Proc. Natl. Acad. Sci. USAVol. 93, pp. 2302-2306, March 1996Neurobiology

Low-calcium-induced enhancement of chemical synaptictransmission from photoreceptors to horizontal cellsin the vertebrate retinaM. PICCOLINO*t, A. L. BYZOVt, D. E. KURENNYI§, A. PIGNATELLI*, F. SAPPIA*, M. WILKINSON§, AND S. BARNES§

*Dipartimento di Biologia, Universita di Ferrara, Ferrara, Italy; tlnstitute for Problems of Information Transmission, Moscow, Russia; and §NeuroscienceResearch Group, University of Calgary, Calgary, AB Canada

Communicated by Torsten N. Wiesel, The Rockefeller University, New York, NY, October 31, 1995 (received for review August 7, 1995)

ABSTRACT According to the classical calcium hypothesisof synaptic transmission, the release of neurotransmitter frompresynaptic terminals occurs through an exocytotic processtriggered by depolarization-induced presynaptic calcium in-flux. However, evidence has been accumulating in the last twodecades indicating that, in many preparations, synaptic trans-mitter release can persist or even increase when calcium isomitted from the perfusing saline, leading to the notion of a"calcium-independent release" mechanism. Our study showsthat the enhancement of synaptic transmission between pho-toreceptors and horizontal cells of the vertebrate retinainduced by low-calcium media is caused by an increase ofcalcium influx into presynaptic terminals. This paradoxicaleffect is accounted for by modifications of surface potential onthe photoreceptor membrane. Since lowering extracellularcalcium concentration may likewise enhance calcium influxinto other nerve cells, other experimental observations of"calcium-independent" release may be reaccommodatedwithin the framework of the classical calcium hypothesiswithout invoking unconventional processes.

Ionic manipulations designed to interfere with presynapticCa>2 influx (reduction of Ca>2 concentration or application oflow-Ca2 medium containing Co2+ or Ni2+) are relativelyineffective in suppressing chemical synaptic transmission fromphotoreceptors to horizontal and bipolar cells in vertebrateretina (1-4). Moreover, block of the horizontal cell (HC)response induced by Co2+ can be relieved by extracellulartransretinal currents which depolarize photoreceptor termi-nals (5). Here we present evidence suggesting that the trans-mission block induced by Co2+, Ni2+, and Zn2+ is to a largeextent accounted for by a depolarizing shift of the Ca2+ currentactivation curve, by means of an interaction with the negativefixed charges on the photoreceptor presynaptic membrane.Presynaptic depolarization with extrinsic currents, or reduc-tion of extracellular Ca2+, relieves the transmission blockbecause these conditions bring presynaptic transmembranepotential to levels where Ca>2 current can still be activated.Our results may give new insights into the problem of so-called"calcium-independent synaptic transmission" (6, 7).

MATERIALS AND METHODSLight Responses in Retinal Eyecups. Eyecups were prepared

from the retina of turtles (Pseudemys scripta elegans) orsalamanders (Ambystoma tigrinum) and continuously super-fused with a modified Ringer saline bubbled continuously witha mixture of 95% 02 and 5% Co2. The composition (in mM)of the saline used in turtle experiments was the following:NaCl, 110; KCI, 2.6; NaHCO3, 22; MgCl2, 2; CaCl2, 2; D-glucose, 10 (pH 7.4). Salamander saline had the following

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composition: NaCl, 95; KCI, 2.5; NaHCO3, 30; MgCl2, 2;CaCl2, 2; D-glucose, 10 (pH 7.6). Intracellular recording of thelight responses induced by 5-mm-diameter white light spots ofvariable intensity and duration were made in HCs (and in otherretinal neurons) by using fine tip microelectrodes preparedwith a Brown-Flaming puller (Sutter Instruments, Novato,CA). The concentration of Ca2> in the nominally zero Ca2>Ringer solution, without EGTA added, was less than 10 ,uM.The pH of the solutions containing EGTA or EDTA wasreadjusted with NaOH. The concentration of Ca2> in mediacontaining CaCl2 in variable proportions and EGTA (2-10,uM) or EDTA (2-5 ,.tM) ranged from 0.45 to 2.4 nM and wasestimated by using the computer program SOL i.d. written byEric Ertel (Hoffmann-La Roche). To keep a normal concen-tration of Mg2+ in media containing EGTA the proportion ofMgCl2 in the perfusing saline was adjusted accordingly. Chem-icals were obtained from Sigma. Bicuculline was used as thesoluble methobromide salt and added to the perfusing mediumjust before application to the retina. Picrotoxin was firstdissolved in slightly basic, hot distilled water and then addedto the Ringer solution. Neutral density filters were used toattenuate light intensity. The flux density of the unattenuatedlight on the retina was about 1.5 x 10-5 utW/tLm2. The HCresponses illustrated in this article were obtained from lumi-nosity HCs of the turtle retina by using light stimuli attenuatedby 2.1 log1o units. Transretinal currents were applied betweentwo silver chloride electrodes positioned at the scleral andvitreous side of the retina, using a technique introduced byTrifonov (8) and modified by Byzov (5) for the purpose ofinducing maintained polarizations of photoreceptor terminals.When the positive electrode is on the sclera these currents leadto a hyperpolarization of the membrane of outer segment andto a depolarization of the membrane of the synaptic terminal.The amplitude of these synaptic terminal depolarizations wasestimated to be less than 10 mV in most of our experiments,and it probably attained 20 mV with the strongest currentsused (see ref. 5 for the details of the method).Ca2+ Channel Currents in Isolated Photoreceptors. Isolated

cone photoreceptors were obtained by trituration of tigersalamander retina after treatment with papain at 10 units/ml(Sigma) for 10-15 min (20-24°C) in a solution of the followingcomposition (mM): 90 NaCl, 2.5 KCl, 3 CaCl2, 8 D-glucose, 10Hepes at pH 7.6. Isolated cells were placed in a recordingchamber (0.5 ml) and superfused continuously with the solu-tion described above. Calcium current, ICa, was recorded fromsingle or double cones by using the perforated patch technique(nystatin, 150 pLg/ml). Fire-polished pipettes were filled withsolution containing (in mM): 100 CsCl, 3.5 MgCl2, 1.5Na2ATP, 1 EGTA, 10 Hepes, pH 7.2. ICa was recorded in thefollowing solution (in mM): 75 NaCl, 2.5 KCl, 5 CsCl, 2 CaCl2,

Abbreviation: HC, horizontal cell.tTo whom reprint requests should be addressed at: Dipartimento diBiologia, Sezione di Fisiologia Generale, Universita di Ferrara, ViaBorsari 46, 44100 Ferrara, Italy.

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10 tetraethylammonium chloride, 8 D-glucose, 10 Hepes at pH7.6. Divalent ions were added or calcium concentration wasreduced as described in the text. Access was achieved a fewminutes after gigaseal formation. Series resistance was com-pensated by 60%, and the voltage was adjusted for theuncompensated resistance during data analysis. To elicit ICa,voltage steps incrementing by 10 mV between -80 mV and+40 mV were applied from a holding potential of -60mV. ICawas measured at the end of the steps and the leak current wassubtracted. Activation curves were then constructed and fittedby the Boltzmann equation to give the half-activation poten-tial, V112, and the slope factor.

RESULTS

Effects ofTransretinal Radial Currents on Divalent Cation-Induced Transmission Block Horizontal cells are second-order retinal neurons which respond to retinal illuminationwith graded hyperpolarizations, the amplitude of which de-pends on the light intensity. These responses result from areduction of the release of the photoreceptor transmitter, veryprobably glutamate, which is maximal in darkness (8). Con-sistent with this view, interfering with the release of photore-ceptor transmitter, or with its postsynaptic action, results inhyperpolarization of the HC membrane in the dark (4, 9-11).We studied the effects of the application of transretinal

currents on the intracellularly recorded light responses of HCsof turtle retina in the presence of Ca2> channel- and synaptictransmission-blocking divalent cations. We found that trans-retinal currents which depolarize photoreceptor synaptic ter-minals (positive electrode on the sclera) could relieve the blockof HC light responses induced by adding to the perfusingmedium Zn> (0.2-1 mM, n = 12; see Fig. 1), Ni> (0.5-2 mM,n = 12) and, as previously reported (5) by Co>2 (1-5 mM, n= 8). Progressively larger current intensities were necessary tocounteract the response block when the divalent cation appli-cation was prolonged, particularly at high concentrations, andeventually only a partial recovery was possible. Neither theapplication of Zn2, Ni>, or Co> nor the electrical stimuliresulted in an important change of response amplitude incones, the main photoreceptors of the turtle retina (n = 18).

Transretinal currents produced only a modest recovery fromthe HC-response block induced by Cd2+ (0.2-1 mM, n = 9) andMg2+ (15-20 mM, n = 11) and could not counteract the blockinduced by low-Ca2+ media (with or without 2-4 mM EGTAadded, n = 6). Moreover, they were also ineffective against the

Control

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FIG. 1. Effect of a sclero-positive transretinal current on theZn2+-induced block of the HC light response. The HC responses(light-induced hyperpolarization, monitored as downward deflections)elicited by 10-ms flashes of white light (illustrated above the potentialtracings inA) were recorded in control conditions (A) and 9 min afteraddition of 0.6 mM ZnCl2 to the control saline (normal Ca2+concentration 2 mM) (B). In the presence of Zn2+ a 30-,uA extracel-lular current was applied from the sclera (positive electrode) to thevitreous side (between the arrows), resulting in a membrane depolar-ization and an almost complete recovery of response amplitude. Thedotted line in B indicates the initial dark potential level in the controlmedium.

block induced by 1-2 mM kynurenic acid (n = 4), a compoundwhich antagonizes the postsynaptic effect of the photoreceptortransmitter, glutamate. These experiments suggested that themain effect of transretinal currents was presynaptic and in-volved a voltage-dependent reversal of the effects of theantagonist divalent cations on photoreceptor Ca2+ current(see also ref. 5). Moreover, it appeared that Zn2+, Ni2+, andCo2+ differed in their blocking action compared with Mg2+and Cd>.

Effects of Low-Ca2+ Media on Divalent Cation-InducedTransmission Block. There is abundant experimental evidencethat the block induced by divalent cations in a variety ofchannels (both voltage-gated and ligand-gated) can be voltagedependent (12-14). It would thus be reasonable to assume thatthe block of Ca2+ current by Zn2+, Ni2+, and Co2+ in photo-receptor terminals is voltage dependent and that the block isrelieved by the depolarization that transretinal currents pro-duce in photoreceptor synaptic endings. However, the mainCa2+ current identified in photoreceptors is of the L type (15,16), and there is no evidence that the block of this currentinduced by Zn>, o>2+, or Ni2>, at the concentrations used inour experiments, can be relieved by presynaptic membranedepolarizations of amplitude comparable to those induced byour transretinal currents. In some preparations, the Ca2+channel block induced by divalent cations can actually increaseas the membrane is depolarized (see, for instance, refs. 17 and18). A possibility was that the HC response block resulted froma low concentration of Zn2+, Co2+, or Ni>2 in the proximity ofthe synapse, before the level of these ions reached valuessimilar to the perfusing saline, and that, moreover, the blockwas due to a mechanism different from a typical channel block.There is evidence that the diffusion time of divalent cations inthe extracellular space of the retina can be very long (19). Aninteresting possibility was that the block of HC light responsesinduced by Zn2+, Ni2+, and Co2+ resulted from a depolarizingshift of the activation curve of photoreceptor Ca2+ current bymeans of an influence on the fixed negative charges present onthe outer surface of the cell membrane (20-22). This possi-bility seemed likely because Zn2+, Ni2+, and Co2+ are verypowerful in neutralizing surface charges and produce largeactivation curve shifts at very low concentrations, while Mg2+,for instance, has a much weaker effect (23). To discriminatebetween a block due to a mechanism of this type and a classicalcompetitive block, we investigated low-Ca2+ effects on thedivalent cation-induced transmission block. A competitiveblock should be potentiated by lowering Ca>, whereas a blockdue to a surface-charge effect should be relieved, to a certainextent, since lowering Ca2+ would bring the activation curve tomore negative potentials. Our experiments carried out in bothturtle and salamander eyecup preparations supported thissecond alternative, showing that nominally Ca2+-free mediacould relieve the HC response block induced by Zn2+ (0.2-0.6mM, n = 22; see Fig. 2), Ni2+ (0.5-2 mM, n = 10), and Co2+(1-3 mM, n = 15). In some experiments carried out in turtleretina the effect of various Ca2+ concentrations was investi-gated on the response block induced by Zn2+, Ni2+, and Co2+.It was found that an almost complete recovery could beobtained by lowering Ca2+ from 2 to 0.1-0.5 mM, in thecontinuous presence of the exogenous cations, and a substan-tial recovery was seen with 1 mM Ca2+ (n = 12).

In contrast to what observed with Zn2+, Ni2+, and Co2+, lowCa2+ produced no detectable recovery of HC light responsesfrom the blocking effect of Mg2+ (15-20 mM, n = 11) and onlya small recovery from the effect of Cd2+ (0.2-1 mM, n = 5),followed by a rapid intensification of the blockade.

Effects of Divalent Cations on Photoreceptor Ca2+ Current.To test the possibility that the action of Zn2+, Ni2+, and o>2+was mediated by surface-charge modifications, we investigatedthe effect of divalent cations on the Ca>2 current in isolatedsalamander cones. As illustrated in Fig. 3, lowering Ca>2 to 0.5

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Zn2+ 0.2 mM0 Ca2+ (nominal)

12 min

20 mV

1-3 sec

FIG. 2. Recovery of the Zn2+-induced HC response block broughtabout by the application of a low-Ca2+ saline. The light responsesinduced by 10-ms light flashes were recorded in control conditions, in0.2 mM ZnCl2 and a normal Ca2+ concentration (2 mM), as well asafter the application of a medium still containing 0.2 mM ZnCl2 butwithout Ca2+ added, as indicated. The numbers near the stimulus tracegive the time in minutes from the application of the relevant testsolution.

mM counteracted the depolarizing shift of the current-voltagerelation induced by 5 ,tM Zn2+ and resulted in an increase ofthe Ca2+ current measured at the foot of the current-voltagecurve, even though peak amplitude was reduced. Since conedark potential in physiological conditions is about -35 mV,transmitter release should thus be potentiated by low-Ca2+media. Similar results were obtained in experiments in whichthe cell was first bathed in a medium containing Zn2+, Ni2+,or Co2+ (5-100 ,uM) and normal Ca2+, and then the perfusingmedium was changed to one containing the same concentra-tions of the exogenous divalent cation and Ca2+ concentrationsreduced to 0.5 or 0.8 mM (n = 29).The ensemble of these experiments supports the hypothesis

that the HC response-blocking effect of Zn2+, Ni2+, or Co2+is largely due to a depolarizing shift of the Ca2+ currentactivation curve. It is worth mentioning here that, as for thetransretinal current experiments, HC responses in eyecup

A

preparations could be restored less easily when low-Ca2+media were applied after long perfusion times with Zn2+, Ni2+,and Co2+. This can be interpreted to mean that the surface-charge effect gradually gives way to a true channel block, as theextracellular concentration of the antagonist divalent cationsreaches increasing levels at the synapse. A true Ca2+ channelblock probably also accounts for the effect of Mg2+ and Cd2+,since the suppression ofHC responses induced by these cationswas not relieved by low-Ca2+ media.

Effects of Low Ca2' in the Absence of Antagonist DivalentCations. The surprising action of low-Ca2+ media in thepresence of some divalent antagonists led us to investigatelow-Ca2+ effects in the absence of exogenous divalent cations.We found that lowering Ca2+ in the perfusing saline resultedin a depolarization of the HC membrane followed by areduction of the light response which could later develop intoa full depolarizing block if the perfusing medium also lackedMg2+. In the presence of Mg2+, however, the initial depolar-izing influence was followed, with prolonged perfusion times(1-2 hr), by a hyperpolarizing shift, and eventually by ahyperpolarizing block (see also refs. 9 and 10), an effectprobably due to a competition of Mg2+ with Ca2+ at low Ca2+levels. Similar effects were observed either by using nominallyCa2+-free solutions or by using media buffered with EGTA(2-10 mM) or EDTA (2-5 mM), the only difference being thefaster time course of modifications in the presence of thebuffers. A hyperpolarizing block was also observed when Mg2+(2-20 mM) or Cd2+ (0.2-1 mM) was applied after the fulldepolarizing block induced by prolonged application of medialacking all divalent cations. Moreover, a similar effect wasinduced by kynurenic acid (1-2 mM), thus indicating that thedepolarization brought about by low-Ca2+, low-Mg2+ mediawas due to an increased release of photoreceptor transmitter.It thus appeared that the effect of low-Ca2+ media is toincrease, not to decrease, the release of photoreceptor trans-mitter, even in the absence of exogenous divalent antagonists.This result can be explained by the hyperpolarizing shift of theCa2+ current activation curve brought about by low-Ca2+media. In isolated salamander cones we found that, due to theshift of the activation curve, lowering extracellular Ca2+ from3 to 0.3 mM resulted in a doubling of Ca2+ current amplitude

B

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OX * -w ~~~~----::---* ..

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2 Ca±Z-- 2 Ca+Zn 4. .,^* 0.5 Ca+Zn

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FIG. 3. Recovery of the Zn2+-induced Ca2+ current activation shift by low Ca2+ in a cone photoreceptor isolated from salamander retina. (A)Leak-subtracted current-voltage relations measured in normal Ca2+ (2 mM; K), normal Ca2+ plus 5 ,uM Zn2+ (O), and in low Ca2+ (0.5 mM)plus 5 ,uM Zn2+ (-). (B) Leak-subtracted currents measured at -30 mV in the same ionic conditions described for A. On average, 5 ,uM Zn2+produced a positive shift of the Boltzmann-fitted activation curve of 3.7 ± 0.3 mV in 2 mM Ca2+, while reduction of Ca2+ to 0.5 mM produceda shift of -5.4 ± 0.8 mV (n = 5) in the presence of Zn2+.

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measured at -40 mV (n = 3). In the intact retina the increaseof Ca2+ current during prolonged application of low-Ca2+media was probably more pronounced, since reducing extra-cellular Ca2+ slowly resulted in a true depolarization of thecone membrane in the dark (see also refs. 1 and 24).

DISCUSSIONOur results indicate that the blocking action of some divalentcations (Zn2+, Ni2+, and Co2+) on transmission betweenphotoreceptors and HCs is largely accounted for by a depo-larizing shift of the presynaptic Ca2+ current activation curvecaused by surface potential modifications. Depolarization ofpresynaptic terminals with transretinal current restores synap-tic transmission because it brings photoreceptor potential backto a range at which Ca2+ current can be modulated by thelight-induced membrane potential changes. Synaptic transmis-sion can also be restored by lowering extracellular Ca , sincethis counteracts the depolarizing shift of the Ca>2 currentactivation curve, thus increasing the release of photoreceptortransmitter. In the absence of Zn2+, Ni2>, or Co2+, prolongedperfusion with low-Ca2+ media may result in a depolarizingblock of HC response. This effect may be in part the conse-quence of a hyperpolarizing shift of the activation curve ofphotoreceptor Ca2+ current, capable of causing an increase oftransmitter release to a point where it cannot be modulated bythe physiological responses of photoreceptors. Surface chargetheory explains our results parsimoniously and can also ac-count for some paradoxical results of previous work. Forinstance Co2+ appeared to be less effective in blocking the lightresponses ofHCs and bipolar cells, when applied in a low-Ca2+medium, than when applied in the presence of a normal Ca2+concentration (1, 10). Moreover, application of low-Ca2+media, with Co2+ or Ni2+ added, resulted in an initial block ofHC response in fish retina followed by a slow recovery (2).According to our interpretation, the first effect would reflectmore the action of the blocking divalent cations in the presenceof Ca>, the second the response recovery due to the slowwashing out of Ca2+ from the extracellular medium.Our experiments suggest that the HC response block

brought about by Cd2+ or high Mg2+ solutions results from aclassical block of Ca2+ channels. The difference between theeffects of the first group of divalent cations (Zn2+, Ni2+, andCo2+) and those of the second group (Cd2+ and Mg2+) couldderive from the relative capability of the various ions toinfluence surface potentials and to block Ca2+ channels. Theions of the first group produce large modifications of surfacepotential at concentrations at which they produce a relativelysmall channel block, while Cd2+ and Mg2+ produce strongchannel blocking at concentrations at which they significantlyaffect the surface potential.Although the surface charge hypothesis can account in a

simple way for the recovery of HC responses brought about byboth transretinal currents and low-Ca2+ media, there are otherpossibilities to consider, particularly with respect to otherpossible sites of divalent cation action. It is known that divalentcations can interfere with the postsynaptic actions of y-ami-nobutyric acid (GABA) in many nerve cells, including pho-toreceptors (see refs. 25 and 26). Thus, it might be that ourresults reflect block of GABAergic input to photoreceptors(reviewed in ref. 27). We ruled this out, however, because wefound in control experiments that the low-Ca2+-induced re-covery ofHC responses after the block induced by Zn2+, Ni2+,or Co2+ was also present in retinae continuously superfusedwith the GABA antagonists picrotoxin (100-250 ,uM, n = 9)or bicuculline (100-200 ,tM, n = 12). Another possibility isthat the effects depended critically on low-Ca2A-induced mod-ifications of photoreceptor responses. It is well known that theamplitude of the response induced by bright flashes in pho-toreceptors increases in low-Ca2 media as a consequence of

modifications of the intracellular level of cyclic GMP, thesecond messenger of phototransduction in vertebrate photo-receptors (24, 28). Even if modifications of photoreceptor lightresponses and membrane potential induced by low Ca2+influence some of the results of our experiments, they cannotbe the main cause of the response recovery after Zn2+, Ni2+,or Co>2 block. Modifications of photoreceptor responsesdeveloped slowly after the application of nominally zero Ca2+media (after about 20-40 min; not shown), whereas HCresponses recovered within about 10 min under the sameconditions (see Fig. 2). Finally, the recovery of blockedtransmission by transretinal currents as shown in Fig. 1 can inno way be attributed to an enhancement of photoreceptor lightresponses.There is, however, an apparent inconsistency with our

explanation, which is based on surface charge modifications, ofthe effects of low-Ca2+ media. As a matter of fact, ourhypothesis that lowering Ca2+ concentration leads to anincrease of transmitter release in the eyecup preparation, as aconsequence of increased Ca2+ influx, seems not applicable toexperiments in which Ca2+ concentration in the perfusingmedium was so low as to reverse the transmembrane Ca2+gradient, as may occur particularly when Ca2+ buffers are used.This argument is based on the assumption that Ca2+ concen-tration in the extracellular space follows rather faithfully theconcentration in the perfusion saline. This appears to beunlikely, however, particularly in relation with the influence ofsurface charge on local ionic concentrations. Surface chargetheory predicts that, due to the negative surface potential, theconcentration of divalent cations in a liquid layer of more than1-nm thickness adjacent to the membrane can be much highernear the membrane than in the bulk solution, and this isparticularly true at low bulk concentrations of the ions (seerefs. 29-31). For instance, at 20°C, in the presence of a bulkconcentration of 1 ,M, the membrane concentration of Ca2+can be larger than 1 mM in the presence of a surface potentialof -90 mV, and it can attain almost 15 mM when surfacepotential is -120 mV. With this consideration in mind, andtaking into account the extreme thinness and tortuosity of theintercellular space in the central nervous system, we can easilyenvision the membrane surface as a high-capacity source ofCa2+, capable of buffering Ca2+ concentration in the extra-cellular space, so as to overcome the effects of foreign Ca2+buffers. Although further experiments are clearly necessary toverify this critical point, there are in the literature severalindications that the equilibration of Ca2+ between the perfus-ing medium and extracellular space is a very slow and incom-plete process (see for instance refs. 32 and 33). Slow diffusionand incomplete equilibration of divalent cations are probablythe reasons why, for instance, in our preparation, Zn2+ or Ni2+influenced HC cell responses only at concentrations of 200-500 ,uM or more, whereas in isolated photoreceptors theymodified L-type Ca2+ current at concentrations as low as 5-50,M. In our experiments application of 50 ,uM Zn2+ even forperiods of 5-10 hr did not result in any substantial reductionof HC responses. Our hypothesis that the increase in trans-mitter release induced by application of perfusing mediacontaining extremely low Ca2+ concentrations is due to in-creased Ca2+ influx in photoreceptors can, moreover, easilyexplain why application of Cd2+ or Mg2+ to these mediaresulted in a reduction of the release. This effect of Cd2+ orMg2+ would be difficult to account for if the release weregenuinely Ca2+ independent.

Since a low Ca2+ concentration presumably increases Ca>2current amplitude at low membrane potentials in other prep-arations besides vertebrate photoreceptors (see refs. 29 and34), one should be very cautious in qualifying as a2+-independent a synaptic process which is not blocked (or is evenpotentiated) by low-Ca2+ media. Indeed, it has been frequentlyreported that "Ca2+-independent" transmitter release is

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blocked by application of Mg2> or Cd2, which argues againsta genuine Ca>2 independence of this process (see refs. 6 and35).The functional peculiarities of transmitter release from

photoreceptors explain the great susceptibility of synaptictransmission at this level to the surface-charge-mediated in-fluence of divalent cations. The Ca>2 current identified inphotoreceptors is activated at potentials positive to about -40mV (see Fig. 3), and, as previously mentioned, photoreceptordark potential is about - 35 mV. Since photoreceptors respondto light with hyperpolarization, a depolarizing shift of the Ca>2current activation curve by only a few millivolts can thus leadto a complete block of Ca>2 influx, and, consequently, oftransmitter release. The impact of a surface charge-mediatedmodification would be less evident at synapses activated bydepolarizing action potentials of large amplitude but would beimportant in local nervous circuits based on graded potentialsor on small-amplitude spike signals (36). Moreover, surfacecharge effects should have a great influence in experimentalconditions in which transmitter release is evoked by small-amplitude graded depolarizations, as for instance, followingthe application of high K+ concentrations or of various phar-macological agents. It is, in fact, from these studies that theidea of a Ca2+-independent transmitter release has beenmainly derived (6, 7, 35).The extracellular Ca>2 in the retina, as well as in other

regions of the nervous system, can change considerably inphysiological conditions (19, 37), and thus transmitter releasecould be normally modulated by Ca>2 concentration changesby means of a surface-charge effect. A similar possibility alsoholds for pH modifications (38-40), since extracellular pHchanges can strongly affect surface potential (41). Finally,Zn>2 is present in many synaptic terminals, including photo-receptor endings, and it could be released into the extracellularspace and thus affect signal transmission (42, 43). On thesegrounds, the fixed charges present on the synaptic membraneof photoreceptors, and of other neurons, could be a target ofdiverse regulatory influences on synaptic transmission at var-ious levels of the nervous system.

We thank Mrs. Claudia Palandri and Mr. Domenico Canino forassistance with computer facilities. This work was supported byFerrara Ricerche, the Human Frontier Science Program, the MedicalResearch Council of Canada, and the Edwin L. and John GustusEndowment. M.W. is a Medical Research Council of Canada Cen-tennial Fellow. S.B. is an Alberta Heritage Foundation Senior Scholarand the Roy Allen Investigator in Visual Sciences.

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