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.

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.

Responses to drugs of abuse and non-drug rewards in leptin deficient ob/ob mice.

RATIONALE: Although leptin receptors are found in hypothalamic nuclei classically associated with homeostatic feeding mechanisms, they are also present in brain regions known to regulate hedonic-based feeding, natural reward processing, and responses to drugs of abuse. The ob/ob mouse is deficient in leptin signaling, and previous work has found altered mesolimbic dopamine signaling and sensitivity to the locomotor activating effects of amphetamine in these mice. OBJECTIVES: We directly assessed responses to three drugs of abuse and non-drug rewards in the leptin-deficient ob/ob mouse. METHODS: Ob/ob mice were tested in assays of sweet preference, novelty seeking, and drug reward/reinforcement. RESULTS: In assays of novelty seeking, novel open field activity and operant sensation seeking were reduced in ob/ob mice, although novel object interaction and novel environment preference were comparable to wild types. We also found that ob/ob mice had specific phenotypes in regard to cocaine: conditioned place preference for 2.5 mg/kg was increased, while the locomotor response to 10 mg/kg was reduced, and cocaine self-administration was the same as wild types. Ob/ob mice also acquired self-administration of the potent opioid remifentanil, but breakpoints for the drug were significantly reduced. Finally, we found significant differences in ethanol drinking in ob/ob mice that correlated negatively with body weight and positively with operant sensation seeking. CONCLUSIONS: In conclusion, ob/ob mice displayed task-specific deficits in novelty seeking and dissociable differences in reward/reinforcement associated with cocaine, remifentanil, and ethanol.

Increased prefrontal cortex neurogranin enhances plasticity and extinction learning.

Increasing plasticity in neurons of the prefrontal cortex (PFC) has been proposed as a possible therapeutic tool to enhance extinction, a process that is impaired in post-traumatic stress disorder, schizophrenia, and addiction. To test this hypothesis, we generated transgenic mice that overexpress neurogranin (a calmodulin-binding protein that facilitates long-term potentiation) in the PFC. Neurogranin overexpression in the PFC enhanced long-term potentiation and increased the rates of extinction learning of both fear conditioning and sucrose self-administration. Our results indicate that elevated neurogranin function within the PFC can enhance local plasticity and increase the rate of extinction learning across different behavioral tasks. Thus, neurogranin can provide a molecular link between enhanced plasticity and enhanced extinction.

The gene desert mammary carcinoma susceptibility locus Mcs1a regulates Nr2f1 modifying mammary epithelial cell differentiation and proliferation.

Genome-wide association studies have revealed that many low-penetrance breast cancer susceptibility loci are located in non-protein coding genomic regions; however, few have been characterized. In a comparative genetics approach to model such loci in a rat breast cancer model, we previously identified the mammary carcinoma susceptibility locus Mcs1a. We now localize Mcs1a to a critical interval (277 Kb) within a gene desert. Mcs1a reduces mammary carcinoma multiplicity by 50% and acts in a mammary cell-autonomous manner. We developed a megadeletion mouse model, which lacks 535 Kb of sequence containing the Mcs1a ortholog. Global gene expression analysis by RNA-seq revealed that in the mouse mammary gland, the orphan nuclear receptor gene Nr2f1/Coup-tf1 is regulated by Mcs1a. In resistant Mcs1a congenic rats, as compared with susceptible congenic control rats, we found Nr2f1 transcript levels to be elevated in mammary gland, epithelial cells, and carcinoma samples. Chromatin looping over ∼820 Kb of sequence from the Nr2f1 promoter to a strongly conserved element within the Mcs1a critical interval was identified. This element contains a 14 bp indel polymorphism that affects a human-rat-mouse conserved COUP-TF binding motif and is a functional Mcs1a candidate. In both the rat and mouse models, higher Nr2f1 transcript levels are associated with higher abundance of luminal mammary epithelial cells. In both the mouse mammary gland and a human breast cancer global gene expression data set, we found Nr2f1 transcript levels to be strongly anti-correlated to a gene cluster enriched in cell cycle-related genes. We queried 12 large publicly available human breast cancer gene expression studies and found that the median NR2F1 transcript level is consistently lower in 'triple-negative' (ER-PR-HER2-) breast cancers as compared with 'receptor-positive' breast cancers. Our data suggest that the non-protein coding locus Mcs1a regulates Nr2f1, which is a candidate modifier of differentiation, proliferation, and mammary cancer risk.

Effective flow cytometric phenotyping of cells using minimal amounts of antibody.

Here we introduce a modified antibody staining method that uses up to 80% less antibody for flow cytometry. We demonstrate this method for the detection of antigens expressed at high, moderate, or low levels in mouse and rat lymphocytes as well as mouse mammary epithelial cells. We obtained reproducibly accurate results for the detection of up to seven parameters for activation induced-proliferation, cell cycle analysis, and phenotyping of cell-surface and intracellular antigens.

Quantification of epithelial cell differentiation in mammary glands and carcinomas from DMBA- and MNU-exposed rats.

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin ß1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin ß3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.