Sarah L. King

Nicotinic Receptors in the Brain: Links between Molecular Biology and Behavior

Molecular cloning has elucidated the sequence of a family of acetylcholine receptor subunits that are activated by nicotine. Subsequent studies on the localization of individual subunits and the physiological properties of nicotinic subunit combinations in vitro, have led to identification of subunit compositions of nicotinic receptors that may function in vivo, as the native receptor. A particular challenge for the field has been to use these molecular data to determine which individual nicotinic receptor subtype is responsible for mediating each of the behavioral effects of nicotine. Human and animal studies have shown that nicotine is reinforcing and likely responsible for the addictive properties of tobacco. In addition, nicotine has been shown to have effects on locomotion, cognition, affect, and pain sensitivity. Recent studies combining the techniques of molecular biology, pharmacology, electrophysiology, and behavioral analysis to analyze knock out mice that lack individual subunits of the nicotinic acetylcholine receptor, have helped identify the role of specific nicotinic subunits in some of these complex behaviors. These studies could ultimately be useful in designing specific nicotinic receptor agonists and antagonists that may have uses in the clinic.

Neuronal nicotinic acetylcholine receptor subunit knockout mice: physiological and behavioral phenotypes and possible clinical implications

Nicotinic acetylcholine receptors (nAChRs) in the muscle, autonomic ganglia, and brain are targets for pharmacologically administered nicotine. Several of the subunits that combine to form neuronal nicotinic receptors have been deleted by knockout or mutated by knockin in mice using homologous recombination. We will review the biochemical, pharmacological, anatomical, physiological, and behavioral phenotypes of mice with genetically altered neuronal nAChR subunits. Clinically relevant mutations in nAChR genes will also be discussed. In addition, some of the signal transduction pathways activated through nAChRs will be described in order to delineate the longer-term changes that might result from persistent activation or inactivation of nAChRs. Genetically manipulated mice have greatly increased our understanding of the subunit composition and physiological properties of nAChRs in vivo. In addition, these mice have provided a model system to determine the molecular basis for many of the pharmacological actions of nicotine on neurotransmitter release and behavior. Genetic manipulations in mice have also elucidated the role of nAChR subunits in various disease states, and suggest several avenues for drug development.

The prototoxin lynx1 acts on nicotinic acetylcholine receptors to balance neuronal activity and survival in vivo.

Nicotinic acetylcholine receptors (nAChRs) affect a wide array of biological processes, including learning and memory, attention, and addiction. lynx1, the founding member of a family of mammalian prototoxins, modulates nAChR function in vitro by altering agonist sensitivity and desensitization kinetics. Here we demonstrate, through the generation of lynx1 null mutant mice, that lynx1 modulates nAChR signaling in vivo. Its loss decreases the EC(50) for nicotine by approximately 10-fold, decreases receptor desensitization, elevates intracellular calcium levels in response to nicotine, and enhances synaptic efficacy. lynx1 null mutant mice exhibit enhanced performance in specific tests of learning and memory. Consistent with reports that mutations resulting in hyperactivation of nAChRs can lead to neurodegeneration, aging lynx1 null mutant mice exhibit a vacuolating degeneration that is exacerbated by nicotine and ameliorated by null mutations in nAChRs. We conclude that lynx1 functions as an allosteric modulator of nAChR function in vivo, balancing neuronal activity and survival in the CNS.

High-affinity nicotinic acetylcholine receptors are required for antidepressant effects of amitriptyline on behavior and hippocampal cell proliferation

BACKGROUND A wide variety of antidepressants act as noncompetitive antagonists of nicotinic acetylcholine receptors (nAChRs), but the relationship between this antagonism and the therapeutic effects of antidepressants is unknown. METHODS Antidepressant properties of the noncompetitive nAChR antagonist mecamylamine in the forced swim test were tested alone and in combination with the tricyclic antidepressant amitriptyline. Mice lacking high-affinity nAChRs were tested in three behavioral models to determine whether these receptors are required for behavioral effects of amitriptyline in common models of antidepressant action. Finally, the brains of wild-type and knockout animals treated with amitriptyline were examined to determine whether high-affinity nAChRs are required for antidepressant-induced increases in hippocampal cell proliferation. RESULTS Inhibition of nAChRs by mecamylamine had antidepressant-like effects in the forced swim test and potentiated the antidepressant activity of amitriptyline when the two drugs were used in combination. Mice lacking high-affinity nAChRs showed no behavioral response to amitriptyline. Finally, after chronic treatment with amitriptyline, nAChR knockout mice did not show the increase in hippocampal cell proliferation seen in wild-type mice. CONCLUSIONS These data support the hypothesis that antagonism of nAChRs is an essential component of the therapeutic action of antidepressants.

An instructive role for patterned spontaneous retinal activity in mouse visual map development.

Complex neural circuits in the mammalian brain develop through a combination of genetic instruction and activity-dependent refinement. The relative role of these factors and the form of neuronal activity responsible for circuit development is a matter of significant debate. In the mammalian visual system, retinal ganglion cell projections to the brain are mapped with respect to retinotopic location and eye of origin. We manipulated the pattern of spontaneous retinal waves present during development without changing overall activity levels through the transgenic expression of β2-nicotinic acetylcholine receptors in retinal ganglion cells of mice. We used this manipulation to demonstrate that spontaneous retinal activity is not just permissive, but instructive in the emergence of eye-specific segregation and retinotopic refinement in the mouse visual system. This suggests that specific patterns of spontaneous activity throughout the developing brain are essential in the emergence of specific and distinct patterns of neuronal connectivity.

Positive effects of cholinergic stimulation favor young APOE epsilon4 carriers.

The potential of putative cognitive-enhancing compounds to improve mental processing both in healthy and vulnerable populations is an area of growing interest to scientific and clinical communities. The possible influence of individual genetic differences on efficacy of these compounds has yet to be considered. We sought to investigate the profile of young-adult apolipoprotein E (APOE) ɛ4 carriers across cognitive domains given that possession of this gene variant increases risk of developing dementia in later life. We also explored whether APOE genotype interacts with the cognitive enhancer, nicotine. A total of 1 mg of the cholinergic agonist nicotine was administered through nasal spray to healthy non-smoking young adults (aged 18–30) with either ɛ3/ɛ3 (N=29) or ɛ4 (at least one ɛ4 allele, N=27) genotype. Participants were matched on age, sex, and IQ in a placebo-controlled, double-blind 2 (drug: placebo, nicotine) × 2 (genotype: ɛ3, ɛ4) between subjects design. Here, we show that, paradoxically, possession of the ɛ4 allele confers a cognitive advantage on tasks mediated by the frontal lobe, and that young carriers of the ɛ4 allele show larger cognitive benefit from procholinergic nicotinic stimulation. These results are the first to show that genetic differences influence the efficacy of a cognitive enhancer.

β2-subunit-containing nicotinic acetylcholine receptors are critical for dopamine-dependent locomotor activation following repeated nicotine administration

Activation of the mesolimbic dopamine system is a critical component underlying addictive behaviors, including smoking. It has been hypothesized that the initial effect of nicotine on the dopamine system is to activate high affinity nicotinic acetylcholine receptors (nAChRs) containing the beta2 subunit, but that these receptors rapidly desensitize and are not critical for ongoing dopaminergic activation. To clarify the role of beta2-subunit-containing (beta2*) nAChRs in activation of the dopamine system and subsequent locomotor activation by repeated nicotine administration, C57BL/6J (B6) mice were administered 200 microg/ml of nicotine in the drinking water and the onset of locomotor activation was measured. B6 mice showed an increase in locomotor activity in response to chronic nicotine which was blocked by oral administration of the dopamine receptor antagonist pimozide. Knockout mice lacking the beta2 subunit of the nAChR did not show locomotor activation in response to chronic nicotine exposure, suggesting that beta2* nAChRs are critical for ongoing activation of the dopamine system by chronic nicotine administration and the resulting locomotor activation in mice.

Cocaine effects on mouse incentive-learning and human addiction are linked to α2 subunit-containing GABAA receptors

Because GABAA receptors containing α2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with α2 gene deletion showed reduced synaptic GABAA receptor-mediated responses. Behaviorally, the deletion abolished cocaine’s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of α2-GABAA receptors (α2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In α2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of α2−GABAA receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

APOE e4 polymorphism in young adults is associated with improved attention and indexed by distinct neural signatures.

The APOE e4 allele, which confers an increased risk of developing dementia in older adulthood, has been associated with enhanced cognitive performance in younger adults. An objective of the current study was to compare task-related behavioural and neural signatures for e4 carriers (e4+) and non-e4 carriers (e4-) to help elucidate potential mechanisms behind such cognitive differences. On two measures of attention, we recorded clear behavioural advantages in young adult e4+ relative to e4-, suggesting that e4+ performed these tasks with a wider field of attention. Behavioural advantages were associated with increased task-related brain activations detected by fMRI (BOLD). In addition, behavioural measures correlated with structural measures derived from a former DTI analysis of white matter integrity in our cohort. These data provide clear support for an antagonistic pleiotropy hypothesis--that the e4 allele confers some cognitive advantage in early life despite adverse consequences in old age. The data implicate differences in both structural and functional signatures as complementary mediators of the behavioural advantage.

Sex differences in anxiety-like behavior and locomotor activity following chronic nicotine exposure in mice

Smoking appears to increase overall levels of stress, despite self-reports that men and women smoke to control symptoms of anxiety. The overall incidence of anxiety disorders is also significantly higher in women. This study examined whether behavioral sensitivity to chronic nicotine varies across sexes in mice. Male and female C57BL/6J mice were exposed chronically to nicotine in the drinking water (50, 100, or 200 microg/ml) and tested for locomotor activation and anxiety-like behavior in the elevated plus maze (EPM). Female mice were less sensitive to the locomotor activating effects of chronic nicotine. Whereas both males and females showed increases in locomotor activity at the highest (200 microg/ml) concentration of nicotine, only males showed locomotor activation at the middle (100 microg/ml) concentration. The decreased sensitivity in females could not be explained by reduced nicotine intake compared to males. In the EPM, nicotine produced an anxiogenic-like response in females, but had no effect in males. Treatment with the high (200 microg/ml) dose of nicotine reduced the amount of time spent in the open arms of the EPM in female, but not male mice. No differences in the anxiogenic-like response to chronic nicotine was observed between beta2-subunit knockout and wildtype mice, suggesting that beta2-subunit containing nicotinic receptors do not mediate the anxiogenic-like response to chronic nicotine in females. This shows that female mice have an anxiogenic-like response to chronic nicotine, but are less sensitive to nicotines psychostimulant properties, which may be related to the increased relapse to smoking following abstinence in women.