A single exposure to the predator odor 2,4,5-trimethylthiazoline causes long-lasting affective behavioral changes in female mice: Modulation by kappa opioid receptor signaling.

The volatile compound 2,4,5-trimethylthiazoline (TMT, a synthetic predator scent) triggers fear, anxiety, and defensive responses in rodents that can outlast the encounter. The receptor systems underlying the development and persistence of TMT-induced behavioral changes remain poorly characterized, especially in females. Kappa opioid receptors regulate threat generalization and fear conditioning and alter basal anxiety, but their role in unconditioned fear responses in females has not been examined. Here, we investigated the effects of the long-lasting kappa opioid receptor antagonist, nor-binalthorphinmine dihydrochloride (nor-BNI; 10 mg/kg), on TMT-induced freezing and conditioned place aversion in female mice. We also measured anxiety-like behavior in the elevated plus maze three days after TMT and freezing behavior when returned to the TMT-paired context ten days after the single exposure. We found that 35µl of 10 % TMT elicited a robust freezing response during a five-minute exposure in female mice. TMT evoked persistent fear as measured by conditioned place aversion, reduced entries into the open arm of the elevated plus maze, and increased general freezing behavior long after TMT exposure. In line with the known role of kappa-opioid receptors in threat generalization, we found that kappa-opioid receptor antagonism increased basal freezing but reduced freezing during TMT presentation. Together, these findings indicate that a single exposure to TMT causes long-lasting changes in fear-related behavioral responses in female mice and highlights the modulatory role of kappa-opioid receptor signaling on fear-related behavioral patterns in females.

Effect of social instability stress in adolescence or adulthood on sensitivity to sucrose concentration in a social context in male and female Long-Evans rats.

Although there is evidence of sex differences in responding to social stress, and that age when stressed matters, females are understudied and adult-stress comparisons are few. Here, we investigated stress effects on reward sensitivity by examining rats' choice of social versus sucrose reward in a continuous spatial allocation design. We predicted social instability stress (SS) in adolescence would result in greater social discounting (spend less time near a novel peer when provided access to sucrose) relative to nonstressed controls (CTLs) and relative to SS in adulthood. All increased sucrose intake as the concentration increased, with no evidence of social discounting. SS males tested soon after the stress had a decrease in intake, whereas those tested long after had an increase in both time near the peer and in intake. CTL and SS females did not differ in intake, although their dose-response curves differed when tested soon after the SS. We also tested whether SS changed the stimulus value of the rat as a social peer; when tested in triads, CTL rats spent similar time in interaction with SS versus CTL rats. In sum, effects of SS on reward sensitivity were greater for males irrespective of administered in adolescence versus adulthood.

Nicotine sensitization (Part 2): Time spent in the centre of an open field sensitizes to repeated nicotine into the drug-free state in female rats.

RATIONALE: Nicotine is initially anxiogenic and becomes anxiolytic after prolonged exposure. The mechanisms that facilitate the shift in anxiety-like behaviour produced by nicotine are unclear. OBJECTIVE: We investigated the change in time spent in the centre of an open field (as a measure anxiety-like behaviour) produced by three intermittent injections of nicotine as part of experiments of locomotor sensitization to nicotine. METHODS: Rats were injected with nicotine (0.4 mg/kg) or saline and immediately placed in the open field arena for 1 h on two consecutive days and again 9 days later. RESULTS: When given saline, time spent in the centre of the arena did not change, whereas repeated nicotine injections increased in time spent in the centre beyond the increase produced by an acute injection of nicotine. Repeated nicotine (and not acute nicotine) also increased time in the centre in a drug-free state when tested 24 h after the last injection. CONCLUSION: Repeated nicotine sensitized the time spent in the centre of an open field with the long-lasting sensitization of this measure of anxiety-like behaviour evident in a drug-free state, in contrast to locomotor sensitization which does not persist in the drug-free state. The results suggest independence of the mechanisms of sensitization that underlie locomotor and anxiolytic effects.

Nicotine sensitization (part 1): estradiol or tamoxifen is required during the induction phase and not the expression phase to enable locomotor sensitization to nicotine in female rats.

RATIONALE: Nicotine sensitization involves two functionally distinct phases: induction and expression. Estradiol enhances nicotine sensitization in female rats, but it is not known whether this enhancement is specific to one or both phases. OBJECTIVES: We investigated the effects of estradiol selectively during the induction and the expression of nicotine sensitization. METHODS: Ovariectomy (OVX) rats were administered E2 during the induction (2 injection days) and/or the expression phase (9 days later) of nicotine sensitization. The selective estrogen receptor modulator tamoxifen (agonist of ERα and ERß, agonist of the g-coupled estradiol receptor GPER1) also was used to elucidate receptor candidates for the effects of E2 on nicotine sensitization. RESULTS: Gonadally intact female rats exhibited expression of nicotine sensitization after a 9-day delay, whereas OVX females did not. Administration of E2 limited to the induction phase of nicotine sensitization rescued expression of nicotine sensitization in OVX females. Tamoxifen during induction did not alter expression of sensitization in gonadally intact female rats, and, like E2, was sufficient to reverse the dampening effects of OVX on expression of sensitization. CONCLUSIONS: The enhancing effects of E2 on nicotine sensitization occur during the induction phase of nicotine sensitization, although require a delay to produce the effects on locomotor activity to nicotine, and may involve non-canonical estrogen pathways (e.g., activation of GPER1).

Adolescent social instability stress leads to immediate and lasting sex-specific changes in the neuroendocrine-immune-gut axis in rats.

Social instability stress (SS; daily 1 h isolation and change of cage partner from postnatal day (P) 30-45) in adolescence produces elevations in corticosterone during the procedure in male and female rats, but no lasting changes in hypothalamic-pituitary-adrenal (HPA) responses to psychological stressors, although deficits in social and cognitive function are evident in adulthood. Here we investigated the effects of SS in corticosterone response to an immune challenge (lipopolysaccharide, LPS, 0.1 mg/kg), on gene expression in the hippocampus, and on gut microbiota, when tested soon- (P46) or long- (P70) after SS. The temporal pattern of corticosterone release after LPS differed between SS and control rats irrespective of the time since SS exposure in females, whereas in males, SS did not alter corticosterone release after LPS. Expression of genes in the hippocampus relevant to immune and HPA function differed between saline-treated SS and control rats depending on sex and time tested, but with lasting consequences of SS in both sexes. LPS-treatment altered hippocampal gene expression, with bigger effects of LPS evident in control than in SS female rats, and the opposite in male rats. Further, effects sometimes depended on the age at time of LPS treatment. SS and control rats differed in both fecal and colon microbiome composition in all but P46 males, and stress history, sex, and age influenced the effects of an immune challenge on the gut microbiome. In sum, adolescent stress history has consequences for immune function into adulthood that may involve effects on the gut microbiome.

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.

Hormone-epigenome interactions in behavioural regulation.

Interactions between hormones and epigenetic factors are key regulators of behaviour, but the mechanisms that underlie their effects are complex. Epigenetic factors can modify sensitivity to hormones by altering hormone receptor expression, and hormones can regulate epigenetic factors by recruiting epigenetic regulators to DNA. The bidirectional nature of this relationship is becoming increasingly evident and suggests that the ability of hormones to regulate certain forms of behaviour may depend on their ability to induce changes in the epigenome. Moreover, sex differences have been reported for several epigenetic modifications, and epigenetic factors are thought to regulate sexual differentiation of behaviour, although specific mechanisms remain to be understood. Indeed, hormone-epigenome interactions are highly complex and involve both canonical and non-canonical regulatory pathways that may permit for highly specific gene regulation to promote variable forms of behavioural adaptation.

The effects of social instability stress and subsequent ethanol consumption in adolescence on brain and behavioral development in male rats.

Excessive drinking in adolescence continues to be a problem, and almost a quarter of young Canadians have reported consuming five or more alcoholic drinks in one occasion in recent surveys. The consequences of such drinking may be more pronounced when commenced in adolescence, given the ongoing brain development during this period of life. Here, we investigated the consequences of 3 weeks' intermittent access to ethanol in mid-adolescence to early adulthood in rats, and the extent to which a stress history moderated the negative consequences of ethanol access. In experiment 1, male rats that underwent adolescent social instability stress (SS; daily 1 h isolation + return to unfamiliar cage partner every day from postnatal day [PND] 30-45) did not differ from control (CTL) rats in intake of 10% ethanol sweetened with 0.1% saccharin (access period; PND 47-66). Ethanol drinking reduced proteins relevant for synaptic plasticity (αCaMKII, ßCaMKII, and PSD-95) in the dorsal hippocampus, and in CTL rats only in the prefrontal cortex (αCaMKII and PSD 95), attenuating the difference between CTL and SS rats in the water-drinking group. In experiment 2, ethanol also attenuated the difference between SS and CTL rats in a social interaction test by reducing social interaction in SS rats; CTL rats, however, had a higher intake of ethanol than did SS rats during the access period. Ethanol drinking reduced baseline and fear recall recovery concentrations of corticosterone relative to those exposed only to water, although there was no effect of either ethanol or stress history on fear conditioning. Ethanol drinking did not influence intake after 9 days of withdrawal; however, ethanol-naïve SS rats drank more than did CTL rats when given a 24-h access in adulthood. These results reveal a complex relationship between stress history and ethanol intake in adolescence on outcomes in adulthood.

Adolescent social stress and social context influence the intake of ethanol and sucrose in male rats soon and long after the stress exposures.

Social instability stress in adolescent rats (SS; postnatal day 30-45, daily 1 hr isolation +new cage partner) alters behavioural responses to psychostimulants, but differences in voluntary consumption of natural and drug rewards are unknown. SS also is associated with an atypical behavioural repertoire, for example reduced social interactions. Here, we investigated whether SS rats differ from control (CTL) rats in ethanol (EtOH) or sucrose intake in experiments involving different social contexts: alone, in the presence of an unfamiliar peer, in the presence of its cage partner, or in competition against its cage partner. SS rats drank more EtOH than CTL rats irrespective of social context, although the effects were driven primarily by those tested soon after the test procedure rather than weeks later in adulthood. SS and CTL rats did not differ in sucrose intake, except in adulthood under conditions of competition for limited access (SS>CTL). Adolescent rats drank more sucrose than adults, in keeping with evidence that adolescents are more sensitive to natural rewards than adult animals. Overall, adolescent SS modified the reward value of EtOH and sucrose, perhaps through stress/glucocorticoids modifying the development of the mesocorticolimbic system.

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.

Predictors of social instability stress effects on social interaction and anxiety in adolescent male rats.

Adolescence is an important phase of development of social behaviors, which may be disrupted by the experience of stressors. We previously reported that exposure to social instability stress in adolescence (SS; postnatal day [PND] 30-45) in rats reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). In experiment 1, we replicated the effect of SS on social interaction and found that the pattern of neural activations based on Fos immunohistochemistry in brain regions during social interactions differed for SS and CTL rats. In experiment 2, we found that individual differences in novelty-seeking behavior on PND 30 and SS exposure were unique predictors of anxiety in the elevated plus maze on PND 46, and interacted to predict social interaction on PND 47; among high novelty-seeking rats, SS and CTL rats do not differ, whereas among low-novelty seeking rats, SS rats engaged in less social interaction than did CTL rats. Thus, high novelty-seeking may be a resilience factor against the effects of social stressors in adolescence.

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.

Social instability stress in adolescent male rats reduces social interaction and social recognition performance and increases oxytocin receptor binding.

Social experiences in adolescence are essential for displaying context-appropriate social behaviors in adulthood. We previously found that adult male rats that underwent social instability stress (SS) in adolescence had reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). Here we determined whether SS altered social recognition and social reward and brain oxytocin and vasopressin receptor density in adolescence. We confirmed that SS rats spent less time interacting with unfamiliar peers than did CTL rats (p=0.006). Furthermore, CTL rats showed a preference for novel over familiar conspecifics in a social recognition test whereas SS rats did not, which may reflect reduced recognition, impaired memory, or reduced preference for novelty in SS rats. The reward value of social interactions was not affected by SS based on conditioned place preference tests and based on the greater time SS rats spent investigating stimulus rats than did CTL rats when the stimulus rat was behind wire mesh (p=0.03). Finally, oxytocin receptor binding density was higher in the dorsal lateral septum and nucleus accumbens shell in SS rats compared with CTL rats (p=0.02, p=0.01, respectively). No effect of SS was found for vasopressin 1a receptor binding density in any of the brain regions analyzed. We discuss the extent to which the differences in social behavior exhibited after social instability in adolescence involve changes in social salience and social competency, and the possibility that changes in oxytocin signaling in the brain underlie the differences in social behavior.