CA1 pyramidal cells in hippocampal slices from humanely killed 12-day rats were whole-cell voltage clamped, usually with potassium gluconate-based internal solution. [Ca2+]i was monitored photometrically with low-affinity indicator fura-2FF. Extracellular photolytic release of L-glutamate was over an area of 200 µm diameter in 1 ms with a near UV flashlamp pulse from nitroindolinyl (NI) or methoxynitroindolinyl (MNI)-L-glutamate (Canepari et al. 2001). Intracellular IP3 was released from the P5 1-(2-nitrophenylethyl) ester of IP3 (Walker et al. 1989). Photolysis calibrations were as described in Canepari et al. (2001). Ionic currents were identified pharmacologically or by ion substitution.
The mGluR response was isolated from the ionotropic response by high concentrations of AMPA-kainate and NMDA-R antagonists, 100 µM NBQX and 400 µM D-AP5 or 200 µM CPP. The mGluR response activated within 1 s after L-glutamate release and was blocked by 1 mM MCPG. L-Glutamate concentrations of 30Ð50 µM resulted in mGluR responses that comprised two components, an early component that reversed at +5 mV and displayed voltage sensitivity, peak current occurring at around -30 mV. The late component reversed at -70 mV. Ion substitution indicated a potassium-selective channel and there was a coincident rise of [Ca2+]i associated with this component. Furthermore, the conductance was blocked by 14 mM TEA, 100 nM apamin, but not 100 nM iberiotoxin, indicating that it is likely to be a small conductance calcium-activated potassium channel.
Antagonists acting selectively at different mGluR-subtypes (CpCCOEt 20 µM, MCCG 1 mM) indicated that the late potassium conductance is linked to activation of a Group I mGluR and the early conductance to a Group II mGluR.
The possibility that IP3-evoked Ca2+ release from stores might underlie the late mGluR1 conductance was tested with photorelease of IP3. This generated a characteristic rise of [Ca2+]i associated with a potassium current that resembled the late mGluR current in time course and amplitude. The reversal potential of this current was -70 mV and it was blocked by antagonists 14 mM TEA and 100 nM apamin. Ion substitution identified a potassium conductance. The apparent affinity for IP3 was K = 12 µM and responses were seen at 1-2 µM, compared with 0.2 µM in autonomic tissues and 9 µM in Purkinje neurones (Ogden & Capiod, 1997). The IP3-evoked [Ca2+]i increase was suppressed but not enhanced by prior [Ca2+]i elevation by depolarisation, as found in Purkinje neurones.
Calcium appears to activate a potassium conductance, possibly due to small-conductance potassium channels. These act to inhibit neuronal excitability, by polarising the membrane towards EK and opposing excitation via fast transmission. Thus the photorelease of IP3 mimics the late component of the mGluR response, both in [Ca2+]i increase and activation of the potassium conductance, indicating that the PI pathway mediates the late inhibitory mGluR1 response.
We thank G. Papageorgiou, J.E.T. Corrie and D.R. Trentham for caged reagents.
All procedures accord with current UK legislation.