OFF-pathway synaptic transmission in the outer retina of the axolotl is mediated by a kainic acid-preferring receptor

Neuroscience Letters, 50 (1984) 7-11

7

Elsevier Scientific Publishers Ireland Ltd. NSL 02871

O F F - P A T H W A Y S Y N A P T I C TRANSMISSION IN THE O U T E R RETINA OF THE A X O L O T L IS M E D I A T E D BY A KAINIC ACID-PREFERRING RECEPTOR

DAVID DVORAK

Department o f Behavioural Biology, Research School o f Biological Sciences, Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601 (Australia) (Received May 8th, 1984; Revised version received and accepted May 24th, 1984)

Key words: retina - transmitter - photoreceptors - bipolar cells - horizontal cells - axolotl

lntracellular recordings were made from OFF-centre bipolar cells and horizontal cells in the superfused axolotl retina eyecup preparation. Bath-applied (+_+_)cis-2,3-piperidine dicarboxylic acid (PDA), 3'-0glutamylglycine (DGG), c-glutamic acid diethyl ester (GDEE), (_+)2-amino-5-phosphonovaleric acid (2-APV) and magnesium ions were assessed as antagonists of the actions of the photoreceptor transmitter. The rank order of antagonist efficacy was PDA > DGG > > 2-APV = GDEE = Mg 2 + . The results indicate that transmission at OFF-pathway synapses in the outer retina of the axolotl is mediated by a kainic acid-preferring receptor.

Vertebrate photoreceptors hyperpolarize in response to light and as a consequence, transmitter release from their synaptic terminals is maximal in darkness [2, 14, 15]. The transmitter depolarizes OFF-centre bipolar cells and horizontal cells, apparently by opening Na ÷ channels [ 15, 16]; however, it hyperpolarizes ON-centre bipolar cells by closing Na ÷ channels or by opening C1- or K ÷ channels [11]. A single photoreceptor terminal can contact dendrites from all three postsynaptic cells, leading to the possibility that one transmitter may be active at second order retinal synapses. The acidic amino acids, z-glutamate and L-aspartate, mimic both the hyperpolarizing and depolarizing actions of the transmitter, but these substances are active in millimolar concentrations which has raised some doubts about their candidacy as photoreceptor transmitters (see for review ref. 6). Recent studies have shown, however, that kainic acid and quisqualic acid, non-endogenous glutamate analogues, are far more potent agonists active in micromolar concentrations [5, 12]. At vertebrate CNS synapses which may involve the excitant amino acids as transmitters three classes of postsynaptic receptor have been defined [9, 17]: one is preferentially activated by kainic acid with cis-2,3-piperidine dicarboxylic acid (PDA) and 3,-D-glutamylglycine (DGG) as antagonists; the second is activated by quisqualic acid and antagonized by glutamic acid diethyl ester (GDEE), and the 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

third is a c t i v a t e d by N - m e t h y l - D - a s p a r t i c acid with 2 - a m i n o - 5 - p h o s p h o n o v a l e r i c acid ( 2 - A P V ) and m a g n e s i u m ions as selective a n t a g o n i s t s . The aim o f the present study was to c h a r a c t e r i z e the type o f r e c e p t o r which m e d i a t e s s y n a p t i c t r a n s m i s s i o n between p h o t o r e c e p t o r s a n d O F F - c e n t r e b i p o l a r cells a n d h o r i z o n t a l cells in the axolotl retina. The effects o f the a b o v e - m e n t i o n e d e x c i t a t o r y a m i n o acid a n t a g o n i s t s on the responses o f these second o r d e r n e u r o n e s were studied a n d it was f o u n d that the t r a n s m i t t e r a p p a r e n t l y acts t h r o u g h a kainic a c i d - p r e f e r r i n g receptor. The d a t a s u p p o r t the conclusions o f a recent s t u d y on the p h a r m a c o l o g y o f O F F - p a t h w a y synaptic t r a n s m i s s i o n in the o u t e r retina o f the chicken [;7]. l n t r a c e l l u l a r r e c o r d i n g s were m a d e f r o m neurones in the s u p e r f u s e d eyecup o f the a x o l o t l , Ambystoma rnexicanum, using techniques similar to those d e s c r i b e d for the m u d p u p p y [3]. The c o m p o s i t i o n o f the Ringer s o l u t i o n was (raM): NaCI, 111 ; KC1, 3.0; CaC12. 1.8; glucose, 11; H E P E S buffer, 5.0; a d j u s t e d to p H 7.8. The stimulus was a 100 ~m d i a m e t e r spot o f light p r o j e c t e d in s h a r p focus on the retinal surface a n d centred in the cell's receptive field. S t i m u l u s wavelength was X - 580 nm, maxi m u m intensity was 2.2 x 1014 p h o t o n s cm 2. s ~, and was a t t e n u a t e d by means o f n e u t r a l density filters. A 30 rain d a r k a d a p t a t i o n p e r i o d p r e c e d e d each r e c o r d i n g , a n d after a stable p e n e t r a t i o n , 3 s light flashes were presented at 25 s intervals. The a n t a g o n i s t s (Tocris Chemicals) were b a t h - a p p l i e d in the f o l l o w i n g c o n c e n t r a t i o n s : ( _ + ) P D A , 2-5 m M ; D G G , 2-5 raM; G D E E , 5 raM; (+_)2-APV, 250 #M-1 m M ; a n d Mg 2 +, 10-50 #M.

CON TROL

PDA, 2 min

PDA, 5 rnin

-20 mV -30 t i

-40i l - 3s

__J--I

__l

I

Fig. 1. Effect of 3 mM PDA on an OFF-centre bipolar celt. The stimulus was a 3 s light flash as indicated by the marker below each record. PDA was bath-applied for 5 rain and caused a marked hyperpolarization and a large decrease in the amplitude of the light response. The reduction in membrane voltage noise during the control light response reflects the decreased level of photoreceptor transmitter present in light. Note that a similar reduction in noise occurred during the hyperpolarizing action of PDA, indicating that the antagonist blocked the postsynaptic effects of the transmitter (see text).

Prior to experiments on second-order cells, the effects of PDA, D G G and 2-AP5 on photoreceptors were examined. Recordings were made from both rods and cones, and in no case was there a significant change in resting potential or light response. It is assumed, therefore, that the effects of the drugs described below were due to interactions with receptors on the postsynaptic membrane. Since the photoreceptor transmitter is released maximally in darkness and has a depolarizing effect on OFF-centre bipolar cells, the expected action of an antagonist would be an increase in the resting membrane potential, i.e. a further hyperpolarization of the membrane in darkness. A more negative resting potential would in turn result in a decrease in the relative amplitude of the hyperpolarizing light response. The effect of 3 mM P D A on the resting membrane potential and light response of an OFF-centre bipolar cell is shown in Fig. 1. In normal solution the resting potential was - 25 mV and increased to about - 40 mV during the light flash. During the flash the amplitude of m e m b r a n e voltage noise decreased; this reduction in noise reflects the decrease in photoreceptor transmitter release and is a characteristic feature of the OFF-centre bipolar cell response [1, 10]. In the presence of PDA, the resting potential increased from - 2 5 mV to - 3 8 mV, and the light response was nearly eliminated. These changes were accompanied by a marked decrease in membrane voltage noise, indicating that the antagonist blocked the postsynaptic actions of the photoreceptor transmitter. In this example, responses of the cell recovered to near control levels 20 min after return to normal solution but intracellular recording conditions were then lost. The long time required for antagonist action and rinse is typical for bath-applied drugs in the eyecup preparation (see refs. 5, 8 and 13) and is presumably due largely to the presence of diffusion barriers within the retina. It proved difficult to record from these cells for longer than 5-10 min, and hence more than one drug could not be tested. Comparison of antagonist effects between cells, however, showed that P D A was always the most potent, followed by DGG. The other agents had little if any effect. It was possible to make long stable recordings from horizontal cells and to compare effects of different drugs on a single cell, as shown in Fig. 2. In these experiments each antagonist was applied for 5 min, and there was a 20-35 min recovery period in normal solution between tests. In the recordings shown in Fig. 2a, the cell's membrane potential in darkness was - 18 mV and increased to - 4 8 mV during illumination. In the presence of DGG (3 mM) the resting potential increased to - 3 0 mV and the light response was reduced by 50%o. P D A (3 raM), however, brought about a much larger increase in resting potential (to - 43 mV) and reduced the light response by about 80%. In Fig. 2b the actions of 2-APV (1 raM) and P D A (3 mM) are compared on another horizontal cell: 2-APV had slight effects but P D A again showed pronounced antagonism. In similar tests, G D E E and Mg 2÷ had minimal effects like 2-APV. Thus, the rank order of antagonist efficacy in blocking the action of the photoreceptor transmitter on both OFF-centre bipolar cells and horizontal cells was

IO

PDA > DGG > > 2-APV = GDEE = Mg* + .

This

finding

is

similar

to

that

demonstrated for kainic acid-preferring receptors in spinal cord and hippocampus [4, 9, 171, which strongly suggests that the physiologically active receptor at OFFpathway synapses in the outer retina of the axolotl may be of the kainic acidpreferring type. A recent study in mudpuppy retina reported similar antagonist actions of PDA on OFF-centre bipolar cells and horizontal cells, but left unanswered the question of the nature of the postsynaptic receptor [ 131: the present results may help clarify this point. In chicken retina, the same rank order of antagonist efficacy as described here has been demonstrated for both the blocking of kainic acidinduced excitotoxicity in second order neurones and the selective elimination of OFF-responses in retinal ganglion cells [7]. It may be, then, that the results described in the present study will prove to be a more general feature of the pharmacology of the outer retina. The presence of a physiologically active, kainic acid-preferring

PDA

DGG

CONTROL

b) I--L._

___J

CONTROL

Fig. 2. Effects of excitatory

1

2 -APV

amino acid antagonists

r

L..-__

PDA

on the resting potential

and light response

of horizon-

tal cells. Each agent was applied for 5 min. and there was a 20-35 min rinse in normal solution between tests to allow for complete recovery of control responses. The actions of 3 mM PDA and DGG in a single cell are compared in a, and the actions of I mM PDA and 2-APV are shown in b.

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