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