Nicotinic Research Articles

Exposure to an environmentally representative mixture of polybrominated diphenyl ethers (PBDEs) alters zebrafish neuromuscular development

Polybrominated diphenyl ethers (PBDEs) are a prevalent group of brominated flame retardants (BFRs) added to several products such as electronics, plastics, and textiles to reduce their flammability. They are reported as endocrine disruptors and neurodevelopmental toxicants that can accumulate in human and wildlife tissues, thus making their ability to leach out of products into the environment a great cause for concern. In this study, zebrafish (Danio rerio) embryos and larvae were exposed to a wide concentration range (1.5, 15, 150 and 300 pM) of a PBDE mixture from one to six days post-fertilization (dpf). Hatching rates, mortality and general morphology were assessed during the exposure period. A delay in hatching was observed at the two highest PBDEs concentrations and mortality rate increased at 6 dpf. By 4 dpf, larvae exposed to 150 pM and 300 pM PBDEs developed an upcurved phenotype. Analysis of motor behavior at 6 dpf revealed that PBDE exposure acutely reduced locomotion. To further analyze these motor deficits, we assessed the neural network density and motor neuron and neuromuscular junctions (NMJ) development by immunostaining and imaging. Acetylated α-tubulin staining revealed a significant loss of neurons in a dose-dependent manner. Synaptic vesicle protein 2 (SV2) and ⍺-bungarotoxin (⍺-BTX) staining revealed a similar pattern, with a significant loss of SV2 and nicotinic acetylcholine receptors, thus preventing the colocalization of presynaptic neurons with postsynaptic neurons. Consistent with these results, the presence of cleaved caspase-3 and acridine orange positive cells showed increased cell death in zebrafish larvae exposed to PBDEs. Our results suggest that exposure to PBDEs leads to deficits in the zebrafish neuromuscular system through neuron death, inducing morphological and motor deficiencies throughout their development. They provide valuable insight into the neurotoxic effects of PBDEs, further highlighting the relevance of the zebrafish model in toxicological studies.

Activation of α7 Nicotinic Acetylcholine Receptor Augments Nerve Growth Factor Action on PCtrk Cells

Although cigarette smoking is known to be a critical risk factor for various organ systems and cancers, accumulating evidence indicates that nicotine – a main constituent of cigarette smoking – can exert neuroprotective effects on neuronal cells through nicotinic acetylcholine receptors (nAChRs). However, the precise molecular mechanisms for nicotinic neuroprotective actions remain to be fully elucidated. In this study, we examine the effects of agonists, such as nicotine and PNU282987, on tropomyosin-related kinase (Trk)-dependent neuroprotective pathways in PC12 cells overexpressing a Trk neurotrophin receptor (PCtrk cells). We found that even considerably higher concentrations (mM range for nicotine and µM range for PN282987) of nAChR agonists exert favorable effects, such as the augmentation of nerve growth factor (NGF)-induced Trk neurotrophin receptor autophosphorylation of tyrosine residues and NGF-induced neurite extension. Moreover, nicotine upregulated reactive oxygen species (ROS) levels in the cells. ROS production was completely cancelled by pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, indicating that the main source of ROS production by nicotine was mitochondria. Furthermore, treatment with nAChR agonists appeared to induce autophagic flux, as evidenced by the upregulation of LC3-II expression in cells. Furthermore, sucrose density ultracentrifugation of nicotine-treated cells clearly disclosed the augmented recruitment of α7nAChR protein into the lipid rafts fraction of the membrane. Intriguingly, a pull-down assay of anti-Trk antibody immunoprecipitates clearly included α7nAChR protein, indicating that Trk and α7nAChR proteins form a complex. These results reveal a new molecular interaction between activated α7nAChR and Trk protein that may serve as a new molecular basis of nicotine-induced neuroprotective action.

Field-evolved resistance to neonicotinoids in the mosquito, Anopheles gambiae, is associated with mutations of nicotinic acetylcholine receptor subunits combined with cytochrome P450-mediated detoxification

New insecticides prequalified for malaria control interventions include modulators of nicotinic acetylcholine receptors that act selectively on different subunits leading to variable sensitivity among arthropods. This study aimed to investigate the molecular mechanisms underlying contrasting susceptibility to neonicotinoids observed in wild populations of two mosquito sibling species. Bioassays and a synergist test with piperonyl butoxide revealed that the sister taxa, Anopheles gambiae and An. coluzzii, from Yaounde, Cameroon, both have the potential to develop resistance to acetamiprid through cytochrome P450-mediated detoxification. However, contrary to An. coluzzii, An. gambiae populations are evolving cross-resistance to several active ingredients facilitated by mutations of nicotinic acetylcholine receptors (nAChRs). We sequenced coding regions on the β1 and α6 nAChR subunits where variants associated with resistance to neonicotinoids or to spinosyns have been found in agricultural pests and detected no mutation in An. coluzzii. By contrast, six nucleotide substitutions including an amino acid change in one of the loops that modulate ligand binding and affect sensitivity were present in the resistant species, An. gambiae. Allele frequency distributions were consistent with the spread of beneficial mutations that likely reduce the affinity of An. gambiae nAChRs for synthetic modulators. Our findings provide critical information for the application and resistance management of nAChR modulators in malaria prevention.

S-(+)-mecamylamine increases the firing rate of serotonin neurons and diminishes depressive-like behaviors in an animal model of stress

Mecamylamine, a noncompetitive blocker of nicotinic acetylcholine receptors (nAChRs), is the racemic mixture of two stereoisomers: S-(+)-mecamylamine (S-mec) and R-(−)-mecamylamine (R-mec), with distinct interactions with α4β2 nAChRs. It has been shown that mecamylamine increases glutamate release and excites serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN). In this study, we separately evaluated the effects of S-mec and R-mec on 5-HT neuron excitability. S-mec (3 μM) increased firing frequency by 40 %, while R-mec (3 μM) raised it by only 22 %. S-mec acts as a positive allosteric modulator on high-sensitivity (HS) α4β2 nAChRs at glutamate terminals, enhancing spontaneous excitatory postsynaptic currents (sEPSCs) in 5-HT neurons. Conversely, R-mec decreased sEPSCs by blocking HS α4β2 nAChRs and reduced GABA-mediated inhibitory currents (sIPSCs) by blocking α7 nAChRs at GABAergic terminals. These mechanisms make S-mec more effective than R-mec in enhancing 5-HT neuron firing. Moreover, combining S-mec with TC-2559, a selective agonist of HS α4β2 nAChRs, increased firing frequency by 65 %, exceeding the effect of S-mec alone. To validate these findings, we evaluated the antidepressant effects of S-mec (1 mg/kg) combined with TC-2559 or RJR-2403, another α4β2 nAChR agonist. This combination successfully reduced depression-like behaviors, suggesting a potential treatment strategy for patients resistant to conventional antidepressants.

Interactive effects of ARRB2 and CHRNA5 genetic polymorphisms on cognitive function in Chinese male methamphetamine use disorder patients.

Both β-arrestin2 and nicotinic acetylcholine receptor (nAChR) have been implicated in cognitive processes, particularly in relation to psychiatric disorders, including addiction. Previous studies have suggested that nAChR may be regulated by β-arrestin2. However, no study has investigated the interaction of β-arrestin2 and nAChR on cognition. We aimed to examine the main and interactive effects of their respective encoding genes, ARRB2 and CHRNA5, on cognitive function in MUD patients. We recruited 559 patients with methamphetamine use disorder (MUD) and 459 healthy controls, assessed their cognitive functioning using the Chinese version of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and genotyped ARRB2 rs1045280 and CHRNA5 rs3829787 polymorphisms in MUD patients. Compared to healthy controls, MUD patients scored significantly lower on all RBANS indexes. Neither ARRB2 rs1045280 nor CHRNA5 rs3829787 had main effects on cognitive function in MUD patients, but there were significant interactive effects between the two single nucleotide polymorphisms (SNPs) on multiple RBANS indexes, including immediate memory, visuospatial/constructional, delayed memory, and total score. In detail, among carriers of CHRNA5 rs3829787 T allele, ARRB2 rs1045280 TT carriers had higher RBANS scores than the C allele carriers, whereas among carriers of CHRNA5 rs3829787 CC genotype, ARRB2 rs1045280 TT carriers performed worse in RBANS. Our study identified for the first time an interactive effect between ARRB2 and CHRNA5 on cognitive function in MUD patients, which would enlarge our knowledge of genetic interaction on cognitive function.

Modulation of Ca2+ oscillation following ischemia and nicotinic acetylcholine receptors in primary cortical neurons by high-throughput analysis.

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2h resulted in an increase in the frequency of Ca2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia.

α9-Containing Nicotinic Acetylcholine Receptors Are Required for RgIA-5474 Attenuation of Chemotherapy-Induced Neuropathic Pain

α9-Containing Nicotinic Acetylcholine Receptors Are Required for RgIA-5474 Attenuation of Chemotherapy-Induced Neuropathic Pain

Targeting the cholinergic anti-inflammatory pathway for type 2 diabetes prevention: A retrospective cohort study.

Chronic inflammation is a key factor in type 2 diabetes mellitus (T2DM) development. The cholinergic anti-inflammatory pathway (CAP) reduces inflammation by activating α7 nicotinic acetylcholine receptors (α7nAChRs) on macrophages, suppressing proinflammatory cytokines. Acetylcholinesterase inhibitors (AChEis), primarily used for Alzheimer's disease (AD), may exert anti-inflammatory effects through the CAP. One AChEi, galantamine, also directly agonizes α7nAChRs, potentially enhancing its anti-inflammatory properties. This study aimed to investigate the association between AChEi use, particularly galantamine, and T2DM risk in AD patients. We conducted a retrospective analysis of Veterans Health Administration (VA) data, examining early- and late-onset AD patients receiving galantamine or other AD medications. Propensity score matching was used to balance groups and minimize confounding. Cox proportional hazard models assessed T2DM risk for galantamine, other AChEis and memantine. A total of 40 065 AD patients were included in the study. Among early-onset AD patients, galantamine use significantly reduced T2DM risk (hazard ratio [HR] = 0.80, 95% confidence interval [CI]: 0.66-0.98). Memantine also showed a protective effect (HR = 0.82, 95% CI: 0.69-1) in this group. Neither galantamine nor memantine influenced T2DM risk in late-onset AD. Other AD medications showed no association with T2DM risk. Galantamine use was associated with a lower risk of T2DM in early-onset AD patients, potentially due to enhanced anti-inflammatory effects through both AChE inhibition and direct α7nAChR agonism. Memantine also demonstrated a protective effect. These findings suggest potential new applications for existing AD medications in T2DM prevention, particularly in early-onset AD patients. Further research, including randomized controlled trials with diverse populations, is needed to confirm these results and the underlying mechanisms.

Assessing the Utility of ColabFold and AlphaMissense in Determining Missense Variant Pathogenicity for Congenital Myasthenic Syndromes

Background/Objectives: Congenital myasthenic syndromes (CMSs) are caused by variants in >30 genes with increasing numbers of variants of unknown significance (VUS) discovered by next-generation sequencing. Establishing VUS pathogenicity requires in vitro studies that slow diagnosis and treatment initiation. The recently developed protein structure prediction software AlphaFold2/ColabFold has revolutionized structural biology; such predictions have also been leveraged in AlphaMissense, which predicts ClinVar variant pathogenicity with 90% accuracy. Few reports, however, have tested these tools on rigorously characterized clinical data. We therefore assessed ColabFold and AlphaMissense as diagnostic aids for CMSs, using variants of the CHRN genes that encode the nicotinic acetylcholine receptor (nAChR). Methods: Utilizing a dataset of 61 clinically validated CHRN variants, (1) we evaluated the possibility of a ColabFold metric (either predicted structural disruption, prediction confidence, or prediction quality) that distinguishes variant pathogenicity; (2) we assessed AlphaMissense’s ability to differentiate variant pathogenicity; and (3) we compared AlphaMissense to the existing pathogenicity prediction programs AlamutVP and EVE. Results: Analyzing the variant effects on ColabFold CHRN structure prediction, prediction confidence, and prediction quality did not yield any reliable pathogenicity indicative metric. However, AlphaMissense predicted variant pathogenicity with 63.93% accuracy in our dataset—a much greater proportion than AlamutVP (27.87%) and EVE (28.33%). Conclusions: Emerging in silico tools can revolutionize genetic disease diagnosis—however, improvement, refinement, and clinical validation are imperative prior to practical acquisition.

Distinct Chrna5 mutations link excessive alcohol use to types I/II vulnerability profiles and IPN GABAergic neurons

Genome wide association and animal studies have implicated genetic variations in CHRNΑ5, encoding the α5 subunit-containing nicotinic acetylcholine receptors (α5*nAChRs), as a risk factor for developing alcohol use disorders (AUDs). To understand how α5*nAChR mutations may influence alcohol (EtOH) drinking behavior, we used a two-bottle choice procedure with intermittent access to alcohol in male and female transgenic mice expressing either the highly frequent human single nucleotide polymorphism (α5SNP/rs16969968) or a deletion of the Chrna5 gene (α5KO). AUDs-related preconsommatory traits (anxiety, sensation-seeking and impulsivity) were assessed with a battery of relevant tasks (elevated-plus maze, novel place preference and step-down inhibitory avoidance). The implication of the α5-expressing IPN GABAergic neurons in AUDs and related behavioral traits was verified using neurospecific lentiviral (LV)-induced reexpression of the α5 subunit in α5KOxGAD-Cre mice. Both α5SNP and α5KO mice showed over-consumption of EtOH, but displayed opposite vulnerability profiles consistent with Cloninger’s subtypes of human AUDs. α5SNP mice showed Type I-like characteristics, i.e., high anxiety, novelty avoidance, whereas α5KOs exhibited Type II-like features such as low anxiety and high impulsivity. LV re-expression of the α5 subunit in IPN GABAergic neurons restored the control of EtOH intake and improved the impulsive phenotype. We demonstrate that the SNP (rs16969968) or null mutation of Chrna5 result in increased volitional EtOH consumption but opposite effects on anxiety, novelty-seeking and impulsive-like behaviors that match Cloninger type I and II of AUDs, including sex-related variations. IPN GABAergic neurons expressing α5*nAChRs play a key role in limiting both EtOH drinking and motor impulsivity.

Cerebral Cortical Vasodilation via Nicotinic Receptors by Heated Tobacco Product Aerosol Extract in Rats

Introduction: Smoking increases the risk of lung cancer due to a number of components of smoke. The use of novel heated tobacco products (HTPs), alternative to conventional combustion cigarettes, has increased in recent years. However, the in vivo biological effects of HTPs are poorly understood. This study aimed to clarify the acute effects of injecting aerosol extract prepared from an HTP on regional cerebral blood flow (rCBF) in rat cortex by comparing them to the effects of injecting smoke extract prepared from conventional combustible cigarettes. Methods: In urethane anesthetized rats, rCBF was measured using laser speckle contrast imaging simultaneously with arterial pressure. Results: Both cigarette smoke extract and HTP aerosol extract, at a dose equivalent to 30 μg nicotine/kg, injected intravenously, increased cortical rCBF without changing arterial pressure. The magnitude and time course of the increased rCBF response to both extracts were similar throughout the cortical area, and the rCBF increases were all abolished by dihydro-β-erythroidine, an α4β2-preferring nicotinic acetylcholine receptor (nAChR) antagonist. Conclusion: In conclusion, our study demonstrated that the effect of injecting aerosol extract prepared from an HTP, an acute increase in cortical rCBF, is mediated via activation of α4β2-like neuronal nAChRs in the brain.

Machine Learning Framework for Conotoxin Class and Molecular Target Prediction

Conotoxins are small and highly potent neurotoxic peptides derived from the venom of marine cone snails which have captured the interest of the scientific community due to their pharmacological potential. These toxins display significant sequence and structure diversity, which results in a wide range of specificities for several different ion channels and receptors. Despite the recognized importance of these compounds, our ability to determine their binding targets and toxicities remains a significant challenge. Predicting the target receptors of conotoxins, based solely on their amino acid sequence, remains a challenge due to the intricate relationships between structure, function, target specificity, and the significant conformational heterogeneity observed in conotoxins with the same primary sequence. We have previously demonstrated that the inclusion of post-translational modifications, collisional cross sections values, and other structural features, when added to the standard primary sequence features, improves the prediction accuracy of conotoxins against non-toxic and other toxic peptides across varied datasets and several different commonly used machine learning classifiers. Here, we present the effects of these features on conotoxin class and molecular target predictions, in particular, predicting conotoxins that bind to nicotinic acetylcholine receptors (nAChRs). We also demonstrate the use of the Synthetic Minority Oversampling Technique (SMOTE)-Tomek in balancing the datasets while simultaneously making the different classes more distinct by reducing the number of ambiguous samples which nearly overlap between the classes. In predicting the alpha, mu, and omega conotoxin classes, the SMOTE-Tomek PCA PLR model, using the combination of the SS and P feature sets establishes the best performance with an overall accuracy (OA) of 95.95%, with an average accuracy (AA) of 93.04%, and an f1 score of 0.959. Using this model, we obtained sensitivities of 98.98%, 89.66%, and 90.48% when predicting alpha, mu, and omega conotoxin classes, respectively. Similarly, in predicting conotoxins that bind to nAChRs, the SMOTE-Tomek PCA SVM model, which used the collisional cross sections (CCSs) and the P feature sets, demonstrated the highest performance with 91.3% OA, 91.32% AA, and an f1 score of 0.9131. The sensitivity when predicting conotoxins that bind to nAChRs is 91.46% with a 91.18% sensitivity when predicting conotoxins that do not bind to nAChRs.

The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.

The alpha 7 nicotinic acetylcholine receptor agonist PHA 568487 dampens inflammation in PBMCs from patients with newly discovered coronary artery disease.

The alpha 7 nicotinic acetylcholine receptor (α7nAChR) regulates inflammation in experimental models and is expressed in human peripheral blood mononuclear cells (PBMCs) and in human atherosclerotic plaques. However, its role in regulating inflammation in patients with cardiovascular disease is unknown. This study aims to investigate whether α7nAChR stimulation can reduce the inflammatory response in PBMCs from patients with newly diagnosed coronary artery disease (CAD). Human PBMCs, extracted from patients with verified CAD (n = 38) and control participants with healthy vessels (n = 38), were challenged in vitro with lipopolysaccharide (LPS) in combination with the α7nAChR agonist PHA 568487. Cytokine levels of the supernatants were analyzed using a multiplex immunoassay. Patients in the CAD group were reexamined after 6 mo. The immune response to LPS did not differ between PBMCs from control and CAD groups. α7nAChR stimulation decreased TNFα in both control and CAD groups. The most pronounced effect of α7nAChR stimulation was observed in patients with CAD at their first visit, where 15 of 17 cytokines were decreased [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-17A, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, and TNFα]. In conclusion, stimulation with α7nAChR agonist PHA 568487 dampens the inflammatory response in human PBMCs. This finding suggests that the anti-inflammatory properties of the α7nAChR may have a role in treating CAD.NEW & NOTEWORTHY The α7nAChR is an important regulator of inflammation; however, its anti-inflammatory function in patients with newly diagnosed coronary artery disease (CAD) remains unclear. We demonstrate that stimulation of α7nAChR with PHA 568487 attenuates the inflammatory response in immune cells extracted from healthy controls and patients with newly diagnosed CAD, with a more pronounced effect observed in patients with CAD. This suggests that the anti-inflammatory properties of α7nAChR may have a role in treating chronic inflammatory diseases.

Alpha-2 nicotinic acetylcholine receptors regulate spectral integration in auditory cortex

IntroductionIn primary auditory cortex (A1), nicotinic acetylcholine receptors (nAChRs) containing α2 subunits are expressed in layer 5 Martinotti cells (MCs)—inhibitory interneurons that send a main axon to superficial layers to inhibit distal apical dendrites of pyramidal cells (PCs). MCs also contact interneurons in supragranular layers that, in turn, inhibit PCs. Thus, MCs may regulate PCs via inhibition and disinhibition, respectively, of distal and proximal apical dendrites. Auditory inputs to PCs include thalamocortical inputs to middle layers relaying information about characteristic frequency (CF) and near-CF stimuli, and intracortical long-distance (“horizontal”) projections to multiple layers carrying information about spectrally distant (“nonCF”) stimuli. CF and nonCF inputs integrate to create broad frequency receptive fields (RFs). Systemic administration of nicotine activates nAChRs to “sharpen” RFs—to increase gain within a narrowed RF—resulting in enhanced responses to CF stimuli and reduced responses to nonCF stimuli. While nicotinic mechanisms to increase gain have been identified, the mechanism underlying RF narrowing is unknown.MethodsHere, we examine the role of α2 nAChRs in mice with α2 nAChR-expressing neurons labeled fluorescently, and in mice with α2 nAChRs genetically deleted.ResultsThe distribution of fluorescent neurons in auditory cortex was consistent with previous studies demonstrating α2 nAChRs in layer 5 MCs, including nonpyramidal somata in layer 5 and dense processes in layer 1. We also observed label in subcortical auditory regions, including processes, but no somata, in the medial geniculate body, and both fibers and somata in the inferior colliculus. Using electrophysiological (current-source density) recordings in α2 nAChR knock-out mice, we found that systemic nicotine failed to enhance CF-evoked inputs to layer 4, suggesting a role for subcortical α2 nAChRs, and failed to reduce nonCF-evoked responses, suggesting that α2 nAChRs regulate horizontal projections to produce RF narrowing.DiscussionThe results support the hypothesis that α2 nAChRs function to simultaneously enhance RF gain and narrow RF breadth in A1. Notably, a similar neural circuit may recur throughout cortex and hippocampus, suggesting widespread conserved functions regulated by α2 nAChRs.

Sex differences in contextual fear conditioning and extinction after acute and chronic nicotine treatment

BackgroundChronic cigarette smokers report withdrawal symptomology, including affective dysfunction and cognitive deficits. While there are studies demonstrating sex specific withdrawal symptomology in nicotine-dependent individuals, literature examining the underlying biological mediators of this is scant and not in complete agreement. Therefore, in this study, we evaluated the sex specific effects of nicotine and withdrawal on contextual fear memory, a hippocampally dependent aspect of cognition that is disrupted in nicotine withdrawal.MethodsMale and female B6/129F1 mice (8–13 weeks old) were used in all experiments. For the acute nicotine experiment, mice received intraperitoneal saline or nicotine (0.5 mg/kg) prior to contextual fear conditioning and test. For the chronic nicotine experiment, mice received nicotine (18 mg/kg/day) or saline for 11 days, then underwent contextual fear conditioning and test. Following the test, mice underwent minipump removal to elicit withdrawal or sham surgery, followed by the fear extinction assay. Bulk cortical tissue was used to determine nicotinic acetylcholine receptor levels via single point [3H]Epibatidine binding assay. Gene expression levels in the dorsal and ventral hippocampus were quantified via RT-PCR.ResultsWe found that female mice had a stronger expression of contextual fear memory than their male counterparts. Further, following acute nicotine treatment, male, but not female, subjects demonstrated augmented contextual fear memory expression. In contrast, no significant effects of chronic nicotine treatment on fear conditioning were observed in either sex. When examining extinction of fear learning, we observed that female mice withdrawn from nicotine displayed impaired extinction learning, but no effect was observed in males. Nicotine withdrawal caused similar suppression of fosb, cfos, and bdnf, our proxy for neuronal activation and plasticity changes, in the dorsal and ventral hippocampus of both sexes. Additionally, we found that ventral hippocampus erbb4 expression, a gene implicated in smoking cessation outcomes, was elevated in both sexes following nicotine withdrawal.ConclusionsDespite the similar impacts of nicotine withdrawal on gene expression levels, fosb, cfos, bdnf and erbb4 levels in the ventral hippocampus were predictive of delays in female extinction learning alone. This suggests sex specific dysfunction in hippocampal circuitry may contribute to female specific nicotine withdrawal induced deficits in extinction learning.

Exploring diabesity pathophysiology through proteomic analysis using Caenorhabditis elegans.

Diabesity, characterized by obesity-driven Type 2 diabetes mellitus (T2DM), arises from intricate genetic and environmental interplays that induce various metabolic disorders. The systemic lipid and glucose homeostasis is controlled by an intricate cross-talk of internal glucose/insulin and fatty acid molecules to maintain a steady state of internal environment. In this study, Caenorhabditis elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients to delve into the mechanistic foundations of diabesity. Various assays were conducted to measure intracellular triglyceride levels, lifespan, pharyngeal pumping rate, oxidative stress indicators, locomotor behavior, and dopamine signaling. Proteomic analysis was also performed to identify differentially regulated proteins and dysregulated KEGG pathways, and microscopy and immunofluorescence staining were employed to assess collagen production and anatomical integrity. Worms raised on diets high in glucose and cholesterol exhibited notably increased intracellular triglyceride levels, a decrease in both mean and maximum lifespan, and reduced pharyngeal pumping. The diabesity condition induced oxidative stress, evident from heightened ROS levels and distinct FT-IR spectroscopy patterns revealing lipid and protein alterations. Furthermore, impaired dopamine signaling and diminished locomotors behavior in diabesity-afflicted worms correlated with reduced motility. Through proteomic analysis, differentially regulated proteins encompassing dysregulated KEGG pathways included insulin signaling, Alzheimer's disease, and nicotinic acetylcholine receptor signaling pathways were observed. Moreover, diabesity led to decreased collagen production, resulting in anatomical disruptions validated through microscopy and immunofluorescence staining. This underscores the impact of diabesity on cellular components and structural integrity in C. elegans, providing insights into diabesity-associated mechanisms.

Ethyl gallate ameliorates diabetes-induced Alzheimer's disease-like phenotype in rats via activation of α7 nicotinic receptors and mitigation of oxidative stress

Cognitive decline, an important comorbidity of type 2 diabetes (T2D), is attributed to oxidative stress and impaired cholinergic signaling in the brain. The α7 nicotinic acetylcholine receptor (α7nAChR) is densely distributed in the hippocampus and cortex, and exerts neuroprotective and procognitive actions. Ethyl gallate (EG), a natural phenolic antioxidant compound, showed high in-silico binding affinity towards α7nAChR and brain penetrability. Therefore, the present study aimed to evaluate the involvement of α7nAChR in the potential of EG to ameliorate T2D-induced Alzheimer's disease-like condition. T2D was induced by intraperitoneal (i.p.) injection of streptozotocin (35 mg/kg) in rats on high-fat diet. Diabetic animals were treated with EG (10 and 20 mg/kg, i.p.) for four weeks, and their learning and memory performance was evaluated by the Morris water maze (MWM). Further, the brains were subjected to biochemical analysis of antioxidants like glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), and oxidative stress marker malonaldehyde (MDA). While diabetic rats showed a significant decline in cognitive performance in the MWM, a substantial improvement was noticed following EG treatment. Further, the diabetes-associated reductions in GSH, SOD, and CAT levels, along with increased MDA contents in the brain, were effectively restored by EG. Interestingly, pre-treatment with α7nAChR antagonist methyllycaconitine (1 mg/kg, i.p.) attenuated the effects of EG on behavioral and biochemical parameters. The results suggest that EG may augment cholinergic signaling in the brain via α7nAChR to mitigate oxidative stress, consequently alleviating T2D-associated dementia. Therefore, EG could be a potential candidate for addressing cognitive impairment comorbid with T2D.

Nicotine-induced CHRNA5 activation modulates CES1 expression, impacting head and neck squamous cell carcinoma recurrence and metastasis via MEK/ERK pathway

The mucosal epithelium of the head and neck region (including the oral cavity, nasal cavity, pharynx, nasopharynx, and larynx) is the primary site exposed to tobacco smoke, and its presence of nicotinic acetylcholine receptors (nAChRs) has been observed in the mucosal epithelial cells of this area. It remains unclear whether HNSC cells can migrate and invade through nAChR signaling. A model of HNSC cells exposed to nicotine is established. Cell proliferation following nicotine exposure is assessed using the CCK-8 assay, while migration and invasion are evaluated through wound healing and Transwell assays. The effects of CHRNA5 knockdown and overexpression are also investigated. Immunofluorescence staining is used to analyze CHRNA5 expression and localization, and clonogenic assays are performed to measure colony proliferation after CHRNA5 knockdown and overexpression. The interaction between CHRNA5 and CES1 is examined using molecular docking, co-immunoprecipitation, and immunofluorescence. Differentially expressed genes are subjected to pathway enrichment analysis, and MEK/ERK protein expression and phosphorylation are validated via western blot. Tumor formation assays are performed in nude mice using sh-CHRNA5 Cal27 cells, followed by western blot and immunohistochemical staining. Additionally, laryngeal and hypopharyngeal cancer tissues are analyzed through immunohistochemistry. Nicotine significantly enhanced the proliferation, migration, and invasion capabilities of head and neck tumor cells, including Cal27, Fadu, HN6, and Tu686 cells, through the expression of CHRNA5. Knockdown of CHRNA5 can reduce cell migration, invasion, and proliferation, whereas nicotine exposure can reverse this trend. Additionally, the mRNA and protein expression of CES1 decreases with the knockdown of CHRNA5, indicating a regulatory relationship between the two. Transcriptomics revealed that the knockdown of CHRNA5 is associated with the MEK/ERK signaling pathway. Further cellular- and tissue-level evidence confirmed that the levels of p-MEK/MEK, p-ERK/ERK, and CES1 decreased following knockdown of CHRNA5, a trend that nicotine can reverse. Nicotine promotes the proliferation, migration, and invasion of HNSC by upregulating CHRNA5 expression. Knockdown of CHRNA5 reduces these effects, which can be reversed by nicotine. Nicotine exposure activates CHRNA5, regulating CES1 expression via the MEK/ERK pathway, contributing to the recurrence and metastasis of head and neck squamous carcinoma.

Proteome of Clothianidin Exposed Honey Bees Reveals a Possible Mechanism Behind Impairment of Sucrose Responsiveness

ABSTRACTNeonicotinoids (NNIs) are the most commonly used insecticides in the world and clothianidin, a type of NNI, is commonly found in honey bee collected pollen. Clothianidin has several negative effects on honey bee health and behaviour, but whether and how it might impact learning and memory remains unclear. Therefore, we exposed honey bee workers to a field‐realistic oral dose of clothianidin for 7 days to assess their sugar responsiveness, and olfactory learning and memory using the proboscis extension response paradigm. Sugar responsiveness impacts important colony‐level traits, such as onset of foraging, that help maintain colony homeostasis as well as impacts learning and memory. We then measured how clothianidin alters protein expression in the brain in an effort to understand the mechanism(s) of clothianidin's effects. Clothianidin‐exposed bees had impaired sugar responsiveness, but no effect was seen on learning and memory. We identified 5069 protein groups and showed that the exposed bees had fewer detectable proteins. The exposed bees also had fewer mitochondrial respiration proteins, as well as reduced levels of nicotinic acetylcholine receptor α7 (nAChRα7) subunit (one of the targets of clothianidin) and reduced levels of acetylcholinesterase and acetylcholinesterase‐2 levels. In the exposed bees, the levels of nAChRα7 subunit positively correlated with sugar responsiveness, providing evidence for a possible mechanical explanation.