mAChR Activation Research Articles

A rapid in vitro assay for evaluating the effects of acetylcholinesterase inhibitors and reactivators in the rat basolateral amygdala.

We established a novel brain slice assay to test the ability of acetylcholinesterase (AChE) reactivators to prevent ACh-induced M1 muscarinic acetylcholine receptor (mAChR) dependent hyperexcitability observed after exposure to the organophosphate (OP)-based AChE inhibitor and sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). Whole-cell patch clamp recordings were used to evaluate the response of pyramidal neurons in the rat basolateral amygdala (BLA) to brief (1 min) bath application of ACh (100 μM), either in control conditions, or after exposure to NIMP ± an AChE reactivator. Bath application of ACh produced atropine- and pirenzepine-sensitive inward currents in voltage clamped BLA pyramidal neurons, and increased the frequency of spontaneous EPSCs, suggesting robust activation of M1 mAChRs. Responses to ACh were increased ~3-5 fold in slices that had been preincubated in NIMP, and these effects were reversed in a concentration dependent manner by exposure to a commercially available AChE reactivator. The current work outlines a simple assay that can be used to evaluate the efficacy of both known and novel AChE reactivators in an area of the limbic system that likely contributes to seizures after acute exposure to OP-based AChE inhibitors.

The dependence of acetylcholine on dynamic changes in the membrane potential and an action potential during spike timing-dependent plasticity induction in the hippocampus.

The hippocampus is an important area for memory encoding and retrieval and is the location of spike timing-dependent plasticity (STDP), a basic phenomenon of learning and memory. STDP is facilitated if acetylcholine (ACh) is released from cholinergic neurons during attentional processes. However, it is unclear how ACh influences postsynaptic changes during STDP induction and determines the STDP magnitude. To address these issues, we obtained patch clamp recordings from CA1 pyramidal neurons to evaluate the postsynaptic changes during stimuli injection in Schaffer collaterals by quantifying baseline amplitudes (i.e., the lowest values elicited by paired pulses comprising STDP stimuli) and action potentials. The results showed that baseline amplitudes were elevated if eserine was applied in the presence of picrotoxin. In addition, muscarinic ACh receptors (mAChRs) contributed more to the baseline amplitude elevation than nicotinic AChRs (nAChRs). Moreover, the magnitude of the STDP depended on the magnitude of the baseline amplitude. However, in the absence of picrotoxin, baseline amplitudes were balanced, regardless of the ACh concentration, resulting in a similar magnitude of the STDP, except under the nAChR alone-activated condition, which showed a larger STDP and lower baseline amplitude induction. This was due to broadened widths of action potentials. These results suggest that activation of mAChRs and nAChRs, which are effective for baseline amplitudes and action potentials, respectively, plays an important role in postsynaptic changes during memory consolidation.

Dorsal hippocampal muscarinic cholinergic receptors orchestrate behavioral and autonomic changes induced by contextual fear retrieval

Re-exposure of rats to a previously fear-conditioned environment arouses great alterations in behavioral and cardiovascular parameters. Pieces of works provide putative evidence for the contribution of the dorsal hippocampus (dHC) to contextual conditioning. dHC gathers massive cholinergic inputs from the basal forebrain, and dHC acetylcholine (ACh) is often described as triggering the retrieval of defensive behavior. ACh acts partially through muscarinic receptors (mAChRs) M1R and M3R subtypes. Hence, activation of mAChRs facilitates autonomic and behavioral responses associated with threats and dangers. Therefore, this study explored the likely involvement of M1R and M3R in rat dHC to establish the behavioral and autonomic changes associated with contextual fear retrieval. Male Wistar rats had stainless steel guide cannula implanted into the dHC before being submitted to contextual fear conditioning (6 footshocks, 1.5 mA, 3 s). A catheter placed within the femoral artery allowed autonomic recordings. A variety of drugs were delivered into the dHC 10 min before contextual re-exposure. The choline reuptake inhibitor hemicholinium induced a decrease of the fear conditioned responses, while did not modify it in non-conditioned animals. The non-selective mAChR antagonist atropine also reduced the fear-conditioned responses, as did the selective M1/M3 mAChRs antagonist fumarate. On the other hand, the M1 selective mAChR antagonist pirenzepine inhibited all the autonomic fear responses without affecting animal freezing. These findings support that cholinergic neurotransmission present in the dHC acts through mAChRs to coordinate the expression of fear evoked by contextual conditioning.

Muscarinic acetylcholine receptors regulate inflammatory responses through arginases 1/2 in zebrafish

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in various effector cells and have been proved to play vital roles in smooth muscle contraction and digestive secretion. However, there are relatively few literatures revealing the roles of mAChRs in inflammatory processes, and its underlying regulatory mechanisms have not been elucidated. Taking the advantages of live imaging of zebrafish, we found that inhibition of mAChRs resulted in increased neutrophils recruitment and proinflammatory cytokines expression, whereas activation of mAChRs led to opposite outcome. Subsequently, we found that mAChRs regulated the expression of arginases (args), and pharmacological intervention of args level could reverse the effects of mAChRs on neutrophils migration and cytokines expression, suggesting that args are important downstream proteins of mAChRs that mediate the regulation of inflammatory response. In this study, we identified args as novel downstream proteins of mAChRs in inflammatory responses, providing additional evidence for system immune regulation of cholinergic receptors.

Abstract 905: Targeting muscarinic acetylcholine receptors in glioma stem like cells

Abstract Primary gliomas arising within the brain remain the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioblastoma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass that are likely responsible for tumor initiation and recurrence of tumors post resection and give rise to drug resistant tumors. Determining a way to suppress these malignant characteristics and/or depleting the GSC population could improve current cancer treatments and the survival of glioma patients. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), found during neural development and also residing in the adult brain. OPCs are prone to malignant transformation and believed to be a cellular origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We investigated the functional characterization of ACh and mAChRs in modulating malignancy in OPC-like cells. We studied cultures of primary mouse OPC-like GSCs. We used the primary cells to culture three-dimensional tumor organoids in vitro to better represent tumor heterogeneity. We also used patient derived xenografts (PDX) of glioma to grow flank xenografts in NSG mice for an in vivo model. Publicly available data and studies in our lab show high levels of expression of CHRM3 (M3mAChR) in glioma patients and in primary OPC like GSCs. A drug screen conducted in the context of multiple sclerosis determined that benztropine (BZT), an anti muscarinic drug targeting M3mAChR, causes normal OPCs to exit the cell cycle, lose stem cell like characteristics, and differentiate. In our lab, electrophysiological studies demonstrated that activation of mAChRs in OPC-like cells from mouse and patient tumors generates rapid (< 1 second) increases in cytosolic calcium. Pharmacologic studies indicated that treatment with FDA approved anti muscarinic benztropine suppresses proliferation in cultured glioma cells. We also observed that serially passaging flank tumors treated with BZT into new host mice slowed down recurrence of new tumors. In order to determine key receptors mediating cholinergic responses in OPC-like cells we performed protein quantification, which displayed lowered phosphorylation of ERK. Electrophysiological studies are being conducted to dissect the mechanisms by which ACh evokes calcium release. These studies improve understanding of how cholinergic microenvironment influences stem-like glioma cells, providing a platform for repositioning available small molecule modifiers for treatment of glioma. Citation Format: Sumyuktha V. Anand, Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, Matthew C. Havrda. Targeting muscarinic acetylcholine receptors in glioma stem like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 905.

Functional adaptation of glial cells at neuromuscular junctions in response to injury.

Synaptic elements from neuromuscular junctions (NMJs) undergo massive morphological and functional changes upon nerve injury. While morphological changes of NMJ‐associated glia in response to injury has been investigated, their functional properties remain elusive. Perisynaptic Schwann cells (PSCs), glial cells at the NMJ, are essential for NMJ maintenance and repair, and are involved in synaptic efficacy and plasticity. Importantly, these functions are regulated by PSCs ability to detect synaptic transmission through, notably, muscarinic (mAChRs) and purinergic receptors' activation. Using Ca2+ imaging and electrophysiological recordings of synaptic transmission at the mouse NMJ, we investigated PSC receptors activation following denervation and during reinnervation in adults and at denervated NMJs in an ALS mouse model (SOD1G37R). We observed reduced PSCs mAChR‐mediated Ca2+ responses at denervated and reinnervating NMJs. Importantly, PSC phenotypes during denervation and reinnervation were distinct than the one observed during NMJ maturation. At denervated NMJs, exogenous activation of mAChRs greatly diminished galectin‐3 expression, a glial marker of phagocytosis. PSCs Ca2+ responses at reinnervating NMJs did not correlate with the number of innervating axons or process extensions. Interestingly, we observed an extended period of reduced PSC mAChRs activation after the injury (up to 60 days), suggesting a glial memory of injury. PSCs associated with denervated NMJs in an ALS model (SOD1G37R mice) did not show any muscarinic adaptation, a phenotype incompatible with NMJ repair. Understanding functional mechanisms that underlie this glial response to injury may contribute to favor complete NMJ and motor recovery.

Nonlinear relationship between CAN current and influx underpins synergistic action of muscarinic and NMDA receptors on bursts induction in midbrain dopaminergic neurons.

Bursting of midbrain dopamine (DA) neurons is believed to represent an important reward signal that instructs and reinforces goal-directed behaviors. In DA neurons, many afferents, including cholinergic and glutamatergic inputs, induce bursting, and it is suggested that a synergy exists between these afferents in bursting induction. However, the underlying mechanisms of the role and the synergy of muscarinic receptors (mAChRs) and NMDA receptors (NMDARs) in bursting induction remain unclear. Present work bestowed analysis using a mathematical model of DA neurons to demonstrate the underlying mechanisms. Activation of mAChRs, leading to rapid translocation of TRPC channels to cell surface, recruited -activated nonspecific (CAN) current ( ), meanwhile NMDARs excitation triggered influx, which induced the positive feedback loop of and , respectively, yielded a robust ramping depolarization with a superimposed high-frequency spiking. In some DA cells, neither NMDARs nor mAChRs induced positive feedback loop unless they were activated simultaneously to induce bursting. Our experimental results verified those theoretical findings. These together unveil the underlying mechanisms of the role and synergy of mAChRs and NMDARs in bursting induction emerge from the nonlinear relationship between influx and . Given the diverse and complex nature of neural circuitry and the DA neuron heterogeneity, our work provides new insights to understand specific afferents, the synergy between those afferents, and the differences in intrinsic excitability to be integrated by the bursting to accurately characterize the dopamine signals in the valances of reward and reinforcement, and a broad spectrum of neuropsychiatric disorders.

Acetylcholine receptor agonists provide cardioprotection in doxorubicin-induced cardiotoxicity via modulating muscarinic M2 and α7 nicotinic receptor expression

The balance between cardiac sympathetic and parasympathetic activities has been intricately linked to mitochondrial function, cellular oxidative status, and immunomodulation in healthy and diseased myocardium. Cardiac autonomic neuropathy, along with the associated mitochondrial and cellular dysfunction, is an important pathophysiological feature of doxorubicin-induced cardiotoxicity (DIC). We tested the hypothesis that autonomic modulation by activation of acetylcholine receptors (AChR) effectively attenuates DIC. Rats were divided into control (0.9% sodium chloride solution) and doxorubicin groups (DOX, 3 mg/kg/d, 6 doses). Rats in the DOX group were equally subdivided into 4 interventional groups and treated for 30 days: vehicle, α7 nicotinic receptor agonist (PNU: PNU-282987, 3 mg/kg/d), muscarinic receptor agonist (BET: bethanechol, 12 mg/kg/d), and combined α7nAChR and mAChR agonists group (COM). Cardiac biochemical and functional analyses were done. The results show that AChR agonists protected the heart against DIC via improving mitochondrial and cardiac function, which was accompanied by reducing mitochondrial oxidative damage, apoptosis, and inflammation. Strikingly, PNU and BET exerted cardioprotection through different molecular pathways. PNU-mediated α7nAChR activation promoted mitochondrial fusion via upregulation of Mfn1-2 and attenuated DOX-induced autophagy. Contrarily, activation of mAChR by BET attenuated mitochondrial fission and mitophagy. The in vitro experiments confirmed the cytoprotective effects of AChR activation in DOX-treated H9c2 cells without compromising the anticancer effect of DOX in cancer cells. In conclusion, α7nAChR and mAChR agonists exerted cardioprotection against DIC via rebalancing autonomic function, improving mitochondrial function, reducing oxidative stress, and decreased cardiomyocyte apoptosis and inflammation, leading to improved cardiac function.

Muscarinic (M1 ) cholinergic receptor activation within the dorsal hippocampus promotes destabilization of strongly encoded object location memories.

Following the initial consolidation process, memories can become reactivated by exposure to a reminder of the original learning event. This can lead to the memory becoming destabilized and vulnerable to disruption or other forms of modification. The memory must then undergo the protein-synthesis dependent process of reconsolidation in order to be retained. However, older and/or stronger memories resist destabilization, but can become labile when reactivated in the presence of salient novelty. We have implicated the neurotransmitter acetylcholine, acting at M1 muscarinic cholinergic receptors (mAChRs) within perirhinal cortex (PRh), in novelty-induced destabilization of remote object memories. It remains unclear, however, whether mAChRs are involved in destabilization of other forms of memory. We hypothesized that the role of M1 mAChRs previously demonstrated for PRh-dependent object memory would extend to hippocampus-dependent spatial memory. Using the object location (OL) task, which relies on the dorsal hippocampus (dHPC), we showed that (a) reactivation-dependent reconsolidation of OL memories requires protein synthesis within the dHPC; (b) destabilization of relatively weak OL memories depends on M1 mAChR activation within the dHPC; (c) salient novelty during reactivation promotes destabilization of resistant strongly encoded OL memories; (d) novelty-induced destabilization of strong OL memories requires activation of mAChRs within the dHPC; and (e) M1 mAChR activation within the dHPC in the absence of novelty during memory reactivation mimics the effect of novelty, destabilizing strongly encoded OL memories. These results implicate ACh acting at M1 mAChRs in the destabilization of dHPC-dependent spatial memories, demonstrating generalizability of this cholinergic function beyond memory for object identity. These findings therefore enhance our understanding of the dynamics of long-term memory storage and suggest implications for the treatment of human conditions such as Alzheimer's disease and aging, which are characterized by behavioral and mnemonic inflexibility.

Oxidative stress and the cholinergic system in non-segmental vitiligo: Effect of narrow band ultraviolet b.

Despite exhaustive research, melanocyte disappearance and the evolution of vitiligo remain enigmatic, and although multi-factorial, oxidative stress appears as a major player. The role of cutaneous cholinergic system in vitiligo pathogenesis has also been reported in some studies. To evaluate and correlate the influence of phototherapy on cutaneous cholinergic system and oxidative stress in vitiligo. Acetyl choline (ACh), its receptors; nicotinic (nAChR) and muscarinic (mAChR); acetylcholine esterase (AChE) and H2 O2 levels were estimated in de-pigmented and re-pigmented lesions of 30 vitiligo patients before and after NB-UVB phototherapy and in 30 controls. ACh and H2 O2 levels were measured by colorimetry. AChE and acetylcholine receptors expression were measured by quantitative real-time PCR. Mean ACh and H2 O2 levels were significantly higher in vitiligo lesions before NB-UVB (P<.001) whereas AChE enzyme level was significantly lower (P<.001) compared to both re-pigmented and control skin. Additionally, mean mAChR was significantly higher and mean nAChR was significantly lower in vitiligo lesions before NB-UVB versus controls and re-pigmented skin (P<.001). Also, H2 O2 and AChE showed negative correlation whereas ACh and mAChR showed significant positive correlation. Although all the studied parameters showed significant changes after treatment and subsequent re-pigmentation, a significant difference continued to exist between all vitiligo skin and controls. Cholinergic system is strongly involved in vitiligo pathogenesis through H2 O2 inhibition of AChE which could be reversed by NB-UVB. Moreover, the strong activation of mAChRs may reflect genetic and/or acquired errors, direct up-regulation by ACh and H2 O2 or both.

Muscarinic Acetylcholine Receptors Modulate Interleukin-6 Production and Immunoglobulin Class Switching in Daudi Cells.

B cells express muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively). Following immunization with ovalbumin, serum immunoglobulin G (IgG) and interleukin (IL)-6 levels were lower in M1 and M5 mAChR double-deficient mice and higher in α7 nAChR-deficient mice than in wild-type mice. This suggests mAChRs participate in the cytokine production involved in B cell differentiation into plasma cells, which induces immunoglobulin class switching from IgM to IgG. However, because these results were obtained with conventional knockout mice, in which all cells in the body were affected, the specific roles of these receptors expressed in B cells remains unclear. In the present study, Daudi B lymphoblast cells were used to investigate the specific roles of mAChRs and nAChR in B cells. Stimulating Daudi cells using Pansorbin cells (heat-killed, formalin-fixed Staphylococcus aureus coated with protein A) upregulated expression of M1-M4 mAChRs and the α4 nAChR subunit. Under these conditions, mAChRs, but not nAChRs, mediated immunoglobulin class switching to IgG. This effect was blocked by scopolamine, a non-selective mAChR antagonist, and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a Gq/11-coupled M1, M3, M5 antagonist. In addition, IL-6 secretion was further enhanced following mAChR activation. Thus, Gq/11-coupled mAChRs expressed in B cells thus appear to contribute to IL-6 production and B cell maturation into IgG-producing plasma cells.

Gymnodimine A and 13-desMethyl Spirolide C Alter Intracellular Calcium Levels via Acetylcholine Receptors.

Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]i). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca]i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca]i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca]i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.

Cholinergic Modulation of Membrane Properties of Calyx Terminals in the Vestibular Periphery

Vestibular nerve afferents are divided into regular and irregular groups based on the variability of interspike intervals in their resting discharge. Most afferents receive inputs from bouton terminals that contact type II hair cells as well as from calyx terminals that cover the basolateral walls of type I hair cells. Calyces have an abundance of different subtypes of KCNQ (Kv7) potassium channels and muscarinic acetylcholine receptors (mAChRs) and receive cholinergic efferent inputs from neurons in the brainstem. We investigated whether mAChRs affected membrane properties and firing patterns of calyx terminals through modulation of KCNQ channel activity. Patch clamp recordings were performed from calyx terminals in central regions of the cristae of the horizontal and anterior canals in 13–26 day old Sprague-Dawley rats. KCNQ mediated currents were observed as voltage sensitive currents with slow kinetics (activation and deactivation), resulting in spike frequency adaptation so that calyces at best fired a single action potential at the beginning of a depolarizing step. Activation of mAChRs by application of oxotremorine methiodide or inhibition of KCNQ channels by linopirdine dihydrochloride decreased voltage activated currents by ∼30%, decreased first spike latencies by ∼40%, resulted in action potential generation in response to smaller current injections and at lower (i.e., more hyperpolarized) membrane potentials, and increased the number of spikes fired during depolarizing steps. Interestingly, some of the calyces showed spontaneous discharge in the presence of these drugs. Together, these findings suggest that cholinergic efferents can modulate the response properties and encoding of head movements by afferents.

Effects mediated by the α7 nicotinic acetylcholine receptor on cell proliferation and migration in rat adipose-derived stem cells.

Adipose-derived stem cells (ASCs) are an attractive source for regenerative medicine as they can be easily isolated, rapidly expandable in culture and show excellent in vitro differentiation potential. Acetylcholine (ACh), one of the main neurotransmitters in central and peripheral nervous systems, plays key roles in the control of several physiological processes also in non-neural tissues. As demonstrated in our previous studies, ACh can contribute to the rat ASCs physiology, negatively modulating ASCs proliferation and migration via M2 muscarinic receptor (mAChR) activation. In the present work we show that rat ASCs also express α7 nicotinic receptors (nAChRs). In particular, we have investigated the effects mediated by the selective activation of α7 nAChRs, which causes a reduction of ASC proliferation without affecting cell survival and morphology, and significantly promotes cell migration via upregulation of the CXCR4 expression. Interestingly, the activation of the α7 nAChR also upregulates the expression of M2 mAChR protein, indicating a cooperation between muscarinic and nicotinic receptors in the inhibition of ASC proliferation.

Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Cholinergic Activation of Primary Human Derived Intestinal Epithelium Does Not Ameliorate TNF-α Induced Injury.

The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity via nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation. A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled via basal stimulation with TNF-α (25ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm2), TEER, lucifer yellow permeability, 70kDa dextran transport, cell morphology, and IL-8 secretion were characterized. Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70kDa dextran but did not ameliorate TNF-α induced paracellular permeability. A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway.

Levetiracetam induction of theta frequency oscillations in rodent hippocampus in vitro.

Levetiracetam (LEV) has been demonstrated to improve cognitive function. Hippocampal theta rhythm (4-12 Hz) is associated with a variety of cognitively related behaviors, such as exploration in both humans and animal models. We investigated the effects of LEV on the theta rhythm in the rat hippocampal CA3 in hippocampal slices in vitro. We found that LEV increased the theta power in a dose-dependent manner. The increase in theta power can be blocked by GABAA receptor (GABAAR) or NMDA receptor (NMDAR) antagonists but not by AMPA receptor antagonist, indicating the involvement of GABAAR and NMDAR in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine or GABA-A agonist THIP, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAAR activation. Furthermore, the increased theta power can be partially reduced by the mACh receptor (mAChR) antagonist atropine but not by nACh receptor antagonists, suggesting that mAChR activation provides excitatory input into local network responsible for LEV-induced theta. Our study demonstrated that LEV induced a novel theta oscillation in vitro, which may have implications in the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

Hippocampal Interneuronal α7 nAChRs Modulate Theta Oscillations in Freely Moving Mice

Muscarinic acetylcholine receptors (mAChRs) are critically involved in hippocampal theta generation, but much less is known about the role of nicotinic AChRs (nAChRs). Here we provide evidence that α7 nAChRs expressed on interneurons, particularly those in oriens lacunosum moleculare (OLM), also regulate hippocampal theta generation. Local hippocampal infusion of a selective α7 nAChR antagonist significantly reduces hippocampal theta power and impairs Y-maze spontaneous alternation performance in freely moving mice. By knocking out receptors in different neuronal subpopulations, we find that α7 nAChRs expressed in OLM interneurons regulate theta generation. Our invitro slice studies indicate that α7 nAChR activation increases OLM neuron activity that, in turn, enhances pyramidal cell excitatory postsynaptic currents (EPSCs). Our study also suggests that mAChR activation promotes transient theta generation, while α7 nAChR activation facilitates future theta generation by similar stimulations, revealing a complex mechanism whereby cholinergic signaling modulates different aspects of hippocampal theta oscillations through different receptor subtypes.

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.

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.