GABAA receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord

Our goal was to test the following hypotheses: 1) GABAA receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABAB receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABAA receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC50] 3 μM) and isoguvacine (EC50 4.5 μM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABAA receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC50] 2 μM) and picrotoxin (IC50 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABAA receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl−, low Ca2+, Ca2+ channel blockers and N-methyl-d-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na+-K+-2Cl− cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABAA receptors. The GABAB agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC50 1.5 μM), whereas the GABAB receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC50 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABAB receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABAA receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABAB receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABAA receptors and inhibiting GABAB receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.