Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

Abstract

A study was made of glycine (Gly) and ␥-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-dayold rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ؎ 35, 346 ؎ 28, and 389 ؎ 22 nA, whereas the GABA-currents (1 mM GABA) were all <40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC 50 for Gly was 611 ؎ 77 M for encephalon, 661 ؎ 28 M for midbrain, and 506 ؎ 18 M for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all Ϸ2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ؎ 3 nM for encephalon, 97 ؎ 4 nM for midbrain, and 72 ؎ 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca 2؉ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription-PCR studies showed differences in the Gly receptor (GlyR) ␣-subunits expressed, whereas the ␤-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca 2؉ could play important functions during brain development.I n all vertebrates the main neurotransmitters responsible for fast inhibitory synaptic transmission are the amino acids glycine (Gly) and ␥-aminobutyric acid (GABA), which act on their specific receptors (1, 2). Molecular cloning indicates that both Gly receptors (GlyR) and GABA receptors (GABAR) have a pentameric subunit structure, arranged around a chloride-selective ion channel. The GlyR is composed of two types of glycosylated integral membrane proteins (␣1-␣4 and ␤) and an associated peripheral membrane protein (1). On the other hand, the GABAR are made up of combinations of ␣-, ␤-, ␥-, ␦-, -, -, or -subunits (3). The density, location, and subunit composition of some neurotransmitter receptors change during development. For example, mammalian fetal skeletal muscle fibers have acetylcholine receptors along their entire surface and the receptors disappear from the nonjunctional areas during development (4), and their subunit composition is also changed (5). Similarly, the amount of GlyR gene translation, assessed by the injection of developing rat cerebral cortex mRNA into Xenopus oocytes, decreases with age, whereas that of GABARs increases (6). Moreover, the type and location of mRNAs coding for GlyR changes ontogenetically (7), and the subunit composition is also changed, because the ␣2-subunit seen by in situ hybridization in embryonic day 14 (E14) midbrain is not found after E19 (8). Additionally, during developm...

Glycine Receptors and Glycinergic Synaptic Transmission in the Deep Cerebellar Nuclei of the Rat: A Patch-Clamp Study

“…A similar switch in glycine receptors was also found in brain stem motoneurons and in the substantia nigra (Mangin et al 2002;Singer et al 1998). In contrast, it has been reported that there is no evidence of a molecular switch in midbrain neurons (Garcia-Alcocer et al 2001). It is notable that subtypes of glycine receptors show different sensitivities to picrotoxin, a blocker of agonist-gated Cl Ϫ channels (Legendre 2001;Mangin et al 2002).…”

“…Therefore the most probable source of glycine as a neurotransmitter on DCN neurons is interneurons of the DCN, which may release pure glycine or glycine and GABA simultaneously. It is relevant in this regard to note the phenomenon of co-localization of GABA and glycine in the same terminal and changes in the neurotransmitter from GABA to glycine or vice versa at certain synapses during development or under some diseased conditions can occur (Gao et al 2001;Garcia-Alcocer et al 2001;Singer and Berger 2000;Sultan et al 2002;Turecek and Trussell 2002;Zhou et al 2002). Additionally, in the cerebellar cortex of the rat, pure and mixed glycinergic and GABAergic transmission has been proved elecrophysiologically in Golgi cells (Dumoulin et al 2001).…”

Developmental expression of glycine receptor subunits in rat cerebellum

“…Furthermore, a functional heterogeneity of GlyRs was found in the developing spinal cord where two different populations of GlyRs were distinguished by the size of their mRNAs and by the functional properties of the receptors expressed (Akagi et al, 1989; Morales et al, 1994). Molecular cloning of the genes encoding the GlyR subunits (α1–4 and β) allowed a determination of their individual contribution to the receptors in the brain and spinal cord as well as in several areas of the embryonic rat brain (Akagi et al, 1989; Charrier et al, 2006; García‐Alcocer et al, 2001).…”

“…In the rat brain embryo, we previously detected mRNA transcripts coding for the β subunit as early as E8 whereas transcripts coding for the α1–3 subunits were expressed from E13 onwards (García‐Alcocer et al, 2001). At developmental stage E13, cerebellar differentiation is incomplete, and the role of GlyRs early during ontogeny is still unknown.…”