decayed in parallel tetraethylammonium (20 mm). 5. scanning a singl

Journal of Physiology (1992), 458, pp. 171-190 171With 10 figuresPrinted in Great Britain

INTRACELLULAR CALCIUM DYNAMICS IN RESPONSE TO ACTIONPOTENTIALS IN BULLFROG SYMPATHETIC GANGLION CELLS

BY MITSUO NOHMI, SHAO-YING HUA AND KENJI KUBA*From the Department of Physiology, Saga Medical School, Saga 849, Japan

(Received 18 November 1991)

SUMMARY

1. Dynamic changes in the intracellular free Ca2" concentration ([Ca2`]j) followingelectrical membrane activity, were recorded from the neurone soma of the excisedbullfrog sympathetic ganglion, using Fura-2 fluorescence and compared with theaccompanying Ca2+-dependent electrical membrane responses.

2. The resting [Ca2+]i was about 100 nm, a value little changed by penetration withan intracellular electrode.

3. A net rise in fluorescence at a wavelength of 340 nm (Ca2+ transient) induced bya single action potential in Ringer solution rose almost in parallel with the initialdecay phase of a slow Ca2+-dependent after-hyperpolarization; decayed in parallelwith the late phase; and increased in amplitude and duration in the presence oftetraethylammonium (20 mM).

4. A Ca2+ transient induced by repetitive action potentials was increasedasymptotically in amplitude and progressively in duration by increasing the numberof spikes, and was slower in time course than the associated Ca2+-dependent K+current.

5. Scanning a single horizontal line across the cytoplasm with an ultraviolet argonion laser (351 nm) and recording Indo-1 fluorescence with a confocal microscopedemonstrated an inward spread of a rise in [Ca2+]i following a tetanus.

6. Both single spike- and tetanus-induced Ca2+ transients were abolished in aCa2+-free solution, while single or repetitive transient rises in [Ca2+]i induced bycaffeine (5-10 mM) were generated under the same conditions.

7. Ryanodine (10-50 aM) did not affect tetanus-induced Ca2+ transients, whereasit blocked completely the caffeine-induced oscillation of [Ca2+]i.

8. Ca2+ transients induced by a tetanus in Ringer solution were independent of theinterval from the preceding tetanus. The amplitude of Ca2+ transients induced by atetanus in the presence of caffeine (5 mM) was equal to, or greater than, thatgenerated in Ringer solution in any of the phases of [Ca2+]i oscillation.

9. It is suggested that under the physiological conditions here, the induction ofaction potentials does not cause the release of Ca2+ in the cells of the freshly excisedbullfrog sympathetic ganglion, and that Ca2+-buffering systems contribute not onlyto lowering a transient rise in [Ca2+]i but also to sustaining an increased [Ca2+]i aftera large Ca2+ load into the cell.

* To whom correspondence should be addressed.MS 8919

M. NOHMI, S.-Y. HUA AND K. KUBA

INTRODUCTION

The origin of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) involvedin various neuronal functions was in general thought to be due to Ca2' entry throughvoltage-dependent Ca2+ channels or transmitter-activated ion channels (cf. Miller,1988). It was previously suggested, however, that in bullfrog sympathetic ganglioncells the release of Ca2+ from intracellular Ca2+ reservoirs contributed to thegeneration of an after-hyperpolarization (AHP) of an action potential, post-tetanichyperpolarization (PTH) and rhythmic membrane hyperpolarizations under somespecial conditions, such as the effect of caffeine or in the intracellular presence ofcertain anions, citrate or acetate (Kuba & Nishi, 1976; Kuba, 1980; Morita, Koketsu& Kuba, 1980; Kuba, Morita & Nohmi, 1983; Nohmi, Kuba & Morita, 1983). Duringthe course of a study analysing the dynamics of [Ca2+]i following electrical membraneactivity in bullfrog sympathetic ganglion cells (Nohmi, Kuba, Ogura & Kudo, 1988),Lipscombe, Madison, Poenie, Reuter, Tsien & Tsien (1988) and Thayer, Hirning &Miller (1988), measuring [Ca2+]i with Fura-2 fluorescence in cultured sympatheticneurones, provided the evidence that the intracellular release of Ca2+ occurred duringmembrane depolarization induced by a high K+ concentration in the absence of anagent (such as caffeine) which enhanced intracellular Ca2+ release. In a subsequentstudy, Thayer & Miller (1990) reported the lack of the involvement of theintracellular release of Ca2+ in the depolarization-induced Ca2+ transient in culturedrat dorsal root ganglion cells. Accordingly, there is still an important question ofwhether the release of Ca2+ from the intracellular store sites takes place by theinduction of action potentials in neurones in sitU, especially under physiologicalconditions; although there is evidence for transmitter-induced intracellular Ca2+release via the inositol 1,4,5-trisphosphate receptor (Sugiyama, Ito & Hirono, 1987;cf. Berridge & Irvine, 1989).

In the present study, we have measured simultaneously, dynamic changes in[Ca2+]i (by recording the fluorescence of a fluorescent Ca2+ indicator, Fura-2,Grynkiewicz, Poenie & Tsien, 1985) and Ca2+-dependent membrane potentials orcurrents (as a reflection of the [Ca2+]i close to the cell membrane) following single orrepetitive action potentials from the neurone soma of the freshly isolated bullfrogsympathetic ganglion and observed the effects of drugs to affect the intracellularCa2+ release. Our objectives were (1) to measure spatial differences in the time courseof a rise in [Ca2+]i after an action potential and (2) to examine the involvement ofthe intracellular Ca2+ release in a rise in [Ca2+]i induced by action potentials underphysiological conditions. The results suggest that the release of Ca2+ fromintracellular reservoirs contributes little to the rise in [Ca2+]i following an actionpotential (Ca2+ transient) in bullfrog sympathetic ganglion cells in freshly isolatedtissue and that intracellular Ca2+-buffering systems not only lower the amplitude ofa Ca2+ transient but also sustain the time course. Part of these results has beenpublished in abstract form (Nohmi & Kuba, 1988).

METHODS

PreparationsBullfrogs (Rana catesbeiana) were killed by pithing. Ninth or tenth lumbar sympathetic ganglia

were isolated as previously described (Kuba et al. 1983) and mounted on the side wall of Sylgard

172

INTRACELLULAR CALCIUM DYNAMICS 173

resin in a recording chamber (set on the stage of an inverted microscope, Olympus IMT-2 or NikonTMD) so as to allow the direct access of an intracellular microelectrode to a ganglion cellsimultaneously with epifluorescence measurement (Nohmi et al. 1988). Only the cells, which werelarge in diameter (presumably B-type neurones; Nishi & Koketsu, 1960) and protruded from the

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Fig. 1. A, schematic diagram for two types of optical systems to record simultaneouslyFura-2 fluorescence and membrane potential (Methods A and B). See text for detail. B,focal depth of the 10 x objective. Fluorescence signals from a droplet of a Fura-2 solution(5 ,UM, 2 ,um in thickness, 50 ,um in diameter) excited at 360 nm were measured at differentlevels of the microscope stage. Fluorescence intensity is in arbitrary units. Positivedisplacement means a decrease in the distance between the objectivTe and the specimen.V, membrn ptnial; 'mX membrane current.

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surface of a ganglion, were used, so that the fluorescence from a single cell could be measured (seebelow, Figs 1 and 6). The ganglion was superfused at room temperature (20-25 °C) with normalRinger solution (mM): NaCl, 115-5; KOl, 2; CaCl2, 1M8; NaHCO3, 2*4. When 20 mM tetra-ethylammonium chloride (TEA) was added, the osmolarity was adjusted by omitting anequimolar amount of NaCl. Ca2+-free Ringer solution was obtained by adding 1 mrM ethyleneglycol-bis-(fl-aminoethylether)(N,N,N',N'-tetraacetic acid (EGTA) to a nominally Ca2+-free Ringersolution. Potassium acetate electrodes (4 M; tip resistance, 30-60 M.Q) were used for intracellularrecording of membrane potential or current (with a single electrode voltage clamp technique:Brennecke &G Lindeman, 1974; Wilson &; Goldner, 1975). Action potentials were antidromicallyelicited by single or repetitive pulses (1~0 ms in duration at 20 Hz).

Loading of fluzorescent Ca2+ probesThe acetoxymethyl ester form of Fura-2, Fura-2 AM (Molecular Probes, Junction City, OR,

USA or Dojin Laboratories, Japan) was dissolved in dimethylsulphoxide (DMSO), to make a .stocksolution (1 mM). Solutions for loading Fura-2 AM (5-7 5 ,UsM) were prepared by vigorously mixingthe stock solution with Ringer solution containing 0*2% surfactant, Cremophor EL (Sigma).

M. NOHMI, S. - Y. HUA AND K. KUBA

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INTRACELLULAR CALCIUM DYNAMICS

Ganglion cells were incubated in the loading solution for 2-3 h at room temperature 20-25 °C in thedark, and then perfused with Ringer solution for 30 min allowing de-esterification of Fura-2 AM inthe cell and its removal from the extracellular space.The loaded intracellular concentration of Fura-2, was estimated to be less than 50,M by

comparing the fluorescence at 360 nm from a droplet (having a similar diameter to those of cells)of a 'pseudocytoplasmic' solution containing (mM): KCl, 120; EGTA, 1; and Pipes, 10 (free Ca2 ,100 nM; pH 7 2) and the known concentration of Fura-2 in liquid paraffin. This value would be lowenough not to cause a buffering effect of Fura-2 on [Ca2+]i (Baylor & Hollingworth 1988), since anAHP of long duration and the associated rise in [Ca2+]i were generated in Fura-2-loaded cells. Themaximum value of [Ca2+]i (720 nM) caused by tetanic stimulation in the bullfrog sympatheticganglion cell was lower than the maximum value (1 1aM) which was measurable with the Fura-2method.

In experiments where Ca2" transients were recorded by a confocal laser-scanning microscope,acetoxymethyl ester of Indo-1, Indo-1 AM (Molecular Probes, Junction City, OR, USA or DojinLaboratories, Japan) was loaded into ganglion cells in freshly isolated tissue. The method forloading was essentially similar to that for Fura-2 AM except that Pluronic F-127 (MolecularProbes) was used as a surfactant.

Measurements of Fura-2 fluorescenceOne of the optical systems used is shown in Fig. 1A. Light from a 150 W xenon arc lamp (Ushio,

Japan) sited in a custom-made light housing (Yamashita Denso, Japan) was passed through a 342,362 or 383 nm interference filter (with bandwidths of 12, 8 or 8 nm, respectively, Asahi SpectraCo., Japan), which are described hereafter as 340, 360 and 380 nm for simplicity, for epifluorescencemeasurement. Fluorescence images were obtained through a 450 nm long-pass exit filter (AsahiSpectra Co.) and an objective (Nikon CF fluor, 10 x (numerical aperture (n.a.) 0 5) or 20 x (n.a.0 75), respectively) with a SIT (Silicon-intensifier-target) camera (C2400-08, Hamamatsu PhotonicsK.K., Japan), monitored on a TV screen (C18460-03, Hamamatsu Photonics K.K.) and stored onvideo tape. At the initial stage of the experiments, fluorescence intensity in the form of a videosignal was measured with a phototransistor (TPS601A, Toshiba) through a glass fibre (1 mm indiameter), one end of which targeted on a small central part (about 1 %) of a single cell image(about 15 mm in diameter for a cell of 50 /tm) on the TV screen (Kudo & Ogura, 1986) which hada y value of unity indicating the linear relationship between the intensity of light emitting fromthe TV screen and video signals. After subtracting the voltage for a dark level, intermittent voltageoutput pulses (60 Hz) from a phototransistor were converted to a continuous voltage output aftertwo step integrations (time constants of 0-2 ms and 2 ms) with sampling-and-holding, and low-passfiltered at 20 Hz (Butterworth, 24 dB/octave). In this method (Method A), excitation filters werealtered manually so that only fluorescence at 340 nm (F340) was recorded during a cellular response(see below). Fluorescence excited at 360 nm (F360) was recorded only at the beginning and end ofthe experiments (see Fig. 3A). The ratio of F340 over F360 (R340/360) was calculated by taking the ratioof F340 (recorded throughout the experiment) to the F360 estimated from linear interpolationbetween the values at the beginning and end of the experiment.

In later stages of the experiments, video signals from the cell image were extracted by a windowcircuit (Method B, Fig. 2). After the subtraction of the dark level, it was converted to a continuousvoltage signal through an integration and sample-hold circuit (Nohmi & Kuba, 1989). In this modeof fluorescence recording, two excitation filters (340 and 380 nm) were intermittently changed at0 5 Hz, and the ratio (R340/380) of fluorescences at 340 and 380 nm (F380) was electronicallycalculated. The methods for processing the video signals and conversion to fluorescence ratios areas follows. Video signals from a SIT camera (Hamamatsu, 2400-08) were clamped at the pedestallevel by a circuit (1; numbers correspond to those in Fig. 2A), and passed through gate controlcircuits; (2 and 8) which selected video signals corresponding to the fluorescence image (monitoredby a superimposer. 9); inscribed to a cell image on a TV screen (dotted circle in Fig. 2B) byhorizontal (H) and vertical (V) gate pulses, (8; that synchronized to the main video signal rate).Selected signals were integrated for each single line of video signals (3) and sampled-and-held withsubtraction of the background fluorescenc.e level (4). Pulsatile voltage signals which consisted oftwo alternate fluorescences excited at different wavelengths (each of which are composed of signalsfrom several video frames) were again integrated (5), and separated into each fluorescence signal(Ft and F2 in Fig. 2A) with the second sample-and-hold circuit (6) which was synchronized withthe rate of a filter exchanger (10), and finally converted to the ratio of fluorescences with a divider

175


(7). The processes from (1) through to (6) are almost common to both Methods A and B except thatthe gate circuit (2) was not used in the Method A. (In Method A, video signals corresponding to thecell fluorescence were selected simply by the position of a glass fibre end.) How video signals wereprocessed at each step of the present system is shown in Fig. 2 C.

In several experiments, two other types of fluorometer were used. One system (CAM-220; JapanSpectroscopic Co., Japan) consisted of two monochrometers, one photomultiplier and an invertedmicroscope (Nikon TMD; objective, CF Fluor 40 x, Method C; see Nohmi, Hua & Kuba, 1992). Inthis system, excitation wavelengths were altered between 340 and 380 nm at 100 or 400 Hz.Fluorescence emitted at wavelengths greater than 491 nm was filtered through a RC (res-istance-capacitance) circuit at 16 Hz, and then their ratio (R340/380) was taken. The other systemwas composed of a confocal laser-scanning system (CLMS: Biorad, MRC-500, UK), an ultravioletargon ion laser (Spectra-Physics, 2025-04, USA; 351 nm, 60 mW) and an inverted microscope(Nikon TMD; objective, CF fluor 40 x ), an achromatic relay lens made of fused silica and fluoriteand a compensation lens in the laser path for chromatic aberration of the objective (cf. Kuba, Hua& Nohmi, 1991, Method D). Technical detail and characteristics will be published later. Fluorescenceof Indo-1 (>395 nm) was measured simultaneously at 410 and 475 nm after separation by adichroic mirror (445 nm long pass).

Fluorescence signals from either type of video-signal conversion devices (Methods A or B) orfrom the photomultiplier (Method C) were stored on FM-tape (NFR-3515, SONY, Japan) or digitaltape after pulse code modulation-processing (RDl 1 T, TEAC, Japan), while image data producedby a confocal microscope (Method D) was stored on a laser disk (JP2020, Japan Personal ComputerCo., Japan). Fluorescence signals (Method A) induced by a single stimulus were summed with anelectronic averager (ATAC-210, Nihon-Kohden, Japan).The 'effective' on and off rate constants for Ca2+ binding of Fura-2-loaded skeletal muscle fibres

were 4-6 times smaller than those in vitro (2-7 x 108 M-1 s-1 and 84-97 s-1 respectively, Jackson,Timmerman, Bagshaw & Ashley, 1987; Kao & Tsien, 1988) due to binding of Fura-2 to thecytoplasmic proteins (Baylor & Hollingworth, 1988). If such a binding occurred in the ganglion cell,the relaxation time constant of Fura-2 reaction for [Ca2+]i of 0-2 and 1,M in the ganglion cell wouldbe 14-100ms. The time course of a 'true' single spike-induced Ca2+ transient, numericallydeconvoluted according to this assumption, however, did not differ much from the time course ofthe recorded Ca2+ transient. This was because the latter had a time course (peak time > 500 ms)much slower than the slowed Fura-2 reaction. Thus, the Method A used in the present study wouldhave faithfully recorded Ca2+ transients induced by a single spike, while all the epifluorescencerecording methods (A, B and C) would be fast enough to record Ca2+ transients induced by atetanus.

Focal depth for objectives used in epifluorescence measurements (Methods A and B) wasmeasured by recording fluorescence from a drop of a Fura-2 solution (5 FM) placed in a plane (lessthan 2,um in thickness and 50 um in diameter) at different levels of the microscope stage (Fig. 1 B).Axial distance required to decrease fluorescence to half the maximum was 50 and 32 4um (in twodirections) for Nikon CF fluor 10 x and 43 and 27um for Nikon CF fluor 20 x The focal depth foran objective (Nikon CF fluor 40 x, Method C) measured with a Fluo-3 fluorescence filled in acapillary (internal diameter <2 Fum) was 20,um in the axial direction away from the objective(Kuba et al. 1991). Accordingly, present epifluorescence systems (Methods A, B and C) recorded theaveraged (or integrated) value of [Ca2+]i of the whole cell cytoplasm, which would therefore mainlyrepresent [Ca2+], values apart from the submembrane space. The focal depth for the confocal laser-scanning microscopy employing an ultraviolet laser was less than 1-5 Fm (Kuba, Hua, Nohmi &Hayashi, 1992), thus providing the sliced image of a ganglion cell (Kuba et al. 1991).The absolute value of [Ca2+]i was estimated by comparing the ratios (R340/360) of Fura-2

fluorescences excited at 340 and 360 nm in a single cell (or R340/380) with those of calibratingsolutions as previously described (Grynkiewicz et al. 1985; Nohmi et al. 1988). These estimates ofthe absolute value of [Ca2+]1 from R340/360 or R340/380 may not necessarily be correct, but would betentative ones for (1) possible binding of Fura-2 to cell constituents as reported for the skeletalmuscles (Baylor & Hollingworth, 1988; Konishi, Olson, Hollingworth & Baylor, 1988) and (2)heterogeneous changes in [Ca2+]i throughout the cell cytoplasm during the course of a Ca2+transient. The [Ca2+]1 values described in this paper, however, would faithfully demonstraterelative changes in [Ca2+]i during a course of Ca2+ transients, since the isosbestic point inwavelength of Fura-2 loaded in the cell remained unchanged (see Fig. 7A). The ratios of Indo-1

176


fluorescences emitted at 410 and 475 nm (R410/475) were converted to absolute [Ca2+]1 values usinga standard calibration curve in vitro for Indo-1 as done for Fura-2 fluorescence.

RESULTS

The basal [Ca2+]i of intact ganglion cells with or without electrode penetrationFigure 3B shows a histogram of resting [Ca2+]i of individual ganglion cells

superfused with normal Ringer solution without penetration. The resting [Ca2+]i was96 nM (± 7 nM S.E.M., n = 31) on average, but varied among cells ranging from 34to 272 nm. These absolute values of the resting [Ca2+]i in the neurone soma of thebullfrog sympathetic ganglia are similar to those reported for the same or other typesof neurones in previous studies (Lipscombe et al. 1988; Nohmi et al. 1988; Thayeret al. 1988), albeit these values may be subject to correction (see Methods).

It is necessary to ensure that the basal [Ca2+]i does not change after penetrationof a ganglion cell with a recording electrode which may cause the cell membraneinjury. If it occurs, the correct comparison of the time course of Ca2+-dependentmembrane responses with that of Ca2+ transients would not be possible. Injury of thecell membrane by electrode penetration may indeed be expected, since thepenetration occasionally results in low resting membrane potential, small inputresistance and short duration ofAHP of an action potential, although these abnormalcell conditions are eventually restored in most cases to normal ones in severalminutes or several tens of minutes (cf. Kuba et al. 1983, see the middle trace of Fig.3A). Only a small transient increase in F340, however, was observed after penetration(top of Fig. 3A), although the resting membrane potential and input resistance weresmall with a short duration of AHP (middle trace, and left-hand side record in thebottom of Fig. 3A). Figure 3C shows a histogram of the resting [Ca2+]i of the cellspenetrated with an electrode, the mean of which was 94 nm (+ 8 nM, n = 36; with arange from 36 to 270 nM) and was not significantly different (P > 0-05) from that ofunpenetrated cells (Fig. 3B). These results indicate that an increase in [Ca2+] due topenetration, if it occurs, may be limited to a localized region of the cytoplasm closeto the site of penetration and was not recordable with the present method (MethodA, see experimental procedures).

Single spike-induced Ca2+ transientA Ca2+ transient produced by a single spike in normal Ringer solution (Fig. 4A)

rose to a maximum in 0-8 s and fell roughly in a single exponential manner with atime constant of 1-6 s. When a Ca2+ transient and the Ca2+-dependent AHP of anaction potential were recorded simultaneously, there was a clear difference in timecourse (Fig. 4A). The major decay phase of AHP paralleled the rising phase of Ca2+transient (inset of Fig. 4A), while the latter decayed in parallel with the late smalldecay phase of AHP (middle trace of Fig. 4A). A net rise in [Ca2+]i at the peak of asingle spike-induced Ca2+ transient was 8-8+ 1-8 nM (n = 4) and the duration at halfthe maximum amplitude (half-duration) was 1-5 + 0-3 s (n = 4).The blockade of a K+ conductance underlying the falling phase of an action

potential by a K+ channel blocker would result in the broadening of an actionpotential and enhance Ca2+ entry. As expected, a Ca2+ transient recorded in thepresence ofTEA (20 mM), a K+ channel blocker, was greater in amplitude and longer

177

178 M. NOHMI, S.-Y. HUA AND K. KUBA

B

A 5 Average = 96-nMF360

1------ -- ~3 =3t||~~~~~~~~ ~~~~~= 31 d hn3

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2 20 mV 5 Average =94 nMC3. n 36

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Fig. 3. The effect of penetration with an electrode on the resting level of [Ca2+],. A,simultaneous recording of fluorescence signal (top) and membrane potential (middle)before, during and after the penetration with an electrode. The time at which a recordingelectrode was penetrated is indicated by a downward arrow. Method A was used.Electrotonic potentials were induced by a hyperpolarizing current pulse (01 nA and30 ms) every 100 ms or 2 s. AHPs of an action potential (denoted 1 and 2) were generatedintermittently by antidromic stimulation. The AHPs on expanded time scale before (1)and after (2), the recovery of the resting potentials are shown at the bottom. Spikeamplitudes were attenuated due to the low frequency response of the pen recorder. B andC, frequency histograms of resting [Ca2+]i obtained from cells penetrated with an electrode(C) or from unpenetrated cells (B).

A 147

R340,,,,, 138 n150R340/360 nM

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15mV

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Fig. 4. A, Ca2+ transients in response to a single action potential and associated AHP. Top,middle and bottom records represent a Ca2+ transient and AHPs in high and lowmagnification. Ninety-eight Ca2+ transients were averaged while the records of AHP arethose of a single sweep. Insets are part of a Ca2+ transient and associated AHP onexpanded time scale. Spike amplitude was attenuated by a long sampling interval (10 msper address). Method A was used. B, Ca2+ transients induced by a single action potentialin the presence of TEA (20 mM) and the associated AHP of an action potential. The Ca2+transient is the average of nine sweeps. Spikes were not seen due to a long samplinginterval (50 ms per address). Method A was used.


in duration (Fig. 4B) than that in normal Ringer solution (Fig. 4A). It reached apeak in about 1 6 s and declined roughly exponentially with a time constant of about6-9 s. The mean net increase in [Ca2+]i at the peak of Ca2+ transient and its half-duration in the presence ofTEA (20 mM) was 25 + 4-9 nM (n = 5) and 8-4+ 0 7 s (n =

A

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Time (s)Fig. 5. A, a tetanus-induced Ca2+ transient (top) and the outward current (bottom)underlying a PTH. After the end of a tetanus (20 Hz, 300 pulses), the membrane potentialwas voltage clamped to the resting potential. The bar indicates the duration of a tetanus.Method A was used. B, comparison of the time course of a net increase in [Ca2+]i (0), andthe associated outward current (@) shown in A. The time in the abscissa begins with theonset of voltage clamping.

5), respectively. These greater amplitudes and the longer duration of a spike-inducedrise in [Ca2+]i in TEA were reflected in a longer AHP (Fig. 4B). The magnitude andtime course of single spike-induced Ca2+ transients in either the presence or absenceof TEA were essentially similar whether a cell was penetrated with an electrode ornot (not shown).

A tetanus-induced Ca2+ transientFigure 5A shows a Ca2+ transient induced by a tetanic stimulation (300 pulses at

20 Hz) and an associated membrane current which was recorded by voltage clampingat the resting potential after the end of the tetanus. The tetanus-induced Ca2+transient continued to grow during the course of repetitive stimuli, remained at aplateau for several tens of seconds and decayed slowly, while the outward currentunderlying a PTH decayed monotonically with a faster time course (Fig. 5A and B).A net increase in [Ca2+]i at the peak of the Ca2+ transient produced by a tetanus of300 pulses at 20 Hz was 196 + 65 nM (n = 18), and the half-duration was 69-6 + 7-2 s(n = 18). Another feature of a tetanus-induced Ca2+ transient was that when thenumber of action potentials was smaller than a certain value (< 100 pulses, 20 Hz),Ca2+ transient rose further, even after the end of the tetanus (not shown). The Ca2+transient under the voltage clamp condition was essentially identical in amplitude


and time course with that recorded from the same cell under the current clampcondition (not shown), suggesting that the rate of Ca2+ buffering in the cell is almostvoltage independent at least in the range of membrane potential measured.

Inward spread of an increased [Ca2+]i produced by a tetanusLarge differences in the time course of a single or repetitive action potential-

induced Ca2+ transient and a corresponding Ca2+-dependent membrane response

Aa b

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10,mFig. 6. Time course of changes in one dimensional distribution of [Ca2+], during and aftertetanic stimulation (10 Hz, 1 s) in normal Ringer solution containing TEA (20 mM) in twodifferent cells (A and B). Image data were obtained by a confocal microscope equippedwith an ultraviolet argon laser (351 nm, 03 mW; Method D). The ganglion cell wasscanned along a single horizontal line (in x-axis) at a fixed position of y-axis shown by ahorizontal line in the x-y scan image (Ab, Bb (for Bd) and Bc (for Be)). The ratio offluorescence at 410 and 475 nm were displayed on the x-t plane (Ac, Bd and Be) inpseudocolour scale that was converted to [Ca2+]i. x-y scanned images (Aa and Ba) offluorescence at 475 nm are shown to demonstrate the location of the nuclei that werestained heavily with Indo-1. A tetanus was given during a period shown by a bracketedline on the left side of the x-t plane images. Indo-1 AM (10 ,LM) was loaded for 3 h at25 °C.

could be accounted for by differences in the time course of changes in [Ca2+]i inperipheral and deeper regions of the cytoplasm. This is because a Ca2+ transientwould represent a change in [Ca2+]i of deeper cytoplasm, while a Ca2+-dependent

180

INTRACELLULAR CALCIUMlf DYNAMICS

membrane response would reflect a change in [Ca2+]i close to the cell membrane (seeMethods and also Discussion). For instance, a slower rate of rise of a single spike-induced Ca2+-transient could result from a delay due to the inward diffusion of theCa2+ entering across the cell membrane and to the Ca2+ buffering during diffusion.Such an inward spread of Ca2+ was indeed demonstrated by a single line-scannedfluorescence image of a ganglion cell using a confocal microscope by Herniindez-Cruz,Sala & Adams (1990) and Kuba et al. (1991). In the present study similar experimentswere carried out, but with a higher fidelity than those of previous studies (seeMethods and below). Figure 6 shows an inward spread ofthe increase in ratio ofIndo-1fluorescences (emitted at 410 and 475 nm) after stimulation of the postganglionicnerve (10 Hz, 1 s) in the presence of TEA (20 mM) in two different cells. Ganglioncells loaded with Indo-1 were scanned repetitively at a single horizontal line in thecytoplasm shown on the horizontal-plane image (x-y scanning, Fig. 6Ab, Bb and Bc),and the results were displayed on an x-t plane (x-t scanning, Fig. 6Ac, Bd and Be).The tetanus caused a rise in [Ca21]i initially at the cytoplasmic region close to the cellmembrane. This increased [Ca2+]i then spread into the deeper region. The velocitiesof a radial spread of 'Ca2+ wave' toward the deeper cytoplasm were variabledepending on regions in the cell as already shown previously (Kuba et al. 1991). Thespeed of Ca2+ wave was much slower in the nucleus than in the 'apparently free'cytosol. For instance, in the cell shown in Fig. 6A, the slope of the 'front line' of Ca2+wave (obtained by fitting a straight line by eye to several different points, showinga roughly half-maximal rise in [Ca2+]i in the x-t scanned image of the cytoplasm),was greater on the right-hand side of the cytoplasm (3-9 #tm s-I versus 15-3 /tm s-' onthe left side), where the existence of the nucleus was indicated apparently from thestrong fluorescence at 475 nm in the x-y plane image (Fig. 6Aa). Likewise, in Fig. 6B,the speed of the Ca2+ wave was much slower in the nucleus (when scanned throughit, 6-9 ,um s-', Fig. 6Bd) than in the non-nuclear regions (16-3 Itm s-', Fig. 6Be). Thevelocity of Ca2+ wave was on average 13-2 + 1-6 tm s-1, n = 18: a range of3-9-27 5 jtm s-'. This value was slightly smaller than that of the previous study(26 ,tm s-' at the normal external Ca2+ concentration; Kuba et al. 1991). Thedifference could be within the range of cell variability and that of variability of theregions scanned, but might also be due to an improved focal depth of CLMS. In theprevious study, fluorescence changes were measured from sliced tissue which wasrelatively thick (about 7 ,um at half-maximum intensity of fluorescence) compared tothat measured in the present study (less than 3 /tm). Measurement from a relativelythicker tissue would have resulted in an overestimate of the speed of 'Ca2+ wave' inthe previous study. If the thickness of the optically sliced cytoplasm is large, thefront line of Ca2` diffusion in the vertical (optical axis) direction would enter thecytoplasmic region (from which fluorescence is measured) earlier than the front lineof Ca2+ diffusion in the horizontal plane, and contribute to the Ca2+ wave measuredin the horizontal plane. The magnitude of the net rise in [Ca2+]i was variable amongdiffering regions of the cytoplasm and cells and averaged 147 + 27 nm, (n = 8).However, there was no significant difference in the magnitude of rise in [Ca21]ibetween the nuclear and non-nuclear regions.

181


The relationships between Ca2+ transient and the number of spikesThe observation that the duration of a tetanus-induced Ca2+ transient (especially

that after the end of a tetanus) was much longer than that of a single spike-evokedCa2+ transient may indicate the involvement of another source of intracellular Ca2+

A 10 pulses 30 100 300300

R3610__ ]700 nM

B C61 mi

E1000 7C 000 100.

c 1001i.

X~~-~t °4 1

U :~tX i10 -- °

1 3 10 30 100 300 1 3 10 30 100 300Pulses Pulses

Fig. 7. A, changes in R3611380 (Ca2+ transients), F361 and F380 induced by a tetanus ofdifferent duration. Note little change in F361 by tetani, indicating that the isosbestic pointof Fura-2 fluorescence remained unchanged even after it was loaded in the cell. MethodC was used. B and C, the relationships between the number of spikes and the amplitudeof Ca2+ transients (B) and between the number of spikes and the half-duration (C). 0,values taken from the records shown in A, *, mean values of the data, bars show + S.E.M.

increase, such as release of Ca2+ from intracellular Ca2+ stores. A greater Ca2+ entrywould recruit Ca2+ release via a Ca2+-induced Ca2+ release mechanism (Endo, Tanaka& Ogawa, 1970; Ford & Podolsky, 1970; Kuba, 1980), thus prolonging the Ca2+transient. If Ca2+ release occurs, it may be reflected in the relationships of theamplitude and duration of Ca2+ transients versus the number of spikes. Both theduration and magnitude of the Ca2+ transient increased almost proportionally to acertain extent, with an increase in the number of spikes (Fig. 7A) similar to theduration of the membrane current underlying the PTH (not shown, see Figs 5 and 6of Tanaka & Kuba (1987)). At a longer tetanus such as those with 100-300 spikes,however, the magnitude tended to approach an asymptote value (Fig. 7A and B).This opposes the idea of the involvement of intracellular Ca2+ release in tetanus-induced Ca2+ transients, as observed in cultured rat dorsal root ganglion cells(Thayer & Miller, 1990). If the release of Ca2+ occurred by Ca2+ entry during atetanus, the amplitude of a tetanus-induced Ca2+ transient would have beenenhanced progressively as the number of spikes was increased, as seen in culturedbullfrog sympathetic ganglion cells (Kuba, Hua & Nohmi, 1990; Hua, Kuba &

182


Nohmi, 1991: see Discussion). The half-duration was not saturable, but increasedprogressively as the duration of a tetanus was lengthened (Fig. 7A and C), indicatingstrong intracellular Ca2+ buffering.

Dependence of spike-induced Ca21 transients on the external Ca2+A Ca2+ transient induced by a single spike in the presence of TEA was abolished

in a Ca2+-free solution (Fig. 8A). The effect appeared in 5 min after superfusion with

AaR~~ ~~~~~I124 n

.UO/360 r 1 05b "o

R340/360-

B

aR<2h i; h,K,,,, 400

R340/360 / ~ s, 220 nMb ------ 120R340/360

30 s

C

1 mM EGTA

10 mM caffeine 10 mM caffeine

] 300170 nM

R34>, = 10~~~ ~ ~ ~ ~ ~ ~ ~ ~-~~100

2 minFig. 8. Effects of a Ca2+-free solution on single spike- and tetanus-induced Ca2l transients(A and B, respectively) and caffeine induced Ca2+ transients (C). A, a single spike-inducedCa2+ transient under the effect of TEA (20 mM) before (a) and during (b) superfusion witha Ca2+-free solution for 5 min, respectively. Nine sweeps were averaged. Method A wasused. B, tetanus-induced Ca2+ transients (20 Hz, 300 pulses) before (a) and during (b)superfusion with a Ca2+-free solution. Method A was used. C, repetitive or single Ca2+transients induced by caffeine (10 mM) before and during superfusion with a Ca2+-freesolution. Method B was used.

the Ca2+-free solution. Under the same conditions, the amplitude and duration ofAHP were markedly reduced as observed in the absence of TEA in the previousstudy (not shown, Kuba et al. 1983). Likewise, a tetanus-induced Ca2+ transient (Fig.8B) as well as a PTH or the underlying outward current (not shown) were abolished5 min after treatment with a Ca2+-free solution. On the other hand, transient or

7 PHY 458


repetitive rises in [Ca2+]i induced by caffeine, which is known to enhance a Ca2+_induced Ca2+ release mechanism, were able to be generated in the same Ca2+-freecondition (Fig. 8C). These caffeine-induced Ca21 transients, however, did not lastlonger, but decreased progressively in amplitude and eventually disappeared in a

A

A[Ca2 ] =80 nM 95 90 80 90 80 75 190~~~1

9

1135 nR340/380 >Vt \,,' 15n_ \ ~~~~~~~~~ - ~~~~100* *@ 0*0*

* 30 pulses for 1 5 s 1 min

B5 mM caffeine

A[Ca2+] =175 nM 135 134 210 140 275 120 160 120

1270

R340/380 -~~~~~~~~~~~~160 nmJ95

*o1 00 pulses for 5 s 2 min

Fig. 9. A, Ca2+ transients induced by a tetanus (20 Hz, 1-5 s) at different intervals. MethodB was used. Note the roughly constant magnitude of net rises in [Ca2+] shown by A[Ca2+]i.B, Ca2+ transients induced by a tetanus (20 Hz, 5 s) before and during the application ofcaffeine 5 mm. Tetani were given at various intervals following a spontaneous rise in[Ca2+]i. Method C was used. Note that tetanus-induced Ca2+ transients in the presence ofcaffeine are almost equivalent to, or larger than those in the absence of caffeine.

Ca2+-free solution (Fig. 8 C). These results suggest that Ca2+ reservoirs endowed witha Ca2+-induced Ca2+ release mechanism retain Ca2+ in the absence of external Ca2+ fora period of at least 8 min and that action potentials alone cannot release Ca2+ fromthis caffeine-sensitive Ca2+ pool in the ganglion cell (in the absence of caffeine).

Tetanus-induced Ca2+ transients are independent of an interval to the precedingtetanus and Ca2+ release

If the release of Ca2+ from Ca2+ reservoirs contributes to a tetanus-induced Ca2+transient, it should depend on the interval to the previous response, as has been shownin caffeine-induced Ca2+-dependent hyperpolarizations (Kuba, 1980). If Ca2+ releaseis involved, a tetanus-induced Ca2+ transient would be smaller when it was generatedat a shorter interval. Figure 9A shows Ca2+ transients induced by a tetanus (20 Hz,30 pulses) at different intervals. The net rise in [Ca2+]i produced by the tetanus wasalmost constant irrespective of when the tetanus was given, except for that inducedduring the early decay phase of the preceding response. This indicates that a Ca2+_buffering system in the ganglion cell restores its original condition in a short periodafter a massive Ca2+ load.A tetanus-induced Ca2+ transient appears to be also independent of the previous

history of Ca2+ release. Tetanus-induced Ca2+ transients were generated at variousintervals to the preceding spontaneous rise in [Ca2+]i during the course of [Ca2+]i

184


oscillation under the influence of caffeine (5 mM). The amplitude and duration of atetanus-induced Ca2+ transient produced under the effect of caffeine were alwaysgreater than that induced in normal Ringer solution, and increased as the intervalfrom a preceding spontaneous Ca2+ transient was lengthened (Fig. 9B). These resultssuggest that a tetanus-induced Ca2+ transient in the presence of caffeine results notonly from Ca2+ entry across the cell membrane but also from intracellular Ca2+release, triggered by Ca2+ entry. The latter component would obviously be responsiblefor the dependence of a tetanus-induced Ca2+ transient on the preceding interval, asalready shown by the analysis of caffeine-induced hyperpolarizing responses (Kuba,1980). In addition, Ca2+ release would be minimal at the shortest interval.Consequently, the fact that the tetanus-induced Ca21 transient induced after theshortest preceding interval under the effect of caffeine was never smaller than thatin Ringer solution, would not favour the possible involvement of Ca2+ release in atetanus-induced Ca2+ transient.

Effects of ryanodine on tetanus-induced Ca2+ transientsRyanodine is known to decrease the amount of Ca2+ released from the sarcoplasmic

reticulum (SR) by opening the Ca2' release channel at the SR membrane at amoderate concentration or by blocking its opening at a high concentration (Sutko,Willerson, Templeton & Besch, 1979; cf. Fill & Coronado, 1988). This alkaloid cantherefore be used to examine whether a Ca2' release channel like that of the SR isinvolved in spike-induced Ca2+ transients in the ganglion cell.Ryanodine was first tested to see if it acts on the Ca2+-induced Ca2+ release

mechanism in the bullfrog ganglion cell. The oscillations of [Ca2+]i induced bycaffeine (3-10 mM) are known to occur as a result of the cyclic activation of the Ca21-induced Ca2+ release mechanism (Kuba, 1980), and were consistently blocked by theapplication of ryanodine in a few minutes (Fig. IOA). The effects were almostirreversible for a period of at least 45 min even after the removal of ryanodine(1O /tM). The basal level of [Ca2+]i was sometimes increased or unchanged by theaction of ryanodine (10 /aM). The rather small effect of ryanodine on basal [Ca2+]i maybe explained by two possible mechanisms: (1) Ca2+ released slowly by the action ofryanodine is quickly pumped out at the cell membrane, or (2) ryanodine blocks theCa2+-release channel (because a relatively high concentration of ryanodine was usedin these experiments).The preceding result indeed proves that the bullfrog sympathetic ganglion cell

possesses a ryanodine-sensitive Ca2+ pool. If Ca2+ is released from this Ca2+ storagesite during or after the generation of action potentials, a tetanus-induced Ca2+transient would be inhibited by ryanodine. However, this was not the case. In themajority of cells (18 out of 25 cells), ryanodine (10-50,M) did not affect Ca2+transients induced by a tetanus (20 Hz, for 1-5 or 5 s, Fig. lOB and C). The amplitudeand half-duration of Ca2+ transients induced by a tetanus (20 Hz) of 1P5 s were102-8 + 5.7 % and 107-1 + 7-4% (n = 4) of the control, respectively, and those by atetanus of 5 s were 104 + 9% and 103 + 9% (n = 18), respectively. In some cells therewere slight reductions in the amplitude and half-duration of Ca2+ transients inducedby a 5s tetanus (20Hz) to 77% (±11%, n= 7) and 64% (±18%, n= 7)respectively under the effect of ryanodine (10 JtM).

7-2

185

M. NOHMI, S. -Y. HUA AND K. KUBA

A5 mM caffeine

g0,M ryanodine 280

-> I' ' '' '' 150 nMR340/3!!,<_. ~'I i ~~~~~~~80

3 min

B1 OUM ryanodine

R340/35 10* 0 0 0 0 0 0 0 0 0 0 0 100*30 pulses for 1-5 s 2 min

C1OUM ryanodine

250R340/38 170 nM

*100 pulses for 5 s 3 minFig. 10. Effects of ryanodine (10 ,UM) on caffeine-induced Ca2+ transients (A) and tetanus-

induced Ca2+ transients (B and C). Method B was used.

DISCUSSION

It is known that a Ca2+-induced Ca2+ release mechanism, initially found in skeletalmuscles (Endo et al. 1970; Ford & Podolsky, 1970), is activated under the influenceof caffeine (Kuba & Nishi, 1976; Kuba, 1980) or some types of intracellular anions(Morita et al. 1980) in the bullfrog ganglion cells, leading to the oscillation of [Ca21]i.Under the same limited conditions, the intracellular release of Ca2+ was suggested tocontribute in part to the generation of a slow after-hyperpolarization (Kuba et al.1983; Nohmi et al. 1983). In line with these findings, it was suggested that a Ca2+_induced Ca2+ release mechanism contributes in part to a rise in [Ca2+]i produced bya high K+-induced depolarization in cultured sympathetic ganglion cells of bullfrogs(Lipscombe et al. 1988) and of rat (Thayer et al. 1988). The present findings, however,negate the involvement of intracellular Ca2+ release in a rise of [Ca2+]i induced byaction potentials in Ringer solution in the postganglionic neurones of freshlyisolated bullfrog sympathetic ganglion. Firstly single spike- or tetanus-induced Ca2+transients were abolished in a Ca2+-free solution, in which caffeine-sensitive Ca2+pools still retained their Ca2+. This indicates that depolarization of the cell membranealone does not release Ca2+ from reservoirs. Secondly, the amplitude of tetanus-induced Ca2+ transient reached an asymptotic value, as the number of actionpotentials increased. This is not consistent with the involvement of a Ca2+-inducedCa2+ release mechanism which produces a facilitatory dependence of a depolarization-induced Ca2+ transient on Ca2+ entry (Hua et al. 1991). Thirdly, the amplitude of atetanus-induced Ca2+ transient was independent of the interval from the precedingtetanus and possibly also from the preceding Ca2+ release induced by caffeine.

186


Finally, ryanodine, an inhibitor of Ca2+ release from caffeine-sensitive Ca2+ stores didnot affect tetanus-induced Ca2+ transients. Accordingly, it appears that the Ca2+transients are fully induced by Ca2` influx across the cell membrane during an actionpotential in bullfrog sympathetic ganglion cells in situ. A similar conclusion was alsomade for cultured dorsal root ganglion cells of rats by Thayer & Miller (1990).

In cultured bullfrog sympathetic ganglion cells, however, we have recentlyobtained evidence indicating the involvement of Ca2+ release in depolarization-induced Ca2+ transients (S.-Y. Hua, M. Nohmi & K. Kuba, unpublished obser-vations; see Kuba et al. 1990; Hua et al. 1991 for preliminary data) as suggested byLipscombe et al. (1988). The reasons for this difference between neurones in cultureand in freshly isolated ganglion tissue could be due to alteration in the properties of'Ca2+ release channels' at the Ca2+ reservoir membrane under culture conditionspossibly due to a change in the mode of gene expression. Alternatively, it may beascribed to differences in experimental procedures causing Ca2+ entry. In the presentexperiments on cells of isolated tissue, the nerve axon was stimulated and the cellcytoplasm was not dialysed. On the other hand, in experiments using culturedneurones, a long-lasting depolarizing pulse was applied to the cell under the wholecell clamp condition in which the cytoplasm was dialysed (see Kuba et al. 1990; Huaet al. 1991), or a K+-induced depolarization was used for Ca2+ entry (Lipscombe et al.1988). How these differences affect the mode of activation of Ca2+ release is nowunder study.

Large differences in time course between Ca2+-dependent membrane responses andCa2+ transients induced by single or repetitive action potentials can be accounted forby simple Ca2+ buffering and radial diffusion of Ca2+ in the cytoplasm. This alsoreasonably explains the dependence of amplitude and duration of Ca2+ transients onthe number of action potentials. This explanation is based on the idea that the Ca2+-dependent after-hyperpolarization and post-tetanic outward current reflect a changein [Ca2+]i close to the cell soma membrane, while the Ca2+ transient recorded from thewhole cytoplasm mainly represents a change in the deeper region of the cytoplasm(see Methods).

There is strong evidence supporting the generation of Ca2+-dependent membrane responses at thesoma membrane. Firstly, the bullfrog paravertebral sympathetic ganglion cell is a monopolarneurone and has no dendrites (Nishi & Koketsu, 1960). Secondly, single-isolated neurones whichhave no axon can produce a Ca2+-dependent slow after-hyperpolarization of an action potentialsimilar to that induced in neurones of freshly isolated tissue (S.-Y. Hua, M. Nohmi & K. Kubaunpublished observation). Thirdly, intracellular injection ofEGTA into the cell body shortened theduration of the after-hyperpolarization (Kuba et al. 1983), while the intracellular injection of Ca2+caused the hyperpolarization of the cell membrane (Kuba & Koketsu, 1978). Fourthly, singlechannel currents of Ca2+-dependent K+ channels were recorded from the cell membrane excisedfrom the cell body (Adams, Constanti, Brown & Clark, 1982; S. Hara & K. Kuba, unpublishedobservations).

A large difference between the rates of rise of [Ca2+]i in the peripheral and deeperregions of the cytoplasm of the cell body was indeed demonstrated by the ratio imageof Indo-1 fluorescence obtained by line-scanning using a CLSM (Fig. 6, cf. Lipscombeet al. 1988; Herniindez-Cruz et al. 1990; Kuba et al. 1991). Furthermore, the imagingwith CLSM demonstrated the slower propagation of the Ca2+ wave in the nucleuscompared to the non-nuclear region although there was no difference in magnitude.

187

M. NOHMI, S. - Y. HUA AND K. KUBA

This is not consistent with the previous findings in the same type of cells(Hernandez-Cruz et al. 1990) and in chick sympathetic neurones (Przywara, Bhave,Bhave, Wakade & Wakade, 1991).

Consequently, [Ca2+]i dynamics following the induction of single or tetanic actionpotentials may take place as follows. Calcium ions entering into the cell during anaction potential are mainly bound by Ca2+-binding proteins and taken up intoorganelles underneath the cell membrane, some of them diffuse into the deeper regionof the cytoplasm, and are similarly bound to proteins and taken up into organellesthere. The calcium ions thus bound and taken up are then dissociated and releasedbecause of a decrease in the cytoplasmic Ca2+ due to eventual extrusion at the cellmembrane. When action potentials are repetitively induced, the amount of Ca21retained in Ca2+-storing organelles increases disproportionately because of the co-operative nature of the Ca2+ uptake mechanism (cf. Carafoli, 1987). After cessationof a large Ca2+ influx, calcium ions overloaded in the organelles as well as Ca2+ boundto proteins in the deeper cytoplasm leak out or are released gradually, leading to asustained rise in [Ca2+]i at a moderate level. This Ca2+-buffering mechanism isobviously relevant for various physiological responses, such as a PTH in thepostsynaptic neurones or various types of plasticities of transmitter release in thepresynaptic terminals (cf. Zucker, 1989; Kuba & Kumamoto, 1990).

We thank Ms. Yamakita for excellent secretarial assistance. This work was supported by aGrant-in-Aid for Scientific Research (03557003 to K. Kuba) and by a Grant-in-Aid for ScientificResearch on Priority Areas of 'Impulse Signalling' (03225101) from the Japanese Ministry ofEducation, Science and Culture.

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