Effects of voluntary adolescent intermittent alcohol exposure and social isolation on adult alcohol intake in male rats.

Initiating alcohol use in adolescence significantly increases the likelihood of developing adult alcohol use disorder (AUD). However, it has been difficult to replicate adolescent alcohol exposure leading to increased adult alcohol intake across differing preclinical models. In the present study, differentially housed male rats (group vs. single cages) were used to determine the effects of voluntary intermittent exposure of saccharin-sweetened ethanol during adolescence on adult intake of unsweetened 20% ethanol. Adolescent male rats were assigned to group- or isolated-housing conditions and underwent an intermittent 2-bottle choice in adolescence (water only or water vs. 0.2% saccharin/20% ethanol), and again in adulthood (water vs. 20% ethanol). Intermittent 2-bottle choice sessions lasted for 24 h, and occurred three days per week, for five weeks. Rats were moved from group or isolated housing to single-housing cages for 2-bottle choice tests and returned to their original housing condition on off days. During adolescence, rats raised in isolated-housing conditions consumed significantly more sweetened ethanol than rats raised in group-housing conditions, an effect that was enhanced across repeated exposures. In adulthood, rats raised in isolated-housing conditions and exposed to sweetened ethanol during adolescence also consumed significantly higher levels of unsweetened 20% ethanol compared to group-housed rats. The effect was most pronounced over the first five re-exposure sessions. Housing conditions alone had little effect on adult ethanol intake. These preclinical results suggest that social isolation stress, combined with adolescent ethanol exposure, may play a key role in adult AUD risk.

Effects of adolescent alcohol exposure via oral gavage on adult alcohol drinking and co-use of alcohol and nicotine in Sprague Dawley rats.

BACKGROUND: Preclinical models simulating adolescent substance use leading to increased vulnerability for substance use disorders in adulthood are needed. Here, we utilized a model of alcohol and nicotine co-use to assess adult addiction vulnerability following adolescent alcohol exposure. METHODS: In Experiment 1, adolescent (PND30) male and female Sprague-Dawley rats received 25% ethanol (EtOH) or a control solution via oral gavage every 8 h, for 2 days. In young adulthood, animals were tested with a 2-bottle choice between H20% and 15% EtOH or 0.2% saccharin/15% EtOH, followed by co-use of oral Sacc/EtOH and operant-based i.v. nicotine (0.03 mg/kg/infusion) self-administration. In Experiment 2, adolescents received control gavage, EtOH gavage, or no-gavage, and were tested in young adulthood in a 2-bottle choice between H20% and 15% EtOH, Sacc/EtOH, or 0.2% saccharin. RESULTS: In Experiment 1, the adolescent EtOH gavage reduced adult EtOH consumption in the 2-bottle choice, but not during the co-use phase. During co-use, Sacc/EtOH served as an economic substitute for nicotine. In Experiment 2, the control gavage increased adult EtOH drinking relative to the no-gavage control group, an effect that was mitigated in the EtOH gavage group. In both experiments, treatment group differences in EtOH consumption were largely driven by males. CONCLUSIONS: EtOH administration via oral gavage in adolescence decreased EtOH consumption in adulthood without affecting EtOH and nicotine co-use. Inclusion of a no-gavage control in Experiment 2 revealed that the gavage procedure increased adult EtOH intake and that including EtOH in the gavage buffered against the effect.

Primed for addiction: A critical review of the role of microglia in the neurodevelopmental consequences of adolescent alcohol drinking.

Alcohol is one of the most widely used recreational substances worldwide, with drinking frequently initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiating alcohol use, often in high doses, and particularly susceptible to alcohol-induced brain changes. Microglia, the brain parenchymal macrophages, have been implicated in mediating some of these effects, though the role that these cells play in the progression from alcohol drinking to dependence remains unclear. Microglia are uniquely positioned to sense and respond to central nervous system insult, and are now understood to exhibit innate immune memory, or "priming," altering their future functional responses based on prior exposures. In alcohol use disorders (AUDs), the role of microglia is debated. Whereas microglial activation can be pathogenic, contributing to neuroinflammation, tissue damage, and behavioral changes, or protective, it can also engage protective functions, providing support and mediating the resolution of damage. Understanding the role of microglia in adolescent AUDs is complicated by the fact that microglia are thought to be involved in developmental processes such as synaptic refinement and myelination, which underlie the functional maturation of multiple brain systems in adolescence. Thus, the role microglia play in the impact of alcohol use in adolescence is likely multifaceted. Long-term sequelae may be due to a failure to recover from EtOH-induced tissue damage, altered neurodevelopmental trajectories, and/or persistent changes to microglial responsivity and function. Here, we review critically the literature surrounding the effects of alcohol on microglia in models of adolescent alcohol misuse. We attempt to disentangle what is known about microglia from other neuroimmune effectors, to which we apply recent discoveries on the role of microglia in development and plasticity. Considered altogether, these studies challenge assumptions that proinflammatory microglia drive addiction. Alcohol priming microglia and thereby perturbing their homeostatic roles in neurodevelopment, especially during critical periods of plasticity such as adolescence, may have more serious implications for the neuropathogenesis of AUDs in adolescents.

Functional Activation of Newborn Neurons Following Alcohol-Induced Reactive Neurogenesis.

Abstinence after alcohol dependence leads to structural and functional recovery in many regions of the brain, especially the hippocampus. Significant increases in neural stem cell (NSC) proliferation and subsequent "reactive neurogenesis" coincides with structural recovery in hippocampal dentate gyrus (DG). However, whether these reactively born neurons are integrated appropriately into neural circuits remains unknown. Therefore, adult male rats were exposed to a binge model of alcohol dependence. On day 7 of abstinence, the peak of reactive NSC proliferation, rats were injected with bromodeoxyuridine (BrdU) to label dividing cells. After six weeks, rats underwent Morris Water Maze (MWM) training then were sacrificed ninety minutes after the final training session. Using fluorescent immunohistochemistry for c-Fos (neuronal activation), BrdU, and Neuronal Nuclei (NeuN), we investigated whether neurons born during reactive neurogenesis were incorporated into a newly learned MWM neuronal ensemble. Prior alcohol exposure increased the number of BrdU+ cells and newborn neurons (BrdU+/NeuN+ cells) in the DG versus controls. However, prior ethanol exposure had no significant impact on MWM-induced c-Fos expression. Despite increased BrdU+ neurons, no difference in the number of activated newborn neurons (BrdU+/c-Fos+/NeuN+) was observed. These data suggest that neurons born during alcohol-induced reactive neurogenesis are functionally integrated into hippocampal circuitry.

Docosahexaenoic acid decreased inflammatory gene expression, but not 18-kDa translocator protein binding, in rat pup brain after controlled cortical impact.

BACKGROUND: Traumatic brain injury is the leading cause of acquired neurologic disability in children. In our model of pediatric traumatic brain injury, controlled cortical impact (CCI) in rat pups, docosahexaenoic acid (DHA) improved lesion volume and cognitive testing as late as postinjury day (PID) 50. Docosahexaenoic acid decreased proinflammatory messenger RNA (mRNA) in microglia and macrophages at PIDs 3 and 7, but not 30. We hypothesized that DHA affected inflammatory markers differentially relative to impact proximity, early and persistently after CCI. METHODS: To provide a temporal snapshot of regional neuroinflammation, we measured 18-kDa translocator protein (TSPO) binding using whole brain autoradiography at PIDs 3, 7, 30, and 50. Guided by TSPO results, we measured mRNA levels in contused cortex and underlying hippocampus for genes associated with proinflammatory and inflammation-resolving states at PIDs 2 and 3. RESULTS: Controlled cortical impact increased TSPO binding at all time points, most markedly at PID 3 and in regions closest to impact, not blunted by DHA. Controlled cortical impact increased cortical and hippocampal mRNA proinflammatory markers, blunted by DHA at PID 2 in hippocampus. CONCLUSION: Controlled cortical impact increased TSPO binding in the immature brain in a persistent manner more intensely with more severe injury, not altered by DHA. Controlled cortical impact increased PIDs 2 and 3 mRNA levels of proinflammatory and inflammation-resolving genes. Docosahexaenoic acid decreased proinflammatory markers associated with inflammasome activation at PID 2. We speculate that DHA's salutary effects on long-term outcomes result from early effects on the inflammasome. Future studies will examine functional effects of DHA on microglia both early and late after CCI.

Translational Research in Nicotine Addiction.

While commendable strides have been made in reducing smoking initiation and improving smoking cessation rates, current available smoking cessation treatment options are still only mildly efficacious and show substantial interindividual variability in their therapeutic responses. Therefore, the primary goal of preclinical research has been to further the understanding of the neural substrates and genetic influences involved in nicotine's effects and reassess potential drug targets. Pronounced advances have been made by investing in new translational approaches and placing more emphasis on bridging the gap between human and rodent models of dependence. Functional neuroimaging studies have identified key brain structures involved with nicotine-dependence phenotypes such as craving, impulsivity, withdrawal symptoms, and smoking cessation outcomes. Following up with these findings, rodent-modeling techniques have made it possible to dissect the neural circuits involved in these motivated behaviors and ascertain mechanisms underlying nicotine's interactive effects on brain structure and function. Likewise, translational studies investigating single-nucleotide polymorphisms (SNPs) within the cholinergic, dopaminergic, and opioid systems have found high levels of involvement of these neurotransmitter systems in regulating the reinforcing aspects of nicotine in both humans and mouse models. These findings and coordinated efforts between human and rodent studies pave the way for future work determining gene by drug interactions and tailoring treatment options to each individual smoker.

Chronic risperidone administration leads to greater amphetamine-induced conditioned place preference.

Risperidone is an atypical antipsychotic drug used increasingly in children to manage symptoms of ADHD and conduct disorder. In rats, developmental risperidone administration is accompanied by increased locomotor activity during adulthood, as well as heightened sensitivity to the locomotor stimulating effects of amphetamine. This study compared sensitivity to the rewarding effects of amphetamine, as measured by conditioned place preference (CPP), between groups of rats administered chronic risperidone (3.0 mg/kg, s.c.) during development (postnatal days 14-42) or adulthood (postnatal days 77-105). Locomotor activity in a novel test cage and amphetamine-induced CPP were measured beginning three and four weeks, respectively, after the final risperidone injection. Female rats administered risperidone early in life were more active than any other group tested. Previous risperidone administration enhanced amphetamine CPP regardless of sex, and this effect appeared more prominent in the developmentally treated group. The density of forebrain dopamine transporters, a primary target of amphetamine, was also quantified in rats administered risperidone early in life and found to be reduced in the medial anterior, posterior, and ventral caudate nucleus. These results suggest that chronic risperidone treatment modifies later locomotor activity and sensitivity to the reinforcing effects of amphetamine, perhaps via a mechanism related to decreased forebrain dopamine transporter density.

Recovery of Hippocampal-Dependent Learning Despite Blunting Reactive Adult Neurogenesis After Alcohol Dependence.

BACKGROUND: The excessive alcohol drinking that occurs in alcohol use disorder (AUD) causes neurodegeneration in regions such as the hippocampus, though recovery may occur after a period of abstinence. Mechanisms of recovery are not clear, though reactive neurogenesis has been observed in the hippocampal dentate gyrus following alcohol dependence and correlates to recovery of granule cell number. OBJECTIVE: We investigated the role of neurons born during reactive neurogenesis in the recovery of hippocampal learning behavior after 4-day binge alcohol exposure, a model of an AUD. We hypothesized that reducing reactive neurogenesis would impair functional recovery. METHODS: Adult male rats were subjected to 4-day binge alcohol exposure and two approaches were tested to blunt reactive adult neurogenesis, acute doses of alcohol or the chemotherapy drug, temozolomide (TMZ). RESULTS: Acute 5 g/kg doses of EtOH gavaged T6 and T7 days post binge did not inhibit significantly the number of Bromodeoxyuridine-positive (BrdU+) proliferating cells in EtOH animals receiving 5 g/kg EtOH versus controls. A single cycle of TMZ inhibited reactive proliferation (BrdU+ cells) and neurogenesis (NeuroD+ cells) to that of controls. However, despite this blunting of reactive neurogenesis to basal levels, EtOH-TMZ rats were not impaired in their recovery of acquisition of the Morris water maze (MWM), learning similarly to all other groups 35 days after 4-day binge exposure. CONCLUSIONS: These studies show that TMZ is effective in decreasing reactive proliferation/neurogenesis following 4-day binge EtOH exposure, and baseline levels of adult neurogenesis are sufficient to allow recovery of hippocampal function.

Long-Term Intranasal Insulin Aspart: A Profile of Gene Expression, Memory, and Insulin Receptors in Aged F344 Rats.

Intranasal insulin is a safe and effective method for ameliorating memory deficits associated with pathological brain aging. However, the impact of different formulations and the duration of treatment on insulin's efficacy and the cellular processes targeted by the treatment remain unclear. Here, we tested whether intranasal insulin aspart, a short-acting insulin formulation, could alleviate memory decline associated with aging and whether long-term treatment affected regulation of insulin receptors and other potential targets. Outcome variables included measures of spatial learning and memory, autoradiography and immunohistochemistry of the insulin receptor, and hippocampal microarray analyses. Aged Fischer 344 rats receiving long-term (3 months) intranasal insulin did not show significant memory enhancement on the Morris water maze task. Autoradiography results showed that long-term treatment reduced insulin binding in the thalamus but not the hippocampus. Results from hippocampal immunofluorescence revealed age-related decreases in insulin immunoreactivity that were partially offset by intranasal administration. Microarray analyses highlighted numerous insulin-sensitive genes, suggesting insulin aspart was able to enter the brain and alter hippocampal RNA expression patterns including those associated with tumor suppression. Our work provides insights into potential mechanisms of intranasal insulin and insulin resistance, and highlights the importance of treatment duration and the brain regions targeted.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease.

Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.

Brain Region Specific Single-Molecule Fluorescence Imaging.

We developed an approach utilizing nanoscale vesicles extracted from brain regions combined with single molecule imaging to monitor how an animal's physiological condition regulates the dynamics of protein distributions in different brain regions. This method was used to determine the effect of nicotine on the distribution of receptor stoichiometry in different mouse brain regions. Nicotine-induced upregulation of α4ß2 nicotinic acetylcholine receptors (nAChRs) is associated with changes in their expression, trafficking, and stoichiometry. The structural assembly of nAChRs has been quantified in cell culture based systems using single molecule techniques. However, these methods are not capable of quantifying biomolecule assembly that takes place in a live animal. Both nicotine-induced upregulation and changes in nAChR stoichiometry differ across brain regions. Our single molecule approach revealed that nicotine acts differentially across brain regions to alter assembly in response to exposure and withdrawal.

Targeting Pathogenic Lafora Bodies in Lafora Disease Using an Antibody-Enzyme Fusion.

Lafora disease (LD) is a fatal childhood epilepsy caused by recessive mutations in either the EPM2A or EPM2B gene. A hallmark of LD is the intracellular accumulation of insoluble polysaccharide deposits known as Lafora bodies (LBs) in the brain and other tissues. In LD mouse models, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Therefore, LBs have become a therapeutic target for ameliorating LD. Herein, we demonstrate that human pancreatic α-amylase degrades LBs. We fused this amylase to a cell-penetrating antibody fragment, and this antibody-enzyme fusion (VAL-0417) degrades LBs in vitro and dramatically reduces LB loads in vivo in Epm2a-/- mice. Using metabolomics and multivariate analysis, we demonstrate that VAL-0417 treatment of Epm2a-/- mice reverses the metabolic phenotype to a wild-type profile. VAL-0417 is a promising drug for the treatment of LD and a putative precision therapy platform for intractable epilepsy.

Binge Alcohol Exposure Transiently Changes the Endocannabinoid System: A Potential Target to Prevent Alcohol-Induced Neurodegeneration.

Excessive alcohol consumption leads to neurodegeneration, which contributes to cognitive decline that is associated with alcohol use disorders (AUDs). The endocannabinoid system has been implicated in the development of AUDs, but little is known about how the neurotoxic effects of alcohol impact the endocannabinoid system. Therefore, the current study investigated the effects of neurotoxic, binge-like alcohol exposure on components of the endocannabinoid system and related N-acylethanolamines (NAEs), and then evaluated the efficacy of fatty acid amide hydrolase (FAAH) inhibition on attenuating alcohol-induced neurodegeneration. Male rats were administered alcohol according to a binge model, which resulted in a transient decrease in [³H]-CP-55,940 binding in the entorhinal cortex and hippocampus following two days, but not four days, of treatment. Furthermore, binge alcohol treatment did not change the tissue content of the three NAEs quantified, including the endocannabinoid and anandamide. In a separate study, the FAAH inhibitor, URB597 was administered to rats during alcohol treatment and neuroprotection was assessed by FluoroJade B (FJB) staining. The administration of URB597 during binge treatment did not significantly reduce FJB+ cells in the entorhinal cortex or hippocampus, however, a follow up "target engagement" study found that NAE augmentation by URB597 was impaired in alcohol intoxicated rats. Thus, potential alcohol induced alterations in URB597 pharmacodynamics may have contributed to the lack of neuroprotection by FAAH inhibition.

The effects of nicotine in the neonatal quinpirole rodent model of psychosis: Neural plasticity mechanisms and nicotinic receptor changes.

Neonatal quinpirole (NQ) treatment to rats increases dopamine D2 receptor sensitivity persistent throughout the animal's lifetime. In Experiment 1, we analyzed the role of α7 and α4ß2 nicotinic receptors (nAChRs) in nicotine behavioral sensitization and on the brain-derived neurotrophic factor (BDNF) response to nicotine in NQ- and neonatally saline (NS)-treated rats. In Experiment 2, we analyzed changes in α7 and α4ß2 nAChR density in the nucleus accumbens (NAcc) and dorsal striatum in NQ and NS animals sensitized to nicotine. Male and female Sprague-Dawley rats were neonatally treated with quinpirole (1mg/kg) or saline from postnatal days (P)1-21. Animals were given ip injections of either saline or nicotine (0.5mg/kg free base) every second day from P33 to P49 and tested on behavioral sensitization. Before each injection, animals were ip administered the α7 nAChR antagonist methyllycaconitine (MLA; 2 or 4mg/kg) or the α4ß2 nAChR antagonist dihydro beta erythroidine (DhßE; 1 or 3mg/kg). Results revealed NQ enhanced nicotine sensitization that was blocked by DhßE. MLA blocked the enhanced nicotine sensitization in NQ animals, but did not block nicotine sensitization. NQ enhanced the NAcc BDNF response to nicotine which was blocked by both antagonists. In Experiment 2, NQ enhanced nicotine sensitization and enhanced α4ß2, but not α7, nAChR upregulation in the NAcc. These results suggest a relationship between accumbal BDNF and α4ß2 nAChRs and their role in the behavioral response to nicotine in the NQ model which has relevance to schizophrenia, a behavioral disorder with high rates of tobacco smoking.

Developmental toxicity of nicotine: A transdisciplinary synthesis and implications for emerging tobacco products.

While the health risks associated with adult cigarette smoking have been well described, effects of nicotine exposure during periods of developmental vulnerability are often overlooked. Using MEDLINE and PubMed literature searches, books, reports and expert opinion, a transdisciplinary group of scientists reviewed human and animal research on the health effects of exposure to nicotine during pregnancy and adolescence. A synthesis of this research supports that nicotine contributes critically to adverse effects of gestational tobacco exposure, including reduced pulmonary function, auditory processing defects, impaired infant cardiorespiratory function, and may contribute to cognitive and behavioral deficits in later life. Nicotine exposure during adolescence is associated with deficits in working memory, attention, and auditory processing, as well as increased impulsivity and anxiety. Finally, recent animal studies suggest that nicotine has a priming effect that increases addiction liability for other drugs. The evidence that nicotine adversely affects fetal and adolescent development is sufficient to warrant public health measures to protect pregnant women, children, and adolescents from nicotine exposure.

Targeting the Cholinergic System for Neuroprotection and/or Enhancement of Functional Recovery Following Neurotrauma.

Development of novel pharmacotherapies for the treatment of traumatic injury to the nervous system has been ongoing for over 40 years. Despite many promising compounds discovered using animal models, no treatments have successfully translated into the clinic. The central dogma in this field is that brain trauma initiates a complex chain of biochemical events leading to secondary brain damage and sustained neurological deficits. The delayed secondary brain injury is likely to result from multiple insults including oxidative stress, mitochondrial dysfunction, breakdown of the blood brain barrier, dysregulated release of glutamate, pro-inflammatory cytokines, and other mediators. However, therapies targeting these systems have generally met with failure in clinical trials. The purpose of this review is to summarize the models used for preclinical neurotrauma research, provide a brief overview of previous failed clinical trials in head and spinal cord injury, and finally, to review involvement of the cholinergic system and discuss implications for future research. Possibilities and pitfalls of targeting the cholinergic system for neuroprotection and/or enhancement of functional recovery are also discussed.

Dietary Docosahexaenoic Acid Improves Cognitive Function, Tissue Sparing, and Magnetic Resonance Imaging Indices of Edema and White Matter Injury in the Immature Rat after Traumatic Brain Injury.

Traumatic brain injury (TBI) is the leading cause of acquired neurologic disability in children. Specific therapies to treat acute TBI are lacking. Cognitive impairment from TBI may be blunted by decreasing inflammation and oxidative damage after injury. Docosahexaenoic acid (DHA) decreases cognitive impairment, oxidative stress, and white matter injury in adult rats after TBI. Effects of DHA on cognitive outcome, oxidative stress, and white matter injury in the developing rat after experimental TBI are unknown. We hypothesized that DHA would decrease early inflammatory markers and oxidative stress, and improve cognitive, imaging and histologic outcomes in rat pups after controlled cortical impact (CCI). CCI or sham surgery was delivered to 17 d old male rat pups exposed to DHA or standard diet for the duration of the experiments. DHA was introduced into the dam diet the day before CCI to allow timely DHA delivery to the pre-weanling pups. Inflammatory cytokines and nitrates/nitrites were measured in the injured brains at post-injury Day (PID) 1 and PID2. Morris water maze (MWM) testing was performed at PID41-PID47. T2-weighted and diffusion tensor imaging studies were obtained at PID12 and PID28. Tissue sparing was calculated histologically at PID3 and PID50. DHA did not adversely affect rat survival or weight gain. DHA acutely decreased oxidative stress and increased anti-inflammatory interleukin 10 in CCI brains. DHA improved MWM performance and lesion volume late after injury. At PID12, DHA decreased T2-imaging measures of cerebral edema and decreased radial diffusivity, an index of white matter injury. DHA improved short- and long-term neurologic outcomes after CCI in the rat pup. Given its favorable safety profile, DHA is a promising candidate therapy for pediatric TBI. Further studies are needed to explore neuroprotective mechanisms of DHA after developmental TBI.

Corticosterone enhances N-methyl-D-aspartate receptor signaling to promote isolated ventral tegmental area activity in a reconstituted mesolimbic dopamine pathway.

Elevations in circulating corticosteroids during periods of stress may influence activity of the mesolimbic dopamine reward pathway by increasing glutamatergic N-methyl-D-aspartate (NMDA) receptor expression and/or function in a glucocorticoid receptor-dependent manner. The current study employed organotypic co-cultures of the ventral tegmental area (VTA) and nucleus accumbens (NAcc) to examine the effects of corticosterone exposure on NMDA receptor-mediated neuronal viability. Co-cultures were pre-exposed to vehicle or corticosterone (CORT; 1 µM) for 5 days prior to a 24 h co-exposure to NMDA (200 µM). Co-cultures pre-exposed to a non-toxic concentration of corticosterone and subsequently NMDA showed significant neurotoxicity in the VTA only. This was evidenced by increases in propidium iodide uptake as well as decreases in immunoreactivity of the neuronal nuclear protein (NeuN). Co-exposure to the NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV; 50 µM) or the glucocorticoid receptor (GR) antagonist mifepristone (10 µM) attenuated neurotoxicity. In contrast, the combination of corticosterone and NMDA did not produce any significant effects on either measure within the NAcc. Cultures of the VTA and NAcc maintained without synaptic contact showed no response to CORT or NMDA. These results demonstrate the ability to functionally reconstitute key regions of the mesolimbic reward pathway ex vivo and to reveal a GR-dependent enhancement of NMDA receptor-dependent signaling in the VTA.

N-acetylcysteine amide confers neuroprotection, improves bioenergetics and behavioral outcome following TBI.

Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.