Juvenile Female Rats Research Articles

Differential role of NMDA receptors in hippocampal-dependent spatial memory and plasticity in juvenile male and female rats.

Early life, or juvenility, stands out as the most pivotal phase in neurodevelopment due to its profound impact over the long-term cognition. During this period, significant changes are made in the brain's connections both within and between different areas, particularly in tandem with the development of more intricate behaviors. The hippocampus is among the brain regions that undergo significant postnatal remodeling, including dendritic arborization, synaptogenesis, the formation of complex spines and neuron proliferation. Given the crucial role of the hippocampus in spatial memory processing, it has been observed that spatial memory abilities continue to develop as the hippocampus matures, particularly before puberty. The N-methyl-d-aspartate (NMDA) type of glutamate receptor channel is crucial for the induction of activity-dependent synaptic plasticity and spatial memory formation in both rodents and humans. Although extensive evidence shows the role of NMDA receptors (NMDAr) in spatial memory and synaptic plasticity, the studies addressing the role of NMDAr in spatial memory of juveniles are sparse and mostly limited to adult males. In the present study, we, therefore, aimed to investigate the effects of systemic NMDAr blockade by the MK-801 on spatial memory (novel object location memory, OLM) and hippocampal plasticity in the form of long-term potentiation (LTP) of both male and female juvenile rats. Our results show the sex-dimorphic role of NMDAr in spatial memory and plasticity during juvenility, as systemic NMDAr blockade impairs the OLM and LTP in juvenile males without an effect on juvenile females. Taken together, our results demonstrate that spatial memory and hippocampal plasticity are NMDAr-dependent in juvenile males and NMDAr-independent in juvenile females. These sex-specific differences in the mechanisms of spatial memory and plasticity may imply gender-specific treatment for spatial memory disorders even in children.

Effects of methylphenidate on mitochondrial dynamics and bioenergetics in the prefrontal cortex of juvenile rats are sex-dependent

Methylphenidate (MPH) is a central nervous system stimulant drug and a first order prescription in the treatment of Attention Deficit Hyperactivity Disorder (ADHD). Although MPH biochemistry in neurodevelopment is not completely understood, studies showed it alters energy metabolism in rat brains. ADHD prevalence during neurodevelopment is related to males and the investigation has been mainly done in these subjects, therefore, little is known about MPH action in females and, consequently, about sexual dimorphism. In the present study we evaluated markers of mitochondrial dynamics (DRP1 and MFN2, fission and fusion, respectively), biogenesis (mtTFA) and bioenergetics (respiratory chain complexes) in prefrontal cortex of male and female juvenile rats submitted to exposure to MPH to better understand MPH effect during postnatal neurodevelopment. ATP and oxidative stress levels were also evaluated. Wistar rats received intraperitoneal injection of MPH (2.0 mg/kg) or control (saline), once a day, from 15th to 45th day of age. Results showed that MPH increased DRP1 and decreased MFN2, as well as increased mtTFA in prefrontal cortex of male rats. In female, MPH decreased NRF1 and increased Parkin, which are mitochondrial regulatory proteins. Respiratory chain complexes (complex I, SDH, complexes III and IV), ATP production and oxidative stress parameters were altered and shown to be sex-dependent. Taken together, results suggest that chronic MPH exposure at an early age in healthy animals changes mitochondrial dynamics, biogenesis and bioenergetics differently depending on the sex of the subjects.

Sex differences in the structure and function of the vasopressin system in the ventral pallidum are associated with the sex-specific regulation of social play behavior in juvenile rats

Vasopressin (AVP) regulates various social behaviors, often in sex-specific ways, including social play behavior, a rewarding behavior displayed primarily by juveniles. Here, we examined whether and how AVP acting in the brain's reward system regulates social play behavior in juvenile rats. Specifically, we focused on AVP signaling in the ventral pallidum (VP), a brain region that is a part of the reward system. First, we examined the organization of the VP-AVP system in juvenile rats and found sex differences, with higher density of both AVP-immunoreactive fibers and AVP V1a receptor (V1aR) binding in males compared to females while females show a greater number of V1aR-expressing cells compared to males. We further found that, in both sexes, V1aR-expressing cells co-express a GABA marker to a much greater extent (approx. 10 times) than a marker for glutamate. Next, we examined the functional involvement of V1aR-expressing VP cells in social play behavior. We found that exposure to social play enhanced the proportion of activated V1aR-expressing VP cells in males only. Finally, we showed that infusion of a specific V1aR antagonist into the VP increased social play behaviors in juvenile male rats while decreasing these behaviors in juvenile female rats. Overall, these findings reveal structural and functional sex differences in the AVP-V1aR system in the VP that are associated with the sex-specific regulation of social play behavior.

Modelling the Effects of Growth and Remodelling on the Density and Structure of Cancellous Bone

A two-stage model is proposed for investigating remodelling characteristics in bone over time and distance to the growth plate. The first stage comprises a partial differential equation (PDE) for bone density as a function of time and distance from the growth plate. This stage clarifies the contributions to changes in bone density due to remodelling and growth processes and tracks the rate at which new bone emanates from the growth plate. The second stage consists of simulating the remodelling process to determine remodelling characteristics. Implementing the second stage requires the rate at which bone moves away from the growth plate computed during the first stage. The second stage is also needed to confirm that remodelling characteristics predicted by the first stage may be explained by a realistic model for remodelling and to compute activation frequency. The model is demonstrated on microCT scans of tibia of juvenile female rats in three experimental groups: sham-operated control, oestrogen deprived, and oestrogen deprived followed by treatment. Model predictions for changes in bone density and remodelling characteristics agree with the literature. In addition, the model provides new insight into the role of treatment on the density of new bone emanating from the growth plate and provides quantitative descriptions of changes in remodelling characteristics beyond what has been possible to ascertain by experimentation alone.

Immature Status Epilepticus Alters the Temporal Relationship between Hippocampal Interictal Epileptiform Discharges and High-frequency Oscillations

The aim was to investigate the long-term effects of a single episode of immature Status Epilepticus (SE) on the excitability of the septal and temporal hippocampus in vitro, by studying the relationship between interictal-like epileptiform discharges (IEDs) and high-frequency oscillations (HFOs; Ripples, Rs and Fast Ripples, FRs). A pentylenetetrazol-induced Status Epilepticus-(SE)-like generalized seizure was induced at postnatal day 20 in 22 male and female juvenile rats, sacrificed >40 days later to prepare hippocampal slices. Spontaneous IEDs induced by Mg2+-free ACSF were recorded from the CA3 area of temporal (T) or septal (S) slices. Recordings were band-pass filtered off-line revealing Rs and FRs and a series of measurements were conducted, with mean values compared with those obtained from age-matched controls (CTRs). In CTR S (vs T) slices, we recorded longer R & FR durations, a longer HFO-IED temporal overlap, higher FR peak power and more frequent FR initiation preceding IEDs (% events). Post-SE, in T slices all types of events duration (IED, R, FR) and the time lag between their onsets (R-IED, FR-IED, R-FR) increased, while FR/R peak power decreased; in S slices, the IED 1st population spike and the FR amplitudes, the R and FR peak power and the (percent) events where Rs or FRs preceded IEDs all decreased. The CA3 IED-HFO relationship offers insights to the septal-to-temporal synchronization patterns; its post-juvenile-SE changes indicate permanent modifications in the septotemporal excitability gradient. Moreover, these findings are in line to region-specific regulation of various currents post-SE, as reported in literature.

Low doses of malathion impair ovarian, uterine, and follicular integrity by altering oxidative profile and gene expression of rats exposed during the peripubertal period.

Malathion serves as a pivotal pesticide in agriculture and the management of the Aedes aegypti mosquito. Despite its widespread use, there is a notable absence of studies elucidating the mechanisms through which malathion may affect the female reproductive system. Consequently, the objective of this investigation was to assess whether exposing juvenile female rats to low doses of malathion during the juvenile and peripubertal periods could compromise pubertal onset, estradiol levels, and the integrity of the ovaries and uterus while also examining the underlying mechanisms of damage. To achieve this, thirty juvenile female rats were subjected to either a vehicle or malathion (10mg/kg or 50mg/kg) between postnatal days 22 and 60, with subsequent verification of pubertal onset. Upon completion of the exposure period, blood samples were collected for estradiol assessment. The ovaries and uterus were then examined to evaluate histological integrity, oxidative stress, and the expression of genes associated with cell proliferation, antiapoptotic responses, and endocrine pathways. Although estradiol levels and pubertal onset remained unaffected, exposure to malathion compromised the integrity and morphometry of the ovaries and uterus. This was evidenced by altered oxidative profiles and changes in the expression of genes regulating the cell cycle, anti-apoptotic processes, and endocrine pathways. Our findings underscore the role of malathion in inducing cell proliferation, promoting cell survival, and causing oxidative damage to the female reproductive system in rats exposed during peripubertal periods.

Evaluation of the diphenyl herbicide, oxyfluorfen, for effects on thyroid hormones in the juvenile rat

Recently, oxyfluorfen, a pre- and post-emergent diphenyl ether herbicide, was identified in our laboratory as an inhibitor of iodide uptake by the sodium iodide symporter (NIS), the first key step in the synthesis of thyroid hormones (THs). This inhibition was observed in vitro, using both a human NIS engineered cell line (hNIS-HEK293T-EPA) and a rat thyroid follicular cell line (FRTL-5). Oxyfluorfen was found to be a potent inhibitor of NIS activity with an EC50 of approximately 2 µM in both cell lines with no observed cytotoxicity at any concentration tested up to 100 μM. The current research tested the hypothesis that oxyfluorfen alters circulating concentrations of THs. This hypothesis was first tested in a pilot study with both juvenile male and female rats exposed to oxyfluorfen for 4 days at 0, 125, 250 and 500 mg/kg/day. Once we identified that this short-term 4-day oxyfluorfen exposure suppressed both total serum thyroxine (T4) and triiodothyronine (T3) at all doses, we tested seven lower concentrations of oxyfluorfen (0.8125 to 62.5 mg/kg day) in an 8-day exposure paradigm to more closely evaluate the dose–response. We found that oxyfluorfen suppressed serum T4 with a LOEL of 3.25 mg/kg/day and T3 with a LOEL 62.5 mg/kg/day. Analytical chemistry of the serum showed an accumulation over time following oral exposure to oxyfluorfen in both the 4- and 8-day groups. Analytical chemistry of the thyroid glands in the 8-day study revealed higher accumulation in the thyroid as compared to the serum (2 to 3- fold at 62.5 mg/kg). No changes in thyroid weight or serum TSH were observed following the 8-day exposure. This study is the first to demonstrate an effect of oxyfluorfen on serum thyroid hormones in the rat. Additional studies are needed to further evaluate the effects on thyroid homeostasis with extended exposures and the potential implications of the observed effects.

Retrieval and savings of contextual fear memories across an extended retention interval in juvenile and adult male and female rats.

Adult rodents exhibit an exceptional ability to retrieve context fear memories across lengthy retention intervals. In contrast, these memories established in younger rodents are susceptible to significant forgetting. The present study aimed to examine the persistence of contextual fear memories established in juvenile and adult Long-Evans male and female rats. Testing 1-day after conditioning, adult males exhibited evidence for greater conditioning than juvenile males, while in females, conditioning did not differ between juvenile and adult rats. In adults, males displayed greater conditioning than females, while in juveniles, males and females reached similar conditioning levels. At the 60-day retention interval, adult sex differences were maintained; however, juvenile rats failed to retrieve this remote contextual fear memory. Next, we examined whether a savings test procedure could recover these remotely established juvenile memories. Following a 60-day retention test, the now adult rats were presented with an additional context-shock pairing to assess the level of savings. While this procedure produced greater conditioning in males than females, the relative savings of this early life memory were similar in males and females. The results of these experiments indicate that adult sex differences in contextual fear memory are maintained across an extended retention interval, while in juveniles, there were no significant sex differences. A novel finding in the present study was that both male and female rats failed to retrieve an initial juvenile memory following an extended retention interval. However, these memories were recovered with a single reminder of the original juvenile experience. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Adolescent food insecurity in female rodents and susceptibility to diet-induced obesity

Food insecurity is defined as having limited or uncertain access to nutritious foods, and adolescent food insecurity is associated with obesity and disordered eating behaviors in humans. We developed a rodent model of adolescent food insecurity to determine whether adolescent food insecurity per se promotes increased susceptibility to diet-induced obesity and altered eating behaviors during adulthood. Female juvenile Wistar rats were singly housed and assigned to three experimental diets: food-secure with standard chow (CHOW), food-secure with a high-fat/sugar Western diet (WD), and food-insecure with WD (WD-FI). Food-secure rats (CHOW and WD) received meals at fixed feeding times (9:00, 13:00, and 16:00). WD-FI rats received meals at unpredictable intervals of the above-mentioned feeding times but had isocaloric amounts of food to WD. We investigated the impact of adolescent food insecurity on motivation for sucrose (Progressive Ratio), approach-avoidance behavior for palatable high-fat food (Approach-Avoidance task), and susceptibility to weight gain and hyperphagia when given an obesogenic choice diet. Secondary outcomes were the effects of food insecurity during development on anxiety-like behaviors (Open Field and Elevated Plus Maze) and learning and memory function (Novel Location Recognition task). Rodents with adolescent food insecurity showed a greater trend of weight gain and significantly increased fat mass and liver fat accumulation on an obesogenic diet in adulthood, despite no increases in motivation for sucrose or high-fat food. These data suggest that adolescent unpredictable food access increases susceptibility to diet-induced fat gain without impacting food motivation or food intake in female rodents. These findings are among a small group of recent studies modeling food insecurity in rodents and suggest that adolescent food insecurity in females may have long-term implications for metabolic physiology later in life.

Repeated mild traumatic brain injury causes sex-specific increases in cell proliferation and inflammation in juvenile rats

Childhood represents a period of significant growth and maturation for the brain, and is also associated with a heightened risk for mild traumatic brain injuries (mTBI). There is also concern that repeated-mTBI (r-mTBI) may have a long-term impact on developmental trajectories. Using an awake closed head injury (ACHI) model, that uses rapid head acceleration to induce a mTBI, we investigated the acute effects of repeated-mTBI (r-mTBI) on neurological function and cellular proliferation in juvenile male and female Long-Evans rats. We found that r-mTBI did not lead to cumulative neurological deficits with the model. R-mTBI animals exhibited an increase in BrdU + (bromodeoxyuridine positive) cells in the dentate gyrus (DG), and that this increase was more robust in male animals. This increase was not sustained, and cell proliferation returning to normal by PID3. A greater increase in BrdU + cells was observed in the dorsal DG in both male and female r-mTBI animals at PID1. Using Ki-67 expression as an endogenous marker of cellular proliferation, a robust proliferative response following r-mTBI was observed in male animals at PID1 that persisted until PID3, and was not constrained to the DG alone. Triple labeling experiments (Iba1+, GFAP+, Brdu+) revealed that a high proportion of these proliferating cells were microglia/macrophages, indicating there was a heightened inflammatory response. Overall, these findings suggest that rapid head acceleration with the ACHI model produces an mTBI, but that the acute neurological deficits do not increase in severity with repeated administration. R-mTBI transiently increases cellular proliferation in the hippocampus, particularly in male animals, and the pattern of cell proliferation suggests that this represents a neuroinflammatory response that is focused around the mid-brain rather than peripheral cortical regions. These results add to growing literature indicating sex differences in proliferative and inflammatory responses between females and males. Targeting proliferation as a therapeutic avenue may help reduce the short term impact of r-mTBI, but there may be sex-specific considerations.

Early-life ozone exposure modulates region-specific gene expression in the developing rat lung

Early-life ozone exposure disrupts normal patterns of lung development, but the molecular determinants underlying these changes are not well understood. This study aimed to elucidate changes in gene expression following episodic ozone exposure to identify potential mechanisms of ozone-mediated impairments in lung development. Rat pups were exposed to either filtered air or ozone (0.5 ppm, 6 hr./day, 5 days/week) from postnatal day (PND) 7–28 (16 dams total with 8 pups each, 4 M & 4 F) and sacrificed at either PND 30–31 or PND 80–84. Lung microdissection isolated major regions for RNA-Seq analysis. Ozone modified inherent differences in gene expression between lung regions in both male and female rat pups, whereas statistically significant changes in gene expression directly attributed to ozone were only identified in females. The greatest number of differentially expressed genes was observed between the distal airways and the parenchyma of ozone-exposed juvenile female rats, with 355 genes being differentially expressed. Genes modulating epithelial-to-mesenchymal transition, cell growth, and adhesion were differentially expressed in the parenchyma of ozone exposed juvenile females, suggesting that episodic ozone exposure may affect branching morphogenesis and lung cell growth. Importantly, our study provides novel targets for future experiments investigating the impact of ozone on lung development.

Prenatal allergic inflammation in rats confers sex-specific alterations to oxytocin and vasopressin innervation in social brain regions

Prenatal exposure to inflammation via maternal infection, allergy, or autoimmunity increases one's risk for developing neurodevelopmental and psychiatric disorders. Many of these disorders are associated with altered social behavior, yet the mechanisms underlying inflammation-induced social impairment remain unknown. We previously found that a rat model of acute allergic maternal immune activation (MIA) produced deficits like those found in MIA-linked disorders, including impairments in juvenile social play behavior. The neuropeptides oxytocin (OT) and arginine vasopressin (AVP) regulate social behavior, including juvenile social play, across mammalian species. OT and AVP are also implicated in neuropsychiatric disorders characterized by social impairment, making them good candidate regulators of social deficits after MIA.We profiled how acute prenatal exposure to allergic MIA changed OT and AVP innervation in several brain regions important for social behavior in juvenile male and female rat offspring. We also assessed whether MIA altered additional behavioral phenotypes related to sociality and anxiety. We found that allergic MIA increased OT and AVP fiber immunoreactivity in the medial amygdala and had sex-specific effects in the nucleus accumbens, bed nucleus of the stria terminalis, and lateral hypothalamic area. We also found that MIA reduced ultrasonic vocalizations in neonates and increased the stereotypical nature of self-grooming behavior. Overall, these findings suggest that there may be sex-specific mechanisms underlying MIA-induced behavioral impairment and underscore OT and AVP as ideal candidates for future mechanistic studies.

Wildtype peers rescue social play and 50-kHz ultrasonic vocalization deficits in juvenile female Cacna1c heterozygous rats.

Healthy brain development depends on early social practices and experiences. The risk gene CACNA1C is implicated in numerous neuropsychiatric disorders, in which key characteristics include deficits in social functioning and communication. Recently, we reported sex-dependent impairments in social behavior and ultrasonic vocalizations (USV) in juvenile heterozygous Cacna1c+/- (HET) rats. Specifically, HET females displayed increases in rough-and-tumble play that eliminated the typically observed sex difference between male and female rats. Interestingly, female wild-type Cacna1c+/+ (WT) pairs also showed a similar increase in social play when housed with HET females, suggesting their behavior may be influenced by HET cage mates. This indicates that the genetic makeup of the social environment related to Cacna1c can influence social play, yet systematic studies are lacking. In the present study, we housed juvenile females in MIXED- or SAME-genotype cages and tested them in a social play paradigm with a same- and opposite-genotype partner. The results show that the early social environment and the genotype of the play partner influence social play and 50-kHz USV emission. Experience with a WT play partner appears necessary for HET females to show comparable levels of play and 50-kHz USV emission. Same-genotype HET pairs played less and emitted fewer 50-kHz USV than same-genotype WT or opposite-genotype pairs; however, we found that the decrease in social play and 50-kHz USV in HET pairs can be rescued by playing with a WT partner. The effect was particularly prominent when the first play partner was WT, as we found it increased play and 50-kHz USV emission in all subsequent interactions with ensuing partners. These findings suggest that the genetic makeup related to the social environment and/or social peers influences social play in Cacna1c+/- haploinsufficient rats. Specifically, our results show that WT peers can rescue behavior and communication alterations in Cacna1c female rats. Our findings have important implications because they show that the genetic makeup of the social environment can divulge phenotypic changes in genetic rat models of neuropsychiatric disorders.

Alteration in social interaction and tactile discrimination of juvenile autistic-like rats following tactile stimulation and whisker deprivation.

Autism spectrum disorder is a developmental disorder that can affect sensory-motor behaviors in the valproic acid (Val) rodent model of autism. Although whisker deprivation (WD) induces plastic changes in the cortical neurons, tactile stimulation (TS) during the neonatal period may reverse it. Here, we investigate the interaction effects of TS and WD on behavioral and histologic features of barrel cortex neurons in juvenile Val-treated. Control (CTL, CTL-TS, CTL-WD, and CTL-TS-WD groups) and Val-treated (Val, Val-TS, Val-WD, and Val-TS-WD groups) rats of both sexes were subjected to behavioral tests of social interaction, and novel texture recognition, and Nissl staining. The TS groups were exposed to sensory stimulation for 15min, three times/day; moreover, all whiskers in the WD groups were trimmed every other day from postnatal days 1 to 21. Both prenatal valproic acid administration and postnatal WD decreased the rats' performance percentage of the Val and CTL-WD groups of both sexes compared with the CTL groups in the social interaction and texture discrimination tests. Following TS, the performance of the Val-TS-WD group increased significantly compared to the Val group (p<.05), whereas the performance of the CTL-TS-WD group rescued to the CTL group. Nissl staining results also revealed the neuron degeneration percentage in the barrel field area of the Val and CTL-WD groups was increased significantly (p<.05) compared with the CTL group. In this regard, TS decreased the neuron degeneration percentage of the Val-TS-WD and the CTL-TS-WD groups, compared with the CTL group, significantly (p<.05). TS in juvenile male and female rats can act as a modulator and compensate for the behavioral and histological consequences of WD and prenatal valproic acid exposure.

Development of social recognition ability in female rats: Effect of pubertal ovarian hormones

The ability to recognize previously encountered conspecifics is crucial for social interaction. This social recognition ability is well characterized in adult rodents of both sexes but remains largely unexplored in juveniles. Using the social discrimination test of social recognition with short intervals (30 min and 1 h), we first found that juvenile female rats do not display a difference in investigation directed toward a novel vs. familiar stimulus rat. Using the social discrimination test with a 30-minute interval, we then showed that social recognition is established by the time of adolescence in female rats. Based on these findings, we hypothesized that social recognition is dependent on the initiation of ovarian hormone release during puberty. To test this, we ovariectomized females prior to puberty and found that prepubertal ovariectomy prevented the development of social recognition ability in adulthood. Administration of estradiol benzoate, 48 h prior to testing, to juvenile females or prepubertally ovariectomized adult females did not restore social recognition, suggesting that ovarian hormones organize the neural circuitry regulating this behavior during adolescence. These findings provide the first evidence of an effect of pubertal development on social recognition ability in female rats and highlight the importance of considering sex and age when interpreting results from behavioral paradigms initially designed for use in adult males.

Ameliorating age-dependent effects of resveratrol on VPA-induced social impairments and anxiety-like behaviors in a rat model of neurodevelopmental disorder

Although anxiety disorders, as well as difficulties in social interaction, are documented in children with autism spectrum disorder (ASD) as a neurodevelopmental disorder, the effectiveness of potential therapeutic procedures considering age and sex differences is under serious discussion. The present study aimed to investigate the effect of resveratrol (RSV) on anxiety-like behaviors and social interaction in juvenile and adult rats of both sex in a valproic acid (VPA)-induced autistic-like model. Prenatal exposure to VPA was associated with increased anxiety, also causing a significant reduction in social interaction in juvenile male subjects. Further administration of RSV attenuated VPA-induced anxiety symptoms in both sexes of adult animals and significantly increased the sociability index in male and female juvenile rats. Taken together, it can be concluded that treatment with RSV can attenuate some of the harsh effects of VPA. This treatment was especially effective on anxiety-like traits in adult subjects of both sexes regarding their performance in open field and EPM. We encourage future research to consider the sex and age-specific mechanisms behind the RSV treatment in the prenatal VPA model of autism.

Sex- and age-specific respiratory alterations induced by prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 in rats.

Cannabis legalization has risen in many countries, and its use during pregnancy has increased. The endocannabinoid system is present in the CNS at early stages of embryonic development, and regulates functional brain maturation including areas responsible for respiratory control, data on the influence of external cannabinoids on the development of the respiratory system and possible consequences during postnatal life are limited. We evaluated the effects of prenatal exposure to synthetic cannabinoid (WIN 55,212-2 [WIN], 0.5 mg·kg-1 ·day-1 ) on the respiratory control system in neonatal (P0, P6-7 and P12-13) and juvenile (P27-28) male and female rats. WIN administration to pregnant rats interfered sex-specifically with breathing regulation of offspring, promoting a greater sensitivity to CO2 at all ages in males (except P6-7) and in juvenile females. An altered hypoxic chemoreflex was observed in P0 (hyperventilation) and P6-7 (hypoventilation) males, which was absent in females. Along with breathing alterations, brainstem analysis showed an increase in the number of catecholaminergic neurons and cannabinoid receptor type 1 (CB1 ) and changes in tissue respiration in the early males. A reduction in pulmonary compliance was observed in juvenile male rats. Preexposure to WIN enhanced spontaneous apnoea and reduced the number of serotoninergic (5-HT) neurons in the raphe magnus nucleus of P0 females. These data demonstrate that excess stimulation of the endocannabinoid system during gestation has prolonged and sex-specific consequences for the respiratory control system.

Fingolimod prevents cognitive impairments following hypoxia-induced neonatal seizure by ameliorating the inflammation and oxidative stress in male and female juvenile rats

Hypoxia-induced neonatal seizure (HINS) is associated with cognitive disorders in later life. Since the neuroinflammation following HINS leads to these deleterious effects, in the current study, the short-term outcomes of Fingolimod (FTY720) treatment as an anti-inflammatory agent was investigated in the rat model of HINS. Seizures were induced in postnatal day (P10) by exposure to 5 % O2 for 15 min. Forty-five minutes after the onset of hypoxia, pups received FTY720 (0.3 mg/kg) or normal saline for 12 consecutive days. Then, behavioral functions of the rats were assessed by commonly used tests. Subsequently, hippocampal tissue sampling at P22-P23 was done for biochemical assessments and real-time PCR. The results showed that FTY720 prevents cognitive impairments and related reduction of malondialehyde (MDA) concentration in the hippocampus of both male and female hypoxic animals. Furthermore, FTY720 administration following HINS could not prevent the decreased brain-derived neurotrophic factor (BDNF) concentration, increased nitric oxide (NO) level and decreased Interleukin-4 (IL-4) gene expression in the hippocampus of both male and female hypoxic animals. Interestingly, FTY720 showed significant interaction with sex in hippocampal Tumor necrosis factor alpha (TNF-α) gene expression and BDNF concentration. Taken together, these results suggest that FTY720 administration in the lactation period can prevent the deleterious effects of HINS on cognition in later life by ameliorating inflammation and oxidative stress in the hippocampus of both male and female juvenile rats.

A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes.

The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation. Sperm collected from a sexually mature male rat the following morning is then pre-cultured. Cumulus cell-oocyte complexes (COCs) are collected from female rats under anesthesia, and COCs are induced into a medium containing concentration-adjusted sperm. Thereafter, oocytes with two pronucleus are selected as fertilized oocytes. Next, fertilized oocytes are transferred into a glass chamber containing CRISPR ribonucleoprotein (RNP) complexes formed from gRNA and Cas9 protein. After electroporation, fertilized oocytes are then immediately transferred to culture medium. The next day, embryos are transferred into the oviduct of pseudopregnant female rats. Using the above method, offspring can be obtained 22days after the day of embryo transfer. In this paper, we outline a method allowing simple and efficient generation of genetically modified rats without the need for technically difficult micromanipulation techniques.

Supplemental Material for Retrieval and Savings of Contextual Fear Memories Across an Extended Retention Interval in Juvenile and Adult Male and Female Rats

Supplemental Material for Retrieval and Savings of Contextual Fear Memories Across an Extended Retention Interval in Juvenile and Adult Male and Female Rats