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Proceedings of The Physiological Society
University of Leeds (2002) J Physiol 544P, S002
Are sympathetic preganglionic neurones (SPNs) directly inhibited by GABAergic neurones in the central region of the rat spinal cord?
S.A. Deuchars, C.J. Milligan and J. Deuchars
School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK
The activity of SPNs is mainly influenced by inputs that originate both supraspinally and at the level of the spinal cord. To date, the contribution of specific groups of local interneurones to SPN activity remains to be elucidated. We utilised in situ hybridisation and electrophysiological studies to determine whether there are local GABAergic neurones that can influence the activity of SPNs.
For in situ hybridisation, rats (150 g) were anaesthetised with Sagatal (60 mg kg-1 I.P.) and perfused transcardially with 4 % paraformaldehyde in 0.1 M phosphate buffer. Spinal cord sections (30 mm) were hybridised with digoxigenin-UTP-labelled GAD65 or GAD67 sense and antisense RNA probes using a modified version of the manufacturer's protocol (www.biochem.roche.com). mRNA for GAD65 and GAD67 was visualised in neurones in a number of regions in the spinal cord but one notable compact group of GAD mRNA positive neurones was located dorsal and lateral to the central canal at the edge of the grey matter.
For electrophysiology, thoracic spinal cord slices (250 mm) were cut from 10-14 day rats anaesthetised with urethane (2 g kg-1, I.P., then humanely killed) and submerged in oxygenated ACSF at room temperature. Whole-cell recordings were obtained from SPNs identified electrophysiologically and histologically. Stimulating electrodes were placed in a position corresponding to the GABAergic neurones in the central region and in the lateral funiculus (lf) for comparison. Drugs were applied to the bathing medium.
Stimulation of the central region elicited IPSPs with short constant latencies in all SPNs tested (n = 12) that were maintained in the presence of the excitatory amino acid antagonist kynurenic acid (500 mM, sufficient to block lf mediated EPSPs). This suggested that the IPSPs were elicited by activating a monosynaptic pathway. Similar IPSPs could not be elicited by stimulating the white matter dorsal to the central region or by stimulation of the contralateral IML or lateral funiculus. Central region evoked IPSPs were always antagonised by application of the GABAA receptor antagonist bicuculline (5 mM; -5.4 ± 0.3 to -0.6 ± 0.1 mV, mean ± S.E.M. n = 7) and no further effect was observed on addition of strychnine. In contrast, IPSPs elicited by lf stimulation were antagonised in part by bicuculline (-5.5 ± 0.9 to 2.2 ± 0.8 mV) and were further blocked by strychnine (2 mM, 2.2 ± 0.8 to 0.1 ± 0.1 mV, n = 6).
These data show that SPNs are directly inhibited by stimulation in the central region of the spinal cord and this may be due to activation of a compact group of GABAergic neurones in this region. This theory is undergoing further investigation in the laboratory.
We thank The British Heart Foundation and The Wellcome Trust.
All procedures accord with current UK legislation.
Where applicable, experiments conform with Society ethical requirements