Department of Physiology, Tokyo Medical and Dental University School of Medicine, Tokyo 113-8519, Japan
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ABSTRACT |
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Momose-Sato, Yoko, Katsushige Sato, Akihiko Hirota, and Kohtaro Kamino. GABA-induced intrinsic light-scattering changes associated with voltage-sensitive dye signals in embryonic brain stem slices: coupling of depolarization and cell shrinkage. J. Neurophysiol. 79: 2208-2217, 1998. We have found new evidence for -aminobutyric acid (GABA)-induced intrinsic optical changes associated with a voltage-sensitive dye signal in the early embryonic chick brain stem slice. The slices were prepared from 8-day-old embryos, and they were stained with a voltage-sensitive dye (NK2761). Pressure ejection of GABA to one site within the preparation elicited optical changes. With 580-nm incident light, two components were identified in the GABA-induced optical change. The first component was wavelength dependent, whereas the second, slower change was independent of wavelength. Comparison with the known action spectrum of the dye indicates that the first component reflects a depolarization of the membrane and that the second, slow component is a light-scattering change resulting from cell shrinkage coupled with the depolarization. Similar optical changes also were induced by glycine, although the amplitude of both the first and second signals was much smaller than for GABA. The optical changes induced by GABA persisted in the presence of picrotoxin and 2-hydroxysaclofen, suggesting that these optical responses include a novel GABA response, which has been termed GABAD in our previous reports.
The neural amino acid Preparations
In the present experiments, we used embryonic chick brain stem slice preparations. Fertilized eggs of white Leghorn chickens were incubated for 8 days in a forced-draft incubator (Type P-03, Showa Incubator Laboratory, Urawa, Japan) at a temperature of 37°C and 60% humidity and were turned once each hour. The brain stems were dissected from the embryos. The pia mater attached to the brain stem was removed carefully using a dissecting microscope. Slices then were prepared by sectioning the embryonic brain stem transversely at the level of the root of the vagus nerve. The thickness of the slice was ~300-400 µm. The slice preparation was attached to the silicone (KE 106LTV; Shin-etsu Chemical, Tokyo) bottom of a simple chamber by pinning it with tungsten wires. The preparation was kept in a bathing solution with the following composition (in mM): 138 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 glucose, and 10 tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.2). The solution was equilibrated with oxygen.
Dye staining
The isolated slice preparation was stained by incubating it for 20 min in a Ringer solution containing 0.2 mg/ml of the voltage-sensitive merocyanine-rhodanine dye NK2761 (Nippon Kankoh Shikiso Kenkyusho, Okayama, Japan) (Kamino et al. 1981 Drug application
Neural responses were elicited by applying a small quantity (~0.5-30 nl) of GABA and other solutions near the nucleus tractus solitarius: drug solutions were pressure ejected on the surface of the preparation, with a Picospritzer (General Valve, Fairfield, NJ) with pressure pulses (20-30 psi) of 10-30 ms duration. The solutions used in the present experiment were as follows: GABA (1-10 mM), glycine (10 mM), high K+ solution (replacement of 100 mM NaCl), hypertonic solution (600 mOsm), and distilled water. These compounds were added to the standard bathing solution.
Optical recording
The optical recording system that we used in this experiment was essentially identical to that described in a previous report (Komuro et al. 1991
GABA-induced optical changes
Figure 2A shows GABA-induced optical changes recorded with three different incident light wavelengths from an 8-day-old embryonic brain stem slice preparation stained with a merocyanine-rhodanine dye, NK2761 (see METHODS). The optical changes were elicited by pressure ejection of small quantities of GABA (10 mM) on the surface of the preparation, and they were recorded simultaneously from multiple sites of the preparation. The nucleus tractus solitarius was covered by the photodiode array. The relative position of the photodiode array on the image of the preparation is drawn on (bottom right) the recordings. Each photodiode element detected transmitted light from a 56 × 56 µm2 area in the preparation. The recordings were made with 580-, 630-, and 700-nm incident light, in a single sweep. The evoked signals appeared to be concentrated in a limited area, spreading from the site to which GABA was applied, and the shapes of the signals varied with the wavelength of the incident light.
Comparison with osmotic responses
To obtain more detailed information about the characteristics of the second signal, we examined osmotic effects on the transmitted light intensity. The results are shown in Fig. 3. These recordings were made at 700 nm on unstained preparations. In this experiment, an intrinsic optical change corresponding to the second signal was evoked by an application of GABA. Similarly, when 600 mOsm (hyperosmotic/2 isotonic) sodium solution was applied, a large optical change (upward deflection of the trace) was caused. On the other hand, when distilled water was applied, a large optical change also was elicited, but its direction was opposite to that induced by GABA or by the hyperosmotic solution.
Effects of external Cl On the basis of the general context of neural GABA-ergic actions (Macdonald and Olsen 1994 Effects of GABA antagonists
We examined the effects of picrotoxin (a noncompetitive blocker for GABAA receptors) on the GABA-induced optical changes at 580 nm (Fig. 4B). In the presence of picrotoxin (200 µM), both the GABA-induced first and second signals were reduced greatly (also see Table 1), suggesting that the optical changes were closely related to GABAA receptors. In Fig. 4B, picrotoxin seemed to affect the potential change much less than the light-scattering change. This result suggests that the linear relationship is not obtained between the depolarization and cell shrinkage.
Comparison with glycine-induced optical changes
We also examined glycine-induced extrinsic and intrinsic changes in transmitted light in stained preparations. In Fig. 5A, glycine-induced optical changes recorded at 580, 630, and 700 nm are shown. When glycine was applied, as with GABA-application, optical changes were observed: a biphasic signal was recorded at 580 nm and a monophasic signal was detected at the 630 and 700 nm. Although the amplitude was smaller than that of the GABA-induced changes, the directions of the changes induced by glycine coincided with those of the GABA-induced change. Furthermore, the glycine-induced changes were eliminated in the presence of strychnine, as shown in Fig. 5B.
Comparison with high K+-induced depolarization
As one possible way to characterize the GABA- and glycine-induced optical changes, we tried to compare them with high K+-induced optical changes in stained preparations. As shown in Fig. 6A, when a high KCl (100 mM) solution was applied to the surface of the preparation in the same manner as GABA application, optical changes were observed. However, the wavelength dependence was different from that of the GABA- and glycine-induced changes. Contrary to the GABA- and glycine-induced optical changes, a biphasic optical signal that was composed of two components was observed at 700 nm. The first signal exhibited a decrease in the transmitted light intensity (upward deflection of the trace), and the second signal appeared in the direction of an increase in the transmitted light intensity (downward deflection of the trace). On the other hand, a monophasic optical change was seen at 630 and 580 nm. These monophasic changes were always in the direction of an increase in the transmitted light intensity as were the second signals at 700 nm. These results indicates that the behavior of the second signals appeared to be opposite in sign to those obtained from the experiments with GABA/glycine, although the high K+-induced first signal has the same sign as the GABA-induced first signal. In Fig. 6B, the first components isolated by subtracting the signals obtained at the isosbestic wavelength (630 nm) from the signals at 580 and 700 nm are also shown. In unstained preparations, the second signal was always observed, but the first signal was not seen even at 700 nm (data not shown).
Spatial spreading
Finally, we also have examined the spatio-temporal profile of the optical change induced by GABA. In Fig. 7A, time courses of six traces of the GABA-induced optical signals are compared. The traces were extracted from optical changes detected simultaneously by six contiguous photodiode elements (J4, J5, J6, I7, I8, and H9), which are indicated by thick lines on the drawing in Fig. 7A. The site to which GABA was applied also is indicated by an open arrowhead-like symbol. The recording was made at 580 nm. This figure shows that both the first and second signals spread progressively from a position J4, decreasing in the amplitude.
The results described in this article provide new evidence that GABA induces depolarization in the neural cell membrane and that the depolarization causes neural cellular shrinkage in the embryonic chick brain stem preparation. The evidence has been obtained, for the first time, by means of simultaneous recordings of intrinsic optical changes and extrinsic voltage-sensitive dye absorption signals.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. As is well known, two classes of GABA receptors, GABAA and GABAB, have been identified. GABAA receptors are ligand-gated chloride channels that are competitively blocked by bicuculline. GABAB receptors regulate potassium and calcium channels through G protein and intracellular second-messenger pathways. They are activated selectively by baclofen and are antagonized by phaclofen and 2-hydroxysaclofen (e.g., for reviews see Misgeld et al. 1995
; Sivilotti and Nistri 1991
). In addition, GABAC receptors, which are both bicuculline and pentobarbital insensitive but are picrotoxin sensitive, have been identified (e.g., for review see Bormann and Feigenspan 1995
).
, 1997b
).
; Grinvald 1985
; Kamino 1991
; Salzberg 1983
; Salzberg et al. 1977
), and the other is that multiple sites of a preparation can be monitored simultaneously (Cohen and Lesher 1986
; Grinvald et al. 1988
; Hirota et al. 1995
). Thus the optical recording technique has provided a unique tool for monitoring neural electrical activity, including postsynaptic potentials, in early developing embryonic nervous systems (for review see Kamino 1990
); indeed, the novel GABA-response was revealed with the optical technique.
, 1997b
), the GABA responses were observed indirectly through the effects on the glutamate-mediated EPSP evoked by vagal stimulus, so it has not been clear whether GABA induces a hyperpolarization or a depolarization in the embryonic chick brain stem preparation. Thus we examined the GABA responses directly and found that GABA induced a depolarization in embryonic brain stem neurons. In the course of this investigation, we found intrinsic optical changes evoked by GABA, and this finding permitted us to analyze the coupling of the GABA-induced depolarization to cell shrinkage.
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Momose-Sato et al. 1995b
; Salzberg et al. 1983
), and the excess (unbound) dye was washed away with dye-free Ringer solution before recording.
). Light from a 300-W tungsten-halogen lamp (Type JC-24V/300W, Kondo Philips, Tokyo) was collimated, rendered quasi-monochromatic with a heat filter (32.5B-76, Olympus Optical, Tokyo) and an interference filter having a transmission maximum at 703 ± 15 nm (mean ± half-width; Asahi Spectra, Tokyo), 580 ± 5 nm, and 630 ± 5 nm (Vacuum Optics Corporation of Japan, Tokyo). The light then was focused on the preparation by means of a bright field condenser with a numerical aperture (NA) matched to that of the microscope objective (S plan Apo, ×10, 0.4 NA). The objective and photographic eyepiece (×2.5) projected a real image of the preparation onto a 12 × 12-element silicon photodiode matrix array (MD-144-4PV; Centronic, Croydon, UK) mounted on an Olympus Vanox microscope (Type AHB-L-1, Olympus Optical, Tokyo). A schematic drawing of the apparatus and an example of the preparation with the photodiode array superimposed are shown in Fig. 1. The magnification of the image was ×25. Each pixel (element) of the array detected light transmitted by a square region (56 × 56 µm2) of the preparation. The output of each detector in the diode array was passed to an amplifier (time constant of AC-coupling = 3 s) via a current-to-voltage converter. The amplified outputs from 127 elements of the detector first were recorded simultaneously on a 128-channel recording system (RP-890 series, NF Electronic Instruments, Yokohama, Japan) and then were passed to a computer (LSI-11/73 system, Digital Equipment, Tewksbury, MA). The 128-channel data recordingsystem is composed of a main processor (RP-891), eight I/O processors (RP-893), a 64K word wave-memory (RP-892), and a videotape recorder. The program for the computer was written in assembly language (Macro-11) called from FORTRAN, under the RT-11 operating system (Version 5.1). The optical recording was carried out in a still chamber without continuous perfusion with Ringer solution, at room temperature, 26-28°C. The incident light was turned off except during the measuring period.
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FIG. 1.
A: schematic diagram of the apparatus used to measure the light intensity transmitted through the slice preparation. See text for additional details. B: action spectra obtained from the action potential-related optical signals (fractional changes in the transmitted light intensity) evoked by a depolarizing square current pulse applied to the vagus nerve in an 8-day-old embryonic chick brain stem preparation stained with a merocyanine-rhodanine dye (NK2761) (also see Momose-Sato et al. 1995b ).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 2.
Optical changes induced by GABA. A: spatial distribution of the -aminobutyric acid (GABA)-induced optical changes. Optical changes were recorded simultaneously from many sites of the brain stem slice preparation dissected from an8-day-old chick embryo, using a 12 × 12-element photodiode array. Recordings were made at 580, 630, and 700 nm and in a single sweep. Site to which GABA was applied is shown by an asterisk together with an open arrow head symbol in each recording. Direction of the arrow to the lower right side of the recording indicates a decrease in transmitted light intensity, and the length of the arrow represents the stated value of the fractional change (= change in the light intensity divided by DC background intensity). Relative position of the photodiode array on the image of the slice preparation is illustrated bottom right. B: enlargements of optical signal characteristics of 3 different wavelengths at which they were obtained. These optical changes were extracted from position G-2 in the recordings shown in Fig. 2A. Thin lines with an arrowhead are placed at the time of the GABA-application. Signal indicated by a solid triangle is the 1st signal, and the signals indicated by open triangles are the 2nd signals. Bottom: control that was obtained by pressure ejection of normal Ringer solution at 700 nm. C: enlargements of the early components isolated by subtracting the signals obtained at the isosbestic wavelength (630 nm) from the signals at 580 and 700 nm. Trace at 630 nm in B was scaled to those at 580 and 700 nm at 0 and 10 s.
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FIG. 3.
Effects of osmotic shock on transmitted light intensity, compared with the GABA-induced intrinsic optical change (top). Middle: change in response to hyperosmotic shock; bottom: change in response to hypoosmotic (application of distilled water) shock. Thin lines with an arrow-head are placed at the time of the application of GABA or of osmotic shock. Optical changes were examined using unstained preparations at700 nm.
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FIG. 4.
A: effects of a low external Cl concentration on the GABA-induced optical change. B: effects of picrotoxin (200 µM) and picrotoxin (200 µM) together with 2-hydroxysaclofen (200 µM) on the GABA-induced optical change. C: effects of a low Cl
concentration on the picrotoxin- and 2-hydroxysaclofen-insensitive component of the GABA-induced optical change. See text for additional details.
) (also see Fig. 1B); further a membrane depolarization causes an increase in absorption (a decrease in transmitted light) at 700 nm and a decrease in absorption (an increase in transmitted light) at 580 nm. From these spectral characteristics, we can conclude reasonably that the first signal is a dye-dependent absorption change that depends on membrane depolarization: the first signal is absent at 630 nm, and, at 700 nm, the first signal is masked by the second signal that might be an intrinsic change. At 630 nm, there is a brief delay, maybe corresponding to the absence of the first signal. We isolated the first component by subtracting the signals obtained at the isosbestic wavelength (630 nm) from the signals at 580 and 700 nm (Fig. 2C). In these traces, the real time course of the depolarization-dependent absorption change is shown clearly.
; Konnerth et al. 1987
). According to this idea, it is likely that the effects of tonicity on the transmitted light intensity in the preparation reflect the light-scattering changes due to hyperosmotic shrinkage and hypoosmotic swelling of neural cells. Thus we suggest that the GABA-induced intrinsic optical change (light-scattering change corresponding to the second signal) results from the increase in the extracellular space due to the shrinkage of the neural cells (also see DISCUSSION).
), the GABA-induced optical changes are expected to be influenced by the extracellular Cl
concentration. Thus we examined the effect of the external Cl
on the optical signals. When NaCl was replaced by Na methanesulfonate, both the first and second signals induced by GABA were reduced markedly. Figure 4A shows GABA-induced optical changes recorded simultaneously from five sites of a preparation in normal Ringer solution (control) and in a low-Cl
Ringer solution in which NaCl was replaced by Na methanesulfonate. In the low-Cl
bathing solution, both of the first and second signals were depressed markedly. Similar results also were obtained with Cl
replacement by NO3
. The result suggests that the GABA-induced first and second signals both depend on extracellular Cl
concentrations. On the other hand, the optical signals were not affected by lowering the external Ca2+ concentration, indicating that Ca2+ activity, which might be decreased by methanesulfonate, does not influence the optical signals (data not shown).
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TABLE 1.
Amplitudes of the first and second components evoked by GABA application
, 1997b
). We have examined whether the picrotoxin- and 2-hydroxysaclofen-insensitive component is dependent on external Cl
. As shown in Fig. 4C, the picrotoxin- and 2-hydroxysaclofen-insensitive component also was reduced markedly in a low-Cl
Ringer solution. This result suggests that the picrotoxin- and 2-hydroxysaclofen-insensitive component is also dependent on extracellular Cl
.
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FIG. 5.
Glycine-induced changes in transmitted light intensity. A: recordings obtained simultaneously from an 8-day-old embryonic preparation, at 580, 630, and 700 nm. B: effects of strychnine (200 µM) on the glycine-induced optical change.
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FIG. 6.
High K+-induced changes in transmitted light intensity. Note that the direction of the 2nd signal is opposite to that of the 2nd signal induced by GABA. See text for additional details.
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FIG. 7.
Spatial spread of the GABA-induced optical changes indicated by waveforms in individual detectors (A) and by a pseudocolor imaging representation (B). Color imagings were constructed from the optical recordings of 12 × 12 matrix array using an interpolation method of "Transform" (Fortner Research LLC, Sterling, VA).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). When applied to dendrites, GABA caused a depolarization with an increase in membrane conductance in CA1 cells in adult guinea pig hippocampal slices (Andersen et al. 1980
) and in the rat hippocampal slice preparation (Alger and Nicoll 1982
). In addition, depolarizing GABA responses were observed recently in the neonatal rat hippocampal preparation (Ben-Ari et al. 1989
; Strata and Cherubini 1994
), in the embryonic chick brain stem (Hyson et al. 1995
), and in the embryonic rat spinal cord (Reichling et al. 1994
; Wu et al. 1992
).
current represents an efflux of the negatively charged Cl
; and then 3) the cell membrane is depolarized. Accordingly, it is suggested that, in comparison with usual adult neurons, the intracellular Cl
concentration is elevated. Indeed, it is known that, in hippocampal slice preparations, increasing the intracellular chloride concentration by injection shifts the reversal potential of the GABA responses in a positive direction (Misgeld et al. 1986
).
bathing solution. From this result, it is speculated that when the external Cl
concentration is lowered, the ECl (EIPSP, Erev) and the Em both shift in the depolarizing direction and that the change in Em is larger than that in the ECl (EIPSP, Erev). Another possible explanation is that reducing Cl
outside might reduce Cl
inside without a steady state change in ECl or Em (Hodgkin and Horowicz 1959
). With lower Cl
on both sides, the putative GABA Cl
channels might have a lower conductance and therefore less current and smaller depolarization.
and K+ driven by a difference of the equilibrium potential for Cl
and the resting potential(ECl
Em). In Fig. 8, one possible sequence for the cell shrinkage caused by membrane depolarization induced by GABA (or glycine) is illustrated. In this figure, a scheme for the cell swelling caused by high K+-induced depolarization also is shown. On the other hand, Cohen, Keynes, and Landowne (1972) have found that a low Cl
solution reduced the (90°) light-scattering signal associated with outward currents in squid giant axons and have discussed it in terms of a transport number effect. It is also possible that such a mechanism is related to our findings.
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FIG. 8.
A possible sequence that might account for the cellular shrinkage coupled with GABA-induced membrane depolarization in the embryonic brain stem preparation. Possible scheme for K+-induced cellular swelling also is shown.
and by Cohen et al. (1968)
. One of the changes in light scattering from axons is thought to represent axonal swelling during activity (Cohen and Keynes 1971
; also for a review, see Cohen 1973
). Using light-scattering measurements, Lipton (1973)
observed that K+-induced membrane depolarization caused swelling of neural cells in guinea pig cerebral cortical slices, and Kamino et al. (1973)
also reported that external K+-induced swelling in nerve-ending particles (synaptosomes). Shrinkage of the volume of the extracellular space, presumably due to cellular swelling, has been shown to occur during evoked activity in the cortex (Dietzel et al. 1980
, 1982
), in the hippocampal slice during high K+ perfusion (McBain et al. 1990
), and in an experimental model of epilepsy (Heinemann and Dietzel 1984
). Furthermore,Salzberg et al. (1985)
have reported light-scattering changes related to excitation-secretion coupling in the mammalian neurohypophysis.
; Kreisman et al. 1995
; MacVicar and Hochman 1991
; Polischuk and Andrew 1996
) and neocortical (Holthoff and Witte 1996
; Holthoff et al. 1994
) slices has been reported. Further, Andrew et al. (1996)
have shown N-methyl-D-aspartate- and kainate-induced intrinsic optical signals resembling dendritic swelling in rat hippocampal slice. We have also shown that in the embryonic chick brain stem slice, glutamate induces cell swelling (Sato et al. 1997
). These observations are regarded as belonging to the category of K+-induced depolarization-related cellular changes, as illustrated in Fig. 6. In contrast, we provide evidence for a second category: the ion-efflux associated with membrane depolarization induced by inhibitory chemical transmitters such as GABA and glycine causes neural cellular shrinkage.
, 1997b
), we have reported evidence for a novel type of GABA response, which is insensitive to antagonists of both GABAA and GABAB receptors, and we have suggested a novel type GABAD receptor. However, because these results were indirect, it has been unclear whether the novel GABAD receptor is dependent on Cl
. Concerning this issue, the results obtained in the present study provide additional important information: the GABAD receptor participates in neural cell shrinkage coupled with membrane depolarization mediated by Cl
. Thus it seems likely that the GABAD receptor contains a component that is functionally similar to the GABAA receptor.
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ACKNOWLEDGEMENTS |
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We thank L. Cohen and B. Salzberg for critical reading of the manuscript and useful comments. We also thank T. Sakai for helpful discussion concerning the manuscript.
This work was supported by grants from the Monbusho of Japan.
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FOOTNOTES |
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Present address of A. Hirota: Dept. of Physiology, Shimane Medical University, Izumo-shi, Shimane 693, Japan.
Address for reprint requests: K. Kamino, Dept. of Physiology, Tokyo Medical and Dental University School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Received 11 September 1997; accepted in final form 12 January 1998.
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REFERENCES |
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