We have characterized a cationic membrane conductance activated by metabotropic glutamatergic and muscarinic cholinergic agonists in CA3 neurons in hippocampal slice cultures using the patch-clamp technique. When the potassium concentration in the superfusing fluid was raised above 5 mM, a biphasic current was observed in cells held at -60 mV in response to stimulation of postsynaptic metabotropic glutamate receptors (mGluRs) with 1S,3R-ACPD (50 microM) or muscarinic receptors with methacholine (MCh, 5 microM). The initial inward component was due to an increase in a cationic membrane conductance as determined by its reversal potential and its sensitivity to changes in extracellular K+ or Na+. The conductance underlying this current displayed no apparent voltage sensitivity over the range -120 to -50 mV. The response was reduced by extracellular application of Ba2+, Cd2+, Mg2+, or TEA, whereas extracellular Cs+ or loading cells with BAPTA or Cs+ did not affect the current. The effects of 1S,3R-ACPD were reversibly inhibited by bath-applied MCPG, an antagonist at mGluRs. Experiments with atropine and pirenzepine indicated that non-M1 muscarinic receptors mediated the MCh-induced current. A decrease in a resting leak potassium conductance (IK,leak) was responsible for the late component of the 1S,3R-ACPD- and MCh-induced response, seen as an outward current in the bathing solution with high K+ concentration. Loading cells with GDP beta S, GTP gamma S, or GTP did not alter the cationic current, while, in the same cells, the reduction in IKleak was abolished or irreversibly activated. Single-channel recordings of cationic channel activity in the cell-attached configuration provided evidence for the requirement of second messengers in coupling these receptors to the cationic channels. The data indicate that in addition to the previously described reduction of IK,leak, IM, and IAHP, both 1S,3R-ACPD and MCh activate a nonselective cationic conductance that is clearly revealed upon elevating external K+ concentration. This current is mediated by activation of metabotropic receptors, although no evidence could be obtained to show an involvement of G-proteins.

1. Effects of 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) acting at metabotropic glutamate receptors (mGluRs), and methacholine (MCh), acting at cholinergic muscarinic receptors, were investigated in CA3 neurones in hippocampal slice cultures using the patch-clamp technique.2. Both 1S,3R-ACPD (10 ,UM) and MCh (0 5 ,UM) activated an inward current associated with a decrease in membrane conductance. The current was observed when the slow calciumdependent after-hyperpolarizing current (IAHP) and the voltage-dependent current (IM)were not activated, reversed close to the reversal potential for K+ (EjK) (E = -92-8+ 10-7 and -89-2 + 8-6 mV for lS,3R-ACPD and MCh, respectively), varied linearly with membrane potential, and thus corresponds to a leak K+ current. 3. The decrease in K+ conductance elicited with 1S,3R-ACPD (50 /SM) was substantially reduced (> 70 %) with bath application of (RS)-a-methyl-4-carboxyphenylglycine (MCPG, 1 mM), a selective mGluR antagonist and was not mimicked by the enantiomer 1R,3S-ACPD (100 /1M). 4. The effects of lS,3R-ACPD and MCh were mediated by activation of G-proteins since no inward current could be elicited in GDP/?S-loaded cells (500 /uM). When cells were dialysed with GTP (100,UM) or GTPyS (250,UM), however, the amplitude of the current was significantly enhanced. 5. These findings provide evidence that G-proteins couple the activation of mGluRs and muscarinic receptors to a decrease in leak K+ conductance.Activation of metabotropic glutamate receptors (mGluRs) and muscarinic cholinergic receptors in the CNS initiates a variety of intracellular processes, such as modulation of second messenger levels and ion channel activity, which are thought to be important in synaptic plasticity and regulation of neuronal function (Nicoll, Malenka & Kauer, 1990;Schoepp & Conn, 1993 METHODSExperiments were performed using organotypic hippocampal slice cultures prepared as previously described (Giihwiler, 1981

1. Desensitization of a metabotropic response was investigated in CA3 pyramidal neurons in hippocampal slice cultures using the patch‐clamp technique. 2. 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylate (1S,3R‐ACPD), an agonist at metabotropic glutamate receptors (mGluRs), and metacholine (MCh), an agonist at muscarinic receptors, induced a cationic current that appears to be activated through a G‐protein‐independent transduction process, as previously shown. Prolonged or repetitive bath application of agonists led to rapid desensitization of the cationic current with a time constant of approximately 20 s. 3. Complete recovery from desensitization was observed within 6 min. 4. These responses mediated by mGluRs and muscarinic receptors cross‐desensitized. 5. Preventing the activation of G‐proteins by loading cells with GDP beta S strongly reduced or suppressed desensitization, and resulted in a sustained inward cationic current. When cells were filled with GTP gamma S to irreversibly activate G‐proteins, the desensitization process was enhanced such that a first application of agonist caused a markedly reduced response. 6. These results show that a cationic current induced by metabotropic agonists in hippocampal pyramidal cells undergoes apparent desensitization and suggests that this process occurs through a G‐protein‐mediated inhibition of the underlying membrane conductance.

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