Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex.

A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

The ultrasonic vocalization (USV) syllable profile during neonatal opioid withdrawal and a kappa opioid receptor component to increased USV emissions in female mice.

RATIONALE: Opioid use during pregnancy can lead to negative infant health outcomes, including neonatal opioid withdrawal syndrome (NOWS). NOWS comprises gastrointestinal, autonomic nervous system, and neurological dysfunction that manifest during spontaneous withdrawal. Variability in NOWS severity necessitates a more individualized treatment approach. Ultrasonic vocalizations (USVs) in neonatal mice are emitted in isolation as a stress response and are increased during opioid withdrawal, thus modeling a negative affective state that can be utilized to test new treatments. OBJECTIVES: We sought to identify the behavioral and USV profile, brainstem transcriptomic adaptations, and role of kappa opioid receptors in USVs during neonatal opioid withdrawal. METHODS: We employed a third trimester-approximate opioid exposure model, where neonatal inbred FVB/NJ pups were injected twice-daily with morphine (10mg/kg, s.c.) or saline (0.9%, 20 ul/g, s.c.) from postnatal day(P) 1 to P14. This protocol induces reduced weight gain, hypothermia, thermal hyperalgesia, and increased USVs during spontaneous morphine withdrawal. RESULTS: On P14, there were increased USV emissions and altered USV syllables during withdrawal, including an increase in Complex 3 syllables in FVB/NJ females (but not males). Brainstem bulk mRNA sequencing revealed an upregulation of the kappa opioid receptor (Oprk1), which contributes to withdrawal-induced dysphoria. The kappa opioid receptor (KOR) antagonist, nor-BNI (30 mg/kg, s.c.), significantly reduced USVs in FVB/NJ females, but not males during spontaneous morphine withdrawal. Furthermore, the KOR agonist, U50,488h (0.625 mg/kg, s.c.), was sufficient to increase USVs on P10 (both sexes) and P14 (females only) in FVB/NJ mice. CONCLUSIONS: We identified an elevated USV syllable, Complex 3, and a female-specific recruitment of the dynorphin/KOR system in increased USVs associated with neonatal opioid withdrawal severity.

The ultrasonic vocalization (USV) syllable profile during neonatal opioid withdrawal and a kappa opioid receptor component to increased USV emissions in female mice.

Rationale: Opioid use during pregnancy can lead to negative infant health outcomes, including neonatal opioid withdrawal syndrome (NOWS). NOWS comprises gastrointestinal, autonomic nervous system, and neurological dysfunction that manifest during spontaneous withdrawal. Variability in NOWS severity necessitates a more individualized treatment approach. Ultrasonic vocalizations (USVs) in neonatal mice are emitted in isolation as a stress response and are increased during opioid withdrawal, thus modeling a negative affective state that can be utilized to test new treatments. Objectives: We sought to identify the behavioral and USV profile, brainstem transcriptomic adaptations, and role of kappa opioid receptors in USVs during neonatal opioid withdrawal. Methods: We employed a third trimester-approximate opioid exposure model, where neonatal inbred FVB/NJ pups were injected twice-daily with morphine (10mg/kg, s.c.) or saline (0.9%, 20 ul/g, s.c.) from postnatal day(P) 1 to P14. This protocol induces reduced weight gain, hypothermia, thermal hyperalgesia, and increased USVs during spontaneous morphine withdrawal. Results: On P14, there were increased USV emissions and altered USV syllables during withdrawal, including an increase in Complex 3 syllables in FVB/NJ females (but not males). Brainstem bulk mRNA sequencing revealed an upregulation of the kappa opioid receptor (Oprk1), which contributes to withdrawal-induced dysphoria. The kappa opioid receptor (KOR) antagonist, nor-BNI (30 mg/kg, s.c.), significantly reduced USVs in FVB/NJ females, but not males during spontaneous morphine withdrawal. Furthermore, the KOR agonist, U50,488h (0.625 mg/kg, s.c.), was sufficient to increase USVs on P10 (both sexes) and P14 (females only) in FVB/NJ mice. Conclusions: We identified an elevated USV syllable, Complex 3, and a female-specific recruitment of the dynorphin/KOR system in increased USVs associated with neonatal opioid withdrawal severity.

Validation studies and multi-omics analysis of Zhx2 as a candidate quantitative trait gene underlying brain oxycodone metabolite (oxymorphone) levels and behavior.

Sensitivity to the subjective reinforcing properties of opioids has a genetic component and can predict addiction liability of opioid compounds. We previously identified Zhx2 as a candidate gene underlying increased brain concentration of the oxycodone (OXY) metabolite oxymorphone (OMOR) in BALB/cJ (J) versus BALB/cByJ (By) females that could increase OXY state-dependent reward. A large structural intronic variant is associated with a robust reduction of Zhx2 expression in J mice, which we hypothesized enhances OMOR levels and OXY addiction-like behaviors. We tested this hypothesis by restoring the Zhx2 loss-of-function in Js (MVKO) and modeling the loss-of-function variant through knocking out the Zhx2 coding exon (E3KO) in Bys and assessing brain OXY metabolite levels and behavior. Consistent with our hypothesis, Zhx2 E3KO females showed an increase in brain OMOR levels and OXY-induced locomotor activity. However, contrary to our hypothesis, state-dependent expression of OXY-CPP was decreased in E3KO females and increased in E3KO males. We also overexpressed Zhx2 in the livers and brains of Js and observed Zhx2 overexpression in select brain regions that was associated with reduced OXY state-dependent learning. Integrative transcriptomic and proteomic analysis of E3KO mice identified astrocyte function, cell adhesion, extracellular matrix properties, and endothelial cell functions as pathways influencing brain OXY metabolite concentration and behavior. These results support Zhx2 as a quantitative trait gene underlying brain OMOR concentration that is associated with changes in OXY behavior and implicate potential quantitative trait mechanisms that together inform our overall understanding of Zhx2 in brain function.

Decreased myelin-related gene expression in the nucleus accumbens during spontaneous neonatal opioid withdrawal in the absence of long-term behavioral effects in adult outbred CFW mice.

Prenatal opioid exposure is a major health concern in the United States, with the incidence of neonatal opioid withdrawal syndrome (NOWS) escalating in recent years. NOWS occurs upon cessation of in utero opioid exposure and is characterized by increased irritability, disrupted sleep patterns, high-pitched crying, and dysregulated feeding. The main pharmacological strategy for alleviating symptoms is treatment with replacement opioids. The neural mechanisms mediating NOWS and the long-term neurobehavioral effects are poorly understood. We used a third trimester-approximate model in which neonatal outbred pups (Carworth Farms White; CFW) were administered once-daily morphine (15 mg/kg, s.c.) from postnatal day (P) day 1 through P14 and were then assessed for behavioral and transcriptomic adaptations within the nucleus accumbens (NAc) on P15. We also investigated the long-term effects of perinatal morphine exposure on adult learning and reward sensitivity. We observed significant weight deficits, spontaneous thermal hyperalgesia, and altered ultrasonic vocalization (USV) profiles following repeated morphine and during spontaneous withdrawal. Transcriptome analysis of NAc from opioid-withdrawn P15 neonates via bulk mRNA sequencing identified an enrichment profile consistent with downregulation of myelin-associated transcripts. Despite the neonatal behavioral and molecular effects, there were no significant long-term effects of perinatal morphine exposure on adult spatial memory function in the Barnes Maze, emotional learning in fear conditioning, or in baseline or methamphetamine-potentiated reward sensitivity as measured via intracranial self-stimulation. Thus, the once daily third trimester-approximate exposure regimen, while inducing NOWS model traits and significant transcriptomic effects in neonates, had no significant long-term effects on adult behaviors.

Decreased myelin-related gene expression in the nucleus accumbens during spontaneous neonatal opioid withdrawal in the absence of long-term behavioral effects in adult outbred CFW mice.

Prenatal opioid exposure is a major health concern in the United States, with the incidence of neonatal opioid withdrawal syndrome (NOWS) escalating in recent years. NOWS occurs upon cessation of in utero opioid exposure and is characterized by increased irritability, disrupted sleep patterns, high-pitched crying, and dysregulated feeding. The main pharmacological strategy for alleviating symptoms is treatment with replacement opioids. The neural mechanisms mediating NOWS and the long-term neurobehavioral effects are poorly understood. We used a third trimester-approximate model in which neonatal outbred pups (Carworth Farms White; CFW) were administered once-daily morphine (15 mg/kg, s.c.) from postnatal day (P) day 1 through P14 and were then assessed for behavioral and transcriptomic adaptations within the nucleus accumbens (NAc) on P15. We also investigated the long-term effects of perinatal morphine exposure on adult learning and reward sensitivity. We observed significant weight deficits, spontaneous thermal hyperalgesia, and altered ultrasonic vocalization (USV) profiles following repeated morphine and during spontaneous withdrawal. Transcriptome analysis of NAc from opioid-withdrawn P15 neonates via bulk mRNA sequencing identified an enrichment profile consistent with downregulation of myelin-associated transcripts. Despite the neonatal behavioral and molecular effects, there were no significant long-term effects of perinatal morphine exposure on adult spatial memory function in the Barnes Maze, emotional learning in fear conditioning, or in baseline or methamphetamine-potentiated reward sensitivity as measured via intracranial self-stimulation. Thus, the once daily third trimester-approximate exposure regimen, while inducing NOWS model traits and significant transcriptomic effects in neonates, had no significant long-term effects on adult behaviors. HIGHLIGHTS: We replicated some NOWS model traits via 1x-daily morphine (P1-P14).We found a downregulation of myelination genes in nucleus accumbens on P15.There were no effects on learning/memory or reward sensitivity in adults.