Traffic-related particulate matter affects behavior, inflammation, and neural integrity in a developmental rodent model.

Recent studies indicate that exposure to airborne particulate matter (PM) is associated with cognitive delay, depression, anxiety, autism, and neurodegenerative diseases; however, the role of PM in the etiology of these outcomes is not well-understood. Therefore, there is a need for controlled animal studies to better elucidate the causes and mechanisms by which PM impacts these health outcomes. We assessed the effects of gestational and early life exposure to traffic-related PM on social- and anxiety-related behaviors, cognition, inflammatory markers, and neural integrity in juvenile male rats. Gestating and lactating rats were exposed to PM from a Boston (MA, USA) traffic tunnel for 5 h/day, 5 days/week for 6 weeks (3 weeks gestation, 3 weeks lactation). The target exposure concentration for the fine fraction of nebulized PM, measured as PM2.5, was 200 µg/m3. To assess anxiety and cognitive function, F1 male juveniles underwent elevated platform, cricket predation, nest building, social behavior and marble burying tests at 32-60 days of age. Upon completion of behavioral testing, multiple cytokines and growth factors were measured in these animals and their brains were analyzed with diffusion tensor MRI to assess neural integrity. PM exposure had no effect on litter size or weight, or offspring growth; however, F1 litters developmentally exposed to PM exhibited significantly increased anxiety (p = 0.04), decreased cognition reflected in poorer nest-organization (p = 0.04), and decreased social play and allogrooming (p = 0.003). MRI analysis of ex vivo brains revealed decreased structural integrity of neural tissues in the anterior cingulate and hippocampus in F1 juveniles exposed to PM (p < 0.01, p = 0.03, respectively). F1 juvenile males exposed to PM also exhibited significantly decreased plasma levels of both IL-18 (p = 0.03) and VEGF (p = 0.04), and these changes were inversely correlated with anxiety-related behavior. Chronic exposure of rat dams and their offspring to traffic-related PM during gestation and lactation decreases social behavior, increases anxiety, impairs cognition, decreases levels of inflammatory and growth factors (which are correlated with behavioral changes), and disrupts neural integrity in the juvenile male offspring. Our findings add evidence that exposure to traffic-related air pollution during gestation and lactation is involved in the etiology of autism spectrum disorder and other disorders which include social and cognitive deficits and/or increased anxiety.

Bedding-generated particulate matter: implications for rodent studies.

OBJECTIVES: Rodents used in scientific research are typically housed in cages containing natural bedding materials. Despite extensive evidence of biological harm from inhaled particulate matter (PM), relatively little work has been performed to measure bedding-generated PM exposure in caged animals used in basic science research. Our objectives were to determine whether bedding-generated PM was present in significant concentrations in rodent cages and to identify the main factors affecting the accumulation and attenuation of bedding-generated PM inside cages. MATERIALS AND METHODS: We measured PM2.5 concentrations in cages containing common bedding materials (pine, aspen, paper, and corncob) with filter top isolator absent or present on the cages. PM2.5 concentrations were monitored with rats inside cages as well as during artificial manipulation of the bedding (designed to simulate rodent activity). RESULTS AND DISCUSSION: Upon rodent digging or mechanical/manual stirring, all four bedding materials produced significant increases in PM2.5 concentrations (as much as 100-200 µg/m3 PM2.5, 50- to 100-fold higher than during periods of no rodent activity), and concentrations in cages fitted with filter tops were an order of magnitude higher than in cages without filter tops. Elevated concentrations were sustained for longer durations in cages with filter tops (5-10 minutes) compared to cages with only bar lids (0-2 minutes). CONCLUSIONS: These results indicate that standard laboratory housing conditions can expose rodents to substantial levels of PM2.5. Bedding-generated PM has potential implications as an environmental agent in rodent studies.

Pain-Suppressed Behaviors in the Red-tailed Hawk (Buteo jamaicensis).

Our ability to provide analgesia in wild and exotic patients is hampered by a lack of species-specific information on effective drugs and protocols. One contributing factor is the difficulty of applying data from traditional laboratory tests of nociception to clinical conditions frequently involving combinations of inflammatory, mechanical, and neuropathic pain. Pain-suppressed behaviors have become a valuable predictor of clinical utility in other species; in this study we extend this framework to red -tailed hawks in a wildlife hospital, in an attempt to develop a new, humane testing method for birds of prey. We scored six behaviors in hawks hospitalized either for orthopedic trauma or for non-painful conditions. These behaviors included: movement about the cage, grooming, head motions, foot shifts, beak clacks, and rouse. Movement, head motions, and beak clacks were all significantly reduced in hawks with recent orthopedic injury, but not in hawks with healed or minor injuries (P<0.05 for all behaviors). However, it should be noted that due to stringent admission criteria, and the difficulties inherent in studying naturally-occuring injury in wild patients, this study only included -subjects in four experimental groups, and this limited our ability to fully investigate confounds within our data. A follow-up experiment was conducted to determine potential effects of buprenorphine, a mu opioid agonist, on the behaviors listed above. Buprenorphine in the absence of pain caused minor, non-significant decreases in most behaviors, and had no effect on head movement frequency. This suggests that head movements in particular may be sensitive to pain but not to sedative side-effects of buprenorphine. Overall, red -tailed hawks with recent orthopedic trauma show consistent and marked red uctions in several normal maintenance behaviors. Head movements, reported for the first time in this study as a potential marker of pain in birds, in particular seem to be insensitive to sedative side effects of buprenorphine, while being a sensitive measu re of affective state in hawks with painful injuries. These behaviors can be scored humanely and with minimal expense, and should be considered for further research on pain and analgesia in avian species.

Environmental enrichment delays pup-induced maternal behavior in rats.

Adult, virgin rats do not spontaneously display maternal behavior when exposed to foster pups. However, continuous daily exposure of the female to foster pups for about 5-7 days can induce a set of maternal behaviors similar to those shown by postpartum dams. Induction latencies depend upon a number of factors, including the stress and anxiety levels of the female. The goal of this study was to attempt to mitigate the likely stressfulness of being singly housed during testing by enriching the rat's home cage environment and to determine if the concomitant environmental change would alter the latency to express maternal behavior. In addition, the effect of varying the number of test pups used for testing was examined. Two groups of virgin Sprague-Dawley rats were first tested on the elevated plus maze after 1 week of exposure to either control (standard housing) or enriched conditions. One week later, maternal behavior testing began using one or three pups. Upon completion of maternal behavior testing, plasma corticosterone concentrations were determined following a mild stressor. The data indicate that enrichment tends to increase anxiety-like behaviors in the elevated plus maze. In addition, enrichment delayed the onset of maternal behavior irrespective of the number of test pups. There were no effects of environmental enrichment on plasma corticosterone levels following exposure to a stressor. These results indicate that what is considered a modestly enriched environment delays the expression of pup-oriented responses and does not apparently reduce stress or improve performance on all behavioral tasks.

Maternal high fat diet and its consequence on the gut microbiome: A rat model.

The biological changes that occur during pregnancy in the female mammal include shifts in hormonal regulation in preparation for parturition and lactation, and changes in energy metabolism. In women, studies have also shown that during pregnancy there is a reduction in bacterial species richness in the gut. In the current experiment rats were used to model the interaction of diet, reproductive status, and intestinal bacterial microbiota during pregnancy and lactation. In Experiment 1 rats were exposed to either standard chow or high-fat chow (60%) and were divided into two groups: unmated (NULL) or mated (RE). In Experiment 2, both NULL and RE rats were exposed to high-fat chow for a 30-day period. High-throughput sequencing of the 16S rRNA gene revealed that pregnancy impacted the gut microbiota in a similar manner to humans. The impact of reproductive status on microbiota composition, however, was stronger in rats fed a high-fat (HF) diet. Diet-induced changes replicated some of the changes observed in humans, such as increasing the Firmicutes/Bacteroidetes ratio. However, in contrast to humans, pregnancy in rats did not increase ß-diversity between microbiota from different animals. These results indicate that during pregnancy in rats, the gut microbiota is altered in a similar manner to that which occurs in women, and that these changes are further exaggerated by exposure to a HF diet. Thus, the rat may allow modelling the effects of consumption of HF food during pregnancy and enable future studies to determine the risks of HF diets during pregnancy and its consequences on the offspring.

Finasteride delays the onset of maternal behavior in primigravid rats.

The inhibitory actions of progesterone on maternal behavior may be due to its metabolite, allopregnanolone. Allopregnanolone has been shown to mediate some cognitive, anxiolytic and sex behavior actions of progesterone. The objectives of the present study were to determine: first, if the administration of finasteride, a 5 alpha-reductase inhibitor, will stimulate fast-latency maternal behavior in primigravid rats and, second, to assess whether finasteride increases anxiety-like behaviors and alters circulating progesterone levels during pregnancy. In Experiment 1, primigravid Sprague-Dawley females received daily injections of either finasteride (50 mg/kg, s.c.) or vehicle (20% ethanol in sesame oil) starting on day 12 of gestation and continuing until parturition. On day 15 of gestation, daily maternal behavior testing began. In contrast to expectation, administration of finasteride significantly delayed the onset of maternal behavior compared to vehicle-treated rats (P < 0.05). In addition, the percentage of animals responding maternally on each test day tended to be lower for the finasteride-treated group (day 19, P = 0.078). In Experiment 2, a separate group of animals received daily finasteride injections starting on day 12 of gestation. On day 15, females were tested on the elevated plus maze and were then implanted with jugular catheters. Blood samples were taken daily (0900-1100 h) on days 17, 18 and 19 of gestation. Finasteride-treated females displayed significantly more anxiety-like behaviors in the elevated plus maze (P < 0.05). In addition, finasteride administration maintained elevated plasma progesterone levels compared to vehicle-treated controls (P = 0.03). These results suggest that the inhibition of maternal behavior by progesterone during pregnancy is not due to its conversion to allopregnanolone.

Neural steroid hormone receptor gene expression in pregnant rats.

Estrogen and progesterone play important roles during pregnancy in stimulating the onset of maternal behavior at parturition. The status of receptor expression of these hormones during pregnancy in neural regions that regulate maternal behavior is unclear. The objective of the present study is to characterize changes in neural gene expression of the estrogen receptors alpha and beta (ERalpha and ERbeta) and the progesterone receptor (PR) during the latter part of pregnancy. Brains from primigravid Sprague-Dawley rats were collected on days 15 and 21 of pregnancy. Micropunches of the olfactory bulb (OB), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BnST), hypothalamus (HYP), medial amygdala (MeA), and the temporal cortex (TCx) were analyzed by real-time RT-PCR (Taqmantrade mark) for levels of gene expression. No changes in either ERalpha or ERbeta mRNA levels were detected in any brain region between days 15 and 21 of pregnancy: however, the MPOA had higher levels of both ERalpha and ERbeta than other brain regions. Progesterone receptor mRNA levels, in contrast, declined significantly in the MPOA, HYP, and TCx, between days 15 and 21 of pregnancy (P < 0.05). In addition, the levels of PR mRNA were significantly higher in the HYP and TCx compared to both the OB and MeA. These data indicate that there is a downregulation of PR prepartum and suggest that this decrease may play a role in the disinhibition of maternal behavior at parturition.

Prolactin receptor gene expression in the forebrain of pregnant and lactating rats.

Prolactin plays a large role in the onset of maternal behavior at parturition. Knowledge of the change in expression of the prolactin receptor in the brain across pregnancy and lactation, however, is limited. Prolactin receptor gene expression was determined by in situ hybridization histochemistry during pregnancy and lactation in rats. Expression of the mRNA for the longform of the prolactin receptor (PRL-R-L) was measured in various forebrain structures in primigravid rats at different stages of pregnancy, in primiparous rats during early, mid-, and late lactation, and in age-matched, nulliparous females in diestrus. Hybridizations were performed using a [33P]-labeled riboprobe specific for the long form of the prolactin receptor mRNA complimentary to 290 bp of the prolactin receptor gene. The following areas of the forebrain were examined: medial preoptic area (MPOA), median preoptic nucleus both dorsal (MePOd) and ventral (MePOv) to the anterior commissure, ventral lateral septum (LSv), and the ventral and principal parts of the bed nucleus of the stria terminalis (BnSTv and BnSTpr, respectively). Overall, the number of cells expressing PRL-R-L mRNA was significantly higher at 2 h postpartum compared to diestrus in all areas examined except the LSv. In addition, there were lower numbers of PRL-R-L cells during all stages of lactation compared to pregnancy. The number of grains per cell in the MPOA and LSv did not change as dramatically as the number of cells expressing PRL-R-L mRNA in those brain regions. These data contribute to the growing body of evidence that the neural lactogenic system changes as a function of female reproductive state. Changes in PRL-R-L mRNA in terms of behavior and endocrine functions are discussed.

Disinhibition of maternal behavior following neurotoxic lesions of the hypothalamus in primigravid rats.

Virgin female rats do not respond maternally to foster pups due to an endogenous neural circuit that actively inhibits the display of maternal behavior. Once pregnant, primigravid rats will continue to avoid foster pups until just prior to or at parturition. Anosmia or lesions of the olfactory tract, medial amygdala, and areas of the hypothalamus will stimulate virgin females to display maternal behavior rapidly, but little is known of the effect of these lesions in primigravid rats. The objective of the present study was to determine if neurotoxic lesions of the dorsomedial (DMH) and ventromedial nuclei (VMH) of the hypothalamus will advance the onset of maternal behavior in primigravid rats. Nulliparous Sprague-Dawley female rats were mated and then on day 8 of gestation bilaterally infused with N-methyl-d-aspartic acid (NMDA; 8 microg/0.2 microl/side) or vehicle directed toward either the DMH or VMH. Beginning on day 15 of gestation until parturition, females were tested daily for maternal responsiveness. DMH and VMH lesions significantly advanced the onset of maternal behavior (5-6 days vs. 0-1 day before parturition) in first-time pregnant rats. These results indicate that the DMH and VMH are involved in the regulation of maternal behavior and may be part of an endogenous neural circuit that inhibits maternal behavior during pregnancy.

Gene Expression Profiling during Pregnancy in Rat Brain Tissue.

The neurophysiological changes that occur during pregnancy in the female mammal have led to the coining of the phrases "expectant brain" and "maternal brain". Although much is known of the hormonal changes during pregnancy, alterations in neurotransmitter gene expression have not been well-studied. We examined gene expression in the ventromedial nucleus of the hypothalamus (VMH) during pregnancy based on the fact that this nucleus not only modulates the physiological changes that occur during pregnancy but is also involved in the development of maternal behavior. This study was designed to identify genes that are differentially expressed between mid- and late-pregnancy in order to determine which genes may be associated with the onset and display of maternal behavior and the development of the maternal brain. A commercially available PCR array containing 84 neurotransmitter receptor and regulator genes (RT2 Profiler PCR array) was used. Brains were harvested from rats on days 12 and 21 of gestation, frozen, and micropunched to obtain the VMH. Total RNA was extracted, cDNA prepared, and SYBR Green qPCR was performed. In the VMH, expression of five genes were reduced on day 21 of gestation compared to day 12 (Chrna6, Drd5, Gabrr2, Prokr2, and Ppyr1) whereas Chat, Chrm5, Drd4, Gabra5, Gabrg2, LOC289606, Nmu5r2, and Npy5r expression was elevated. Five genes were chosen to be validated in an additional experiment based on their known involvement in maternal behavior onset. This experiment confirmed that gene expression for both the CCK-A receptor and the GABAAR γ2 receptor increases at the end of pregnancy. In general, these results identify genes possibly involved in the establishment of the maternal brain in rats and indicate possible new genes to be investigated.