Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording

Abstract

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the currentclamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0-9.5 mV) and suppressed the action potentials. In the voltageclamp mode (V H , −70 mV), the application of NA produced an outward current that was blocked by Cs + and GDP-β-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre-and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC 50 , 20 µM), and the reversal potential was −88 mV. The outward current was mimicked by an α 2 -adrenoceptor agonist, clonidine, and suppressed by an α 2 -adrenoceptor antagonist, yohimbine, but not by α 1 -and β-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K + channels through α 2 -adrenoceptors, thereby producing an antinociceptive effect.

Direct GABAergic and Glycinergic Inhibition of the Substantia Gelatinosa from the Rostral Ventromedial Medulla Revealed byIn VivoPatch-Clamp Analysis in Rats

“…The time necessary for the solution to flow from the stopcock to the surface of the spinal cord was ϳ5 s. The drugs applied were 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Tocris Cookson, St. Louis, MO), DL-2-amino-5-phosphonovaleric acid (APV) (Sigma, St. Louis, MO), strychnine (Sigma), bicuculline (Sigma), methysergide maleate salt (Sigma), ketanserin (Sigma), ondansetron (LKT Laboratories, St. Paul, MN), and RS 23597-190 hydrochloride (Tocris Cookson). The effectiveness of the drugs applied in the same way under in vivo conditions was confirmed in our previous study (Sonohata et al, 2004).…”

“…Because the glutamate antagonists (CNQX and APV) affected neither the amplitude nor frequency of IPSCs during the RVM-ES, we concluded that the RVM-ES-eIPSCs were mediated by monosynaptic GABA A and/or glycinergic receptors. Our previous studies have confirmed that drugs perfused on the surface of the spinal cord act equally on SG neurons, which are located within a range of 30 to 150 m, as do those administered by bath application in slice preparations but not through systemic circulation (Furue et al, 1999;Sonohata et al, 2004).…”

“…The methods used for the in vivo patch-clamp recording were similar to those described previously (Furue et al, 1999;Narikawa et al, 2000;Sonohata et al, 2004). Under urethane anesthesia, oxygen was supplied through a nose cone.…”

“…Under urethane anesthesia, oxygen was supplied through a nose cone. Artificial ventilation of the pneumothorax was not performed, as reported previously (Sonohata et al, 2004). If a withdrawal reflex to the noxious stimuli appeared, an additional dose of urethane was given during surgery and the data collection period.…”

<b>Noradrenergic effects in rat sacral autonomic nucleus using in vitro slice patch-clamp r</b><b>ecordings </b>

“…Any delays of first firing to current injections were considered as a delayed firing types of neurons, and other cells were considered as regular firing types. In some cases, the recorded neurons were anatomically identified neurons (29,33) and some populations of interneurons (10), whereas it directly hyperpolarizes the majority of spinal dorsal horn neurons (24,26). A number of experiments have shown the involvement of NA in pelvic organ function and have indicated its multiple actions.…”