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{"project":"2_test","denotations":[{"id":"15061865-9247267-12857767","span":{"begin":506,"end":508},"obj":"9247267"},{"id":"15061865-11323267-12857768","span":{"begin":1090,"end":1092},"obj":"11323267"},{"id":"15061865-11323267-12857769","span":{"begin":4252,"end":4254},"obj":"11323267"}],"text":"Methods\n\nSubjects\nThe sixteen, 8–10 week old, 25–30 g mice (8 per genotype) used in this study were the congenic offspring of breeders that were descendants of the original F2 hybrid (129/Sv × C57BL/6J; Kelly et al., 1997) that was backcrossed to inbred C57Bl/6J stock (Jackson Laboratories, ME). This breeding strategy was repeated for 20 successive generations with the gender of the donor/mutant alternating with each generation. All experimental subjects were genotyped by PCR as previously described [23]. All testing occured randomly across estrous cycles. The Department of Comparative Medicine at Oregon Health and Science University approved all protocols, and all animals were maintained in accordance to National Institutes of Health's Guide for the Care and Use of Laboratory Animals.\n\nExperimental procedures\n\nOdor discrimination training and testing\nA 2-odor discrimination paradigm was chosen because it permits a measure of attentional focus in goal-directed behavior. Odor discrimination was chosen as the particular task because it is a highly robust behavior in rodents [18]. To minimize experimental error, the experimenter was blind to genotype during testing. Mice were mildly food deprived to approximately 85% ad libitum weight. Dopamine D2R-/- and D2R+/+ mice (n = 8 per group) were initially trained to ambulate the length of a polyvinyl mouse cage (30-cm) to retrieve ~30-mg of Pico® fat supplemented rodent food located in a small plastic cup (3-cm) attached to the floor of the testing apparatus with Velcro. Each subject received five trials, and the latency to locate and start to consume the food pellet was recorded manually with a standard hand-held stopwatch. Twenty-four hours later subjects were trained to ambulate towards and dig through the same 3-cm dish filled with sterile sand to locate a food pellet buried underneath the sand. Subjects received 10 trials. If a mouse failed to locate a food pellet within 10-min of the first trial, an additional food pellet was dropped on top of the dish. This manipulation usually ensured that a subject would dig through the sand to locate the original pellet and learn this task.\nThe next day, mice were presented to the testing apparatus that now contained 2 dishes, each filled with different scented sand. One odor signaled that a food reinforcer was buried in that particular dish (S+) while the other odor signaled that no reinforcer was contained within that particular dish (S-). The sand was scented by adding 0.80 grams of either cinnamon or dill weed to 98.8 grams of autoclaved playground sand purchased from a local farm and garden store. Finally, 0.40 grams of finely ground food was mixed with the scented sand in order to mask any odor emitted by the food reinforcer in the S+ dish. This final mixture did not appear to be palatable to the subjects. A trial terminated when the mouse: dug in the correct dish and consumed the hidden pellet, or began to dig in the incorrect dish. Digging was considered the actual physical displacement of sand with the forepaws and/or snout. Placing forelimbs on either of the dishes and sniffing was not considered digging and was not penalized or scored. Each completed trial was separated by a 20-sec inter-trial-interval (ITI). Preliminary experiments demonstrated that an extended time-out period following an incorrect response was not required to ensure goal-directed behavior in the wild-type or mutant mice for this particular task (data not shown). Therefore, the 20-sec ITI was used to separate all trials (successful and unsuccessful). The S+ and S- scents were counterbalanced across subjects in order to control for potential scent bias (pilot data suggest there are no biases, data not shown). Following accurate discrimination, defined as 8 correct retrievals across 10 consecutive trials, reversal trials began.\n\nReversal learning measurement\nWe next investigated the contribution of dopamine D2Rs to reversal learning behavior by inverting the reinforcement contingencies – now the former S+ signaled no reinforcer and the previous S- now indicated reinforcement availability. This manipulation permits an evaluation of the ability to inhibit responses to previously reinforced discriminative stimuli [18]. The number of trials necessary to establish 8 out of 10 accurate responses was our definition of successful reversal learning.\n\nData analysis\nThe latency to retrieve a pellet from an empty dish, the latency to dig through a dish filled with unscented sand, and the number of errors of commission committed across the reversal sessions were analyzed with 2-way analyses of variance (genotype × trials). The number of trials necessary to attain criterion performance in odor discrimination (8 correct responses across 10 consecutive trials) and errors committed while learning the discrimination tasks were separated as individual testing phases (e.g. odor discrimination and reversal learning) and analyzed separately with unpaired 2-tailed t-tests. Significance was established at p \u003c 0.05. Errors committed during reversal trials were further categorically separated as errors of commission (failing to withhold responding to S-) and errors of omission (failing to dig in either dish after 3-min had expired). Categorical errors and number of commission errors committed during the first reversal learning session were analyzed between groups with nonparametric Mann-Whitney U tests."}

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PMC:400732 / 22594-28035 JSON TXT