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.

The Endocannabinoid System and Invertebrate Neurodevelopment and Regeneration.

Cannabis has long been used for its medicinal and psychoactive properties. With the relatively new adoption of formal medicinal cannabis regulations worldwide, the study of cannabinoids, both endogenous and exogenous, has similarly flourished in more recent decades. In particular, research investigating the role of cannabinoids in regeneration and neurodevelopment has yielded promising results in vertebrate models. However, regeneration-competent vertebrates are few, whereas a myriad of invertebrate species have been established as superb models for regeneration. As such, this review aims to provide a comprehensive summary of the endocannabinoid system, with a focus on current advances in the area of endocannabinoid system contributions to invertebrate neurodevelopment and regeneration.

Adolescent CB1 receptor antagonism influences subsequent social interactions and neural activity in female rats.

We previously demonstrated that repeated exposure to the CB1 receptor antagonist/inverse agonist AM251 in adolescence (PND 30-44) increased social interactions in female rats when tested 48 h after the final exposure to the antagonist. Here, we investigated whether the increased sociality would be present after a longer drug washout period (5 days) in both male and female rats (experiment 1), and sought to identify candidate brain regions that may explain the observed differences in social behaviours between AM251 and vehicle-treated female rats (experiment 2). While drug-free, adolescent AM251 treatment increased social interactions in females and not in males. AM251 female rats had increased neural activity (as measured by the expression of early growth response protein-1; EGR-1) in the nucleus accumbens shell and cingulate gyrus of the medial prefrontal cortex, with no observed differences in EGR-1 expression in the dorsal hippocampus, nucleus accumbens core, or prelimbic and infralimbic subdivisions of the medial prefrontal cortex relative to vehicle rats. Together, these results demonstrate a sex-specific role of adolescent endocannabinoid signalling in the normative development of social behaviours and provide further support for adolescence as a vulnerable period for the effects of altered endocannabinoid signalling.

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.

Sex-specific effects of CB1 receptor antagonism and stress in adolescence on anxiety, corticosterone concentrations, and contextual fear in adulthood in rats.

There is a paucity of research regarding the role of endogenous cannabinoid signalling in adolescence on brain and behaviour development. We previously demonstrated effects of repeated CB1 receptor antagonism in adolescence on socioemotional behaviours and neural protein expression 24-48 h after the last drug administration in female rats, with no effect in males. Here we investigate whether greater effects would be manifested after a lengthier delay. In Experiment 1, male and female rats were administered either 1 mg / kg of the CB1 receptor-selective antagonist AM251, vehicle (VEH), or did not receive injections (NoINJ) daily on postnatal days (PND) 30-44 either alone (no adolescent confinement stress; noACS), or in tandem with 1 h ACS. On PND 70, adolescent AM251 exposure reduced anxiety in an elevated plus maze in males, irrespective of ACS, with no effects in females. On PND 73, there were no group differences in either sex in plasma corticosterone concentrations before or after 30 min of restraint stress, although injection stress resulted in higher baseline concentrations in males. Brains were collected on PND 74, with negligible effects of either AM251 or ACS on protein markers of synaptic plasticity and of the endocannabinoid system in the hippocampus and medial prefrontal cortex. In Experiment 2, rats from both sexes were treated with vehicle or AM251 on PND 30-44 and were tested for contextual fear conditioning and extinction in adulthood. AM251 females had greater fear recall than VEH females 24 h after conditioning, with no group differences in within- or between-session fear extinction. There were no group differences in long-term extinction memory, although AM251 females froze more during a reconditioning trial compared with VEH females. There were no group differences on any of the fear conditioning measures in males. Together, these findings indicate a modest, sex-specific role of CB1 receptor signalling in adolescence on anxiety-like behaviour in males and conditioned fear behaviour in females.

Effects of CB1 receptor antagonism and stress exposures in adolescence on socioemotional behaviours, neuroendocrine stress responses, and expression of relevant proteins in the hippocampus and prefrontal cortex in rats.

Little is known about the consequences of altered endocannabinoid signalling in adolescence. We hypothesized that CB1 receptor antagonism (AM251, 1 mg/kg) and stress exposures (1 h confinement stress) in adolescence (daily, postnatal days 30-44) would interact to increase neuroendocrine stress responses and anxiety when investigated a minimum of 24 h after drug and stress treatments; these treatment effects were independent of each other. Changes in homecage behaviour and in weight gain confirmed that both males and females were sensitive to the treatments. Nevertheless, in males, repeated AM251 administration was without effect on any of the measures investigated in days post-treatment. Males had reduced corticosterone release to the repeated stress and had increased GAD67 expression in the ventral hippocampus under baseline conditions. In females, AM251 also reduced weight gain and increased stereotypic behaviours in the homecage; these same females showed increased sociality, reduced CB1 receptor expression in the dorsal hippocampus, and increased GAD67 expression in the prefrontal cortex. Further, females exposed to repeated stress had enhanced recovery to baseline corticosterone concentrations after stress. The inclusion of a non-injected comparison group also revealed stress of injection effects in both sexes that otherwise would have been masked. Together, the findings demonstrate effects of CB1 receptor antagonism and stress that were more evident in females than males, suggesting that females may be more vulnerable to the consequences of disrupted endocannabinoid signalling during adolescence.

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.

Intracellular signalling and plasma hormone profiles associated with the expression of unconditioned and conditioned fear and anxiety in female rats.

There is considerable overlap in the neural regions and intracellular signalling pathways implicated in anxiety and fear, although less is known in females. Here, we investigated whether unconditioned and conditioned fear are associated with distinct patterns of expression of extracellular signal-regulated kinase-1 and -2 (ERK1/2), protein kinase B (Akt), and calcineurin (CaN) (proteins that are key regulators of the expression of and/or memory processes of fear and anxiety) in the dorsal and ventral hippocampus, medial prefrontal cortex, and amygdala (important regions in neural fear circuitry) of adult female rats, and used a multivariate approach to find patterns of signalling that might discriminate between the different states of fear. To isolate fear to the conditioned cue from generalized fear to the test context, rats were conditioned to an auditory tone (i.e. tone paired with footshock) and twenty-four hours later exposed to a novel context in the presence or absence of the conditioned cue. A third group that was exposed to the conditioning context without undergoing fear conditioning was included to control for unconditioned responses to the testing procedures, which are anxiogenic. A discriminate function analysis and MANOVA determined that hippocampal signalling best discriminated the three groups from each other. The addition of values for plasma concentrations of corticosterone and progesterone (as indices of activation of the hypothalamic-pituitary-adrenal stress axis) to statistical analyses increased the separation of the three groups. There was high degree of association among the three signalling molecules in the four brain regions within each group. There was an absence of the associations between the medial prefrontal cortex and the amygdala in the cued fear recall group that were strong for the non-conditioned group. These results demonstrated unique neuronal and hormonal signalling profiles associated with unconditioned, generalized, and conditioned fear expression in females and highlight the importance of including appropriate comparisons to best discriminate between these different emotional states.

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.

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.

Peer pressures: social instability stress in adolescence and social deficits in adulthood in a rodent model.

Studies in animal models generate and test hypotheses regarding developmental stage-specific vulnerability that might inform research questions about human development. In both rats and humans, peer relationships are qualitatively different in adolescence than at other stages of development, and social experiences in adolescence are considered important determinants of adult social function. This review describes our adolescent rat social instability stress model and the long-lasting effects social instability has on social behaviour in adulthood as well as the possible neural underpinnings. Effects of other adolescent social stress experiences in rats on social behaviours in adulthood also are reviewed. We discuss the role of hypothalamic-pituitary-adrenal (HPA) function and glucocorticoid release in conferring differential susceptibility to social experiences in adolescents compared to adults. We propose that although differential perception of social experiences rather than immature HPA function may underlie the heightened vulnerability of adolescents to social instability, the changes in the trajectory of brain development and resultant social deficits likely are mediated by the heightened glucocorticoid release in response to repeated social stressors in adolescence compared to in adulthood.

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.