Transcriptome profiling of the ventral pallidum reveals a role for pallido-thalamic neurons in cocaine reward.

Psychostimulant exposure alters the activity of ventral pallidum (VP) projection neurons. However, the molecular underpinnings of these circuit dysfunctions are unclear. We used RNA-sequencing to reveal alterations in the transcriptional landscape of the VP that are induced by cocaine self-administration in mice. We then probed gene expression in select VP neuronal subpopulations to isolate a circuit associated with cocaine intake. Finally, we used both overexpression and CRISPR-mediated knockdown to test the role of a gene target on cocaine-mediated behaviors as well as dendritic spine density. Our results showed that a large proportion (55%) of genes associated with structural plasticity were changed 24 h following cocaine intake. Among them, the transcription factor Nr4a1 (Nuclear receptor subfamily 4, group A, member 1, or Nur77) showed high expression levels. We found that the VP to mediodorsal thalamus (VP → MDT) projection neurons specifically were recapitulating this increase in Nr4a1 expression. Overexpressing Nr4a1 in VP → MDT neurons enhanced drug-seeking and drug-induced reinstatement, while Nr4a1 knockdown prevented self-administration acquisition and subsequent cocaine-mediated behaviors. Moreover, we showed that Nr4a1 negatively regulated spine dynamics in this specific cell subpopulation. Together, our study identifies for the first time the transcriptional mechanisms occurring in VP in drug exposure. Our study provides further understanding on the role of Nr4a1 in cocaine-related behaviors and identifies the crucial role of the VP → MDT circuit in drug intake and relapse-like behaviors.

Individual differences in stereotypy and neuron subtype translatome with TrkB deletion.

Motor stereotypies occurring in early-onset neuropsychiatric diseases are associated with dysregulated basal ganglia direct-pathway activity. Disruptions in network connectivity through impaired neuronal structure have been implicated in both rodents and humans. However, the neurobiological mechanisms leading to direct-pathway neuron disconnectivity in stereotypy remain poorly understood. We have a mouse line with Tropomyosin receptor kinase B (TrkB) receptor deletion from D1-expressing cells (D1-Cre-flTrkB) in which a subset of animals shows repetitive rotations and head tics with juvenile onset. Here we demonstrate these behaviors may be associated with abnormal direct-pathway activity by reducing rotations using chemogenetic inhibition of dorsal striatum D1-medium spiny neurons (D1-MSNs) in both juvenile and young-adult mice. Taking advantage of phenotypical differences in animals with similar genotypes, we then interrogated the D1-MSN specific translatome associated with repetitive behavior by using RNA sequencing of ribosome-associated mRNA. Detailed translatome analysis followed by multiplexed gene expression assessment revealed profound alterations in neuronal projection and synaptic structure related genes in stereotypy mice. Examination of neuronal morphology demonstrated dendritic atrophy and dendritic spine loss in dorsal striatum D1-MSNs from mice with repetitive behavior. Together, our results uncover phenotype-specific molecular alterations in D1-MSNs that relate to morphological adaptations in mice displaying stereotypy behavior.

Adaptations in Nucleus Accumbens Neuron Subtypes Mediate Negative Affective Behaviors in Fentanyl Abstinence.

BACKGROUND: Opioid discontinuation generates a withdrawal syndrome marked by increased negative affect. Increased symptoms of anxiety and dysphoria during opioid discontinuation are significant barriers to achieving long-term abstinence in opioid-dependent individuals. While adaptations in the nucleus accumbens are implicated in opioid abstinence syndrome, the precise neural mechanisms are poorly understood. Additionally, our current knowledge is limited to changes following natural and semisynthetic opioids, despite recent increases in synthetic opioid use and overdose. METHODS: We used a combination of cell subtype-specific viral labeling and electrophysiology in male and female mice to investigate structural and functional plasticity in nucleus accumbens medium spiny neuron (MSN) subtypes after fentanyl abstinence. We characterized molecular adaptations after fentanyl abstinence with subtype-specific RNA sequencing and weighted gene co-expression network analysis. We used viral-mediated gene transfer to manipulate the molecular signature of fentanyl abstinence in D1-MSNs. RESULTS: Here, we show that fentanyl abstinence increases anxiety-like behavior, decreases social interaction, and engenders MSN subtype-specific plasticity in both sexes. D1-MSNs, but not D2-MSNs, exhibit dendritic atrophy and an increase in excitatory drive. We identified a cluster of coexpressed dendritic morphology genes downregulated selectively in D1-MSNs that are transcriptionally coregulated by E2F1. E2f1 expression in D1-MSNs protects against loss of dendritic complexity, altered physiology, and negative affect-like behaviors caused by fentanyl abstinence. CONCLUSIONS: Our findings indicate that fentanyl abstinence causes unique structural, functional, and molecular changes in nucleus accumbens D1-MSNs that can be targeted to alleviate negative affective symptoms during abstinence.

Transcriptomic profiling of reward and sensory brain areas in perinatal fentanyl exposed juvenile mice.

Use of the synthetic opioid fentanyl increased ~300% in the last decade, including among women of reproductive ages. Adverse neonatal outcomes and long-term behavioral disruptions are associated with perinatal opioid exposure. Our previous work demonstrated that perinatal fentanyl exposed mice displayed enhanced negative affect and somatosensory circuit and behavioral disruptions during adolescence. However, little is known about molecular adaptations across brain regions that underlie these outcomes. We performed RNA sequencing across three reward and two sensory brain areas to study transcriptional programs in perinatal fentanyl exposed juvenile mice. Pregnant dams received 10 µg/ml fentanyl in the drinking water from embryonic day 0 (E0) through gestational periods until weaning at postnatal day 21 (P21). RNA was extracted from nucleus accumbens (NAc), prelimbic cortex (PrL), ventral tegmental area (VTA), somatosensory cortex (S1) and ventrobasal thalamus (VBT) from perinatal fentanyl exposed mice of both sexes at P35. RNA sequencing was performed, followed by analysis of differentially expressed genes (DEGs) and gene co-expression networks. Transcriptome analysis revealed DEGs and gene modules significantly associated with exposure to perinatal fentanyl in a sex-wise manner. The VTA had the most DEGs, while robust gene enrichment occurred in NAc. Genes enriched in mitochondrial respiration were pronounced in NAc and VTA of perinatal fentanyl exposed males, extracellular matrix (ECM) and neuronal migration enrichment were pronounced in NAc and VTA of perinatal fentanyl exposed males, while genes associated with vesicular cycling and synaptic signaling were markedly altered in NAc of perinatal fentanyl exposed female mice. In sensory areas from perinatal fentanyl exposed females, we found alterations in mitochondrial respiration, synaptic and ciliary organization processes. Our findings demonstrate distinct transcriptomes across reward and sensory brain regions, with some showing discordance between sexes. These transcriptome adaptations may underlie structural, functional, and behavioral changes observed in perinatal fentanyl exposed mice.

Mitochondria-Related Nuclear Gene Expression in the Nucleus Accumbens and Blood Mitochondrial Copy Number After Developmental Fentanyl Exposure in Adolescent Male and Female C57BL/6 Mice.

The potency of the synthetic opioid fentanyl and its increased clinical availability has led to the rapid escalation of use in the general population, increased recreational exposure, and subsequently opioid-related overdoses. The wide-spread use of fentanyl has, consequently, increased the incidence of in utero exposure to the drug, but the long-term effects of this type of developmental exposure are not yet understood. Opioid use has also been linked to reduced mitochondrial copy number in blood in clinical populations, but the link between this peripheral biomarker and genetic or functional changes in reward-related brain circuitry is still unclear. Additionally, mitochondrial-related gene expression in reward-related brain regions has not been examined in the context of fentanyl exposure, despite the growing literature demonstrating drugs of abuse impact mitochondrial function, which subsequently impacts neuronal signaling. The current study uses exposure to fentanyl via dam access to fentanyl drinking water during gestation and lactation as a model for developmental drug exposure. This perinatal drug-exposure is sufficient to impact mitochondrial copy number in circulating blood leukocytes, as well as mitochondrial-related gene expression in the nucleus accumbens (NAc), a reward-related brain structure, in a sex-dependent manner in adolescent offspring. Specific NAc gene expression is correlated with both blood mitochondrial copy number and with anxiety related behaviors dependent on developmental exposure to fentanyl and sex. These data indicate that developmental fentanyl exposure impacts mitochondrial function in both the brain and body in ways that can impact neuronal signaling and may prime the brain for altered reward-related behavior in adolescence and later into adulthood.