptors regulate long-term potentiation at hippocampal CA3 pyramidal cell synapses in an input-specific fashion

Muscarinic receptors have long been known as crucial players in hippocampus-dependent learning and memory, but our understanding of the cellular underpinnings and the receptor subtypes involved lags well behind. This holds in particular for the hippocampal CA3 region, where the mechanisms of synaptic plasticity depend on the type of afferent input. Williams and Johnston (Williams S, Johnston D. Science 242: 84-87, 1988; Williams S, Johnston D. J Neurophysiol 64: 1089-1097, 1990) demonstrated muscarinic depression of mossy fiber (MF) long-term potentiation (LTP) through a presynaptic site of action and Maeda et al. (Maeda T, Kaneko S, Satoh M. Brain Res 619: 324-330, 1993) proposed a bidirectional modulation of MF LTP by muscarinic receptor subtypes. Since then, this issue, as well as muscarinic regulation of plasticity at associational/commissural (A/C) fiber-CA3 synapses has remained largely neglected, not least because of the lack of highly selective ligands for the different muscarinic receptor subtypes. In the present study, we performed field potential and whole cell recordings from the hippocampal CA3 region of M(2) receptor knockout mice to determine the role of M(2) receptors in short-term and long-term plasticity at A/C and MF inputs to CA3 pyramidal cells. At the A/C synapse, M(2) receptors promoted short-term facilitation and LTP. Unexpectedly, M(2) receptors mediated the opposite effect on LTP at the MF synapse, which was significantly reduced, most likely involving a depressant effect of M(2) receptors on adenylyl cyclase activity in MF terminals. Our data demonstrate that cholinergic projections recruit M(2) receptors to redistribute the gain of LTP in CA3 pyramidal cells in an input-specific manner.     

Intact coupling of M1 receptors and preserved M2 and M4 receptors in the cortex in progressive supranuclear palsy: contrast with other dementias

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterised clinically by motor and cognitive symptoms. Cholinergic dysfunction is thought to be responsible for much of the cognitive symptomatology. To date, however, cholinergic replacement therapies have been ineffective. We used receptor specific radioligand autoradiography to measure M1, M2, and M4 receptor density, and the functional status of the principal cortical subtype, M1, in the frontal cortex in post-mortem brain tissue of PSP patients (n = 14). Results were compared to normal controls (n = 17) and patients with dementia with Lewy bodies (DLB, n = 12) and Alzheimer's disease (AD, n = 15). In PSP there were no changes in M1, M2, or M4 muscarinic receptor densities or M1 coupling. DLB cases showed a non-significant increase in M1 receptors. In AD there was a reduction in M1 receptors and coupling in most frontal cortical areas which reached significance, compared to DLB, for M1 receptors in the cingulate (p < 0.05). We conclude from this first systematic study of cortical muscarinic receptors in PSP that functioning cortical muscarinic receptors are preserved. A further, larger trial of cholinergic therapy, such as an M1 agonist, may be warranted.     

Muscarinic acetylcholine receptor-dependent induction of persistent synaptic enhancement in rat hippocampus in vivo

Presynaptic terminal autoinhibitory muscarinic acetylcholine (ACh) receptors are predominantly of the M2/M4 subtypes and antagonists at these receptors may facilitate cognitive processes by increasing ACh release. The present study examined the ability of the M2/M4 muscarinic ACh receptor antagonist N,N'-bis [6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octane diamine tetrahydrochloride (methoctramine) to induce and modulate synaptic plasticity in the CA1 area of the hippocampus in urethane-anesthetized rats. Both methoctramine and another M2/M4 antagonist, {11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} (AF-DX 116), caused a rapid onset and persistent increase in baseline synaptic transmission after i.c.v. injection. Consistent with a requirement for activation of non-M2 receptors by endogenously released ACh, the M1/M3 receptor selective antagonists 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and 4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H-thieno[3,4-b][1,5]benzodiazepin-10-one dihydrochloride (telenzepine) prevented the induction of the persistent synaptic enhancement by methoctramine. The requirement for cholinergic activation was transient and independent of nicotinic ACh receptor stimulation. The synaptic enhancement was inhibited by the prior induction of long-term potentiation (LTP) by high frequency stimulation but induction of the synaptic enhancement by methoctramine before high frequency stimulation did not inhibit LTP. Unlike high frequency stimulation-evoked LTP, the synaptic enhancement induced by methoctramine appeared to be NMDA receptor-independent. The present studies provide evidence for the rapid induction of a persistent potentiation at hippocampal glutamatergic synapses by endogenous ACh in vivo following disinhibition of inhibitory M2 muscarinic autoreceptors.     

Effects of neonatal handling on the basal forebrain cholinergic system of adult male and female rats

Neonatal handling is an early experience which results in improved function of the hypothalamic-pituitary-adrenal axis, increased adaptability and coping as a response to stress, as well as better cognitive abilities. In the present study, we investigated the effect of neonatal handling on the basal forebrain cholinergic system, since this system is known to play an important role in cognitive processes. We report that neonatal handling results in increased number of choline-acetyl transferase immunopositive cells in the septum/diagonal band, in both sexes, while no such effect was observed in the other cholinergic nuclei, such as the magnocellular preoptic nucleus and the nucleus basalis of Meynert. In addition, neonatal handling resulted in increased M1 and M2 muscarinic receptor binding sites in the cingulate and piriform cortex of both male and female rats. A handling-induced increase in M1 muscarinic receptor binding sites was also observed in the CA3 and CA4 (fields 3 and 4 of Ammon's horn) areas of the hippocampus. Furthermore, a handling-induced increase in acetylcholinesterase staining was found only in the hippocampus of females. Our results thus show that neonatal handling acts in a sexually dimorphic manner on one of the cholinergic parameters, and has a beneficial effect on BFCS function, which could be related to the more efficient and adaptive stress response and the superior cognitive abilities of handled animals.     

Age-related differences in brain choline acetyltransferase, cholinesterases and muscarinic receptor sites in two strains of rats

The age-related changes in choline acetyltransferase (ChAT), cholinesterases (ChE) and muscarinic receptor sites (measured as Bmax of 3H-QNB binding) were evaluated in the cerebral cortex, hippocampus and striatum of Fischer 344 and Wistar male rats at the ages of 3 and 24 months. In the aged Fischer rats there was a significant decline of ChAT (except the hippocampus), ChE and muscarinic receptor densities in the regions analyzed. In the aged Wistar rats cortical and hippocampal ChAT as well as cortical muscarinic receptors remained constant while striatal ChAT, hippocampal and striatal muscarinic receptors decreased significantly; ChE were reduced in all regions analyzed. Factorial analysis of variance (2 strains x 2 ages ANOVA) showed significant strain-related differences in ChAT and muscarinic receptor sites in the three brain areas (about 1.5 times higher levels in the Fischer rats). The same analysis showed significant interactions between strain and age for ChAT and muscarinic receptors in the cerebral cortex, but not in the hippocampus and striatum; no interactions were found for ChE in the regions analyzed. This means that cortical ChAT and muscarinic receptors behave differently in aging in the two strains of rats, i.e., their alterations are strain-specific. Conversely, all other age-related changes (or lack of them for hippocampal ChAT) cannot be considered strain-specific. Moreover, an additional group of 33-month Wistar rats showed a significant decline of cortical muscarinic receptors with respect to 24 month rats but not of other markers in any area. The data underscore the need to consider genotype in the assessment of age-related cholinergic deficits in animal models.     

Muscarinic receptor subtypes are differentially distributed in the rat cochlea

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Galpha(q/11)-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Galpha(i) and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters' cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype.From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters' cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.     

The distribution of muscarinic M1 receptors in the human hippocampus

The muscarinic M1 receptor plays a significant role in cognition, probably by modulating information processing in key regions such as the hippocampus. To understand how the muscarinic M1 receptor achieves these functions in the hippocampus, it is critical to know the distribution of the receptor within this complex brain region. To date, there are limited data on the distribution of muscarinic M1 receptors in the human hippocampus which may also be confounded because some anti-muscarinic receptor antibodies have been shown to lack specificity.Initially, using Western blotting and immunohistochemistry, we showed the anti-muscarinic M1 receptor antibody to be used in our study bound to a single 62 kDa protein that was absent in mice lacking the muscarinic M1 receptor gene. Then, using immunohistochemistry, we determined the distribution of muscarinic M1 receptors in human hippocampus from 10 subjects with no discernible history of a neurological or psychiatric disorder.Our data shows the muscarinic M1 receptor to be predominantly on pyramidal cells in the hippocampus. Muscarinic M1 receptor positive cells were most apparent in the deep polymorphic layer of the dentate gyrus, the pyramidal cell layer of cornu ammonis region 3, the cellular layers of the subiculum, layer II of the presubiculum and layer III and V of the parahippocampal gyrus. Positive cells were less numerous and less intensely stained in the pyramidal layer of cornu ammonis region 2 and were sparse in the molecular layer of the dentate gyrus as well as cornu ammonis region 1. Although immunoreactivity was present in the granular layer of the dentate gyrus, it was difficult to identity individual immunopositive cells, possibly due to the density of cells.This distribution of the muscarinic M1 receptors in human hippocampus, and its localisation on glutamatergic cells, would suggest the receptor has a significant role in modulating excitatory hippocampal neurotransmission.     

Differential expression of muscarinic subtype mRNAs after exposure to neurotoxic pesticides

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

M1 and M4 receptors modulate hippocampal pyramidal neurons

Acetylcholine (ACh), acting at muscarinic ACh receptors (mAChRs), modulates the excitability and synaptic connectivity of hippocampal pyramidal neurons. CA1 pyramidal neurons respond to transient ("phasic") mAChR activation with biphasic responses in which inhibition is followed by excitation, whereas prolonged ("tonic") mAChR activation increases CA1 neuron excitability. Both phasic and tonic mAChR activation excites pyramidal neurons in the CA3 region, yet ACh suppresses glutamate release at the CA3-to-CA1 synapse (the Schaffer-collateral pathway). Using mice genetically lacking specific mAChRs (mAChR knockout mice), we identified the mAChR subtypes responsible for cholinergic modulation of hippocampal pyramidal neuron excitability and synaptic transmission. Knockout of M1 receptors significantly reduced, or eliminated, most phasic and tonic cholinergic responses in CA1 and CA3 pyramidal neurons. On the other hand, in the absence of other G(q)-linked mAChRs (M3 and M5), M1 receptors proved sufficient for all postsynaptic cholinergic effects on CA1 and CA3 pyramidal neuron excitability. M3 receptors were able to participate in tonic depolarization of CA1 neurons, but otherwise contributed little to cholinergic responses. At the Schaffer-collateral synapse, bath application of the cholinergic agonist carbachol suppressed stratum radiatum-evoked excitatory postsynaptic potentials (EPSPs) in wild-type CA1 neurons and in CA1 neurons from mice lacking M1 or M2 receptors. However, Schaffer-collateral EPSPs were not significantly suppressed by carbachol in neurons lacking M4 receptors. We therefore conclude that M1 and M4 receptors are the major mAChR subtypes responsible for direct cholinergic modulation of the excitatory hippocampal circuit.     

Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.     

Aging and brain cholinergic muscarinic receptor subtypes: an autoradiographic study in the rat

Cholinergic M1 and M2 muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated QNB in the presence of pirenzepine or carbachol. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most thalamic and hypothalamic nuclei. M1 and M2 receptors showed small but significant decreases in cortical regions and in the striatum. The largest effects were seen in M2 receptors of the ventral forebrain cholinergic nuclei where binding was reduced by up to 40%. No similar reductions were seen in the M1 receptor population in these regions. The results suggest that both muscarinic receptor subtypes show an anatomically selective pattern of decrease with age, with the M2 receptor subtype in the basal forebrain nuclei being specially vulnerable to the effects of aging.     

Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice

Blockade of cholinergic neurotransmission by muscarinic receptor antagonists produces profound deficits in attention and memory. However, the antagonists used in previous studies bind to more than one of the five muscarinic receptor subtypes. Here we examined memory in mice with a null mutation of the gene coding the M1 receptor, the most densely distributed muscarinic receptor in the hippocampus and forebrain. In contrast with previous studies using nonselective pharmacological antagonists, the M1 receptor deletion produced a selective phenotype that included both enhancements and deficits in memory. Long-term potentiation (LTP) in response to theta burst stimulation in the hippocampus was also reduced in mutant mice. M1 null mutant mice showed normal or enhanced memory for tasks that involved matching-to-sample problems, but they were severely impaired in non-matching-to-sample working memory as well as consolidation. Our results suggest that the M1 receptor is specifically involved in memory processes for which the cortex and hippocampus interact.