Nicotinic Responses Research Articles

Cocaine potently blocks neuronal α3β4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3β4-nAChR expressed in native SH-SY5Y cells. α3β4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 μM. Kinetic analysis showed that cocaine accelerated α3β4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 μM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-βS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 μM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3β2β3-nAChRs and α4β2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3β4-nAChRs expressed in SH-SY5Y cells.

Ghrelin modulates morphine-nicotine interaction in avoidance memory: Involvement of CA1 nicotinic receptors

Ghrelin is a stomach-derived hormone which regulates appetite and energy balance in the body. Recent studies show that ghrelin has been linked to the learning and memory process. Ghrelin also modulates reward properties of addictive drugs. However, the involvement of ghrelin in cognitive effects of addictive drugs has not been examined yet. The goal of present study is to examine the effect of intra-CA1 administration of ghrelin on morphine response for avoidance task alone or in combination with nicotine. Here, we also investigated the role of hippocampal nicotinic cholinergic receptors in possible interaction of the drugs in adult male Wistar rats. Results showed that subcutaneous administration of morphine immediately after training impaired memory in the test day and induced amnesia, while intra-CA1 pre-injection of ghrelin prevented amnesic effect of morphine and improved memory. Also, systemic administration of nicotine five min prior to morphine administration dose-dependently inhibited morphine-induced amnesia. The results showed that intra-CA1 injection of an ineffective dose of ghrelin (0.03 nmol/µl) potentiated the nicotine (0.2 mg/kg, s.c.) response on amnesia induced by morphine. This stimulatory effect was inhibited by mechamylamine, a non-competitive nicotinic receptor antagonist. Moreover, post-training administration of drugs (ghrelin, nicotine and mecamylamine) alone had no effect on memory consolidation. In conclusion, present study suggests the significant role of ghrelin in morphine-related memory and its interactive effect with nicotine in avoidance task via CA1 nicotinic receptors.

Mouse strain-specific acute respiratory effects of nicotine unrelated to nicotine metabolism

Plethysmograph measurement of respiratory phenotypes provides a highly sensitive means to study nicotine response in experimental model animals. We measured average respiratory frequency, tidal volume, minute volume and inspiratory time in C3H/HeJ and C57BL/6J mice subcutaneously administered 0.35 and 0.70 mg/kg nicotine. Both mouse strains showed significantly altered respiratory and locomotion phenotypes relative to saline-injected controls when administered the higher dose, but only C57BL/6J responded to the lower nicotine dose. Respiratory and locomotion phenotypes rarely differed significantly by sex. To investigate whether the strain-specific differences in nicotine sensitivity were related to differences in clearance, we followed up by measuring nicotine clearance in C3H/HeJ and C57BL/6J mice (0.35 mg/kg subcutaneous) and found sex differences in both strains, but no difference between strains.

Nitric oxide blockade in mediodorsal thalamus impaired nicotine/ethanol-induced memory retrieval in rats via inhibition of prefrontal cortical pCREB/CREB signaling pathway.

Reciprocal connections between the mediodorsal thalamic nucleus (MD) and the prefrontal cortex (PFC) are important for memory processes. Since the co-abuse of nicotine and ethanol affects memory formation, this study investigated the effect of nitric oxide inhibition in the MD on memory retrieval induced by co-administration of nicotine and ethanol. Subsequently, western blot analysis was used to evaluate how this change would alter the PFC pCREB/CREB signaling pathway. Male Wistar rats were bilaterally cannulated into the MD and the memory retrieval was measured by passive avoidance task. Intraperitoneal (i.p.) administration of ethanol (1 g/kg, i.p) 30 min before the test impaired memory retrieval and caused ethanol-induced amnesia. Subcutaneous (s.c.) administration of nicotine (0.05-0.2 mg/kg, s.c.) prevented ethanol-induced amnesia and improved memory retrieval. Intra-MD microinjection of a nitric oxide synthase (NOS) inhibitor, L-NAME (0.5-1 μg/rat) inhibited the improving effect of nicotine (0.2 mg/kg, s.c.) on ethanol-induced amnesia, while intra-MD microinjection of a precursor of nitric oxide, l-arginine (0.25-1 μg/rat), potentiated such effect. Noteworthy, intra-MD microinjection of the same doses of L-NAME or l-arginine by itself had no effect on memory retrieval. Furthermore, intra-MD microinjection of L-NAME (0.05, 0.1 and 0.3 μg/rat) reversed the l-arginine improving effect on nicotine response. Successful memory retrieval significantly increased the p-CREB/CREB ratio in the PFC tissue. Ethanol-induced amnesia, however, decreased this ratio in the PFC while the co-administration of nicotine and ethanol increased the PFC CREB signaling. Interestingly, the inhibitory effect of L-NAME and the potentiating effect of l-arginine on nicotine response were associated with the decrease and increase of the PFC p-CREB/CREB ratio respectively. It can be concluded that MD-PFC connections are involved in the combined effects of nicotine and ethanol on memory retrieval. The mediodorsal thalamic NO system possibly mediated this interaction via the pCREB/CREB signaling pathways in the PFC.

Response to Reduced Nicotine Content in Vulnerable Populations: Effect of Menthol Status.

In this study, we investigated potential effects of being a menthol smoker on response to reduced nicotine content (RNC) cigarettes in smokers especially vulnerable to smoking. Participants were 169 smokers (61 menthol and 108 non-menthol smokers) with comorbid mental illness, substance use disorder, or socioeconomic disadvantage. Participants completed a double-blind study assessing addiction potential, withdrawal/craving, and compensatory smoking across 4 research cigarettes varying in nicotine content from very low levels to commercial levels (0.4, 2.4, 5.2, 15.8mg/g of tobacco). Repeated measures analysis of variance was used to examine potential moderating effects of menthol status. Statistically significant effects of nicotine dose were noted across measures, with higher doses producing greater economic demand and relief from withdrawal/craving. The relationships between nicotine dose and response to RNC cigarettes do not differ by menthol status. Results of this study suggest menthol does not have a differential impact on response to RNC cigarettes across measures of economic demand, withdrawal/craving, or smoking topography. These results suggest that any potential beneficial effects of RNC cigarettes should extend to menthol smokers including those especially vulnerable to smoking.

Analysis of whole genome-transcriptomic organization in brain to identify genes associated with alcoholism

Alcohol exposure triggers changes in gene expression and biological pathways in human brain. We explored alterations in gene expression in the Pre-Frontal Cortex (PFC) of 65 alcoholics and 73 controls of European descent, and identified 129 genes that showed altered expression (FDR < 0.05) in subjects with alcohol dependence. Differentially expressed genes were enriched for pathways related to interferon signaling and Growth Arrest and DNA Damage-inducible 45 (GADD45) signaling. A coexpression module (thistle2) identified by weighted gene co-expression network analysis (WGCNA) was significantly correlated with alcohol dependence, alcohol consumption, and AUDIT scores. Genes in the thistle2 module were enriched with genes related to calcium signaling pathways and showed significant downregulation of these pathways, as well as enrichment for biological processes related to nicotine response and opioid signaling. A second module (brown4) showed significant upregulation of pathways related to immune signaling. Expression quantitative trait loci (eQTLs) for genes in the brown4 module were also enriched for genetic associations with alcohol dependence and alcohol consumption in large genome-wide studies included in the Psychiatric Genetic Consortium and the UK Biobank’s alcohol consumption dataset. By leveraging multi-omics data, this transcriptome analysis has identified genes and biological pathways that could provide insight for identifying therapeutic targets for alcohol dependence.

Genetic variants in nicotinic receptors and smoking cessation in Parkinson's disease

BackgroundNegative associations between smoking and Parkinson's disease (PD) are well documented. While common biases may not explain this association, some studies have suggested reverse causality and ease of quitting might be an early sign of PD, possibly related to a reduced nicotinic response. We investigated nicotinic receptor (nAChR) genetics to add to our understanding of possible biologic mechanisms underlying the smoking-PD relationship. MethodsWe relied on 612 patients and 691 controls enrolled in the PEG (Parkinson's Environment and Gene) study for whom we obtained information on smoking and quitting ease through interviews. Genotyping in the nAChR genes, i.e. CHRNA5-A3-B4 and CHRNB3-A6 gene regions that have been linked to smoking or quitting behaviors, were based on blood and saliva DNA samples. We assessed associations with logistic regression assuming logit-additive allelic effects and used product terms for genetic allele status and smoking or quitting assessing interactions. ResultsAs expected, we observed negative associations between smoking and PD that were strongest for current followed by former smokers. In former smokers, high quitting difficulty was negatively associated with PD risk (extremely hard vs. easy: OR = 0.62 [0.39–0.99], p = 0.05), meaning those who developed PD were able to quit smoking with less difficulty than controls. The CHRNA3 rs578776-A allele predicted quitting difficulty in smoking controls (OR = 0.53 [0.32–0.91], p = 0.02), but not in smoking PD patients (OR = 1.09 [0.61–1.95], p = 0.77). ConclusionOur study further corroborates previous findings that ease of quitting may be an early sign of PD onset related to a loss of nicotinic response in prodromal stages.

Pharmacogenomics – From Large Collaborations To Implementation and Drug Discovery

Overall Abstract Introduction Recent genetic studies have identified variants in specific gene regions showing not only evidence of association but also biological mechanisms underlying the association. The goal of this symposium is to demonstrate how human genetic association findings can be translated into improved understanding of biological mechanism, disease prognosis, and treatment response using examples across different psychiatric disorders such as mood disorders, schizophrenia, eating disorder, and addiction. Methods A variety of methods are used including randomized treatment trials, large genome wide association studies or meta-analyses, bioinformatics, and epigenetics. Results The first talk by Dr. Schulze is on genotype-phenotype relationships in bipolar and related traits. He will demonstrate how applying the friendly data sharing approach spanning five continents has helped pave the way for a better understanding of the genetic basis of response to lithium treatment, serving as an example for successful precision medicine research. The 2nd talk by Dr. Muller is on current evidence, evidence adjudication, implementation challenges of pharmacogenetics testing for antidepressants and antipsychotics use. He will present a review of the evidence that was considered in formulating that guidance, along with a summary of the main recommendations and associated ethical issues. He will also discuss the kinds of evidence still needed to incorporate genetic testing in clinical decision-making. The 3rd talk by Dr. Breen is on translation of genetic findings from large GWAS studies in Psychiatric Genomics Consortium (PGC) into druggable genomes. He will discuss how discovery of the biological pathways can not only reveal etiological pathways but also lead to drug discovery. The 4th talk by Dr. Chen demonstrates the significant genetic variants that predict nicotine dependence, smoking cessation, and response to cessation pharmacotherapy. These data suggest that genetic risks may predict smoking cessation outcomes and moderate the effect of pharmacological treatments. Some pharmacogenetic findings have been replicated in meta-analyses of multiple smoking cessation trials. The variation in efficacy between smokers with different genetic markers suggests that personalized smoking cessation pharmacotherapy based upon genotype could maximize the efficiency of such treatment while minimizing side effects. As a result, these genetic markers are useful to predict clinical predictor such as the number needed to treat (NNT) and the number needed to harm. Conclusion Different psychiatric disorders serve as successful examples in how genetic findings in human association studies inform biological experiments to uncover the underlying mechanisms and treatments tailored to individual genetic background. Each presentation represents unique and complementary research paradigms in understanding how genomics inform personalized medicine by linking the biology and treatment in different psychiatric disorders. Learning Objectives 1) human genetic studies inform the biological mechanisms of psychiatric disorders, 2) pharmacogenetic studies inform personalized treatments with maximized efficacy and minimized risk of side effects.

Pathways To Precision Medicine In Smoking Cessation Treatments

Introduction: Cigarette smoking is highly addictive and prevalent among individuals with mental illness. Modern genetic research has identified robust genetic influences on nicotine dependence. An important step in translating these genetic findings is to identify the genetic factors affecting smoking cessation in order to enhance current smoking cessation treatments. MethodsWe investigated the significant genetic variants that predict nicotine dependence, smoking cessation, and response to cessation pharmacotherapy using both large cohort studies of smokers and randomized controlled trials. ResultsWe find that genetic risks may predict prognosis and response to pharmacological treatments. For prognosis, the nicotinic receptor genotypes predict delayed quitting and earlier lung cancer diagnoses after adjusting for smoking quantity in large meta-analyses of studies of lung cancer and nicotine dependence. For treatment response, some pharmacogenetic findings have been replicated in meta-analyses or multiple smoking cessation trials for both pharmacodynamic markers (e.g., CHRNA5) and pharmacokinetic marker (e.g., CYP2A6). The variation in efficacy between smokers with different genetic markers supports the notion that personalized smoking cessation intervention based upon genotype could maximize the efficiency of such treatment while minimizing side effects, thus influencing the number needed to treat (NNT) and the number needed to harm. ConclusionCurrent evidence strongly suggests that cessation failure and cessation pharmacotherapy effectiveness are modulated by biomarkers such as nicotinic receptor genotypes, nicotine metabolism ratio (NMR), or polygenic risk scores for nicotine dependence. These findings strengthen the case for further research on genetics of smoking cessation success, and the development and rigorous testing of treatments for smokers with different genetic risk profiles. Learning Objectives1) human genetic studies can inform the biological mechanisms of nicotine dependence and health consequences such as lung cancer, 2) pharmacogenetic studies may inform personalized treatments for tobacco use disorder, possibly increasing efficacy and reducing risk of side effects.

Screening and evaluation of ethanolic extract from Casuarina equisetifolia inflorescence on isolated chick rectum, frog rectum and frog rectus abdominus muscle for identification of muscarinic and nicotinic receptor’s action

The present experiments were undertaken to justify the use of an ethanolic extract from inflorescence of Casuarina equisetifolia, Family: Casuarinaceae, influencing the nicotine responses on isolated chick rectum and frog rectum (Smooth Muscles) and frog rectus abdominus muscle (Skeletal Muscle). The isolated tissues were mounted in organ bath filled with physiological solution and was suitably aerated. After equilibration, responses were taken to different doses of nicotine (log doses) till a ceiling response was obtained. A sub-maximal dose of nicotine was selected and responses to this dose was taken and ensured that there is reproducibility of response. The drum was allowed to move for 1min., different concentrations of extracts into the organ baths were added and allowed to act for 1min without flushing the baths, then the sub-maximal dose of nicotine was added and allowed to act for 1min. this procedure was repeated (without extract) till the original response was obtained. The inference drawn from these experiments, the ethanolic extract of inflorescence of Casuarina equisetifolia antagonised the action of nicotine on isolated chick rectum, relaxed the effect of nicotine on frog rectum and it potentiated the effect of nicotine on frog rectus abdominus muscle. The nicotine receptors of rectus abdominus is activated, perhaps by the prevention of hydrolysis of acetylcholine by the extract. Based on the results obtained from the isolated chick rectum the ethanolic extract is having antinicotinic activity and it may act on the nicotinic acetylcholine receptors (nAChRs) as well as muscarinic acetylcholine receptors (mAChRs) on other isolated tissues. The extract might contain ganglionic blocking activity or non-specific activity or membrane stabilising activity also.&#x0D; Keywords: Nicotine, Ethanolic extract, Inflorescence of Casuarina equisetifolia, Isolated Chick Rectum, Isolated Frog Rectum and Isolated Frog Rectus Abdominus Muscle.

Sex differences in the nicotinic excitation of principal neurons within the developing hippocampal formation.

The hippocampal formation (HF) plays an important role to facilitate higher order cognitive functions. Cholinergic activation of heteromeric nicotinic acetylcholine receptors (nAChRs) within the HF is critical for the normal development of principal neurons within this brain region. However, previous research investigating the expression and function of heteromeric nAChRs in principal neurons of the HF is limited to males or does not differentiate between the sexes. We used whole-cell electrophysiology to show that principal neurons in the CA1 region of the female mouse HF are excited by heteromeric nAChRs throughout postnatal development, with the greatest response occurring during the first two weeks of postnatal life. Excitability responses to heteromeric nAChR stimulation were also found in principal neurons in the CA3, dentate gyrus, subiculum, and entorhinal cortex layer VI (ECVI) of young postnatal female HF. A direct comparison between male and female mice found that principal neurons in ECVI display greater heteromeric nicotinic passive and active excitability responses in females. This sex difference is likely influenced by the generally more excitable nature of ECVI neurons from female mice, which display a higher resting membrane potential, greater input resistance, and smaller afterhyperpolarization potential of medium duration (mAHP). These findings demonstrate that heteromeric nicotinic excitation of ECVI neurons differs between male and female mice during a period of major circuitry development within the HF, which may have mechanistic implications for known sex differences in the development and function of this cognitive brain region.

Transcriptomic Characterization of the Human Habenula Highlights Drug Metabolism and the Neuroimmune System.

Due to size and accessibility, most information about the habenula is derived from rodent studies. To better understand the molecular signature of the habenula we characterized the genes that have high expression in the habenula. We compared anatomical expression profiles of three normal adult human brains and four fetal brains. We used gene set enrichment analyses to determine if genes annotated to specific molecular functions, cellular components, and biological processes are enriched in the habenula. We also tested gene sets related to depression and addiction to determine if they uniquely involve the habenula. As expected, we observed high habenular expression of GPR151, nicotinic cholinergic receptors, and cilia-associated genes (medial division). Genes identified in genetic studies of smoking and associated with nicotine response were enriched in the habenula. Genes associated with major depressive disorder did not have enriched expression in the habenula but genes negatively correlated with hedonic well-being were, providing a link to anhedonia. We observed enrichment of genes associated with diseases that are comorbid with addictions (hematopoiesis, thrombosis, liver cirrhosis, pneumonia, and pulmonary fibrosis) and depression (rheumatoid arthritis, multiple sclerosis, and kidney disease). These inflammatory diseases mark a neuroimmune signature that is supported by genes associated with mast cells, acute inflammatory response, and leukocyte migration. We also found enrichment of cytochrome p450 genes suggesting the habenula is uniquely sensitive to endogenous and xenobiotic compounds. Our results suggest the habenula receives negative reward signals from immune and drug processing molecules. This is consistent with the habenular role in the “anti-reward” system and suggests it may be a key bridge between autoimmune disorders, drug use, and psychiatric diseases.

Effects of acute and chronic nicotine on catecholamine neurons of the nucleus of the solitary tract.

Nicotine is an addictive drug that has broad effects throughout the brain. One site of action is the nucleus of the solitary tract (NTS), where nicotine initiates a stress response and modulates cardiovascular and gastric function through nicotinic acetylcholine receptors (nAChRs). Catecholamine (CA) neurons in the NTS influence stress and gastric and cardiovascular reflexes, making them potential mediators of nicotine's effects; however nicotine's effect on these neurons is unknown. Here, we determined nicotine's actions on NTS-CA neurons by use of patch-clamp techniques in brain slices from transgenic mice expressing enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). Picospritzing nicotine both induced a direct inward current and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in NTS-CA neurons, effects blocked by nonselective nAChR antagonists TMPH and MLA. The increase in sEPSC frequency was mimicked by nAChRα7 agonist AR-R17779 and blocked by nAChRα7 antagonist MG624. AR-R17779 also increased the firing of TH-EGFP neurons, an effect dependent on glutamate inputs, as it was blocked by the glutamate antagonist NBQX. In contrast, the nicotine-induced current was mimicked by nAChRα4β2 agonist RJR2403 and blocked by nAChRα4β2 antagonist DHβE. RJR2403 also increased the firing rate of TH-EGFP neurons independently of glutamate. Finally, both somatodendritic and sEPSC nicotine responses from NTS-CA neurons were larger in nicotine-dependent mice that had under gone spontaneous nicotine withdrawal. These results demonstrate that 1) nicotine activates NTS-CA neurons both directly, by inducing a direct current, and indirectly, by increasing glutamate inputs, and 2) NTS-CA nicotine responsiveness is altered during nicotine withdrawal.

Early-adolescent male C57BL/6J and DBA/2J mice display reduced sensitivity to acute nicotine administration

BackgroundThe initial response to nicotine is an important predictor of subsequent use. Multiple factors may alter this response including genetics and age of first use. Here we investigated the influence of age, genetics, and their interaction on nicotine sensitivity. We then examined whether these factors influence the relationship between initial behavioral responses and voluntary nicotine consumption in adulthood. MethodsWe measured initial nicotine responses, including nicotine-induced locomotor depression and hypothermia following an acute intraperitoneal injection (0, 0.5, or 1 mg/kg), during early-adolescence, middle-adolescence, late-adolescence, or adulthood. Thirty-five days after the initial testing, mice were assessed for voluntary oral nicotine consumption. ResultsEarly-adolescent mice were more resistant to nicotine-induced hypothermia and locomotor depression than later ages, further hypothermia was influenced by genetics. In the DBA/2J strain, early-adolescent mice were insensitive to nicotine-induced hypothermia, but this response developed at later ages. In contrast, C57BL/6J mice were sensitive at all ages, but sensitivity increased across developmental age. There was little evidence of a relationship between initial behavioral response and choice nicotine consumption. ConclusionBy understanding how age of exposure and genetics influence initial nicotine behavioral responses, we have a greater understanding of factors that make adolescents differentially sensitive to the effects of this drug.

Genetic variation in CHRNA7 and CHRFAM7A is associated with nicotine dependence and response to varenicline treatment.

The role of nicotinic acetylcholine receptors (nAChR) in nicotine dependence (ND) is well established; CHRNA7, encoding the α7 subunit, has a still uncertain role in ND, although it is implicated in a wide range of neuropsychiatric conditions. CHRFAM7A, a hybrid gene containing a partial duplication of CHRNA7, is possibly involved in modulating α7 nAChR function. The aim of this study was to investigate the role of CHRNA7 and CHRFAM7A genetic variants in ND and to test the hypothesis that α7 nAChR variation may modulate the efficacy of varenicline treatment in smoking cessation. We assessed CHRNA7 and CHRFAM7A copy number, CHRFAM7A exon 6 ∆2 bp polymorphism, and sequence variants in the CHRNA7 proximal promoter in an Italian sample of 408 treatment-seeking smokers. We conducted case-control and quantitative association analyses using two smoking measures (cigarettes per day, CPD, and Fagerström Test for Nicotine Dependence, FTND). Next, driven by the hypothesis that varenicline may exert some of its therapeutic effects through activation of α7 nAChRs, we restricted the analysis to a subgroup of 142 smokers who received varenicline treatment. The CHRNA7 promoter variant rs28531779 showed association with both smoking quantitative measures (FNTD p = 0.026, β = 0.89, 95% CI 0.11-1.67; CPD p = 0.006, β = 4.82 95% CI 1.42-8.22). Moreover, in the varenicline-treated subgroup we observed association of CHRFAM7A copy number with 6 months smoking abstinence (p = 0.035, OR = 3.18, 95% CI = 1.09-9.30). Thus, our study points to a possible role of genetic variation in CHRNA7 and CHRFAM7A in tobacco addiction mechanisms and response to varenicline treatment.

Postnatal development of cholinergic input to the thalamic reticular nucleus of the mouse.

The thalamic reticular nucleus (TRN), a shell-like structure comprised of GABAergic neurons, gates signal transmission between thalamus and cortex. While TRN is innervated by axon collaterals of thalamocortical and corticothalamic neurons, other ascending projections modulate activity during different behavioral states such as attention, arousal, and sleep-wake cycles. One of the largest arise from cholinergic neurons of the basal forebrain and brainstem. Despite its integral role, little is known about how or when cholinergic innervation and synapse formation occurs. We utilized genetically modified mice, which selectively express fluorescent protein and/or channelrhodopsin-2 in cholinergic neurons, to visualize and stimulate cholinergic afferents in the developing TRN. Cholinergic innervation of TRN follows a ventral-to-dorsal progression, with nonvisual sensory sectors receiving input during week 1, and the visual sector during week 2. By week 3, the density of cholinergic fibers increases throughout TRN and forms a reticular profile. Functional patterns of connectivity between cholinergic fibers and TRN neurons progress in a similar manner, with weak excitatory nicotinic responses appearing in nonvisual sectors near the end of week 1. By week 2, excitatory responses become more prevalent and arise in the visual sector. Between weeks 3-4, inhibitory muscarinic responses emerge, and responses become biphasic, exhibiting a fast excitatory, and a long-lasting inhibitory component. Overall, the development of cholinergic projections in TRN follows a similar plan as the rest of sensory thalamus, with innervation of nonvisual structures preceding visual ones, and well after the establishment of circuits conveying sensory information from the periphery to the cortex.

Similar nicotinic excitability responses across the developing hippocampal formation are regulated by small-conductance calcium-activated potassium channels.

The hippocampal formation forms a cognitive circuit that is critical for learning and memory. Cholinergic input to nicotinic acetylcholine receptors plays an important role in the normal development of principal neurons within the hippocampal formation. However, the ability of nicotinic receptors to stimulate principal neurons across all regions of the developing hippocampal formation has not been determined. We show in this study that heteromeric nicotinic receptors mediate direct inward current and depolarization responses in principal neurons across the hippocampal formation of the young postnatal mouse. These responses were found in principal neurons of the CA1, CA3, dentate gyrus, subiculum, and entorhinal cortex layer VI, and they varied in magnitude across regions with the greatest responses occurring in the subiculum and entorhinal cortex. Despite this regional variation in the magnitude of passive responses, heteromeric nicotinic receptor stimulation increased the excitability of active principal neurons by a similar amount in all regions. Pharmacological experiments found this similar excitability response to be regulated by small-conductance calcium-activated potassium (SK) channels, which exhibited regional differences in their influence on neuron activity that offset the observed regional differences in passive nicotinic responses. These findings demonstrate that SK channels play a role to coordinate the magnitude of heteromeric nicotinic excitability responses across the hippocampal formation at a time when nicotinic signaling drives the development of this cognitive brain region. This coordinated input may contribute to the normal development, synchrony, and maturation of the hippocampal formation learning and memory network. NEW & NOTEWORTHY This study demonstrates that small-conductance calcium-activated potassium channels regulate similar-magnitude excitability responses to heteromeric nicotinic acetylcholine receptor stimulation in active principal neurons across multiple regions of the developing mouse hippocampal formation. Given the importance of nicotinic neurotransmission for the development of principal neurons within the hippocampal formation, this coordinated excitability response is positioned to influence the normal development, synchrony, and maturation of the hippocampal formation learning and memory network.

Adolescent Social Stress Increases Anxiety-like Behavior and Alters Synaptic Transmission, Without Influencing Nicotine Responses, in a Sex-Dependent Manner

Early-life stress is a risk factor for comorbid anxiety and nicotine use. Because little is known about the factors underlying this comorbidity, we investigated the effects of adolescent stress on anxiety-like behavior and nicotine responses within individual animals. Adolescent male and female C57BL/6J mice were exposed to chronic variable social stress (CVSS; repeated cycles of social isolation + social reorganization) or control conditions from postnatal days (PND) 25–59. Anxiety-like behavior and social avoidance were measured in the elevated plus-maze (PND 61–65) and social approach-avoidance test (Experiment 1: PND 140–144; Experiment 2: 95–97), respectively. Acute nicotine-induced locomotor, hypothermic, corticosterone responses, (Experiment 1: PND 56–59; Experiment 2: PND 65–70) and voluntary oral nicotine consumption (Experiment 1: PND 116–135; Experiment 2: 73–92) were also examined. Finally, we assessed prefrontal cortex (PFC) and nucleus accumbens (NAC) synaptic transmission (PND 64–80); brain regions that are implicated in anxiety and addiction. Mice exposed to adolescent CVSS displayed increased anxiety-like behavior relative to controls. Further, CVSS altered synaptic excitability in PFC and NAC neurons in a sex-specific manner. For males, CVSS decreased the amplitude and frequency of spontaneous excitatory postsynaptic currents in the PFC and NAC, respectively. In females, CVSS decreased the amplitude of spontaneous inhibitory postsynaptic currents in the NAC. Adolescent CVSS did not affect social avoidance or nicotine responses and anxiety-like behavior was not reliably associated with nicotine responses within individual animals. Taken together, complex interactions between PFC and NAC function may contribute to adolescent stress-induced anxiety-like behavior without influencing nicotine responses.

Simultaneous Determination of Tobacco Alkaloids, Tobacco-Specific Nitrosamines, and Solanesol in Consumer Products Using UPLC–ESI-MS/MS

The US Alcohol and Tobacco Tax and Trade Bureau (TTB) is responsible for collecting Federal excise taxes on tobacco products. Tobacco products in the USA may fall into several taxable categories including cigars, cigarettes, snuff, chewing tobacco, pipe tobacco, and roll-your-own. The existence of these taxable categories means that the TTB is also responsible for the determination of proper tax classification. Not only does proper classification determine the amount of tax owed, but comprehensive classification procedures must also determine if a consumer product is subject to the tobacco excise tax. Since a product must contain tobacco to be subject to the excise tax, laboratory methods that test for the presence of tobacco can provide useful information to ascertain the taxable status of a product. To test for the presence of chemical markers associated with tobacco, an analytical method was developed that permits the simultaneous determination of nicotine and related alkaloids, tobacco-specific nitrosamines (TSNA), and solanesol in methanolic extracts of tobacco. The method utilizes ultra performance liquid chromatography with electrospray ionization–tandem mass spectrometric detection (UPLC–ESI-MS/MS) and was optimized for the analysis of nicotine, cotinine, nornicotine, anatabine, myosmine, anabasine, isonicoteine, nornicotyrine, nicotyrine, N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosonornicotine (NNN) and solanesol. The analytical method was designed to attenuate the instrument response of nicotine, which is overwhelming, to permit simultaneous analysis of all analytes.

Novel CYP2A6 diplotypes identified through next-generation sequencing are associated with in-vitro and in-vivo nicotine metabolism.

Smoking patterns and cessation rates vary widely across smokers and can be influenced by variation in rates of nicotine metabolism [i.e. cytochrome P450 2A6 (CYP2A6), enzyme activity]. There is high heritability of CYP2A6-mediated nicotine metabolism (60-80%) owing to known and unidentified genetic variation in the CYP2A6 gene. We aimed to identify and characterize additional genetic variants at the CYP2A6 gene locus. A new CYP2A6-specific sequencing method was used to investigate genetic variation in CYP2A6. Novel variants were characterized in a White human liver bank that has been extensively phenotyped for CYP2A6. Linkage and haplotype structure for the novel single nucleotide polymorphisms (SNPs) were assessed. The association between novel five-SNP diplotypes and nicotine metabolism rate was investigated. Seven high-frequency (minor allele frequencies ≥6%) noncoding SNPs were identified as important contributors to CYP2A6 phenotypes in a White human liver bank (rs57837628, rs7260629, rs7259706, rs150298687 (also denoted rs4803381), rs56113850, rs28399453, and rs8192733), accounting for two times more variation in in-vitro CYP2A6 activity relative to the four established functional CYP2A6 variants that are frequently tested in Whites (CYP2A6*2, *4, *9, and *12). Two pairs of novel SNPs were in high linkage disequilibrium, allowing us to establish five-SNP diplotypes that were associated with CYP2A6 enzyme activity (rate of nicotine metabolism) in-vitro in the liver bank and in-vivo among smokers. The novel five-SNP diplotype may be useful to incorporate into CYP2A6 genotype models for personalized prediction of nicotine metabolism rate, cessation success, and response to pharmacotherapies.