Incubation of palatable food craving is associated with brain-wide neuronal activation in mice.

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.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 h) enriched for inducible transcription factors and later changes (4 h) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 h, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 h after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

MultipleXed Population Selection and Enrichment single nucleus RNA sequencing (XPoSE-seq) enables sample identity retention during transcriptional profiling of rare populations.

Single nucleus RNA-sequencing is critical in deciphering tissue heterogeneity and identifying rare populations. However, current high throughput techniques are not optimized for rare target populations and require tradeoffs in design due to feasibility. We provide a novel snRNA pipeline, MulipleXed Population Selection and Enrichment snRNA-sequencing (XPoSE-seq), to enable targeted snRNA-seq experiments and in-depth transcriptomic characterization of rare target populations while retaining individual sample identity.

Inactivation of the infralimbic cortex decreases discriminative stimulus-controlled relapse to cocaine seeking in rats.

Persistent susceptibility to cue-induced relapse is a cardinal feature of addiction. Discriminative stimuli (DSs) are one type of drug-associated cue that signal drug availability (DS+) or unavailability (DS-) and control drug seeking prior to relapse. We previously established a trial-based procedure in rats to isolate DSs from context, conditioned stimuli, and other drug-associated cues during cocaine self-administration and demonstrated DS-controlled cocaine seeking up to 300 abstinence days. The behavioral and neural mechanisms underlying trial-based DS-control of drug seeking have rarely been investigated. Here we show that following discrimination training in our trial-based procedure, the DS+ and DS- independently control the expression and suppression of cocaine seeking during abstinence. Using microinjections of GABAA + GABAB receptor agonists (muscimol + baclofen) in medial prefrontal cortex, we report that infralimbic, but not prelimbic, subregion of medial prefrontal cortex is critical to persistent DS-controlled relapse to cocaine seeking after prolonged abstinence, but not DS-guided discriminated cocaine seeking or DS-controlled cocaine self-admininstration. Finally, using ex vivo whole-cell recordings from pyramidal neurons in the medial prefrontal cortex, we demonstrate that the disruption of DS-controlled cocaine seeking following infralimbic cortex microinjections of muscimol+baclofen is likely a result of suppression of synaptic transmission in the region via a presynaptic mechanism of action.