Paul D. Gardner

Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area

Nicotine activation of nicotinic acetylcholine receptors (nAChRs) within the dopaminergic (DAergic) neuron-rich ventral tegmental area (VTA) is necessary and sufficient for nicotine reinforcement. In this study, we show that rewarding doses of nicotine activated VTA DAergic neurons in a region-selective manner, preferentially activating neurons in the posterior VTA (pVTA) but not in the anterior VTA (aVTA) or in the tail VTA (tVTA). Nicotine (1 μM) directly activated pVTA DAergic neurons in adult mouse midbrain slices, but had little effect on DAergic neurons within the aVTA. Quantification of nAChR subunit gene expression revealed that pVTA DAergic neurons expressed higher levels of α4, α6, and β3 transcripts than did aVTA DAergic neurons. Activation of nAChRs containing the α4 subunit (α4* nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: nicotine failed to activate pVTA DAergic neurons in α4 knockout animals; in contrast, pVTA α4* nAChRs were selectively activated by nicotine in mutant mice expressing agonist-hypersensitive α4* nAChRs (Leu9′Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by α4* nAChRs and were significantly larger in pVTA neurons than in aVTA neurons. Infusion of an α6* nAChR antagonist into the VTA blocked activation of pVTA DAergic neurons in WT mice and in Leu9′Ala mice at nicotine doses, which only activate the mutant receptor indicating that α4 and α6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA through α4α6* nAChRs. These receptors represent novel targets for smoking-cessation therapies.

Activation of α4* nAChRs is Necessary and Sufficient for Varenicline-Induced Reduction of Alcohol Consumption

Recently, the smoking cessation therapeutic varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to reduce alcohol consumption. However, the mechanism and nAChR subtype(s) involved are unknown. Here we demonstrate that varenicline and alcohol exposure, either alone or in combination, selectively activates dopaminergic (DAergic) neurons within the posterior, but not the anterior, ventral tegmental area (VTA). To gain insight into which nAChR subtypes may be involved in the response to alcohol, we analyzed nAChR subunit gene expression in posterior VTA DAergic neurons. Ethanol-activated DAergic neurons expressed higher levels of α4, α6, and β3 subunit genes compared with nonactivated neurons. To examine the role of nicotinic receptors containing the α4 subunit (α4* nAChRs) in varenicline-induced reduction of alcohol consumption, we examined the effect of the drug in two complementary mouse models, a knock-out line that does not express the α4 subunit (α4 KO) and another line that expresses α4* nAChRs hypersensitive to agonist (Leu9′Ala). While varenicline (0.1–0.3 mg/kg, i.p.) reduced 2% and 20% alcohol consumption in wild-type (WT) mice, the drug did not significantly reduce consumption in α4 KO animals. Conversely, low doses of varenicline (0.0125–0.05 mg/kg, i.p.) that had little effect in WT mice dramatically reduced ethanol intake in Leu9′Ala mice. Infusion of varenicline into the posterior, but not the anterior VTA was sufficient to reduce alcohol consumption. Together, our data indicate that activation of α4* nAChRs is necessary and sufficient for varenicline reduction of alcohol consumption.

Sp1 and Sp3 Regulate Expression of the Neuronal Nicotinic Acetylcholine Receptor β4 Subunit Gene

Neuronal nicotinic acetylcholine receptors play important roles in signal transduction within the nervous system. The receptors exist in a variety of functionally distinct subtypes that are determined by their subunit structures. The subunits are encoded by 11 genes, α2–α9 and β2–β4. Three of the genes, α3, α5, and β4, are tightly clustered, and their encoded proteins make up the predominant receptor subtype in the peripheral nervous system. The tight linkage of the genes suggests there may be a common regulatory mechanism underlying their expression. However, although their expression patterns significantly overlap, they are not identical, indicating that independent regulatory mechanisms must also exist. Our studies have focused upon the gene encoding the β4 subunit for which we have identified several transcriptional regulatory elements. One of these elements, E2, specifically interacts with the general transcription factor Sp1. Here we show that another member of the Sp family of factors, Sp3, can specifically interact with E2 whereas two other members, Sp2 and Sp4, cannot. Co-transfection experiments indicate that Sp3 can transactivate a β4 promoter/reporter gene construct and, furthermore, that Sp1 and Sp3 can transactivate the β4 reporter construct synergistically. The transactivation is dependent upon an intact E2 and may involve direct interactions between Sp1 and Sp3.

Differential Effects of Heterogeneous Nuclear Ribonucleoprotein K on Sp1- and Sp3-mediated Transcriptional Activation of a Neuronal Nicotinic Acetylcholine Receptor Promoter

The neuronal nicotinic acetylcholine receptor gene family consists of 11 members, α2–α9 and β2–β4. Three of the genes, those encoding the α3, α5, and β4 subunits, are clustered tightly within the genome. These three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system. The genomic proximity of the three genes suggests a regulatory mechanism ensuring their coordinate expression. However, it is likely that gene-specific regulatory mechanisms are also functioning because the expression patterns of the three genes, although similar, are not identical. Previously we identified regulatory elements within the β4 promoter region and demonstrated that these elements interact specifically with nuclear proteins. One of these elements, E1, interacts with the regulatory factor Purα as well as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and Sp3. Because E1 and E2 are immediately adjacent to one another, we postulated that the proteins that bind to the elements interact to regulate β4 gene expression. Here we report the identification of the 65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K and demonstrate that it affects the transactivation of β4 promoter activity by Sp1 and Sp3 differentially.

Nicotine persistently activates ventral tegmental area dopaminergic neurons via nicotinic acetylcholine receptors containing α4 and α6 subunits

Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brains mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by α-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the α6 subunit. α-conotoxin MII[H9A;L15A] also significantly reduced the long-lasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either α4 or α6 nAChR subunits. Conversely, selective activation of nAChRs containing the α4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to α-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing α4 and α6 subunits.

The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: Dual role in nicotine addiction and lung cancer

More than 1 billion people around the world smoke, with 10 million cigarettes sold every minute. Cigarettes contain thousands of harmful chemicals including the psychoactive compound, nicotine. Nicotine addiction is initiated by the binding of nicotine to nicotinic acetylcholine receptors, ligand-gated cation channels activated by the endogenous neurotransmitter, acetylcholine. These receptors serve as prototypes for all ligand-gated ion channels and have been extensively studied in an attempt to elucidate their role in nicotine addiction. Many of these studies have focused on heteromeric nicotinic acetylcholine receptors containing α4 and β2 subunits and homomeric nicotinic acetylcholine receptors containing the α7 subunit, two of the most abundant subtypes expressed in the brain. Recently however, a series of linkage analyses, candidate-gene analyses and genome-wide association studies have brought attention to three other members of the nicotinic acetylcholine receptor family: the α5, α3 and β4 subunits. The genes encoding these subunits lie in a genomic cluster that contains variants associated with increased risk for several diseases including nicotine dependence and lung cancer. The underlying mechanisms for these associations have not yet been elucidated but decades of research on the nicotinic receptor gene family as well as emerging data provide insight on how these receptors may function in pathological states. Here, we review this body of work, focusing on the clustered nicotinic acetylcholine receptor genes and evaluating their role in nicotine addiction and lung cancer.

Nicotinic acetylcholine receptor-mediated mechanisms in lung cancer

Despite the known adverse health effects associated with tobacco use, over 45 million adults in the United States smoke. Cigarette smoking is the major etiologic factor associated with lung cancer. Cigarettes contain thousands of toxic chemicals, many of which are carcinogenic. Nicotine contributes directly to lung carcinogenesis through the activation of nicotinic acetylcholine receptors (nAChRs). nAChRs are ligand-gated ion channels, expressed in both normal and lung cancer cells, which mediate the proliferative, pro-survival, angiogenic, and metastatic effects of nicotine and its nitrosamine derivatives. The underlying molecular mechanisms involve increases in intracellular calcium levels and activation of cancer signal transduction pathways. In addition, acetylcholine (ACh) acts as an autocrine or paracrine growth factor in lung cancer. Other neurotransmitters and neuropeptides also activate similar growth loops. Recent genetic studies further support a role for nAChRs in the development of lung cancer. Several nAChR antagonists have been shown to inhibit lung cancer growth, suggesting that nAChRs may serve as valuable targets for biomarker-guided lung cancer interventions.

Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety during nicotine withdrawal.

Increased anxiety is a predominant withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here, we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signaling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a meso-interpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knock-down of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signaling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms

BACKGROUND Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes. RESULTS Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naive mice and activated nonsomatostatin neurons in the IPN. CONCLUSIONS Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Conserved Chromosome 2q31 Conformations Are Associated with Transcriptional Regulation of GAD1 GABA Synthesis Enzyme and Altered in Prefrontal Cortex of Subjects with Schizophrenia

Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS-50kbLoop was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS-50kbLoop was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS-55kbLoop, the murine homolog to GAD1-TSS-50kbLoop, is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS-55kbLoop and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.