Timing matters: Sex differences in inflammatory and behavioral outcomes following repetitive blast mild traumatic brain injury.

BACKGROUND: Repetitive blast-related mild traumatic brain injury (mTBI) caused by exposure to high explosives is increasingly common among warfighters as well as civilians. While women have been serving in military positions with increased risk of blast exposure since 2016, there are few published reports examining sex as a biological variable in models of blast mTBI, greatly limiting diagnosis and treatment capabilities. As such, here we examined outcomes of repetitive blast trauma in female and male mice in relation to potential behavioral, inflammatory, microbiome, and vascular dysfunction at multiple timepoints. METHODS: In this study we utilized a well-established blast overpressure model to induce repetitive (3x) blast-mTBI in both female and male mice. Acutely following repetitive exposure, we measured serum and brain cytokine levels, blood-brain barrier (BBB) disruption, fecal microbial abundance, and locomotion and anxiety-like behavior in the open field assay. At the one-month timepoint, in female and male mice we assessed behavioral correlates of mTBI and PTSD-related symptoms commonly reported by Veterans with a history of blast-mTBI using the elevated zero maze, acoustic startle, and conditioned odorant aversion paradigms. RESULTS: Repetitive blast exposure resulted in both similar (e.g., increased IL-6), and disparate (e.g., IL-10 increase only in females) patterns of acute serum and brain cytokine as well as gut microbiome changes in female and male mice. Acute BBB disruption following repetitive blast exposure was apparent in both sexes. While female and male blast mice both exhibited acute locomotor and anxiety-like deficits in the open field assay, only male mice exhibited adverse behavioral outcomes that lasted at least one-month. DISCUSSION: Representing a novel survey of potential sex differences following repetitive blast trauma, our results demonstrate unique similar yet divergent patterns of blast-induced dysfunction in female vs. male mice and highlight novel targets for future diagnosis and therapeutic development.

The dynorphin/kappa opioid receptor mediates adverse immunological and behavioral outcomes induced by repetitive blast trauma.

BACKGROUND: Adverse pathophysiological and behavioral outcomes related to mild traumatic brain injury (mTBI), posttraumatic stress disorder (PTSD), and chronic pain are common following blast exposure and contribute to decreased quality of life, but underlying mechanisms and prophylactic/treatment options remain limited. The dynorphin/kappa opioid receptor (KOR) system helps regulate behavioral and inflammatory responses to stress and injury; however, it has yet to be investigated as a potential mechanism in either humans or animals exposed to blast. We hypothesized that blast-induced KOR activation mediates adverse outcomes related to inflammation and affective behavioral response. METHODS: C57Bl/6 adult male mice were singly or repeatedly exposed to either sham (anesthesia only) or blast delivered by a pneumatic shock tube. The selective KOR antagonist norBNI or vehicle (saline) was administered 72 h prior to repetitive blast or sham exposure. Serum and brain were collected 10 min or 4 h post-exposure for dynorphin A-like immunoreactivity and cytokine measurements, respectively. At 1-month post-exposure, mice were tested in a series of behavioral assays related to adverse outcomes reported by humans with blast trauma. RESULTS: Repetitive but not single blast exposure resulted in increased brain dynorphin A-like immunoreactivity. norBNI pretreatment blocked or significantly reduced blast-induced increase in serum and brain cytokines, including IL-6, at 4 h post exposure and aversive/anxiety-like behavioral dysfunction at 1-month post-exposure. CONCLUSIONS: Our findings demonstrate a previously unreported role for the dynorphin/KOR system as a mediator of biochemical and behavioral dysfunction following repetitive blast exposure and highlight this system as a potential prophylactic/therapeutic treatment target.

One Is Not Enough: Understanding and Modeling Polysubstance Use.

Substance use disorder (SUD) is a chronic, relapsing disease with a highly multifaceted pathology that includes (but is not limited to) sensitivity to drug-associated cues, negative affect, and motivation to maintain drug consumption. SUDs are highly prevalent, with 35 million people meeting criteria for SUD. While drug use and addiction are highly studied, most investigations of SUDs examine drug use in isolation, rather than in the more prevalent context of comorbid substance histories. Indeed, 11.3% of individuals diagnosed with a SUD have concurrent alcohol and illicit drug use disorders. Furthermore, having a SUD with one substance increases susceptibility to developing dependence on additional substances. For example, the increased risk of developing heroin dependence is twofold for alcohol misusers, threefold for cannabis users, 15-fold for cocaine users, and 40-fold for prescription misusers. Given the prevalence and risk associated with polysubstance use and current public health crises, examining these disorders through the lens of co-use is essential for translatability and improved treatment efficacy. The escalating economic and social costs and continued rise in drug use has spurred interest in developing preclinical models that effectively model this phenomenon. Here, we review the current state of the field in understanding the behavioral and neural circuitry in the context of co-use with common pairings of alcohol, nicotine, cannabis, and other addictive substances. Moreover, we outline key considerations when developing polysubstance models, including challenges to developing preclinical models to provide insights and improve treatment outcomes.

Cocaine reward and memory after chemogenetic inhibition of distinct serotonin neuron subtypes in mice.

RATIONALE: We probed serotonin neurons, those denoted by their developmental gene expression as r2Hoxa2-Pet1 (experiment 1) and Drd1a-Pet1 (experiment 2), for differential modulation of cocaine reward and memory as revealed by the expression and development of conditioned place preference (CPP) in transgenic mice. OBJECTIVES: To query roles in CPP, we inhibited neurons cell autonomously in vivo by activating the transgenically expressed, synthetic DREADD receptor hM4Di (Di) with the exogenous ligand clozapine-N-oxide (CNO). METHODS: To examine CPP expression, mice were conditioned using behaviorally active doses of cocaine (10.0 or 17.8 mg/kg) vs. saline followed by CPP assessment, first without neuron inhibition (post-conditioning session 1), and then with CNO-mediated neuron inhibition (post-conditioning session 2), followed by 4 more post-conditioning sessions. To examine CPP development, we administered CNO during conditioning sessions and then assayed CPP across 6 post-conditioning sessions. RESULTS: In r2Hoxa2-Pet1-Di mice, post-conditioning CNO administration did not impact cocaine CPP expression, but after CNO administration during conditioning, cocaine CPP (17.8 mg/kg) persisted across post-conditioning sessions compared with that in controls, suggesting a deficit in extinguishing cocaine memory. Drd1a-Pet1-Di mice, prior to CNO-Di-triggered neuronal inhibition, unexpectedly expressed heightened cocaine CPP (10.0 and 17.8 mg/kg) compared with controls, and this basal phenotype was transiently blocked by acute post-conditioning CNO administration and persistently blocked by repeated CNO administration during conditioning. CONCLUSION: Cocaine reward and memory likely map to distinct serotonergic Pet1 neuron subtypes. r2Hoxa2-Pet1 neurons normally may limit the durability of cocaine memory, without impacting initial cocaine reward magnitude. Drd1a-Pet1 neurons normally may help to promote cocaine reward.

The impact of cocaine and heroin drug history on motivation and cue sensitivity in a rat model of polydrug abuse.

RATIONALE: Comorbid use of heroin and cocaine is highly prevalent among drug users and can greatly increase addiction risk. Nonetheless, little is known regarding how a multi-drug history impacts motivation and cue responsivity to individual drugs. OBJECTIVE: We used behavioral-economic procedures to examine motivation to maintain drug consumption and tests of drug-seeking to drug-associated cues to assess sensitivity to heroin and cocaine-associated cues in rats that had a self-administration history of heroin, cocaine, or both drugs. RESULTS: Unexpectedly, we found that groups with a polydrug history of heroin and cocaine did not have higher levels of motivation or cue-induced reinstatement of drug-seeking for either cocaine or heroin compared to single drug groups. Nonetheless, we did find drug-specific differences in both economic price and cue sensitivity. Specifically, demand elasticity was lower for cocaine compared to heroin in animals with a single drug history, but not with polydrug groups. In addition, cocaine demand was predictive of the degree of cue-induced reinstatement of drug-seeking for cocaine following extinction, whereas heroin demand was predictive of the degree of reactivity to a heroin-associated cue. Furthermore, although cue reactivity following the initial self-administration phase did not differ across cues and drug history, reactivity to both heroin and cocaine cues was greater during subsequent heroin use compared to cocaine use, and this enhanced reactivity to heroin cues persisted during forced abstinence. CONCLUSIONS: These results indicate that there is a greater motivation to maintain cocaine consumption, but higher sensitivity to drug-associated cues with a history of heroin use, suggesting that cocaine and heroin may drive continued drug use through different behavioral processes.

Blockade of α2-adrenergic receptors in prelimbic cortex: impact on cocaine self-administration in adult spontaneously hypertensive rats following adolescent atomoxetine treatment.

RATIONALE: Research with the spontaneously hypertensive rat (SHR) model of attention deficit/hyperactivity disorder demonstrated that chronic methylphenidate treatment during adolescence increased cocaine self-administration established during adulthood under a progressive ratio (PR) schedule. Compared to vehicle, chronic atomoxetine treatment during adolescence failed to increase cocaine self-administration under a PR schedule in adult SHR. OBJECTIVES: We determined if enhanced noradrenergic transmission at α2-adrenergic receptors within prefrontal cortex contributes to this neutral effect of adolescent atomoxetine treatment in adult SHR. METHODS: Following treatment from postnatal days 28-55 with atomoxetine (0.3 mg/kg) or vehicle, adult male SHR and control rats from Wistar-Kyoto (WKY) and Wistar (WIS) strains were trained to self-administer 0.3 mg/kg cocaine. Self-administration performance was evaluated under a PR schedule of cocaine delivery following infusion of the α2-adrenergic receptor antagonist idazoxan (0 and 10-56 µg/side) directly into prelimbic cortex. RESULTS: Adult SHR attained higher PR break points and had greater numbers of active lever responses and infusions than WKY and WIS. Idazoxan dose-dependently increased PR break points and active lever responses in SHR following adolescent atomoxetine vs. vehicle treatment. Behavioral changes were negligible after idazoxan pretreatment in SHR following adolescent vehicle or in WKY and WIS following adolescent atomoxetine or vehicle. CONCLUSIONS: α2-Adrenergic receptor blockade in prelimbic cortex of SHR masked the expected neutral effect of adolescent atomoxetine on adult cocaine self-administration behavior. Moreover, greater efficacy of acute idazoxan challenge in adult SHR after adolescent atomoxetine relative to vehicle is consistent with the idea that chronic atomoxetine may downregulate presynaptic α2A-adrenergic autoreceptors in SHR.

Methylphenidate treatment beyond adolescence maintains increased cocaine self-administration in the spontaneously hypertensive rat model of attention deficit/hyperactivity disorder.

Past research with the spontaneously hypertensive rat (SHR) model of attention deficit/hyperactivity disorder showed that adolescent methylphenidate treatment enhanced cocaine abuse risk in SHR during adulthood. The acquisition of cocaine self-administration was faster, and cocaine dose-response functions were shifted upward under fixed-ratio and progressive ratio schedules compared to adult SHR that received adolescent vehicle treatment or to control strains that received adolescent methylphenidate treatment. The current study determined if extending treatment beyond adolescence would ameliorate long-term consequences of adolescent methylphenidate treatment on cocaine abuse risk in adult SHR. Treatments (vehicle or 1.5mg/kg/day oral methylphenidate) began on postnatal day 28. Groups of male SHR were treated with vehicle during adolescence and adulthood, with methylphenidate during adolescence and vehicle during adulthood, or with methylphenidate during adolescence and adulthood. The group receiving adolescent-only methylphenidate was switched to vehicle on P56. Cocaine self-administration began on postnatal day 77, and groups receiving methylphenidate during adolescence and adulthood were treated either 1-h before or 1-h after daily sessions. At baseline under a fixed-ratio 1 schedule, cocaine self-administration (2h sessions; 0.3mg/kg unit dose) did not differ among the four treatment groups. Under a progressive ratio schedule (4.5h maximum session length; 0.01-1.0mg/kg unit doses), breakpoints for self-administered cocaine in SHR receiving the adult methylphenidate treatment 1-h pre-session were not different from the vehicle control group. However, compared to the vehicle control group, breakpoints for self-administered cocaine at the 0.3 and 1.0mg/kg unit doses were greater in adult SHR that received adolescent-only methylphenidate or received methylphenidate that was continued into adulthood and administered 1-h post-session. These findings suggest that extending methylphenidate treatment beyond adolescence does not ameliorate explicitly the long-term consequences of adolescent methylphenidate treatment. Pre-session methylphenidate may mask temporarily the detection of an increase in cocaine self-administration following chronic methylphenidate treatment.