Muscarinic acetylcholine receptor subtype mRNA expression and ligand binding in the aged rat forebrain

Previous studies indicate that a 20-30% decline in muscarinic acetylcholine receptor binding occurs in localized areas of rat brain during aging. In this study, reduced [3H]-quinuclidinyl benzilate binding was observed in striata from 24-25-month-old rats relative to 5-6-month-old animals using homogenate binding assays. To determine if the decline in receptor concentration occurs as a result of decreased receptor synthesis, the expression of the m1, m3, and m4 muscarinic receptor mRNAs as well as [3H]-QNB binding were determined in adjacent sections of young and old male rats using in situ hybridization and in vitro receptor autoradiography respectively. A significant decline in collective muscarinic receptor binding as assessed by [3H]-QNB was observed in the caudate putamen, olfactory tubercle, nucleus accumbens, and several frontal and parietal cortical areas. The only difference observed in muscarinic mRNA expression for any of the three subtypes examined was a decline in m1 hybridization in the olfactory tubercle. The results of this study demonstrate that the regional brain areas displaying age-related decreases in receptor binding do not correlate with those areas showing a decrease in muscarinic receptor expression. Apparently, the decline in muscarinic acetylcholine receptor density with age does not result from a decline in receptor gene expression.     

Muscarinic cholinergic receptor subtypes in the hippocampus of aged rats.

In spite of the suggestion of impaired muscarinic function in adult-onset cognitive disorders, data on the expression of muscarinic receptors in the hippocampus as a function of age are inconsistent. One reason may be that the majority of investigations were unable to differentiate the five brain muscarinic receptors subtypes. In this study, using a protocol based on a combination of both kinetic and equilibrium binding approaches, we have assessed the expression and the density of M1-M5 muscarinic cholinergic receptors in the hippocampus of Fisher 344 rats aged 6, 15 and 22 months. An age-related decrease of the density of M1 receptor was found in pyramidal neurons of the CA1 subfield. In this area, other subtypes of muscarinic receptors were unchanged with the exception of a loss of M2 receptor in the radial layer. In the CA3 subfield, receptor changes involved M2, M3 and M5 subtypes, whereas in the dentate gyrus, the main changes affected M1 and M2 receptors of the granular layer and M2 and M3 receptors of the molecular layer. The above findings indicate that analysis of age-related changes of different muscarinic cholinergic receptors might represent a useful contribution to identifying the basis of cholinergic neurotransmission impairment in adult-onset cognitive dysfunction.     

Muscarinic receptor activation attenuates D2 dopamine receptor mediated inhibition of acetylcholine release in rat striatum: indications for a common signal transduction pathway

In the present investigations, we used a superfusion system to study the effect of simultaneous activation of D2 dopamine receptors and so-called muscarinic "autoreceptors" on the K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. Activation of D2 receptors with the selective agonist LY 171555 (0.01-1 microM) clearly decreased the evoked release of [3H]acetylcholine. This effect was markedly attenuated in the presence of either the selective muscarinic receptor agonist oxotremorine (3 microM) or the cholinesterase inhibitor physostigmine (1 microM). Conversely, D2 receptor activation with LY 171555 (1 microM) completely abolished the muscarinic receptor mediated inhibition of evoked [3H]acetylcholine release induced by oxotremorine (0.03-10 microM). These results show that the inhibitory effects of D2 dopamine receptor and muscarinic receptor activation on striatal acetylcholine release are non-additive and therefore are interdependent processes. In addition, we investigated some aspects of the signal transduction mechanism by which the muscarinic receptor mediates inhibition of K(+)-evoked in vitro release of [3H]acetylcholine from rat striatal tissue slices. It appeared that the effect of muscarinic receptor activation was not significantly influenced either by a lowering of the extracellular Ca2+ concentration from the usual 1.2-0.12 mM or by an increase of the intracellular cyclic adenosine-3',5'-monophosphate content. However, increasing extracellular K+ strongly decreased the inhibition of evoked [3H]acetylcholine release mediated by activation of muscarinic receptors. This set of results indicates that the muscarinic "autoreceptor" mediates the decrease of depolarization induced [3H]acetylcholine release from rat striatum to a large extent through stimulation of K+ efflux (opening of K+ channels) in a cyclic adenosine-3',5'-monophosphate independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of kainic acid lesions on calcium uptake and dopamine release in nerve endings isolated from rat striata

Rats were injected intrastriatally with kainic acid and the viability of dopaminergic terminals two days following the injection was determined by comparing voltage dependent calcium uptake and dopamine release in isolated nerve endings. Evoked dopamine release remained normal following the lesion, but the initial rate of potassium stimulated calcium entry decreased by approximately 1/3. These results suggest that the excitotoxic lesion caused by acute intrastriatal injection of kainic acid results in retention of functional dopaminergic terminals.     

Muscarinic cholinergic receptor binding in rat mast cells

Specific [³H]-QNB binding was present in isolated, purified, intact rat mast cells and in crude membrane preparations. The binding is saturable, time- and temperature-dependent. Cholinergic agents inhibit selectively the binding: atropine is the most effective of the antagonists while oxotremorine is the most potent of the muscarinic agonists. It is concluded that rat mast cells are provided with muscarinic cholinergic receptors.     

Muscarinic agonist receptor subtypes in aging rat brain

Whole brain homogenates from rats aged 6 months (young) and 24 months (old) showed a decline with age of the pre-synaptic cholinergic marker, choline acetyltransferase, and also of total specific binding sites for the muscarinic antagonist L(?)quinuclidinyl benzilate (L-QNB). However, neither the proportion nor the inhibition constants of high and low affinity muscarinic agonist binding sites (defined by displacement of L-QNB binding with carbachol) changed with age. These findings may be relevant to the central cholinergic deficit reported to be associated with cognitive impairment in aging man.     

Muscarinic receptors depress GABAergic synaptic transmission in rat midbrain dopamine neurons

The effects of muscarine and nicotine on evoked and spontaneous release of GABA were studied using intracellular and whole-cell patch-clamp recordings from rat midbrain dopamine neurons in an in vitro slice preparation. Muscarine (30 microM) reversibly depressed the pharmacologically isolated inhibitory postsynaptic potential evoked by local electrical stimulation. The maximal inhibition of the inhibitory postsynaptic potential amplitude was 39.6+/-5%. This depressant effect of muscarine was blocked by the M3/M1 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (100 nM), but was slightly affected by the M1/M3 receptor antagonist pirenzepine (1 microM). In addition, muscarine decreased the frequency of the miniature synaptic currents without any effect on their amplitude. Moreover, muscarine did not change the GABA-induced hyperpolarization, indicating that its effect on the inhibitory postsynaptic potential is mediated by presynaptic receptors. On the contrary, the cholinergic agonist nicotine did not change the frequency or the amplitude of the spontaneous glutamatergic and GABAergic synaptic currents.Our data indicate that a prevalent activation of presynaptic M3 muscarinic receptors inhibits the GABA-mediated synaptic events, while the activation of nicotinic receptors does not affect the release of glutamate and GABA on midbrain dopamine neurons.     

NMDA receptor blockade augmented nicotine-evoked dopamine release from rat prefrontal cortex slices

Amyloid beta (Abeta), a peptide family produced and deposited in neurons and endothelial cells (EC), is found at subnanomolar concentrations in the plasma of healthy individuals. Simple conformational changes produce a form of Abeta, Abeta42, which creates toxic plaque in the brains of Alzheimer's patients. Oxidative stress induced blood brain barrier degeneration has been proposed as a key factor for Abeta42 toxicity, but cannot account for lack of injury from the same peptide in healthy tissues. We hypothesized that cell state mediates Abeta effect. Thus, we examined the viability of aortic EC, vascular smooth muscle cells (SMC) and epithelial cells (EPI) in different states in the presence of Abeta secreted from transfected Chinese hamster ovary cells (CHO). Abeta was more toxic to all cell types when they were subconfluent. Subconfluent EC sprouted and SMC and EPI were inhibited by Abeta. Confluent EC were virtually resistant to Abeta and suppressed Abeta production by Abeta+CHO. Products of subconfluent EC overcame this resistant state, stimulating the production and toxicity of Abeta42. Confluent EC overgrew approximately 35% beyond their quiescent state in the presence of Abeta conditioned in media from subconfluent EC. These findings imply that Abeta42 may well be even more cytotoxic to cells in injured or growth states and potentially explain the variable and potent effects of this protein. One may now need to consider tissue and cell state in addition to local concentration of and exposure duration to Abeta. The specific interactions of Abeta and EC in a state-dependent fashion may help understand further the common and divergent forms of vascular and cerebral toxicity of Abeta and the spectrum of AD.     

Somatostatin modulates dopamine release in rat retina

The aim of the present study was to determine the possible role of somatostatin as a modulator of dopamine release in rat retina. Basal release of dopamine, and how this is influenced by somatostatin receptor (sst) selective ligands, was examined ex vivo in rat retinal explants. Dopamine levels were quantified by high-pressure liquid chromatography (HPLC) with electrochemical detection. Basal levels of dopamine were measured over 120 min of tissue incubation and found to be 1.17+/-0.35 ng/ml. Somatostatin (10(-6), 10(-5), 10(-4)M) increased dopamine levels in a concentration-dependent manner, while the sst(2) antagonist CYN154806 (10(-4)M) reversed its actions. BIM23014 (sst(2) agonist) increased dopamine levels in a statistically significant manner only at the concentration of 10(-5)M. The sst(1) agonist L797.591 (10(-5), 10(-4)M) also increased dopamine levels, while activation of the sst(3) receptor (sst(3) agonist, L796.778, 10(-4)M) had no effect. These data substantiate a neuromodulatory role for sst(1) and sst(2) somatostatin receptors in the retina and show for the first time somatostatin's influence on dopamine release.     

Muscarinic inhibition of endogenous glutamate release from rat hippocampus synaptosomes

The effects of acetylcholine (ACh) on the depolarization-evoked release of endogenous glutamic acid (Glu) have been studied using synaptosomes prepared from rat hippocampus and depolarized in superfusion with 15 mM KCl. Acetylcholine inhibited Glu release in a concentration-dependent way. The natural agonist was particularly effective causing 50% inhibition of Glu release at 10 microM in the absence of acetylcholinesterase (AChE) inhibitors. The inhibitory effect of ACh on the K+-evoked release of Glu was antagonized by the selective muscarinic receptor antagonist atropine but not by the nicotinic receptor antagonist mecamylamine. The data represent the first demonstration that muscarinic receptors located on Glu axon terminals in rat hippocampus may modulate the release of Glu.     

Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence

Release of ³H-ACh from isolated nerve endings of rat hippocampus was evoked by incubation in Krebs-Ringer's buffer containing 25 or 35 mM potassium. The release was Ca²⁺-dependent and could be inhibited by Mg²⁺ (20 mM). The muscarinic antagonist, atropine (10^-10–10^-6 M), enhanced ³H-ACh-release. The muscarinic agonist, carbachol (10^-5–10^-3 M) inhibited ³H-ACh release via interaction with muscarinic receptors: this effect could be blocked by atropine (10^-6 M). The presence of the feed-back regulation of ³H-ACh release in a cell-free preparation provides further evidence that the presynaptic regulation is exerted by muscarinic autoreceptors localized on the cholinergic nerve ending itself. The feed back inhibition of the ³H-ACh release does not require the presence of intact neurons or neuronal loops as tetrodotoxin (2.5. 10^-6 M) does not affect the above results.     

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.     

Muscarinic antagonists attenuate neurotensin-stimulated accumbens and striatal dopamine metabolism.

The effect of scopolamine and atropine upon the increase in extracellular 3,4-dihydroxyphenylacetic acid induced by central injection of neurotensin was examined in the nucleus accumbens and the striatum of anaesthetized rats using in vivo differential pulse voltammetry with carbon fibre electrodes. Scopolamine (1 and 3 mg/kg, i.p.) and atropine (20 micrograms, i.c.v.) did not alter the 3,4-dihydroxyphenylacetic acid level in the nucleus accumbens or the striatum, measured for 60 min after administration. Neurotensin (10 micrograms, i.c.v.) increased the 3,4-dihydroxyphenylacetic acid peak height in both regions. Pretreatment with scopolamine (1 mg/kg) 15 min before neurotensin injection blocked the increase in extracellular 3,4-dihydroxyphenylacetic acid in the striatum but not in the nucleus accumbens whilst scopolamine (3 mg/kg) partially attenuated the effect of neurotensin in the nucleus accumbens and blocked the increase in 3,4-dihydroxyphenylacetic acid in the striatum. Atropine partially attenuated the effect produced by neurotensin in the nucleus accumbens and blocked the increase in 3,4-dihydroxyphenylacetic acid induced by the peptide in the striatum. However, the increase in extracellular 3,4-dihydroxyphenylacetic acid induced by haloperidol (1 mg/kg, s.c.) was not altered by scopolamine (1 mg/kg) or atropine. Also, the increase in dopamine metabolism in the nucleus accumbens and the striatum after centrally injected haloperidol (10 micrograms, i.c.v.) was not altered by atropine (20 micrograms, i.c.v.). Together, the results demonstrate a functional interaction between muscarinic antagonists and neurotensin on in vivo dopamine metabolism in the nucleus accumbens and the striatum but with a greater effect in the latter region.     

Postnatal development of dopamine receptor expression in rat peripheral blood lymphocytes.

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [3H]SCH 23390 and the dopamine D2-like receptor agonist [3H]7-OH-DPAT were used as radioligands. The affinity (K(d)) of [3H]SCH 23390 or of [3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density (B(max)) of [3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B(max) value of [3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.     

The modulation of striatal dopamine release correlates with water-maze performance in aged rats

Cluster analysis of performance during acquisition of a place-learning task in the water maze distinguished between subpopulations of aged rats (25-27 months) classified as moderately (AMI) or severely impaired (ASI) in comparison with young adults (3-5 months). Using a slice-superfusion device, electrically or nicotine-evoked release of dopamine from striatum was assessed in the presence of GR-55,562 (5-HT(1B) receptor antagonist), methiotepin (mixed 5-HT(1/2) receptor antagonist) and/or sulpiride (D(2)/D(3) receptor antagonist). The main neuropharmacological results demonstrated age-related alterations in the 5-HT(1B)- and D(2)/D(3)-mediated modulation of electrically evoked striatal dopamine release. Regression analyses indicated a possible contribution of such alterations to the age-related behavioural deficits: the larger the deficit, the weaker the electrically evoked release under 5-HT(1B) and D(2)/D(3) receptor blockade. Extending our recent report on the modulation of striatal acetylcholine release in aged rats [Cassel et al., 2007. Neurobiol. Aging 28, 1270-1285], these new findings make dopaminergic and serotonergic functional alterations potential candidates to participate in age-related deficits in the water maze, most probably in interaction with formerly described cholinergic dysfunctions.     

Muscarinic ACh receptor activation causes transmitter release from isolated frog vestibular hair cells

In the frog, vestibular efferent fibers innervate only type-II vestibular hair cells. Through this direct contact with hair cells, efferent neurons are capable of modifying transmitter release from hair cells onto primary vestibular afferents. The major efferent transmitter, acetylcholine (ACh), is known to produce distinct pharmacological actions involving several ACh receptors. Previous studies have implicated the presence of muscarinic ACh receptors on vestibular hair cells, although, surprisingly, a muscarinic-mediated electrical response has not been demonstrated in solitary vestibular hair cells. This study demonstrates that muscarinic receptors can evoke transmitter release from vestibular hair cells. Detection of this release was obtained through patch-clamp recordings from catfish cone horizontal cells, serving as glutamate detectors after pairing them with isolated frog semicircular canal hair cells in a two-cell preparation. Although horizontal cells alone failed to respond to carbachol, application of 20 microM carbachol to the two-cell preparation resulted in a horizontal cell response that could be mimicked by exogenous application of glutamate. All of the horizontal cells in the two-cell preparation responded to 20 microM CCh. Furthermore, this presumed transmitter release persisted in the presence of d-tubocurarine at concentrations that block all known hair cell nicotinic ACh receptors. The effect on the detector cell, imparted by the carbachol application to the hair cell-horizontal cell preparation, was blocked both by 2-amino-5-phosphonopentanoic acid, a selective N-methyl-D-aspartate antagonist, and the muscarinic antagonist, atropine. Thus vestibular hair cells from the frog semicircular canal can be stimulated to release transmitter by activating their muscarinic receptors.     

P2 receptor-mediated inhibition of dopamine release in rat neostriatum

Axon terminal nucleotide P2 receptors mediating an inhibition of transmitter release have, so far, been detected in various sympathetically innervated tissues,(8,27) and on central noradrenergic,(14,26) glutamatergic(15) and serotonergic neurons. (28) We have now investigated the effect of ATP and related nucleotides on the release of endogenous dopamine from slices of rat neostriatum using fast cyclic voltammetry. Mutual interactions between the two neurotransmitters have been observed previously: ATP and related nucleotides induce a release of dopamine in PC12 pheochromocytoma cells, a frequently used model for sympathetic neurons;(10,22) they also increase the dopamine concentration in rat brain measured by in vivo microdialysis(16,32) and stimulate the uptake of dopamine by rat striatal synaptosomes.(3) Dopamine, in contrast, facilitates activation of ligand-gated cation channels (i. e. P2X(2) receptors) by ATP.(11,20) Here, we show that ATP and two of its analogues decrease the electrically evoked release of endogenous dopamine in rat neostriatum. The inhibitory effect of ATP is blocked by the P2 receptor antagonists suramin, reactive blue 2 and cibacron blue 3GA. Suramin, in addition, partly prevents the attenuation of dopamine release evoked by a single stimulus that follows a brief train of high-frequency pulses.These findings suggest the existence of release-inhibiting P2 receptors on dopaminergic nerve terminals and indicate that dopaminergic transmission in rat neostriatum might be modulated by an endogenous P2 receptor ligand, presumably ATP.