Muscarinic Activation Research Articles

Neuromodulation of Persistent Activity and Working Memory Circuitry in Primate Prefrontal Cortex by Muscarinic Receptors

Neuromodulation by acetylcholine plays a vital role in shaping the physiology and functions of cerebral cortex. Cholinergic neuromodulation influences brain-state transitions, controls the gating of cortical sensory stimulus responses, and has been shown to influence the generation and maintenance of persistent activity in prefrontal cortex. Here we review our current understanding of the role of muscarinic cholinergic receptors in primate prefrontal cortex during its engagement in the performance of working memory tasks. We summarize the localization of muscarinic receptors in prefrontal cortex, review the effects of muscarinic neuromodulation on arousal, working memory and cognitive control tasks, and describe the effects of muscarinic M1 receptor stimulation and blockade on the generation and maintenance of persistent activity of prefrontal neurons encoding working memory representations. Recent studies describing the pharmacological effects of M1 receptors on prefrontal persistent activity demonstrate the heterogeneity of muscarinic actions and delineate unexpected modulatory effects discovered in primate prefrontal cortex when compared with studies in rodents. Understanding the underlying mechanisms by which muscarinic receptors regulate prefrontal cognitive control circuitry will inform the search of muscarinic-based therapeutic targets in the treatment of neuropsychiatric disorders.

Dysregulation of epithelial ion transport and neurochemical changes in the colon of a parkinsonian primate

The pathological changes underlying gastrointestinal (GI) dysfunction in Parkinson’s disease (PD) are poorly understood and the symptoms remain inadequately treated. In this study we compared the functional and neurochemical changes in the enteric nervous system in the colon of adult, L-DOPA-responsive, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmoset, with naïve controls. Measurement of mucosal vectorial ion transport, spontaneous longitudinal smooth muscle activity and immunohistochemical assessment of intrinsic innervation were each performed in discrete colonic regions of naïve and MPTP-treated marmosets. The basal short circuit current (Isc) was lower in MPTP-treated colonic mucosa while mucosal resistance was unchanged. There was no difference in basal cholinergic tone, however, there was an increased excitatory cholinergic response in MPTP-treated tissues when NOS was blocked with L-Nω-nitroarginine. The amplitude and frequency of spontaneous contractions in longitudinal smooth muscle as well as carbachol-evoked post-junctional contractile responses were unaltered, despite a decrease in choline acetyltransferase and an increase in the vasoactive intestinal polypeptide neuron numbers per ganglion in the proximal colon. There was a low-level inflammation in the proximal but not the distal colon accompanied by a change in α-synuclein immunoreactivity. This study suggests that MPTP treatment produces long-term alterations in colonic mucosal function associated with amplified muscarinic mucosal activity but decreased cholinergic innervation in myenteric plexi and increased nitrergic enteric neurotransmission. This suggests that long-term changes in either central or peripheral dopaminergic neurotransmission may lead to adaptive changes in colonic function resulting in alterations in ion transport across mucosal epithelia that may result in GI dysfunction in PD.

Effects of Ruscus extract on muscarinic receptors: Is there a role for endothelium derived relaxing factors on macromolecular permeability protection and microvascular diameter changes?

Protective effects of Ruscus extract on macromolecular permeability depend on its capacity to stimulate muscarinic receptors on endothelial cells and induce the release of endothelium derived relaxing factors (EDRFs). To investigate if these effects depend only on activation of muscarinic receptors or if EDRFs release are also necessary. We have also investigated the participation of Ruscus extract on muscarinic-induced release of EDRFs on microvascular diameters. Hamsters were treated daily during two weeks with Ruscus extract (50, 150 and 450 mg/kg/day) and then macromolecular permeability induced by histamine and arteriolar and venular diameters after cyclooxygenase (COX) and nitric oxide synthase (NOS) inhibitors: indomethacin and Nω-Nitro-L-arginine (LNA), respectively applied topically at 10-8M, 10-6M and 10-4M were observed on the cheek pouch preparation. Ruscus extract decreased macromolecular permeability in a dose-dependent fashion and did not affect microvascular diameters. NOS and COX inhibitors enhanced its effect on microvascular permeability. NOS inhibition reduced arteriolar diameter and COX blocking decreased arteriolar and venular diameters at the lowest dose and increased them at higher doses of Ruscus extract. The protective effect of Ruscus extract on macromolecular permeability seems to be mediated only via muscarinic receptors. Muscarinic activation attenuated vasoconstrictive tone through cyclooxygenase-independent endothelium derived relaxing factors.

Influence of RGS6 on Inhibitory G Protein Signaling in Mouse Sinoatrial Nodal Cells

IntroductionM2 muscarinic (M2R) activation of G protein‐gated inwardly rectifying K+ (GIRK) channels in sinoatrial nodal (SAN) cells is a key mediator of vagal/parasympathetic slowing of heart rate. The strength of M2R‐GIRK signaling is negatively regulated by Regulator of G protein Signaling 6 (RGS6). Adenosine, used clinically to diagnose and treat certain arrhythmias, exerts these actions through A1 adenosine receptor (A1R) activation of GIRK channels. The extent to which RGS6 regulates A1R‐GIRK signaling, however, is unclear. Here, we investigated the impact of RGS6 on A1R‐GIRK and M2R‐GIRK signaling in mouse SAN cells.Methods and ResultsWe measured the efficacy and potency of M2R‐ and A1R‐GIRK signaling in adult SAN cells from wild‐type and RGS6−/− mice using whole‐cell patch‐clamp electrophysiology. As previously reported, SAN cells from RGS6−/− mice displayed prolonged channel deactivation kinetics and increased channel sensitivity to the non‐selective cholinergic agonist carbachol (CCh), relative to wild‐type counterparts. In contrast, we observed a striking increase in the amplitude of the GIRK response evoked by adenosine (Ado) in RGS6−/− SAN cells compared to wild‐type controls, with no impact on channel deactivation kinetics or sensitivity. Further recordings in wild‐type SAN cells revealed that M2R activation completely occludes the Ado‐induced response, whereas Ado application activates only a smaller fraction of GIRK channels. Both CCh‐ and Ado‐ induced currents were abolished in SAN cells treated by pertussis toxin (PTX), indicating these responses are mediated by Gi/o G proteins. Utilizing a bioluminescence resonance energy transfer (BRET) assay in transfected HEK cells, we found that RGS6 prefers Gαo as a substrate for over Gαi. Additionally, we found that while A1R does not discriminate between Gαo and Gαi, M2R displays a clear preference for Gαo. We next employed a Cre/flox strategy to remove Gαo from atrial tissue, including SAN cells. Loss of Gαo yielded significantly blunted CCh‐induced responses, which also displayed prolonged activation and deactivation kinetics. While Ado‐induced responses had prolonged deactivation kinetics, activation kinetics and response amplitude were unaffected.ConclusionsThese results suggest that RGS6 negatively regulates A1R‐GIRK signaling distinct from its influence on M2R‐GIRK signaling in mouse SAN cells likely due to differing utilization of Gao by M2R and A1R.Support or Funding InformationThis work was supported by NIH grants R01 HL105550 and F31 HL139090.

Muscarinic-Dependent miR-182 and QR2 Expression Regulation in the Anterior Insula Enables Novel Taste Learning.

In a similar manner to other learning paradigms, intact muscarinic acetylcholine receptor (mAChR) neurotransmission or protein synthesis regulation in the anterior insular cortex (aIC) is necessary for appetitive taste learning. Here we describe a parallel local molecular pathway, where GABAA receptor control of mAChR activation causes upregulation of miRNA-182 and quinone reductase 2 (QR2) mRNA destabilization in the rodent aIC. Damage to long-term memory by prevention of this process, with the use of mAChR antagonist scopolamine before novel taste learning, can be rescued by local QR2 inhibition, demonstrating that QR2 acts downstream of local muscarinic activation. Furthermore, we prove for the first time the presence of endogenous QR2 cofactors in the brain, establishing QR2 as a functional reductase there. In turn, we show that QR2 activity causes the generation of reactive oxygen species, leading to modulation in Kv2.1 redox state. QR2 expression reduction therefore is a previously unaccounted mode of mAChR-mediated inflammation reduction, and thus adds QR2 to the cadre of redox modulators in the brain. The concomitant reduction in QR2 activity during memory consolidation suggests a complementary mechanism to the well established molecular processes of this phase, by which the cortex gleans important information from general sensory stimuli. This places QR2 as a promising new target to tackle neurodegenerative inflammation and the associated impediment of novel memory formation in diseases such as Alzheimer’s disease.

Adaptive short-term associative conditioning in the pancreatic β-cell.

This study associates cholinergic stimulation of the pancreatic β‐cell electrical activity with a short‐term memory phenomenon. Glucose pulses applied to a basal glucose concentration induce depolarizing waves which are used to estimate the evolution of the β‐cell glucose sensitivity. Exposure to carbamoylcholine (carbachol) increases the size of the glucose‐induced depolarizing waves. This change appears after carbachol withdrawal and implies a temporal potentiation of sensitivity (TPS) lasting up to one hour. TPS induction requires the simultaneous action of carbachol and glucose. The substitution of glucose with the secretagogues glyceraldehyde or 2‐ketoisocaproate mimics glucose‐induced TPS, while palmitate does not. TPS is not produced if the membrane is kept hyperpolarized by diazoxide. Glucose can be replaced by tolbutamide, suggesting a role of depolarization and a subsequent increase in intracellular calcium concentration. A role for kinases is suggested because staurosporine prevents TPS induction. Cycloheximide does not impair TPS induction, indicating that de novo protein synthesis is not required. The fact that the two inputs acting simultaneously produce an effect that lasts up to one hour without requiring de novo protein synthesis suggests that TPS constitutes a case of short‐term associative conditioning in non‐neural tissue. The convergence of basal glucose levels and muscarinic activation happens physiologically during the cephalic phase of digestion, in order to later absorb incoming fuels. Our data reveals that the role of the cephalic phase may be extended, increasing nutrient sensitivity during meals while remaining low between them.

Breakdown of phospholipids and the elevated nitric oxide are involved in M3 muscarinic regulation of acetylcholine secretion in the frog motor synapse

Previously, we found that muscarine downregulates the acetylcholine release at the frog neuromuscular junction acting via M3 muscarinic receptors. Here, the molecular mechanisms underlying the inhibitory effect of muscarine on the quantal secretion of acetylcholine were studied. Inhibition of phospholipase C (with U-73122) prevented the reduction of evoked neurotransmitter release induced by muscarine. Interruption of synthesis of phosphatidylinositol 3-phosphate by the inhibitor of phosphoinositide-3-kinase (wortmannin) did not affect the depressant action of muscarine but precluded the restoration of secretion after removal of muscarine from the bathing solution. The effect of muscarine was not significantly modified by the blockade of endocannabinoid receptors (with AM 281), but it was abolished by the inhibitor of nitric oxide synthase (L-NAME) as well as extracellular nitric oxide (NO) chelator (hemoglobin). Moreover, muscarine increased NO-sensitive dye fluorescence in junctional region, which was prevented by the M3 receptor antagonist 4-DAMP. The data obtained indicate that the attenuation of acetylcholine release mediated by muscarine is associated with a change in the activity of both lipid-metabolizing enzymes and NO synthases.

Muscarinic Modulation of SK2-Type K+ Channels Promotes Intrinsic Plasticity in L2/3 Pyramidal Neurons of the Mouse Primary Somatosensory Cortex.

Muscarinic acetylcholine receptors (mAChRs) inhibit small-conductance calcium-activated K+ channels (SK channels) and enhance synaptic weight via this mechanism. SK channels are also involved in activity-dependent plasticity of membrane excitability (“intrinsic plasticity”). Here, we investigate whether mAChR activation can drive SK channel-dependent intrinsic plasticity in L2/3 cortical pyramidal neurons. Using whole-cell patch-clamp recordings from these neurons in slices prepared from mouse primary somatosensory cortex (S1), we find that brief bath application of the mAChR agonist oxotremorine-m (oxo-m) causes long-term enhancement of excitability in wild-type mice that is not observed in mice deficient of SK channels of the SK2 isoform. Similarly, repeated injection of depolarizing current pulses into the soma triggers intrinsic plasticity that is absent from SK2 null mice. Intrinsic plasticity lowers spike frequency adaptation and attenuation of spike firing upon prolonged activation, consistent with SK channel modulation. Depolarization-induced plasticity is prevented by bath application of the protein kinase A (PKA) inhibitor H89, and the casein kinase 2 (CK2) inhibitor TBB, respectively. These findings point toward a recruitment of two known signaling pathways in SK2 regulation: SK channel trafficking (PKA) and reduction of the calcium sensitivity (CK2). Using mice with an inactivation of CaMKII (T305D mice), we show that intrinsic plasticity does not require CaMKII. Finally, we demonstrate that repeated injection of depolarizing pulses in the presence of oxo-m causes intrinsic plasticity that surpasses the plasticity amplitude reached by either manipulation alone. Our findings show that muscarinic activation enhances membrane excitability in L2/3 pyramidal neurons via a downregulation of SK2 channels.

Muscarinic modulation of M and h currents in gerbil spherical bushy cells.

Descending cholinergic fibers innervate the cochlear nucleus. Spherical bushy cells, principal neurons of the anterior part of the ventral cochlear nucleus, are depolarized by cholinergic agonists on two different time scales. A fast and transient response is mediated by alpha-7 homomeric nicotinic receptors while a slow and long-lasting response is mediated by muscarinic receptors. Spherical bushy cells were shown to express M3 receptors, but the receptor subtypes involved in the slow muscarinic response were not physiologically identified yet. Whole-cell patch clamp recordings combined with pharmacology and immunohistochemistry were performed to identify the muscarinic receptor subtypes and the effector currents involved. Spherical bushy cells also expressed both M1 and M2 receptors. The M1 signal was stronger and mainly somatic while the M2 signal was localized in the neuropil and on the soma of bushy cells. Physiologically, the M-current was observed for the gerbil spherical bushy cells and was inhibited by oxotremorine-M application. Surprisingly, long application of carbachol showed only a transient depolarization. Even though no muscarinic depolarization could be detected, the input resistance increased suggesting a decrease in the cell conductance that matched with the closure of M-channels. The hyperpolarization-activated currents were also affected by muscarinic activation and counteracted the effect of the inactivation of M-current on the membrane potential. We hypothesize that this double muscarinic action might allow adaptation of effects during long durations of cholinergic activation.

Neurons, Receptors, and Channels.

Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal excitability and ion channel function, largely, but not exclusively, using sympathetic neurons as test objects. Studies include effects of centrally active drugs on sympathetic transmission; neuronal action and neuroglial uptake of GABA in the ganglia and brain; the action of muscarinic agonists on sympathetic neurons; the action of bradykinin on neuroblastoma-derived cells; and the identification of M-current as a target for muscarinic action, including experiments to determine its distribution, molecular composition, neurotransmitter sensitivity, and intracellular regulation by phospholipids and their hydrolysis products. Techniques used include electrophysiological recording (extracellular, intracellular microelectrode, whole-cell, and single-channel patch-clamp), autoradiography, messenger RNA and complementary DNA expression, antibody injection, antisense knockdown, and membrane-targeted lipidated peptides. I finish with some recollections about my scientific career, funding, and changes in laboratory life and pharmacology research over the past 60 years.

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

Articles That May Change Your Practice: Sugammadex

Neuromuscular blocking agents (NMBAs) are typically used to facilitate tracheal intubation and mechanical ventilation and provide appropriate conditions for surgery. Prompt reversal of neuromuscular blockage is desired in some situations. Until recently, acetylcholinesterase inhibitors such as neostigmine were the only reversal agents available. These agents are often associated with bradycardia, bronchospasm, and other side effects because of their muscarinic activity. Anticholinergic agents used to relieve these muscarinic side effects are themselves associated with other unacceptable side effects. Russell D. MacDonald, MD, MPH, FCFP, FRCPC, is the medical director at Ornge Transport Medicine; medical director at Toronto Paramedic Services; an associate professor in the Faculty of Medicine at the University of Toronto; and an attending staff member at Sunnybrook Health Sciences Centre in Toronto, Ontario, Canada. He can be reached at .

Betel Nut (areca) and Smokeless Tobacco Use in Myanmar

Background: Betel nut (areca) is the world’s fourth most commonly used addictive substance. Arecoline, a muscarinic agonist in areca, is also a partial agonist for the addiction-related high-affinity brain nicotine receptors. In many countries, smokeless tobacco is commonly mixed with areca. Objective: We sought to evaluate the knowledge of self-harm, and addiction associated betel quid use in an unban population. Methods: We conducted a survey study of 200 betel quid users in Yangon, Myanmar, and a survey of betel quid vendors to determine the relative amounts of areca and tobacco in the available quids. Results: The data determined that a large majority of the survey subjects (84%) used tobacco with their areca. Users had a general awareness that betel chewing was “a bad habit” (85%) and 80% were aware of the cancer risks. Understanding areca addiction remains a challenge since, aside from the strong muscarinic activity of arecoline stimulating salivation, overt neurologic effects are difficult for even the users to identify. Fifty eight percent of the respondents indicated that chewing betel quid had effects like drinking coffee, and 55.5% indicated that it had effects like drinking alcohol. Data obtained from the quid vendors indicated that 75% added tobacco in equal amounts to areca. Conclusion: The concomitant use of nicotine and areca indicates that betel quid addiction includes a significant component of nicotine dependence. However, the additional activities of areca, including the muscarinic effects of arecoline, indicate that potential cessation therapies should optimally address other factors as well.

Synthesis and Pharmacological Activity of 2-(Diethylamino)Ethyl 9-Hydroxy-9H-Fluorene-9-Carboxylate Hydrochloride

New fluorenecarboxylic acid derivatives (structural analogs of amizil) were synthesized. A compound with low toxicity (LD50 = 45 ± 9.9 mg/kg) and antidepressant and antianxiety effects was identified. The central and peripheral muscarinic anticholinergic properties of 2-dimethylaminoethyl 9-hydroxyfluorene-9-carboxylate were studied using an arecoline tremor model. Apharmacological analysis showed that the new amizil structural analog exhibited pronounced central muscarinic anticholinergic activity and did not possess peripheral muscarinic anticholinergic effects. The advantages of the tested compound over amizil, amitriptyline that is widely used in the clinic, and the benzodiazepine tranquilizer diazepam were discussed. The results indicated that 2-diethylaminoethyl 9-hydroxyfluorene-9-carboxylate was promising for further development for the treatment of depressive and anxiety disorders.

GIRK Channel Activation Contributes to the Muscarinic Modulation of XII Motoneuron Excitability in Neonatal Mice in Vitro

Sleep specific decreases in airway muscle tone, and particularly the genioglossus muscle of the tongue (primary tongue protruder), have been causally implicated in obstructive sleep apnea. During REM (rapid eye movement) sleep, decreased excitability of the XII motoneurons that innervate the genioglossus muscle may be due to a loss of excitatory noradrenergic modulation, or due to activation of muscarinic acetylcholine receptors that may act via G‐protein coupled inward rectifying potassium (GIRK) channels. GIRK channel activation results in membrane hyperpolarization and thus could mediate a decrease in excitability of XII motoneurons during REM sleep. Here we test the hypothesis that muscarine acts via GIRK channels to inhibit inspiratory burst amplitude of XII motoneurons in vitro. Using immunohistochemical techniques, our preliminary data indicate that XII motoneurons express GIRK1, GIRK2, and GIRK3 isoforms. Next, using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day, P0–5), we investigated the effects of GIRK channel and muscarinic acetycholine receptor activation on XII inspiratory burst characteristics including burst amplitude and burst period. Local application of muscarine (30 s, 100 mM) to the XII motor nucleus had the net effect of increasing XII inspiratory burst amplitude, although local pre‐application of the GIRK channel antagonist Tertiapin Q (TQ, 60 s, 100 nM) led to two different responses. In P0–2 mice (n = 4), muscarine increased burst amplitude to 120% of control (range 105–133%) while pre‐application of TQ followed by local application of muscarine led to a similar 115% (range 104–123%) increase in amplitude, suggesting that muscarine does not activate GIRK channels in P0–2 mice. In P3–5 mice (n = 4), however, local application of muscarine increased burst amplitude to 135% (range 122–156%) of baseline, and pre‐application of TQ to block GIRK channel activation further increased the response to muscarine to 160% (range 130–170%) of baseline amplitude. These results indicate that muscarine acts at GIRK channels to inhibit XII motoneuron excitability in P3–5 mice, but also acts at other channels to enhance XII motoneuron excitability. Preliminary data suggests this effect is specific to muscarinic activation of GIRK channels because bath application of TQ (10 nM) in P3–5 mice resulted in no change in inspiratory burst amplitude (mean: 102% of baseline, range 90–109%, after 30 min in TQ, n = 3). In contrast, bath application of the GIRK channel agonist ML297 (10 mM) resulted burst amplitude decrease to 80–88% of baseline in 2 out of 3 P3–5 neonatal mice and an increase in burst amplitude to 120% of baseline in a third prep. Burst period increased to 179% of baseline (range: 128–263%) indicating that GIRK channels are likely also present in preBötzinger Complex where inspiratory rhythm is generated. These data support the hypothesis that activation of muscarinic receptors in XII motoneurons leads to GIRK channel activation in P3–5 neonatal mice, which may be one mechanism through which muscarine mediates a decrease in XII motoneuron excitability in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic activity in hippocampus and entorhinal cortex is crucial for spatial and fear memory retrieval.

Hippocampus and entorhinal cortex are key players of learning and memory. Despite their established role in memory processes, the contribution of muscarinic receptor activity in these brain regions during memory retrieval remains elusive. This study was aimed to assess the role of hippocampal CA1 and medial entorhinal cortex muscarinic receptors in memory retrieval. Mice were implanted with bilateral cannulas in the hippocampus CA1 and medial entorhinal cortex. After recovery they were trained for Morris water maze test, novel object recognition test and contextual fear conditioning. Scopolamine was infused 10 min prior to retrieval test. Pre-test scopolamine infusion in hippocampal CA1 and medial entorhinal cortex significantly reduced overall exploration of objects (p<0.001). Similarly, pre-retrieval inactivation dorsal hippocampal CA1 and medial entorhinal cortex muscarinic activity caused significant impairment of spatial and fear memories retrieval (p<0.05). These findings showed vital role of muscarinic activity in retrieving hippocampal and entorhinal cortex dependent memories and suggest a possible target for treating retrograde amnesia.

Simultaneous activation of muscarinic and GABAB receptors as a bidirectional target for novel antipsychotics

Recent preclinical studies point to muscarinic and GABAB receptors as novel therapeutic targets for the treatment of schizophrenia. This study was aimed to assess the role of muscarinic and GABAB receptor interactions in animal models of schizophrenia, using positive allosteric modulators (PAMs) of GABAB receptor (GS39783), muscarinic M4 (VU0152100) and M5 (VU0238429) receptor, and partial allosteric agonist of M1 receptor (VU0357017).DOI-induced head twitches, social interaction and novel object recognition tests were used as the models of schizophrenia. Analyses of DOI-induced increases in sEPSCs (spontaneous excitatory postsynaptic currents) were performed as complementary experiments to the DOI-induced head twitch studies. Haloperidol-induced catalepsy and the rotarod test were used to examine the adverse effects of the drugs.All three activators of muscarinic receptors were active in DOI-induced head twitches. When administered together with GS39783 in subeffective doses, only the co-administration of VU0152100 and GS39783 was effective. The combination also reduced the frequency but not the amplitude of DOI-induced sEPSCs. Neither VU0357017 nor VU0238429 were active in social interaction test when given alone, and also the combination of VU0152100 and GS39783 failed to reverse MK-801-induced deficits observed in this test. All muscarinic activators when administered alone or in combination with GS39783 reversed the MK-801-induced disruption of memory in the novel object recognition test, and their actions were blocked by specific antagonists. None of the tested compounds or their combinations influenced the motor coordination of the animals. The compounds had no effect on haloperidol-induced catalepsy and did not induce catalepsy when administered alone. Pharmacokinetic analysis confirmed lack of possible drug-drug interactions after combined administration of GS39783 with VU0357017 or VU0152100; however, when the drug was co-administered with VU0238429 its ability to pass the blood-brain barrier slightly decreased, suggesting potential drug-drug interactions.Our data show that modulation of cholinergic and GABAergic systems can potentially be beneficial in the treatment of the positive and cognitive symptoms of schizophrenia without inducing the adverse effects typical for presently used antipsychotics.

Gender dependent contribution of muscarinic receptors in memory retrieval under sub-chronic stress

Stress induces retrograde amnesia in humans and rodents. Muscarinic antagonism under normal physiological conditions causes gender dependent impairment in episodic memory retrieval. We aimed to explore the gender dependent role of muscarinic receptors in memory retrieval under sub-chronic stress condition. Male and female mice were trained for Morris water maze test and contextual fear conditioning, followed by 3 h restraint stress per day for five days. Stress was either given alone or in combination with a daily subcutaneous injection of scopolamine (1 mg/kg) or donepezil (1 mg/kg). Control mice were given saline without any stress. Sub-chronic stress (induced for five days) impaired spatial memory retrieval in males (P < 0.005) but not in females (P > 0.05). Stress induced spatial memory recall deficit in male mice was independent of muscarinic receptor activity (P > 0.05). However, stress induced contextual fear memory recall impairment was reversed by donepezil treatment in male (P < 0.005) and female (P < 0.0001) mice. These findings suggest that differential role of muscarinic activity in retrieving different types of memories under stress depends on gender of subjects.

Identification of Mepenzolate Derivatives With Long-Acting Bronchodilatory Activity.

The standard treatment for chronic obstructive pulmonary disease is a combination of anti-inflammatory drugs and bronchodilators. We recently found that mepenzolate bromide (MP), an antagonist for human muscarinic M3 receptor (hM3R), has both anti-inflammatory and short-acting bronchodilatory activities. To obtain MP derivatives with longer-lasting bronchodilatory activity, we synthesized hybrid compounds based on MP and two other muscarinic antagonists with long-acting bronchodilatory activity glycopyrronium bromide (GC) and aclidinium bromide (AD). Of these three synthesized hybrid compounds (MP-GC, GC-MP, MP-AD) and MP, MP-AD showed the highest affinity for hM3R and had the longest lasting bronchodilatory activity, which was equivalent to that of GC and AD. Both MP-GC and MP-AD exhibited an anti-inflammatory effect equivalent to that of MP, whereas, in line with GC and AD, GC-MP did not show this effect. We also confirmed that administration of MP-AD suppressed elastase-induced pulmonary emphysema in a mouse model. These findings provide important information about the structure-activity relationship of MP for both bronchodilatory and anti-inflammatory activities.

Lithium modulates the muscarinic facilitation of synaptic plasticity and theta-gamma coupling in the hippocampal-prefrontal pathway

Mood disorders are associated to functional unbalance in mesolimbic and frontal cortical circuits. As a commonly used mood stabilizer, lithium acts through multiple biochemical pathways, including those activated by muscarinic cholinergic receptors crucial for hippocampal-prefrontal communication. Therefore, here we investigated the effects of lithium on prefrontal cortex responses under cholinergic drive. Lithium-treated rats were anesthetized with urethane and implanted with a ventricular cannula for muscarinic activation, a recording electrode in the medial prefrontal cortex (mPFC), and a stimulating electrode in the intermediate hippocampal CA1. Either of two forms of synaptic plasticity, long-term potentiation (LTP) or depression (LTD), were induced during pilocarpine effects, which were monitored in real time through local field potentials. We found that lithium attenuates the muscarinic potentiation of cortical LTP (<20 min) but enhances the muscarinic potentiation of LTD maintenance (>80 min). Moreover, lithium treatment promoted significant cross-frequency coupling between CA1 theta (3–5 Hz) and mPFC low-gamma (30–55 Hz) oscillations. Interestingly, lithium by itself did not affect any of these measures. Thus, lithium pretreatment and muscarinic activation synergistically modulate the hippocampal-prefrontal connectivity. Because these alterations varied with time, oscillatory parameters, and type of synaptic plasticity, our study suggests that lithium influences prefrontal-related circuits through intricate dynamics, informing future experiments on mood disorders.