Chronic voluntary alcohol consumption causes persistent cognitive deficits and cortical cell loss in a rodent model methods
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Adult male Long Evans rats were obtained from a commercial supplier (Animal Resources Centre, Perth, Australia). All procedures were approved by the Florey Animal Ethics Committee (project number 15-075) and followed the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and are reported in compliance with the ARRIVE guidelines. Rats were introduced to the facility at 8 weeks of age and were acclimatized to their living conditions for 2 weeks prior to commencement of scheduled alcohol access. All rats were maintained on a reverse 12/12 light-dark cycle (lights off at 0700 h) within a specific pathogen free environment. They were housed in open-top cages with aspen bedding, equipped with tunnels and nesting material. During acclimatization, and for the first 20 weeks of alcohol access all rats were pair-housed. Following this, r...
Schedule of alcohol access
Experimental timeline is shown in Fig.. Following acclimatization, rats were kept in the facility for 26 weeks, during which time they had access to either alcohol (20% v/v in tap water) or a calorie-matched solution of maltodextrin (Bulk Nutrients, Tasmania, Australia) under two bottle choice conditions in their home cage (one bottle water, one bottle alcohol/maltodextrin solution). Maltodextrin was also dissolved in tap water, at a concentration that was calculated weekly based on the mean volume of alcohol consumed by the Alcohol group relative to the mean volume of alcohol consumed by the calorie-matched group (group Maltodextrin). This ensured that liquid caloric intake remained equivalent between the two groups. Figure 1 Basic timeline for all experiments. ( A ) Touchscreen chamber with discrimination and reversal task ( B ) and 5-choice serial reaction time task ( C ).
Schedule of alcohol access
Access to alcohol/maltodextrin was on an intermittent schedule such that it was available for 3 × 24-hour periods per week. This has been shown to produce high drinking rates in Long Evans rats. However, in Experiment 2 the rats' initial drinking was initially lower than 4 g/kg/session. To overcome this problem, the alcohol was temporarily sweetened for 6 weeks from week 12 (1 week at 0.5% maltodextrin, 1 week at 1% maltodextrin, 4 weeks at 5% maltodextrin). The concentration of maltodextrin solution for the Maltodextrin control group were adjusted accordingly. Food and water were available ad libitum throughout the experiment, except during behavioral testing when rats were restricted to 85% of free-feeding body weight.
Experiment 1: Pairwise Discrimination and reversal
Following withdrawal from alcohol (2 days), all rats (Maltodextrin n = 8, Alcohol n = 12 per group) were placed on food restrictions to reduce body weight to 85% of free-feeding weight. During this time, they were handled at least 3 times, and were familiarized with the sugar pellet reward used for cognitive training (approximately 15 pellets were made available daily in their home cage). 7 Days following the last day of alcohol access, behavioral testing began. Training sessions took place 6 days a week (Monday-Saturday).
Experiment 1: Pairwise Discrimination and reversal
Rats were first subjected to a series of pretraining sessions. In the initial stage they were habituated to the chambers with 30-minute sessions in which 10 pellets were placed in the magazine. Once all 10 pellets were consumed within 30 minutes (1-2 sessions), rats proceeded to initial touch training, where rats learned to associate appearance of a stimulus with reward delivery. In each trial, an image (randomly selected from the catalogue) appeared in one of two windows on the touchscreen. Offset of the image coincided with a 2 second tone (3KHz), illumination of magazine light and delivery of 1 sucrose pellet. If the rat touched the image, 3 pellets rather than 1 were dispensed. The next trial was initiated when the rat collected the pellet from the magazine. Once the rat was able to earn 60 pellets within 60 minutes, they moved onto instrumental training. H...
Experiment 1: Pairwise Discrimination and reversal
In the pairwise discrimination task, 2 novel images were presented in each trial - "fan" and "marble" (Fig. ). These same images were used throughout discrimination and reversal, in a counterbalanced manner to serve as conditioned stimulus (CS)+ and CS-. Both images appeared on the screen simultaneously, and responses to the correct image (CS+) triggered delivery of a sugar pellet, together with illumination of the magazine light and presentation of tone cue. Responses to the incorrect image (CS-) resulted in a timeout period followed by correction trials (identical in configuration to the trial where incorrect response was made) until the rat made a correct response. The criterion for achieving this task was attaining 60 trials in 60 minutes with ≥80% correct, 2 days in a row.
Experiment 1: Pairwise Discrimination and reversal
Once the pairwise discrimination task was acquired, rats passed immediately into the reversal task. Here images were reversed, so previous CS+ became CS- and vice versa. The reversal task was otherwise exactly as the pairwise discrimination task and continued until all rats had achieved criterion of 60 trials/session with ≥80% correct, 2 days in a row. Data were further split into early and late reversal sessions, since for the initial sessions rats would continue to respond in accordance with rules learned previously. The split was defined as the point where performance first reached 50% correct. Responding during these distinct phases is differentially dependent on dopamine release in the striatum. Previous work has defined early reversal as the first session in which contingencies are reversed, mid reversal as the first trial after subjects first attain a criterion o...
Experiment 2: 5-choice serial reaction time task
In the 5-choice (5-CSRTT) task itself the stimulus (white square light) was only presented for a limited time (stimulus duration), and responses to the cued window had to take place within a set time frame (limited hold time). There were four possible outcomes for each trial. A correct trial occurred when the rat responded in time to the window in which the stimulus appeared. An incorrect trial occurred when the rat responded in time, but to one of the other 4 windows. An omission trial was recorded if the rat failed to touch the screen at all within the limited hold time. Finally, a premature trial occurred where rats touched the screen before the onset of the stimulus. Premature trials were punished as for incorrect and omission trials however they did not count towards the total trials for the session. Duration and hold times were gradually reduced as the task progressed, according...
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Adult male Long Evans rats were obtained from a commercial supplier (Animal Resources Centre, Perth, Australia). All procedures were approved by the Florey Animal Ethics Committee (project number 15-075) and followed the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and are reported in compliance with the ARRIVE guidelines. Rats were introduced to the facility at 8 weeks of age and were acclimatized to their living conditions for 2 weeks prior to commencement of scheduled alcohol access. All rats were maintained on a reverse 12/12 light-dark cycle (lights off at 0700 h) within a specific pathogen free environment. They were housed in open-top cages with aspen bedding, equipped with tunnels and nesting material. During acclimatization, and for the first 20 weeks of alcohol access all rats were pair-housed. Following this, rats were single-housed for the remainder of the study. All behavioral tasks were carried out in abstinence. Prior to commencement of behavioral tasks rats were placed on food restrictions (body weight maintained at 85% of free feeding weight) and acclimatized to handling.
Experimental timeline is shown in Fig.. Following acclimatization, rats were kept in the facility for 26 weeks, during which time they had access to either alcohol (20% v/v in tap water) or a calorie-matched solution of maltodextrin (Bulk Nutrients, Tasmania, Australia) under two bottle choice conditions in their home cage (one bottle water, one bottle alcohol/maltodextrin solution). Maltodextrin was also dissolved in tap water, at a concentration that was calculated weekly based on the mean volume of alcohol consumed by the Alcohol group relative to the mean volume of alcohol consumed by the calorie-matched group (group Maltodextrin). This ensured that liquid caloric intake remained equivalent between the two groups. Figure 1 Basic timeline for all experiments. ( A ) Touchscreen chamber with discrimination and reversal task ( B ) and 5-choice serial reaction time task ( C ).
Access to alcohol/maltodextrin was on an intermittent schedule such that it was available for 3 × 24-hour periods per week. This has been shown to produce high drinking rates in Long Evans rats. However, in Experiment 2 the rats' initial drinking was initially lower than 4 g/kg/session. To overcome this problem, the alcohol was temporarily sweetened for 6 weeks from week 12 (1 week at 0.5% maltodextrin, 1 week at 1% maltodextrin, 4 weeks at 5% maltodextrin). The concentration of maltodextrin solution for the Maltodextrin control group were adjusted accordingly. Food and water were available ad libitum throughout the experiment, except during behavioral testing when rats were restricted to 85% of free-feeding body weight.
Following withdrawal from alcohol (2 days), all rats (Maltodextrin n = 8, Alcohol n = 12 per group) were placed on food restrictions to reduce body weight to 85% of free-feeding weight. During this time, they were handled at least 3 times, and were familiarized with the sugar pellet reward used for cognitive training (approximately 15 pellets were made available daily in their home cage). 7 Days following the last day of alcohol access, behavioral testing began. Training sessions took place 6 days a week (Monday-Saturday).
Rats were first subjected to a series of pretraining sessions. In the initial stage they were habituated to the chambers with 30-minute sessions in which 10 pellets were placed in the magazine. Once all 10 pellets were consumed within 30 minutes (1-2 sessions), rats proceeded to initial touch training, where rats learned to associate appearance of a stimulus with reward delivery. In each trial, an image (randomly selected from the catalogue) appeared in one of two windows on the touchscreen. Offset of the image coincided with a 2 second tone (3KHz), illumination of magazine light and delivery of 1 sucrose pellet. If the rat touched the image, 3 pellets rather than 1 were dispensed. The next trial was initiated when the rat collected the pellet from the magazine. Once the rat was able to earn 60 pellets within 60 minutes, they moved onto instrumental training. Here, offset of the image, and delivery of cues and reward was only initiated when the animal touched the screen. Criterion again was 60 trials in 60 minutes, after which rats passed into punish incorrect training where touches to the blank window during stimulus presentation initiated a 5̴...
In the pairwise discrimination task, 2 novel images were presented in each trial - "fan" and "marble" (Fig. ). These same images were used throughout discrimination and reversal, in a counterbalanced manner to serve as conditioned stimulus (CS)+ and CS-. Both images appeared on the screen simultaneously, and responses to the correct image (CS+) triggered delivery of a sugar pellet, together with illumination of the magazine light and presentation of tone cue. Responses to the incorrect image (CS-) resulted in a timeout period followed by correction trials (identical in configuration to the trial where incorrect response was made) until the rat made a correct response. The criterion for achieving this task was attaining 60 trials in 60 minutes with ≥80% correct, 2 days in a row.
Once the pairwise discrimination task was acquired, rats passed immediately into the reversal task. Here images were reversed, so previous CS+ became CS- and vice versa. The reversal task was otherwise exactly as the pairwise discrimination task and continued until all rats had achieved criterion of 60 trials/session with ≥80% correct, 2 days in a row. Data were further split into early and late reversal sessions, since for the initial sessions rats would continue to respond in accordance with rules learned previously. The split was defined as the point where performance first reached 50% correct. Responding during these distinct phases is differentially dependent on dopamine release in the striatum. Previous work has defined early reversal as the first session in which contingencies are reversed, mid reversal as the first trial after subjects first attain a criterion of 50% trial correct in a session, and late reversal as the first session after the session in which they attain ≥80% correct for the first time,. We examined latency to reach each stage and responding in the following session. Dependent measures included days to criterion, total trials and c...
In the 5-choice (5-CSRTT) task itself the stimulus (white square light) was only presented for a limited time (stimulus duration), and responses to the cued window had to take place within a set time frame (limited hold time). There were four possible outcomes for each trial. A correct trial occurred when the rat responded in time to the window in which the stimulus appeared. An incorrect trial occurred when the rat responded in time, but to one of the other 4 windows. An omission trial was recorded if the rat failed to touch the screen at all within the limited hold time. Finally, a premature trial occurred where rats touched the screen before the onset of the stimulus. Premature trials were punished as for incorrect and omission trials however they did not count towards the total trials for the session. Duration and hold times were gradually reduced as the task progressed, according to the parameters shown in Table. In order to pass from stage to stage, rats had to achieve 60 trials in 60 minutes, at ≥80% correct. Once they had passed stage 5, they were held with food restrictions but not tested while the other rats completed training. They then received 2 s...
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