Perinatal Protein Restriction Impacts Nuclear O-GalNAc Glycosylation in Cells of Liver and Brain Structures of the Rat.

BACKGROUND: Post-translational modifications are key factors in the modulation of nuclear protein functions controlling cell physiology and an individual's health. OBJECTIVES: This study examined the influence of protein restriction during the perinatal period on the nuclear O-N-acetylgalactosamine (O-GalNAc) glycosylation of cells from the liver and parts of the brain in the rat. METHODS: Pregnant Wistar rats were divided into 2 groups on day 14 of pregnancy and fed ad libitum 1 of 2 isocaloric diets containing 24% (well-fed) or 8% (protein-restricted diet) casein until the end of the experiment. Male pups were studied after weaning at 30 d of life. Animals and their organ/tissues (liver, cerebral cortex, cerebellum and hippocampus) were weighed. Cell nuclei were purified, and the presence in nucleus and cytoplasm of all factors required for the initiation of O-GalNAc glycan biosynthesis, i.e., the sugar donor (UDP-GalNAc), enzyme activity (ppGalNAc-transferase) and the glycosylation product (O-GalNAc glycans), were evaluated by western blotting, fluorescent microscopy, enzyme activity, enzyme-lectin sorbent assay and mass spectrometry. RESULTS: The perinatal protein deficit reduced progeny weight, as well as the cerebral cortex and cerebellum weight. UDP-GalNAc levels in the cytoplasm and nuclei of the liver, the cerebral cortex, cerebellum, or hippocampus were not affected by the perinatal dietary protein deficits. However, this deficiency affected the ppGalNAc-transferase activity localized in the cerebral cortex and hippocampus cytoplasm as well as in the liver nucleus, thus reducing the "writing" ppGalNAc-transferase activity of O-GalNAc glycans. In addition, liver nucleoplasm from protein-restricted offspring revealed a significant reduction in the expression of O-GalNAc glycans on important nuclear proteins. CONCLUSIONS: Our results report an association between the consumption of a protein-restricted diet by the dam and her progeny with the modulation in the offspring' liver nuclei O-GalNAc glycosylation, which may ultimately regulate nuclear protein functions.

Decrease of lymphoproliferative response by amphetamine is mediated by dopamine from the nucleus accumbens: influence on splenic met-enkephalin levels.

Despite the mesocorticolimbic dopaminergic pathway being one of the main substrates underlying stimulating and reinforcing effects induced by psychostimulant drugs, there is little information regarding its role in their effects at the immune level. We have previously demonstrated that acute exposure to amphetamine (5 mg/kg, i.p.) induced an inhibitory effect on the splenic T-cell proliferative response, along with an increase in the methionine(met)-enkephalin content at limbic and immune levels, 4 days after drug administration. In this study, we investigated if a possible dopamine mechanism underlies these amphetamine-induced effects by administering D1 and D2 dopaminergic antagonists or a dopaminergic terminal neurotoxin before the drug. Pre-treatment with either SCH-23390 (0.1 mg/kg, i.p.) or raclopride (0.1 mg/kg, i.p.), a D1 or D2 dopaminergic receptor antagonist, respectively, abrogated the effects of amphetamine on the lymphoproliferative response and on met-enkephalin levels of the spleen. The amphetamine-induced increase in limbic met-enkephalin content was suppressed by SCH-23390 but not by raclopride pre-treatment. Finally, an intra-accumbens 6-hydroxy-dopamine injection administered 2 weeks previously prevented amphetamine-induced effects on the lymphoproliferative response and on met-enkephalin levels in the prefrontal cortex and spleen. These findings strongly suggest that D1 and D2 dopaminergic receptors are involved in amphetamine-induced effects at immune level as regards the lymphoproliferative response and the changes in spleen met-enkephalin content, whereas limbic met-enkephalin levels were modulated only by the D1 dopaminergic receptors. In addition, this study showed that a mesolimbic component modulated amphetamine-induced effects on the immune response, as previously shown at a behavioral level.

Perinatal protein deprivation facilitates morphine cross-sensitization to cocaine and enhances ΔFosB expression in adult rats.

Previous studies have indicated that neural changes induced by early nutritional insult cause an altered response to pharmacological treatments, including addictive drugs. This study evaluates the influence of perinatal protein malnutrition in developing cross-sensitization to cocaine-induced rewarding effects in animals pre-exposed to morphine. Different groups of well-nourished (C-rats) and protein-deprived animals (D-rats) were treated twice a day for three days with increasing doses of morphine or with saline. After 3days, the incentive motivational effects of cocaine were assessed in a Conditioned Place Preference paradigm in both groups. In saline pre-treated animals, dose-response curves to cocaine revealed a conditioning effect in D-rats at doses of 5, 7.5 and 10mg/kg, while this effect was observed in C-rats only with 10 and 15mg/kg. Furthermore, when animals of both groups were pre-treated with escalating doses of morphine, cross-sensitization to the conditioning effect of cocaine was elicited only in D-rats with low doses of cocaine (5 and 7.5mg/kg). In contrast, under the same experimental conditions, C-rats show no cross-sensitization. To correlate this differential rewarding response with a molecular substrate linked to the behavioral changes observed after repeated drug exposure, ΔFosB expression was assessed in different brain regions. D-rats showed a significant increase in this transcription factor in the nucleus accumbens, amygdala and medial prefrontal cortex. These results demonstrated that perinatal protein deprivation facilitates rewarding effects and the development of cross-sensitization to cocaine, which correlates with an upregulation of ΔFosB in brain areas related to the reward circuitry.

Pharmacological reactivity to cocaine in adult rats undernourished at perinatal age: behavioral and neurochemical correlates.

The influence of neuronal alterations induced by early undernutrition on the stimulant effect of cocaine was assessed in adult rats submitted to a protein deprivation schedule at perinatal age. To evaluate the sensitization phenomenon induced by repeated cocaine administration, different groups of control (C) and deprived (D) rats received a daily injection of cocaine (5, 10 or 15 mg/kg, i.p.) for 16 days. Behavioral parameters were assessed every two days in an open-field. Dose-response curves obtained with different doses of cocaine used revealed a shift to the left in the locomotor activity curves of D rats compared to controls. Thus, D animals showed a clear behavioral sensitization to the lower dose of cocaine, whereas this phenomenon was only observed in C rats for the higher dose used. To correlate this differential development of sensitization with neurochemical parameters, we assessed extracellular dopamine (DA) levels in nucleus accumbens (core and shell) and in the dorsal caudate-putamen, using a microdialysis technique. A challenge with cocaine in cocaine pre-exposed animals produced a different increase in DA output only in nucleus accumbens "core" of D animals. Comparable DA levels were observed in nucleus accumbens shell and in dorsal caudate-putamen of both groups. These results demonstrate that D rats had a lower threshold developing a progressive behavioral sensitization following repeated cocaine administration, as well as higher responsiveness of the nucleus accumbens (core) expressed by increased DA release.

Exogenous GM1 ganglioside increases accumbal BDNF levels in rats.

Gangliosides are compounds that are abundant throughout the CNS, participating actively in neuroplasticity. We previously described that exogenous GM1 ganglioside pretreatment enhances the rewarding properties of cocaine, evidenced by a lower number of sessions and/or dosage necessary to induce conditioned place preference (CPP). Since GM1 pretreatment did not modify cocaine's pharmacokinetic parameters, we suspected that the increased rewarding effect found might be mediated by BDNF, a neurotrophic factor closely related to cocaine addiction. This study was performed to investigate the possibility that GM1 may induce changes in BDNF levels in the nucleus accumbens (NAc), a core structure in the brain's reward circuitry, of rats submitted to three conditioning sessions with cocaine (10 mg/kg, i.p.). The results demonstrate that GM1 administration, which showed no rewarding effect by itself in the CPP, induced a significant increase of BDNF protein levels in the NAc, which may account for the increased rewarding effect of cocaine shown in the CPP paradigm.

Locus coeruleus activity in perinatally protein-deprived rats: effects of fluoxetine administration.

We have previously described an increased locus coeruleus activity in perinatally protein-deprived rats. Since locus coeruleus dysfunction has been involved in different types of anxiety disorders and considering the modulating action of serotonergic transmission on locus coeruleus activity, we assessed the effect of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on locus coeruleus activity as measured by the firing rate and the number of spontaneously active cells/track. Repeated fluoxetine administration reduced locus coeruleus activity in both control and protein-deprived rats, although the reduction was greater in protein-deprived rats. Dose-response curves for the inhibitory effect of clonidine showed subsensitivity of alpha2-adrenergic autoreceptors in protein-deprived rats, a phenomenon reversed by fluoxetine treatment. Dose-response curves for the inhibitory effect of 2,5-dimethoxy-4-iodoamphetamine (DOI) were similar in both groups of rats. Following fluoxetine administration, subsensitivity to this effect developed in control but not in protein-deprived rats. Extracellular noradrenaline level in the prefrontal cortex, as measured by microdialysis procedure, was higher in protein-deprived rats compared to controls, and this difference was reduced after fluoxetine administration. A challenge with yohimbine increased the extracellular noradrenaline level in control but not in protein-deprived rats, suggesting subsensitivity of alpha2-adrenergic autoreceptors in early protein malnourished animals. These results stress the complexity of plastic changes induced by early protein malnutrition and sustain the hypothesis that perinatally protein-deprived rats may represent a useful animal model for screening antipanic agents.

GM1 ganglioside enhances the rewarding properties of cocaine in rats.

GM1 pretreatment enhanced the rewarding properties of cocaine as assessed in the conditioned place preference paradigm. This effect was shown by the lower dosage of cocaine necessary to induce conditioning compared with rats receiving cocaine alone, as well as by the fewer number of sessions necessary to induce place preference. GM1 pretreatment did not modify the plasma level of cocaine, but it induced a significant increase in the brain cocaine level compared with animals receiving cocaine alone. In order to evaluate the possibility that GM1 pretreatment may alter the pharmacokinetic parameters of cocaine, the brain and plasma esterase activities, the plasma bound/free cocaine ratio and the brain blood barrier permeability to i.v. Evans Blue administration were assessed. None of these parameters was modified by the GM1 administration. In addition, GM1 (100microM) did not alter the dopamine transporter inhibition induced by cocaine (10(-7)-10(-5)M), as determined by the uptake of [(3-)H]-dopamine in the microsacs of nucleus accumbens. In conclusion, GM1 pretreatment, which did not have any effect per se, increased the rewarding effect of cocaine, a phenomenon correlated with a significant increase in the brain cocaine levels. The different pharmacokinetic parameters evaluated, as well as the inhibitory effect of cocaine on the dopamine transporter, were not modified by GM1, but it modifies the brain cocaine disposition. Thus, the mechanisms by which GM1 enhanced the rewarding effects of cocaine merit further study.