Brain Research, 120 (1977) 67-83

67

© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

S P R E A D I N G DEPRESSION IN ISOLATED CARP R E T I N A

HARUHIRO HIGASHIDA*, MANABU SAKAK1BARA and GENYO MITARAI Research Institute of Environmental Medicine, Nagoya University, Nagoya 464 (Japan}

(Accepted May 10th, 1976)

SUMMARY (1) Spreading depression (SD) could be elicited in isolated carp retina by KC1 application, the concomitants of which were similar to those described in other vertebrates. (2) The threshold for generating SD was greatly reduced by brief immersion of the retina in low CI- Ringer's solutions. The properties of SD waves were almost the same with treated and untreated retinas, except for intervals. (3) Extracellular negative potential shifts during SD, averaging 4.6 mV in amplitude and 27 sec in duration, were recorded in whole retinal layers with the maximum amplitude about at the inner plexiform layer. (4) The P m potential of the local electroretinogram was virtually unaffected by SD. (5) Both L- and C-type S-potentials could be evoked with increase of 20-40 in amplitude around the peak of slow membrane depolarizations (mean value of maximal amplitude 5.8 mV) during SD in horizontal cells. (6) Increase in spike number was observed in both on- and off-center ganglion cells before and after the spike cessation during SD in the untreated retina. However, the off-discharges, which were a unique response to light in the immersed retina, only decreased during SD.

I NTRODUCTION Spreading depression (SD) 4,17, first found by Le~o in rabbit cortex 15, has also been identified in the vertebrate retina of several species: amphibian 7, toad 7 (see also ref. 19), bullfrog 9,t0,2a, chickenlS-20, 34, and pigeon 2s. Although the fish retina has * To whom correspondence should be addressed. The author's present address: Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md. 20014, U.S.A.

68 basically the same structure as in other vertebrates38, 39, and SD would presumably occur, it has never been clearly demonstrated (see refs. 4 and 19). The present study, therefore, was undertaken to establish whether it is possible to elicit SD in fish retina. Local application of KC1 solution induced spreading slow potential changes in the isolated carp retina, but SD could be elicited only rarely in untreated retinas. Thus, a consistent SD was obtained with the support of C1- deficient solution, as proposed by Hanawa et al.9,1° and Le~,o 16. Many extracellular potential changes during SD were recorded and compared in treated and untreated retinas, and intracellular and extracellular observations of horizontal and ganglion cells were made during SD. METHODS Experiments were performed on retinas isolated from carp (Cyprh~us carpio) of about 25 cm in body length. The carp was dark-adapted for over 2 h before enucleation of the eye under dim white light. Retinas of about 5-6 m m in radius were isolated from the pigment epithelium and mounted receptor-side up on the 1 ~ a g a r Ringer layer in a small moist chamber at room temperature under a continuous stream of moist 98 ~ 02, 2 ~ COs (Fig. 1A). In the first series, the isolated retinas were given a 30 min recovery period before recording. In the second, to reduce the threshold in generation of SD, the just isolated retina was immediately immersed in the low C1Ringer's solutions for 20 sec in the chamber, and then the excess fluid in the chamber was completely drained. This procedure was repeated at least 3 times, and then retinas were left for a given 30 min recovery period before recording. Three kinds of low C1- medium were used: 6.2 m M CI- and hypotonic solution (I); 3 m M C1- and hypotonic solution (II); and 3 m M C1- and isotonic solution (III) (see Table I). Every effort was taken to minimize retinal trauma during preparation. Glass microelectrodes of 2-50 M ~ resistance, filled with 2.5 M NaCI or potassium citrate, were used to record the intraretinal DC-potential, intraretinal electroretinogram (ERG), and S-potentials of horizontal cells. Glass-ensheathed platinum-

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Fig. 1. A: arrangement for recording of intraretinal potentials with microelectrodes in isolated carp retina. Retina (R) with the vitreous (V) was placed receptor-side up on agar-Ringer layer (A) in a small chamber. B: diagram illustrating the progress of SD in retina. Estimated potential changes of SD are presented by heavy lines. Arrow denotes direction of propagation. The filled circle and dotted one indicate sites of KCI application and spot illumination, respectively. Recordings were obtained from the illuminated position with the opening about 1-6 mm from the stimulating site.

69 TABLE I Composition and osmolarity o f low CI- Ringer's solution

All concentrations and osmolarities are expressed in terms of mM and mOsM, respectively. Reagents

Na2SO4 KC1 CaCI~ Ca-Gluconate MgSO4 NaHCOa NaH2PO4 Glucose Sucrose Osmolarity§

Solution (I) *

65 2.6 1.8 --2.4 ---168

Solution (II) **

Solution (Ill) **

55 3 -2

55 3 -2

1

1

20 3 20 -202

20 3 20 55 262

* Titrated to pH 7.2 with H2504. ** Buffered at pH 7.2 by airing with a gas mixture of 98Y0 02 and 2~o CO2. § The osmolarities of normal-chloride solutions analogous to solutions (I) and (II) above, containing 130 and 110 mM NaCI instead of Na~SO4, respectively, were 256 and 283 mOsM, respectively. The osmolarity was measured with an Advanced osmometer (Advanced Instruments Inc., 3L) by one of the authors (H.H.) in the Laboratory of Biochemical Genetics, NHLI, National Institutes of Health, U.S.A. iridium electrodes with a tip diameter of 5-20/*m were used for recording extraceUular spikes of ganglion cells, and at the same time served for recording of extracellular slow potentia! changes at the same position. A circular Ag-AgC1 wire [embedded in agar layer served as a reference electrode (Fig. 1A). The signal of ganglion cell discharges and slow potential changes recorded with metal electrodes was led through a preamplifier (Nihonkoden MZ-4) to a dual beam oscilloscope (Iwatsu DS-5016). For measuring the conduction velocity of SD and intra- and extracellular potential changes of horizontal cells during SD, simultaneous recordings with 2 glass microelectrodes were made through 2 preamplifiers (W-P Inst. M 701, and a high input-impedance, 25-gain amplifier made by M. S.). All responses of DC-potentials displayed on the oscilloscope were simultaneously recorded on a continuously running rectilinear penwriter (Sanei Rectigraph 85). On some occasions, the data were also recorded on magnetic tape with a T E A C F M tape recorder. Part of the spike count was done manually on the X-ray films photographed from the oscilloscope, and part by minicomputer (Hitachi 10-II). A white light (500 W xenon arc lamp) was passed through a series of neutral density filters to reduce the intensity in logarithmic steps (300 to 5/~W/sq. cm) and then focused on the retinal surface in the form of various spots ranging from 0.3 to 12 m m in diameter. To determine response types of S-potentials in horizontal cells, monochromatic blue and red lights were used with interference filters of 453 and 641 nm, respectively. Light flashes of 500 msec duration were delivered every 0.24).5 sec. A tungsten lamp was used to maintain a constant light-adapted background (10 # W / sq. cm) to measure ganglion cell activities of treated (solution I) and untreated retinas, and these cells showed clear center-surround organization ~.

70 To produce SD, about 2-5 #1 of 2 % (270 mM)-25 ~ (3.3 M) KCI solution was applied in drops on the periphery of the retinal surface (receptor side) by means of a micropipette, with the opening about i-6 mm from the recording site. The KCI solution once applied spread over the retinal surface, usually forming a 1-3 mm band in good preparations and distinguished by the milky pink color of the tissue 7. Since it was difficult to elicit SD when the retinal surface was wet, application of solution was done 1.5-4 h after the dissection. It has been demonstrated earlier that a single KCI application commonly causes recurrent SDs in the retina 30 and the cortexa,11,12 , 17,31. To distinguish the SD in the present experiment from phenomena produced merely by KC1 diffusion with no contribution of cellular activities, its occurrence was only considered when recurrent negative potential changes were well developed. Cases were excluded in which only a single potential wave similar to SD was identifiable. RESULTS In 170 retinas isolated from 112 carp, more than 200 KCI applications were made on the retinal surface, and SD potential changes were examined at nearly 800 electrode locations. Potential changes of the SD type could be elicited only 6 times out of 105 untreated retinas, but 31 times out of 50 in those immersed in the low C1- Ringer's solutions in Table I (Table II). Even though types of solutions were varied, SD potential changes produced in the treated retinas was much higher than in untreated ones. The latency time from the application of KC1 to the first evoked potential changes ranged from 0.6 to 38 min, with a mean value of 10 ± 9 min (q- S.D.). In 4 cases in both types of retina, latency was over 20 min. Once the first potential change was generated, the recurrent potential changes were recorded spontaneously and continuously at every place in the retina for over 6.6 h in maximum. The mean observation time of recurrent SD waves was 167 ± 117 min in 37 cases in which it was elicited. However, it is still unclear how the SD wave developed in the carp retina. To confirm the specificity of CI- in the treating medium, KCl-evoking SD was tested in 10 retinas treated with a normal Ringer's solution (containing 130 m M NaC1) and 5 treated with a low Na + solution (replacing 1 I0 m M choline chloride with 55 m M Na2SO4 only from the solution (II)), but none of them showed SD.

Properties of extracellular slow potential changes of SD In Table II, results of extracellular slow potential changes of SD observed between different types of the retina are summarized quantitatively. The average amplitude of negative potential shifts ranged from --3.6 to --5.2 mV for various types of the retina. The duration of those potential shifts whose mean value was calculated at 24, 28, and 29 sec for the 4 types of retina, respectively, was rather shorter than that reported for SD in amphibian retinal No significant difference in amplitude and duration of potential changes of SD was observed between treated and untreated retinas (Table II). The average amplitudes in a total of 1105 negative potential shifts and the duration in a total of 1082 were 4.2 mV and 27 sec, respectively. The small positive potential shifts were also observed both before and after the

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Number of caseswhich was elicited

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Number of tested retinas

Types of retinas

Characteristics of the extracellular slow potential changes during SD*

TABLE

27

2 4 4- 5 n = 107 28 4- 8§ n-- 572 2 4 4- 4 n = 305

2 9 4- 7 n = 98

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Fig. 2. Intraretinal depth profile of extracellular slow potential shifts during SD in the retina immersed in the low CI- Ringer's solution (I). Potential measurements were made both advancing and retracing electrode at various depths of the retina showing serial SD. Depth reading was made by the micromanipulator indication. A: specimen responses at indicated depth from retinal surface of receptor side. In this and all subsequent figures, negativity is displayed as a downward deflection. B: amplitude of negative potential shift is plotted against the depth for A series. Each filled circle indicates amplitude of one negative potential shift, and the curve corresponds to the mean amplitude at each depth.

negative potential shifts in some cases (Figs. 2, 3, 7, and 9), though no such observation was made by Gouras on amphibian retinal The magnitude of the second positive potential shift was about twice that of the first reaction, which averaged less than 1 mV (average value 0.6 mV, in Table II). The positive-negative-positive potential shift during SD produced in the carp was similar to that observed in the cortex of the rabbit 11,12, except for the smaller amplitude and shorter duration in the fish retina. In order to obtain further information about the origin of the negative potential shift during SD, it was recorded at different depths in 3 retinas immersed in the low CI- and hypotonic solution (I). The recording was continued for a while before the start of the experiments to confirm the generation of SD waves after application of 25 ~ KCI solution. Then the initial contact of the electrode with the retinal surface of the receptor side was monitored with a loud speaker and a recording glass microelectrode was successively inserted downward into the retina from the receptor side to the ganglion cell layer. Recordings were obtained at 50 # m intervals for about l0 min. The depth was checked by the readout on a digital micromanipulator (Narushige SM-20) and no histological analysis was made. Until the electrode tip reached a region of 250 # m from the receptor side, negative potential shifts with almost the same amplitude of about - - 3 mV were recorded, with the maximum amplitude of about - - 7 mV at a level of 200 # m (Fig. 2). Then, at a region 300 #m, where the electrode seemed to penetrate the inner limiting membrane into the vitreous layer, it abruptly decrease in amplitude, and assumed a potential almost equal to that of the reference electrode in the agar layer under the vitreous. The potential distribution is presented

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Fig. 3. Changes of peak to peak intervals of successive potential changes of SD waves repeatedly appeared more than 33 times in untreated retina ( 0 ) and that treated with solution (I) ((3). The nth interval is the time between the nth and the (n+l)th SD waves. Upper: recordings of extracellular potential changes of 1.5 min duration of indicated number's wave in the untreated retina. Lower: recordings of the first and 30th potential changes in the treated retina. Voltage calibration: 14 mV as a scale of 2 min in the vertical. graphically in Fig. 2B as an intraretinal laminar profile o f the negative potential shift o f SD. Fig. 3 indicates changes in the peak to peak intervals o f 2 typical cases in which the serial SD potential changes were recorded over 30 times. In the untreated retina, the first negative potential change was evoked at 3.3 min after application, and the intervals gradually increased f r o m 4 to 9 min, as was observed in SD o f cephalopod retina by Schad6 and Collewijn 3°. The amplitude grew till the 10th negative potential shift, and then decreased gradually. W h e n immersed in solution (I), however, the first one was elicited at 8 rain after application, and the intervals decreased f r o m 4-5 to 1-2 min. The amplitude o f the negative reaction was not changed during 35 recurrent SDs. In this manner, consequently, the potential change occurred frequently every 1-2 min in the retina immersed in the hypotonic solutions (Table II). A n example is seen in Fig. 2A. The retina treated with the isotonic solution (III), however, showed no intervals o f less than 2 min. The average

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Time(rain) Fig. 5. Intracellular recordings from a horizontal cell showing a L-type S-potential (lower) and extracellular DC-potential record (upper) during one episode of SD in the retina immersed in the low CIand hypotonic solution (II). Blue light of continuous intensity gave hyperpolarizing S-potentials and local ERG. The recordings were obtained at 268 min from the start of the initially evoked SD. interval time of 5.3 min (Table II) was a mean value between untreated retinas and those immersed in the hypotonic solutions (I and II). The conduction velocity was measured in 2 types of the retina treated with the solutions (II and III). Two glass microelectrodes, with the tips about 0.13-2.3 mm apart, were located with the opening about 1-4 mm from the stimulating site through the retina at a depth of 50-150/~m from the receptor side. As SD waves propagated, these electrodes recorded the appearance of those potential changes at each electrode with retardation in the arrival of SD, as shown in Fig. 4. The conduction rate ranged from 1.6 to 6.8 mm/min (average 3.9 mm/min) in cases treated with the hypotonic solution (II), and from 1.2 to 6.3 mm/min (average 3.5 mm/min) in ones immersed in the isotonic solution (III), respectively, which indicates that the osmolarity of the solution has no effect on the propagation rate. The speed of the propagation of SD in the immersed carp retina was apparently greater than in amphibian retina (1 mm/ min) 7 or in cephalopod retina (2 mm/min) 8°, but agreed with findings that the propagation rate of SD was hastened by the dilution of NaCI concentration 15,20.

Intraretinal local ERG during SD When microelectrodes penetrated the retina from the receptor surface, a potential that was almost rectangular in shape and positive in polarity was elicited by the focal illumination of the retina, as shown in Fig. 4. The positive-going slow potential showed a maximum amplitude at the receptor layer, and decreased in the inmost retinal layer, but did not reverse the polarity of the response, a phenomenon which was identified as the conventional PHI component in the intraretinal local electroretinogram25,2a, s2. Fig. 4 indicates the time courses of changes in these positive potentials during the negative potential shift of SD recorded with 2 glass microelectrodes at the receptor layer in the retina treated with the isotonic solution (Ill). The positiveevoked potentials of about 1 mV, obtained by the focal circular spot of 1 mm diameter and 500 msec duration, could be observed even during negative potential changes of 1.2 and 2.3 mV, respectively. The amplitude of those positive-going reactions during SD showed an increase of about 30 ~ over the SD before, which was frequently observed when fine glass microelectrodes were used for recording (cf. Figs. 5 and 6).

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Fig. 6. Intracellular recording from C-type horizontal cells (lower) and extracellular DC-potential record (upper) during one episode of SD in the retina immersed in the solution (II). Red light of constant intensity gave depolarizing S-potentials and local ERG. The recordings were obtained at 185 min after the first SD.

Horizontal cells during SD Intracellular recordings were obtained from horizontal cells in the retina immersed in the solution (II), whose responses in carp retina were identified according to the physiological and morphological criteria established by Mitarai et al. z2. Reliable observations were made on 82 horizontal cells located at a region 50-150/~m from the receptor side showing a membrane potential level in dark (resting membrane potential) of between --15 and --60 mV, with a mean value of 32 4- 15 mV. In experiments about 2-6 h after the dissection, the membrane potential in dark of over - - 4 0 mV was frequently recorded. S-potentials of horizontal cells in immersed retinas showed the same spatial summating characteristics and response-intensity curves for different sizes and intensity of spot light as those usually observed in the untreated ones. In the L-type horizontal cell shown in Fig. 5, a dark membrane potential level o f - - 5 6 mV was first recorded and the amplitudes of hyperpolarizing S-potentials to blue and red lights of 1 mm diameter were 13 and 2 mV, respectively. With an invasion of SD, the cell showed a slow membrane depolarization of 10 mV, and around the peak of the membrane depolarization, hyperpolarizing S-potentials temporarily increased their amplitude as much as 20-30 ~ . The change of membrane potential in C-type horizontal cells during SD was, in general identical with that of L-type ones. Its time course resembling extracellular potential changes by SD, the dark membrane potential level depolarized from --62 to i 5 7 mV at the peak value, and then repolarized beyond the initial level (Fig. 6). The red depolarizing S-potential of 7 mV (Fig. 6) and blue hyperpolarizing one of 29 mV (not illustrated) were also evoked with an increase of about 30 ~ in amplitude during SD. The maximal depolarization amplitude of 45 horizontal cells with a mean membrane potential level o f - - 3 7 mV ranged from 1 to 23 mV, and the mean value of 5.8 4- 4.6 mV was the same for the average amplitude of simultaneously recorded extracellular negative potential shifts (5.6 4- 3.0 m Y ) .

Ganglion cell unit discharges during SD Extracellular unit activities were recorded from about 300 ganglion cells in

76 I

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Fig. 7. Examples of firing pattern of an on-center ganglion cell activity during SD with their relationship to the concomitant extracellular slow potential changes indicated. Upper deflection of signal indicates duration of photic stimulation of 0.4 mm in diameter. A: on-discharges before invasion of SD. B: increased firings before spike depression. C: spike depression during SD. various regions of the retina with metal electrodes under a constant background light. The area-threshold method av was employed to investigate the size of the receptive field. O f the 55 units tested in untreated carp retinas, 8 (15 ~ ) were classified as oncenter units, 20 (36 ~ ) as on-off cells, and 27 (49 ~ ) as off-center units, which was comparable to those figures obtained in the frog by Barlow 1 for distribution by type of response. Records of ganglion cells in the retina immersed in solution (I) under light adaptation showed that the on-discharge was abolished but the off-discharge remained for spot illumination, as described by Miller and Dacheux 21. In 28 units tested by various sizes of spot illumination, all showed the off-discharges and none displayed on-responses. Changes in firing pattern of ganglion cells by SD were investigated in 6 units in each of 6 untreated retinas and 20 units in 7 treated with the solution (I). All 26 cells encountered showed some changes during SD as follows. In 2 units of 2 untreated retinas and 5 units in 5 treated ones which showed spontaneous and sustained discharges without close correlation between the photic stimulation and spike discharges, a sharp increase in the number of spikes was recorded before and after depression (not illustrated), just as found by GourasL The frequency of spikes during light on and off was quite the same throughout the SD time course. SD changes were recorded in 3 on-center units in 3 untreated retinas. Fig. 7 shows changes in spikes of an on-center unit during the second SD corresponding to

77

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Fig. 8. Examples of response pattern of an off-center ganglion unit at each stage of SD indicated in Fig. 9. Upper deflection of the uppermost indicates duration of illumination. Upward and downward deflections of baselines at onset and offset of photic stimulation indicate the so-called surface potential (Pro component of intraretinal ERG) isolated by the same electrode for the spike measurement recorded via RC-coupled amplifier. This potential was not suppressed even when spikes depressed (C), a finding identical to results in Fig. 4. A: some sustained discharges appeared at light off, with no spike at light on before SD invasion. B: increase in number of spikes at light off and 3 spikes even at light on is seen in the initial phase of SD. D: spike train after gradual recover from depression. Note that larger spikes generated during illumination though small burst discharges were generated at light off. recurrent SD in the untreated retina presented in Fig. 3. During the rest phase between the first and second SD, on-discharges with 5-15 spikes were evoked in response to spot light of 0.4 mm in diameter during 500 msec with a few spontaneous spikes during the cessation of light for 2.8 sec (Fig. 7A). When the SD wave invaded the unit, since spontaneous discharges increased in number during light-off and spikes during light-on did not, almost an equal number of spikes was generated during light on and off (Fig. 7B) before and after the complete depression of spikes for 9 sec (C). In other units, just before the spike cessation, spikes during illumination were rather smaU in number as compared to increased burst discharges during light off. Only in an off-center unit shown in Figs. 8 and 9 was there observed a time course during SD as a response to spot illumination of 0.8 mm diameter. At first the cell showed sustained spikes when the light was off and no spikes when the light was on (Fig. 8A), before the onset of SD. These firing patterns were changed into spikes newly generated even at light on with vanishing of the on-inhibition and a number of spikes at light off (B). Fig. 8D is a recording when spikes recovered from complete cessation

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Fig. 9. Time course of slow potential changes, spike frequencyat light on (©) and off (•), and latency between the onset of illumination and the first spike generated during light stimulation (A) in the off-center unit shown in Fig. 8. (C). While small discharges appeared at light off, on the contrary, larger spikes were generated during illumination (D). Frequency of spikes at both light on and off was increased at the same rate before the spike depression for 20 sec and decreased to the initial level after regeneration of burst discharges (Fig. 9). The latency when the first spike was generated after the onset of the light during illumination, decreased with a sigmoid curve from 300 to 100 msec, and increased inversely after depression (Fig. 9). In the recording of Fig. 8, the spike discharges and slow potential evoked by illumination, which are presented by the same upward and downward deflection of base lines recorded via a CR coupled amplifier (time constant 0.03 sec), were isolated by the same electrode. The slow potential, which may be the PIII component of the intraretinaI E R G , did not disappear during the negative potential shift, even though spikes had ceased (Fig. 8C), similar to the results shown in Fig. 4. In the immersed retina the behavior of firings during SD was observed in 15 units having responses to light. One unit with the off-sustained discharges showed only decrease in number with 2 newly generated spikes during light on by invasion of SD (Fig. 10), which was a quite different change in firing pattern during SD from the offsustained cell in the untreated retina. The other 14 units showing the off-transient discharges (not illustrated) also manifested a unilateral decrease in number of evoked spikes without any enhancement in spontaneous spikes during SD. DISCUSSION Despite the frequent use of fish in the retinal investigationla,22,25,a2,aa, a9, SD in fish retina has not been demonstrated (see refs. 4 and 19). However, the present experimental results in isolated carp retina clearly indicate that SD can be observed in the retina of fish as in other vertebrates?,9,z0,18-20, 2a,2s,a4. In order to elicit SD, stimulation was made not by illumination of the retina superfused with the low Cl- solution10,20, 2a, but by the local application of concentrated KC1 solution. In the superfused retina, slow depressive potential has been reported to be rapidly evoked in the whole retina following the b wave, so one may

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Fig. 10. Slow potential changes and spike frequency time histogram of off-sustained discharges in the retina immersed in the solution (I). Upper: an example of spike train for 5.5 sec by CRO, whose recording period corresponds to bar in the lower. The upward signal of the uppermost row indicates duration of light stimuli of 0.6 mm diameter. Lower: frequency histogram of spikes during cessation of illumination, and slow potential changes isolated by the same electrode measured extracellular spikes. well wonder if slow depressive potential propagation is absolutely the same as in SD. In this series of experiments, it was possible to observe SD propagated from the stimulating to the recording site, as seen in Fig. 4. " The rate of SD produced was only 6 ~ in all trials involving untreated retinas, and when it actually was produced, a long latency of 20-40 min from stimulation to initiation was necessary on several occasions, suggesting that the carp retina obviously has a very high threshold. This might be one reason why it has not been found up to now in fish retina. Based on the results of Hanawa et al. in frog retinag, 10, namely, that the slow depressive potential could easily be evoked by photic and mechanical stimulation when 15 m M CI- modified Conway's solution was applied on the receptor side, 3 types of the low C1- Ringer's solution (Table I) for immersion of the retina caused considerable decrease in the threshold for producing SD (Table II). Since properties of amplitude, duration, and conduction rate of the slow negative potential shift of SD were very close between 4 different types of treated and untreated retinas, except for the intervals (Table II), SD evoked in the 4 types could be identified as basically the same in all. F r o m the results that SD could not be evoked in 15 retinas treated with the normal Ringer's and the low N a ÷ solution, it was concluded that SD production rate depends especially on the extracellular chloride concentration of the tissue. The significant decrease in the threshold for eliciting SD in retina immersed in low C1solution might be explained as follows: (1) The shift in C1- equilibrium potential to a more depolarized value of some elements could predispose the retina to increased excitability, a view supported by the observations that seizure discharges are readily induced in the cortex in Cl--free medium27, 29. (2) Since SD is thought to cause the influx of N a +, CI-, and water in neuronsll, 35, and the influx of C1- coupled with K +

80 exhibiting Michaelis-Menten kinetics in glial cells ~, the immersion in the low C1solution may serve as a preliminary for the same low [C1-]0 state as that generated during SD. (3) The lack of Ca 2-- regulatory mechanism because of increase of insoluble CaSO4 caused by sulfate ions in the medium, might have some effects on elicitation of SD, although this is probably negligible9,10. In retinas immersed in 2 hypotonic solutions, shorter intervals of about 2 min in average were obtained (Table II). As the duration of every negative potential shift remained unaltered in successive SDs (Table II, Figs. 2 and 3), the decrease in intervals might indicate a shortening in the resting phase of the tissue between SDs due to the effect of the hypotonic medium; the water may facilitate the further increase of intracellular water and swelling of neurons and neuroglial cells accompanying a reduction of the extracellular compartment 3,33, and these conditions are similar to those considered to prevail during SD 35. Because the extracellular negative potential shift during SD was recorded from the retinal surface of the receptor side and not from the vitreous in the depth-voltage study (Fig. 2), it can be assumed that SD occurred throughout every retina layer from the receptor to the inner limiting membrane. The potential maximum in the negative potential shift was recorded at a region about 200/zm from the receptor side (Fig. 2), which is near the inner plexiform layer, to judge from the histological results in carp retina22,3s, 39. In the experiments involving light scattering, Martins-Ferreira and Oliveira Castro have observed the maximum change in the same layer is. Recently, Mori et al. have shown that the SD potential maximum was in the plexiform layer or more vitreous side in frog retina 23. These results suggest that the inner plexiform layer might serve as the current sink of the negative potential shift in the retina during SD. It was of interest that SD did not suppress the local ERG (Fig. 4). As the positive response evoked by focal light has been identified as the P u t component of Granit25, 2s, mass potentials arising from receptors for the most part 3z, the above findings suggest that receptor potentials may be less affected by SD, though this is not conclusive. Hanawa et al. 10 and Ogden and Wylie 28 have also reported similar results that the a wave in the E R G was most resistant to SD associated with depression of the b wave during SD. Therefore, it is quite conceivable that the slight increase in amplitude of the positive responses of the summated mass potential (ERG) during SD is caused by virtually no contribution of the negative potential (i.e., depression of the PH component as observed in frog l0 and pigeon 2s retinas) rather than by the movement of the recording positions with tissue swelling 19 and by the real increase of receptor potentials. The above estimation seems to be supported by the fact that this increase was seldom observed in cases where metal electrodes, which can record from the wider field area, were used. This is the first report on the behavior of horizontal cells during SD. Changes of membrane potentials in horizontal cells during SD which showed slow membrane depolarizations with a time course resembling the extracellular SD, have the same polarity as in neuronsS,6,11, 31 and neuroglial cells 11A2,14,31 in the cortex, and Mtiller ceils in the retina 23, but different from receptor cells (Mori et al., Jap. J. Physiol., in preparation). Judging from the usual membrane potential level o f - - 2 0 to --40 mV,

81 membrane potentials of the horizontal cells in Figs. 5 and 6 were extremely large, though slow depolarizations are shown clearly. Horizontal cells showing membrane potentials lower than --40 mV in fact depolarized during SD. The amplitude of membrane depolarizations during SD in horizontal cells (mean value 5.8 mV)approached amplitude value of extracellular negative potential changes (mean value 5.6 mV). These facts indicate that the horizontal cell, the secondary neuron, contributes to the electrogenesis of extracellular field potentials of SD in the retina, as well as Miiller cells2~. Isolation of the PII component in the local ERG is so difficult that no direct evidence of changes of the potential during SD was obtained in this experiment, because the amplitude of the Piu component was great and the negative potential of the PII was not recorded in every retina layer of the carp 25. However, considering the changes of the intraretinal local ERG (Fig. 4), it is highly possible that PIt component might be suppressed during SD in the carp retina, as well as frog 1° and pigeon 2s retinas. If this were true, since S-potentials could be evoked by spot light during SD (Figs. 5 and 6), it could be postulated that S-potentials of horizontal cells would contribute almost nothing to the electrogenesis of the PII component (b wave) of the ERG. As suggested by Kaneko and Shimazaki 13 and Nelson 28, Na ÷ and K + significantly contribute to the membrane potential level of horizontal cells in dark and light, respectively, so horizontal cell membrane depolarizations during SD (Figs. 5 and 6) may be caused by the increase of the sodium permeability of the cell membrane and the extracellular concentration of potassium around the cells. Our recent results have shown that the membrane resistance of 3 horizontal cells decreased with membrane depolarizations by SD measured through the bridge circuit (unpublished results). On the potassium movement during SD in the retina, Mori et al. reported that the peak level of external potassium concentration was about 30-35 mM and 10 mM (estimated from Fig. 1 of the reference 23) at the inner plexiform layer and at the horizontal cell layer, respectively, during slow depressive potential in frog retina 23. As observed intracellularly in cortical neurons 5,6,11, increase and depression of spontaneous firing discharges seen in units with no close relationship between firings and photic stimuli, on- and off-center units (Figs. 7 and 8) during SD, may be caused by membrane depolarizations of the units. No increase in spike number of on-discharges during SD in the on-center units (Fig. 7A and B) may be explained by the fact that on-discharges superimposed on a sustained slow depolarization a6 will not be evoked further with slow depolarizations by SD, (i.e., the ceiling effect). With spikes newly produced during illumination in the off-center unit (Fig. 8), the proper membrane potential level for firing might be created by mutual balance between the hyperpolarization caused by illumination a8 and the gradual membrane depolarization by SD. This seems to be supported by the fact that the latency of spikes during illumination was observed to change regularly, so as to form a sigmoid curve (Fig. 9). Usually, neurons have been found to show burst discharges before and after the spike depression 4-6,11,12,17,24,al, as seen in untreated retinas in the experiment (Figs. 7 and 8). However, interestingly enough, our experimental results in the immersed

82 retina reveal that all units showing off-discharges generated n o burst discharge d u r i n g SD (Fig. 10). Even t h o u g h it remains obscure why on-exciting mechanisms by light disappeared in the treated retina, the above findings might at least indicate that med i a t i o n of SD could n o t be provoked in some n e u r o n s in the retina immersed in the low C1- solution, which would conflict in part with the SD hypothesis of Grafstein 8. ACKNOWLEDGEMENTS We would like to t h a n k S. Takagi for his technical assistance and J. M. Shields for his help in preparing the manuscript.

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