No abstract

1. Whole-cell recordings were made from cultured cerebellar granule neurons using perforated patch clamp techniques. The primary cultures were prepared using 6-to 9-day-old Sprague-Dawley rats. 2. Neurons in culture for less than 48 h possessed resting membrane potentials of -29 mV.However, neurons in culture for 7 days had much more hyperpolarized resting membrane potentials (-89 mV). Over the same period, these neurons developed an additional component of outward current. 3. This non-inactivating current was activated by depolarization, exhibited outward rectification and reversed close to the potassium equilibrium potential. The kinetics of activation and deactivation were very rapid.4. Muscarine ((+)-muscarine chloride) reversibly inhibited the current with an ECQ0 of 0-17 /M. The inhibition by muscarine was unaffected by pre-incubation for 17-20 h with 120 jug ml-' pertussis toxin.5. The current and its inhibition by muscarine were unaffected by 100 /SM Cd2+. In Ca2+-free conditions, the current was significantly larger than in 0 5 mm Ca2+, but inhibition by 10 /M muscarine was significantly reduced. 6. The standing outward current was not obviously affected by 50/uM 5-HT, 50 ,UM noradrenaline, 50 /uM 2-chloroadenosine or 5 mm tetraethylammonium. It was reduced by 10 /LM La3+, 10 ,uM Zn2+ and 1 mm Ba2+.7. Muscarinic agonists increased the input resistance of neurons and shifted the zero current level in the depolarized direction when voltage clamped. This enhanced excitability was evident under current clamp, where 10 /LM muscarine depolarized granule neurons such that action potentials became evident.

Tetanic stimulation of parallel fibres (PFs) produces a slow EPSP (sEPSP) or slow EPSC (sEPSC) in Purkinje neurones (PNs), mediated by type 1 metabotropic glutamate receptors (mGluR1). The conductance change underlying the sEPSP was investigated with rapid photolytic release of L‐glutamate from nitroindolinyl (NI)‐caged glutamate with ionotropic glutamate receptors blocked, and showed a slow mGluR1‐activated cation channel. In cerebellar slices rapid photolytic release (t1/2 < 0.7 ms) of 7‐70 μM L‐glutamate on PNs voltage clamped at −65 mV activated first a transient inward current, peaking in 8 ms, followed by a slow inward current with time course similar to the PF sEPSP, peaking at −1 nA in 700 ms. The initial current was inhibited by 300 μM threo‐hydroxyaspartate (THA) and did not reverse as the potential was made positive up to +50 mV, suggesting activation of electrogenic glutamate uptake. The slow current was inhibited reversibly by 1 mM (R,S)‐MCPG or the non‐competitive mGluR1 antagonist CPCCOEt (20 μM), indicating activation of metabotropic type 1 glutamate receptors. The mGluR current was associated with increases of input conductance and membrane current noise, and reversed close to 0 mV, indicating activation of channels permeant to Na+ and K+. The sEPSC was not blocked by Cd2+, Co2+, Mg2+ or Gd3+ ions, by the inhibitor of hyperpolarisation‐activated current (IH) ZD7288, or by the purinoceptor inhibitor PPADS. Activation was not affected by inhibitors of phospholipase C (PLC) or protein kinase C (PKC), nor mimicked by photorelease of InsP3 or Ca2+. The results show that mGluR1 in PNs produces a slow activation of cation‐permeable ion channels which is not mediated by PLC activation, Ca2+ release from stores, or via the activation of PKC.

The effects of the trivalent cation, lanthanum (La3+) on voltage-dependent K+ conductances were studied in rat isolated cerebellar granule neurons under whole-cell voltage-clamp conditions. La3+ at low micromolar concentrations caused a pronounced enhancement in the outward current evoked by depolarising steps from -50 mV, with the apparent recruitment of an inactivating component. The steady-state inactivation curve for the transient outward current, evoked by depolarising steps from -140 mV, was shifted by approximately 40 mV in the depolarising direction by 10 microM La3+, with a slight increase in the slope factor. The kinetics of activation and inactivation were slowed in the presence of La3+. A shift of 10 mV in the depolarising direction was seen for the activation curve of the delayed rectifier current in the presence of 10 microM La3+. These results indicate that La3+ has a potent effect on the gating characteristics of voltage-activated K+ currents. This effect cannot be explained by surface charge considerations.