Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.     

Biochemical and behavioral evidence for muscarinic autoreceptors in the CNS

Muscarinic autoreceptors of the M2 subclass were examined in rat forebrain using a number of different methodologies, including receptor autoradiography and image analysis, regulation of acetylcholine release, phosphoinositide turnover, low-Km GTP hydrolysis, and behavioral analysis. The relatively minor population of M2 receptors in coronal sections was visualized by autoradiography and image analysis using [3H]quinuclidinyl benzilate in the presence of a concentration of pirenzepine that blocked most of M1 (and M4) receptors. The highest densities of M2 receptors in forebrain regions were found in the outer layers of the cortex, CA1 region of the hippocampus and striatum. The M2-, but not M1-selective antagonists were able to block the oxotremorine-induced attenuation of acetylcholine release in forebrain synaptosomes. Low concentrations of the M2-selective antagonist gallamine increased phosphoinositide turnover, which is thought to be an M1 postsynaptic response in the forebrain, in brain slices by a Ca2(+)-dependent mechanism. The M2-selective agonist oxotremorine produced a substantial stimulation of low-Km GTPase in cortical membranes, suggesting that M2 forebrain receptors are efficiently coupled to G-proteins in the cortex. Behavioral signs of cholinergic stimulation were observed after intracerebroventricular injections of M2-, but not M1-selective antagonists. It is suggested that a minor population of forebrain M2 receptors regulates acetylcholine release by a mechanism that includes coupling through G-proteins presynaptically at synapses for which the postsynaptic response involves phosphoinositide turnover. Selective blockade of these receptors produces both biochemical and behavioral signs of acetylcholine release.     

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

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

Dose dependent inhibition of REM sleep in normal volunteers by biperiden, a muscarinic antagonist

We tested the effect of biperiden (2, 4, and 8 mg per os 30 min before bedtime) on the polygraphically recorded sleep of normal volunteers (n = 8). Biperiden is a cholinergic, muscarinic receptor antagonist that may preferentially block the M1 receptor subtype. Compared with placebo, biperiden significantly prolonged rapid eye movement (REM) latency and suppressed REM sleep time and REM percentage in a dose-dependent manner. These REM suppressing effects of biperiden are similar to those previously reported with scopolamine and other nonselective muscarinic receptor antagonists. Because of uncertainties about the pharmacological specificity of biperiden, further studies are needed to determine the mechanism of action and the role of M1 receptors subtypes in the regulation of REM sleep.     

The effects of aging on muscarinic receptor/G-protein coupling in the rat hippocampus and striatum

In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age which has been traced to events early in the signal transduction pathway. Our laboratory has therefore focussed on investigations at this level. The current experiments investigate the effects of age on G-protein/receptor interactions by using competitive binding assays to measure the ability of GppNHp to decrease the proportion of receptors bound to G-proteins in the absence and the presence of added Mg2+. L-[3H]Quinuclidinyl benzilate was used as a nonselective ligand and [3H]pirenzepine as an M1 selective ligand. We find that: (1) muscarinic receptors and G-proteins in the striatum appear to become loosely coupled with age, with no change in Mg2+ sensitivity. (2) M1-receptor/G-protein complexes in the hippocampus display increased sensitivity to the presence of Mg2+ with age, with those from old but not young tissue requiring added Mg2+ in order to uncouple. This effect, however, may not be M1 specific.     

Spinal nerve ligation increases alpha2-adrenergic receptor G-protein coupling in the spinal cord

Intrathecal and epidural administration of the alpha2-adrenergic receptor agonist clonidine in humans results in analgesia to both acute nociceptive and chronic neuropathic pain. The potency of clonidine increases with hypersensitivity to mechanical stimuli after nerve injury, although the reasons for this change are unknown. In the present study, we tested the hypothesis that peripheral nerve injury alters either spinal alpha2-adrenergic receptor-mediated G-protein activity or alpha2-adrenergic receptor number. Rats were randomized to left spinal nerve ligation (SNL) or sham surgery. Tactile hypersensitivity in the hindpaw was confirmed and lumbar spinal cords were removed for binding assays. To examine agonist-induced G-protein coupling, [35S]GTP gamma S binding experiments were performed in spinal cord membranes and sections using norepinephrine as an alpha2-adrenergic agonist. SNL was associated with an increase in maximal efficacy, but not potency, of norepinephrine-stimulated [35S]GTP gamma S binding in dorsal horn. SNL had no effect on basal [35S]GTP gamma S binding or on muscarinic cholinergic-stimulated [35S]GTP gamma S binding. [35S]GTP gamma S autoradiography showed that this increase in alpha2-adrenergic-activated G-proteins occurred both ipsilateral and contralateral to SNL surgery. SNL did not alter total alpha2-adrenergic receptor number or affinity to [3H]-rauwolscine binding, and displacement studies with the alpha2A-adrenergic antagonist BRL44408 revealed that most of the binding was associated with the alpha2A-adrenergic subtype. These data suggest that the increased potency of clonidine in neuropathic pain could reflect increased efficiency of G-protein coupling from spinal alpha2-adrenergic receptors.     

Identification of direct competition for, and indirect influences on, striatal muscarinic cholinergic receptors: in vivo [3H]quinuclidinyl benzilate binding in rats.

[3H]Quinuclidinyl benzilate (QNB) binds to specific muscarinic receptors of rat striatum, in vivo. The binding is saturable and displaceable by muscarinic drugs. Clozapine and thioridazine are unique antipsychotic agents with low liability for extrapyramidal side-effects, and both displaced ONB, while several other neuroleptics did not. In addition to this apparent direct competition for cholinergic receptors, morphine and amphetamine increased ONB binding by indirect influences on muscarinic receptors. In vivo QNB binding not only confirms in vitro findings, but it also detects indirect, probably transsynaptic, alterations of muscarinic cholinergic receptor dynamics.     

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

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

Muscarinic cholinergic receptors on intact human lymphocytes. Properties and subclass characterization

Saturable specific binding of tritiated N-methyl-scopolamine (3H-NMS) can be demonstrated on intact circulating human lymphocytes, with an average KD of 7 nmol/liter and an average density of about 15 fmol/10(6) cells. Specific 3H-NMS binding can be inhibited by cholinergic antagonists and agonists, is highly stereospecific for the enantiomers of the cholinergic antagonist quinuclidinyl benzilate (QNB), and is modulated by the stable guanosine triphosphate (GTP) analog GppNHp in a fashion similar to the specific binding of the same radioligand to rat heart membranes. The results indicate the presence of muscarinic cholinergic receptors on intact human lymphocytes, with a predominance of the M2 subtype. As central muscarinic cholinergic receptors have been reported to play an important role in the pathogenesis of affective disorders, the lymphocyte might represent a suitable model for the study of muscarinic receptor functions in humans.     

Attenuation of muscarinic receptor-G-protein interaction in Alzheimer disease

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [³H]PIP₂ hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease. Whether this defect reflects an alteration in the M1 muscarinic receptor, its respective guanine nucleotide binding (G) protein or both is not known. This study compares the number and both basal and muscarinic receptor-mediated function of G-proteins in synaptosomal membranes from cerebral cortical samples of age-matched control subjects and Alzheimer disease patients. Immuno-blotting with anti-Gα_q/11 and anti-Gβ antibodies demonstrated no alteration in the number of these G-protein subunits in Alzheimer disease. Basal [³⁵S]GTP_γS binding and hydrolysis of [γ-³²P]GTP by high-affinity GTPase also were not significantly altered in Alzheimer disease compared to control membrane samples. However, muscarinic agonist-stimulated GTP_γS binding and GTP hydrolysis were significantly reduced (80–100%) in Alzheimer disease cortical samples. Diminished agonist-stimulated GTP_γS binding and GTP hydrolysis corre-related with the loss of guanine nucleotide-sensitive, high-affinity agonist binding (K _L/K _H) ratio) to the M1 receptor subtype. These data provide further evidence for the loss of muscarinic receptor-G protein coupling in Alzheimer disease and support the hypothesis that muscarinic receptor-mediated cortical activation may be compromised in Alzheimer disease.     

The relative selectivity of anticholinergic drugs for the M1 and M2 muscarinic receptor subtypes

Anticholinergic drugs are used to treat a number of neurologic disorders, including parkinsonism, vestibular disturbances, and dystonia. Traditionally, these drugs have been thought to act in similar fashion, as competitive antagonists at a single class of muscarinic receptors, and not to differ significantly in their therapeutic efficacy. Recently, however, pharmacologic studies have shown that the novel antagonist pirenzepine is capable of recognizing heterogeneity among muscarinic receptors; high-affinity pirenzepine sites have been classified as M1 sites and low-affinity sites as M2. This study examined whether the anticholinergics currently available for treatment of neurologic symptoms have selectivity for these subtypes and whether they differ in their degree of selectivity; the study showed that these drugs do demonstrate selectivity. All had greater affinity for the M1 site, indicated by higher affinity for rat forebrain membranes, where M1 predominates, than hindbrain preparations, where M2 predominates. The degree of selectivity varied greatly; some compounds, such as ethopropazine, had little M1 selectivity, whereas others, such as scopolamine, trihexyphenidyl, and biperiden, were quite selective, like pirenzepine. It is unknown whether these differences in selectivity have any immediate therapeutic implications. However, these results support the emerging concept of muscarinic receptor subtypes and the prospect of developing more selective agents, with enhanced therapeutic efficacy.     

Radioligand binding assay of M1-M5 muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes.

Analysis of lymphocyte muscarinic cholinergic receptors using quantitative techniques such as radioligand binding assay is made difficult due to the low density of these sites and the lack of subtype-specific selectivity of most available muscarinic ligands. In this study, a combined kinetic and equilibrium labeling technique recently developed for brain tissue was used for labeling the five muscarinic cholinergic receptor subtypes in intact human peripheral blood lymphocytes. No specific muscarinic M1 receptor binding was detectable in human peripheral blood lymphocytes using [3H]-pirenzepine as a ligand. Labeling of M2-M5 muscarinic receptors using [3H]N-methyl-scopolamine (NMS) by occluding various receptor subtypes with muscarinic antagonist and mamba venom resulted in the labeling of M2-M5 receptors in brain as well as in human peripheral blood lymphocytes. The relative density of different receptor subtypes was M3 > M5 > M4 > M2. The development of a reproducible technique for assaying muscarinic cholinergic receptor subtypes expressed by human peripheral blood lymphocytes may contribute to clarify their role in lymphocyte function.     

Biperiden for treatment of somnambulism in adolescents and adults with or without epilepsy: Clinical observations

BackgroundSleepwalking in adolescents and adults may lead to serious injuries and require treatment. Anecdotal treatment recommendations include benzodiazepines (which also work in focal seizures of the frontal lobe that are an important differential diagnosis), imipramine and amitriptyline.MethodsWe assessed in a follow-up study of 4 years (medium, range: 2–7 years) the usefulness of the antiparkinsonian drug biperiden (Akineton©), an acetylcholine antagonist with high affinity for muscarinic M1-type receptors, in four consecutive cases of arousal disorder with sleepwalking and confusional behavior in adolescents and adults with or without epilepsy who did not respond to diazepam, clonazepam or amitriptyline.FindingsThe adjunctive use of biperiden was associated with reduction or remission of sleepwalking episodes in four consecutive treatment-refractory cases of arousal disorder with sleepwalking and confusional behavior. In contrast, biperiden showed no effect in a patient with REM behavioral disorder.InterpretationAlthough our observations do not and cannot establish the efficacy or safety of biperiden, it may be useful to consider biperiden for treatment of sleepwalking, if needed. A putative cholinergic mechanism of arousal disorders, including sleepwalking, provides a reasonable hypothesis why the anticholinergic agent biperiden might work. Evidence for efficacy and safety from randomized controlled trials is needed to confirm our preliminary observations.     

Acetylcholine Muscarinic M2 Receptor Stimulated [35S]GTPγS Binding Shows Regional Selective Changes in Alzheimer's Disease Postmortem Brain

Oxotremorine-M stimulated [³⁵S]GTPγS binding was used to assess acetylcholine muscarinic M2 receptor mediated G-protein function in superior frontal cortical, superior temporal cortical and hippocampal membranes from a series of Alzheimer's disease and matched control subjects. No significant differences were seen in basal [³⁵S]GTPγS binding between the groups. The maximal level of oxotremorine-M stimulated [³⁵S]GTPγS binding over basal was significantly increased in Alzheimer's disease superior temporal cortex, suggesting an enhanced muscarinic M2 receptor-G-protein coupling efficiency in this region. In contrast, the maximal level of oxotremorine-M stimulated [³⁵S]GTPγS binding over basal was unaltered in Alzheimer's disease superior frontal cortex and significantly reduced in Alzheimer's disease hippocampus. Western immunoblotting using antisera towards the α-subunits of those G-protein types known to couple muscarinic receptors, revealed that G_qαand G_iα, but not G_oα, levels were significantly reduced in Alzheimer's disease superior temporal cortex. Neither G_qα, G_iαnor G_oαlevels were significantly altered in Alzheimer's disease superior frontal cortex or hippocampus. These results suggest that the efficacy of muscarinic M₂receptor G-protein coupling shows regional selective changes in Alzheimer's disease postmortem brain with deficits occurring only in a region that shows severe pathology.     

Effective G-protein coupling of Y2 receptors along axonal fiber tracts and its relevance for epilepsy

Neuropeptide Y (NPY)-Y2 receptors are G-protein coupled receptors and, upon activation, induce opening of potassium channels or closing of calcium channels. They are generally presynaptically located. Depending on the neuron in which they are expressed they mediate inhibition of release of NPY and of the neuron's classical transmitter GABA, glutamate or noradrenaline, respectively. Here we provide evidence that Y2 receptor binding is inhibited dose-dependently by GTPγS along Schaffer collaterals, the stria terminalis and the fimbria indicating that Y2 receptors are functionally coupled to G-proteins along these fiber tracts. Double immune fluorescence revealed coexistence of Y2-immunoreactivity with β-tubulin, a marker for axons in the stria terminalis, but not with synaptophysin labeling presynaptic terminals, supporting the localization of Y2 receptors along axonal tracts. After kainic acid-induced seizures in rats, GTPγS-induced inhibition of Y2 receptor binding is facilitated in the Schaffer collaterals but not in the stria terminalis. Our data indicate that Y2 receptors are not only located at nerve terminals but also along fiber tracts and are there functionally coupled to G-proteins.     

Loss of stimulatory effect of guanosine triphosphate on [(35)S]GTPgammaS binding correlates with Alzheimer's disease neurofibrillary pathology in entorhinal cortex and CA1 hippocampal subfield

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G-proteins) couple many different cell surface receptor types to intracellular effector mechanisms. Uncoupling between receptors and G-proteins and between G-proteins and adenylyl cyclase (AC) and phospholipase C (PLC) has been described for Alzheimer's disease (AD) brain. However, there is little information on whether altered G-protein signaling in AD is just an end-stage phenomenon or is important for the progression of disease pathology. Here we used [(35)S]GTPgammaS autoradiography to study G-protein distribution in sections of entorhinal cortex and hippocampus from 23 cases staged for neurofibrillary changes and amyloid deposits according to Braak and Braak (Acta Neuropathol. [1991] 82:239-259). We also studied the effects of GTP, which has been found to increase [(35)S]GTPgammaS binding in an Mg(2+)-dependent manner. Results show that the ability of GTP (3 microM) to stimulate [(35)S]GTPgammaS binding declined significantly with staging for neurofibrillary changes in the entorhinal cortex (P < 0.05, ANOVA) and CA1 subfield of the hippocampus (P < 0.05, ANOVA). No significant changes were seen for [(35)S]GTPgammaS binding in the absence of GTP. Our results suggest a decrease in G-protein GTP hydrolysis, which correlates with the progression of AD neurofibrillary changes, in the regions most affected by this pathology. These alterations appear to occur prior to stages corresponding to clinical disease and could lead to an impaired regulation of several signaling systems in AD brain.     

Neurotransmitter receptor-mediated activation of G-proteins in brains of suicide victims with mood disorders: selective supersensitivity of alpha(2A)-adrenoceptors

Abnormalities in the density of neuroreceptors that regulate norepinephrine and serotonin release have been repeatedly reported in brains of suicide victims with mood disorders. Recently, the modulation of the [(35)S]GTPgammaS binding to G-proteins has been introduced as a suitable measure of receptor activity in postmortem human brain. The present study sought to evaluate the function of several G-protein coupled receptors in postmortem brain of suicide victims with mood disorders. Concentration-response curves of the [(35)S]GTPgammaS binding stimulation by selective agonists of alpha(2)-adrenoceptors, 5-HT(1A) serotonin, mu-opioid, GABA(B), and cholinergic muscarinic receptors were performed in frontal cortical membranes from 28 suicide victims with major depression or bipolar disorder and 28 subjects who were matched for gender, age and postmortem delay. The receptor-independent [(35)S]GTPgammaS binding stimulation by mastoparan and the G-protein density were also examined. The alpha(2A)-adrenoceptor-mediated stimulation of [(35)S]GTPgammaS binding with the agonist UK14304 displayed a 4.6-fold greater sensitivity in suicide victims than in controls, without changes in the maximal stimulation. No significant differences were found in parameters of 5-HT(1A) serotonin receptor and other receptor-mediated [(35)S]GTPgammaS binding stimulations. The receptor-independent activation of G-proteins was similar in both groups. Immunoreactive densities of G(alphai1/2)-, G(alphai3)-, G(alphao)-, and G(alphas)-proteins did not differ between suicide victims and controls. In conclusion, alpha(2A)-adrenoceptor sensitivity is increased in the frontal cortex of suicide victims with mood disorders. This receptor supersensitivity is not related to an increased amount or enhanced intrinsic activity of G-proteins. The new finding provides functional support to the involvement of alpha(2)-adrenoceptors in the pathogenesis of mood disorders.     

Nigrostriatal dopamine release modulated by mesopontine muscarinic receptors

The present study investigated the regulation of substantia nigra pars compacta (SNc) dopamine neuronal activity by pedunculopontine (PPT) cholinergic neurons. Changes in dopamine efflux following chemical activation or blockade of muscarinic acetylcholine receptors in the PPT were measured at stearate-carbon paste electrodes in the striatum of urethane (1.5 g/kg) anaesthetized male rats using in vivo chronoamperometry (30 s sampling rate). Intra-PPT infusions of a mixed muscarinic/nicotinic (carbachol 8 microg/microl) or M2/4-selective muscarinic (oxotremorine 0.5 microg/microl) receptor agonist attenuated striatal dopamine efflux, whereas a non-selective (scopolamine 100 microg/microl) or M2/4-selective (methoctramine 50 microg/microl) muscarinic receptor antagonist enhanced striatal dopamine efflux. These results suggest that M2/4 muscarinic receptors in the mesopontine tonically influence SNc basal dopamine cell activity and striatal dopamine release.     

Decreased muscarinic M1 receptor gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin-induced diabetic rats

The brain neurotransmitters' receptor activity and hormonal pathways control many physiological functions in the body. Acetylcholine (ACh), a major neurotransmitter from autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The objective of the present study was to investigate the changes in the total muscarinic and muscarinic M1 receptor ([(3)H]quinuclidinyl benzilate; QNB) binding and gene expression in the hypothalamus, brainstem, and pancreatic islets of streptozotocin (STZ)-induced diabetic and insulin-treated diabetic rats. In the hypothalamus and brainstem, total muscarinic receptor numbers were increased in diabetic rats with increase in affinity. Hypothalamic and brainstem muscarinic M1 receptors number were decreased in STZ diabetic rats with increase in affinity. In the pancreatic islets, muscarinic M1 receptors of diabetic rats were decreased, with a decrease in affinity. In all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR data also showed a decrease in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. Thus our results suggest that insulin modulates binding parameters and gene expression of total and muscarinic M1 receptors.     

Laminar organization and age-related loss of cholinergic receptors in temporal neocortex of rhesus monkey

Using in vitro receptor autoradiography, the distributions of cholinergic muscarinic [3H-N-methyl scopolamine (NMS), 3H-pirenzepine (PZ), and 3H-oxotremorine-M (OXO-M)] and nicotinic [3H-acetylcholine (ACh)] receptors were mapped in the temporal cortices of rhesus monkeys (Macaca mulatta) ranging from 2-22 years of age. Although high-affinity 3H-PZ, low-affinity 3H-NMS binding (M1 sites) and high-affinity 3H-OXO-M, high-affinity 3H-NMS binding (M2 sites) occurred across all layers of the temporal neocortex, the laminar distribution of M1 and M2 receptor binding sites was different. M1 muscarinic receptor binding was concentrated in layers II and III, whereas M2 muscarinic receptor binding was greatest in layers IV and V. The concentration of both muscarinic (M1 and M2) and nicotinic receptor binding sites declined with increasing age, and decrements were uniform across all cortical layers. This investigation provides evidence for a decrease in cholinergic receptor binding with age in temporal cortices of rhesus monkeys. Moreover, these changes appear to precede previously reported age-associated memory deficits and neuropathological changes that occur in this species.