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Nicotine can enhance contextual learning while ethanol impairs some forms of learning. Nicotine can overcome some of the impairing effects of ethanol when the two drugs are co-administered. The specific brain nicotinic acetylcholine receptors (nAChRs) that mediate nicotine''s effects on contextual learning and whether any of ethanol''s actions are mediated by nAChRs are unknown. The potential roles of nAChRs in contextual and cued fear conditioning as well as the effects of nicotine, ethanol, or co-administration of nicotine and ethanol were examined in wild type and homozygous null mutant mice from α7, β2, β3, and β4 mouse lines at 24 h after training. Nicotine was given prior to training and testing, whereas ethanol was given only before training. Nicotine enhanced contextual learning in both α7 wild types and mutants when mice were trained at 0.17 mA, but not 0.35 mA. Mutants lacking the α7 subunit were less sensitive to the memory impairing effects of ethanol trained at 0.35 mA. β2 Null mutants receiving saline showed a small, but significant, impairment in contextual learning compared with wild type littermates when the shock stimulus was 0.35 mA. β2 Null mutant mice also did not respond to the cognitive enhancing effects of nicotine alone, or after ethanol administration. β3 and β4 null mutants did not differ from wild types either after saline or any of drug treatments, These results show that β2-containing nAChRs, but not β3- or β4-containing receptors, mediate the enhancing effects of nicotine on contextual learning and confirm previous studies implicating β2 in other forms of learning. A new role for α7 nAChRs in regulating sensitivity to the cognitive disrupting effects of ethanol is proposed. [Copyright &y& Elsevier]

Abstract: Kainic acid (KA) is a well-known excitatory, neurotoxic substance. In mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the CA3 pyramidal neurons. In the present study, the possible role of nicotinic acetylcholine receptors (nAchRs) in hippocampal cell death induced by KA (0.1μg) administered i.c.v. was examined. Methyllycaconitine (MC; nAchRs antagonist, 20μg) attenuated KA-induced CA3 pyramidal cell death. KA increased immunoreactivities (IRs) of phorylated extracellular signal-regulated kinase (p-ERK; at 30min), p-CaMK II (at 30min), c-Fos (at 2h), c-Jun (at 2h), glial fibrillary acidic protein (GFAP at 1 day), and the complement receptor type 3 (OX-42; at 1 day) in hippocampal area. MC attenuated selectively KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. Our results suggest that p-CaMK II may play as an important regulator responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Reactive astrocytes, which was meant by GFAP IR, and activated microglia, which was meant by OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA-induced excitotoxicity. Furthermore, it is implicated that niconitic receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice. [Copyright &y& Elsevier]

The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 μM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed Kd values of 1.9±1.1 and 3.6±1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, α3β4* nAChRs. Although the intracellular nAChRs represent ∼70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation. [Copyright &y& Elsevier]

We examined the role of nucleus isthmi in enhancing intracellular calcium concentrations in retinotectal fibers in the frog optic tectum in vitro. The intracellular calcium levels were measured using the fluorescent calcium-sensitive dye, Calcium Green-1 3000 mw dextran conjugate (CG-1), which was injected into one optic nerve. Electrical stimulation of the labeled optic nerve alone increased tectal CG-1 fluorescence whereas electrical stimulation of nucleus isthmi alone had no effect on CG-1 fluorescence. Electrical stimulation of the nucleus isthmi ipsilateral to the labeled tectum, followed by electrical stimulation to the optic nerve can enhance calcium uptake more than a double pulse stimulation of the optic nerve alone. Maximum enhancement of the calcium signal by nucleus isthmi occurs when optic nerve stimulation follows the ipsilateral nucleus isthmi stimulation by 10 ms. These results suggest that nucleus isthmi input can facilitate retinotectal neurotransmission, and the mechanism could be used to allow the frog to attend to a single prey stimulus in an environment of several prey stimuli. [Copyright &y& Elsevier]

The mechanisms that regulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), synthesis, transport, targeting and surface expression are of fundamental importance to understand the molecular basis of fast excitatory neurotransmission and synaptic plasticity in the mammalian CNS. An area of intense current interest is how AMPARs are directed to the correct locations in the neuron as and when required. This is a multi-layered problem, which involves complex spatio-temporal coordination of multiple protein interactions. Considerable progress has been achieved in identifying a number of proteins that bind directly to AMPAR subunits and the functional consequences of blocking some of these interactions have been determined. This review highlights recent developments in the field. [Copyright &y& Elsevier]

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels involved in fast synaptic transmission. Recently mutations in nAChR genes have been reported in nocturnal frontal lobe epilepsy. We performed molecular analysis on the human neuronal nAChR α6 subunit (CHRNA6) gene, a member of nAChR gene family, to understand its role in disease. Genomic analysis revealed that the gene consisted of six exons with an estimated size of 16 kb. We mapped the CHRNA6 gene to chromosome 8p11.21–11.22 by fluorescence in situ hybridization, the same putative region responsible for adolescent-onset idiopathic generalized epilepsy. Examination of the 5′-regulatory region failed to identify either a TATA box or GC-rich sequences, but did highlight tandem Alu sequences, located between 910 and 370 bp upstream from a potential cap site. Analyses of transcriptional activity, performed using nested deletions of the 5′-upstream region, showed that the downstream Alu repeat and another element(s) in the promoter region, function as negative regulators. Further analyses of the tandem Alu repeats, examined by fusing them to a different ion channel gene promoter, confirmed their role as transcriptional repressors regardless of their orientation and copy number. These data may explain the limited expression of the CHRNA6 gene in the brain. [Copyright &y& Elsevier]

Amyloid β1–42, a major component of amyloid plaques, binds with exceptionally high affinity to the α7 nicotinic acetylcholine receptor and accumulates intracellularly in neurons of Alzheimer’s disease brains. In this study, we investigated the possibility that this binding plays a key role in facilitating intraneuronal accumulation of amyloid β1–42. Consecutive section immunohistochemistry and digital imaging were used to reveal the spatial relationship between amyloid β1–42 and the α7 receptor in affected neurons of Alzheimer’s disease brains. Results showed that neurons containing substantial intracellular accumulations of amyloid β1–42 invariably express relatively high levels of the α7 receptor. Furthermore, this receptor is highly co-localized with amyloid β1–42 within neurons of Alzheimer’s disease brains. To experimentally test the possibility that the binding interaction between exogenous amyloid β1–42 and the α7 receptor facilitates internalization and intracellular accumulation of amyloid β1–42 in Alzheimer’s disease brains, we studied the fate of exogenous amyloid β1–42 and its interaction with the α7 receptor in vitro using cultured, transfected neuroblastoma cells that express elevated levels of this receptor. Transfected cells exhibited rapid binding, internalization and accumulation of exogenous amyloid β1–42, but not amyloid β1–40. Furthermore, the rate and extent of amyloid β1–42 internalization was related directly to the α7 receptor protein level, since (1) the rate of amyloid β1–42 accumulation was much lower in untransfected cells that express much lower levels of this receptor and (2) internalization was effectively blocked by α-bungarotoxin, an α7 receptor antagonist. As in neurons of Alzheimer’s disease brains, the α7 receptor in transfected cells was precisely co-localized with amyloid β1–42 in prominent intracellular aggregates. Internalization of amyloid β1–42 in transfected cells was blocked by phenylarsine oxide, an inhibitor of endocytosis.We suggest that the intraneuronal accumulation of amyloid β1–42 in Alzheimer’s disease brains occurs predominantly in neurons that express the α7 receptor. In addition, internalization of amyloid β1–42 may be facilitated by the high-affinity binding of amyloid β1–42 to the α7 receptor on neuronal cell surfaces, followed by endocytosis of the resulting complex. This provides a plausible explanation for the selective vulnerability of neurons expressing the α7 receptor in Alzheimer’s disease brains and for the fact that amyloid β1–42 is the dominant amyloid β peptide species in intracellular accumulations and amyloid plaques. [Copyright &y& Elsevier]

Nicotinic acetylcholine receptors (nAChRs) play an important role in homeostasis and respiratory diseases. Controversies regarding the association between COVID-19 hospitalizations and smoking suggest that nAChRs may contribute to SARS-CoV-2 respiratory syndrome. We recently detailed the expression and localization of all nAChR subunits in the human lung. Since virus association with nAChRs has been shown in the past, we hypothesize that nAChR subunits act as SARS-CoV-2 Spike co-receptors. Based on sequence alignment analysis, we report domains of high molecular similarities in nAChRs with the binding domain of hACE2 for SARS-CoV-2 Spike protein. This hypothesis supported by in silico pilot data provides a rational for the modelling and the in vitro experimental validation of the interaction between SARS-CoV-2 and the nAChRs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Elsevier Ltd. All rights reserved.)

This study is evaluating the effects of ethanol and nicotine exposure during pregnancy and lactation on placenta histology and follicular atresia in the first-generation (f1) mice pups. The experimental groups were 5 groups of NMRI pregnant mice, including: control, vehicle (received normal saline) ethanol (3 g/kg/day, 20 % v/v intraperitoneally), nicotine (1 mg/kg/day, subcutaneously), and ethanol plus nicotine which received both. Pregnant animals in each group were then divided into two groups, one group for examining the placenta that was treated for 18 days and the other group for the ovary of one-day-old (PND1) and fifty-six-day-old (PND56) female offspring who were treated for 42 days (during intrauterine development and lactation). After the autopsy procedure, histopathological and morphometrical observations were done. Data revealed that the exposed mice had a significant change in the placenta morphometry and histology as well as a marked increase in the number of ovarian TUNEL positive cells on postnatal days 1 and 56. Therefore, maternal exposure to alcohol and nicotine during developmental and lactation periods could lead to changes in the placenta properties as well as an increase in the apoptotic ovarian follicles in f1 mice pups., Competing Interests: The authors declare no conflict of interest., (© 2021 Published by Elsevier B.V.)

SARS-CoV-2 infection was announced as a pandemic in March 2020. Since then, several scientists have focused on the low prevalence of smokers among hospitalized COVID-19 patients. These findings led to our hypothesis that the Nicotinic Cholinergic System (NCS) plays a crucial role in the manifestation of COVID-19 and its severe symptoms. Molecular modeling revealed that the SARS-CoV-2 Spike glycoprotein might bind to nicotinic acetylcholine receptors (nAChRs) through a cryptic epitope homologous to snake toxins, substrates well documented and known for their affinity to the nAChRs. This binding model could provide logical explanations for the acute inflammatory disorder in patients with COVID-19, which may be linked to severe dysregulation of NCS. In this study, we present a series of complexes with cholinergic agonists that can potentially prevent SARS-CoV-2 Spike glycoprotein from binding to nAChRs, avoiding dysregulation of the NCS and moderating the symptoms and clinical manifestations of COVID-19. If our hypothesis is verified by in vitro and in vivo studies, repurposing agents currently approved for smoking cessation and neurological conditions could provide the scientific community with a therapeutic option in severe COVID-19., Competing Interests: The authors report no declarations of interest., (© 2020 The Authors. Published by Elsevier B.V.)

devTOX quick Predict (devTOX qP ) is a metabolomics biomarker-based assay that utilises human induced pluripotent stem (iPS) cells to screen for potential early stage embryonic developmental toxicity in vitro. Developmental toxicity potential is assessed based on the assay endpoint of the alteration in the ratio of key unrelated biomarkers, ornithine and cystine (o/c). This work aimed to compare the developmental toxicity potential of tobacco-containing and tobacco-free non-combustible nicotine products to cigarette smoke. Smoke and aerosol from test articles were produced using a Vitrocell VC10 smoke/aerosol exposure system and bubbled into phosphate buffered saline (bPBS). iPS cells were exposed to concentrations of up to 10% bPBS. Assay sensitivity was assessed through a spiking study with a known developmental toxicant, all -trans- retinoic acid (ATRA), in combination with cigarette smoke extract. The bPBS extracts of reference cigarettes (1R6F and 3R4F) and a heated tobacco product (HTP) were predicted to have the potential to induce developmental toxicity, in this screening assay. The bPBS concentration at which these extracts exceeded the developmental toxicity threshold was 0.6% (1R6F), 1.3% (3R4F), and 4.3% (HTP) added to the cell media. Effects from cigarette smoke and HTP aerosol were driven largely by cytotoxicity, with the cell viability and o/c ratio dose-response curves crossing the developmental toxicity thresholds at very similar concentrations of added bPBS. The hybrid product and all the electronic cigarette (e-cigarette) aerosols were not predicted to be potential early developmental toxicants, under the conditions of this screening assay., Competing Interests: The mybluTM devices used in this study were manufactured by Fontem Ventures B.V., a wholly owned subsidiary of Imperial Brands. All the authors were employed by Imperial Brands PLC or subsidiaries at time of writing, these were Simms, L., Rudd, K., Czekala, L., Yu, F., Chapman, F., Trelles-Sticken, E., Wieczorek, R., Bode, L., Stevenson, M., Walele, T., (© 2020 The Authors.)