Male mice of the G42 line (CB6-Tg(Gad1-EGFP)G42Zjh/J; Jackson Laboratories, Bar Harbor, ME, USA RRID:IMSR_JAX:007677) were used for all experiments. Littermates were assigned to one of four groups: Saline-control (SAL-SAL), ketamine followed by saline (KET-SAL), ketamine followed by NAC (KET-NAC), or saline followed by NAC (SAL-NAC). Starting on PND 5, all animals received subcutaneous injections of either saline or NAC (0.1% in saline; 10 μl/g bodyweight) until they were weaned at PND 21. In addition, on PND 7, 9 and 11 all animals received subcutaneous injections of either saline, or a subanesthetic dose of ketamine (30 mg/kg; Ketathesia HCl, Henry Schein) to induce NMDAR dysfunction. After weaning, animals in the NAC groups continued to receive NAC (0.1%) in their drinking water until the end of the experiments. All experiments were performed using adult animals (PND 70–120). Figure 1 shows a graphical summary of the experimental time course. We used different cohorts of animals for the different behavioral tasks: one cohort of animals was used for the spontaneous alternation task, novel object recognition, and the social interaction task. For these experiments the number of animals in each treatment group ranged between 8–10 mice. Three other cohorts of animals were tested separately either on the Response Discrimination and Shift-to-Visual-Cue Discrimination (group sizes of 8–10 animals), for their locomotor response following an amphetamine challenge in an open field (group sizes of 13–17 animals), or for PPI (group sizes of 11–14), respectively. Prior to all behavioral tasks animals were handled for 2 weeks in their home cages for 5 min each day. In addition, 3 days before the experiments animals were also handled in the room where the experiments were performed. On the day of testing, animals were transferred to the behavioral room at least 30 min before testing began. Experimenters were blind to the treatment of experimental animals throughout testing and analysis. All procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas at Dallas.

Procedures followed those previously described (Jeevakumar et al., 2015), which are modified from Floresco et al. (2006). Mice were gradually food restricted to 85% of their free-feeding weight over 2 weeks. The task was performed over the course of 6 days. In brief, mice were habituated to a plus maze over 3 days and learned to explore all four arms to retrieve food rewards. On day 4, the maze was converted to a T-maze and the propensity of mice to turn left or right (turn bias) was assessed. On day 5, the mice were trained on a response discrimination that required them to always turn to one side (against their turn bias). Finally, on day 6 mice had to shift strategies and turn towards a previously ignored visual cue to obtain the reward. Figure 2A illustrates the experimental setup on the different days of training.

Testing took place in white wooden plus maze (each arm is 10 × 34 × 15 cm, with a 10 × 10 cm center area) under low ambient illumination. The arms were labeled East, West, South and North for reference. On days 4–6, the maze was converted into a T-maze by blocking off one of the arms with a divider, and additionally a visual cue (vertical black and white stripes on a 13 × 10 cm plastic sheet) was placed alternately near the entrance of one of the two choice arms in a pseudorandom manner (see below). During all days, reward pellets (Cheerio bits) were placed around the outside of the maze in order to prevent animals from using olfactory cues to infer the location of the reward.

On the first day of habituation, four reward pellets (1/8th Cheerios bits) were placed in each of the arms of the plus maze. Animals were placed into the center of the maze and were allowed to freely explore the maze for 15 min. If a mouse consumed all 16 pellets before the end of the 15 min habituation period, it was briefly placed in a holding cage while the maze was rebaited, and then the mouse was placed back into the maze until the end of the 15 min period. On the second day of habituation, arms were baited with two pellets each, and on the third day of habituation only one food pellet was placed at the end of each arm. To reach habituation criterion, animals were required to consume all four food pellets at least four times within the 15 min period. All animals in this study reached this criterion on the third habituation day.

On the following day, the animals’ turn bias was determined. Therefore, the plus maze was converted into a T-maze by blocking off one of the arms. Mice were placed in the stem arm and allowed to turn left or right to obtain a food pellet. After the mouse consumed the reward, it was returned to the stem arm and allowed to make another choice. If the mouse chose the same arm as on the initial choice, it was returned to the stem arm until it chose the other arm and consumed the food pellet. The direction of the initial turn chosen four or more times over seven trials was considered the turn bias.

On the next day, mice were trained on a response discrimination task that required them to always turn into only one arm (left or right, chosen opposite to the direction of their turn bias) to obtain the food reward. The location of the stem arm was pseudorandomly rotated among three arms (East, West and South) to prevent mice from using an allocentric spatial strategy to locate the food. A visual cue was placed close to the entrance of one of the choice arms. Placement of this cue into the right or left arm varied pseudorandomly to balance the frequency of occurrences in each arm across blocks of 12 consecutive trials. Similarly, the order of the stem arms alternated pseudorandomly in a balanced fashion across blocks of 12 trials. Training continued until the mouse made nine correct choices over 10 consecutive trials. When animals achieved this acquisition criterion, a probe trial was administered. In the probe trial the previously unused fourth arm (North) was used as a stem arm. If the mice performed the probe trial correctly, Response Discrimination training was completed. If an incorrect turn occurred, response training continued until the mouse made another five consecutive correct choices, and then another probe trial was administered.

On the final day, mice were trained to shift their strategy to now follow the visual cue in order to obtain food rewards. The location of the visual cue and the position of the start arm were again varied pseudorandomly so that their frequency was balanced across blocks of 12 consecutive trials. The training and response criteria for the Shift-to-Visual-Cue Discrimination were identical to those during Response Discrimination.

For each of the two test days, we analyzed the total number of trials to criterion and the number of probe trials required to reach criterion. For the Shift-to-Visual-Cue Discrimination, errors were scored as entries into arms that did not contain the visual cue, and they were further broken down into three subcategories to determine whether the animals’ treatment altered the ability to either shift from the previously learned strategy (perseverative errors), or to maintain the new strategy after perseveration had ceased (regressive errors, or never-reinforced errors). In order to detect shifts in the strategies animals used, trials were separated into consecutive blocks of four trials each. A perseverative error occurred when a mouse made the same egocentric response as required during the Response Discrimination, but which was opposite to the direction of the arm containing the visual cue. Six of every 12 consecutive trials required the mouse to respond in this manner. A perseverative error was scored when the mouse entered the incorrect arm on three or more trials per block of 4 trials. Once the mouse made less than three perseverative errors in a block, all subsequent errors of the same type were now scored as regressive errors (because at this point the mouse was following an alternative strategy at least half of the time). So-called never-reinforced errors were scored when a mouse entered the incorrect arm on trials where the visual cue was placed on the same side that the mouse had been trained to enter on the previous day.

Mice were placed into the stem arm of a T-maze and a central partition wedge was placed in between the horizontal goal arms. Mice were left to choose between the left and the right open goal arm. Once a mouse had entered an arm it was confined to this arm for 30 s by blocking off the entrance of the arm. After 30 s, the barrier was removed and the animal was placed back into the stem arm and allowed to once more choose between the left and right arm. Each animal completed a total of six trials with a 20 min interval between trials. The maze was cleaned with 70% ethanol after each trial. Each trial was timed and the direction of the turns was recorded to calculate a percentage of spontaneous alternations between left and right arm entries.

Mice were first habituated for 2 days to an empty open chamber (39 × 19 × 30.5 cm) in 10 min sessions. On the third day, the 10 min habituation session was followed by a training trial during which mice were placed back into the chamber and were allowed to investigate two wooden objects for 3 min. Animals were then placed into their home cage for 2 min before they were placed back into the chamber which now contained one familiar object from the training trial and one novel wooden object. Animals again were allowed to investigate both objects for 2 min. The objects were cleaned with 20% ethanol and the chamber was cleaned with 70% ethanol between all trials, and configurations of the objects during each trial were changed for each animal in all treatment groups. Both the training trial and novel trial were recorded by an overhead camera and the amount of time the mice spent investigating the objects during both the training trial and the novel object trial were analyzed. In order to assess whether animals recognized the novel object as such we calculated a “recognition index” which is the percentage of time spent investigating the novel object over the total investigation time for both objects.

Experimental mice and two stimulus mice were housed individually for 3 days prior to the task. On test day, the experimental mice were placed in a new cage for 1 h prior to experimentation. A stimulus mouse was placed in a custom cylindrical cage (height 20 cm, steel bars separated by 1 cm, acrylic base and lid) and introduced into the cage of the experimental mouse for four consecutive trials. Trials lasted 1 min each and were separated by 10 min intervals during which the stimulus mouse was returned to its home cage. For the fifth trial, a second stimulus mouse was placed in the custom cage and introduced to the experimental animal’s cage for 1 min. The cylindrical cages were cleaned with 20% ethanol after each exposure. All trials were video recorded and interaction times (defined as sniffing and investigation at close proximity) were scored.

Mice were placed inside a 60 × 60 cm open field and their behavior was video-recorded over a 30 min baseline period using an overhead camera and two side mounted cameras. After the baseline period, mice were given an i.p. injection of amphetamine (4 mg/kg d-amphetamine hemisulfate; Sigma, St. Louis, MO, USA) in saline. They were placed in their home cage for 10 min before they were returned to the open field for an additional 60 min. The total distance traveled was measured using a custom-written MATLAB program (Rodent tracker, Vulintus, Plano, TX, USA). In order to assess changes in stereotypic behavior, three 1-min clips of baseline activity and 20 1-min clips of post-injection activity (selected at 3 min intervals) were rated by two investigators using a 1–9 scale: 1—asleep: lying down, eyes closed; 2—inactive: lying down, eyes open; 3—in-place activities: normal grooming or chewing cage litter; 4—normal: alert, active moving about cage, sniffing, rearing; 5—hyperactive: running, movement characterized by rapid changes in position (jerky), 6—slow patterned: repetitive exploration of the cage at normal level of activity; 7—Fast patterned: repetitive exploration of the cage with hyperactivity; 8—restricted: remaining in same place in cage with fast repetitive head and/or foreleg movement (includes licking, chewing and gnawing stereotypies); and 9—dyskinetic-reactive: backing up, jumping, seizures abnormally maintained postures, dyskinetic movements (Ellinwood and Balster, 1974).

Mice were tested in an acrylic cage (9 × 5.5 × 4.5 cm) with a wire mesh bottom contained within an insulated startle box (67 × 67 × 67 cm). The cage was mounted on a startle platform (Lafayette Instrument Co., Lafayette, IN, USA) that uses a piezoelectric transducer to generate a continuous record of activity measured in volts. Startle tones were generated using a real-time processor (RP2.1, Tucker-Davis Technologies, Alachua, FL, USA) and presented by an overhead speaker (Optimus Bullet Horn Tweeter) positioned above the cage offset 20 cm from the center of the cage. Mice were habituated to the startle box twice a day for 2 days in 5 min sessions with 65 dB white noise playing in the background. On day 3, an input-output curve of the startle response was recorded. Mice were placed into the startle box and given a 5-min acclimation period during which only the 65 dB background noise was played. Following the acclimation period, the animals were presented with a startle tone (20 ms white noise) every 20 s, starting at 75 dB and increasing in loudness by 5 dB until a maximum of 115 dB was reached, totaling 10 startle presentations. The peak-to-peak voltage generated by the startle platform within 500 ms following the startle tone was recorded for each startle presentation. These points were then normalized for each animal using the following equation: xi = xi−xminxmax−xmin. On day 4, mice were placed into the startle box and given a 5-min acclimation period during which only the 65 dB background noise was played. This was immediately followed by 20 presentations of the startle tone (20 ms, 115 dB) by itself in order to establish the baseline startle for the animal. Animals were then presented with a prepulse tone (4 ms white noise; 75 dB or 85 dB) followed by a startle tone with a 30 ms interstimulus interval. Trials were pseudorandomly ordered by prepulse intensity, and each prepulse intensity was presented 10 times each with a 20 s intertrial interval. Peak-to-peak voltage startle responses were recorded for each presentation. To analyze PPI, prepulse trials were sorted by prepulse intensity and then the startle response was averaged across all 10 trials for each intensity. PPI was calculated by dividing the prepulse startle over the baseline startle for each animal and subtracting this from 100%. Animals with low baseline startle, or animals that were moving during most of the test period were excluded. A total of 11 (3 SAL-SAL; 3 KET-SAL; 2 SAL-NAC; 3 KET-NAC) out of 51 animals were excluded based on these criteria.

Differences between groups were compared using one-way ANOVAs or two-way mixed ANOVAs as indicated. Post hoc analyses using Bonferroni correction were used to determine specific group differences. For all measures the data is presented as mean ± standard error of the mean (SEM). An alpha level of p < 0.05 was considered significant.

Deficits in cognitive flexibility mediated by the PFC represent a significant disability associated with schizophrenia and several other neurological and neuropsychiatric disorders (Pantelis et al., 1999; Nieuwenstein et al., 2001). In order to test the effects of NAC on deficits in cognitive flexibility induced by perinatal ketamine treatment, we studied differences in the acquisition of a Response Discrimination, followed by a Shift-to-Visual Cue-Discrimination (Floresco et al., 2006; Jeevakumar et al., 2015). This task required the animals to shift between two strategies and in the second discrimination to focus on a previously ignored stimulus in order to receive a reward (Figure 2A). A one-way ANOVA revealed that animals in all treatment groups were able to acquire the Response Discrimination strategy at the same rate (F_(3,32) = 0.811, p = 0.497; Figure 2B). In contrast, drug treatment significantly affected the number of trials required to shift to the visual cue strategy (F_(3,32) = 9.267, p < 0.001; Figure 2B). Post hoc comparisons using Bonferroni correction showed that KET-SAL treated animals took significantly more trials compared with controls, while both NAC treated groups performed comparable to controls. A separate ANOVA also revealed an effect of treatment on total number of errors committed during the Shift-to-Visual-Cue Discrimination (F_(3,32) = 11.113, p < 0.001; Figure 2C). Post hoc comparisons showed that KET-SAL animals committed significantly more errors than all other treatment groups. These errors were further broken down into perseverative, regressive and never reinforced errors. While there was not a significant effect of treatment on perseverative errors (Persev: F_(3,32) = 0.834, p = 0.485; Figure 2D), there was a significant effect on both regressive and never reinforced errors (Reg: F_(3,32) = 8.610, p < 0.001; NR: F_(3,32) = 9.592, p < 0.001; Figure 2D). The majority of animals required only one probe trial to reach criterion and accordingly there were no significant differences in the number of probe trials between treatment groups during either Response Discrimination (F_(3,32) = 0.620, p = 0.607) or Shift-to-Visual-Cue Discrimination (F_(3,32) = 0.658, p = 0.584; data not shown). Taken together, these results show that ketamine treatment did not interfere with the animals’ ability to acquire the first strategy, but resulted in a selective impairment in performing the shift to a visual cue-based strategy. Importantly, animals treated with NAC exhibit normal cognitive flexibility, learning the shift to the visual cue strategy at the same rate as controls.

When exploring their environment, drug-naïve rodents show a strong tendency to alternate entries into the arms of a maze. In order to measure treatment-induced changes in spatial learning and short term memory, we measured the frequency of spontaneous alternations between entries into a left or right goal-arm during free-choice trials in a T-maze (Figure 3). A one-way ANOVA showed a significant effect of drug treatment on spontaneous alternation (F_(3,31) = 3.506, p = 0.027). Post hoc comparisons revealed that KET-SAL animals showed significantly fewer alternations than animals in the SAL-SAL group (Figure 3B). In contrast, mice in the KET-NAC and SAL-NAC groups showed similar patterns of alternation as the SAL-SAL group, indicating that co-administration of NAC was able to protect against ketamine-induced deficits in spatial learning and memory. There were no differences between treatment groups in the average time to completion (F_(3,31) = 0.781, p = 0.514; Figure 3C), making it unlikely that ketamine-induced differences in the alternation pattern reflect an unspecific deficit in overall activity.

Next, we tested how developmental ketamine treatment affects the ability of mice to distinguish between a familiar and a novel object (Figure 4). We first introduced animals to two objects during the training phase and scored the investigation time for each of the two objects. A two-way mixed ANOVA with treatment as the between-subjects factor and the two objects as the within-subjects factor revealed no significant difference in investigation time between treatment groups (F_(3,32) = 2.588, p = 0.070) and no difference between objects (F_(1,32) = 3.945, p = 0.056; Figure 4B). After a 2 min delay, animals were then exposed to one of the familiar objects and a novel object, and we again scored investigation times for each of the two objects. As expected, there was a significant difference in investigation time between the familiar and novel objects (F_(1,32) = 81.364, p < 0.001l) suggesting an overall preference for the novel object across all groups. However, this was qualified by an interaction between treatment group and object preference (F_(3,32) = 4.545, p = 0.009). Further analysis of simple main effects revealed that this interaction was due to the KET-SAL group, which showed no significant preference for the novel object (Figure 4C). For each animal we also calculated a recognition index, which normalized the novel investigation time to a percent of the total investigation time. A one-way ANOVA revealed a significant effect of treatment on this recognition index (F_(3,32) = 7.306, p = 0.001; Figure 4D). Post hoc comparisons revealed that KET-SAL animals showed significantly lower recognition indices than animals in the other three treatment groups. On the other hand the recognition index of NAC-treated animals did not differ significantly from that of mice in the SAL-SAL control group, indicating that NAC was able to prevent deficits in novel object recognition that result from developmental ketamine-treatment.

Next, we used a social interaction task to assess sociability and preference for social novelty, which may be relevant to negative symptoms of schizophrenia and possibly to some features of depression. We have previously shown that perinatal ketamine treatment induces deficits in social interaction, leading to greatly reduced interaction times of the KET-SAL mice with the stimulus mice across all exposures (Jeevakumar et al., 2015). Here we replicate these findings and examine whether N-acetylcysteine treatment can prevent these social deficits (Figure 5). A two-way mixed ANOVA, with drug-treatment as the between-groups factor and trial number as the within-group factor revealed a significant main effect of treatment (F_(3,36) = 12.965, p < 0.001; Figure 5B) and a significant interaction between trial number and treatment (F_(12,144) = 2.709, p = 0.003) on social interaction. Compared to animals in the other three treatment groups ketamine-treated mice interacted significantly less with the first stimulus mouse during trials 1 and 2. With repeated exposures of a now familiar mouse interaction times continued to decease in the other three treatment groups, and by trial 3 and 4 interaction times were similar across all groups. However, upon exposure to a novel stimulus mouse, interaction times significantly increased again in all treatment groups, except the KET-SAL group, indicating deficits in social interaction in ketamine-treated animals (Figure 4B). Importantly, KET-NAC animals showed normal patterns and durations of interaction times that were indistinguishable from SAL-SAL controls, indicating that NAC prevented the development of ketamine-induced deficits in social interaction.

Prepulse inhibition (PPI) of the startle response is a measure of sensorimotor gating that is reduced in patients with schizophrenia (Braff and Geyer, 1990) and various rodent models of the disease (Lipska et al., 1995; Young et al., 2009; Powell et al., 2012b), including models that induce NMDAR dysfunction during development (Wedzony et al., 2008; Lyall et al., 2009). In humans, PPI deficits correlate with distractibility (Karper et al., 1996), and with quantitative measures of thought disorder (Perry and Braff, 1994). In order to test the effects of perinatal ketamine treatment and NAC on sensorimotor gating, we first measured startle responses over a range of increasing startle pulse intensities to ensure that ketamine or NAC treatment did not affect the baseline startle response. Then we tested PPI under two different prepulse conditions (10 or 20 dB over background noise). There were no significant differences in either the normalized input-output curve of startle responses (F_(3,36) = 0.287, p = 0.835; Figure 6A) or baseline startle responses to the startle pulse alone during PPI (F_(3,36) = 1.412, p = 0.255), indicating that animals in all treatment groups startled to the same extent. In contrast, a one-way ANOVA with PPI as the dependent variable and treatment as the between-groups factor revealed a significant effect of treatment on PPI for the 10 dB condition (F_(3,36) = 5.298, p = 0.004). Bonferroni corrected post hoc tests revealed a significant effect between SAL-SAL and KET-SAL, as well as SAL-NAC and KET-SAL animals (Figure 6B). However, even though there was clear trend, the KET-NAC and KET-SAL groups did not differ statistically, indicating that NAC failed to fully protect against the ketamine-induced deficits in PPI. When the prepulse was more salient (20 dB condition), mice in all groups exhibited comparable PPI (F_(3,36) = 0.449, p = 0.719). Our data suggests that perinatal ketamine treatment results in a long-lasting deficit in PPI, which can be partly recovered by concomitant NAC application.

Dysregulation of subcortical dopamine function plays an important role in the pathophysiology of schizophrenia, and psychostimulants like amphetamine, which enhance dopamine transmission, can exacerbate psychotic symptoms in schizophrenic subjects (Breier et al., 1997; Abi-Dargham et al., 1998). For this reason, the effects of psychotropic drugs on locomotor activity have widely been used to model positive symptoms of schizophrenia in rodents (Geyer and Markou, 1995; van den Buuse, 2010). Here we tested whether treatment with NAC can prevent the increased sensitivity to d-amphetamine that is seen following perinatal ketamine treatment (Jeevakumar et al., 2015). We measured both changes in overall locomotor activity (Figure 7A), as well as stereotypic behavior (Figure 7B). We first assessed overall locomotor activity as the total distance traveled. A two-way mixed ANOVA with total distance traveled as the dependent variable, drug treatment as the between-groups factor, and time as the within-groups factor revealed a significant main effect of time (F_(22,1034) = 26.989, p < 0.001) on overall locomotor activity; however, we found no significant main effect of treatment (F_(3,47) = 1.843, p = 0.152) and no interaction effect of time and treatment (F_(66,1034) = 1.134, p = 0.223). Although there was no significant main effect of treatment on total distance traveled across the four experimental groups, KET-SAL animals showed a trend towards reduced amphetamine-induced locomotor activity. Amphetamine-induced reductions in locomotor behavior may reflect increased repetitive movement in place, characterized by nearly continuous repetitive head movements, forelimb movements, sniffing, licking or biting (Ellinwood and Balster, 1974; Robinson and Becker, 1986). In order to explore this possibility, we rated stereotypic movements on a scale from 1 to 9 (Ellinwood and Balster, 1974) across the same time bins shown in Figure 6A. We found a significant main effect of time (F_(22,1232) = 158.616, p < 0.001), as well as a significant main effect of drug treatment (F_(3,56) = 4.844, p = 0.005) on stereotypic behavior. Both of these main effects were qualified by a significant interaction effect of time and treatment (F_(66,1232) = 1.314, p = 0.050). Further analysis of simple main effects revealed that KET-SAL animals exhibited increased stereotypy compared to SAL-SAL and KET-NAC animals across several time points following amphetamine injection, without significant differences in baseline activity (Figure 7B). These results indicate that developmental ketamine administration increases the sensitivity to psychostimulants that stimulate the subcortical dopamine system, leading to increased stereotypic motor behavior in mice. Co-treatment with NAC prevented emergence of these symptoms.