Nicotinic Acetylcholine Receptor Subunit Research Articles

Myrtus communis subsp. communis improved cognitive functions in ovariectomized diabetic rats.

The purpose of this study was to investigate the possible effects of Myrtus communis subsp. communis (MC) on cognitive impairment in ovariectomized diabetic rats. Female Sprague-Dawley rats were divided into 5 groups consisting of 15 rats each; Control (C), Diabetes (D), Ovariectomy and diabetes (OVX+D), Ovariectomy, diabetes and donepezil (OVX+D+Don), Ovariectomy, diabetes and Myrtus communis subsp. communis (OVX+D+MC). Blood glucose measurements were made at the beginning and end of the experiments. The animals underwent the novel object recognition test (NORT) and their performance was evaluated. In hippocampal tissues; amyloid beta (Aβ) and neprilysin levels, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT) activities, polysialylated neural cell adhesion molecule (PSA-NCAM), α7 subunit of neuronal nicotinic acetylcholine receptor (α7-nAChR) and brain derived neurotrophic factor (BDNF) gene expressions were examined. Animals with ovariectomy and diabetes showed increased levels of blood glucose, AChE activity and Aβ levels, and decreased neprilysin levels, ChAT activity, α7-nAChR, PSA-NCAM and BDNF gene expressions in parallel with a decrease in NORT performance score. On the other hand, in the MC-treated OVX+D group, there was a significant decrease observed in blood glucose levels and AChE activities while there was improvement in NORT performances and an increase in hippocampal ChAT activity, neprilysin levels, α7-nAChR, PSA-NCAM and BDNF expressions. These results suggest that MC extract could improve cognitive and neuronal functions with its anticholinesterase and antihyperglycemic properties.

β4-Nicotinic Receptors Are Critically Involved in Reward-Related Behaviors and Self-Regulation of Nicotine Reinforcement.

Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster, coding for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. β4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that β4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. β4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, β4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and β4KO self-administered more than WT mice, whereas β4-overexpressing mice avoided nicotine injections. Viral expression of β4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of β4KO mice revealed dose- and region-dependent differences: β4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas β4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional β4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of β4*nAChRs in the MHb-IPN. These data indicate that β4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENT Human genetic studies have provided strong evidence for a relationship between variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and nicotine addiction. Yet, little is known about the role of β4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of β4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the β4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the β4 subunit into either the MHb or IPN restored a "stop" signal on nicotine self-administration. These results suggest that β4*nAChRs provide a promising novel drug target for smoking cessation.

Memantine treatment prevents okadaic acid induced neurotoxicity at the systemic and molecular levels

The present study was designed to investigate the effects of okadaic acid intracerebroventricular (ICV) injection on memory function and expression level of α7 subunit of nicotinic acetylcholine receptor (nAChR) and NR2B subunit of NMDA glutamate receptors in the hippocampus, as well as effect of the antidementic drug memantine on okadaic acid induced changes at systemic and molecular levels in rats. Okadaic acid was dissolved in artificial cerebrospinal fluid (aCSF) and injected ICV 200 ng/10 μl. Vehicle control received 10 μl of aCSF ICV bilaterally. Control and okadaic acid injected rats were divided into two subgroups: treated i.p. with saline or memantine (5 mg/kg daily for 13 days starting from the day of okadaic acid injection). Rats were trained in the dual-solution plus-maze task that can be solved by using place or response strategies. The Western immunoblotting was used to determine relative amount of hippocampal receptors protein levels. Obtained data revealed that okadaic acid ICV injected rats were severely impaired at acquiring the place version of the maze accompanied by significant decline in hippocampal α7 subunit of nACh receptors protein levels. Memantine treatment can prevent okadaic acid induced impairment of hippocampal-dependent spatial memory and accompanied by modulation of the expression level of α7 subunit of nACh and NR2B subunit of NMDA receptors in the hippocampus. Thus, our results support the presumption that α7 nACh receptors may play an important role in the cognitive enhancer effects of memantine and emphasize the role of cholinergic-glutamatergic interactions in memory.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, “silent” nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.

Cholinergic α5 nicotinic receptor is involved in the proliferation and invasion of human prostate cancer cells.

Nicotinic acetylcholine receptor (nAChR) subunit α5 (α5-nAChR) is involved in tumor cell proliferation, inhibition of apoptosis, progression of metastasis, and induction of angiogenesis in certain solid tumors. However, the role of α5-nAChR in prostate cancer cell growth and metastasis is unclear. In the present study, the role of α5-nAChR in cell proliferation, migration, invasion and apoptosis was investigated by silencing the expression levels of α5-nAChR in the prostate cancer cell lines DU145 and PC3. A siRNA oligonucleotide targeting α5-nAChR was designed. The cell proliferation of DU145 and PC3 cell lines was analyzed by the Cell Counting Kit-8 (CCK-8) assay. Cell migratory and invasive activities were determined using wound healing and Transwell assays, respectively. Western blot analysis was used to quantify α5-nAChR, p-AKT and p-ERK1/2 levels in DU145 and PC3 cells. Knockdown of α5-nAChR was associated with decreased cell proliferation, migration, invasion and increased apoptosis. In addition, decreased phosphorylation levels of AKT and ERK1/2 were revealed following α5-nAChR knockdown in DU145 and PC3 cells compared with those observed in the scramble control samples. The expression levels of the apoptosis-related proteins were altered following silencing of α5-nAChR. In summary, the data indicated that α5-nAChR was involved in the proliferation and invasion of human prostate cancer cells.

A study of the differences in sensitivities to rocuronium between the facial muscles and limb skeletal muscles and facial muscle-evoked myogenic potential monitoring

Objective To test the differences in sensitivities to rocuronium between facial nerve- and somatic nerve-innervated muscles, investigate whether the different muscle sensitivities to rocuronium are related to the nicotinic acetylcholine receptor (nAChR) subunits, and explore the relationship between the differences in nAChR expression among these muscles and the agrin/low-density lipoprotein receptor-related protein 4 (Lrp4)/muscle-specific kinase (MuSK) and neuregulin1 (NRG1)/epidermal growth factor receptor (ErbB) signaling pathways. Methods The left sided orbicularis oris and gastrocnemius were obtained from male Sprague-Dawley rats. Their pharmacodynamic responses to rocuronium were detected in vitro, and the values of 50% inhibitory concentration (IC50) were obtained according to dose-response curves. The levels of nAChR α1, β1, δ, e, and γ subunits were assessed by immunofluorescence. The mRNA and protein amounts of agrin, Lrp4, MuSK, matrix metalloproteinase (MMP3), NRG1, ErbB2, ErbB3, and ErbB4 were assessed by reverse transcription polymerase chain reaction(RT-PCR) and Western blot. Results The IC50 value of the orbicularis oris to rocuronium was significantly higher than that of the gastrocnemius. The orbicularis oris presented a higher density of nAChR subunits in unit cross-sectional area than the gastrocnemius. The mRNA and protein amounts of NRG1β,ErbB2, ErbB3, and ErbB4 were higher in the orbicularis oris than those in the gastrocnemius. Meanwhile, no differences were observed in the mRNA or protein levels of agrin, Lrp4, MuSK, or MMP3. Conclusions Facial nerve-innervated muscle is less sensitive to skeletal muscular relaxants than somatic nerve-innervated muscle, which may be related to the density of nAChR subunits in unit cross-sectional area. The higher expression of nAChR subunits in the orbicularis oris may be related to the NRG1/ErbB signaling pathway, rather than the agrin/Lrp4/MuSK signaling pathway. Key words: Facial muscles; Skeletal muscles; Facial nerve; Evoked myogenic potentials; Rocuronium; Nicotinic acetylcholine receptor

Loop-mediated isothermal amplification for the detection of R81T mutation in nAChR with crude genomic DNA extracted from individual Myzus persicae

Myzus persicae is an important insect pest worldwide and is mainly controlled by chemical insecticides. The escalating use of neonicotinoid insecticides has resulted in resistance development. Arginine-to-threonine substitution (R81T) in the D loop region of the nicotinic acetylcholine receptor (nAChR) subunit β1 has been associated with imidacloprid resistance in M. persicae. A simple and fast method to detect the frequency and distribution of this mutation in field populations of M. persicae is important for effective pest management. A modified buffer was used to lyse the individual M. persicae to obtain crude genomic DNA as a template for LAMP reactions. Four sets of primers were designed to target a 641-bp fragment of nAChR in M. persicae. With the crude gDNA and special primers, the concentrations of reaction components, time and temperature were optimized. Therefore, the LAMP for the detection of the R81T mutation was processed at 60 °C for 45 min followed by 80 °C for 10 min to stop the reaction using a common hot bath. Four field-collected populations of M. persicae were used to confirm the application of LAMP for detecting the R81T mutation (both RR and RS genotypes) based on HNB visualization and gel electrophoresis. A rapid, visual and efficient LAMP was successfully developed to distinguish between wild and mutated individuals with crude gDNA as a template extracted from individual M. persicae. This provides an easy and quick method for detecting R81T mutations in field populations of M. persicae for resistance management.

Neurobehavioral Comorbidities in Pentylenetetrazol-kindled Mice and Role of the α4β2 Nicotinic Acetylcholine Receptor

Patients with epilepsy are often affected by not only seizures but also a variety of cognitive and psychiatric comorbidities that further impair their quality of life. However, it is unclear whether epilepsy is associated with psychic function. The aim of the present study was to clarify the effects of kindling-induced epileptic seizures on psychic functioning, using behavioral pharmacological tests. Pentylenetetrazol (PTZ)-kindled mice displayed impaired motor coordination (in the rotarod test), and social approach impairment (in the three-chamber social test) compared with vehicle mice. Intraperitoneal ABT-418 treatment (0.05 mg/kg) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 subunit of the α4β2 nicotinic acetylcholine receptor in the piriform cortex was significantly decreased in PTZ-kindled mice. In contrast, expression of the synaptic adhesion molecule neuroligin 3 (NLG3) was significantly higher in the piriform cortex of PTZ-kindled mice compared with vehicle mice. Collectively, our findings suggest that attention deficit/hyperactivity disorder (ADHD)-like or autistic-like behavioral abnormalities associated with epilepsy are closely linked to downregulation of the α4 subunit of the α4β2 receptor and upregulation of NLG3 in the mouse piriform cortex. In summary, this study indicates that ABT-418 is a good candidate for the treatment of patients with epilepsy complicated by psychiatric symptoms such as autism and ADHD.

Warming-promotion needling improves learning-memory ability by up-regulating expression of hippocampal nicotinic acetylcholine receptor subunit in rats with vascular dementia

To investigate the mechanism of Warming-promotion needling underlying improvement of vascular dementia (VD) by observing its effect on learning-memory ability and expression of hippocampal nicotinic acetylcholine receptor(nAChR)subunit proteins and mRNAs in rats with VD. Sixty male SD rats were randomized into normal control, model, medication, warming-promotion needling(WPN), uniform reinforcing-reducing needling (URRN), and needle-twirling groups (n=10 in each group). The VD model was established via repeated occlusion and reperfusion of the bilateral common carotid arteries. Rats of the medication group were fed with Nimodipine twice a day for 14 days. For rats of the 3 acupuncture groups, WPN, or URRN or needle-twirling was applied to"Dazhui"(GV14),"Baihui"(GV20) and"Shuigou"(GV26) for 30 min, once a day for 14 days. The learning-memory ability was detected by Morris water maze tests, and the expression levels of nAChR α4, α7 and β2 subunit proteins and mRNAs in the hippocampus were detected by Western blot and real-time PCR, separately. Compared with the normal group, the escape latency of Morris water maze tests was significantly prolonged (P<0.05), and the number of the original safe-platform quadrant crossing was significantly reduced in the model group (P<0.05). The expression levels of nAChR α4, α7, β2 subunit proteins and mRNAs (except β2 mRNA) in the hippocampus were significantly decreased in the model group relevant to the normal group (P<0.05). Following the interventions, the increased escape latency, and the reduced safe-platform quadrant crossing times, and the down-regulated expression levels of hippocampal nAChR α4, α7, β2 subunit proteins and mRNAs in the medication, WPN, URRN and needle-twirling groups were all reversed (P<0.05). The effects of WPN were significantly superior to those of URRN and needle-twirling in up-regulating nAChR α4, α7, β2 subunit proteins and mRNAs (P<0.05). The warming-promotion needling can improve the learning and memory ability in VD rats, which may be related to its effects in up-regulating the expression of nAChR su-bunits in the hippocampus.

Identification of nicotinic acetylcholine receptor subunits in different lung cancer cell lines and the inhibitory effect of alpha-conotoxin TxID on lung cancer cell growth

Lung cancer is an aggressive tumor with high incidence and mortality rate. There was growing evidence supporting that nicotinic acetylcholine receptors (nAChRs) play vital role inlung cancer development. In this study, the expression of α3, α4, α5, α6, α7, α9, α10, β2, β3, β4 nAChR subunits on protein and mRNA level were studied in A549, NCI–H1299, NCI–H1688, DMS114 and normal human embryonic lung fibroblast (HEL) cell lines by real-time quantitative PCR (qPCR) and Western blot assay respectively. The results indicated that most of these nAChR subunits were expressed in these five cell lines. Compared with normal cells, the expression of α3 and β4 nAChR subunits were upregulated in A549 and NCI–H1299. Thus, we treated A549 and NCI–H1299 with an antagonist α-conotoxin TxID which potently and selectively blocks α3β4 nAChRs. TxID treatment could inhibit A549 and NCI–H1299 cell growth and enhance the inhibitory effect of adriamycin when treated simultaneously. To sum up, our study identified the expression of nAChR subunits in different lung cells and the anti-tumor effect of α-conotoxin TxID, which may provide novel strategies for lung cancer therapy.

Using genomic profiling for understanding and improving response to smoking cessation treatment.

This review focuses on the implementation of genomic discoveries associated with smoking behaviors and smoking-related illnesses to improve prevention efforts and smoking cessation. Large-scale studies have discovered genomic variation that contributes to smoking heaviness and smoking cessation. Variation in the α5 nicotinic acetylcholine receptor subunit (CHRNA5) and the primary nicotine metabolizing gene (CYP2A6) strongly contribute to smoking behaviors, lung cancer, and chronic obstructive pulmonary disease. The analytic and clinical validity of these genes is clearly established, and evidence of clinical utility for risk stratification is growing. Equity will be a critical issue facing the translation of genomic findings into improved health because the majority of genomic studies have been conducted in European ancestry populations. Now is the time to increase the state of readiness for implementation of genomic profiling and we must address issues of increasing healthcare disparities that may arise with this new genomic technology.

Nicotinic Acetylcholine Receptor Subunit Alpha-5 Promotes Radioresistance via Recruiting E2F Activity in Oral Squamous Cell Carcinoma.

Radiotherapy is commonly used to treat patients with oral squamous cell carcinoma (OSCC), but a subpopulation of OSCC patients shows a poor response to irradiation treatment. Therefore, identifying a biomarker to predict the effectiveness of radiotherapy in OSCC patients is urgently needed. In silico analysis of public databases revealed that upregulation of CHRNA5, the gene encoding nicotinic acetylcholine receptor subunit alpha-5, is extensively detected in primary tumors compared to normal tissues and predicts poor prognosis in OSCC patients. Moreover, CHRNA5 transcript level was causally associated with the effective dose of irradiation in a panel of OSCC cell lines. Artificial silencing of CHRNA5 expression enhanced, but nicotine reduced, the radiosensitivity of OSCC cells. Gene set enrichment analysis demonstrated that the E2F signaling pathway is highly activated in OSCC tissues with high levels of CHRNA5 and in those derived from patients with cancer recurrence after radiotherapy. CHRNA5 knockdown predominantly suppressed E2F activity and decreased the phosphorylation of the Rb protein; however, nicotine treatment dramatically promoted E2F activity and increased Rb phosphorylation, which was mitigated after CHRNA5 knockdown in OSCC cells. Notably, the signature combining increased mRNA levels of CHRNA5 and the E2F signaling gene set was associated with worse recurrence-free survival probability in OSCC patients recorded to be receiving radiotherapy. Our findings suggest that CHRNA5 is not only a useful biomarker for predicting the effectiveness of radiotherapy but also a druggable target to enhance the cancericidal effect of irradiation on OSCC.

Abstract P176: Familial Autonomic Ganglionopathy Caused by Rare CHRNA3 Genetic Variants. Novel Genetic Cause of Neurogenic Orthostatic Hypotension

Patients with autonomic failure developed severe neurogenic orthostatic hypotension (nOH). There are only two reported genetic conditions that cause nOH, dopamine β-hydroxylase deficiency, and CYB561 deficiency; both affect norepinephrine synthesis resulting in an isolated sympathetic failure. Objective: To determine molecular basis of a new Mendelian cause of familial nOH with impaired cardiovagal function, and miosis associated with sympathetic (SNS) and parasympathetic (PNS) failure. Method and Results: The proband developed disabling nOH with poor compensatory increase in heart rate at 39 yrs of age. Plasma norepinephrine levels were abnormally low. His sister had onset of nOH in her late teens. Their parents and brother were healthy. Whole exome sequencing of DNAs from the proband and his affected sister showed that both were compound heterozygotes for c.907_908delCT (p.L303Dfs*115) /c.688G&gt;A (p.D230N) variants in the acetylcholine receptor, neuronal nicotinic, alpha 3 subunit (CHRNA3) gene. CHRNA3 is a subunit of nicotinic acetylcholine receptors (nAChRs) that regulates blood pressure through modulation of synaptic transmission in the autonomic ganglia. The autonomic ganglia regulates both SNS and PNS activity. The frameshift (fs) and missense CHRNA3 variants were inherited from the sibs’ mother and father, respectively, and their non-affected brother was a nl/p.D230N heterozygote. The fs variant is obviously pathogenic and the p.D230N variant is predicted to be damaging (SIFT)/probably damaging (PolyPhen2). Furthermore, we predict from structural modeling that the p.D230N variant lies on the interface between CHRNA3 and other nAChR subunits and that it would destabilize the nAChR pentameric complex. In humans, the presence of acquired nAChR autoantibodies against the alpha 3 subunit is associated with nOH and non-reactive pupils. Conclusions: We report sibs affected with NOH and miosis who are compound heterozygotes forCHRNA3 variants that co-segregate with and have predicted effects on nAChR structure that suggest they likely cause this family’s SNS and PNS failure.

Sex differences in cholinergic systems in the interpeduncular nucleus following nicotine exposure and withdrawal

The medial habenula-interpeduncular nucleus (MHb-IPN) pathway modulates negative affective states produced by nicotine withdrawal. Sex differences in the contribution of acetylcholine (ACh) systems in this pathway have not been explored. Thus, this study assessed ACh levels and gene expression of α- and β-containing nicotinic acetylcholine receptor (nAChR) subunits in the IPN of female and male rats following nicotine treatment and withdrawal. Rats were prepared with a pump that delivered nicotine for 14 days, and naïve controls received a sham surgery. In Study 1, rats were prepared with a probe in the IPN, and ACh levels were measured following saline and then mecamylamine administration. In Study 2, separate groups of naïve control or nicotine-treated rats received saline or mecamylamine and physical signs and anxiety-like behavior were assessed using elevated plus maze (EPM) procedures. The IPN was then dissected and mRNA levels were assessed using RT-qPCR methods. Nicotine treatment increased ACh levels to a larger extent in females than males. Nicotine withdrawal produced a similar increase in physical signs; however, females displayed greater anxiety-like behavior than males. In females, gene expression of α5 increased following nicotine treatment and withdrawal. In males, α7 increased following nicotine treatment and α2 and α3 increased during nicotine withdrawal. Both females and males displayed an increase in β3 and β4 during nicotine withdrawal. In females, anxiety-like behavior was correlated with α4, α5, and β2 gene expression in the IPN. These results suggest that sex differences in withdrawal are modulated via cholinergic systems in the IPN.

Assessing nicotine dependence using an oral nicotine free-choice paradigm in mice

Models to assess the addictive-like properties of nicotine in mice are limited. Therefore, we aimed to characterize and validate an addiction index by using an oral nicotine free-choice paradigm in mice.Adult C57BL/6J, DBA/2J, or genetically modified mice carrying deletions for nicotinic acetylcholine receptor (nAChR) subunits, (n = 8–10/sex/group) were given a choice of water or nicotine (10–960 μg/ml) solution using a two-bottle free-choice (2BC) paradigm. In general, oral nicotine intake and preference were higher in female mice compared to males. Absence of nicotine led to withdrawal, and intermittent access resulted in an escalation in consumption and greater nicotine withdrawal than continuous exposure. Additionally, oral nicotine consumption increased nucleus accumbens tyrosine hydroxylase levels. While β2 and α6 KO mice showed a significant decrease in nicotine intake, deletion of α5 nAChRs increased nicotine consumption at high concentrations. Deletion of the α7 subunit altered the observed sex difference in nicotine consumption, with females consuming less than males. The α4β2 partial agonist varenicline decreased oral nicotine consumption. Although addition of quinine to the nicotine solution lowered nicotine intake, mice primed with nicotine did not lower their intake after quinine addition. Nicotine deprivation followed by re-exposure showed increased nicotine consumption, and DBA/2J mice consumed less nicotine compared to C57BL/6J.We validated the mouse 2BC paradigm to study nicotine's addictive-like properties including nicotine intake, preference, withdrawal, and escalation of nicotine consumption during binge drinking or after reinstatement of a deprivation period.

Nicotinic acetylcholine receptor subunit expression in the gastrocnemius and in the orbicularis oris before and after facial nerve injury in rats

ABSTRACTObjectives: To observe the expression of nicotinic acetylcholine receptor (AChR) subunits in normal orbicularis oris and gastrocnemius muscles and to explore the relationships between the expression of AChR subunits and the severity of facial nerve injury.Methods: Gene and protein expression of AChR subunits in the orbicularis oris and gastrocnemius muscles of male Sprague–Dawley rats was measured by reverse transcription polymerase chain reaction and western blotting, respectively, 1–90 days after graded facial nerve injury.Results: Expression of ε-AChR in the normal orbicularis oris was significantly higher than that in the gastrocnemius, whereas no γ subunit expression was observed. Expression of α, β, δ, ε, and γ subunits was upregulated in the orbicularis oris and was positively correlated with the degree of facial nerve injury.Discussion: We demonstrated the higher expression of the AChR subunits in the orbicularis oris, compared to gastrocnemius muscles. The differences in expression of these subunits between muscles innervated by the facial nerve and somatic nerves and the correlation of AChR subunit expression with the degree of facial nerve injury yield insights into the sensitivity to muscle relaxants during intraoperative facial nerve monitoring.

Capsaicin inhibits the function of α7-nicotinic acetylcholine receptors expressed in Xenopus oocytes and rat hippocampal neurons.

Capsaicin is a naturally occurring alkaloid derived from Chili peppers fruits. Using the two-electrode voltage-clamp technique in Xenopus oocyte expression system, actions of capsaicin on the functional properties of α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor were investigated. Ion currents activated by ACh (100 μM) were reversibly inhibited with an IC50 value of 8.6 μM. Inhibitory actions of capsaicin was independent of membrane potential. Furthermore, Ca2+-dependent Cl- channels expressed endogenously in oocytes were not involved in inhibitory actions of capsaicin. In addition, increasing the ACh concentrations could not reverse the inhibitory effects of capsaicin. Importantly, specific binding of [125I] α-bungarotoxin remained unaltered by capsaicin suggesting that its effect is noncompetitive. Whole cell patch-clamp technique was performed in CA1 stratum radiatum interneurons of rat hippocampal slices. Ion currents induced by choline, a selective-agonist of α7-receptor, were reversibly inhibited by 10 min bath application of capsaicin (10 μM). Collectively, results of our investigation indicate that the function of the α7-nACh receptor expressed in Xenopus oocytes and in hippocampal interneurons are inhibited by capsaicin.

Nicotinic acetylcholine receptor subunit α7-knockout mice exhibit degraded auditory temporal processing.

The CHRNA7 gene that encodes the α7-subunit of the nicotinic acetylcholine receptor (α7-nAChR) has been associated with some autism spectrum disorders and other neurodevelopmental conditions characterized, in part, by auditory and language impairment. These conditions may include auditory processing disorders that represent impaired timing of neural activity, often accompanied by problems understanding speech. Here, we measure timing properties of sound-evoked activity via the auditory brainstem response (ABR) of α7-nAChR knockout mice of both sexes and wild-type colony controls. We find a significant timing delay in evoked ABR signals that represents midbrain activity in knockouts. We also examine spike-timing properties of neurons in the inferior colliculus, a midbrain nucleus that exhibits high levels of α7-nAChR during development. We find delays of evoked responses along with degraded spiking precision in knockout animals. We find similar timing deficits in responses of neurons in the superior paraolivary nucleus and ventral nucleus of the lateral lemniscus, which are brainstem nuclei thought to shape temporal precision in the midbrain. In addition, we find that other measures of temporal acuity including forward masking and gap detection are impaired for knockout animals. We conclude that altered temporal processing at the level of the brainstem in α7-nAChR-deficient mice may contribute to degraded spike timing in the midbrain, which may underlie the observed timing delay in the ABR signals. Our findings are consistent with a role for the α7-nAChR in types of neurodevelopmental and auditory processing disorders and we identify potential neural targets for intervention.NEW & NOTEWORTHY Disrupted signaling via the α7-nicotinic acetylcholine receptor (α7-nAChR) is associated with neurodevelopmental disorders that include impaired auditory processing. The underlying causes of dysfunction are not known but a common feature is abnormal timing of neural activity. We examined temporal processing of α7-nAChR knockout mice and wild-type controls. We found degraded spike timing of neurons in knockout animals, which manifests at the level of the auditory brainstem and midbrain.

Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors.

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data.

Modulation of "Protective" Nicotine Perception and Use Profile by Flavorants: Preliminary Findings in E-cigarettes.

Characterizing flavors are widely available in e-cigarettes and motivate initiation and continued use. Flavors may enhance appeal and facilitate development of addiction to tobacco products through modulation of tobacco products' reinforcing or aversive actions. Palatable flavors (eg, fruit) may increase appeal through primary reinforcing properties. Menthol's cooling and anesthetic effects may increase appeal by counteracting nicotine's aversive effects. Genetics provide a method for modeling individual differences in sensitivity to nicotine's effects. A common polymorphism, rs16969968, encoded in the α5 nicotinic acetylcholine receptor subunit gene (CHRNA5), is a well-recognized marker for smoking risk and reduces sensitivity to nicotine aversiveness. This pilot study tested how flavors impacted e-cigarette appeal and self-administration. In a single testing day, cigarette smokers (N = 32; 94% menthol-smokers) self-administered e-cigarettes containing e-liquids differing in nicotine level (0 mg/mL, 24 mg/mL) and flavor (unflavored, menthol, fruit-flavored) within directed and ad libitum e-cigarette paradigms. Subjective drug effects, number of puffs, rs16969968 genotype, plasma nicotine, and menthol glucuronide levels were collected. Menthol partially ameliorated nicotine aversiveness; fruit did not. In nicotine's absence, fruit flavor increased self-reported preference and ad libitum use relative to menthol-containing or unflavored e-liquids. Individuals with high-smoking-risk rs16969968 genotype (N = 7) reported greater craving alleviation following directed administration of nicotine-containing e-liquids, showed a trend rating nicotine-containing e-liquids as less harsh, and self-administered more nicotine during ad libitum compared to individuals with low-smoking-risk genotype (N = 23). While menthol countered aversiveness of nicotine-containing e-liquids, fruit flavor increased appeal of nicotine-free e-liquids. These preliminary findings suggest menthol and fruit flavor increase e-cigarettes' appeal through distinct mechanisms. This study provides a detailed characterization of the effects of flavors (unflavored, menthol, fruit), nicotine (0 mg/mL, 24 mg/mL) and their interactions on the subjective drug effects and ad libitum self-administration of e-cigarettes. Genetics were used to assess these effects in higher-smoking-risk (diminished sensitivity to nicotine aversiveness) and lower-risk groups. Findings could inform impact of regulation of flavors or nicotine in e-cigarettes, and their impacts on vulnerable sub-populations.