Development of serotonin, substance P and thyrotrophin-releasing hormone in mouse medullary raphe grown in organotypic tissue culture: developmental regulation by serotonin

Abstract

Substance P (SP) and thyrotrophin-releasing hormone (TRH) are co-localized with serotonin (5-HT) in cells of the medullary raphe nuclei. In order to examine the factors that control development of multiple neurotransmitters within individual brain nuclei, we have grown presumptive raphe nuclei in organotypic tissue culture, an environment in which mammalian embryonic brain is easily accessible and manipulable. Tissue was obtained from E13 mice. A discrete midline segment of the rhombencephalon was dissected intact or was separated into ‘rostral’ (RR) and ‘medullary’ (MR) fragments. Tissue was explanted onto collagen coverslips and grown for up to two weeks in Maximow depression chambers. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, was barely detectable at explantation. During the first week in culture, however, TPH activity increased 7-fold. After two weeks, TPH activity increased almost 2.5-fold above the one-week level. Immunocytochemical analysis of the cultures confirmed a widespread distribution of 5-HT-positive cells and fibers throughout the explant. SP, monitored by radioimmunoassay, was detected after two days in culture, and attained a level of111.7 ± 9.8 pg/culture after two weeks. TRH activity was similarly elevated after two weeks in vitro. Therefore, developmental increases in TPH, SP, and TRH occurred in culture, mimicking the condition in vivo. RR and MR fragments, when grown apart on separate coverslips, developed 1.57–2.26 times the TPH activity that developed in the undivided piece. Inclusion of 1 μM pargyline in the fragments restored TPH to control levels. The effect of pargyline was blocked by methiothepin, suggesting autoreceptor-mediated regulation. By contrast, SP activity was additive, the amount of SP present in the whole piece being equal to the sum of the RR and MR fragments. Pargyline did not affect the development of TRH or SP. These data suggest that factors (particularly autoreceptor-mediated interactions) affecting the development of 5-TH in the cultures do not act simultaneously to affect peptide development.