Adolescent Rat Brain Research Articles

Intermittent ethanol vapor exposure enhances ferroptosis following repeated mild traumatic brain injury in the frontal lobe of adolescent rats; potential role of mitochondrial dysregulation

Adolescents involved in sports have increased risk of traumatic brain injuries (TBI), and frequently participate in binge alcohol consumption in comparison to non-athletic peers. Recovery from a TBI requires substantial energy, and effcient metabolic activity, which depend on intact mitochondria structure and function. Alcohol impairs mitochondrial function, including mitochondrial dynamics and metabolism); that may ultimately lead to cell death. Among cell death pathways, the iron-dependent programmed cell death known as ferroptosis; which has been shown to be predominant in TBI is especially sensitive to changes in mitochondrial energy production. Whether alcohol affects TBI-associated alterations in mitochondrial function and promotes ferroptosis is not known. The aim of this study was to determine how alcohol contributes to repeated-mild TBI (rmTBI)-induced alterations in mitochondrial function (dynamics & metabolism) and ferroptosis signaling in the prefrontal cortex of adolescent male rats. Adolescent male Wistar rats were randomly assigned to i) Sham + Air (control), ii) Sham + ethanol (EtOH), iii) rmTBI + Air, or iv) rmTBI + EtOH. Starting on postnatal day 45, the animals received 3 days of intermittent EtOH vapor exposure (14 hrs on/10 hrs off). Twenty-four hours after the final EtOH exposure, animals were given an mTBI or sham procedure. This sequence was repeated for four cycles. Seven days after the final rmTBI, animals were euthanized, and brains excised for analysis, specifically the frontal lobe. Our preliminary results demonstrate that mitochondrial fusion protein, OPA1, expression was not altered in the PFC by either rmTBI or by EtOH exposure alone. However, rmTBI+EtOH resulted in a significant increase in OPA1 expression. EtOH exposure produced a significant increase in COX-IV (a subunit of ETC complex IV) expression. However, COX-IV expression was not significantly different between rmTBI or rmTBI + EtOH and controls. Mitochondrial complex I activity was not altered by rmTBI, EtOH, or rmTBI + EtOH. Expression of transferrin receptor (TfR1), a specific marker of ferroptosis was significantly increased by rmTBI and EtOH this effect was further exacerbated in the rmTBI + EtOH group. Overall, our study shows that the combination of rmTBI with EtOH exposure affects OPA1-mediated mitochondrial fusion. Additionally, exposure to EtOH increases complex IV expression. Moreover, both rmTBI and EtOH exposure independently triggered signaling pathways associated with ferroptosis, and this effect was synergistically increased when with rmTBI+ EtOH. These findings underscore the intricate interplay between EtOH exposure and rmTBI, influencing crucial mitochondrial proteins and markers of ferroptosis in the frontal lobe of the adolescent rat brain. Further research is needed to fully comprehend the underlying mechanisms and implications of these observations. This research was supported by NIH/NIAAA R01 AA025792 (PEM, NWG) & T32 AA007577 (PEM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dopamine and Norepinephrine Tissue Levels in the Developing Limbic Brain Are Impacted by the Human CHRNA6 3'-UTR Single-Nucleotide Polymorphism (rs2304297) in Rats.

We previously demonstrated that a genetic single-nucleotide polymorphism (SNP, rs2304297) in the 3' untranslated region (UTR) of the human CHRNA6 gene has sex- and genotype-dependent effects on nicotine-induced locomotion, anxiety, and nicotine + cue-induced reinstatement in adolescent rats. This study aims to investigate how the CHRNA6 3'-UTR SNP influences dopaminergic and noradrenergic tissue levels in brain reward regions during baseline and after the reinstatement of drug-seeking behavior. Naïve adolescent and adult rats, along with those undergoing nicotine + cue reinstatement and carrying the CHRNA6 3'-UTR SNP, were assessed for dopamine (DA), norepinephrine (NE), and metabolites in reward pathway regions. The results reveal age-, sex-, and genotype-dependent baseline DA, NE, and DA turnover levels. Post-reinstatement, male α6GG rats show suppressed DA levels in the Nucleus Accumbens (NAc) Shell compared to the baseline, while nicotine+ cue-induced reinstatement behavior correlates with neurotransmitter levels in specific brain regions. This study emphasizes the role of CHRNA6 3'-UTR SNP in the developmental maturation of the dopaminergic and noradrenergic system in the adolescent rat brain, with tissue levels acting as predictors of nicotine + cue-induced reinstatement.

Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency

Aminoacylase 1 (ACY1) deficiency is a rare genetic disorder that affects the breakdown of short-chain aliphatic N-acetylated amino acids, leading to the accumulation of these amino acid derivatives in the urine of patients. Some of the affected individuals have presented with heterogeneous neurological symptoms such as psychomotor delay, seizures, and intellectual disability. Considering that the pathological mechanisms of brain damage in this disorder remain mostly unknown, here we investigated whether major metabolites accumulating in ACY1 deficiency, namely N-acetylglutamate (NAG) and N-acetylmethionine (NAM), could be toxic to the brain by examining their in vitro effects on important mitochondrial properties. We assessed the effects of NAG and NAM on membrane potential, swelling, reducing equivalents, and Ca2+ retention capacity in purified mitochondrial preparations obtained from the brain of adolescent rats. NAG and NAM decreased mitochondrial membrane potential, reducing equivalents, and calcium retention capacity, and induced swelling in Ca2+-loaded brain mitochondria supported by glutamate plus malate. Notably, these changes were completely prevented by the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP and by ruthenium red, implying the participation of MPT and Ca2+ in these effects. Our findings suggest that NAG- and NAM-induced disruption of mitochondrial functions involving MPT may represent relevant mechanisms of neuropathology in ACY1 deficiency.

Efeitos dos exercícios físicos sobre a neurogênese do cérebro de ratos adolescentes

Introduction. The neurogenesis of the brain of young rats can be affected by several factors in their development, with the practice of physical exercises being an important factor in the neurogenesis of nervous tissue. Objective. To investigate the effects of different physical exercise protocols of medium and high intensity on neurogenesis in the brain of adolescent rats. Method. The experimental study, where three groups of animals were used: a control group (RC), a group that performed medium-intensity physical exercise (R1) and a group that performed high-intensity physical exercise (R2). Each group with 8 animals, totaling 24 animals. The progressive resisted physical exercise program on the vertical ladder was carried out for 4 weeks, 5 days a week with a duration varying between 30 and 45 minutes. After the end of the experiments, the animals were euthanized for removal of the brain and subsequent processing, staining using the Nissl method and histomorphometric analysis by counting neurons in the dentate gyrus of the hippocampus and subventricular zone. Results. The data demonstrated that there was no significant difference in the area and height of the apex of the hippocampal dentate gyrus between the groups (p=0.2474 and p=0.3337; respectively). The results of the length of the apex of the hippocampal dentate gyrus showed significant differences (p=0.0172). Conclusion. The practice of medium and high intensity progressive resisted physical exercises did not modify the morphology and neuronal density in the hippocampal dentate gyrus and subventricular zone of young rats.

Neurotoxicity of Chlorpyrifos, Carbofuran, and Cypermethrin in Adolescent Rats’ Brain

Chronic exposure to insecticides, even at low levels, has led to chronic neurotoxicity. Adolescent brain is still undergoing important developments, including in the hippocampus. This study investigated neurotoxicity effects of subacute exposure to chlorpyrifos, carbofuran and cypermethrin in the brain of adolescent rats. This study was performed at the Histology, Biochemistry, and Animal House Laboratory of Jember University from November to December 2021. Subjects were divided into five groups: normal, control, chlorpyrifos, carbofuran, and cypermethrin. Short-term memory was evaluated by Y maze test and tissue damage was evaluated by histological examination. Brain MDA levels were determined by thiobarbituric acid reactive substance method to evaluate the oxidative stress effect on the organ. This study showed spontaneous alternation in the Y maze test and the number of pyramidal neurons in hippocampus decreased in the cypermethrin group. There was a significant elevation of brain MDA level in carbofuran and cypermethrin groups. Thus, cypermethrin causes more severe neurotoxicity than carbofuran and chlorpyrifos. Improvements in the insecticide application management are urgently needed to prevent neurotoxicity.

Tannic Acid and Tea Prevents the Accumulation of Lead and Cadmium in the Lungs, Heart and Brain of Adolescent Male Wistar Rats-Possible Therapeutic Option.

Simple SummaryPolyphenols, including tannic acid, have strong antioxidant properties. They chelate prooxidant toxic metals and are known as pro-oxidant/antioxidant balance agents. Tea aids detoxification of the body by inhibiting absorption and facilitating excretion of toxic metals from the body. Since the tea infusions acted more effectively than the isolated TA, it can be assumed that the protective effect of teas on the organism against toxic metals should be considered in the context of the cumulative effect of various active substances present in the infusions.The protective effect of tannic acid and tea solutions on the lungs, hearts and brains of adolescent Wistar rats exposed to Pb and Cd was studied. Metals were administered with feed (7 mg Cd and 50 mg Pb/kg). Two experiments were carried. Experiment 1 aimed to determine the level of tannic acid (TA), most effectively reducing the adverse impact of Pb and Cd on the organs of adolescent rats (aged 5 weeks, weighing 169.3 ± 14.7 g) during combined exposure. TA was administered with drink (0, 0.5, 1, 1.5, 2 or 2.5% solutions). In Experiment 2, adolescent rats (aged 6 weeks, weighing 210.6 ± 12.1 g) received an aqueous solutions of black, green, red or white teas. TA and teas had a positive effect on reducing the accumulation of Cd in the organs. The results obtained suggest that long-term continuing administration of TA increases its effectiveness as a chelator for Pb. A 2% TA and white tea solution proved to be the most effective. In the analyzed tissues, increased activity of SOD and CAT was recorded as a result of the use of the TA and teas; thus, they can efficiently prevent the prooxidant effect of toxic metals.

Alcohol binge drinking in adolescence and psychological profile: Can the preclinical model crack the chicken-or-egg question?

During adolescence, internal and external factors contribute to engaging with alcohol binge drinking (ABD), putting at risk the neurodevelopment of brain regions crucial for emotional control and stress coping. This research assessed the prevalence of ABD in late adolescent students of Southern Italy and characterized their psychological profile and drinking motives. Translational effects of alcohol binge drinking in the animal model were also studied. Seven hundred and fifty-nine high school students of both sexes (aged 18–20) were recruited. Alcohol Use Disorder Identification Test-Consumption (AUDIT-C), Drinking Motives Questionnaire-Revised Short Form, Millon Clinical Multiaxial Inventory-Third Ed., State-Trait Anxiety Inventory, Connor-Davidson Resilience Scale, and Basic Self-Esteem Scale identified alcohol habits, drinking motives, and psychopathological profile. Eighty-five percentage of the students drank alcohol and 28% of them engaged in ABD; AUDIT-C correlated with enhancement, coping, and conformity motives. ABD was related to a greater likelihood of presenting clinical syndromes and personality disorders, as well as low resilience and self-esteem. Thereafter, in the pre-clinical model, adolescent male rats were exposed to alcohol (3.5 g/kg) in an intermittent binge-like paradigm and tested during prolonged abstinence. Rats were evaluated for anxiety-like behavior, motivated behaviors, resilience, and stress response following a psychosocial challenge. Binge-like alcohol-exposed adolescent rats displayed high integrated z-score for social- and novelty-induced anxiety, altered motivation-driven output, decreased resilience, and a blunted HPA axis response to psychosocial stress, with respect to respective controls. Our data confirm that ABD is the chosen pattern of drinking in a significant percentage of high school students in Southern Italy, and highlights AUDIT-C score as a relevant parameter able to predict the occurrence of affective disturbances. The evidence from the preclinical model shows that ABD produces detrimental consequences in the adolescent rat brain, resulting in negative affect, emotional dysregulation, and aberrant stress response, pointing to decreasing excessive alcohol drinking as a primary goal for the global act for brain health.

P.0418 Neonatal dexamethasone prevents an increase of pro-inflammatory cytokines expression after stress in the brains of adolescent and young adult rats

P.0418 Neonatal dexamethasone prevents an increase of pro-inflammatory cytokines expression after stress in the brains of adolescent and young adult rats

Chronic developmental lead exposure increases μ-opiate receptor levels in the adolescent rat brain

Childhood lead (Pb2+) intoxication is a global public health problem best known for producing deficits in learning and poor school performance. Human and preclinical studies have suggested an association between childhood Pb2+ intoxication and proclivity to substance abuse and delinquent behavior. While environmental factors have been implicated in opioid addiction, less is known about the role of exposure to environmental pollutants on the brain opioid system.Opioid receptors are involved in the biological effects of opioids and other drugs of abuse. In this study, we examine the effect of chronic developmental Pb2+ exposure (1500 ppm in the diet) on μ-opioid receptor (MOR) levels in the rat brain using [3H]-d-Ala2-MePhe4-Gly-ol5 enkephalin ([3H]-DAMGO) quantitative receptor autoradiography at different developmental stages (juvenile, early-adolescent, late adolescent and adult) in male and female rats.Our results indicate that chronic developmental Pb2+ exposure increases the levels of [3H]-DAMGO specific binding to MOR in juvenile and early adolescent Pb2+-exposed male and female rat brain with no changes in late-adolescent (PN50) and minor changes in Pb2+-exposed adult male rats (PN120). Specifically, at PN14, Pb2+-exposed males had an increase in MOR binding in the lateral posthalamic nuclei (LPTN), and Pb2+-exposed females had increased MOR binding in LPTN, medial thalamus, and hypothalamus. At PN28, Pb2+-exposed males had increased MOR levels in the striatum, stria medullaris of the thalamus, LPTN, medial thalamus, and basolateral amygdala, while Pb2+-exposed females showed an increase in nucleus accumbens core, LPTN, and medial thalamus. No changes were detected in any brain region of male and female rats at PN50, and at PN120 there was a decrease in MOR binding of Pb2+-exposed males in the medial thalamus.Our findings demonstrate age and gender specific effects of MOR levels in the rat brain as a result of chronic developmental Pb2+ exposure. These results indicate that the major changes in brain MOR levels were during pre-adolescence and early adolescence, a developmental period in which there is higher engagement in reward and drug-seeking behaviors in humans.In summary, we show that chronic exposure to Pb2+, an ubiquitous and well-known environmental contaminant and neurotoxicant, alters MOR levels in brain regions associated with addiction circuits in the adolescent period, these findings have important implications for opioid drug use and abuse.

Nicotine Exposure Along with Oral Contraceptive Treatment in Female Rats Exacerbates Post-cerebral Ischemic Hypoperfusion Potentially via Altered Histamine Metabolism.

Smoking-derived nicotine (N) and oral contraceptives (OCs) synergistically exacerbate ischemic brain damage in the female, and the underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial electron transport chain function. Because mitochondria play an important role in cellular metabolism, we investigated the global metabolomic profile of brains of adolescent and adult female Sprague-Dawley rats exposed to N with or without OC (N+/-OC). Rats were randomly exposed to saline or N+/-OC for 16-21days followed by random allocation into two cohorts. The first cohort was used to characterize the cortical metabolome. Pathway enrichment analysis showed a significant increase in several histamine metabolites including 1-methylhistamine, 1-methyl-4-imidazoleacetate, and 1-ribosyl-imidazleacetate, along with carnosine and homocarnosine in adolescent and adult animals treated with N and N+OC in relation to respective saline controls, which may be reflective of altered histamine metabolism with nicotine treatment. We also observed reduced levels of the neurotransmitters N-acetyl-aspartyl-glutamate (NAAG), gamma-aminobutyrate (GABA), and N-methyl-GABA in N+OC treatment in adolescent animals. The second cohort underwent bilateral carotid artery occlusion and hypotension followed by cerebral blood flow (CBF) assessment a day later. Autoradiographic images of the brain 24h after ischemic episodes showed severe reduction in cortical and hippocampal local CBF in N+/-OC-exposed rats compared with saline treated. Because GABA and histamine are critical for CBF maintenance, altered metabolism of these neurotransmitters may be responsible for observed severe post-ischemic hypoperfusion, which in turn exacerbates ischemic brain damage.

Simultaneous nicotine and oral contraceptive exposure alters brain energy metabolism and exacerbates ischemic stroke injury in female rats

Smoking-derived nicotine (N) and oral contraceptives (OC) synergistically exacerbate ischemic brain damage in the females and underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial function owing to a defect in the activity of cytochrome c oxidase. Here, we investigated the global metabolomic profile of brains of adolescent female Sprague-Dawley rats exposed to N ± OC. Rats were randomly exposed to saline or N + /−OC for 16–21 days followed by random allocation into two cohorts. One cohort underwent transient middle cerebral artery occlusion and histopathology was performed 30 days later. From the second cohort, cortical tissues were collected for an unbiased global metabolomic profile. Pathway enrichment analysis showed significant decrease in glucose, glucose 6-phosphate and fructose-6-phosphate, along with a significant increase in pyruvate in the N + /−OC exposed groups when compared to saline (p < 0.05), suggesting alterations in the glycolytic pathway which were confirmed by Western blot analyses of glycolytic enzymes. Infarct volume quantification showed a significant increase following N alone or N + OC as compared to saline control. Because glucose metabolism is critical for brain physiology, altered glycolysis deteriorates neural function, thus exacerbating ischemic brain damage.

Acute Dilation of Venous Sinuses in Animal Models of Mild Traumatic Brain Injury Detected Using 9.4T MRI.

Mild traumatic brain injury (mTBI) is a debilitating but extremely common form of brain injury that affects a substantial number of people each year. mTBI is especially common in children and adolescents. Our understanding of mTBI pathophysiology is limited, and there is currently no accepted marker for disease severity. A potential marker for disease severity may be cerebrovascular dysfunction. Recent findings have implicated cerebrovascular alteration as an important component of mTBI and suggest it contributes to the development of persistent, long-term symptoms. In this paper, we conducted two studies to investigate whether mTBI affects venous drainage patterns in the central nervous system using alterations in the size of venous sinuses as a marker of changes in drainage. Using a closed head vertical weight-drop model and a lateral impact injury model of mTBI, we imaged and quantified the size of three major draining vessels in the adolescent rat brain using 9.4T MRI. Areas and volumes were quantified in the superior sagittal sinus and left and right transverse sinuses using images acquired from T2w MRI in one study and post-gadolinium T1w MRI in another. Our results indicated that the three venous sinuses were significantly larger in mTBI rats as compared to sham rats 1-day post injury but recovered to normal size 2 weeks after. Acutely enlarged sinuses post-mTBI may indicate abnormal venous drainage, and this could be suggestive of a cerebrovascular response to trauma.

The implications of hippocampal neurogenesis in adolescent rats after status epilepticus: a novel role of notch signaling pathway in regulating epileptogenesis.

Seizure-induced neurogenesis has a widely recognized pro-epileptogenic function. Given the critical role of Notch signaling during the maintenance and neurogenesis of neural stem cells, we hypothesized that Notch may affect epileptogenesis and its progression through its role in neurogenesis in the adolescent rat brain. We used the lithium-pilocarpine-induced epilepsy model in adolescent Sprague-Dawley rats in order to evaluate hippocampal neurogenesis and epileptogenesis following the onset of status epilepticus (SE). We used western blotting analyses and qPCR to measure levels of Notch signaling at different phases after seizures and immunofluorescence to detect the proliferation and differentiation of neural stem cells after seizure. Following the administration of DAPT, a Notch γ-secretase inhibitor, into the lateral ventricles, we observed a suppression of abnormal neurogenesis in the acute phase and a reduction of gliosis in the chronic phase after SE. Accordingly, the frequency and duration of spontaneous seizures in chronic phase were decreased. Our results clarify the basic concept regarding the involvement of Notch signaling in the regulation of hippocampal neurogenesis and epileptogenesis, thereby potentially offering a novel and alternative treatment for epilepsy.

Free Radical Scavengers Prevent Argininosuccinic Acid-Induced Oxidative Stress in the Brain of Developing Rats: a New Adjuvant Therapy for Argininosuccinate Lyase Deficiency?

Tissue accumulation and high urinary excretion of argininosuccinate (ASA) is the biochemical hallmark of argininosuccinate lyase deficiency (ASLD), a urea cycle disorder mainly characterized by neurologic abnormalities, whose pathogenesis is still unknown. Thus, in the present work, we evaluated the in vitro and in vivo effects of ASA on a large spectrum of oxidative stress parameters in brain of adolescent rats in order to test whether disruption of redox homeostasis could be involved in neurodegeneration of this disorder. ASA provoked in vitro lipid and protein oxidation, decreased reduced glutathione (GSH) concentrations, and increased reactive oxygen species generation in cerebral cortex and striatum. Furthermore, these effects were totally prevented or attenuated by the antioxidants melatonin and GSH. Similar results were obtained by intrastriatal administration of ASA, in addition to increased reactive nitrogen species generation and decreased activities of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. It was also observed that melatonin and N-acetylcysteine prevented most of ASA-induced in vivo pro-oxidant effects in striatum. Taken together, these data indicate that disturbance of redox homeostasis induced at least in part by high brain ASA concentrations per se may potentially represent an important pathomechanism of neurodegeneration in patients with ASLD and that therapeutic trials with appropriate antioxidants may be an adjuvant treatment for these patients.

Physiologically Oriented Interactions of Nutrients and Toxicants (POINT) Model of Mercury-Selenium Interactions During Fetal Development (FS08-08-19)

ObjectivesThis study used POINT models to assess the effects of differing levels of MeHg exposures on selenium (Se) distributions in adolescent rat brain tissues and the effects of maternal rat MeHg exposures on Se distribution and functions in their brains and those of their offspring as a means of predicting interactive effects of MeHg and Se in human epidemiological studies. The effects of dietary MeHg and Se intakes on activities of thioredoxin reductase (TXNRD) and glutathione peroxidase (GPX1) in brain tissues in adult and fetal rats are known to be indicative of Hg-dependent disruptions of Se homeostasis and physiology. MethodsUsing the previously developed Physiologically Oriented Interactions of Nutrients and Toxicants (POINT) model, the toxicokinetic and toxicodynamics of MeHg that had previously been observed in laboratory rat feeding studies were reanalyzed to create a predictive model of how dietary MeHg exposures accumulate and distribute into adult as well as maternal/fetal tissues and use inhibition of selenoenzyme activities to indirectly assess Se availability. ResultsApplying retention coefficients to predict tissue Hg and Se concentrations provided toxicokinetic and toxicodymanic influences of MeHg on Se distributions and functions as well as Se effects on MeHg distributions. In adolescent rats, brain Se was significantly related to dietary Se (F = 42.0; P < 0.0001) and but not to MeHg in the diet. Brain Hg was strongly related to dietary MeHg (F = 248.9; P < 0.0001), but not dietary Se. Brain Se availability was strongly related to dietary MeHg (F = 111.6; P < 0.0001), but not to diet Se. Brain GPx activity was significantly associated with brain Se availability (F = 13.8; P < 0.001) and brain Se contents (F = 7.4; P = 0.01), but not to brain Hg. Brain TRx activity was significantly associated with brain Se availability (F = 48.2; P < 0.00001) and brain Hg contents (F = 25.8; P < 0.0001). Fetal brain was particularly sensitive to maternal MeHg and Se intakes, providing a highly consistent relationship between calculated [HgSe] and [Hg][Se] (F = 652; P < 0.000001). ConclusionsPOINT Models provide reliable predictions of tissue Hg and Se relationships that reflect associated risks vs. benefits Funding SourcesThis work was funded by the United States Environmental Protection Agency (U.S. E.P.A.) and the National Oceanic and Atmospheric Administration (NOAA).

Abstract TP124: Nicotine Alters Brain Energy Metabolism and Exacerbates Ischemic Brain Damage

Background: Smoking-derived nicotine (N) and oral contraceptives (OC) are known to synergistically magnify the risk and severity of cerebral ischemia in females. The underlying pathological mechanism remains elusive. Our studies have shown that N toxicity is exacerbated by OC via altered mitochondrial function, which involved a defect in activity of cytochrome c oxidase, the terminal enzyme of the electron transport chain. However, the effects of impaired mitochondrial function on brain metabolism remain to be investigated. To understand the impact in brain metabolisms, in the current study we investigated the global metabolomic profile of brains of adolescent and adult female rats exposed to N +/- OC. Methods: Six and twelve weeks old Sprague-Dawley female rats were randomly (n = 8/group) exposed to either saline, N (4.5 mg/kg) +/- OC for 16-21 days. At the end of the treatment, brain tissue was harvested for metabolomic analysis (performed by Metabolon Inc.) The metabolomic profile was complemented with western blot analysis and enzyme activity measurements. Results: Pathway enrichment analysis showed significant changes in energy metabolism (glycolysis and TCA cycle) and neurotransmitters in both adolescent and adult rats exposed to N, OC and N+OC in relation to saline treatment. The changes were more pronounced in adolescent rats with a significant decrease in glucose, glucose 6-phosphate, fructose-6-phosphate along with a significant increase in pyruvate in N and N+OC exposed groups when compared to saline (p&lt;0.05), suggesting alterations in the glycolytic pathway. Western blot analyses of glycolytic enzymes support the observed metabolic changes. Conclusion: Nicotine and N+OC exposure increased brain glycolysis in an age-dependent manner. Since glucose metabolism is critical for brain physiology, altered glycolysis deteriorates neural function thus exacerbating ischemic brain damage. Moreover, significant decrease in the neuroactive peptide GABA was observed in young female rats treated with N+OC when compared to saline group. Discerning the exact effects of N +/- OC on overall brain metabolism and the molecular mechanisms affecting mitochondrial function at different ages will open a new window for future therapeutic intervention.

Imidacloprid Impacts on Neurobehavioral Performance, Oxidative Stress, and Apoptotic Events in the Brain of Adolescent and Adult Rats.

Currently, imidacloprid (IMI) is the first insecticide and the second agrochemical highly applied all over the world. Here, we report on the impacts of IMI on neurobehavioral performance, oxidative stress, and apoptotic changes in the brain in either adult or adolescent rats. Forty male rats (adult and adolescent) were allocated to four groups. IMI groups were orally given 1 mg IMI/kg b.wt. dissolved in corn oil, whereas the controls were orally administered corn oil daily for 60 days. The obtained results demonstrated that IMI exposure resulted in less exploratory activity, deficit sensorimotor functions, and high depression. Levels of neurotransmitter including serotonin, gamma-aminobutyric acid, and dopamine were significantly reduced. Oxidative damage of brain tissues was evident following IMI exposure represented by the high levels of protein carbonyl, 8-hydroxyguanosine, and malondialdehyde, but total antioxidant capacity was reduced. Histopathological investigations of the brain tissues of IMI treated group revealed varying degrees of degeneration of the neuron. The immunohistochemical evaluation revealed a strong presence of glial fibrillary acidic protein (GFAP) and Bax positive cells, but a low expression of Bcl-2. These injurious impacts of IMI were very prominent in the adult rats than in the adolescent rats. Conclusively, exposure to IMI even at very low concentration could induce multiple neurobehavioral aberrations and neurotoxic impacts, especially in adults.

In Vitro Neurochemical Assessment of Methylphenidate and Its "Legal High" Analogs 3,4-CTMP and Ethylphenidate in Rat Nucleus Accumbens and Bed Nucleus of the Stria Terminalis.

3,4-dichloromethylphenidate (3,4-CTMP) and ethylphenidate are new psychoactive substances and analogs of the attention deficit medication methylphenidate. Both drugs have been reported on online user fora to induce effects similar to cocaine. In the UK, 3,4-CTMP appeared on the drug market in 2013 and ethylphenidate has been sold since 2010. We aimed to explore the neurochemical effects of these drugs on brain dopamine and noradrenaline efflux. 3,4-CTMP and ethylphenidate, purchased from online vendors, were analyzed using gas chromatography and mass spectroscopy to confirm their identity. Drugs were then tested in adolescent male rat brain slices of the nucleus accumbens and stria terminalis for effects on dopamine and noradrenaline efflux respectively. Fast cyclic voltammetry was used to measure transmitter release. Methylphenidate (10 μM) increased evoked dopamine and noradrenaline efflux by 4- and 2-fold, respectively. 3,4-CTMP (0.1 and 1 μM) increased evoked dopamine and noradrenaline efflux by ~6-fold and 2-fold, respectively. Ethylphenidate (1 μM) doubled evoked dopamine and noradrenaline efflux in both cases. 3,4-CTMP's effect on dopamine efflux was greater than that of methylphenidate, but ethylphenidate appears to be a weaker dopamine transporter inhibitor. Experiments using the dopamine D2 antagonist haloperidol, the noradrenaline α2 receptor antagonist yohimbine, the dopamine transporter inhibitor GBR12909 and the noradrenaline transporter inhibitor desipramine confirmed that we were measuring dopamine in the accumbens and noradrenaline in the ventral BNST. All three psychostimulant drugs, through their effects on dopamine efflux, may have addictive liability although the effect of 3,4-CTMP on dopamine suggests that it might be most addictive and ethylphenidate least addictive.

Chronic Δ⁸-THC Exposure Differently Affects Histone Modifications in the Adolescent and Adult Rat Brain.

Adolescence represents a vulnerable period for the psychiatric consequences of delta9-tetrahydrocannabinol (Δ9-THC) exposure, however, the molecular underpinnings of this vulnerability remain to be established. Histone modifications are emerging as important epigenetic mechanisms involved in the etiopathogenesis of psychiatric diseases, thus, we investigated the impact of chronic Δ9-THC exposure on histone modifications in different brain areas of female rats. We checked histone modifications associated to both transcriptional repression (H3K9 di- and tri-methylation, H3K27 tri-methylation) and activation (H3K9 and H3K14 acetylation) after adolescent and adult chronic Δ9-THC exposure in the hippocampus, nucleus accumbens, and amygdala. Chronic exposure to increasing doses of Δ9-THC for 11 days affected histone modifications in a region- and age-specific manner. The primary effect in the adolescent brain was represented by changes leading to transcriptional repression, whereas the one observed after adult treatment led to transcriptional activation. Moreover, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the Δ9-THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to Δ9-THC adverse effects.

Influence of Pre-reproductive Maternal Enrichment on Coping Response to Stress and Expression of c-Fos and Glucocorticoid Receptors in Adolescent Offspring

Environmental enrichment (EE) is an experimental setting broadly used for investigating the effects of complex social, cognitive, and sensorimotor stimulations on brain structure and function. Recent studies point out that parental EE experience, even occurring in the pre-reproductive phase, affects neural development and behavioral trajectories of the offspring. In the present study we investigated the influences of pre-reproductive EE of female rats on maternal behavior and adolescent male offspring's coping response to an inescapable stressful situation after chronic social isolation. For this purpose female Wistar rats were housed from weaning to breeding age in enriched or standard environments. Subsequently, all females were mated and housed in standard conditions until offspring weaning. On the first post partum day (ppd 1), mother-pup interactions in undisturbed conditions were recorded. Further, after weaning the male pups were reared for 2 weeks under social isolation or in standard conditions, and then submitted or not to a single-session Forced Swim Test (FST). Offspring's neuronal activation and plastic changes were identified by immunohistochemistry for c-Fos and glucocorticoid receptors (GRs), and assessed by using stereological analysis. The biochemical correlates were measured in the hippocampus, amygdala and cingulate cortex, structures involved in hypothalamic-pituitary-adrenocortical axis regulation. Enriched dams exhibited increased Crouching levels in comparison to standard reared dams. In the offspring of both kinds of dams, social isolation reduced body weight, decreased Immobility, and increased Swimming during FST. Moreover, isolated offspring of enriched dams exhibited higher levels of Climbing in comparison to controls. Interestingly, in the amygdala of both isolated and control offspring of enriched dams we found a lower number of c-Fos immunopositive cells in response to FST and a higher number of GRs in comparison to the offspring of standard dams. These results highlight the profound influence of a stressful condition, such as the social isolation, on the brain of adolescent rats, and underline intergenerational effects of maternal experiences in regulating the offspring response to stress.