Striatal transcriptome changes linked to drug-induced repetitive behaviors

Abstract

Disruptive or excessive repetitive motor patterns (stereotypies) are cardinal symptoms in numerous neuropsychiatric disorders. Stereotypies are also evoked by psychomotor stimulants such as amphetamine. The acquisition of motor sequences is paralleled by changes in activity patterns in the striatum, and stereotypies have been linked to abnormal plasticity in these reinforcement-related circuits. Here, we designed experiments in mice to identify transcriptomic changes that underlie striatal plasticity occurring alongside the development of drug-induced stereotypic behavior. We identified three schedules of amphetamine treatment inducing different degrees of stereotypy and used bulk RNAseq to compare striatal gene expression changes among groups of mice treated with the different drug-dose schedules and vehicle-treated, cage-mate controls. Mice were identified as naïve, sensitized, or tolerant to drug-induced stereotypy. All drug-treated groups exhibited expression changes in genes that encode members of the extracellular signal-regulated kinase (ERK) cascades known to regulate psychomotor stimulant responses. In the sensitized group with the most prolonged stereotypy, we found dysregulation of 20 genes that were not changed in other groups. Gene set enrichment analysis indicated highly significant overlap with genes regulated by neuregulin 1 (Nrg1). Nrg1 is known to be a schizophrenia and autism susceptibility gene that encodes a ligand for Erb-B receptors, which are involved in neuronal migration, myelination, and cell survival, including that of dopamine-containing neurons. Stimulant abuse is a risk factor for schizophrenia onset, and these two disorders share behavioral stereotypy phenotypes. Our results raise the possibility that drug-induced sensitization of the Nrg1 signaling pathway might underlie these links.

Keywords: amphetamine; restricted repetitive behavior; sensitization; stereotypy; tolerance.

FIGURE 1

Timeline of drug treatments, behavioral assessments and tissue collection.

FIGURE 2

Sensitization to amphetamine-induced early-phase and late-phase locomotion occurs at different rates. (a) Measurements of distance traveled across the last saline-treatment day and following amphetamine-treatment days. Each row shows one day with averaged distance traveled measurements in 5 min bins. Injection occurred at time = 0. The average duration of confined locomotion, between the early-phase and late-phase locomotion periods, diminishes with prolonged treatments but recurs on the challenge day. (b) Average distance traveled before and after D-amphetamine injection on day 1 (magenta), day 2 (black), and day 7 (green). There was significantly less mid-phase locomotion on treatment days 2 and 7 than on day 1 (*P < 0.005 at every point marked in the comparisons between day 1 and day 7 and between day 1 and day 2). By day 7, the mice showed increased late-phase locomotion, relative to day 1 (^†P < 0.005 at every point marked in the comparison between day 1 and day 7). (c) On the challenge day (orange), mice maintained early-phase sensitization, based on their high levels of locomotion within 5 min of drug injection (*P = 8 x 10⁻⁶ by 2-tailed, paired t-test compared to day 1 data shown in panel b and File S1). During the late-phase locomotion period, however, the mice showed a diminished locomotor response on challenge day than on day 21 (blue) (^†P < 0.03). Averages and +SEM across mice are shown (n = 12 mice).

FIGURE 3

Heat maps of distance traveled by each mouse, illustrating inter-animal variability in sensitization rate. D-amphetamine treatment days 1–21 are shown (day 16 data from some of the mice were lost).

FIGURE 4

Model of distance traveled data shows a significant increase in late-phase locomotion beginning on D-amphetamine treatment day 9. (a) Distance traveled in the 80–85 min time bin across days. The raw data from each of the 12 mice are plotted as gray dots joined by lines, with estimates of individual fit to distance traveled (light purple) and group median estimate with 90% credible intervals (blue). (b) Day-by-day comparison of the state-space fit to the group estimates of distance traveled shown in A. Each point on the surface represents the probability that the group estimate on the day shown on the x-axis is higher than the day shown on the y-axis. Light colored surface indicates very low probability, and blue highlight indicates P < 0.005. Conversely, dark color shows that the day on the x-axis is higher than the day on the y-axis near P = 1, and red highlight means P > 0.995.

FIGURE 5

Mice show sensitization to confined stereotypy by day 2 of D-amphetamine injection and tolerance by day 21 of treatment. Plots show rating of behaviors during the 80–82 min post-injection time period for locomotion (a; *P = 0.02, ^†P = 0.008, **P = 0.01, ^††P = 0.01, by 2-tailed, paired t-tests), any confined stereotypy (b; *P = 0.01, ^†P = 0.008, **P = 0.004, ^††P = 0.005), and confined sniffing or licking at the wall (c; *P = 0.02, ^†P = 0.003). Averages and +SEM across mice are shown, with individual mice identified by the symbol X.

FIGURE 6

Mice show a preferred location for stereotypy across continuous treatment. (a) Example of stationary vs. circling behavior of a single mouse on D-amphetamine treatment days 1, 7 and 21 and on challenge day. An angular coordinate deflection from −180° to +180° (vertical lines) represents a full revolution around the cage. (b) For each mouse, the coordinate position data, either before (pre) or after (post) drug injection was plotted against the average coordinate position during that time interval across all days and the average correlations were plotted. Beginning on the second day of drug treatment, there was a high correlation across days for the post-injection period, reflecting a relatively constant favored location. The correlation for the pre-injection period was low, indicating that the mice did not have a highly preferred location before drug injection. Averages and +SEM across mice are shown (n = 12 mice).

FIGURE 7

The heat maps for gene expression changes show the sharpest differences on high-stereotypy day 7, and progressively lower proportions of upregulated genes across all days. Each column shows results from one mouse for genes that were significantly different in group comparisons between saline-treated mice and mice treated with D-amphetamine for 1 day (a; modest behavioral response), 7 days (b; prolonged stereotypy response) and 21 days (c; strong behavioral response with curtailed stereotypy period) . Genes with RPKM < 1 in all samples, or that were changed in any pair-wise comparisons among saline-treatment groups, were removed.