Individual differences in the conditioned and unconditioned rat 50-kHz ultrasonic vocalizations elicited by repeated amphetamine exposure

Abstract

Rationale: Adult rats often produce 50-kHz ultrasonic vocalizations (USVs), particularly the frequency-modulated varieties, in appetitive situations. These calls are thought by some to reflect positive affective states and the reinforcing value of drugs such as amphetamine and cocaine.

Objective: The objective of this study was to determine whether the number of unconditioned 50-kHz USVs elicited by amphetamine predicts the development and/or magnitude of drug-conditioned motivation.

Methods: In three experiments, we recorded USVs before and after injections of 1 mg/kg amphetamine (i.v. or i.p.) administered once per session. Rats were categorized as "high callers" or "low callers" according to individual differences in the number of 50-kHz USVs elicited by their first amphetamine injection. We examined the conditioned appetitive behavior and conditioned place preference (CPP) that emerged in high and low callers after repeated pairings of amphetamine with specific contexts. We also examined whether amphetamine-induced calling was affected by treatment within an unfamiliar (test chamber) versus familiar (home cage) context.

Results: Within an unfamiliar environment, the high callers consistently produced more amphetamine-induced 50-kHz USVs than the low callers. Compared to the low callers, high callers showed significantly greater amphetamine CPP as well as enhanced conditioned 50-kHz USVs and locomotor activity during anticipation of amphetamine. Individual differences were stable when amphetamine was administered in test chambers, but when it was administered in home cages, low callers showed an increase in 50-kHz calling that matched the high callers.

Conclusions: These findings suggest that individual differences in drug-induced USVs can reveal environment-sensitive traits involved in drug-related appetitive motivation.

Fig. 1

Fifty-kilohertz USVs in high callers (n=8), low callers (n=8), and the saline-treated control group (n=9) in experiment 1. Figure shows 50-kHz USVs per 10 min (mean+SEM) produced during habituation/baseline sessions (h1–h4) and amphetamine sessions (1–15). a During the anticipatory period (minutes 0–10 of sessions), the high callers made significantly more conditioned 50-kHz USVs than the low callers and controls. b In the first 10 min of the post-injection period (minutes 11–20 of sessions), high callers produced more amphetamine-induced 50-kHz USVs than the low callers and controls in all 15 amphetamine sessions. There were no significant differences between the low callers and controls at any point

Fig. 2

Individual differences in the number of 50-kHz USVs elicited by amphetamine in experiment 1. Figures show calls counted in 30-s bins (mean+SEM) during sessions 1, 5, 10, and 15. All sessions began with a waiting period in the conditioning chambers, with i.v. infusions of amphetamine (or saline in the control group, n=9) delivered at 10 min. a Amphetamine-treated rats were categorized as high callers (n=8) or low callers (n=8) based on the number of post-amphetamine 50-kHz USVs made in session 1. b–d In the first 5 min post-injection (minutes 11–15 of sessions), the high callers showed a greater increase in 50-kHz USVs relative to controls in sessions 5, 10, and 15 compared to session 1. *p<0.05 (within-subjects contrasts). The high callers also showed an increase in anticipatory 50-kHz USVs during the 30-s illumination of the cue light that preceded infusions (indicated by the shaded bar), and this effect was greater in sessions 5, 10, and 15 compared to session 1. ⁺⁺p<0.01; ⁺⁺⁺p<0.001 (within-subjects contrasts)

Fig. 3

FM and flat 50-kHz USVs (mean+SEM) produced by high callers (n=8), low callers (n=8), and saline control rats (n=9) in experiment 1. Samples of 50-kHz USVs were taken from sessions 1, 5, 10, and 15. a–c Top panels, Anticipatory 50-kHz USVs produced during the 30-s illumination of the cue light. There were no significant differences between FM and flat calls in any group during this period. d–f Bottom panels, 50-kHz USVs in the first 5 min post-injection (minutes 11–15 of sessions). In the high callers, amphetamine elicited more FM calls than flat calls. When later sessions were compared to the first session, the high callers showed a significant increase in FM calls, but not flat calls. *p<0.05 (paired t tests)

Fig. 4

Locomotor activity in high callers (n=8), low callers (n=8), and saline control rats (n=8) in experiment 1. Figure shows counts of photobeam breakages (mean+SEM) across habituation/baseline sessions (h1–h4) and amphetamine sessions (1–15). a In the last 30 s of the anticipatory period, conditioned locomotor activity was significantly greater in high callers than saline-treated rats. b When locomotor activity was counted for the entire anticipatory period (minutes 0–10 of sessions), there were no significant group differences. c During the post-injection period (minutes 11–30 of sessions), both high- and low-caller groups showed significant amphetamine-induced increases in activity compared to saline-treated rats

Fig. 5

Fifty-kilohertz USVs per 10 min (mean+SEM) produced by high callers (n=6) and low callers (n=6) in experiment 2. Left panels, Calls made in habituation/baseline sessions (h1–h4) and amphetamine sessions (1–3) that took place in conditioning chambers. a Anticipatory calls made in the pre-injection period (minutes 0–10 of sessions). b Calls made in the post-injection period (minutes 11–20 of sessions), when high callers made significantly more 50-kHz USVs than low callers. Right panels, Calls produced during habituation/baseline sessions (h1–h2) and amphetamine sessions (1–15) within the home cages. c In the pre-injection period, both high and low callers showed a significant increase in anticipatory calls across sessions. d In the post-injection period, both high and low callers made significantly more 50-kHz USVs in amphetamine sessions than baseline sessions

Fig. 6

Fifty-kilohertz USVs per 10 min (square-root-transformed values) made by individual high and low callers in experiment 1 (Exp1 High and Exp1 Low) and experiment 2 (Exp2 High and Exp2 Low). Post-amphetamine USVs were counted in minutes 11–20 of session 1; anticipatory USVs were counted in minutes 0–10 of session 15. In rats from experiment 2, this refers to home cage sessions 1 and 15, in which low callers vocalized at rates similar to high callers. Overall, there was a significant positive correlation between amphetamine-induced calls and anticipatory calls (r=0.622, p<0.001, n=28). Correlations were also significant when each experiment was considered separately (experiment 1: r=0.503, p<0.05, n=16; experiment 2: r=0.669, p<0.05, n=12)

Fig. 7

In the test for amphetamine conditioned place preference (experiment 3), high callers (n=10) spent more time in the CS+ side of the chamber than low callers (n=10) and saline-treated controls (n=10). Top graphs show 50-kHz USVs made by high callers (a) and low callers (b) counted in 5-min bins (mean+SEM) during the two saline sessions (S1 and S2) and two amphetamine sessions (A1 and A2). c When baseline sessions were compared to test sessions, high callers showed a significant increase in the percent of time spent in the CS+ side compared to saline controls (**p<0.01), whereas low callers did not differ significantly from controls. d There was a significant correlation between the number of 50-kHz USVs per 30 min produced in the first amphetamine session (log-transformed values) and the percent of time spent in the amphetamine-paired side during CPP testing (p<0.05)

Fig. 8

Correlations between 22- and 50-kHz USVs associated with amphetamine. Points represent individual amphetamine-treated high and low callers from experiment 1 (Exp1 High and Exp1 Low) and experiment 2 (Exp2 High and Exp2 Low) that made 22-kHz USVs in at least one session. Values represent counts of 50- and 22-kHz USVs per 10 min (square-root-transformed) averaged across all amphetamine sessions in which 22-kHz USVs were produced. a During the anticipatory period (minutes 0–10 of sessions), there was a significant positive correlation between 50- and 22-kHz calls (r=0.746, p<0.01, n=11). b During the first 10 min of the post-injection period (minutes 11–20 of sessions), there was a significant negative correlation between 50- and 22-kHz calls (r=−0.698, p<0.05, n=10)