Maria Luisa Malosio

Perinatal morphine exposure alters peptidergic development in the striatum

It has been reported that perinatal exposure to opiates affects mRNA synthesis, body growth and brain development in mammals, including humans. We have observed that morphine administration in drinking water during the perinatal period alters peptide development in the striatum of the rat. There is a marked increase in substance P and met‐enkephalin content, the latter is maintained even at 30 days postnatally. The transient increase or earlier maturation of substance P content is correlated by a more precocious axon terminal organization as revealed by immunocytochemical staining. The increased metenkephalin content is correlated by a higher abundance of preproenkephalin A mRNA and this correlation is particularly evident at 15 days postnatally. At earlier times both northern blotting and in situ hybridization techniques fail to show any significant difference between control and morphine exposed rats, likely because the peptide content is not very different in the two groups or at least the gap is not as wide as at later times.

Tyrosine phosphorylation induced by integrin-mediated adhesion of retinal neurons to laminin.

Integrin α6β1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to understand some of the intracellular mechanisms which allow integrin‐mediated neurite extension on laminin in primary neuronal cultures. Both integrin‐mediated adhesion to laminin and antibody‐induced integrin clustering resulted in the increased tyrosine phosphorylation of a 120 kDa polypeptide which was identified as the focal adhesion kinase. The kinetics of phosphorylation and dephopsphorylation of this kinase were dramatically different in neurons plated on laminin, than in neurons in which the receptors were clustered with anti‐integrin antibodies. To look at possible interactions of the focal adhesion kinase with integrins, we made use of sucrose velocity gradients, which have allowed the identification of a large complex containing the α6β1 laminin receptor. Analysis of the gradients showed that the focal adhesion kinase was not associated with the integrin receptors under these experimental conditions, while about 26% of the c‐Src kinase codistributed with the integrin receptor complex, and showed a molecular size and a distribution similar to that of a 59 kDa phosphoprotein co‐migrating with the α6β1 receptor. Our results suggest that integrin‐induced tyrosine phosphorylation is an early intracellular event during neuronal adhesion, and that the integrin‐mediated increase in tyrosine phosphorylation of the focal adhesion kinase is not sufficient per se for the induction of neurite outgrowth. Furthermore, our data indicate that Src kinase may be involved in integrin‐mediated neuronal interactions with laminin.

In situ hybridization study of myelin protein mRNA in rats with an experimental diabetic neuropathy

Distribution of protein zero (P0) and myelin basic protein (MBP) mRNAs in the sciatic nerve from rats with alloxan-induced diabetes was analyzed at two different time points using in situ hybridization. Some animals of each diabetic group were treated with insulin. Densitometric quantitation of silver clusters revealed that 5 weeks after diabetes induction P0 mRNA only is significantly increased, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels and slightly counteracts increased P0 mRNA at both stages of diabetes. An increase in MBP mRNA is observed in chronic diabetic animals only, and is unaltered by the normoglycemic effect of insulin. The increased transcript levels of P0 and MBP suggest that Schwann cells can modulate gene expression of myelin-specific proteins in response to diabetic-induced metabolic derangement. Such a change may represent a higher turnover of myelin proteins as an attempt by the Schwann cells to repair the diabetes-induced nerve damage. The observed pattern of transcript amount is only slightly influenced by insulin treatment.

Myelin protein transcripts increase in experimental diabetic neuropathy

A Northern blot analysis of P0 and MBP myelin protein transcripts in the sciatic nerve from rats with alloxan-induced diabetes at two different time points is described. After 5 weeks of diabetes induction, only P0 mRNA is significantly increased by 39%, while at 14 weeks both P0 and MBP mRNA contents are markedly higher than controls. Insulin treatment normalizes glycemia levels, partially counteracts P0 mRNA increase at both stages of diabetes and delays MBP mRNA increase present only in chronic animals. We suggest that increased transcript levels of P0 and MBP in Schwann cells may represent a higher turnover of myelin sheath specific proteins in diabetic syndrome, as attempt to repair the hyperglycemia-induced nerve damage, which is partially prevented by insulin treatment.

Perinatal exposure to morphine: reactive changes in the brain after 6-hydroxydopamine

The effects of neonatal 6-hydroxydopamine treatment on the brain of control rats and of rats perinatally exposed to morphine were examined. Noradrenaline levels were increased in the pons-medulla, mesencephalon and caudate of 8-week-old control rats lesioned with neonatal 6-hydroxydopamine; perinatal morphine treatment prevented such an increase. In the caudate, there was a loss of dopamine and an increase of serotonin following the neurotoxic lesion; exposure to perinatal morphine prevented the serotonin increase. Brain expression of synapsin I mRNA was particularly abundant in cerebral cortex, hippocampus, dentate gyrus and olfactory bulb. In perinatal morphine-treated rats, the expression of synapsin I mRNA was significantly reduced; interestingly, the neonatal treatment with 6-hydroxydopamine normalized its expression. Therefore, brain-reactive neurochemical changes triggered by 6-hydroxydopamine were suppressed by perinatal morphine exposure whereas the association of morphine exposure and 6-hydroxydopamine lesion promoted the normal mRNA expression of the synaptic marker synapsin I.

Exposure to perinatal morphine promotes developmental changes in rat striatum

This study shows that perinatal exposure to morphine promotes developmental changes (up to 8 months of life) in the striatum by up‐regulating concentrations of substance P and met‐enkephalin with changes of prometenkephalin A mRNA expression at the day of birth only. Dopamine metabolism (up to 60 days) is also increased as suggested by the reduced concentrations of dopamine and increased content of 3,4‐dihydroxyphenylacetic acid. Tyrosine hydroxylase mRNA expression is selectively reduced only in the substantia nigra by perinatal morphine. Serotonin content is reduced only during the early postnatal days and is unaffected thereafter. Supplementation of naltrexone to morphine‐exposed rats prevents monoaminergic and neuropeptidergic changes in the striatum, which directly implicates opioid receptors in the developmental changes caused by morphine. The data suggest that perinatal morphine may inhibit met‐enkephalin release, causing accumulation of the peptide without corresponding changes in specific mRNA. Dopamine release may also be increased as indicated by a higher metabolism and consequent reduction of tyrosine hydroxylase mRNA expression in the substantia nigra.