Tetrodotoxin does not block the axonal transmission of S-potentials in goldfish retina

Neuroscience Letters, 49 (1984) 233-238

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Elsevier Scientific Publishers Ireland Ltd.

NSL 02793

T E T R O D O T O X I N DOES N O T BLOCK T H E A X O N A L T R A N S M I S S I O N OF S - P O T E N T I A L S IN G O L D F I S H R E T I N A

M.B.A. DJAMGOZ l and W.K. STELL 2

1Department of Pure and Applied Biology, Imperial College, London, SW7 2BB (U.K.) and 2Department of Anatomy and Lions" Sight Centre, The University of Calgary, Calgary, Alberta, T2N 4N1 (Canada) (Received March 15th, 1984; Revised version received April 9th, 1984; Accepted April 10th, 1984)

Key words." retina - S-potential - horizontal cell - axon terminal - regenerative activity - tetrodotoxin

Light-evoked responses (S-potentials) have been recorded from the somata and the axon terminals of horizontal cells of goldfish retinas. Data obtained in the controlled states of the retinas and following tetrodotoxin (TTX) application show that TTX does not affect the quantitative relationships of the two sets of response potential. It is concluded that TTX does not influence the axonal transmission of S-potentials

Cone-connected horizontal cells (HCs) of teleost retinas are structurally unique in that their axons form greatly expanded cylindrical terminals (Cajal's 'internal horizontal cells' [1]) [13]. These axon terminals (HC-ATs) are located at proximal levels of the inner nuclear layer, and do not contact any photoreceptors [13]. HCATs are sufficiently large to be impaled with microelectrodes, and have been shown to generate light-evoked responses (S-potentials) [3, 6, 15, 17]. The sites of recording have been determined in several cases by intracellular dye injection [3, 6, 17]. In goldfish retina, in which HC axons are some 0.5/zm in diameter and 200 #m long, Stell [13] showed that signal decay of more than 80°70 would be expected from cable theory when S-potentials spread from soma (HC-S) to the axon terminal*. However, it has been shown repeatedly by intracellular recording that S-potentials generated by HC-ATs and HC-S have very similar amplitudes [6, 8, 17]. One difference between the two is that the HC-ATs seem to be electrically coupled more strongly than are the somata [6]. The physiological and anatomical evidence, taken together, suggest that cone-driven S-potentials spread from the HC soma, where they are generated post-synaptically by photoreceptor input(s), to the HC-ATs via

*In his original calculation Stell [13] used an assumed value of HC membrane resistance to estimate the length constant. Since then, this parameter has been measured directly [5, 16l, but this does not affect SteWs [13] original conclusion. Stell [13] also assumed, improperly, infinite cable length, but consideration of the HC axon as a finite cable would only make its length constant even shorter [10].

0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

234 the axon by some regenerative mechanism. Recent electrophysiological studies on isolated teleostean H C s have shown that H C membranes are capable of generating 'slow' action potentials through voltage-sensitive Na and Ca channels [5, 11, 12, 16]. These studies have raised the possibility that the axonal transmission of Spotentials may involve sodium- a n d / o r calcium-dependent regenerative activity. We have tested the possibility that voltage-sensitive sodium channels are involved. Our approach was made possible by a unique neurophysiological tool, tetrodotoxin (TTX), which is a well known, highly specific blocker of voltage-sensitive sodium channels (see ref. 9 for a review). We found that T T X does not affect the axonal transmission of S-potentials and conclude, therefore, that TTX-sensitive voltagegated sodium channels are not involved in this process. Experiments were carried out on isolated retinas of c o m m o n goldfish, Carassius auratus. A dark-adapted fish was killed, and a retina was dissected out. The retina was placed with the photoreceptors upward in a recording chamber, surrounded by tissue paper soaked in distilled water to minimize dehydration, and supplied with moist oxygen. Microelectrodes were fabricated from 1.2 m m diameter glass capillary tubing with an internal fibre and filled with 3 M KCI. Those with DC resistances in the range 100-150 Mf~ were used. The microelectrode was connected to an electrophysiological probe system (WP1 Model M701), the output of which was displayed simultaneously on an analogue storage oscilloscope and a chart recorder. The retina was stimulated by diffuse light flashes (300 ms duration, presented once every 2 s). HC responses were identified and classified by their relative response amplitudes to red, green and blue test light stimuli (650, 500 and 430 nm, respectively) of approximately equal quantum content. Measurements were made mostly on the photopic red-sensitive Ll-type S-potentials, which are generated by the axon-bearing H I - t y p e cone HCs [3, 14, 17]. In a separate set of experiments, ganglion cell activity was recorded extracellularly using tungsten microelectrodes and an AC-coupled amplifier. Spike activity was photographed on Polaroid film from the oscilloscope screen, and was also monitored continuously on an audio amplifier. T T X (Sigma) at 4-18 #m freshly dissolved in oxygenated goldfish Ringer [3] was applied to the receptor surface of the isolated retina as a single 100/~1 drop from a microlitre syringe. Assuming that the volume of the retina and the adhering vitreous body was 25 ~1, and that the drop diffused uniformly throughout, the final intra-retinal concentration of T T X used was in the range 3-14 ~M. The actual effective concentration of T T X in the retina was probably lower than these values as some of the applied solution would flow over the retina into the recording chamber. Since application of T T X by this method whilst recording intracellularly from an HC invariably caused the displacement of the microelectrode, measurements were taken in the control states of 6 retinas, averaged, and compared with average data obtained subsequently in the same retinas following T T X treatment. In contrast, application of the drops during extracellular recording from the ganglion cells did not dislodge the electrodes, and the effect of T T X could be followed during continuous recordings from single units.

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During a microelectrode penetration into the retina from the photoreceptor side, 2-4 S-potential units were impaled in succession, as shown originally by Kaneko [6]. In the 6 retinas used, a total of 42 microelectrode penetrations were performed in their control states, and 46 penetrations following T T X application. Different types of S-potential were encountered in a characteristic sequence, penetration after penetration. These observations are summarized in Table I. In both control and TTX-treated conditions, 89% or more of the units impaled first in the H C layer gave L,-type responses. In 89% or more of these cases (79% or more of all penetrations) a second Ll-type unit was encountered deeper in the same penetration. Further, in almost every such penetration, in which two L~-type units were found in succession, at least one other unit (usually an H2 cell generating biphasic C-type responses) was also impaled in between the two Ll-type units. In summary, in 77% of all penetration in control states and 90% in TTX-treated states of the retinas, 2 Ll-units were found in the same penetration with another unit in between. Kaneko [6] showed definitively under practically identical conditions that the Ll-type responses recorded nearest the photoreceptors arise from H C somata, whereas those recorded at deeper levels within the inner nuclear layer arise from H C axon terminals. Our observations show, therefore, that S-potentials can be recorded from HC-ATs in TTX-treated retinas as frequently as in the controls, i.e. T T X does not interfere with S-potential transmission from HC-S to HC-AT. The quantitative relationship between the HC-S and H C - A T responses (V~ and Va, respectively), elicited by a mid-range test stimulus (650 nm, approximately 6 g W / c m 2) and recorded in the s a m e retinal penetration, was determined (Fig. 1). Although the absolute amplitudes varied considerably from penetration to penetration Vs and Vat were approximately equal for each pair of units recorded in the same penetration. This was equally true for control and TTX-treated retinas. The dependence of HC-S and H C - A T response levels on the illumination level of the stimulus over the f u l l operating range was also investigated (Fig. 2). The relative 'response versus stimulus intensity' functions were identical for HC-S and HC-ATs, whether in control or TTX-treated retinas. We also examined qualitatively the

TABLE l NUMBERS OF H1 AND OTHER HC TYPES PENETRATIONS INTO ISOLATED RETINAE Type of observation

H I , first unit impaled 2 H l s in one penetration 2 H l s and other unit(s) in between

ENCOUNTERED

IN MICROELECTRODE

% Of times observation made Control (n = 42)

TTX-treated (n = 46)

89 79 77

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VslmVl Fig. 1. The relationship of the horizontal cell axon terminal response (V~) to soma response (V,) elicited by a mid-range, 650 nm test stimulus (diffuse light of approximately 6 t~W/cm2). Only pairs of units found in the same penetration have been included. Closed circles denote data points obtained in the control states of the retinas; the open circles are from TTX-treated retinas. The straight line denotes the equality V~, V,. Using linear regression analysis, the closed and open circles can be fitted with separate straight lines of slopes 0.73 and 0.96; correlation coefficients 0.83 and 0.98, respectively, but these have been omitted for clarity. Inset: the waveform of a typical S-potentialk (upper trace); lower trace denotes the presentation of the light flash. The maximum levels of response were used to measure V~,, and V~s.

response waveforms at all the intensities used and could not observe any obvious difference induced by TTX treatment. The three sets of data presented in this paper show that in TTX-treated retinas, compared with the controls: (i) HC-AT responses are not encountered any less frequently; (ii) HC-S and HC-AT responses elicited by the same test stimulus, and recorded at the same retinal location, are nearly equal in amplitude; and (iii) the operating ranges and the profiles of relative 'response vs light intensity' relationships for HC-S and H C - A T responses are very similar in both cases. Taken together, the data show that TTX does not affect, in any way, the transmission of the Lt-type responses from the somata to the axon terminals of the H l-type HCs. Further, since two C-type S-potentials were frequently impaled at greatly different depths in the same retinal penetration in TTX-treated retinas as in controls, TTX may also not affect the axonal transmission of S-potentials in H2-type HCs. Since application of TTX to the retina had no appreciable effect on HCs, we were

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obliged to test the possibility that the toxin was simply not penetrating the retina to the depth of the HCs. We tested this hypothesis by applying TTX whilst recording extracellularly from the ganglion cells, which are known to generate conventional, sodium-dependent (Hodgkin-Huxley type) spikes [7]. In all 4 ganglion cells tested, concentrations of TTX comparable to those applied in the HC experiments blocked all spike activity completely within 3-7 rain of the application. This effect did not appear to reverse within 40 min, the longest period tested. We also observed no obvious structural difference between Ringer- and TTX-treated retinas fixed for electron microscopy, embedded in epoxy resin, sectioned at 1 #m, and examined in the light microscope. It is clear, then, that we have been able to record the light-evoked responses of both cone-driven horizontal cell somata and axon terminals; that conduction of these responses from somata to axon terminals is practically without decrement; and that TTX, applied in a concentration sufficient to block regenerative action potentials in ganglion cells, has no discernable effect on axonal propagation o f horizontal cell responses. One possible explanation of these observations would be that goldfish cone HCs lack regenerative sodium channels. This conclusion would be consistent with the result o f Tachibana [16] who failed to find a TTX-sensitive membrane current in isolated cone HCs of goldfish retina. Shingai and Christensen [11, 12], on the other hand, showed that a TTX-sensitive membrane current does exist in catfish HCs. If goldfish HCs also possess hitherto undiscovered, voltage-sensitive sodium channels, then it would follow that their functioning is unrelated to the ax-

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onal transmission of S-potentials, assuming that these channels are TTX-sensitive (a few TTX-insensitive sodium channels are known to exist and have been listed by Hagiwara [2]). Another possibility is that the axonal transmission of S-potentials, in fact, depends on regenerative calcium currents, which have been found in both goldfish and catfish HCs [5, 11, 12, 16]. This possibility would be more difficult to investigate in the intact retina, however, since blockage of calcium channels by conventional means, such as treatment with divalent cations, would also result in the interruption of the photoreceptor input(s) to the HCs and subsequent suppression of all S-potentials. M.B.A.D. thanks the University of Calgary for the award of a Visiting Scholarship, which enabled this study to be carried out in the Department of Anatomy. We also thank Drs. F. Quandt and R. Shingai for discussions. 1 Cajal, S., R a m o n y, La reline des vertebres, La Cellule, 9 (1893) 17-257. 2 Hagiwara, S., Calcium channel, Ann. Rev. Neurosci., 4 (1981) 69-125. 3 Hashimoto, Y., Kato, A., Inokuchi, M. and Watanabe, K., Re-examination of horizontal cells in the carp retina with Procion yellow electrode, Vision Res., 16 (1976) 25-29. 4 lshida, A. and Fain, G.L., D-Aspartate potentiates the effects of L-glutamate on horizontal cells in goldfish retina, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 5890-5894. 5 Johnston, D. and Lam, D.M.K., Regenerative and passive m e m b r a n e properties of isolated horizontat cells from a teleost retina, Nature (Lond.), 292 (1981) 451-454. 6 Kaneko, A., Physiological and morphological identification of horizontal, bipolar and amacrine cells in goldfish retina, J. Physiol. (Lond.), 207 (1970) 623-633. 7 Murakami, M. and Shigematsu, Y., Duality of conduction mechanism in bipolar cells of the frog retina, Vision Res., 10 (1970) 1-10. 8 Naka, K.I., Marmarelis, P.Z. and Chan, R.Y., Morphological and functional identification of catfish retinal neurones. Ill. Functional identification, J. Neurophysiol., 38 (1975) 92-131. 9 Narahashi, T., Chemicals as tools in the study of excitable membranes, Physiol. Rev., 54 (1974) 813-889. 10 Nelson, R., kiitzov, A.V., Kolb, H. and Gouras, P., Horizontal cells in cat retina with independent dendritic systems, Science, 189 (1975) 137-139. I1 Shingai, R. and Christensen, B.N., Voltage clamp studies in isolated horizontal cells, A.R.V.O. Abstracts, (1983) 179. 12 Shingai, R. and Christensen, B.N., Sodium and calcium currents measured in isolated catfish horizontal cells under voltage clamp, Neuroscience, 10 (1983) 893-897. 13 Stell, W.K., Horizontal cell axons and axon terminals in goldfish retina, .I. comp. Neurol., 159 (1975) 503-520. 14 Stell, W+K., Kretz, R. and Lightfoot, D+O., Horizontal cell connectivity in goldfish. In B+D. Drujan and M. Laufer, (Eds.), The S-Potential, Alan Liss, New York, 1982, pp. 51-75. 15 Svaetichin, G., The cone action potential, Acta physiol, scand., 29 (1953) 565-600. 16 Tachibana, M., Membrane properties of solitary horizontal cells isolated from goldfish retina, J. Physiol. (l+ond.), 321 (1981) 141-161. 17 Weiler, R. and Zettler, I7., The axon-bearing horizontal cells in the leleost retina are functional as well as structural units, Vision Res., 19 (1979) 25-29.