Achim Berthele

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

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.

Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation

Metabotropic glutamate receptors are thought to play a role in the development and maintenance of spinal hyperexcitability resulting in hyperalgesia and pain. In this study we have used in situ hybridization to investigate the distribution of metabotropic glutamate receptors mGluR1-7 messenger RNA in the rat spinal cord in a model of inflammatory hyperalgesia. Hyperalgesia was induced in nine-day-old rats by exposure of the left hindpaw to an ultraviolet light source. Lumbar portions of spinal cords were removed from control and ultraviolet-treated animals. In situ hybridization with specific oligonucleotide probes was used to localize metabotropic glutamate receptor messenger RNAs. mGluR1, 3-5 and 7 subtype messenger RNA was detected in the gray matter of the spinal cord with distribution being specific for the different subtypes. A significant increase in the expression of mGluR3 messenger RNA was seen in cells of the dorsal laminae in both sides of the lumbar spinal cord. This increase was most pronounced in laminae II, III and IV but gradually decreased and disappeared by the third day of inflammation. In parallel with this, behavioural experiments revealed mechanical hyperalgesia in both hindlimbs after ultraviolet irradiation. There was no change in mGluR3 messenger RNA expression in the thoracic segments. No changes have been detected in the levels of expression of mGluR 1,2,4,5,7 subtype messenger RNA in spinal cords taken from hyperalgesic animals. These observations show that during ultraviolet irradiation induced inflammation, the synthesis of mGluR3 messenger RNA is altered suggesting that regulation of metabotropic glutamate receptor expression may be instrumental in plastic changes within the spinal cord during the development of hyperalgesia and pain.

Differential expression of rat and human type 1 metabotropic glutamate receptor splice variant messenger RNAs

The type I metabotropic glutamate receptor (mGlu1) messenger RNA and protein are known to be widely expressed in rat brain, but knowledge of the regional expression of splice variants other than mGlu1a is limited. Probes were designed for in situ hybridization that specifically recognize each of the carboxy-terminal splice variants mGlu1a, -1b, -1c and -1d. The novel rat mGlu1d sequence was obtained by polymerase chain reaction and the predicted protein is highly homologous to the human sequence but contains both conservative and radical substitutions and is slightly longer (912 vs 908 amino acids). Each rat mGlu1 splice variant messenger RNA was found in a unique expression pattern. The messenger RNA encoding mGlu1a was abundant in cerebellar Purkinje cells and in mitral and tufted cells of the olfactory bulb. Strong expression was also detected in hippocampal interneurons, and neurons of the thalamus and substantia nigra, while moderate expression was found in colliculi and cerebellar granule cells. The mGlu1b messenger RNA was strongly expressed in Purkinje cells, hippocampal pyramidal neurons, dentate gyrus granule cells and lateral septum, and moderately expressed in striatal, superficial cortical and cerebellar granule neurons. The mGlu1d messenger RNA was expressed in all regions where mGlu1a and -1b were detected; abundant in Purkinje cells, mitral and tufted cells, and hippocampal principal neurons and interneurons, strong in thalamus and substantia nigra, and moderate in lateral septum, cortex, striatum and colliculi. Human mGlu1 splice variant expression in the cerebellum matched that found for the rat. No specific signal was found with a probe capable of hybridizing to the rat mGlu1c splice junction, although another probe designed against a more 3 sequence of mGlu1c gave strong signals in the cerebellum and hippocampus, and moderate signals in thalamus and colliculi. It is concluded that mGlu1d messenger RNA is widely expressed, that mGlu1a and -1b messenger RNAs are expressed in almost complementary patterns and that formation of the mGlu1c splice junction is a rare event.

Cellular and Subcellular Distribution of NMDAR1 Splice Variant mRNA in the Rat Lumbar Spinal Cord

The regional distribution of alternatively spliced messenger RNA encoding the N‐methyl‐D‐aspartate (NMDA) receptor R1 subunit (NMDAR1) variants was examined by in situ hybridization in the rat lumbar spinal cord. Splice‐specific oligonucleotide probes [recognizing full‐length mRNA (NMDAR1‐1), deletion exon 21 (NMDAR1‐2), deletion exon 22 (NMDAR1‐3), combined deletion exons 21 and 22 (NMDAR1‐4) and mRNA which lacks (NMDAR1‐a) or contains exon 5 (NMDAR1‐b)] detected marked differences in abundance and distribution of N‐ and C‐terminal spliced variants. The NMDAR1‐a, NMDAR1‐2 and NMDAR1‐4 mRNAs were evenly distributed throughout all laminae of the dorsal and ventral horns. In the superficial dorsal horn NMDAR1‐b mRNA was preferentially detected in laminae II inner and III, while NMDAR1‐1 mRNA was restricted to laminae I to III. Large neurons in laminae IV and V contained mainly NMDAR1‐a, NMDAR1‐2 and NMDAR1‐4 mRNAs and occasionally NMDAR1‐b. The NMDAR1‐3 variant was only detected in very low abundance, being restricted to occasional cells in lamina I and II. In the ventral horn, motor neurons showed strong signals for NMDAR1‐a, NMDAR1‐b, NMDAR1‐2 and NMDAR1‐4 mRNAs. Serial sectioning through large motor neurons permitted the detection of multiple splice variants in single neurons. Analysis of the subcellular distribution of the mRNAs revealed that the NMDAR1‐1 mRNA was almost exclusively found in the cell nucleus, NMDAR1‐a mRNA was largely in the cytoplasm, while all other splice variants showed a homogeneous distribution between nucleus and cytoplasm. Comparison of the in situ hybridization images with functional analyses of heteromeric recombinant receptors will be necessary to ascertain whether splice variants of the NMDAR1 receptor subunit can account for some of the known electrophysiological properties of spinal cord neurons under physiological and pathophysiological conditions.

Flip and Flop variants of AMPA receptors in the rat lumbar spinal cord

The expression of eight messenger RNA splice forms encoding the Flip and Flop variants of AMPA receptor subunits GluR‐A to ‐D in the rat lumbar spinal cord was examined by in situ hybridization using specific oligonucleotides. In the dorsal horn (laminae I, II and III) the predominant mRNA was GluR‐B Flip. Much lower levels of GluR‐A Flip were found in lamina I and in superficial parts of lamina II outer. In the ventral horn, motor neurons expressed mainly GluR‐B Flip, GluR‐C Flip and Flop, and GluR‐D Flip. Serial sectioning through large motor neurons indicated that a given cell contained, for example, both GluR‐C Flip and Flop splice types.

Differential expression of group I metabotropic glutamate receptors in rat spinal cord somatic and autonomic motoneurons: Possible implications for the pathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of somatic, but not autonomic, motoneurons. The reason for this selective vulnerability is unknown. The pathogenesis of ALS is thought to involve glutamatergic excitotoxic mechanisms. While overactivation of ionotropic glutamate receptors may trigger excitotoxicity, we have previously shown that stimulation of group I metabotropic glutamate receptors (mGluRs) can exert neuroprotective effects on cultured motoneurons. Using in situ hybridization, we found a differential expression of group I mGluRs (mGluR1 and 5) in rat spinal cord. Autonomic motoneurons from the sacral parasympathetic Onufs nucleus and thoracic sympathetic neurons, which are spared in ALS, express high levels of mGluR5, while somatic motoneurons do not. In addition, mGluR1 mRNA is found only in smaller somatic motoneurons, which seem to be less vulnerable in ALS. Thus, differential mGluR expression might provide a possible clue to the selective vulnerability of different motoneuronal subpopulations in ALS.

Involvement of glutamatergic neurotransmission and protein kinase C in spinal plasticity and the development of chronic pain

Publisher Summary This chapter focuses on the study that induces monoarthritis in rats and investigates the expression patterns of ionotropic glutamate receptor subunit messenger RNA (mRNA), as well as the binding intensity and the laminar distribution of protein kinase C (PKC) in the spinal cord dorsal horn. Molecular cloning studies have demonstrated that ionotropic glutamate receptor constituents belong to the same gene family. Recombinant expression studies have showed that native glutamate receptors are supposed to be heterooligomeric assemblies of five subunits. Protein phosphorylation of ligand-gated ion channels appears to be another major mechanism to regulate the properties of the diverse types of glutamate activated receptor channels. As the functional properties of neurons are critically determined by their individual receptor subunit composition, the chapter investigates the distribution of ionotropic glutamate receptor subunit mRNA in spinal cord neurons. It may be assumed that a differential distribution of glutamate receptor subunits may be tailored to fit the neurons individual response characteristics that are required to participate in particular spinal neuronal circuits.

Axotomy of the sciatic nerve differentially affects expression of metabotropic glutamate receptor mRNA in adult rat motoneurons

Previous studies indicated that axotomy exposes motoneurons to glutamatergic excitotoxic stress and protection from glutamatergic overactivation might be crucial for survival. Depending on the experimental model and the subtype involved, activation of metabotropic glutamate receptors (mGluRs) may either enhance excitotoxicity or exert protective effects. To investigate a possible involvement of mGluRs in neuronal rescue mechanisms after axotomy we have monitored the distribution of mGluR mRNA with in situ hybridization in adult rat motoneurons 1, 2, 3, and 4 weeks after sciatic nerve transection. Motoneurons in sham-operated control animals expressed mGluR 1, 4, and 7 mRNA. The mGluR1 mRNA signal was reduced to 49.6+/-6.9% as compared to the contralateral side 2 weeks after axotomy and 31.2+/-8.3% after 4 weeks. The mGluR4 signal declined to 22.1+/-5.1% after 1 week and 10.2+/-1.6% after 2 weeks, remaining stable thereafter. During the entire observation period the mRNA for mGluR7 was not significantly altered. Axotomy did not change the overall number of motoneurons on the ipsi- or contralateral side. The differential regulation of mGluR subtypes may be part of an adaptive cell program that helps to rescue adult motoneurons from excitotoxic cell death during the stress induced by peripheral denervation.