Inés Ibañez-Tallon

A role for LYNX2 in anxiety-related behavior

Anxiety disorders are the most prevalent mental disorders in developed societies. Although roles for the prefrontal cortex, amygdala, hippocampus and mediodorsal thalamus in anxiety disorders are well documented, molecular mechanisms contributing to the functions of these structures are poorly understood. Here we report that deletion of Lynx2, a mammalian prototoxin gene that is expressed at high levels in anxiety associated brain areas, results in elevated anxiety-like behaviors. We show that LYNX2 can bind to and modulate neuronal nicotinic receptors, and that loss of Lynx2 alters the actions of nicotine on glutamatergic signaling in the prefrontal cortex. Our data identify Lynx2 as an important component of the molecular mechanisms that control anxiety, and suggest that altered glutamatergic signaling in the prefrontal cortex of Lynx2 mutant mice contributes to increased anxiety-related behaviors.

Reexposure to nicotine during withdrawal increases the pacemaking activity of cholinergic habenular neurons

Significance According to the World Health Organization, tobacco consumption causes the death of close to 6 million people each year, yet successful attempts to quit smoking are very rare. The present study identifies a group of neurons in the brain that respond differently to nicotine after a period of abstinence, suggesting that altered activity of these neurons may contribute to difficulties with smoking cessation. The discovery of genetic variants in the cholinergic receptor nicotinic CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula–interpeduncular axis as a critical relay circuit in the control of nicotine dependence. Although clear roles for α3, β4, and α5 receptors in nicotine aversion and withdrawal have been established, the cellular and molecular mechanisms that participate in signaling nicotine use and contribute to relapse have not been identified. Here, using translating ribosome affinity purification (TRAP) profiling, electrophysiology, and behavior, we demonstrate that cholinergic neurons, but not peptidergic neurons, of the medial habenula (MHb) display spontaneous tonic firing of 2–10 Hz generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels and that infusion of the HCN pacemaker antagonist ZD7288 in the habenula precipitates somatic and affective signs of withdrawal. Further, we show that a strong, α3β4-dependent increase in firing frequency is observed in these pacemaker neurons upon acute exposure to nicotine. No change in the basal or nicotine-induced firing was observed in cholinergic MHb neurons from mice chronically treated with nicotine. We observe, however, that, during withdrawal, reexposure to nicotine doubles the frequency of pacemaking activity in these neurons. These findings demonstrate that the pacemaking mechanism of cholinergic MHb neurons controls withdrawal, suggesting that the heightened nicotine sensitivity of these neurons during withdrawal may contribute to smoking relapse.

Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption.

The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.

Suppression of peripheral pain by blockade of voltage-gated calcium 2.2 channels in nociceptors induces RANKL and impairs recovery from inflammatory arthritis in a mouse model

A hallmark of rheumatoid arthritis (RA) is the chronic pain that accompanies inflammation and joint deformation. Patients with RA rate pain relief as the highest priority; however, few studies have addressed the efficacy and safety of therapies directed specifically toward pain pathways. The ω‐conotoxin MVIIA (ziconotide) is used in humans to alleviate persistent pain syndromes, because it specifically blocks the voltage‐gated calcium 2.2 (CaV2.2) channel, which mediates the release of neurotransmitters and proinflammatory mediators from peripheral nociceptor nerve terminals. The aims of this study were to investigate whether blockade of CaV2.2 can suppress arthritis pain, and to examine the progression of induced arthritis during persistent CaV2.2 blockade.

Retrograde inhibition by a specific subset of interpeduncular α5 nicotinic neurons regulates nicotine preference

Significance The CHRNA5-A3-B4 gene cluster, which encodes the α5, α3, and β4 nicotinic acetylcholine receptor subunits, has been genetically associated with high risk of developing nicotine dependence. Here we show that a specific α5 nicotinic receptor population in the midbrain interpeduncular nucleus (IPN) responds to chronic nicotine by increasing the expression of genes that regulate feedback inhibition of the medial habenula, the major source of input to the IPN. Inhibiting neurotransmitter release from this population of α5+ neurons or reducing expression of one of these genes, Nos1, blocks the rewarding effects of nicotine. Our data identify molecular mechanisms that may explain the genetic link between CHRNA5 and smoking predisposition in humans. Repeated exposure to drugs of abuse can produce adaptive changes that lead to the establishment of dependence. It has been shown that allelic variation in the α5 nicotinic acetylcholine receptor (nAChR) gene CHRNA5 is associated with higher risk of tobacco dependence. In the brain, α5-containing nAChRs are expressed at very high levels in the interpeduncular nucleus (IPN). Here we identified two nonoverlapping α5+ cell populations (α5-Amigo1 and α5-Epyc) in mouse IPN that respond differentially to nicotine. Chronic nicotine treatment altered the translational profile of more than 1,000 genes in α5-Amigo1 neurons, including neuronal nitric oxide synthase (Nos1) and somatostatin (Sst). In contrast, expression of few genes was altered in the α5-Epyc population. We show that both nitric oxide and SST suppress optically evoked neurotransmitter release from the terminals of habenular (Hb) neurons in IPN. Moreover, in vivo silencing of neurotransmitter release from the α5-Amigo1 but not from the α5-Epyc population eliminates nicotine reward, measured using place preference. This loss of nicotine reward was mimicked by shRNA-mediated knockdown of Nos1 in the IPN. These findings reveal a proaddiction adaptive response to chronic nicotine in which nitric oxide and SST are released by a specific α5+ neuronal population to provide retrograde inhibition of the Hb-IPN circuit and thereby enhance the motivational properties of nicotine.

Smoking-Related Genes and Functional Consequences

As the leading preventable cause of cancer and death, nicotine use and dependence have been the subject of a multitude of genetic studies in the past decade, ranging from candidate gene studies to genome-wide association studies (GWAS) to prospective studies. The genetics of nicotine addiction, smoking, and cancer are multifactorial, as would be expected from a complex behavior such as cigarette smoking. The combined heritability based on twin studies is estimated at 50–75 % (Li, Am J Med Sci 326(4): 168–73, 2003; Hall et al. Tob Control 11(2): 119–24, 2002; Lessov et al. Psychol Med 34(5): 865–79, 2004.; Lessov-Schlaggar et al. Int J Epidemiol 35(5): 1278–85, 2006; Maes et al. Psychol Med 34(7): 1251–61, 2004), with a large number of genes contributing to a small amount of risk individually. Some genes contribute to the quantity of nicotine used, while another set is associated with the duration of nicotine use, and yet another set is linked with severity of nicotine dependence. Associated “risky” genes comprise genes encoding for nicotinic acetylcholine receptor (nAChR) subunits, but also include genes like the bitter taste receptor. Still others have been associated with initiation and with success, or lack thereof, in cessation. In this chapter we review the current findings in the genetics of smoking, focusing on those studies that have linked nicotinic acetylcholine receptors (nAChR) to nicotine addiction, and further discuss how mutations in these receptors alter their function.