A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus.

Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition-binding studies revealed that the α3ß2* and α6ß2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6ß2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely ß2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by ß3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxin MII-sensitive α3α4ß2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4ß2* subtype nAChRs.

The promises and perils of psychedelic pharmacology for psychiatry.

Psychedelic drugs including psilocybin, N,N'-dimethyltryptamine (DMT) and lysergic acid diethylamide (LSD) are undergoing a renaissance as potentially useful drugs for various neuropsychiatric diseases, with a rapid onset of therapeutic activity. Notably, phase II trials have shown that psilocybin can produce statistically significant clinical effects following one or two administrations in depression and anxiety. These findings have inspired a 'gold rush' of commercial interest, with nearly 60 companies already formed to explore opportunities for psychedelics in treating diverse diseases. Additionally, these remarkable phenomenological and clinical observations are informing hypotheses about potential molecular mechanisms of action that need elucidation to realize the full potential of this investigative space. In particular, despite compelling evidence that the 5-HT2A receptor is a critical mediator of the behavioural effects of psychedelic drugs, uncertainty remains about which aspects of 5-HT2A receptor activity in the central nervous system are responsible for therapeutic effects and to what degree they can be isolated by developing novel chemical probes with differing specificity and selectivity profiles. Here, we discuss this emerging area of therapeutics, covering both controversies and areas of consensus related to the opportunities and perils of psychedelic and psychedelic-inspired therapeutics. We highlight how basic science breakthroughs can guide the discovery and development of psychedelic-inspired medications with the potential for improved efficacy without hallucinogenic or rewarding actions.

Increased nicotinic acetylcholine receptor protein underlies chronic nicotine-induced up-regulation of nicotinic agonist binding sites in mouse brain.

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of α4ß2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. Tissue sections were prepared, and binding of [(125)I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to ß2*-nAChR sites, [(125)I]monoclonal antibody (mAb) 299 to α4 nAChR subunits, and [(125)I]mAb 270 to ß2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express α4ß2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant ß2*-nAChR without α4 subunits, relatively less increase in mAb 270 binding to ß2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [(125)I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [(125)I]epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in α4ß2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.

Acetylcholine-stimulated [3H]GABA release from mouse brain synaptosomes is modulated by alpha4beta2 and alpha4alpha5beta2 nicotinic receptor subtypes.

Nicotinic acetylcholine receptor (nAChR) agonists stimulate the release of GABA from GABAergic nerve terminals, but the nAChR subtypes that mediate this effect have not been elucidated. The studies reported here used synaptosomes derived from the cortex, hippocampus, striatum, and thalamus of wild-type and alpha4-, alpha5-, alpha7-, beta2-, and beta4-null mutant mice to identify nAChR subtypes involved in acetylcholine (ACh)-evoked GABA release. Null mutation of genes encoding the alpha4 or beta2 subunits resulted in complete loss of ACh-stimulated [(3)H]GABA release in all four brain regions. In contrast, alpha5 gene deletion exerted a small but significant decrease in maximal ACh-evoked [(3)H]GABA release in hippocampus and striatum, with a more profound effect in cortex. Acetylcholine-stimulated [(3)H]GABA release from thalamic synaptosomes was not significantly affected by alpha5 gene deletion. No effect was detected in the four brain regions examined in alpha7- or beta4-null mutant mice. Further analysis of ACh-evoked [(3)H]GABA release revealed biphasic concentration-response relationships in the four brain regions examined from all wild-type animals and in alpha5 null mutant mice. Moreover, a selective reduction in the maximum response of the high-affinity component was apparent in alpha5-null mutant mice. The results demonstrate that alpha4beta2-type nAChRs are critical for ACh-stimulated [(3)H]GABA release from all four brain regions examined. In addition, the results suggest that alpha5-containing receptors on GABAergic nerve terminals comprise a fraction of the high ACh-sensitivity component of the concentration-response curve and contribute directly to the ability of nicotinic agonists to evoke GABA release in these regions.

Differential effects of withdrawal from intermittent and continuous nicotine exposure on reward deficit and somatic aspects of nicotine withdrawal and expression of α4ß2* nAChRs in Wistar male rats.

BACKGROUND: Chronic nicotine exposure produces neuroadaptations in brain reward systems and α4ß2 nicotinic acetylcholine receptors (nAChRs) in the corticolimbic brain areas. We previously demonstrated opposite effects of nicotine exposure delivered by self-administration or pumps on brain reward thresholds that can be attributed to the different temporal pattern and contingency of nicotine exposure. We investigated the effects of these two factors on reward thresholds and somatic signs during nicotine withdrawal, and on nAChRs binding in corticolimbic brain areas. METHODS: The intracranial self-stimulation procedure was used to assess reward thresholds in rats prepared with pumps delivering various doses of nicotine continuously or intermittently. Separate group of rats were randomly exposed to nicotine via pumps (non-contingent) or nicotine self-administration (contingent) to determine [125I]-epibatidine binding at α4ß2* nAChRs. RESULTS: Withdrawal from continuous non-contingent nicotine exposure led to significant elevations in thresholds and increases in somatic signs in rats, while there was no significant effect of withdrawal from intermittent non-contingent nicotine exposure at the same doses. nAChRs were upregulated during withdrawal from continuous non-contingent nicotine exposure. α4ß2* nAChRs were upregulated in the ventral tegmental area and prelimbic cortex during withdrawal from non-contingent intermittent exposure and in the nucleus accumbens during withdrawal from contingent intermittent nicotine exposure to the same dose. CONCLUSIONS: During non-contingent nicotine exposure, the temporal pattern of nicotine delivery differentially affected thresholds and somatic signs of withdrawal. Upregulation of α4ß2* nAChRs was brain site-specific and depended on both temporal pattern and contingency of nicotine exposure.

Nuclear roles for cilia-associated proteins.

Cilia appear to be derived, evolutionarily, from structures present in the ancestral (pre-ciliary) eukaryote, such as microtubule-based vesicle trafficking and chromosome segregation systems. Experimental observations suggest that the ciliary gate, the molecular complex that mediates the selective molecular movement between cytoplasmic and ciliary compartments, shares features with nuclear pores. Our hypothesis is that this shared transport machinery is at least partially responsible for the observation that a number of ciliary and ciliogenesis-associated proteins are found within nuclei where they play roles in the regulation of gene expression, DNA repair, and nuclear import and export. Recognizing the potential for such nuclear roles is critical when considering the phenotypic effects that arise from the mutational modification of ciliary proteins.

Immunolabeling demonstrates the interdependence of mouse brain alpha4 and beta2 nicotinic acetylcholine receptor subunit expression.

Immunolabeling of beta2 and alpha4 subunits was quantitated in brain sections (14 mum) using [(125)I]mAb 270 and [(125)I]mAb 299, respectively. Specificity was demonstrated by signal loss in beta2(-/-) and alpha4(-/-) brain sections, respectively. Even mild paraformaldehyde fixation severely affected immunolabeling, so this study used unfixed sections. Immunolabeling autoradiography was used to map and quantitate the effects of beta2 and alpha4 subunit-null mutations on their putative partner subunits' protein expression. [(125)I]mAb 299 labeling was nearly eliminated in beta2(-/-) sections, although dorsal interpeduncular nucleus (IPN) retained a faint signal. Therefore, alpha4 subunit expression is almost universally beta2-dependent. In contrast, alpha4-null mutation effects on [(125)I]mAb 270 immunolabeling varied widely among brain regions. In corticothalamic regions, [(125)I]mAb 270 labeling was eliminated. However, in habenulopeduncular regions, alpha4 genotype had no effect. Other (predominantly dopaminergic and optic tract) nuclei also retained reduced [(125)I]mAb 270 labeling in alpha4(-/-) sections. Thus, although most beta2 subunit protein expression is alpha4-dependent, this dependence is not universal. Presumably, residual beta2 subunits are found in non-alpha4* subtypes. Together, these results show that immunolabeling is applicable to reliable, quantitative investigations of neuronal nAChRs, and that subunit-null mutants can be appropriate controls for such experiments. In situ mRNA hybridization was also performed to determine if altered mRNA transcription mediated the interdependence of alpha4 and beta2 subunit expression. alpha4-Null mutation did not affect beta2 mRNA expression, nor did beta2 genotype affect alpha4 mRNA expression. Consequently, it seems that the two subunits' effects on each other's expression are mediated at the protein, rather than gene expression level.

Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex.

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the ß2 subunit (ß2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the ß2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

Presynaptic GABAB autoreceptor regulation of nicotinic acetylcholine receptor mediated [(3)H]-GABA release from mouse synaptosomes.

Activation of nicotinic acetylcholine receptors (nAChRs) can elicit neurotransmitter release from presynaptic nerve terminals. Mechanisms contributing to cell-and-terminal specific regulation of nAChR-mediated neurotransmitter exocytosis are not fully understood. The experiments discussed here examine how activation of GABAB auto- and hetero-receptors suppress nAChR-mediated release of [(3)H]-GABA and [(3)H]-dopamine ((3)H-DA) from mouse striatal synaptosomes. Activation of presynaptic GABAB receptors with (R)-baclofen decreased both [(3)H]-GABA and [(3)H]-DA release evoked by potassium depolarization. However, when nAChRs were activated with ACh to evoke neurotransmitter release, (R)-baclofen had no effect on [(3)H]-DA release, but potently inhibited ACh-evoked [(3)H]-GABA release. Inhibition of nAChR-evoked [(3)H]-GABA release by (R)-baclofen was time sensitive and the effect was lost after prolonged exposure to the GABAB agonist. The early inhibitory effect of GABAB activation on ACh-evoked [(3)H]-GABA release was partially attenuated by antagonists of the phosphatase, calcineurin. Furthermore, antagonists of protein kinase C (PKC) prevented the time-dependent loss of the inhibitory (R)-baclofen effect on [(3)H]-GABA release. These results suggest that α4ß2*-nAChRs present on GABAergic nerve terminals in the striatum are subject to functional regulation by GABAB autoreceptors that is apparently cell-type specific, since it is absent from DAergic striatal nerve terminals. In addition, the functional modulation of α4ß2*-type nAChRs on striatal GABAergic nerve terminals by GABAB autoreceptor activation is time-sensitive and appears to involve opposing actions of calcineurin and PKC.

Exploring the nicotinic acetylcholine receptor-associated proteome with iTRAQ and transgenic mice.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and ß2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between α4ß2-containing (α4ß2(∗)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4ß2(∗) nAChRs in a genedose dependent pattern by immunopurifying ß2(∗) nAChRs from mice that differ in α4 and ß2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the ß2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of α4ß2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a ß2(∗) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4ß2 nAChRs and their downstream signaling mechanisms.

Morphine dependence and withdrawal induced changes in cholinergic signaling.

Cholinergic signaling is thought to be involved in morphine dependence and withdrawal, but the specific mechanisms involved remain unclear. The current study aimed to identify alterations in the cholinergic system that may contribute to the development of morphine dependence and withdrawal. Acetylcholinesterase (AChE) activity and [³H]-epibatidine binding were evaluated in order to determine if morphine dependence and withdrawal induces alterations in cholinergic signaling or expression of high affinity nicotinic acetylcholine receptors (nAChRs) in the midbrain (MB), medial habenula (MHb) and interpeduncular nucleus (IPN). The effect of cholinergic signaling through nAChRs on morphine-withdrawal induced jumping behavior was then determined. Lastly, the contribution of ß4-containing nAChRs receptors in the MHb to morphine-withdrawal induced jumping behavior and neuronal activity as indicated by c-fos expression was assessed. Chronic morphine administration decreased AChE activity in MB and MHb, an effect that was no longer present following precipitated withdrawal. Morphine dependent mice showed increased nicotinic acetylcholine receptor (nAChR) levels in MB. Further, nicotine (0.4 mg/kg) and lobeline (3 mg/kg) decreased jumping behavior while mecamylamine (1 mg/kg) had no effect. Knock-down of ß4 subunit-containing nAChRs in the MHb attenuated c-fos activation, but did not decrease morphine withdrawal-induced jumping. Thus, morphine withdrawal induces cholinergic signaling in the MHb, but this does not appear to be responsible for the effects of cholinergic drugs on somatic signs of opiate withdrawal, as measured by jumping behavior.

Changes in the cholinergic system between bipolar depression and euthymia as measured with [123I]5IA single photon emission computed tomography.

BACKGROUND: The cholinergic system is substantially altered in individuals with major depression and is partially restored when depression remits. We quantified the availability of ß2-subunit-containing nicotinic acetylcholine receptors (ß2*-nAChR) in subjects with bipolar disorder. METHODS: Twenty-five subjects with bipolar disorder (15 depressed, 10 euthymic) and 25 sex- and age-matched control subjects had a [(123)I]5IA-85380 single photon emission computed tomography scan to quantify ß2*-nAChR VT/fP (total volume of distribution, corrected for individual differences in metabolism and protein binding of the radiotracer). Average VT/fP was compared between groups and correlated with clinical characteristics. Postmortem analysis of ß2*-nAChRs was conducted using equilibrium binding with [(125)I]5IA in subjects with bipolar disorder and matched control subjects. RESULTS: We showed significantly lower ß2*-nAChR availability (20%-38%) in subjects with bipolar depression compared with euthymic and control subjects across all brain regions assessed (frontal, parietal, temporal, and anterior cingulate cortex, hippocampus, amygdala, thalamus, striatum). The postmortem binding study in which endogenous acetylcholine was washed out did not show a statistically significant difference in ß2*-nAChR number in temporal cortex of the bipolar depressed and control groups (15% difference; p = .2). CONCLUSIONS: We show that the alteration in the cholinergic system observed during a depressive episode appears to resolve during euthymia. We suggest that lower VT/fP observed in vivo may be due to a combination of higher endogenous acetylcholine levels during depression, which could compete with radiotracer binding to the receptor in vivo, and lower receptor number in bipolar depression. Identification of differences in cholinergic signaling in subjects with bipolar depression may improve our understanding of its etiology and reveal new treatment targets.

Deletion of the alpha7 nicotinic receptor subunit gene results in increased sensitivity to several behavioral effects produced by alcohol.

BACKGROUND: The finding that most people with alcoholism are also heavy smokers prompted several research groups to evaluate the effects of ethanol on neuronal nicotinic acetylcholine receptor (nAChR) function. Data collected in vitro indicate that physiologically relevant concentrations of ethanol inhibit the functional activation of homomeric alpha7 nAChRs, which are one of the most abundant nAChR subtypes expressed in the mammalian brain. The studies outlined here used alpha7 gene knockout (null mutant) mice to evaluate the potential role of alpha7 nAChRs in modulating selected behavioral and physiological effects produced by ethanol. METHODS: Current evidence indicates that many responses to ethanol are not genetically correlated. Therefore, the authors measured the effects of acute administration of ethanol on several behaviors that are altered by both ethanol and nicotine: two tests of locomotor activity, acoustic startle, prepulse inhibition of acoustic startle, and body temperature. Ethanol-induced durations of loss of righting reflex and ethanol elimination rates were also determined. These studies used null mutant (alpha7(-/-)) and wild-type (alpha7(-/-)) mice. RESULTS: Relative to alpha7(+/+) mice, alpha7(-/-) mice were more sensitive to the activating effects of ethanol on open-field activity, ethanol-induced hypothermia, and duration of loss of the righting response. Deletion of the alpha7 gene did not influence the effects of ethanol on Y-maze crossing or rearing activities, acoustic startle, or prepulse inhibition of startle. Gene deletion did not alter ethanol metabolism. CONCLUSIONS: These results indicate that some but not all of the behavioral effects of ethanol are mediated in part by effects on nAChRs that include the alpha7 subunit and may help to explain the robust association between alcohol consumption and the use of tobacco.

Alpha 4 beta 2* nicotinic acetylcholine receptors modulate the effects of ethanol and nicotine on the acoustic startle response.

BACKGROUND: Ethanol modulates the functional activity of alpha4beta2 neuronal nicotinic cholinergic receptors (nAChR) when measured in vitro, but the potential role of alpha4beta2 nAChRs in regulating behavioral effects of ethanol is unknown. Recently, Tritto et al. (Tritto T, Stitzel JA, Marks MJ, Romm E, Collins AC (2002) Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes. Pharmacogenetics 12:197-208) reported that a polymorphism (A529T) in the alpha4 nAChR subunit gene is associated with variability in nicotine's effects on startle in the LSxSS recombinant inbred (RI) strains. Ethanol also alters the acoustic startle response. Thus, we evaluated the potential role of alpha4beta2 nAChRs in modulating ethanol's effects on acoustic startle. METHODS: The effects of ethanol on acoustic startle were determined in the LSxSS RI strains. In addition, the effects of ethanol and nicotine were also measured in alpha4 gain of function and beta2 null mutant mice. The beta2 mutants do not express the major variant of alpha4 nAChRs, alpha4beta2. RESULTS: An association between the alpha4 A529T polymorphism and ethanol's effects on startle was found in the LSxSS RI strains; those strains that express the A529 variant of alpha4 were more sensitive to ethanol-induced depression of startle. The alpha4 gain of function mutants were more sensitive to the effects of both nicotine and ethanol and the beta2 null mutants were less sensitive to both drugs. CONCLUSIONS: alpha4beta2-containing nAChRs may play important roles in modulating the effects of both ethanol and nicotine on the acoustic startle response. We suggest that nAChR subunit genes should be evaluated as potential contributors to both alcoholism and tobacco abuse.