Dissociable dorsal medial prefrontal cortex ensembles are necessary for cocaine seeking and fear conditioning in mice.

The dmPFC plays a dual role in modulating drug seeking and fear-related behaviors. Learned associations between cues and drug seeking are encoded by a specific ensemble of neurons. This study explored the stability of a dmPFC cocaine seeking ensemble over two weeks and its influence on persistent cocaine seeking and fear memory retrieval. In the first series of experiments, we trained TetTag mice in cocaine self-administration and tagged strongly activated neurons with EGFP during the initial day 7 cocaine seeking session. Subsequently, a follow-up seeking test was conducted two weeks later to examine ensemble reactivation between two seeking sessions via c-Fos immunostaining. In the second series of experiments, we co-injected viruses expressing TRE-cre and a cre-dependent inhibitory PSAM-GlyR into the dmPFC of male and female c-fos -tTA mice to enable "tagging" of cocaine seeking ensemble or cued fear ensemble neurons with an inhibitory chemogenetic receptors. Then we investigated their contribution to subsequent cocaine seeking and fear recall during inhibition of the tagged ensemble by administering uPSEM792s (0.3 mg/kg), a selective ligand for PSAM-GlyR. In both sexes, there was a positive association between the persistence of cocaine seeking and the proportion of reactivated EGFP+ neurons within the dmPFC. More importantly, inhibition of the cocaine seeking ensemble suppressed cocaine seeking, but not recall of fear memory, while inhibition of the fear ensemble reduced conditioned freezing but not cocaine seeking. The results demonstrate that cocaine and fear recall ensembles in the dmPFC are stable, but largely exclusive from one another.

Comparison of online to face-to-face instruction for anatomy review in a third-year clinical course.

OBJECTIVE: This project compared student learning and satisfaction of an anatomy review delivered by a face-to-face lecture (F2FL) and an online learning module (OLM) for third-year doctor of chiropractic students. METHODS: This cohort study compared student learning and satisfaction of a pediatric spinal anatomy review delivered via F2FL (cohort 1, n = 23) and OLM (cohort 2, n = 18) in 2 successive 2019 (pre-COVID) course offerings. Previously validated pre- and post-tests were given. Students completed a survey assessing delivery, comfort with online learning and online learning technology, and preference of F2FL vs OLM of review material. Pre- and post-test results were assessed using repeated-measures analysis of variance. RESULTS: Testing results showed an improvement with both groups (F2FL 53.7%, p < .001 vs OLM 51.8%, p < .001), with no significant difference between the F2FL and OLM groups (p = .53; p = .82). The survey showed: 83.3% of OLM students felt the online method was effective, and 88.9% of the OLM students would prefer online reviews or have no preference between online or face-to-face; meanwhile, 80% of the F2FL group thought the lecture engaging/effective, whereas 60% of the F2FL group would have preferred to have the material presented online. CONCLUSION: The OLM was found to be as effective as the F2FL for the content assessed. The majority of students would prefer the online method for future anatomy review content presented in the course. This strategy could be applied to provide review materials in other clinical courses, allowing material to be developed and given by content experts while freeing valuable in-class time.

Environmental Enrichment during Abstinence Reduces Oxycodone Seeking and c-Fos Expression in a Subpopulation of Medial Prefrontal Cortex Neurons.

BACKGROUND: Several preclinical studies have demonstrated that environmental enrichment (EE) during abstinence reduces drug seeking for psychostimulant and opioid drugs. Drug seeking is dependent on activity within the dorsomedial prefrontal cortex, and enrichment has been able to reduce drug seeking-associated increases in c-Fos in this region. In this study, we tested the hypothesis that EE during abstinence from oxycodone self-administration would reduce drug seeking and c-Fos immunoreactivity within the prefrontal cortex in a cell-type specific manner. METHODS: Male rats self-administered oxycodone in two-hours sessions for three weeks, then underwent an initial drug seeking test under extinction conditions after one week of forced abstinence. Following this test, rats received either EE or remained individually housed in their home cage, then a second drug seeking test, with tissue collection immediately afterward. RESULTS: Compared to rats in standard housing, environmentally enriched rats had lower oxycodone seeking. In the prelimbic and infralimbic prefrontal cortices, the number of c-Fos+ cells was reduced, and this reduction was predominantly in inhibitory cells neurons, as evidenced by a reduction in the proportion of c-Fos+ cells in GAD+, but not CamKII+ cells. There was also a robust positive relationship between the number of c-Fos+ cells and persistence of oxycodone seeking in both the PrL and IL. CONCLUSIONS: These findings further support the effectiveness of enriched environments to reduce reactivity to drug-associated stimuli and contexts and provide a potential mechanism by which this occurs.

The effects of sleep restriction during abstinence on oxycodone seeking: Sex-dependent moderating effects of behavioral and hypothalamic-pituitary-adrenal axis-related phenotypes.

During opioid use and abstinence, sleep disturbances are common and are thought to exacerbate drug craving. In this study, we tested the hypothesis that sleep restriction during abstinence from oxycodone self-administration would increase drug seeking during extinction and footshock reinstatement tests. We also performed behavioral phenotyping to determine if individual variation in responses to stressors and/or pain are associated with oxycodone seeking during abstinence, as stress, pain and sleep disturbance are often co-occurring phenomena. Sleep restriction during abstinence did not have selective effects on oxycodone seeking for either sex in extinction and footshock reinstatement tests. Some phenotypes were associated with drug seeking; these associations differed by sex and type of drug seeking assessment. In female rats, pain-related phenotypes were related to high levels of drug seeking during the initial extinction session. In male rats, lower anxiety-like behavior in the open field was associated with greater drug seeking, although this effect was lost when correcting for oxycodone intake. Adrenal sensitivity prior to oxycodone exposure was positively associated with footshock reinstatement in females. This work identifies sex-dependent relationships between HPA axis function and opioid seeking, indicating that HPA axis function could be a therapeutic target for the treatment of opioid use disorder, with tailored approaches based on sex. Sleep disturbance during abstinence did not appear to be a major contributing factor to opioid seeking.

Sleep restriction during opioid abstinence affects the hypothalamic-pituitary-adrenal (HPA) axis in male and female rats.

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.

Chronicity of repeated blast traumatic brain injury associated increase in oxycodone seeking in rats.

Numerous epidemiological studies have found co-morbidity between non-severe traumatic brain injury (TBI) and substance misuse in both civilian and military populations. Preclinical studies have also identified this relationship for some misused substances. We have previously demonstrated that repeated blast traumatic brain injury (rbTBI) increased oxycodone seeking without increasing oxycodone self-administration, suggesting that the neurological sequelae of traumatic brain injury can elevate opioid misuse liability. Here, we determined the chronicity of this effect by testing different durations of time between injury and oxycodone self-administration and durations of abstinence. We found that the subchronic (four weeks), but not the acute (three days) or chronic (four months) duration between injury and oxycodone self-administration was associated with increased drug seeking and re-acquisition of self-administration following a 10-day abstinence. Examination of other abstinence durations (two days, four weeks, or four months) revealed no effect of rbTBI on drug seeking at any of the abstinence durations tested. Together, these data indicate that there is a window of vulnerability after TBI when oxycodone self-administration is associated with elevated drug seeking and relapse-related behaviors.

Effects of photon irradiation in the presence and absence of hindlimb unloading on the behavioral performance and metabolic pathways in the plasma of Fischer rats.

Introduction: The space environment astronauts experience during space missions consists of multiple environmental challenges, including microgravity. In this study, we assessed the behavioral and cognitive performances of male Fisher rats 2 months after sham irradiation or total body irradiation with photons in the absence or presence of simulated microgravity. We analyzed the plasma collected 9 months after sham irradiation or total body irradiation for distinct alterations in metabolic pathways and to determine whether changes to metabolic measures were associated with specific behavioral and cognitive measures. Methods: A total of 344 male Fischer rats were irradiated with photons (6 MeV; 3, 8, or 10 Gy) in the absence or presence of simulated weightlessness achieved using hindlimb unloading (HU). To identify potential plasma biomarkers of photon radiation exposure or the HU condition for behavioral or cognitive performance, we performed regression analyses. Results: The behavioral effects of HU on activity levels in an open field, measures of anxiety in an elevated plus maze, and anhedonia in the M&M consumption test were more pronounced than those of photon irradiation. Phenylalanine, tyrosine, and tryptophan metabolism, and phenylalanine metabolism and biosynthesis showed very strong pathway changes, following photon irradiation and HU in animals irradiated with 3 Gy. Here, 29 out of 101 plasma metabolites were associated with 1 out of 13 behavioral measures. In the absence of HU, 22 metabolites were related to behavioral and cognitive measures. In HU animals that were sham-irradiated or irradiated with 8 Gy, one metabolite was related to behavioral and cognitive measures. In HU animals irradiated with 3 Gy, six metabolites were related to behavioral and cognitive measures. Discussion: These data suggest that it will be possible to develop stable plasma biomarkers of behavioral and cognitive performance, following environmental challenges like HU and radiation exposure.

A Preclinical Rodent Model for Repetitive Subconcussive Head Impact Exposure in Contact Sport Athletes.

Repetitive subconcussive head impact exposure has been associated with clinical and MRI changes in some non-concussed contact sport athletes over the course of a season. However, analysis of human tolerance for repeated head impacts is complicated by concussion and head impact exposure history, genetics, and other personal factors. Therefore, the objective of the current study was to develop a rodent model for repetitive subconcussive head impact exposure that can be used to understand injury mechanisms and tolerance in the human. This study incorporated the Medical College of Wisconsin Rotational Injury Model to expose rats to multiple low-level head accelerations per day over a 4-week period. The peak magnitude of head accelerations were scaled from our prior human studies of contact sport athletes and the number of exposures per day were based on the median (moderate exposure) and 95th percentile (high exposure) number of exposures per day across the human sample. Following the exposure protocol, rats were assessed for cognitive deficits, emotional changes, blood serum levels of axonal injury biomarkers, and histopathological evidence of injury. High exposure rats demonstrated cognitive deficits and evidence of anxiety-like behaviors relative to shams. Moderate exposure rats did not demonstrate either of those behaviors. Similarly, high exposure rats had histopathological evidence of gliosis [i.e., elevated Iba1 intensity and glial fibrillary acidic protein (GFAP) volume relative to shams] in the basolateral amygdala and other areas. Blood serum levels of neurofilament light (NFL) demonstrated a dose response relationship with increasing numbers of low-level head acceleration exposures with a higher week-to-week rate of NFL increase for the high exposure group compared to the moderate exposure group. These findings demonstrate a cumulative effect of repeated low-level head accelerations and provide a model that can be used in future studies to better understand mechanisms and tolerance for brain injury resulting from repeated low-level head accelerations, with scalable biomechanics between the rat and human.

Repeated blast mild traumatic brain injury and oxycodone self-administration produce interactive effects on neuroimaging outcomes.

Traumatic brain injury (TBI) and drug addiction are common comorbidities, but it is unknown if the neurological sequelae of TBI contribute to this relationship. We have previously reported elevated oxycodone seeking after drug self-administration in rats that received repeated blast TBI (rbTBI). TBI and exposure to drugs of abuse can each change structural and functional neuroimaging outcomes, but it is unknown if there are interactive effects of injury and drug exposure. To determine the effects of TBI and oxycodone exposure, we subjected rats to rbTBI and oxycodone self-administration and measured drug seeking and several neuroimaging measures. We found interactive effects of rbTBI and oxycodone on fractional anisotropy (FA) in the nucleus accumbens (NAc) and that FA in the medial prefrontal cortex (mPFC) was correlated with drug seeking. We also found an interactive effect of injury and drug on widespread functional connectivity and regional homogeneity of the blood oxygen level dependent (BOLD) response, and that intra-hemispheric functional connectivity in the infralimbic medial prefrontal cortex positively correlated with drug seeking. In conclusion, rbTBI and oxycodone self-administration had interactive effects on structural and functional magnetic resonance imaging (MRI) measures, and correlational effects were found between some of these measures and drug seeking. These data support the hypothesis that TBI and opioid exposure produce neuroadaptations that contribute to addiction liability.

Comparison of prefrontal cortex sucrose seeking ensembles engaged in multiple seeking sessions: Context is key.

Encoding of memories, including those associated with prior drug or reward, is thought to take place within distinct populations of neurons, termed ensembles. Neuronal ensembles for drug- and reward-seeking have been identified in regions of the medial prefrontal cortex, but much of our understanding of these ensembles is based on experiments that take place in a single reward-associated environment and measure ensemble encoding over short durations of time. In contrast, reward seeking behavior is evident across different reward-associated environments and persists over time. Using TetTag mice and Fos immunohistochemistry, we examined the relationship between persistent sucrose-seeking and ensemble encoding in mice that undergo seeking sessions in the same or different sucrose self-administration contexts 2 weeks apart. We found that prelimbic (PrL) and anterior cingulate cortex ensembles tagged in the first seeking session were highly sensitive to the context in which a second seeking session took place: reactivation of these ensembles was reduced in the same context but elevated in a distinct sucrose self-administration context. Correlational analyses revealed that ensemble reactivation in the PrL was proportional to the persistence of sucrose seeking behavior across sessions in differing ways in female mice. In the same context, reactivation was proportional to the persistence of non-reinforced operant responses, whereas in a distinct context, reactivation was proportional to the persistence of non-reinforced head entries into the sucrose receptacle. This study underlines the importance of the medial prefrontal cortex importance in maintaining a reward-seeking ensemble over time and identifies context-dependent changes in behavioral correlates of ensemble reactivation.

Does Traumatic Brain Injury Cause Risky Substance Use or Substance Use Disorder?

There is a high co-occurrence of risky substance use among adults with traumatic brain injury (TBI), although it is unknown if the neurologic sequelae of TBI can promote this behavior. We propose that to conclude that TBI can cause risky substance use, it must be determined that TBI precedes risky substance use, that confounders with the potential to increase the likelihood of both TBI and risky substance use must be ruled out, and that there must be a plausible mechanism of action. In this review, we address these factors by providing an overview of key clinical and preclinical studies and list plausible mechanisms by which TBI could increase risky substance use. Human and animal studies have identified an association between TBI and risky substance use, although the strength of this association varies. Factors that may limit detection of this relationship include differential variability due to substance, sex, age of injury, and confounders that may influence the likelihood of both TBI and risky substance use. We propose possible mechanisms by which TBI could increase substance use that include damage-associated neuroplasticity, chronic changes in neuroimmune signaling, and TBI-associated alterations in brain networks.

Effects of 5-Ion Beam Irradiation and Hindlimb Unloading on Metabolic Pathways in Plasma and Brain of Behaviorally Tested WAG/Rij Rats.

A limitation of simulated space radiation studies is that radiation exposure is not the only environmental challenge astronauts face during missions. Therefore, we characterized behavioral and cognitive performance of male WAG/Rij rats 3 months after sham-irradiation or total body irradiation with a simplified 5-ion mixed beam exposure in the absence or presence of simulated weightlessness using hindlimb unloading (HU) alone. Six months following behavioral and cognitive testing or 9 months following sham-irradiation or total body irradiation, plasma and brain tissues (hippocampus and cortex) were processed to determine whether the behavioral and cognitive effects were associated with long-term alterations in metabolic pathways in plasma and brain. Sham HU, but not irradiated HU, rats were impaired in spatial habituation learning. Rats irradiated with 1.5 Gy showed increased depressive-like behaviors. This was seen in the absence but not presence of HU. Thus, HU has differential effects in sham-irradiated and irradiated animals and specific behavioral measures are associated with plasma levels of distinct metabolites 6 months later. The combined effects of HU and radiation on metabolic pathways in plasma and brain illustrate the complex interaction of environmental stressors and highlights the importance of assessing these interactions.

Within-animal comparisons of novelty and cocaine neuronal ensemble overlap in the nucleus accumbens and prefrontal cortex.

Novelty seeking is a personality trait associated with an increased vulnerability for substance abuse. In rodents, elevated novelty seeking has been shown to be a predictor for elevated drug self-administration and compulsive use. While previous studies have shown that both novelty and drugs of abuse have actions within similar mesocorticolimbic regions, little is known as to whether the same neural ensembles are engaged by these two stimuli. Using the TetTag mouse model and Fos immunohistochemistry, we measured neurons engaged by novelty and acute cocaine exposure, respectively in the prefrontal cortex (PFC) and nucleus accumbens (NAc). While there was no significant impact of novelty exposure on the size of the EGFP+ ensemble, we found that cocaine engaged significantly more Fos+ neurons in the NAc, while stress increased the size of the Fos+ ensemble in the PFC. Analysis of ensemble reactivation was specific to the emotional valence of the second stimuli. We found that a greater proportion of the EGFP+ ensemble was reactivated in the groups that paired novelty with a positive (cocaine) or neutral (saline) experience in the NAc, while the novelty/stress paired groups exhibited significantly less ensemble overlap in the PFC. However, only in the NAc shell was this increase in ensemble overlap specific to those exposed to both novelty and cocaine. This suggests that the NAc shell, but not the NAc core or PFC, may play an important role in general reward processing by engaging a similar network of neurons.

Gender Analysis for One Health: Theoretical Perspectives and Recommendations for Practice.

One health emphasizes the interdependent health of humans, animals, and their shared environments and shows promise as an integrated, equitable transdisciplinary approach to important ecohealth issues. Notably, research or programming explicitly examining the intersection of gender and one health is limited, although females represent half of the human population and play important roles in human and animal health around the world. Recognizing these gaps, scholars from the University of Wisconsin-Madison in collaboration with United States Department of Agriculture convened a consultative workshop, "Women and One Health," in 2016. This paper outlines the workshop methods and highlights outcomes toward shared terminology and integration of frameworks from one health, gender analysis, and women in agriculture. Further, recommendations for education, policy, and service delivery at the intersection of women's empowerment and one health are offered as important efforts toward the dual goals of gender equality and sustainable health of humans, animals, and their shared ecosystems.

Repeated blast model of mild traumatic brain injury alters oxycodone self-administration and drug seeking.

Each year, traumatic brain injuries (TBI) affect millions worldwide. Mild TBIs (mTBI) are the most prevalent and can lead to a range of neurobehavioral problems, including substance abuse. A single blast exposure, inducing mTBI alters the medial prefrontal cortex, an area implicated in addiction, for at least 30 days post injury in rats. Repeated blast exposures result in greater physiological and behavioral dysfunction than single exposure; however, the impact of repeated mTBI on addiction is unknown. In this study, the effect of mTBI on various stages of oxycodone use was examined. Male Sprague Dawley rats were exposed to a blast model of mTBI once per day for 3 days. Rats were trained to self-administer oxycodone during short (2 h) and long (6 h) access sessions. Following abstinence, rats underwent extinction and two cued reinstatement sessions. Sham and rbTBI rats had similar oxycodone intake, extinction responding and cued reinstatement of drug seeking. A second group of rats were trained to self-administer oxycodone with varying reinforcement schedules (fixed ratio (FR)-2 and FR-4). Under an FR-2 schedule, rbTBI-exposed rats earned fewer reinforcers than sham-exposed rats. During 10 extinction sessions, the rbTBI-exposed rats exhibited significantly more seeking for oxycodone than the sham-injured rats. There was a positive correlation between total oxycodone intake and day 1 extinction drug seeking in sham, but not in rbTBI-exposed rats. Together, this suggests that rbTBI-exposed rats are more sensitive to oxycodone-associated cues during reinstatement than sham-exposed rats and that rbTBI may disrupt the relationship between oxycodone intake and seeking.

Mitochondria-Targeted Honokiol Confers a Striking Inhibitory Effect on Lung Cancer via Inhibiting Complex I Activity.

We synthesized a mitochondria-targeted honokiol (Mito-HNK) that facilitates its mitochondrial accumulation; this dramatically increases its potency and efficacy against highly metastatic lung cancer lines in vitro, and in orthotopic lung tumor xenografts and brain metastases in vivo. Mito-HNK is >100-fold more potent than HNK in inhibiting cell proliferation, inhibiting mitochondrial complex ?, stimulating reactive oxygen species generation, oxidizing mitochondrial peroxiredoxin-3, and suppressing the phosphorylation of mitoSTAT3. Within lung cancer brain metastases in mice, Mito-HNK induced the mediators of cell death and decreased the pathways that support invasion and proliferation. In contrast, in the non-malignant stroma, Mito-HNK suppressed pathways that support metastatic lesions, including those involved in inflammation and angiogenesis. Mito-HNK showed no toxicity and targets the metabolic vulnerabilities of primary and metastatic lung cancers. Its pronounced anti-invasive and anti-metastatic effects in the brain are particularly intriguing given the paucity of treatment options for such patients either alone or in combination with standard chemotherapeutics.

Effects of Mild Blast Traumatic Brain Injury on Cognitive- and Addiction-Related Behaviors.

Traumatic brain injury (TBI) commonly results in cognitive and psychiatric problems. Cognitive impairments occur in approximately 30% of patients suffering from mild TBI (mTBI), and correlational evidence from clinical studies indicates that substance abuse may be increased following mTBI. However, understanding the lasting cognitive and psychiatric problems stemming from mTBI is difficult in clinical settings where pre-injury assessment may not be possible or accurate. Therefore, we used a previously characterized blast model of mTBI (bTBI) to examine cognitive- and addiction-related outcomes. We previously demonstrated that this model leads to bilateral damage of the medial prefrontal cortex (mPFC), a region critical for cognitive function and addiction. Rats were exposed to bTBI and tested in operant learning tasks several weeks after injury. bTBI rats made more errors during acquisition of a cue discrimination task compared to sham treated rats. Surprisingly, we observed no differences between groups in set shifting and delayed matching to sample, tasks known to require the mPFC. Separate rats performed cocaine self-administration. No group differences were found in intake or extinction, and only subtle differences were observed in drug-primed reinstatement 3-4 months after injury. These findings indicate that bTBI impairs acquisition of a visual discrimination task and that bTBI does not significantly increase the ability of cocaine exposure to trigger drug seeking.

Acute Clinical Predictors of Symptom Recovery in Emergency Department Patients with Uncomplicated Mild Traumatic Brain Injury or Non-Traumatic Brain Injuries.

There is a subset of patients with mild traumatic brain injury (mTBI) who report persistent symptoms that impair their functioning and quality of life. Being able to predict which patients will experience prolonged symptom recovery would help clinicians target resources for clinical follow-up to those most in need, and would facilitate research to develop precision medicine treatments for mTBI. The purpose of this study was to investigate the predictors of symptom recovery in a prospective sample of emergency department trauma patients with either mTBI or non-mTBI injuries. Subjects were examined at several time points from within 72 h to 45 days post-injury. We quantified and compared the value of a variety of demographic, injury, and clinical assessment (symptom, neurocognitive) variables for predicting self-reported symptom duration in both mTBI (n = 89) and trauma control (n = 73) patients. Several injury-related and neuropsychological variables assessed acutely (< 72 h) post-injury predicted symptom duration, particularly loss of consciousness (mTBI group), acute somatic symptom burden (both groups), and acute reaction time (both groups), with reasonably good model fit when including all of these variables (area under the receiver operating characteristic curve [AUC] = 0.76). Incorporating self-reported litigation involvement modestly increased prediction further (AUC = 0.80). The results highlight the multifactorial nature of mTBI recovery, and injury recovery more generally, and the need to incorporate a variety of variables to achieve adequate prediction. Further research to improve this model and validate it in new and more diverse trauma samples will be useful to build a neurobiopsychosocial model of recovery that informs treatment development.