Identification of kainic and quisqualic acid receptors on inner retinal cells of the salamander Ambystoma mexicanum

European Journal of Pharmacology, 184 (1990) 143-150

143

Elsevaer EJP 51422

Identification of kainic and quisqualic acid receptors on inner retinal cells of the salamander A mbystoma mexicanum J a n B F V a n der Valk

1, I a n

G. M o r g a n a n d D a v i d R. D v o r a k

Visual Sctences Group, Centre for Vzsual Sctences and Research School of Btologtcal Scwnces, Austrahan Natmnal Unwerstty, G P 0 Box 475, Canberra Ctty, A C T 2601, Austraha

Received 8 February 1990, rexqsed MS received 25 April 1990, accepted 8 May 1990

The presence of kamlc (K.A) and qulsquallc acid (QA) receptors on inner retinal neurones of the axolotl Ambystoma mexwanum has been studied using intracellular recording techmques In the presence of CoC12, wluch

blocks neurotransnutter release, KA and QA depolarized the membrane The minimum concentration of KA that induced a response was 1 #M and a maximum response was obtained with 10/~M (EC50 3 #M) The operating range of QA was between 0 5 and 5/~M with an ECs0 of 1 #M These data show that tuner retinal cells of the axolotl axe sensitive to KA and QA Cls-2,3-pIpendme dicarboxyhc acid (PDA, 3 mM) completely blocked responses to 5 gtM KA, but not those reduced by 2/~M QA Tins suggests that the KA- and QA-sensltive receptors on inner retinal cells of the salamander are pharmacologically different and that PDA can be a valuable tool in chstingmshmg KA- and QA-sensllave receptors on these neurones Kalmc acid, Qmsquahc acid, ClS-2,3-Plpendme dlcarboxyhc acid (PDA), Retma, (Salamander), (Electrophyslology)

1. Introduction The excitatory ammo acids aspartate and glutamate have been shown to act at at least three distinct types of receptors, known as kalmc acid (KA)-, qulsquallc acid (QA)- and N-methyl-Daspartate (NMDA)-preferrmg receptors These receptors are distinguished by the relatwe potenoes of these non-endogenous glutamate and aspartate analogues and by the actmn of antagomsts In a d d m o n there are 2-ammo-4-phosphonobutync acid (2AP4) receptors, wluch in the outer retina are postsynaptlc and mediate hyperpolarlzlng re-

i Present address Research Institute of Toxacology, Sectaon Neurotoxacology, P O Box 80 176, NL-3508 TD Utrecht, The Netherlands Correspondence to J Van der Valk, Research Institute of Toracology, Secuon Neurotoxacology, P O Box 80 176, NL3508 TD Utrecht, The Netherlands

sponses to 2AP4 and K A Non-selective antagonists hke os-2,3-plpendme dlcarboxyhc acid (PDA) and kynuremc acid generally block all glutamate receptors So far, selectwe and potent antagonists have been found only for the N M D A - p r e f e m n g receptor The n o n - N M D A receptors are potently and competitively blocked by 6,7-dlmtroqulnoxahne-2,3-dlone (DNQX) and 6cyano-7-nltroqumoxahne-2,3-dxone (CNQX) (Honor6 et al, 1988) Evidence has been presented that m the inner retina the three types of excitatory amino acid receptors are present (Slaughter and Miller, 1983a,b, Lukaslewlcz and McReynolds, 1985, Bloomfield and Dowhng, 1985b, Coleman et al, 1986, Atzenham et al, 1988) In salamander retina the N M D A - p r e f e m n g receptor ~s involved in the generation of sustained phases of light responses (Lukaslewlcz and McReynolds, 1985) The role of KA- and QA-preferrlng receptors in generating

0014-2999/90/$03 50 © 1990 Elsevier Science Pubhshers B V (BiomedicalDivision)

144

the hght response is more ddfIcult to study since both receptors are also present on presynaptlc elements m the outer retina (Shiells et al, 1981, Slaughter and Mdler, 1981, Slaughter and Miller, 1983c, Rowe and Ruddock, 1982, Massey and Mdler, 1987) We have investigated the sensltxvaty of the inner retinal cells of the axolotl to the glutamate agonlsts KA and QA as well as to the antagomst PDA using intracellular recordings in order to characterlze glutamate receptors on these cells

2. Materials and methods

Intracellular recordings have been made from inner retinal neurones m the eyecup preparation of the salamander, Ambystoma mextcanum The preparation and recordmg techniques have been described m detail before (Belgum et al, 1982) The eyecup preparatmn was continuously superfused at a rate of 1 m l / m l n with oxygenated Ranger solution of the following composition (mM) NaC1 111, KC1 3 0, CalC12 3 6, glucose 11, HEPES buffer 5, adjusted to p H 7 4 The bathing soluUon m the eyecup could be qmckly changed from standard Ringer to Ranger with known concentratxons of COC12, QA (Cambndge Research Bmchenucals), KA (Sigma) or PDA (Tocris Neuramin) without changing the flow rate and fired level at the site of the preparatmn Electrodes were pulled on a modtfied Livingstone-type Narlshlge horizontal electrode puller Electrodes were filled with 4 M potassmm acetate and had a reststance between 500-800 MI2 The hght stimulus was a 600 /Lm spot of hght positioned m the centre of the cell's receptwe field The unattenuated hght intensity was 3 54 × 10 I5 photons cm -2 s -1 at 525 nm Recordmgs were made from tuner retinal neurones, winch were 1denUded by their ablhty to generate action potentmls or by their transient responses to hght stimulation ON- and OFFcentre neurones were further identified by the sustained depolarization or hyperpolanzatlon, respectively, of the membrane to fllurmnaUon of the centre of the receptive field O N - O F F neurones responded with transient depolarlzatmns at hght

on and hght off Experiments were performed at room temperature (19-21 ° C)

3. Results

Ringer solutions contalmng 10 # M KA or 10 /~M QA had strong depolarizing effects on all inner retinal ceils studied These effects were reversible on return to normal Ranger solution To determine the operating concentrations of KA and QA the responses to increasing concentrations of the drugs were recorded lntracellularly To avoid presynaptlc effects the experiments were performed m the presence of the synapuc transmission blocker CoC12 It is now well established that polyvalent metal ions such as magnesium, cobalt and cadmium block the voltage-gated calcium channel, and thus inhibit transmitter release from presynaptlc terminals by blocking calcium reflux (Godfraand et al, 1986) Apphcatlon of 4 mM CoC12 in the dark always resulted in complete loss of the hght response and m hyperpolarlzatlon of the dark potential, accompanied by a decrease in input conductance All cells responded with a depolarization to non-saturating doses of KA and QA m the presence of COC12

3 1 Dose-response functwn for KA The results of a typical experiment c a m e d out on an ON-responding mner retinal neurone are shown m fig 1 Apphcatlon of 4 m M CoC12 resulted m a hyperpolanzatlon and gradual ehmlnatlon of the hght response (fig 1A) Subsequent apphcatlon of 1 # M KA had no effect on the membrane potential (fig 1B) Voltage dasplacements, winch were the result of injecting short current steps in order to monitor input conductance (G1) chances (see arrow) did not change, lndacatmg that 1 # M KA did not noticeably change G 1 In the presence of 5 # M KA (fig 1C) the membrane depolarized by 28 mV, winch was associated with a small increase m G, In response to 10 ~tM K A the membrane depolarized 26 mV (fig 1D) During tins response a hyperpolarlzang dip of 3 mV occurred which was accompanied by a slgmflcant conductance decrease (see arrow) The

145

A

The concentration

4 mM Co ?.-+

10 m Y [ _ _ 1 mm

B 1 jJM KA

10 mV~..._ 1 mm

of KA ellcltlng a half maxa-

m u m r e s p o n s e , ECs0, w a s e s U m a t e d f r o m t h e H i l l plot Tins was obtained by pooling and averaging depolarizations reduced by given concentrations o f K A f o r all u m t s r e c o r d e d f r o m T h e e s t i m a t e d ECs0 o f K A w a s 3 / ~ M T h e t h e o r e t i c a l c o n c e n t r a tion-effect curve based on the data obtained from t h e H i l l p l o t is s h o w n m fig 2 as a d o t t e d c u r v e For companson, n o r m a h z e d d a t a f r o m t h e SLX neurones for winch sufficient data was obtamed to normalize are plotted as the sohd hne The theor e u c a l c u r v e fits t i n s h m l t e d set o f d a t a q m t e w e l l Wltinn experimental error

3 2 Dose-responsefunctton for QA 5 .UM KA 10 m v L _ 1 mm

10 ,uM KA

I n t h e p r e s e n c e o f 4 m M C o 2+ t h e m e m b r a n e of an ON-OFF n e u r o n e h y p e r p o l a n z e d a n d its h g h t r e s p o n s e d i s a p p e a r e d (fig 3 A ) T h e r e s p o n s e to 0 5 /zM QA consisted of a depolanzatlon of a b o u t 2 m V (fig 3 B ) I n t h e p r e s e n c e o f 5 / ~ M Q A t h e m e m b r a n e d e p o l a r i z e d 14 m V , w i n c h w a s 70%

100 10 mV[__

1 mln Fig 1 Dose-dependent response of an ON-centre cell to apphed KA (A) In the presence of Co 2+ (4 mM) to block synapuc transrmsslon, the cell hyperpolanzed and the hght response disappeared (B) Superfuslon with 1 /tM KA had no effect on the membrane potentml Neither did it change the mput conductance (G,) (C) In the presence of 5 / t M KA the cell depolarized 28 mV assocmted with a small decrease m the input resistance (D) 10 /LM KA depolarized the cell 26 mV followed by a small hyperpolanzmg dip (arrow) dunng winch the resistance markedly decreased All responses to KA were reversible In these and following figures an upward deflectmn m the line above a trace indicates hght ON and OFF The downward deflections in a trace are voltage drops produced by current steps of - 0 04 nA used to momtor input conductance changes (arrow m B) The heavy line below each trace ln&cates the presence of the test substance in the superfusate All drug experiments were performed in the presence of Co2 ÷ (4 mM) winch mbablts neurotranstmtter release responses to increasing concentrauons of KA, slmdar to those described above, were recorded from 18 n e u r o n e s (16 O N - O F F , o n e O F F - c e n t r e a n d one ON-centre)

i iIt

50

III liltIt

......

----'-=05

1

I

I

I

i

5

10

25

100

Fig 2 Dose-response curve of KA The solid curve is based on experiments from wtuch sufficient data could be obtained to normahze (n = 6) The dotted theoretical curve is fitted with a slope factor of 3 9 and an ECs0 of 3 #M, wbJch were estimated from the Hall plot, based on all data In tlus and fig 4 the responses to the agomsts were recorded in the presence of 4 mM Co2+ On the vertical axts is set the percentage ratio of the response to the maxamum response On the horizontal axas ,s set the concentrataon of the test substance The verttcal bars represent the S E

146

A

I"1

I"1

4 mM Co 2+

rl

t"l

L

10 mV[~ los

B

TI "1 I- I-I 1 0 5 ~M QA

1 -| 1 10 m V |

lO

1 ;uM QA

10 mV[__--

10s Fig 3 Dose-dependent response of an ON-OFF cell to apphed QA (A) CoCI 2 (4 mM) hyperpolanzed the cell dunng which the hght response dtsappeared gradually (B) Apphcatlon of 0 5 /~M QA depolanzed the membrane by about 2 mV (C) Superfusmn voth 1/~M QA depolarized the membrane 14 mV wluch was 70% of the maxamum response of tlus neurone to apphed QA

3 3 Effect of PDA on KA- and QA-mduced responses Further characterization of the KA- and QAsensitive receptors took place by studying the effect of 3 m M P D A on the responses evoked by non-saturating concentrations of K A and QA m the presence of 4 m M Co 2 ÷ In the presence of 4 m M Co 2÷ the m e m b r a n e of an O N - O F F cell hyperpolanzed and the light response decreased gradually (fig 5A) Bath apphcatlon of 3 m M P D A had no effect on the membrane potentaal or G,, mchcatlng that 3 m M P D A had no agomst effects A d d m o n of 5 # M K A in the presence of P D A had no effect on the m e m b r a n e potenUal or G, (fig 5B) The blockang effect of P D A was reverszble since 5 /xM K A depolarized the membrane after P D A was washed from the system (fig 5C) Smular results have been obtained m sxx O N - O F F responding neurones The experiments were repeated for QA m five O N - O F F cells In the presence of Co 2 ÷ the membrane hyperpolanzed and the hght response dzsappeared (fig. 6A) Figure 6B shows the effect of

100

,"" _

_......

I

iI

of the m a x t m u m response (fig 3C) Slmdar responses to increasing concentrataons of QA m the presence of 4 m M CoCI 2 were recorded m stx neurones (four O N - O F F and two ON-centre) Ltke the experiments with KA, the response to 10/~M QA consisted of a depolarization, followed by a small hyperpolanzataon wtuch was associated with a szgmficant decrease m G,. The dotted curve m fig 4 represents the theoretical concentration-effect relataon based on these data (n = 6) The estimated ECs0 was 1 /~M The sohd line connects the data points and follows the theoretical curve closely These results indicate that tuner retinal neurones are also sensitive to QA

50

'

0 1

05

1

5

10

25

Fig 4 Dose-response curve of QA The dotted ]me zs based on a slope factor of 3 5 and a half ma~mum concentration of 1 /~M, esumated from the Hdl plot The sobd hne connects the means of the experimental data at each concentrataon On the verucal ayas is set the percentage ratio of the response to the maxnnum response On the horizontal ax~s is set the concentratmn of the test substance

147

A

4. Discussion

4mM Co 2 +

10 mV 1 1 mln

5 pM K A

I n this s t u d y K A a n d Q A were shown to be p o t e n t excltants of i n n e r retinal n e u r o n e s I n add m o n , it was d e m o n s t r a t e d with n o n - s a t u r a t i n g c o n c e n t r a t i o n s of K A a n d QA, that QA- a n d K A - s e n s m v e receptors were present o n m n e r retin a l n e u r o n e s of the s a l a m a n d e r a n d that these receptors are p h a r m a c o l o g i c a l l y disunct, b y the different b l o c k i n g actions of P D A

4 1 Charactertsttcs of the QA- and KA-sensmve receptors on mner retmal neurones

3 mM PDA 10 mV I _

1 mln

C 5 ~JM K A

I n the p r e s e n t study, Q A evoked excitation at lower c o n c e n t r a t i o n s t h a n K A Thas f i n d i n g is consistent with o b s e r v a t i o n s of Lukaslewlcz a n d M c R e y n o l d s (1985) a n d C o l e m a n a n d Miller

A 10 mV /

1 mm Fig 5 PDA blocks the KA-mduced response of an ON-OFF cell (A) Effect of 4 mM Co2+ (B) In the presence of 3 mM PDA, 5 ~tM KA dtd not change the membrane potentaal or conductance (C) When PDA was washed from the eyecup, 5 btM KA evoked a large depolanzang response, demonstratmg the reverslbdlty of the antagomstm action of PDA

4 mM Co 2 +

10 mVl_" 3s

m

2 /JM QA

3 mM PDA

a d d m o n of 2 /~M Q A to the Ringer solution c o n t a i n i n g 3 m M P D A a n d 4 m M Co 2+ T h e m e m b r a n e depolarized 20 m V a n d fast t r a n s i e n t depolarizations occurred s u p e r i m p o s e d o n this response All tested n e u r o n e s showed a depolarmation associated with splke-hke transients i n response to Q A m the presence of P D A T h e m a g m tude of the transients was d~fferent m each neurone (see fig 7B) b u t m n o cell did P D A block Q A - m d u c e d responses T h e effect of P D A o n the Q A - m d u c e d response was reversible as Is s h o w n m figs 6C a n d 7C These results indicate that 3 m M P D A does n o t effectively block the response to n o n - s a t u r a t i n g concentralaons of Q A

10 mV I 20 s

2 /JM Q A 10 rnV

I 20 s

Fig 6 Effect of 3 mM PDA on the response of QA m an ON-OFF cell (A) Effect of 4 mM Co2+ (B) In the presence of 3 mM PDA the response to apphed QA (2 ~tM) consisted of a 20 mV depolarization which was associated wtth small potential fluctuations (C) After PDA was nnsed from the eyecup the same concentration of QA depolarized the membrane 40 mV

148

A

4 mM Co~ +

20 mV L 20 s

2 ~M QA 3 mM PDA 1o

mvl

2 ~JM QA I0 mV[__ 30 s

Fig 7 Effect of 3 mM PDA on the response of QA m an ON-OFF cell (A) Effect of 4 mM Co2+ (B) The response to 2 /~M QA m the presence of 3 mM PDA consisted of a depolanzauon of 5 mV on top of winch large splke-hke transients were found (C) After PDA was nnsed from the eyecup QA evoked a depolanzauon of 20 mV

(1989) m m u d p u p p y retina In rabbit K A appears to be more potent than QA (Bloomfield and Dowhng, 1985b, Massey and Miller, 1988) This could be a species chfference, but difference m experimental design :s a more apparent explanation Bloomfield and Dowlmg (1985b), tested K A and QA without bloclong synaptlc transmission KA, but not QA, had a strong depolanzlng effect on hyperpolanzlng bipolar cells (Bloomfield and Dowhng, 1985a) The recorded response of inner retinal cells to K A apphcatlon could therefore be composed of presynaptlc excitation by the O F F bipolar cells and the dxrect postsynapt:c excitation by KA, making thas drug apparently more potent than QA The experiments performed m this study showed that the concentrations for K A and QA that evoked a half maximum response (ECs0) were 3 and 1 ~ M respectwely These values are 5-10 tlmes lower than those used in a number of other

studies on the role of the excitatory amino acids m synaptlc transrmsslon m the inner retina (Slaughter and Mdler, 1983a,b, Bloomfield and Dowhng, 1985b, Lukaslew:cz and McReynolds, 1985, Coleman et al, 1986) It has been observed that K A and QA can brad to other excitatory amino acid receptors at tugh concentrations (Honor6 and N:elsen, 1982, Foster and Fagg, 1984) It is very hkely that at saturating concentrations binding and physiological effects of K A and QA wtll be less specific and that results obtained with such concentrations wdl be difficult to interpret 4 2 Antagontsttc acttons of PDA on the KA- and QA-mduced responses This study has shown that PDA does not have direct excitatory effects on tuner retinal neurones This as m contrast to the weak partml agomst effect of P D A m rat hlppocampus (Colhngrldge et a l , 1983) and cat spinal cord (Davies and Watkms, 1981) This difference rmght be due to a difference m experimental preparation but can also be explamed by a presynaptlc effect, since the partial agomstlc effects were observed m the absence of C o C I 2 leaving neurotransnuss~on intact P D A tested against non-saturating concentrations of agomsts blocked the KA-evoked depolarization completely but antagomzed only partially the effect of QA These results indicate that the KA- and QA-sensltlVe receptors on inner retinal cells are pharmacologically different Slrmlar concluslons have been drawn from experiments using the antagonist kynurenlc acid (Coleman et al, 1986), although presynaptlc blocking action of kynuremc acid on second order neurones could not be excluded since the experiments w~th the antagonist have not been performed m the presence of a neurotransrmtter release blocker There are several reasons to expect that KA- and QApreferring receptors are pharmacologically distract It has been shown that a KA-sensltlVe receptor can be preferentially blocked by D-y-glutamylglycme (Davies and Watklns, 1981) and that a QA-sensltlVe receptor can be preferenually blocked by glutamlc acid dlethylester (Dawes and Watlons, 1979, McLennan and Lodge, 1979), although these antagonists were not as selective m other studies

149 (Peet et a l , 1983, R o w e a n d R u d d o c k , 1982) R e c e n t l y it has b e e n suggested that the new c o m pound 6,7-dxchloro-3-hydroxy-2-qumoxahnecarb o x y h c acxd is a relative selectxve antagonxst at K A - a n d N M D A - s e n s l U v e receptors ( F r e y et a l , 1988) R a d l o h g a n d b i n d i n g a u t o r a d l o g r a p h y studles have shown that the K A - a n d Q A - s e n s l t w e receptors show d l s t m c u v e o r g a m z a t l o n s m the b r a i n ( M o n a g h a n a n d C o t m a n , 1982, M o n a g h a n et a l , 1984, R a t n b o w et a l , 1984, a n d see C o t m a n et a l , 1987) This s t u d y has n o t b e e n r e p e a t e d with higher or lower c o n c e n t r a t i o n s of P D A a n d conclusions a b o u t the degree of selecUvaty of P D A t o w a r d s the K A - s e n s l t w e r e c e p t o r c a n n o t b e m a d e as tlus c o u l d be concentration-dependent T h e efficacy of a n t a g o m s m of P D A n u g h t n o t b e a b s o l u t e a n d m a y b e greater against the K A - t h a n against the Q A - s e n s l t l v e r e c e p t o r T h e r e is i n d e e d evidence t h a t P D A b l o c k s the responses to Q A in o t h e r p a r t s of the central nervous system (Davies et a l , 1981, Blrley et a l , 1982, Boksa et a l , 1989) F u t u r e studies should establish the m i n i m u m conc e n t r a t l o n of P D A that b l o c k s the K A - s e n s l t w e r e c e p t o r since P D A m i g h t b e a v a l u a b l e tool to distinguish K A - s e n s m v e ( a n d N M D A - p r e f e r r l n g ) receptors f r o m those sensitive to Q A I n a d d i t i o n , the b l o c k i n g abilities of tugher doses of P D A on the Q A - a n d K A - s e n s l t l v e receptors should b e s t u d i e d to d e t e r m i n e w h e t h e r ~ts selectlxqty is absolute I n the p r e s e n t study, ~t was shown t h a t the Q A - e v o k e d responses m the presence of P D A were a c c o m p a m e d b y d e p o l a r i z i n g transients T h e s e transxents were n o t p r e s e n t in the a b s e n c e of P D A , neither were they o b s e r v e d d u n n g experim e n t s with K A a n d n o e x p l a n a t i o n could be f o u n d for these effects T h e q u e s t i o n that arises is w h e t h e r P D A acts as a p a r t m l a g o m s t u n d e r these conchtlons T h e transients c o u l d have b e e n the r e a s o n for the r e p o r t e d i n c r e a s e d firing rate of rat lupp o c a m p a l n e u r o n e s d u n n g slmtlar e x p e r i m e n t s ( C o l h n g n d g e et a l , 1983) I n the h l p p o c a m p u s sm'ular d e p o l a r i z i n g transients a s s o c m t e d with the response to N M D A have been r e p o r t e d ( M c l e n n a n et a l , 1987) These transients were Ca2+-de p e n d e n t since t h e y were b l o c k e d b y C o 2÷ T h e d e p o l a n z a n g transients in the p r e s e n t s t u d y were

b a s i c a l l y different since they a p p e a r e d m the presence of C o 2÷ T h e s e different m e m b r a n e events a c t w a t e d b y e x c i t a t o r y a n u n o acids d l u s t r a t e the complexaty of the r e c e p t o r - c h a n n e l c o m p l e x a n d m i g h t b e a further d l s t m c t w e p r o p e r t y b e t w e e n receptor types

Acknowledgements The authors wishes to thank Dr Henk Vllverberg for comments on the manuscnpt Jan Van der Valk was a reclp~ent of an A N U Ph D Scholarstup

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