Adolescent high fat diet alters the transcriptional response of microglia in the prefrontal cortex in response to stressors in both male and female mice.

Both obesity and high fat diets (HFD) have been associated with an increase in inflammatory gene expression within the brain. Microglia play an important role in early cortical development and may be responsive to HFD, particularly during sensitive windows, such as adolescence. We hypothesized that HFD during adolescence would increase proinflammatory gene expression in microglia at baseline and potentiate the microglial stress response. Two stressors were examined, a physiological stressor [lipopolysaccharide (LPS), IP] and a psychological stressor [15 min restraint (RST)]. From 3 to 7 weeks of age, male and female mice were fed standard control diet (SC, 20% energy from fat) or HFD (60% energy from fat). On P49, 1 h before sacrifice, mice were randomly assigned to either stressor exposure or control conditions. Microglia from the frontal cortex were enriched using a Percoll density gradient and isolated via fluorescence-activated cell sorting (FACS), followed by RNA expression analysis of 30 genes (27 target genes, three housekeeping genes) using Fluidigm, a medium throughput qPCR platform. We found that adolescent HFD induced sex-specific transcriptional response in cortical microglia, both at baseline and in response to a stressor. Contrary to our hypothesis, adolescent HFD did not potentiate the transcriptional response to stressors in males, but rather in some cases, resulted in a blunted or absent response to the stressor. This was most apparent in males treated with LPS. However, in females, potentiation of the LPS response was observed for select proinflammatory genes, including Tnfa and Socs3. Further, HFD increased the expression of Itgam, Ikbkb, and Apoe in cortical microglia of both sexes, while adrenergic receptor expression (Adrb1 and Adra2a) was changed in response to stressor exposure with no effect of diet. These data identify classes of genes that are uniquely affected by adolescent exposure to HFD and different stressor modalities in males and females.

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.

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.

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.

Housing and testing in mixed-sex rooms increases motivation and accuracy during operant testing in both male and female mice.

Operant behavior tasks are widely used in neuroscience research, but little is known about how variables such as housing and testing conditions affect rodent operant performance. We have previously observed differences in operant performance in male and female mice depending on whether mice were housed and tested in rooms containing only one sex versus rooms containing both sexes. Here, male and female mice in either single-sex or mixed sex housing rooms were trained on fixed ratio 1 (FR1) and progressive ratio (PR) tasks. For both sexes, animals in the mixed sex room had more accurate performance in FR1 and were more motivated in the PR task. We then moved the single sex housed animals to the mixed sex room and vice versa. Animals that started in mixed sex housing had no change to PR, but both sexes who started in single sex housing were more motivated after the switch. Additionally, the females that moved into single-sex housing performed less accurately in FR1. We conclude that housing and testing conditions can affect performance on FR1 and PR tasks. As these tasks are commonly used as training steps to more complex tasks, housing and testing conditions should be carefully considered during experiment design and reported in publications.

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.

Treading water: mixed effects of high fat diet on mouse behavior in the forced swim test.

Diet is an environmental factor with significant potential to affect the brain and behavior in both positive and negative ways. Work in animals is necessary to understand this relationship and how it may apply to mental health in humans. One area which has been investigated extensively is whether diet, specifically a high fat diet (HFD), can alter behavior in tasks, such as the forced swim test (FST) that assess stress coping. Therefore, we sought to analyze the literature regarding the effect of HFD on performance in the FST to determine whether there was a consistent effect of HFD on stress coping behavior. We conducted a Google Scholar search of English-language articles with the following terms: high fat diet, obesity, forced swim test, depression like behavior, mouse. Thirty studies from twenty-five publications are included in this survey. Fifteen studies were found where HFD had no effect on FST, 4 where HFD decreased immobility, and 11 where HFD increased immobility. Experimental details in these studies varied widely, including differences in the diet, mice, and experimental design. Additionally, we analyzed thirteen studies that performed the tail-suspension test (TST) after HFD, with six studies finding no change due to HFD and 7 reporting that HFD increased immobility. Further, 6 of these studies used both FST and TST with largely similar results in the two tasks, indicating concordance between the two tests of stress-coping behavior. We conclude that due to widely varying experimental details across studies no consistent effect of high fat diet on stress coping behavior can be determined at this point.