Adenosine is an important neuromodulator in the CNS. Its actions are mediated via G-protein-coupled receptors classified as A1, A2_A, A2_B and A3. When administered intrathecally, agonists at the A1R (Koh et al. 1996) and A2_AR (Koh et al. 2000) decrease blood pressure and heart rate. Deuchars et al. (2001) determined the presence and function of A1Rs in the IML but since the neuronal circuitry mediating the A2_AR effect is unclear we studied the distribution and role of A2_ARs in the spinal cord.

For immunohistochemical studies, rats (100-150 g) were injected intraperitoneally with 0.1 ml 1 % Fluorogold and 7 days later were humanely killed by Sagatal (60 mg kg^-1 I.P.) and perfused transcardially with 4 % paraformaldehyde/0.1-0.5 % glutaraldehyde. Thoracic spinal cord slices (50 mm) were cut and incubated in anti-A2_AR antibody (Santa Cruz, Biotechnology) followed by a Cy3 conjugated secondary antibody. A2_AR immunoreactivity was observed throughout the spinal cord. Intense punctate staining was observed in a compact region of the IML. These punctate structures, suggestive of terminals, apposed Fluorogold labelled sympathetic preganglionic neurones (SPNs). For electro-physiology, thoracic spinal cord slices (250 mm) from terminally urethane-anaesthetised (2 g kg^-1 I.P.) 10- to 12-day-old rats were submerged in ACSF equilibrated with 95 % O₂ and 5 % CO₂ at 20°C. Whole-cell recordings were made from SPNs and interneurones in the IML, identified by electrophysiology and histology. Bath application of the A2_AR agonist CGS-21680 (1 mM) had no significant effect (Student’s paired t test) on EPSPs evoked by stimulation of the lateral funiculus but significantly enhanced the amplitude of bicuculline and strychnine sensitive IPSPs (control 5.1 ± 0.6 mV, CGS-21680 7.2 ± 0.8 mV, mean ± S.E.M., n = 19, P ▓le│ 0.005). This increase was blocked by the selective A2_AR antagonist, ZM-241385 (1 mM, n = 6). Application of CGS-21680 changed the paired pulse ratio (n = 6) of evoked IPSPs, but had no effect on input resistance suggesting a presynaptic site of action.

This study indicates that A2_ARs are found in the IML where their activation selectively increases inhibitory transmitter release from terminals presynaptic to IML neurones. These results suggest A2_ARs may play a vital role in the control of sympathetic outflow and provide evidence for a way in which activation of inhibitory A1Rs and/or excitatory A2_ARs causes similar cardiovascular responses.

This work was funded by the British Heart Foundation and the School of Biomedical Sciences, University of Leeds.

All procedures accord with current UK legislation.