Evaluation of tyrosine hydroxylase-like immunoreactive chick embryo pontine neuroblasts differentiation following administration of cysteamine

AIMS: In our previous studies, we characterized a population of somatostatin (SOM) immunoreactive (ir) pontine neuroblasts that localizes close to the neuroblasts ir for the tyrosine hydroxylase (TH) during the embryonic development, suggesting a possible paracrine role exherted by SOM (1). In this study, we treated the embryos with cisteamine (CSH), a known SOM depleting agent, and characteriZed the modifications on SOM and TH ir neuroblasts in order to evaluate the possible interactions among the two populations. MATERIALS AND METHODS: Leghorn chick embryos from E (embryonic day) 10 were treated twice a day with a CSH or a saline solution applied to the vascularized chorioallantoic membrane. 10 μm thick paraffin serial sections were obtained from embryo brains at E 13, E 15, E 17 and E 19 and then incubated with either primary mouse anti-TH or rat anti-SOM antibodies according to ABC technique. The sections were digitized and the images of positive neuroblasts were analized to obtain morphocytometric parameters, optical density (O.D.) and cell counts. Protein lysates obtained from pons of embryo brains at E 13, E 15, E 17 and E 19 were separated by SDS gel electrophoresis, blotted on nitrocellulose membrane and incubated with mouse anti-TH antibody. TH positive bands were revealed by chemiluminescence and quantified by densitometric analysis. RESULTS AND DISCUSSION: First, the distribution of TH ir neuroblasts corresponded with that of neurons belonging to the nucleus of the locus coeruleus and locus subcoeruleus. In saline treated embryos, there was not a marked increase of the number of TH ir neuroblasts from E13 to E19, suggesting that the massive proliferation of this population of cells was already almost completed. In the embryos treated with CSH, the cell counts of TH ir neuroblasts showed higher values compared to saline, in particular at E15. On the other hand, as expected, the number of SOM ir cells of CSH - treated embryos resulted generally lowered; in particular, the lowest values resulted at E15. Taken together, those data suggested a tonic inhibitory effect of SOM ir neuroblasts on TH ir ones during the embryonic development. By means of western blot we also evaluated the expression of TH during the embryonic stages: we observed a massive expression of the enzyme already at early embryonic stages, suggesting a possible morphogenetic role exherted by SOM on the early expression of the catecholamines of the pontine region.