Necessity and recruitment of cue-specific neuronal ensembles within the basolateral amygdala during appetitive reversal learning.

Through Pavlovian appetitive conditioning, environmental cues can become predictors of food availability. Over time, however, the food, and thus the value of the associated cues, can change based on environmental variations. This change in outcome necessitates updating of the value of the cue to appropriately alter behavioral responses to these cues. The basolateral amygdala (BLA) is critical in updating the outcomes of learned cues. However, it is unknown if the same BLA neuronal ensembles that are recruited in the initial associative memory are required when the new cue-outcome association is formed during reversal learning. The current study used the Daun02 inactivation method that enables selective targeting and disruption of activated neuronal ensembles in Fos-lacZ transgenic rats. Rats were implanted with bilateral cannulas that target the BLA and underwent appetitive discriminative conditioning in which rats had to discriminate between two auditory stimuli. One stimulus (CS+) co-terminated with food delivery, and the other stimulus was unrewarded (CS-; counterbalanced). Rats were then tested for CS+ or CS- memory retrieval and infused with either Daun02 or a vehicle solution into the BLA to inactivate either CS+ or CS- neuronal ensembles that were activated during that test. To assess if the same neuronal ensembles are necessary to update the value of the new association when the outcomes are changed, rats underwent reversal learning: the CS+ was no longer followed by food (reversal CS-, rCS-), and the CS- was now followed by food (reversal CS+; rCS+). The group that received Daun02 following CS+ session showed a decrease in conditioned responding and increased latency to the rCS- (previously CS+) during the first session of reversal learning, specifically during the first trial. This indicates that the neuronal ensemble that was activated during the recall of the CS+ memory was the same neuronal ensemble needed for learning the new outcome of the same CS, now rCS-. Additionally, the group that received Daun02 following CS- session was slower to respond to the rCS+ (previously CS-) during reversal learning. This indicates that the neuronal ensemble that was activated during the recall of the CS- memory was the same neuronal ensemble needed for learning the new outcome of the same CS. These results demonstrate that different neuronal ensembles within the BLA mediate memory recall of CS+ and CS- cues and reactivation of each cue-specific neuronal ensemble is necessary to update the value of that specific cue to respond appropriately during reversal learning. These results also indicate substantial plasticity within the BLA for behavioral flexibility as both groups eventually showed similar terminal levels of reversal learning.

Fos-expressing neuronal ensemble in rat ventromedial prefrontal cortex encodes cocaine seeking but not food seeking in rats.

Neuronal ensembles in ventromedial prefrontal cortex (vmPFC) play a role in both cocaine and palatable food seeking. However, it is unknown whether similar or different vmPFC neuronal ensembles mediate food and cocaine seeking. Here, we used the Daun02 inactivation procedure to assess whether the neuronal ensembles mediating food and cocaine seeking can be functionally distinguished. We trained male and female Fos-LacZ rats to self-administer palatable food pellets and cocaine on alternating days for 18 days. We then exposed the rats to a brief nonreinforced food- or cocaine-seeking test to induce Fos and ß-gal in neuronal ensembles associated with food or cocaine seeking, respectively and infused Daun02 into vmPFC to ablate the ß-gal-expressing ensembles. Two days later, we tested the rats for food or cocaine seeking under extinction conditions. Although inactivation of the food-seeking ensemble did not influence food or cocaine seeking, inactivation of the cocaine-seeking ensemble reduced cocaine seeking but not food seeking. Results indicate that the neuronal ensemble activated by cocaine seeking in vmPFC is functionally separate from the ensemble activated by food seeking.

Separate vmPFC Ensembles Control Cocaine Self-Administration Versus Extinction in Rats.

Recent studies suggest that the ventral medial prefrontal cortex (vmPFC) encodes both operant drug self-administration and extinction memories. Here, we examined whether these opposing memories are encoded by distinct neuronal ensembles within the vmPFC with different outputs to the nucleus accumbens (NAc) in male and female rats. Using cocaine self-administration (3 h/d for 14 d) and extinction procedures, we demonstrated that vmPFC was similarly activated (indexed by Fos) during cocaine-seeking tests after 0 (no-extinction) or 7 extinction sessions. Selective Daun02 lesioning of the self-administration ensemble (no-extinction) decreased cocaine seeking, whereas Daun02 lesioning of the extinction ensemble increased cocaine seeking. Retrograde tracing with fluorescent cholera toxin subunit B injected into NAc combined with Fos colabeling in vmPFC indicated that vmPFC self-administration ensembles project to NAc core while extinction ensembles project to NAc shell. Functional disconnection experiments (Daun02 lesioning of vmPFC and acute dopamine D1-receptor blockade with SCH39166 in NAc core or shell) confirm that vmPFC ensembles interact with NAc core versus shell to play dissociable roles in cocaine self-administration versus extinction, respectively. Our results demonstrate that neuronal ensembles mediating cocaine self-administration and extinction comingle in vmPFC but have distinct outputs to the NAc core and shell that promote or inhibit cocaine seeking.SIGNIFICANCE STATEMENT Neuronal ensembles within the vmPFC have recently been shown to play a role in self-administration and extinction of food seeking. Here, we used the Daun02 chemogenetic inactivation procedure, which allows selective inhibition of neuronal ensembles identified by the activity marker Fos, to demonstrate that different ensembles for cocaine self-administration and extinction memories coexist in the ventral mPFC and interact with distinct subregions of the nucleus accumbens.

Circuit and Synaptic Plasticity Mechanisms of Drug Relapse.

High rates of relapse to drug use during abstinence is a defining feature of human drug addiction. This clinical scenario has been studied at the preclinical level using different animal models in which relapse to drug seeking is assessed after cessation of operant drug self-administration in rodents and monkeys. In our Society for Neuroscience (SFN) session entitled "Circuit and Synaptic Plasticity Mechanisms of Drug Relapse," we will discuss new developments of our understanding of circuits and synaptic plasticity mechanisms of drug relapse from studies combining established and novel animal models with state-of-the-art cellular, electrophysiology, anatomical, chemogenetic, and optogenetic methods. We will also discuss the translational implications of these new developments. In the mini-review that introduces our SFN session, we summarize results from our laboratories on behavioral, cellular, and circuit mechanisms of drug relapse within the context of our session.

Selective Inhibition of Amygdala Neuronal Ensembles Encoding Nicotine-Associated Memories Inhibits Nicotine Preference and Relapse.

BACKGROUND: Nicotine craving and relapse often occurs after reactivation of nicotine reward memories. We recently developed a memory retrieval-reconsolidation interference procedure in which reactivating nicotine reward memories by acute exposure to nicotine (the unconditioned stimulus [UCS]) and then pharmacologically interfering with memory reconsolidation decreased relapse to nicotine seeking in rats and nicotine craving in smokers. Here, we investigated underlying mechanisms. METHODS: In the first series of experiments, we trained rats for nicotine-induced conditioned place preference (CPP) or nicotine self-administration and ventricularly microinjected them with the protein synthesis inhibitor anisomycin immediately after UCS-induced memory retrieval. In the second series of experiments, we used tyramide-amplified immunohistochemistry-fluorescence in situ hybridization to examine neural ensembles in the basolateral amygdala (BLA) reactivated by nicotine conditioned stimulus- or UCS-induced memory retrieval. We then used the Daun02 chemogenetic inactivation procedure to selectively inhibit the nicotine UCS-reactivated BLA neuronal ensembles. RESULTS: Ventricular injections of the anisomycin immediately after nicotine UCS memory retrieval inhibited subsequent nicotine CPP and relapse to operant nicotine seeking after short or prolonged abstinence. More important, within BLA, distinct neuronal ensembles encoded pavlovian CPP and operant self-administration reward memories and nicotine (the UCS) injections in the home cage reactivated both neuronal ensembles. Daun02 chemogenetic inactivation of the nicotine-reactivated ensembles inhibited both nicotine CPP and relapse to nicotine seeking. CONCLUSIONS: Results demonstrate that the nicotine UCS-induced memory retrieval manipulation reactivates multiple nicotine reward memories that are encoded by distinct BLA neuronal ensembles that play a role in nicotine preference and relapse.

Role of Dorsomedial Striatum Neuronal Ensembles in Incubation of Methamphetamine Craving after Voluntary Abstinence.

We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral striatum (DLS) and dorsomedial striatum (DMS) in this incubation. We trained rats to self-administer palatable food pellets (6 d, 6 h/d) and methamphetamine (12 d, 6 h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/d) for 19 d. We used in situ hybridization to measure the colabeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization colabeling results, we tested the causal role of DMS D1 and D2 family receptors, and DMS neuronal ensembles in "incubated" methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively. Methamphetamine seeking was higher after 21 d of voluntary abstinence than after 1 d (incubation of methamphetamine craving). The incubated response was associated with increased Fos expression in DMS but not in DLS; Fos was colabeled with both Drd1 and Drd2 DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. In Fos-lacZ transgenic rats, selective inactivation of relapse test-activated Fos neurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21. Results demonstrate a role of DMS dopamine D1 and D2 receptors in the incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation. SIGNIFICANCE STATEMENT: In human addicts, abstinence is often self-imposed and relapse can be triggered by exposure to drug-associated cues that induce drug craving. We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we used classical pharmacology, in situ hybridization, immunohistochemistry, and the Daun02 inactivation procedure to demonstrate a critical role of dorsomedial striatum neuronal ensembles in this new form of incubation of drug craving.

Daun02 Inactivation of Behaviorally Activated Fos-Expressing Neuronal Ensembles.

Learned associations about salient experiences (e.g., drug exposure, stress) and their associated environmental stimuli are mediated by a minority of sparsely distributed, behaviorally activated neurons coined 'neuronal ensembles.' For many years, it was not known whether these neuronal ensembles played causal roles in mediating learned behaviors. However, in the last several years the 'Daun02 inactivation technique' in Fos-lacZ transgenic rats has proved very useful in establishing causal links between neuronal ensembles that express the activity-regulated protein Fos and learned behaviors. Fos-expressing neurons in these rats also express the bacterial protein ß-galactosidase (ß-gal) in strongly activated neurons. When the prodrug Daun02 is injected into the brains of these rats 90 min after a behavior (e.g., drug-seeking) or cue exposure, then Daun02 is converted into daunorubicin by ß-gal, which selectively inactivates Fos- and ß-gal-expressing neurons that were activated 90 min before the Daun02 injection. This unit presents protocols for breeding the Fos-lacZ rats and conducting appropriate Daun02 inactivation experiments. © 2016 by John Wiley & Sons, Inc.

Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories.

UNLABELLED: In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in "neuronal ensembles." Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area. SIGNIFICANCE STATEMENT: A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.

Losing Control: Excessive Alcohol Seeking after Selective Inactivation of Cue-Responsive Neurons in the Infralimbic Cortex.

Loss of control over drinking is a key deficit in alcoholism causally associated with malfunction of the medial prefrontal cortex (mPFC), but underlying molecular and cellular mechanisms remain unclear. Cue-induced reinstatement of alcohol seeking activates a subset of mPFC neurons in rats, identified by their common expression of the activity marker cFos and comprised of both principal and interneurons. Here, we used cFos-lacZ and pCAG-lacZ transgenic rats for activity-dependent or nonselective inactivation of neurons, respectively, which by their lacZ encoded ß-galactosidase activity convert the inactive prodrug Daun02 into the neurotoxin daunorubicin. We report that activity-dependent ablation of a neuronal ensemble in the infralimbic but not the prelimbic subregion induced excessive alcohol seeking. The targeted neuronal ensemble was specific for the cue-induced response because stress-induced reinstatement was not affected in these animals. Importantly, nonselective inactivation of infralimbic neurons, using pCAG-lacZ rats, was without functional consequence on the cue-induced reinstatement task. Thus, inhibitory control over alcohol seeking is exerted by distinct functional ensembles within the infralimbic cortex rather than by a general inhibitory tone of this region on the behavioral output. This indicates a high level of functional compartmentation within the rat mPFC whereat many functional ensembles could coexist and interact within the same subregion. SIGNIFICANCE STATEMENT: Hebb's (1949) idea of memories as being represented in local neuronal networks is supported by identification of transiently stable activity patterns within subgroups of neurons. However, it is difficult to link individual networks to specific memory tasks, for example a learned behavior. By a novel approach of activity-dependent ablation, here we identify a specific neuronal ensemble located in the infralimbic subregion of the medial prefrontal cortex that controls a seeking response for alcohol in rats. Our data demonstrate that functional output depends on specific neuronal ensembles within a given brain region rather than on the global activity of that region, which raises important questions about the interpretation of numerous earlier experiments using site-directed silencing or stimulation for elucidating brain function.

Using c-fos to study neuronal ensembles in corticostriatal circuitry of addiction.

Learned associations between drugs and environment play an important role in addiction and are thought to be encoded within specific patterns of sparsely distributed neurons called neuronal ensembles. This hypothesis is supported by correlational data from in vivo electrophysiology and cellular imaging studies in relapse models in rodents. In particular, cellular imaging with the immediate early gene c-fos and its protein product Fos has been used to identify sparsely distributed neurons that were strongly activated during conditioned drug behaviors such as drug self-administration and context- and cue-induced reinstatement of drug seeking. Here we review how Fos and the c-fos promoter have been employed to demonstrate causal roles for Fos-expressing neuronal ensembles in prefrontal cortex and nucleus accumbens in conditioned drug behaviors. This work has allowed identification of unique molecular and electrophysiological alterations within Fos-expressing neuronal ensembles that may contribute to the development and expression of learned associations in addiction.

Role of nucleus accumbens shell neuronal ensembles in context-induced reinstatement of cocaine-seeking.

Environmental contexts previously associated with drug use provoke relapse to drug use in humans and reinstatement of drug seeking in animal models of drug relapse. We examined whether context-induced reinstatement of cocaine seeking is mediated by activation of context-selected nucleus accumbens neurons. We trained rats to self-administer cocaine in Context A and extinguished their lever-pressing in a distinct Context B. On test day, reexposure to the cocaine-associated Context A reinstated cocaine seeking and increased expression of the neural activity marker Fos in 3.3% of accumbens shell and 1.6% of accumbens core neurons. To assess a causal role for these activated neurons, we used the Daun02 inactivation procedure to selectively inactivate these neurons. We trained c-fos-lacZ transgenic rats to self-administer cocaine in Context A and extinguished their lever-pressing in Context B. On induction day, we exposed rats to either Context A or a novel Context C for 30 min and injected Daun02 or vehicle into accumbens shell or core 60 min later. On test day, 3 d after induction day, the ability of Context A to reinstate cocaine seeking and increase neuronal activity in accumbens shell was attenuated when Daun02 was previously injected after exposure to Context A. Daun02 injections after exposure to the novel Context C had no effect on context-induced reinstatement of cocaine seeking despite much greater numbers of Fos-expressing neurons induced by Context C. Daun02 injections in accumbens core had no effect. Our data suggest that context-induced reinstatement of cocaine seeking is mediated by activation of context-selected accumbens shell but not core neuronal ensembles.