Pirenzepine, the prototype M1 muscarinic receptor antagonist, is an important compound for investigating the functional significance of M1 receptors at the integrated level of behavior but may have limitations imposed by its physical chemistry. Like the nonselective antagonist methylatropine, pirenzepine is highly hydrophilic and crosses the blood-brain-barrier with difficulty. We compared methylatropine with pirenzepine, given intraperitonealy, as antagonists of the behavioral effects of peripheral or central muscarinic activation. Lever-press responses of male Sprague-Dawley rats were maintained under a schedule requiring 10 responses for each food delivery. Administration of oxotremorine or the quaternary analog oxotremorine-M decreased rates of responding by at least 90%. Both methylatropine and pirenzepine antagonized the behavioral effects of oxotremorine-M; maximum reversal was 70%. Although methylatropine was about 30 times more potent than pirenzepine as an antagonist of the peripheral muscarinic activity of oxotremorine-M, it was inactive as an antagonist of oxotremorine when given in doses up to 153 mumol/kg. Pirenzepine, however, reversed oxotremorine-induced behavioral effects, with a maximum antagonism of 50%. These results suggest that pirenzepine interacts with central muscarinic receptors when administered systemically without producing marked behavioral effects of its own. Systemically administered pirenzepine may thus be a useful tool in further investigations of the relevance of M1 receptors to behavioral function.

Repetitive behaviors with restricted interests is one of the core criteria for the diagnosis of autism spectrum disorder (ASD). Current pharmacotherapies that target the dopaminergic or serotonergic systems have limited effectiveness in treating repetitive behaviors. Previous research has demonstrated that administration of muscarinic cholinergic receptor (mAChR) antagonists can exacerbate motor stereotypies while mAChR agonists reduce stereotypies. The present study determined whether the mAChR agonist, oxotremorine affected repetitive behaviors in the BTBR T+ tf/J (BTBR) mouse model of autism. To test the effects of oxotremorine on repetitive behaviors, marble burying and grooming behavior were measured in BTBR mice and compared to that in C57BL/6J (B6) mice. The effects of oxotremorine on locomotor activity was also measured. Thirty minutes before each test, mice received an intraperitoneal (ip) injection of saline, 0.001 mg or 0.01 mg of oxotremorine methiodide. Saline- treated BTBR mice exhibited increased marble burying and self-grooming behavior compared to that of saline-treated B6 mice. Oxotremorine significantly reduced marble burying and self-grooming behavior in BTBR mice, but had no significant effect in B6 mice. In addition, oxotremorine did not affect locomotor activity in BTBR mice, but significantly reduced locomotor activity in B6 mice at the 0.01 mg dose. These findings demonstrate that activation of mAChRs reduces repetitive behavior in the BTBR mouse and suggest that treatment with a mAChR agonist may be effective in reducing repetitive behaviors in ASD.

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aβ1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aβ1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aβ1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

Organophosphates (OPs) inhibit acetylcholinesterase (AChE) activity causing cholinergic stimulation in the central nervous system (CNS). Cholinergic systems are crucial in electroencephalogram (EEG) generation and regulation of behavior; however, little is known about how OP exposure affects the EEG and behavioral states. We recorded EEG, core temperature and motor activity before and after exposure to the OP pesticide chlorpyrifos (CHP) in adult female rats implanted with telemetric transmitters. The recording and reference electrodes were placed in the occipital and frontal bones, respectively. The animals received CHP, 25 mg/kg, p.o., or oxotremorine (OX), 0.2 mg/kg, s.c. CHP led to a significant increase in delta (0.1-3.5 Hz), slow theta (4-6.5 Hz), gamma 2 (35.5-50 Hz), reduction in fast theta (7-8.5 Hz), alpha/sigma (9-14 Hz), beta 1 (14.5-24 Hz), beta 2 (24.5-30 Hz) and gamma 1 (30.5-35 Hz) powers, slowing of peak frequencies in 1-9 Hz range, hypothermia and decrease in motor activity. The drop in 7-14 Hz was associated with cholinergic suppression of sleep spindles. Changes in behavioral state were characterized by dramatic diminution of sleep postures and exploring activity and prolongation of quiet waking. There was recovery in all bands in spite of continued inhibition of AChE activity [44,45] in rats exposed to CHP. OX-induced EEG and behavioral alterations were similar to CHP except there was no increase in delta and the onset and recovery were more rapid. We did not find a correlation between the EEG and core temperature alterations. Overall, changes in EEG (except in delta band) and behavior following CHP were attributable to muscarinic stimulation. Cortical arousal together with increased quiet waking and decreased sleep after CHP occurred independently from inhibition of motor activity and lowering of core temperature.

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) play essential roles in dopamine (DA) transmission in the striatum. It is suggested that a link exists between muscarinic acetylcholine receptors (mAChRs) and DA/DARPP-32 signaling, but the molecular mechanisms mediating this relationship have not been elucidated. The predominant mAChRs subtypes in the striatum are M1 and M4. In this study, we investigated the functions of these two receptors, particularly M4, in regulating cAMP production and DARPP-32 phosphorylation in rat striatal medium spiny neurons (MSNs). We used time-resolved fluorescence resonance energy transfer, immunofluorescence confocal microscopy, and western blot assays. In cultured intact MSNs, we confirmed that muscarinic M1 and M4 receptors were highly expressed. Notably, M4 receptors were co-expressed with D1 receptors in only a portion of the cultured MSNs. The nonselective muscarinic agonist oxotremorine M (OX) slightly enhanced cAMP production, but this effect was independent of M1 or M4 receptors. However, OX directly participated in DARPP-32 phosphorylation, phosphorylating DARPP-32 at Thr75 (the CDK5 site) and concomitantly de-phosphorylating DARPP-32 at Thr34 (the PKA site) in virtually cultured MSNs, whereas APO phosphorylated DARPP-32 at both Thr34 and Thr75. The OX-induced time-dependent increase in DARPP-32 phosphorylation at Thr75 was accompanied by increased p35 and CDK5 activity. Specifically, elevated immunoreactivity for phospho-DARPP-32-Thr75 and p35 was detected in M4 receptor-expressing MSNs. Both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3, an M4 receptor-selective antagonist, decreased the OX-induced DARPP-32-Thr75 phosphorylation in MSNs. These results indicate that the M4 muscarinic receptor plays a critical role in modulating phosphorylation of DARPP-32-Thr75 in MSNs. The results suggest that M4 receptor activation acts antagonistically with dopamine D1-like receptors within the striatum, and indicate that M4 receptors may be a potential target for the treatment of Parkinson's disease and other relevant central nervous system disorders.

The differentiation of neural progenitors is a complex process that integrates different signals to drive transcriptional changes, which mediate metabolic, electrophysiological, and morphological cellular specializations. Understanding these adjustments is essential within the framework of stem cell and cancer research and therapy. Human neuroblastoma SH-SY5Y cells, widely used in neurobiology research, can be differentiated into neuronal-like cells through serum deprivation and retinoic acid (RA) supplementation. In our study, we observed that the differentiation process triggers the expression of Heat Shock Protein 70 (HSP70). Notably, inhibition of HSP70 expression by KNK437 causes a dramatic increase in cell death. While undifferentiated SH-SY5Y cells show a dose-dependent decrease in cell survival following exposure to hydrogen peroxide (H2O2), differentiated cells become resistant to H2O2-induced cell death. Interestingly, the differentiation process enhances the expression of SOD1 protein, and inhibition of HSP70 expression counteracts this effect and increases the susceptibility of differentiated cells to H2O2-induced cell death, suggesting that the cascade HSP70-SOD1 is involved in promoting survival against oxidative stress-dependent damage. Treatment of differentiated SH-SY5Y cells with Oxotremorine-M (Oxo), a muscarinic acetylcholine receptor agonist, enhances the expression of HSP70 and SOD1 and counteracts tert-Butyl hydroperoxide-induced cell death and reactive oxygen species (ROS) generation. It is worth noting that co-treatment with KNK437 reduces SOD1 expression and Oxo-induced protection against oxidative stress damage, suggesting the involvement of HSP70/SOD1 signaling in this beneficial effect. In conclusion, our findings demonstrate that manipulation of the HSP70 signal modulates SH-SY5Y differentiation and susceptibility to oxidative stress-dependent cell death and unravels novel mechanisms involved in Oxo neuroprotective functions. Altogether these data provide novel insights into the mechanisms underlying neuronal differentiation and preservation under stress conditions.

1. Radioligand binding experiments indicate that the affinity of muscarinic receptors for their agonists may be enhanced by allosteric modulators. We have now investigated if brucine can enhance the inhibitory effects of muscarinic receptor agonists on the electrically evoked release of [3H]acetylcholine ([3H]ACh) from superfused slices of rat striatum. 2. The evoked release of [3H]ACh was inhibited by all agonists tested (i.e., furmethide, oxotremorine-M, bethanechol and oxotremorine). 3. Brucine enhanced the inhibitory effects of furmethide, oxotremorine-M and bethanechol on the evoked [3H]ACh release without altering the inhibitory effect of oxotremorine. 4. Alcuronium was applied for comparison and found to diminish the inhibitory effect of furmethide on the evoked [3H]ACh release. 5. The results demonstrate that it is possible both to enhance and diminish the functional effects of muscarinic receptor agonists by allosteric modulators. 6. The direction of the observed effects of brucine and alcuronium on [3H]ACh release fully agrees with the effects of these modulators on the affinities of human M4 receptors for furmethide, oxotremorine-M, bethanechol and oxotremorine, as described by Jakubik et al. (1997). This supports the view that the presynaptic muscarinic receptors responsible for the autoinhibition of ACh release in rat striatum belong to the M4 muscarinic receptor subtype.

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl(-) concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl(-) channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca(2+)-activated Cl(-) and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

Extensive evidence indicates that glucocorticoid hormones act in a variety of brain regions to enhance the consolidation of memory of emotionally motivated training experiences. We previously reported that corticosterone, the major glucocorticoid in the rat, administered into the dorsal striatum immediately after inhibitory avoidance training dose-dependently enhances memory consolidation of this training. There is also abundant evidence that the intrinsic cholinergic system of the dorsal striatum is importantly involved in memory consolidation of inhibitory avoidance training. However, it is presently unknown whether these two neuromodulatory systems interact within the dorsal striatum in the formation of long-term memory. To address this issue, we first investigated in male Wistar rats whether the muscarinic receptor agonist oxotremorine administered into the dorsal striatum immediately after inhibitory avoidance training enhances 48 h retention of the training. Subsequently, we examined whether an attenuation of glucocorticoid signaling by either a systemic administration of the corticosterone-synthesis inhibitor metyrapone or an intra-striatal infusion of the glucocorticoid receptor (GR) antagonist RU 38486 would block the memory enhancement induced by oxotremorine. Our findings indicate that oxotremorine dose-dependently enhanced 48 h retention latencies, but that the administration of either metyrapone or RU 38486 prevented the memory-enhancing effect of oxotremorine. In the last experiment, corticosterone was infused into the dorsal striatum together with the muscarinic receptor antagonist scopolamine immediately after inhibitory avoidance training. Scopolamine blocked the enhancing effect of corticosterone on 48 h retention performance. These findings indicate that there are mutual interactions between glucocorticoids and the striatal cholinergic system in enhancing the consolidation of memory of inhibitory avoidance training.

Here, we investigated intrinsic spinal cord mechanisms underlying the physiological requirement for autonomic and somatic motor system coupling. Using an in vitro spinal cord preparation from newborn rat, we demonstrate that the specific activation of muscarinic cholinergic receptors (mAchRs) (with oxotremorine) triggers a slow burst rhythm in thoracic spinal segments, thereby revealing a rhythmogenic capability in this cord region. Whereas axial motoneurons (MNs) were rhythmically activated during both locomotor activity and oxotremorine-induced bursting, intermediolateral sympathetic preganglionic neurons (IML SPNs) exhibited rhythmicity solely in the presence of oxotremorine. This somato-sympathetic synaptic drive shared by MNs and IML SPNs could both merge with and modulate the locomotor synaptic drive produced by the lumbar motor networks. This study thus sheds new light on the coupling between somatic and sympathetic systems and suggests that an intraspinal network that may be conditionally activated under propriospinal cholinergic control constitutes at least part of the synchronizing mechanism.

Kv12.1 K+ channels are expressed in several brain areas, but no physiological function could be attributed to these subunits so far. As genetically-modified animal models are not available, identification of native Kv12.1 currents must rely on characterization of distinct channel properties. Recently, it was shown in Xenopus laevis oocytes that Kv12.1 channels were modulated by membrane PI(4,5)P2. However, it is not known whether these channels are also sensitive to physiologically-relevant PI(4,5)P2 dynamics. We thus studied whether Kv12.1 channels were modulated by activation of phospholipase C β (PLCβ) and found that they were insensitive to receptor-triggered depletion of PI(4,5)P2. Thus, Kv12.1 channels add to the growing list of K+ channels that are insensitive to PLCβ signaling, although modulated by PI(4,5)P2 in Xenopus laevis oocytes.

We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg(-1)) 2-24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC(50) value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 microM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M(2) function is enhanced following streptozotocin treatment.

Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations, using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm-generating networks in an emerging genetically tractable locomotor system.NEW & NOTEWORTHY Using a combination of pharmacology, electrophysiology, and Ca2+ imaging, we find that signaling through mACh receptors plays a critical role in rhythmogenesis in different regions of the Drosophila larval CNS. mAChR-dependent rhythm generators reside in distal regions of the larval CNS and provide functional substrates for central pattern-generating networks (CPGs) underlying headsweep behavior and forward locomotion. This provides new insights into locomotor CPG operation in soft-bodied animals that navigate over terrain.

In the ocean, the crab Cancer borealis is subject to daily and seasonal temperature changes. Previous work, done in the presence of descending modulatory inputs, had shown that the pyloric rhythm of the crab increases in frequency as temperature increases but maintains its characteristic phase relationships until it "crashes" at extremely high temperatures. To study the interaction between neuromodulators and temperature perturbations, we studied the effects of temperature on preparations from which the descending modulatory inputs were removed. Under these conditions, the pyloric rhythm was destabilized. We then studied the effects of temperature on preparations in the presence of oxotremorine, proctolin, and serotonin. Oxotremorine and proctolin enhanced the robustness of the pyloric rhythm, whereas serotonin made the rhythm less robust. These experiments reveal considerable animal-to-animal diversity in their crash stability, consistent with the interpretation that cryptic differences in many cell and network parameters are revealed by extreme perturbations.

The intrathecal administration of pertussis toxin (PTX) not only blocks the antinociceptive effects of the muscarinic cholinergic receptor agonist oxotremorine administered systemically, but also produces a long-lasting thermal allodynia in mice. The purpose of the present studies was to determine both the antinociceptive effects in normal mice and the antiallodynic effects in PTX-treated mice of systemically administered muscarinic cholinergic receptor agonists and cholinesterase inhibitors. In normal mice, antinociceptive effects were tested using a 55 degrees C water-bath tail-flick test. In mice treated 7 days previously with PTX (0.3 microg i.t.), antiallodynic effects were tested using a 45 degrees C water-bath tail-flick test. The nonselective high-efficacy muscarinic agonists oxotremorine, H-TZTP (3-(1,2, 5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine oxalate), and methylthio[2.2.1], (exo (+)3-(3-methylthio-1,2, 5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane oxalate), as well as vedaclidine, a mixed M(2)/M(4) muscarinic receptor partial agonist and M(1)/M(3)/M(5) muscarinic receptor antagonist, the nonselective partial agonists RS86 and pilocarpine, and the cholinesterase inhibitors physostigmine and tacrine all produced dose-related antinociception. Oxotremorine, H-TZTP and methylthio[2.2.1] produced dose-related reversals of PTX-induced thermal allodynia whereas vedaclidine produced a partial reversal and RS86 and pilocarpine, as well as physostigmine and tacrine, failed to reverse the allodynia. The present results provide further evidence that decrements in PTX-sensitive G(i/o)-protein functioning may be involved in initiating and/or maintaining some persistent or neuropathic pain states. Moreover, the present results suggest that muscarinic receptor agonists such as vedaclidine may be useful in the treatment of persistent pain states that are due at least in part to dysfunction of inhibitory second messenger systems.

Disorders characterized by disturbed cholinergic signaling, such as schizophrenia, exhibit impaired performance on measures of real-world cost/benefit decision-making. Whether the cholinergic system contributes to the choice deficits observed is currently unknown. We therefore determined the effects of broad-acting agonists and antagonists at the nicotinic and muscarinic receptor on decision making, as measured by the rodent gambling task (rGT). Given the anatomical and functional connectivity of the cholinergic and dopaminergic systems, we also sought to modulate amphetamine's previously reported effect on rGT performance via the cholinergic system. Male rats were trained on the rGT, during which animals chose from four different options. The optimal strategy on the rGT is to favor options associated with smaller immediate rewards and less punishment/loss. Impulsive action was also measured by recording the number of premature responses made. Performance on the rGT was assessed following acute treatment with the muscarinic receptor agonist oxotremorine, the muscarinic receptor antagonist scopolamine, nicotine, and the nicotinic receptor antagonist mecamylamine. Similar to the effect produced by amphetamine, muscarinic receptor antagonism with scopolamine (0.1mg/kg) impaired decision making, albeit to a lesser degree. Prior muscarinic agonism with oxotremorine was unable to attenuate amphetamine's effects on rGT performance. Oxotremorine, nicotine, and mecamylamine did not affect the choice profile. We therefore conclude that modulation of the muscarinic, but not nicotinic, receptor system can affect decision making under conditions of risk and uncertainty. Such findings contribute to a broader understanding of the cognitive deficits observed in disorders in which cholinergic signaling is compromised.

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of β-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic β cells.

1. The modulation of high-voltage-activated (HVA) Ca2+ channels by acetylcholine (ACh) was studied in the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats by use of the nystatin perforated patch recording configuration under voltage-clamp conditions. 2. ACh inhibited the HVA Ca2+ currents in a concentration- and voltage-dependent manner. 3. The inhibition was mimicked by a muscarinic agonist, oxotremorine. Pirenzepine and methoctramine produced parallel shifts to the right in the ACh concentration-response curves. Schild analysis of the ACh concentration-ratios yield pA2 values for pirenzepine and methoctramine of 6.85 and 8.57, respectively, suggesting the involvement of an M2 receptor. 4. Nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC reduced the HVA I(Ca) by 16.8, 59.2 and 6.3%, respectively. A current insensitive to all of these Ca2+ antagonists, namely 'R-type', was also observed. The results indicated the existence of L-, N-, P/Q-, and R-type Ca2+ channels. 5. The ACh-sensitive current component was markedly reduced in the presence of omega-conotoxin-GVIA, but not with both nifedipine and omega-conotoxin-MVIIC. ACh also inhibited the R-type HVA I(Ca) remaining in saturating concentrations of nifedipine, omega-conotoxin-GVIA and omega-conotoxin-MVIIC. 6. The inhibitory effect of ACh was prevented by pretreatment with pertussis toxin. 7. It was concluded that ACh selectively reduces both the N- and R-type Ca2+ channels, by activating pertussis toxin sensitive G-protein through the M2 muscarinic receptor in paratracheal ganglion cells.