Non-selective Muscarinic Agonist Research Articles

Effects of oxotremorine on convulsions in mice induced by scopolamine and food intake after fasting.

Antimuscarinic administration and food intake cause convulsions in mice and rats after fasting for 48h or less. Increased M1 and M2 muscarinic receptor expression in brain regions during fasting, and reversal of changes by refeeding may contribute to these seizures. Since receptor expression is regulated in response to agonist stimulation, this study investigated effects of nonselective muscarinic receptor agonist oxotremorine on convulsions in fasted animals. Mice deprived of food for 24h were given oxotremorine during (0.1mg/kg, twice daily, s.c.) or after (0.05 or 0.1mg/kg, i.p.) fasting. Fasted animals were treated with saline or scopolamine (i.p.) and observed for 30min for the convulsions after being allowed to eat ad libitum. Oxotremorine administration during fasting produced no significant effect on convulsion development. Incidence and onset of convulsions, and seizure stages were indifferent between the scopolamine and oxotremorine - scopolamine groups. However, oxotremorine (0.1mg/kg) administration after fasting reduced incidence of convulsions. Resulting from an agonist-antagonist interaction at M1 and/or M2 muscarinic receptors, oxotremorine administered after fasting exhibited an anticonvulsant activity. Oxotremorine administration during fasting was expected to suppress seizure development via inhibition of receptor expression. Results did not confirm this expectation and suggested that muscarinic receptor expression was either not affected or not related to the convulsions. Food intake after fasting, and food deprivation have been shown to induce cholinergic hyperexcitability. Although contrary to our hypothesis, future research may investigate whether increased expression of muscarinic receptors mediate or contribute to an increase in cholinergic activity.

Muscarinic receptor activation preferentially inhibits rebound in vulnerable dopaminergic neurons.

Dopaminergic subpopulations of the substantia nigra pars compacta (SNc) differentially degenerate in Parkinson's disease and are characterized by unique electrophysiological properties. The vulnerable population expresses a T-type calcium channel-mediated afterdepolarization (ADP) and shows rebound activity upon release from inhibition, whereas the resilient population does not have an ADP and is slower to fire after hyperpolarization. This rebound activity can trigger dopamine release in the striatum, an important component of basal ganglia function. Using whole-cell patch clamp electrophysiology on ex vivo slices from adult mice of both sexes, we find that muscarinic activation with the non-selective muscarinic agonist Oxotremorine inhibits rebound activity more strongly in vulnerable vs resilient SNc neurons. Here, we show that this effect depends on the direct activation of muscarinic receptors on the SNc dopaminergic neurons. Through a series of pharmacological and transgenic knock-out experiments, we tested whether the muscarinic inhibition of rebound was mediated through the canonical rebound-related ion channels: T-type calcium channels, hyperpolarization-activated cation channels (HCN), and A-type potassium channels. We find that muscarinic receptor activation inhibits HCN-mediated current (Ih) in vulnerable SNc neurons, but that Ih activity is not necessary for the muscarinic inhibition of rebound activity. Similarly, we find that Oxotremorine inhibits rebound activity independently of T-type calcium channels and A-type potassium channels. Together these findings reveal new principles governing acetylcholine and dopamine interactions, showing that muscarinic receptors directly affect SNc rebound activity in the midbrain at the somatodendritic level and differentially modify information processing in distinct SNc subpopulations.

Muscarinic Modulation of Synaptic Transmission and Short-Term Plasticity in the Dorsal and Ventral Hippocampus

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Development and characterization of niosomes loaded mucoadhesive biodegradable ocular inserts for extended release of pilocarpine HCl

Abstract Background Pilocarpine HCl is a non-selective muscarinic receptor agonist that is prescribed for the treatment of glaucoma. The use of pilocarpine conventional eye drops is associated with several side effects, such as loss of visual acuity, and the need for several applications due to rapid drainage away via the nasolacrimal duct, especially for elderly people. Such adverse effects can lead to low patient compliance and poor clinical outcomes. Therefore, the aim of this project was to develop, optimise and characterise a biodegradable pilocarpine HCl ocular insert using niosomes as a drug delivery vehicle. To achieve that, various polymers such as hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), and a blend of both were investigated to prepare the ocular inserts using solvent casting technique. The niosomes of pilocarpine HCl were prepared using span-60 and cholesterol by thin film hydration method. The produced noisome-loaded ocular inserts were characterised using various analytical techniques, including Fourier Transform Infrared (FTIR), X-ray Diffractions (XRD), thermal analysis, particle size analysis, weight and content uniformity, surface pH and drug release profile, among others. Results The results indicated that drug-free ocular inserts of the two polymers (HPMC + PVA) were better than single polymer-based ocular inserts (HPMC or PVA alone). The formed niosomes demonstrated good entrapment efficiency of 49.7% ± 7.0, with an average particle size of 325.7 ± 3.5 nm. The FTIR analysis showed no interaction between the compositions of niosomes. Four optimal formulations with various co-polymer ratios and pilocarpine content were further evaluated. Pilocarpine-containing niosomes-loaded ocular inserts provided uniformity in pilocarpine content (89–96%), with 34.8% moisture content and an average pH of 7. The release profile of niosomes-loaded inserts demonstrated an initial burst release within 2 h ranging from 26.54% (T4) to 41.22% (T2), and continuous sustained release for the next 24 h (68.32 ± 5.11% (T4) to 82.11 ± 6.01% (T2)). Conclusions This work successfully optimised biodegradable ocular inserts containing slow-release pilocarpine HCl encapsulated in niosomes for the treatment of glaucoma without dose dumping, resulting in a user-friendly drug delivery system. Graphical abstract

6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease.

Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions. This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

Hypotonicity-Induced Increase in Duodenal Mucosal Permeability Is Regulated by Cholinergic Receptors in Rats

BackgroundThe role of cholinergic receptors in the regulation of duodenal mucosal permeability in vivo is currently not fully described.AimsTo elucidate the impact of nicotinic and muscarinic acetylcholine receptor signaling in response to luminal hypotonicity (50 mM NaCl) in the proximal small intestine of rat.MethodsThe effect on duodenal blood-to-lumen clearance of 51Cr-EDTA (i.e., mucosal permeability) and motility was studied in the absence and presence of nicotinic and muscarinic receptor agonists and antagonists, a sodium channel blocker (tetrodotoxin), and after bilateral cervical vagotomy.ResultsRats with duodenal contractions responded to luminal hypotonicity by substantial increase in intestinal permeability. This response was absent in animals given a non-selective nicotinic receptor antagonist (mecamylamine) or agonist (epibatidine). Pretreatment with tetrodotoxin reduced the increase in mucosal permeability in response to luminal hypotonicity. Further, the non-selective muscarinic receptor antagonist (atropine) and agonist (bethanechol) reduced the hypotonicity-induced increase in mucosal permeability, while vagotomy was without an effect, suggesting that local enteric reflexes dominate. Finally, neither stimulating nor blocking the α7-nicotinic receptor had any significant effects on duodenal permeability in response to luminal hypotonicity, suggesting that this receptor is not involved in regulation of duodenal permeability. The effect of the different drugs on mucosal permeability was similar to the effect observed for duodenal motility.ConclusionsA complex enteric intramural excitatory neural reflex involving both nicotinic and muscarinic receptor subtypes mediates an increase in mucosal permeability induced by luminal hypotonicity.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease

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.Graphical 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.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Detection of pentylenetetrazol-induced seizure activity in the 19-21 Hz beta range using a magnetic coil induction method.

A force transducer or automatic scoring system is not sufficient to detect small or fine seizure activity. To improve previous assessments of epileptic behavior, a novel coil method was developed to detect an early behavioral marker for epileptic seizures. The present study used the γ-aminobutyric acid (GABA) receptor antagonist pentylenetetrazol (PTZ) to induce seizure activity and epileptic behavior in mice. A coil method was used to detect motor seizures consisting of small amplitude 19-21 Hz muscle contractions. Seizure activity in the 19-21 Hz range detected by the coil method was positively correlated with generalized clonic seizures with a kangaroo posture after PTZ administration. GABA receptor agonist valproic acid and ethosuximide decreased PTZ-induced 19-21 Hz seizure activity. The pattern of the amplitude ratio (%) of 19-21 Hz seizure activity after administration of the GABAA/C receptor antagonist picrotoxin was similar to the group that was treated with PTZ but different from the group that was treated with the nonselective muscarinic receptor agonist pilocarpine. The coil method detected 19-21 Hz seizure activity after PTZ administration. However, the force transducer method did not detect 19-21 Hz seizure activity. The coil method was more sensitive than the force transducer method for detecting epileptic behaviors. The findings may indicate a novel behavioral marker that can be detected by the coil method to reveal epileptic seizures, thus improving our understanding of the brain mechanisms of action and specific brain waves that are associated with PTZ-induced 19-21 Hz seizure activity.

Novel Insights Into Muscarinic and Purinergic Responses in Primary Cultures of Rat Lacrimal Gland Myoepithelial Cells.

PurposeThe functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally.MethodsRat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1–M5) and examined functionally regarding intracellular [Ca2+] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied.ResultsExpression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10−11–10−3 M) did not cause a significant increase in intracellular [Ca2+]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10−8–10−3 M) caused a concentration-dependent increase in intracellular [Ca2+] that could be blocked by the P2 antagonists PPADS and suramin.ConclusionsPrimary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca2+] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.

Acute Inhibitory Effects of Antidepressants on Lacrimal Gland Secretion in the Anesthetized Rat.

PurposePatients that medicate with antidepressants commonly report dryness of eyes. The cause is often attributed to the anticholinergic properties of the drugs. However, regulation of tear production includes a substantial reflex-evoked component and is regulated via distinct centers in the brain. Further, the anticholinergic component varies greatly among antidepressants with different mechanisms of action. In the current study it was wondered if acute administration of antidepressants can disturb production of tears by affecting the afferent and/or central pathway.MethodsTear production was examined in vivo in anesthetized rats in the presence or absence of the tricyclic antidepressant (TCA) clomipramine or the selective serotonin reuptake inhibitor (SSRI) escitalopram. The reflex-evoked production of tears was measured by challenging the surface of the eye with menthol (0.1 mM) and cholinergic regulation was examined by intravenous injection with the nonselective muscarinic agonist methacholine (1–5 µg/kg).ResultsAcute administration of clomipramine significantly attenuated both reflex-evoked and methacholine-induced tear production. However, escitalopram only attenuated reflex-evoked tear production, while methacholine-induced production of tears remained unaffected.ConclusionsThis study shows that antidepressants with different mechanisms of action can impair tear production by attenuating reflex-evoked signaling. Further, antimuscarinic actions are verified as a likely cause of lacrimal gland hyposecretion in regard to clomipramine but not escitalopram. Future studies on antidepressants with different selectivity profiles and mechanisms of action are required to further elucidate the mechanisms by which antidepressants affect tear production.

Additive effects of intravenous and intravesical application of vibegron, a β3-adrenoceptor agonist, on bladder function in rats with bladder overactivity.

To examine the effects of intravenous and intravesical application of vibegron, a new β3-adrenoceptor (β3-AR) agonist, on bladder function in rats with oxotremorine methiodide (oxo-M: a nonselective muscarinic receptor agonist)-induced bladder overactivity. Cystometry was performed in conscious female rats with intravesical instillation of oxo-M (200μM). In oxo-M-treated rats, vehicle or vibegron (1 and 10mg/kg) was cumulatively applied intravenously at 30-min intervals. In other groups of rats, oxo-M + vehicle or oxo-M + vibegron (10, 100μM, and 1mM) was cumulatively instilled intravesically at 60-min intervals followed by intravenous application of vibegron (10mg/kg). Expression of β3-ARs in the bladder was also evaluated using immunohistochemical staining. Intravenous application of vibegron (10mg/kg) significantly increased bladder capacity (1.3 times) and decreased baseline, threshold, and maximal voiding pressure compared with vehicle. Next, intravesical application of vibegron (1mM) significantly increased threshold pressure and bladder capacity (1.2 times) compared with vehicle. Combined treatments of intravesical (1mM) and intravenous (10mg/kg) application of vibegron induced a significantly larger degree of increases in bladder capacity (1.4 times) compared with vehicle. In addition, β3-ARs were expressed throughout the rat bladder, mainly in the urothelium. These results suggest that vibegron excreted in urine as an unchanged compound can induce the additive inhibitory effects on bladder overactivity possibly through urothelial β3-AR activation, which inhibits the afferent limb of micturition reflex rather than the efferent function as evidenced by the increases in threshold pressure and bladder capacity without affecting bladder contractile function after intravesical vibegron application.

PD24-02 ADDITIVE EFFECTS OF INTRAVENOUS AND INTRAVESICAL APPLICATION OF VIBEGRON, A β 3 -ADRENOCEPTOR AGONIST, ON BLADDER FUNCTION IN RATS WITH BLADDER OVERACTIVITY

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II (PD24)1 Apr 2020PD24-02 ADDITIVE EFFECTS OF INTRAVENOUS AND INTRAVESICAL APPLICATION OF VIBEGRON, A β3-ADRENOCEPTOR AGONIST, ON BLADDER FUNCTION IN RATS WITH BLADDER OVERACTIVITY Akira Furuta*, Yasuyuki Suzuki, Taro Igarashi, Yusuke Koike, Shin Egawa, and Naoki Yoshimura Akira Furuta*Akira Furuta* More articles by this author , Yasuyuki SuzukiYasuyuki Suzuki More articles by this author , Taro IgarashiTaro Igarashi More articles by this author , Yusuke KoikeYusuke Koike More articles by this author , Shin EgawaShin Egawa More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000881.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Approximately 20% of oral intake of vibegron, a new β3-adrenoceptor agonist, is excreted into urine in the unchanged form. Therefore, we examined the effects of intravesical application of vibegron on bladder function in rats with oxotremorine methiodide (oxo-M: a nonselective muscarinic acetylcholine receptor agonist) -induced bladder overactivity. METHODS: Cystometry was performed in conscious female rats with intravesical instillation of oxo-M (200μM). In oxo-M-treated rats, vehicle or vibegron (1, 10mg/kg) was cumulatively administered intravenously at 30-minute intervals. In another group of rats, vehicle or vibegron (10, 100μM, 1mM) was cumulatively instilled intravesically at 60-minute intervals followed by intravenously application of vibegron (10mg/kg). Time-dependent changes in bladder function via cumulative intravenous or intravesical application of vehicle were also exmained. In addition, expression of β3-adrenoceptors (ARs) in the rat bladder was evaluated using immunohistochemical staining. RESULTS: Intravesical instillation of oxo-M induced bladder overactivity as evidenced by decreases in bladder capacity with increases in baseline, threshold and maximal voiding pressure. In oxo-M-treated rats, intravenous application of vibegron (10mg/kg) significantly increased bladder capacity (1.2 times) and decreased baseline, threshold and maximal voiding pressure compared with vehicle. Next, intravesical application of vibegron (1mM) significantly increased threshold pressure and bladder capacity (1.2 times) compared with vehicle. Combined treatments of intravesical (1mM) and intravenous (10mg/kg) application of vibegron induced a significantly larger rate of increases in bladder capacity (1.4 times) compared with vehicle although there were no significant time-dependent changes in bladder function. In addition, β3-ARs were expressed throughout the rat bladder, mainly in the urothelium. CONCLUSIONS: These results suggest that vibegron excreted into urine as the unchanged form induces the additive inhibitory effects on urinary frequency possibly by activating urothelial β3-ARs, which inhibits the afferent limb of micturition reflex rather than the efferent function evident as the increases in threshold pressure and bladder capacity without affecting bladder contractile function. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e532-e533 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akira Furuta* More articles by this author Yasuyuki Suzuki More articles by this author Taro Igarashi More articles by this author Yusuke Koike More articles by this author Shin Egawa More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement PDF downloadLoading ...

The muscarinic agonist pilocarpine modifies cocaine-reinforced and food-reinforced responding in rats: comparison with the cholinesterase inhibitor tacrine.

Activation of muscarinic receptors in the brain antagonizes the actions of cocaine, blocking both its discriminative stimulus and reinforcing properties. Pilocarpine is a nonselective muscarinic agonist that is used clinically, but has not been well characterized for its actions during cocaine-reinforced behavior. This study evaluated its effects on cocaine-reinforced and food-reinforced behaviors in rats, using the cholinesterase inhibitor tacrine as a comparator. Intraperitoneal pilocarpine or tacrine at doses of 1.0 mg/kg or more attenuated self-administration of low-dose cocaine (0.1 mg/kg injection) but also increased oral movements. Pilocarpine was less potent than tacrine in decreasing responding supported by low or intermediate amounts of liquid food. Combined treatment with pilocarpine and tacrine was more effective than either compound alone in attenuating self-administration of intermediate-dose cocaine. At a low (0.66 mg/kg) dose which did not modify reinforced responding, pilocarpine increased nonspecific behavior (sniffing, rearing, and activity) in cocaine-reinforced but not in food-reinforced animals; with greater doses increasing cholinergic or gastrointestinal signs. These effects were most consistently correlated with changes in reinforcement in rats responding for cocaine relative to food-reinforced animals. Overall, pilocarpine exhibited modest selectivity for attenuating self-administration of low-dose cocaine without affecting a nondrug reinforcer.

The role of dopamine D1- and D2-like receptors related to muscarinic M1 receptors in impulsive choice in high-impulsive and low-impulsive rats

The non-selective muscarinic receptor agonist oxotremorine-M has been found to decrease impulsive choice in high-impulsive (HI) rats and increase impulsive choice in low-impulsive (LI) rats, but little is known about the muscarinic M1 receptor agonist N-desmethylclozapine (NDMC). This study investigated effects of NDMC on impulsive choice, and the effect of co-administration of NDMC with the dopamine D1-like receptor antagonist SCH 23390 or D2-like receptor antagonist raclopride on impulsive choice in HI and LI rats, characterized by basal levels of impulsive choice in a delay-discounting task. The results revealed that NDMC (1 and 2 mg/kg) significantly increased impulsive choice in HI, but not LI rats. SCH 23390 significantly promoted impulsive choice in HI rats at 0.01 mg/kg, and in LI rats at 0.0075 and 0.01 mg/kg. Moreover, SCH 23390 (0.005 and 0.0075 mg/kg) significantly inhibited the increase in impulsive choice induced by NDMC (1 mg/kg) in HI rats, whereas the increase in impulsive choice produced by SCH 23390 (0.0075 mg/kg) was significantly reversed by NDMC (1 mg/kg) in LI rats. Raclopride (0.04, 0.08, and 0.12 mg/kg) did not affect choice in both HI and LI rats, but significantly antagonized the increase in impulsive choice induced by NDMC (1 mg/kg) in HI rats. These findings suggest that D1- and D2-like receptors might be involved in different effects of the M1 receptor agonist on impulsive choice between HI and LI rats.

Central muscarinic and LPBN mechanisms on sodium intake

Central cholinergic activation stimulates water intake, but also NaCl intake when the inhibitory mechanisms are blocked with injections of moxonidine (α2 adrenergic/imidazoline agonist) into the lateral parabrachial nucleus (LPBN). In the present study, we investigated the involvement of central M1 and M2 muscarinic receptors on NaCl intake induced by pilocarpine (non-selective muscarinic agonist) intraperitoneally combined with moxonidine into the LPBN or by muscimol (GABAA agonist) into the LPBN. Male Holtzman rats with stainless steel cannulas implanted bilaterally in the LPBN and in the lateral ventricle were used. Pirenzepine (M1 muscarinic antagonist, 1 nmol/1 μl) or methoctramine (M2 muscarinic antagonist, 50 nmol/1 μL) injected intracerebroventricularly (i.c.v.) reduced 0.3 M NaCl and water intake in rats treated with pilocarpine (0.1 mg/100 g of body weight) injected intraperitoneally combined with moxonidine (0.5 nmol/0.2 μL) into the LPBN. In rats treated with muscimol (0.5 nmol/0.2 μL) into the LPBN, methoctramine i.c.v. also reduced 0.3 M NaCl and water intake, however, pirenzepine produced no effect. The results suggest that M1 and M2 muscarinic receptors activate central pathways involved in the control of water and sodium intake that are under the influence of the LPBN inhibitory mechanisms.

Heat shock protein (Hsp) regulation by muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

The cholinergic system plays a crucial role in modulating in the central nervous system physiological responses such as neurogenesis, neuronal differentiation, synaptic plasticity, and neuroprotection. In a recent study, we showed that Oxotremorine-M, a non-selective muscarinic acetylcholine receptor agonist, is able to transactivate the fibroblast growth factor receptor and to produce a significant increase in the hippocampal primary neurite outgrowth. In the present study we aimed to explore in the rat hippocampus the possible effect of acute or chronic treatment with Oxotremorine-M on some heat shock proteins (Hsp60, Hsp70, Hsp90) and on activation of related transcription factor heat shock factor 1 (HSF1). Following single injection of Oxotremorine-M (0.4 mg/kg) all Hsps examined were significantly increased in at least one of the time points studied (24, 48, and 72 hr). Treatment with Oxotremorine-M significantly increased the level of phosphorylated HSF1 in all time points studied, without change of protein levels. Similar pattern of Hsps changes was obtained following chronic Oxotremorine-M treatment (0.2 mg/kg) for 5 days. Surprisingly, following chronic treatment for 10 days no changes were observed in Hsps. The muscarinic acetylcholine receptor antagonist scopolamine (1 mg/kg) was able to completely block Oxotremorine-M effects on Hsps. In conclusion, considering the function of Hsps in protecting neuronal cells from deleterious proteotoxic stress, for example, protein mis-folding and aggregation, the results obtained indicate that muscarinic acetylcholine receptor activation may have implications in potential treatment of neurodegenerative disorders linked to protein aggregation, such as Alzheimer disease.

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.

Role of muscarinic receptors in the contraction of jejunal smooth muscle in the horse: An in vitro study

Nonselective antimuscarinic drugs are clinically useful in several pathologic conditions of horses, but, blocking all muscarinic receptor (MR) subtypes, may cause several side effects. The availability of selective antimuscarinic drugs could improve therapeutic efficacy and safety.We aimed to enlighten the role of different MR subtypes by evaluating the effects of nonselective, and selective M1, M2 and M3 MR antagonists on the contractions of horse jejunum.Segments of circular muscle of equine jejunum, were put into organ baths, connected to isotonic transducers, and the effects on ACh concentration-response curves, and on electrical field stimulation (EFS)-evoked contractions of intestinal preparations, induced by nonselective or selective MR antagonists, compared to pre-drug level, were studied. Atropine (nonselective MR antagonist), pirenzepine (selective M1 antagonist), and p-FHHSiD (selective M3 antagonist) competitively antagonized ACh (pA2=9.78±0.21; 7.14±0.25 and 7.56±0.17, respectively). Methoctramine (selective M2 antagonist) antagonized ACh in a concentration-unrelated fashion; however, it competitively antagonized carbachol, a nonselective muscarinic agonist (pA2=6.42±0.23). Atropine dose-dependently reduced EFS-evoked contractions, reaching a maximal effect of −45.64±6.54%; the simultaneous block of neurokinin receptors, almost completely abolished the atropine-insensitive contractions. p-FHHSiD dose-dependently reduced EFS-induced contractions, while pirenzepine caused a minor decrease. Methoctramine, ineffective up to 10−7M, enhanced the contractions at 10−6M; the block of neurokinin receptors abolished the increase of contraction. Cholinergic contractions of horse jejunum are mainly mediated by M3 receptors; M2 selective antagonists seem to scarcely affect cholinergic, and to enhance neurokininergic contractions of equine jejunum, thus their use entails a lower risk of causing intestinal hypomotility, compared to nonselective drugs.

Peri-adolescent asthma symptoms cause adult anxiety-related behavior and neurobiological processes in mice

Human and animal studies have shown that physical challenges and stressors during adolescence can have significant influences on behavioral and neurobiological development associated with internalizing disorders such as anxiety and depression. Given the prevalence of asthma during adolescence and increased rates of internalizing disorders in humans with asthma, we used a mouse model to test if and which symptoms of adolescent allergic asthma (airway inflammation or labored breathing) cause adult anxiety- and depression-related behavior and brain function. To mimic symptoms of allergic asthma in young BALB/cJ mice (postnatal days [P] 7–57; N=98), we induced lung inflammation with repeated intranasal administration of house dust mite extract (most common aeroallergen for humans) and bronchoconstriction with aerosolized methacholine (non-selective muscarinic receptor agonist). Three experimental groups, in addition to a control group, included: (1) “Airway inflammation only”, allergen exposure 3 times/week, (2) “Labored breathing only”, methacholine exposure once/week, and (3) “Airway inflammation+Labored breathing”, allergen and methacholine exposure. Compared to controls, mice that experienced methacholine-induced labored breathing during adolescence displayed a ∼20% decrease in time on open arms of the elevated plus maze in early adulthood (P60), a ∼30% decrease in brainstem serotonin transporter (SERT) mRNA expression and a ∼50% increase in hippocampal serotonin receptor 1a (5Htr1a) and corticotropin releasing hormone receptor 1 (Crhr1) expression in adulthood (P75). This is the first evidence that experimentally-induced clinical symptoms of adolescent asthma alter adult anxiety-related behavior and brain function several weeks after completion of asthma manipulations.