Journal of Neuroscience Methods, 32 (1990) 75-79
75
Elsevier NSM 01056
A simple perfusion system for measuring endogenous retinal dopamine release C a n d a c e J. G i b s o n Department of Pathology, University of Western Ontario, London, Ont. N6A 5C1 (Canada)
(Received 25 August 1989) (Revised version received 11 December 1989) (Accepted 18 December 1989)
Key words: Retina; Dopamine; Release; Potassium; Superperfusion; Light
A simple superperfusion apparatus for measuring endogenous dopamine (DA) release from rat retina is described. DA release is stimulated by increasing levels of potassium (K) in the superfusion medium and this K-stimulated release is calcium dependent. Exposure of dark-adapted retinae to light also increases endogenous DA release. This system should prove useful in determining what factors may control endogenous DA release in the retina.
Dopamine is considered a retinal "neurotransmitter" with an important role in regulating sensitivity to light (Dowling, 1986). DA and its synthetic enzyme, tyrosine hydroxylase, have been located histochemically in a sub-population of amacrine cells and in the interplexiform cells of the teleost retina and most mammalian retina (Ehinger, 1982; Dowling, 1986). DA modulates information flow in the retina by decreasing responsiveness of horizontal cells to light stimuli and by decreasing electrical coupling between adjacent horizontal cells (Dowling, 1986). The factors (light; presynaptic inputs) which normally regulate endogenous DA release are still unknown. Radioactively labelled DA can be taken up by these retinal cells and subsequently released by light stimulation (Bauer et al., 1980; Ehinger, 1982) or high K (Thomas et al., 1978; Bauer et al., 1980; Ofori et al., 1986). Recent studies investigating release of 3H-DA from striatal tissue slices indi-
Correspondence: Dr. C.J. Gibson, PhD, Department of Pa-
thology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
cate that there may be differences between release of exogenous labelled DA and the endogenous transmitter (Herdon et al., 1985). Few studies to date have investigated the release of endogenous D A from the retina (Godley and Wurtman, 1988) possibly due to the lack of sufficiently sensitive techniques for measuring the low levels of DA released from the retina (particularly during basal release). We have developed a simple in vitro perfusion system for the investigation of endogenous transmitter release from rat retina using a sensitive coulometric detector system for D A in the perfusate. Endogenous retinal DA is released upon exposure to fight and by exposure to increasing levels of potassium in the perfusion medium. Potassium-stimulated release of DA is calcium dependent. This simple perfusion system allows for a rapid survey of the effects of various compounds on retinal D A release and will allow one to determine what substances may modulate dopamine release normally in the retina. If a sensitive detection system is available the endogenous release of other "putative' retinal neurotransmitters may also be measured.
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
76
Methods
The perfusion system consisted of a peristaltic Manostat cassette pump (Canlab, Don Mills, Ontario) connected to tissue chambers made from inverted MiUipore Swinnex filters (SX00 013 00; Millipore Corporation, Bedford, MA). The original white filter discs were removed and replaced by a piece of nylon mesh (Wire Cloth Enterprises, Inc., Pittsburgh, PA) affixed with Epoxy resin. A second piece of nylon mesh was cut to fit the inner dimensions of the filter and was kept in position with a silicone gasket (SX00 013 01; Millipore Corporation, Bedford, MA). The chambers were connected to the pump by decreasing sizes of Tygon or polypropylene (PPE) tubing. During perfusion experiments the chambers were seated in a plastic rack placed in a water bath at 37 ° C. Two to 4 chambers were used in each experiment. The Krebs' perfusion buffer (final pH 7.4) was made up fresh each time using filtered HPLC grade water (Nanopure, Barnstead-Sybron Corp., Rexdale, Ont.), equilibrated with 95% 02/5% CO2, and contained 143 mM Na +, 3.1 mm K ÷, 1.15 m M C a 2+, 1.2 mM Mg 2+, 23 mM NaHCO3-, 125 mM CI-, and 6 mM glucose (Ames and Nesbett, 1981). In experiments in which the potassium concentration was varied, as K was increased an equivalent amount of Na was removed. Pargyline (20 #M; Sigma Chemical Co., St. Louis, MO) was routinely added to the buffers; without its addition to the perfusion medium, basal levels of DA release were often below the limits of detectability. All chemicals used were analytical reagent grade. Rats (male Sprague-Dawley, 300 g) were killed by decapitation, the eye removed and placed in a small plastic dish on ice. A single incision was made through the sdera, the lens removed and the retina removed, in whole, and placed in 1 ml Krebs buffer on ice. Each retina was placed flat on the nylon mesh floor of the chamber and the perfusion begun (0.5 ml/min). Retina were allowed to equilibrate for 30 rain before starting to collect the perfusate. Three baseline collections were made (15 rain each) before switching to the high potassium buffer. Samples were collected every 15 re.in into vials containing 0.5 ml 1.0 N
perchloric acid with 1 mM ascorbate and EDTA and 100 or 250 pg dihydroxybenzylamine(DHBA, Sigma Chemical Co., St. Louis, MO) added as an internal standard (the perfusate took 1 rain to travel from the chamber to the acidified collection vial). Samples were concentrated on alumina columns (Felice et al., 1978) and DA and DHBA eluted with 300 #1 0.5 N acetic acid. Standards made up in 7.5 ml Krebs buffer were run through the same column procedure and the values for DA calculated from the D A / D H B A ratio in this external standard curve. The assay was linear in the range of 5-5000 pg and recoveries of DHBA and DA from the column were routinely between 50 and 60%. Five pg represented twice baseline and was considered to be the limit of detectability in this system. At the end of each perfusion the retina was removed and placed into 0.5 ml 0.1 N perchloric acid containing 0.1 mM ascorbate and EDTA with 500 pg DHBA added as an internal standard; sonicated, centrifuged and the supernatant run through the column procedure with an appropriate set of standards (250-2000 pg). The tissue pellet was dissolved in 0.1 N NaOH and aliquots assayed for protein content by the method of Lowry et al. (1951). The HPLC system consisted of a BAS dual-piston pump, Rheodyne 7125 manual injector, Altex Ultrasphere I.P. reverse phase column (5 gm; 4.6 × 150 mm) connected to an ESA coulometric detector (Scientific Products & Equipment Ltd., Rexdale, Ontario). DHBA and DA were detected on detector 2 in the reduced form (-0.28 V) with detector 1 set at +0.05 V and the guard cell at +0.35 V. The mobile phase was 0.05 M sodium acetate with 0.1 mM EDTA and 0.32 mM sodium octyl sulfate, pH 5, with 10% methanol (HPLC grade, Fisher Scientific, Don Mills, Ontario) and was pumped at a flow rate of 1.5 ml/min. In the light stimulation experiment rats were killed 2 h after lights out during the dark phase of the cycle, retina removed and transferred to the chambers under a red light. Two baseline collections were made before exposure to a bright white light (75 W) placed 12 inches above the chambers. Data are expressed as pg DA released per retina per 15 rain fraction; pg DA released per mg
77 r e t i n a l p r o t e i n p e r 15 m i n f r a c t i o n o r as a f r a c t i o n a l release ( [ e v o k e d D A (i.e. h i g h K m i n u s b a s e l i n e ) / f i n a l tissue D A p l u s e v o k e d D A ] × ]00%).
150
"~~ii
~,2oo
\
Results
A t y p i c a l p e r f u s i o n p r o f i l e is i l l u s t r a t e d in Fig. 1. Basal D A r e l e a s e a v e r a g e d 12 + 5 p g p e r r e t i n a o r 17 + 5 p g p e r m g p r o t e i n . U p o n s t i m u l a t i o n w i t h 40 m M K, D A r e l e a s e was s i g n i f i c a n t l y e l e v a t e d b y 696% o v e r b a s e l i n e , r e p r e s e n t i n g a b o u t 9.1% o f t o t a l tissue D A . P o t a s s i u m e v o k e d release w a s d o s e d e p e n d e n t (Fig. 2), d o u b l i n g as K was i n c r e a s e d f r o m 25 to 55 m M . S i m i l a r results w e r e o b t a i n e d w h e t h e r d a t a w e r e e x p r e s s e d p e r retina, p e r m g p r o t e i n o r as a f r a c t i o n a l release, a l t h o u g h e x p r e s s i o n o f d a t a as a f r a c t i o n a l release g a v e
\
I00
15 25 40 ~
15
~40
~
~l
40 55
mMK*
Fig. 2. Variation of retinal DA release with increasing potassium concentration. Each bar represents the mean _+SD from 4 retinae. Stippled bars represent the average of three 15-min baseline collections. Open bars represent DA release during the fourth 15-min fraction and exposure to the K concentration indicated on the abscissa. Data are expressed as pg DA released per retina (left panel); pg DA released per mg protein (middle panel) or as a fractional release (right panel). DA release in the presence of 25, 40 and 55 mM K was significantly different from baseline by one-way ANOVA.
m o r e c o n s i s t e n t results as r e t i n a l D A c o n t e n t o f t e n v a r i e d f r o m e x p e r i m e n t to e x p e r i m e n t . P o t a s s i u m s t i m u l a t e d release was c a l c i u m d e p e n d e n t ; t h e r e was n o r e l e a s e o f D A b y 40 m M K w h e n c a l c i u m was o m i t t e d f r o m the m e d i u m (14 +_ 3 p g / r e t i n a b a s e l i n e v e r s u s 11 + 4 p g / r e t i n a w i t h 40 m M K). S t i m u l a t i o n w i t h light s i g n i f i c a n t l y i n c r e a s e d D A release b y a n a v e r a g e o f 30% o v e r b a s e l i n e and DA could be subsequently released by potass i u m s t i m u l a t i o n ( T a b l e I). T h e s e i n c r e a s e s w e r e s i g n i f i c a n t w h e t h e r e x p r e s s e d p e r r e t i n a o r as a
100
t~
\ 5o
TABLE I RELEASE OF DA BY LIGHT OR POTASSIUM
I
2
3
FRACTION
4
5
NO,
Fig. 1. Dopamine release in perfused rat retina. Three 15-rain baseline collections were made before switching to high potassium Krebs' buffer (40 mM" stippled bar). Data represent the mean _+SD from 4 retinae and are expressed as pg DA released per retina per 15 rain fraction.
Dark
Light (pg DA/retina/15 min)
40 mM K
22.0+8.0
25.4+7.8 *
148±60 *
Retinae were removed and transferred to perfusion chambers under red light. Two 15-min baseline collections were made in the dark prior to exposure to white light and subsequently high potassium, n = 6, mean + S.D. * P < 0.05 significantly different from dark control, paired t-test.
78
fractional release (1.8 + 0.6% (dark); 2.3 + 0.7% (light) and 10.4 + 5.5% (40 mM K)).
Discussion Dopamine has long been recognized as a transmitter in retina with a role in modulating the sensitivity to fight. Release of exogenously labelled dopamine has also been described following perfusion with high levels of potassium (Thomas et al., 1978; Sarthy and Lain, 1979; Ofori et al., 1986) or with pulses of light (Kramer, 1971; Bauer et al., 1980). Release of endogenous DA may be different from that of exogenously labelled dopamine due (1) to the existence o f multiple pools of releasable DA; (2) the ability of the perfused retina to increase DA synthesis in a readily releasable form; or (3) due to uptake of trace doses of labelled compound into cells which do not normally synthesize or contain the transmitter but which will release it when stimulated (Herdon et al., 1985). Thus, measurement of the endogenous transmitter directly is preferable and is now possible with the current sensitivity of electrochemical detection systems. Release of endogenous DA in rabbit retina following light or potassium stimulation has recently been reported by Godley and Wurtman (1988). Use of the rat retina is considerably cheaper; more readily available; and allows the use of intact whole retina rather than retinal pieces. Stimulation of the retina by light effectively releases DA (by a maximum of 60% over baseline) but this effect is much smaller than that seen with exposure to potassium (for example 400% at 25 mm K). This difference has also been reported for the release of labelled DA (Kramer, 1971; Bauer et al., 1980; Godley and Wurtman, 1988) and may be due to the fact that stimulation by fight does not involve a direct neuroanatomical connection of photoreceptor cells to the retinal DA amacrine or interplexiform cell; whereas release induced by potassium would involve a direct effect on the amacrine ceil via depolarization of the membrane. Physiological depolarization of these cells may also involve an influx oLpotassium of far less magnitude as has been used in these studies (15-55
mM K) or in other published studies (50 mM K). The response to potassium appears to be a graded response in whole retina thus lower levels of potassium evoke lower levels of DA release (Fig. 2). This superfusion system provides a useful model for determining what factors may normally influence the endogenous release of retinal DA with greater ease, sensitivity and less expense than previously reported systems.
Acknowledgements I would like to thank Dr. Joseph Hirsch for his many helpful suggestions and discussions. Funding was provided by the Canadian Medical Research Council.
References 1 Ames, A., Ill and Nesbett, F.B. (1981) In vitro retina as an experimental model of the central nervous system. J. Neurochem., 37: 867-877. 2 Bauer, B., Ehinger, B. and Aberg, L. (1980) [3H]Dopamine release from the rabbit retina. Arch. Kiln. Ophthalmol., 215: 71-78. 3 Dowiingo J.E. (1986) Dopamine: a retinal neuromodulator? Trends Neurol. Sci., 9: 236-240. 4 Ehinger, B. (1982) Neurotransmitter system in the retina. Retina, 2: 305-321. 5 Felice, L.J., Felice, J.D. and Kissinger, P.T. (1978) Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. J. Neurochem., 31: 1461-1465. 6 Godley, B.F. and Wurtman, R.J. (1988) Release of endogenous dopamine from the superfuso:l rabbit retina in vitro: effect of light stimulation. Brain Res., 452: 393-395. 7 Herdon, H., Strupish, J. and Naborski, S.R, (1985) Differences between the release of radiolabelled and endogenous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis inhibition. Brain Res., 348: 309-320. 8 Kramer, S.G. (1971) Dopamine; a retinal neurotransmitter. I. Retinal uptake, storage and light-stimulated release of 3H-dopamine in vivo. Invest. Opthalmol., 10: 438-452. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. 10 Ofori, S., Magistretti, P.J. and Schorderet, M. (1986) Investigation of dopamine content, synthesis and release in the
75
Elsevier NSM 01056
A simple perfusion system for measuring endogenous retinal dopamine release C a n d a c e J. G i b s o n Department of Pathology, University of Western Ontario, London, Ont. N6A 5C1 (Canada)
(Received 25 August 1989) (Revised version received 11 December 1989) (Accepted 18 December 1989)
Key words: Retina; Dopamine; Release; Potassium; Superperfusion; Light
A simple superperfusion apparatus for measuring endogenous dopamine (DA) release from rat retina is described. DA release is stimulated by increasing levels of potassium (K) in the superfusion medium and this K-stimulated release is calcium dependent. Exposure of dark-adapted retinae to light also increases endogenous DA release. This system should prove useful in determining what factors may control endogenous DA release in the retina.
Dopamine is considered a retinal "neurotransmitter" with an important role in regulating sensitivity to light (Dowling, 1986). DA and its synthetic enzyme, tyrosine hydroxylase, have been located histochemically in a sub-population of amacrine cells and in the interplexiform cells of the teleost retina and most mammalian retina (Ehinger, 1982; Dowling, 1986). DA modulates information flow in the retina by decreasing responsiveness of horizontal cells to light stimuli and by decreasing electrical coupling between adjacent horizontal cells (Dowling, 1986). The factors (light; presynaptic inputs) which normally regulate endogenous DA release are still unknown. Radioactively labelled DA can be taken up by these retinal cells and subsequently released by light stimulation (Bauer et al., 1980; Ehinger, 1982) or high K (Thomas et al., 1978; Bauer et al., 1980; Ofori et al., 1986). Recent studies investigating release of 3H-DA from striatal tissue slices indi-
Correspondence: Dr. C.J. Gibson, PhD, Department of Pa-
thology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
cate that there may be differences between release of exogenous labelled DA and the endogenous transmitter (Herdon et al., 1985). Few studies to date have investigated the release of endogenous D A from the retina (Godley and Wurtman, 1988) possibly due to the lack of sufficiently sensitive techniques for measuring the low levels of DA released from the retina (particularly during basal release). We have developed a simple in vitro perfusion system for the investigation of endogenous transmitter release from rat retina using a sensitive coulometric detector system for D A in the perfusate. Endogenous retinal DA is released upon exposure to fight and by exposure to increasing levels of potassium in the perfusion medium. Potassium-stimulated release of DA is calcium dependent. This simple perfusion system allows for a rapid survey of the effects of various compounds on retinal D A release and will allow one to determine what substances may modulate dopamine release normally in the retina. If a sensitive detection system is available the endogenous release of other "putative' retinal neurotransmitters may also be measured.
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
76
Methods
The perfusion system consisted of a peristaltic Manostat cassette pump (Canlab, Don Mills, Ontario) connected to tissue chambers made from inverted MiUipore Swinnex filters (SX00 013 00; Millipore Corporation, Bedford, MA). The original white filter discs were removed and replaced by a piece of nylon mesh (Wire Cloth Enterprises, Inc., Pittsburgh, PA) affixed with Epoxy resin. A second piece of nylon mesh was cut to fit the inner dimensions of the filter and was kept in position with a silicone gasket (SX00 013 01; Millipore Corporation, Bedford, MA). The chambers were connected to the pump by decreasing sizes of Tygon or polypropylene (PPE) tubing. During perfusion experiments the chambers were seated in a plastic rack placed in a water bath at 37 ° C. Two to 4 chambers were used in each experiment. The Krebs' perfusion buffer (final pH 7.4) was made up fresh each time using filtered HPLC grade water (Nanopure, Barnstead-Sybron Corp., Rexdale, Ont.), equilibrated with 95% 02/5% CO2, and contained 143 mM Na +, 3.1 mm K ÷, 1.15 m M C a 2+, 1.2 mM Mg 2+, 23 mM NaHCO3-, 125 mM CI-, and 6 mM glucose (Ames and Nesbett, 1981). In experiments in which the potassium concentration was varied, as K was increased an equivalent amount of Na was removed. Pargyline (20 #M; Sigma Chemical Co., St. Louis, MO) was routinely added to the buffers; without its addition to the perfusion medium, basal levels of DA release were often below the limits of detectability. All chemicals used were analytical reagent grade. Rats (male Sprague-Dawley, 300 g) were killed by decapitation, the eye removed and placed in a small plastic dish on ice. A single incision was made through the sdera, the lens removed and the retina removed, in whole, and placed in 1 ml Krebs buffer on ice. Each retina was placed flat on the nylon mesh floor of the chamber and the perfusion begun (0.5 ml/min). Retina were allowed to equilibrate for 30 rain before starting to collect the perfusate. Three baseline collections were made (15 rain each) before switching to the high potassium buffer. Samples were collected every 15 re.in into vials containing 0.5 ml 1.0 N
perchloric acid with 1 mM ascorbate and EDTA and 100 or 250 pg dihydroxybenzylamine(DHBA, Sigma Chemical Co., St. Louis, MO) added as an internal standard (the perfusate took 1 rain to travel from the chamber to the acidified collection vial). Samples were concentrated on alumina columns (Felice et al., 1978) and DA and DHBA eluted with 300 #1 0.5 N acetic acid. Standards made up in 7.5 ml Krebs buffer were run through the same column procedure and the values for DA calculated from the D A / D H B A ratio in this external standard curve. The assay was linear in the range of 5-5000 pg and recoveries of DHBA and DA from the column were routinely between 50 and 60%. Five pg represented twice baseline and was considered to be the limit of detectability in this system. At the end of each perfusion the retina was removed and placed into 0.5 ml 0.1 N perchloric acid containing 0.1 mM ascorbate and EDTA with 500 pg DHBA added as an internal standard; sonicated, centrifuged and the supernatant run through the column procedure with an appropriate set of standards (250-2000 pg). The tissue pellet was dissolved in 0.1 N NaOH and aliquots assayed for protein content by the method of Lowry et al. (1951). The HPLC system consisted of a BAS dual-piston pump, Rheodyne 7125 manual injector, Altex Ultrasphere I.P. reverse phase column (5 gm; 4.6 × 150 mm) connected to an ESA coulometric detector (Scientific Products & Equipment Ltd., Rexdale, Ontario). DHBA and DA were detected on detector 2 in the reduced form (-0.28 V) with detector 1 set at +0.05 V and the guard cell at +0.35 V. The mobile phase was 0.05 M sodium acetate with 0.1 mM EDTA and 0.32 mM sodium octyl sulfate, pH 5, with 10% methanol (HPLC grade, Fisher Scientific, Don Mills, Ontario) and was pumped at a flow rate of 1.5 ml/min. In the light stimulation experiment rats were killed 2 h after lights out during the dark phase of the cycle, retina removed and transferred to the chambers under a red light. Two baseline collections were made before exposure to a bright white light (75 W) placed 12 inches above the chambers. Data are expressed as pg DA released per retina per 15 rain fraction; pg DA released per mg
77 r e t i n a l p r o t e i n p e r 15 m i n f r a c t i o n o r as a f r a c t i o n a l release ( [ e v o k e d D A (i.e. h i g h K m i n u s b a s e l i n e ) / f i n a l tissue D A p l u s e v o k e d D A ] × ]00%).
150
"~~ii
~,2oo
\
Results
A t y p i c a l p e r f u s i o n p r o f i l e is i l l u s t r a t e d in Fig. 1. Basal D A r e l e a s e a v e r a g e d 12 + 5 p g p e r r e t i n a o r 17 + 5 p g p e r m g p r o t e i n . U p o n s t i m u l a t i o n w i t h 40 m M K, D A r e l e a s e was s i g n i f i c a n t l y e l e v a t e d b y 696% o v e r b a s e l i n e , r e p r e s e n t i n g a b o u t 9.1% o f t o t a l tissue D A . P o t a s s i u m e v o k e d release w a s d o s e d e p e n d e n t (Fig. 2), d o u b l i n g as K was i n c r e a s e d f r o m 25 to 55 m M . S i m i l a r results w e r e o b t a i n e d w h e t h e r d a t a w e r e e x p r e s s e d p e r retina, p e r m g p r o t e i n o r as a f r a c t i o n a l release, a l t h o u g h e x p r e s s i o n o f d a t a as a f r a c t i o n a l release g a v e
\
I00
15 25 40 ~
15
~40
~
~l
40 55
mMK*
Fig. 2. Variation of retinal DA release with increasing potassium concentration. Each bar represents the mean _+SD from 4 retinae. Stippled bars represent the average of three 15-min baseline collections. Open bars represent DA release during the fourth 15-min fraction and exposure to the K concentration indicated on the abscissa. Data are expressed as pg DA released per retina (left panel); pg DA released per mg protein (middle panel) or as a fractional release (right panel). DA release in the presence of 25, 40 and 55 mM K was significantly different from baseline by one-way ANOVA.
m o r e c o n s i s t e n t results as r e t i n a l D A c o n t e n t o f t e n v a r i e d f r o m e x p e r i m e n t to e x p e r i m e n t . P o t a s s i u m s t i m u l a t e d release was c a l c i u m d e p e n d e n t ; t h e r e was n o r e l e a s e o f D A b y 40 m M K w h e n c a l c i u m was o m i t t e d f r o m the m e d i u m (14 +_ 3 p g / r e t i n a b a s e l i n e v e r s u s 11 + 4 p g / r e t i n a w i t h 40 m M K). S t i m u l a t i o n w i t h light s i g n i f i c a n t l y i n c r e a s e d D A release b y a n a v e r a g e o f 30% o v e r b a s e l i n e and DA could be subsequently released by potass i u m s t i m u l a t i o n ( T a b l e I). T h e s e i n c r e a s e s w e r e s i g n i f i c a n t w h e t h e r e x p r e s s e d p e r r e t i n a o r as a
100
t~
\ 5o
TABLE I RELEASE OF DA BY LIGHT OR POTASSIUM
I
2
3
FRACTION
4
5
NO,
Fig. 1. Dopamine release in perfused rat retina. Three 15-rain baseline collections were made before switching to high potassium Krebs' buffer (40 mM" stippled bar). Data represent the mean _+SD from 4 retinae and are expressed as pg DA released per retina per 15 rain fraction.
Dark
Light (pg DA/retina/15 min)
40 mM K
22.0+8.0
25.4+7.8 *
148±60 *
Retinae were removed and transferred to perfusion chambers under red light. Two 15-min baseline collections were made in the dark prior to exposure to white light and subsequently high potassium, n = 6, mean + S.D. * P < 0.05 significantly different from dark control, paired t-test.
78
fractional release (1.8 + 0.6% (dark); 2.3 + 0.7% (light) and 10.4 + 5.5% (40 mM K)).
Discussion Dopamine has long been recognized as a transmitter in retina with a role in modulating the sensitivity to fight. Release of exogenously labelled dopamine has also been described following perfusion with high levels of potassium (Thomas et al., 1978; Sarthy and Lain, 1979; Ofori et al., 1986) or with pulses of light (Kramer, 1971; Bauer et al., 1980). Release of endogenous DA may be different from that of exogenously labelled dopamine due (1) to the existence o f multiple pools of releasable DA; (2) the ability of the perfused retina to increase DA synthesis in a readily releasable form; or (3) due to uptake of trace doses of labelled compound into cells which do not normally synthesize or contain the transmitter but which will release it when stimulated (Herdon et al., 1985). Thus, measurement of the endogenous transmitter directly is preferable and is now possible with the current sensitivity of electrochemical detection systems. Release of endogenous DA in rabbit retina following light or potassium stimulation has recently been reported by Godley and Wurtman (1988). Use of the rat retina is considerably cheaper; more readily available; and allows the use of intact whole retina rather than retinal pieces. Stimulation of the retina by light effectively releases DA (by a maximum of 60% over baseline) but this effect is much smaller than that seen with exposure to potassium (for example 400% at 25 mm K). This difference has also been reported for the release of labelled DA (Kramer, 1971; Bauer et al., 1980; Godley and Wurtman, 1988) and may be due to the fact that stimulation by fight does not involve a direct neuroanatomical connection of photoreceptor cells to the retinal DA amacrine or interplexiform cell; whereas release induced by potassium would involve a direct effect on the amacrine ceil via depolarization of the membrane. Physiological depolarization of these cells may also involve an influx oLpotassium of far less magnitude as has been used in these studies (15-55
mM K) or in other published studies (50 mM K). The response to potassium appears to be a graded response in whole retina thus lower levels of potassium evoke lower levels of DA release (Fig. 2). This superfusion system provides a useful model for determining what factors may normally influence the endogenous release of retinal DA with greater ease, sensitivity and less expense than previously reported systems.
Acknowledgements I would like to thank Dr. Joseph Hirsch for his many helpful suggestions and discussions. Funding was provided by the Canadian Medical Research Council.
References 1 Ames, A., Ill and Nesbett, F.B. (1981) In vitro retina as an experimental model of the central nervous system. J. Neurochem., 37: 867-877. 2 Bauer, B., Ehinger, B. and Aberg, L. (1980) [3H]Dopamine release from the rabbit retina. Arch. Kiln. Ophthalmol., 215: 71-78. 3 Dowiingo J.E. (1986) Dopamine: a retinal neuromodulator? Trends Neurol. Sci., 9: 236-240. 4 Ehinger, B. (1982) Neurotransmitter system in the retina. Retina, 2: 305-321. 5 Felice, L.J., Felice, J.D. and Kissinger, P.T. (1978) Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography. J. Neurochem., 31: 1461-1465. 6 Godley, B.F. and Wurtman, R.J. (1988) Release of endogenous dopamine from the superfuso:l rabbit retina in vitro: effect of light stimulation. Brain Res., 452: 393-395. 7 Herdon, H., Strupish, J. and Naborski, S.R, (1985) Differences between the release of radiolabelled and endogenous dopamine from superfused rat brain slices: Effects of depolarizing stimuli, amphetamine and synthesis inhibition. Brain Res., 348: 309-320. 8 Kramer, S.G. (1971) Dopamine; a retinal neurotransmitter. I. Retinal uptake, storage and light-stimulated release of 3H-dopamine in vivo. Invest. Opthalmol., 10: 438-452. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. 10 Ofori, S., Magistretti, P.J. and Schorderet, M. (1986) Investigation of dopamine content, synthesis and release in the
