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 beta2-subunit of the nicotinic acetylcholine receptor (nAChR) by stereotaxically injecting a lentiviral vector into the VTA of mice carrying beta2-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 beta2-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.

Targeted in vivo expression of nicotinic acetylcholine receptors in mouse brain using lentiviral expression vectors.

Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.

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

RATIONALE: Nicotine addiction is characterized by two distinct behaviors, chronic compulsive self-administration and the induction of a withdrawal syndrome upon cessation of nicotine consumption. OBJECTIVE: To examine if these two processes rely on beta2-containing nicotinic receptors--beta2*nAChRs--we analyzed the behavior of mice lacking these receptors in the two situations. RESULTS: First, we showed that, in contrast to wild-type (WT) mice, beta2-knockout (beta2-/-) mice exhibit no intra-ventral tegmental area (VTA) nicotine self-administration, whereas their ability to self-administer morphine is intact. However, beta2-/- mice showed some sensitivity to locomotor effects of nicotine, implying an effect of the drug on other nicotinic subtypes. Then, we observed that beta2-/- mice exhibited a normal nicotine withdrawal syndrome, i.e., increased levels of rearing and jumping upon precipitated withdrawal. Thus, the beta2*nAChRs are not involved in the behaviors induced by cessation of nicotine consumption. CONCLUSION: Taken 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.

Anxiogenic-like effects limit rewarding effects of cocaine in balb/cbyj mice.

Previous studies have reported intravenous cocaine self-administration behavior in several strains of mice with the exception of BALB/cByJ, a strain considered a mouse model of high emotional reactivity. The present experiments further investigated acquisition of self-administration in BALB/cByJ mice using a low dose and a habituation session. Following evidence of an initial drug-seeking behavior, we observed a progressive decline of intravenous self-administration. Pretreatment with diazepam (0.5 mg/kg, IP), reinstated cocaine-maintained responding. To test the hypothesis that injections directly into a reward-relevant brain region might support consistent cocaine-seeking behavior, BALB/cByJ mice implanted in the nucleus accumbens (NAc) or the caudate-putamen nucleus (CPu) were trained to discriminate between the arm enabling a microinjection of cocaine (30 pmol/50 nl or 150 pmol/50 nl) and the neutral arm of a Y-maze. Only NAc subjects exhibited a spatial discrimination toward the cocaine-reinforced arm and the D2 antagonist, sulpiride (50 mg/kg, IP) eliminated intra-NAc cocaine self-administration. However, after several days of cocaine self-injection, animals developed an approach/avoidance-like behavior between the start box and the reinforced arm. This behavior was suppressed by systemic diazepam (1 mg/kg, IP) pretreatment. We conclude that: (1) medio-ventral NAc is involved both in the rewarding (via a D2 dopaminergic mechanism) and aversive effects of cocaine in mice; and (2) anxiolytic pretreatment (diazepam) indirectly enhanced the reinforcing properties of cocaine in BALB/cByJ, suggesting that emotionality can act as a protective mechanism against stimulant abuse.

Effect of clonidine and phentolamine on self-stimulation behavior in the dorsal and ventral regions of the lateral hypothalamus in mice.

The effects of IP injections of increasing doses of clonidine and phentolamine on self-stimulation behavior (ICSS) in the dorsal and ventral regions of the lateral hypothalamus were studied in C57BL/6 mice. Clonidine specifically stimulates alpha-noradernergic receptors and has two distinct effects on ICSS. Ventral hypothalamic ICSS is immediately depressed at low doses (5 microgram/kg) whereas dorsal ICSS exhibits a discrete increase. At higher doses, ICSS is perturbed at both sites. Phentolamine is a specific blocking agent of alpha-noradrenergic receptors and depresses dorsal ICSS more than that of the ventral region. These observations clearly indicate a noradrenergic influence in the dorsal region of the hypothalamus.

Effects of Lilly 110140 (flouxetine) on self-stimulation behavior in the dorsal and ventral regions of the lateral hypothalamus in the mouse.

The effects of Lilly 110141 (fluoxetine) injected IP on self-stimulation behavior triggered in the dorsal and ventral regions of the lateral hypothalamus were studied in C57BL/6 mice strain. Lilly 110140 inhibits serotonin reuptake. It depresses dorsal self-stimulation at a low dose (5 mg/kg) and improves ventral sel-stimulation at a high dose (20 mg/kg). The changes brought about by the injection of the 5 mg/kg dose support the proposed inhibitory role of serotonin on self-stimulation and also suggest that the fibers communicating with certain serotonergic neurons are localized primarily in the dorsal region of the lateral hypothalamus.

Anatomical and pharmacological specificity of the rewarding effect elicited by microinjections of morphine into the nucleus accumbens of mice.

RATIONALE: The involvement of nucleus accumbens (NAc) in initiating opiate-induced reward has been difficult to demonstrate in rats, and has not been studied in mice. OBJECTIVES: To determine whether a reward-sensitive strain of mice (BALB/c) would self-administer morphine directly into the NAc or sub-regions of the dorsal striatum. METHODS: BALB/c mice were unilaterally implanted with a guide-cannula above either the NAc, the anterior caudate putamen, or the posterior caudate putamen. On each experimental day, a stainless-steel injection cannula was inserted into the guide cannula to test the capacity for morphine self-administration (6.5 pmol or 65 pmol/50 nl) using a spatial discrimination task in a Y maze. RESULTS: Only the ventro-medial NAc group discriminated between the arm enabling a microinjection of morphine and the neutral arm. Once self-administration had been acquired, the effects of a pretreatment with two doses of the opiate antagonist naloxone (0.4 mg/kg or 4 mg/kg) were tested. Both doses slightly disrupted self-administration on the first 2 days. Only subjects receiving the 4-mg/kg dose exhibited an extinction of self-administration, related to an increasing number of jump attempts; none of the other opiate withdrawal-associated signs were observed. Self-administration was reinstated when naloxone was replaced with saline. CONCLUSIONS: (1) Medio-ventral NAc is involved in acute rewarding effects of opiates in mice. (2) Neither anterior nor posterior dorsal striatum seem to participate in these effects. (3) NAc is involved in jumping caused by naloxone-induced extinction, a behavior presumably revealing an aversive state associated with the unexpected suppression of reward.

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

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 pretreatment 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.

Self-stimulation behavior can be elicited from various 'aversive' brain structures.

Electrical stimulation applied to various brain regions of the negative motivational system: the dorsal part of the mesencephalic central gray area (CG) the medial hypothalamus (MH), the medial lemniscus (ML), the lateral tegmentum (LT) and the reticular formation (RF), produces vigorous escape responses in mice. In spite of their highly aversive consequences, stimulation of all these regions also elicits self-stimulation behavior. This paradoxical approach response was clearly observed when the animals were placed in a Y-maze where they could successively trigger and turn off continuous electrical stimulation. In effect, mice stimulated in 'aversive' structures, similarly to animals stimulated in the lateral hypothalamus (LH), were able to discriminate between the reinforced arm and the non-reinforced arm of the Y-maze in order to self-administer the stimulation. When the mice were placed in a lever press box which delivered 0.2 s of electrical stimulation, an evident self-stimulation behavior was observed in LH and in some animals implanted in RF or MH. On the other hand, the very low response rates recorded in CG, LT, ML and in other RF or MH implanted mice do not permit a description of the motivational properties of these different stimulation sites. These results show that stimulation of brain structures of the negative reinforcement system has an approach component which, however, shows up clearly only in certain experimental situations.

A comparative study of self-administration of morphine into the amygdala and the ventral tegmental area in mice.

BALB/c mice were unilaterally implanted with a guide-cannula, the tip of which was positioned 1.5 mm above either the amygdala (AMY) or the ventral tegmental area (VTA). On each experimental day, a stainless-steel injection cannula was inserted into these structures in order to compare the self-administration of two doses of morphine (5 ng or 50 ng) in independent groups using a spatial discrimination task in a Y-maze. During the acquisition phase, both AMY and VTA injected mice showed a regular self-administration response at the two doses used. The latency to trigger the injection was short, particularly in the VTA group. Subcutaneous injection of naloxone (4 mg/kg) in trained mice reduced the number of self-administrations to a level near to chance in both groups, which suggests that the drug-seeking behavior observed is effectively dependent on an opiate receptor-mediated mechanism. However the rate of extinction was more rapid in AMY than in VTA injected mice. The 'perseveration' response exhibited by the VTA group during the withdrawal precipitated by naloxone may probably be due to the strong motivational and/or rewarding effect of morphine when injected in this brain structure during acquisition.

Differential involvement of the lateral and medial divisions of the septal area on spatial learning processes as revealed by intracranial self-administration of morphine in mice.

The present study was conducted to investigate the effects of morphine self-administration into either the medial or lateral divisions of the septal region on spatial discrimination abilities in mice. To this end, BALB/c mice received a dose of 5 ng/50 nl of morphine sulfate, via a stainless steel injection cannula, inserted into either the medial septal area (MS) or the lateral septal nucleus (LS), when they are close to the end of one of the two choice arms of a Y-maze (acquisition period). In these conditions a discrimination between the reinforced arm and the neutral arm is acquired in MS as well as in LS mice. However both acquisition and subsequent extinction (vehicle only available) was more rapid in the LS group than in the MS group. Moreover, during the extinction period, numerous escape attempts from the Y-maze were observed for MS mice. When the dose of morphine was raised to 50 ng the pattern of acquisition was unchanged in the LS group. In contrast animals of the MS group learned to avoid the arm where the higher dose of morphine was delivered thus allowing us to conclude that in MS animals the drug effect became aversive at this higher dose. This possibility was directly investigated in a second experiment by closing the access to the neutral arm. Thus, for a 5-ng dose the rewarding effect of morphine was demonstrated in both MS and LS groups by the decrease of self-administration latencies which, furthermore, were notably lower than in the discrimination situation. Moreover, with the dose of 50 ng of morphine the latencies were identical for the two groups and at their lowest value thus indicating that morphine had similar appetitive effects in MS as well as in LS mice in this situation. Thus, the previously observed deficit of MS subjects, including escape attempts, disappeared when the dose of morphine was raised and the experimental context simplified. These results which demonstrate differential functional properties of these two septal divisions are discussed in terms of conflict resulting from the strongly appetitive effects of the morphine which induces, in parallel, deleterious consequences on cognitive processing in MS self-injected mice.

Dose-dependent effects of morphine differentiate self-administration elicited from lateral hypothalamus and mesencephalic central gray area in mice.

BALB/c mice were unilaterally implanted with a guide-cannula, the tip of which was positioned 1 mm above either the lateral hypothalamus (LH) or the mesencephalic central gray area (CG). On each experimental day, a stainless-steel injection cannula was inserted into the LH or the CG and self-administration of two doses of morphine (50 and 5 ng) was compared in the two brain structures using a spatial discrimination task in a Y-maze. At the dose of 50 ng, mice injected into the LH rapidly discriminated the reinforced arm from the neutral arm of the maze in order to self-administer morphine. In contrast, at this same dose, mice of the CG group do not show any regular self-administration behavior. At the dose of 5 ng, both LH and CG injected mice show a regular self-administration response. The rate of discrimination was similar in the two groups. When naloxone (5 ng) was mixed with morphine (5 ng), the number of self-administrations progressively decreased in both brain areas. This decrease was both larger and more rapid in CG than in LH. Marked signs of physical dependence (escapes from the maze) were observed in the two groups during this phase. Finally, when morphine alone (5 ng) was again made available, a regular self-administration response reappeared in the two brain structures. These data suggest (1) that morphine has reinforcing effects in both LH and CG and (2) that in these two brain structures self-injection of this drug is dependent on an opiate receptor mediated mechanism.

A Y-maze test reveals the positively reinforcing properties of electrical stimulation of the mesencephalic central gray area.

Animals stimulated in the dorsal part of the mesencephalic central gray area (CG) are able, as are animals stimulated in the lateral hypothalamus, to discriminate between the reinforced and the non-reinforced arm of an Y-maze to trigger a continuous electrical stimulation. This result clearly demonstrates that stimulation of the dorsal part of CG has an appetitive component.

Central catecholamine metabolism and hypothalamic self-stimulation behaviour in two inbred strains of mice.

These experiments were conducted in order to better understand the role of catecholaminergic neurons in intracranial self-stimulation behaviour (ICSS) elicited from the lateral hypothalamus (LH). Two strains of mice which differ in their ICSS rate and their thresholds were studied. In a first time, we compared the catecholamine (CA) content and activity in various parts of the brain including cell bodies and nerve terminals of the man CA bundle. We used for this determination a high performance liquid chromatographic separation and an electrochemical detection. We observed that the BALB/c strain is distinguished by a higher CA activity than the DBA/2 strain. This correlates with a higher ICSS response rate in the BALB/c strain. The effects of stimulation on CA metabolism were then investigated, the electrodes being implanted specifically either in the dorsal or the ventral part of the LH and the biochemical data obtained analysed separately. Significant enhances of CA turn-over (TO) were noted in nerve terminals as hippocampus, cortex and accumbens. These results provide further evidence for the involvement of dorsal noradrenergic bundle and mesolimbic dopaminergic bundle in LH ICSS. Stimulation in the dorsal part of the LH produced the higher ICSS rate and seemed to induce a large variation of the CA TO. We noted also that the CA metabolism was always more altered in the DBA/2 than in the BALB/c strain, which is surprising in regard to the behaviour and remains unclearly explained.

Self-stimulation of an 'aversive' brain structure: the mesencephalic central gray area.

Four experiments were performed in BALB/c mice implanted either in the lateral hypothalamus (LH) or in the dorsal part of the mesencephalic central gray area (CG) or both in LH and CG. In the first 3 experiments the animals were placed in a Y-maze where they could successively trigger and turn off continuous electrical stimulation. Expt. 1 showed that mice stimulated in CG (like animals stimulated in LH) are able to discriminate between the reinforced and the non-reinforced arm of the Y-maze in order to self-administer the stimulation. This behavior was observed whether the experimental situation was a simple spatial discrimination or a more complex light-dark discrimination. During the second experiment, the animals could choose between stimulation of low intensity (in one arm) or high intensity (in the other arm). We observed that, like LH animals, CG implanted mice chose the higher stimulation, despite its strong aversive component. On the other hand, Expt. 3 showed that when the animals could chose between CG stimulation, whose intensity was progressively increased, and LH stimulation whose intensity remained constant (low) they preferred the latter. When the mice were placed in a lever-press box where they could obtain 0.2 s of electrical stimulation (last experiment), LH animals showed vigorous self-stimulation behavior, whereas CG animals showed only a weak response rate. These results show that, in spite of its aversiveness, dorsal CG stimulation has an appetitive component whose magnitude may vary under different experimental conditions.

Lesion of the temporo-ammonic perforant path facilitates self-stimulation of the lateral entorhinal cortex in mice.

The effect of a lesion of the perforant path (PP) on self-stimulation (SS) of the lateral entorhinal cortex (LEC) was tested in mice between 8 and 21 days after surgery. The current intensities tested ranged between 0 and 80 microA (peak to peak 100 Hz sine-wave). The PP lesion led to a two-fold increase in SS rates at intensities above 30 microA without affecting the baseline SS rates (0 microA) and SS threshold (30 microA). The lesion also led to a significant increase in LEC after-discharge (AD) threshold and eliminated behavioral convulsions during SS testing. The suppression of AD by i.p. Na phenobarbital injection (10 mg/kg) led to a similar increase in SS rates in sham-lesioned mice; there was no difference in PP-lesioned animals. These results might be interpreted as evidence in favor of an independence of the neuronal processes mediating entorhinal and hippocampal reward-related behaviors.

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.

Dissociation of limbic structures by pharmacological effects of diazepam on electrical self-stimulation in the mouse.

The effect of diazepam was tested on self-stimulation (SS) in 21 mice implanted with a bipolar electrode in the lateral hypothalamus (LH), the dorsolateral hippocampus (HPC) or the lateral entorhinal cortex (LEC). Diazepam, injected i.p. in doses of 0.5, 1 and 2 mg/kg, significantly increased SS rates with electrodes in LH while 4 and 8 mg/kg of diazepam had no significant effect. At low doses, similar increases were seen in mice with LEC electrodes but high doses produced a significant suppression. HPC animals showed an almost total suppression of SS beginning at 2 mg/kg of diazepam; lower doses had no significant effect. The results indicate that entorhinal and hippocampal SS are at least partly independent phenomena; in addition, the suppression of SS by moderate doses of diazepam remains specific to the HPC among the brain structures studied to date.

Approach responses for mesencephalic central gray stimulation are facilitated by D-amphetamine or food deprivation.

The effects of either injection of D-amphetamine or food deprivation on approach and escape responses produced by continuous electrical stimulation of either the lateral hypothalamus (LH) or the dorsal part of the mesencephalic central gray area (CG), were studied in the DBA/2 strain of mice using the shuttle-box technique. Both D-amphetamine and food deprivation decreased approach latency for LH as well as for CG stimulation. Changes in escape latency were observed only for LH stimulation and only during the food deprivation test. These results suggest the involvement of positive reinforcement circuits along with dorsal CG activation.

Hypothalamic self-stimulation and operant activity in the mottled mutant mouse.

Some behavioural effects of the mottled mutation were studied in the variegated heterozygote female. In mutants, compared to normals, operant activity was increased, whereas hypothalamic self-stimulation was always decreased. These results are discussed relative to the excepted central catecholaminergic disturbances resulting from the mutant gene.