Analgesic effect of a cholinergic agonist (carbachol) in a sural nerve ligation-induced hypersensitivity mouse model.

OBJECTIVES: Neuropathic pain is characterized by long-lasting, intractable pain. Sciatic nerve ligation is often used as an animal model of neuropathic pain, and the spared nerve injury (SNI) model, in which the common peroneal nerve (CPN) and tibial nerve (TN) are ligated, is widely used. In the present study, we evaluated the analgesic effect of a cholinergic agonist, carbachol, on a neuropathic pain model prepared by sural nerve (SN) ligation in mice. METHODS: The SN was tightly ligated as a branch of the sciatic nerve. Mechanical and thermal allodynia, and hyperalgesia were assessed using von Frey filaments and heat from a hot plate. The analgesic effects of intracerebroventricularly-administered morphine and carbachol were compared. RESULTS: SN ligation resulted in a significant decrease in pain threshold for mechanical stimulation 1 day after ligation. In response to thermal stimulation, allodynia was observed at 50°C and hyperalgesia at 53 and 56°C 3 days after ligation. Content of thiobarbituric acid reactive substances (TBARS) in the spinal cord increased significantly at 6 and 12 h after ligation. Acetylcholine content of the spinal cord also increased at 5 and 7 days after ligation. Intracerebroventricular administration of carbachol at 7 days after ligation produced a marked analgesic effect against mechanical and thermal stimuli, which was stronger and longer-lasting than morphine at all experimental time points. CONCLUSION: These findings suggest that cholinergic nerves are involved in allodynia and hyperalgesia of the SN ligation neuropathic pain model.

Cognitive-Enhancing Effects of Acetylcholine Receptor Agonists in Group-Housed Cynomolgus Monkeys Who Drink Ethanol.

The cognitive impairments that are often observed in patients with alcohol use disorder (AUD) partially contribute to the extremely low rates of treatment initiation and adherence. Brain acetylcholine receptors (AChR) mediate and modulate cognitive and reward-related behavior, and their distribution can be altered by long-term heavy drinking. Therefore, AChRs are promising pharmacotherapeutic targets for treating the cognitive symptoms of AUD. In the present study, the procognitive efficacy of two AChR agonists, xanomeline and varenicline, were evaluated in group-housed monkeys who self-administered ethanol for more than 1 year. The muscarinic AChR antagonist scopolamine was used to disrupt performance of a serial stimulus discrimination and reversal (SDR) task designed to probe cognitive flexibility, defined as the ability to modify a previously learned behavior in response to a change in reinforcement contingencies. The ability of xanomeline and varenicline to remediate the disruptive effects of scopolamine was compared between socially dominant and subordinate monkeys, with lighter and heavier drinking histories, respectively. We hypothesized that subordinate monkeys would be more sensitive to all three drugs. Scopolamine dose-dependently impaired performance on the serial SDR task in all monkeys at doses lower than those that produced nonspecific impairments (e.g., sedation); its potency did not differ between dominant and subordinate monkeys. However, both AChR agonists were effective in remediating the scopolamine-induced deficit in subordinate monkeys but not in dominant monkeys. These findings suggest xanomeline and varenicline may be effective for enhancing cognitive flexibility in individuals with a history of heavy drinking. SIGNIFICANCE STATEMENT: Procognitive effects of two acetylcholine (ACh) receptor agonists were assessed in group-housed monkeys who had several years' experience drinking ethanol. The muscarinic ACh receptor agonist xanomeline and the nicotinic ACh receptor agonist varenicline reversed a cognitive deficit induced by the muscarinic ACh receptor antagonist scopolamine. However, this effect was observed only in lower-ranking (subordinate) monkeys and not higher-ranking (dominant monkeys). Results suggest that ACh agonists may effectively remediate alcohol-induced cognitive deficits in a subpopulation of those with alcohol use disorder.

An in vitro analog of learning that food is inedible in Aplysia: decreased responses to a transmitter signaling food after pairing with transmitters signaling failed swallowing.

An in vitro analog of learning that a food is inedible provided insight into mechanisms underlying the learning. Aplysia learn to stop responding to a food when they attempt but fail to swallow it. Pairing a cholinergic agonist with an NO donor or histamine in the Aplysia cerebral ganglion produced significant decreases in fictive feeding in response to the cholinergic agonist alone. Acetylcholine (ACh) is the transmitter of chemoreceptors sensing food touching the lips. Nitric oxide (NO) and histamine (HA) signal failed attempts to swallow food. Reduced responses to the cholinergic agonist after pairing with NO or HA indicate that learning partially arises via a decreased response to ACh in the cerebral ganglion.

Cholinergic modulation shifts the response of CA1 pyramidal cells to depolarizing ramps via TRPM4 channels with potential implications for place field firing.

A synergistic combination of in vitro electrophysiology and multicompartmental modeling of rat CA1 pyramidal neurons identified TRPM4 channels as major drivers of cholinergic modulation of the firing rate during a triangular current ramp, which emulates the bump in synaptic input received while traversing the place field. In control, fewer spikes at lower frequencies are elicited on the down-ramp compared to the up-ramp due to long-term inactivation of the NaV channel. The cholinergic agonist carbachol (CCh) removes or even reverses this spike rate adaptation, causing more spikes to be elicited on the down-ramp than the up-ramp. CCh application during Schaffer collateral stimulation designed to simulate a ramp produces similar shifts in the center of mass of firing to later in the ramp. The non-specific TRP antagonist flufenamic acid and the TRPM4-specific blockers CBA and 9-phenanthrol, but not the TRPC-specific antagonist SKF96365, reverse the effect of CCh; this implicates the Ca2+-activated nonspecific cation current, ICAN, carried by TRPM4 channels. The cholinergic shift of the center of mass of firing is prevented by strong intracellular Ca2+ buffering but not by antagonists for IP3 and ryanodine receptors, ruling out a role for known mechanisms of release from intracellular Ca2+ stores. Pharmacology combined with modeling suggest that [Ca2+] in a nanodomain near the TRPM4 channel is elevated through an unknown source that requires both muscarinic receptor activation and depolarization-induced Ca2+ influx during the ramp. Activation of the regenerative inward TRPM4 current in the model qualitatively replicates and provides putative underlying mechanisms for the experimental observations.

Are Voltage Sensors Really Embedded in Muscarinic Receptors?

Unexpectedly, the affinity of the seven-transmembrane muscarinic acetylcholine receptors for their agonists is modulated by membrane depolarization. Recent reports attribute this characteristic to an embedded charge movement in the muscarinic receptor, acting as a voltage sensor. However, this explanation is inconsistent with the results of experiments measuring acetylcholine binding to muscarinic receptors in brain synaptoneurosomes. According to these results, the gating of the voltage-dependent sodium channel (VDSC) acts as the voltage sensor, generating activation of Go-proteins in response to membrane depolarization, and this modulates the affinity of muscarinic receptors for their cholinergic agonists.

Phospholipase C-ε defines a PACAP-stimulated pathway for secretion in the chromaffin cell.

Adrenomedullary chromaffin cells respond to splanchnic (sympathetic) nerve stimulation by releasing stress hormones into the circulation. The signal for hormone secretion is encoded in the neurotransmitters - especially acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP) - that are released into the splanchnic-chromaffin cell synapse. However, functional differences in the effects of ACh and PACAP on the chromaffin cell secretory response are not well defined. Here, selective agonists of PACAP receptors or nicotinic and muscarinic acetylcholine receptors were applied to chromaffin cells. The major differences in the effects of these agents were not on exocytosis, per se, but rather on the steps upstream of exocytosis. In almost every respect, the properties of individual fusion events triggered by PACAP and cholinergic agonists were similar. On the other hand, the properties of the Ca2+ transients evoked by PACAP differed in several ways from those evoked by muscarinic and nicotinic receptor stimulation. A defining feature of the PACAP-stimulated secretory pathway was its dependence on signaling through exchange protein directly activated by cAMP (Epac) and PLCε. However, the absence of PLCε did not disrupt Ca2+ transients evoked by cholinergic agonists. Accordingly, inhibition of Epac activity did not disrupt secretion triggered by acetylcholine or specific agonists of muscarinic and nicotinic receptors. Thus, PACAP and acetylcholine stimulate chromaffin cell secretion via separate and independent pathways. This feature of stimulus-secretion coupling may be important for sustaining hormone release from the adrenal medulla under conditions associated with the sympathetic stress response.

Allergic inflammation disrupts epithelial electrogenic electrolyte transport through cholinergic regulation in the mouse colon.

Intestinal transport of electrolytes is regulated by the enteric nervous system. Acetylcholine (ACh) is considered the most important neurotransmitter for electrolyte transport in the colon. However, electrolyte transport regulated by ACh is not fully understood in the colon. We investigated the regulation of electrogenic electrolyte transport by cholinergic agonists in the mouse colon by measuring the short-circuit current (Isc) using an Ussing chamber system. Muscarinic stimulation induced transient electrogenic Cl- secretion, and nicotinic stimulation induced electrogenic K+ secretion to the apical side in the normal mouse colon, and these effects were reduced in the colon of mice with food allergy (FA). Administration of prednisolone to mice with FA suppressed mild inflammation in the colon and allergic symptoms and thereby ameliorated the disruption of electrogenic electrolyte transport induced not only by cholinergic pathway activation but also by electrical field stimulation and intracellular cAMP signaling pathway activation in the colon. These results suggest that the electrogenic electrolyte transport function in the colon is impaired by FA-induced colonic inflammation and that the suppression of inflammation ameliorates the dysfunction of electrogenic electrolyte transport in the colon of mice with FA.

The nonopioid cholinergic agonist GTS-21 mitigates morphine-induced aggravation of burn injury pain together with inhibition of spinal microglia activation in young rats.

BACKGROUND: Repetitive opioid use does not always alleviate basal pain, procedural pain, or both after burn injury. Mitigation of burn injury-site pain can be achieved by GTS-21 stimulation of α7-acetylcholine nicotinic receptors (α7AChRs) and reduced microglia activation in rat. We tested the hypothesis that morphine exaggerates burn injury-site pain and GTS-21 alleviates both morphine-induced aggravated burn injury pain and microglia activation. METHODS: Young rats with dorsal paw burn injury or sham-burn received intraperitoneal saline, morphine, GTS-21, or a combination twice daily for 14 days. Ipsilateral plantar pain thresholds were tested every other day before morning drugs from days 0-20. Spinal microglia activation, evidenced as pain-transducer (tumour necrosis factor-α [TNF-α], interleukin [IL]-6, IL-1ß, nuclear factor kappa B [NF-κB], Toll-like receptor 4 [TLR4]) expression, was examined using immunohistochemistry and immunoblot. In cultured microglia, morphine-induced cytokine expression was measured (quantitative polymerase chain reaction/enzyme-linked immunosorbent assay [qPCR/ELISA]). RESULTS: Morphine aggravated allodynia at day 5 in sham-burn (P=0.039, n=8-11) but significantly aggravated burn injury site allodynia by day 3 (P=0.010, n=8-11). Microgliosis paralleled nociceptive behaviour changes where burn injury with morphine had highest microgliosis compared with burn injury, morphine alone, or controls (number of cells per field [SD]: 33.8 [2.4], 18.0 [4.1], 8.2 [1.9], and 4.8 [2.0], respectively; P<0.001, n=4-5]. GTS-21 reversed the morphine-induced pain component in sham-burn and burn injury rats together with reduced microgliosis and spinal pain-transducer expression (TNF-α, IL-6, IL-1ß, NF-κB, and TLR4). Morphine-exposed microglial cells showed increased cytokine expression, which was mitigated by GTS-21. CONCLUSIONS: Morphine or burn injury alone increases pain together with microgliosis and pain-transducer expression. Morphine administration augments burn injury-site nociception sooner and aggravated spinal microgliosis and inflammatory pain-transducer expression. GTS-21 has the potential to treat morphine-induced pain in burn injury.

An extracellular scaffolding complex confers unusual rectification upon an ionotropic acetylcholine receptor in C. elegans.

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at -60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering-defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.

Sex differences in rat renal arterial responses following exercise training.

During aerobic exercise, hemodynamic alterations occur. Although blood flow in skeletal muscle arteries increases, it decreases in visceral vessels because of mesenterial vasoconstriction. However, maintaining renal blood flow during intensive sport is also a priority. Our aim was to investigate the changes of vascular reactivity and histology of isolated renal artery of male and female rats in response to swim training. Wistar rats were distributed into four groups: male sedentary (MSed), male trained (MTr), female sedentary (FSed), and female trained (FTr). Trained animals underwent a 12-wk-long intensive swimming program. Vascular function of isolated renal artery segments was examined by wire myography. Phenylephrine-induced contraction was lower in FSed than in MSed animals, and it was decreased by training in male but not in female animals. Inhibition of cyclooxygenases by indomethacin reduced contraction in both sedentary groups, and in MTr but not in FTr animals. Inhibition of nitric oxide production increased contraction in both trained groups. Acetylcholine induced relaxation was similar in all experimental groups showing predominant NO-dependency. Elastin and smooth muscle cell actin density was reduced in female rats after aerobic training. This study shows that, as a result of a 12-wk-long training, there are sex differences in renal arterial responses following exercise training. Swimming moderates renal artery vasoconstriction in male animals, whereas it depresses elastic fiber and smooth muscle actin density in females.NEW & NOTEWORTHY We provided the first detailed analysis of the adaptation of the renal artery after aerobic training in male and female rats. As a result of a 12-wk-long training program, the pharmacological responses of renal arteries changed only in male animals. In phenylephrine-induced contraction, cyclooxygenase-mediated vasoconstriction mechanisms lost their significance in female rats, whereas NO-dependent relaxation became a significant contraction reducing factor in both sexes. Early structural changes, such as reduced elastin and smooth muscle cell actin evolves in females.

Retinoic acid attenuates nuclear factor kappaB mediated induction of NLRP3 inflammasome.

BACKGROUND: Acetylcholine (ACh), a neurotransmitter and a part of the cholinergic system, can modify immune responses. Expression of acetylcholine receptors (AChR) in immune cells, including macrophages, leads to modulation of their function. Inflammasomes are part of the innate immune system and have been linked to a variety of inflammatory diseases. The NLRP3/ASC/caspase-1/IL-1 axis has emerged as a critical signaling pathway in inflammation process initiation. The role of ACh in modulating inflammasomes in macrophages remains relatively under-explored. METHODS: The effect of AChR agonist carbachol on inflammasome expression was investigated using murine and human macrophages. Cell lysates were assessed by western blot for protein analysis. Immunofluorescence studies were used to study the translocation of p65. The experiments were conducted in the presence of NF-ĸB inhibitor, AChR antagonists, and retinoic acid (RA) to study the role of NF-ĸB, ACh receptors, and RA, respectively. RESULTS: We found that carbachol increased the expression of NLRP3 inflammasome (NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18). The treated cells also showed an increase in NF-ĸB activation. The effect of carbachol was diminished by NF-ĸB inhibitor and atropine, a mAChR antagonist. The addition of RA also significantly reduced the effect of carbachol on NLRP3 inflammasomes. CONCLUSIONS: Our current study suggests that carbachol induces NLRP3 inflammasome activation through mAChR and NF-ĸB, and that RA abolishes the inflammatory response. It reveals the potentials of co-administration of RA with cholinergic drugs to prevent inflammatory responses during cholinergic medications.

Acetylcholine prioritises direct synaptic inputs from entorhinal cortex to CA1 by differential modulation of feedforward inhibitory circuits.

Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway.

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

Metaplastic Reinforcement of Long-Term Potentiation in Hippocampal Area CA2 by Cholinergic Receptor Activation.

Hippocampal CA2, an inconspicuously positioned area between the well-studied CA1 and CA3 subfields, has captured research interest in recent years because of its role in social memory formation. However, the role of cholinergic inputs to the CA2 area for the regulation of synaptic plasticity remains to be fully understood. We show that cholinergic receptor activation with the nonselective cholinergic agonist, carbachol (CCh), triggers a protein synthesis-dependent and NMDAR-independent long-term synaptic depression (CCh-LTD) at entorhinal cortical (EC)-CA2 and Schaffer collateral (SC)-CA2 synapses in the hippocampus of adult male Wistar rats. The activation of muscarinic acetylcholine receptors (mAChRs) is critical for the induction of CCh-LTD with the results suggesting an involvement of M3 and M1 mAChRs in the early facilitation of CCh-LTD, while nicotinic AChR activation plays a role in the late maintenance of CCh-LTD at CA2 synapses. Remarkably, we find that CCh priming lowers the threshold for the subsequent induction of persistent long-term potentiation (LTP) of synaptic transmission at EC-CA2 and the plasticity-resistant SC-CA2 pathways. The effects of such a cholinergic-dependent synaptic depression on subsequent LTP at EC-CA2 and SC-CA2 synapses have not been previously explored. Collectively, the results demonstrate that CA2 synaptic learning rules are regulated in a metaplastic manner, whereby modifications triggered by prior cholinergic stimulation can dictate the outcome of future plasticity events. Moreover, the reinforcement of LTP at EC inputs to CA2 following the priming stimulus coexists with concurrent sustained CCh-LTD at the SC-CA2 pathway and is dynamically scaled by modulation of SC-CA2 synaptic transmission.SIGNIFICANCE STATEMENT The release of the neuromodulator acetylcholine is critically involved in processes of hippocampus-dependent memory formation. Cholinergic afferents originating in the medial septum and diagonal bands of Broca terminating in the hippocampal area CA2 might play an important role in the modulation of area-specific synaptic plasticity. Our findings demonstrate that cholinergic receptor activation induces an LTD of synaptic transmission at entorhinal cortical- and Schaffer collateral-CA2 synapses. This cholinergic activation-mediated LTD displays a bidirectional metaplastic switch to LTP on a future timescale. This suggests that such bidirectional synaptic modifications triggered by the dynamic modulation of tonic cholinergic receptor activation may support the formation of CA2-dependent memories given the increased hippocampal cholinergic tone during active wakefulness observed in exploratory behavior.

Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice.

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.

Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

Efficacy of Combining Varenicline and Naltrexone for Smoking Cessation and Drinking Reduction: A Randomized Clinical Trial.

OBJECTIVE: Pharmacological treatments that can concomitantly address cigarette smoking and heavy drinking stand to improve health care delivery for these highly prevalent co-occurring conditions. This superiority trial compared the combination of varenicline and naltrexone against varenicline alone for smoking cessation and drinking reduction among heavy-drinking smokers. METHODS: This was a phase 2 randomized double-blind clinical trial. Participants (N=165) who were daily smokers and drank heavily received either 2 mg/day of varenicline plus 50 mg/day of naltrexone or 2 mg/day of varenicline plus matched placebo pills for 12 weeks. Primary outcomes were 7-day point prevalence of nicotine abstinence (bioverified by a breath CO reading ≤5 ppm) at the 26-week follow-up and number of drinks per drinking day during the 12-week treatment phase. RESULTS: Smoking abstinence at week 26 was significantly higher in the varenicline plus placebo condition than in the varenicline plus naltrexone condition (N=37 [45.1%] compared with N=22 [26.5%]). For drinks per drinking day, there was a medication effect favoring the combination of varenicline and naltrexone over varenicline alone across the 12-week treatment phase, although it did not meet the significance threshold. CONCLUSIONS: These findings suggest that smoking cessation and drinking reduction can be concomitantly targeted with pharmacotherapy and that while varenicline alone may be sufficient as a smoking cessation aid in heavy-drinking smokers, the combination of varenicline and naltrexone may confer benefits with regard to drinking outcomes, particularly during the 12-week period of active medication treatment.

Activation of KCNQ (KV7) K+ channels in enteric neurons inhibits epithelial Cl- secretion in mouse distal colon.

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).

Cholinergic manipulations affect sensory responses but not attentional enhancement in macaque MT.

BACKGROUND: Attentional modulation in the visual cortex of primates is characterized by multiplicative changes of sensory responses with changes in the attentional state of the animal. The cholinergic system has been linked to such gain changes in V1. Here, we aim to determine if a similar link exists in macaque area MT. While rhesus monkeys performed a top-down spatial attention task, we locally injected a cholinergic agonist or antagonist and recorded single-cell activity. RESULTS: Although we confirmed cholinergic influences on sensory responses, there was no additional cholinergic effect on the attentional gain changes. Neither a muscarinic blockage nor a local increase in acetylcholine led to a significant change in the magnitude of spatial attention effects on firing rates. CONCLUSIONS: This suggests that the cellular mechanisms of attentional modulation in the extrastriate cortex cannot be directly inferred from those in the primary visual cortex.

Inhibition of Matrix Metalloproteinase 9 Activity Promotes Synaptogenesis in the Hippocampus.

Information coding in the hippocampus relies on the interplay between various neuronal ensembles. We discovered that the application of a cholinergic agonist, carbachol (Cch), which triggers oscillatory activity in the gamma range, induces the activity of matrix metalloproteinase 9 (MMP-9)-an enzyme necessary for the maintenance of synaptic plasticity. Using electrophysiological recordings in hippocampal organotypic slices, we show that Cch potentiates the frequency of miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs, respectively) in CA1 neurons and this effect is MMP-9 dependent. Interestingly, though MMP-9 inhibition prevents the potentiation of inhibitory events, it further boosts the frequency of excitatory mEPSCs. Such enhancement of the frequency of excitatory events is a result of increased synaptogenesis onto CA1 neurons. Thus, the function of MMP-9 in cholinergically induced plasticity in the hippocampus is to maintain the fine-tuned balance between the excitatory and the inhibitory synaptic transmission.