Neuronal Acetylcholine Receptors Research Articles

Binding and gating of neuronal acetylcholine receptors associated with nicotine withdrawal

Binding and gating of neuronal acetylcholine receptors associated with nicotine withdrawal

Regulation of Src family kinases by muscarinic acetylcholine receptors in heterologous cells and neurons.

Five muscarinic acetylcholine (mACh) receptor subtypes are divided into two classes: the M1 class (M1, M3, and M5) and the M2 class (M2 and M4). The former is coupled to Gq proteins, while the latter is coupled to Gi/o proteins. Accumulating evidence indicates that mACh receptors play a significant role in the regulation of the Src family kinase (SFK), a subfamily of non-receptor tyrosine kinases. mACh receptors exert their roles in a subtype-dependent fashion and preferentially target Src and Fyn, two members of SFKs that are expressed in the brain and enriched at synaptic sites. While the M1 receptor positively modulates SFK activity, the M4 receptor inhibits it. By modulating SFKs, mACh receptors are actively involved in the regulation of expression and function of a variety of receptors, structural proteins, and signaling molecules. In particular, the M4 receptor and the dopamine D1 receptor are coexpressed in striatonigral projection neurons of the striatum. Gi/o-coupled M4 and Gq-coupled D1 receptors antagonistically regulate SFK activity, thereby forming a dynamic balance controlling glutamate receptor activity, excitability of neurons, and synaptic plasticity. In summary, mACh receptors play a crucial role in regulating SFK activity in heterologous cells and neurons.

Cajanus cajan (L) Millsp. seed extract ameliorates scopolamine-induced amnesia through increase in antioxidant defense mechanisms and cholinergic neurotransmission.

Decline in cholinergic function and oxidative/nitrosative stress play a central role in Alzheimer's disease (AD). Previous quantitative HPLC profiling analysis has revealed the presence of Pinostrobin, formononetin, vitexin and other neuroprotective flavonoids in Cajanus cajan seed extract. This study was designed to investigate the protective action of Cajanus cajan ethanol seed extract (CC) on learning and memory functions using scopolamine mouse model of amnesia. Materials and methods: Adult mice were pretreated with CC (50, 100, or 200mg/kg, p.o) or vehicle (10ml/kg, p.o) for 16 days consecutively. Scopolamine, a competitive muscarinic cholinergic receptor antagonist (1mg/kg, i.p.) was given an hour after CC pretreatment from days 3 to 16. The mice were subjected to behavioural tests from day 11 (open field test (OFT)/ Y-maze test (YMT) and Morris water maze task (MWM) from days 12-16. Animals were euthanized 1h after behavioral test on day 16 and discrete brain regions isolated for markers of oxidative stress and cholinergic signaling. Molecular docking analysis was undertaken to predict the possible mechanism(s) of CC-induced anti-amnesic action. pre-administration of CC significantly reversed working memory and learning deficits caused by scopolamine in YMT and MWM tests, respectively. Moreover, CC prevented scopolamine-induced oxidative and nitrosative stress radicals in the hippocampus evidenced in significant increase in glutathione (GSH) level, superoxide dismutase (SOD) and catalase (CAT) activities with a marked decrease in malondialdehyde (MDA) production, as well as significant inhibition of hippocampal scopolamine-induced increase in acetylcholinesterase activity by CC. The molecular docking analysis showed that out of the 19 compounds, the following had the highest binding affinity; Pinostrobin (-8.7 Kcal/mol), friedeline (-7.5kCal/mol), and lupeol (-8.2 Kcal/mol), respectively, to neuronal muscarinic M1 acetylcholine receptor, α7 nicotinic acetylcholine receptor and amyloid beta peptide binding pockets, which further supports the ability of CC to enhance neuronal cholinergic signaling and possible inhibition of amyloid beta aggregation. This study showed that Cajanus cajan seeds extract improved working memory and learning through enhancement of cholinergic signaling, antioxidant capacity and reduction in amyloidogenesis.

Prolonged nicotine exposure reduces aversion to the drug in mice by altering nicotinic transmission in the interpeduncular nucleus.

Nicotine intake is likely to result from a balance between the rewarding and aversive properties of the drug, yet the individual differences in neural activity that control aversion to nicotine and their adaptation during the addiction process remain largely unknown. Using a two-bottle choice experiment, we observed considerable heterogeneity in nicotine-drinking profiles in isogenic adult male mice, with about half of the mice persisting in nicotine consumption even at high concentrations, whereas the other half stopped consuming. We found that nicotine intake was negatively correlated with nicotine-evoked currents in the interpeduncular nucleus (IPN), and that prolonged exposure to nicotine, by weakening this response, decreased aversion to the drug, and hence boosted consumption. Lastly, using knock-out mice and local gene re-expression, we identified b4-containing nicotinic acetylcholine receptors of IPN neurons as molecular and cellular correlates of nicotine aversion. Collectively, our results identify the IPN as a substrate for individual variabilities and adaptations in nicotine consumption.

Intravenous Immunoglobulin Therapy for Autoimmune Dysautonomia in children: A Case Series (P10-5.013)

<h3>Objective:</h3> To assess the efficacy of intravenous immunoglobulin (IVIG) therapy in Autoimmune Dysautonomia <h3>Background:</h3> IVIG has been successfully used in the past for pain management and Autoimmune Gastric Dysmotility Disorder (AGID) of adult patients with suspected autoimmune etiology. However, the literature on efficacy of IVIG in children is scarce. <h3>Design/Methods:</h3> A retrospective review of AGID patients treated with IVIG was conducted. All symptoms were reviewed. <h3>Results:</h3> Patient 1 was positive for IgM TS-HDS Ab. Patients 2 and 3 were positive for Neuronal Acetylcholine Receptor Ganglionic (alpha-3) Ab. Patients 1 and 2 had Small Fiber Neuropathy diagnosis. Age at diagnosis of patients 1, 2, and 3 were 14, 17.25, and 16.5 years, respectively. Duration of treatment for patient 3 was 6 months, and 1 year for patients 1 and 2. Post treatment, patient 1 did not require venting of J tube; though still dependent on Total Parenteral Nutrition (TPN). Patient 1 is no longer dependent on enemas, as distal colonic and antroduodenal contractions have normalized. Esophageal dysmotility remains unchanged. Abdominal pain and distention, Postural Orthostatic Tachycardia Syndrome (POTS), and neuropathic pain improved. Patient 2 presented with intractable vomiting and POTS. She was dependent on Nasogastric tube (NG) feedings due to vomiting and was on cyproheptadine, erythromycin, bethanechol, amitriptyline, and prucalopride. Post treatment, vomiting resolved and all but prucalopride were discontinued. Colonic motility has not been re-assessed, however constipation, abdominal distention, and pain resolved. Polyethylene glycol and Bisacodyl were discontinued. Likewise, her POTS resolved. Patient 3 had hyperadrenergic POTS, persistent vomiting, and constipation, suffering a 4 kg weight loss. Following treatment, her vomiting and constipation improved, regaining 3.5 kg. Activity levels and orthostatic dizziness improved, and midodrine and fludrocortisone were discontinued. <h3>Conclusions:</h3> IVIG is an effective treatment for autoimmune dysautonomia in patients with positive IgM TS-HDS Ab and N-AchR Ganglionic (alpha-3) Ab. <b>Disclosure:</b> Mr. Bolufer has nothing to disclose. Dr. Prakash has nothing to disclose.

Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venoms

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action.

The KCNQ channel inhibitor XE991 suppresses nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglion neurons.

XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is reportedly a potent and selective Kv7 (KCNQ) channel inhibitor. This study aimed to evaluate how XE991 affects nicotinic responses in intracardiac ganglion neurons. We studied how the KCNQ channel inhibitor XE991 could affect nicotinic responses in acutely isolated rat intracardiac ganglion neurons using a perforated patch-clamp recording configuration and Ca2+ imaging. XE991 reversibly and concentration-dependently inhibited the nicotine (10μM)-induced current with an IC50 of 14.4μM. The EC50 values for nicotine-induced currents in the absence and presence of 10μM XE991 were 8.7 and 12.0μM, respectively. Because XE991 suppressed the maximum response of the nicotine concentration-response curve, the inhibitory effect of this drug appears to be noncompetitive. In addition, linopirdine reduced the amplitude of 10µM nicotine-induced currents with an IC50 value of 16.9μM. The inorganic KCNQ channel inhibitor Ba2+ affected neither the nicotine-induced current nor the inhibitory effect of XE991 on the nicotinic response. The KCNQ activator flupirtine at a concentration of 10μM slightly but markedly inhibited the nicotine-induced current. Finally, XE991 inhibited the nicotine-induced elevation of intracellular calcium concentration and the nicotine-induced firing of action potentials. We propose that XE991 inhibits nicotinic acetylcholine receptors in intracardiac ganglion neurons, which in turn attenuate nicotine-induced neuronal excitation.

A cholinergic medial septum input to medial habenula mediates generalization formation and extinction of visual aversion.

Generalization of visual aversion is a critical function of the brain that supports survival, but the underlying neurobiological mechanisms are unclear. We establish a rapid generalization procedure for inducing visual aversion by dynamic stripe images. By using fiber photometry, apoptosis, chemogenetic and optogenetic techniques, and behavioral tests, we find that decreased cholinergic neurons' activity in the medial septum (MS) leads to generalization loss of visual aversion. Strikingly, we identify a projection from MS cholinergic neurons to the medial habenula (MHb) and find that inhibition of the MS→MHb cholinergic circuit disrupts aversion-generalization formation while its continuous activation disrupts subsequent extinction. Further studies show that MS→MHb cholinergic projections modulate the generalization of visual aversion possibly via M1 muscarinic acetylcholine receptors (mAChRs) of downstream neurons coreleasing glutamate and acetylcholine. These findings reveal that the MS→MHb cholinergic circuit is a critical node in aversion-generalizationformation and extinction and potentially provides insight into the pathogenesis of affective disorders.

Learning Ability and Hippocampal Transcriptome Responses to Early and Later Life Environmental Complexities in Dual-Purpose Chicks.

Simple SummaryThe hippocampus region of birds is a pivotal area for learning and memory. Early-life conditions can have a long-lasting impact on animals. However, the influence of early-life and later-life environments on animals’ health and welfare has not been well-studied. This study addresses the impact of early-life enrichment and the later environment on the learning ability and hippocampal responses in chicks. We found that the early-life environmental complexity did not prepare better for learning ability in response to life challenges in the future. This study indicates that perches and litter materials, which enriched early-life conditions, were conducive to improved stress responses later in life in terms of neural- and immune-related gene expression and functional pathways. This can be attributed to the “silver spoon” effect. However, the enrichment through litter materials alone in early life does not improve the hippocampal plasticity in later stressed environments. In addition, early-life barren conditions that match later-life conditions have beneficial impacts on neural development, supporting the match and mismatch theory. This study helps us to understand the relationship between early- and later-life environments from the perspective of animal neural- and immune-related development. It also has the potential to guide the treatment of mental problems and personal health in humans based on the interactions between the early-life and later-life environments. In this study, we hypothesized that complex early-life environments enhance the learning ability and the hippocampal plasticity when the individual is faced with future life challenges. Chicks were divided into a barren environment group (BG), a litter materials group (LG), and a perches and litter materials group (PLG) until 31 days of age, and then their learning abilities were tested following further rearing in barren environments for 22 days. In response to the future life challenge, the learning ability showed no differences among the three groups. In the hippocampal KEGG pathways, the LG chicks showed the downregulation of neural-related genes neuronal growth regulator 1 (NEGR1) and neurexins (NRXN1) in the cell adhesion molecules pathway compared to the BG (p < 0.05). Immune-related genes TLR2 in Malaria and Legionellosis and IL-18 and IL18R1 in the TNF signaling pathway were upregulated in the LG compared to in the BG (p < 0.05). Compared to the BG, the PLG displayed upregulated TLR2A in Malaria (p < 0.05). The PLG showed upregulated neural-related gene, i.e., neuronal acetylcholine receptor subunit alpha-7-like (CHRNA8) in the nicotine addiction pathway and secretagogin (SCGN) gene expression, as compared to the LG (p < 0.05). In conclusion, early-life environmental complexities had limited effects on the learning ability in response to a future life challenge. Early-life perches and litter materials can improve neural- and immune-related gene expression and functional pathways in the hippocampus of chicks.

In Silico Analysis of Potential Nicotine Addiction Treatment by Cinnamomum verum Phytochemicals against nAChRα3 and nAChRα7

Nicotine addiction has a direct effect on the occurrence of smoking-related diseases. The receptor that can increase absorption is the neuronal acetylcholine receptor alpha-3 (nAChRα3) and alpha-7 (nAChRα7). This in silico study was conducted to determine the effect of Cinnamomum verum to overcome nicotine addiction by inhibiting the target protein nAChRα3 and nAChRα7. Two hundred and eighty-one phytochemicals Cinnamomum verum were screened into nine ligands by Swiss Adme and PyRx. Molecular visualization and docking analyzes were performed using Avogadro, AutoDock 4.2., and Biovia Discovery Studio 2016. The docking results showed that 2 of 9 ligands in 4zk4 and 6 of 9 ligands in 3sq9 had hydrogen bonds. Sesquiterpenes is the compound with the highest binding affinity in two proteins. However, in 4zk4, the highest affinity with H-bond is linalool. Phenols is the second ligand that effectively binds amino acids. Phytochemicals in Cinnamomum verum potentially reduce addiction to nicotine by inhibiting the receptor and improving the neuroinflammation due to nicotine. The sesquiterpenes is the primary ligand that binds to the 4zk4 and 3sq9.

Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.

Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.

Loss of α7 nicotinic acetylcholine receptors in GABAergic neurons causes sex-dependent decreases in radial glia-like cell quantity and impairments in cognitive and social behavior.

The dentate gyrus (DG) is a unique brain structure in that neurons can be generated postnatally and integrated within existing circuitry throughout life.The maturation process of these newly generated neurons (granule cells) is modulated by nicotinic acetylcholine receptors (nAChRs) through a variety of mechanisms such as neural stem pool proliferation, cell survival, signal modulation, and dendritic integration. Disrupted nAChR signaling has been implicated in neuropsychiatric and neurodegenerative disorders, potentially via alterations in DG neurogenesis. GABAergic interneurons are known to express nAChRs, predominantly the α7 subtype, and have been shown to shape development, integration, and circuit reorganization of DG granule cells. Therefore, we examined histological and behavioral effects of knocking out α7 nAChRs in GABAergic neurons. Deletion of α7 nAChRs resulted in a reduction of radial glia-like cells within the subgranular zone of the DG and a concomitant trend towards decreased immature neurons, specifically in male mice, as well as sex-dependent changes in several behaviors, including social recognition and spatial learning. Overall, these findings suggest α7 nAChRs expressed in GABAergic neurons play an important role in regulating the adult neural stem cell pool and behavior in a sex-dependent manner. This provides important insight into the mechanisms by which cholinergic dysfunction contributes to the cognitive and behavioral changes associated with neurodevelopmental and neurodegenerative disorders.

Rab43 GTPase directs postsynaptic trafficking and neuron-specific sorting of G protein–coupled receptors

G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.

In vitro analysis of the activity of human monoamine oxidase type B (hMAOB), treated with the cyanotoxin anatoxin-a: supposed factor of neurodegenerative diseases

In this study, we investigated the hypothesis that an additional source of free radicals may be hydrogen peroxide formed by monoamino oxidase (MAO) -catalyzed deamination of catecholamines. Also, increased MAO-B activity in the brain has been linked to the development of some neurodegenerative diseases. The toxicant we used to treat recombinant human MAO-B enzyme is the cyanotoxin аnatoxin-a. For anatoxin-a is known that it’s an agonist of neuronal acetylcholine receptors with 20 times greater affinity to them compared to the natural neurotransmitter. In this study, we analyzed the effect of anatoxin at various selected concentrations on the activity of recombinant human MAO-B enzyme. The method we use is to analyze the activity of human MAO-B with the fluorimetric reagent Amplex UltraRed and the substrate tyramine hydrochloride. The aim of this study is to analyze the effect of anatoxin-a on the hMAO-B enzyme activity and its influence as a factor for the development and progression of neurodegenerative diseases.

The potential role of micro-RNA-211 in the pathogenesis of sleep-related hypermotor epilepsy

Sleep-related hypermotor epilepsy (SHE) is a rare epileptic syndrome characterized by epileptic seizures which occur during the non-rapid eye movement (NREM) stage of sleep. It manifests with hypermotor semiology resembling violent limb movements and an asymmetric tonic-dystonic posture. The genes which are responsible for the autosomal dominant form of SHE (ADSHE) and whose function is to code the sub-unit of the neuronal acetylcholine receptor are well known. Considering that ADSHE is a prototype of SHE, it is thought that the dysfunction of the cortico-subcortical cholinergic network, which regulates the cycle of sleep, has a key role in the epileptogenesis of this syndrome. Namely, studies to date, have shown that the hypercholinergic activity is sufficient for the development of epileptic seizures, even though the exact mechanism remains to be elucidated. NREM parasomnias are sleep disorders that are the most difficult to differentiate from SHE due to a similar clinical presentation. Considering the clinical similarities, NREM occurrence and probable genetic connection, it is considered that fundamentally, both of these conditions share a common pathophysiological mechanism i.e. cholinergic dysfunction. The main difference between SHE and NREM parasomnias are the genuine epileptic seizures that are responsible for the semiology in SHE. These genuine seizures are not present in NREM parasomnias. Why this is so, remains to be elucidated. Considering that animal studies have shown that dynamic changes and the decreased levels of microRNA-211 contribute to epileptic seizures and to changes in cholinergic pathways, our hypothesis is that epileptic seizures and the development of epileptogenesis in SHE are a consequence of cholinergic dysfunction and decreased levels of microRNA-211 as opposed to NREM parasomnias where there is a stable level of microRNA-211, preventing epileptogenesis despite the cholinergic system dysfunction.

METHYLCYTISINE ALCALOID POTENTIALLY ACTIVE AGAINST DENGUE VIRUS: A MOLECULAR DOCKING STUDY AND ELECTRONIC STRUCTURAL CHARACTERIZATION

Dengue fever is a serious disease acquired from the Aedes aegypti mosquito present in tropical and subtropical regions, deeply impacting the population's quality of life. Its control requires combating the virus, and the use of substances that do not cause damage to the environment is of fundamental importance. The present work was carried out in silico to perform the structural-electronic characterization of the alkaloid Methylcytisine, a tricyclic quinolizidine alkaloid that has insecticidal activities, identifying the molecular boundary orbitals and descriptors of global chemical reactivity and assessing the inhibitory potential of methylcytisine on NS5 methyltransferase enzyme dengue virus, as well as identifying possible biological targets in humans. Methylcytisine was geometrically optimized through semi-empirical quantum calculations with thermodynamically more stable conformation, characterizing its structure (atoms, angles and bonds) and its reactivity descriptors. The analysis of the molecular docking simulations showed that methylcytisine is coupled in the same active site of the NS5 enzyme methyltransferase DENV, very similar to the complexed ligand S-adenosyl-L-homocysteine. The intermolecular interactions found for the complex formed and the distance values of the enzyme residues, indicate that methylcytisine has potential application as a new inhibitor of the dengue virus, however it has a high possibility of interaction with human neuronal acetylcholine receptors.

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats

To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord. The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time. The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (n=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed (P &lt; 0.01), and spontaneous discharge frequency was obviously reduced (P &lt; 0.01, n=12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures (P &lt; 0.01, n=6). etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

An Integrated Genomic Strategy to Identify CHRNB4 as a Diagnostic/Prognostic Biomarker for Targeted Therapy in Head and Neck Cancer.

Although many studies have shown the association between smoking and the increased incidence and adverse prognosis of head and neck squamous cell carcinoma (HNSCC), the mechanisms and pharmaceutical targets involved remain unclear. Here, we integrated gene expression signatures, genetic alterations, and survival analyses to identify prognostic indicators and therapeutic targets for smoking HNSCC patients, and we discovered that the FDA-approved drug varenicline inhibits the target for cancer cell migration/invasion. We first identified 18 smoking-related and prognostic genes for HNSCC by using RNA-Seq and clinical follow-up data. One of these genes, CHRNB4 (neuronal acetylcholine receptor subunit beta-4), increased the risk of death by approximately threefold in CHRNB4-high expression smokers compared to CHRNB4-low expression smokers (log rank, p = 0.00042; hazard ratio, 2.82; 95% CI, 1.55–5.14), former smokers, and non-smokers. Furthermore, we examined the functional enrichment of co-regulated genes of CHRNB4 and its 246 frequently occurring copy number alterations (CNAs). We found that these genes were involved in promoting angiogenesis, resisting cell death, and sustaining proliferation, and contributed to much worse outcomes for CHRNB4-high patients. Finally, we performed CHRNB4 gene editing and drug inhibition assays, and the results validate these observations. In summary, our study suggests that CHRNB4 is a prognostic indicator for smoking HNSCC patients and provides a potential new therapeutic drug to prevent recurrence or distant metastasis.

Altered Expression of Nicotinic Receptors in Perinatal Life Related to Prenatal Exposure to Toxics—An Overview of the Research Carried Out on This Topic at the “Lino Rossi” Research Center of the Milan University

The article aims to underline the impact of nicotine and pesticides on neuronal α7-nicotinic acetylcholine receptors expression in brainstem regions receiving cholinergic projections, given their fundamental role during the neuronal development. The in-depth histopathological/immunohistochemical examination of the autonomic nervous system performed at the “Lino Rossi” Research Center of the Milan University on a wide group of sudden unexpected fetal and infant deaths, highlighted the frequent hypodevelopment of brainstem structures checking the vital functions associated to altered expression of α7-nicotinic acetylcholine receptors and smoke absorption in pregnancy. A dysregulation of the catecholamine system was also observed in the cerebellar cortex of the same cases. However, in a not negligible percentage of sudden deaths with altered expression of α7-nicotinic receptors, the mothers never smoked but lived in rural areas. Specific analytical procedures showed the presence of agricultural pesticides in cerebral cortex samples of these victims. Therefore, it is possible to believe that the exposition to pesticides during pregnancy can produce the same harmful effects as nicotine on the nicotinic acetylcholine receptors. Moreover, alterations of α7-nicotinic acetylcholine receptors receptor expression were also detected in the lungs of many sudden perinatal death victims, allowing to consider even these findings as possible consequence of maternal exposure to toxic factors.

Acetylcholine and Its Receptors in Honeybees: Involvement in Development and Impairments by Neonicotinoids.

Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.