Perinatal morphine but not buprenorphine affects gestational and offspring neurobehavioral outcomes in mice.

Within the national opioid epidemic, there has been an increase in the number of infants exposed to opioids in utero. Additionally, opioid agonist medications are the standard of care for women with opioid use disorder during pregnancy. Buprenorphine (BUP), a partial µ -opioid receptor agonist, has been successful in improving gestational and neonatal outcomes. However, in utero exposure has been linked to childhood cognitive and behavioral problems. Therefore, we sought to compare offspring cognitive and behavioral outcomes after prenatal exposure to a clinically relevant low dose of BUP compared to morphine (MO), a full µ -opioid receptor agonist and immediate metabolite of heroin. We used a mouse model to assess gestational and offspring outcomes. Mouse dams were injected once daily s.c. with saline (SAL, n = 12), MO (10 mg/kg, n = 15), or BUP (0.1 mg/kg, n = 16) throughout pre-gestation, gestation, and lactation until offspring were weaned on postnatal day (P)21. Offspring social interaction and exploratory behavior were assessed, along with executive function via the touchscreen 5 choice serial reaction time task (5CSRTT). We then quantified P1 brain gene expression in the frontal cortex and amygdala (AMG). Perinatal MO but not BUP exposure decreased gestational weight gain and was associated with dystocia. In adolescent offspring, perinatal MO but not BUP exposure increased social exploration in males and grooming behavior in females. In the 5CSRTT, male MO exposed offspring exhibited increased impulsive action errors compared to male BUP offspring. In the AMG of P1 MO exposed offspring, we observed an increase in gene expression of targets related to activity of microglia. Importantly, both MO and BUP caused acute hyperlocomotion in the dams to a similar degree, indicating that the selected doses are comparable, in accordance with previous dose comparisons on analgesic and reward efficacy. These data suggest that compared to MO, low dose BUP improves gestational outcomes and has less of an effect on the neonatal offspring brain and later adolescent and adult behavior.

Molecular neurobiology of loss: a role for basolateral amygdala extracellular matrix.

Psychological loss is a common experience that erodes well-being and negatively impacts quality of life. The molecular underpinnings of loss are poorly understood. Here, we investigate the mechanisms of loss using an environmental enrichment removal (ER) paradigm in male rats. The basolateral amygdala (BLA) was identified as a region of interest, demonstrating differential Fos responsivity to ER and having an established role in stress processing and adaptation. A comprehensive multi-omics investigation of the BLA, spanning multiple cohorts, platforms, and analyses, revealed alterations in microglia and the extracellular matrix (ECM). Follow-up studies indicated that ER decreased microglia size, complexity, and phagocytosis, suggesting reduced immune surveillance. Loss also substantially increased ECM coverage, specifically targeting perineuronal nets surrounding parvalbumin interneurons, suggesting decreased plasticity and increased inhibition within the BLA following loss. Behavioral analyses suggest that these molecular effects are linked to impaired BLA salience evaluation, leading to a mismatch between stimulus and reaction intensity. These loss-like behaviors could be rescued by depleting BLA ECM during the removal period, helping us understand the mechanisms underlying loss and revealing novel molecular targets to ameliorate its impact.

Early life cancer and chemotherapy lead to cognitive deficits related to alterations in microglial-associated gene expression in prefrontal cortex.

Children that survive leukemia are at an increased risk for cognitive difficulties. A better understanding of the neurobiological changes in response to early life chemotherapy will help develop therapeutic strategies to improve quality of life for leukemia survivors. To that end, we used a translationally-relevant mouse model consisting of leukemic cell line (L1210) injection into postnatal day (P)19 mice followed by methotrexate, vincristine, and leucovorin chemotherapy. Beginning one week after the end of chemotherapy, social behavior, recognition memory and executive function (using the 5 choice serial reaction time task (5CSRTT)) were tested in male and female mice. Prefrontal cortex (PFC) and hippocampus (HPC) were collected at the conclusion of behavioral assays for gene expression analysis. Mice exposed to early life cancer + chemotherapy were slower to progress through increasingly difficult stages of the 5CSRTT and showed an increase in premature errors, indicating impulsive action. A cluster of microglial-related genes in the PFC were found to be associated with performance in the 5CSRTT and acquisition of the operant response, and long-term changes in gene expression were evident in both PFC and HPC. This work identifies gene expression changes in PFC and HPC that may underlie cognitive deficits in survivors of early life exposure to cancer + chemotherapy.

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Perinatal Morphine Exposure Leads to Sex-Dependent Executive Function Deficits and Microglial Changes in Mice.

Children exposed prenatally to opioids are at an increased risk for behavioral problems and executive function deficits. The prefrontal cortex (PFC) and amygdala (AMG) regulate executive function and social behavior and are sensitive to opioids prenatally. Opioids can bind to toll-like receptor 4 (TLR4) to activate microglia, which may be developmentally important for synaptic pruning. Therefore, we tested the effects of perinatal morphine exposure on executive function and social behavior in male and female mouse offspring, along with microglial-related and synaptic-related outcomes. Dams were injected once daily subcutaneously with saline (n = 8) or morphine (MO; 10 mg/kg; n = 12) throughout pregestation, gestation, and lactation until offspring were weaned on postnatal day 21 (P21). Male MO offspring had impairments in attention and accuracy in the five-choice serial reaction time task, while female MO offspring were less affected. Targeted gene expression analysis at P21 in the PFC identified alterations in microglial-related and TLR4-related genes, while immunohistochemical analysis in adult brains indicated decreased microglial Iba1 and phagocytic CD68 proteins in the PFC and AMG in males, but females had an increase. Further, both male and female MO offspring had increased social preference. Overall, these data demonstrate male vulnerability to executive function deficits in response to perinatal opioid exposure and evidence for disruptions in neuron-microglial signaling.

Rates of substance and polysubstance use through universal maternal testing at the time of delivery.

OBJECTIVE: To report substance and polysubstance use at the time of delivery. STUDY DESIGN: A cross-sectional study was performed on mothers consented for universal drug testing (99%) during hospital admission at six delivery hospitals in Cincinnati, Ohio. Mass spectrometry urinalysis detected positivity rates of 46 substances. Rates of positive drug tests for individual and common co-occurring substances measured were reported. RESULTS: 2531 maternal samples were tested (88%) and 33% contained cotinine, 11.3% THC, 7.2% opioids, 3.8% cocaine, and 1.9% methamphetamines. Polysubstance use prevalence was as high as 15%. Among mothers testing positive for methadone or buprenorphine, 93% also tested positive for cotinine and 39% tested positive for a third substance in addition to cotinine. CONCLUSIONS: Substance use at delivery is more prevalent than previously reported. Many mothers testing positive for opioids also test positive for other substances, which may increase overdose risk and exacerbate neonatal opioid withdrawal syndrome (NOWS).

Translationally relevant mouse model of early life cancer and chemotherapy exposure results in brain and small intestine cytokine responses: A potential link to cognitive deficits.

Survivors of acute lymphoblastic leukemia (ALL), the most common childhood cancer, are at increased risk for long-term cognitive problems, including executive function deficits. The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients and is closely associated with cognitive deficits. To address how early life cancer chemotherapy leads to cognitive deficits, we developed a translationally relevant mouse model of leukemia survival that exposed mice to leukemic cells and chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) in early life. Male and female mice were tested several weeks later using novel object recognition (recognition memory) and 5-choice serial reaction time task (executive function). Gene expression of proinflammatory, white matter and synapse-associated molecules was assessed in the prefrontal cortex and small intestine both acutely after chemotherapy and chronically after cognitive testing. Early life cancer-chemotherapy exposure resulted in recognition memory and executive function deficits in adult male mice. Prefrontal cortex expression of the chemokine Ccl2 was increased acutely, while small intestine expression of the proinflammatory cytokine tumor necrosis factor-alpha was elevated both acutely (both sexes) and chronically (males only). Inflammation in the small intestine was correlated with prefrontal cortical proinflammatory and synaptic gene expression changes, as well as to executive function deficits. Collectively, these data indicate that the current protocol results in a robust mouse model in which to study cognitive deficits in leukemia survivors, and suggest that small intestine inflammation may represent a novel contributor to adverse CNS consequences of early life chemotherapy.

Improving translational relevance: The need for combined exposure models for studying prenatal adversity.

Prenatal environmental adversity is a risk factor for neurodevelopmental disorders (NDDs), with the neuroimmune environment proposed to play a role in this risk. Adverse maternal exposures are associated with cognitive consequences in the offspring that are characteristics of NDDs and simultaneous neuroimmune changes that may underlie NDD risk. In both animal models and human studies the association between prenatal environmental exposure and NDD risk has been shown to be complex. Maternal overnutrition/obesity and opioid use are two different examples of complex exposure epidemics, each with their own unique comorbidities. This review will examine maternal obesity and maternal opioid use separately, illustrating the pervasive comorbidities with each exposure to argue a need for animal models of compound prenatal exposures. Many of these comorbidities can impact neuroimmune function, warranting systematic investigation of combined exposures to begin to understand this complexity. While traditional approaches in animal models have focused on modeling a single prenatal exposure or second exposure later in life, a translational approach would begin to incorporate the most prevalent co-occurring prenatal exposures. Long term follow-up in humans is extremely challenging, so animal models can provide timely insight into neurodevelopmental consequences of complex prenatal exposures. Animal models that represent this translational context of comorbid exposures behind maternal obesity or comorbid exposures behind maternal opioid use may reveal potential synergistic neuroimmune interactions that contribute to cognitive consequences and NDD risk. Finally, translational co-exposure models can identify concerning exposure combinations to guide treatment in complex cases, and identify high risk children starting in the prenatal period where early interventions improve prognosis.

Voluntary alcohol consumption is increased in female, but not male, oxytocin receptor knockout mice.

INTRODUCTION: The oxytocin (Oxt) system, while typically associated with the neural regulation of social behaviors, also plays a role in an individual's vulnerability to develop alcohol use disorders (AUD). In humans, changes to the Oxt system, due to early life experience and/or genetic mutations, are associated with increased vulnerability to AUD. While a considerable amount is known about Oxt's role in AUD in males, less is known or understood, about how Oxt may affect AUD in females, likely due to many clinical and preclinical studies of AUD not directly considering sex as a biological variable. This is unfortunate given that females are more vulnerable to the effects of alcohol and have increased alcohol consumption, as compared to males. Therefore, in the current study we wanted to determine whether genetic disruption of the Oxt receptor (Oxtr), that is, Oxtr knockout (-/-) mice, affected stress-induced alcohol consumption in males and females. We hypothesized that genetic disruption of the Oxtr would result in increased stress-induced alcohol consumption in both males and females compared to wild-type (+/+) controls. Though, we predicted that these disruptions might be greater in female Oxtr -/- mice. METHODS: To test this hypothesis, a two-bottle preference test was utilized along with the forced swim test (FST), and pre- and poststress alcohol consumption and preference measured within each sex (males and females were run separately). As a follow-up experiment, a taste preference test, to control for possible genotypic differences in taste, was also performed. RESULTS: In males, we found no significant genotypic differences in alcohol consumption or preference. However, in females, we found that genetic disruption of the Oxtr resulted in a greater consumption of alcohol both pre- and poststress compared to controls. CONCLUSION: These data suggest that in females, disruptions in Oxt signaling may contribute to increased vulnerability to alcohol-associated addiction.

Differential impact of stress and environmental enrichment on corticolimbic circuits.

Stress exposure can produce profound changes in physiology and behavior that can impair health and well-being. Of note, stress exposure is linked to anxiety disorders and depression in humans. The widespread impact of these disorders warrants investigation into treatments to mitigate the harmful effects of stress. Pharmacological treatments fail to help many with these disorders, so recent work has focused on non-pharmacological alternatives. One of the most promising of these alternatives is environmental enrichment (EE). In rodents, EE includes social, physical, and cognitive stimulation for the animal, in the form of larger cages, running wheels, and toys. EE successfully reduces the maladaptive effects of various stressors, both as treatment and prophylaxis. While we know that EE can have beneficial effects under stress conditions, the morphological and molecular mechanisms underlying these behavioral effects are still not well understood. EE is known to alter neurogenesis, dendrite development, and expression of neurotrophic growth factors, effects that vary by type of enrichment, age, and sex. To add to this complexity, EE has differential effects in different brain regions. Understanding how EE exerts its protective effects on morphological and molecular levels could hold the key to developing more targeted pharmacological treatments. In this review, we summarize the literature on the morphological and molecular consequences of EE and stress in key emotional regulatory pathways in the brain, the hippocampus, prefrontal cortex, and amygdala. The similarities and differences among these regions provide some insight into stress-EE interaction that may be exploited in future efforts toward prevention of, and intervention in, stress-related diseases.

Adolescent microglia play a role in executive function in male mice exposed to perinatal high fat diet.

In humans, excessive gestational weight gain during pregnancy is associated with an increased risk for executive function deficits in the offspring. Our previous work has confirmed this finding in mice, as offspring from dams fed a 60% high fat (HF) diet during breeding, gestation, and lactation demonstrate impulsive-like behavior in the 5 choice serial reaction time task (5CSRTT). Because the prefrontal cortex (PFC), which plays a key role in executive function, undergoes substantial postnatal adolescent pruning and microglia are actively involved in synaptic refinement, we hypothesized that microglia may play a role in mediating changes in brain development after maternal HF diet, with a specific focus on microglial activity during adolescence. Therefore, we treated male and female offspring from HF or control diet (CD) dams with PLX3397-formulated diet (PLX) to ablate microglia during postnatal days 23-45. After PLX removal and microglial repopulation, adult mice underwent testing to evaluate executive function. Adolescent PLX treatment did increase the control male dropout rate in learning the basic FR1 task, but otherwise had a minimal effect on behavior in control offspring. In males, HF offspring learned faster and performed better on a simple operant task (fixed ratio 1) without an effect of PLX. However, in HF offspring this increase in FR1 responding was associated with more impulsive errors in the 5CSRTT while PLX eliminated this association and decreased impulsive errors specifically in HF offspring. This suggests that adolescent PLX treatment improves executive function and particularly impulsive behavior in adult male HF offspring, without an overall effect of perinatal diet. In females, maternal HF diet impaired reversal learning but PLX had no effect on performance. We then measured gene expression in adult male PFC, nucleus accumbens (NAC), and amygdala (AMG), examining targets related to synaptic function, reward, and inflammation. Maternal HF diet increased PFC synaptophysin and AMG psd95 expression. PFC synaptophysin expression was correlated with more impulsive errors in the 5CSRTT in the HF offspring only and PLX treatment eliminated this correlation. These data suggest that adolescent microglia may play a critical role in mediating executive function after perinatal high fat diet in males.

Prefrontal Cortex Regulates Chronic Stress-Induced Cardiovascular Susceptibility.

Background The medial prefrontal cortex is necessary for appropriate appraisal of stressful information, as well as coordinating visceral and behavioral processes. However, prolonged stress impairs medial prefrontal cortex function and prefrontal-dependent behaviors. Additionally, chronic stress induces sympathetic predominance, contributing to health detriments associated with autonomic imbalance. Previous studies identified a subregion of rodent prefrontal cortex, infralimbic cortex (IL), as a key regulator of neuroendocrine-autonomic integration after chronic stress, suggesting that IL output may prevent chronic stress-induced autonomic imbalance. In the current study, we tested the hypothesis that the IL regulates hemodynamic, vascular, and cardiac responses to chronic stress. Methods and Results A viral-packaged small interfering RNA construct was used to knockdown vesicular glutamate transporter 1 (vGluT1) and reduce glutamate packaging and release from IL projection neurons. Male rats were injected with a vGluT1 small interfering RNA-expressing construct or GFP (green fluorescent protein) control into the IL and then remained as unstressed controls or were exposed to chronic variable stress. IL vGluT1 knockdown increased heart rate and mean arterial pressure reactivity, while chronic variable stress increased chronic mean arterial pressure only in small interfering RNA-treated rats. In another cohort, chronic variable stress and vGluT1 knockdown interacted to impair both endothelial-dependent and endothelial-independent vasoreactivity ex vivo. Furthermore, vGluT1 knockdown and chronic variable stress increased histological markers of fibrosis and hypertrophy. Conclusions Knockdown of glutamate release from IL projection neurons indicates that these cells are necessary to prevent the enhanced physiological responses to stress that promote susceptibility to cardiovascular pathophysiology. Ultimately, these findings provide evidence for a neurobiological mechanism mediating the relationship between stress and poor cardiovascular health outcomes.

Functional disruption of stress modulatory circuits in a model of temporal lobe epilepsy.

Clinical data suggest that the neuroendocrine stress response is chronically dysregulated in a subset of patients with temporal lobe epilepsy (TLE), potentially contributing to both disease progression and the development of psychiatric comorbidities such as anxiety and depression. Whether neuroendocrine dysregulation and psychiatric comorbidities reflect direct effects of epilepsy-related pathologies, or secondary effects of disease burden particular to humans with epilepsy (i.e. social estrangement, employment changes) is not clear. Animal models provide an opportunity to dissociate these factors. Therefore, we queried whether epileptic mice would reproduce neuroendocrine and behavioral changes associated with human epilepsy. Male FVB mice were exposed to pilocarpine to induce status epilepticus (SE) and the subsequent development of spontaneous recurrent seizures. Morning baseline corticosterone levels were elevated in pilocarpine treated mice at 1, 7 and 10 weeks post-SE relative to controls. Similarly, epileptic mice had increased adrenal weight when compared to control mice. Exposure to acute restraint stress resulted in hypersecretion of corticosterone 30 min after the onset of the challenge. Anatomical analyses revealed reduced Fos expression in infralimbic and prelimbic prefrontal cortex, ventral subiculum and basal amygdala following restraint. No differences in Fos immunoreactivity were found in the paraventricular nucleus of the hypothalamus, hippocampal subfields or central amygdala. In order to assess emotional behavior, a second cohort of mice underwent a battery of behavioral tests, including sucrose preference, open field, elevated plus maze, 24h home-cage monitoring and forced swim. Epileptic mice showed increased anhedonic behavior, hyperactivity and anxiety-like behaviors. Together these data demonstrate that epileptic mice develop HPA axis hyperactivity and exhibit behavioral dysfunction. Endocrine and behavioral changes are associated with impaired recruitment of forebrain circuits regulating stress inhibition and emotional reactivity. Loss of forebrain control may underlie pronounced endocrine dysfunction and comorbid psychopathologies seen in temporal lobe epilepsy.

Adolescent environmental enrichment prevents behavioral and physiological sequelae of adolescent chronic stress in female (but not male) rats.

The late adolescent period is characterized by marked neurodevelopmental and endocrine fluctuations in the transition to early adulthood. Adolescents are highly responsive to the external environment, which enhances their ability to adapt and recover from challenges when given nurturing influences, but also makes them vulnerable to aberrant development when exposed to prolonged adverse situations. Female rats are particularly sensitive to the effects of chronic stress in adolescence, which manifests as passive coping strategies and blunted hypothalamo-pituitary adrenocortical (HPA) stress responses in adulthood. We sought to intervene by exposing adolescent rats to environmental enrichment (EE) immediately prior to and during chronic stress, hypothesizing that EE would minimize or prevent the long-term effects of stress that emerge in adult females. To test this, we exposed male and female rats to EE on postnatal days (PND) 33-60 and implemented chronic variable stress (CVS) on PND 40-60. CVS consisted of twice-daily unpredictable stressors. Experimental groups included: CVS/unenriched, unstressed/EE, CVS/EE and unstressed/unenriched (n = 10 of each sex/group). In adulthood, we measured behavior in the open field test and forced swim test (FST) and collected blood samples following the FST. We found that environmental enrichment given during the adolescent period prevented the chronic stress-induced transition to passive coping in the FST and reversed decreases in peak adrenocortical responsiveness observed in adult females. Adolescent enrichment had little to no effect on males or unstressed females tested in adulthood, indicating that beneficial effects are specific to females that were exposed to chronic stress.

Loss of Environmental Enrichment Elicits Behavioral and Physiological Dysregulation in Female Rats.

Chronic stress drives behavioral and physiological changes associated with numerous psychiatric disease states. In rodents, the vast majority of chronic stress models involve imposition of external stressors, whereas in humans stress is often driven by internal cues, commonly associated with a sense of loss. We previously exposed groups of rats to environmental enrichment (EE) for a protracted period (1 month), followed by removal of enrichment (ER), to induce an experience of loss in male rats. ER enhanced immobility in the forced swim test (FST), led to hypothalamic pituitary adrenal (HPA) axis hypoactivity, and caused hyperphagia relative to continuously enriched (EE), single-housed (Scon) and pair-housed (Pcon) groups, most of which were reversible by antidepressant treatment (Smith et al., 2017). Here, we have applied the same approach to study enrichment loss in female rats. Similar to the males, enrichment removal in females led to an increase in the time spent immobile in the FST and increased daytime food intake compared to the single and pair-housed controls. Unlike males, ER females showed decreased sucrose preference, and showed estrus cycle-dependent HPA axis hyperactivity to an acute restraint stress. The increase in passive coping (immobility), anhedonia-like behavior in the sucrose preference test and HPA axis dysregulation suggest that enrichment removal produces a loss phenotype in females that differs from that seen in males, which may be more pronounced in nature.

Behavioral and physiological consequences of enrichment loss in rats.

Significant loss produces the highest degree of stress and compromised well-being in humans. Current rodent models of stress involve the application of physically or psychologically aversive stimuli, but do not address the concept of loss. We developed a rodent model for significant loss, involving removal of long-term access to a rewarding enriched environment. Our results indicate that removal from environmental enrichment produces a profound behavioral and physiological phenotype with depression-like qualities, including helplessness behavior, hypothalamo-pituitary-adrenocortical axis dysregulation and overeating. Importantly, this enrichment removal phenotype was prevented by antidepressant treatment. Furthermore, the effects of enrichment removal do not occur following relief from chronic stress and are not duplicated by loss of exercise or social contact.

Divergent effects of repeated restraint versus chronic variable stress on prefrontal cortical immune status after LPS injection.

Previous work from our group has shown that chronic homotypic stress (repeated restraint - RR) increases microglial morphological activation in the prefrontal cortex (PFC), while chronic heterotypic stress (chronic variable stress - CVS) produces no such effect. Therefore, we hypothesized that stressor modality would also determine the susceptibility of the PFC to a subsequent inflammatory stimulus (low dose lipopolysaccharide (LPS)). We found that RR, but not CVS, increased Iba-1 soma size in the PFC after LPS injection, consistent with microglial activation. In contrast, CVS decreased gene expression of proinflammatory cytokines and Iba-1 in the PFC under baseline conditions, which were not further affected by LPS. Thus, RR appears to promote microglial responses to LPS, whereas CVS is largely immunosuppressive. The results suggest that neuroimmune changes caused by CVS may to some extent protect the PFC from subsequent inflammatory stimuli. These data suggest that modality and/or intensity of stressful experiences will be a major determinant of central inflammation and its effect on prefrontal cortex-mediated functions.