Phytolacca americana

The task was based on delayed reinforcement choice tasks that have been described before [73 (link),74 (link)]. The session began in darkness with the levers retracted; this was designated the intertrial state. Trials began at 40-s intervals; the format of a single trial is shown in Figure 2. Each trial began with the illumination of the houselight and the traylight. The rat was required to make a nosepoke response, ensuring that it was centrally located at the start of the trial (latency to poke was designated the initiation latency). If the rat did not respond within 10 s of the start of the trial, the operant chamber was reset to the intertrial state until the next trial began and the trial was scored as an omission. If the rat was already nosepoking when the trial began, the next stage followed immediately. Upon a successful nosepoke, the traylight was extinguished and one or both levers were extended. One lever was designated the Large/Uncertain lever, the other the Small/Certain lever (counterbalanced left/right). The latency to choose a lever was recorded. (If the rat did not respond within 10 s of lever presentation, the chamber was reset to the intertrial state until the next trial and the trial was scored as an omission.) When a lever was chosen, both levers were retracted and the houselight was switched off. Choice of the Small lever caused the certain delivery of one pellet; choice of the Large lever caused the delivery of 4 pellets with a particular probability (see below). When reinforcement was delivered, the traylight was switched on. Multiple pellets were delivered 0.5 s apart. If the rat collected the pellets before the next trial began, then the traylight was switched off and the time from delivery of the first pellet until a nosepoke occurred was recorded as the collection latency. If the rat did not collect the food within 10 s of its delivery, the operant chamber entered the intertrial state, though collection latencies were still recorded up to the start of the next trial. The chamber was then in the intertrial state and remained so until the next trial. There was no mechanism to remove uneaten pellets, but failure to collect the reward was an extremely rare event. The large-reinforcer probability was varied systematically across the session as follows. A session consisted of 5 blocks, each comprising 16 trials in which only one lever was presented (8 trials for each lever, randomized in pairs) followed by 10 free-choice trials. The probability that the large reinforcer was delivered, given that the Large lever had been chosen (p_reinforcer), varied across blocks: it was initially 1, 0.5, 0.25, 0.125, and 0.0625, respectively, for each block. As trials began every 40 s and there were 130 trials per session, the total session length was ~87 minutes; subjects received one session per day. Choice ratios (percentage choice of the large reinforcer, for each trial block) were calculated using only choice trials on which the subject responded.

Some rats were also subjected to a 40-min test for conditioned reinforcement. The data from these tests have been reported previously [9] (link), [25] (link), but are reanalyzed here. On the day after Pavlovian training, as described above, rats were placed into the conditioning chambers, but these were reconfigured. The food magazine was removed and the lever was now positioned in its place, in the center of the wall. Two nose-poke ports (2 cm diameter; 2 cm above the floor), equipped with photocells, were added, one on each side of the lever. One port was designated “Active” and the other “Inactive”. When a rat made a nose poke into the Active port the lever was extended into the chamber (and illuminated) for 2–4 s. Nose pokes into the Inactive port had no consequence This test occurred under extinction conditions, in that no food pellets were delivered. The number of responses into the ‘Active’ and ‘Inactive’ ports were recorded.

Mice were trained to self-administer a 20% alcohol solution in operant-conditioning chambers (ENV-307A, Med Associates Inc., St Albans, VT, USA) prior to starting alcohol exposure. Briefly, the chambers contained two nose-poke holes, located on one wall of the chamber 3 cm above a grid floor, fitted with a photocell and a yellow cue light, and a steel dish into which reinforcers were delivered from a syringe pump (Thomsen and Caine, 2005 (link); Bornebusch et al., 2019 (link)). All chambers were individually enclosed in sound-attenuating boxes equipped with a light and a ventilation fan. A nose-poke in the active hole triggered a syringe pump to deliver 30 μL of the reinforcer and the cue light to turn on. Nose-pokes in the inactive hole were recorded but had no scheduled consequences. Training sessions consisted of different solutions, starting with Nutridrink Compact Protein Vanilla (Nutricia, Amsterdam, Netherlands), then Calogen unflavored fat emulsion (Nutricia, Amsterdam, Netherlands), Calogen with 10% alcohol, Calogen with 20% alcohol and eventually water with 20% alcohol.
Training sessions used a fixed ratio (FR) 1 schedule of reinforcement, with a post-reinforcer timeout of 20 s, running for 1 h daily until criteria were met, i.e., a minimum of 20 reinforcers were earned within one session. Experimental sessions consisted of a progressive ratio (PR) schedule of reinforcement running for a maximum of 6 h per day. PR sessions terminated when no reinforcers were administered for 1 h (i.e., 1 h limited hold), at which time the number of reinforcers earned was recorded as the breaking point and the session ended. PR sessions consisted of a PR1 training period (response requirement 1, 2, 3 etc.), then, an exponential progression starting at 3 responses and increasing by a factor of 0.115 log units (response requirement 3, 4, 6, 7, 10, 13, 16, 21, 28, 36 etc.) which was used as the experimental schedule of reinforcement. The reinforcer-delivery cup was examined at the end of the sessions for any solution left unconsumed; in all the PR experimental sessions, all solution was consumed.

Risk attitude was tested in female and male rats using a probabilistic decision-making task. After magazine training was completed, rats were trained on an operant fixed ratio (FR) schedule to a criterion of >23 presses out of 20 total trials where one pellet was delivered following the depression of the left or right lever. Rats then underwent auto-shaping, where the rats were required to first nose-poke the magazine tray to begin the trail where the intertrial interval was increased from 0 to 15 s, the time to perform the trail initiating the poke was decreased to 10 s, and the intertrial interval was increased to 30 s.
Detailed methods for these and the following tasks can be found in previous publications (Nasrallah et al., 2009 (link), 2011 (link); Clark et al., 2012 (link); Schindler et al., 2014 (link)). During the task, rats were presented with two levers flanking the magazine tray where one lever represented the certain lever (low-risk) and the other the uncertain lever (high-risk). The low-risk lever was associated with a certain (1.00) delivery of two sucrose pellets and the uncertain lever was associated with the probabilistic (1.00, 0.75, 0.50, 0.25, and 0.00) delivery of four sucrose pellets. Each session consisted of 24 forced trials followed by 24 free choice trials where each probability presented on a different day decreased in descending order with an intertrial interval of 45 s. During the forced choice trials and following the trial initiation, a single lever would extend and the pressing of that lever resulted in the illumination of the tray light signaling the delivery of the associated reward based on the certainty of that lever and probability of that day; whereas following trial initiation during the free choice trials, both levers were extended with a total of 10 s for that rat to choose between the two levers.
After the probabilistic decision-making was completed, female control and ethanol rats underwent anesthetized surgeries with fast-scan cyclic voltammetry to measure pedunculopontine tegmental nucleus (PPT) and medial forebrain bundle (MFB) stimulated dopamine release in the nucleus accumbens (NAcc) as follows.

For a recorded afferent fiber, its mechanosensitive receptive field in the hindpaw glabrous skin was first searched using a glass rod. Poking with the glass rod at the mechanosensitive receptive field of the recorded afferent fiber would result in the detection of APs by the recording electrode. In the present study, all data were collected from mechanosensitive receptive sites, i.e., mechanoreceptors in the hindpaw glabrous skin. Once a mechanoreceptor was identified, mechanical stimulation was applied to the same receptive field with a force-calibrated mechanical indenter (300C-I, Aurora Scientific Inc., Ontario, Canada) to determine mechanical thresholds. The tip size of the indenter was 0.8 mm in diameter. The indenter was connected to a Digidata 1550B Digitizer to allow generating ramp-and-hold mechanical stimulation using the pClamp 11 software. Prior to the application of mechanical stimulation, the tip of the indenter was lowered to the surface of the receptive field with a 10-mN force and then the 10-mN force was canceled to 0 so that the tip of the indenter was just in contact with the receptive field surface. Under the force control module, ramp-and-hold mechanical stimuli were applied to the mechanoreceptor of the glabrous skin. The step force commanders were calibrated by applying indenter at finger tips, paw pads and other areas of plantar skin, and the actual forces after the calibration were used in experiments. The ramp-and-hold force steps were at 0, 5, 30, and 80 mN. The duration of the ramp (dynamic phase) was 10 ms, and the duration of the holding (static phase) was 0.98 s. The minimal force at which AP impulses was elicited was defined as intender mechanical threshold of the mechanoreceptors. In a different set of experiments mechanical stimulation was applied using von Frey filaments to vertically poke the glabrous skin. The von Frey mechanical thresholds were determined by mechanical stimulation with von Frey filaments (0.08 ~ 6 g) onto mechanoreceptors.
To determine whether a mechanoreceptor was from Nav1.8^ChR2-positive or Nav1.8^ChR2-negative afferent fibers, the same mechanosensitive receptive field was stimulated by a blue LED light (Thorlab; M455L4, 455 nm) to test light sensitivity. A mechanoreceptor was from Nav1.8^ChR2-positive afferent fibers if light stimulation evoked impulses. Otherwise, the mechanoreceptor was from light-insensitive or Nav1.8^ChR2-negative afferent fibers. The blue Light was applied through a 40 × objective to a mechanoreceptor with a 1-s light stimulation pulse at the intensity of 50 mW. Afferent impulses evoked by mechanical and light stimulation were recorded using the pressure-clamped single-fiber recordings, and signals were amplified by the Multiclamp 700B amplifier and sampled at 25 kHz with band path filter between 0.1 Hz and 3 kHz on AC recording mode.