The initial acute effect of a drug (i.e., positive hedonic response), including psychostimulants, is opposed or counteracted by longer-duration homeostatic changes in brain systems recruited by drug administration, thereby resulting in a longer-duration negative affective state upon withdrawal that is thought to contribute to the relapse and maintenance of addiction [4, 5, 46]. In the context of AMPH, this negative state appears even after initial acute AMPH administration and is accompanied by changes in both affective behavior and DA system activity [13].

Here we show that withdrawal from acute AMPH results in an anxiety-like state involving increases in anxiety-like behavior (i.e., less open arm entries, less time spent in open arms) and decreases in social motivation (i.e., reduced social cage sniff time). Increases in anxiety-like behavior are also observed in rats during short-term (24–48 h) withdrawal from chronic cocaine [21, 47] and AMPH [10]. In humans, acute withdrawal from psychostimulants is also accompanied by increased anxiety symptoms [17,18,19]. Collectively, these data suggest that both acute and repeated psychostimulant administration result in an increased anxiety-like state during the early withdrawal period. Our finding of social motivation impairment in AMPH-withdrawn rats, as indexed by lower levels of social sniff (i.e., cage) time, is consistent with prior reports, indicating suppression of rat social behaviors following acute AMPH administration [48, 49] and decreased motivation for natural reinforcers in animals undergoing AMPH-W [50, 51]. Since both high anxiety and low social motivation were present in AMPH-withdrawn rats, changes in social approach/social cage time likely reflect the anxiety state of the rat, suggesting that reducing the anxiety induced by AMPH-W may correct the increased anxiety-like behavior and the social behavior deficit observed.

Diazepam, a drug known for its robust anti-anxiety effects, reversed the affective dysregulation induced by acute AMPH-W. Acute diazepam administration increased the time spent in the open arms in the EPM only in AMPH-withdrawn rats. Diazepam reduced the number of open arm entries and the total number of arm entries in AMPH-withdrawn and non-withdrawn (i.e., control) rats, suggesting that the drug exerted a sedative/hypolocomotor effect. This is consistent with a prior study indicating reduced locomotor activity in the EPM at higher doses in adult male Sprague Dawley rats [52]. Indeed, diazepam effects on locomotion were present in both AMPH-withdrawn and non-withdrawn rats, although a difference in open arm time was only found in diazepam treated rats undergoing AMPH-W, suggesting that effects of diazepam on locomotor activity did not drive the anxiolytic behavioral effects exerted by diazepam in this study. This is also consistent with a reduction in the total number of crossings in the social approach test in both diazepam-treated groups, in which the increase in social cage time was restricted to AMPH-withdrawn animals treated with diazepam. Thus, although diazepam reduced general locomotor activity in AMPH-withdrawn and non-withdrawn rats in both behavioral tests, the anxiolytic effects were specific to the AMPH-withdrawn group. Importantly, diazepam increased social cage time in AMPH-withdrawn rats and prevented the percentage decrease from baseline in social cage time observed in AMPH-withdrawn rats treated with vehicle while having no effect on SAL controls in these measures, suggesting a selective effect on social dysregulation induced by AMPH-W. Moreover, diazepam also resulted in an increase from baseline in social cage time only in AMPH-withdrawn animals. Taken together, these data suggest that diazepam not only neutralized the decrease in social motivation following withdrawal from acute AMPH but also enhanced social motivation. This agrees with several preclinical studies that have highlighted the ability of diazepam to positively modulate anxiety-like behavior and social impairments in rodent models of schizophrenia and autism, respectively [36, 53]. For example, diazepam: (i) increases time spent in the social zone in N-methyl-d-aspartate receptor-deficient mice, which normally show deficits in affiliative behaviors and reduced sociability [53]; (ii) increases time spent in the proximal social zone of the social-approach avoidance test in a high anxiety strain (F-344) of rats [54]; (iii) abolishes social avoidance induced by prior footshock exposure [37]; and (iv) facilitates social dominance in high anxious rats, which frequently show reduced social competitiveness and a subordinate status [55]. Altogether, these data indicate a link between anxiety and alterations within the social domain and suggest that reductions in social motivation can serve as a measure of anxiety in a social context, which is consistent with diazepam’s anxiolytic effect on social behavior following AMPH-W, which was specific to the high anxiety group (i.e., AMPH-withdrawn rats).

Consistent with a prior study [13], we observed an attenuation of VTA population activity (i.e., DA cells/track) during withdrawal from acute AMPH. Thus motivational changes in behavior (i.e., anxiety, social dysfunction) during AMPH-W likely reflect opponent process-like changes (i.e., downregulation) in the reward circuitry initially activated by acute drug exposure. Reductions in VTA population activity have also been observed during short-term withdrawal (24 h) from an escalating-dose, binge-like regimen of cocaine [56] and psychostimulant withdrawal results in marked reductions of mesolimbic DA release in rats [57]. Drug abstinence in psychostimulant users is associated with dopaminergic alterations including changes in DA release, transporter, and receptor availability [8]. Thus drug withdrawal-induced decreases in reward-related dopaminergic neurotransmission may reflect a within-system neuroadaptation and contribute significantly to the negative motivational state associated with drug abstinence, thereby making the mesolimbic DA system a possible target for modulating the negative affective state induced by drug withdrawal.

Diazepam reversed VTA DA hypofunction induced by AMPH-W and had a specific effect on VTA population activity following acute AMPH-W as it normalized the attenuation in the number of active DA cells, while having no effect on SAL controls. Amphetamine-withdrawn rats treated with diazepam did not exhibit the reduction in VTA population activity observed in vehicle-treated AMPH-withdrawn rats and instead had numbers of spontaneously active DA neurons that were comparable to the vehicle and diazepam-treated control (i.e., nonwithdrawn) groups. Diazepam has also been shown to attenuate neural alterations, as measured by Fos immunoreactivity, in response to chronic social defeat stress across multiple brain regions: prefrontal cortices, limbic structures, and midbrain structures including the VTA [58], suggesting that diazepam can reverse stress-induced patterns of neural activation. Notably, when placed in the context of our past work, the DA downregulation induced by AMPH-W is comparable to that observed in rats following exposure to other stressors (CMS, learned helplessness) [14, 16]. The decrease in VTA population activity in helpless rats is reversed by systemic ketamine administration, which normalizes synaptic activity within the ventral hippocampal–nucleus accumbens pathway to restore DA population activity [16]. Similarly, the reduction in VTA population activity induced by withdrawal from acute AMPH is also reversed by ketamine [13], implicating restored activity within this pathway as a potential mechanism. Since ketamine administration did not reverse increased immobility induced by withdrawal from acute AMPH, this suggested that VTA DA neuron activity and FST immobility are correlated but not interdependent and raised the possibility that different processes may be present during acute AMPH-W. Importantly, DA downregulation induced by withdrawal from acute AMPH is reversed by inactivation of the basolateral amygdala (BLA), and BLA hyperactivity is observed in rodent models of depression and schizophrenia involving increased anxiety [14, 36]. Moreover, diazepam reduces anxiety-like behavior and normalizes amygdala hyperactivity in the methylazoxymethanol acetate (MAM) model of schizophrenia [36]. Taken together, these data suggest that diazepam may normalize the anxiety-like state and social deficit in AMPH-withdrawn rats via inhibitory actions in the VTA, as diazepam mimicked the previously reported effects of BLA inactivation on VTA population activity [13]. A recent study identified the VTA as a key site of action for diazepam’s enhancing effects on social competitiveness and dominance in high anxiety rodents [55]. Thus it is possible that diazepam reduces increased anxiety-like behavior and enhances motivated social behavior in AMPH-withdrawn rats through actions on the VTA, although this is a hypothesis that remains to be tested.

Finally, given that diazepam prevents a subset of the negative affective state symptoms resulting from acute withdrawal from a single dose of AMPH, future studies could examine whether these findings extend to repeated (i.e., chronic) use and whether they have implications for future drug-seeking and/or self-stimulation behavior. Because transition from drug use to addiction is thought to result, at least in part, from the need to alleviate dysphoric states that emerge during drug withdrawal [4], if diazepam is effective at reducing the anxiety-like state following withdrawal from chronic use, then this may help break the cycle. However, this is a possibility that remains to be tested. In addition, stress exposure (i.e., social defeat) alters the mesolimbic DA system, including VTA DA activity, and increases the risk for addiction and escalated psychostimulant self-administration [33, 59, 60]. It would be interesting to see whether diazepam could circumvent or ameliorate the effects of stress on subsequent drug consumption.

In sum, the present data suggest that acute withdrawal from a single dose of AMPH is sufficient to induce an anxiety-like state characterized by enhanced anxiety-like behavior and social avoidance. These behavioral changes are accompanied by reduced activity within dopaminergic neurons of the VTA, suggesting that these neurons may play an important role in the affective dysregulation induced by AMPH-W. We tested whether increasing activity within this pathway (in conjunction with decreasing anxiety) would reverse the adverse behavioral effects precipitated by AMPH-W and found that the negative affective state resulting from AMPH-W can be reversed by diazepam, which circumvents the decrease in tonic DA neuron firing that we propose underlies the negative withdrawal state. These data suggest that VTA DA neurons may be an appropriate target for modulating the negative affective state-induced withdrawal from acute AMPH.