Abundant extrasynaptic expression of α3ß4-containing nicotinic acetylcholine receptors in the medial habenula-interpeduncular nucleus pathway in mice.

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and ß4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3ß4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and ß4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and ß4 subunits disappeared in α5ß4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that α3ß4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3ß4-containing nAChRs in the crucial pathway of nicotine dependence.

Investigating CHRNA5, CHRNA3, and CHRNB4 variants in the genetic landscape of substance use disorder in Jordan.

BACKGROUND: Substance use disorder (SUD) is a complex illness that can be attributed to the interaction between environmental and genetic factors. The nicotinic receptor gene cluster on chromosome 15 has a plausible association with SUD, particularly with nicotine dependence. METHODS: This study investigated 15 SNPs within the CHRNA5, CHRNA3, and CHRNB4 genes. Sequencing was used for genotyping 495 Jordanian males with SUD and 497 controls matched for age, gender, and descent. RESULTS: Our findings revealed that none of the tested alleles or genotypes were correlated with SUD. However, our analysis suggests that the route of substance use was linked to rs1051730 (P value = 0.04), rs8040868 (P value = 0.01) of CHRNA3, and rs16969968 (P value = 0.03) of CHRNA5. Additionally, a correlation was identified between rs3813567 of the CHRNB4 gene and the age at substance use onset (P value = 0.04). CONCLUSIONS: Variants in CHRNA5, CHRNA3, and CHRNB4 may interact with SUD features that can influence the development and progression of the disorder among Jordanians.

Spermatogonial stem cells in the 129 inbred strain exhibit unique requirements for self-renewal.

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species.

Bulk segregant mapping and transcriptome analyses reveal the molecular mechanisms of spinetoram resistance in Spodoptera frugiperda.

The evolution of resistance to insecticides poses a significant threat to pest management programs. Understanding the molecular mechanisms underlying insecticide resistance is essential to design sustainable pest control and resistance management programs. The fall armyworm, Spodoptera frugiperda, is an important insect pest of many crops and has a remarkable ability to evolve resistance to insecticides. In this study, we employed bulk segregant analysis (BSA) combined with DNA and RNA sequencing to characterize the molecular basis of spinetoram resistance in S. frugiperda. Analysis of genomic data derived from spinetoram selected and unselected bulks and the spinetoram-resistant and susceptible parental strains led to the identification of a three-nucleotide deletion in the gene encoding the nicotinic acetylcholine receptor α6 subunit (nAChR α6). Transcriptome profiling identified the upregulation of few genes encoding detoxification enzymes associated with spinetoram resistance. Thus, spinetoram resistance in S. frugiperda appears to be mediated mainly by target site insensitivity with a minor role of detoxification enzymes. Our findings provide insight into the mechanisms underpinning resistance to spinetoram in S. frugiperda and will inform the development of strategies to control this highly damaging, globally distributed crop pest.

Identifying and assessing a prognostic model based on disulfidptosis-related genes: implications for immune microenvironment and tumor biology in lung adenocarcinoma.

Introduction: Lung cancer, with the highest global mortality rate among cancers, presents a grim prognosis, often diagnosed at an advanced stage in nearly 70% of cases. Recent research has unveiled a novel mechanism of cell death termed disulfidptosis, which is facilitated by glucose scarcity and the protein SLC7A11. Methods: Utilizing the least absolute shrinkage and selection operator (LASSO) regression analysis combined with Cox regression analysis, we constructed a prognostic model focusing on disulfidptosis-related genes. Nomograms, correlation analyses, and enrichment analyses were employed to assess the significance of this model. Among the genes incorporated into the model, CHRNA5 was selected for further investigation regarding its role in LUAD cells. Biological functions of CHRNA5 were assessed using EdU, transwell, and CCK-8 assays. Results: The efficacy of the model was validated through internal testing and an external validation set, with further evaluation of its robustness and clinical applicability using a nomogram. Subsequent correlation analyses revealed associations between the risk score and infiltration of various cancer types, as well as oncogene expression. Enrichment analysis also identified associations between the risk score and pivotal biological processes and KEGG pathways. Our findings underscore the significant impact of CHRNA5 on LUAD cell proliferation, migration, and disulfidptosis. Conclusion: This study successfully developed and validated a robust prognostic model centered on disulfidptosis-related genes, providing a foundation for predicting prognosis in LUAD patients.

Aspartic acid mutagenesis of αO-Conotoxin GeXIVA isomers reveals arginine residues crucial for inhibition of the α9α10 nicotinic acetylcholine receptor.

The two most active disulfide bond isomers of the analgesic αO-conotoxin GeXIVA, namely GeXIVA[1, 2] and GeXIVA[1, 4], were subjected to Asp-scanning mutagenesis to determine the key amino acid residues for activity at the rat α9α10 nicotinic acetylcholine receptor (nAChR). These studies revealed the key role of arginine residues for the activity of GeXIVA isomers towards the α9α10 nAChR. Based on these results, additional analogues with 2-4 mutations were designed and tested. The analogues [T1A,D14A,V28K]GeXIVA[1, 2] and [D14A,I23A,V28K]GeXIVA[1, 4] were developed and showed sub-nanomolar activity for the α9α10 nAChR with IC50 values of 0.79 and 0.38 nM. The latter analogue had exceptional selectivity for the α9α10 receptor subtype over other nAChR subtypes and can be considered as a drug candidate for further development. Molecular dynamics of receptor-ligand complexes allowed us to make deductions about the possible causes of increases in the affinity of key GeXIVA[1, 4] mutants for the α9α10 nAChR.

Alpha9alpha10 knockout mice show altered physiological and behavioral responses to signals in masking noise.

Medial olivocochlear (MOC) efferents modulate outer hair cell motility through specialized nicotinic acetylcholine receptors to support encoding of signals in noise. Transgenic mice lacking the alpha9 subunits of these receptors (α9KOs) have normal hearing in quiet and noise, but lack classic cochlear suppression effects and show abnormal temporal, spectral, and spatial processing. Mice deficient for both the alpha9 and alpha10 receptor subunits (α9α10KOs) may exhibit more severe MOC-related phenotypes. Like α9KOs, α9α10KOs have normal auditory brainstem response (ABR) thresholds and weak MOC reflexes. Here, we further characterized auditory function in α9α10KO mice. Wild-type (WT) and α9α10KO mice had similar ABR thresholds and acoustic startle response amplitudes in quiet and noise, and similar frequency and intensity difference sensitivity. α9α10KO mice had larger ABR Wave I amplitudes than WTs in quiet and noise. Other ABR metrics of hearing-in-noise function yielded conflicting findings regarding α9α10KO susceptibility to masking effects. α9α10KO mice also had larger startle amplitudes in tone backgrounds than WTs. Overall, α9α10KO mice had grossly normal auditory function in quiet and noise, although their larger ABR amplitudes and hyperreactive startles suggest some auditory processing abnormalities. These findings contribute to the growing literature showing mixed effects of MOC dysfunction on hearing.

α5-nAChR/ADAM10 signaling mediates nicotine-related cutaneous melanoma progression via STAT3 activation.

Skin cutaneous melanoma (SKCM) is the skin malignancy with the highest mortality rate, and its morbidity rate is on the rise worldwide. Smoking is an independent marker of poor prognosis in melanoma. The α5-nicotinic acetylcholine receptor (α5-nAChR), one of the receptors for nicotine, is involved in the proliferation, migration and invasion of SKCM cells. Nicotine has been reported to promote the expression of a disintegrin and metalloproteinase 10 (ADAM10), which is the key gene involved in melanoma progression. Here, we explored the link between α5-nAChR and ADAM10 in nicotine-associated cutaneous melanoma. α5-nAChR expression was correlated with ADAM10 expression and lower survival in SKCM. α5-nAChR mediated nicotine-induced ADAM10 expression via STAT3. The α5-nAChR/ADAM10 signaling axis was involved in the stemness and migration of SKCM cells. Furthermore, α5-nAChR expression was associated with ADAM10 expression, EMT marker expression and stemness marker expression in nicotine-related mice homograft tissues. These results suggest the role of the α5-nAChR/ADAM10 signaling pathway in nicotine-induced melanoma progression.

Potency and Target Surface Interaction of Diazinoyl Nicotinic Insecticides.

Structure-activity relationships of diazinoyl nicotinic insecticides (diazinoyl isomers and 5- or 6-substituted pyrazin-2-oyl analogues) are considered in terms of affinity to the insect nicotinic acetylcholine receptor (nAChR) and insecticidal activity against the imidacloprid-resistant brown planthopper. Among the test compounds, 3-(6-chloropyridin-3-ylmethyl)-2-(pyrazinoyl)iminothiazoline shows the highest potency in nAChR affinity and insecticidal activity. Aplysia californica acetylcholine binding protein (AChBP) mutants (Y55W + Q57R and Y55W + Q57T) are utilized to compare molecular recognition of nicotinic insecticides with diverse pharmacophores. N-nitro- or N-cyanoimine imidacloprid or acetamiprid, respectively, exhibits a high affinity to these AChBP mutants at a similar potency level. Intriguingly, the pyrazin-2-oyl analogue has a higher affinity to AChBP Y55W + Q57R than that to Y55W + Q57T, thereby indicating that pyrazine nitrogen atoms contact Arg57 guanidinium and Trp55 indole NH. Furthermore, nicotine prefers AChBP Y55W + Q57T over Y55W + Q57R, conceivably suggesting that the protonated nicotine is repulsed by Arg57 guanidinium, consistent with its inferior potency to insect nAChR.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Functions and pharmacology of α2ß2 nicotinic acetylcholine receptors; in and out of the shadow of α4ß2 nicotinic acetylcholine receptors.

Although α2 was the first neuronal nicotinic acetylcholine receptor (nAChR) receptor subunit to be cloned, due to its low level of expression in rodent brain, its study has largely been neglected. This study provides a comparison of the α2 and α4 structures and their functional similarities, especially in regard to the existence of low and high sensitivity forms based on subunit stoichiometry. We show that the pharmacological profiles of the low and high sensitivity forms of α2ß2 and α4ß2 receptors are very similar in their responses to nicotine, with high sensitivity receptors showing protracted responses. Sazetidine A, an agonist that is selective for the high sensitivity α4 receptors also selectively activates high sensitivity α2 receptors. Likewise, α2 receptors have similar responses as α4 receptors to the positive allosteric modulators (PAMs) desformylflustrabromine (dFBr) and NS9283. We show that the partial agonists for α4ß2 receptors, cytisine and varenicline are also partial agonists for α2ß2 receptors. Studies have shown that levels of α2 expression may be much higher in the brains of primates than those of rodents, suggesting a potential importance for human therapeutics. High-affinity nAChR have been studied in humans with PET ligands such as flubatine. We show that flubatine has similar activity with α2ß2 and α4ß2 receptors so that α2 receptors will also be detected in PET studies that have previously presumed to selectively detect α4ß2 receptors. Therefore, α2 receptors need more consideration in the development of therapeutics to manage nicotine addiction and declining cholinergic function in age and disease.

Computational Modeling Oriented Substructure Splicing Application in the Identification of Thiazolidine Derivatives as Potential Low Honeybee Toxic Neonicotinoids.

With the aim of identifying novel neonicotinoid insecticides with low bee toxicity, a series of compounds bearing thiazolidine moiety, which has been shown to be low bee toxic, were rationally designed through substructure splicing strategy and evaluated insecticidal activities. The optimal compounds A24 and A29 exhibited LC50 values of 30.01 and 17.08 mg/L against Aphis craccivora, respectively. Electrophysiological studies performed on Xenopus oocytes indicated that compound A29 acted on insect nAChR, with EC50 value of 50.11 µM. Docking binding mode analysis demonstrated that A29 bound to Lymnaea stagnalis acetylcholine binding protein through H-bonds with the residues of D_Arg55, D_Leu102, and D_Val114. Quantum mechanics calculation showed that A29 had a higher highest occupied molecular orbit (HOMO) energy and lower vertical ionization potential (IP) value compared to the high bee toxic imidacloprid, showing potentially low bee toxicity. Bee toxicity predictive model also indicated that A29 was nontoxic to honeybees. Our present work identified an innovative insecticidal scaffold and might facilitate the further exploration of low bee toxic neonicotinoid insecticides.

Sugar-coated survival: N-glycosylation as a unique bearded dragon venom resistance trait within Australian agamid lizards.

In the ongoing evolutionary arms race between predators and prey, adaptive innovations often trigger a reciprocal response. For instance, the emergence of α-neurotoxins in snake venom has driven prey species targeted by these snakes to evolve sophisticated defense mechanisms. This study zeroes in on the particular motifs within the orthosteric sites of post-synaptic nicotinic acetylcholine receptors (nAChR) that confer resistance to α-neurotoxins, often through structural alterations of nAChR. This research examined Australian agamid lizards, a primary prey group for Australian elapid snakes, which are subject to predatory selection pressures. We previously showed that Pogona vitticeps (Central bearded dragon) was resistant to α-neurotoxic snake venoms through a steric hindrance form resistance evolving within the nAChR orthosteric, specifically through the 187-189NVT motif resulting in the presence of N-glycosylation, with the branching carbohydrate chains impeding the binding by the neurotoxins. This adaptive trait is thought to be a compensatory mechanism for the lizard's limited escape capabilities. Despite the significance of this novel adaptation, the prevalence and evolutionary roots of such venom resistance in Australian agamids have not been thoroughly investigated. To fill this knowledge gap, we undertook a comprehensive sequencing analysis of the nAChR ligand-binding domain across the full taxonomical diversity of Australian agamid species. Our findings reveal that the N-glycosylation resistance mechanism is a trait unique to the Pogona genus and absent in other Australian agamids. This aligns with Pogona's distinctive morphology, which likely increases vulnerability to neurotoxic elapid snakes, thereby increasing selective pressures for resistance. In contrast, biolayer interferometry experiments with death adder (Acanthophis species) venoms did not indicate any resistance-related binding patterns in other agamids, suggesting a lack of similar resistance adaptations, consistent with these lineages either being fast-moving, covered with large defensive spines, or being arboreal. This research not only uncovers a novel α-neurotoxin resistance mechanism in Australian agamids but also highlights the complex dynamics of the predator-prey chemical arms race. It provides a deeper understanding of how evolutionary pressures shape the interactions between venomous snakes and their prey.

Pathogenic residue insertion in neuronal nicotinic receptor alters intra- and inter-subunit interactions that tune channel gating.

We describe molecular-level functional changes in the α4ß2 nicotinic acetylcholine receptor by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single-channel currents reveal the primary effect is to stabilize the open channel state, while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4)2(ß2)3 and (α4)3(ß2)2, indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intrasubunit and intersubunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together, the results reveal a novel structural region of the α4ß2 nicotinic acetylcholine receptor in which interhelical interactions tune the stability of the open channel state.

Ion transport in muscle acetylcholine receptor maintained by conserved salt bridges between the pore and lipid membrane.

Pores through ion channels rapidly transport small inorganic ions along their electrochemical gradients. Here, applying single-channel electrophysiology and mutagenesis to the archetypal muscle nicotinic acetylcholine receptor (AChR) channel, we show that a conserved pore-peripheral salt bridge partners with those in the other subunits to regulate ion transport. Disrupting the salt bridges in all five receptor subunits greatly decreases the amplitude of the unitary current and increases its fluctuations. However, disrupting individual salt bridges has unequal effects that depend on the structural status of the other salt bridges. The AChR ε- and δ-subunits are structurally unique in harboring a putative palmitoylation site near each salt bridge and bordering the lipid membrane. The effects of disrupting the palmitoylation sites mirror those of disrupting the salt bridges, but the effect of disrupting either of these structures depends on the structural status of the other. Thus, rapid ion transport through the AChR channel is maintained by functionally interdependent salt bridges linking the pore to the lipid membrane.

Single-cell quantitative expression of nicotinic acetylcholine receptor mRNA in rat hippocampal interneurons.

Hippocampal interneurons are a very diverse population of cells. Using single-cell quantitative PCR to analyze rat CA1 hippocampal interneurons, we quantified neuronal nicotinic acetylcholine receptor (nAChR) mRNA subunit expression and detailed possible nAChR subtype combinations for the α2, α3, α4, α5, α7, ß2, ß3, and ß4 subunits. We also compared the expression detected in the stratum oriens and the stratum radiatum hippocampal layers. We show that the majority of interneurons in the CA1 of the rat hippocampus contain detectable levels of nAChR subunit mRNA. Our results highlight the complexity of the CA1 nAChR population. Interestingly, the α3 nAChR subunit is one of the highest expressed subunit mRNAs in this population, while the α4 is one of the least likely subunits to be detected in CA1 interneurons. The ß2 nAChR subunit is the highest expressed beta subunit mRNA in these cells. In addition, Pearson's correlation coefficient values are calculated to identify significant differences between the nAChR subunit combinations expressed in the CA1 stratum oriens and the stratum radiatum. Statistical analysis also indicates that there are likely over 100 different nAChR subunit mRNA combinations expressed in rat CA1 interneurons. These results provide a valid avenue for identifying nAChR subtype targets that may be effective hippocampus-specific pharmacological targets.

The Cloning and Characterization of a Three-Finger Toxin Homolog (NXH8) from the Coralsnake Micrurus corallinus That Interacts with Skeletal Muscle Nicotinic Acetylcholine Receptors.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve-diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response.

Acetylcholine is produced in the spleen in response to vagus nerve activation; however, the effects on antibody production have been largely unexplored. Here, we use a chronic vagus nerve stimulation (VNS) mouse model to study the effect of VNS on T-dependent B cell responses. We observed lower titers of high-affinity IgG and fewer antigen-specific germinal center (GC) B cells. GC B cells from chronic VNS mice exhibited altered mRNA and protein expression suggesting increased apoptosis and impaired plasma cell differentiation. Follicular dendritic cell (FDC) cluster dispersal and altered gene expression suggested poor function. The absence of acetylcholine-producing CD4+ T cells diminished these alterations. In vitro studies revealed that α7 and α9 nicotinic acetylcholine receptors (nAChRs) directly regulated B cell production of TNF, a cytokine crucial to FDC clustering. α4 nAChR inhibited coligation of CD19 to the B cell receptor, presumably decreasing B cell survival. Thus, VNS-induced GC impairment can be attributed to distinct effects of nAChRs on B cells.