Muscarinic receptors in basal ganglia in dementia with Lewy bodies,Parkinson's disease and Alzheimer's disease

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.     

Effects of aging and cholinergic deafferentation on putative muscarinic cholinergic receptor subtypes in rat cerebral cortex

Despite a 34% decrease in the activity of choline acetyltransferase (ChAT) in the rat cerebral cortex following lesions of the nucleus basalis, there were no changes in the Bmax of the antagonist ligands [3H]quinuclidinyl benzilate ((-)-[3H]QNB) or (-)-[3H]N-methylscopolamine ((-)-[3H]NMS). Furthermore, this treatment produced no significant change in the proportions or affinities of muscarinic receptors having high and low affinity for pirenzepine or (-)-NMS. These data indicate that putative M2 muscarinic receptors are not restricted to ChAT-containing neurons in rat cerebral cortex. In senescent compared to mature rats there was no significant loss of ChAT activity although a significant reduction in the Bmax of both (-)-[3H]QNB and (-)-[3H]NMS binding was observed. However, no changes in the competition of pirenzepine or (-)-NMS for the remaining (-)-[3H]QNB binding sites were observed. Therefore, there is no evidence for any differential regulation of either putative muscarinic receptor subtype in response to cholinergic deafferentation or as a function of the natural aging process.     

Cholinergic innervation and topographical organization of muscarinic binding sites in rat brain: a comparative autoradiographic study

Employing [3H]hemicholinium-3 ([3H]HC), [3H]pirenzepine([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB), autoradiographic binding studies were performed to identify and quantitate the localization of high-affinity choline carriers, M1-subtype of muscarinic binding sites and a mixed population of M1- and M2-subtypes of muscarinic binding sites, respectively, in 38 anatomically defined areas of rat brain. Labelling of adjacent brain sections with [3H]HC, [3H]PZ and [3H]QNB revealed different topographical binding patterns. [3H]HC binding, which is supposed to reflect cholinergic innervation, was dense in the nucleus accumbens, olfactory tubercle, caudate putamen, basolateral amygdaloid nucleus and the interpeduncular nucleus. Moderate but heterogeneous binding was found in thalamic, hypothalamic, hippocampal and cortical areas. Maximal [3H]PZ binding was observed in the nucleus accumbens, olfactory tubercle and in discrete substructures of the hippocampus, e.g. CA1 and dentate gyrus. Binding to other hippocampal and cortical areas was intermediate, whilst minor binding was found in thalamic, hypothalamic and brain stem areas. The binding of [3H]QNB was more evenly distributed over the brain as compared to that of [3H]PZ. [3H]QNB clearly exceeded the binding of [3H]PZ in the thalamus, hypothalamus and brain stem. A relationship was found between the topography patterns of the [3H]PZ and [3H]QNB binding sites. However, some brain areas showed preference for one of the two ligands, pointing to a distinct localization of M1- and M2-subtypes of muscarinic binding sites. Although M1 sites appeared to predominate in the basal ganglia, hippocampus and cortex, some heterogeneity was observed indicative of the minor occurrence of M2 sites within these structures. There was no relationship between the density of the presumed cholinergic innervation and the binding capacity of either of the muscarinic sites in the various brain areas. However, a relationship was found between M2-selectivity and [3H]HC binding, pointing to a possible presynaptic localization of the M2-sites. In addition, it is suggested that distinct cholinergic cell groups might project their fibres to brain areas containing particular subsets of postsynaptic muscarinic binding sites.     

Ontogeny of M1 and M2 muscarinic binding sites in the striatum of the cat: relationships to one another and to striatal compartmentalization

The ontogeny of striatal M1 and M2 muscarinic cholinergic binding sites was studied autoradiographically in cats ranging in age from embryonic day 40 to postnatal day six. Direct labeling with [3H]pirenzepine revealed M1 sites, and M2 sites were labeled with [3H]N-methylscopolamine in the presence of pirenzepine. In serial tissue sections, distributions of striatal M1 and M2 sites were compared to one another and to patterns of acetylcholinesterase staining and tyrosine hydroxylase-like immunoreactivity. The younger fetal material demonstrated heterogeneous distributions for both subtypes of muscarinic binding sites, with patches of dense binding corresponding to islands of dopaminergic nigrostriatal innervation. For both M1 and M2 binding, lateral to medial and caudal to rostral density gradients were present in the patches and in the surrounding matrix. During fetal development and into the perinatal period, overall muscarinic binding increased, but more so in the matrix than in the patches. By postnatal day six striatal M2 binding appeared nearly homogeneous. M1 binding, however, was slightly more concentrated in patches than in matrix. The patches of elevated M1 binding were still present at maturity, and corresponded to striosomes. These findings suggest that the ontogenetic regulation of muscarinic binding sites is influenced by location relative to striatal compartments, and that expression of M1 and M2 binding site subtypes is differentially regulated.     

Alterations in cortical muscarinic receptors following cholinotoxin (AF64A) lesion of the rat nucleus basalis magnocellularis.

Cortical choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), muscarinic receptors and sodium-dependent, high-affinity, choline uptake (SDHACU) sites were examined in the rat brain following unilateral stereotaxic injection of the cholinotoxin, AF64A, into the nucleus basalis magnocellularis (NBM). Injection of AF64A resulted in a significant loss of presynaptic cholinergic markers in the cortex without alteration in TH and TPH activity. The binding to SDHACU sites was reduced to background values in the NBM and increased in the central amygdala (Ce) and cortex. The increase in cortical [3H]QNB binding was the result of a change in muscarinic receptor number (BMAX) and not a change in receptor affinity (KD). Examination of muscarinic receptor subtypes demonstrated a reduction of M1 receptor binding in the cortex and NBM without any alteration in the Ce. Non-M1 binding was significantly increased in all the laminae of the cortex and in the Ce, but decreased in the NBM. These data suggest that there exists a population of M1 receptors on NBM projections to the cortex and that NBM projections influence a population of postsynaptic receptors in the cortex and Ce which are not of the M1 subtype.     

Comparative distribution of binding of the muscarinic receptor ligands pirenzepine,AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.     

A minority of muscarinic receptors mediate rabbit tracheal smooth muscle contraction.

To enhance our understanding of cholinergic mechanisms and muscarinic receptors in bronchoconstriction, we have characterized the muscarinic receptor subtypes in rabbit tracheal smooth muscle using radioligand binding and functional assays. The Kd for [3H]quinuclidinyl benzilate ([3H](-)QNB) binding determined from saturation isotherms was 12.6 x/divided by 1.1 pM (geometric mean x/divided by SEM), and the Bmax was 269 +/- 7 fmol/mg protein (arithmetic mean +/- SEM). Competitive inhibition studies with the muscarinic antagonists pirenzepine (PZ), 11[[2-[(diethylamino)-methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), and hexahydrosiladifenidol (HHSiD) demonstrated heterogeneity of muscarinic receptor subtypes in rabbit tracheal smooth muscle. PZ bound with low affinity to a single receptor site, indicative of an absence of M1 receptors. AF-DX116 (M2 selective) bound with high affinity to approximately 83% of muscarinic binding sites, and 4-DAMP and HHSiD (M3 antagonists) bound with high affinity to approximately 24 and 28% of muscarinic binding sites, respectively. Additionally, direct binding studies with [3H]4-DAMP demonstrated high-affinity binding with 23% of muscarinic binding sites. Thus, the majority of muscarinic receptors in rabbit tracheal smooth muscle bound with high affinity to an M2-selective antagonist, and the remaining receptor sites bound with high affinity to M3 antagonists. The inhibitory effects of atropine, PZ, AF-DX116, and 4-DAMP on methacholine-induced contraction of rabbit tracheal rings were compared. 4-DAMP was a potent inhibitor of methacholine-induced contraction, but PZ and AF-DX116 demonstrated low potency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional distribution of muscarinic and nicotinic cholinergic receptor binding activities in the human brain

The distribution of nicotinic and muscarinic receptor subtypes was examined in human brain tissue obtained at autopsy from neurologically normal adult (50-60 years) individuals. Membrane preparations from 15 brain regions were examined for nicotinic (L-[3H]nicotine) binding, both M1 and M2 muscarinic receptor binding (distinguished on the basis of pirenzepine affinity) and high (H) and low (L) affinity muscarinic agonist binding (distinguished on the basis of carbachol displacement). Total muscarinic receptor binding sites were relatively high in striatal and cortical areas, where both M1 subtype and agonist binding type L predominated, compared with thalamic, nigral and cerebellar regions and spinal cord, where the M2 subtype and agonist binding type H predominated. Nicotinic receptor binding sites (predominantly high affinity, measured at low ligand concentrations) did not parallel any of the muscarinic subtypes, being concentrated in thalamic, neocortical and striatal regions. Scatchard analysis indicated the presence of both high and low affinity nicotinic sites, the numbers of the latter generally exceeding the former by over one order of magnitude. Neither muscarinic nor nicotinic receptor binding sites were closely related to the distribution of the cholinergic neuronal marker, choline acetyltransferase, suggesting that individual cholinergic pathways may be distinguished by the relative proportion of the different types of cholinergic receptors present.     

Alterations of the muscarinic cholinergic (mACh) receptors in the striatum of the MPTP-induced parkinsonian model in mice: in vitro quantitative autoradiographical analysis

The alteration of muscarinic cholinergic (mACh) receptors in the striatum of mice after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was examined using a [3H]quinuclidinyl benzilate [( 3H]QNB) in vitro labeling macro-autoradiographic technique and mACh receptor concentration was quantitatively analyzed using a computer analysis system. Two weeks after cessation of MPTP administration, the striatal mACh receptors were significantly increased. In the subchronic phase, at 6 weeks, the striatal mACh receptors were significantly decreased but recovered to the normal level by the treatment with L-dihydroxyphenylalanine (L-DOPA) for two weeks before sacrifice. These findings indicated that the striatal mACh receptors are strongly regulated by the nigrostriatal dopaminergic function.     

Effects of nucleus basalis lesion on muscarinic receptor subtypes

The cholinergic system in the central nervous system is an important component of the neural circuitry of learning, memory and cognition. A decline of cholinergic innervation in the human brain is a characteristic feature of dementia of Alzheimer's type. In this study, changes in cholinergic markers were studied after a unilateral lesion of the nucleus basalis magnocellularis (nbM). Acetylcholinesterase (AChE) histochemistry showed a loss of cortical AChE-containing neurons, and choline acetyltransferase (ChAT) immunohistochemistry demonstrated a loss of cholinergic cells in nbM. The localizations of muscarinic Ml and M2 receptors using [³H]pirenzepine ([³H]PZ) and [³H]AF-DX 384, respectively, were studied by quantitative autoradiography 1, 2, 4 and 6 weeks following unilateral ibotenic acid lesion of nbM. A significant decrease in [³H]PZ binding sites was observed at postlesion week 1 in the parietal and temporal cortices. The decrease in [³H]AF-DX 384 binding sites on the lesioned side was observed throughout frontal, parietal and temporal cortices after postlesion week 1, with a significant increase after 6 weeks, possibly as result of loss of presynaptic receptors and upregulation of postsynaptic ones. Moreover, laminar distribution after nbM lesion shows that M1 and M2 receptor binding sites are more affected in superficial layers (I,II,III) than in the deep layers (IV,V,VI), depending on ligand, postlesion period and cortical region. Furthermore, nbM lesion causes a higher deficit of M2 receptors than of M1 receptors. These data suggest the existence of a presynaptic population as well as a postsynaptic population of M1 and M2 receptors which are differently affected after unilateral nbM lesion.     

Autoradiographic localization of muscarinic cholinergic receptors in visual areas of cat brain: variations in sensitivity of N-[3H]methylscopolamine binding sites to carbachol and pirenzepine

The distribution of muscarinic acetylcholine receptors (MChRs) was studied in visual areas of cat brain using in vitro quantitative autoradiography with 1 nM N-[3H]methylscopolamine ([3H]NMS) as a radioligand. The highest density of [3H]NMS binding was observed in lamina A of the lateral geniculate nucleus (LGN) and in layer II/III of the visual cortex. The lowest binding was seen in the stratum griseum intermediale of the superior colliculus (SC). The comparison of inhibition of [3H]NMS binding by 100 microM carbachol and 300 nM pirenzepine showed that the SC and LGN contain predominantly M2 sites. M1 sites constitute the main population of MChRs in the cortical areas studied.     

The binding of cholinesterase inhibitors tacrine (tetrahydroaminoacridine) and 7-methoxytacrine to muscarinic acetylcholine receptors in rat brain in the presence of eserine

Cholinesterase inhibitor tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is known to interfere with the binding of specific ligands to muscarinic receptors with unusually steep binding inhibition curves. We investigated whether the concentration dependence of the inhibition of binding is associated with the inhibitory effect of tacrine on the activity of cholinesterases, and compared the effect of tacrine with that of 7-methoxytacrine. Tacrine was found to inhibit the specific binding of [3H]quinuclidinyl benzilate (QNB) in rat brain cortex with IC50 values of 11 microM both in the absence and in the presence of 100 microM eserine, which had been added to ensure complete inhibition of cholinesterases at all concentrations of tacrine; in the cerebellum, the IC50 value was 10 microM in the absence and 14 microM in the presence of eserine; Hill slope factors (nH) were in the range of 1.55-1.79 and were not significantly affected by the presence of eserine. 7-Methoxytacrine inhibited the binding of [3H]QNB with an IC50 value of 2.3 microM in the cortex and of 2.6 microM in the cerebellum. The results indicate that the degree and the steep course of the inhibition of [3H]QNB binding to M1 and M2 muscarinic receptors by tacrine do not depend on its inhibitory effect on cholinesterases, and that 7-methoxytacrine is likely to interfere with the function of muscarinic receptors 4-5 times more strongly than tacrine.     

Cholinergic markers in the plasticity of murine barrel field

Cholinergic muscarinic receptor binding and acetylcholinesterase (AChE) histochemistry were studied in the barrel cortex of adult, vibrissae deprived and vibrissae denervated mice. In the control barrel field muscarinic receptors labeled with [3H]quinuclidinyl benzilate ([3H]QNB) showed a higher density in the granular cortex and a higher accumulation of label in the barrels. AChE staining revealed a punctuated pattern corresponding to the barrels in the upper part of layer IV and a reverse-image pattern of staining showing only the walls of barrels in the lower part of layer IV. Neonatal denervation of rows of vibrissae lowered both binding of [3H]QNB to the tissue in the shrunken rows and AChE activity in the denervated rows of barrels. Deprivation and late denervation produced no effects on either pattern, or intensity, of [3H]QNB labeling and AChE staining. These observations suggest that the changes in cholinergic markers are related to the altered morphological structure and not to the abnormal functioning of the barrel cortex which received reduced sensory input from the vibrissae.     

Muscarinic cholinergic receptor binding in bovine retina

Using [³H] quinuclidinyl benzylate ([³H] QNB) muscarinic cholinergic receptors have been demonstrated in crude membrane fractions of bovine retina. Specific [³H] QNB binding is saturable with a K_D of 0.5 nM and a maximal number of muscarinic agonists and antagonists for displacing specific [³H] QNB binding closely parallel the affinities for muscarinic receptors in rat brain and guinea pig ileum. The findings may explain atropine sensitive effects of muscarinic agonists on the electroretinogram and on retinal cells in vitro.     

Expression of muscarinic cholinergic receptors during T cell maturation in the thymus

Thymocytes at various stages of their ontogeny have been studied in relation to their ability to bind [3H]quinuclidinyl benzilate [( 3H]QNB), a specific radioligand of the muscarinic cholinergic receptors. [3H]QNB-specific binding to thymocytes from 15-19-day fetal, newborn and adult thymuses of mice and rats was compared and correlated. Our experiments showed that the kinetics of [3H]QNB binding to thymocytes at 37 degrees C was similar to that of the lymph node lymphocytes (LNL) with maximum after 5 min of incubation and subsequent decrease to 10% of the maximum after 90 min of incubation. Maximal binding for the entire thymocyte population was twice lower than for the cortisone-resistant thymocytes (CRT) or for LNL. Binding of [3H]QNB carried out at 4 degrees C resulted in disappearance of the maximum, but did not alter the difference between CRT and entire thymocyte population. Depletion of CRT detectable [3H]QNB-specific binding to thymocytes until 18th day of gestation but the maximal binding increased up to 20% at the day 19 and reached 90% of adult level on the third day after birth. Moreover, carbamylcholine (a muscarinic agonist) treatment in vivo induced a significant decrease in [3H]QNB binding to the thymocytes. We thus suggest that a subpopulation of thymocytes bearing muscarinic receptors in the periphery acquired these receptors in the thymus as one of the last steps of their maturation. We cannot exclude the possibility that cholinergic stimulation might trigger these lymphocytes to leave the thymus.     

Light-microscopic distribution and parasagittal organisation of muscarinic receptors in rabbit cerebellar cortex

Recent studies on the effects of intrafloccular injections of muscarinic agonists and antagonists on compensatory eye movements in rabbit, indicate that muscarinic receptors may play a modulatory role in the rabbit cerebellar circuitry. It was previously demonstrated by Neustadt et al. (1988), that muscarinic receptors in rabbit cerebellar cortex are distributed into alternating longitudinal zones of very high and very low receptor density. In the present study, the zonal and cellular distribution of muscarinic receptors in the rabbit cerebellar cortex is investigated in detail using in vitro ligand autoradiography with the non-selective high-affinity antagonist [³H]quinuclidinyl benzilate (QNB), and the M2-specific antagonist [³H]AF-DX384, and immunocytochemistry with a monoclonal antibody specific for the cloned m2 muscarinic receptor protein. [³H]QNB and [³H]AF-DX384 binding sites and m2-immunoreactivity had similar overall distributions: dense labeling occurred in the dendritec arbors of a subset of Purkinje cells that are organized into parasagittal bands. A high level of muscarinic receptor labeling was also observed in a thin substratum of the molecular layer immediately above the Purkinje cell layer of the vestibulo-cerebellar lobules, i.e. the nodulus, the ventral uvula and the flocculus. Labeling in this stratum was associated with densely packed fibres, which were putatively identified as parallel fibres. Also Golgi cells, which were localized in part in the molecular layer, and a subset of mossy fibre rosettes, primarily concentrated in lobule VI, were immunoreactive for the m2 receptor. The parasagittal bands of labeled Purkinje cell dendrites were most prominent in the anterior lobe (lobules I–V), in crus 1 and 2, in the flocculus, the ventral paraflocculus and the rostral folium of the nodulus. In other lobules, only infrequent Purkinje cells contained muscarinic receptors. The parasagittal organisation of muscarinic receptors differed from that of zebrin I, a Purkinje cell-specific protein which is often used as a marker of parasagittal parcelation of the cerebellar cortex. In the anterior lobe, however, there was a partial correspondence between muscarinic receptor and zebrin I bands. In the flocculus the distribution of muscarinic-receptor-positive Purkinje cells was related to the distinct white matter compartments as revealed with acetylcholinesterase (AChE) histochemistry. Muscarinic receptor-containing Purkinje cells were located primarily in the floccular zone 1, which is implicated in the control of eye movements about a horizontal axis. In order to relate the distribution of muscarinic receptor labeling to that of cholinergic nerve terminals, [³H]QNB binding sites and sodium-dependent [³H]hemicholinium-3 binding were compared. Sodium-dependent [³H]hemicholinium-3 binding sites mainly occurred in the granule cell layer of the vestibulo-cerebellum, which corresponds well with the distribution of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). However, sodium-dependent [³H]hemicholinium binding complemented, rather than co-localized with, muscarinic receptors which were primarily distributed in the molecular layer of the lobules of the vestibulo-cerebellar lobules. Their functional significance is puzzling, since their distribution does not correspond to that of markers of cholinergic innervation.     

Pharmacology, distribution and development of muscarinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens

Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5-9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes.These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for excitatory subtypes.     

Cholinergic muscarinic and nicotinic binding by human lymphocytes: differences between peripheral blood cells and cultivated cell lines

Kinetic and equilibrium studies of [3H]QNB and [3H]nicotine binding to human peripheral blood lymphocytes have been performed. The calculated number of muscarinic binding sites is 6 x 10(4) per cell, and of nicotinic binding sites is 2 x 10(3) per cell. The conditions for routine estimations of muscarinic and nicotinic receptors on human lymphocytes have been established. Significant differences in the distribution of muscarinic and nicotinic binding sites among normal PBL and malignant cell lines have been described.     

Presynaptic muscarinic receptors in guinea pig superior cervical ganglion

This study characterizes the presynaptic muscarinic cholinergic receptors associated with the modulation of the electrically-evoked acetylcholine output from guinea pig superior cervical ganglion preincubated with [3H]choline. The M1-selective agonist pilocarpine had no effect while carbachol and oxotremorine strongly decreased the evoked outflow of tritium. Atropine increased such evoked release of [3H]acetylcholine whereas the M1-selective antagonist pirenzepine was ineffective. Moreover, atropine but not pirenzepine antagonized the inhibitory effect of carbachol. These results suggest that the guinea-pig superior cervical ganglion is equipped with presynaptic inhibitory muscarinic receptors of the M2 subtype.     

Chronic treatment with simvastatin upregulates muscarinic M1/4 receptor binding in the rat brain

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.