In the present study we report 50 kHz USVs emitted during the course of a typical cocaine self-administration study using a standard cocaine dose. In addition, we assessed comparable affective states associated with a non-drug reward (sucrose). Interestingly, we found that the number of 50 kHz USVs on the first day of cocaine self-administration was negatively correlated with the number of days required for acquisition of stable self-administration behavior. This demonstrates that spontaneous, untrained 50 kHz USVs may be a sensitive predictor of the rat’s vulnerability to attain sustained cocaine self-administration. While both the number of active nose-poke responses and the number of cocaine rewards on the first day of acquisition showed similar trends, these were not significant, suggesting that USV emission upon initial cocaine experience is a better predictor of the rate of self-administration acquisition. This finding supports those made in humans wherein individuals who rate their first experience with cocaine as a positive subjective experience are more likely to develop cocaine dependence and/or lifetime cocaine use (Lambert et al., 2006
The number of 50 kHz USVs increased from the beginning to end of acquisition training in the cocaine self-administration experiment. USV production is dose-dependent (Barker et al., 2010
); thus, USVs may have increased as the number of cocaine rewards increased from the beginning to the end of self-administration. Alternatively, USVs may have sensitized from the beginning to end of acquisition training. 50 kHz USVs sensitize with repeated administration of the same dose of non-contingent cocaine (Mu et al., 2009
; Williams and Undieh, 2010
), amphetamine (Ahrens et al., 2009
), and methylphenidate (Panksepp et al., 2002
). The increase in 50 kHz USVs may reflect an increase of the incentive motivational properties of cocaine since USVs have been characterized as motivational markers (Burgdorf et al., 2000
; Knutson et al., 1998
; Sales, 1972a
). However, no increase in the total number of USVs was observed from the beginning to the end of sucrose self-administration acquisition despite an increase in sucrose rewards. These findings suggest that the increase in USVs observed over the 2 hr sessions may not generalize to non-drug rewards.
Very few 22 kHz USVs were observed in either cocaine- or sucrose-self-administering rats. 22 kHz USVs are emitted during withdrawal following cocaine binges, but this effect is not detectable unless additional mildly aversive stimuli such as air puffs are applied to the rat (Barros and Miczek, 1996
; Mutschler et al., 2001
; Mutschler and Miczek, 1998a
). Furthermore, Barker et al. (2010)
reported 22 kHz USV emissions during cocaine self-administration, but the vast majority were less than 0.3 sec. While both short (<0.3 sec) and long (>0.3 sec) 22 kHz USV have been reported (Brudzynski et al., 1993
; Brudzynski et al., 1991
; Sales, 1972a
), only the long 22 kHz USVs have been associated with negative affect (Brudzynski, 2001
; Knutson et al., 2002
; Van der Poel and Miczek, 1991
). Thus, only 22 kHz USVs over 0.3 sec may reflect negative affect. Based on this standard, 22 kHz USVs collectively accounted for less than 1% of the calls observed in all of our experiments. Indeed, such scarcity of 22 kHz USVs upon cocaine exposure is also reported in several other studies (Ahrens et al., 2009
; Barker et al., 2010
; Williams and Undieh, 2010
During extinction, 50 kHz USVs decreased in rats exposed to cocaine self-administration but not in rats exposed to sucrose self-administration. During the first day of cocaine extinction, USV levels were similar to those found at the end of acquisition only during the first 5 min. However, after the first 5 min, the number of USVs dropped to the level observed during the first day of acquisition. This decrease in USVs may reflect a rapid decrease in positive affect during extinction. However, at no point did USVs in the cocaine or sucrose experiments drop to the level of non-treated naïve animals, suggesting that an environment paired with rewarding stimuli continues to elicit a modestly elevated positive affective state even in the absence of the reward.
Interestingly, although animals demonstrated a rapid decrease in 50 kHz USVs during the first extinction session, several more days were required for the animals’ nose-poke responses to decrease to extinction levels. This result suggests that rats continued to attempt to obtain cocaine by nose-poke responding even though they may no longer have maintained the previously-associated positive affective state. This is reasonable from the perspective that the USVs were not an operant for the receipt of cocaine, and hence the affective measure should have declined much more rapidly than the instrumental responding, which may have become a learned behavioral habit (Everitt et al., 2001
50 kHz USVs also reflect anticipation of both natural and drug rewards (Burgdorf et al., 2000
; Knutson et al., 1998
; Ma et al., 2010
). Panksepp & Moskal (2008)
proposed that USV production demonstrates reward expectancy resulting from the activation of an ethologically-characterized expectancy/seeking system (Panksepp et al., 2002
). Recently, Ma et al. (2010)
reported that anticipatory 50 kHz USVs increased during a 10 min period prior to the start of each cocaine self-administration session between the beginning and end of acquisition training regardless of whether the drug was administered contingently or non-contingently, and this anticipatory USV production was greater after a 2-day forced abstinence period (Maier et al., 2010a
). Indeed, in the present study, most USVs were emitted at the start of the self-administration sessions. This may signify that most of the USVs observed were in anticipation of the drug reward, rather than just in response to the cocaine itself, suggesting that USVs may reflect an anticipatory state. Another possibility is that the start of the training session elicits a greater level of positive affect. This may result from the alleviation of negative affect experienced by the separation of the animal from cocaine between training sessions. However, this possibility is mitigated by the fact that during the first 5 min of the first day of extinction, the number of USVs was roughly equal to that found at the end of acquisition, suggesting that USVs in the first 5 min of the first extinction day represent an anticipatory state based on prior experience. Likewise, in the sucrose experiment, there was no change in the total number of USVs throughout acquisition, and the increase in USVs was constrained largely to the first 5 min of testing. In other words, the majority of the USVs emitted appear to have shifted toward the beginning of the session as conditioning proceeded, perhaps in anticipation of reward.
The production of USVs is closely linked to dopamine availability in the nucleus accumbens. Amphetamine injection into the nucleus accumbens elicits 50 kHz USVs (Burgdorf et al., 2001a
; Thompson et al., 2006
). Dopamine depletion in the striatum decreases 50 kHz frequency-modulated USVs induced by female odor in males (Ciucci et al., 2009
), and by play or rewarding electrical brain stimulation (Burgdorf et al., 2007
). Furthermore, dopamine antagonists decrease 50 kHz USVs induced by cocaine, while apomorphine (a D1/D2 agonist) elicits USVs (Williams and Undieh, 2010
). The increase in 50 kHz USVs during cue- and cocaine-induced reinstatement in the cocaine experiment may therefore reflect increased dopamine levels in the nucleus accumbens (Kiyatkin and Stein, 1996
; Neisewander et al., 1996
; Phillips et al., 2003
; Weiss et al., 2000
). This suggests that the 50-kHz USVs may index the amount of endogenous dopamine release during behavioral tasks.
There are also some qualifications that need to be highlighted. First, the failure of the two types of rewards--cocaine and sucrose--to parallel each other in all behavioral measures suggests that the rewarding properties (presumably affective) of conventional rewards and drug rewards are somehow distinct for animals. In this context, it is especially interesting that with sucrose reward, no decrease in the number of USVs was observed in animals during extinction. It is possible that extinction from sucrose does not decrease the positive expectancy of reward as much as extinction from cocaine, perhaps because cocaine withdrawal induces a stronger negative affective state than does termination of access to sucrose. Alternatively, the two rewards may be qualitatively different at the affective level. Second, it should be noted that the methodology for the cocaine and sucrose experiments was not entirely parallel. We did not observe cue-induced reinstatement of active nose-poke responding in the sucrose experiment so it may be expected that USVs would not be elevated during this reinstatement, suggesting that the cue light did not become a salient stimulus when paired with sucrose in the present study. In addition, during the final reinstatement session, rats in the sucrose experiment were allowed to nose-poke for sucrose pellets while rats in the cocaine experiment were given a non-contingent cocaine injection but did not receive cocaine upon nose-poke responding. Therefore, we must constrain our interpretation to the idea that reinstatement by the unconditioned stimulus, whether contingent sucrose or non-contingent cocaine, elevated 50 kHz USVs. Finally, the response requirements for cocaine self-administration were increased (FR1→FR5) during the acquisition phases, while response requirements did not increase in the sucrose experiment (FR1 only). Although there is the potential for response requirements to alter USVs, we chose to balance the number of active nose-poke responses during the acquisition phases. Third, 50 kHz USVs have been classified into several distinct categories (Wright et al., 2010
). Although these categories of USVs may represent specific affective states, that proposition remains to be tested. Clearly, additional studies are required to link these several different types of USVs with distinct behavioral or affective profiles. Here we focused on changes in the total number of 50 kHz USVs. Of note, repeated amphetamine injection leads to sensitization of “trill” USVs, but a similar pattern of sensitization is observed when assessing the total number of 50 kHz USVs (Ahrens et al., 2009
). Still, further analyses of the different sub-types of 50 kHz USVs may allow for a greater understanding of affective states involved in self-administration and reinstatement.
In summary, changes in 50 kHz USVs may reflect the dynamic affective state of animals during self-administration of rewards including drugs of abuse. Such a direct non-invasive measure of affect in behaving animals may serve as a sensitive tool in bridging addiction-related behavioral outputs with their corresponding emotional/motivational states.
Browning et al.
- Measured 50 kHz ultrasonic vocalizations (USVs) during cocaine self-administration
- USVs are a spontaneous (untrained) index of affective responses to cocaine
- 50 kHz USVs are a dynamic index that quantifies reinforcing efficacy of cocaine
- USVs may provide a gauge of underlying affective processes mediating addiction
- Ability to measure affect in animals may link preclinical models to human addiction