nAChR Currents Research Articles

Conductance properties of the α9α10 nicotinic acetylcholine receptor of neonatal mouse inner and outer hair cells

In the developing cochlea, just before the onset of hearing on postnatal day 12, the medial olivocochlear efferent axons in synaptic contact with the inner hair cells (IHCs) start withdrawing and new efferent synaptic connections are formed on the outer hair cells (OHCs), thereby progressing towards the adult pattern of medial olivocochlear efferent innervation. The synapses are inhibitory, calcium influx through the α9α10 nicotinic acetylcholine receptors (nAChRs) driving opening of calcium-dependent potassium channels. The nAChRs appear to function similarly in IHCs and OHCs, although with probable kinetic differences. Our aim was to assess their functional similarity in the neonatal mouse cochlea by making whole-cell recordings from both hair cell types between postnatal day 7 and 10 when nAChRs are expressed. ACh was applied to voltage-clamped hair cells by pressure-ejection from a pipette. The cells were dialysed with a Cs+-based solution designed to eliminate calcium-dependent potassium currents. There were differences in amplitude, voltage-sensitivity and reversal potential of the nAChR currents between IHCs and OHCs. There was also some indication that IHC nAChRs have slower activation and desensitization kinetics, although the relatively slow ACh application limited interpretation of this result. These differences, particularly concerning the reversal potential, might indicate the presence of different auxiliary protein subunits of the α9α10 receptor in neonatal IHCs and OHCs.

Abstract Mo135: Correlation of Leptin Resistance With Attenuation of Cardiac Vagal Activation in Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) is one of the most prominent diseases impacting individuals worldwide. A leading cause of death for T2DM patients is MI-related ventricular arrhythmia, which is associated with cardiac vagal nerve dysfunction. As T2DM patients exhibit leptin resistance, and leptin signaling has been implicated in heart rate regulation, we set out to characterize changes in cardiac vagal ganglionic neuron activity during development of T2DM and determine leptin signaling’s role in cardiac vagal dysfunction. In this study, T2DM was induced in Sprague-Dawley rats by feeding a high fat diet (HFD) plus a low dose streptozotocin injection. To characterize cardiac dysfunction, we measured arterial baroreflex sensitivity and found that 12 wks post-T2DM induction, rats experienced a diminished baroreflex response (gain max 0.62 ± 0.04 bpm/mm Hg T2DM vs. 2.21 ± 0.14 bpm/mm Hg sham). To determine if the change in baroreflex sensitivity is due to altered cardiac vagal nerve activity, we measured nAChR currents and cell excitability in intracardiac ganglion (ICG) neurons and found that T2DM rat cardiac vagal neurons exhibited reduced nAChR current density (213.9 ± 25.6 pA/pF T2DM vs. 375.5 ± 44.3 pA/pF sham) and action potential frequency (9.1 ± 0.5 spikes/s T2DM vs. 19.9 ± 1.4 spikes/s sham). Furthermore, acetylcholine release from cardiac vagal nerve terminals was attenuated in T2DM rats (0.4 ± 0.16 nM T2DM vs. 1.07 ± 0.12 nM sham). To determine the role of leptin signaling in the decrease of cardiac vagal nerve activity, we examined leptin and leptin receptor levels in ICG at 4, 8, and 12 wks post-T2DM induction. Western blots revealed elevated ICG leptin levels at 4, 8, and 12 wks post-T2DM induction (8.9 ± 0.7, 9.1 ± 1.3, and 11.7 ± 2.1 times sham level, respectively). qRT-PCR revealed reduced levels of leptin receptor transcript at 12 wks post-T2DM induction (48 ± 12% of sham). A reduction of leptin receptor protein in ICG neurons at 12 wks post-T2DM induction was observed using IF microscopy. These data demonstrate that T2DM reduces cardiac vagal nerve activity resulting in diminished control of cardiac functions such as the baroreflex. Furthermore, leptin resistance may play a role in this diminished cardiac function through reductions in leptin receptor levels.

BNC210, a negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor, demonstrates anxiolytic- and antidepressant-like effects in rodents

This work describes the characterization of BNC210 (6-[(2,3-dihydro-1H-inden-2-yl)amino]-1-ethyl-3-(4-morpholinylcarbonyl)-1,8-naphthyridin-4(1H)-one), a selective, small molecule, negative allosteric modulator (NAM) of α7 nicotinic acetylcholine receptors (α7 nAChR). With the aim to discover a non-sedating, anxiolytic compound, BNC210 was identified during phenotypic screening of a focused medicinal chemistry library using the mouse Light Dark (LD) box to evaluate anxiolytic-like activity and the mouse Open Field (OF) (dark) test to detect sedative and/or motor effects. BNC210 exhibited anxiolytic-like activity with no measurable sedative or motor effects. Electrophysiology showed that BNC210 did not induce α7 nAChR currents by itself but inhibited EC80 agonist-evoked currents in recombinant GH4C1 cell lines stably expressing the rat or human α7 nAChR. BNC210 was not active when tested on cell lines expressing other members of the cys-loop ligand-gated ion channel family. Screening over 400 other targets did not reveal any activity for BNC210 confirming its selectivity for α7 nAChR. Oral administration of BNC210 to male mice and rats in several tests of behavior related to anxiety- and stress- related disorders, demonstrated significant reduction of these behaviors over a broad therapeutic range up to 500 times the minimum effective dose. Further testing for potential adverse effects in suitable rat and mouse tests showed that BNC210 did not produce sedation, memory and motor impairment or physical dependence, symptoms associated with current anxiolytic therapeutics. These data suggest that allosteric inhibition of α7 nAChR function may represent a differentiated approach to treating anxiety- and stress- related disorders with an improved safety profile compared to current treatments.

Synthesis and characterization of αM-conotoxin SIIID, a reversible human α7 nicotinic acetylcholine receptor antagonist

α-Conotoxins, a group of small marine peptide toxins that target nAChRs with high potency and selectivity, are valuable pharmacological tools and potential drug leads. In this study, we reported the synthesis and physiological functions of a novel αM-superfamily conotoxin SIIID (CCGEGSSCPKYFKNNFICGCC) from a fish-hunting Conus striatus. Three SIIID isomers with different cystine connectivities were synthesized by solid-phase polypeptide synthesis and confirmed by mass spectrometry. Patch clamp experiments on HEK293 cells expressing nAChR subtypes showed that 1 μM SIIID (1–4, 2–5, 3–6) inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents by 48.45%, which was higher than 5.08% of SIIID (1–5, 2–4, 3–6) and 9.57% of SIIID (1–6, 2–4, 3–5). Further study on the most active SIIID isomer showed that 10 μM SIIID inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents by 76.33% but had no obvious effect on acetylcholine induced human α3β4 nAChR currents. In addition, SIIID inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents with an IC50 value of 880.71 ± 271.91 nM, and this inhibition was reversible. Patch clamp experiments on rat DRG neurons showed that 10 μM SIIID had <15% inhibitory effects on sodium, potassium and calcium currents. Our results suggested that SIIID would be a promising neuropharmacology tool for the study of human α7 nAChR and its related diseases.

Cell-Genotype Specific Effects of Mecp2 Mutation on Spontaneous and Nicotinic Acetylcholine Receptor-Evoked Currents in Medial Prefrontal Cortical Pyramidal Neurons in Female Rett Model Mice

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene. Random X-inactivation produces a mosaic of mutant (MT) and wild-type (WT) neurons in female Mecp2+/− (het) mice. Many RTT symptoms are alleviated by increasing activity in medial prefrontal cortex (mPFC) in RTT model mice (Howell et al., 2017). Using a GFP-MeCP2 fusion protein to distinguish WT from MT pyramidal neurons in mPFC we found cell autonomous (cell genotype specific) and non-autonomous effects of MeCP2 deficiency on spontaneous excitatory/inhibitory balance, nicotinic acetylcholine receptor (nAChR) currents and evoked activity. MT Layer 5 and 6 (L5, L6) neurons of male nulls, and MT L6 of het mice had reduced spontaneous excitatory synaptic input compared to WT in wild-type male (WTm), female (WTf) and het mice. Inhibitory synaptic charge in MT L6 equaled WT in 2–4-month hets. At 6–7 months inhibitory charge in WT in het slices was increased compared to both MT in het and WT in WTf; however, in hets the excitatory/inhibitory charge ratio was still greater in WT compared to MT. nAChR currents were reduced in L6 of nulls and MT L6 in het slices compared to WT neurons of het, WTm and WTf. At 2–4 months, ACh perfusion increased frequency of inhibitory currents to L6 neurons equally in all genotypes but increased excitatory inputs to MT and WT in hets less than WT in WTfs. Unexpectedly ACh perfusion evoked greater sustained IPSC and EPSC input to L5 neurons of nulls compared to WTm.

Nicotine Evoked Currents in Human Primary Sensory Neurons

Sensory neuron nicotinic acetylcholine receptors (nAChRs) contribute to pain associated with tissue injury. However, there are marked differences between rats and mice with respect to both the properties and distribution of nAChR currents in sensory neurons. Because both species are used to understand pain signaling in humans, we sought to determine whether the currents present in either species was reflective of those present in human sensory neurons. Neurons from the L4/L5 dorsal root ganglia were obtained from adult male and female organ donors. Nicotine evoked currents were detected in 40 of 47 neurons (85%). In contrast with the naïve mouse, in which almost all nAChR currents are transient, or the rat, in which both mouse-like transient and more slowly activating and inactivating currents are detected, all the currents in human DRG neurons were slow, but slower than those in the rat. Currents were blocked by the nAChR antagonists mecamylamine (30 µmol/L), but not by the TRPA1 selective antagonist HC-030031 (10 µmol/L). Single cell polymerase chain reaction analysis of nicotinic receptor subunit expression in human DRG neurons are consistent with functional data indicating that receptor expression is detected 85 ± 2.1% of neurons assessed (n = 48, from 4 donors). The most prevalent coexpression pattern was α3/β2 (95 ± 4% of neurons with subunits), but α7 subunits were detected in 70 ± 3.4% of neurons. These results suggest that there are not only species differences in the sensory neuron distribution of nAChR currents between rodent and human, but that the subunit composition of the channel underlying human nAChR currents may be different from those in the mouse or rat. PerspectiveThe properties and distribution of nicotine evoked currents in human sensory neurons were markedly different from those previously observed in mice and rats. These observations add additional support to the suggestion that human sensory neurons may be an essential screening tool for those considering moving novel therapeutics targeting primary afferents into clinical trials.

A dominant role for the beta 4 nicotinic receptor subunit in nicotinic modulation of glomerular microcircuits in the mouse olfactory bulb

Nicotinic acetylcholine receptors (nAChRs) regulate information transfer across the main olfactory bulb by instituting a high-pass intensity filter allowing for the filtering out of weak inputs. Excitation-driven inhibition of the glomerular microcircuit via GABA release from periglomerular cells appears to underlie this effect of nAChR activation. The multiplicity of nAChR subtypes and cellular locations raises questions about their respective roles in mediating their effects on the glomerular output. In this study, we address this issue by targeting heteromeric nAChRs using receptor knockouts (KOs) for the two dominant nAChR β-subunit genes known to be expressed in the central nervous system. KOs of the β2-nAChR subunit did not affect nAChR currents from mitral cells (MCs) but attenuated those from the external tufted (ET) cells. In slices from these animals, activation of nAChRs still effectively inhibited excitatory postsynaptic currents (EPSCs) and firing on MCs evoked by the olfactory nerve (ON) stimulation, thereby indicating that the filter mechanism was intact. On the other hand, recordings from β4-KOs showed that nAChR responses from MCs were abolished and those from ET cells were attenuated. Excitation-driven feedback was abolished as was the effect of nAChR activation on ON-evoked EPSCs. Experiments using calcium imaging showed that one possible consequence of the β2-subunit activation might be to alter the time course of calcium transients in juxtaglomerular neurons suggesting a role for these receptors in calcium signaling. Our results indicate that nAChRs containing the β4-subunit are critical in the filtering of odor inputs and play a determinant role in the cholinergic modulation of glomerular output. NEW & NOTEWORTHY In this study, using receptor gene knockouts we examine the relative contributions of heteromeric nAChR subtypes located on different cell types to this effect of receptor activation. Our results demonstrate that nAChRs containing the β4-subunit activate MCs resulting in feedback inhibition from glomerular interneurons. This period of inhibition results in the selective filtering of weak odor inputs providing one mechanism by which nAChRs can enhance discrimination between two closely related odors.

Human nicotinic receptors in chromaffin cells: characterization and pharmacology.

During the last 10years, we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for recording of α7 nAChR currents, something that had previously not been achieved in non-human species. The use of α-conotoxins allowed us to characterize non-α7 nAChR subtypes and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3β4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxβ2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage-dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on catecholamine release in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.

Sigma-1 receptor ligands inhibit catecholamine secretion from adrenal chromaffin cells due to block of nicotinic acetylcholine receptors.

Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100nM to 1μM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈50% block at 100nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.

Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2.

Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments.

Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace 3H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism.Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

Pro-cognitive activity in rats of 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

α7 nicotinic acetylcholine receptors (α7 nAChRs) may represent useful targets for cognitive improvement. The aim of this study is to compare the pro-cognitive activity of selective α7-nAChR ligands, including the partial agonists, DMXBA and A-582941, as well as the positive allosteric modulator, 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). The attentional set-shifting task (ASST) and the novel object recognition task (NORT) in rats, were used to evaluate the pro-cognitive activity of each ligand [i.e., PAM-2 (0.5, 1.0, and 2.0 mg·kg(-1) ), DMXBA and A-582941 (0.3 and 1.0 mg·kg(-1) )], in the absence and presence of methyllycaconitine (MLA), a selective competitive antagonist. To determine potential drug interactions, an inactive dose of PAM-2 (0.5 mg·kg(-1) ) was co-injected with inactive doses of either agonist - DMXBA: 0.1 (NORT); 0.3 mg·kg(-1) (ASST) or A-582941: 0.1 mg·kg(-1) . PAM-2, DMXBA, and A-582941 improved cognition in a MLA-dependent manner, indicating that the observed activities are mediated by α7 nAChRs. Interestingly, the co-injection of inactive doses of PAM-2 and DMXBA or A-582941 also improved cognition, suggesting drug interactions. Moreover, PAM-2 reversed the scopolamine-induced NORT deficit. The electrophysiological results also support the view that PAM-2 potentiates the α7 nAChR currents elicited by a fixed concentration (3 μM) of DMXBA with apparent EC50 = 34 ± 3 μM and Emax = 225 ± 5 %. Our results support the view that α7 nAChRs are involved in cognition processes and that PAM-2 is a novel promising candidate for the treatment of cognitive disorders.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

Intracardiac Ganglion Neuron Dysfunction in Type 2 Diabetes

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptor (nAChR) mediates synaptic transmission in intracardiac ganglion (ICG) and regulates the excitability of postganglionic neurons. We hypothesize that changes in the responsiveness of ICG neuronal excitability and postsynaptic nAChR to nicotine (a nAChR agonist) might be involved in attenuated cardiac vagal tone in type 2 diabetic rats. Type 2 diabetic rats were induced by a combination of both high‐fat diet and low‐dose streptozotocin (30 mg/kg i.p.) injection. As an index of cardiac vagal tone, changes of heart rate in response to vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In Langendorff‐perfused hearts, there was no significant different on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole‐cell patch‐clamp data showed that nicotine increases action potential frequency and Ca2+ currents in ICG neurons from sham and T2DM rats, which was completely abolished by hexamethonium (a nAChR antagonist). T2DM reduced the responsiveness of ICG neruonal excitability and Ca2+ channel to nicotine. Additionally, T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. Data from immunofluorescence staining studies showed that T2DM did not alter the protein expression of nAChR subunits in ICG neurons. These results suggest that the low sensitivity of ICG neurons to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.

Nicotinic Acetylcholine Receptors and Cardiac Vagal activity in Rats with Type 2 Diabetes

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in the intracardiac ganglia (ICG) and regulate the excitability of postganglionic parasympathetic neurons. We hypothesized that changes in the functional responsiveness of postsynaptic nAChRs to nicotine (a nAChR agonist) might be involved in attenuated cardiac parasympathetic (vagal) activity in type 2 diabetic rats. A rat model of T2DM was induced by a combination of both high-fat diet and injection of low-dose streptozotocin (30 mg/kg, i.p.). As an index of cardiac vagal function, changes of heart rate in response to graded vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In isolated Langendorff-perfused hearts, there was no significant difference on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole-cell patch-clamp data showed that T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. However, the data from immunofluorescence staining showed that there is no significant difference in the protein expression of ?3 and ?4 nAChR subunits in ICG neurons of sham and T2DM rats. These results suggest that the low sensitivity of nAChR channels to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.

Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freund’s adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4− while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1μM) produced a transient (1min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery.

Research tool: Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches.

There is much interest in α7 nicotinic acetylcholine receptors (nAChRs) in CNS function since they are found throughout peripheral tissues as well as being highly expressed in brain regions implicated in attention, learning and memory. As such, the role of these receptors in many aspects of CNS function and disease is being actively investigated. To date, only one null mouse model (A7KO) is available which is non-conditional and constitutive. Since α7 nAChRs are present on neurons and glia (including astrocytes), as well as being developmentally regulated, there is an unmet need for the technical capability to control α7 nAChR gene expression. Therefore we have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7(LBDEx4007Ehs)) which we refer to as floxed α7 nAChR conditional knockout or α7nAChR(flox). We validated the chosen approach by mating α7nAChR(flox) with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChR(flox), GFAP-A7KO and appropriate littermate controls performed equally in our standard Rodent In Vivo Assessment Core battery to assess general health, locomotion, emotional and cognitive behaviours. Neither α7nAChR(flox) nor GFAP-A7KO exhibited significant differences from littermate controls in any of the baseline behavioural assessments we conducted, similar to the 'first generation' non-conditional A7KO mice. We also determined that α7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from α7nAChR(flox) and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of α7 nAChRs by demonstrating that choline-induced α7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of α7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the α7nAChR(flox) and wild-type (WT) mice. Thus, we have in vivo and in vitro evidence that the Chrna7 exon 4 targeting strategy does not alter behavioural, cognitive, or electrophysiological properties compared to WT and that Cre-mediated excision is an effective approach to delete α7 nAChR expression in a cell-specific manner.

Alterations in the cholinergic system of brain stem neurons in a mouse model of Rett syndrome.

Rett syndrome is an autism-spectrum disorder resulting from mutations to the X-linked gene, methyl-CpG binding protein 2 (MeCP2), which causes abnormalities in many systems. It is possible that the body may develop certain compensatory mechanisms to alleviate the abnormalities. The norepinephrine system originating mainly in the locus coeruleus (LC) is defective in Rett syndrome and Mecp2-null mice. LC neurons are subject to modulation by GABA, glutamate, and acetylcholine (ACh), providing an ideal system to test the compensatory hypothesis. Here we show evidence for potential compensatory modulation of LC neurons by post- and presynaptic ACh inputs. We found that the postsynaptic currents of nicotinic ACh receptors (nAChR) were smaller in amplitude and longer in decay time in the Mecp2-null mice than in the wild type. Single-cell PCR analysis showed a decrease in the expression of α3-, α4-, α7-, and β3-subunits and an increase in the α5- and α6-subunits in the mutant mice. The α5-subunit was present in many of the LC neurons with slow-decay nAChR currents. The nicotinic modulation of spontaneous GABAA-ergic inhibitory postsynaptic currents in LC neurons was enhanced in Mecp2-null mice. In contrast, the nAChR manipulation of glutamatergic input to LC neurons was unaffected in both groups of mice. Our current-clamp studies showed that the modulation of LC neurons by ACh input was reduced moderately in Mecp2-null mice, despite the major decrease in nAChR currents, suggesting possible compensatory processes may take place, thus reducing the defects to a lesser extent in LC neurons.

Competitive Inhibition of the Nondepolarizing Muscle Relaxant Rocuronium on Nicotinic Acetylcholine Receptor Channels in the Rat Superior Cervical Ganglia

A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

Modulation of nicotinic receptor channels by adrenergic stimulation in rat pinealocytes

Melatonin secretion from the pineal gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs, by activating nicotinic cholinergic receptors (nAChR), have been suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat pinealocytes. Acetylcholine or the selective nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) activated large nAChR currents in whole cell patch-clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked by a β-adrenergic receptor agonist, isoproterenol, and blocked by a β-adrenergic receptor antagonist, propranolol. Increasing intracellular cAMP levels using membrane-permeable 8-bromoadenosine (8-Br)-cAMP or 5,6-dichlorobenzimidazole riboside-3',5'-cyclic monophosphorothioate (cBIMPS) also reduced nAChR activity, mimicking the effects of NE and isoproterenol. Further, removal of ATP from the intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of nAChR by adrenergic stimulation. After the downmodulation by NE, nAChR channels mediated a smaller Ca(2+) influx and less membrane depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates nicotinic cholinergic signaling.