Nicotine enhances alcohol intake and dopaminergic responses through β2* and β4* nicotinic acetylcholine receptors

Alcohol and nicotine are the most widely co-abused drugs. Both modify the activity of dopaminergic (DA) neurons of the Ventral Tegmental Area (VTA) and lead to an increase in DA release in the Nucleus Accumbens, thereby affecting the reward system. Evidences support the hypothesis that distinct nicotinic acetylcholine receptors (nAChRs), the molecular target of acetylcholine (ACh) and exogenous nicotine, are also in addition implicated in the response to alcohol. The precise molecular and neuronal substrates of this interaction are however not well understood. Here we used in vivo electrophysiology in the VTA to characterise acute and chronic interactions between nicotine and alcohol. Simultaneous injections of the two drugs enhanced their responses on VTA DA neuron firing and chronic exposure to nicotine increased alcohol-induced DA responses and alcohol intake. Then, we assessed the role of β4 * nAChRs, but not β2 * nAChRs, in mediating acute responses to alcohol using nAChR subtypes knockout mice (β2−/− and β4−/− mice). Finally, we showed that nicotine-induced modifications of alcohol responses were absent in β2−/− and β4−/− mice, suggesting that nicotine triggers β2* and β4 * nAChR-dependent neuroadaptations that subsequently modify the responses to alcohol and thus indicating these receptors as key mediators in the complex interactions between these two drugs.


Drugs
For electrophysiological recordings, alcohol was prepared as a 20% (v/v) solution in a sterile 0.9% NaCl solution from Ethanol 96% (EMPROVE® exp Ph Eur, BP, Merck Millipore) and injected in a volume proportional to the dose (20 to 120µl). Injected doses were: 125mg/kg, 250mg/kg, 500mg/kg and 750mg/kg. Nicotine hydrogen tartrate salt (Sigma-Aldrich) was freshly dissolved in sterile 0.9% NaCl solution and pH adjusted to 7.4 with NaOH and injected at the dose of 30 µg/kg (free base). Before drugs administration, control saline injections were performed in different final volumes (10, 40 and 120 µl). As no statistical difference was found between saline injections at different volumes solution (t.test: 10 vs 40: t=-0.2, df=22.5, p=0.8; 10 vs 120: t=0.03, df=13.8, p=0.9; 40 vs 120: t=0.2, df=13.5, p=0.8) (example for WT mice is shown in Fig. S5c), data were pooled and used as unique control injection. For pharmacological neuron identification, quinpirole hydrochloride and eticlopride hydrochloride (Tocris Bioscience), respectively the agonist and antagonist of dopaminergic D2 receptors, were dissolved in sterile 0.9% NaCl and intravenously injected at the dose of 1mg/kg in a final volume of 10µl.
In osmotic mini-pumps, nicotine was infused at the dose of 10mg/kg/d (free base). For intake experiment, alcohol-drinking solutions were presented as 3, 6, 10 and 15% (v/v) solutions in mineral water.

In vivo electrophysiology
8-16 weeks old male (25 to 30 g) C57Bl/6J wild-type (WT), β2-/-and β4-/-mice were deeply anaesthetized with chloral hydrate (8%), 400 mg/kg i.p., supplemented as required to maintain optimal anesthesia throughout the experiment. The scalp was opened and a whole was drilled in the skull above the location of the VTA. The saphenous vein was catheterized for intravenous administration of drugs. Extracellular recording electrodes were constructed from 1.5 mm O.D. / 1.17 mm I.D. borosilicate glass tubing (Harvard Apparatus) using a vertical electrode puller (Narishige). Under microscopic control, the tip was broken to obtain a diameter of approximately 1 µm. The electrodes were filled with a 0.5% NaCl solution containing 1.5% of neurobiotin tracer (AbCys) yielding impedances of 6-9 MΩ. Electrical signals were amplified by a high-impedance amplifier (Axon Instruments) and monitored audibly through an audio monitor (A.M. Systems Inc.). The signal was digitized, sampled at 25 kHz and recorded on a computer using Spike2 software (Cambridge Electronic Design) for later analysis. The electrophysiological activity was sampled in the central region of the VTA (coordinates: between 3.1 to 4 mm posterior to Bregma, 0.3 to 0.7 mm lateral to midline, and 4 to 4.8 mm below brain surface). Individual electrode tracks were separated from one another by at least 0.1 mm in the horizontal plane. Spontaneously active DAergic neurons were identified on the basis of previously established electrophysiological criteria (see main text, methods section). After a baseline recording of at least 5 minutes, a saline solution (0.9% sodium chloride) was injected into the saphenous vein, and after another 5 minutes, injection of alcohol and/or nicotine hydrogen tartrate were administered via the same route.
Successive injections were performed after the neuron returned to its baseline, or when the firing activity returned stable for at least 3 minutes. For combined alcohol + nicotine injections, the two saphenous veins were catheterized and the two drugs concomitantly injected in the two veins. D2 receptors pharmacological identification was performed on the last neuron of the day ( Supplementary Fig. S5b). Quinpirole hydrochloride in saline solution (1mg/kg) was injected intravenously, followed 5 minutes later by eticlopride hydrochloride (1mg/kg). Once D2-R pharmacology was applied, no further neurons were recorded and the animal was discarded.

Immunocytochemical identification of recorded neurons
When possible, neurons were electroporated and neurobiotin was expulsed from the electrode using positive current pulses as already described (Eddine et al, 2015). The mouse was then killed and the brain post-fixed in 4% paraformaldehyde. 60µm slices were cut on a vibratome.
Fluorescence immunohistochemistry was performed as follows: free-floating VTA brain sections were incubated 1 hour at 4°C in a blocking solution of PBS containing 3% BSA (Bovine serum albumine) and 0.2% Triton X-100 and then overnight at 4°C in PBS containing primary antibodies at appropriate dilution, 1,5% BSA and 0.2% Triton X-100. The next day sections were rinsed with PBS and then incubated 3 hours at room temperature with secondary antibodies in a solution of 1,5% BSA and 0.2% Triton X-100 in PBS. After three rinses in PBS, slices were wet-mounted using Prolong Gold Antifade Reagent (Invitrogen).

Two bottle choice procedure
Mice were housed individually in standard cages equipped with two bottles, containing either alcohol or mineral water, to which they had continuous free access. Each bottle was attached to a precision weighing sensor mounted to the cage lid and interfaced to a computer that automatically measured the amount of liquid consumed over time (TSE systems, Germany).
Quantity of alcohol or water ingested was recorded every minute. Food (standard pellets for rodents) was available ad libitum. Mice were subjected to a 4-days habituation period in which only water was presented in the two bottles. From the 5th day, mice were exposed to progressively increasing concentrations of ethanol (within 18 days) under the free-choice procedure adapted from Kelaï et al. (2008). Mice were offered 3% ethanol (v/v) versus water for 4 days, then 6% ethanol for the next 4 days, then 10% ethanol for the next 5 days and finally animals had access to 15% ethanol for the last 5 days. Mice were weighed every 4 days and bottles positions changed every two or three days to control for position preference.

Surgical implantation of mini-pumps
Mice were slightly anesthetized with a ketamine (1.5%) and xylazine (0.05%) combination in PBS. Alzet® osmotic mini-pumps (mod. 2004; release rate: 0,25µL/h; duration 28±2 days) were implanted subcutaneously (s.c.) between the two scapulae, parallel to the spine. They delivered nicotine (10mg/kg/d) or vehicle (saline solution). Electrophysiological experiments were carried out between the 22nd and the 26th day after the implantation, thus before the end of the delivery duration to avoid the appearance of withdrawal or abstinence.