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.

Perinatal Opioid Exposure Results in Persistent Hypoconnectivity of Excitatory Circuits and Reduced Activity Correlations in Mouse Primary Auditory Cortex.

Opioid use by pregnant women results in neonatal opioid withdrawal syndrome (NOWS) and lifelong neurobehavioral deficits including language impairments. Animal models of NOWS show impaired performance in a two-tone auditory discrimination task, suggesting abnormalities in sensory processing in the auditory cortex. To investigate the consequences of perinatal opioid exposure on auditory cortex circuits, we administered fentanyl to mouse dams in their drinking water throughout gestation and until litters were weaned at postnatal day (P)21. We then used in vivo two-photon Ca2+ imaging in adult animals of both sexes to investigate how primary auditory cortex (A1) function was altered. Perinatally exposed animals showed fewer sound-responsive neurons in A1, and the remaining sound-responsive cells exhibited lower response amplitudes but normal frequency selectivity and stimulus-specific adaptation (SSA). Populations of nearby layer 2/3 (L2/3) cells in exposed animals showed reduced correlated activity, suggesting a reduction of shared inputs. We then investigated A1 microcircuits to L2/3 cells by performing laser-scanning photostimulation (LSPS) combined with whole-cell patch-clamp recordings from A1 L2/3 cells. L2/3 cells in exposed animals showed functional hypoconnectivity of excitatory circuits of ascending inputs from L4 and L5/6 to L2/3, while inhibitory connections were unchanged, leading to an altered excitatory/inhibitory balance. These results suggest a specific reduction in excitatory ascending interlaminar cortical circuits resulting in decreased activity correlations after fentanyl exposure. We speculate that these changes in cortical circuits contribute to the impaired auditory discrimination ability after perinatal opioid exposure.SIGNIFICANCE STATEMENT This is the first study to investigate the functional effects of perinatal fentanyl exposure on the auditory cortex. Experiments show that perinatal fentanyl exposure results in decreased excitatory functional circuits and altered population activity in primary sensory areas in adult mice. These circuit changes might underlie the observed language and cognitive deficits in infants exposed to opioids.

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.

Perinatal Fentanyl Exposure Leads to Long-Lasting Impairments in Somatosensory Circuit Function and Behavior.

One consequence of the opioid epidemic are lasting neurodevelopmental sequelae afflicting adolescents exposed to opioids in the womb. A translationally relevant and developmentally accurate preclinical model is needed to understand the behavioral, circuit, network, and molecular abnormalities resulting from this exposure. By employing a novel preclinical model of perinatal fentanyl exposure, our data reveal that fentanyl has several dose-dependent, developmental consequences to somatosensory function and behavior. Newborn male and female mice exhibit signs of withdrawal and sensory-related deficits that extend at least to adolescence. As fentanyl exposure does not affect dams' health or maternal behavior, these effects result from the direct actions of perinatal fentanyl on the pups' developing brain. At adolescence, exposed mice exhibit reduced adaptation to sensory stimuli, and a corresponding impairment in primary somatosensory (S1) function. In vitro electrophysiology demonstrates a long-lasting reduction in S1 synaptic excitation, evidenced by decreases in release probability, NMDA receptor-mediated postsynaptic currents, and frequency of miniature excitatory postsynaptic currents (mEPSCs), as well as increased frequency of miniature inhibitory postsynaptic currents (mIPSCs). In contrast, anterior cingulate cortical neurons exhibit an opposite phenotype, with increased synaptic excitation. Consistent with these changes, electrocorticograms (ECoGs) reveal suppressed ketamine-evoked γ oscillations. Morphologic analysis of S1 pyramidal neurons indicate reduced dendritic complexity, dendritic length, and soma size. Further, exposed mice exhibited abnormal cortical mRNA expression of key receptors involved in synaptic transmission and neuronal growth and development, changes that were consistent with the electrophysiological and morphologic changes. These findings demonstrate the lasting sequelae of perinatal fentanyl exposure on sensory processing and function.SIGNIFICANCE STATEMENT This is the first study to show that exposure to fentanyl in the womb results in behavioral, circuitry, and synaptic effects that last at least to adolescence. We also show, for the first time, that this exposure has different, lasting effects on synapses in different cortical areas.

Enduring consequences of perinatal fentanyl exposure in mice.

Opioid use by pregnant women is an understudied consequence associated with the opioid epidemic, resulting in a rise in the incidence of neonatal opioid withdrawal syndrome (NOWS) and lifelong neurobehavioral deficits that result from perinatal opioid exposure. There are few preclinical models that accurately recapitulate human perinatal drug exposure and few focus on fentanyl, a potent synthetic opioid that is a leading driver of the opioid epidemic. To investigate the consequences of perinatal opioid exposure, we administered fentanyl to mouse dams in their drinking water throughout gestation and until litters were weaned at postnatal day (PD) 21. Fentanyl-exposed dams delivered smaller litters and had higher litter mortality rates compared with controls. Metrics of maternal care behavior were not affected by the treatment, nor were there differences in dams' weight or liquid consumption throughout gestation and 21 days postpartum. Twenty-four hours after weaning and drug cessation, perinatal fentanyl-exposed mice exhibited signs of spontaneous somatic withdrawal behavior and sex-specific weight fluctuations that normalized in adulthood. At adolescence (PD 35), they displayed elevated anxiety-like behaviors and decreased grooming, assayed in the elevated plus maze and sucrose splash tests. Finally, by adulthood (PD 55), they displayed impaired performance in a two-tone auditory discrimination task. Collectively, our findings suggest that perinatal fentanyl-exposed mice exhibit somatic withdrawal behavior and change into early adulthood reminiscent of humans born with NOWS.

Adolescent fluoxetine history impairs spatial memory in adult male, but not female, C57BL/6 mice.

BACKGROUND: Epidemiological reports indicate that mood-related disorders are common in the adolescent population. The prevalence of juvenile major depressive disorder has resulted in a parallel increase in the prescription rates of fluoxetine (FLX) within this age group. Although such treatment can last for years, little is known about the enduring consequences of adolescent antidepressant exposure on memory-related performance. METHODS: We exposed separate groups of adolescent (postnatal day [PD] 35) male and female C57BL/6 mice to FLX (20 mg/kg) for 15 consecutive days (PD35-49). Three weeks after FLX exposure (PD70), we assessed learning and memory performance on a single-day training object novelty recognition test, or a spatial memory task on the Morris water maze (MWM). RESULTS: We found that FLX pretreatment did not influence performance on either the object novelty recognition task or the MWM, 24 h after training. Conversely, 48 h post spatial-training on the MWM, FLX pretreated male mice spent significantly less time on the quadrant of the missing platform during a standard probe trial. No differences in MWM performance were observed in the adult female mice pretreated with FLX. LIMITATIONS: A limitation of this study is that normal adolescent mice (i.e., non-stressed) were evaluated for memory-related behavior three weeks after antidepressant exposure. Thus, it is possible that FLX pre-exposure in combination with animal models for the study of depression may yield different results. CONCLUSION: Together, these results demonstrate enduring spatial memory-related deficiencies after pre-exposure to FLX during adolescence in male, but not female, C57BL/6 mice.

Vicarious Social Defeat Stress Induces Depression-Related Outcomes in Female Mice.

BACKGROUND: Stress is a prevailing risk factor for mood-related illnesses, wherein women represent the majority of those affected by major depression. Despite the growing literature suggesting that affective disorders can arise after a traumatic event is vicariously experienced, this relationship remains understudied in female subjects at the preclinical level. Thus, the objective of the current investigation was to examine whether exposure to emotional and/or psychological stress (ES) mediates depression-related outcomes in female mice. METHODS: Female C57BL/6 mice (8 weeks old, null parity) vicariously experienced the defeat bout of a male conspecific, by a male CD1 aggressor, for 10 consecutive days. Twenty-four hours after the last stress exposure, female mice were tested in the social interaction, sucrose preference, tail suspension, or elevated plus maze tests. Furthermore, we examined whether ketamine and chlordiazepoxide, pharmacological agents used to treat mood-related disorders in the clinical population, would reverse the ES-induced social dysfunction. RESULTS: When compared with control mice, female mice exposed to ES displayed decreased social behavior and preference for sucrose, along with increased immobility in the tail suspension test. Also, they displayed higher levels of blood serum corticosterone, as well as decreased body weight. Lastly, the ES-induced avoidance-like phenotype was ameliorated by both ketamine and chlordiazepoxide. CONCLUSIONS: Our data indicate that female mice exposed to ES display a behavioral and physiologic profile that mimics symptoms of depression in the clinical population. As such, this experimental model may be adopted to examine vicarious stress-induced mood-related disorders, as well as pharmacological antidepressant response, in a sex-specific manner.

Social defeat stress induces depression-like behavior and alters spine morphology in the hippocampus of adolescent male C57BL/6 mice.

Social stress, including bullying during adolescence, is a risk factor for common psychopathologies such as depression. To investigate the neural mechanisms associated with juvenile social stress-induced mood-related endophenotypes, we examined the behavioral, morphological, and biochemical effects of the social defeat stress model of depression on hippocampal dendritic spines within the CA1 stratum radiatum. Adolescent (postnatal day 35) male C57BL/6 mice were subjected to defeat episodes for 10 consecutive days. Twenty-four h later, separate groups of mice were tested on the social interaction and tail suspension tests. Hippocampi were then dissected and Western blots were conducted to quantify protein levels for various markers important for synaptic plasticity including protein kinase M zeta (PKMζ), protein kinase C zeta (PKCζ), the dopamine-1 (D1) receptor, tyrosine hydroxylase (TH), and the dopamine transporter (DAT). Furthermore, we examined the presence of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor subunit GluA2 as well as colocalization with the post-synaptic density 95 (PSD95) protein, within different spine subtypes (filopodia, stubby, long-thin, mushroom) using an immunohistochemistry and Golgi-Cox staining technique. The results revealed that social defeat induced a depression-like behavioral profile, as inferred from decreased social interaction levels, increased immobility on the tail suspension test, and decreases in body weight. Whole hippocampal immunoblots revealed decreases in GluA2, with a concomitant increase in DAT and TH levels in the stressed group. Spine morphology analyses further showed that defeated mice displayed a significant decrease in stubby spines, and an increase in long-thin spines within the CA1 stratum radiatum. Further evaluation of GluA2/PSD95 containing-spines demonstrated a decrease of these markers within long-thin and mushroom spine types. Together, these results indicate that juvenile social stress induces GluA2- and dopamine-associated dysregulation in the hippocampus - a neurobiological mechanism potentially underlying the development of mood-related syndromes as a consequence of adolescent bullying.