Nutritional Supplementation Reduces Lesion Size and Neuroinflammation in a Sex-Dependent Manner in a Mouse Model of Perinatal Hypoxic-Ischemic Brain Injury.

Perinatal hypoxia-ischemia (HI) is a major cause of neonatal brain injury, leading to long-term neurological impairments. Medical nutrition can be rapidly implemented in the clinic, making it a viable intervention to improve neurodevelopment after injury. The omega-3 (n-3) fatty acids docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), uridine monophosphate (UMP) and choline have previously been shown in rodents to synergistically enhance brain phospholipids, synaptic components and cognitive performance. The objective of this study was to test the efficacy of an experimental diet containing DHA, EPA, UMP, choline, iodide, zinc, and vitamin B12 in a mouse model of perinatal HI. Male and female C57Bl/6 mice received the experimental diet or an isocaloric control diet from birth. Hypoxic ischemic encephalopathy was induced on postnatal day 9 by ligation of the right common carotid artery and systemic hypoxia. To assess the effects of the experimental diet on long-term motor and cognitive outcome, mice were subjected to a behavioral test battery. Lesion size, neuroinflammation, brain fatty acids and phospholipids were analyzed at 15 weeks after HI. The experimental diet reduced lesion size and neuroinflammation specifically in males. In both sexes, brain n-3 fatty acids were increased after receiving the experimental diet. The experimental diet also improved novel object recognition, but no significant effects on motor performance were observed. Current data indicates that early life nutritional supplementation with a combination of DHA, EPA, UMP, choline, iodide, zinc, and vitamin B12 may provide neuroprotection after perinatal HI.

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU).

INTRODUCTION: In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. MATERIAL & METHODS: 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. RESULTS: In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. CONCLUSION: This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition.

Juvenile antioxidant treatment prevents adult deficits in a developmental model of schizophrenia.

Abnormal development can lead to deficits in adult brain function, a trajectory likely underlying adolescent-onset psychiatric conditions such as schizophrenia. Developmental manipulations yielding adult deficits in rodents provide an opportunity to explore mechanisms involved in a delayed emergence of anomalies driven by developmental alterations. Here we assessed whether oxidative stress during presymptomatic stages causes adult anomalies in rats with a neonatal ventral hippocampal lesion, a developmental rodent model useful for schizophrenia research. Juvenile and adolescent treatment with the antioxidant N-acetyl cysteine prevented the reduction of prefrontal parvalbumin interneuron activity observed in this model, as well as electrophysiological and behavioral deficits relevant to schizophrenia. Adolescent treatment with the glutathione peroxidase mimic ebselen also reversed behavioral deficits in this animal model. These findings suggest that presymptomatic oxidative stress yields abnormal adult brain function in a developmentally compromised brain, and highlight redox modulation as a potential target for early intervention.

Time-dependent decreases in nucleus accumbens AMPA/NMDA ratio and incubation of sucrose craving in adolescent and adult rats.

RATIONALE AND OBJECTIVE: There is evidence that cue-induced sucrose seeking progressively increases after cessation of oral sucrose self-administration (incubation of sucrose craving) in both adolescent and adult rats. The synaptic plasticity changes associated with this incubation at different age groups are unknown. We assessed whether incubation of sucrose craving in rats trained to self-administer sucrose as young adolescents, adolescents, or adults is associated with changes in 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/N-methyl-D-aspartate (NMDA) ratio (a measure of postsynaptic changes in synaptic strength) in nucleus accumbens. METHODS: Three age groups initiated oral sucrose self-administration training (10 days) on postnatal day (P) 35 (young adolescents), P42 (adolescents), or P70 (adults). They were then tested for cue-induced sucrose seeking (assessed in an extinction test) on abstinence days 1 and 21. Separate groups of rats were trained to self-administer sucrose or water (a control condition), and assessed for AMPA/NMDA ratio in nucleus accumbens on abstinence days 1-3 and 21. RESULTS: Adult rats earned more sucrose rewards, but sucrose intake per body weight was higher in young adolescent rats. Time-dependent increases in cue-induced sucrose seeking (incubation of sucrose craving) were more pronounced in adult rats, less pronounced in adolescents, and not detected in young adolescents. On abstinence day 21, but not days 1-3, AMPA/NMDA ratio in nucleus accumbens were decreased in rats that self-administered sucrose as adults and adolescents, but not young adolescents. CONCLUSIONS: Our data demonstrate age-dependent changes in magnitude of incubation of sucrose craving and nucleus accumbens synaptic plasticity after cessation of sucrose self-administration.

Prefrontal cortical kappa-opioid receptor modulation of local neurotransmission and conditioned place aversion.

Kappa-opioid receptors (KORs) are important for motivation and other medial prefrontal cortex (mPFC)-dependent behaviors. Although KORs are present in the mPFC, their role in regulating transmission in this brain region and their contribution to KOR-mediated aversion are not known. Using in vivo microdialysis in rats and mice, we demonstrate that intra-mPFC administration of the selective KOR agonist U69,593 decreased local dopamine (DA) overflow, while reverse dialysis of the KOR antagonist nor-Binaltorphimine (nor-BNI) enhanced mPFC DA overflow. Extracellular amino-acid levels were also affected by KORs, as U69,593 reduced glutamate and GABA levels driven by the glutamate reuptake blocker, l-trans-pyrrolidine-2,4-dicarboxylate. Whole-cell recordings from mPFC layer V pyramidal neurons revealed that U69,593 decreased the frequency, but not amplitude, of glutamatergic mini EPSPs. To determine whether KOR regulation of mPFC DA overflow was mediated by KOR on DA terminals, we utilized a Cre recombinase-driven mouse line lacking KOR in DA neurons. In these mice, basal DA release or uptake was unaltered relative to controls, but attenuation of mPFC DA overflow by local U69,593 was not observed, indicating KOR acts directly on mPFC DA terminals to locally inhibit DA levels. Conditioning procedures were then used to determine whether mPFC KOR signaling was necessary for KOR-mediated aversion. U69,593-mediated conditioned place aversion was blocked by intra-mPFC nor-BNI microinjection. These findings demonstrate that mPFC KORs negatively regulate DA and amino-acid neurotransmission, and are necessary for KOR-mediated aversion.

Early social experience is critical for the development of cognitive control and dopamine modulation of prefrontal cortex function.

Social experiences during youth are thought to be critical for proper social and cognitive development. Conversely, social insults during development can cause long-lasting behavioral impairments and increase the vulnerability for psychopathology later in life. To investigate the importance of social experience during the juvenile and early adolescent stage for the development of cognitive control capacities, rats were socially isolated from postnatal day 21 to 42 followed by re-socialization until they reached adulthood. Subsequently, two behavioral dimensions of impulsivity (impulsive action in the five-choice serial reaction time task (5-CSRTT) and impulsive choice in the delayed reward task) and decision making (in the rat gambling task) were assessed. In a separate group of animals, long-lasting cellular and synaptic changes in adult medial prefrontal cortex (PFC) pyramidal neurons were determined following social isolation. Juvenile and early adolescent social isolation resulted in impairments in impulsive action and decision making under novel or challenging circumstances. Moreover, socially isolated rats had a reduced response to enhancement of dopaminergic neurotransmission (using amphetamine or GBR12909) in the 5-CSRTT under challenging conditions. Impulsive choice was not affected by social isolation. These behavioral deficits were accompanied by a loss of sensitivity to dopamine of pyramidal neurons in the medial PFC. Our data show long-lasting deleterious effects of early social isolation on cognitive control and its neural substrates. Alterations in prefrontal cognitive control mechanisms may contribute to the enhanced risk for psychiatric disorders induced by aberrations in the early social environment.

The Yin and Yang of Nicotine: Harmful during Development, Beneficial in Adult Patient Populations.

Nicotine has remarkably diverse effects on the brain. Being the main active compound in tobacco, nicotine can aversively affect brain development. However, it has the ability to act positively by restoring attentional capabilities in smokers. Here, we focus on nicotine exposure during the prenatal and adolescent developmental periods and specifically, we will review the long-lasting effects of nicotine on attention, both in humans and animal models. We discuss the reciprocal relation of the beneficial effects of nicotine, improving attention in smokers and in patients with neuropsychiatric diseases, such as schizophrenia and attention deficit/hyperactivity disorder, vs. nicotine-related attention deficits already caused during adolescence. Given the need for research on the mechanisms of nicotine's cognitive actions, we discuss some of the recent work performed in animals.

Adolescent nicotine exposure transiently increases high-affinity nicotinic receptors and modulates inhibitory synaptic transmission in rat medial prefrontal cortex.

Adolescence is a critical developmental period during which most adult smokers initiate their habit. Adolescents are more vulnerable than adults to nicotine's long-term effects on addictive and cognitive behavior. We investigated whether adolescent nicotine exposure in rats modifies expression of nicotinic acetylcholine receptors (nAChRs) in medial prefrontal cortex (mPFC) in the short and/or long term, and whether this has functional consequences. Using receptor binding studies followed by immunoprecipitation of nAChR subunits, we showed that adolescent nicotine exposure, as compared with saline, caused an increase in mPFC nAChRs containing α4 or ß2 subunits (24 and 18%, respectively) 24 h after the last injection. Nicotine exposure in adulthood had no such effect. This increase was transient and was not observed 5 wk following either adolescent or adult nicotine exposure. In line with increased nAChRs expression 1 d after adolescent nicotine exposure, we observed a 34% increase in amplitude of nicotine-induced spontaneous inhibitory postsynaptic currents in layer II/III mPFC pyramidal neurons. These effects were transient and specific, and observed only acutely after adolescent nicotine exposure, but not after 5 wk, and no changes were observed in adult-exposed animals. The acute nicotine-induced increase in α4ß2-containing receptors in adolescents interferes with the normal developmental decrease (37%) of these receptors from early adolescence (postnatal day 34) to adulthood (postnatal day 104) in the mPFC. Together, this suggests that these receptors play a role in mediating the acute rewarding effects of nicotine and may underlie the increased sensitivity of adolescents to nicotine.

Lasting synaptic changes underlie attention deficits caused by nicotine exposure during adolescence.

Tobacco smoking and nicotine exposure during adolescence interfere with prefrontal cortex (PFC) development and lead to cognitive impairments in later life. The molecular and cellular underpinnings of these consequences remain elusive. We found that adolescent nicotine exposure induced lasting attentional disturbances and reduced mGluR2 protein and function on presynaptic terminals of PFC glutamatergic synapses. Restoring mGluR2 activity in vivo by local infusion of a group II mGluR agonist in adult rats that received nicotine as adolescents rescued attentional disturbances.

Development of the motivational system during adolescence, and its sensitivity to disruption by nicotine.

The brain continues to develop during adolescence, and exposure to exogenous substances such as nicotine can exert long-lasting adaptations during this vulnerable period. In order to fully understand how nicotine affects the adolescent brain it is important to understand normal adolescent brain development. This review summarizes human and animal data on brain development, with emphasis on the prefrontal cortex, for its important function in executive control over behavior. Moreover, we discuss how nicotine exposure during adolescence can disrupt brain development bearing long-term consequences on executive cognitive function in adulthood.

Changes in molecular composition of rat medial prefrontal cortex synapses during adolescent development.

Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)-based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34-P78), with an over-representation of cell-membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44-P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non-stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.

Dopamine receptor D1/D5 gene expression in the medial prefrontal cortex predicts impulsive choice in rats.

A neuropsychological hallmark of attention deficit/hyperactivity disorder (ADHD) is the reduced ability to tolerate delay of reinforcement, leading to impulsive choice. Genetic association studies have implicated several genes involved in dopaminergic neurotransmission in ADHD. In this study, we investigated whether differences in the expression level of these dopamine-related genes of rats predict the individual level of impulsive choice. Among all frontostriatal brain regions tested, only in the medial prefrontal cortex (mPFC), we observed significant positive correlations between impulsive choice and transcript levels of the dopamine receptor D(1), the dopamine receptor D(5) and calcyon. Local mPFC infusions of the D(1)/D(5) receptor antagonist SCH 23390 and agonist SKF 38393 resulted in increased impulsive choice, in agreement with the idea that endogenous receptor D(1)/D(5) stimulation in the mPFC promotes the choice of large delayed rewards. Together, these data indicate that this class of dopamine receptors in the mPFC plays a pivotal role in impulsive choice, and aberrancies thereof might contribute to ADHD symptomatology.

Long-lasting cognitive deficits resulting from adolescent nicotine exposure in rats.

Adolescence is a developmental period, during which the brain and particularly medial prefrontal cortical (mPFC) regions thereof have not fully matured. Because epidemiological data have suggested that adolescent nicotine use may result in disturbances in cognitive function in adulthood, we investigated the long-term effects of adolescent nicotine exposure in rats. Male Wistar rats were exposed to either nicotine (three times daily, 0.4 mg/kg s.c.) or saline for 10 days during (postnatal day (PND) 34-43) or following (PND 60-69) adolescence. After 5 weeks during adulthood, separate groups of animals were tested in operant paradigms taxing attention and distinct measures of impulsivity. Visuospatial attention and impulsive action were tested in the five-choice serial reaction time task, whereas impulsive choice was assessed in the delayed reward task. Our data show that adolescent, but not postadolescent, nicotine exposure affects cognitive performance in adulthood and results in diminished attentional performance and increments in impulsive action, while leaving impulsive choice intact. This altered cognitive performance appeared to be associated with enhanced releasability of dopamine in the mPFC. Together, these data suggest that adolescence is a time window during which the brain is vulnerable to long-lasting cognitive disturbances resulting from nicotine exposure.