The Medial Orbitofrontal Cortex-Basolateral Amygdala Circuit Regulates the Influence of Reward Cues on Adaptive Behavior and Choice.

Adaptive reward-related decision making requires accurate prospective consideration of the specific outcome of each option and its current desirability. Often this information must be inferred based on the presence of predictive environmental events. The basolateral amygdala (BLA) and medial orbitofrontal cortex (mOFC) are two key nodes in the circuitry supporting such outcome expectations, but very little is known about the function of direct connections between these regions. Here, in male rats, we first anatomically confirmed the existence of bidirectional, direct projections between the mOFC and BLA and found that BLA projections to mOFC are largely distinct from those to lateral OFC (lOFC). Next, using pathway-specific chemogenetic inhibition and the outcome-selective Pavlovian-to-instrumental transfer and devaluation tests, we interrogated the function of the bidirectional mOFC-BLA connections in reward-directed behavior. We found evidence that the mOFC→BLA pathway mediates the use of environmental cues to understand which specific reward is predicted, information needed to infer which action to choose, and how desirable that reward is to ensure adaptive responses to the cue. By contrast, the BLA→mOFC pathway is not needed to use the identity of an expected reward to guide choice but does mediate adaptive responses to cues based on the current desirability of the reward they predict. These functions differ from those we previously identified for the lOFC-BLA circuit. Collectively, the data reveal the mOFC-BLA circuit as critical for the cue-dependent reward outcome expectations that influence adaptive behavior and decision making.SIGNIFICANCE STATEMENT To make good decisions we evaluate how advantageous a particular course of action would be. This requires understanding what rewarding outcomes can be expected and how desirable they currently are. Such prospective considerations are critical for adaptive decision making but disrupted in many psychiatric diseases. Here, we reveal that direct connections between the medial orbitofrontal cortex and basolateral amygdala mediate these functions. These findings are especially important in light of evidence of dysfunction in this circuit in substance use disorder and mental illnesses marked by poor decision making.

The Firing of Theta State-Related Septal Cholinergic Neurons Disrupt Hippocampal Ripple Oscillations via Muscarinic Receptors.

The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.

Group II muscarinic acetylcholine receptors attenuate hepatic injury via Nrf2/ARE pathway.

Parasympathetic nervous system dysfunction is common in patients with liver disease. We have previously shown that muscarinic acetylcholine receptors (mAchRs) play an important role in the regulation of hepatic fibrosis and that the receptor agonists and antagonists affect hepatocyte proliferation. However, little is known about the impact of the different mAchR subtypes and associated signaling pathways on liver injury. Here, we treated the human liver cell line HL7702 with 10 mmol/L carbon tetrachloride (CCL4) to induce hepatocyte damage. We found that CCL4 treatment increased the protein levels of group I mAchRs (M1, M3, M5) but reduced the expression of group II mAchRs (M2, M4) and activated the Nrf2/ARE and MAPK signaling pathways. Although overexpression of M1, M3, or M5 led to hepatocyte damage with an intact Nrf2/ARE pathway, overexpression of M2 or M4 increased, and siRNA-mediated knockdown of either M2 or M4 decreased the protein levels of Nrf2 and its downstream target genes. Moreover, CCL4 treatment increased serum ALT levels more significantly, but only induced slight changes in the expression of mAchRs, NQO1 and HO1, while reducing the expression of M2 and M4 in liver tissues of Nrf2-/- mice compared to wild type mice. Our findings suggest that group II mAchRs, M2 and M4, activate the Nrf2/ARE signaling pathway, which regulates the expression of M2 and M4, to protect the liver from CCL4-induced injury.

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice.

Mutations that lead to Huntington's disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M4 subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M4 positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M4 PAMs could provide a therapeutic strategy for the treatment of HD.

Impaired functional organization in the visual cortex of muscarinic receptor knock-out mice.

Acetylcholine modulates maturation and neuronal activity through muscarinic and nicotinic receptors in the primary visual cortex. However, the specific contribution of different muscarinic receptor subtypes in these neuromodulatory mechanisms is not fully understood. The present study evaluates in vivo the functional organization and the properties of the visual cortex of different groups of muscarinic receptor knock-out (KO) mice. Optical imaging of intrinsic signals coupled to continuous and episodic visual stimulation paradigms was used. Retinotopic maps along elevation and azimuth were preserved among the different groups of mice. However, compared to their wild-type counterparts, the apparent visual field along elevation was larger in M2/M4-KO mice but smaller in M1-KO. There was a reduction in the estimated relative receptive field size of V1 neurons in M1/M3-KO and M1-KO mice. Spatial frequency and contrast selectivity of V1 neuronal populations were affected only in M1/M3-KO and M1-KO mice. Finally, the neuronal connectivity was altered by the absence of M2/M4 muscarinic receptors. All these effects suggest the distinct roles of different subtypes of muscarinic receptors in the intrinsic organization of V1 and a strong involvement of the muscarinic transmission in the detectability of visual stimuli.

Effects of selective activation of M1 and M4 muscarinic receptors on object recognition memory performance in rats.

Acetylcholine signaling through muscarinic receptors has been shown to benefit memory performance in some conditions, but pan-muscarinic activation also frequently leads to peripheral side effects. Drug therapies that selectively target M1 or M4 muscarinic receptors could potentially improve memory while minimizing side effects mediated by the other muscarinic receptor subtypes. The ability of three recently developed drugs that selectively activate M1 or M4 receptors to improve recognition memory was tested by giving Long-Evans rats subcutaneous injections of three different doses of the M1 agonist VU0364572, the M1 positive allosteric modulator BQCA or the M4 positive allosteric modulator VU0152100 before performing an object recognition memory task. VU0364572 at 0.1 mg/kg, BQCA at 1.0 mg/kg and VU0152100 at 3.0 and 30.0 mg/kg improved the memory performance of rats that performed poorly at baseline, yet the improvements in memory performance were the most statistically robust for VU0152100 at 3.0 mg/kg. The results suggested that selective M1 and M4 receptor activation each improved memory but that the likelihood of obtaining behavioral efficacy at a given dose might vary between subjects even in healthy groups depending on baseline performance. These results also highlighted the potential of drug therapies that selectively target M1 or M4 receptors to improve memory performance in individuals with impaired memory.

Role of various subtypes of muscarinic cholinergic receptors in the development of posthemorrhagic abnormalities in systemic and portal circulation in rats.

The experiments employing high-frequency ultrasonic technique and selective blockers of M1, M3, and M4 muscarinic cholinergic receptors pirenzepine, 4-DAMP, and tropicamide, respectively, revealed individual roles of these receptors in the development of severe posthemorrhagic hypotension in rats with low or high individual resistance to circulatory hypoxia. The study showed that M1 and M4 muscarinic receptors are involved in shock-limiting and shock-activating processes, respectively, while M3 receptors exert no effect on the course of posthemorrhagic abnormalities in systemic and hepatic portal circulation and on the posthemorrhagic lifespan. Poor resistance of the cardiovascular system to circulatory hypoxia during shock development is considered to be dysregulatory pathology.

Diabetic neuropathy enhances voltage-activated Ca2+ channel activity and its control by M4 muscarinic receptors in primary sensory neurons.

Painful neuropathy is one of the most serious complications of diabetes and remains difficult to treat. The muscarinic acetylcholine receptor (mAChR) agonists have a profound analgesic effect on painful diabetic neuropathy. Here we determined changes in T-type and high voltage-activated Ca(2+) channels (HVACCs) and their regulation by mAChRs in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathy. The HVACC currents in large neurons, T-type currents in medium and large neurons, the percentage of small DRG neurons with T-type currents, and the Cav3.2 mRNA level were significantly increased in diabetic rats compared with those in control rats. The mAChR agonist oxotremorine-M significantly inhibited HVACCs in a greater proportion of DRG neurons with and without T-type currents in diabetic than in control rats. In contrast, oxotremorine-M had no effect on HVACCs in small and large neurons with T-type currents and in most medium neurons with T-type currents from control rats. The M(2) and M(4) antagonist himbacine abolished the effect of oxotremorine-M on HVACCs in both groups. The selective M(4) antagonist muscarinic toxin-3 caused a greater attenuation of the effect of oxotremorine-M on HVACCs in small and medium DRG neurons in diabetic than in control rats. Additionally, the mRNA and protein levels of M(4), but not M(2), in the DRG were significantly greater in diabetic than in control rats. Our findings suggest that diabetic neuropathy potentiates the activity of T-type and HVACCs in primary sensory neurons. M(4) mAChRs are up-regulated in DRG neurons and probably account for increased muscarinic analgesic effects in diabetic neuropathic pain.

Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders.

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer's disease (AD) and other disorders involving impaired cognitive function. More recent evidence indicates that mAChR activators might also have utility in treating psychosis and other symptoms associated with schizophrenia and other central nervous system (CNS) disorders. Efforts to develop mAChR subtype-selective agonists have been hampered by difficulty in achieving high selectivity for individual mAChR subtypes important for CNS function (M(1) and M(4)) and adverse effects due to activation of peripheral mAChRs (especially M(2) and M(3)). Major advances have now been achieved in the discovery of allosteric agonists and positive allosteric modulators of M(1) and M(4) that show greater selectivity for individual mAChR subtypes than do previous mAChR agonists. Early studies indicate that these allosteric mAChR activators have properties needed for optimization as potential clinical candidates and have robust effects in animal models that predict efficacy in the treatment of AD, schizophrenia and related disorders.

Revealing a compulsive phenotype in cholinergic M4-/- mice depends on the inter-trial interval initiation settings in a five choice serial reaction time task.

BACKGROUND: Muscarinic acetylcholine receptor 4 (M4) modulates dopaminergic neurotransmission and is a target for novel treatments of schizophrenia, cognitive deficits, and addiction. Impulsive and compulsive behaviors are key traits of addiction, yet the importance of M4 receptor signaling to these traits is poorly understood. We investigated impulsive action and compulsivity by measuring premature and perseverative responses in the five choice serial reaction time task (5CSRTT). Furthermore, we hypothesized that inter-trial interval (ITI) initiation settings affected training durations and test performances in these experiments. METHODS: M4-/- and wildtype mice were trained and tested on two versions of the 5CSRTT with different ITI initiation settings. One setting, the head-in condition, allowed the ITI to start while the mouse's head remained in the reward receptacle (magazine). The other setting, the head-out condition, required the mouse to remove its head from the magazine to initiate the ITI. RESULTS AND DISCUSSION: We did not observe differences in premature or perseverative responses in M4-/- mice in either condition, but found evidence of reward-related compulsive behavior in M4-/- mice. In the head-in condition, M4-/- mice were slower to acquire the 5CSRTT, had more omissions, and had longer correct response latencies than wildtype mice. In the head-out condition, genotypes did not differ in training, but M4-/- mice showed small decreases in accuracy. Our findings demonstrate that ITI initiation settings contribute to different training durations and tested behaviors in M4-/- mice, suggesting ITI initiation settings are an important consideration for the general use of the 5CSRTT.

Lack of M4 muscarinic receptors in the striatum, thalamus and intergeniculate leaflet alters the biological rhythm of locomotor activity in mice.

The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR -/- mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

M4 muscarinic receptors are involved in modulation of neurotransmission at synapses of Schaffer collaterals on CA1 hippocampal neurons in rats.

All five subtypes of muscarinic acetylcholine receptors (mAChR; M(1)-M(5)) are expressed in the hippocampus, where they are involved both in cognitive functions and in synaptic plasticity, such as long-term potentiation (LTP). Muscarinic toxins (MTs) are small proteins from mamba snake venoms that display exquisite discrimination between mAChRs. MT1 acts as an agonist at M(1) and an antagonist at M(4) receptors, with similar affinities for both. MT3, the most selective antagonist available for M(4) receptors, infused into the CA1 region immediately after training caused amnesia in the rat, indicating the participation of M(4) receptors in memory consolidation. Our goal was to investigate the participation of M(4) receptor in neurotransmission at the hippocampal Schaffer collaterals-CA1 synapses. Two different preparations were used: 1) field potential recordings in freshly prepared rat hippocampal slices with high-frequency stimulation to induce potentiation and 2) whole-cell voltage clamp in cultured hippocampal organotypic slices with paired stimuli. In preparation 1, a dose of MT3 that was previously shown to cause amnesia blocked LTP; the nonselective antagonist scopolamine blocked LTP without affecting basal transmission, although it was depressed with higher concentration. In preparation 2, basal transmission was decreased and LTP induction was prevented by an MT3 concentration that would bind mainly to M(4) receptors. Although M(1) receptors appeared to modulate transmission positively at these excitatory synapses, M(1) activation concomitant with M(4) blockade (by MT1) only allowed a brief, short-term potentiation. Accordingly, M(4) blockade by MT3 strongly supports a permissive role of M(4) receptors and suggests their necessary participation in synaptic plasticity at these synapses.

Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors.

To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by M4 and inhibited by M3. Additional studies showed that M4 increases expression of "migratory" integrins alpha5beta1, alphaVbeta5, and alphaVbeta6, whereas M3 up-regulates "sedentary" integrins alpha2beta1 and alpha3beta1. Inhibition of migration by M3 was mediated through Ca2+-dependent guanylyl cyclase-cyclic GMP-protein kinase G signaling pathway. The M4 effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclase-cyclic AMP-protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for M3 and M4 regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.

In Vivo Modulation of Hippocampal Excitability by M4 Muscarinic Acetylcholine Receptor Activator: Implications for Treatment of Alzheimer's Disease and Schizophrenic Patients.

Abnormal hippocampal activity has been linked to impaired cognitive performance in Alzheimer's disease and schizophrenia, leading to a hypothesis that normalization of this activity may be therapeutically beneficial. Our work suggests that one approach for hippocampal normalization may be through activation of the M4 muscarinic acetylcholine receptor. We used a brain penetrant M4 muscarinic acetylcholine receptor selective activator, PT-3763, to show dose-dependent attenuation of field potentials in Schaffer collateral (CA3-CA1) and recurrent associational connections (CA3-CA3) ex vivo in hippocampal slices. In vivo, systemic administration of PT-3763 led to attenuation of glutamate release in CA3 as measured by amperometry and to a dose-dependent decrease in population CA1 pyramidal activity as measured by fiber photometry. This decrease in population activity was also evident with a localized administration of the compound to the recorded site. Finally, PT-3763 reversed scopolamine-induced deficit in Morris water maze. Our results suggest that M4 muscarinic acetylcholine receptor activation may be a suitable therapeutic treatment in diseases associated with hyperactive hippocampal activity.

New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD (designer receptor exclusively activated by a designer drug).

The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.

A role for protein kinase A and protein kinase M zeta in muscarinic acetylcholine receptor-initiated persistent synaptic enhancement in rat hippocampus in vivo.

Antagonists at presynaptic muscarinic autoreceptors increase endogenous acetylcholine (ACh) release and enhance cognition but little is known regarding their actions on plasticity at glutamatergic synapses. Here the mechanisms of the persistent enhancement of hippocampal excitatory transmission induced by the M2/M4 muscarinic ACh receptor antagonist methoctramine were investigated in vivo. The persistent facilitatory effect of i.c.v. methoctramine in the CA1 region of urethane-anesthetized rats was mimicked by gallamine, an M2 receptor antagonist, supporting a role for this receptor subtype. Neither the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino phosphonopentanoic acid (d-AP5) and memantine, nor the metabotropic glutamate receptor subtype 1a antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) significantly affected the methoctramine-induced persistent synaptic enhancement, indicating a lack of requirement for these glutamate receptors. The selective kinase inhibitors Rp-adenosine-3', 5'-cyclic monophosphorothioate (Rp-cAMPS) and the myrostylated pseudosubstrate peptide, Myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH (ZIP), were used to investigate the roles of protein kinase A (PKA) and the atypical protein kinase C, protein kinase Mzeta (PKM zeta), respectively. Remarkably, pretreatment with either agent prevented the induction of the persistent synaptic enhancement by methoctramine and post-methoctramine treatment with Rp-cAMPS transiently reversed the enhancement. These findings are strong evidence that antagonism of M2 muscarinic ACh receptors in vivo induces an NMDA receptor-independent persistent synaptic enhancement that requires activation of both PKA and PKM zeta.

Effects of muscarinic M1 and M4 acetylcholine receptor stimulation on extinction and reinstatement of cocaine seeking in male mice, independent of extinction learning.

RATIONALE: Stimulating muscarinic M1/M4 receptors can blunt reinforcing and other effects of cocaine. A hallmark of addiction is continued drug seeking/craving after abstinence and relapse. OBJECTIVES: We tested whether stimulating M1 and/or M4 receptors could facilitate extinction of cocaine seeking, and whether this was mediated via memory consolidation. METHODS: Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1 mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding extinguished to ≤30% of cocaine-reinforced responding. Immediately after each extinction session, mice received saline, the M1/M4 receptor-preferring agonist xanomeline, the M1 receptor-selective allosteric agonist VU0357017, the M4 receptor-selective positive allosteric modulator VU0152100, or VU0357017 + VU0152100. In additional experiments, xanomeline was administered delayed after the session or in the home cage before extinction training began. In the latter group, reinstatement of responding by a 10-mg/kg cocaine injection was also tested. RESULTS: Stimulating M1 + M4 receptors significantly expedited extinction from 17.2 sessions to 8.3 using xanomeline or 7.8 using VU0357017 + VU0152100. VU0357017 alone and VU0152100 alone did not significantly modify rates of extinction (12.6 and 14.6 sessions). The effect of xanomeline was fully preserved when administered delayed after or unpaired from extinction sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. CONCLUSIONS: These findings show that M1/M4 receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests that M1/M4 receptor stimulation modulated or reversed some neurochemical effects of cocaine exposure.

Activation of muscarinic M4 receptor augments NGF-induced pro-survival Akt signaling in PC12 cells.

Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). Recent studies have demonstrated that GPCRs can modulate signals elicited by receptor tyrosine kinases (RTK) and vice versa. Here, we examined the activity of pro-survival Akt kinase, in response to stimulation by muscarinic acetylcholine receptors (mAChRs) and co-activation with the nerve growth factor (NGF) receptor in PC12 cells endogenously expressing Gi-coupled M4 mAChR and Gq-coupled M1 and M5 mAChRs. Western blotting analysis using a phosphospecific anti-Akt antibody revealed a dose- and time-dependent increase in Akt phosphorylation in cells stimulated with mAChR specific agonist carbachol (CCh). Co-stimulation with CCh and NGF resulted in augmentation of Akt activity in a pertussis toxin (PTX)-sensitive manner, suggesting that M4 mAChR, but not M1 and M5 mAChRs, was associated with this synergistic Akt activation. The use of transducin as a Gbetagamma scavenger indicated that Gbetagamma subunits rather than Galphai/o acted as the signal transducer. Additional experiments showed that CCh treatment augmented NGF-induced phosphorylation and degradation of the Akt-regulated translation regulator tuberin. This augmentation was also inhibited by PTX pre-treatment or overexpression of transducin. Finally, co-stimulation of PC12 cells with CCh and NGF resulted in enhancement of cell survival. This is the first study that demonstrates the augmentation effect between M4 mAChR and NGF receptor, and the regulatory role of mAChR on tuberin.

The M4 muscarinic acetylcholine receptor plays a key role in the control of murine hair follicle cycling and pigmentation.

Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in M4R KO mice, compared to age-matched wild type controls. On day 17, when mice enter the first hair growth cycle, the KO mice still showed a slightly retarded catagen phase. Subsequently, hair follicles of the KO mice stayed in a highly significantly prolonged telogen phase, while wild type mice had already far progressed in the hair cycle by entry into anagen. Most strikingly, the M4R KO mice did not engage in follicular melanogenesis and failed to produce pigmented hair shafts. The current pilot study suggests that the M4R plays a fundamental role in the control of the murine hair follicle cycling and is an essential signaling element in the control of hair follicle pigmentation.

M(4) muscarinic receptors and locomotor activity regulation.

M(4) muscarinic receptors (M(4) MR) represent a subfamily of G-protein coupled receptors serving a substantial role in spontaneous locomotor activity regulation, cognition and modulation of cholinergic system. With increasing body of literature discussing the role of M(4) MR some controversies arose. Thus, we try here to summarize the current evidence regarding the M(4) MR, with the special focus on their role in Locomotor activity control. We review the molecular function of M(4) MR in specific brain areas implicated in locomotor regulation, and shortly in other CNS processes that could be connected to locomotor activity. We also focus on brain areas implicated in locomotor activity biorhythm changes like suprachiasmatic nucleus, subparaventricular zone posterior hypothalamic area, striatum and thalamus. Gender-related aspects and differences in locomotor activity in males and females are discussed further.