SUMMARY1. A voltage-clamp study was made of some properties of the non-synaptic hyperpolarization-activated C1-conductance recently described in Aplysia neurones loaded with Cl-ions (Chesnoy-Marchais, 1982). The experiments were performed on an identified family of neurones, which present cholinergic responses allowing an easy measurement of the equilibrium potentials of Cl-(EC1) and K+ ions (EK).2. The Cl-selectivity ofthe hyperpolarization-activated conductance was deduced from four observations: (1) the extrapolated reversal potential of the hyperpolarization-activated current, Er, was close to the reversal potential ofthe cholinergic Cl-response, which is the equilibrium potential for Cl-ions, EC1. 3. The steady-state Cl-conductance (ass) increases steeply with hyperpolarization.The kinetics of activation and deactivation are exponential and are characterized by the same voltage-dependent time constant (r), of the order of a few seconds or fractions of seconds. The curves g.8(V) and r( V) can both be fitted by a two-state model in which the rate constants are exponential functions of the membrane potential (e-fold change for 12-16 mV).4. The Cl-current is much more affected by changes of the intracellular Clconcentration than predicted simply from the change in Cl-driving force. Both the conductance and the time constant of activation are strongly modified. Modifications of the extracellular Cl-concentration do not always alter the amplitude of the hyperpolarization-activated Cl-current, but systematically affect its kinetics.5. The hyperpolarization-activated current is abolished after prolonged exposure of the cell to an artificial sea water where N03-ions replace Cl-ions, as well as after intracellular injections of N03-ions.6. Increasing the external pH shifts the g.(V) and r(V) curves to the left.Lowering the external pH has reverse but less pronounced effects. 7. In cells which were not loaded with Cl-ions and did not present the hyperpolarization-activated Cl-current, this current could be detected if the D. CHESNO Y-MARCHAIS hyperpolarizing jump was preceded by short depolarizing pulses. In cells which were loaded with Cl-ions, the Cl-current became larger after a short depolarizing pulse.In the presence of extracellular C02+ ions, depolarizing pulses no longer increased the Cl-current.8. The Cl-current is not affected by extracellularly applied DIDS (4,4'-diisothiocyano-2,2'-disulphonic acid stilbene), but is markedly reduced by intracellular injection of DIDS.9. Extracellular Cs+ ions, which have been reported to block some cationic hyperpolarization-activated inward currents, do not reduce the hyperpolarizationactivated Cl-current. High concentrations of Cs+ produce complex effects which are probably due to an increased synaptic activity, but the hyperpolarization-activatedCl-current persists after complete substitution of the extracellular and intracellular monovalent cations by Cs+.

We have evaluated the influence of the secretory phenotype of presynaptic boutons on the accumulation of postsynaptic glycine receptors (GlyRs), type A GABA receptors (GABA(A)Rs), and gephyrin clusters. The cellular distribution of these components was analyzed on motoneurons cultured either alone or with glycinergic and/or GABAergic neurons. In motoneurons cultured alone, we observed gephyrin clusters at nonsynaptic sites and in front of cholinergic boutons, whereas glycine and GABA(A) receptors formed nonsynaptic clusters. These receptors are functionally and pharmacologically similar to those found in cultures of all spinal neurons. Motoneurons receiving GABAergic innervation from dorsal root ganglia neurons displayed postsynaptic clusters of gephyrin and GABA(A)Rbeta but not of GlyRalpha/beta subunits. In motoneurons receiving glycinergic and GABAergic innervation from spinal interneurons, gephyrin, GlyRalpha/beta, and GABA(A)Rbeta formed mosaics at synaptic loci. These results indicate that (1) the transmitter phenotype of the presynaptic element determines the postsynaptic accumulation of specific receptors but not of gephyrin and (2) the postsynaptic accumulation of gephyrin alone cannot account for the formation of GlyR-rich microdomains.

SUMMARY1. The whole-cell voltage-clamp mode of the patch-clamp technique was used to investigate the presence of voltage-gated inward currents in osteoblasts from newborn rat calvaria.2. In K+-free solutions, three kinds of inward currents could be activated by depolarization: a voltage-gated Na+ current and two different types of Ca21 currents.3. The Na+ current was activated by depolarization above -40 mV in all the cells. It was reduced by half by 10 nM-TTX (tetrodotoxin).4. In an isotonic Ba2+ external solution containing TTX, and with a Cs-EGTA internal solution buffered at pCa 8, depolarizing jumps induced both a transient Ba21 current and a sustained Ba21 current. The relative proportions of these two currents varied greatly among cells.5. The transient and sustained Ba2+ currents differ with respect to their time course and their voltage dependence.6. The depolarization-activated inward currents were also observed under more physiological conditions, in the presence of only 2 mM-external Ca2+ and with a K+ internal solution buffered at pCa 7.7. A few records obtained in current clamp showed that it is possible to induce action potentials in osteoblasts.

The sensitivity of various types of recombinant glycine receptors (GlyRs) to ICS-205,930 was studied by fast perfusion in Xenopus laevis oocytes. This compound has previously been shown to potentiate glycine responses in rat spinal neurons between 10 nM and 1 microM, independently of its 5-HT(3) antagonist properties. In contrast, submicromolar concentrations of ICS-205,930 failed to affect responses of homomeric GlyRs formed from human alpha1 or alpha2 subunits, and micromolar concentrations (1-20 microM) acted differentially on the two types of homomeric receptors, potentiating the responses to glycine (10-20 microM) of alpha1 homomeric GlyRs and inhibiting the responses of alpha2 homomeric GlyRs. GlyRs beta subunits markedly influenced the modulations induced by ICS-205,930. In oocytes expressing alpha1/beta or alpha2/beta heteromeric GlyRs, low concentrations of ICS-205,930 (20 nM-1 microM) induced a potentiation of glycine responses that was counteracted by an inhibitory effect at higher concentrations. Thus, GlyRs beta subunits reduce by 2 orders of magnitude the concentration range potentiating alpha1-containing GlyRs and are required for potentiation of alpha2-containing GlyRs. These results reveal a new high-affinity potentiating site on GlyRs, to which beta subunits participate. The difference in ICS sensitivity between alpha1 and alpha2 GlyRs cannot be explained by their difference in TM2 segment and extracellular domains partly conserved between glycine and 5-HT(3) receptors are probably involved in the interaction of some 5-HT(3) antagonists with GlyRs.

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SUMMARY1. Two kinds of single channels, carrying inward currents even above the Na and Cl ion equilibrium potentials, were observed in outside-out patches from Aplysia neurones bathed in K-free internal and external solutions. The channel carrying the larger elementary current has been studied in detail.2. When the internal solution contained mainly CsCl, this channel usually inactivated during the first minutes following isolation of the membrane patch. However, when the internal solution contained NaCl instead of CsCl, the channel remained functional during several hours, thus allowing the present study.3. Na-Tris, NaCl-mannitol and Ca-Ba external substitution experiments showed that the channel studied is much more permeable to divalent cations than to sodium ions. 12. The possible relationships between this channel and previously described 'Ca channels' and 'slow inward currents' are discussed.

During whole-cell recording of rat osteoblastic cells with high-Cl- internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl- channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs+ ions but is blocked in a voltage-dependent manner by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) 1 mM and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mM. Cd2+ ions are potent blockers of this current, blocking completely above 300 microM. The amplitude of the Cl- current activated by a given hyperpolarization increases during the first 10-20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl- channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl- current. However, this hyposmolarity-induced Cl- current and the hyperpolarization-activated Cl- current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl- current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl- current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.

SUMMARY1. A voltage clamp study was made of the K+ permeability increases produced in certain identifiable neurones of the cerebral ganglion of Aplysia by any one of three distinct agonists (carbachol, histamine and dopamine).2. The three K+ responses involve three distinct receptors, as shown by the selective effects of reversible antagonists (Gruol & Weinreich, 1979a) as well as by the differential, irreversible effects of trimethyloxonium (TMO) ions.3. Prolonged exposure of the neurones to one of the agonists reduces the response to the same agonist (desensitization) and, over the low concentration range, doubling the concentration of an agonist leads to supra-linear summation (potentiation).4. Prolonged exposure of the neurones to one of the agonists also reduces the response to the other agonists (cross-desensitization) and combined application of two agonists reveals cross-potentiation.5. The time course of desensitization (onset and decay) was the same for the histamine and carbachol responses and, except at very high concentration, was indistinguishable from that of cross-desensitization. Likewise, potentiation was similar in the two agonist systems and did not differ significantly from cross-potentiation.6. The results can be interpreted by assuming that the responses to the three agonists involve specific steps followed by common reaction steps, and that some of the common reaction steps control both potentiation and desensitization.7. The responses to carbachol and histamine differ in their voltage sensitivity. This suggests that one or more of the specific steps are voltage-sensitive.8. Although an increase of the intracellular Ca2+ concentration can itself open K+ channels, and also inhibit the responses to the three agonists, an increase of internal Ca2+ does not appear to play an important role either in the development of the response or in the desensitization process.