Stéphanie Pons

Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors

Worldwide, 100 million people are expected to die this century from the consequences of nicotine addiction, but nicotine is also known to enhance cognitive performance. Identifying the molecular mechanisms involved in nicotine reinforcement and cognition is a priority and requires the development of new in vivo experimental paradigms. The ventral tegmental area (VTA) of the midbrain is thought to mediate the reinforcement properties of many drugs of abuse. Here we specifically re-expressed the β2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying β2-subunit deletions. We demonstrate the efficient re-expression of electrophysiologically responsive, ligand-binding nicotinic acetylcholine receptors in dopamine-containing neurons of the VTA, together with the recovery of nicotine-elicited dopamine release and nicotine self-administration. We also quantified exploratory behaviours of the mice, and showed that β2-subunit re-expression restored slow exploratory behaviour (a measure of cognitive function) to wild-type levels, but did not affect fast navigation behaviour. We thus demonstrate the sufficient role of the VTA in both nicotine reinforcement and endogenous cholinergic regulation of cognitive functions.

Crucial Role of α4 and α6 Nicotinic Acetylcholine Receptor Subunits from Ventral Tegmental Area in Systemic Nicotine Self-Administration

The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the β2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different α subunit partners of β2 (i.e., α4 and α6), the homo-pentameric α7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7–52.6 μg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the β2, α4, α6 and α7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, β2-VEC mice with the β2 subunit re-expressed exclusively in the VTA, α4-VEC mice with selective α4 re-expression in the VTA, α6-VEC mice with selective α6 re-expression in the VTA, and α7-KO mice promptly self-administer nicotine intravenously, whereas β2-KO, β2-VEC in the substantia nigra, α4-KO and α6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of α4β2- and α6β2-subunit containing nicotinic receptors (α4β2*- and α6β2*-nAChRs), but not α7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.

Nicotine upregulates its own receptors through enhanced intracellular maturation.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smokers brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4beta2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to act inside the cell and to increase the amount of beta subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4beta2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

Hierarchical Control of Dopamine Neuron-Firing Patterns by Nicotinic Receptors

Nicotine elicits dopamine release by stimulating nicotinic acetylcholine receptors (nAChRs) on dopaminergic neurons. However, a modulation of these neurons by endogenous acetylcholine has not been described. We recorded, in vivo, the spontaneous activity of dopaminergic neurons in the VTA of anaesthetized wt and nAChR knockout mice and their response to nicotine injections. Deleting alpha7 or beta2 subunits modified the spontaneous firing patterns, demonstrating their direct stimulation by endogenous acetylcholine. Quantitative analysis further revealed four principal modes of firing, each depending on the expression of particular nAChR subunits and presenting unique responses to nicotine. The prominent role of the beta2 subunit was further confirmed by its selective lentiviral reexpression in the VTA. These data suggest a hierarchical control of dopaminergic neuron firing patterns by nAChRs: activation of beta2*-nAChR switches cells from a resting to an excited state, whereas activation of alpha7*-nAChRs finely tunes the latter state but only once beta2*-nAChRs have been activated.

Distinct contributions of nicotinic acetylcholine receptor subunit α4 and subunit α6 to the reinforcing effects of nicotine

Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit α4- and subunit α6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the α4 subunit, but not the α6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits α4 and α6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that α4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing α4 and α6 subunits, underlies nicotine self-administration and its long-term maintenance.

Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement

Smoking is the most important preventable cause of mortality and morbidity worldwide. This nicotine addiction is mediated through the nicotinic acetylcholine receptor (nAChR), expressed on most neurons, and also many other organs in the body. Even within the ventral tegmental area (VTA), the key brain area responsible for the reinforcing properties of all drugs of abuse, nicotine acts on several different cell types and afferents. Identifying the precise action of nicotine on this microcircuit, in vivo, is important to understand reinforcement, and finally to develop efficient smoking cessation treatments. We used a novel lentiviral system to re-express exclusively high-affinity nAChRs on either dopaminergic (DAergic) or γ-aminobutyric acid-releasing (GABAergic) neurons, or both, in the VTA. Using in vivo electrophysiology, we show that, contrary to widely accepted models, the activation of GABA neurons in the VTA plays a crucial role in the control of nicotine-elicited DAergic activity. Our results demonstrate that both positive and negative motivational values are transmitted through the dopamine (DA) neuron, but that the concerted activity of DA and GABA systems is necessary for the reinforcing actions of nicotine through burst firing of DA neurons. This work identifies the GABAergic interneuron as a potential target for smoking cessation drug development.

Interplay of β2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion

Acetylcholine (ACh) is a known modulator of the activity of dopaminergic (DAergic) neurons through the stimulation of nicotinic ACh receptors (nAChRs). Yet, the subunit composition and specific location of nAChRs involved in DA-mediated locomotion remain to be established in vivo. Mice lacking the β2 subunit of nAChRs (β2KO) display striking hyperactivity in the open field, which suggests an imbalance in DA neurotransmission. Here, we performed the selective gene rescue of functional β2*-nAChRs in either the substantia nigra pars compacta (SNpc) or the ventral tegmental area (VTA) of β2KO mice. SNpc rescued mice displayed normalization of locomotor activity, both in familiar and unfamiliar environments, whereas restoration in the VTA only rescued exploratory behavior. These data demonstrate the dissociation between nigrostriatal and mesolimbic β2*-nAChRs in regulating unique locomotor functions. In addition, the site-directed knock-down of the β2 subunit in the SNpc by RNA interference caused hyperactivity in wild-type mice. These findings highlight the crucial interplay of nAChRs over the DA control of spontaneous locomotion.

Nicotine differentially activates inhibitory and excitatory neurons in the dorsal spinal cord

&NA; Nicotinic agonists have well‐documented antinociceptive properties when administered subcutaneously or intrathecally in mice. However, secondary mild to toxic effects are observed at analgesic doses, as a consequence of the activation of the large family of differentially expressed nicotinic receptors (nAChRs). In order to elucidate the action of nicotinic agonists on spinal local circuits, we have investigated the expression and function of nAChRs in functionally identified neurons of neonate mice spinal cord. Molecular markers, amplified at the single‐cell level by RT‐PCR, distinguished two neuronal populations in the dorsal horn of the spinal cord: GABAergic/glycinergic inhibitory interneurons, and calbindin (CA) or NK1 receptor (NK1‐R) expressing, excitatory interneurons and projection neurons. The nicotinic response to acetylcholine of single cells was examined, as well as the pattern of expression of nAChR subunit transcripts in the same neuron. Beside the most expressed subunits &agr;4, &bgr;2 and &agr;7, the &agr;2 subunit transcript was found in 19% of neurons, suggesting that agonists targeting &agr;2* nAChRs may have specific actions at a spinal level without major supra‐spinal effects. Both inhibitory and excitatory neurons responded to nicotinic stimulation, however, the nAChRs involved were markedly different. Whereas GABA/glycine interneurons preferentially expressed &agr;4&agr;6&bgr;2* nAChRs, &agr;3&bgr;2&agr;7* nAChRs were preferentially expressed by CA or NK1‐R expressing neurons. Recorded neurons were also classified by firing pattern, for comparison to results from single‐cell RT‐PCR studies. Altogether, our results identify distinct sites of action of nicotinic agonists in circuits of the dorsal horn, and lead us closer to an understanding of mechanisms of nicotinic spinal analgesia.

Nicotine consumption is regulated by a human polymorphism in dopamine neurons

Smoking is the most important preventable cause of morbidity and mortality worldwide. Recent genome-wide association studies highlighted a human haplotype on chromosome 15 underlying the risk for tobacco dependence and lung cancer. Several polymorphisms in the CHRNA3-CHRNA5-CHRNB4 cluster coding for the nicotinic acetylcholine receptor (nAChR) α3, α5 and β4 subunits were implicated. In mouse models, we define a key role in the control of sensitivity to nicotine for the α5 subunit in dopaminergic (DAergic) neurons of the ventral tegmental area (VTA). We first investigated the reinforcing effects of nicotine in drug-naive α5−/− mice using an acute intravenous nicotine self-administration task and ex vivo and in vivo electrophysiological recordings of nicotine-elicited DA cell activation. We designed lentiviral re-expression vectors to achieve targeted re-expression of wild-type or mutant α5 in the VTA, in general, or in DA neurons exclusively. Our results establish a crucial role for α5*-nAChRs in DAergic neurons. These receptors are key regulators that determine the minimum nicotine dose necessary for DA cell activation and thus nicotine reinforcement. Finally, we demonstrate that a single-nucleotide polymorphism, the non-synonymous α5 variant rs16969968, frequent in many human populations, exhibits a partial loss of function of the protein in vivo. This leads to increased nicotine consumption in the self-administration paradigm. We thus define a critical link between a human predisposition marker, its expression in DA neurons and nicotine intake.

Prefrontal nicotinic receptors control novel social interaction between mice

Social behavior is a defining mammalian feature that integrates emotional and motivational processes with external rewarding stimuli. It is thus an appropriate readout for complex behaviors, yet its neuronal and molecular bases remain poorly understood. In this study, we investigated the role of the mouse prefrontal area, particularly the involvement of β2‐subunit nicotinic receptors (β2∗‐nAChRs) in a paradigm of social behavior with concurrent motivations. We previously observed that mice lacking β2∗‐nAChRs (β2‐/‐) display increased time in social contact and exaggerated approach movements toward the novel conspecific. Here, combining behavioral analysis, localized brain lesions, and lentiviral gene rescue, we found that c‐Fos expression is specifically activated in the prelimbic (PrL) area of the prefrontal cortex (PFC) of mice exposed to a novel conspecific; lesions of the PrL area in wild‐type mice produce the same social pattern as in β2‐/‐ mice; and virally mediated reexpression of the β2‐subunit in the PrL area of β2‐/‐ mice rescues behavioral components in the social interaction task up to normal levels. Together, these data reveal that social interactions particularly mobilize the PrL area of the mouse PFC and that the presence of functional PrL β2∗‐nAChRs is necessary for this integrated behavior to emerge.—Avale, M. E., Chabout, J., Pons, S., Serreau, P., De Chauont, F., Olivo‐Marin, J‐C., Bourgeois, J‐P., Maskos, U., Changeux, J‐P., Granon, S. Prefrontal nicotinic receptors control novel social interaction between mice. FASEB J. 25, 2145‐2155 (2011). www.fasebj.org