Muscarinic Cholinergic Agonist Research Articles

Cholinergic and behavior-dependent beta and gamma waves are coupled between olfactory bulb and hippocampus.

Olfactory oscillations may enhance cognitive processing through coupling with beta (β, 15-30 Hz) and gamma (γ, 30-160 Hz) activity in the hippocampus (HPC). We hypothesize that coupling between olfactory bulb (OB) and HPC oscillations is increased by cholinergic activation in control rats and is reduced in kainic-acid-treated epileptic rats, a model of temporal lobe epilepsy. OB γ2 (63-100 Hz) power was higher during walking and immobility-awake (IMM) compared to sleep, while γ1 (30-57 Hz) power was higher during grooming than other behavioral states. Muscarinic cholinergic agonist pilocarpine (25 mg/kg ip) with peripheral muscarinic blockade increased OB power and OB-HPC coherence at β and γ1 frequency bands. A similar effect was found after physostigmine (0.5 mg/kg ip) but not scopolamine (10 mg/kg ip). Pilocarpine increased bicoherence and cross-frequency coherence (CFC) between OB slow waves (SW, 1-5 Hz) and hippocampal β, γ1 and γ2 waves, with stronger coherence at CA1 alveus and CA3c than CA1 stratum radiatum. Bicoherence further revealed a nonlinear interaction of β waves in OB with β waves at the CA1-alveus. Beta and γ1 waves in OB or HPC were segregated at one phase of the OB-SW, opposite to the phase of γ2 and γ3 (100-160 Hz) waves, suggesting independent temporal processing of β/γ1 versus γ2/γ3 waves. At CA1 radiatum, kainic-acid-treated epileptic rats compared to control rats showed decreased theta power, theta-β and theta-γ2 CFC during baseline walking, decreased CFC of HPC SW with γ2 and γ3 waves during baseline IMM, and decreased coupling of OB SW with β and γ2 waves at CA1 alveus after pilocarpine. It is concluded that β and γ waves in the OB and HPC are modulated by a slow respiratory rhythm, in a cholinergic and behavior-dependent manner, and OB-HPC functional connectivity at β and γ frequencies may enhance cognitive functions.

Transdermal iontophoretic application of l-NAME is available in sweating research induced by heat stress in young healthy adults

Iontophoretic transdermal administration of NG-nitro-l-arginine methyl ester hydrochloride [l-NAME, a nitric oxide synthase (NOS) inhibitor] has been used as a non-invasive evaluation of NOS-dependent mechanisms in human skin. However, the availability has yet to be investigated in sweating research. Prior observations using invasive techniques (e.g., intradermal microdialysis technique) to administer l-NAME have implicated that NOS reduces sweating induced by heat stress but rarely influences the response induced by the administration of cholinergic muscarinic receptor agonists. Therefore, we investigated whether the transdermal iontophoretic administration of l-NAME modulates sweating similar to those prior observations. Twenty young healthy adults (10 males, 10 females) participated in two experimental protocols on separate days. Before each protocol, saline (control) and 1% l-NAME were bilaterally administered to the forearm skin via transdermal iontophoresis. In protocol 1, 0.001% and 1% pilocarpine were iontophoretically administered at l-NAME-treated and untreated sites. In protocol 2, passive heating was applied by immersing the lower limbs in hot water (43 °C) until the rectal temperature increased by 0.8 °C above baseline. The sweat rate was continuously measured throughout both protocols. Pilocarpine-induced sweat rate was not significantly different between the control and l-NAME-treated sites in both pilocarpine concentrations (P ≥ 0.316 for the treatment effect and interaction of treatment and pilocarpine concentration). The sweat rate during passive heating was attenuated at the l-NAME-treated site relative to the control (treatment effect, P = 0.020). Notably, these observations are consistent with prior sweating studies administrating l-NAME into human skin using intradermal microdialysis techniques. Based on the similarity of our results with already known observations, we conclude that transdermal iontophoresis of l-NAME is a valid non-invasive technique for the investigation of the mechanisms of sweating related to NOS during heat stress.

The Partial M1 Muscarinic Cholinergic Receptor Agonist, CDD-0102A, Differentially Modulates Glutamate Efflux in Striatal Subregions during Stereotyped Motor Behavior in the BTBR Mouse Model of Autism.

The BTBR T+ Itpr3tf/J (BTBR) mouse displays elevated repetitive motor behaviors. Treatment with the partial M1 muscarinic receptor agonist, CDD-0102A, attenuates stereotyped motor behaviors in BTBR mice. The present experiment investigated whether CDD-0102A modifies changes in striatal glutamate concentrations during stereotyped motor behavior in BTBR and B6 mice. Using glutamate biosensors, change in striatal glutamate efflux was measured during bouts of digging and grooming behavior with a 1 s time resolution. Mice displayed both decreases and increases in glutamate efflux during such behaviors. Magnitude of changes in glutamate efflux (decreases and increases) from dorsomedial and dorsolateral striatum were significantly greater in BTBR mice compared to those of B6 mice. In BTBR mice, CDD-0102A (1.2 mg/kg) administered 30 min prior to testing significantly reduced the magnitude change in glutamate decreases and increases from the dorsolateral striatum and decreased grooming behavior. Conversely, CDD-0102A treatment in B6 mice potentiated glutamate decreases and increases in the dorsolateral striatum and elevated grooming behavior. The findings suggest that activation of M1 muscarinic receptors modifies glutamate transmission in the dorsolateral striatum and self-grooming behavior.

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo.

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc-Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.

Muscarinic cholinergic modulation of cardiovascular variables in spinal cord injured rats

Spinal cord injury (SCI) leads not only to major impairments in sensorimotor control but also to dramatic dysregulation of autonomic functions including major cardiovascular disturbances. Consequently, individuals with SCI endure daily episodic hypo/hypertension and are at increased risk for cardiovascular disease. Several studies have suggested that an intrinsic spinal coupling mechanism between motor and sympathetic neuronal networks exist and that propriospinal cholinergic neurons may be responsible for a synchronized activation of both somatic and sympathetic outputs. We therefore investigated in the present study, the effect of cholinergic muscarinic agonists on cardiovascular parameters in freely moving adult rats after SCI. Female Sprague-Dawley rats were implanted with radiotelemetry sensors for long-term in vivo monitoring of blood pressure (BP). From BP signal, we calculated heart rate (HR) and respiratory frequency. We first characterized the physiological changes occurring after a SCI performed at the T3-T4 level in our experimental model system. We then investigated the effects on BP, HR and respiration, of the muscarinic agonist oxotremorine using one variant that crossed the blood brain barrier (Oxo-S) and one that does not (Oxo-M) in both Pre- and Post-SCI animals. After SCI, both HR and respiratory frequency increased. BP values exhibited an immediate profound drop before progressively increasing over the three-week post-lesion period but remained below control values. A spectral analysis of BP signal revealed the disappearance of the low frequency component of BP (0.3-0.6Hz) referred to as Mayer waves after SCI. In Post-SCI animals, central effects mediated by Oxo-S led to an increase in HR and MAP, a slowdown in respiratory frequency and to an increased power in the 0.3-0.6Hz frequency band. This study unravels some of the mechanisms by which muscarinic activation of spinal neurons could contribute to partial restoration of BP after SCI.

M1/M4-Preferring Muscarinic Cholinergic Receptor Agonist Xanomeline Reverses Wake and Arousal Deficits in Nonpathologically Aged Mice.

Degeneration of the cholinergic basal forebrain is implicated in the development of cognitive deficits and sleep/wake architecture disturbances in mild cognitive impairment (MCI) and Alzheimer's disease (AD). Indirect-acting muscarinic cholinergic receptor agonists, such as acetylcholinesterase inhibitors (AChEIs), remain the only FDA-approved treatments for the cognitive impairments observed in AD that target the cholinergic system. Novel direct-acting muscarinic cholinergic receptor agonists also improve cognitive performance in young and aged preclinical species and are currently under clinical development for AD. However, little is known about the effects of direct-acting muscarinic cholinergic receptor agonists on disruptions of sleep/wake architecture and arousal observed in nonpathologically aged rodents, nonhuman primates, and clinical populations. The purpose of the present study was to provide the first assessment of the effects of the direct-acting M1/M4-preferring muscarinic cholinergic receptor agonist xanomeline on sleep/wake architecture and arousal in young and nonpathologically aged mice, in comparison with the AChEI donepezil, when dosed in either the active or inactive phase of the circadian cycle. Xanomeline produced a robust reversal of both wake fragmentation and disruptions in arousal when dosed in the active phase of nonpathologically aged mice. In contrast, donepezil had no effect on either age-related wake fragmentation or arousal deficits when dosed during the active phase. When dosed in the inactive phase, both xanomeline and donepezil produced increases in wake and arousal and decreases in nonrapid eye movement sleep quality and quantity in nonpathologically aged mice. Collectively, these novel findings suggest that direct-acting muscarinic cholinergic agonists such as xanomeline may provide enhanced wakefulness and arousal in nonpathological aging, MCI, and AD patient populations.

Hippocampal cholinergic receptors and the mTOR participation in fear-motivated inhibitory avoidance extinction memory

Evidence has demonstrated the hippocampal cholinergic system and the mammalian target of rapamycin (mTOR) participation during the memory formation of aversive events. This study assessed the role of these systems in the hippocampus for the extinction memory process by submitting male Wistar rats to fear-motivated step-down inhibitory avoidance (IA). The post-extinction session administration of the nicotinic and muscarinic cholinergic receptor antagonists, mecamylamine and scopolamine, respectively, both at doses of 2 µg/µl/side, and rapamycin, an mTOR inhibitor (0.02 µg/µl/side), into the CA1 region of the dorsal hippocampus, impaired the IA extinction memory. Furthermore, the nicotinic and muscarinic cholinergic receptor agonists, nicotine and muscarine, respectively, had a dose-dependent effect on the IA extinction memory when administered intra-CA1, immediately after the extinction session. Nicotine (0.6 µg/µl/side) and muscarine (0.02 µg/µl/side), respectively, had no effect, while the higher doses (6 and 2 µg/µl/side, respectively) impaired the IA extinction memory. Interestingly, the co-administration of muscarine at the lower dose blocked the impairment that was induced by rapamycin. This effect was not observed when nicotine at the lower dose was co-administered. These results have demonstrated the participation of the cholinergic receptors and mTOR in the hippocampus for IA extinction, and that the cholinergic agonists had a dose-dependent effect on the IA extinction memory. This study provides insights related to the behavioural aspects and the neurobiological properties underlying the early stage of fear-motivated IA extinction memory consolidation and suggests that there is hippocampal muscarinic receptor participation independent of mTOR in this memory process.

Xanomeline and Trospium: Potential substitute for conventional antipsychotics.

Dear Madam, Schizophrenia is a chronic mental illness with a global prevalence of 24 million people (1). With paucity of researches mounting the exact prevalence of schizophrenia in Pakistan, the treatment of this illness becomes even harder to combat. This condition is typically managed with antipsychotics, which work by blocking the D2 dopamine receptors but recent advances in research also suggest that the muscarinic cholinergic system is also involved in its pathophysiology (2). The drug Xanomeline is an oral muscarinic cholinergic receptor agonist that activates M1 and M4 receptors. Xanomeline however causes side effects like vomiting, nausea, diarrhoea, sweating and hypersalivation. To bypass these effects, Trospium was added, which acts by blocking peripheral muscarinic cholinergic receptors. (3, 4). To observe the reduction of psychosis in schizophrenics, a double-blinded clinical trial was done with promising results: reduction of both, positive and negative symptoms of Schizophrenia, good tolerance and no evidence of weight gain (4). Side effects included constipation, nausea, dry mouth, dyspepsia, and vomiting although the incidence of these effects reduced over the course of the trial. However, there is a dearth of research when it comes to comparison of this combination drug (called KARXT) with the usual antipsychotics. Therefore, further research regarding the safety of KARXT, and its efficacy needs to be explored for potential amelioration of symptoms. Moreover, studies comparing the efficacy of this drug with the commonly used antipsychotics need to be explored in order to effectively assess the advantage and potential replacement benefits of this drug.

Effects of the Partial M1 Muscarinic Cholinergic Receptor Agonist CDD-0102A on Stereotyped Motor Behaviors and Reversal Learning in the BTBR Mouse Model of Autism.

BackgroundAutism spectrum disorders (ASD) are a set of neurodevelopmental disorders marked by a lack of social interaction, restrictive interests, and repetitive behaviors. There is a paucity of pharmacological treatments to reduce core ASD symptoms. Various lines of evidence indicate that reduced brain muscarinic cholinergic receptor activity may contribute to an ASD phenotype.MethodsThe present experiments examined whether the partial M1 muscarinic receptor agonist, 5-(3-ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A), alleviates behavioral flexibility deficits and/or stereotyped motor behaviors in the BTBR mouse model of autism. Behavioral flexibility was tested using a reversal learning test. Stereotyped motor behaviors were measured by eliciting digging behavior after removal of nesting material in a home cage and by measuring repetitive grooming.ResultsCDD-0102A (0.2 and 0.6 mg/kg but not 1.2 mg/kg) injected prior to reversal learning attenuated a deficit in BTBR mice but did not affect performance in B6 mice. Acute CDD-0102A treatment (1.2 and 3 mg/kg) reduced self-grooming in BTBR mice and reduced digging behavior in B6 and BTBR mice. The M1 muscarinic receptor antagonist VU0255035 (3 mg/kg) blocked the effect of CDD-0102A on grooming behavior. Chronic treatment with CDD-0102A (1.2 mg/kg) attenuated self-grooming and digging behavior in BTBR mice. Direct CDD-0102A infusions (1 µg) into the dorsal striatum reduced elevated digging behavior in BTBR mice. In contrast, CDD-0102A injections in the frontal cortex were not effective.ConclusionsThe results suggest that treatment with a partial M1 muscarinic receptor agonist may reduce repetitive behaviors and restricted interests in autism in part by stimulating striatal M1 muscarinic receptors.

Nerve Growth Factor: A Dual Activator of Noradrenergic and Cholinergic Systems of the Rat Ovary.

The functioning of the ovary is influenced by the autonomic system (sympathetic and cholinergic intraovarian system) which contributes to the regulation of steroid secretion, follicular development, and ovulation. There is no information on the primary signal that activates both systems. The nerve growth factor (NGF) was the first neurotrophic factor found to regulate ovarian noradrenergic neurons and the cholinergic neurons in the central nervous system. The aim of this study was to determine whether NGF is one of the participating neurotrophic factors in the activation of the sympathetic and cholinergic system of the ovary in vivo and its role in follicular development during normal or pathological states. The administration of estradiol valerate (a polycystic ovary [PCO] phenotype model) increased norepinephrine (NE) (through an NGF-dependent mechanism) and acetylcholine (ACh) levels. Intraovarian exposure of rats for 28 days to NGF (by means of an osmotic minipump) increased the expression of tyrosine hydroxylase and acetylcholinesterase (AChE, the enzyme that degrades ACh) without affecting enzyme activity but reduced ovarian ACh levels. In vitro exposure of the ovary to NGF (100 ng/ml for 3 h) increased both choline acetyl transferase and vesicular ACh transporter expression in the ovary, with no effect in ACh level. In vivo NGF led to an anovulatory condition with the appearance of follicular cysts and decreased number of corpora lutea (corresponding to noradrenergic activation). To determine whether the predominance of a NE-induced polycystic condition after NGF is responsible for the PCO phenotype, rats were exposed to an intraovarian administration of carbachol (100 μM), a muscarinic cholinergic agonist not degraded by AChE. Decreased the number of follicular cysts and increased the number of corpora lutea, reinforcing that cholinergic activity of the ovary participates in controlling its functions. Although NGF increased the biosynthetic capacity for ACh, it was not available to act in the ovary. Hence, NGF also regulates the ovarian cholinergic system, implying that NGF is the main regulator of the dual autonomic control. These findings highlight the need for research in the treatment of PCO syndrome by modification of locally produced ACh as an in vivo regulator of follicular development.

Muscarinic Cholinergic Receptor Agonist and Peripheral Antagonist for Schizophrenia

BackgroundThe muscarinic receptor agonist xanomeline has antipsychotic properties and is devoid of dopamine receptor–blocking activity but causes cholinergic adverse events. Trospium is a peripherally restricted muscarinic receptor antagonist that reduces peripheral cholinergic effects of xanomeline. The efficacy and safety of combined xanomeline and trospium in patients with schizophrenia are unknown.MethodsIn this double-blind, phase 2 trial, we randomly assigned patients with schizophrenia in a 1:1 ratio to receive twice-daily xanomeline–trospium (increased to a maximum of 125 mg of xanomeline and 30 mg of trospium per dose) or placebo for 5 weeks. The primary end point was the change from baseline to week 5 in the total score on the Positive and Negative Syndrome Scale (PANSS; range, 30 to 210, with higher scores indicating more severe symptoms of schizophrenia). Secondary end points were the change in the PANSS positive symptom subscore, the score on the Clinical Global Impression–Severity (CGI-S) scale (range, 1 to 7, with higher scores indicating greater severity of illness), the change in the PANSS negative symptom subscore, the change in the PANSS Marder negative symptom subscore, and the percentage of patients with a response according to a CGI-S score of 1 or 2.ResultsA total of 182 patients were enrolled, with 90 assigned to receive xanomeline–trospium and 92 to receive placebo. The PANSS total score at baseline was 97.7 in the xanomeline–trospium group and 96.6 in the placebo group. The change from baseline to week 5 was −17.4 points with xanomeline–trospium and −5.9 points with placebo (least-squares mean difference, −11.6 points; 95% confidence interval, −16.1 to −7.1; P<0.001). The results for the secondary end points were significantly better in the xanomeline–trospium group than in the placebo group, with the exception of the percentage of patients with a CGI-S response. The most common adverse events in the xanomeline–trospium group were constipation, nausea, dry mouth, dyspepsia, and vomiting. The incidences of somnolence, weight gain, restlessness, and extrapyramidal symptoms were similar in the two groups.ConclusionsIn a 5-week trial, xanomeline–trospium resulted in a greater decrease in the PANSS total score than placebo but was associated with cholinergic and anticholinergic adverse events. Larger and longer trials are required to determine the efficacy and safety of xanomeline–trospium in patients with schizophrenia. (Funded by Karuna Therapeutics and the Wellcome Trust; ClinicalTrials.gov number, NCT03697252.)

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

The regulatory G protein signaling complex, Gβ5–R7, promotes glucose- and extracellular signal–stimulated insulin secretion

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5-R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gβ5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Effects of Carbachol on the Excitatory Muscarinic Cholinergic Receptors in Urinary Bladder and Plasma Inflammatory Mediators in Anesthetized Rats

To observe the effects of muscarinic cholinergic receptor agonist carbachol on urinary bladder function and plasma inflammatory mediators in anesthetized rats. A total of 20 rats were randomly divided into carbachol group and solvent control group, 10 rats in each group. The detection of bladder wall thickness and histological observation were conducted by light microscopy, the changes of urinary bladder function and plasma inflammatory mediators in rats were compared between the two groups. The maximum bladder capacity, static bladder pressure, compliance, bladder wet weight and bladder wall thickness in the carbachol group were significantly higher than those in the solvent control group. Light microscopic evaluation revealed that the detrusor cells in the solvent control group appeared to be in order in terms of size, arrangement and shape, while in the carbachol group the detrusor cells exhibited hypertrophy together with disordered arrangement, diverse morphology and vacuolar degeneration of muscle fiber. The levels of TNF-α and IL-10 in plasma of the carbachol group were significantly lower than those of the solvent control group. In anesthetized rats, the stimulation of muscarinic cholinergic receptors can significantly reduce urinary frequency, increase the volume of bladder storage and decrease systemic inflammatory response.

M4-muscarinic acetylcholine receptor into the pedunculopontine tegmental nucleus mediates respiratory modulation of conscious rats

The pedunculopontine tegmental nucleus (PPTg) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. The PPTg is considered an important nucleus in the mesopontine region with considerably cholinergic input to the ventral respiratory column. In addition, recent studies indicate that cholinergic innervation of the ventral respiratory column may play an important role in modulation of breathing. Here, we investigated the cholinergic stimulation of the PPTg and the changes in breathing output in conscious rats. Male Wistar rats (280–350 g, N = 5–12/group) with unilateral stainless steel cannula implanted into the PPTg were used. Respiratory parameters (tidal volume (VT), respiratory frequency (fR) and ventilation (VE)) were analyzed by whole body plethysmography. In unrestrained awake rats, unilateral injection of the cholinergic muscarinic agonist carbachol (10 mM–100 nL) in the PPTg decreased fR, and increase VT, without changing VE. The changes in fR and VT elicited by carbachol into the PPTg are abolished by previous blockade of the M4 muscarinic cholinergic receptors tropicamide into the PPTg. No significant changes in fR and VT elicited by carbachol were observed after blockade of the M1 and/or M3 muscarinic cholinergic receptors pirenzepine or 4-DAMP into the PPTg. Our data suggest that the changes in fR and VT produced by muscarinic cholinergic stimulation of PPTg is presumably mediated through a Gi-coupled M4 muscarinic receptors.

Medial septal cholinergic mediation of hippocampal theta rhythm induced by vagal nerve stimulation.

BackgroundElectrical vagal nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. This technique also remains under investigation as a specific treatment of patients with Alzheimer’s disease. Recently we discovered that VNS induced hippocampal formation (HPC) type II theta rhythm, which is involved in memory consolidation. In the present study, we have extended our previous observation and addressed the neuronal substrate and pharmacological profile of HPC type II theta rhythm induced by VNS in anesthetized rats.MethodsMale Wistar rats were implanted with a VNS cuff electrode around the left vagus nerve, a tungsten microelectrode for recording the HPC field activity, and a medial septal (MS) cannula for the injection of a local anesthetic, procaine, and muscarinic agents. A direct, brief effect of VNS on the HPC field potential was evaluated before and after medial-septal drug injection.ResultsMedial septal injection of local anesthetic, procaine, reversibly abolished VNS-induced HPC theta rhythm. With the use of cholinergic muscarinic agonist and antagonists, we demonstrated that medial septal M1 receptors are involved in the mediation of the VNS effect on HPC theta field potential.ConclusionThe MS cholinergic M1 receptor mechanism integrates not only central inputs from the brainstem synchronizing pathway, which underlies the production of HPC type II theta rhythm, but also the input from the vagal afferents in the brain stem.

High pH-Sensitive Store-Operated Ca2+ Entry Mediated by Ca2+ Release-Activated Ca2+ Channels in Rat Odontoblasts.

Odontoblasts play a crucial role in dentin formation and sensory transduction following the application of stimuli to the dentin surface. Various exogenous and endogenous stimuli elicit an increase in the intracellular free calcium concentration ([Ca2+]i) in odontoblasts, which is mediated by Ca2+ release from intracellular Ca2+ stores and/or Ca2+ influx from the extracellular medium. In a previous study, we demonstrated that the depletion of Ca2+ stores in odontoblasts activated store-operated Ca2+ entry (SOCE), a Ca2+ influx pathway. However, the precise biophysical and pharmacological properties of SOCE in odontoblasts have remained unclear. In the present study, we examined the functional expression and pharmacological properties of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE and evaluated the alkali sensitivity of SOCE in rat odontoblasts. In the absence of extracellular Ca2+, treatment with thapsigargin (TG), a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, induced an increase in [Ca2+]i. After [Ca2+]i returned to near-resting levels, the subsequent application of 2.5 mM extracellular Ca2+ resulted in an increase in [Ca2+]i which is a typical of SOCE activation. Additionally, application of 2-methylthioadenosine diphosphate trisodium salt (2-MeSADP), a P2Y1,12,13 receptor agonist, or carbachol (CCh), a muscarinic cholinergic receptor agonist, in the absence of extracellular Ca2+, induced a transient increase in [Ca2+]i. The subsequent addition of extracellular Ca2+ resulted in significantly higher [Ca2+]i in 2-MeSADP- or CCh-treated odontoblasts than in untreated cells. SOCE, that is activated by addition of extracellular Ca2+ in the TG pretreated odontoblasts was then suppressed by Synta66, BTP2, or lanthanum, which are CRAC channel inhibitors. Treatment with an alkaline solution enhanced SOCE, while treatment with HC030031, a TRPA1 channel antagonist, inhibited it. The amplitude of SOCE at pH 9 in the presence of HC030031 was higher than that at pH 7.4 in the absence of HC030031. These findings indicate that CRAC channel-mediated alkali-sensitive SOCE occurs in odontoblasts. SOCE is mediated by P2Y and muscarinic-cholinergic receptors, which are activated by endogenous ligands in odontoblasts.

Myotropic Effects of Cholinergic Muscarinic Agonists and Antagonists in the Beetle Tenebrio molitor L.

In mammals, the cholinergic nervous system plays a crucial role in neuronal regulation of physiological processes. It acts on cells by two types of receptors - nicotinic and muscarinic receptors. Both signal transmission pathways also operate in the central and peripheral cholinergic nervous system of insects. In our pharmacological experiments, we studied the effects of two muscarinic agonists (carbachol, pilocarpine) and two muscarinic antagonists (atropine, scopolamine) on the muscle contractile activity of visceral organs in the beetle, Tenebrio molitor. Both antagonists, when injected to haemolymph at concentration 10-5 M, caused delayed and prolonged cardioinhibitory effects on heart contractility in ortho- and antidromic phases of heart activity in T. molitor pupa what was observed as negative chrono- and inotropic effects. Agonist of muscarinic receptors - carbachol evoked opposite effect and increased contraction rate but only in antidromic phase. Pilocarpine, the second agonist induced weak negative chronotropic effects in the antiand orthodromic phases of heart activity. However, neither agonists had an effect on semi-isolated beetle heart in vitro. Only atropine at the highest tested concentrations slightly decreased the frequency of myocardial contractions. These suggest the regulation of heart activity by muscarinic system indirectly. The tested compounds also affected the contractility of the oviduct and hindgut, but the responses of these organs were varied and depended on the concentration of the applied compounds. These pharmacological experiments suggest the possible modulation of insect visceral muscle contractility by the cholinergic nervous system and indirectly indicate the presence of muscarinic receptor(s) in the visceral organs of the beetle T. molitor.

Heat Stress and Agonists of Muscarinic Cholinergic Receptors Modulate Sensitivity of Nicotinic Cholinergic Receptors in Soil Nematode Caenorhabditis elegans.

We studied the effect of moderate heat stress (30oC) and muscarinic cholinergic receptor agonists arecoline and pilocarpine on sensitivity of the behavior of the nematode Caenorhabditis elegans of N2 line to the action of the agonist of nicotinic cholinergic receptor agonist levamisole. Heat stress and muscarinic cholinergic receptor agonists increased the sensitivity of swimming induced by mechanical stimulation to levamisole (32-64 μM), which manifested in dyscoordination of locomotor muscles during swimming and complete loss of ability to swim. Combined exposure to heat stress and muscarinic cholinergic receptor agonists revealed their synergism in the influence on sensitivity of swimming behavior to levamisole: heating to 30oC potentiated the effect of arecoline and arecoline potentiated the effect of heat stress. It is assumed, that the effect of heat stress on the sensitivity of nicotinic receptors is mediated by its effect on muscarinic receptors.

Cost-effectiveness of glaucoma management with monotherapy medications in Egypt.

Glaucoma is a serious chronic ophthalmic disease since it causes irreversible visual disability if untreated can lead to blindness. Treatment options include medications (classified into five major classes of drugs which are muscarinic cholinergic agonists, alpha-2 adrenergic agonists, beta-1 adrenergic antagonists, prostaglandins [PGs], and carbonic anhydrase inhibitors); use of laser therapy or conventional surgery. Pharmacoeconomic analysis helps in choosing among this variety of treatments. There is a great need for such analysis in Egypt since undergoing of it in different countries or societies may produce different results. This work aimed to compare cost-effectiveness of bimatoprost 0.03% once daily versus brimonidine 0.2% twice daily and timolol 0.5% twice daily as monotherapy treatment in Egyptian patients with open-angle glaucoma or ocular hypertension. Clinical data revealed that all treatments decreased intraocular pressure (IOP) significantly but bimatoprost 0.03% showed the highest efficacy (27.7% decrease in IOP from baseline), while timolol 0.5% reduced IOP by 22.5% then brimonidine 0.2% which decreased IOP by 20.8%. From the cost-effectiveness view, it would be preferable to initiate treatment with timolol in case of absence of any contraindications. PG analog can be used as add-on therapy in low responder patients or as alternative treatment in case of presence of contraindication to use of beta blockers.