A rodent "self-report" measure of methamphetamine craving? Rat ultrasonic vocalizations during methamphetamine self-administration, extinction, and reinstatement

Abstract

Rats emit ultrasonic vocalizations (USVs) in a variety of contexts, and it is increasingly clear that USVs reflect more complex information than mere positive and negative affect states. We sought to examine USVs in a common model of addiction and relapse, the self-administration/reinstatement paradigm, in order to gain insight into subjective states experienced by rats during various types of methamphetamine seeking. We measured three subtypes of "50kHz" USVs [flats, trills, and non-trill frequency modulated (FM) USVs], as well as long and short duration "22kHz" USVs, during self-administration and extinction training, and during reinstatement elicited by cues, a methamphetamine prime, cues+prime, or the pharmacological stressor yohimbine. During self-administration and extinction, rats emitted many flats and FMs, (and short duration "22kHz" USVs on day 1 of self-administration), but few trills. In contrast, methamphetamine priming injections potently enhanced FMs and trills, and trill production was correlated with the degree of methamphetamine+cue-elicited reinstatement. Cues alone yielded increases only in flat USVs during reinstatement, though a subset of rats displaying strong cue-induced reinstatement also emitted long duration, aversion-related "22kHz" USVs. Although yohimbine administration caused reinstatement, it did not induce "22kHz" USVs in methamphetamine-experienced or methamphetamine-naïve rats (unlike footshock stress, which did induce long duration "22kHz" USVs). These findings demonstrate heterogeneity of rat USVs emitted during different types of methamphetamine seeking, and highlight their potential usefulness for gaining insight into the subjective states of rats in rodent models of drug addiction and relapse.

Figure 1

Examples of “50kHz” (flat, FM, and trills) and “22kHz” (long and short) USVs, captured from screen shots of spectrographs displayed at 490Hz with a time resolution of 0.5ms.

Figure 2

(A) Mean (+SEM) lever responding during the first and last days of methamphetamine self-administration (LEFT), extinction (MIDDLE) and reinstatement by methamphetamine-paired cues (Cue), methamphetamine-prime (Meth), cue+methamphetamine (Cue+Meth), or yohimbine (Yoh; RIGHT). Active lever presses are shown in black bars, while inactive levers are shown in grey bars. Significant differences in responding between self-administration and extinction days (#), significant reinstatement relative to the last day of extinction (*), difference in reinstatement behavior compared to Yoh day (+), and differences between Cue+Meth and other reinstatement groups (@) are indicated (p<0.05; n=15; repeated measures ANOVA; ps < 0.05). (B) Time course of active lever pressing within each reinstatement session (cue, methamphetamine, cue+methamphetamine, yohimbine), and the last day of extinction training (Ext 7) are displayed in 15min bins for these 2hr sessions.

Figure 3

(A) Mean (± SEM) “50kHz” USVs during the first and last days of methamphetamine self-administration and extinction days 1 and 7. *Decrease from self-admin day 1 to 15 in flats, n=14, one-way ANOVA, p<0.05. (B) Mean (± SEM) “22kHz” USVs during the first and last days of methamphetamine self-administration and extinction days 1 and 7. *Decrease from self-administration day 1 to 15 in short “22kHz” USVs, n=14, one-way ANOVA, p<0.05. (C) Mean (± SEM) “50kHz” and “22kHz” USVs during extinction day 7 (Ext.), cue reinstatement (Cue), methamphetamine prime (Meth), cues + methamphetamine (Cue+Meth), or yohimbine (Yoh). *Increase from extinction levels, ns=11–13, one-way ANOVA, p<0.05; **p<0.01. (D) Pearson correlation between trills (X-axis) and methamphetamine seeking (active lever presses in first 30min of reinstatement session; Y-axis) on cue+methamphetamine test day. (E) Proportion of all USVs emitted on each test day that were of each subtype. Mean (± SEM) proportion of USVs emitted by each rat that were of each USV subtype [short “22kHz,” long “22kHz,” trill, non-trill frequency modulated (FM), and flat]. #Difference between self-administration day 15 and extinction day 1, ns=11–14, one-way ANOVA, p<0.05. *Difference between reinstatement day and extinction day 7, ns=11–13, one-way ANOVA, p<0.05

Figure 4

Percentage of all observed long (A) and short “22kHz” USVs (B) emitted during each of the behavioral contexts tested here: Methamphetamine self-administration days 1&15 (SA Day 1 &15), extinction days 1&7 (Ext Day 1&7), and cue, methamphetamine (Meth), cue+methamphetamine (C+M), and yohimbine (Yoh), reinstatement (Reinst). (C) Pearson correlation between active lever (AL) pressing (1st 30min of cued reinstatement session) and the number of long “22kHz” USVs emitted. (D) Mean (± SEM) long and short “22kHz” USVs/30min emitted after yohimbine during reinstatement testing in methamphetamine-experienced animals (Meth Animals), after yohimbine in drug-naïve animals, or after 15min of foot shock in drug naïve animals (right).

Figure 5

(A) Frequency (kHz) of “50kHz” and “22kHz” USVs during each reinstatement test as a function of time (min) in cue, methamphetamine (Meth), yohimbine, or cue+methamphetamine (Meth+Cue) tests. (B) Histogram showing total occurrences of USVs emitted at various frequency ranges during reinstatement tests. The total number of long “22kHz,” short “22kHz,” flat, trill, and non-trill frequency modulated USVs emitted by rats during reinstatement tests are plotted based upon their mean frequencies falling into 5kHz bins ranging from 18kHz to 100kHz (X-Axis).