Kainate responses of leech Retzius neurons in situ and in vitro

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5-20 microM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 microM kainate. In RCs in situ the membrane responses to 5 microM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 microM kainate were similar to the membrane response to 20 microM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca2+]i) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca2+]i, indicating that the Ca2+ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca2+]i occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca2+ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 microM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca2+ and high-Mg2+ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2-7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca2+ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified.