Konomi Koyano

The role of GABAergic inputs for coincidence detection in the neurones of nucleus laminaris of the chick

1 Synaptic inputs to nucleus laminaris (NL) neurones were studied in a brainstem slice preparation of chick embryos (E15‐20) using the whole‐cell patch clamp technique. NL neurones are third order auditory neurones and are proposed to behave as coincidence detectors concerned with interaural timing discrimination. 2 Under voltage clamp conditions, electrical stimuli applied to either ventral or dorsal dendritic layers evoked EPSCs. These fast currents decayed with a time constant of 1.1 ms near the resting potential, reversed close to 0 mV, and were blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 20 μM) or 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX, 20 μM). Coincident or near coincident stimulation of the ventral and the dorsal dendritic layers increased the probability of action potential generation (response probability). 3 In the presence of CNQX (40 μM) other postsynaptic currents (PSCs) were observed, which reversed close to the equilibrium potential for chloride (ECl), and were reversibly blocked by bicuculline (20 μM) and, therefore, were mediated by GABAA receptors. Spontaneous GABAergic PSCs were inward going near the resting membrane potential immediately after starting whole‐cell recording with a low Cl− (5 mM, ECl= ‐90 mV) pipette medium, but became outward‐going with time. This indicates that GABAergic inputs may generate depolarizing potentials in intact NL neurones. 4 Local GABA (10 μM) application reduced both the EPSP and EPSC amplitude and shortened the EPSP decay time constant (from 5.3 to 2.1 ms), while the EPSC decay time constant was not affected (from 1.3 to 1.2 ms). These GABA effects were mostly due to the shunting conductance of the postsynaptic GABAA receptors. 5 Depolarizing current injections combined with electrical stimuli to a unilateral axon bundle simulated bilateral synaptic inputs. Response probability increased with decreased interstimulus intervals, while local GABA (10 μM) application to the soma narrowed the time dependence of the response probability. 6 These results suggest that GABAergic inputs to NL neurones may serve to improve coincidence detection of the bilateral excitatory inputs through an increase in membrane conductance.

Binding of ω-conotoxin to receptor sites associated with the voltage-sensitive calcium channel

Abstract The binding of radioiodinated ω-conotoxin GVIA, a probable Ca channel antagonist, to synaptic plasma membranes of rat brain was examined. Two kinds of specific binding sites were found with apparent dissociation constants of 10 pM and 0.5 nM and maximum binding capacities of 0.5 and 3.4 pmol/mg prot., respectively. The binding of the toxin was not affected by high concentrations of Ca antagonists or an agonist, indicating distinct binding sites of the toxin from those of these drugs. Divalent and trivalent metal ions strongly inhibited the binding. The order of their inhibitory potencies was similar to that for inhibition of the Ca current through certain Ca channels. These results suggest that the binding sites of ω-conotoxin GVIA are functionally related to the Ca 2+ -binding site postulated to be in the pore of the Ca channel.

Long-term potentiation of transmitter release induced by repetitive presynaptic activities in bull-frog sympathetic ganglia.

Long‐lasting potentiation of transmitter release induced by repetitive presynaptic activities in bull‐frog sympathetic ganglia was studied by recording intracellularly fast excitatory post‐synaptic potentials (fast e.p.s.p.s.). Following a brief period of post‐tetanic potentiation or depression (less than 10 min), the amplitude of the fast e.p.s.p. was potentiated for a period between several tens of minutes and more than 2 h in response to tetanic stimulation of the preganglionic nerve in twenty‐one out of twenty‐eight cells. Quantal analysis revealed that this long‐term potentiation of the fast e.p.s.p. (l.t.p.) was accompanied by an increase in quantal content m (in nine out of twenty‐one cells), quantal size (four cells) or both (eight cells). The increased quantal content (presynaptic l.t.p.) declined exponentially (ten cells) or decayed gradually to a certain enhanced level which lasted several hours. In contrast, the increased quantal size grew with a relatively long latency (10‐25 min) and remained relatively constant for at least 2 h. The magnitude of presynaptic l.t.p. increased with increased duration of the presynaptic tetanus (33 Hz) from 2 to 5 s. No l.t.p. was elicited by a 1‐s tetanus, whereas the time course appears to be independent of the tetanus duration and the magnitude of l.t.p. There was a positive correlation between the magnitude of presynaptic l.t.p. and the pre‐tetanic quantal content up to m = 3, but the former deviated from linear regression when the value of the latter exceeded 3. No l.t.p. occurred when quantal content was less than 0.5. A tetanus (33 Hz, 10 s) applied in Ca2+‐free solution elicited no presynaptic l.t.p., while the same tetanus in normal Ringer solution produced a large presynaptic l.t.p. Presynaptic l.t.p. was enhanced in magnitude at low temperature (8‐10 degrees C). These results demonstrate the existence of a use‐dependent, long‐term potentiation of transmitter release in bull‐frog sympathetic ganglia. Several possible mechanisms are discussed in terms of Ca2+‐buffering mechanisms of the presynaptic nerve terminals.

Effects of synthetic ω-conotoxin on synaptic transmission

Abstract The effects of chemically synthesized ω-conotoxin GVIA (a neurotoxic peptide from Conus geographus ) on synaptic transmission at the bullfrog sympathetic ganglion, frog neuromuscular junction and electric organ of the ray, Narke japonica , were studied. The synthetic toxin irreversibly suppressed synaptic transmission at these synapses by arresting the release of transmission from the nerve terminals without showing postsynaptic effects. This action of the toxin was effectively antagonized by high concentrations of extracellular Ca 2+ . The synthetic toxin irreversibly blocked the Ca 2+ -dependent action potential of bullfrog sympathetic ganglion cells. These results suggest that ω-conotoxin GVIA blocks synaptic transmission by interfering with the Ca 2+ influx through the voltage-sensitive Ca 2+ channel of the nerve terminal. These results indicate that the chemically synthesized ω-conotoxin GVIA acts exactly like the natural ω-conotoxin GVIA. Thus, the synthetic toxin can be used in place of the natural toxin as a useful probe for the voltage-sensitive Ca 2+ channel in the nervous system.

Differential effects of apamin on Ca2+-dependent K+ currents in bullfrog sympathetic ganglion cells

In B-type neurones of bullfrog sympathetic ganglia, apamin (10 nM) suppressed the Ca2+-dependent K+ current (IAH) involved in the afterhyperpolarization of an action potential, while it did not affect the Ca2+-dependent K+ current (Ic) underlying the spike repolarization. IAH was further separated into two exponential components which were differentially affected by apamin, voltage and alterations in Ca2+ influx, suggesting the existence of 3 different types of Ca2+-dependent K+ channel in bullfrog sympathetic neurones.

A patch-clamp study on the muscarine-sensitive potassium channel in bullfrog sympathetic ganglion cells.

1. A voltage‐independent K+ channel was characterized and effects of muscarine were studied in cultured bullfrog sympathetic ganglion cells using the cell‐attached patch‐clamp configuration. 2. Three types of single‐channel current were recorded from 2‐ to 10‐day‐old cultured cells in the presence of tetraethylammonium (2‐20 mM), tetrodotoxin (1‐2 microM), Cd2+ (0.1 mM) and apamin (20 nM). 3. The most frequently observed channel was a voltage‐independent K+ channel which was open at the resting membrane potential and had a conductance of 52.6, 78.9 and 114.9 pS at a [K+]o of 2, 40 and 100 mM, respectively. This channel was designated background K+ channel. 4. Two other channel types were observed less frequently. One had a conductance of 26 pS (external K+, 118 mM) and a long open time of several seconds at the resting membrane potential. The second channel had a smaller conductance (20 pS) and displayed a voltage‐dependent activation. 5. The open probability of the background K+ channel varied between patches, ranging from 0.0005 to 0.486. The open time distribution was fitted by a single exponential with a time constant of 0.51 ms. Both of these parameters were independent of the membrane potential. The closed time distribution consisted of at least four exponentials having time constants of 0.17, 3.7, 120 ms and several seconds. 6. Muscarine (10‐20 microM) applied to the membrane outside the patch pipette reversibly enhanced the activity of the background K+ channel. This effect was associated with an increase in the open probability, which resulted from an increase in the mean open time concomitant with a decrease in the mean closed time. Muscarine did not change the single‐channel conductance of this channel. 7. The effects of muscarine were blocked by atropine (1 microM). 8. It is concluded that there exists a muscarine‐sensitive, voltage‐independent K+ channel in cultured bullfrog ganglion cells. This K+ channel appears to contribute to the generation of the resting membrane potential and underlie the slow inhibitory postsynaptic potential of these neurones in situ.

Pharmacological evidence for two types of Ca2+-dependent K+-conductance in bullfrog sympathetic ganglion cells.

The effect of apamin, a bee venom toxin, on the action potential and the spike after hyperpolarization was studied in bullfrog sympathetic ganglion cells. Apamin reduced the duration of the afterhyperpolarization but did not affect the maximum rates of rise and fall of Na+- and Ca2+-dependent action potentials. In the presence of apamin and Co2+, the maximum rate of fall of the action potential was decreased, and the spike duration was prolonged. These results suggest that at least two types of Ca2+-dependent K+-conductance co-exist in bullfrog sympathetic ganglion cells.

A muscarine-activated voltage-independent K+ channel in cultured bullfrog sympathetic neurones

In cultured bullfrog sympathetic neurones, cell-attached patch clamp revealed a voltage-independent K+ channel having a conductance of 46-113 pS at an external K+ of 2-120 mM. Muscarine (10-20 microM), applied to the cell membrane outside a recording pipette, increased its open probability and mean open time in an atropine-sensitive manner. This muscarine-activated K+ channel could underlie some of the muscarinic inhibitory postsynaptic potentials in both central and peripheral nervous systems.

An Increased Basal Calcium Hypothesis for Long-Term Potentiation of Transmitter Release in Bullfrog Sympathetic Ganglia

Long-term potentiation (LTP) of synaptic transmission, a basis for learning and memory (cf. Tsukahara, 1981), occurs in response to conditioning stimuli in various neuronal elements at both central and peripheral synapses. In bullfrog sympathetic ganglia, there are two types of long-term potentiation of transmitter release, one induced by conditional tetanic stimulation of the preganglionic nerve through a Ca2+ -dependent mechanism (presynaptic LTP, pre-LTP; Koyano et al., 1985), and the other generated by the action of epinephrine through a cAMP-dependent mechanism (epinephrine-induced LTP, adrLTP; Kuba et al., 1981; Kuba and Kumamoto, 1986). We describe here novel mechanisms of these LTPs in which a rise in the basal level of the intracellular free Ca2+ ([Ca2+]i) in the presynaptic terminal plays an important role.

316 Developmental change of release probability of neurotransmitter in medial nucleus of the trapezoid body in rat

Hiromu Yawo’, Shuichi Saheki2 Norepinephrine (NE) potentiated the transmitter release from the giant presynaptic terminal of chick ciliary ganglion through a receptor pharmacologically different from alphaand beta-adrenergic receptors (1) which may’activate cGMP-protein kinase G cascade. The mechanism how NE activates guanylyl cyclase was investigated. The NEdependent potentiation was not affected by either NO-synthase inhibitor, N”nitro-L-arginine methyl ester (L-NAME, 100microM) or NO-scavenger, hemoglobin (30microM). Moreover, the NO-donors, neither sodium nitroprusside (100microM) nor (&)-(E)-4-Methyl-P-[(E)-hydroxyiminol-5-nitro-8methoxyS-hexenamide (NORl, POmicroM) did not potentiate the transmitter release even in the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyxanthine (IBMX). Therefore, the NO-sensitive soluble guanylyl cyclase appears not to be expressed in the giant presynaptic terminals of embryonic chick ciliary ganglion. It is concluded that the novel adrenergic receptor different from both alphaand beta-adrenergic receptors activate guanylyl cyclase without involving NO. 1. Yawo, H. (1996) J. Physiol. Lond. 493:385-391.