Maternal environmental enrichment protects neonatal brains from hypoxic-ischemic challenge by mitigating brain energetic dysfunction and modulating glial cell responses.

There is evidence that maternal milieu and changes in environmental factors during the prenatal period may exert a lasting impact on the brain health of the newborn, even in case of neonatal brain hypoxia-ischemia (HI). The present study aimed to investigate the effects of maternal environmental enrichment (EE) on HI-induced energetic and metabolic failure, along with subsequent neural cell responses in the early postnatal period. Male Wistar pups born to dams exposed to maternal EE or standard conditions (SC) were randomly divided into Sham-SC, HI-SC, Sham-EE, and HI-EE groups. Neonatal HI was induced on postnatal day (PND) 3. The Na+,K+-ATPase activity, mitochondrial function and neuroinflammatory related-proteins were assessed at 24 h and 48 h after HI. MicroPET-FDG scans were used to measure glucose uptake at three time points: 24 h post-HI, PND18, and PND24. Moreover, neuronal preservation and glial cell responses were evaluated at PND18. After HI, animals exposed to maternal EE showed an increase in Na+,K+-ATPase activity, preservation of mitochondrial potential/mass ratio, and a reduction in mitochondrial swelling. Glucose uptake was preserved in HI-EE animals from PND18 onwards. Maternal EE attenuated HI-induced cell degeneration, white matter injury, and reduced astrocyte immunofluorescence. Moreover, the HI-EE group exhibited elevated levels of IL-10 and a reduction in Iba-1 positive cells. Data suggested that the regulation of AKT/ERK1/2 signaling pathways could be involved in the effects of maternal EE. This study evidenced that antenatal environmental stimuli could promote bioenergetic and neural resilience in the offspring against early HI damage, supporting the translational value of pregnancy-focused environmental treatments.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Early-life stress affects behavioral and neurochemical parameters differently in male and female juvenile Wistar rats.

Neonatal handling is an early life stressor that leads to behavioral and neurochemical changes in adult rats in a sex-specific manner and possibly affects earlier stages of development. Here, we investigated the effects of neonatal handling (days 1-10 after birth) on juvenile rats focusing on biochemical parameters and olfactory memory after weaning. Male neonatal handled rats performed more crossings on the hole-board task, increased Na+ /K+ -ATPase activity in the olfactory bulb, and decreased acetylcholinesterase activity in the hippocampus versus non-handled males. Female neonatal handled animals increased the number of rearing and nose-pokes on the hole-board task, decreased glutathione peroxidase activity, and total thiol content in the hippocampus versus non-handled females. This study reinforces that early life stress affects behavioral and neurochemical parameters in a sex-specific manner even before the puberty onset.

Chronic mild hyperhomocysteinemia induces anxiety-like symptoms, aversive memory deficits and hippocampus atrophy in adult rats: New insights into physiopathological mechanisms.

In the last decade, increased homocysteine levels have been implicated as a risk factor for neurodegenerative and psychiatric disorders. We have developed an experimental model of chronic mild hyperhomocysteinemia (HHcy) in order to observe metabolic impairments in the brain of adult rodents. Besides its known effects on brain metabolism, the present study sought to investigate whether chronic mild HHcy could induce learning/memory impairments associated with biochemical and histological damage to the hippocampus. Adult male Wistar rats received daily subcutaneous injections of homocysteine (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th day of life or saline solution (Controls). After injections, anxiety-like and memory tests were performed. Following behavioral analyses, brains were sliced and hippocampal volumes assessed and homogenized for redox state assessment, antioxidant activity, mitochondrial functioning (chain respiratory enzymes and ATP levels) and DNA damage analyses. Behavioral analyses showed that chronic mild HHcy may induce anxiety-like behavior and impair long-term aversive memory (24 h) that was evaluated by inhibitory avoidance task. Mild HHcy decreased locomotor and/or exploratory activities in elevated plus maze test and caused hippocampal atrophy. Decrease in cytochrome c oxidase, DNA damage and redox state changes were also observed in hippocampus of adult rats subjected to mild HHcy. Our findings show that chronic mild HHcy alters biochemical and histological parameters in the hippocampus, leading to behavioral impairments. These findings might be considered in future studies aiming to search for alternative strategies for treating the behavioral impairments in patients with mild elevations in homocysteine levels.

Effect of Sciatic Nerve Transection on acetylcholinesterase activity in spinal cord and skeletal muscles of the bullfrog Lithobates catesbeianus / Efeito da Secção do Nervo Isquiático sobre a atividade da acetilcolinesterase em medula espinal e músculos esqueléticos da rã-touro Lithobates catesbeianus

Abstract Sciatic nerve transection (SNT), a model for studying neuropathic pain, mimics the clinical symptoms of "phantom limb", a pain condition that arises in humans after amputation or transverse spinal lesions. In some vertebrate tissues, this condition decreases acetylcholinesterase (AChE) activity, the enzyme responsible for fast hydrolysis of released acetylcholine in cholinergic synapses. In spinal cord of frog Rana pipiens, this enzyme's activity was not significantly changed in the first days following ventral root transection, another model for studying neuropathic pain. An answerable question is whether SNT decreases AChE activity in spinal cord of frog Lithobates catesbeianus, a species that has been used as a model for studying SNT-induced neuropathic pain. Since each animal model has been created with a specific methodology, and the findings tend to vary widely with slight changes in the method used to induce pain, our study assessed AChE activity 3 and 10 days after complete SNT in lumbosacral spinal cord of adult male bullfrog Lithobates catesbeianus. Because there are time scale differences of motor endplate maturation in rat skeletal muscles, our study also measured the AChE activity in bullfrog tibial posticus (a postural muscle) and gastrocnemius (a typical skeletal muscle that is frequently used to study the motor system) muscles. AChE activity did not show significant changes 3 and 10 days following SNT in spinal cord. Also, no significant change occurred in AChE activity in tibial posticus and gastrocnemius muscles at day 3. However, a significant decrease was found at day 10, with reductions of 18% and 20% in tibial posticus and gastrocnemius, respectively. At present we cannot explain this change in AChE activity. While temporally different, the direction of the change was similar to that described for rats. This similarity indicates that bullfrog is a valid model for investigating AChE activity following SNT.

Resumo A transecção do nervo isquiático (SNT), um modelo para estudar dor neuropática, simula os sintomas clínicos do "membro fantasma", uma condição dolorosa que ocorre nos humanos após amputação ou secção completa da medula espinal. Essa condição muda a atividade da acetilcolinesterase (AChE), a enzima responsável pela rápida hidrólise da acetilcolina liberada nas sinapses colinérgicas, em alguns tecidos de vertebrados. Em medula espinal de rã Rana pipiens, a atividade da AChE não foi significativamente alterada nos primeiros dias após a secção da raiz ventral, outro modelo para o estudo da dor neuropática. Uma questão ainda não respondida é se a SNT diminui a atividade da AChE na medula espinal de rã Lithobates catesbeianus, uma espécie que vem sendo usada como modelo em estudos da dor neuropática induzida por SNT. Como cada modelo animal é criado a partir de metodologia específica, e seus resultados tendem a variar com pequenas mudanças na metodologia de indução da dor, o presente estudo avaliou a atividade da AChE em medula espinal lombossacral de rã-touro Lithobates catesbeianus, adultos, machos, 3 e 10 dias após a completa SNT. Como há diferenças temporais na maturação de placas motoras em músculos esqueléticos de ratos, nosso estudo ainda demonstrou, na rã-touro, os efeitos da SNT sobre a atividade da AChE nos músculos esqueléticos tibial posticus, um músculo postural, e gastrocnêmio, um músculo frequentemente usado em estudos do sistema motor. A atividade da AChE não mudou significativamente na medula espinal aos 3 e 10 dias após a SNT. Nos músculos, a atividade não alterou significativamente aos 3 dias após a lesão, mas reduziu de forma significativa aos 10 dias após a SNT. Aos 10 dias, a diminuição foi 18% no músculo tibial posticus e 20% no gastrocnêmio. No momento, nós não temos explicação para essa mudança na atividade da AChE. Embora temporalmente diferente, o sentido da mudança é similar ao que é descrito em ratos. Esta similaridade torna a rã-touro um modelo válido para se estudar questões ainda não respondidas da SNT sobre a AChE.

Are the consequences of neonatal hypoxia-ischemia dependent on animals' sex and brain lateralization?

Hypoxia-ischemia on 3-day-old rats (HIP3) allows the investigation of HI damage in the immature brain. HIP3 is characterized for neurological disabilities caused by white matter injury. This study investigates the relationship between animals' sex and injured hemisphere on HIP3 consequences. Male and female Wistar rats had their right or left common carotid artery occluded under halotane anesthesia and exposed to 8% O2 for 1.5 h. Control rats received sham surgery and exposure to 1.5 h of room air in isolation of their mothers. Sex and injured hemisphere influence in Na+/K+ -ATPase activity 24h after lesion: females and the right brain hemispheres showed decreased enzymatic activity after HIP3. Cognitive impairment was observed in step-down inhibitory avoidance, in which females HIP3 left injured were the most damaged. Histological analysis showed a trend to white matter damage in females left injured without hemispherical nor hippocampal volume decrease in HIP3 rats at postnatal day 21. However, at PND90, hemisphere and sex effects were noted in hemispherical volume and myelination: left brain hemisphere and the females evidenced higher histological damage. Our results points to an increased resistance of male rats and right brain hemisphere to support the impairment caused in Na+/K+ -ATPase activity early after HIP3, and evidencing more discrete behavioral impairments and histological damage at adulthood. Present data adds new evidence of distinct effects of brain lateralization and sex vulnerability on biochemical, behavioral and histological parameters after hypoxia-ischemia.

Mild hyperhomocysteinemia alters extracellular adenine metabolism in rat brain.

Since homocysteine (Hcy) is considered a risk factor to cerebral diseases and adenine nucleotides are important molecules to brain normal function, in the present study we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in rat cerebral cortex. The levels of ATP, ADP, AMP and adenosine (Ado) in cerebrospinal fluid (CSF) of adult rats also were evaluated by high-performance liquid chromatography. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) was administered subcutaneously from the 30th to the 60th day of life. Control rats received saline solution in the same volumes. Results showed that Hcy significantly decreased nucleotide hydrolysis in the synaptosomal fraction and increased E-NTPDase1 and ecto-5'-nucleotidase transcripts in rat cerebral cortex. ATP levels were significantly increased, while Ado decreased in CSF of Hcy-treated rats. These findings suggest that the unbalance in ATP and Ado levels may be, at last in part, involved in the cerebral toxicity of mild hyperhomocysteinemia.

Isolation stress during the prepubertal period in rats induces long-lasting neurochemical changes in the prefrontal cortex.

Social isolation during postnatal development leads to behavioral and neurochemical changes, and a particular susceptibility of the prefrontal cortex to interventions during this period has been suggested. In addition, some studies showed that consumption of a palatable diet reduces some of the stress effects. Therefore, our aim is to investigate the effect of isolation stress in early life on some parameters of oxidative stress and energy metabolism (Na(+),K(+)-ATPase activity, respiratory chain enzymes activities and mitochondrial mass and potential) in prefrontal cortex of juvenile and adult male rats. We also verified if the consumption of a palatable diet during the prepubertal period would reduce stress effects. The results showed that, in juvenile animals, isolation stress increased superoxide dismutase and Complex IV activities and these effects were still observed in the adulthood. An interaction between stress and diet was observed in catalase activity in juveniles, while only the stress effect was detected in adults, reducing catalase activity. Access to a palatable diet increased Na(+),K(+)-ATPase activity in juveniles, an effect that was reversed after removing this diet. On the other hand, isolation stress induced a decreased activity of this enzyme in adulthood. No effects were observed on glutathione peroxidase, total thiols and free radicals production, as well as on mitochondrial mass and potential. In conclusion, isolation stress in the prepubertal period leads to long-lasting changes on antioxidant enzymes and energetic metabolism in the prefrontal cortex of male rats, and a palatable diet was not able to reverse these stress-induced effects.

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

L-NAME administration prevents the inhibition of nucleotide hydrolysis by rat blood serum subjected to hyperargininemia.

The main objective of the present study was to evaluate the in vivo and in vitro effect of Arg on serum nucleotide hydrolysis. The action of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the effects produced by Arg was also examined. Sixty-day-old rats were treated with a single or a triple (with an interval of 1 h between each injection) intraperitoneal injection of saline (group I), Arg (0.8 g/kg) (group II), L-NAME (2.0 mg/kg or 20 mg/kg) (group III) or Arg (0.8 g/kg) plus L-NAME (2.0 mg/kg or 20 mg/kg) (group IV) and were killed 1 h later. The present results show that a triple Arg administration decreased ATP, ADP and AMP hydrolysis. Simultaneous injection of L-NAME (20 mg/kg) prevented such effects. Arg in vitro did not alter nucleotide hydrolysis. It is suggested that in vivo Arg administration reduces nucleotide hydrolysis in rat serum, probably through nitric oxide or/and peroxynitrite formation.

Inhibition of energy metabolism by 2-methylacetoacetate and 2-methyl-3-hydroxybutyrate in cerebral cortex of developing rats.

Mitochondrial beta-ketothiolase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are inherited neurometabolic disorders affecting isoleucine catabolism. Biochemically, beta-ketothiolase deficiency is characterized by intermittent ketoacidosis and urinary excretion of 2-methyl-acetoacetate (MAA), 2-methyl-3-hydroxybutyrate (MHB) and tiglylglycine (TG), whereas in MHBD deficiency only MHB and tiglylglycine accumulate. Lactic acid accumulation and excretion are also observed in these patients, being more pronounced in MHBD-deficient individuals, particularly during acute episodes of decompensation. Patients affected by MHBD deficiency usually manifest severe mental retardation and convulsions, whereas beta-ketothiolase-deficient patients present encephalopathic crises characterized by metabolic acidosis, vomiting and coma. Considering that the pathophysiological mechanisms responsible for the neurological alterations of these disorders are unknown and that lactic acidosis suggests an impairment of energy production, the objective of the present work was to investigate the in vitro effect of MAA and MHB, at concentrations varying from 0.01 to 1.0 mmol/L, on several parameters of energy metabolism in cerebral cortex from young rats. We observed that MAA markedly inhibited CO2 production from glucose, acetate and citrate at concentrations as low as 0.01 mmol/L. In addition, the activities of the respiratory chain complex II and succinate dehydrogenase were mildly inhibited by MAA. MHB, at 0.01 mmol/L and higher concentrations, strongly inhibited CO2 production from all tested substrates, as well as the respiratory chain complex IV activity. The other activities of the respiratory chain were not affected by these metabolites. The data indicate a marked blockage in the Krebs cycle and a mild inhibition of the respiratory chain caused by MAA and MHB. Furthermore, MHB inhibited total and mitochondrial creatine kinase activities, which was prevented by the use of the nitric-oxide synthase inhibitor L-NAME and glutathione (GSH). These data indicate that the effect of MHB on creatine kinase was probably mediated by oxidation or other modification of essential thiol groups of the enzyme by nitric oxide and other by-products derived from this organic acid. In contrast, MAA did not affect creatine kinase activity. Taken together, these observations indicate that aerobic energy metabolism is inhibited by MAA and to a greater extent by MHB, a fact that may be related to lactic acidaemia occurring in patients affected by MHBD and beta-ketothiolase deficiencies. If the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to the neurological dysfunction found in these disorders.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

BACKGROUND: Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. MATERIALS AND METHODS: In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. RESULTS: We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. CONCLUSION: Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

In vivo and in vitro effect of imipramine and fluoxetine on Na+, K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10 percent) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27 percent) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80 percent) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70 percent) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45 percent) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria

ATP diphosphohydrolase (apyrase)(EC3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylaninemia induced by subcutaneous administration of 5.2 µmol phenylalanine/g body weight (twice a day) plus 0.9 µmol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATp (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalnine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ATP diphosphohrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria