Peripuberty Is a Sensitive Period for Prefrontal Parvalbumin Interneuron Activity to Impact Adult Cognitive Flexibility.

INTRODUCTION: Developmental windows in which experiences can elicit long-lasting effects on brain circuitry and behavior are called "sensitive periods" and reflect a state of heightened plasticity. The classic example of a sensitive period comes from studies of sensory systems, like the visual system, where early visual experience is required for normal wiring of primary visual cortex and proper visual functioning. At a mechanistic level, loss of incoming visual input results in a decrease in activity in thalamocortical neurons representing the affected eye, resulting in an activity-dependent reduction in the representation of those inputs in the visual cortex and loss of visual perception in that eye. While associative cortical regions like the medial prefrontal cortex (mPFC) do not receive direct sensory input, recent findings demonstrate that changes in activity levels experienced by this region during defined windows in early development may also result in long-lasting changes in prefrontal cortical circuitry, network function, and behavior. For example, we recently demonstrated that decreasing the activity of mPFC parvalbumin-expressing (PV) interneurons during a period of time encompassing peripuberty (postnatal day P14) to adolescence (P50) led to a long-lasting decrease in their functional inhibition of pyramidal cells, as well as impairments in cognitive flexibility. While the effects of manipulating mPFC PV interneuron activity were selective to development, and not adulthood, the exact timing of the sensitive period for this manipulation remains unknown. METHODS: To refine the sensitive period in which inhibiting mPFC PV cell activity can lead to persistent effects on prefrontal functioning, we used a chemogenetic approach to restrict our inhibition of mPFC PV activity to two distinct windows: (1) peripuberty (P14-P32) and (2) early adolescence (P33-P50). We then investigated adult behavior after P90. In parallel, we performed histological analysis of molecular markers associated with sensitive period onset and offset in visual cortex, to define the onset and offset of peak-sensitive period plasticity in the mPFC. RESULTS: We found that inhibition of mPFC PV interneurons in peripuberty (P14-P32), but not adolescence (P33-P50), led to an impairment in set-shifting behavior in adulthood manifest as an increase in trials to reach criterion performance and errors. Consistent with a pubertal onset of sensitive period plasticity in the PFC, we found that histological markers of sensitive period onset and offset also demarcated P14 and P35, respectively. The time course of expression of these markers was similar in visual cortex. CONCLUSION: Both lines of research converge on the peripubertal period (P14-P32) as one of heightened sensitive period plasticity in the mPFC. Further, our direct comparison of markers of sensitive period plasticity across the prefrontal and visual cortex suggests a similar time course of expression, challenging the notion that sensitive periods occur hierarchically. Together, these findings extend our knowledge about the nature and timing of sensitive period plasticity in the developing mPFC.

Sex steroid priming for growth hormone stimulation testing in children and adolescents with short stature: A systematic review.

OBJECTIVE: Growth hormone stimulation testing (GHST) is used to diagnose growth hormone deficiency (GHD) in children. As sex steroids impact on anterior pituitary function, there is concern around the efficacy of GHST in peripubertal children, where endogenous sex steroid levels are low. Sex steroid priming before GHST is thought to improve test efficacy in these children, however evidence to support its use in clinical practice is limited. In this systematic review, we addressed the following research questions: Does priming increase GH stimulation test efficacy in peripubertal children? Does priming identify those who would benefit most from treatment in terms of final height? Is there evidence for an optimal sex-steroid priming regimen? DESIGN, PATIENTS, MEASUREMENTS: The study was registered with PROSPERO and conducted according to PRISMA guidelines. We searched Medline, Cochrane-Library, Scopus, EMBASE and Web-of-Science and included all studies that included GHST in both primed and unprimed children. A GH cut-off of 7 µg/L was used as a threshold for GHD. Study quality was assessed using the Risk-Of-Bias in Non- Randomized Studies (ROBINS-I) tool or the revised Cochrane risk-of-bias tool for Randomised trials. RESULTS: Fifteen studies met our inclusion criteria, of which 4/15 (27%) were randomised control trials. The majority (9/15) of the studies indicated that priming increases growth hormone response upon GHST in peripubertal children, increasing test specificity. Two studies investigated final height after treatment based on the results of primed versus unprimed GHST. These results indicate that growth hormone treatment based on results of a primed GHST improve outcomes compared with treatment based on an unprimed test. CONCLUSION: Sex-steroid priming increases the growth hormone response during GHST, resulting in fewer patients meeting the threshold required for a diagnosis of GHD. Unnecessary GH treatment may be avoided in some patients without a detrimental effect on final height. Numerous sex-steroid priming regimens have been used in clinical practice and the majority appear to be effective, but an optimal regimen has not been determined.

Biobehavioral mechanisms underlying testosterone and mood relationships in peripubertal female adolescents.

The pubertal transition is characterized by pronounced sex hormone fluctuation, refinement of affective neural circuitry, and an increased risk of depression in female adolescents. Sex hormones, including testosterone, exert modulatory effects on frontal-limbic brain networks and are associated with emotion dysregulation and depressive symptoms. Weekly changes in hormones predict affective symptoms in peripubertal female adolescents, particularly in the context of stress; however, the biobehavioral mechanisms underlying hormone change and mood relationships during the pubertal transition have yet to be determined and was the objective of the present study. Forty-three peripubertal female adolescents (ages 11-14) collected 8-weekly salivary hormone (estrone, testosterone) samples and mood assessments to evaluate hormone-mood relationships, followed by a biobehavioral testing session with psychosocial stress and EEG. Within-person correlations between weekly hormone changes and corresponding mood were performed to determine individual differences in mood sensitivity to weekly hormone change. Increased frontal theta activity indexing emotion reactivity, reduced cortisol reactivity, and reduced vagal efficiency predicted the strength of the relationship between testosterone and mood. Further, testosterone-sensitivity strength was associated with the enhancement of negative affect following stress testing. Results identify divergent frontal theta and stress responses as potential biobehavioral mechanisms underlying mood sensitivity to peripubertal testosterone fluctuation.

Pre-pubertal exposure to ibuprofen impairs sperm parameters in male adult rats and compromises the next generation.

Ibuprofen is one of the most commonly prescribed anti-inflammatory drugs in pediatric practice. This drug inhibits the cyclooxygenase enzyme, reducing the production of prostaglandin, an important mediator on male reproductive function. We examined if pre-pubertal treatment with ibuprofen in male rats can affect the reproductive parameters of these animals in adult life and on their descendants. Male rats (23 days old) received ibuprofen (0; 2.4; 7.2 or 14.3 mg/kg/day), per gavage, from postnatal day (PND) 23 to 53. At sexual maturity, treated males were placed with untreated females for obtaining the next generation (F1). The highest dose of ibuprofen interfered in sexual behavior and reduced the fertility potential of these animals in adulthood. Additionally, the ibuprofen treatment altered the sperm quantity and quality, as evidenced by a decrease in sperm motility and in the daily sperm production in the testis. Testosterone levels were also reduced by pre-pubertal treatment. The paternal treatment with this drug also influenced the reproductive outcomes of progeny. The male offspring from males treated exhibited acceleration in sperm transit time in the epididymis and the number and volume of Leydig cell nuclei were decreased, while the estrous cyclicity was displayed and the fertility potential reduced in the female offspring. The pre-pubertal ibuprofen-treatment caused negative reproductive impacts in adulthood, compromising sperm quality and quantity, as well as interfered in the reproductive outcomes of the next generation.

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex.

Adverse life experiences increase the lifetime risk to several stress-related psychopathologies, such as anxiety or depressive-like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography-mass spectrometry (LC-MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu were upregulated, while Leu-enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, peptide PHI-27, somatostatin-28 (AA1-12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain-area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression-associated behaviors.

Differentiating between Testicular Toxicity and Sexual Immaturity in Ortho-phthalaldehyde Inhalation Toxicity Studies in Rats and Mice.

Early deaths of young or juvenile animals (before sexual maturation is achieved) in routine regulatory safety studies present pathologists and toxicologists with the challenge of interpreting findings in the male reproductive tract. Additionally, the advent of toxicity testing regulations has resulted in a growing need for the use of juvenile animals in toxicology studies. Here, we present the reproductive toxicity findings from a 13-week inhalation toxicity study with ortho-phthalaldehyde (OPA) in male rats and mice as a case example for working through this challenging task. In this study with OPA, survival was significantly reduced in the two highest exposure concentrations of OPA tested. Early deaths and histopathological lesions in the testes and epididymides were generally also limited to these two highest exposure groups. Therefore, there was concern that peripubertal morphological features could be a confounding factor for the histopathological evaluation of exposure-related testicular and epididymal findings. Although it can be difficult to differentiate exposure-related effects from the normal morphological features defining peripubertal changes in the testes and epididymides in animals that die early in a toxicity study, the use of age-matched controls in this case study with OPA provided a reference and aided in the differentiation of these effects.

Exposure to phthalates is associated with lipid profile in peripubertal Mexican youth.

Animal models indicate that endocrine disrupting chemicals (EDCs) affect circulating lipid concentrations by interfering with hepatic fatty acid oxidation. Little is known of the relationship between EDC exposure and lipid profile in humans. We measured bisphenol A (BPA) and 9 phthalate metabolites in maternal urine collected at up to three time points during pregnancy as a measure of in utero exposure, and in the child's urine at 8-14 years as a measure of concurrent, peripubertal exposure among 248 participants of a Mexico City pre-birth cohort. We used linear regression to examine relations of BPA and phthalate exposure with peripubertal serum lipids, while also adjusting for child age, sex, and specific gravity. While in utero EDC exposure was not associated with lipid profile, higher concurrent levels of mono-3-carboxypropyl phthalate (MCPP), monoethyl phthalate (MEP), and dibutyl phthalate metabolites (DBP) corresponded with lower total cholesterol and low-density lipoprotein (LDL-C) in boys; e.g., an interquartile range increment in MCPP corresponded with 7.4% (2.0%, 12.8%) lower total cholesterol and 12.7% (3.8%, 21.6%) lower LDL-C. In girls, higher urinary di-2-ethylhexyl phthalate metabolites (ΣDEHP) correlated with lower LDL-C (-7.9% [-15.4%, -0.4%]). Additional longitudinal research is needed to determine whether these associations persist beyond adolescence.

Fish pollutants MeHg and Aroclor cause permanent structural damage in male gonads and kidneys after prepubertal exposure.

This study investigated whether or not prepubertal exposure to the fish contaminants methylmercury (MeHg) and the polychlorinated bisphenol Aroclor in low doses interferes with the histomorphometry of the testes, epididymis, liver and kidneys in rats. Wistar male rats, 21 days old, were allocated into the following: control (n = 17, received corn oil), MeHg (n = 17, received MeHg at 0.5 mg/kg/day), Aroclor (n = 17, received Aroclor at 1.0 mg/kg/day), low mix (n = 18, received MeHg at 0.05 mg/kg/day and Aroclor at 0.1 mg/kg/day), high mix (n = 18, received MeHg at 0.5 mg/kg/day and Aroclor at 1.0 mg/kg/day). Dosing continued from post natal day (PND) 23 to 53, by gavage. Euthanasia was performed on PND 53; or, after an interval of 62 days without exposure to chemicals, on PND 115. The degree of maturation of the seminiferous epithelium was delayed in chemical-exposed groups and testicular interstitial oedema was observed at adulthood. The pattern of male gonad organization was changed in the Aroclor group on PND 53 and in all treated groups at adulthood. The animals from Aroclor, low mix and high mix groups showed a reduction in the number of Sertoli cells. Histological evidence of renal injury was observed in all chemical-exposed groups in both ages. A probable target for MeHg and Aroclor in the reproductive system was Sertoli cells, in which possible dysfunctions could be linked to the other testicular alterations. Curiously, the main deleterious effects were late outcomes, along with the absence of synergistic interaction of MeHg and Aroclor in the parameters investigated. In conclusion, fish pollutants MeHg and Aroclor caused permanent structural damage in male gonads and kidneys after prepubertal exposure, without showing clear chemical interactions.

Chlormequat chloride induces hepatic steatosis by promoting mTOR/SREBP1 mediated lipogenesis via AMPK inhibition.

Chlormequat chloride (CCC), a widely used plant growth regulator, is a choline analogue that has been shown to have endocrine-disrupting effects. Previous studies have shown that maternal exposure to CCC could induce hyperlipidemia and growth disruption in rat offspring. This study aims to further investigate the effects of peripubertal exposure to CCC on pubertal development and lipid homeostasis, as well as the underlying mechanisms. In vivo, male weanling rats were exposed to CCC (0, 20, 75 and 200 mg/kg bw/day) from post-natal day 21-60 via daily oral gavage. The results in rats showed that 75 mg/kg CCC treatment induced hepatic steatosis, predominantly microvesicular steatosis with a small amount of macrovesicular steatosis, in rat livers and 200 mg/kg CCC treatment induced liver damage including inflammatory infiltration, hepatic sinusoidal dilation and necrosis. In vitro, HepG2 cells were treated with CCC (0, 30, 60, 120, 240 and 480 µg/mL) for 24 h. And the results showed that CCC above 120 µg/mL induced an increase in triglyceride and neutral lipid levels of HepG2 cells. Mechanism exploration revealed that CCC treatment promoted the activation of mTOR/SREBP1 signalling pathway and inhibited activation of AMPK in both in vivo rat livers and in vitro HepG2 cells. Treatment with AMPK activator Acadesine (AICAR) could alleviate the lipid accumulation in HepG2 cells induced by CCC. Collectively, the present results indicate that CCC might induce hepatic steatosis by promoting mTOR/SREBP1 mediated lipogenesis via AMPK inhibition.

Methods for characterizing ovarian and adrenal hormone variability and mood relationships in peripubertal females.

Peripubertal females are at elevated risk for developing affective illness compared to males, yet biological mechanisms underlying this sex disparity are poorly understood. Female risk for depression remains elevated across a woman's reproductive lifespan, implicating reproductive hormones. A sensitivity to normal hormone variability during reproductive transition events (e.g., perimenopause) precipitates affective disturbances in susceptible women; however, the extent of hormone variability during the female pubertal transition and whether vulnerability to peripubertal hormone flux impacts affective state change in peripubertal females has not been studied. 52 healthy peripubertal females (ages 11-14) provided 8 weekly salivary samples and mood ratings. 10 salivary ovarian and adrenal hormones (e.g., estrone, testosterone, dehydroepiandrosterone (DHEA)) were analyzed weekly for 8 weeks using an ultrasensitive assay to characterize the female peripubertal hormone environment and its association with affective state. Hormone variability indices, including standard deviation, mean squared and absolute successive differences of the 8 weekly measurements were analyzed by menarche status. Within-person partial correlations were computed to determine the strength of the relationship between weekly change in hormone level and corresponding mood rating for each participant. As expected, results indicated that hormone variability was greater for post- relative to pre-menarchal females and with advancing pubertal development, yet pregnenolone-sulfate and aldosterone did not differ by menarche status. Mood sensitivity to changes in estrone was exhibited by 57% of participants, whereas 37% were sensitive to testosterone and 6% were sensitive to DHEA changes. The present results offer novel evidence that a substantial proportion of peripubertal females appear to be mood sensitive to hormone changes and may inform future investigations on the biological mechanisms underlying hormone-induced affect dysregulation in peripubertal females.

Protein Restriction in the Peri-Pubertal Period Induces Autonomic Dysfunction and Cardiac and Vascular Structural Changes in Adult Rats.

Perturbations to nutrition during critical periods are associated with changes in embryonic, fetal or postnatal developmental patterns that may render the offspring more likely to develop cardiovascular disease in later life. The aim of this study was to evaluate whether autonomic nervous system imbalance underpins in the long-term hypertension induced by dietary protein restriction during peri-pubertal period. Male Wistar rats were assigned to groups fed with a low protein (4% protein, LP) or control diet (20.5% protein; NP) during peri-puberty, from post-natal day (PN) 30 until PN60, and then all were returned to a normal protein diet until evaluation of cardiovascular and autonomic function at PN120. LP rats showed long-term increased mean arterial pressure (p = 0.002) and sympathetic arousal; increased power of the low frequency (LF) band of the arterial pressure spectral (p = 0.080) compared with NP animals. The depressor response to the ganglion blocker hexamethonium was increased in LP compared with control animals (p = 0.006). Pulse interval variability showed an increase in the LF band and LF/HF ratio (p = 0.062 and p = 0.048) in LP animals. The cardiac response to atenolol and/or methylatropine and the baroreflex sensitivity were similar between groups. LP animals showed ventricular hypertrophy (p = 0.044) and increased interstitial fibrosis (p = 0.028) compared with controls. Reduced protein carbonyls (PC) (p = 0.030) and catalase activity (p = 0.001) were observed in hearts from LP animals compared with control. In the brainstem, the levels of PC (p = 0.002) and the activity of superoxide dismutase and catalase (p = 0.044 and p = 0.012) were reduced in LP animals, while the levels of GSH and total glutathione were higher (p = 0.039 and p = 0.038) compared with NP animals. Protein restriction during peri-pubertal period leads to hypertension later in life accompanied by sustained sympathetic arousal, which may be associated with a disorganization of brain and cardiac redox state and structural cardiac alteration.

Maternal intake of omega-3 and omega-6 polyunsaturated fatty acids during mid-pregnancy is inversely associated with linear growth.

This study investigates relations of maternal N-3 and N-6 polyunsaturated fatty acids (PUFA) intake during pregnancy with offspring body mass index (BMI), height z-score and metabolic risk (fasting glucose, C-peptide, leptin, lipid profile) during peripuberty (8-14 years) among 236 mother-child pairs in Mexico. We used food frequency questionnaire data to quantify trimester-specific intake of N-3 alpha-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); N-6 linoleic acid and arachidonic acid (AA); and N-6:N-3 (AA:EPA+DHA), which accounts for the fact that the two PUFA families have opposing effects on physiology. Next, we used multivariable linear regression models that accounted for maternal education and parity, and child's age, sex and pubertal status, to examine associations of PUFA intake with the offspring outcomes. In models where BMI z-score was the outcome, we also adjusted for height z-score. We found that higher second trimester intake of EPA, DHA and AA were associated with lower offspring BMI and height z-score. For example, each 1-s.d. increment in second trimester EPA intake corresponded with 0.25 (95% CI: 0.03, 0.47) z-scores lower BMI and 0.20 (0.05, 0.36) z-scores lower height. Accounting for height z-score in models where BMI z-score was the outcome attenuated estimates [e.g., EPA: -0.16 (-0.37, 0.05)], suggesting that this relationship was driven by slower linear growth rather than excess adiposity. Maternal PUFA intake was not associated with the offspring metabolic biomarkers. Our findings suggest that higher PUFA intake during mid-pregnancy is associated with lower attained height in offspring during peripuberty. Additional research is needed to elucidate mechanisms and to confirm findings in other populations.

Peripubertal serum dioxin concentrations and subsequent sperm methylome profiles of young Russian adults.

BACKGROUND: The association of exposure to endocrine disrupting chemicals in the peripubertal period with subsequent sperm DNA methylation is unknown. OBJECTIVE: We examined the association of peripubertal serum 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentrations with whole-genome bisulfite sequencing (WGBS) of sperm collected in young adulthood. METHODS: The Russian Children's Study is a prospective cohort of 516 boys who were enrolled at 8-9 years of age and provided semen samples at 18-19 years of age. WGBS of sperm was conducted to identify differentially methylated regions (DMR) between highest (n = 4) and lowest (n = 4) peripubertal TCDD groups. RESULTS: We found 52 DMRs that distinguished lowest and highest peripubertal serum TCDD concentrations. One of the top scoring networks, "Cellular Assembly and Organization, Cellular Function and Maintenance, Carbohydrate Metabolism", identified estrogen receptor alpha as its central regulator. CONCLUSION: Findings from our limited sample size suggest that peripubertal environmental exposures are associated with sperm DNA methylation in young adults.

Effects of adverse early-life events on aggression and anti-social behaviours in animals and humans.

We review the impact of early adversities on the development of violence and antisocial behaviour in humans, and present three aetiological animal models of escalated rodent aggression, each disentangling the consequences of one particular adverse early-life factor. A review of the human data, as well as those obtained with the animal models of repeated maternal separation, post-weaning social isolation and peripubertal stress, clearly shows that adverse developmental conditions strongly affect aggressive behaviour displayed in adulthood, the emotional responses to social challenges and the neuronal mechanisms activated by conflict. Although similarities between models are evident, important differences were also noted, demonstrating that the behavioural, emotional and neuronal consequences of early adversities are to a large extent dependent on aetiological factors. These findings support recent theories on human aggression, which suggest that particular developmental trajectories lead to specific forms of aggressive behaviour and brain dysfunctions. However, dissecting the roles of particular aetiological factors in humans is difficult because these occur in various combinations; in addition, the neuroscientific tools employed in humans still lack the depth of analysis of those used in animal research. We suggest that the analytical approach of the rodent models presented here may be successfully used to complement human findings and to develop integrative models of the complex relationship between early adversity, brain development and aggressive behaviour.

CRHR1 links peripuberty stress with deficits in social and stress-coping behaviors.

Stressful life events during childhood and adolescence are important risk factors for the development of psychopathologies later in life. The corticotropin releasing hormone (CRH) and the CRH receptor 1 (CRHR1) have been implicated in the link between early life adversity and adult anxiety and depression, with rodent studies identifying the very early postnatal period as highly susceptible to this programming. Here, we investigated whether stress exposure during the peripubertal period - comprising juvenility and puberty - is effective in inducing long-lasting changes in the expression of CRHR1 and CRHR2 in the hippocampus and amygdala, and whether treating animals with a CRHR1 antagonist following stress exposure could reverse behavioral alterations induced by peripuberty stress. We show that peripuberty stress leads to enhanced expression of the Crhr1, but not Crhr2, gene in the hippocampal CA1 and the central nucleus of the amygdala, in association with social deficits in the social exploration test and increased stress-coping behaviors in the forced swim test. Treatment with the CRHR1 antagonist NBI30775 (10 mg/kg) daily for 1 week (from P43 to P49), immediately following peripuberty stress exposure, prevented the occurrence of those psychopathological behaviors at adulthood. These findings highlight peripuberty as a period of plasticity for the enduring modulation of the CRHR1 system and support a growing body of data implicating the CRHR1 system in the programming effects of early life stress on eventual psychopathology. They also support recent evidence indicating that temporarily tackling CRHR1 during development might represent a therapeutic opportunity to correct behavioral trajectories linking early stress to adult psychopathology.