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The effects of GABA and glycine on horizontal cells of the rabbit retina
Pergamon
PII: S0042-6989(96)00145-9
Vision Res., Vol. 36, No. 24, pp. 3987-3995, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0042-6989/96 $15.00 + 0.00
The Effects of GABA and Glycine on Horizontal Cells of the Rabbit Retina ROMAN BLANCO,*t CECILIA F. VAQUERO,* PEDRO DE LA VILLA*
Intraceilular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3/aM GABA but none to glycine, even at 100 #M. Dose-response relationship for GABA gave ECso values around 10/aM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted C l - equilibrium potential. Partial replacement of intracellular C i - with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30/aM), a GABAA agonist mimicked the effect of GABA, but baclofen (30/aM), a GABAn agonist and cis-aminocaprionic acid (30/aM), a GABAc agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 I~M) and picrotoxin (100/aM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes. Copyright © 1996 Elsevier Science Ltd
GABA
Glycine
Horizontalcells
Intracellular recording
INTRODUCTION It is now well documented that lateral inhibition in the outer retina is mediated by horizontal cells (HCs). HCs respond to light with graded hyperpolarizations which are transmitted through inhibitory feedback chemical synapses to cones and bipolar cells (Baylor et al., 1971). HCs regulate the cone and bipolar cell light responses by modulating their response kinetics and mediating the surround responses which constitute their centre-surround receptive field (Yang & Wu, 1989, 1993; Wu, 1994). In the mammalian retina it has been shown that HCs are responsible for the centre-surround organization of the ganglion cell receptive field (Mangel, 1991). In the outer plexifoml layer (OPL) of the retina, the role of GABA and glycine is not yet fully understood. Recent results suggest that the cellular distribution, functional characteristics and possible physiological role of GABA receptors in tlhe retina might be more diverse than previouslY thought (Djamgoz, 1995). In the retina of lower vertebrates, G A B A acts at a least three pharmacologically distinct sites, termed GABAA, GABAB, and GABAc receptors. The GABAA receptor, which is a ionotropic receptor, gate,s a C1- channel, is antagonized by bicuculline and is modulated by benzodiazepines *Department of Physiology and Pharmacology, University of Alcal;i de Henares, Madrid, Spain. tTo whom all correspondence should be addressed [Email [email protected]].
Patch-clamp Rabbitretina
(Ishida, 1992). The GABAa receptor seems to control calcium and potassium conductances via G-proteins [see Slaughter (1995) for review]. The GABAc receptor also gates C1- channels and can be blocked by picr0toxin but is insensitive to bicuculline and baclofen; it is activated by the agonist CACA (cis-4-aminocrotonic acid) (Feigenspan et al., 1993). Several different subunits (~Z1-6, ill-4, 71-3 and 6) have been associated with GABAA receptors, whereas GABAc is associated to expression of "p" subunit. To date, GABAA and GABAc receptors have only been described on HC of cold-blooded vertebrates retinas (Qian & Dowling, 1994; Dong et al., 1994). GABA has been shown to modulate the HC light responses. The adaptation-induced change in the HC response rise time (HCRRT) is mediated by GABA in the amphibian retina (Yang & Wu, 1989, 1993). It is known that GABA is released continuously in darkness from HC, and it slows the HCRRT through GABAA receptors (Kamermans & Werblin, 1992). It has been also shown that GABA induces HC depolarization in the amphibian retina (Stockton & Slaughter, 1991). In the mammalian retina, it has also been noted that GABA slows the HCRRT (Frumkes & Nelson, 1995). However, it is not known whether in the mammalian retina GABAA receptors are located in HC, or/and in other retinal neurons which influence the HC light responses through chemical synapses. On the other hand, glycine has also been shown to play
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an important role as an inhibitory neurotransmitter in the retina [reviewed by Marc (1989)]. In this study we examined the action of glycine on HC with a view to understanding its role in the external mammalian retina. Dissociated HC have been the object of numerous electrophysiological studies on cold-blooded vertebrates retinas (Ishida et el., 1984; Lasater, 1986; Perlman et el., 1989; O'Dell & Christensen, 1989; Zhou et el., 1993) but very few have been published in HC of mammalian retinas (Ueda et el., 1992; L6hrke & Hofmann, 1994; Blanco et el., 1996). Since diversity in neural organization is generally greater for lower vertebrates, mammalian retina is considered to be a more suitable preparation for understanding the function of the human retina. Our electrophysiological study examines the GABAergic and glycinergic action in distal mammalian retina and their receptors on HC. We have examined the properties of GABAA-receptor channels in dissociated axonless horizontal cells (ALHC) and investigated whether these cells also express functional glycine receptors. Our results demonstrate that ALHC possess GABAA but not glycine receptors. Portions of the present study were presented previously in abstract form (Blanco et el., 1995).
METHODS
Intracellular recordings
Two-month-old rabbits were anesthetized with urethane (1.5 g/kg i.p.) (Dacheux & Miller, 1981; Miller et el., 1981). All the experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) on experimental animals. The eye was enucleated, hemisected and everted over a Teflon plug, which fits into an oxygenation chamber. Superfusate flowed by gravity (23 ml/min) onto the surface of the retina. This extracellular solution was made according to the formula of Ames and Nesbett (1981): NaCI, 120 mM; KC1, 5 mM; NaHCO3, 25 mM; CaC12, 2 mM; MgSO4, 1 mM; Nail2_ PO4, 0.1 mM; Na2HPO4, 0.8 mM; glucose, 10 mM. All solutions were bubbled with 95% O2-5%COe yielding a pH of 7.4 at 37°C. Drug solutions were added to the superfusate and adjusted to this pH with NaOH or HCI as required. As judged by physiological criteria, the rabbit retina was well maintained under these conditions for 24 hr and intracellular recordings could be held through solution changes. Drugs were introduced at controlled concentrations and in combination as required. The dead space in the perfusion system was minimal and drug effects occurred within 20-40 sec. Intracellular recordings were obtained with microelectrodes pulled from 1.2-mm Omega-dot glass on a horizontal puller (Brown-Flaming). Electrodes were filled with 3 M potassium acetate and the resistance was typically between 100 and 400 Mfl. Cell impalement was facilitated by adjusting the negative capacitance in the electrode headstage. Voltage traces were monitored with an oscilloscope. ALHC were identified from their
physiological responses and/or by iontophoretic application of carboxifluorescein 5% [see Villa et el. (1991)]. Results were recorded on a pen recorder and computer hard-disk for later analysis. The retinal preparation was stimulated by a dual-beam photostimulator. A centred light beam whose intensity and wavelength could be adjusted by neutral-density filters and interference filters, was provided by a quartz halogen source (Villa et el., 1994). In most experiments described, light spots of 600800/2m in diameter were used.
Patch-clamp
Recordings were made from dissociated HC of the retina from 2-month-old rabbits. Dissociated cells were obtained as previously described (Ueda et al., 1992; Villa et el., 1995). Briefly, rabbits were anesthesized with uretane and their eyes rapidly removed. The retina was stripped from the pigment epithelium, divided into pieces and incubated in a standard solution containing (mM): NaC1, 135; KC1, 5; CaC1, 2; MgC12 1; HEPES 5; glucose 10; pH 7.4, adjusted with NaOH, which contained papain (25 U/ml) and 2.0 mM cysteine. The retina was incubated in this solution for 20-30 min and then placed in a standard solution. The retina was dissociated into individual neurons by repeatedly passing the tissue through a Pasteur pipette. Using this procedure, a single class of HC, the ALHC, could be prepared and identified by their morphological characteristics. The cells were placed in a standard solution filled chamber mounted on the stage of an inverted phase-contrast microscope (Nikon TMD, Tokyo, Japan). During recordings, the cells were continuously perfused with a standard solution. Drugs were applied to the cell via the perfusion medium. The drugs used both in patch-clamp and intracellular recordings experiments, were all acquired from Sigma Chemical. Whole-cell recordings were made at room temperature (22-25°C) using a patch-clamp amplifier (RK-300, Biologic instruments). Patch pipettes were pulled from thickwalled glass (Clark Electromedical GC150F) to a resistance of 8-15 MQ when filled with a pipette solution containing (mM): NaCl, 10; KC1, 110; CaCl, 0.5; MgC12 1; HEPES 10; EGTA 5. In some experiments, the intracellular C1- was replaced by equimolar isothetionate. The capacitive component was electrically compensated, although incompletely, perhaps due to cell morphology. Electrode series resistance ranged from 10 to 20 M~. The voltage error produced by these series resistances (series resistance x recorded current) was not compensated as it was <5% of the holding potential. The junction potential at the electrode tip was neglected because it was small ( < - 3 mV) under the present conditions. Whole-cell responses to perfusion-applied agonist were analysed from records digitized at 0.57 kHz (Intel 80486-based personal computer; VCAN 3.1.) (Dempster, 1994).
GABA RECEPTORS ON HORIZONTAL CELLS
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A
'
GABA
100 uM
mv
I
25 see
B GABA 100 pM
'mVI
-I
1 sec
FIGURE 1. (A) Intracellular data showing the effect of 100 #M GABA on the HC voltage responses to 0.5 sec light steps (800 pm dia and intensity of 2.71 x 1014 photons cm - 2 sec- 1 at 500 nm) as indicated by the square-wave pulses at the bottom of the voltage trace. Tile dark bar below the recording indicates the time application of GABA. Note that the effect of GABA is both to depolarize the cell and decrease the HC light response. (B) Horizontal cell recordings from the same experiment are shown at a expanded time scale [arrows in (A)]. We see how GABA decreases the HC light response in addition to slowing down the horizontal cell response rise time (HCRRT).
RESULTS
Intracellular recordings Intracellular recordings were obtained from a total of 30 HCs in the perflmed rabbit retina. The resting membrane potential varied from cell to cell, from - 2 0 to - 4 0 mV. All recorded cells corresponded to ALHC, according to the shape of their light response (Bloomfield & Miller, 1982). In some cases (n = 5) we confirmed the origin of light responses by iontophoretic injection of carboxifluoresecein 5%, after recording their physiological responses (Villa et al., 1991); and in all these cases we found them to be ALHC. Figure 1 shows the effects of GABA on HC voltage responses to 0.5 sec light steps. In the presence of 100 #M GABA, the HC w a s depolarized and the light responses amplitude was reduced. GABA seemed to
reduce both the cone and rod responses and slowed down the HCRRT. Figure 2 shows the effects of various concentrations of glycine on HC light responses. In the presence of both 60 and 100 ~M glycine, the HC light responses amplitudes were diminished or abolished, respectively. Glycine seemed to reduce the cone input the strongest and did not slow down the HCRRT. These experiments did not definitively identify the distal GABAergic (and glycinergic) target. Its location on either photoreceptor cells, HCs, both cell types or other cells (e.g. interplexiform cells) remained in doubt. We, therefore decided to perform additional experiments on isolated HCs using whole cell patch-clamp.
Patch-clamp Patch-clamp recordings were obtained from a total of
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A 2 mln/~/ 10mVI
a5 sec-
GLYC 60 uM
~~~1~~2
m l n ~ 6
GLYC 100 IJM
rain~T~7.,~.~
10mVi
25sec
B
~ ~V' Control/Relovery . , j R
5°v1
1 sec
FIGURE 2. (A) Effects of various concentrations of glycine on the HC voltage responses to the same light stimuli (see legend of Fig. 1). Glycine, at 60/tM, diminished the HC light responses amplitude and, at 100/~M, completely abolished the cell light responses. (B) Horizontal cell recordings from the same experiment are shown at an expanded time scale [arrows in (A), upper trace]. Note that glycine does not slow down the time of the HC light-induced response.
45 isolated HCs. Dissociated A L H C were identified on the basis of their characteristic morphology (Fig. 3). These cells responded to the application of G A B A with inward currents when clamped at negative potentials as expected for the activation of C I - channels under conditions where ERev for chloride is around 0 m V [Fig. 4(A)]. The responses to G A B A developed rapidly and desensitized to a steady-state level. The extent of desensitization was variable from cell to cell. In all cases, the responses declined rapidly when agonist was removed. The repeated application of G A B A did not lead to a progressive decrease in the peak amplitude of
the current nor changes in the time-dependent desensitization ( G A B A responses tested for >1 hr; n = 15). To characterize further the G A B A receptors in these cells we examined their concentration-response relationships [Fig. 4(A)]. The lowest concentration producing a detectable current was 3 ~M for GABA. Maximum response was obtained for G A B A concentrations up to 100/~M. For each cell, the peak currents amplitudes evoked by various concentrations of each agonists were normalized to those evoked by 100 pM G A B A and pooled data were plotted against agonist concentration. With increasing concentrations of G A B A the whole-cell
GABA RECEPTORS ON HORIZONTAL CELLS
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FIGURE 3. Microphotograph of an ALHCs dissociated from the rabbit retina. Calibration bar 40/~m.
A
GABA 3 pM
,oA]
GABA 10 pM
GABA 30 pM
GABA 100 pM
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B
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,m,
O.1
0.2
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1
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[GABA] (/aM)
100
10
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[GABA] (MM)
FIGURE 4. Whole cell patch-clamp recordings of GABA induced-currents in ALHCs. GABA was applied by superfusion. (A) Currents induced by GABA (3, 10, 30 and 100 #M) were recorded from an ALHC maintained at a holding voltage of - 70 mV. Bars above each current trace indicate the timing of drug application and numbers above the bar correspond to the GABA concentration (/tM). Note that increasing concentrations of GABA produce a faster desensitization of the inward current. (B) Dose-response curve of the GABA induced current averaged from five cells. Responses have been normalized with respect to the maximun response for GABA (100/~M). Line drawn by eye. (C) Logarithmic plot of data from (B). Analysis of the data from five cells yielded a Hill coefficient of 1.5 and an ECs0 around 5 ~M for GABA.
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A Vh(mY)
GABA ( 3 0 u M )
-60 -40
C
-20
i
0
[ C l I j = ~
20
,~ ~ ,
'/a r
40 -100 - 8 0 ] ~
B
4oo pA
, 400
I
I
I
I
20 40 60 8o mV -200 -400
pA
< | 20
4~' [CI]l=121 mM -600 | 40
! 80 m V
-200
- -400 - -600
FIGURE5. (A) Reversalpotentialof GABAresponsesin standardbath and pipette solutions. Responsesof a single HCs to brief application of 30 pM GABA at each of the holding potentials indicated (Vh). Traces have been arbitrarilyseparated vertically for clarity. (B) Peak amplitude of the responses shown in (A) are plotted against holding potential. (C) Current-voltage relationships of the GABA induced currents recorded from two different cells, as measured by ramp depolarization.Reversal potential of 30pM GABA response is shown for one cell perfused with control chloride intracellular solution ( [ C l - ] i = 121 mM) and for another cell in low-chloride pipette solution ( [ C 1 - ] i = 20 raM). When pipette contained control solution (Ea = - 3 mV), the GABAresponsereversesnear - 5 mV; in the low-chloridepipette,the GABAresponsereversesat -48 mV (Ecl= -50 mY). currents increased in amplitude in a sigmoidal fashion until saturation was apparent at 100 #M [Fig. 4(B)]. Analysis of the data from five cells yielded a Hill coefficient of 1.3 and an ECso around 10 pM for GABA [Fig. 4(C)]. The Hill coefficient suggested that the binding of more than one GABA molecule is required for receptor activation. Desensitization was apparent at GABA concentrations >3 #M. Because of the modest speed of agonist application in the present study, desensitization will take place during the rising phase of current responses. In a series of experiments, GABA induced current was recorded in cells clamped at different holding potentials. When the bath and electrode solutions contained equal chloride concentrations, the responses to GABA reversed in polarity near 0 mV [Fig. 5(A and B)], close to the equilibrium potential C I - (Ecl). In order to test the effect of C1- on GABA induced currents, we recorded from HC with pipette solution containing different chloride ion concentrations. For these experiments, intracellular chloride was replaced by equimolar isothetionate. Voltage ramps (from - 90 to +60 mV, 80 mV/sec) were commanded before and during the GABA application (10-30/~M GABA). Current-voltage relationships were analysed after subtracting the current obtained in the absence of the agonist from that obtained during the response to GABA. In each condition, three or four ramps
were applied and the resulting currents averaged. For low intracellular chloride concentration, the corresponding reversal potential shifted to more negative values. An example of this is illustrated in Fig. 5(C) which shows that in low intracellular chloride concentration, the corresponding reversal potentials shifted by very nearly the same amount ( - 4 8 mV) as did the Ect ( - 5 0 mV). These results suggest that GABA increases a chlorideselective conductance in HCs. To confirm from which GABA subtype receptors GABA responses arose, we examined the effects of the GABA agonists muscimol, baclofen and CACA and the GABA antagonists bicuculline and picrotoxin (Fig. 6). All results presented here were obtained in at least three different ALHC. Muscimol (30 #M), a GABAA agonist always mimicked the effect of GABA (n = 5), but baclofen (30#M), a GABAB agonist and CACA (30 #M), a GABAc agonist did not elicit any effect on ALHC. Bicuculline methyiodide (10/~M), a GABAA selective antagonist and picrotoxin (100 pM), an ionotropic GABA receptor antagonist, reversibly antagonized completely the GABA-evoked currents. Glycine (100/~M - 1 mM), did not evoke any effect on the HCs tested (n = 5), as we show in Fig. 7. In this figure we compare the GABA-evoked current before after applying glycine on a HC.
GABA RECEPTORS ON HORIZONTAL CELLS GABA
GABA
MUSCIMOL
GABA
GABA
GLYCINE
GABA i
ii
Wt:
250 A
GABA
BACLOFEN
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10 •
FIGURE 7. Glycine (100 pM) did not elicit any current on ALHC compared to the inward currents elicited by GABA (30 #M) applied before and after glycine. Cell clamped at Vh = - 60 mV. GABA
CACA
mmmmmmm
GABA
m
los
GABA
GA,,~,~.BIC
GAB~
GABA
GABA
p•_S00 10s
FIGURE 6. Pharmacology of GABA receptors in ALHCs. Currents induced by different agonists/antagonists are shown in horizontal series. In each series, control (left column), test (middle) and recovery (right) are shown. GABA concentration was 30 #M for the three upper rows; muscimol, 30 pM; baclofen, 30 pM; CACA, 100/.tM. GABA concentration was 3 pM for the lower two rows; Bicuculline methyiodide (BIC) (10/.tM) and picrotoxin (PTX) (100 pM), were applied together with GABA (13 pM). In all experiments, cell were clamped at Vh = - 60 mV.
DISCUSSION Our study extends observations made previously in lower vertebrates to the mammalian retina. We have shown that GABA activates a chloride conductance via GABAA receptors in isolated ALHCs of the rabbit retina. At the same time we did not find any evidence for GABAc receptors, GABA transporter currents or glycine-activated chloride conductances in these cells. These results suggest that the effects of GABA on HC are mediated by GABAA receptors and that the depolarization and the changes :in the light response rise time observed in these cells could be explained by the mediation of the GABAA receptors in the HC membrane. While GABA certainly slows the light response rise time, the mechanism of this action is not clear. The GABAergic positive feedback loop model has previously been suggested by Kamermans (Kamermans & Werblin, 1992) but direct evidence suggesting the evidence of the
loop in the mammalian retina is not available. In this model, the GABA transporter plays a prominent role while we have found no evidence for a GABA transporter in ALHC of the rabbit retina. We might note that in the catfish retina the presence or absence of GABA transporter currents have been observed in cone- and rod-driven HCs, respectively (Dong et al., 1994). Several pieces of evidence indicate that GABA is the neurotransmitter released by HCs in the mammalian retina (Vardi et al., I994). On the other hand, data published on the GABA receptor localization in the mammalian retina are somehow confusing. Detection of GABA receptors in the mammalian outer retina by immunocytochemistry has proved to be difficult (Brecha & Weigmann, 1990). At present, several studies have reported GABA-like immunoreactivity in different cell types of the outer retina of mammals. The localization of GABAA receptors has been demonstrated on the synaptic terminal of cones (Hughes et al., 1989; Brecha & Weigmann, 1990), on HC dendrites (Greferath et al., 1994b, 1995) and upon the dendrites of bipolar cells within the triad invaginations of cones (Vardi et al., 1992; Griinert & W~issle, 1990, 1994). Our intracellular recordings indicate that 100/~M GABA is required in the bath to induce changes in HC light responses. This concentration is high compared with that needed to elicit responses in dissociated mammalian HCs. The need for a higher bath concentration of GABA in the whole retina preparation is probably due to the existence of high affinity uptake systems in the retina (e.g. Miiller cells) suggesting that the concentration achieved by exogenous application of GABA in the synaptic cleft is probably lowered. However, the lack of evidence for GABA uptake in HC of the mammalian retina is significant, particularly when compared to the positive evidence in cone HCs of nonmammal vertebrates (Dong et al., 1994). Our findings that the GABA current is C1- mediated and entirely suppressed by bicuculline argue that GABA transporters may not be present on mammalian ALHCs. It remains possible, however, that the isolation procedure could in some way have removed them because of their location at the tip of the synaptic invaginations. In the retina, glycine has been shown to play an important role as an inhibitory neurotransmitter (Karschin & W/issle, 1990). In the mammalian retina
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glycinergic receptors have been localized in bipolar, amacrine and ganglion cells but not in HCs (Marc, 1989; Yazulla & Studholme, 1991; Greferath et al., 1994a, 1995). It is possible that glycine receptors might be present which are not recognized by the antibodies used until now in immunochemical studies (J~iger & W~issle, 1987). In our intracellular recordings, a striking feature of the action of glycine in the intact retina was that it diminished the amplitudes of HC responses to light. However, when the effect of glycine was tested on voltage-clamped isolated cells, it failed to induce any current activation. The absence of glycine receptors in dissociated HC reflects, in our oppinion, the actual glycine receptor distribution in situ. It is not likely that the absence of glycine effect has been caused by loss of glycine receptors on HC during the dissociation process (Gilbertson et al., 1991), since GABA responses are always present on the same recorded HC. Although we do not have a clear explanation for the in situ effect of glycine application, we suggest that the changes in HC light responses may be mediated by interplexiform cells sensitive to glycine. The physiological role of this glycinergic pathway in the mammalian retina is presently not fully understood. We could argue, therefore, that glycinergic interplexiform cells may mediate feedback interactions from the inner plexiform layer to the OPL, similar to the well known effects of dopaminergic interplexiform cells in the retina of coldblooded vertebrates.
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retina I: Effects of GABAa agonists. Visual Neuroscience, 12, 641651. Gilbertson, T. A., Borges, S. & Wilson, M. (1991). The effects of glycine and GABA on isolated horizontal cells from the salamander retina. Journal of Neurophysiology, 66, 2002-2013. Greferath, U., Brandstatter, J. H., W/issle, H., Kirsch, J., Kuhse, J. & Gr~inert, U. (1994a). Differential expression of glycine receptor subunits in the retina of the rat: A study using immunohistochemistry and in situ hybridization. Visual Neuroscience, 11,721-729. Greferath, U., Griinert, U., Fritschy, J. M., Stephenson, A., Mohler, H. & W/issle, H. (1995). GABAA receptor subunits have differential distributions in the rat retina: In situ hybridization and immunohistochemistry. Journal of Comparative Neurology, 353, 553-571. Greferath, U., Griinert, U., Mtiller, F. & W~issle, H. (1994b). Localization of GABAA receptors in the rabbit retina. Cell Tissue Research, 276, 295-307. Griinert, U., Martin, P. R. & W/issle, H. (1994). Immunocytochemical analysis of bipolar cells in the macaque monkey retina. Journal of Comparative Neurology, 348, 607-627. Griinert, U. & W/issle, H. (1990). GABA-Iike immunoreactivity in the macaque monkey retina: A light and electron microscopy study. Journal of Comparative Neurology, 297, 509-514. Hughes, T. E., Carey, R. G., Vitorica, J., de Bias, A. L. & Karten, H. L. (1989). Immunocytochemical localization of GABAA receptors in the retina of the new world primate Saimiri sciureus. Visual Neuroscience, 2, 565-581. Ishida, A. T. (1992). The physiology of GABAA receptors in retinal neurons. Progress in Brain Research, 90, 29--45. Ishida, A. T., Kaneko, A. & Tachibana, M. (1984). Responses of solitary retinal horizontal cells from Carassius auratus to Lglutamate and related amino acids. Journal of Physiology (London), 348, 255-270. J~iger, J. & W~issle, H. (1987). Localization of glycine uptake and receptors in the cat retina. Neuroscience Letters, 75, 147-151. Kamermans, M. & Werblin, F. (1992). GABA-mediated positive autofeedback loop controls horizontal cell kinetics in tiger salamander retina. Journal of Neuroscience, 12, 2451-2463. Karschin, A. & W~issle, H. (1990). Voltage- and transmitter-gated currents in isolated rod bipolar cells of rat retina. Journal of Neurophysiology, 63, 860-876. Lasater, E. M. (1986). Ionic currents of cultured horizontal cells isolated from white perch retina. Journal of Neurophysiology, 55, 499-513. L6hrke, S. & Hofmann, H. D. (1994). Voltage-gated currents of rabbit A- and B-type horizontal cells in retinal monolayer cultures. Visual
Neuroscience, 11,369-378. Mangel, S. C. (1991). Analysis of the horizontal cell contribution to the receptive field surround of ganglion cells in the rabbit retina. Journal of Physiology (London), 442, 211-234. Marc, R. E. (1989). The role of glycine in the mammalian retina. Progress in Retinal Research, 8, 67-107. Miller, R. F., Frumkes, T. E., Slaughter, M. & Dacheux, R. F. (1981). Physiological and pharmacological basis of GABA and glycine action on neurons of mudpuppy retina I. Receptors, horizontal cells, bipolars and G-cells. Journal of Neurophysiology, 45, 743-763. O'Dell, T. J. & Christensen, B. N. (1989). Horizontal cells isolated from catfish retina contain two types of excitatory amino acid receptors. Journal of Neurophysiology, 61, 1097-1109. Perlman, I., Knapp, A. G. & Dowling, J. E. (1989). Responses of isolated white perch horizontal cells to changes in the concentration of photoreceptor transmitter agonists. Brain Research, 487, 16-25. Qian, H. H. & Dowling, J. E. (1994). Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina. Journal of Neuroscience, 14, 4299-4307. Slaughter, M. M. (1995). GABAB receptors in the vertebrate retina. Progress in Retinal Eye Research, 14, 293-312. Stockton, R. A. & Slaughter, M. M. (1991). Depolarizing actions of GABA and glycine on amphibian retinal horizontal cells. Journal of Neurophysiology, 65, 680-691. Ueda, I., Kaneko, A. & Kaneda, M. (1992). Voltage-dependent ionic
GABA RECEPTORS ON HORIZONTAL CELLS currents in solitary horizontal cells isolated from cat retina. Journal of Neurophysiology, 68, 1143-1150. Vardi, N., Kaufman, D. L. & Sterling, P. (1994). Horizontal cells in cat and monkey retina express different isoforms of glutamic acid decarboxylase. Visual Neuroscience, 11,135-142. Vardi, N., Masarachia, P. & Sterling, P. (1992). Immunoreactivity to GABAA receptor in the outer plexiform layer of the cat retina. Journal of Comparative Neurology, 320, 394-397. Villa, P., Bedmar, M. D. & Bar6n, M. (1991). Studies on rod horizontal cell S-potential in dependence of the dark/light adapted state: A comparative study in Cyprinius carpio and Scyliorhinus canicula retinas. Vision Research, 31, 425-436. Villa, P., Kurahashi, T. & Kaneko, A. (1995). L-Glutamate-induced responses and cGMP-activated channels in three subtypes of retinal bipolar cells dissociated from the cat. Journal of Neuroscience, 15, 3571-3582. Villa, P., Mazo, M., Recio, M., Mafianes, F. & Blanco, R. (1994). Computer-driven optical sysl:em for light stimulation in physiological experiments of retinal cells. Measurement Science and Technology, 6, 67-71.
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Wu, S. M. (1994). Synaptic transmission in the outer retina. Annual Review in Physiology, 56, 141-168. Yang, X. L. & Wu, S. M. (1989). Effects of prolonged light exposure, GABA, and glycine on horizontal cell responses in tiger salamander retina. Journal of Neurophysiology, 61, 1025-1035. Yang, X. L. & Wu, S. M. (1993). Effects of GABA on horizontal cells in the tiger salamander retina. Vision Research, 33, 1339-1344. Yazulla, S. & Studholme, K. M. (1991). Glycinergic interplexiform ceils make synaptic contact with amacrine cell bodies in goldfish retina. Journal of Comparative Neurology, 310, 1-20. Zhou, Z. M. J., Fain, G. L. & Dowling, J. E. (1993). The excitatory and inhibitory amino acid receptors on horizontal cells isolated from the white perch retina. Journal of Neurophysiology, 70, 8-19.
Acknowledgements---This work was supported by Grants from the Spanish Government: Fondo de Investigaciones Sanitarias (FIS) Grants 93/5358, 95/5001 to R. Blanco and Direcci6n General de Investigaci6n Cientffica y T6cnica (DGICYT) Projects PB 92/0048, PB 94/0369 to P. Villa. We would like to thank Dr J. E. G. Downing for comments on the manuscript and English corrections
PII: S0042-6989(96)00145-9
Vision Res., Vol. 36, No. 24, pp. 3987-3995, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0042-6989/96 $15.00 + 0.00
The Effects of GABA and Glycine on Horizontal Cells of the Rabbit Retina ROMAN BLANCO,*t CECILIA F. VAQUERO,* PEDRO DE LA VILLA*
Intraceilular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3/aM GABA but none to glycine, even at 100 #M. Dose-response relationship for GABA gave ECso values around 10/aM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted C l - equilibrium potential. Partial replacement of intracellular C i - with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30/aM), a GABAA agonist mimicked the effect of GABA, but baclofen (30/aM), a GABAn agonist and cis-aminocaprionic acid (30/aM), a GABAc agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 I~M) and picrotoxin (100/aM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes. Copyright © 1996 Elsevier Science Ltd
GABA
Glycine
Horizontalcells
Intracellular recording
INTRODUCTION It is now well documented that lateral inhibition in the outer retina is mediated by horizontal cells (HCs). HCs respond to light with graded hyperpolarizations which are transmitted through inhibitory feedback chemical synapses to cones and bipolar cells (Baylor et al., 1971). HCs regulate the cone and bipolar cell light responses by modulating their response kinetics and mediating the surround responses which constitute their centre-surround receptive field (Yang & Wu, 1989, 1993; Wu, 1994). In the mammalian retina it has been shown that HCs are responsible for the centre-surround organization of the ganglion cell receptive field (Mangel, 1991). In the outer plexifoml layer (OPL) of the retina, the role of GABA and glycine is not yet fully understood. Recent results suggest that the cellular distribution, functional characteristics and possible physiological role of GABA receptors in tlhe retina might be more diverse than previouslY thought (Djamgoz, 1995). In the retina of lower vertebrates, G A B A acts at a least three pharmacologically distinct sites, termed GABAA, GABAB, and GABAc receptors. The GABAA receptor, which is a ionotropic receptor, gate,s a C1- channel, is antagonized by bicuculline and is modulated by benzodiazepines *Department of Physiology and Pharmacology, University of Alcal;i de Henares, Madrid, Spain. tTo whom all correspondence should be addressed [Email [email protected]].
Patch-clamp Rabbitretina
(Ishida, 1992). The GABAa receptor seems to control calcium and potassium conductances via G-proteins [see Slaughter (1995) for review]. The GABAc receptor also gates C1- channels and can be blocked by picr0toxin but is insensitive to bicuculline and baclofen; it is activated by the agonist CACA (cis-4-aminocrotonic acid) (Feigenspan et al., 1993). Several different subunits (~Z1-6, ill-4, 71-3 and 6) have been associated with GABAA receptors, whereas GABAc is associated to expression of "p" subunit. To date, GABAA and GABAc receptors have only been described on HC of cold-blooded vertebrates retinas (Qian & Dowling, 1994; Dong et al., 1994). GABA has been shown to modulate the HC light responses. The adaptation-induced change in the HC response rise time (HCRRT) is mediated by GABA in the amphibian retina (Yang & Wu, 1989, 1993). It is known that GABA is released continuously in darkness from HC, and it slows the HCRRT through GABAA receptors (Kamermans & Werblin, 1992). It has been also shown that GABA induces HC depolarization in the amphibian retina (Stockton & Slaughter, 1991). In the mammalian retina, it has also been noted that GABA slows the HCRRT (Frumkes & Nelson, 1995). However, it is not known whether in the mammalian retina GABAA receptors are located in HC, or/and in other retinal neurons which influence the HC light responses through chemical synapses. On the other hand, glycine has also been shown to play
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an important role as an inhibitory neurotransmitter in the retina [reviewed by Marc (1989)]. In this study we examined the action of glycine on HC with a view to understanding its role in the external mammalian retina. Dissociated HC have been the object of numerous electrophysiological studies on cold-blooded vertebrates retinas (Ishida et el., 1984; Lasater, 1986; Perlman et el., 1989; O'Dell & Christensen, 1989; Zhou et el., 1993) but very few have been published in HC of mammalian retinas (Ueda et el., 1992; L6hrke & Hofmann, 1994; Blanco et el., 1996). Since diversity in neural organization is generally greater for lower vertebrates, mammalian retina is considered to be a more suitable preparation for understanding the function of the human retina. Our electrophysiological study examines the GABAergic and glycinergic action in distal mammalian retina and their receptors on HC. We have examined the properties of GABAA-receptor channels in dissociated axonless horizontal cells (ALHC) and investigated whether these cells also express functional glycine receptors. Our results demonstrate that ALHC possess GABAA but not glycine receptors. Portions of the present study were presented previously in abstract form (Blanco et el., 1995).
METHODS
Intracellular recordings
Two-month-old rabbits were anesthetized with urethane (1.5 g/kg i.p.) (Dacheux & Miller, 1981; Miller et el., 1981). All the experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) on experimental animals. The eye was enucleated, hemisected and everted over a Teflon plug, which fits into an oxygenation chamber. Superfusate flowed by gravity (23 ml/min) onto the surface of the retina. This extracellular solution was made according to the formula of Ames and Nesbett (1981): NaCI, 120 mM; KC1, 5 mM; NaHCO3, 25 mM; CaC12, 2 mM; MgSO4, 1 mM; Nail2_ PO4, 0.1 mM; Na2HPO4, 0.8 mM; glucose, 10 mM. All solutions were bubbled with 95% O2-5%COe yielding a pH of 7.4 at 37°C. Drug solutions were added to the superfusate and adjusted to this pH with NaOH or HCI as required. As judged by physiological criteria, the rabbit retina was well maintained under these conditions for 24 hr and intracellular recordings could be held through solution changes. Drugs were introduced at controlled concentrations and in combination as required. The dead space in the perfusion system was minimal and drug effects occurred within 20-40 sec. Intracellular recordings were obtained with microelectrodes pulled from 1.2-mm Omega-dot glass on a horizontal puller (Brown-Flaming). Electrodes were filled with 3 M potassium acetate and the resistance was typically between 100 and 400 Mfl. Cell impalement was facilitated by adjusting the negative capacitance in the electrode headstage. Voltage traces were monitored with an oscilloscope. ALHC were identified from their
physiological responses and/or by iontophoretic application of carboxifluorescein 5% [see Villa et el. (1991)]. Results were recorded on a pen recorder and computer hard-disk for later analysis. The retinal preparation was stimulated by a dual-beam photostimulator. A centred light beam whose intensity and wavelength could be adjusted by neutral-density filters and interference filters, was provided by a quartz halogen source (Villa et el., 1994). In most experiments described, light spots of 600800/2m in diameter were used.
Patch-clamp
Recordings were made from dissociated HC of the retina from 2-month-old rabbits. Dissociated cells were obtained as previously described (Ueda et al., 1992; Villa et el., 1995). Briefly, rabbits were anesthesized with uretane and their eyes rapidly removed. The retina was stripped from the pigment epithelium, divided into pieces and incubated in a standard solution containing (mM): NaC1, 135; KC1, 5; CaC1, 2; MgC12 1; HEPES 5; glucose 10; pH 7.4, adjusted with NaOH, which contained papain (25 U/ml) and 2.0 mM cysteine. The retina was incubated in this solution for 20-30 min and then placed in a standard solution. The retina was dissociated into individual neurons by repeatedly passing the tissue through a Pasteur pipette. Using this procedure, a single class of HC, the ALHC, could be prepared and identified by their morphological characteristics. The cells were placed in a standard solution filled chamber mounted on the stage of an inverted phase-contrast microscope (Nikon TMD, Tokyo, Japan). During recordings, the cells were continuously perfused with a standard solution. Drugs were applied to the cell via the perfusion medium. The drugs used both in patch-clamp and intracellular recordings experiments, were all acquired from Sigma Chemical. Whole-cell recordings were made at room temperature (22-25°C) using a patch-clamp amplifier (RK-300, Biologic instruments). Patch pipettes were pulled from thickwalled glass (Clark Electromedical GC150F) to a resistance of 8-15 MQ when filled with a pipette solution containing (mM): NaCl, 10; KC1, 110; CaCl, 0.5; MgC12 1; HEPES 10; EGTA 5. In some experiments, the intracellular C1- was replaced by equimolar isothetionate. The capacitive component was electrically compensated, although incompletely, perhaps due to cell morphology. Electrode series resistance ranged from 10 to 20 M~. The voltage error produced by these series resistances (series resistance x recorded current) was not compensated as it was <5% of the holding potential. The junction potential at the electrode tip was neglected because it was small ( < - 3 mV) under the present conditions. Whole-cell responses to perfusion-applied agonist were analysed from records digitized at 0.57 kHz (Intel 80486-based personal computer; VCAN 3.1.) (Dempster, 1994).
GABA RECEPTORS ON HORIZONTAL CELLS
3989
A
'
GABA
100 uM
mv
I
25 see
B GABA 100 pM
'mVI
-I
1 sec
FIGURE 1. (A) Intracellular data showing the effect of 100 #M GABA on the HC voltage responses to 0.5 sec light steps (800 pm dia and intensity of 2.71 x 1014 photons cm - 2 sec- 1 at 500 nm) as indicated by the square-wave pulses at the bottom of the voltage trace. Tile dark bar below the recording indicates the time application of GABA. Note that the effect of GABA is both to depolarize the cell and decrease the HC light response. (B) Horizontal cell recordings from the same experiment are shown at a expanded time scale [arrows in (A)]. We see how GABA decreases the HC light response in addition to slowing down the horizontal cell response rise time (HCRRT).
RESULTS
Intracellular recordings Intracellular recordings were obtained from a total of 30 HCs in the perflmed rabbit retina. The resting membrane potential varied from cell to cell, from - 2 0 to - 4 0 mV. All recorded cells corresponded to ALHC, according to the shape of their light response (Bloomfield & Miller, 1982). In some cases (n = 5) we confirmed the origin of light responses by iontophoretic injection of carboxifluoresecein 5%, after recording their physiological responses (Villa et al., 1991); and in all these cases we found them to be ALHC. Figure 1 shows the effects of GABA on HC voltage responses to 0.5 sec light steps. In the presence of 100 #M GABA, the HC w a s depolarized and the light responses amplitude was reduced. GABA seemed to
reduce both the cone and rod responses and slowed down the HCRRT. Figure 2 shows the effects of various concentrations of glycine on HC light responses. In the presence of both 60 and 100 ~M glycine, the HC light responses amplitudes were diminished or abolished, respectively. Glycine seemed to reduce the cone input the strongest and did not slow down the HCRRT. These experiments did not definitively identify the distal GABAergic (and glycinergic) target. Its location on either photoreceptor cells, HCs, both cell types or other cells (e.g. interplexiform cells) remained in doubt. We, therefore decided to perform additional experiments on isolated HCs using whole cell patch-clamp.
Patch-clamp Patch-clamp recordings were obtained from a total of
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al.
A 2 mln/~/ 10mVI
a5 sec-
GLYC 60 uM
~~~1~~2
m l n ~ 6
GLYC 100 IJM
rain~T~7.,~.~
10mVi
25sec
B
~ ~V' Control/Relovery . , j R
5°v1
1 sec
FIGURE 2. (A) Effects of various concentrations of glycine on the HC voltage responses to the same light stimuli (see legend of Fig. 1). Glycine, at 60/tM, diminished the HC light responses amplitude and, at 100/~M, completely abolished the cell light responses. (B) Horizontal cell recordings from the same experiment are shown at an expanded time scale [arrows in (A), upper trace]. Note that glycine does not slow down the time of the HC light-induced response.
45 isolated HCs. Dissociated A L H C were identified on the basis of their characteristic morphology (Fig. 3). These cells responded to the application of G A B A with inward currents when clamped at negative potentials as expected for the activation of C I - channels under conditions where ERev for chloride is around 0 m V [Fig. 4(A)]. The responses to G A B A developed rapidly and desensitized to a steady-state level. The extent of desensitization was variable from cell to cell. In all cases, the responses declined rapidly when agonist was removed. The repeated application of G A B A did not lead to a progressive decrease in the peak amplitude of
the current nor changes in the time-dependent desensitization ( G A B A responses tested for >1 hr; n = 15). To characterize further the G A B A receptors in these cells we examined their concentration-response relationships [Fig. 4(A)]. The lowest concentration producing a detectable current was 3 ~M for GABA. Maximum response was obtained for G A B A concentrations up to 100/~M. For each cell, the peak currents amplitudes evoked by various concentrations of each agonists were normalized to those evoked by 100 pM G A B A and pooled data were plotted against agonist concentration. With increasing concentrations of G A B A the whole-cell
GABA RECEPTORS ON HORIZONTAL CELLS
3991
FIGURE 3. Microphotograph of an ALHCs dissociated from the rabbit retina. Calibration bar 40/~m.
A
GABA 3 pM
,oA]
GABA 10 pM
GABA 30 pM
GABA 100 pM
-j
"----,1,'-"
30 see
B
C 1
A
0.8
X
m
E
m
0.6 0.4
,m,
O.1
0.2
-2
0 0.1
1
10
[GABA] (/aM)
100
10
100
[GABA] (MM)
FIGURE 4. Whole cell patch-clamp recordings of GABA induced-currents in ALHCs. GABA was applied by superfusion. (A) Currents induced by GABA (3, 10, 30 and 100 #M) were recorded from an ALHC maintained at a holding voltage of - 70 mV. Bars above each current trace indicate the timing of drug application and numbers above the bar correspond to the GABA concentration (/tM). Note that increasing concentrations of GABA produce a faster desensitization of the inward current. (B) Dose-response curve of the GABA induced current averaged from five cells. Responses have been normalized with respect to the maximun response for GABA (100/~M). Line drawn by eye. (C) Logarithmic plot of data from (B). Analysis of the data from five cells yielded a Hill coefficient of 1.5 and an ECs0 around 5 ~M for GABA.
3992
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A Vh(mY)
GABA ( 3 0 u M )
-60 -40
C
-20
i
0
[ C l I j = ~
20
,~ ~ ,
'/a r
40 -100 - 8 0 ] ~
B
4oo pA
, 400
I
I
I
I
20 40 60 8o mV -200 -400
pA
< | 20
4~' [CI]l=121 mM -600 | 40
! 80 m V
-200
- -400 - -600
FIGURE5. (A) Reversalpotentialof GABAresponsesin standardbath and pipette solutions. Responsesof a single HCs to brief application of 30 pM GABA at each of the holding potentials indicated (Vh). Traces have been arbitrarilyseparated vertically for clarity. (B) Peak amplitude of the responses shown in (A) are plotted against holding potential. (C) Current-voltage relationships of the GABA induced currents recorded from two different cells, as measured by ramp depolarization.Reversal potential of 30pM GABA response is shown for one cell perfused with control chloride intracellular solution ( [ C l - ] i = 121 mM) and for another cell in low-chloride pipette solution ( [ C 1 - ] i = 20 raM). When pipette contained control solution (Ea = - 3 mV), the GABAresponsereversesnear - 5 mV; in the low-chloridepipette,the GABAresponsereversesat -48 mV (Ecl= -50 mY). currents increased in amplitude in a sigmoidal fashion until saturation was apparent at 100 #M [Fig. 4(B)]. Analysis of the data from five cells yielded a Hill coefficient of 1.3 and an ECso around 10 pM for GABA [Fig. 4(C)]. The Hill coefficient suggested that the binding of more than one GABA molecule is required for receptor activation. Desensitization was apparent at GABA concentrations >3 #M. Because of the modest speed of agonist application in the present study, desensitization will take place during the rising phase of current responses. In a series of experiments, GABA induced current was recorded in cells clamped at different holding potentials. When the bath and electrode solutions contained equal chloride concentrations, the responses to GABA reversed in polarity near 0 mV [Fig. 5(A and B)], close to the equilibrium potential C I - (Ecl). In order to test the effect of C1- on GABA induced currents, we recorded from HC with pipette solution containing different chloride ion concentrations. For these experiments, intracellular chloride was replaced by equimolar isothetionate. Voltage ramps (from - 90 to +60 mV, 80 mV/sec) were commanded before and during the GABA application (10-30/~M GABA). Current-voltage relationships were analysed after subtracting the current obtained in the absence of the agonist from that obtained during the response to GABA. In each condition, three or four ramps
were applied and the resulting currents averaged. For low intracellular chloride concentration, the corresponding reversal potential shifted to more negative values. An example of this is illustrated in Fig. 5(C) which shows that in low intracellular chloride concentration, the corresponding reversal potentials shifted by very nearly the same amount ( - 4 8 mV) as did the Ect ( - 5 0 mV). These results suggest that GABA increases a chlorideselective conductance in HCs. To confirm from which GABA subtype receptors GABA responses arose, we examined the effects of the GABA agonists muscimol, baclofen and CACA and the GABA antagonists bicuculline and picrotoxin (Fig. 6). All results presented here were obtained in at least three different ALHC. Muscimol (30 #M), a GABAA agonist always mimicked the effect of GABA (n = 5), but baclofen (30#M), a GABAB agonist and CACA (30 #M), a GABAc agonist did not elicit any effect on ALHC. Bicuculline methyiodide (10/~M), a GABAA selective antagonist and picrotoxin (100 pM), an ionotropic GABA receptor antagonist, reversibly antagonized completely the GABA-evoked currents. Glycine (100/~M - 1 mM), did not evoke any effect on the HCs tested (n = 5), as we show in Fig. 7. In this figure we compare the GABA-evoked current before after applying glycine on a HC.
GABA RECEPTORS ON HORIZONTAL CELLS GABA
GABA
MUSCIMOL
GABA
GABA
GLYCINE
GABA i
ii
Wt:
250 A
GABA
BACLOFEN
3993
10 •
FIGURE 7. Glycine (100 pM) did not elicit any current on ALHC compared to the inward currents elicited by GABA (30 #M) applied before and after glycine. Cell clamped at Vh = - 60 mV. GABA
CACA
mmmmmmm
GABA
m
los
GABA
GA,,~,~.BIC
GAB~
GABA
GABA
p•_S00 10s
FIGURE 6. Pharmacology of GABA receptors in ALHCs. Currents induced by different agonists/antagonists are shown in horizontal series. In each series, control (left column), test (middle) and recovery (right) are shown. GABA concentration was 30 #M for the three upper rows; muscimol, 30 pM; baclofen, 30 pM; CACA, 100/.tM. GABA concentration was 3 pM for the lower two rows; Bicuculline methyiodide (BIC) (10/.tM) and picrotoxin (PTX) (100 pM), were applied together with GABA (13 pM). In all experiments, cell were clamped at Vh = - 60 mV.
DISCUSSION Our study extends observations made previously in lower vertebrates to the mammalian retina. We have shown that GABA activates a chloride conductance via GABAA receptors in isolated ALHCs of the rabbit retina. At the same time we did not find any evidence for GABAc receptors, GABA transporter currents or glycine-activated chloride conductances in these cells. These results suggest that the effects of GABA on HC are mediated by GABAA receptors and that the depolarization and the changes :in the light response rise time observed in these cells could be explained by the mediation of the GABAA receptors in the HC membrane. While GABA certainly slows the light response rise time, the mechanism of this action is not clear. The GABAergic positive feedback loop model has previously been suggested by Kamermans (Kamermans & Werblin, 1992) but direct evidence suggesting the evidence of the
loop in the mammalian retina is not available. In this model, the GABA transporter plays a prominent role while we have found no evidence for a GABA transporter in ALHC of the rabbit retina. We might note that in the catfish retina the presence or absence of GABA transporter currents have been observed in cone- and rod-driven HCs, respectively (Dong et al., 1994). Several pieces of evidence indicate that GABA is the neurotransmitter released by HCs in the mammalian retina (Vardi et al., I994). On the other hand, data published on the GABA receptor localization in the mammalian retina are somehow confusing. Detection of GABA receptors in the mammalian outer retina by immunocytochemistry has proved to be difficult (Brecha & Weigmann, 1990). At present, several studies have reported GABA-like immunoreactivity in different cell types of the outer retina of mammals. The localization of GABAA receptors has been demonstrated on the synaptic terminal of cones (Hughes et al., 1989; Brecha & Weigmann, 1990), on HC dendrites (Greferath et al., 1994b, 1995) and upon the dendrites of bipolar cells within the triad invaginations of cones (Vardi et al., 1992; Griinert & W~issle, 1990, 1994). Our intracellular recordings indicate that 100/~M GABA is required in the bath to induce changes in HC light responses. This concentration is high compared with that needed to elicit responses in dissociated mammalian HCs. The need for a higher bath concentration of GABA in the whole retina preparation is probably due to the existence of high affinity uptake systems in the retina (e.g. Miiller cells) suggesting that the concentration achieved by exogenous application of GABA in the synaptic cleft is probably lowered. However, the lack of evidence for GABA uptake in HC of the mammalian retina is significant, particularly when compared to the positive evidence in cone HCs of nonmammal vertebrates (Dong et al., 1994). Our findings that the GABA current is C1- mediated and entirely suppressed by bicuculline argue that GABA transporters may not be present on mammalian ALHCs. It remains possible, however, that the isolation procedure could in some way have removed them because of their location at the tip of the synaptic invaginations. In the retina, glycine has been shown to play an important role as an inhibitory neurotransmitter (Karschin & W/issle, 1990). In the mammalian retina
R. BLANCO et al.
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glycinergic receptors have been localized in bipolar, amacrine and ganglion cells but not in HCs (Marc, 1989; Yazulla & Studholme, 1991; Greferath et al., 1994a, 1995). It is possible that glycine receptors might be present which are not recognized by the antibodies used until now in immunochemical studies (J~iger & W~issle, 1987). In our intracellular recordings, a striking feature of the action of glycine in the intact retina was that it diminished the amplitudes of HC responses to light. However, when the effect of glycine was tested on voltage-clamped isolated cells, it failed to induce any current activation. The absence of glycine receptors in dissociated HC reflects, in our oppinion, the actual glycine receptor distribution in situ. It is not likely that the absence of glycine effect has been caused by loss of glycine receptors on HC during the dissociation process (Gilbertson et al., 1991), since GABA responses are always present on the same recorded HC. Although we do not have a clear explanation for the in situ effect of glycine application, we suggest that the changes in HC light responses may be mediated by interplexiform cells sensitive to glycine. The physiological role of this glycinergic pathway in the mammalian retina is presently not fully understood. We could argue, therefore, that glycinergic interplexiform cells may mediate feedback interactions from the inner plexiform layer to the OPL, similar to the well known effects of dopaminergic interplexiform cells in the retina of coldblooded vertebrates.
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Acknowledgements---This work was supported by Grants from the Spanish Government: Fondo de Investigaciones Sanitarias (FIS) Grants 93/5358, 95/5001 to R. Blanco and Direcci6n General de Investigaci6n Cientffica y T6cnica (DGICYT) Projects PB 92/0048, PB 94/0369 to P. Villa. We would like to thank Dr J. E. G. Downing for comments on the manuscript and English corrections