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. 2019 Jan-Dec:15:1744806919875026.
doi: 10.1177/1744806919875026.

Differential expression of mGluRs in rat spinal dorsal horns and their modulatory effects on nocifensive behaviors

Masamichi Okubo  1 , Hiroki Yamanaka  1 , Kimiko Kobayashi  1 , Koichi Noguchi  1
Affiliations

Differential expression of mGluRs in rat spinal dorsal horns and their modulatory effects on nocifensive behaviors

Masamichi Okubo et al. Mol Pain. 2019 Jan-Dec.

Abstract

Glutamate is a neurotransmitter present in most excitatory synapses in the nervous system. It also plays a key role in the spinal cord’s physiological excitatory circuit and is involved in pathological neurotransmissions such as those observed in inflammatory and neuropathic pain conditions. The actions of glutamate are mediated by different types of ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Although expressions of iGluRs are well studied, those of mGluRs are not fully elucidated in the spinal cord. In this study, we examined the expressions of mGluRs (mGluR1-8) and investigated which mGluR subtypes can modulate pain transmission in the dorsal horn of the spinal cord using an inflammatory pain model. Reverse transcription-polymerase chain reaction revealed that mGluR mRNAs, except for mGluR2 and 6, were detected in the spinal cord. Double labeling analysis, in situ hybridization histochemistry with immunohistochemistry, was used to examine the distribution of each mGluR in neurons or glial cells in the lamina I–II of the spinal dorsal horn. mGluR1, 5, and 7 were generally, and 4 and 8 were frequently, expressed in neurons. mGluR3 was expressed not only in neurons but also in oligodendrocytes. We next examined the distribution of mGluR4 and 8 were expressed in excitatory or inhibitory neurons. Both mGluR4 and 8 were preferentially expressed in inhibitory neurons rather than in excitatory neurons. Furthermore, intrathecal delivery of CPPG((RS)-α-cyclopropyl-4-phosphonophenylglycine), an antagonist for mGluR 4 and 8, attenuated nocifensive behaviors and the increase in fos-positive-excitatory neurons of the dorsal horn induced by intraplantar injection of formalin. These findings suggest that mGluR4 and 8, which are preferentially expressed in inhibitory neurons, may play roles in the modulation of pain transmission in the spinal dorsal horn.

Keywords: Glutamate; dorsal horn; formalin; metabotropic glutamate receptor; nociception; spinal cord.

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Figures

Figure 1.
Figure 1.
Expression patterns of a series of mGluRs in the spinal cord. Electrophoresis images show mRNA expressions of mGluRs in L4–L5 of the spinal cord (a) and mGluR2 and 6 in the cortex for positive control (b). Different gene primers produced different lengths of PCR products: 776 bp (mGluR1), 655 bp (mGluR2), 699 bp (mGluR3), 454 bp (mGluR4), 861 bp (mGluR5), 713 bp (mGluR6), 490 bp (mGluR7), 658 bp (mGluR8), and 271 bp (GAPDH). (c) The drawing indicates the lamina structure of the half of spinal cord for dark field ISHH images. Dark field images (upper panels) show the expression pattern of Group I (d), Group II (e), and Group III (f) mGluRs in the spinal cord. Lower panels show higher magnification bright field images of the dorsal horn (lamina I–II) at the upper panels. Sections were counterstained by hematoxylin-eosin. Aggregation of grains indicates positive cells in ISHH. Scale bar = 500 μm in upper panels and 12.5 μm in lower panels (d–f).
Figure 2.
Figure 2.
Percentages of each mGluR colocalization with neurons in the lamina I–II of the spinal dorsal horn. (a–f) Double labeling ISHH study of each mGluR mRNA with immunohistochemistry of NeuN in the lamina I–II. Panels show colocalization of Group I (a and b), Group II (c), and Group III (d–f) mGluR mRNAs (aggregation of grains) with NeuN (brown staining). Aggregation of grains indicates the positive cells in ISHH. Brown-stained cells indicate IHC-positive cells. The percentages of colocalization of each mGluR mRNA-expressing cells in NeuN-positive cells (g). NeuN-positive cells in each mGluR mRNA-expressing cells (h). Sections were counterstained with hematoxylin. Scale bar = 10 μm in (a) to (f). Data are presented as mean ± standard error of the mean (n = 4 in (g) and (h)).
Figure 3.
Figure 3.
Double labeling analyses of mGluR3 mRNA with glial cell markers. ISHH of mGluR3 mRNA and IHC of GFAP (a), Iba1 (b), and Olig2 (c). Solid arrowheads indicate single-labeled cells using ISHH, open arrowheads indicate single-immunostained cells, and arrows indicate double-labeled cells. Sections were counterstained by hematoxylin. Scale bar = 12.5 μm.
Figure 4.
Figure 4.
Percentages of colocalization of mGluR3, 4, and 8 with lmx1b- or pax2-positive cells in lamina I–II. Representative images show colocalization of mGluR3 (left column), 4 (middle column), and 8 (right column) mRNAs with lmx1b (a) or pax2 (c) immunoreactive cells. Percentage of colocalization of each mGluR-expressing cells with lmx1b (b) or pax2 immunoreactive cells (d). Solid arrowheads indicate single-labeled cells using ISHH, open arrowheads indicate single-immunostained cells, and arrows indicate double-labeled cells. Sections were counterstained with hematoxylin. Scale bar = 12.5 μm. Data are presented as mean ± standard error of the mean (n = 4).
Figure 5.
Figure 5.
Inhibition of Group III mGluRs suppressed fos expressions in lmx1b-positive cells and nocifensive behaviors. Rats were pretreated with CPPG (500 nmol), L-AP4 (100 nmol), or PBS intrathecally 5 min before formalin (1.5% in 50 μl PBS) injection. Time course (a) and cumulative data (b) of the number of flinching after formalin injection with CPPG, L-AP4, or PBS pretreatment were measured. The number counted per 5-min interval in the initial hour postinjection period in (a). (b) The cumulative number of flinching during phase I (0–5 min) and phase II (5–60 min) after formalin injection. (c) Representative images of fos (upper lane, green) and lmx1b (middle lane, red) immunoreactive cells in the dorsal horn of controls (left column), vehicle-treated (second column), CPPG-treated (third column), and L-AP4-treated (right column) animals. (d) Bar graphs show the quantitative number of total fos-expressing cells (left), and that of fos-expressing cells in lmx1b-positive (middle) or lmx1b-negative cells (right) in the dorsal horn. Solid arrowheads indicate the merged cells. ctr: control; veh: vehicle. #<0.05 versus ctr, § <0.05 versus veh. Scale bar = 12.5 μm. Data are presented as mean ± standard error of the mean (n = 5–6). CPPG: (RS)-α-cyclopropyl-4-phosphonophenylglycine; L-AP4: L-(+)-2-amino-4-phosphonobutyric acid; n.s.: not significant.
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