Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.

Addiction is characterized by a lack of insight into the likely outcomes of one's behavior. Insight, or the ability to imagine outcomes, is evident when outcomes have not been directly experienced. Using this concept, work in both rats and humans has recently identified neural correlates of insight in the medial and orbital prefrontal cortices. We found that these correlates were selectively abolished in rats by cocaine self-administration. Their abolition was associated with behavioral deficits and reduced synaptic efficacy in orbitofrontal cortex, the reversal of which by optogenetic activation restored normal behavior. These results provide a link between cocaine use and problems with insight. Deficits in these functions are likely to be particularly important for problems such as drug relapse, in which behavior fails to account for likely adverse outcomes. As such, our data provide a neural target for therapeutic approaches to address these defining long-term effects of drug use.

There has been a marked increase in the availability of synthetic drugs designed to mimic the effects of marijuana. These cannabimimetic drugs, sold illicitly as 'Spice' and related products, are associated with serious medical complications in some users. In vitro studies suggest that synthetic cannabinoids in these preparations are potent agonists at central cannabinoid CB1 receptors (CB1Rs), but few investigations have delineated their cellular effects, particularly in comparison with the psychoactive component of marijuana, Δ9 -tetrahydrocannabinol (Δ9 -THC). We compared the ability of three widely abused synthetic cannabinoids and Δ9 -THC to alter glutamate release and long-term potentiation in the mouse hippocampus. JWH-018 was the most potent inhibitor of hippocampal synaptic transmission (EC50 ~15 nM), whereas its fluoropentyl derivative, AM2201, inhibited synaptic transmission with slightly lower potency (EC50 ~60 nM). The newer synthetic cannabinoid, XLR-11, displayed much lower potency (EC50 ~900 nM) that was similar to Δ9 -THC (EC50 ~700 nM). The effects of all compounds occurred via activation of CB1Rs, as demonstrated by reversal with the selective antagonist/inverse agonist AM251 or the neutral CB1R antagonist PIMSR1. Moreover, AM2201 was without effect in the hippocampus of transgenic mice lacking the CB1R. Hippocampal slices exposed to either synthetic cannabinoids or Δ9 -THC exhibited significantly impaired long-term potentiation (LTP). We find that, compared with Δ9 -THC, the first-generation cannabinoids found in Spice preparations display higher potency, whereas a recent synthetic cannabinoid is roughly equipotent with Δ9 -THC. The disruption of synaptic function by these synthetic cannabinoids is likely to lead to profound impairments in cognitive and behavioral function.

Clinical trials in Parkinson's disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts.

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.

The discovery of functionally biased and physiologically beneficial ligands directed toward G-protein coupled receptors (GPCRs) has provided the impetus to design dopamine D2 receptor (D2R) targeted molecules that may be therapeutically advantageous for the treatment of certain neuropsychiatric or basal ganglia related disorders. Here we describe the synthesis of a novel series of D2R agonists linking the D2R unbiased agonist sumanirole with privileged secondary molecular fragments. The resulting ligands demonstrate improved D2R affinity and selectivity over sumanirole. Extensive in vitro functional studies and bias factor analysis led to the identification of a novel class of highly potent Go-protein biased full D2R agonists with more than 10-fold and 1000-fold bias selectivity toward activation of specific G-protein subtypes and β-arrestin, respectively. Intracellular electrophysiological recordings from midbrain dopamine neurons demonstrated that Go-protein selective agonists can elicit prolonged ligand-induced GIRK activity via D2Rs, which may be beneficial in the treatment of dyskinesias associated with dopamine system dysfunction.

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.

Development of self-regulatory competencies during adolescence is partially dependent on normative brain maturation. Here, we report that adolescent rats as compared to adults exhibit impulsive and compulsive-like behavioral traits, the latter being associated with lower expression of mRNA levels of the immediate early gene zif268 in the anterior insula cortex (AIC). This suggests that underdeveloped AIC function in adolescent rats could contribute to an immature pattern of interoceptive cue integration in decision making and a compulsive phenotype. In support of this, we report that layer 5 pyramidal neurons in the adolescent rat AIC are hypoexcitable and receive fewer glutamatergic synaptic inputs compared to adults. Chemogenetic activation of the AIC attenuated compulsive traits in adolescent rats supporting the idea that in early stages of AIC maturity there exists a suboptimal integration of sensory and cognitive information that contributes to inflexible behaviors in specific conditions of reward availability.

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.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks that include fatalities. Many SCRAs exhibit much higher efficacy and potency, compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC), at the cannabinoid receptor 1 (CB1R), a G protein-coupled receptor involved in modulating neurotransmitter release. In this study, we investigated structure activity relationships (SAR) of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer (BRET) assays, we identified a few of SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting β-arrestin than the reference CB1R full agonist CP55940. Importantly, adding a methyl group at the head moiety of 5F-MMB-PICA yielded 5F-MDMB-PICA, an agonist exhibiting a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by a functional assay of the effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R bound with either of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA, and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks, including fatalities. Many SCRAs exhibit much higher efficacy and potency compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC) at the cannabinoid receptor 1 (CB1R), leading to dramatic differences in signaling levels that can be toxic. In this study, we investigated the structure-activity relationships of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer assays, we identified a few SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting β-arrestin than the reference CB1R full agonist CP55940. Importantly, the extra methyl group on the head moiety of 5F-MDMB-PICA, as compared to that of 5F-MMB-PICA, led to a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by the functional effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R models bound with both of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of the structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

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.

The lateral habenula (LHb) is involved in reward, aversion, addiction and depression through descending interactions with several brain structures, including the ventral tegmental area (VTA). The VTA provides reciprocal inputs to LHb, but their actions are unclear. Here we show that the majority of rat and mouse VTA neurons innervating LHb coexpress markers for both glutamate signaling (vesicular glutamate transporter 2; VGluT2) and GABA signaling (glutamic acid decarboxylase; GAD, and vesicular GABA transporter; VGaT). A single axon from these mesohabenular neurons coexpresses VGluT2 protein and VGaT protein and, surprisingly, establishes symmetric and asymmetric synapses on LHb neurons. In LHb slices, light activation of mesohabenular fibers expressing channelrhodopsin2 driven by VGluT2 (Slc17a6) or VGaT (Slc32a1) promoters elicits release of both glutamate and GABA onto single LHb neurons. In vivo light activation of mesohabenular terminals inhibits or excites LHb neurons. Our findings reveal an unanticipated type of VTA neuron that cotransmits glutamate and GABA and provides the majority of mesohabenular inputs.

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.