In previous work, we have shown that light-adaptive pigment granule dispersion can be induced in vitro by treating retinal pigment epithelium (RPE) isolated from bluegill retina with acetylcholine or its analog carbachol and that these agents act through muscarinic receptors to induce pigment granule dispersion. RPE is a monolayer of tissue found between the neural retina and the choroid. In fish, RPE has long apical projections enmeshed with the distal part of photoreceptors, reaching down to the level of their nuclei. The RPE disperses melanin pigment granules into the apical projections to shield light-sensitive photoreceptor outer segments from photobleaching when fish are under bright-light conditions. During development, RPE begin to respond to light at 5days post-fertilization, raising the question of whether responsiveness is correlated to receptor expression. Here, we isolate, clone and sequence chrm-odd receptor genes in zebrafish, characterize them phylogenetically and observe their expression in the eyes of the zebrafish at different developmental stages using RT-PCR and immunofluorescence microscopy. We find that zebrafish express six unique chrm-odd receptor subtypes: chrm1a, chrm1b, chrm3a, chrm3b, chrm5a and chrm5b - and these receptors are differentially expressed during development. Our phylogenetic analysis confirms the assignments of chrm1b and chrm5b, isolated here, as well as other muscarinic receptor genes and their duplicates and suggests previously described muscarinic receptors may need to be reclassified. Differences between the expression patterns of ostensibly duplicated genes raise the possibility that subtle differences between the duplicates may enable refined regulation of specific developmental events.

The present experiments determined whether temporary inactivation or blockade of muscarinic cholinergic receptors in the dorsomedial striatum affects acquisition or reversal learning of a response discrimination. Testing occurred in a modified cross-maze across two consecutive sessions. In the acquisition phase, a rat learned to make a turn to the left or to the right for 10 consecutive correct choices. In the reversal learning phase, a rat learned to turn in the opposite direction as required during acquisition for 10 consecutive correct choices. Experiment 1 investigated the effects of the local anesthetic, 2% bupivacaine, infused into the dorsomedial striatum on acquisition and reversal learning. Experiment 2 examined the effects of the muscarinic cholinergic antagonist, scopolamine injected into the dorsomedial striatum on acquisition and reversal learning. Bupivacaine infusions did not impair acquisition, but did impair reversal learning of the response discrimination. Analysis of the errors indicated that the deficit was not due to perseveration of the previously learned strategy, but to an inability to learn the new strategy. Bilateral injections of scopolamine, 1 or 8 microg/side, did not affect acquisition. Infusions of scopolamine at 8 microg, but not 1 microg, produced a reversal learning deficit. The scopolamine-induced deficit resulted from an inability to learn the new strategy. The results suggest that the dorsomedial striatum is important for behavioral flexibility and that activation of muscarinic cholinergic receptors in this region may facilitate the learning of situationally adaptive response patterns.

Carbachol, a non-specific cholinergic agonist, when administered intraventricularly or directly into the suprachiasmatic nucleus (SCN), causes phase-dependent shifts in circadian rhythms of wheel-running activity in rodents. The cholinergic receptor subtype involved in mediating these carbachol-induced phase shifts, however, remains uncertain. In order to investigate this issue we injected carbachol into the SCN through indwelling cannulas at circadian times (CT) 6, 14 and 22 in Syrian hamsters (Mesocricetus auratus) maintained in constant darkness. Carbachol elicited large phase advances at CT 6 (69.8 +/- 15.7 min; mean +/- S.E.M.) and CT 22 (83.9 +/- 24.8 min) and phase delays at CT 14 (59.7 +/- 18 min). We attempted to block the carbachol-induced phase shifts at these three phases using specific antagonists of nicotinic and muscarinic receptors. Mecamylamine, a nicotinic receptor antagonist, did not block carbachol-induced phase shifts at any of the phases tested. Atropine, a muscarinic receptor antagonist, blocked carbachol-induced phase shifts at CT 6 (-11.6 +/- 4.8 min; Mean phase shift +/- S.E.M.) and CT 22 (-20 +/- 6.6 min), suggesting that carbachol mediates its phase-shifting effects at these phases through muscarinic receptors.

Functional and pharmacological studies have suggested that there are muscarinic receptors (mAChRs) on the endothelial cells of major cerebral arteries, while recent immunological studies indicate that there are no mAChRs on the endothelium of brain capillaries. This difference may be because the distribution of mAChR on the endothelium varies with the type of vessel. This paper examines the distribution of mAChR on the vascular endothelium along intraparenchymal blood vessels in the rat brain by immunolabelling and laser confocal microscopy. Sections were immunostained by combinations of an anti-mAChR antibody (M35) with antibodies to endothelial (anti-GLUT1), or to smooth muscle markers (anti-actin). Antibody labellings were detected with fluorescent second antibodies. Most of the penetrating vessels bore mAChR immunolabelling which coincided over almost all the vessel surface with endothelial labelling. The mAChR immunolabelling was less widespread over the endothelium on the medium sized vessels (diameter < 50 microm) and only 50% of these vessels had mAChR staining on the endothelium. There was no mAChR immunostaining on the endothelium of the capillaries. In contrast with the basilar artery, there was no mAChR immunolabelling on the smooth muscle layer of the intracortical vessels. These data indicate that the intensity of mAChR immunolabelling decreases along the vascular tree from large conducting vessels to capillaries.

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

The mitogen-activated protein kinase (MAPK), especially its extracellular signal-regulated kinase (ERK) subfamily, is a group of kinases enriched in the mammalian brain. While ERK is central to cell signaling and neural activities, the regulation of ERK by transmitters is poorly understood. In this study, the role of acetylcholine in the regulation of ERK was investigated in adult rat striatum in vivo. We focused on muscarinic M1 and M4 receptors, two principal muscarinic acetylcholine (mACh) receptor subtypes in the striatum. A systemic injection of the M1-preferring antagonist telenzepine did not alter ERK phosphorylation in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. Similarly, telenzepine did not affect ERK phosphorylation in the medial prefrontal cortex (mPFC), hippocampus, and cerebellum. Moreover, telenzepine had no effect on the ERK phosphorylation induced by dopamine stimulation with the psychostimulant amphetamine. In contrast to telenzepine, the M4-preferring antagonist tropicamide consistently increased ERK phosphorylation in the striatum and mPFC. This increase was rapid and transient. Tropicamide and amphetamine when coadministered at subthreshold doses induced a significant increase in ERK phosphorylation. These results demonstrate that mACh receptors exert a subtype-specific modulation of ERK in striatal and mPFC neurons. While the M1 receptor antagonist has no effect on ERK phosphorylation, M4 receptors inhibit constitutive and dopamine-stimulated ERK phosphorylation in these dopamine-innervated brain regions.

Here we studied muscarinic receptors in the gerbil thalamus at 8 different ages - from 6 to 36 months - using receptor and functional autoradiography. The pharmacological profile inhibiting [(3)H]N-methyl scopolamine ([(3)H]NMS) binding with 50 and 200 nM pirenzepine, 30 nM pFHHSiD and 100 nM AF-DX 116 revealed the predominance of the M(2) muscarinic subtype in the thalamic nuclei studied, mainly in the anteroventral, anteromedial and paraventricular thalamic nuclei. These data correlated with the highest [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTP gamma S) binding induced in these nuclei by the muscarinic agonist oxotremorine in functional autoradiographic assays. Significant aging-dependent increases in the functional response in these three nuclei were observed, but only the anteroventral and anteromedial thalamic nuclei showed aging-dependent increases in [(3)H]NMS binding. Since these nuclei exert relevant functions, in which cholinergic pathways are involved and acetylcholine release is reported to decrease during aging, we suggest that the anteroventral and anteromedial thalamic nuclei would play critical roles in the cholinergic transmission that require compensatory mechanisms during the aging process and that are not observed in other thalamic nuclei.

In a working memory task with three-panel runway paradigm, cysteamine, a depletor of somatostatin, at 100 or 200 mg/kg i.p. given 24 h before testing, had no effect on the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). Cysteamine at 100 mg/kg caused a significant reduction in somatostatin-like immunoreactivity in the rat brain, including the hippocampus and cerebral cortex. Working memory errors were significantly increased by scopolamine, a muscarinic receptor antagonist, at 0.32 mg/kg i.p. given 20 min before testing, whereas errors were not affected by the 0.1 mg/kg dose. Combined administration of 100 mg/kg cysteamine and 0.1 mg/kg scopolamine significantly increased the number of working memory errors. However, cysteamine at 100 mg/kg and scopolamine at 0.1 mg/kg had no effect on reference memory errors, whether they were administered alone or in combination. These results suggest that depletion of brain somatostatin aggravates working memory deficits induced by blockade of muscarinic receptors.

Neuropeptide Y (NPY) participates in the control of several functions in the nervous system. NPYergic neurons present in brain areas involved in cognitive processes are linked to ascending projections of the cholinergic system, a finding that suggests a role for acetylcholine in the control of these cells. In the present study, the effect of the activation of cholinergic muscarinic receptors on the expression of the human NPY gene was assessed. The SH-SY5Y neuroblastoma cell line was used as an in vitro model of human neurons; NPY mRNA levels were evaluated by Northern blot analysis. The results indicate that: (a) the expression of the human NPY gene in SH-SY5Y cells is stimulated by the cholinergic muscarinic agonist, carbachol; (b) this effect is mediated by the M3 muscarinic receptor subtype, as indicated by the inhibitory effect of the M3 antagonist 4-DAMP; (c) protein kinase C (PKC) activation plays an important role in the induction of NPY gene expression in this system, as suggested by experiments with the PKC activator, TPA, and the PKC inhibitor, GF 109203X; (d) the stimulation of NPY mRNA levels by TPA and by carbachol in SH-SY5Y cells requires de novo synthesis of RNA and protein. In conclusion, the present study shows that the activation of PKC-coupled muscarinic receptors as the M3 subtype positively modulates the expression of the human NPY gene in SH-SY5Y neuroblastoma cells, via PKC-related mechanisms.

Activation of muscarinic m1 receptors which are coupled to the phosphoinositide (PI) second messenger transduction system is the initial objective of cholinergic replacement therapy in Alzheimer's disease. Thus, we evaluated the ability of the selective muscarinic receptor agonist (SMRA) xanomeline to stimulate in vivo phosphoinositide (PI) hydrolysis and compared it to a number of direct acting muscarinic agonists, two cholinesterase inhibitors and a putative m1 agonist/muscarinic m2 antagonist. Using a radiometric technique, it was determined that administration of xanomeline robustly stimulated in vivo PI hydrolysis and the effect was blocked by muscarinic antagonists, demonstrating mediation by muscarinic receptors. The non-selective muscarinic agonists pilocarpine, oxotremorine, RS-86, S-aceclidine, but not the less active isomer R-aceclidine, also effectively stimulated PI hydrolysis in mice. Amongst the putative m1 agonists, thiopilocarpine, hexylthio-TZTP as well as xanomeline effectively stimulated PI hydrolysis, but milameline, WAL 2014, SKB 202026 and PD 142505 did not significantly alter PI hydrolysis. Furthermore, WAL 2014 and SKB 202026 inhibited agonist-induced PI stimulation, suggesting that they act as antagonists at PI-coupled receptors in vivo. The cholinesterase inhibitors, tacrine and physostigmine, and the mixed muscarinic m1 agonist/m2 antagonist LU25-109 did not activate in vivo PI hydrolysis. Xanomeline, hexylthio-TZTP and thiopilocarpine were relatively free of cholinergic side effects, whereas milameline, WAL 2014 and SKB 202026 produced non-selective effects. Therefore, these data demonstrate that xanomeline selectively activates in vivo PI hydrolysis, consistent with activation of biochemical processes involved in memory and cognition and xanomeline's beneficial clinical effects on cognition in Alzheimers patients.

Hippocampal levels of fatty acids extracted from phosphatidylinositol 4,5-bisphosphate (PIP2), free fatty acids (FFA), and lactate were measured after central fluid percussion traumatic brain injury (TBI) in rats. At 5 min after injury, there was a decrease in fatty acids extracted from PIP2 suggesting a decrease in PIP2. At the same time point, total FFA increased in saline-treated TBI rats. Levels of arachidonic acid were significantly decreased in PIP2, while at the same time arachidonic and stearic acids increased in FFA in saline-treated TBI rats. No significant alterations in PIP2-derived fatty acids or FFA were observed at 20 min after TBI. Hippocampal concentrations of lactate were significantly elevated at 5 and 20 min after injury in saline-treated rats. In general, these alterations were blunted by preinjury administration of the muscarinic antagonist, scopolamine. These results suggest that the PIP2 signal transduction pathway is activated in the hippocampus at the onset of central fluid percussion TBI and that the enhanced phospholipase C-catalyzed phosphodiestric breakdown of PIP2 is a major mechanism of liberation of FFA in these sites immediately after such injury. The blunting of PIP2 and FFA alterations in animals treated with scopolamine suggests that activation of muscarinic receptors significantly contributes to the phospholipase C (PLC) signal transduction pathophysiology in TBI. The attenuation of lactate accumulation in scopolamine-treated rats suggests that TBI-induced muscarinic receptor activation also contributes to increased glycolytic metabolism and/or ionic imbalances.

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [3H]pirenzepine or [3H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [3H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [3H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [3H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.

A significant body of evidence suggests that the development and maintenance of elevated blood pressure in the spontaneously hypertensive rat (SHR), a genetic model for essential hypertension, is due at least partly to a central hyper-cholinergic state. For example, this strain responds with an exaggerated pressor response to pharmacological stimulation of central muscarinic receptors in certain brain regions compared to normotensive Wistar Kyoto rats (WKY). At least part of the enhanced response to central muscarinic receptor stimulation in SHR is due to the altered expression of post-synaptic receptors. In the present study, the reverse transcriptase-polymerase chain reaction and autoradiographic techniques were used to estimate the relative levels of mRNA and density of receptor binding sites for the five subtypes of muscarinic receptors within the rostral ventrolateral medulla (RVL) of SHR and WKY. Adult (12-week-old) SHR exhibited an increase in the levels of both M2 muscarinic mRNA, and M2 receptor binding sites in RVL compared to age-matched normotensive WKY. Similarly, 4-week-old pre-hypertensive SHR exhibited increased levels of M2 mRNA in whole medulla oblongata, and an increase in the number of binding sites for M2 receptors in the RVL. Since the RVL is known to integrate tonic cholinergic sympathoexcitatory input, these results suggest that the increased expression of M2 muscarinic receptors in this region represents one neurochemical correlate for the maintenance of excessive central efferent sympathetic nervous activity in the SHR. Since the neurochemical change precedes the development of hypertension, the altered medullary M2 receptor expression may play a role as an initiating or predisposing factor for the development of hypertension in SHR.

A recently introduced technique of [gamma-35S]GTP autoradiography was used to localize and characterize muscarinic receptor-dependent activation of G-proteins in tissue sections of the chick optic tectum, a brain region with relatively high expression of G-protein-coupled receptors for the neurotransmitter acetylcholine. Within the highly stratified tectal structure, the bulk of muscarinic receptor-mediated [gamma-35S]GTP signal was localized to the stratum griseum et fibrosum superficiale with considerably lower binding responses in other tectal layers. Quantitative comparison of [gamma-35S]GTP binding responses in tectal sections and membranes revealed a close match between the two tissue preparations for the response elicited by the cholinergic agonist carbachol, its dose-dependent reversal with the non-selective muscarinic antagonist atropine, its approximately 100-fold sensitivity towards blockade with M1-type (pirenzepine) over M2-type (gallamine) muscarinic antagonists, as well as absolute requirement for micromolar concentrations of GDP (EC50 approximately 10 microM) for the receptor-mediated [gamma-35S]GTP response. The pharmacological profile is consistent with that of cm4, a recently cloned chicken homolog of the mammalian m4 muscarinic acetylcholine receptor. Moreover, the strict GDP-dependence of the binding response suggest activation of Gi/o, the inhibitory class of G-proteins. These data provide the first functional characterization of the chick tectal muscarinic receptors. A close match between [gamma-35S]GTP responses in membranes and tissue sections strongly suggest that [gamma-35S]GTP autoradiography offers great potential for studies on G-protein-mediated signaling, with particular use within anatomically restricted regions that are not readily approached with more conventional techniques. It is anticipated that [gamma-35S]GTP autoradiography should greatly facilitate studies on signaling capacity of an individual receptor subtype whilst in its native cellular environment.

Cholinergic modulation of spontaneous GABAergic currents (mIPSC) was studied using whole-cell patch methods in mouse accessory olfactory bulb slices. Carbachol (above 100 microM) administration produced an increase in the mIPSC frequency in mitral cells, but did not affect the responses of mitral cells to GABA. The carbachol effect persisted in the presence of combined ionotropic and metabotropic glutamatergic receptor antagonists. The carbachol effect was reduced by the muscarinic receptor type-1 and -4 (M1 and M4) antagonist pirenzepine (10 microM), but not by the M2 and M4 antagonist himbacine (10 microM). The KCNQ/Kv7 potassium channel openers retigabine (80 microM) and diclofenac (300 microM) blocked the carbachol action, while the KCNQ potassium channel blocker XE-911 (20 microM) increased the mIPSC frequency. XE-911's action persisted in the presence of glutamate receptor blockers. In the presence of carbachol, mIPSCs were abolished by Ni (200 microM), while being insensitive to the calcium channel blocker nimodipine (30 microM), suggesting a role for R-type calcium channels in the GABA release. These results suggest that carbachol closed KCNQ channels by stimulating M1 receptors on granule cell dendrites, and the resulting depolarized and unstable membrane promoted calcium influx, thus increasing the GABA release. The possible role of acetylcholine in facilitating formation of a pheromone memory in mice is also discussed.

It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep.

Treatment of intact rats with the full D(1) dopamine agonist A-77636 induced Fos-like immunoreactivity in the medial and, to a lesser extent, the lateral portions of the striatum. Pretreatment with the muscarinic antagonist scopolamine hydrobromide (1.5-6 mg/kg) potentiated the response to A-77636 and eliminated the mediolateral staining gradient seen after A-77636 alone. Similar effects were not produced by scopolamine methylbromide, which fails to cross the blood-brain barrier, demonstrating that the actions of scopolamine were centrally mediated. The effects of scopolamine were further compared to those of the D(2)-like dopamine agonist quinpirole using a factorial design in which subjects were pretreated with either scopolamine, quinpirole, or a combination of the two drugs before receiving A-77636. Pretreatment with either scopolamine or quinpirole increased staining in the lateral striatum, but the combination of the two drugs was no more effective than was quinpirole alone. Pretreatment with quinpirole, but not scopolamine, resulted in a markedly "patchy" pattern of staining and actually suppressed staining in the region between patches in the medial striatum. These findings demonstrate that there are both differences and similarities between the effects of scopolamine and quinpirole on D(1) agonist-induced Fos expression and suggest that although inhibition of cholinergic neurons may be one of the mechanisms through which the effects of quinpirole are produced, other factors must also contribute.

Activation of muscarinic receptors located in bladder sensory pathways is generally considered to be the primary contributor for driving the pathogenesis of neurogenic detrusor overactivity following spinal cord injury. The present study is undertaken to examine whether moxibustion improves neurogenic detrusor overactivity via modulating the abnormal muscarinic receptor pathway.