Regulation of glycine receptors by embryonic rat spinal cord neurons during development in vitro.

Abstract

This work addresses developmental regulation of neurotransmitter receptors in the vertebrate central nervous system (CNS). Glycine receptors (GlyR) play a major role in inhibitory neurotransmission in the spinal cord. Changes in the types of GlyRs being expressed by embryonic rat spinal cord neurons are examined during development in vitro. Spinal cord neurons are cultured at the fourteenth day of gestation, prior to receiving afferent input. Previous work demonstrated a delayed expression of the adult-type GlyR α subunits. Reverse transcriptase-polymerase chain reaction demonstrates the presence of mRNA for GlyR α2 subunits by these neurons at early times in culture. The presence of GlyR α2 subunits are confirmed by immunofluorescence microscopy with a new α2 subunit specific antibody. These subunits appear by the first day in culture and exhibit a diffuse subcellular distribution. During the course of these experiments, populations of embryonic rats were found to differ in the subtypes of GlyR they expressed at early times during development. The expression of functional GlyRs is investigated in two populations of embryonic rats using whole-cell patch clamp recordings. The GlyR antagonist, strychnine, is used as a tool to distinguish between some forms of the GlyR. The two populations are similar in their onset of responsiveness to glycine and in the ion-dependence of the glycine-induced current. The strychnine-sensitivity of responses to glycine differs between the two populations. Neurons from the first population of rats exhibit a developmentally regulated increase in the sensitivity to strychnine, while the strychnine-sensitivity of responses to glycine by neurons from the second population remains high throughout development in culture. These results suggest that two populations differ in the type of functional GlyR they express during early development in culture. The relatively low sensitivity to strychnine exhibited during the first few days in culture by neurons from the first population of rats cannot be accounted for by changes in sensitivity to glycine or by non-specific cross-activation of γ-aminobutyric acid receptors (GABARs). Neurons from the first population undergo a gradual change from the predominant expression of a strychnine-insensitive GlyR to some form(s) of strychnine-sensitive GlyR.