Research report
Distribution and developmental changes in metabotropic glutamate receptor messenger RNA expression in the rat lumbar spinal cord

https://doi.org/10.1016/S0165-3806(98)00156-4Get rights and content

Abstract

Using in situ hybridisation, the regional distribution of primary transcripts and splice variants of all metabotropic glutamate receptor subtypes (mGluR) currently known to be expressed in the spinal cord have been studied in the lumbar enlargement of the rat spinal cord. In adult animals, the messenger RNA of the mGluR subtypes 1, 5, 3, 4 and 7 were differentially expressed. The transcripts of mGluR1 and 5 were most abundant with mGluR5 messenger RNA being concentrated in the superficial dorsal horn. In contrast, the mGluR2 transcript was not detectable with the sensitivity of the method. Secondly, age related changes (postnatal days 1, 7, 12, 21) in the postnatal expression of mGluR1–5 and 7 transcripts have been investigated. mGluR1 and 7 messenger RNA showed a general decrease in spinal expression from postnatal day 1 to day 21. Quantitative densitometry showed high mGluR3 and 5 messenger RNA levels especially in the superficial dorsal horn at birth, however these levels decreased with age. In addition to changes in density, the regional distribution of mGluR3 messenger RNA was altered with postnatal development. Up to postnatal day 12, mGluR3 messenger RNA expression was almost exclusively restricted to the spinal grey matter, but with postnatal day 21 a strong additional expression in the white matter occurred. Distribution of mGluR4 messenger RNA showed little change in the dorsal horn, however motoneuronal expression emerged during development. These changes may suggest different roles for mGluRs in the maturation of spinal transmission of the rat nervous system.

Introduction

There is substantial information regarding the distribution of individual metabotropic glutamate receptor (mGluR) subtype mRNA and their splice variant expression in the mammalian brain. Metabotropic glutamate receptors are divided into three groups [48]based on second messenger coupling, their amino acid homology and agonist interactions. Group I comprises mGluR1 and 5, group II mGluR2 and 3, and group III mGluR4, 6, 7 and the recently cloned mGluR8. Functionally, it is accepted that the mGluR family is involved directly in neurotransmission in some cases but predominantly contributes to the acute or long-lasting modulation of synaptic transmission in the central nervous system 12, 35. Thus it is not surprising that changes in mGlu receptor function have been suggested to underlie changes during development and neuropathological conditions 6, 9, 40, 57.

The presence of mGluR mRNAs in the adult rat spinal cord has already been demonstrated by in situ hybridisation 42, 45, 46, 55, but in comparison to the rat brain, the differential regional distribution of mGluRs in the spinal cord has not yet been investigated in greater detail [11].

The functional roles of spinal mGluRs are currently a matter of intense research. There is mounting evidence that mGluRs are involved in the spinal processing of somatosensory information, especially contributing to the development of spinal hyperexcitability following noxious stimulation 4, 5, 8, 39. It is well known that both sensory and motor functions in the spinal cord undergo an intensive maturation process after birth in the rat 13, 15, 16, 17, 18, 19, 33, 54, and it has been suggested that receptor–ligand systems could be selectively regulated during this maturation [26]. Changes in different transmitter systems such as ionotropic glutamate receptors, GABAA and glycine 24, 30, 34, 62have already been described during spinal development.

Concerning mGluRs, knowledge about their expression in the central nervous system has mainly been derived from studies in the adult animal, but there is evidence that the pattern of distribution is age dependent. Catania et al. [10]have described changes in the distribution of the mGluRs in the developing brain, and changes in mGluR expression have been associated with functional consequences during development, e.g., synaptogenesis. However, there is no similar study to date concerning changes of mGluR expression in the spinal cord during development.

This present study addresses these issues by using in situ hybridisation with oligonucleotide probes to characterise the mRNA expression of mGluR subtypes in the lumbar spinal cord and their changes during the course of postnatal development.

Section snippets

Tissue preparation

Lumbar portions (L4–5 segments) of spinal cords were removed from terminally anaesthetised (Enflurane), non-perfusion-fixed Sprague–Dawley rat pups on postnatal (PN) days 1, 7, 12 and 21 (n=3 per group) and from adult animals (10 weeks; n=6). Blocks of spinal cord were rapidly frozen on dry ice. Sections (14 μm) were cut on a cryostat at −20°C, and thaw-mounted onto poly-l-lysine coated glass slides. The sections were fixed in 4% paraformaldehyde and stored in 96% ethanol until hybridisation.

In situ hybridisation

Distribution of mGluR subtype mRNA in the adult lumbar spinal cord, expression of splice variants

mGluR1, 5, 3, 4, 7 subtypes showed mRNA expression in the lumbar spinal cord (Fig. 1). The expression of all mGluR1 (1a, 1b, 1c, 1d), mGluR5 (5a, 5b), mGluR4 (4a, 4b), and mGluR7 (7a, 7b) splice variants were investigated. In the spinal cord, the differences between splice variant expression patterns of mGluR1 and 5 were more marked in comparison to mGluR4 and 7. mGluR2 showed very low mRNA expression only in the superficial dorsal horn, which was barely detectable with the methods used here.

Discussion

This study outlines a broad spectrum analysis of mGluR subtype mRNA expression in the lumbar spinal cord of adult rats and during postnatal development (PN days 1–21).

Acknowledgements

AB and TRT were supported by SFB 391 and SJB was supported by the Physiological Society and a BBSRC CASE award. The first two authors contributed equally to this work.

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