It is well established that presynaptic inhibition of primary afferents involves the activation of GABAA receptors located on presynaptic terminals. However, the source of GABA remains unknown.
In an integrated preparation of the spinal cord of the adult turtle, we evoked dorsal root potentials (DRP) by stimulating one dorsal root and recording another one. In the presence of a low concentration of tetrodotoxin (100nM) nerve impulses are abolished centrally but remain in nociceptive afferents. Under these conditions, a DRP generated by a non-spiking microcircuit remained. In the presence of blockers for the GABA transporters 1 & 3 (GAT), the DRP was strongly reduced. Since GAT3 is mainly expressed in astrocytes, our results suggest that these glial cells are part of the microcircuit that controls the activity of primary afferents. Addition of the potent chloride channel blocker NPPB also inhibited the DRP, suggesting that GABA could be released through a chloride conductance.
In a thick slice preparation from the spinal cord, we loaded superficial astrocytes with sulforhodamine 101 and the calcium indicator Oregon-green BAPATA-AM. The calcium signal of double stained cells was monitored with a 2-photon microscope. Stimulation of primary afferents evoked a transient increase in calcium concentration in a subset of cells. The response disappeared after addition of CNQX. This showed that primary afferents activate astrocytes.
In a thin slice preparation, we recorded astrocytes with the whole cell patch clamp technique. We found that the membrane of astrocytes expressed a NPPB sensitive current. We are currently investigating if this current is partly carried by GABA.