Evidence suggesting the presence of GluR6-containing kainate receptors on neonatal rat motoneurones J.C.A. More, H.M. Troop & D.E. Jane. MRC Centre for Synaptic Plasticity, Dept. of Pharmacology, University of Bristol, Bristol BS8 1TD.

Print abstract Search PubMed for: More JCA Troop HM Jane DE

We have previously reported that kainate-evoked responses can be seen on neonatal rat motoneurones in the presence of the AMPA receptor antagonist GYKI53655 and (S)-3-(4-carboxybenzyl)willardiine (UBP282), which blocks both AMPA and GluR5-containing kainate receptors (More et al., 2002). The aim of this study is to use pharmacological and immunohistochemical techniques to provide evidence that at least part of this kainate-evoked response is due to the activation of GluR6-containing kainate receptors.

Recordings were made from ventral roots of hemisected spinal cords from 2-5 day old rats, in the presence of 100 nM tetrodotoxin (More et al., 2002). 1 min applications of kainate (2 µM) and AMPA (0.7 µM) were made in control conditions, in the presence of 200 µM UBP282 and in the combined presence of UBP282 and CNQX (50 µM) (antagonists pre-incubated for 30 min). For binding studies, HEK293 cells were transiently transfected with GluR6 cDNA then membranes harvested 2 days later as described by Swanson et al. (1997). Membranes were incubated with 10 nM [³H]kainate in 10 mM HEPES buffer in the presence of competing ligand for 1 h at 4 °C, followed by vacuum filtration. All results are expressed as mean ± s.e.m.

Immunohistochemistry was performed using 8 µm cross-sectional slices from the lumbar region of 5 day old rat spinal cords which had been embedded in paraffin wax after formalin fixation. Localisation studies were carried out as described previously (Miller et al., 2001) using antibodies obtained from Santa Cruz Biotech. Final antibody dilutions in antiserum were: GluR6/7 (goat polyclonal; 1:1500), GluR7 (goat polyclonal 1:750). Antibody incubations were carried out overnight at 4 °C. The primary antibody was detected using biotinylated secondary antibody then visualisation was carried out using a diaminobenzidine chromagen.

In the presence of 200 µM UBP282 responses to kainate and AMPA were reduced to 62 ± 7 and 4 ± 2 % of control values, respectively (n=3). Then, in the additional presence of 50 µM CNQX, the responses to both kainate and AMPA were abolished (n=3). In radioligand binding studies using GluR6-transfected HEK293 cells, CNQX gave an IC₅₀ value of 1.32 ± 0.09 µM (n=3) for the displacement of [³H]kainate binding whereas UBP282 had an IC₅₀ > 1 mM (n=3).

When immunohistochemistry was carried out using the GluR6/7 antibody, light microscopic analysis showed strong staining around the motoneurones in the lamina IX region of the ventral horn whereas when the GluR7 antibody was used, this type of staining was not observed (n=6). These results imply that GluR6-containing kainate receptors are present on these motoneurones.

As UBP282 is inactive at GluR6 yet blocks GluR5-containing kainate receptors and the AMPA receptors on motoneurones, the residual response to kainate in the presence of this antagonist may be due to the activation of GluR6-containing kainate receptors. These preliminary results are consistent with the immunohistochemical analysis, which showed the localisation of GluR6 protein on the motoneurones in lamina IX of neonatal rat spinal cords.

This work was supported by the MRC and Tocris Cookson Ltd.

More, J.C.A., Troop, H.M. & Jane, D.E. (2002) Br. J. Pharmacol 137, 1125-1133.
Miller, J.C., O'Neill, M.J. & Jane, D.E. (2001) Br. J. Pharmacol. 133, 190P.
Swanson, G.T., Gereau IV, R.W., Green, T. et al. (1997) Neuron 19, 913-926.