Sex differences in the effects of mild traumatic brain injury and progesterone treatment on anxiety-like behavior and fear conditioning in rats.

Mild traumatic brain injuries (mild TBIs) commonly occur in young adults of both sexes, oftentimes in high-stress environments. In humans, sex differences have been observed in the development of post-concussive anxiety and PTSD-like behaviors. Progesterone, a sex steroid that has neuroprotective properties, restores cognitive function in animal models following more severe TBI, but its effectiveness in preventing the psychological symptoms associated with mild TBI has not been evaluated. Using a model of mild TBI that pairs a social stressor (social defeat) with weight drop, male and naturally estrous-cycling female rats were treated with 4 mg/kg progesterone or vehicle once daily for 5 days after injury. Behavioral measures, including elevated plus maze (EPM), contextual fear conditioning, and novel object recognition (NOR) were assessed following progesterone treatment. Anxiety-like behavior was increased by mild TBI in male rats, with a smaller effect seen in female rats in the diestrus phase at the time of EPM testing. In contrast, mild TBI impaired fear learning in female rats in estrus at the time of fear acquisition. Progesterone treatment failed to attenuate post-mild TBI anxiety-like behavior in either sex. Furthermore, progesterone increased fear conditioning and impaired NOR discrimination in male rats, independent of TBI status. Overall, both sex and estrous cycle contributed to psychological outcomes following mild TBI, which were not ameliorated by post-TBI progesterone. This suggests sex steroids play an important role as a moderator of the expression of mild TBI-induced psychological symptoms, rather than as a potential treatment for their underlying etiology.

GABAA Receptor and Serotonin Transporter Expression Changes Dissociate Following Mild Traumatic Brain Injury: Influence of Sex and Estrus Cycle Phase in Rats.

Mild traumatic brain injuries (mild TBIs) can affect both males and females, but females are more likely to report long-term psychological complications, including changes in mood and generalized anxiety. Additionally, reproductive cycle phase has been shown to affect mild TBI symptom expression within females. These variances may result from sex differences in mild TBI-induced alterations to neurotransmission in brain regions that influence mood and emotion, possibly mediated by sex steroids. The hippocampus and amygdala are implicated in stress responses and anxiety, and within these regions, gamma-aminobutyric acid (GABA) and serotonin modulate output and behavioral expression. Metabolites of progesterone can allosterically enhance GABAergic signaling, and sex steroids are suggested to regulate the expression of the serotonin transporter (SERT). To determine how mild TBI might alter GABA receptor and SERT expression in males and females, immunocytochemistry was used to quantify expression of the alpha-1 subunit of the GABAA receptor (α1-GABAA), SERT, and a neuronal marker (NeuN) in the brains of adult male and naturally-cycling female rats, both with and without mild TBI, 17 days after injury. Mild TBI altered the expression of α1-GABAA in the amygdala and hippocampus in both sexes, but the direction of change observed depended on sex and reproductive cycle phase. In contrast, mild TBI had little effect on SERT expression. However, SERT expression differed between sexes and varied with the cycle phase. These findings demonstrate that regulation of neurotransmission following mild TBI differs between males and females, with implications for behavioral outcomes and the efficacy of therapeutic strategies.

Prenatal exposure to alcohol impairs responses of cerebral arterioles to activation of potassium channels: Role of oxidative stress.

BACKGROUND: Potassium channels play an important role in the basal tone and dilation of cerebral resistance arterioles in response to many stimuli. However, the effect of prenatal alcohol exposure (PAE) on specific potassium channel function remains unknown. The first goal of this study was to determine the influence of PAE on the reactivity of cerebral arterioles to activation of ATP-sensitive potassium (KATP ) and BK channels. Our second goal was to determine whether oxidative stress contributed to potassium channel dysfunction of cerebral arterioles following PAE. METHODS: We fed Sprague-Dawley dams a liquid diet with or without alcohol (3% EtOH) for the duration of their pregnancy (21 to 23 days). We examined in vivo responses of cerebral arterioles in control and PAE male and female offspring (14 to 16 weeks after birth) to activators of potassium channels (Iloprost [BK channels] and pinacidil [KATP channels]), before and following inhibition of oxidative stress with apocynin. RESULTS: We found that PAE impaired dilation of cerebral arterioles in response to activation of potassium channels with iloprost and pinacidil, and this impairment was similar in male and female rats. In addition, treatment with apocynin reversed the impaired vasodilation to iloprost and pinacidil in PAE rats to levels observed in control rats. This effect of apocynin also was similar in male and female rats. CONCLUSIONS: PAE induces dysfunction in the ability of specific potassium channels to dilate cerebral arterioles which appears to be mediated by an increase in oxidative stress. We suggest that these alterations in potassium channel function may contribute to the pathogenesis of cerebral vascular abnormalities and/or behavioral/cognitive deficits observed in fetal alcohol spectrum disorders.

Chronic administration of glucocorticoid receptor ligands increases anxiety-like behavior and selectively increase serotonin transporters in the ventral hippocampus.

Organic cation transporter-3 (OCT3) is widely distributed in the brain with high expression in portions of the stress axis. These high capacity, polyspecific transporters function in monoamine clearance and are sensitive to the stress hormone corticosterone. In rats, withdrawal from chronic amphetamine increases OCT3 expression in specific limbic brain regions involved anxiety and stress responses, including the ventral hippocampus, central nucleus of amygdala (CeA) and dorsomedial hypothalamus. (DMH). Previous studies show that glucocorticoid receptor (GR) agonists increase OCT1 mRNA and OCT2 mRNA expression in non-neural tissues. Thus, we hypothesized that corticosterone increases OCT3 expression in the brain by activating GRs. Male Sprague-Dawley rats were pre-treated daily with the GR antagonist mifepristone (20 mg/kg; sc.) or vehicle followed 45 min later by injections of corticosterone or vehicle for 2 weeks. Corticosterone treatment significantly increased OCT3 expression in the ventral hippocampus and increased anxiety-like behavior. However, these effects were not blocked by mifepristone. Interestingly, treatment with mifepristone alone reduced plasma corticosterone levels and increased serotonin transporter and GR expression in the ventral hippocampus but did not significantly affect OCT3 expression or behavior. No treatment effects on OCT3, serotonin transporter or GR expression were observed in the DMH, CeA or dorsal hippocampus. Our findings suggest that corticosterone increases OCT3 expression in the ventral hippocampus by a mechanism independent of GRs, and that mifepristone and corticosterone can act in an independent manner to affect HPA axis-related physiological and behavioral parameters.

Constrictor responses of cerebral resistance arterioles in male and female rats exposed to prenatal alcohol.

While it is known that dilation of cerebral arterioles to NOS-dependent agonists is impaired in rats exposed to prenatal alcohol, no studies have examined the influence of prenatal alcohol on constrictor response of cerebral arterioles. Our goal was to determine whether constrictor responses of cerebral resistance arterioles are altered by prenatal exposure to alcohol and if any changes differed as a function of age or sex. We fed Sprague-Dawley rat dams a liquid diet with or without alcohol (3% ethanol) for the duration of their pregnancy. We then examined reactivity of cerebral arterioles to thromboxane (U-46619; 0.01 and 0.1 µM), arginine vasopressin (0.1 and 1 nM), and angiotensin II (1 and 10 µM) in four groups of offspring: control male and female, and prenatal alcohol male and female at two different ages (adolescent: 4-6 weeks old and adult: 14-16 weeks old). Constriction of cerebral arterioles to U-46619 and AVP were similar in male and female rats regardless of exposure to prenatal alcohol and age. Similarly, adolescent male and female rats showed no difference to angiotensin II following prenatal exposure to alcohol. However, alcohol-exposed females exhibited an unexpected dilation to the high concentration of angiotensin II in adulthood, which was absent in males. We suggest that the findings from these studies may have implications regarding the susceptibility of the brain to cerebral ischemic damage. We speculate that impaired vasodilation, coupled with preserved vasoconstriction, can lead to a scenario favoring a decrease in cerebral blood flow during times of increased metabolic demand.

Rosiglitazone restores nitric oxide synthase-dependent reactivity of cerebral arterioles in rats exposed to prenatal alcohol.

BACKGROUND: Prenatal exposure to alcohol leads to a greater incidence of many cardiovascular-related diseases, presumably via a mechanism that may involve increased oxidative stress. An agonist of peroxisome proliferator-activated receptor gamma (PPARγ; rosiglitazone) has been shown to suppress alcohol-induced neuroinflammation and oxidative stress. The goal of this study was to determine whether acute and chronic treatment with rosiglitazone could restore or prevent impaired nitric oxide synthase (NOS)-dependent responses of cerebral arterioles in male and female adult (14-16 weeks old) rats exposed to alcohol in utero. METHODS: We fed Sprague-Dawley dams a liquid diet with or without 3% ethanol for the duration of their pregnancy (21-23 days). In the first series of studies, we examined the reactivity of cerebral arterioles to eNOS- (ADP), nNOS-dependent (NMDA), and NOS-independent agonists in male and female adult rats before and during acute (1 hour) topical application of rosiglitazone (1 µM). In a second series of studies, we examined the influence of chronic treatment with rosiglitazone (3 mg/kg/day in drinking water for 2-3 weeks) on the responses of cerebral arterioles in male and female adult rats exposed to alcohol in utero. RESULTS: We found that in utero exposure to alcohol similarly reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in male and female adult rats. In addition, acute treatment of the male and female adult rats with rosiglitazone similarly restored this impairment in cerebral vascular function to that observed in controls. We also found that chronic treatment with rosiglitazone prevented impaired vascular function in male and female adult rats that were exposed to alcohol in utero. CONCLUSIONS: PPARγ activation may be an effective and relevant treatment to reverse or prevent cerebral vascular abnormalities associated with prenatal exposure to alcohol.

The stalk-eyed fly as a model for aggression - is there a conserved role for 5-HT between vertebrates and invertebrates?

Serotonin (5-HT) has largely been accepted to be inhibitory to vertebrate aggression, whereas an opposing stimulatory role has been proposed for invertebrates. Herein, we argue that critical gaps in our understanding of the nuanced role of 5-HT in invertebrate systems drove this conclusion prematurely, and that emerging data suggest a previously unrecognized level of phylogenetic conservation with respect to neurochemical mechanisms regulating the expression of aggressive behaviors. This is especially apparent when considering the interplay among factors governing 5-HT activity, many of which share functional homology across taxa. We discuss recent findings using insect models, with an emphasis on the stalk-eyed fly, to demonstrate how particular 5-HT receptor subtypes mediate the intensity of aggression with respect to discrete stages of the interaction (initiation, escalation and termination), which mirrors the complex behavioral regulation currently recognized in vertebrates. Further similarities emerge when considering the contribution of neuropeptides, which interact with 5-HT to ultimately determine contest progression and outcome. Relative to knowledge in vertebrates, much less is known about the function of 5-HT receptors and neuropeptides in invertebrate aggression, particularly with respect to sex, species and context, prompting the need for further studies. Our Commentary highlights the need to consider multiple factors when determining potential taxonomic differences, and raises the possibility of more similarities than differences between vertebrates and invertebrates with regard to the modulatory effect of 5-HT on aggression.

Sex differences in anxiety-like behaviors in rats.

The use of animal models for behavioral and pharmaceutical testing is employed in many different fields of research but often relies solely on male animals. When females are included, the existing literature frequently offers inconsistent results regarding the effects of sex and/or estrous cycle on anxiety-like behaviors. Our current study sought to establish baseline or normative behaviors in three commonly employed tests of anxiety-like behavior, and determine any sex or cycle differences. Anxiety-like behaviors in male and naturally-cycling female Sprague-Dawley rats were assessed using elevated plus maze, open field, and a social interaction/avoidance paradigm. Female rats were examined once daily to determine their stage of estrous. Results from the elevated plus maze but not the open field showed that female rats spent significantly more time in open areas than did male rats; however, there was no effect of estrous cycle stage. The social avoidance test revealed that female rats spent significantly more time in the interaction zone with an empty wire mesh cage (novel object), but there was no sex difference in time spent with an age- and sex- matched target rat. Females often exhibited greater locomotion as compared to males in social and non-social tests, but this was not related to primary anxiety measures in these tests. Overall, our findings indicate that outcomes differ in tests of anxiety-like behaviors, highlighting sex differences in the expression of anxiety-like behaviors that depend on the test employed. Importantly, the lack of estrous cycle effects suggest that for these anxiety-based tests, female Sprague-Dawley rats could be collapsed across the cycle phases to facilitate the inclusion of females in future behavioral experiments.

Sex differences in aggression: Differential roles of 5-HT2, neuropeptide F and tachykinin.

Despite the conserved function of aggression across taxa in obtaining critical resources such as food and mates, serotonin's (5-HT) modulatory role on aggressive behavior appears to be largely inhibitory for vertebrates but stimulatory for invertebrates. However, critical gaps exist in our knowledge of invertebrates that need to be addressed before definitively stating opposing roles for 5-HT and aggression. Specifically, the role of 5-HT receptor subtypes are largely unknown, as is the potential interactive role of 5-HT with other neurochemical systems known to play a critical role in aggression. Similarly, the influence of these systems in driving sex differences in aggressive behavior of invertebrates is not well understood. Here, we investigated these questions by employing complementary approaches in a novel invertebrate model of aggression, the stalk-eyed fly. A combination of altered social conditions, pharmacological manipulation and 5-HT2 receptor knockdown by siRNA revealed an inhibitory role of this receptor subtype on aggression. Additionally, we provide evidence for 5-HT2's involvement in regulating neuropeptide F activity, a suspected inhibitor of aggression. However, this function appears to be stage-specific, altering only the initiation stage of aggressive conflicts. Alternatively, pharmacologically increasing systemic concentrations of 5-HT significantly elevated the expression of the neuropeptide tachykinin, which did not affect contest initiation but instead promoted escalation via production of high intensity aggressive behaviors. Notably, these effects were limited solely to males, with female aggression and neuropeptide expression remaining unaltered by any manipulation that affected 5-HT. Together, these results demonstrate a more nuanced role for 5-HT in modulating aggression in invertebrates, revealing an important interactive role with neuropeptides that is more reminiscent of vertebrates. The sex-differences described here also provide valuable insight into the evolutionary contexts of this complex behavior.

Negative consequences of early-life adversity on substance use as mediated by corticotropin-releasing factor modulation of serotonin activity.

Early-life adversity is associated with increased risk for substance abuse in later life, with women more likely to report past and current stress as a mediating factor in their substance use and relapse as compared to men. Preclinical models of neonatal and peri-adolescent (early through late adolescence) stress all support a direct relationship between experiences of early-life adversity and adult substance-related behaviors, and provide valuable information regarding the underlying neurobiology. This review will provide an overview of these animal models and how these paradigms alter drug and alcohol consumption and/or seeking in male and female adults. An introduction to the corticotropin-releasing factor (CRF) and serotonin systems, their development and their interactions at the level of the dorsal raphe will be provided, illustrating how this particular stress system is sexually dimorphic, and is well positioned to be affected by stressors early in development and throughout maturation. A model for CRF-serotonin interactions in the dorsal raphe and how these influence dopaminergic activity within the nucleus accumbens and subsequent reward-associated behaviors will be provided, and alterations to the activity of this system following early-life adversity will be identified. Overall, converging findings suggest that early-life adversity has long-term effects on the functioning of the CRF-serotonin system, highlighting a potentially important and targetable mediator linking stress to addiction. Future work should focus on identifying the exact mechanisms that promote long-term changes to the expression and activity of CRF receptors in the dorsal raphe. Moreover, it is important to clarify whether similar neurobiological mechanisms exist for males and females, given the sexual dimorphism both in CRF receptors and serotonin indices in the dorsal raphe and in the behavioral outcomes of early-life adversity.

Enhanced dopamine D2 autoreceptor function in the adult prefrontal cortex contributes to dopamine hypoactivity following adolescent social stress.

Adult psychiatric disorders characterized by cognitive deficits reliant on prefrontal cortex (PFC) dopamine are promoted by teenage bullying. Similarly, male Sprague-Dawley rats exposed to social defeat in mid-adolescence (P35-39) show impaired working memory in adulthood (P56-70), along with decreased medial PFC (mPFC) dopamine activity that results in part from increased dopamine transporter-mediated clearance. Here, we determined if dopamine synthesis and D2 autoreceptor-mediated inhibition of dopamine release in the adult mPFC are also enhanced by adolescent defeat to contribute to later dopamine hypofunction. Control and previously defeated rats did not differ in either DOPA accumulation following amino acid decarboxylase inhibition (NSD-1015 100 mg/kg ip.) or total/phosphorylated tyrosine hydroxylase protein expression, suggesting dopamine synthesis in the adult mPFC is not altered by adolescent defeat. However, exposure to adolescent defeat caused greater decreases in extracellular dopamine release (measured using in vivo chronoamperometry) in the adult mPFC upon local infusion of the D2 receptor agonist quinpirole (3 nM), implying greater D2 autoreceptor function. Equally enhanced D2 autoreceptor-mediated inhibition of dopamine release is seen in the adolescent (P40 or P49) mPFC, which declines in control rats by adulthood. However, this developmental decrease in autoreceptor function is absent following adolescent defeat, suggesting retention of an adolescent-like phenotype into adulthood. Current and previous findings indicate adolescent defeat decreases extracellular dopamine availability in the adult mPFC via both enhanced inhibition of dopamine release and increased dopamine clearance, which may be viable targets for improving treatment of cognitive deficits seen in neuropsychiatric disorders promoted by adolescent stress.

Carbamoylated erythropoietin modulates cognitive outcomes of social defeat and differentially regulates gene expression in the dorsal and ventral hippocampus.

Cognitive deficits are widespread in psychiatric disorders and frequently as debilitating as the affective component. Widely prescribed antidepressants for treating depressive disorders have limited efficacy in normalizing cognitive function. Erythropoietin (Epo) has been shown to improve cognitive function in schizophrenia and treatment resistant depressed patients. However, the potent elevation of red blood cell counts by Epo can cause hematological complications in non-anemic patients. We investigated a chemically engineered, posttranslational modification of Epo, carbamoylation, which renders it non-erythropoietic. We conducted mass-spectrometry-based peptide mapping of carbamoylated Epo (Cepo) and tested its ability to improve cognitive function after social defeat stress. Gene expression analysis in discrete brain regions was performed to obtain mechanistic insight of Cepo action. Cepo reversed stress-induced spatial working memory deficits while affecting long-term (24 h) novel object recognition in these rats. Contextual fear conditioning following defeat was enhanced by Cepo, but attenuated in controls. However, Cepo improved fear extinction in all rats compared to vehicle treatment. Cepo induced differential gene expression of BDNF, VGF, Arc, TH. and neuritin in the mPFC and discrete hippocampal subfields, with strongest induction in the dorsal hippocampus. Analysis of gene-brain region-behavior interactions showed that Cepo-induced neurotrophic mechanisms influence cognitive function. Carbamoylated erythropoietin can be developed as a therapeutic neurotrophic agent to treat cognitive dysfunction in neuropsychiatric diseases. Due to its distinct mechanism of action, it is unlikely to cross react with the activity of currently prescribed small molecule drugs and can be used as an add-on biologic drug.

Impact of juvenile chronic stress on adult cortico-accumbal function: Implications for cognition and addiction.

Repeated exposure to stress during childhood is associated with increased risk for neuropsychiatric illness, substance use disorders and other behavioral problems in adulthood. However, it is not clear how chronic childhood stress can lead to emergence of such a wide range of symptoms and disorders in later life. One possible explanation lies in stress-induced disruption to the development of specific brain regions associated with executive function and reward processing, deficits in which are common to the disorders promoted by childhood stress. Evidence of aberrations in prefrontal cortex and nucleus accumbens function following repeated exposure of juvenile (pre- and adolescent) organisms to a variety of different stressors would account not only for the similarity in symptoms across the wide range of childhood stress-associated mental illnesses, but also their persistence into adulthood in the absence of further stress. Therefore, the goal of this review is to evaluate the current knowledge regarding disruption to executive function and reward processing in adult animals or humans exposed to chronic stress over the juvenile period, and the underlying neurobiology, with particular emphasis on the prefrontal cortex and nucleus accumbens. First, the role of these brain regions in mediating executive function and reward processing is highlighted. Second, the neurobehavioral development of these systems is discussed to illustrate how juvenile stress may exert long-lasting effects on prefrontal cortex-accumbal activity and related behavioral functions. Finally, a critical review of current animal and human findings is presented, which strongly supports the supposition that exposure to chronic stress (particularly social aggression and isolation in animal studies) in the juvenile period produces impairments in executive function in adulthood, especially in working memory and inhibitory control. Chronic juvenile stress also results in aberrations to reward processing and seeking, with increased sensitivity to drugs of abuse particularly noted in animal models, which is in line with greater incidence of substance use disorders seen in clinical studies. These consequences are potentially mediated by monoamine and glutamatergic dysfunction in the prefrontal cortex and nucleus accumbens, providing translatable therapeutic targets. However, the predominant use of male subjects and social-based stressors in preclinical studies points to a clear need for determining how both sex differences and stressor heterogeneity may differentially contribute to stress-induced changes to substrates mediating executive function and reward processing, before the impact of chronic juvenile stress in promoting adult psychopathology can be fully understood.

Restraint stress differentially regulates inflammation and glutamate receptor gene expression in the hippocampus of C57BL/6 and BALB/c mice.

The inbred mouse strains, C57BL/6 and BALB/c have been used widely in preclinical psychiatric research. The differences in stress susceptibility of available strains has provided a useful platform to test pharmacological agents and behavioral responses. Previous brain gene profiling efforts have indicated that the inflammation and immune response gene pathway is the predominant gene network in the differential stress response of BALB/c and C57BL/6 mice. The implication is that a composite stress paradigm that includes a sequence of extended, varied and unpredictable stressors induces inflammation-related genes in the hippocampus. We hypothesized that the regulation of inflammation genes in the brain could constitute a primary stress response and tested this by employing a simple stress protocol, repeated exposure to the same stressor for 10 days, 2 h of restraint per day. We examined stress-induced regulation of 13 proinflammatory cytokine genes in male BALB/c and C57BL/6 mice using quantitative PCR. Elevated cytokine genes included tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), interleukin 10 (IL10), tumor necrosis factor (TNF) super family members and interleukin 1 receptor 1 (IL1R1). In addition, we examined restraint stress-induced regulation of 12 glutamate receptor genes in both strains. Our results show that restraint stress is sufficient to elevate the expression of inflammation-related genes in the hippocampus of both BABLB/c and C57BL/6 mice, but they differ in the genes that are induced and the magnitude of change. Cell types that are involved in this response include endothelial cells and astrocytes. Lay summary Repeated exposure to a simple restraint stress altered the activities of genes involved in inflammation and the functions of the excitatory neurotransmitter, glutamate. These changes in the hippocampus of the mouse brain showed differences that were dependent on the strain of mice and the length of the stress exposure. The effects of stress on activity of these genes may lead to alterations in behavior.

Amphetamine Withdrawal Differentially Increases the Expression of Organic Cation Transporter 3 and Serotonin Transporter in Limbic Brain Regions.

Amphetamine withdrawal increases anxiety and stress sensitivity related to blunted ventral hippocampus (vHipp) and enhances the central nucleus of the amygdala (CeA) serotonin responses. Extracellular serotonin levels are regulated by the serotonin transporter (SERT) and organic cation transporter 3 (OCT3), and vHipp OCT3 expression is enhanced during 24 hours of amphetamine withdrawal, while SERT expression is unaltered. Here, we tested whether OCT3 and SERT expression in the CeA is also affected during acute withdrawal to explain opposing regional alterations in limbic serotonergic neurotransmission and if respective changes continued with two weeks of withdrawal. We also determined whether changes in transporter expression were confined to these regions. Male rats received amphetamine or saline for two weeks followed by 24 hours or two weeks of withdrawal, with transporter expression measured using Western immunoblot. OCT3 and SERT expression increased in the CeA at both withdrawal timepoints. In the vHipp, OCT3 expression increased only at 24 hours of withdrawal, with an equivalent pattern seen in the dorsomedial hypothalamus. No changes were evident in any other regions sampled. These regionally specific changes in limbic OCT3 and SERT expression may partially contribute to the serotonergic imbalance and negative affect during amphetamine withdrawal.

Differential effects of glucocorticoid and mineralocorticoid antagonism on anxiety behavior in mild traumatic brain injury.

Mild traumatic brain injuries (TBIs) comprise three-quarters of all TBIs occurring in the United States annually, and psychological symptoms arising from them can last years after injury. One commonly observed symptom following mild TBI is generalized anxiety. Most mild TBIs happen in stressful situations (sports, war, domestic violence, etc.) when glucocorticoids are elevated in the brain at the time of impact, and glucocorticoids have negative effects on neuronal health following TBI. Therefore, blocking glucocorticoid receptors might prevent emergence of anxiety symptoms post-injury. Adult male rats received mifepristone (20mg/kg) or spironolactone (50mg/kg) to block glucocorticoid and mineralocorticoid receptors, respectively, 40min prior to being exposed to acute social defeat stress followed immediately by mild TBI. In defeated rats with concomitant mild TBI, mifepristone restored time spent in the open arms of an elevated plus maze to control levels, demonstrating for the first time that glucocorticoid receptors play a critical role in the development of anxiety after mild TBI. Future treatments could target these receptors, alleviating anxiety as a major side effect in victims of mild TBI sustained in stressful situations.

Mild Traumatic Brain Injury with Social Defeat Stress Alters Anxiety, Contextual Fear Extinction, and Limbic Monoamines in Adult Rats.

Mild traumatic brain injury (mTBI) produces symptoms similar to those typifying posttraumatic stress disorder (PTSD) in humans. We sought to determine whether a rodent model of stress concurrent with mTBI produces characteristics of PTSD such as impaired contextual fear extinction, while also examining concurrent alterations to limbic monoamine activity in brain regions relevant to fear and anxiety states. Male rats were exposed to social stress or control conditions immediately prior to mTBI induction, and 6 days later were tested either for anxiety-like behavior using the elevated plus maze (EPM), or for contextual fear conditioning and extinction. Brains were collected 24 h after EPM testing, and tissue from various limbic regions analyzed for content of monoamines, their precursors and metabolites using HPLC with electrochemical detection. Either social defeat or mTBI alone decreased time spent in open arms of the EPM, indicating greater anxiety-like behavior. However, this effect was enhanced by the combination of treatments. Further, rats exposed to both social defeat and mTBI exhibited greater freezing within extinction sessions compared to all other groups, suggesting impaired contextual fear extinction. Social defeat combined with mTBI also had greater effects on limbic monoamines than either insult alone, particularly with respect to serotonergic effects associated with anxiety and fear learning. The results suggest social stress concurrent with mTBI produces provides a relevant animal model for studying the prevention and treatment of post-concussive psychobiological outcomes.

Amphetamine withdrawal differentially affects hippocampal and peripheral corticosterone levels in response to stress.

Amphetamine withdrawal is associated with heightened anxiety-like behavior, which is directly driven by blunted stress-induced glucocorticoid receptor-dependent serotonin release in the ventral hippocampus. This suggests that glucocorticoid availability in the ventral hippocampus during stress may be reduced during amphetamine withdrawal. Therefore, we tested whether amphetamine withdrawal alters either peripheral or hippocampal corticosterone stress responses. Adult male rats received amphetamine (2.5mg/kg, ip) or saline for 14 days followed by 2 weeks of withdrawal. Contrary to our prediction, microdialysis samples from freely-moving rats revealed that restraint stress-induced corticosterone levels in the ventral hippocampus are enhanced by amphetamine withdrawal relative to controls. In separate groups of rats, plasma corticosterone levels increased immediately after 20min of restraint and decreased to below stress-naïve levels after 1h, indicating negative feedback regulation of corticosterone following stress. However, plasma corticosterone responses were similar in amphetamine-withdrawn and control rats. Neither amphetamine nor stress exposure significantly altered protein expression or enzyme activity of the steroidogenic enzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) or hexose-6-phosphate dehydrogenase (H6PD) in the ventral hippocampus. Our findings demonstrate for the first time that amphetamine withdrawal potentiates stress-induced corticosterone in the ventral hippocampus, which may contribute to increased behavioral stress sensitivity previously observed during amphetamine withdrawal. However, this is not mediated by either changes in plasma corticosterone or hippocampal steroidogenic enzymes. Establishing enhanced ventral hippocampal corticosterone as a direct cause of greater stress sensitivity may identify the glucocorticoid system as a novel target for treating behavioral symptoms of amphetamine withdrawal.

Adolescent social defeat alters N-methyl-D-aspartic acid receptor expression and impairs fear learning in adulthood.

Repeated social defeat of adolescent male rats results in adult mesocortical dopamine hypofunction, impaired working memory, and increased contextual anxiety-like behavior. Given the role of glutamate in dopamine regulation, cognition, and fear and anxiety, we investigated potential changes to N-methyl-D-aspartic acid (NMDA) receptors following adolescent social defeat. As both NMDA receptors and mesocortical dopamine are implicated in the expression and extinction of conditioned fear, a separate cohort of rats was challenged with a classical fear conditioning paradigm to investigate whether fear learning is altered by adolescent defeat. Quantitative autoradiography was used to measure 3H-MK-801 binding to NMDA receptors in regions of the medial prefrontal cortex, caudate putamen, nucleus accumbens, amygdala and hippocampus. Assessment of fear learning was achieved using an auditory fear conditioning paradigm, with freezing toward the auditory tone used as a measure of conditioned fear. Compared to controls, adolescent social defeat decreased adult NMDA receptor expression in the infralimbic region of the prefrontal cortex and central amygdala, while increasing expression in the CA3 region of the hippocampus. Previously defeated rats also displayed decreased conditioned freezing during the recall and first extinction periods, which may be related to the observed decreases and increases in NMDA receptors within the central amygdala and CA3, respectively. The alteration in NMDA receptors seen following adolescent social defeat suggests that dysfunction of glutamatergic systems, combined with mesocortical dopamine deficits, likely plays a role in the some of the long-term behavioral consequences of social stressors in adolescence seen in both preclinical and clinical studies.

Assessment strategies and fighting patterns in animal contests: a role for serotonin?

Accurate assessment of the probability of success in an aggressive confrontation with a conspecific is critical to the survival and fitness of the individuals. Various game theory models have examined these assessment strategies under the assumption that contests should favor the animal with the greater resource-holding potential (RHP), body size typically being the proxy. Mutual assessment asserts that an individual can assess their own RHP relative to their opponent, allowing the inferior animal the chance to flee before incurring unnecessary costs. The model of self-determined persistence, however, assumes that an individual will fight to a set personal threshold, independent of their opponent's RHP. Both models have been repeatedly tested using size as a proxy for RHP, with neither receiving unambiguous support. Here we present both morphological and neurophysiological data from size-matched and mismatched stalk-eyed fly fights. We discovered differing fighting strategies between winners and losers. Winners readily escalated encounters to higher intensity and physical contact and engaged in less low-intensity, posturing behaviors compared with losers. Although these fighting strategies were largely independent of size, they were associated with elevated levels of 5-HT. Understanding the neurophysiological factors responsible for mediating the motivational state of opponents could help resolve the inconsistencies seen in current game theory models. Therefore, we contend that current studies using only size as a proxy for RHP may be inadequate in determining the intricacies of fighting ability and that future studies investigating assessment strategies and contest outcome should include neurophysiological data.