Repeated injections of cocaine and morphine in laboratory rats cause a variety of molecular neuroadaptations in the cAMP signaling pathway in nucleus accumbens and ventral tegmental area. Here we report similar neuroadaptations in postmortem tissue from the brains of human smokers and former smokers. Activity levels of two major components of cAMP signaling, cAMP-dependent protein kinase A (PKA) and adenylate cyclase, were abnormally elevated in nucleus accumbens of smokers and in ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of the catalytic subunit of PKA were correspondingly higher in the ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of other candidate neuroadaptations, including glutamate receptor subunits, tyrosine hydroxylase, and other protein kinases, were within normal range. These findings extend our understanding of addiction-related neuroadaptations of cAMP signaling to tobacco smoking in human subjects and suggest that smoking-induced brain neuroadaptations can persist for significant periods in former smokers.

Drug addiction is a chronic relapsing disorder of compulsive drug use. Studies of the neurobehavioral factors that promote drug relapse have yet to produce an effective treatment. Here we take a different approach and examine the factors that suppress-rather than promote-relapse. Adapting Pavlovian procedures to suppress operant drug response, we determined the anti-relapse action of environmental cues that signal drug omission (unavailability) in rats. Under laboratory conditions linked to compulsive drug use and heightened relapse risk, drug omission cues suppressed three major modes of relapse-promotion (drug-predictive cues, stress, and drug exposure) for cocaine and alcohol. This relapse-suppression is, in part, driven by omission cue-reactive neurons, which constitute small subsets of glutamatergic and GABAergic cells, in the infralimbic cortex. Future studies of such neural activity-based cellular units (neuronal ensembles/memory engram cells) for relapse-suppression can be used to identify alternate targets for addiction medicine through functional characterization of anti-relapse mechanisms.

Learned associations between environmental stimuli and rewards drive goal-directed learning and motivated behavior. These memories are thought to be encoded by alterations within specific patterns of sparsely distributed neurons called neuronal ensembles that are activated selectively by reward-predictive stimuli. Here, we use the Fos promoter to identify strongly activated neuronal ensembles in rat prelimbic cortex (PLC) and assess altered intrinsic excitability after 10 d of operant food self-administration training (1 h/d). First, we used the Daun02 inactivation procedure in male FosLacZ-transgenic rats to ablate selectively Fos-expressing PLC neurons that were active during operant food self-administration. Selective ablation of these neurons decreased food seeking. We then used male FosGFP-transgenic rats to assess selective alterations of intrinsic excitability in Fos-expressing neuronal ensembles (FosGFP+) that were activated during food self-administration and compared these with alterations in less activated non-ensemble neurons (FosGFP-). Using whole-cell recordings of layer V pyramidal neurons in an ex vivo brain slice preparation, we found that operant self-administration increased excitability of FosGFP+ neurons and decreased excitability of FosGFP- neurons. Increased excitability of FosGFP+ neurons was driven by increased steady-state input resistance. Decreased excitability of FosGFP- neurons was driven by increased contribution of small-conductance calcium-activated potassium (SK) channels. Injections of the specific SK channel antagonist apamin into PLC increased Fos expression but had no effect on food seeking. Overall, operant learning increased intrinsic excitability of PLC Fos-expressing neuronal ensembles that play a role in food seeking but decreased intrinsic excitability of Fos- non-ensembles.SIGNIFICANCE STATEMENT Prefrontal cortex activity plays a critical role in operant learning, but the underlying cellular mechanisms are unknown. Using the chemogenetic Daun02 inactivation procedure, we found that a small number of strongly activated Fos-expressing neuronal ensembles in rat PLC play an important role in learned operant food seeking. Using GFP expression to identify Fos-expressing layer V pyramidal neurons in prelimbic cortex (PLC) of FosGFP-transgenic rats, we found that operant food self-administration led to increased intrinsic excitability in the behaviorally relevant Fos-expressing neuronal ensembles, but decreased intrinsic excitability in Fos- neurons using distinct cellular mechanisms.

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.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

We previously demonstrated a role of piriform cortex (Pir) in relapse to fentanyl seeking after food choice-induced voluntary abstinence. Here, we used this model to further study the role of Pir and its afferent projections in fentanyl relapse. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 d (6 h/day). We assessed relapse to fentanyl seeking after 12 voluntary abstinence sessions, achieved through a discrete choice procedure between fentanyl and palatable food (20 trials/session). We determined projection-specific activation of Pir afferents during fentanyl relapse with Fos plus the retrograde tracer cholera toxin B (injected into Pir). Fentanyl relapse was associated with increased Fos expression in anterior insular cortex (AI) and prelimbic cortex (PL) neurons projecting to Pir. We next used an anatomical disconnection procedure to determine the causal role of these two projections (AI→Pir and PL→Pir) in fentanyl relapse. Contralateral but not ipsilateral disconnection of AI→Pir projections decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, contralateral but not ipsilateral disconnection of PL→Pir projections modestly decreased reacquisition but not relapse. Fluorescence-activated cell sorting and quantitative PCR data showed molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse. Finally, we found minimal or no sex differences in fentanyl self-administration, fentanyl versus food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in nonreinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence.SIGNIFICANCE STATEMENT We previously showed a role of Pir in fentanyl relapse after food choice-induced voluntary abstinence in rats, a procedure mimicking human abstinence or a significant reduction in drug self-administration because of the availability of alternative nondrug rewards. Here, we aimed to further characterize the role of Pir in fentanyl relapse by investigating the role of Pir afferent projections and analyzing molecular changes in relapse-activated Pir neurons. We identified dissociable roles of two Pir afferent projections (AI→Pir and PL→Pir) in relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after voluntary abstinence. We also characterized molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed "incubation of craving." Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

Adaptive behavior depends on the delicate and dynamic balance between acquisition and extinction memories. Disruption of this balance, particularly when the extinction of memory loses control over behavior, is the root of treatment failure of maladaptive behaviors such as substance abuse or anxiety disorders. Understanding this balance requires a better understanding of the underlying neurobiology and its contribution to behavioral regulation.

Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.

Conflicting evidence exists regarding the role of infralimbic cortex (IL) in the environmental control of appetitive behavior. Inhibition of IL, irrespective of its intrinsic neural activity, attenuates not only the ability of environmental cues predictive of reward availability to promote reward seeking, but also the ability of environmental cues predictive of reward omission to suppress this behavior. Here we report that such bidirectional behavioral modulation in rats is mediated by functionally distinct units of neurons (neural ensembles) that are concurrently localized within the same IL brain area but selectively reactive to different environmental cues. Ensemble-specific neural activity is thought to function as a memory engram representing a learned association between environment and behavior. Our findings establish the causal evidence for the concurrent existence of two distinct engrams within a single brain site, each mediating opposing environmental actions on a learned behavior.

Mouse models of social behavior fail to account for volitional aspects of social interaction, and current neurobiological investigation of social behavior is performed almost exclusively using C57BL/6J mice, the background strain of most transgenic mice. Here, we introduce a mouse model of operant social self-administration and choice, using a custom-made apparatus.

Acute cocaine can inhibit catecholamine biosynthesis by regulating the enzymatic activity of tyrosine hydroxylase via alterations in the phosphorylation state of the enzyme. The mechanisms underlying acute cocaine-dependent regulation of tyrosine hydroxylase phosphorylation have not been determined. In this study, 0, 15 or 30 mg/kg cocaine was administered intraperitoneally to rats and the phosphorylation state of tyrosine hydroxylase in the brain was examined using antibodies specific for the phosphorylated forms of serine-19, -31 and -40 in tyrosine hydroxylase. In the caudate and nucleus accumbens, cocaine dose-dependently decreased the levels of phosphorylated serine-19, -31 and -40. In the ventral tegmental area, the levels of phosphorylated serine-19, but not serine-31 and -40, were decreased by 15 and 30 mg/kg cocaine. In the amygdala, the levels of phosphorylated serine-19, but not serine-31 or -40, were decreased. The functional effects of these alterations in phosphorylation state were assessed by measuring tyrosine hydroxylase activity in vivo (accumulation of DOPA after administration of the decarboxylase inhibitor NSD-1015). Acute administration of 30 mg/kg cocaine significantly decreased l-DOPA production in caudate and accumbens but not in amygdala. These data suggest that the phosphorylation of serine-31 or -40, but not serine-19, is involved in the regulation of tyrosine hydroxylase activity by acute cocaine.

Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.

Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6 h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons.

The orbitofrontal cortex (OFC) is a brain region involved in higher-order decision-making. Rodent studies show that cocaine self-administration (CSA) reduces OFC contribution to goal-directed behavior and behavioral strategies to avoid drug intake. This change in OFC function persists for many weeks after cocaine withdrawal, suggesting involvement in the process of addiction. The mechanisms underlying impaired OFC function by cocaine are not well-understood. However, studies implicate altered OFC serotonin (5-HT) function in disrupted cognitive processes during addiction and other psychiatric disorders. Thus, it is hypothesized that cocaine impairment of OFC function involves changes in 5-HT signaling, and previous work shows that 5-HT1A and 5-HT2A receptor-mediated effects on OFC pyramidal neurons (PyNs) are impaired weeks after cocaine withdrawal. However, 5-HT effects on other contributors to OFC circuit function have not been fully investigated, including the parvalbumin-containing, fast-spiking interneurons (OFCPV), whose function is essential to normal OFC-mediated behavior. Here, 5-HT function in naive rats and those withdrawn from CSA were evaluated using a novel rat transgenic line in which the rat parvalbumin promoter drives Cre-recombinase expression to permit identification of OFCPV cells by fluorescent reporter protein expression. We find that whereas CSA altered basal synaptic and membrane properties of the OFCPV neurons in a sex-dependent manner, the effects of 5-HT on these cells were unchanged by CSA. These data suggest that the behavioral effects of dysregulated OFC 5-HT function caused by cocaine experience are primarily mediated by changes in 5-HT signaling at PyNs, and not at OFCPV neurons.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. Discriminative stimuli (DSs) are unique in that they exert stimulus-control over both drug taking and drug seeking behavior and are difficult to extinguish. For this reason, incubation of the excitatory effects of DSs that signal drug availability, not yet examined in preclinical studies, could be relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds promise for drug relapse treatments.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.

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.

Understanding the neurobiology of complex behaviors requires measurement of activity in the discrete population of active neurons, neuronal ensembles, which control the behavior. Conventional neuroimaging techniques ineffectively measure neuronal ensemble activity in the brain in vivo because they assess the average regional neuronal activity instead of the specific activity of the neuronal ensemble that mediates the behavior. Our functional molecular photoacoustic tomography (FM-PAT) system allows direct imaging of Fos-dependent neuronal ensemble activation in Fos-LacZ transgenic rats in vivo. We tested four experimental conditions and found increased FM-PAT signal in prefrontal cortical areas in rats undergoing conditioned fear or novel context exposure. A parallel immunofluorescence ex vivo study of Fos expression found similar findings. These findings demonstrate the ability of FM-PAT to measure Fos-expressing neuronal ensembles directly in vivo and support a mechanistic role for the prefrontal cortex in higher-order processing of response to specific stimuli or environmental cues.

High relapse rate is a key feature of opioid addiction. In humans, abstinence is often voluntary due to negative consequences of opioid seeking. To mimic this human condition, we recently introduced a rat model of incubation of oxycodone craving after electric barrier-induced voluntary abstinence. Incubation of drug craving refers to time-dependent increases in drug seeking after cessation of drug self-administration. Here, we used the activity marker Fos, muscimol-baclofen (GABAa + GABAb receptor agonists) global inactivation, Daun02-selective inactivation of putative relapse-associated neuronal ensembles, and fluorescence-activated cell sorting of Fos-positive cells and quantitative polymerase chain reaction to demonstrate a key role of vSub neuronal ensembles in incubation of oxycodone craving after voluntary abstinence, but not homecage forced abstinence. We also used a longitudinal functional magnetic resonance imaging method and showed that functional connectivity changes in vSub-related circuits predict opioid relapse after abstinence induced by adverse consequences of opioid seeking.