Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development

Akagi, Hiroyuki; Hirai, Keiko; Hishinuma, Fumio

doi:10.1016/0014-5793(91)80383-e

Cited by 86 publications

(39 citation statements)

References 16 publications

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“…Our estimate of a long value for newborn L-GlyRs is in good agreement with that obtained for the a2 subunit (19) cloned from the L-GlyR mRNA fraction of young rats (21); this suggests strongly that the properties ofnewborn L-GlyRs are due to the a2 subunit. A similar variety ofGlyRs probably occurs in the human nervous system since, as shown earlier (13) (4,22) or in situ hybridization (23).…”

Section: Methodssupporting

confidence: 77%

Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.

Morales

Nguyen

Miledi

1994

Proc. Natl. Acad. Sci. U.S.A.

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The properties of glycine receptors (GlyRs) from newborn and adult rat spinal cord were studied in Xenopus oocytes injected with whole mRNA or the heavy (H) or light (L) mRNA fractions encig their respective GlyRs. Mean open times and conductances of channels gated by H-or L-GlyRs were determined by noise analysis or voltage jumps. quickly frozen in liquid nitrogen and stored at -800C. Newborn rats (postnatal days 0-3) were deeply anaesthetized with ether and decapitated and spinal cords were dissected as above.mINA Isolation and Size Fractionation. mRNA preparation and size separation on sucrose gradients were as described (2). Briefly, tissues were homogenized in phenol-saturated homogenization buffer (200 mM Tris-HCl/50 mM NaCl/10 mM EDTA/0.5% SDS, pH 9) supplemented with heparin (1 mg/ml). The mixture was shaken for 5 min and centrifuged for 10 min at 15,300 x g. Nucleic acids were isolated in the aqueous phase by several extractions with chloroform and phenol/chloroform, 1:1 (wt/vol). Total RNA was separated by selective precipitation in 3 M NaCl and washed three times with 3 M sodium acetate (pH 6). Poly(A)+ mRNA was purified by oligo(dT) chromatography, precipitated, washed in 70% ethanol, and concentrated to 1 pg/gl in diethyl pyrocarbonate-treated water. About 100 gg of adult or newborn mRNA was size fractionated on a sucrose gradient as described (8). A rRNA gradient was run in parallel to give the positions of the 18S and 28S rRNAs. Thirty fractions were collected, ethanol-precipitated, dried in vacuum, and resuspended in 10-12 ;4 of diethyl pyrocarbonate-treated water.Oocyte Injection and Recording. Oocytes were injected with 50 nl of whole (W) mRNA or the H-or L-GlyR fraction and kept at 15-16'C in a modified Barth's solution (with Nystatin at 50 units/ml and gentamicin at 0.1 mg/ml). Two days after injection, oocytes were treated with collagenase (0.5 mg/ml) to remove the surrounding layers (9) and recordings were performed at room temperature (20-240C)

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Section: Methodssupporting

confidence: 77%

Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.

Morales

Nguyen

Miledi

1994

Proc. Natl. Acad. Sci. U.S.A.

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“…A "neonatal" GlyR isoform (containing ␣2 subunits with a molecular mass of 49 kDa) prevails in the spinal cord of fetal and early postnatal rats and accounts for 70% of the total receptor protein (Becker et al, 1988;Akagi et al, 1991). In contrast, "adult" GlyR isoforms (containing ␣1 subunits with a molecular weight of 48 kDa) are present in the spinal cord of mature animals (Becker et al, 1988).…”

mentioning

confidence: 89%

Localization of rat glycine receptor α1 and α2 subunit transcripts in the developing auditory brainstem

Piechotta

Weth²,

Harvey

et al. 2001

J of Comparative Neurology

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Glycine receptors (GlyRs), aside from GABA(A) receptors, mediate fast postsynaptic inhibition in the mammalian nervous system. Spatial and temporal expression of the genes encoding ligand-binding alpha subunits (alpha1-alpha4) and the structural beta subunit leads to the formation of various GlyR isoforms. Currently, the idea of the GlyRs containing the "adult" alpha1 subunit replacing those with the "neonatal" alpha2 subunit during early postnatal development predominates. Here, we describe the patterns of expression of the GlyR alpha1 and alpha2 subunit genes in the rat auditory brainstem between postnatal day (P) 0 and P20, by using both nonradioactive and radioactive in situ hybridization. We show that the alpha1 subunit mRNA appears throughout the auditory brainstem during the first 8 postnatal days, which resembles the time of onset described within the spinal cord. In the rostral auditory nuclei (nuclei of the lateral lemniscus and inferior colliculus), the alpha1 subunit transcript appears later (P8) than in the caudal nuclei (cochlear nuclear complex and superior olivary complex; P0). Surprisingly, we found that low levels of the alpha2 subunit transcript are present in the auditory brainstem at birth and persist throughout the period analyzed. However, alpha2 subunit mRNA is present at high levels in other neonatal brainstem structures, such as cranial motor nuclei. Therefore, we conclude that the changes in GlyR composition in the auditory brainstem deviate from the classic alpha2 to alpha1 subunit switch observed in spinal cord. Our data suggest that genes for other GlyR subunits (e.g., alpha3 and alpha4) may be expressed during early development in the auditory brainstem.

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“…PMBA was about 60 times less potent than strychnine in the spinal cord of newborn rats, and similar inhibitory ratios of PMBA to strychnine have been obtained in other in vitro electrophysiological experiments. Xenopus oocytes injected with cRNA encoding rat glyR responded to glycine (Akagi et al, 1991), and the inward current induced by glycine was effectively blocked by PMBA (IC,0:0.5,UM) and strychnine (IC50: 0.01 JLM) (personal communication from Dr H. Akagi).…”

Section: Electrophysiological Studies In Isolated Spinal Cordsmentioning

confidence: 99%

A novel antagonist, phenylbenzene ω‐phosphono‐α‐amino acid, for strychnine‐sensitive glycine receptors in the rat spinal cord

Saitoh

Ishida

Maruyama

et al. 1994

British J Pharmacology

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1 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine (PMBA) is a novel glycine antagonist at strychnine-sensitive receptors. The chemical structure of PMBA, possessing both a glycine moiety and a phosphono group, is quite different from that of strychnine.2 In the spinal motoneurone of newborn rats, glycine (100 gmM-1 mM) induced depolarizing responses in a concentration-dependent manner. PMBA effectively inhibited depolarizing responses to glycine and other agonists, such as taurine and P-alanine. The dose-response curves for glycine were shifted to the right in an almost parallel manner (pA2 value: 5.30 ± 0.23, n = 5) by PMBA which was about 60 times less potent than strychnine (pA2 value: 7.08 ± 0.21, n = 5) as a glycine antagonist. 3 PMBA (1-100 1AM) did not interact with modulatory glycine sites on N-methyl-D-aspartate (NMDA) receptors, which suggests a high selectivity of PMBA for strychnine-sensitive glycine receptors. At considerably high concentrations (0.1 mM-I mM), PMBA depressed responses to GABA (pA2 value: 3.57±0.24, n=3). 4 PMBA inhibited the binding of [3H]-strychnine to synaptosomes from adult rat spinal cords; the IC50 values of PMBA, glycine and strychnine were 8 ± 2, 9 + 3 and 0.08 ± 0.04 JM, respectively (n = 5) for[3H]-strychnine (4.8 nM). 5 PMBA is a central excitant drug with relatively high potency and selectivity and should be useful as a pharmacological probe for analysing the mechanisms underlying physiological functions of glycine receptors.

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Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development

Cited by 86 publications

References 16 publications

Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.

Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.

Localization of rat glycine receptor α1 and α2 subunit transcripts in the developing auditory brainstem

A novel antagonist, phenylbenzene ω‐phosphono‐α‐amino acid, for strychnine‐sensitive glycine receptors in the rat spinal cord

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