Proceedings of The Physiological Society

Trinity College Dublin (2003) J Physiol 551P, C50


GluR1 localisation and responses to 5-fluorowillardiine in sympathetic preganglionic neurones of rat thoracic spinal cord

Susan A. Deuchars *, Ruth E. Brooke * and Ida Llewellyn-Smith †

* School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK and † Flinders University, South Bedford, Australia

AMPA receptors are composed of GluR1-4 subunits that form homomers or heteromeric complexes of any two of these four subunits. The pharmacology of these receptors is dependent on the subunit composition and one agonist, 5-fluorowillardiine (5-F-Will) has a much higher affinity for GluR1 (Ki 14.7 nM) and 2 (Ki 25.1 nM) than GluR4 (305 nM). Here we sought to determine whether the GluR1 subunit is localised and functional in sympathetic regions of the spinal cord.

We first used immunohistochemistry to determine whether GluR1 receptor subunits were located in the spinal cord. Male Wistar rats (200 g) were injected intraperitoneally with Fluorogold (0.1 ml of 1 % in 0.9 % NaCl, Fluorochrome Int.) 5-7 days prior to being humanely killed by anaesthetizing with sodium pentobarbitone (60 mg kg-1, I.P.) and perfusing transcardially with 4 % paraformaldehyde in 0.1 M phosphate buffer. Vibrating microtome sections (50 µm) of upper thoracic spinal cord (T2-T5) were incubated in rabbit anti-GluR1 (1:150, Chemicon) for 24 h prior to visualisation with Cy3-conjugated donkey anti-rabbit (1 : 800, Jackson Immunoresearch). GluR1 immunoreactivity was detected in approximately 50 % of Fluorogold-labelled sympathetic preganglionic neurones (SPNs) in the intermediolateral cell column (IML) while fluorogold-negative neurones in the IML and lamina X were also immunopositive.

Using visualised whole cell patch clamp techniques the responses of identified SPNs in the IML to different concentrations of 5-F-Will were determined. Rats (12-15 days) were anaesthetized with urethane (2 g kg-1, I.P.) and after removal of the upper thoracic spinal cord were humanely killed by decapitation. Slices (250 µm) were superfused with oxygenated aCSF (composition (mM): NaCl (124); NaHCO3 (26); KCl (3); MgSO4 (2); NaH2PO4 (2.5); CaCl2 (2); glucose (10)) at room temperature. Eight of 21 neurones depolarised to bath applied 15-30 nM 5-F-Will (13.8 ± 2.2 mV; mean ± S.E.M.) indicating that these neurones contained GluR1/2 subunits. The remaining 13 SPNs were unaffected by this concentration but were depolarised by 300 nM 5-F-Will (20.2 ± 2.5 mV) consistent with the presence of GluR4 but not the GluR1/2 subunits. Since parallel investigations indicate that GluR2-immunoreactive SPNs are located more caudally and the proportion of SPNs responding to 15-30 nM 5-F-Will corresponds with the percentage of GluR1-immunopositive SPNs, the current data suggest that GluR1 subunits are indeed present and functional in SPNs of the upper thoracic spinal cord.

We acknowledge the support of the British Heart Foundation.

Where applicable, experiments conform with Society ethical requirements