Endocannabinoid system contributions to sex-specific adolescent neurodevelopment.

With an increasing number of countries and states adopting legislation permitting the use of cannabis for medical purposes, there is a growing interest among health and research professionals into the system through which cannabinoids principally act, the endocannabinoid system (ECS). Much of the seminal research into the ECS dates back only 30 years and, although there has been tremendous development within the field during this time, many questions remain. More recently, investigations have emerged examining the contributions of the ECS to normative development and the effect of altering this system during important critical periods. One such period is adolescence, a unique period during which brain and behaviours are maturing and reorganizing in preparation for adulthood, including shifts in endocannabinoid biology. The purpose of this review is to discuss findings to date regarding the maturation of the ECS during adolescence and the consequences of manipulations of the ECS during this period to normative neurodevelopmental processes, as well as highlight sex differences in ECS function, important technical considerations, and future directions. Because most of what we know is derived from preclinical studies on rodents, we provide relevant background of this model and some commentary on the translational relevance of the research in this area.

Age-dependent regulation by androgens of gene expression in the anterior hypothalamus and stress-induced release of adrenal hormones in adolescent and adult male rats.

Adolescents show greater and/or more prolonged activation of the hypothalamic-pituitary-adrenal axis in response to stressors than adults, although the basis for such an age difference is not understood. We investigated developmental shifts in the regulation of HPA function by testosterone using androgen replacement in orchiectomised (OCX) pre-pubertal and post-pubertal adolescent rats and in adults, as well as using inhibitors of testosterone synthesis in non-operated rats. The expected dampening effect of testosterone in adult OCX rats did not meet statistical significance in all of the three experiments. Nevertheless, in each, adolescents had higher post-stress concentrations of corticosterone compared to adults despite similar concentrations of testosterone. The effect of testosterone was in the opposite direction in post-pubertal adolescents compared to that in adults, with testosterone replacement leading to increased rather than lower corticosterone concentration. Testosterone replacement decreased arginine vasopressin and corticotrophin-releasing hormone immune-reactive cell counts in the paraventricular nucleus at all ages. In a fourth experiment, we provide evidence that the basis of the age difference in corticosterone release is because of a greater conversion of testosterone to oestradiol in adolescents and a greater conversion of testosterone to dihydrotestosterone in adults: aromatase inhibition had little effect in adults and attenuated the age difference by decreasing stress-induced corticosterone release in adolescents. By contrast, 5α-reductase inhibition or an androgen receptor antagonist had little effect in adolescents and attenuated the age difference by increasing stress-induced corticosterone release in adults. In the adrenal gland, adolescents had reduced 5α-reductase and androgen receptor gene expression. There also were age differences in the regulation of hypothalamic mRNA expression of androgen receptors, oestrogen receptors and aromatase by testosterone. In sum, the results suggest that developmental shifts in the synthesis of testosterone and the regulation of gene expression are factors with respect to age differences in corticosterone release in response to stressors.

Effects of long-term dietary administration of estrogen receptor-beta agonist diarylpropionitrile on ovariectomized female ICR (CD-1) mice.

Diarylpropionitrile (DPN) is an estrogen receptor-ß-specific agonist that has been linked to neuroprotection, preserving cognitive function with age, the suppression of anxiety-like behaviors, inhibition of cancer growth, and other positive properties. We hypothesized that DPN may have pro-longevity properties. DPN was administered via feed at a dose corresponding to approximately 3 mg/kg/day to ovariectomized female mice beginning at 7 months of age. Mice were followed for the duration of their lifespans while monitoring body mass, aspects of behavior, learning, memory, and frailty. DPN-treated mice gained more body mass over the first 2 years of age (17 months of the study). A test of voluntary running behavior at 24 months of age behavior revealed no deficits in DPN-treated mice, which were as likely as control mice to engage in extended bouts of wheel running, and did so at higher average speeds. DPN administration had anxiolytic-like effects when measured using an elevated plus maze at 9 months of age. A mouse frailty index was used to assess age-related changes. The correlation between age and frailty differed between control and DPN-treated mice. Overall, dietary DPN administration had some beneficial effects on the aging phenotype of ovariectomized female mice with few significant detrimental effects.

Changes in collegiate ice hockey player anthropometrics and aerobic fitness over 3 decades.

Over the past several decades, an increased emphasis on fitness training has emerged among collegiate ice hockey teams, with the objective of improving on-ice performance. However, it is unknown if this increase in training has translated over time into changes in the anthropometric and fitness profiles of collegiate ice hockey players. The purposes of this study were to describe anthropometric (height, weight, body mass index (BMI), percent body fat (%fat)) and aerobic fitness (peak oxygen consumption) characteristics of collegiate ice hockey players over a period of 36 years and to evaluate whether these characteristics differ among player positions. Anthropometric and physiologic data were obtained through preseason fitness testing of players (N = 279) from a National Collegiate Athletic Association Division I men's ice hockey team from the years 1980 through 2015. Changes over time in the anthropometric and physiologic variables were evaluated via regression analysis using linear and polynomial models, and differences among player positions were compared via ANOVA (p < 0.05). Regression analysis revealed that a cubic model best predicted changes in mean height (R2 = 0.65), weight (R2 = 0.77), and BMI (R2 = 0.57), whereas a quadratic model best fit change in %fat by year (R2 = 0.30). Little change was observed over time in the anthropometric characteristics. Defensemen were significantly taller than forwards (184.7 ± 12.1 vs. 181.3 ± 5.9 cm) (p = 0.007), and forwards had a higher relative peak oxygen consumption compared with defensemen (58.7 ± 4.7 vs. 57.2 ± 4.4 mL·kg-1·min-1) (p = 0.032). No significant differences were observed in %fat or weight by position. Although average player heights and weights fluctuated over time, increased emphasis on fitness training did not affect the athletes' relative aerobic fitness. Differences in height and aerobic fitness levels were observed among player positions.

The effects of ovarian hormones on stressor-induced hormonal responses, glucocorticoid receptor expression and translocation, and genes related to receptor signaling in adult female rats.

Estradiol potentiates hypothalamic-pituitary-adrenal activity and delays the return of glucocorticoid secretion to baseline after a stressor exposure in female rats; we investigated whether estradiol effects involve actions on glucocorticoid receptor (GR) translocation and expression of receptor co-chaperones. In Experiment 1 intact females and ovariectomized (OVX) females were treated for four days with vehicle (VEH), 17ß-estradiol benzoate (EB), or EB and progesterone (EB + P). Samples were taken from rats in the home cage (baseline) or after 30 min of restraint stress in a plastic restrainer (post-restraint) (n = 10/group). OVX-VEH treatment reduced baseline and post-restraint plasma concentrations of corticosterone versus all other treatments. Western blots indicated that OVX-VEH treated rats had greater hippocampal cytosolic GR expression than other treatments. Stress increased hippocampal nuclear GR expression, but without treatment differences. In Experiment 2 OVX rats were treated daily with VEH, EB, or EB + P (n = 8/group). OVX-VEH rats showed a lower stimulation of corticosterone secretion by restraint stress than other treatments and OVX-EB + P treated rats had lower concentrations than the OVX-EB group, suggesting progesterone mitigated estradiol effects. Quantitative polymerase chain reaction experiments indicated that stress increased Fkbp5 mRNA in the ventral hippocampus, with no effect of stress or treatment on Nr3c1 (GR), Nr3c2 (MR), Fkbp4, Bag1, or Ncoa1 (SRC-1) expression. Thus, the hypothesis is that estradiol effects on negative feedback are mediated by altered expression of receptor co-chaperones or co-modulators in the hippocampus was not supported. Estradiol may blunt feedback by limiting the availability of cytosolic GR protein.

Translational relevance of rodent models of hypothalamic-pituitary-adrenal function and stressors in adolescence.

Elevations in glucocorticoids that result from environmental stressors can have programming effects on brain structure and function when the exposure occurs during sensitive periods that involve heightened neural development. In recent years, adolescence has gained increasing attention as another sensitive period of development, a period in which pubertal transitions may increase the vulnerability to stressors. There are similarities in physical and behavioural development between humans and rats, and rats have been used effectively as an animal model of adolescence and the unique plasticity of this period of ontogeny. This review focuses on benefits and challenges of rats as a model for translational research on hypothalamic-pituitary-adrenal (HPA) function and stressors in adolescence, highlighting important parallels and contrasts between adolescent rats and humans, and we review the main stress procedures that are used in investigating HPA stress responses and their consequences in adolescence in rats. We conclude that a greater focus on timing of puberty as a factor in research in adolescent rats may increase the translational relevance of the findings.

Glucocorticoid receptor translocation and expression of relevant genes in the hippocampus of adolescent and adult male rats.

We investigated whether pre-pubertal (postnatal day [P] 35) and post-pubertal adolescent (P45) and adult (P75) male rats differed in stressor-induced hormonal responses and in glucocorticoid receptor (GR) translocation because it has been proposed that negative feedback is maturing in adolescence and may be a basis for the prolonged activation of the HPA axis in adolescents compared with adults. The three age groups did not differ at baseline in plasma corticosterone or progesterone concentrations, and P35 had lower concentrations of testosterone than did both P45 and P75 rats, which did not differ. After 30min of restraint stress, plasma concentrations of corticosterone and progesterone increased to a greater extent in the adolescents than in the adults. Whereas restraint stress increased concentrations of testosterone in adult males, concentrations decreased in adolescents. In all three age groups, restraint stress reduced GR expression in the cytosol and increased expression in the nucleus within the hippocampus, and the increase in nuclear GR was greater in pre-pubertal adolescents compared with adults. In a separate set of rats we investigated age differences in hippocampal mRNA expression of corticosteroid receptors (MR and GR) and of chaperones (FKBP5, FKBP4, BAG-1), which are known to modulate their activity, at baseline and after restraint stress. Restraint stress decreased the expression of GR and increased the expression of FKBP5 mRNA, and age was not a significant factor. Higher expression of FKBP4 mRNA was found at P35 than at P75. Most research of HPA function in adolescent rats has involved pre-pubertal rats; the present findings indicate that despite their increase in gonadal function, responses to stressors in P45 rats are more like those of pre-pubertal than adult rats. The greater stressor-induced GR translocation in pre-pubertal adolescents parallels their greater release of corticosterone in response to stressors, which may contribute to the enhanced sensitivity of adolescent rats to the effects of chronic stress exposures compared with adults.

Sex and stress steroids in adolescence: Gonadal regulation of the hypothalamic-pituitary-adrenal axis in the rat.

This review provides an overview of the current understanding of the role of the hypothalamic-pituitary-gonadal (HPG) axis in regulating the hypothalamic-pituitary-adrenal (HPA) axis response to stressors. HPA function is influenced by both organizational (programming) and activational effects of gonadal hormones. Typically, in adult rats, estradiol increases and androgens decrease the HPA response to stressors, thereby contributing to sex differences in HPA function, and sensitivity of the HPA axis to gonadal steroids is in part determined by exposure to these hormones in early development. Although developmental differences in HPA function are well characterized, the extent to which gonadal steroids contribute to age differences in HPA function is not well understood. Deficits in the understanding of the relationships between the HPA and HPG axes are greatest for the adolescent period of development. The critical outstanding questions are, when do gonadal hormones begin to regulate HPA function in adolescence, and what mechanisms precipitate change in sensitivity of the HPA axis to the HPG axis at this stage of life.

Differential effects of CB1 receptor agonism in behavioural tests of unconditioned and conditioned fear in adult male rats.

We investigated the effects of the highly selective CB1 receptor agonist ACEA and the CB1 receptor antagonist/inverse agonist AM251 on two behavioural tests of unconditioned fear, the elevated plus maze (EPM) and open field test (OFT), as well as on the recall and extinction of a conditioned auditory fear. Both ACEA and AM251 increased anxiety-like behaviour in the EPM and OFT. There was no effect of either drug on recall of the conditioned fear, and ACEA enhanced and AM251 impaired fear extinction. Further, though both the low (0.1 mg/kg) and high (0.5 mg/kg) dose of ACEA facilitated fear extinction, the low dose attenuated, and the high dose potentiated, fear induced corticosterone release suggesting independent effects of the drug on fear and stress responses. Although the extent to which cannabinoids are anxiogenic or anxiolytic has been proposed to be dose-dependent, these results indicate that the same dose has differential effects across tasks, likely based in differences in sensitivities of CB1 receptors to the agonist in the neural regions subserving unconditioned and conditioned fear.

Effects of social context on endocrine function and Zif268 expression in response to an acute stressor in adolescent and adult rats.

There is a paucity of studies comparing social buffering in adolescents and adults, despite their marked differences in social behavior. We investigated whether greater effects of social buffering on plasma corticosterone concentrations and expression of Zif268 in neural regions after an acute stressor would be found in adolescent than adult rats. Samples were obtained before and after 1h of isolation stress and after either 1 or 3h of recovery back in the colony with either a familiar or unfamiliar cage partner. Adolescent and adult rats did not differ in plasma concentrations of corticosterone at any time point. Corticosterone concentrations were higher after 1h isolation than at baseline (p<0.001), and rats with a familiar partner during the recovery phase had lower corticosterone concentrations than did rats with an unfamiliar partner (p=0.02). Zif268 immunoreactive cell counts were higher in the arcuate nucleus in both age groups after isolation (p=0.007) and in the paraventricular nucleus of adolescents than adults during the recovery phase irrespective of partner familiarity. There was a significant decrease in immunoreactive cell counts after 1h isolation compared to baseline in the basolateral amygdala, central nucleus of the amygdala, and in the pyramidal layer of the hippocampus (all p<0.05). An effect of partner familiarity on Zif268 immunoreactive cell counts was found in the granule layer of the dentate gyrus irrespective of age (higher in those with a familiar partner, p=0.03) and in the medial prefrontal cortex in adolescents (higher with an unfamiliar partner, p=0.02). Overall, the acute stress and partner familiarity produced a similar pattern of results in adolescents and adults, with both age groups sensitive to the social context.

Effects of stressors in adolescence on learning and memory in rodent models.

This article is part of a Special Issue "Puberty and Adolescence". Learning and memory is affected by a myriad of factors, including exposure to stressors and the corresponding rise in circulating glucocorticoids. Nevertheless, the effects of stressors depend on the sex, species, the type of stressor used, the duration of exposure, as well as the developmental time-point in which stressors are experienced. Effects of stress in adolescence, however, have received less attention than other developmental periods. In adolescence, the hypothalamic-pituitary-adrenal axis and brain regions involved in learning and memory, which also richly express corticosteroid receptors, are continuing to develop, and thus the effects of stress exposures would be expected to differ from those in adulthood. We conclude from a review of the available literature in animal models that hippocampal function is particularly sensitive to adolescent stressors, and the effects tend to be most evident several weeks after the exposure, suggesting stressors alter the developmental trajectory of the hippocampus.

Effects of social instability stress in adolescence on long-term, not short-term, spatial memory performance.

There is evidence that exposure to stressors in adolescence leads to lasting deficits on hippocampal-dependent tasks, but whether medial prefrontal cortical function is also impaired is unknown. We previously found that rats exposed to social instability stress in adolescence (SS; daily 1h isolation and subsequent change of cage partner between postnatal days 30 and 45) had impaired memory performance on a Spatial Object Location test and in memory for fear conditioning context, tasks that depend on the integrity of the hippocampus. Here we investigated whether impaired performance would be evident after adolescent SS in male rats on a different test of hippocampal function, spatial learning and memory in the Morris water maze (MWM) and on a working memory task for which performance depends on the integrity of the medial prefrontal cortex, the Delayed Alternation task (DAT). During MWM testing, SS rats showed greater improvements in performance across trials within days compared to control (CTL) rats, but showed less retention of learning between days (48 h) compared to CTL rats. Similarly, SS rats had impaired long-term memory in the Spatial Object Location test after a long delay (240 min), but not after shorter delays (15 or 60 min) compared to CTL rats. No group differences were observed on the DAT, which assessed working memory across brief delays (5-90 s). Thus, deficits in memory performance after chronic social stress in adolescence may be limited to long-term memory.

Social instability stress in adolescence increases anxiety and reduces social interactions in adulthood in male Long-Evans rats.

We investigated the effects of social instability stress (daily 1-hr isolation, change of cage partner, postnatal day 30-45) in adolescence in male rats on open field exploration and social behavior in adulthood. Social stressed rats had longer latencies to enter the center of an open field and then took longer to approach an object placed in the center of the field. When another rat was placed in the open field, stressed rats spent less time in social interaction than control rats, particularly when paired with another stressed, rather than a control, rat. The groups did not differ in social approach tests (when a stimulus rat was separated by wire mesh) nor in novel object exploration (when controlling for open field anxiety). The results suggest social stress in adolescence increases open field anxiety while maintaining exploratory behavior, and alters social interactions in adulthood.

Deficits in male sexual behavior in adulthood after social instability stress in adolescence in rats.

There is increasing evidence that exposure to stressors in adolescence has long-lasting effects on emotional and cognitive behavior, but little is known as to whether reproductive functions are affected. We investigated appetitive and consummatory aspects of sexual behavior in male rats that were exposed to chronic social instability stress (SS, n=24) for 16 days in mid-adolescence compared to control rats (CTL, n=24). Over five sexual behavior test sessions with a receptive female, SS rats made fewer ejaculations (p=0.02) and had longer latencies to ejaculation (p=0.03). When only data from rats that ejaculated in the fifth session were analyzed, SS rats (n=18) had reduced copulatory efficiency (more mounts and intromissions before ejaculation) compared to CTL rats (n=19) (p=0.004), and CTL rats were twice as likely as SS rats to make more than one ejaculation in the fifth session (p=0.05). Further, more CTL (14/24) than SS (5/25) rats ejaculated in four or more sessions (p=0.05). SS rats had lower plasma testosterone concentrations than CTL rats (p=0.05), but did not differ in androgen receptor, estrogen receptor alpha, or Fos immunoreactive cell counts in the medial preoptic area. The groups did not differ in a partner preference test administered between the fourth and fifth sexual behavior session. The results suggest that developmental history contributes to individual differences in reproductive behavior, and that stress exposures in adolescence may be a factor in sexual sluggishness.

Active N-Ras and B-Raf inhibit anoikis by downregulating Bim expression in melanocytic cells.

B-Raf and N-Ras proteins are often activated in melanoma, yet their roles in producing inherent survival signals are not fully understood. In this study, we investigated how N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis induced by detachment from the extracellular matrix (anoikis). We found that expression of constitutively active N-RAS(Q61K) and B-RAF(V600E) downregulated the proapoptotic Bim protein in an immortalized melanocyte cell line. Bim is one of the main proapoptotic mediators of anoikis. Western blot analysis showed that detachment increased Bim expression in melanocytes, and Annexin V staining indicated that detachment induced cell death significantly in melanocytes. Blocking Bim expression by using RNAi vectors or by expressing N-RAS(Q61K) significantly inhibited anoikis in melanocytes. In summary, this report indicates that N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis in part by downregulating Bim expression, suggesting that Bim is a possible treatment target for overriding melanoma's inherent defenses against cell death.

Natural killer T cells in families of patients with systemic lupus erythematosus: their possible role in regulation of IGG production.

OBJECTIVE: To determine whether there is a link between the frequency of natural killer T (NKT) cells and high levels of IgG in patients with systemic lupus erythematosus (SLE) and their relatives. METHODS: Blood samples were obtained from patients with SLE, their first-degree relatives, patients with rheumatoid arthritis (RA), and healthy control subjects. The frequency of NKT cells (defined as CD56+ T cells) was expressed as a percentage of total blood lymphocytes. Plasma levels of total IgG and IgM, and IgG antibodies to double-stranded DNA (dsDNA) were determined. RESULTS: The frequency of NKT cells was lower in patients with SLE than in control subjects. No such decrease was observed in the relatives of patients with SLE or in patients with RA. High levels of IgG were observed in both patients with SLE and their relatives, while low levels of IgM were observed in these same groups. In relatives of patients with SLE, an inverse correlation between the frequency of NKT cells and IgG levels was observed. Moreover, raised levels of IgG in patients with SLE and their relatives and high levels of IgG anti-dsDNA in patients were associated with low frequencies of NKT cells. CONCLUSION: These results suggest that NKT cells have an important role in the regulation of IgG production, although NKT cells with invariant T cell receptors may not necessarily be involved. NKT cells in the setting of SLE could lack the cytokine stimulus from NK or other cells that is needed to exert control on IgG production. Enhancement of NKT cell activity may provide a novel basis for therapy in SLE.

Relationship between physiological profiles and on-ice performance of a National Collegiate Athletic Association Division I hockey team.

Ice hockey is a game that relies heavily on both aerobic and anaerobic energy production systems as players perform in various game situations. However, we found no studies evaluating the relationship between a player's physical condition and individual success in games throughout a competitive hockey season. The purpose of this study was to determine the relationship between a player's aerobic fitness (VO(2)max), blood lactate, and percent body fat to his total minutes played during a season (Tmin) and net scoring chances (SCn). Players' (N = 29) preseason VO(2)max, lactate at the fourth stage of an incremental treadmill test (Lac 4), and percent body fat values from the 1999- 2001 National Collegiate Athletic Association Division I hockey seasons were archived and retrieved for this study. The players' Tmin and SCn were used as the on-ice performance variables and were compared with their fitness measures. Lactate at 4th treadmill stage (r = 0.41, p < 0.03) and percent body fat (r = 0.39, p < 0.03) but not VO(2)max (r = 0.20, p < 0.24) were significantly related to Tmin. Both Lac 4 and percent body fat were entered into a stepwise regression model that accounted for 25% of the variance in Tmin among players (p < 0.02). Both VO(2)max (r = 0.41, p < 0.03) and Lac 4 (r = 0.33, p < 0.05) were significantly related to the players' SCn, but percent body fat was not (r = 0.10, p < 0.57). Only VO(2)max significantly predicted the players' SCn, accounting for 17% of the variance. These findings suggest a relationship between a player's conditioning level and on-ice performance. Our results support the value of implementing seasonal physiological testing, which will help strength and conditioning coaches make individualized modifications to a player's fitness regimens in an effort to improve specific physiological attributes.