Effects of external calcium on horizontal cells in the superfused goldfish retina

Neuroscience Research, Suppl. 6 (1987) S 147- S 164

S 147

0168-0102/87/$03.30 © 1987 Elsevier Scientific Publishers Ireland Ltd. EFFECTS OF EXTERNAL CALCIUM ON HORIZONTAL CELLS IN THE SUPERFUSED GOLDFISH RETINA

JOHN S. ROWE

Department of Neurosciences, McMaster University, 1200 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada. INTRODUCTION The retina has been described as a "natural brain slice" (1) since it can be removed from the eye without damage and maintained in vitro where it still responds normally to light stimuli.

Teleost retinae can be maintained at room

temperature under moist oxygen. This method, though used successfully in a number of studies (e.g.2,3), is restrictive when a pharmacological approach must be adopted because chemicals must be sprayed onto the retina. Spraying has major drawbacks; the exact retinal concentration of chemical is usually in doubt, sequences and combinations of chemicals are difficult to apply, and removal of chemicals is dubious. Retinal superfusion overcomes these problems but imposes the requirement that the basic perfusate must adequately mimic the normal retinal extracellular fluid. One early finding was that, for teleost retinae, changes in the concentration of calcium (Ca) in the perfusate had profound effects on the electrical activity of horizontal cells (4), a result which prompted the experiments on the actions of Ca in distal retina of goldfish reviewed here. Photoreceptors respond to light flashes with hyperpolarizing responses which may be recorded by microelectrode (5) and which appear to be the end product of similar transduction processes in rods and cones (see 6 for recent review). The hyperpolarizing signal is modified by the voltage-dependent conductances of inner segment membrane (7) and, as suggested by Trifonov (8), results in a reduction in the efflux of transmitter from photoreceptor terminals that is graded with light intensity (9-12). L-glutamate is the most likely transmitter candidate (2,4,13). Horizontal cells (HCs) have synaptic glutamate channels with a reversal potential of about -3mY (14,15). The hyperpolarizing S-potentials of HCs first recorded by Svaetichin (16) result from a decrease in the synaptic conductance as transmitter efflux declines together with the efflux of potassium through non-synaptic voltage-gated conductances on the HC membrane (14). Calcium plays a number of roles in the distal retina. It is important for the transduction process, though not as the intracellular messenger as originally envisaged by Yoshikami and Hagins (17). Rather it appears to modulate the cyclic nucleotide cascade possibly regulating photoreceptor activity via a negative feedback loop (18). Calcium also regulates transmitter release from photoreceptors, a tonic dark Ca current is presumably reduced in light by photoreceptor hyperpolarization (19). Consequently, cobalt, high magnesium or low calcium treatments block synaptic transmission (9-II).

Presented at the 9th Taniguchi International Symposium on Visual Science, November 24-28, 1986.

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The effects on HC activity of varying Ca from 0.01 pM to 5 mM have been investigated in isolated, perfused goldfish retina. The results of these experiments are reviewed here. Perhaps the most striking result occurs when calcium is buffered

to approx. 0.5 pM; a depolarizing component appeals in HC responses

to light flashes. The possible sites of origin of, and mechanisms underlying, the depolarizing component are discussed.

METHODS Retinae were removed from the excised eyes of freshly-killed goldfish which had been dark-adapted for about 30 min immediately prior to dissection. The isolated retina was placed on a Nucleopore filter disc which was placed on conventional filter paper for several minutes to partially absorb vitreous humour before being transferred to the recording chamber. Once in the chamber the retina was maintained under a gravity-fed perfusate flowing at 4-6 ml/min. A six-way valve permitted selection of one of six perfusates. The base solution consisted of llOmM NaCI, 2.bmM KCI, 20 mM NaHCO3, 1.2 mM MgSO,, and 20 mM glucose continuously bubbled with 95% Oz, 5% COz and maintained at 20°C and pH 7.2. For reasons that will become obvious later the "normal" perfusate, i.e. the initial and final solution for each experiment, usually contained 50 ~M CaCI2. When lower concentrations were required EGTA-buffered solutions were used to which 1 mM EGTA plus a calculated quantity of CaCI2 were added followed by pH adjustment. An apparent association constant for calcium with EGTA at 20°C and pH 7.2 of 6.83 (20) was used to derive values for added CaClx. Free calcium values and quantities of other chemicals added are given in the figure captions. Glass micropipettes filled with 2.5 M KCI (resistance 40-80 MO), pulled on a Brown and Flaming type puller, were used in conjunction with conventional electronics to record S-potentials. Alternating 350 msec flashes of 540 nm and 640 nm near monochromatic light were employed for initial identification of horizontal cell type. When necessary, cell identity and response characteristics could be further tested with series of a) equal quantal stimuli (400 - 720 nm in 20 nm steps), b) intensities and c) aperture sizes, all generated by a computercontrolled optical system. RESULTS The high calcium range Adding more than 50-100 UM Ca to the perfusate hyperpolarizes all classes of goldfish HCs. Figure IA shows a typical result for an HI (luminosity) type HC exposed briefly to 2 mM Ca. The dark resting potential (DRP) hyperpolarized and the amplitude of the light-evoked response declined. The response to Ca is

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Fig. i. Effects of Ca, Mg and IBMX on goldfish HCs. The intracellularly recorded HC membrane potential is displayed as a function of time, with downward displacement of the membrane potential corresponding to hyperpolarization. Chemicals were added to the perfusate for periods indicated by the solid bars beneath the recordings. A. Recording from an HI HC stimulated with 350 msec flashes of 640 nm, 0.67 pW/mm = light. For this experiment only no Ca or Mg was added to the perfusate except for the periods indicated. B° HI HC stimulated with alternating 640 nm and 540 nm light flashes. Upward deflections of the lower trace represent 640 nm stimuli and downward deflections 540 nm, 0.5 pW/mm = stimuli. C. Rod HC exposed to 1 mM Ca and stimulated with 540 nm, 1.89 nW/mm 2 flashes.

graded, the DRP falling almost linearly as log [Ca]o rises until, at approximately 2 mM Ca, the DRP approaches the equilibrium potential for potassium (Ek) at about -75 mV (11,21) and the decline ceases (22). These results are similar to those reported for roach (Rutilus rutilus) retina (4,23) except that, in the roach, 2 mM Ca routinely abolished all light-evoked activity while in the goldfish hyperpolarizing responses,

albeit of reduced amplitude, are still recorded.

The DRP of HCs in perfused roach retina was only found to match DRPs of cells recorded in isolated retina maintained under moist oxygen if Ca was omitted from the perfusate. In goldfish, HC DRPs in isolated, perfused retina only match those recorded in vivo if perfusate Ca is 50-100 pM (22) and, consequently,

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50 ~M was selected as the initial perfusate [Ca] for the experiments to be described. The effect of elevated Ca is quite distinct from that of elevated Mg. Only slight hyperpolarization is produced by 2 mM Mg (Fig. IA), much higher concentrations being required to demonstrate the synaptic blocking action of Mg (9,11). Changing the pH buffer has been reported to alter the effect of Ca on photoreceptors (24) but changing from a bicarbonate-buffered to a HEPES buffered medium did not prevent HC hyperpolarization by Ca nor did the addition of taurine which mimics the effects of lowering [Ca]o on retinomotor movements (25). To date only the addition to the perfusate of isobutyl-methylxanthine

(IBMX), a

potent phosphodiesterase inhibitor, has been found to largely reverse the effects of elevated Ca (Fig. IB). The addition of 1 mM cobalt (Co) in conjunction with 1 mM Ca and 100 pM IBMX caused complete synaptic blockage indicating that the depolarizing effect of IBMX is not a direct action on HCs but rather an effect mediated at the photoreceptor level (26). The view that Ca and IBMX are acting on the photoreceptors is reinforced by intracellular recordings from the giant cone receptors of walleye (Stizostedium vitreum vitreum) retina which also exhibit DRP hyperpolarization and reduction of response amplitude in 1 mM Ca and by recordings of the glutamate-isolated photoreceptor component of the goldfish ERG which also declines

as

[Ca]o rises.

The sensitivity of HCs to light has been examined in 50 ~M-2 IM Ca media. With sensitivity defined as the intensity required to produce a half-maximal response, no consistent differences could be found among the different Ca levels though at 2 mM the maximal response amplitude was generally only 10-20% of the amplitude recorded in 50 pM Ca (27). The goldfish rod HC appears to be more sensitive to the effects of external Ca than do the cone HCs with 1 mM Ca frequently being sufficient to abolish all light-evoked activity (Fig IC). The depolarizing responses of the H2 and H3 (colour) type HCs were not found to be of maximal amplitude in 50 ~M Ca. These cells behaved like HI and rod HCs with respect to the hyperpolarization of the DRP and the declining amplitude of the hyperpolarizing S-potentials with rising [Ca]o (Fig. 2A).

The amplitude of

the depolarizing component, however, was usually maximal in 100-500 ~M Ca. For H3 cells the increase in the depolarizing response to 640 nm flashes with increasing Ca was often striking (Fig. 2B). Equal quantal spectral scans for H3 cells in 50 pM Ca (Fig. 2C) and in 500 pM Ca (Fig. 2D) show the effect clearly. The depolarizing responses and hyperpolarizing responses to long-wave stimuli are suppressed or absent in the 50 pM Ca perfusate.

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Fig. 2. A. H2 HC exposed to an ascending series of Ca concentrations. Alternating 640 nm and 540 nm stimuli as for Fig. IB in this and in subsequent figures unless otherwise noted. Cell was initially identified by a scan of equal quantal stimuli from 400 to 720 nm. B. H3 HC initially perfused with 50 wM Ca then with 500 wM Ca. C and D. Equal quantal scans from 400 to 720 nm on an H3 HC in 50 wM Ca and 500 wM Ca respectively.

The low calcium range Initial attempts to reduce Ca below basal levels by adding small quantities of EGTA to the perfusate gave inconsistent results, presumably because of inconsistency in the Ca level achieved by this method. Repeatable results were obtained when 1 mM EGTA plus calculated quantities of Ca were added to achieve accurate free Ca values. Buffering Ca to i0 or 5 pM produced no marked changes in HC activity from that recorded in 50 pM Ca. Buffering to 3 pM generally depolarized the HC and reduced the amplitude of the S-potentials, results similar to those reported for Eugerres retina by Laufer (28) who attributed the depolarization of the DRP to an increase in HC membrane permeability to sodium. Reducing [Ca]o to 0.5-1 wM produced a very striking change in HC electrical activity (Fig. 3). As the [Ca] fell the DRP hyperpolarized and achieved a new stable level usually well above Ek (see below). The normal responses of the cell to light flashes (Fig.3A) decreased in amplitude and depolarizing transients appeared at stimulus on and off (Fig.3B). The depolarizing transients increase in amplitude and the hyperpolarizing response diminished (Fig. 3C) until, finally, the response to light was either purely depolarizing or else depolarizing with a residual hyperpolarizing notch (Fig.3D). The characteristics

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of the depolarizing response are summarized in the next section. Approximately 75% of cells tested showed some evidence of a depolarizing component in 0.5 pM Ca. Those that did not exhibited decreased amplitude S-potentials with a variable degree of DRP hyperpolarization. With a further decrease in [Ca]o to 0.i pM DRP hyperpolarization became more pronounced (Fig. 4A) consistent with blockage of a Ca-dependent synapse at this level of Ca. Residual responses to light were most frequently depolarizing with larger depolarizing responses visible as [Ca]o fell to and rose from 0.i uM. The potent glutamate analogue, quisqualic acid (QA) was added to the perfusate to test the viability of the postsynaptic receptors in 0.1 pM Ca (Fig.4A). Normal, vigorous responses to QA were always recorded supporting the view that the action of low Ca is to block the synapse and not to impair the HC's ability to respond to transmitter. At still lower values of [Ca]o, approximately 0.02 pM and below, the DRP reached a depolarized plateau with no light-evoked activity visible (Fig. 4B). The depolarization is outwardly similar to that achieved by adding glutamate or its analogues to the perfusate but can be distinguished from this state by the application of kynurenic acid (KYN), a potent glutamate blocker (29) effective

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at the fish photoreceptor to HC synapse (30, see also Fig. 8A), which has no effect on the depolarized plateau. No further changes in HC activity were observed with further decreases in [Ca].

Characteristics of the depolarizing response component The depolarizing response to light could only he observed over a narrow range of [Ca]o. Above 1-2 wM Ca it was never observed. Below approximately 0.I pM Ca it was still present but was of small amplitude. A value of 0.5 pM Ca (i mM EGTA + 0.75 mM Ca) was found to be optimal for eliciting the depolarizing response. All four classes of goldfish HC produced depolarizing responses in 0.5 pM Ca (Fig. 5). If H2 or H3 type colour cells exhibited normal depolarizing responses in 50 wM Ca these rapidly disappeared as the [Ca] fell. The response of the H3 cell shown in Figure 5C is typical for this class of cell. The DRP first depolarized and the depolarizing response to a 640 nm stimulus disappeared. Subsequently the DRP hyperpolarized and the cell began ~roducing depolarizing responses to the 540 nm stimulus that had previously elicited strong hyperpolarizing S-potentials, the 640 nm stimulus produced no response.

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It quickly became apparent that the waveform of the response finally achieved in low Ca was highly dependent on the stimulus intensity. Figure 6C shows a HC in 0.5 ~M Ca stimulated with 640 nm flashes of increasing intensity. Dim stimuli elicited pure depolarizing responses (e.g. Fig. 6D). The hyperpolarizing component returned with increasing intensity (e.g. Fig. 6E). The largest depolarizing responses so far observed in 0.5 pM Ca had an amplitude of 14 mV. Adjusting the stimulus intensity provided a convenient method of eliminating the hyperpolarizing response thereby facilitating observation of the depolarizing response component (e.g. as in Fig. 5C). It also permitted examination of the spectral characteristics of the depolarizing response. For HI cells the depolarizing response (Fig. 6B) appeared to reach a maximum at shorter wavelengths than did the hyperpolarizing responses of an HI cell in 50 pM Ca (Fig.6A).

Depolarizing responses were found to occur for all HC classes over

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Figure 7 shows results for a cell believed to be a blue cone because of a) the absence of response to 10 ~M kainic acid, a potent glutamate analogue that depolarizes all second-order neurons (32), h) its lack of spatial summation and c) its superficial location in the retina. This cell continued to produce purely hyperpolarizing responses in 0.5 ~M Ca. Figure 7E shows a spectral scan for an H3 HC in 0.5 ~M Ca, in contrast to the presumed photoreceptor

(Fig. 7D), it depolarized

in the blue spectral region. HCs in 0.5 pM Ca continue to respond, with no reduction in sensitivity, to exogenously applied glutamate, aspartate, kainate and quisqualate. Responses in all cases were similar to that shown for quisqualate in conjunction with

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Ca in Figure 4A. In addition, the glutamate blocker, kynurenic acid, exerted its usual action, hyperpolarizing HCs and blocking the depolarizing responses (Fig. 8B). Clearly, cone photoreceptors maintain considerable tonic release of transmitter when perfused with 0.5 ~M Ca, Folic acid, previously shown to block fish photoreceptor to HC transmission (23), was also an effective blocker of the

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depolarizing responses. N-methyl aspartic acid (NMDA) and 4-amino phosphonobutyric acid (APB) had no effect on DRP or on depolarizing responses. GABA had a potentiated depolarizing effect on DRPs of HCs in low Ca (33) but baclofen and musicimol were without activity and neither the depolarizing response nor the response to GABA were blocked by bicuculline or picrotoxin. A

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Attempts have been made to block the depolarizing response by modifying the action of known conductances. Cobalt cannot be used in conjunction with EGTA to block Ca conductances because it binds strongly to EGTA and releases Ca. Magnesium can be used since Ng binds to EGTA I05. s less strongly than Ca. Omitting the 1.2 mM Mg normally present in the perfusate had little effect on the depolarizing responses (Fig. 9A) nor did the use of EDTA instead of EGTA as buffer. However, increasing Mg to i0 mM (which requires a slight adjustment to added Ca to maintain the 0.5 pM value) caused an initial membrane depolarization (Fig. 9B), surprising in view of the normal hyperpolarizing effect of i0 mM Mg

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(11), followed by membrane hyperpolarization. Depolarizing responses were blocked by 10 mM Mg but small hyperpolarizing responses could still be obtained by increasing the stimulus intensity. Experiments in which Ca was buffered to nanomolar levels and 30-50 pM strontium (St) added to the perfusate resulted in enhanced depolarizing responses as large as 20 mV (Fig. 10). This condition was, however, unstable, the DRP depolarized and the response amplitude soon diminished.

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External TEA, which blocks voltage-gated K conductances (for review see 34) increased the amplitude of the depolarizing component and reduced the hyperpolarizing component (Fig. 9D). Also, when a cell failed to produce depolarizing responses in 0.5 pM Ca the addition of TEA frequently revealed these (Fig. 9C) suggesting that the depolarizing component may be masked by powerful K conductances.

DISCUSSION

The high calcium range Both the DRP hyperpolarization which occurs as [Ca]o rises above 100 pM and the reversal of this effect by IBMX appear to be mediated at the photoreceptor level. Thus, in high calcium media, the HCs remain able to respond to transmitter but

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are hyperpolarized because photoreceptor transmitter effects

o u t p u t i s r e d u c e d . The

of e x t e r n a l Ca on p h o t o r e c e p t o r s a r e w e l l documented ( e . g .

Calcium e n t e r s t h e p h o t o r e c e p t o r s a s p a r t of t h e d a r k c u r r e n t elevated

[Ca]o l e a d s t o e l e v a t e d

[Ca]l.

In t u r n ,

[Ca], a p p e a r s t o m o d u l a t e t h e

c y c l i c n u c l e o t i d e c a s c a d e , p o s s i b l y by s t i m u l a t i n g l i g h t - a c t i v a t e d diesterase

(PDE) a c t i v i t y

or by i n h i b i t i n g

[cyclic-GMP] and h e n c e t h e d a r k c u r r e n t polarize

( 6 ) . The p h o t o r e c e p t o r would t h u s h y p e r -

i n t h e r e l e a s e of t r a n s m i t t e r

The p u z z l i n g a s p e c t of t h e h i g h Ca r e s u l t s retina

phospho-

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i n a C a - d e p e n d e n t manner w i t h IBMX i n h i b i t i n g

events reflected

35-37).

(38) so t h a t

t h i s by b l o c k i n g PDE (39),

to second-order neurons. i s t h a t t h e DRPs of HCs i n p e r f u s e d

o n l y match t h o s e r e c o r d e d i n v i v o when Ca i s 50-100 pM w h i l e a t t h e 1-2 mM

l e v e l s r e g a r d e d a s normal f o r e x t r a c e l l u l a r light-evoked activity

s p a c e HCs a r e h y p e r p o l a r i z e d and

i s s u p p r e s s e d or a b s e n t . Assuming t h a t t h e 1-2 mM r e s u l t s

a r e n o t t h e normal b e h a v i o u r of HCs, two e x p l a n a t i o n s may be a d v a n c e d . F i r s t , is possible that a naturally

it

o c c u r i n g s u b s t a n c e e q u i v a l e n t t o IBMX c o u l d be

f l u s h e d away by t h e p e r f u s a t e or t h a t c y c l i c GMP i s f l u s h e d out of p h o t o r e c e p t o r s during perfusion creating

an a r t i f i c i a l l y

r e d u c i n g e x t e r n a l Ca below normal l e v e l s . r e c e p t o r membrane i s r e l a t i v e l y HC a c t i v i t y indicating

remains stable

seems u n l i k e l y s i n c e p h o t o -

impermeable t o c y c l i c GMP (40) and a l s o b e c a u s e

f o r l o n g p e r i o d s when t h e p e r f u s a t e c o n t a i n s 50 pM Ca

t h a t t h e p h o t o r e c e p t o r c y c l i c GMP l e v e l can r e m a i n s t a b l e d e s p i t e

continuous perfusion. subretinal

low l e v e l which must be compensated by The l a t t e r

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is possible that photoreceptors reside in a

compartment w i t h a [Ca] l e s s t h a n 1-2 mM. However, C a - s e n s i t i v e

electrode measurements of subetinal Ca in excised carp eye-cups were in the 1.2-1.4 mM range (41). How there could be any rod HC function in vivo if these values are correct remains to be resolved. The depolarizing responses of H2 and H3 cells reach maximum amplitude at higher Ca values than the hyperpolarizing S-potentials. Possibly the feedback synapses thought to underlie the depolarizing responses (3,42-44) may require a higher level of Ca than the photoreceptor to HC synapse. This is consistent with the abolition of depolarizing responses which occurs when [Ca]o falls below i0 pM but not with the ability of HI HCs to release GABA, upon which feedback may depend (43-45), via a Ca-independent mechanism (46). Alternatively, the depolarizing responses may increase in amplitude simply because the DRP falls with increasing [Ca]o, moving away from the reversal potential for the feedback mechanism underlying the depolarizing responses.

The low c a l c i u m r a n g e The p h o t o r e c e p t o r t o HC s y n a p s e a p p e a r s to be C a - d e p e n d e n t a s p r e v i o u s l y reported

(9-12)

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low v a l u e s of

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external Ca, complete blockage not occuring until [Ca]o is 0.05-0.1 ~M. An implicit assumption here is that Ca at the synapse falls to the perfusate value, certainly other agents active at the synapse such as Co appear to access the synaptic region easily. Also, the reduction of HC activity could be due to impaired transduction as well as reduced transmitter efflux in low Ca. While sequestration of internal Ca by EGTA depolarizes turtle cones and increases their response to bright lights (47), ERG recordings from goldfish retina show the photoreceptor component declining in amplitude when [Ca]o falls below 0.5 ~M (48) The depolarized plateau reached at 0.02 pM Ca and below was not due to activation of postsynaptic receptor sites but, since Ca is essential for membrane integrity (49), may be due to a general increase in membrane permeability.

The depolarizing response component Depolarizing responses of HCs in low Ca media have been recorded in other species of freshwater teleost and from turtle (50) as well as in goldfish. Three questions regarding these responses require answers. First, what is their site of origin? They could originate at the photoreceptor level with HCs continuing to function normally but now receiving increased levels of transmitter during light stimulation. On the other hand they could originate at the HC membrane as an altered response to the usual decrease of transmitter during stimulation. Second, what mechanism underlies the depolarizing responses? Third, is the mechanism active at normal Ca levels but masked by other activity or is it activated only when Ca is low? Obviously it will be difficult to argue that these responses have any functional significance if the mechanism operates solely in a Ca range attainable only by adding EGTA. These questions are discussed in the light of currently available evidence below. A number of studies confirm the results presented here that reducing [Ca]o to 0.5-1 pM only causes the hyperpolarizing responses of photoreceptors to increase in amplitude (11,I0,51). Photoreceptors only produce depolarizing responses (36,52) if external Na is replaced by choline and both Ca and Mg are reduced to low levels. Therefore it seems reasonable to suggest that a normal hyperpolarizing response is conducted from outer segment to inner segment. The signal is modified by a number of voltage-gated conductances in inner segment membrane and synaptic region (7) with a Ca conductance likely being ultimately responsible for transmitter release (19). However, the tonic release of transmitter in 0.5 pM Ca that can be blocked by kynurenic acid or by a further reduction of [Ca]o indicates a continuing inward current of Ca into the photoreceptor terminal. Aiso, restoration of the hyperpolarizing component by high intensity stimuli suggests that a reduction of transmitter release by light can still occur. In addition, cesium (Cs) application should block anomalous

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rectifiers at photoreceptor (7) and HC level (53-55) but had little effect on depolarizing responses (56), chloride substitution rules out photoreceptor chloride conductances (56), and TEA and 4-AP treatments exclude a role for the K delayed rectifier again at both photoreceptor and HC levels (53-55). In short there is, at present, no evidence that the depolarizing responses originate at the photoreceptor level. There is also no evidence that the depolarizing responses are due to feedback from HCs to photoreceptors. Not only do low Ca depolarizing responses always begin before the hyperpolarizing component but also they occur in different spectral regions from those in which normal feedback-mediated depolarizations occur. Furthermore, unlike the normal depolarizing responses (3,43,44) the low Ca responses cannot be blocked by GABA antagonists. By elimination, therefore, the HC membrane must be regarded as the most likely site of origin for the depolarizing responses. Two glutamate conductances are located on the HC membrane. In the presence of glutamate one conductance increases due to the activation of glutamate channels (15,57,58), the other decreases due to a blockage of the K-current through the anomalous rectifier (15,53,57). It is, however, difficult to interpret the depolarizing responses in terms of these two conductances. Cesium experiments rule out the anomalous rectifier and the glutamate channels appear to be functionally normal in 0.5 ~M Ca since glutamate and its analogues effectively depolarize HCs in low Ca while kynurenic acid exerts its normal synaptic blocking action. It is possible to construct a novel HC membrane component with the requisite characteristics to explain the results. The effectiveness of kynurenic and folic acids in blocking depolarizing responses suggests that another glutamate channel may be responsible. If this conductance were opened by glutamate its effect would be to oppose the normal glutamate conductance, hence, since the response to glutamate is normal in low Ca, a channel whose conductance is reduced by glutamate may be a better candidate. Since depolarizing responses are not observed during other treatments which should reduce the synaptic glutamate concentration (e.g. high Ca or Co application) it is necessary to add the property that Ca inactivates the novel channel. The other alternative, namely that low Ca impairs the normal glutamate channels, does not seem tenable in view of the continued effectiveness of glutamate and its analogues. Having introduced a novel channel it is also possible to speculate on the ionic current it may carry. Chloride has been ruled out by substitution experiments. Potassium efflux would give hyperpolarizing responses. Tests to discover if sodium plays a role have yet to be performed. The enhancement of the depolarizing responses by Sr and the blockage of depolarizing responses by Mg suggests a role for Ca itself, though one that is difficult to reconcile with the reduced driving

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force on Ca in low Ca media. However, it may be noted that a number of examples of Ca channels whose conductances are decreased by neurotransmitters have been found in other systems (for review see 59) and also that the Ca conductance in goldfish HC membrane does appear to be inactivated by Ca (60). The model presented above requires a channel that is closed by both glutamate and Ca. Bow such a channel could contribute to visual processing in vivo remains a mystery. It is clear, however, that conditions exist under which HCs produce depolarizing responses to light thereby providing an exception to the scheme of photoreceptor to HC transmission proposed by Trifonov (8).

ACKNOWLEDGEMENTS This work was supported by the Medical Research Council of Canada. The results with strontium were obtained in collaboration with Dr J. Kleinschmidt. J.S.R. wishes to thank K. Hankinson, B. Holt and A. Gloster for help with experiments.

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