Colocalization of muscarinic acetylcholine receptors and protein kinase C gamma in rat parietal cortex

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKC gamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKC gamma. Conversely, nearly all PKC gamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 microns in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKC gamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKC gamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKC gamma, a candidate key enzyme in cortical learning and memory mechanisms.     

Intrinsic GABAergic neurons in the rat central extended amygdala

The present study examined the distribution, morphology, and connections of gamma-aminobutyric acid-immunoreactive (GABA-IR) neurons in the three principal components of the central extended amygdala: the central amygdaloid nucleus, the bed nucleus of the stria terminalis (BNST) and the sublenticular substantia innominata. In the central nucleus, large numbers of GABA-IR neurons were identified in the lateral, lateral capsular, and ventral subdivisions, though in the medial subdivision, GABA-IR neurons were only present at very caudal levels. Combined immunocytochemistry-Golgi impregnation revealed that GABA-IR neurons in the lateral central nucleus were medium-sized spiny neurons that were morphologically similar to GABAergic neurons in the striatum. Injections of horseradish peroxidase into the bed nucleus of the stria terminalis labeled a major proportion of the GABA-IR neurons in the central nucleus. In the bed nucleus, the majority of GABA-IR neurons were located in the anterolateral subdivision, ventral part of the posterolateral subdivision and the parastrial subdivision. GABA-IR neurons in the anterolateral bed nucleus were of the typical medium-sized spiny type. Injections of horseradish peroxidase into the central nucleus labeled a few GABA-IR neurons in the posterior part of the anterolateral bed nucleus. GABA-IR neurons were identified in the sublenticular substantia innominata and medial shell of the nucleus accumbens and contributed to the continuum of GABA-IR extending from the central nucleus to the bed nucleus. Injections of horseradish peroxidase (HRP) into the central nucleus, but not the BNST, labeled a few GABA-IR neurons in the substantia innominata. The data point to GABA-IR neurons being a characteristic feature of the central extended amygdala and that GABA-IR neurons participate in the long intrinsic connections linking the major components of this structure. Since lesions of the stria terminalis and basolateral amygdaloid nucleus failed to deplete GABA-IR terminals in the central nucleus, the role of GABA in local and short intrinsic connections in the central extended amygdala is discussed. Further, physiological findings implicating the intrinsic GABAergic system of the central extended amygdala in the tonic inhibition of brainstem efferents are reviewed.     

Expression of acetylcholinesterase messenger RNA in human brain: an in situ hybridization study

The distribution of messenger RNA coding for acetylcholinesterase was studied in human post mortem brain and rhesus monkey by in situ hybridization histochemistry and compared to the distribution of acetylcholinesterase activity. Acetylcholinesterase messenger RNA had--similar to acetylcholinesterase enzymatic activity--a widespread distribution in human bain. Acetylcholinesterase messenger RNA positive cells corresponded to perikarya rich in acetylcholinesterase activity in most but not all regions. Examples for mismatches included the inferior olive and human cerebellar cortex. The presence of hybridization signals in cerebral cortex and an enrichment in layer III and V of most isocortical areas confirmed that perikaryal acetylcholinesterase in cerebral cortex is of postsynaptic origin and not derived from cholinergic projections. In striatum the expression of high levels of acetylcholinesterase messenger RNA was restricted to a small population of large striatal neurons. In addition, low levels of expression were found in most medium sized striatal neurons. Cholinergic neurons tended to express high levels of acetylcholinesterase messenger RNA whereas in cholinoceptive neurons the levels were moderate to low. However, some noncholinergic neurons like dopaminergic cells in substantia nigra, noradrenergic cells in locus coeruleus, serotoninergic cells in raphé dorsalis, GABAergic cells in thalamic reticular nucleus, granular cells in cerebellar cortex and pontine relay neurons expressed levels comparable to cholinergic neurons in basal forebrain. It is suggested that neurons expressing high levels of acetylcholinesterase messenger RNA may synthesize acetylcholinesterase for axonal transport whereas neurons with an expression of acetylcholinesterase confined to somatodendritic regions tend to contain lower levels of acetylcholinesterase messenger RNA.     

Localization of the m2 muscarinic acetylcholine receptor protein and mRNA in cortical neurons of the normal and cholinergically deafferented rhesus monkey

The m2 muscarinic acetylcholine receptor in the cerebral cortex has traditionally been thought of as an autoreceptor located on cholinergic fibers that originate from neurons in the nucleus basalis of Meynert. We now provide evidence for widespread localization of the m2 receptor in noncholinergic neurons and fibers of the cerebral cortex. The cellular and subcellular distribution of the m2 receptor protein and mRNA were examined in normal monkeys and in monkeys in which the cortical cholinergic afferents were selectively lesioned by injection of the specific immunotoxin, anti-p75NTR-saporin into the nucleus basalis. Both in normal and immunolesioned monkeys, the m2 mRNA and protein were localized in pyramidal and nonpyramidal neurons. In pyramidal neurons, membrane-associated receptor immunoreactivity was found exclusively in dendritic spines receiving asymmetric synapses, indicating that the m2 receptor may modulate excitatory neurotransmission at these sites. In nonpyramidal neurons, the m2 immunoreactivity was present along the cytoplasmic surface of membranes in cell bodies, dendrites and axons. Both in pyramidal and nonpyramidal neurons of normal and lesioned monkeys, the m2 receptor was located peri- and extra-synaptically, suggesting that it may be contacted by acetylcholine via volume transmission. The localization of the m2 receptor in cortical neurons and the sparing of m2 immunoreactivity in lesioned monkeys indicates that the m2 receptor is synthesized largely within the cortex and/or is localized to noncholinergic terminals of either intrinsic or extrinsic origin. These findings open the possibility that the loss of the m2 receptor in Alzheimer's disease may in part be due to degenerative changes in m2 positive neurons of the cortex rather than entirely due to the loss of autoreceptors.     

Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons

Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1-7) cells induced prominent peaks in GnRH release, each followed by a rapid decrease, a transient plateau, and a decline to below basal levels. The complex profile of GnRH release suggested that acetylcholine (ACh) acts through different cholinergic receptor subtypes to exert stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G(alpha q/11) immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G(alpha i3) was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein alpha-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective transduction pathways. These results indicate that ACh modulates GnRH release from hypothalamic neurons through both M1 and M2 muscarinic receptors. These receptor subtypes are coupled to Gq and Gi proteins that respectively influence the activities of PLC and adenylyl cyclase/ion channels, with consequent effects on neurosecretion.     

CNS GABA neurons express the mu-opioid receptor: immunocytochemical studies

It has been proposed that mu-opioid receptors excite neurons in hippocampus and nucleus raphe dorsalis (NRD) by decreasing GABAergic tone. In the present study, we examined whether immunocytochemical evidence of interaction between GABAergic neurons and the mu-opioid receptor could be found in the CNS. Portions of rat brain were sectioned and stained for GABA and for the cloned mu-opioid receptor (MOR1) using two-color immunofluorescence. Neurons double-labeled for GABA and MOR1 were present in hippocampus and NRD, as well as in olfactory bulb, dorsal lateral periaqueductal gray matter, nucleus raphe medianis, nucleus raphe obscurus, and the spinal trigeminal nucleus and tract. We conclude that expression of the mu-opioid receptor by GABAergic neurons is common in the rat CNS.     

Recognition and management of childhood cardiac arrhythmias

Axonal connections between the amygdala and the hypothalamic paraventricular nucleus were examined by combined anterograde-retrograde tract tracing. Iontophoretic injections of the retrograde tracer Fluorogold were placed in the paraventricular nucleus, and the anterograde tracer PHA-L in the ipsilateral central or medial amygdaloid nuclei. Single and double-label immunohistochemistry were used to detect tracers. Single label anterograde and retrograde tracing suggest limited evidence for direct connections between the central or medial amygdala and the paraventricular nucleus. In general, scattered PHA-L-positive terminals were seen in autonomic subdivisions of the paraventricular nucleus (lateral parvocellular, dorsal parvocellular and ventral medial parvocellular subnuclei) following central or medial amygdaloid nucleus injection. Double-label studies indicate that central and medial amygdaloid nucleus efferents contact paraventricular nucleus-projecting cells in several forebrain nuclei. In the case of central nucleus injections, PHA-L positive fibers occasionally contacted Fluorogold-labeled neurons in the anteromedial, ventromedial and preoptic subnuclei of the bed nucleus of the stria terminalis. Overall, such contacts were quite rare, and did not occur in the bed nucleus of the stria terminalis regions showing greatest innervation by the central amygdaloid nucleus. In contrast, medial amygdala injections resulted in a significantly greater overlap of PHA-L labeling and Fluorogold-labeled neurons, with axosomatic appositions observed in medial divisions of the bed nucleus of the stria terminalis, anterior hypothalamic area and preoptic area. The results provide anatomical evidence that a substantial proportion of amygdaloid connections with hypophysiotrophic paraventricular nucleus neurons are likely multisynaptic, relaying in different subregions of the bed nucleus of the stria terminalis and hypothalamus.     

A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.     

Evidence for the colocalization of estrogen receptor-beta mRNA and estrogen receptor-alpha immunoreactivity in neurons of the rat forebrain

Estrogen receptor beta (ER beta) mRNA is expressed in several rat brain regions where ER alpha is abundant. In vitro studies have shown that ER alpha and ER beta can heterodimerize and that the activity of this complex may be different than an ER alpha or ER beta homodimer complex. The purpose of the present study was to ascertain if ER alpha and ER beta are co-expressed by certain neuronal populations using a double label in situ hybridization/immunocytochemistry method. The results revealed that neurons in the bed nucleus of the stria terminalis, medial amygdala and preoptic area contain both ERs, with the vast majority of the neurons being double labeled. In other brain regions including the arcuate nucleus, cortical amygdaloid nuclei and ventromedial nucleus, only a few double-labeled cells were detected, while neurons in the paraventricular nucleus, supraoptic nucleus, and cerebral cortex expressed only ER beta mRNA. The results of these double label experiments provide the first evidence that ER alpha and ER beta coexist in neurons under in vivo conditions and suggest that estrogens may differentially modulate the activity of certain neuronal populations depending on whether the cells expresses ER alpha, ER beta or both ERs.     

Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans

Acetylcholine acts as a neurotransmitter in the central and peripheral nervous systems in humans. However, recent experiments demonstrate a widespread expression of the cholinergic system in non-neuronal cells in humans. The synthesizing enzyme choline acetyltransferase, the signalling molecule acetylcholine, and the respective receptors (nicotinic or muscarinic) are expressed in epithelial cells (human airways, alimentary tract, epidermis). Acetylcholine is also found in mesothelial, endothelial, glial, and circulating blood cells (platelets, mononuclear cells), as well as in alveolar macrophages. The existence of non-neuronal acetylcholine explains the widespread expression of muscarinic and nicotinic receptors in cells not innervated by cholinergic neurons. Non-neuronal acetylcholine appears to be involved in the regulation of important cell functions, such as mitosis, trophic functions, automaticity, locomotion, ciliary activity, cell-cell contact, cytoskeleton, as well as barrier and immune functions. The most important tasks for the future will be to clarify the multiple biological roles of non-neuronal acetylcholine in detail and to identify pathological conditions in which this system is up- or down-regulated. This could provide the basis for the development of new therapeutic strategies to target the non-neuronal cholinergic system.     

Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-alpha-bungarotoxin receptor autoradiography and in situ hybridization of alpha 7 nAChR subunit mRNA

The cochlear nucleus (CN) is the first site in the central nervous system (CNS) for processing auditory information. Acetylcholine in the CN is primarily extrinsic and is an important neurotransmitter in efferent pathways thought to provide CNS modulation of afferent signal processing. Although muscarinic acetylcholine receptors have been studied in the CN, the role of nicotinic receptors has not. We examined the distribution of one nicotinic acetylcholine receptor subtype, the alpha-bungarotoxin receptor (alpha Bgt), in the CN. Quantitative autoradiography was used to localize receptors and in situ hybridization was used to localize alpha 7 mRNA in CN neurons that express the alpha Bgt receptor. Binding sites for alpha Bgt are abundant in the anterior ventral, posterior ventral, and dorsal divisions of the CN, and receptor density is low in the granule cell layer and interstitial nucleus. Heterogeneity in CN subregions is described. Four distinct patterns of alpha Bgt binding were observed: (1) binding over and around neuronal cell bodies, (2) receptors locally surrounding neurons, (3) dense punctate binding in the dorsal CN (DCN) not associated with neuronal cell bodies, and (4) diffuse fields of alpha Bgt receptors prominent in the DCN molecular layer, a field underlying the granule cell layer and in the medial sheet. The perikaryial receptors are abundant in the ventral CN (VCN) and are always associated with neurons expressing mRNA for the receptor. Other neurons in the VCN also express alpha 7 mRNA, but without alpha Bgt receptor expression associated with the cell body. In general, alpha Bgt receptor distribution parallels cholinergic terminal distribution, except in granule cell regions rich in cholinergic markers but low in alpha Bgt receptors. The findings indicate that alpha Bgt receptors are widespread in the CN but are selectively localized on somata, proximal dendrites, or distal dendrites depending on the specific CN subregion. The data are consistent with the hypothesis that descending cholinergic fibers modulate afferent auditory signals by regulating intracellular Ca2+ through alpha Bgt receptors.     

Ultrastructural localization of P2X3 receptors in rat sensory neurons

We used isolated IgG antibodies selective for P2X3 receptors to study the ultrastructural distribution of these receptors in rat sensory neurons. In trigeminal ganglia, P2X3 receptor immunoreactivity occurred in small and large nerve cell bodies and their processes. Endoplasmic reticulum and Golgi apparatus were heavily stained; cytoplasmic matrix was faintly to moderately stained. In synaptic glomeruli in lamina II of cervical dorsal horn, P2X3 receptor-immunoreactive core terminals were postsynaptic to unlabelled vesicle-containing dendrites and axons. In the nucleus of the solitary tract, receptor-positive boutons synapsed on dendrites and cell bodies and had complex synaptic relationships with other axon terminals and vesiculated dendrites. These observations identify sites from which ATP could be released to influence sensory signalling within the central nervous system.     

Relationship of thalamic basal forebrain projection neurons to the peptidergic innervation of the midline thalamus

To better understand the input-output organization of the midline thalamus, we compared the distribution of its peptidergic and monoaminergic afferents, which were visualized by using immunocytochemistry, with the distribution of neurons projecting to different basal forebrain structures, which were mapped using retrograde fluorescent tracers. Serotonin and most of the peptides were found throughout paraventricular thalamic nucleus (PV) and in other midline and intralaminar nuclei (type 1 pattern). Neuropeptide Y, alpha MSH and the catecholamine synthetic enzymes were largely restricted to dorsolateral PV (type 2 pattern). Vasopressin was found in dorsomedial PV and intermediodorsal nucleus in a pattern complementary to the type 2 distribution (type 3 pattern). Neurons projecting to accumbens core were present in paraventricular, intermediodorsal, and other midline nuclei. Neurons projecting to accumbens shell and to central amygdaloid nucleus were found in dorsal PV. The peptidergic zones were only loosely correlated with the distribution of different classes of projection neurons. The type 2 pattern overlapped best with neurons projecting to accumbens shell, and to a lesser extent to central amygdaloid nucleus, while the type 3 pattern overlapped best with neurons projecting to core of accumbens. This partial overlap suggests that some brainstem and hypothalamic nuclei preferentially affect different basal forebrain targets through the midline thalamus, and may allow, for example, information about stress to specifically influence accumbens shell and central amygdaloid nucleus. Nevertheless, most of the peptidergic afferents (type 1 pattern) to midline thalamus cover neurons projecting throughout the basal forebrain, which suggests that all of these neurons receive a variety of brainstem and hypothalamic inputs.     

Ultrastructural features of tyrosine-hydroxylase-immunoreactive afferents and their targets in the rat amygdala

Interrelations of tyrosine-hydroxylase-immunoreactive afferent fibres with neuronal elements were studied in central, basal and intercalated nuclei of the rat amygdaloid complex. Comparison with dopamine-beta-hydroxylase-immunoreacted and phenylethanolamine-N-methyltransferase-immunoreacted parallel sections indicated that the tyrosine-hydroxylase immunoreaction labelled preferentially dopaminergic axons. At the electron-microscopic level, the majority of tyrosine-hydroxylase-immunoreactive axons possessed small boutons containing small clear vesicles and contacting dendrites, spines or somata of amygdala neurons, forming mostly symmetric synapses. They were often directly apposed to or in the vicinity of unlabelled terminals synapsing on the same structure. Synaptic density was highest in the central lateral part of the central nucleus. In the central and basal nuclei labelled axons synapsed preferentially on small dendrites and dendritic spines, and on somata of a few neurons. A detailed study of the neuronal ultrastructure showed that innervated somata possessed the differential characteristics displayed by the predominant neuron types in the medial and central lateral central nucleus and resembled the typical projection neurons in the basal nuclei. In the paracapsular intercalated cell groups the majority of neurons possessed intense perisomatic innervation by immunoreactive terminals. The results suggest that tyrosine-hydroxylase-immunoreactive, predominantly dopaminergic amygdaloid afferent fibres preferentially modulate the effect of extrinsic inputs into neurons of the central and basal nuclei, while a nonselective regulation is exerted upon the output of paracapsular intercalated neurons. It is suggested that this innervation pattern may be important for the coordinated integration of extrinsic and intraamygdaloid connections and thus for balanced output of the structure.     

Postnatal expression of H1-receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of L-histidine decarboxylase (HDC) in the two pools was revealed. H1-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1-receptor expression. Systemic kainic acid administration induced increased expression of H1-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1-CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1-receptor, is involved in the regulation of seizure activity in the brain.     

Clumsiness in children: a defect of kinaesthetic perception?

The distribution of type I interleukin-1 receptor (IL-1R1) mRNA in the rat brain was examined by in situ hybridization technique. IL-1R1 mRNA was expressed in several brain regions including the anterior olfactory nucleus, medial thalamic nucleus, posterior thalamic nucleus, basolateral amygdaloid nucleus, ventromedial hypothalamic nucleus, arcuate nucleus, median eminence, mesencephalic trigeminal nucleus, motor trigeminal nucleus, facial nucleus and Purkinje cells of the cerebellum. Furthermore, we identified neuronal expression of IL-1R1 mRNA using simultaneous detection (double in situ hybridization) of IL-1R1 mRNA with neuron specific enolase mRNA. In addition to the expression in neuronal cells, IL-1R1 mRNA was also expressed on the vascular walls and the epithelial cells of the choroid plexus and the ventricles. These findings suggest the possibility that IL-1 produces its multiple effects on the central nervous system through the actions not only on neuronal cells but also on endothelial and epithelial cells.     

The M5 (m5) receptor subtype: fact or fiction?

By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological functions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of m5-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3H]NMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the m1-m4 receptor subtypes, to selectively label the m5 receptor subtype. This m5-selective labeling approach, along with those developed for the other four receptor subtypes, has permitted for the first time a comparison of the relative expression levels and anatomical localizations of the five muscarinic receptor subtypes in the brain. The distribution profile of the m5 receptor is distinct from the other four subtypes and is enriched in the outer layers of the cortex, specific subfields of the hippocampus, caudate putamen, olfactory tubercle and nucleus accumbens. These studies have also demonstrated that the levels of m5 receptor protein expression are apparently higher and more widespread than anticipated from previous in situ hybridization and immunoprecipitation studies. Taken together, the results suggest a unique and potentially physiologically important role for the m5 receptor subtype in modulating the actions of acetylcholine in the brain.     

Neurotransmitter receptor plasticity in aging

Neurotransmitter receptor plasticity is an important part of the compensatory processes by which the central nervous system adapts to pathological insult, long-term exposure to drugs or neuronal loss with advanced age. Receptor plasticity can be manifest as changes in the number of receptors (i.e., up- or down-regulation), changes in expression of mRNA for discrete receptor proteins, or alterations in receptor coupling to signal transduction systems. Evidence exists for impaired plasticity of neurons in the aged brain, which results in decreased ability to adjust to changes in their environment. However, such data are highly dependent on the neurotransmitter examined, the stimulus for receptor regulation and the animal model chosen for study. For example, senescent rats show an age-related impairment of muscarinic receptor up- or down-regulation after long-term exposure to cholinergic drugs. Thus, young rats exposed to chronic (three weeks) intracerebroventricular infusions of methylatropine or oxotremorine exhibit compensatory changes in the density of muscarinic receptors in frontal cortex and hypothalamus. In contrast, 3H-QNB binding is unaltered in the same brain regions of identically treated senescent rats. Similar observations of impaired muscarinic receptor plasticity in senescent animals have been confirmed by other investigators. Age-related differences in coupling of brain muscarinic receptors to G-proteins and in muscarinic receptor-stimulated phosphoinositide hydrolysis have also been reported. Interestingly, neuropeptides such as neurotensin, cholecystokinin and VIP can potentiate carbachol-stimulated phosphoinositide hydrolysis in frontal cortex of both young and aged rats. This adds another level at which cholinergic neurotransmission may be modulated in senescent animals. Potential age-related differences in the effects of chronic drug treatments or experimental brain lesions on muscarinic receptor coupling to second messenger systems or on expression of mRNA for particular muscarinic receptors are currently unknown. Hence, it is possible that senescent animals may show additional deficiencies in plasticity of muscarinic receptor mediated signal transduction or expression of muscarinic receptors subtypes.     

Muscarinic receptor antagonism of the nucleus accumbens core causes avoidance to flavor and spatial cues.

Pharmacological blockade of muscarinic receptors in the nucleus accumbens reduces food intake and instrumental behaviors that are reinforced by food delivery. Nucleus accumbens muscarinic antagonism may specifically suppress the hedonic or reinforcing effects of food, thus blocking its capacity to direct behavior. Alternatively, muscarinic receptor blockade may cause a negative hedonic state that interferes with appetitive learning and food intake. In these experiments, rats received infusions of scopolamine methyl bromide (10 μg/0.5 μl) into the nucleus accumbens core, following exposure to a novel flavor of liquid diet (Experiment 1) or prior to being placed into a place preference apparatus (Experiment 2). In both experiments, nucleus accumbens muscarinic receptor antagonism caused subsequent avoidance of the paired cue (flavor or spatial location). This effect was specific to cholinergic manipulation; no conditioned taste avoidance was observed after pairing the novel flavor with nucleus accumbens core antagonism of N-methyl-D-aspartate, dopamine D?, or opioid receptors (Experiment 3). These experiments confirm previous reports of a critical role for striatal acetylcholine in modulating goal-directed behaviors, but suggest caution when interpreting behavioral effects of pharmacological manipulation of striatal acetylcholine. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT2 receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1-1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1-15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03-0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT2 receptors has additive effects in the suppression of thalamocortical oscillations in aged rats.