Regulation Of Nicotinic Receptors Research Articles

Chrna5 and lynx prototoxins identify acetylcholine super-responder subplate neurons

Attention depends on cholinergic excitation of prefrontal neurons but is sensitive to perturbation of α5-containing nicotinic receptors encoded by Chrna5. However, Chrna5-expressing (Chrna5+) neurons remain enigmatic, despite their potential as a target to improve attention. Here, we generate complex transgenic mice to probe Chrna5+ neurons and their sensitivity to endogenous acetylcholine. Through opto-physiological experiments, we discover that Chrna5+ neurons contain a distinct population of acetylcholine super-responders. Leveraging single-cell transcriptomics, we discover molecular markers conferring subplate identity on this subset. We determine that Chrna5+ super-responders express a unique complement of GPI-anchored lynx prototoxin genes (Lypd1, Ly6g6e, and Lypd6b), predicting distinct nicotinic receptor regulation. To manipulate lynx regulation of endogenous nicotinic responses, we developed a pharmacological strategy guided by transcriptomic predictions. Overall, we reveal Chrna5-Cre mice as a transgenic tool to target the diversity of subplate neurons in adulthood, yielding new molecular strategies to manipulate their cholinergic activation relevant to attention disorders.

Bidirectional sex-dependent regulation of α6 and β3 nicotinic acetylcholine receptors by protein kinase Cε.

Nicotine and alcohol are the most commonly abused substances worldwide and are frequently coabused. Nicotinic acetylcholine receptors (nAChRs) containing the α6 and β3 subunits are expressed in neural reward circuits and are critical for nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKCε), which impact transcription of α6 and β3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKCε-/- mice, who also consume less nicotine and alcohol compared with wild-type (WT) littermates. Using RT-qPCR, we show that female PKCε-/- mice have higher expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior as female but not male PKCε-/- mice exhibit locomotor hypersensitivity to low-dose (0.25 mg/kg i.p.) nicotine. Female PKCε-/- mice show no differences in alcohol-induced sedation in the loss-of-righting reflex assay (4.0 g/kg i.p.) compared with WT littermates, whereas male PKCε-/- mice have enhanced sedation compared with WT mice. Female PKCε-/- mice also show reduced immobility time in response to varenicline (1.0 mg/kg i.p.) compared with WT littermates in the tail suspension test, and this effect was absent in male mice. Additionally, we found that female PKCε-/- mice show altered alcohol and nicotine consumption patterns in chronic voluntary two-bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKCε, highlighting the importance of studying both sexes in preclinical animal models.

Sex Differences in Nicotinic Receptor Regulation of Local Nucleus Accumbens Circuitry underlying Motivated Behavior

Sex is a critical biological variable in neurological conditions characterized by abnormalities in motivation and reward processing – e.g. anxiety, schizophrenia, depression, and substance use disorder. In both human and animal studies, females exhibit faster reward learning and increased reward‐associated cue responses, highlighting the need for preclinical investigation of reward learning and motivation in female subjects. The neural control of reward and motivation is dependent upon the mesolimbic dopamine pathway connecting the ventral tegmental area (VTA) to the nucleus accumbens (NAc), where dopamine release in the NAc causally controls motivation and reward seeking. A particularly potent regulator of dopamine release is cholinergic (ChAT) interneurons signaling through pentameric nicotinic acetylcholine receptors (nAChRs) located on dopamine terminals. In fact, ChAT interneurons can elicit dopamine release directly via the α4β2 subunit containing nAChRs. Further, nAChRs have been shown to directly modulate tonic‐to‐phasic dopamine release, a process that is vital to encode reward information and drive behavior – highlighting the importance of nAChRs in dopaminergic control of behavior. Previous work has shown that Mecamylamine, a non‐selective nAChR antagonist, typically reduces tonic dopamine release, while enhancing or not changing phasic release in males; however, we show here, in females it reduces dopamine release across all frequencies. This suggests that females require more potent regulation of dopamine terminals by nicotinic systems to drive dopamine release, yet, it is unclear how subunit specific nAChRs differentially regulate this process in females. To understand how these differences in nAChR regulation of dopamine release could be driven by subunit specific neurochemical mechanisms we took a multipronged approach to outlining sex‐differences in nAChR regulation of VTA dopamine terminals in the NAc. Using fast‐scan cyclic voltammetry paired with pharmacology we measured subsecond dopamine kinetics in male and female mice using the selective α4β2 nAChR antagonist, DHβE, to determine the specific role of the α4β2 subunits on sex‐differences in nAChR modulation of tonic‐to‐phasic dopamine release. We found that, in males, DHβE had a trending effect toward reducing tonic dopamine release while not effecting phasic release. Contrary to this, DHβE decreased phasic dopamine release while not having a significant effect on tonic release in females. This data suggests an α4β2 nAChR‐dependent gating mechanism for the adaptation of activity‐dependent dopamine release in females, which likely acts to drive enhanced motivation. Moving forward it will be critical to directly link these sex‐differences to reward processing and reinforcement learning for the development of sex‐specific pharmacotherapies to treat a variety of debilitating neurological disorders.Support or Funding InformationFunding was provided by startup funds from V.U. School of Medicine, Department of Pharmacology and the National Institutes of Health (NIH) (to E.S.C.). Funds from the National Institute of Drug Abuse (NIDA) DA042111 and DA048931, the Brain and Behavior Research Foundation, Whitehall Foundation, and the Edward J Mallinckrodt Jr. Foundation (E.S.C) also supported this work.

Nicotine content and abstinence state have different effects on subjective ratings of positive versus negative reinforcement from smoking

Despite the well-known adverse health consequences of smoking, approximately 20% of US adults smoke tobacco cigarettes. Much of the research on smoking reinforcement and the maintenance of tobacco smoking behavior has focused on nicotine; however, a number of other non-nicotine factors are likely to influence the reinforcing effects of smoked tobacco. A growing number of studies suggest that non-nicotine factors, through many pairings with nicotine, are partially responsible for the reinforcing effect of smoking. Additionally, both clinical studies and preclinical advances in our understanding of nicotinic receptor regulation suggest that abstinence from smoking may influence smoking reinforcement. These experiments were conducted for 2 reasons: to validate a MRI-compatible cigarette smoking device; and to simultaneously investigate the impact of nicotine, smoking-associated conditioned reinforcers, and smoking abstinence state on subjective ratings of smoking reinforcement. Participants smoked nicotine and placebo cigarettes through an fMRI compatible device in an overnight-abstinent state or in a nonabstinent state, after having smoked a cigarette 25minutes prior. Outcome measures were within-subject changes in physiology and subjective ratings of craving and drug effect during the smoking of nicotine or placebo cigarettes on different days in both abstinence states. Cigarette type (nicotine vs. placebo) had a significant effect on positive subjective ratings of smoking reinforcement (“High”, “Like Drug”, “Feel Drug”; nicotine>placebo). In contrast, abstinence state was found to have significant effects on both positive and negative ratings of smoking reinforcement (“Crave”, “Anxiety”, “Irritability”; abstinence>nonabstinence). Interaction effects between abstinence and nicotine provide clues about the importance of neuroadaptive mechanisms operating in dependence, as well as the impact of conditioned reinforcement on subjective ratings of smoking-induced high.

Does smoking reduce the risk of Parkinson’s disease through stimulation of the ubiquitin–proteasome system?

Parkinson's disease is a neurodegenerative condition characterized by tremor, rigidity, bradykinesia, and postural instability. Smoking is an inverse risk factor for Parkinson's disease, although the mechanism for this apparent neuroprotection is not definitively established. Smoking consistently upregulates nicotinic acetylcholine receptor levels in various brain regions known to be involved in Parkinson's disease. The ubiquitin-proteasome system--the system that tags and removes unwanted, misfolded, or damaged proteins from cells--regulates nicotinic receptor levels. The ubiquitin-proteasome system has also been implicated in Parkinson's disease, with aberrant activity identified in both sporadic and familial forms of the disease. The involvement of the ubiquitin-proteasome system in nicotinic receptor regulation and Parkinson's disease pathology suggests a link between the two, which forms the basis of the present hypothesis. Specifically, this paper considers the hypothesis that smoking reduces the risk of Parkinson's disease through the upregulation of nicotinic cholinergic receptors in key brain regions involved in Parkinson's disease. This receptor upregulation is hypothesized to increase activity of the ubiquitin-proteasome system, which is believed to prevent neurodegeneration caused by the accumulation of misfolded or damaged proteins or other consequences of inadequate protein sequestration and/or degradation. This hypothesis is supported by evidence documenting the upregulation of nicotinic receptors in the brains of smokers, neuroprotective effects of nicotine, reduced activity of the ubiquitin-proteasome in Parkinson's disease, and increased activity of the ubiquitin-proteasome system in animals exposed to chronic nicotine. Additional research is needed to test several predictions of the hypothesis, including increased activity of the ubiquitin-proteasome system in key brain regions of smokers.

P2-057: Characterization and differential regulation of nicotinic receptors in cholinergic SN56 cells

P2-057: Characterization and differential regulation of nicotinic receptors in cholinergic SN56 cells

Reversal of isolation-rearing-induced PPI deficits by an α7 nicotinic receptor agonist

The alpha7 subtype of the nicotinic receptor plays an important role in auditory sensory gating. Schizophrenics show deficient sensory gating and abnormalities in the number and regulation of nicotinic receptors. Prepulse inhibition (PPI) deficits exhibited by isolation-reared rats are thought to model the sensorimotor gating deficits seen in schizophrenia. To examine the role of nicotinic alpha7 receptors in the isolation-rearing rat model, we tested whether the selective alpha7 receptor agonist (R)-N-(1-Azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (compound A) could reverse isolation-rearing-induced PPI deficits, and investigated alpha7 receptor RNA expression in the hippocampus, prefrontal cortex, cerebellum, nucleus accumbens and thalamus, and alpha7 receptor protein expression in the hippocampus of isolation- and group-reared rats. Rats reared in isolation or groups of five from weaning were tested in the PPI paradigm under conditions of variable inter-stimulus interval (ISI) (pulse = 110 dB/50 ms; prepulse = 75 dB/30 ms; ISI = 30, 100 and 300 ms) 30 min following administration of compound A (3.2-10 mg/kg i.p.). Alpha7 receptor expression was measured by TaqMan RT-PCR (total RNA) and autoradiography (protein). Isolation-rearing-induced PPI deficits were attenuated by both doses of compound A at 100-ms ISI and by 10 mg/kg at 300-ms ISI. Expression of alpha7 receptor RNA and protein was unaltered in isolation-reared rats. Although altered alpha7 receptor expression may not underlie the phenotype of isolation-reared rats, the activation of these receptors may be of benefit in re-establishing efficient gating function.

The Biology of Tobacco and Nicotine: Bench to Bedside

Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss "The Biology of Nicotine and Tobacco: Bench to Bedside." Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.

Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia.

To analyze the effects of nicotine on palatal fusion inhibition in vitro and determine if nicotine modulated transforming growth factor beta3 or phosphatidylinositol-3 kinase signaling. A second objective was to determine the localization and regulation of nicotinic receptors in the medial edge epithelia (MEE) during palatal fusion. Palatal shelves from embryonic day (E) 13.5 mice were cultured in serum free media and treated with 0, 0.06, 0.6, or 6 mM nicotine, nicotinic receptor antagonist alpha-bungarotoxin, or the combination of nicotine and alpha-bungarotoxin. Tissues harvested at 72 h were analyzed for epithelial-mesenchymal transformation (EMT) and fusion. MEE samples collected at 20 h were analyzed for phosphorylated Akt-Ser473, phosphorylated Smad2, and nicotinic receptors. Nicotine inhibited palatal fusion in vitro in a dose dependent manner. Activated Akt-Ser473 was greater in control MEE than in nicotine treated tissues; while there was no difference in activated Smad2 between groups. The alpha7 subunit of nicotinic receptor was expressed in MEE during palate fusion and increased in nicotine treated tissues. Alpha-bungarotoxin did not rescue the nicotine treated palates. Nicotine treatment had no effect on Smad2, but caused a down regulation of the PI-3 kinase pathway that may have contributed to inhibiting palatal fusion in vitro.

Executive and social behaviors under nicotinic receptor regulation.

Nicotine enhances several cognitive and psychomotor behaviors, and nicotinic antagonists cause impairments in tasks requiring cognitive effort. To explore the contribution of nicotinic receptors to complex cognitive functions, we developed an automated method to investigate sequential locomotor behavior in the mouse and an analysis of social behavior. We show that, in the beta2-/- mutant, the high-order spatiotemporal organization of locomotor behavior, together with conflict resolution and social interaction, is selectively dissociated from low-level, more automatic motor behaviors. Such deficits in executive functions resemble the rigid and asocial behavior found in some psychopathological disorders such as autism and attention deficit hyperactivity disorder.

Nicotinic receptor expression following nicotine exposure via maternal milk.

Studies have shown nicotine is excreted into maternal milk, so that suckling offspring would be a target of the drug during the pre-weaning period. Since nicotine exposure leads to an upregulation of neuronal nicotinic receptors, this study examines the hypothesis that nicotine delivered via maternal milk is capable of altering neuronal nicotinic receptor regulation in the drug-exposed rat pups. The present study showed that postnatal nicotine exposure via maternal milk was sufficient to induce an upregulation in brain nicotinic receptors similar to that seen in adults that smoke. Such exposure may result in altered neuronal development and synaptic activity and structure, potentially leading to long-term behavioral, learning, and memory deficits.

The effect of nicotine and haloperidol co-treatment on nicotinic receptor levels in the rat brain

Genetic and biological data have suggested a role for the neuronal nicotinic acetylcholine receptors in the neuropathophysiology of schizophrenia. Studies in human postmortem brain demonstrate dose-dependent increases in nicotinic receptor binding in normal smokers. We found this upregulation to be reduced in schizophrenic smokers, many of whom had taken typical neuroleptics during their lifetime. The present study examined the hypothesis that typical antipsychotic drug treatment might modulate nicotinic receptor upregulation in a rat model. Nicotine, administered alone or in combination with haloperidol, increased both high and low affinity neuronal nicotinic receptors in a region specific manner. Haloperidol had no generalized effect on basal levels of nicotinic receptor binding or nicotine induced upregulation of nicotinic receptors. However, haloperidol attenuated high affinity nicotinic receptor upregulation in thalamus and low affinity receptor upregulation in hippocampus. These results suggest that haloperidol is not likely to affect nicotinic receptor regulation by smoking in most brain regions.

Regulation of nicotinic receptors in the brain of mice withdrawn from chronic oral nicotine treatment.

The effect of nicotine withdrawal on regional regulation of brain nicotinic receptors was studied in mice after chronic administration of nicotine in the drinking water for 2, 4 or 7 weeks. Two weeks of chronic nicotine administration did not alter the binding of [3H]-nicotine in the midbrain, cortex or cerebellum of the mice, while after both 4-and 7-week treatments a significant increase in the specific [3H]-nicotine binding was observed in cortical and midbrain membranes. In the midbrain, the [3H]-nicotine binding was increased by about 40% in mice withdrawn for 48-72 h from the 4-week chronic nicotine treatment and in mice withdrawn for 48 h from the 7-week treatment. The [3H]-nicotine binding was significantly increased (by 55-65%) in the cortex at 48 h and 72 h after withdrawal from 4-week chronic nicotine and it was even somewhat more increased (by 72-66%) after 7-week treatment. The cortical [3H]-nicotine binding was not altered at 24 h after the 4-week treatment, but in mice withdrawn for 24 h from the 7-week treatment it was increased by 116%. The increases in [3H]-nicotine binding returned to control levels within 1 week after withdrawal. None of the studied treatments affected the [3H]-nicotine binding in the cerebellum. Tolerance towards nicotine-induced locomotor depression was only found in mice withdrawn for 24 h from the 7-week chronic nicotine administration. These findings suggest that at least 4-week chronic nicotine administration in the drinking water is needed before any upregulation of nicotinic receptors can be observed. Furthermore, in our experiments the increase in the [3H]-nicotine binding was seen before behavioural tolerance could be demonstrated. The differences between brain regions in the time course of nicotinic receptor upregulation may reflect variations in nicotinic receptor subunits and their sensitivity to chronic nicotine treatment.

Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine.

1. Chronic administration of nicotinic agonists in vivo increases the density of brain nicotinic binding sites. It has been proposed that this up-regulation results from agonist-induced functional blockade of nicotinic receptors. This hypothesis was tested by examining post mortem [3H]-nicotine and [125I]-alpha-bungarotoxin ([125I]-alpha BTX) binding following treatment in vivo with the quasi-irreversible and insurmountable CNS nicotinic blocker chlorisondamine, given either alone or in combination with chronic nicotine administration. 2. In rats that had not received chlorisondamine pretreatment, chronic nicotine administration (0.6 mg kg-1 s.c., twice daily for 12 days) increased [3H]-nicotine binding density (Bmax) in forebrain tissue sections by 19%, with no change in the apparent dissociation constant (KD). Chlorisondamine (10 mg kg-1, s.c.), given once prior to the chronic treatment phase, neither increased [3H]-nicotine binding by itself, nor altered the extent of nicotine-induced up-regulation. Nevertheless, chlorisondamine pretreatment resulted in a persistent blockade of CNS nicotinic receptors, as demonstrated by complete block of acute locomotor responses to nicotine. 3. In a second experiment, [3H]-nicotine and [125I]-alpha BTX binding was measured in tissue homogenates prepared from several brain regions. In the absence of chlorisondamine pretreatment, chronic nicotine administration (1 mg kg-1 s.c., twice daily for 12 days) increased the Bmax of [3H]-nicotine binding in the cerebral cortex (by 34%), striatum (by 28%), midbrain (by 16%) and hippocampus (by 36%); KD was unchanged. As before, this up-regulation was neither mimicked nor blocked by chlorisondamine pretreatment (10 mg kg-1, s.c., given twice), despite persistent blockade of acute locomotor responses to nicotine. Chronic nicotine treatment also increased the Bmax (but not KD) of [125I]-alpha BTX binding in cerebral cortex (by 35%), hippocampus (by 46%) and midbrain (by 35%). Chlorisondamine altered neither Bmax nor KD when given alone, but significantly attenuated the nicotine-induced up-regulation of toxin binding sites in midbrain, with a similar trend in the other two regions.4. The finding that chronic receptor blockade neither mimicked nor blocked the agonist-induced up-regulation of [3H]-nicotine binding sites suggests that up-regulation of these receptors is not determined by their functional status. In contrast, it appears that chronic nicotine-induced up-regulation of[125I]-alpha BTX binding sites may result from receptor activation.

Inhibition of nicotinic receptor mediated ion fluxes in rat sympathetic ganglia by BGT II-S1 a potent phospholipase

The mechanism of action of the bungarotoxin fraction II-S1 (BGT II-S1), which copurifies with α-bungarotoxin (α-BGT) and inhibits nicotinic transmission, has been further characterized. BGT II-S1 (1 μM) inhibited the carbachol (100 μM) or nicotine (50 μM) stimulated uptake of [ 3H]agmatine into rat sympathetic ganglia by 73% and 52%, respectively. These responses were inhibited 90% by d-tubocurarine (100 μM), but unaffected by α-BGT (1 μM) or atropine (10 μM), suggesting that BGT II-S1 affects nicotinic function at a postsynaptic site. Binding of physiologically active [ 125I]BGT II-S1 could be demonstrated to intact sympathetic ganglia; however, the binding could not be displaced by nicotinic agents, suggesting that BGT II-S1 is not interacting at the receptor. Because some neurotoxins produce their effect at the synapse through a phospholytic action, the phospholipase activity of BGT II-S1 was determined. The results demonstrate that BGT II-S1 is a very potent calcium dependent phospholipase. In addition, conditions which abolished the toxin's phospholytic activity presented its effects on nicotinic transmission and on nicotinic receptor mediated ion fluxes. These include irreversible inhibition of enzymic activity by treatment of BGT II-S1 with p-bromophenacylbromide, as well as reversible inhibition of the phospholipase by substitution of Ba 2+ or Sr 2+ ions for Ca 2+ ions in the physiological medium. Thus, in rat sympathetic ganglia, BGT II-S1 blocks the nicotinic receptor mediated movement of ions across the membrane. This is probably not due to a direct interaction at the nicotinic acetylcholine recognition site; rather, it may be an ion channel associated effect which is mediated by alterations in the phospholipid environment of the receptor complex or of the membrane. Although BGT II-S1 also has presynaptic actions, in a cultured system of postsynaptic cells, it could prove a useful tool to study the role of phospholipids in neuronal nicotinic receptor regulation.