Stress and Rodent Models of Drug Addiction: Role of VTA-Accumbens-PFC-Amygdala Circuit

Behavioral Sensitization

As reviewed in detail elsewhere [35–37], behavioral sensitization is a long-term consequence of repeated exposure to a drug of abuse that is defined by an augmented locomotor response to a drug challenge. Briefly, the neuroadaptive changes that mediate behavioral sensitization to the effects of abused drugs have been postulated as a possible mechanism contributing to increased drug use [35;36]. Repeated intermittent administration of cocaine, amphetamine, morphine, phencyclidine (PCP), nicotine, and also ethanol can result in a progressive augmentation of the behavioral effects of the drug, particularly of locomotor activity [38–50]; [Fig. 1a]. Facilitated drug taking behavior can follow previous exposure to psychostimulants [51–55].

In humans, sensitized behaviors following extended repetitive stimulant drug administration closely resemble symptoms of schizophrenia and may include psychotic symptoms of paranoia, ideas of reference (i.e., perception that irrelevant or innocuous phenomena in the world refer to them directly or have special personal significance), and auditory and visual hallucinations in otherwise healthy individuals [56;57]. However, sensitization is not always prominently exhibited by humans, and the applicability of behavioral sensitization to the human condition is confounded by retrospective reports from individuals who experienced psychosis following drug binges. Additionally, imaging studies using PET and fMRI techniques have generally shown a profile in limbic regions of reduced rather than augmented responses in cocaine addicted patients compared to controls to intravenous methylphenidate, a drug that, like cocaine, causes an increase in synaptic dopamine [58]. Still, it is a phenomenon that appears useful in studying the neural changes that occur with repeated drug or stress administration and that may contribute to the escalation of drug taking.

Exposure to stress can induce cross-sensitization to psychostimulants [Table 1], as indicated by an augmented locomotor response to a later psychostimulant challenge [59]. For example, repeated tail-pinch stress in rats results in behavioral sensitization to a later injection of amphetamine [60]. Sensitized behavioral responses to psychostimulants have also been observed after repeated foot shock [61;62], restraint [63], food restriction [30], social defeat stress [64;50]; [Fig. 1b] and maternal separation [29]. Many stress manipulations, including handling stress, swim stress, foot-shock and social defeat stress, increase the metabolic responses of dopamine neurons and change dopamine activity in the mesocorticolimbic system [61; 65–67], particularly in the NAC and prefrontal cortex (PFC) [68–75], in a way similar to drugs of abuse (reviewed in detail in [26]). The mesocorticolimbic dopamine system connects stress to subsequent escalated drug self-administration through changes in neural plasticity in this circuit [26;76].

Conditioned place preference

As discussed in detail elsewhere [77–79], place preference or aversion is based on a Pavlovian conditioning procedure that is most often used with rodents to study the positive (rewarding) or negative (aversive) motivational effects of a particular stimulus. One of the earliest demonstrations of place preference was the observation by Olds and Milner [80] in which rats stimulated through an intracranial electrode would return to the location in which they received the stimulation. In brief, the standard procedure pairs a distinctive environmental cue (positive conditioned stimulus, CS+) with a significant event (unconditioned stimulus, US). In a simple version of the place preference paradigm, animals experience two distinct neutral environments that are subsequently paired spatially and temporally with distinct drug states (the US). The animal is later given an opportunity to choose, in the absence of the drug state, to enter and explore either environment, and the time spent in either environment is considered an index of the rewarding effect of the drug (the US). The animal’s choice to spend more time in the drug-paired environment is assumed to be an expression of the positively rewarding experience within that environment. The main advantages of conditioned place preference (CPP) are: (1) it tests animals in a drug-free state, relying on the memory of previous associations, (2) it is sensitive to both reward and aversion, (3) it is methodologically simple, (4) protocols are generally brief (ca. 2 weeks), and (5) locomotor activity can be concurrently measured, although changes in locomotor activity may confound preference measurements [78;79].

From a pharmacological perspective, one of the most serious disadvantages of this methodology is that it is difficult to produce graded dose-effect curves, with most drugs showing little effect across a range of low doses before suddenly showing a maximal effect across a range of higher doses, leaving many pharmacological questions unanswered [78;79]. Another disadvantage is dealing with a bias to one side of the apparatus, where the animal will prefer one of the two distinct contexts before conditioning occurs [77]. Despite these limitations, CPP provides good preliminary information about the rewarding effect of the environmental context associated with a drug stimulus, and the influence of stress experiences on this effect can be examined. The CPP technique is relatively inexpensive, non-invasive, and simple to use. Although the CPP method does not directly measure drug reinforcement, the concordance between CPP and self-administration studies is fairly good [81].

Results using various stressors and CPP have been intriguing, with an emerging trend towards enhancement of CPP [Table 1]. A single session of inescapable tail shock stress enhances morphine CPP [82;83], up to 200% at high doses, but not cocaine, amphetamine or ethanol CPP [82;84]. This effect of electric tail shock stress on morphine CPP is seen only when it is given 6–7 days prior to CPP training and not when given earlier (14–15 days). Repeated forced-swim stress preceding cocaine conditioning nearly doubles the subsequent place preference response in male C57Bl/6 mice [85]. Results from studies using food deprivation stress are mixed, depending on the drug used. One study showed that 3 days of food restriction greatly increases the rewarding motivational effects of morphine, 2–3 fold above the sated condition [86]. In another study, chronic food restriction to 80% of free-feeding body weight modestly increases cocaine CPP for a medium dose (5 mg/kg), but not for a lower (2.5 mg/kg) or higher (10 mg/kg) doses, highlighting the difficulty in using the CPP methodology for studying dose-dependent shifts in drug response [87]. Mild chronic food restriction of 15 g/day (ca. 90% of free-feeding body weight) enhances amphetamine CPP for a moderate dose (0.85 mg/kg), but decreases preference for two higher doses (1.7 and 3.4 mg/kg) [88]. Contrary to what is found using tail shock stress and food deprivation, social defeat stress has not been studied in a systematic manner in terms of its effect on CPP. In one study housed rats in pairs for at least 16 weeks -- enough time for the social hierarchy to be determined – only dominant, and not subordinate, rats demonstrated CPP for morphine [89], suggesting the important influence of social status on morphine-induced place conditioning. In a later study, social defeat stress-exposed C57Bl/6 mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place-preference for the drug-paired chamber over the responses of unstressed mice [90]. While CPP is enhanced by stress in most studies, it is only seen under specific stress and drug conditions.

Intravenous self-administration

The gold standard for modeling essential features of drug abuse in the laboratory is the intravenous self-administration methodology. Laboratory animals can be surgically prepared with intravenous catheters that permit automated drug injections [91]. Nearly all drugs that are addictive in humans are self-administered by laboratory animals, and drugs that are not self-administered by laboratory animals are generally not addictive in humans. Notable exceptions are LSD-like hallucinogens, although hallucinogens have not been found to cause physical or psychological dependence [92;93]. I.V. self-administration is used to study the neurobiological basis of drug taking and seeking, and to screen new psychoactive medications for possible abuse liability. This last application helps the pharmaceutical industry minimize the risk that new medications will be abused by humans.

As an individual rodent learns to self-administer drugs, there are time- and experience-dependent changes in the frequency and intensity of behavior. Thus, acquisition is a process with the essential feature of an increase in drug use over time. Indeed, acquisition is just one example of self-administration behavior in transition. Others include the escalation of drug use during extended access or “binge” conditions as physical dependence develops [94] or as brain reward systems change [64;95–100], the change in self-administration behavior when the pharmacological effects of the drug are blocked or removed (i.e., extinction, withdrawal, or compensatory increases in drug intake due to blockade of drug’s actions), and the reacquisition of behavior after a period of abstinence or extinction of previous drug taking. Both exposure to foot shock stress and social stress (e.g., [101] and [102] can facilitate acquisition of drug self-administration. Other pharmacological and environmental factors that influence acquisition are dose (e.g., [103]and [104]), availability of alternative reinforcement (e.g., [105]), sex (e.g., [106]and [107]), rat strain (e.g., [108]), developmental stage (e.g., [109]) and the presence of drug-paired sensory stimuli (e.g., [110]). Self-administration behavior during transition periods may provide unique information about individual differences in drug use that are not evident during stable drug-taking, and numerous factors, particularly specific stresses, can enhance acquisition of drug taking.

Studies using self-administration sessions of continuous access to the drug for an extended period of time or a progressive ratio schedule highlight different aspects of drug taking behavior. “Binge” self-administration in rodents is thought to mimic the compulsive drug taking behavior of human addicts. Unlike limited access conditions, prolonged access or “binges” in pre-clinical studies reveal a pattern of self-administration behavior similar to that by the compulsive drug user [111–113]. Specifically, rodent models of prolonged access to cocaine indicate an apparent shift from controlled, regulated drug intake to out-of- control or dysregulated intake [96;114]. Clinical literature suggests that the transition from recreational drug intake to compulsive drug seeking behavior is a hallmark of addiction [115]. Self-administration models of continuous access “binges” allow the animal to control the rate of consumption [113]. However, unlimited access to cocaine can, after several days or weeks, degenerate into severe toxicity resulting eventually in death [91;116;117]. While the behavior of animals given unlimited access models some important clinical features of cocaine use by human addicts, some limitations must be placed on the amount of drug the animal can administer to avoid toxicity. Some have studied the effect of binge intake by limiting access to a single session of 72 h [96] and, most often, less. Access has been examined over longer periods by limiting daily sessions to, for example, 6 [95;118] or 10 h [119;120]. Another procedure is the use of continuous discrete trials over the course of several days, as described and reviewed in detail by Olsen and Roberts in this special issue [121]. Due to the higher cumulative dose and different kinetics of binge self-administration protocols, they can produce some changes in gene expression not seen with noncontingent cocaine administration or limited access self-administration [122].

Progressive ratio schedules of reinforcement are thought to measure an animal’s motivation to self-administer a particular drug [123]. However, an equal interpretation for the progressively higher behavioral output expended in progressive ratio tests could be that of resistance to extinction (i.e. a resistance to cease responding in the absence of reward). In the simplest case, the first behavioral response is reinforced, and each subsequent reinforcement is delivered after meeting a doubling in behavioral demand. An example of this type of PR schedule would be 1, 2, 4, 8, 16, etc. The requirement increases until the behavioral demand eventually becomes so large that the subject fails to meet the response criterion for the next reinforcement within a criterion time of 30 or 60 min. The last completed ratio requirement after the subject stops being reinforced is defined as the “breaking point” or “break point.” Dynamic patterns of behavior are maintained by PR schedules of drug delivery. These response patterns reflect not only the influence of ratio requirement, but also the effects of increasing blood and brain concentrations of the self-administered drug, which can impact operant behavior by producing motor-behavior disruption or stimulation, temporary satiation, or other effects. The influence of accumulating blood levels of drug may be particularly substantial at the beginning of a session and soon thereafter, when several small-sized ratios can be completed in rapid succession [124]. The break point may be an indicator of the motivation to continue a self-administration bout, which is different from the motivation to initiate drug self-administration [121;125].

Several reports assessed the effect of intermittent shock on opiate (morphine and heroin) and cocaine i.v. self-administration during acquisition and daily limited access (i.e., maintenance) phases. Female rats trained to self-administer morphine for 3 weeks increased their response rate until they self-administered lethal doses of the drug when each lever press resulted in a brief mild shock to the leg. A possible reason for this result is the association of morphine delivery with pain relief [126]. In another study, rats received ten minutes of mild intermittent foot shock exposure before each of four daily heroin self-administration sessions [127]. Following acquisition of heroin-reinforced behavior (100 micrograms/kg per infusion), during which no group differences emerged, animals were placed on a progressive ratio schedule of reinforcement. Animals exposed to foot shock before each drug session had higher rates of lever pressing for heroin and achieved higher final ratios on the progressive ratio schedule than animals in the control group at the higher doses of heroin. Thus, under the conditions of this experiment, exposure to mild intermittent stress appeared to enhance the reinforcing effects of heroin, where animals responded at higher rates for the drug after foot shock. Under certain conditions, intermittent shock can increase cocaine self-administration behavior. Rats observing another rat receiving foot shock just prior to five daily sessions initiated cocaine self-administration reinforced by a very low dose that was insufficient for control rats or rats exposed to the shock to initiate cocaine self-administration (0.031 mg/kg/infusion) [128]. In another study, [129], rats given foot shock during sessions of food self-administration, just prior to the cocaine self-administration sessions showed enhanced acquisition of cocaine self-administration. This finding was extended, showing foot shock enhancing acquisition of cocaine self-administration, regardless of whether or not shocks were given contingently or non-contingently with lever pressing behavior [130]. Under most conditions, brief intermittent foot shocks facilitate the acquisition of cocaine self-administration and can increase responding for heroin under a progressive ratio schedule of reinforcement.

Several studies showed that acute food deprivation or restriction (24-hr deprivation of food or restriction of feeding amount to 5–8 g per day in rats) or chronic food restriction (i.e., multiple days or weeks of limited access to food) significantly increased the initiation and maintenance of i.v. self-administration of opiates and psychomotor stimulants. Chronic restricted feeding to 80% of free-feeding weight accelerated the acquisition of heroin and methadone self-administration [131;132], increased heroin intake during the maintenance phase [131], and increased the initiation of amphetamine self-administration over a range of unit doses (0.05–0.8 mg/kg) [133]. These findings have been extended to the initiation and maintenance of cocaine and amphetamine self-administration [134–138]. Using food restriction of 8–12 g of food either every third day or every day, rats showed increased cocaine self-administration behavior during the maintenance phase ([139]; [140–142], but see [134]). Also, 20 g of food per day accelerated acquisition of cocaine self-administration [143]. Daily food restriction, under most conditions, facilitates heroin, amphetamine and cocaine taking behavior during acquisition and daily limited access periods, and this may be attributed to neural changes that occur in the mesocorticolimbic dopamine system in response to stress [144].

Brief social stress can produce enduring neural sensitization as expressed through immediate early gene activation in the mesocorticolimbic circuit, and this genomic change due to stress may play a role in the transition to compulsive drug taking. Using a protocol of 4 intermittent defeat episodes over the course of 10 days in rats, repeated social defeat stress resulted in a decline in basal zif268 immediate early gene expression in the prefrontal cortex and an increase in zif268 expression in the central and medial amygdala for as long as 60 days after the last defeat episode [145]. Defeated rats also showed sensitized Fos labeling in the VTA and amygdala 60 days after the last stress experience [146]. In rats that previously experienced social defeat, exposure to olfactory, visual and auditory cues increased accumbens dopamine release; previously defeated rats also acquired intravenous cocaine self-administration in half the time non-stressed rats did [102]. Defeat stress-induced sensitization increased cocaine self-administration and intake [64]. Rats that experienced four social defeat episodes increased drug intake when compared to non-stressed control animals during a 24-hour continuous access session [64;145]; [Fig. 2]. Defeat stress-sensitized rats, but not mice, also achieved higher “break points” to a low dose of cocaine when placed on a progressive ratio schedule of reinforcement [64; 147; 148]. Recent studies point to the role of NMDA receptor activation in the VTA in the induction of behavioral sensitization and in prompting heightened cocaine intake during the binge [50;97].

This escalation in cocaine self-administration during a 24-hour binge due to previous defeat stress experience can be attributed to the activity of the extracellular signal-regulated kinase (ERK) pathway, which when activated by repeated cocaine administration, can promote the downstream activation of the transcription factor cAMP response element binding protein (CREB) and enhance immediate early gene expression of c-fos and zif268 [149;150], genes also activated in response to repeated social defeat [145;146]. Using the MEK (mitogen-activated protein (MAP) kinase kinase; the immediate upstream activator kinase of ERK) inhibitor U0126 to prevent the phosphorylation of ERK in the VTA before each social defeat protected against the development of social defeat stress-induced sensitization and stress-induced increases in cocaine intake during the binge [151], highlighting the critical role of the VTA in the potentiation of cocaine self-administration due to social defeat. Therefore, the cascade of molecular and intracellular events that occur in the VTA in response to brief social defeat episodes can modulate subsequent intravenous cocaine taking behavior, particularly during a 24-hour continuous access binge.

Reinstatement

Clinical and epidemiological evidence documents the relapsing nature of drug addiction, and insights into the factors that promote and trigger relapse require adequate experimental models. There are two main versions of the reinstatement model that are employed with the aim to approximate the clinical phenomenon of relapse to drug abuse; one based on the operant self-administration procedure, and the other on the classical conditioned place preference procedure. In the last decade, the use of the latter version has become more widespread due to its ease of implementation, and the results obtained mostly complement those obtained in self-administration studies. It has been observed that the conditioned place preference induced by opioids, psychostimulants, nicotine, ethanol and other drugs of abuse can be extinguished and reinstated by drug priming or exposure to stressful events.

Over 25 years ago, de Wit and Stewart [152;153] used a reinstatement procedure in which the effect of acute non-contingent exposure to drug or non-drug stimuli on reinstatement of drug seeking was determined following training for drug self-administration and subsequent extinction of the drug-reinforced behavior. Non-contingent priming injections of the self-administered drug or related drugs reinstate lever-pressing behavior in rats with a history of cocaine or heroin self-administration. Based on these data, and those from earlier studies [154–156], de Wit and Stewart suggested that the reinstatement procedure may approximate an animal model of drug relapse. The reinstatement procedure has also been suggested as a model of drug-induced craving [157], although this subjective description can only be inferred. The appeal of the reinstatement model for basic scientists and clinicians derives from the observations that factors reported to reinstate drug seeking in laboratory animals also provoke relapse and craving in humans. These factors include re-exposure to drug or drug-associated cues ([158–161]; reviewed in detail by Fuchs et al. in this issue [162]) and exposure to certain stressors [3;163]. Both drugs and some types of stressors can reinstate drug seeking in laboratory animals even following prolonged withdrawal periods [164]. Furthermore, intracranial drug injections of morphine or amphetamine into the ventral tegmental area or the nucleus accumbens reinstate heroin or cocaine seeking, respectively [165;166], and conditioned cues previously paired with drug infusions reinstate morphine or cocaine seeking in rhesus monkeys or rats when extinction of lever pressing was conducted in the absence of these cues [167;168;156]. The most significant criticism of the early reinstatement model is that human addicts do not experience extinction of the drug habit, a condition in which the individual must actively learn that a response is no longer reinforced and inhibit the behavior leading to the drug delivery. Recent methods limit extinction learning, with extinction sessions and reinstatement testing conducted in the same day [157;164;169], as opposed to prolonged periods of extinction training over multiple daily sessions [154;156].

Most stress-induced reinstatement studies implement intermittent low-intensity foot shock as a form of stress because of its ease of use and its reliability in the reinstatement of drug seeking. This reinstating effect of mild foot shock [170;171] generalized to acute food deprivation but did not extend to restraint stress [172]. Transient inhibition of the central amygdala (CeA), NAC shell, NAC core, VTA, and the dorsal PFC blocked the effect of foot shock stress to reinstate lever pressing previously associated with cocaine delivery [173]. However, inhibition of the basolateral amygdala, mediodorsal nucleus of the thalamus, or the ventral prefrontal cortex had no effect on drug-seeking behavior. These data suggest that foot shock stress activates limbic circuitry of the CeA that, via the VTA, activates motor output circuitry responsible for producing lever press responding. Consistent with this proposal, the D1/D2 dopamine receptor antagonists fluphenazine and SCH 23390 blocked foot shock-induced reinstatement when infused into the PFC [173] or NAC [174], respectively. Further, inhibition of the NAC shell blocked the foot shock-induced increase in dopamine within the PFC and concomitantly blocked reinstatement responding [173]. Inactivation of the PFC was also shown to block stress-induced glutamate release within the NAC core while concurrently inhibiting reinstatement responding [173]. Taken together, these data suggest that foot shock activates limbic circuitry in the CeA, which in turn activates a VTA dopamine projection to the PFC. The rise in dopamine within the PFC initiates reinstatement via a glutamatergic projection to the NAC core.

Additional evidence points to the role of corticotropin-releasing factor (CRF) receptor activation in mesocorticolimbic areas, particularly the VTA and bed nucleus of the stria terminalis (BNST) [175–177]. Intracerebroventricular (i.c.v.) administration of the nonselective CRF antagonists α-helical CRF and d-Phe-CRF and systemic injections of the specific CRF1 receptor antagonist CP-154,526 attenuate foot shock-induced reinstatement of heroin, cocaine, and alcohol seeking [178–181]. I.c.v. and intraperitoneal (i.p.) blockade of the CRF receptors and specifically the CRF1 receptor similarly attenuates foot shock-induced reinstatement of morphine- and cocaine-conditioned place preference [182;183]. Foot shock reinstates cocaine seeking in cocaine-experienced rats by inducing corticotropin-releasing factor (CRF) and glutamate release in the VTA and thus activating VTA dopaminergic neurons [175]. Foot shock-induced VTA glutamate release, dopamine activation and reinstatements are blocked by VTA administration of alpha-helical CRF, a nonselective CRF receptor antagonist [175] as well as by infusion of the selective CRF2 receptor antagonist antisauvagine-30 into the VTA, while infusion of selective CRF1 receptor antagonists NBI-27914 or R121919 had no effect, suggesting that reinstatement of cocaine seeking and increases in dopamine and glutamate release in the VTA due to mild foot shock stress are primarily dependent on CRF2 receptor activation in the VTA [184]. The suggestion that CRF2 receptors are critical is not easily reconciled with: (1) the foot shock-induced reinstatement of morphine and cocaine conditioned place preference studies using i.c.v and systemic administration of CRF1 receptor antagonists [182;183] and (2) the findings that subcutaneous injections of a selective CRF1 receptor antagonist block reinstatement of cocaine, heroin, and alcohol seeking induced by footshock stress [179;181]. Moreover, CRF has a substantial weaker affinity for CRF2 receptors than for CRF1 receptors [185]. One of the possibilities may be that the CRF1 receptor-mediated effects are expressed in other brain regions that are also immunoreactive for CRF and CRF receptors [184]. Indeed, it has been shown that direct injections of CRF antagonists, including a selective CRF1 receptor antagonist, into the BNST are sufficient to block foot shock-induced reinstatements of either cocaine or morphine seeking [176] [186], where CRF1 and CRF2 receptors have both been identified.

Reliable reinstatement of heroin and cocaine seeking after exposure to acute, 24-hr food deprivation [172;187] was previously shown to be controlled by a leptin-dependent mechanism. Food deprivation-induced reinstatement of heroin seeking was completely blocked by i.c.v. administration of leptin [188]. However, leptin administration had no effect on foot shock- or heroin priming-induced reinstatement of heroin seeking [188], suggesting that the neural mechanisms that underlie food deprivation-induced reinstatement may be dissociable from those involved in reinstatement induced by foot shock stress or drug priming [189]. However, a later study showed that both foot shock-induced reinstatement of drug seeking [178] and reinstatement induced by acute food deprivation [190] are mediated by common stress-related neural pathways, with i.c.v. administration of CRF receptor antagonists dose-dependently attenuating both foot shock- and food deprivation-induced reinstatement of heroin seeking and adrenalectomy, which removes circulating corticosterone, affecting neither. Acute 1-day food deprivation stress also reinstates previously extinguished cocaine seeking, and in this case, this was attenuated by adrenalectomy [187], extending the finding that adrenalectomy attenuates foot shock-induced reinstatement of cocaine seeking [180]. While there is some overlap, there are differences in the way food deprivation and foot shock reinstate heroin- and cocaine-seeking behavior, particularly regarding the involvement of circulating corticosterone.

The only study to date using social defeat stress as the trigger for reinstatement has been with morphine CPP [191]. Adult male OF1 mice were conditioned with 10, 20, or 40 mg/kg of morphine or saline. Only morphine-conditioned animals acquired CPP, and all mice underwent extinction sessions. A single encounter with an aggressive resident male mouse was effective in reinstating morphine CPP. However, while an odor cue associated with defeat stress has been shown to reinstate alcohol drinking [192], reinstated cocaine or heroin seeking in rats that previously self-administered these drugs intravenously has yet to be established.