Ontogeny of GABAB receptor subunit expression and function in the rat spinal cord

doi:10.1016/S0006-8993(03)02534-4

Brain Research

Volume 972, Issues 1–2, 16 May 2003, Pages 197-206

https://doi.org/10.1016/S0006-8993(03)02534-4 Get rights and content

Abstract

Little is known about the chronology of expression, cellular localization and function of GABA_B subunits in the developing rat spinal cord. In the present study, in situ hybridization, immunohistochemistry and quantitative RT-PCR analysis were used to examine this issue. At embryonic day 18, in situ hybridization reveals that all three transcripts, GABA_B(1a), GABA_B(1b), and GABA_B(2), are present throughout the gray matter. At postnatal day (PN) 2, while overall expression appears to decrease, it becomes more highly concentrated in motoneurons of the ventral horn. By PN 7, distinct subpopulations of cells expressing the transcripts become heavily expressed in motoneurons. Immunohistochemical analysis revealed that, unlike mRNA, GABA_B(1) protein is more highly concentrated in the dorsal horn as compared to the motoneurons. Analysis using RT-PCR demonstrates that in spinal cord GABA_B(1a) mRNA expression remains constant throughout development, GABA_B(1b) increases from PN 2 to adult, and GABA_B(2) decreases from PN 2 to adult. The distribution of functional GABA_B receptors, as measured by baclofen-stimulated [³⁵S]GTPγS binding, in the spinal cord during development generally follows the distribution of subunit expression, being widely distributed throughout the gray matter in embryonic spinal cord slices and becoming more concentrated in the dorsal horn during postnatal development, similar to the distribution of subunit proteins as measured by immunohistochemistry. These findings suggest that spinal cord GABA_B(1a), GABA_B(1b), and GABA_B(2) transcripts are differentially regulated during development with the chronology of this expression suggesting that GABA_B receptor subunits, in addition to forming functional GABA_B receptors, may have a trophic function or participate in synaptogenesis.

Introduction

A unique feature of the γ-aminobutyric acid-B (GABA_B) receptor is its heterodimeric structure [4], [5]. Inasmuch as heterodimerization of GABA_B(1) and GABA_B(2) subunits appears to be a prerequisite for receptor function, it would seem reasonable to assume that the expression of these proteins would occur in tandem to maintain the requisite stoichiometry. However, reports indicate this is not the case, with differences noted in the regional distribution of these subunits throughout the central nervous system [3], [5], [7], [8], [9], [10], [11]. Moreover, differential modifications in GABA_B mRNA have been noted in the rat spinal cord in response to pain [9], and the temporal expression of these subunits varies during development, with GABA_B(2) being virtually undetectable in adult rat spinal cord [5]. Indeed, it has been reported that GABA_B receptor activity, as measured by baclofen-stimulated [³⁵S]GTPγS binding in spinal cord membranes, declines dramatically during development, being absent in adult tissue [10]. While the decline in GABA_B receptor function can be attributed to the reduction in GABA_B(2) subunits over time, these findings are curious in light of the fact that other spinal cord responses to GABA_B receptor activation are robust in adult rats [9]. Thus, there is a lack of concordance between the anatomical and biochemical data on the one hand, and the functional results on the other.

The present study was undertaken to address this issue using a variety of techniques to study GABA_B subunit mRNA and protein, as well as receptor function in the developing rat spinal cord. The results indicate a differential rate of expression of mRNA and protein for GABA_B(1a) and GABA_B(1b) splice variants, and GABA_B(2), differences in the anatomical localization of these subunits and a lack of concordance between the levels of mRNA and protein. The data also reveal that while GABA_B(2) production diminishes during development, this protein is present in the adult spinal cord and that baclofen-stimulated [³⁵S]GTPγS binding can be detected in spinal cord slices, as opposed to membrane preparations. These findings are consistent with the notion that GABA_B subunits may serve multiple functions in spinal cord tissue.

Section snippets

Animals

Adult males and pregnant Sprague–Dawley rats (Sasco, Omaha, NE, USA) were housed individually on a 12-h light/dark cycle with food and water ad libitum. On embryonic day (ED) 18 the dam was anesthetized with pentobarbital (50 mg/kg) and the embryos removed and immediately decapitated. Postnatal day (PN) 2, 7, 15, and adult (>PN 28) rats were sacrificed by decapitation.

Following removal, spinal cords were immediately placed on dry ice or in 4% paraformaldehyde, with the latter subsequently

GABA_B receptor subunit expression

Expression of GABA_B(1a), GABA_B(1b) and GABA_B(2) is detectable throughout the spinal cord gray matter at ED 18, with little mRNA observed in the white matter (Fig. 1, Fig. 2, Fig. 3). However, at all ages examined, the expression of GABA_B(2) is less robust than for either of the GABA_B(1) subunits. During the first week after birth, mRNA expression for all three subunits appears to diminish, with neurons containing transcripts forming distinct subpopulations (Fig. 1, Fig. 2, Fig. 3). For example,

Discussion

As a heteromer, the GABA_B receptor is a structurally unique metabotropic site [4]. While heterodimerization appears to be absolutely required for receptor function, reports indicate that the subunit constituents are differentially expressed during development [7] and in response to pain [9]. Moreover, it has been reported that GABA_B receptor function in the spinal cord declines during maturation, possibly because of a reduction in expression of the GABA_B(2) subunit [10]. These and other data

Acknowledgements

This research was supported by the University of Missouri Research Board (UMRB). The authors would like to thank Dr. G.K.Y. Ng for the GABA_B receptor antibodies, Dr. Jeff Price for his assistance in the RT-PCR, and Jennifer Crane for her technical assistance with the immunohistochemistry.

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Research reportOntogeny of GABAB receptor subunit expression and function in the rat spinal cord

Abstract

Introduction

Section snippets

Animals

GABAB receptor subunit expression

Discussion

Acknowledgements

Dev. Brain Res.

Neuroscience

Neuroscience

Mol. Cell. Neurosci.

Neuropharmacology

Life Sci.

Mol. Cell. Neurosci.

Research report
Ontogeny of GABA_B receptor subunit expression and function in the rat spinal cord

GABA_B receptor subunit expression