Pierre Cazala

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.

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.

Reinforcing effects of nicotine microinjections into the ventral tegmental area of mice: Dependence on cholinergic nicotinic and dopaminergic D1 receptors

We used an intracranial self-administration (ICSA) procedure to assess the involvement of the ventral tegmental area (VTA) nicotinic receptors in the rewarding effects of nicotine. We then challenged intra-VTA nicotine self-administration via systemic or local injections of dopamine (DA)-D1 and nicotinic receptor antagonists. C57BL/6J mice were stereotaxically implanted unilaterally with a guide cannula above the VTA. After 1 week of recovery, mice were allowed to discriminate between two arms of a Y-maze over seven daily sessions, one arm being reinforced by intracranial nicotine microinjection. Mice exhibited nicotine self-administration at both doses tested, i.e. 10 ng (21.6 pmol) and 100 ng (216 pmol)/50-nl injection. In contrast, mice receiving a 216-pmol nicotine dose 0.8 mm above VTA performed at chance level. Once the ICSA response was acquired, systemic pretreatment with the DA-D1 receptor antagonist SCH 23390 (25 microg/kg i.p.) or co-infusion of the nAChR antagonist DHbetaE with nicotine disrupted ICSA. Replacement of SCH 23390 by vehicle, or withdrawal of DHbetaE from nicotine/DHbetaE mixed solutions led to recovery of intra-VTA nicotine self-administration. We conclude that nicotinic receptors in the VTA, presumably alpha4beta2 nAChRs are critically to mediate the rewarding effects of nicotine and that DA-D1 receptors are also directly implicated.

Long-term effects of chronic nicotine exposure on brain nicotinic receptors

Chronic nicotine exposure results in long-term homeostatic regulation of nicotinic acetylcholine receptors (nAChRs) that play a key role in the adaptative cellular processes leading to addiction. However, the relative contribution of the different nAChR subunits in this process is unclear. Using genetically modified mice and pharmacological manipulations, we provide behavioral, electrophysiological, and pharmacological evidence for a long-term mechanism by which chronic nicotine triggers opposing processes differentially mediated by β2*- vs. α7*nAChRs. These data offer previously undescribed insights into the understanding of nicotine addiction and the treatment of several human pathologies by nicotine-like agents chronically acting on β2*- or α7*nAChRs.

Genetic dissociation of two behaviors associated with nicotine addiction: Beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome

RationaleNicotine addiction is characterized by two distinct behaviors, chronic compulsive self-administration and the induction of a withdrawal syndrome upon cessation of nicotine consumption.ObjectiveTo examine if these two processes rely on β2-containing nicotinic receptors—β2*nAChRs—we analyzed the behavior of mice lacking these receptors in the two situations.ResultsFirst, we showed that, in contrast to wild-type (WT) mice, β2-knockout (β2−/−) mice exhibit no intra-ventral tegmental area (VTA) nicotine self-administration, whereas their ability to self-administer morphine is intact. However, β2−/− mice showed some sensitivity to locomotor effects of nicotine, implying an effect of the drug on other nicotinic subtypes. Then, we observed that β2−/− mice exhibited a normal nicotine withdrawal syndrome, i.e., increased levels of rearing and jumping upon precipitated withdrawal. Thus, the β2*nAChRs are not involved in the behaviors induced by cessation of nicotine consumption.ConclusionTaken together, the present data demonstrated a genetic dissociation of two distinct behavioral patterns associated with nicotine addiction. They further suggested that independent molecular mechanisms underlie these two aspects, offering the possibility of controlling them separately.

Self-administration of morphine into the lateral hypothalamus in the mouse

BALB/c mice were chronically and unilaterally implanted with a guide cannula, the tip of which was positioned 1 mm above the lateral hypothalamus (LH). On each experimental day, a stainless-steel injection cannula was inserted into the LH, and self-administration of morphine or vehicle in this brain area was studied by using a spatial discrimination test in a Y-maze. In a first experiment, we observed that when mice had access to morphine (0.1 microgram by injection) they rapidly discriminated the reinforced arm from the neutral arm of the maze in order to self administer, with increasing frequency, the drug into the LH. In contrast when only vehicle was present, the two arms were no longer discriminated. In a second experiment we compared the effects of 3 doses of morphine (0.1 microgram, 0.05 microgram and 0.025 microgram by injection); optimal discrimination was obtained with the lowest dose used. In a third experiment we observed that subcutaneous injections of naloxone (4 mg/kg) progressively reduced the number of self-administrations of morphine into the LH, a result which suggests that this response is dependent on an opiate receptor mechanism.

Self-administration of the GABAA antagonist bicuculline into the ventral tegmental area in mice: dependence on D2 dopaminergic mechanisms

Abstract BALB/c mice were unilaterally implanted with a guide cannula, the tip of which was positioned 1.5 mm above the ventral tegmental area (VTA). On each day of the experimental period, a stainless steel injection cannula was inserted into the VTA in order to study the eventual self-administration of a low dose (1.5 ng/50 nl) of bicuculline, a GABAA-antagonist, using a spatial discrimination task in a Y maze. Mice rapidly discriminated between the arm enabling a micro-injection of bicuculline and the neutral arm of the maze, and robust self-administration of this GABAergic antagonist was observed. Once this self-administration response for bicuculline had been fully acquired, the systemic injection of the dopaminergic D2 antagonist sulpiride (50 mg/kg), 30 min before the test, produced a rapid extinction of the self-administration response. Moreover, if this same sulpiride pre-treatment was given during the initial acquisition period mice did not discriminate between the two arms of the Y-maze. These data demonstrate the dopamine D2 dependence of this bicuculline self-administration behavior, and confirm that GABAergic interneurons and/or inputs normally transynaptically inhibit neuronal activity in the mesocorticolimbic dopamine system.

Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice.

RationaleNicotine is the main addictive component of tobacco and modifies brain function via its action on neuronal acetylcholine nicotinic receptors (nAChRs). The mesolimbic dopamine (DA) system, where neurons of the ventral tegmental area (VTA) project to the nucleus accumbens (ACb), is considered a core site for the processing of nicotine’s reinforcing properties. However, the precise subtypes of nAChRs that mediate the rewarding properties of nicotine and that contribute to the development of addiction remain to be identified.ObjectivesWe investigated the role of the nAChRs containing the α7 nicotinic subunit (α7*nAChRs) in the reinforcing properties of nicotine within the VTA and in the nicotine-induced changes in ACb DA outflow in vivo.MethodsWe performed intra-VTA self-administration and microdialysis experiments in genetically modified mice lacking the α7 nicotinic subunit or after pharmacological blockade of α7*nAChRs in wild-type mice.ResultsWe show that the reinforcing properties of nicotine within the VTA are lower in the absence or after pharmacological blockade of α7*nAChRs. We also report that nicotine-induced increases in ACb DA extracellular levels last longer in the absence of these receptors, suggesting that α7*nAChRs regulate the action of nicotine on DA levels over time.ConclusionsThe present results reveal new insights for the role of α7*nAChRs in modulating the action of nicotine within the mesolimbic circuit. These receptors appear to potentiate the reinforcing action of nicotine administered into the VTA while regulating its action over time on DA outflow in the ACb.

Hypothalamic self-stimulation in three inbred strains of mice

Abstract Male mice from 3 inbred strains (DBA/2 Orl, BALB/c Orl and C57 BL/6 Orl) were implanted with a bipolar electrode in the hypothalamus and tested for self-stimulation. The 3 strains differed in performances: the BALB/c mice showed higher scores than the DBA/2 mice which themselves showed higher rates than the C57 BL/6 mice. These strains also differed in frequency, threshold and the nature of seizures suspending their self-stimulation behavior. Moreover, for each strain, a comparison between dorsal and ventral hypothalamus implantation was made. In the 3 strains dorsal implantation gave higher self-stimulation scores than ventral. On the other hand the seizures occured more frequently with ventral implantations.

Brain Regional Fos Expression Elicited by the Activation of μ - but not δ -Opioid Receptors of the Ventral Tegmental Area: Evidence for an Implication of the Ventral Thalamus in Opiate Reward

Both μ-opioid receptors (MORs) and δ-opioid receptors (DORs) are expressed in the ventral tegmental area (VTA) and are thought to be involved in the addictive properties of opiates. However, their respective contributions to opiate reward remain unclear. We used intracranial self-administration (ICSA) to study the rewarding effects of morphine microinjections into the VTA of male and female MOR−/− and DOR−/− mice. In brains of mice tested for intra-VTA morphine self-administration, we analyzed regional Fos protein expression to investigate the neural circuitry underlying this behavior. Male and female WT and DOR−/− mice exhibited similar self-administration performances, whereas knockout of the MOR gene abolished intra-VTA morphine self-administration at all doses tested. Naloxone (4 mg/kg) disrupted this behavior in WT and DOR mutants, without triggering physical signs of withdrawal. Morphine ICSA was associated with an increase in Fos within the nucleus accumbens, striatum, limbic cortices, amygdala, hippocampus, the lateral mammillary nucleus (LM), and the ventral posteromedial thalamus (VPM). This latter structure was found to express high levels of Fos exclusively in self-administering WT and DOR−/− mice. Abolition of morphine reward in MOR−/− mice was associated with a decrease in Fos-positive neurons in the mesocorticolimbic dopamine system, amygdala, hippocampus (CA1), LM, and a complete absence within the VPM. We conclude that (i) VTA MORs, but not DORs, are critical for morphine reward and (ii) the role of VTA-thalamic projections in opiate reward deserves to be further explored.