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Modification of electroretinograms in dopamine-depleted retinas
Brattt Research, ~45 ! !q85 ) 1S~- i ~)1
186
Elsevier
BRE 21090
Modifioation of electroreti~rams in dopamine-depleted retinas MARK C. CITRON l, LYNDA ERINOFF 1, DENNIS W. RICKMANl and NICHOLAS C. BRECHA2-4 lChildrens Hospital of Los Angeles, University of Southern California, Department of Neurology, Los Angeles, CA 90054, 2Centerfor Ulcer Research and Education, Veterans Administration Center-Wadsworth, Los Angeles, CA 90073, 3Jules Stein Eye Institute, Department of Medicine and 4Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted May 14th, 1985) Key words: electroretinogram - - dopamine - - amacrine cell - - oscillatory potential - - b-wave - retina - - frog - - tyrosine hydroxylase-like immunoreactivity
The functional role of dopamine in frog retina was examined in a combined neurochemical, immunohistochemical and electrophysiological study. Dopamine and serotonin are the primary monoamines present in the retina and they are localized to amacrine cells which have distinct morphologies. Intravitreal injection of 6-hydroxydopamine was found to produce a selective depletion of retinal dopamine content and elimination of tyrosine hydroxylase-like immunoreactivity. Electroretinograms from 6-hydroxydopaminetreated retinas demonstrated enhanced oscillatory potentials and a lengthening of the b-wave implicit time compared to vehicle control retinas; both of these changes in the electroretinogram were reversed by the dopamine agonist apomorphine. These observations support earlier suggestions that dopamine-containing amacrine cells are part of a retinal feedback system that generates oscillatory potentials and plays a role in light adaptation.
The predominant catecholamine in the vertebrate retina is dopamine ( D A ) 2. Biochemical studies have demonstrated significant concentrations of D A in all retinas as well as the existence of its biosynthetic enzyme, tyrosine hydroxylase, high affinity D A uptake mechanisms, and specific D A receptor sites. Anatomical studies have demonstrated the presence of catecholamine fluorescent, tyrosine hydroxylaselike (TH) immunoreactive, and DA-accumulating amacrine and/or interplexiform cells. Tyrosine hydroxylase activity as well as D A release are increased in response to lightS,9A2. Furthermore. this increase in D A synthesis appears to be directly related to light intensity 16. These neurochemical and anatomical data strongly suggest that D A plays a functional role in the retina. however electrophysiological evidence for this role is sparse and contradictory. That is, studies on the effects of D A and dopaminergic drugs on the electroretinogram ( E R G ) have reported conflicting results. Apomorphine H, a D A agonist, and reserpine 8. a
monoamine depleter, diminished the b-wave amplitude in rabbit E R G : while chlorpromazine 1°, a neuroleptic with D A receptor blocking activity, increased b-wave amplitude. In contrast, reserpine 7 and L - D O P A 19. a D A precursor, had no effect on b-wave amplitude in cat E R G . Oscillatory potentials, another component of the E R G . are low amplitude wavelets superimposed on the b-wave that are thought to be generated in the inner retina 13.21. Reserpine decreased oscillatory potentials in cat 7 and rabbits and L - D O P A reversed this effect. However. direct application of D A to m u d p u p p y retina decreased oscillatory potentials 20. The present study was directed towards a better understanding of the functional role of D A in the frog retina. This study analyzed the effects of 6-hydroxydopamine ( 6 - O H D A ) - i n d u c e d D A depletion on the E R G recorded from a frog eyecup preparation. The effect of apomorphine, a D A agonist which stimulates presumed retinal postsynaptic D A receptors14,18, on the E R G of 6-OHDA-treated retinas was
Correspondence: M. C. Citron. Childrens Hospital of Los Angeles. Neurology Research. PO Box 54700. Los Angeles. CA 90054. U.S.A.
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
187 determined. We assessed the specificity of 6 - O H D A treatment by determining D A and serotonin concentrations and the presence of TH and serotonin-like immunoreactivity. Frogs (Rana pipiens) were anesthetized with a 0.7-1.0 ml injection of 30 mM ethyl m-aminobenzoate (Tricane, Sigma) in frog Ringers into the dorsal lymph sac before the intravitreal administration of 6O H D A (Sigma). The following injection protocol was used: on two successive days, 100 ~tg 6 - O H D A (free base) in 5 ~tl 0.9% saline with 0.1% ascorbate was injected into the right eye using a 26-gauge needle; the left eye was injected with an equal volume of the vehicle. Following at least a 1-week survival period the frogs were pithed, the eyes were removed, and the retinas were analyzed using neurochemical, immunohistochemical or electrophysiological techniques as described below. Monoamines were extracted from isolated retinas by a modification of the method of Shellenberger and Gordon 17 (alumina adsorption of catecholamines and solvent extraction of serotonin) with dihydroxybenzylamine and N-methyl serotonin used as internal standards. The samples were then analyzed by high performance liquid chromatography with electrochemical detection 4. The limits of detection of this assay are 100 pg for monoamines and 300 pg for dihydroxyphenylacetic acid (DOPAC). Protein content of the retinas was determined by the Bradford~ method (Bio-Rad, Richmond, CA). Normal, vehicle control, and 6-OHDA-treated retinas were immersion-fixed in 4% paraformaldehyde, 0.1 M D,L-lysine and 0.01 M sodium periodate in 0.1 M phosphate buffer for 2 h at room temperature. The retinas were washed overnight in 25% sucrose in 0.1 M phosphate buffer at 4 °C until processing using standard immunohistochemical techniques 3. Retinas were either sectioned perpendicular to the vitreal surface at 10-16 ~tM with a cryostat and mounted onto gelatin-coated slides or treated freefloating. Retinal sections or whole retinas were washed in 0.1 M phosphate buffer or 0.5 M Tris-HC1 buffer (pH 7.4). After incubation in diluted primary antiserum directed to tyrosine hydroxylase (1:250) or serotonin (1:500) (0.5-1.0% Triton X-100 in buffer) for 24-96 h at 4 °C, they were washed in buffer for 30-60 min. Retinal sections were then incubated in
IgG-FITC (1:50) ( D A K O ) for 2 h at room temperature, washed in buffer and then coverslipped with a glycerol-phosphate buffer (9:1) mixture. Whole retinas were incubated in IgG (1:50) ( D A K O ) at 4 °C, washed in buffer and subsequently incubated in peroxidase-antiperoxidase (1:50) (DAKO). The retinas were then washed, preincubated in 3,3'-diaminobenzidine HCI (DAB) (50 mg/100 ml) for 30 min and incubated in DAB with 0.01% H202 for an additional 30 min. Whole retinas were then washed, mounted, dehydrated and coverslipped. Specificity of the antisera was assessed by substituting normal rabbit serum in place of the primary antiserum. Since control experiments cannot exclude the possibility that the immunoreactive material is similar but not identical to tyrosine hydroxylase or serotonin, the staining observed is referred to as THor serotonin-like. ERGs were recorded from an eyecup preparation taken from a light adapted frog (6.5 ktW/cm 2 room illumination) using an LSI-11/23 computer with a 12bit data-acquisition module at a digitizing rate of 1 ms. The vitreous was removed prior to recording and the retina was maintained in humidified 100% oxygen at room temperature. The stimulus light was a quartz halogen lamp (GE no. DNE) which produced an intensity of 30 ~W/cm 2 (2.3 x 1030 photons/(cm 2' s) at 473 nm)) in the plane of the retina though a fiber-optic guide. The duration of the flash was 10 ms and was controlled by an electronic shutter (Vincent Associates, 310-B). Ambient illumination during the experiment was 0.03 ktW/cm 2. A silver ball electrode was placed on the retinal surface halfway between the optic nerve head and the periphery. One E R G was recorded every 4 s, and, typically, 30-50 E R G s were recorded. The eyecup was then washed with a drop of frog Ringers and an additional 30-50 E R G s recorded. The Ringers was then replaced by a 1 ~M, 10 ktM, 100 ,uM or 1.0 mM solution of freshly prepared apomorphine (Sigma) in Ringers and a final 30-50 ERGs were recorded. Following completion of the E R G recordings, the retinas were removed from the eyecup and frozen at -80 °C for subsequent monoamine determination. Each group of 30-50 E R G s were averaged to create the averaged E R G used for identifying the amplitude of the b-wave peak and its implicit time (time from stimulus to b-wave peak). The bin in the aver-
188 aged E R G with the largest positive digital value was judged to be the b-wave peak. This bin was then used to determine the b-wave implicit time with 1 ms resolution. Single unit responses of horizontal cells to 0.5 s flashes of the same stimulus as used for the E R G s were r e c o r d e d with a glass m i c r o e l e c t r o d e filled with 2.0 M KC1. Response waveforms were r e c o r d e d to assess the viability of the o u t e r retina. The concentration of D A in vehicle control retinas was 15.5 + 3.9 ng/mg protein (mean _+ S . D . , n = 28), which did not differ from D A content m e a s u r e d in normal retinas. No o t h e r catecholamines or D O P A C were detected. Immunohistochemical studies demonstrated the presence of T H immunoreactive cells in the normal and vehicle control retinas, l m m u n o r e active s o m a t a were present in the proximal inner nuclear layer (INL) near or at the b o r d e r of the inner plexiform layer (IPL) and their processes were observed only in the IPL suggesting they are amacrine cells. T H immunoreactive processes f o r m e d a prominent continuous plexus in lamina 1 of the IPL adjacent to the I N L and a less p r o m i n e n t , discontinuous plexus in lamina 5. Processes were also sparsely distributed in o t h e r regions of the IPL. In addition, occasional immunoreactive s o m a t a were located in the ganglion cell layer, and these cells gave rise to processes that ramified in the IPL, suggesting that they are displaced amacrine cells. In retinal whole mounts, ellipsoidal T H i m m u n o r e active somata were distributed in all regions of the retina. These cells measured 12.1 _+ 1.4~m by 14.0 _+ 1.5 ~ m in diameter; the mean area was 133.1 ,urn 2 with a range of 84.1 to 176.3 ktm2 (mean + S . D . , n = 15). I m m u n o r e a c t i v e cells gave rise to 3 or 4 primary processes, the majority of which ramified in lamina 1. These processes in turn gave rise to secondary processes that also ramified in lamina 1 (Fig. 1A) and a p p e a r e d to form rings a r o u n d unstained somata. In addition, the primary processes gave rise to thick processes that passed through the I P L to lamina 5 of the IPL (Fig. 1B). The density of processes in lamina 5 was less than that seen in lamina 1. Serotonin content of vehicle control and 6O H D A - t r e a t e d retinas was 69.7 + 34.3 and 55.0 +_ 19.7 ng/mg protein (mean + S . D . , n -- 28), respectively, and did not differ from each other or normal retinas. Serotonin-like immunoreactive amacrine
Fig. 1. TH immunoreactivity in a retinal whole mount preparation focused at lamina 1 (A) and at lamina 5 (B) of the IPL. A: TH immunoreactive somata (arrows) in the proximal INL at the border of the IPL. B: coarse immunoreactive processes distributed in lamina 5 of the IPL. Arrows in B show the position of the somata illustrated in A. Calibration bat = 25 gem.
cells were also distributed in all regions of the retina. I m m u n o r e a c t i v e s o m a t a were located in the I N L at the b o r d e r of the IPL and processes were distributed across all laminae of the IPL. These observations are consistent with another r e p o r t of serotonin-like immunoreactivity in the frog retina 15. Neurochemical studies d e m o n s t r a t e d that D A was selectively d e p l e t e d by intravitreal injection o f 6O H D A . Different dosage regimens of 6 - O H D A (2 x 200/~g/10/~1, 2 x 100 ag/10 #l, and 2 x 100/~g/5/~l)
189 TABLE I i,
Effects of intravitreal 6-hydroxydopamine treatment (2 times 100/~g/5/~l) on retinal dopamine and serotonin content Postinjection Survival
n
3days 7 1 week 7 2weeks 4 2-3weeks 10
I:
!!,
Dopamine +_S.D. (ng/mg protein)
Serotonin + S . D . (ng/mg protein)
Control
Treated
Control
Treated
il~i::i
i!
( Retina
15.4 +_ 4.2 16.1 +_ 5.8 16. l_+4.0 14.4+-3.1
1.5 +_ 4.0* 4.2 + 5.3* 1.4+-1.0" 1.0+ 1.8"
58.3 +_ 16.9 95.6+57.1 76.1 +_20.3 54.0+ 12.9
43.9 _+ 13.7 59.8+20.6 67.4+_ 14.6 57.4+23.1
'',f,,'t'i\!! , , ]iii,,~ , ',
i!',, ..... ~', ',~ ',~, ~
6-HDA
I', i"
Apomorphine
( 100 ,urn )
-~181R03
)
Treated
( Retina-~181R01
)
* P < 0.05
w e r e i n v e s t i g a t e d , a n d t w o d o s e s o f 100 g g 6 - O H D A in 5 B1 i n j e c t e d o n s u c c e s s i v e d a y s w e r e f o u n d to p r o duce greater than 70% depletion of retinal DA while s e r o t o n i n c o n t e n t w a s u n c h a n g e d c o m p a r e d to v e h i cle c o n t r o l s ( T a b l e I). D A c o n t e n t o f t h e r e t i n a w a s r e d u c e d as e a r l y as 3 d a y s a f t e r 6 - O H D A
administra-
t i o n a n d D A levels s h o w e d n o signs of r e c o v e r y u p to 3 weeks after treatment.
Fig. 3. The effect of apomorphine on ERG shown in Fig. 2. Curve from 6-OHDA-treated eve (solid line) is the same as in Fig. 2. The retina was treated with 100uM apomorphine and 30 more ERGs were averaged (dashed line). Calibration - 100 ms, 100 ~V.
lmmunohistochemical studies demonstrated that 7 days a f t e r 6 - O H D A
t r e a t m e n t (2 x 1 0 0 / ~ g / 5 / d ) all
T H i m m u n o r e a c t i v i t y w a s e l i m i n a t e d in t h e r e t i n a . T h i s t r e a t m e n t d i d n o t h a v e a n o t i c e a b l e effect, at least at t h e light m i c r o s c o p i c level, o n e i t h e r r e t i n a l h i s t o l o g y o r t h e d i s t r i b u t i o n of s e r o t o n i n - l i k e i m m u -
ij i
noreactivity.
Jl
Control I
( Retina
Ii
II iI i I
ii iJ i
,!!
All of t h e r e t i n a s u s e d in t h e E R G
,
6-HDA
i I
i, '
' ~, I
I
ii
Ii
(Retina
~181L01
scribed below were analyzed for DA and serotonin
)
content. Only 6-OHDA-treated
Treated #
181R01
)
r e t i n a s w i t h D A de-
p l e t i o n s o f g r e a t e r t h a n 7 0 % w e r e c h o s e n for e l e c t r o p h y s i o l o g i c a l analysis. N o d i f f e r e n c e s w e r e s e e n be-
i I
s t u d i e s de-
I
t w e e n t h e E R G s of n o r m a l a n d v e h i c l e c o n t r o l retinas. In v e h i c l e c o n t r o l r e t i n a s t h e m e a n a - w a v e a m -
"Li
~l 1t 1
p l i t u d e was 0.167 + 0.07 m V a n d t h e b - w a v e w a v e , . . . . . . . . .
o,,
a m p l i t u d e was 0.209 _+ 0.07 m V ( m e a n + S . D . , n = 20). In 6 - O H D A - t r e a t e d
r e t i n a s t h e a m p l i t u d e of t h e
a - w a v e was d e c r e a s e d to 0.092 + 0.06 m V a n d t h e
II I I
a m p l i t u d e of t h e b - w a v e was d e c r e a s e d to 0.104 _+ 0.09 m V ( m e a n + S . D . , n = 20, P ~< 0.01). N o r m a l b-
Fig. 2. The effect of retinal DA depletion on frog ERG recorded from the surface of the eyecup. Each curve is the average of 30 ERGs recorded from the control eye (solid line) and 6OHDA-treated eye (dashed line) of the same frog. The duration of the stimulus flash was 10 ms. Note increased amplitude of oscillatory potentials and increased b-wave implicit time after DA depletion. Calibration = 100 ms, 100 uV.
w a v e i m p l i c i t t i m e w a s 68.5 + 9.39 m s S . D . In 6OHDA-treated
r e t i n a s t h e t i m e to t h e b - w a v e p e a k
i n c r e a s e d to 105.1 + 32.5 m s ( m e a n _+ S . D . , n = 20, P ~< 0.01). T h e t i m e c o u r s e o f t h e a - w a v e r e m a i n e d u n c h a n g e d . A n i n c r e a s e in t h e b - w a v e i m p l i c i t t i m e was o b s e r v e d in all D A d e p l e t e d r e t i n a s t e s t e d (n -
I9(t 20). The oscillatory potentials were increased in 14 out of 20 6-OHDA-treated retinas relative to control. Horizontal cell responses were recorded from the same retinas used for the E R G analysis. In contrast to the amplitude changes in the E R G of 6 - O H D A treated retinas, the number of horizontal cells encountered was the same as in normal retinas and these horizontal cells showed typical hyperpolarizations in response to a full-field stimulus. Representative E R G s from each eye of a frog are shown in Figs. 2 and 3. The vehicle control retina (solid line) had an a-wave implicit time of 20 ms and was followed by a b-wave that peaked at 52 ms (Fig. 2). The D A depleted retina (broken line) showed larger oscillatory waves than the control, and the implicit time had increased to 62 ms. The reversal of the effects of D A depletion by the D A agonist, apomorphine (100/,M) are shown in Fig. 3. The implicit time of the b-wave decreased to approach values measured in the vehicle control retina; apomorphine treatment decreased b-wave implicit time in the D A depleted retina to only 5 ms greater than that of the vehicle control. The heightened oscillatory potentials were also decreased by apomorphine. Table I1 presents the change in b-wave implicit time in 6 - O H D A - t r e a t e d retinas before and after treatment with apomorphine. Only 10 and 100 ,uM concentrations of apomorphine produced a significant decrease in b-wave implicit time. In addition, the application of 100/~M apomorphine significantly decreased the b-wave implicit time of vehicle control retinas from 60.2 to 52.9 _+ 21.1 ms (mean _+ S.D., n = 6, P < 0.05, paired t-test). A p o m o r p h i n e reversed the increased oscillatory potential in 9 of 14 retinas. Oscillatory potentials in control retinas were too small to detect any decrease caused by apomorphine. A p o m o r p h i n e was without effect on the a- or
b-wave amplitudes of either the control ~r treated retinas. These studies demonstrate that D A is the principal catecholamine in Rana pipiens; we were unable to detect epinephrine, norepinephrine or D O P A C with our assay suggesting that they are either at low levels or not present. Serotonin levels were several fold higher than D A levels. In contrast, Osborne 14 reported approximately equal concentrations of these monoamines in the frog retina. 6 - O H D A treatment resulted in a selective and long-lasting depletion of retinal D A , but no changes were seen in serotonin levels. Immunohistochemical studies support these general observations. T H immunoreactivity is present in wide-field amacrine cells whose processes are distributed primarily to lamina 1. No immunoreactivity is present 7 days after 6 - O H D A treatment. In contrast, serotonin-like immunoreactive amacrine cells, as well as general retinal histology, appear to be unaffected by the 6 - O H D A treatment. These observations suggest that the 6 - O H D A treatment regime had a specific effect only upon dopaminergic amacrine cells in the frog retina. This is the first electrophysiological study to use the frog eyecup preparation to examine the effects of 6 - O H D A induced D A depletion. Depletions were verified both neurochemically and histochemically. The physiological effects of this treatment were examined using an eyecup preparation which has the advantage of enabling localized drug application, thus avoiding the complications of systemic drug administration. Studies on the role of D A in retinal function have produced conflicting dataT,S,lO,n, t920. It is difficult to compare the present results to those of other studies since there are marked methodological and species differences. The effects of D A depletion on the E R G were to in-
TABLE II Effect of apomorphine on b-wave implicit time in 6-hydroxydopamine treated retinas Apomorphine concentration
b-wave implicit time (ms) Before
After
% Change
1 ,uM (n = 4) 10#M (n = 5) t00~tM (n = 6) 1 mM (n = 5)
85.17 _+26.30 118.67 + 27.65 119.56 + 37.47 98.93 + 31.02
77.34 + 19.28 87.20 + 23.43 84.89 + 21.18 95.73 _+24.92
9.20 26.52* 29.00* 3.23
* P < 0.05, paired t-test
% DA Depletion
84.25 + 87.40 + 90.83 + 92.20 +
18.30 10.88 6.68 14.81
191 crease both the size of the oscillatory potentials and the b-wave implicit time and these effects were reversed by apomorphine. There are apparently no other reports of changes in b-wave implicit time in the literature, although we note that Figs. 1 and 4 of H e m p e P showed increases in b-wave implicit time in reserpine treated rabbits.
hanced oscillatory potentials in D A depleted retinas which are similar to effects produced by increasing stimulus intensity8,13, 2o,21. These results provide further evidence for such a role for amacrine cells. O u r morphological observations that T H immunoreactive amacrine cells have widely-spreading processes
Although a- and b-wave amplitudes were de-
in the IPL are also consistent with this role. However the exact synaptic relationships that these amacrine
creased, horizontal cell responses remained unchanged. In view of the apparently normal retinal
cells have in this hypothesized amacrine cell feedback system is u n k n o w n .
morphology, lack of change in serotonin-like immunoreactive cells, unchanged serotonin content and
This work was supported by N I H Grants EY04711
normal horizontal cell electrophysiology, we con-
and EY00250 to M.C.C. and EY04067, AM17328 and an Alfred P. Sloan fellowship to N.C.B. We
clude that the changes we observed in the E R G s from depleted retinas are due to D A depletion. Amacrine cells are thought to be involved in feedback circuits responsible for initiating oscillatory potentials j3.20, and also to play a role in light adaptation('. Dopaminergic amacrine cells have been implicated in both of these processes8,20. We report en-
1 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 2 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P. C. Emson (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 85-121. 3 Brecha, N. C., Oyster, C. W. and Takahashi, E. S., Identification and characterization of tyrosine hydroxylase immunoreactive amacrine cells, Invest. Ophthalmol. Vis. Sci., 25 (1984) 66-70. 4 Co, C., Smith, J. E. and Lane, J. D., Use of a single compartment LCEC cell in the determinations of biogenic amine content and turnover, Pharmacol. Biochem. Behav., 16 (1982) 641-646. 5 Cohen, J., Hadjiconstantinou, M. and Neff, N. H., Activation of dopamine-containing amacrine cells of retina: lightinduced increase of acidic dopamine metabolites, Brain Research, 260 (1983) 125-127. 6 Dowling, J. E., The site of visual adaptation, Science, 155 (1967) 273-279. 7 Gutierrez, C. O. and Spiguel, R. D., Oscillatory potentials of the cat retina: effects of adrenergic drugs, Life Sci., 13 (1973) 991-999. 8 Hempel, F. G., Modification of the rabbit electroretinogram by reserpine, Ophthal. Res., 4 (1972/73) 65-75. 9 luvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. 10 Jagadesh, J. M., Lee, H. C. and Salazar-Bookaman, M., Influence of chlorpromazine on the rabbit electroretinogram, Invest. Ophthalmol. Vis. Sci., 19 (1980) 1449-1456. 11 Jagadesh, J. M. and Sanchez, R., Effects of apomorphine on the rabbit electroretinogram, Invest. Ophthalmol. Vis.
thank Linda Schultz and Geoff R u b i n for assisting with the m o n o a m i n e analysis, Drs. A. W. T a n k and N. Weiner for the antisera directed to tyrosine hydroxylase, and Dr. T. E. Ogden for helpful comments on this manuscript.
Sci., 21 (1981)620-625. 12 Kramer, S. G., Dopamine in Retinal Transmission. In S. L. Bonting (Ed.), Transmitters in the Visual Process, Pergamon Press, Oxford, 1976, pp. 165-198. 13 Ogden, T. E., The oscillatory wave of the primate electroretinogram, Vision Res., 13 (1973) 1059-1074. 14 Osborne, N. N., Binding of 3H-ADTN, a dopamine agonist, to membranes of the bovine retina, Cell. Mol. Neurobiol., 1 (1981) 167-174. 15 Osborne, N. N., Nesselhut, T., Nicholas, D. A., Patel, S. and Cuello, A. C., Serotonin-containing neurons in vertebrate retinas, J. Neurophysiol., 39 (1982) 1519-1528. 16 Proll, M. A., Kamp, C. W. and Morgan, W. M., Use of liquid chromatography with electrochemistry to measure effects of varying intensities of white light on DOPA accumulation in rat retinas, Life Sci., 30 (1982) 11-19. 17 Shellenberger, M. K. and Gordon, J. H., A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas, Anal. Biochem., 39 (1971) 356-372. 18 Spano, P. F., Govoni, S., Hofmann, M., Kumakura, K. and Trabucchi, M., Physiological and pharmacological influences on dopaminergic receptors in the retina. In E. Costa and G. L. Gessa (Eds.), Advances in Biochemical Psychopharmacology, Vol. 16, Raven Press, New York, 1977, pp. 307-310. 19 Straschill, M. and Perwein, J., The inhibition of retinal ganglion cells by catecholamines and gamma-aminobutyric acid, Pfliigers Arch., 312 (1969)45-54. 20 Wachtmeister, L. and DoMing, J. E., The oscillatory potentials of the mudpuppy retina, Invest. Ophthalmol. Vis. Sci., 17 (1978) 1176-1188. 21 Yonemura, D. and Hatta, M., Studies of the minor components of the frog's electroretinogram, Jap. J. Physiol., 16 (1966) 11-22.
186
Elsevier
BRE 21090
Modifioation of electroreti~rams in dopamine-depleted retinas MARK C. CITRON l, LYNDA ERINOFF 1, DENNIS W. RICKMANl and NICHOLAS C. BRECHA2-4 lChildrens Hospital of Los Angeles, University of Southern California, Department of Neurology, Los Angeles, CA 90054, 2Centerfor Ulcer Research and Education, Veterans Administration Center-Wadsworth, Los Angeles, CA 90073, 3Jules Stein Eye Institute, Department of Medicine and 4Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.)
(Accepted May 14th, 1985) Key words: electroretinogram - - dopamine - - amacrine cell - - oscillatory potential - - b-wave - retina - - frog - - tyrosine hydroxylase-like immunoreactivity
The functional role of dopamine in frog retina was examined in a combined neurochemical, immunohistochemical and electrophysiological study. Dopamine and serotonin are the primary monoamines present in the retina and they are localized to amacrine cells which have distinct morphologies. Intravitreal injection of 6-hydroxydopamine was found to produce a selective depletion of retinal dopamine content and elimination of tyrosine hydroxylase-like immunoreactivity. Electroretinograms from 6-hydroxydopaminetreated retinas demonstrated enhanced oscillatory potentials and a lengthening of the b-wave implicit time compared to vehicle control retinas; both of these changes in the electroretinogram were reversed by the dopamine agonist apomorphine. These observations support earlier suggestions that dopamine-containing amacrine cells are part of a retinal feedback system that generates oscillatory potentials and plays a role in light adaptation.
The predominant catecholamine in the vertebrate retina is dopamine ( D A ) 2. Biochemical studies have demonstrated significant concentrations of D A in all retinas as well as the existence of its biosynthetic enzyme, tyrosine hydroxylase, high affinity D A uptake mechanisms, and specific D A receptor sites. Anatomical studies have demonstrated the presence of catecholamine fluorescent, tyrosine hydroxylaselike (TH) immunoreactive, and DA-accumulating amacrine and/or interplexiform cells. Tyrosine hydroxylase activity as well as D A release are increased in response to lightS,9A2. Furthermore. this increase in D A synthesis appears to be directly related to light intensity 16. These neurochemical and anatomical data strongly suggest that D A plays a functional role in the retina. however electrophysiological evidence for this role is sparse and contradictory. That is, studies on the effects of D A and dopaminergic drugs on the electroretinogram ( E R G ) have reported conflicting results. Apomorphine H, a D A agonist, and reserpine 8. a
monoamine depleter, diminished the b-wave amplitude in rabbit E R G : while chlorpromazine 1°, a neuroleptic with D A receptor blocking activity, increased b-wave amplitude. In contrast, reserpine 7 and L - D O P A 19. a D A precursor, had no effect on b-wave amplitude in cat E R G . Oscillatory potentials, another component of the E R G . are low amplitude wavelets superimposed on the b-wave that are thought to be generated in the inner retina 13.21. Reserpine decreased oscillatory potentials in cat 7 and rabbits and L - D O P A reversed this effect. However. direct application of D A to m u d p u p p y retina decreased oscillatory potentials 20. The present study was directed towards a better understanding of the functional role of D A in the frog retina. This study analyzed the effects of 6-hydroxydopamine ( 6 - O H D A ) - i n d u c e d D A depletion on the E R G recorded from a frog eyecup preparation. The effect of apomorphine, a D A agonist which stimulates presumed retinal postsynaptic D A receptors14,18, on the E R G of 6-OHDA-treated retinas was
Correspondence: M. C. Citron. Childrens Hospital of Los Angeles. Neurology Research. PO Box 54700. Los Angeles. CA 90054. U.S.A.
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
187 determined. We assessed the specificity of 6 - O H D A treatment by determining D A and serotonin concentrations and the presence of TH and serotonin-like immunoreactivity. Frogs (Rana pipiens) were anesthetized with a 0.7-1.0 ml injection of 30 mM ethyl m-aminobenzoate (Tricane, Sigma) in frog Ringers into the dorsal lymph sac before the intravitreal administration of 6O H D A (Sigma). The following injection protocol was used: on two successive days, 100 ~tg 6 - O H D A (free base) in 5 ~tl 0.9% saline with 0.1% ascorbate was injected into the right eye using a 26-gauge needle; the left eye was injected with an equal volume of the vehicle. Following at least a 1-week survival period the frogs were pithed, the eyes were removed, and the retinas were analyzed using neurochemical, immunohistochemical or electrophysiological techniques as described below. Monoamines were extracted from isolated retinas by a modification of the method of Shellenberger and Gordon 17 (alumina adsorption of catecholamines and solvent extraction of serotonin) with dihydroxybenzylamine and N-methyl serotonin used as internal standards. The samples were then analyzed by high performance liquid chromatography with electrochemical detection 4. The limits of detection of this assay are 100 pg for monoamines and 300 pg for dihydroxyphenylacetic acid (DOPAC). Protein content of the retinas was determined by the Bradford~ method (Bio-Rad, Richmond, CA). Normal, vehicle control, and 6-OHDA-treated retinas were immersion-fixed in 4% paraformaldehyde, 0.1 M D,L-lysine and 0.01 M sodium periodate in 0.1 M phosphate buffer for 2 h at room temperature. The retinas were washed overnight in 25% sucrose in 0.1 M phosphate buffer at 4 °C until processing using standard immunohistochemical techniques 3. Retinas were either sectioned perpendicular to the vitreal surface at 10-16 ~tM with a cryostat and mounted onto gelatin-coated slides or treated freefloating. Retinal sections or whole retinas were washed in 0.1 M phosphate buffer or 0.5 M Tris-HC1 buffer (pH 7.4). After incubation in diluted primary antiserum directed to tyrosine hydroxylase (1:250) or serotonin (1:500) (0.5-1.0% Triton X-100 in buffer) for 24-96 h at 4 °C, they were washed in buffer for 30-60 min. Retinal sections were then incubated in
IgG-FITC (1:50) ( D A K O ) for 2 h at room temperature, washed in buffer and then coverslipped with a glycerol-phosphate buffer (9:1) mixture. Whole retinas were incubated in IgG (1:50) ( D A K O ) at 4 °C, washed in buffer and subsequently incubated in peroxidase-antiperoxidase (1:50) (DAKO). The retinas were then washed, preincubated in 3,3'-diaminobenzidine HCI (DAB) (50 mg/100 ml) for 30 min and incubated in DAB with 0.01% H202 for an additional 30 min. Whole retinas were then washed, mounted, dehydrated and coverslipped. Specificity of the antisera was assessed by substituting normal rabbit serum in place of the primary antiserum. Since control experiments cannot exclude the possibility that the immunoreactive material is similar but not identical to tyrosine hydroxylase or serotonin, the staining observed is referred to as THor serotonin-like. ERGs were recorded from an eyecup preparation taken from a light adapted frog (6.5 ktW/cm 2 room illumination) using an LSI-11/23 computer with a 12bit data-acquisition module at a digitizing rate of 1 ms. The vitreous was removed prior to recording and the retina was maintained in humidified 100% oxygen at room temperature. The stimulus light was a quartz halogen lamp (GE no. DNE) which produced an intensity of 30 ~W/cm 2 (2.3 x 1030 photons/(cm 2' s) at 473 nm)) in the plane of the retina though a fiber-optic guide. The duration of the flash was 10 ms and was controlled by an electronic shutter (Vincent Associates, 310-B). Ambient illumination during the experiment was 0.03 ktW/cm 2. A silver ball electrode was placed on the retinal surface halfway between the optic nerve head and the periphery. One E R G was recorded every 4 s, and, typically, 30-50 E R G s were recorded. The eyecup was then washed with a drop of frog Ringers and an additional 30-50 E R G s recorded. The Ringers was then replaced by a 1 ~M, 10 ktM, 100 ,uM or 1.0 mM solution of freshly prepared apomorphine (Sigma) in Ringers and a final 30-50 ERGs were recorded. Following completion of the E R G recordings, the retinas were removed from the eyecup and frozen at -80 °C for subsequent monoamine determination. Each group of 30-50 E R G s were averaged to create the averaged E R G used for identifying the amplitude of the b-wave peak and its implicit time (time from stimulus to b-wave peak). The bin in the aver-
188 aged E R G with the largest positive digital value was judged to be the b-wave peak. This bin was then used to determine the b-wave implicit time with 1 ms resolution. Single unit responses of horizontal cells to 0.5 s flashes of the same stimulus as used for the E R G s were r e c o r d e d with a glass m i c r o e l e c t r o d e filled with 2.0 M KC1. Response waveforms were r e c o r d e d to assess the viability of the o u t e r retina. The concentration of D A in vehicle control retinas was 15.5 + 3.9 ng/mg protein (mean _+ S . D . , n = 28), which did not differ from D A content m e a s u r e d in normal retinas. No o t h e r catecholamines or D O P A C were detected. Immunohistochemical studies demonstrated the presence of T H immunoreactive cells in the normal and vehicle control retinas, l m m u n o r e active s o m a t a were present in the proximal inner nuclear layer (INL) near or at the b o r d e r of the inner plexiform layer (IPL) and their processes were observed only in the IPL suggesting they are amacrine cells. T H immunoreactive processes f o r m e d a prominent continuous plexus in lamina 1 of the IPL adjacent to the I N L and a less p r o m i n e n t , discontinuous plexus in lamina 5. Processes were also sparsely distributed in o t h e r regions of the IPL. In addition, occasional immunoreactive s o m a t a were located in the ganglion cell layer, and these cells gave rise to processes that ramified in the IPL, suggesting that they are displaced amacrine cells. In retinal whole mounts, ellipsoidal T H i m m u n o r e active somata were distributed in all regions of the retina. These cells measured 12.1 _+ 1.4~m by 14.0 _+ 1.5 ~ m in diameter; the mean area was 133.1 ,urn 2 with a range of 84.1 to 176.3 ktm2 (mean + S . D . , n = 15). I m m u n o r e a c t i v e cells gave rise to 3 or 4 primary processes, the majority of which ramified in lamina 1. These processes in turn gave rise to secondary processes that also ramified in lamina 1 (Fig. 1A) and a p p e a r e d to form rings a r o u n d unstained somata. In addition, the primary processes gave rise to thick processes that passed through the I P L to lamina 5 of the IPL (Fig. 1B). The density of processes in lamina 5 was less than that seen in lamina 1. Serotonin content of vehicle control and 6O H D A - t r e a t e d retinas was 69.7 + 34.3 and 55.0 +_ 19.7 ng/mg protein (mean + S . D . , n -- 28), respectively, and did not differ from each other or normal retinas. Serotonin-like immunoreactive amacrine
Fig. 1. TH immunoreactivity in a retinal whole mount preparation focused at lamina 1 (A) and at lamina 5 (B) of the IPL. A: TH immunoreactive somata (arrows) in the proximal INL at the border of the IPL. B: coarse immunoreactive processes distributed in lamina 5 of the IPL. Arrows in B show the position of the somata illustrated in A. Calibration bat = 25 gem.
cells were also distributed in all regions of the retina. I m m u n o r e a c t i v e s o m a t a were located in the I N L at the b o r d e r of the IPL and processes were distributed across all laminae of the IPL. These observations are consistent with another r e p o r t of serotonin-like immunoreactivity in the frog retina 15. Neurochemical studies d e m o n s t r a t e d that D A was selectively d e p l e t e d by intravitreal injection o f 6O H D A . Different dosage regimens of 6 - O H D A (2 x 200/~g/10/~1, 2 x 100 ag/10 #l, and 2 x 100/~g/5/~l)
189 TABLE I i,
Effects of intravitreal 6-hydroxydopamine treatment (2 times 100/~g/5/~l) on retinal dopamine and serotonin content Postinjection Survival
n
3days 7 1 week 7 2weeks 4 2-3weeks 10
I:
!!,
Dopamine +_S.D. (ng/mg protein)
Serotonin + S . D . (ng/mg protein)
Control
Treated
Control
Treated
il~i::i
i!
( Retina
15.4 +_ 4.2 16.1 +_ 5.8 16. l_+4.0 14.4+-3.1
1.5 +_ 4.0* 4.2 + 5.3* 1.4+-1.0" 1.0+ 1.8"
58.3 +_ 16.9 95.6+57.1 76.1 +_20.3 54.0+ 12.9
43.9 _+ 13.7 59.8+20.6 67.4+_ 14.6 57.4+23.1
'',f,,'t'i\!! , , ]iii,,~ , ',
i!',, ..... ~', ',~ ',~, ~
6-HDA
I', i"
Apomorphine
( 100 ,urn )
-~181R03
)
Treated
( Retina-~181R01
)
* P < 0.05
w e r e i n v e s t i g a t e d , a n d t w o d o s e s o f 100 g g 6 - O H D A in 5 B1 i n j e c t e d o n s u c c e s s i v e d a y s w e r e f o u n d to p r o duce greater than 70% depletion of retinal DA while s e r o t o n i n c o n t e n t w a s u n c h a n g e d c o m p a r e d to v e h i cle c o n t r o l s ( T a b l e I). D A c o n t e n t o f t h e r e t i n a w a s r e d u c e d as e a r l y as 3 d a y s a f t e r 6 - O H D A
administra-
t i o n a n d D A levels s h o w e d n o signs of r e c o v e r y u p to 3 weeks after treatment.
Fig. 3. The effect of apomorphine on ERG shown in Fig. 2. Curve from 6-OHDA-treated eve (solid line) is the same as in Fig. 2. The retina was treated with 100uM apomorphine and 30 more ERGs were averaged (dashed line). Calibration - 100 ms, 100 ~V.
lmmunohistochemical studies demonstrated that 7 days a f t e r 6 - O H D A
t r e a t m e n t (2 x 1 0 0 / ~ g / 5 / d ) all
T H i m m u n o r e a c t i v i t y w a s e l i m i n a t e d in t h e r e t i n a . T h i s t r e a t m e n t d i d n o t h a v e a n o t i c e a b l e effect, at least at t h e light m i c r o s c o p i c level, o n e i t h e r r e t i n a l h i s t o l o g y o r t h e d i s t r i b u t i o n of s e r o t o n i n - l i k e i m m u -
ij i
noreactivity.
Jl
Control I
( Retina
Ii
II iI i I
ii iJ i
,!!
All of t h e r e t i n a s u s e d in t h e E R G
,
6-HDA
i I
i, '
' ~, I
I
ii
Ii
(Retina
~181L01
scribed below were analyzed for DA and serotonin
)
content. Only 6-OHDA-treated
Treated #
181R01
)
r e t i n a s w i t h D A de-
p l e t i o n s o f g r e a t e r t h a n 7 0 % w e r e c h o s e n for e l e c t r o p h y s i o l o g i c a l analysis. N o d i f f e r e n c e s w e r e s e e n be-
i I
s t u d i e s de-
I
t w e e n t h e E R G s of n o r m a l a n d v e h i c l e c o n t r o l retinas. In v e h i c l e c o n t r o l r e t i n a s t h e m e a n a - w a v e a m -
"Li
~l 1t 1
p l i t u d e was 0.167 + 0.07 m V a n d t h e b - w a v e w a v e , . . . . . . . . .
o,,
a m p l i t u d e was 0.209 _+ 0.07 m V ( m e a n + S . D . , n = 20). In 6 - O H D A - t r e a t e d
r e t i n a s t h e a m p l i t u d e of t h e
a - w a v e was d e c r e a s e d to 0.092 + 0.06 m V a n d t h e
II I I
a m p l i t u d e of t h e b - w a v e was d e c r e a s e d to 0.104 _+ 0.09 m V ( m e a n + S . D . , n = 20, P ~< 0.01). N o r m a l b-
Fig. 2. The effect of retinal DA depletion on frog ERG recorded from the surface of the eyecup. Each curve is the average of 30 ERGs recorded from the control eye (solid line) and 6OHDA-treated eye (dashed line) of the same frog. The duration of the stimulus flash was 10 ms. Note increased amplitude of oscillatory potentials and increased b-wave implicit time after DA depletion. Calibration = 100 ms, 100 uV.
w a v e i m p l i c i t t i m e w a s 68.5 + 9.39 m s S . D . In 6OHDA-treated
r e t i n a s t h e t i m e to t h e b - w a v e p e a k
i n c r e a s e d to 105.1 + 32.5 m s ( m e a n _+ S . D . , n = 20, P ~< 0.01). T h e t i m e c o u r s e o f t h e a - w a v e r e m a i n e d u n c h a n g e d . A n i n c r e a s e in t h e b - w a v e i m p l i c i t t i m e was o b s e r v e d in all D A d e p l e t e d r e t i n a s t e s t e d (n -
I9(t 20). The oscillatory potentials were increased in 14 out of 20 6-OHDA-treated retinas relative to control. Horizontal cell responses were recorded from the same retinas used for the E R G analysis. In contrast to the amplitude changes in the E R G of 6 - O H D A treated retinas, the number of horizontal cells encountered was the same as in normal retinas and these horizontal cells showed typical hyperpolarizations in response to a full-field stimulus. Representative E R G s from each eye of a frog are shown in Figs. 2 and 3. The vehicle control retina (solid line) had an a-wave implicit time of 20 ms and was followed by a b-wave that peaked at 52 ms (Fig. 2). The D A depleted retina (broken line) showed larger oscillatory waves than the control, and the implicit time had increased to 62 ms. The reversal of the effects of D A depletion by the D A agonist, apomorphine (100/,M) are shown in Fig. 3. The implicit time of the b-wave decreased to approach values measured in the vehicle control retina; apomorphine treatment decreased b-wave implicit time in the D A depleted retina to only 5 ms greater than that of the vehicle control. The heightened oscillatory potentials were also decreased by apomorphine. Table I1 presents the change in b-wave implicit time in 6 - O H D A - t r e a t e d retinas before and after treatment with apomorphine. Only 10 and 100 ,uM concentrations of apomorphine produced a significant decrease in b-wave implicit time. In addition, the application of 100/~M apomorphine significantly decreased the b-wave implicit time of vehicle control retinas from 60.2 to 52.9 _+ 21.1 ms (mean _+ S.D., n = 6, P < 0.05, paired t-test). A p o m o r p h i n e reversed the increased oscillatory potential in 9 of 14 retinas. Oscillatory potentials in control retinas were too small to detect any decrease caused by apomorphine. A p o m o r p h i n e was without effect on the a- or
b-wave amplitudes of either the control ~r treated retinas. These studies demonstrate that D A is the principal catecholamine in Rana pipiens; we were unable to detect epinephrine, norepinephrine or D O P A C with our assay suggesting that they are either at low levels or not present. Serotonin levels were several fold higher than D A levels. In contrast, Osborne 14 reported approximately equal concentrations of these monoamines in the frog retina. 6 - O H D A treatment resulted in a selective and long-lasting depletion of retinal D A , but no changes were seen in serotonin levels. Immunohistochemical studies support these general observations. T H immunoreactivity is present in wide-field amacrine cells whose processes are distributed primarily to lamina 1. No immunoreactivity is present 7 days after 6 - O H D A treatment. In contrast, serotonin-like immunoreactive amacrine cells, as well as general retinal histology, appear to be unaffected by the 6 - O H D A treatment. These observations suggest that the 6 - O H D A treatment regime had a specific effect only upon dopaminergic amacrine cells in the frog retina. This is the first electrophysiological study to use the frog eyecup preparation to examine the effects of 6 - O H D A induced D A depletion. Depletions were verified both neurochemically and histochemically. The physiological effects of this treatment were examined using an eyecup preparation which has the advantage of enabling localized drug application, thus avoiding the complications of systemic drug administration. Studies on the role of D A in retinal function have produced conflicting dataT,S,lO,n, t920. It is difficult to compare the present results to those of other studies since there are marked methodological and species differences. The effects of D A depletion on the E R G were to in-
TABLE II Effect of apomorphine on b-wave implicit time in 6-hydroxydopamine treated retinas Apomorphine concentration
b-wave implicit time (ms) Before
After
% Change
1 ,uM (n = 4) 10#M (n = 5) t00~tM (n = 6) 1 mM (n = 5)
85.17 _+26.30 118.67 + 27.65 119.56 + 37.47 98.93 + 31.02
77.34 + 19.28 87.20 + 23.43 84.89 + 21.18 95.73 _+24.92
9.20 26.52* 29.00* 3.23
* P < 0.05, paired t-test
% DA Depletion
84.25 + 87.40 + 90.83 + 92.20 +
18.30 10.88 6.68 14.81
191 crease both the size of the oscillatory potentials and the b-wave implicit time and these effects were reversed by apomorphine. There are apparently no other reports of changes in b-wave implicit time in the literature, although we note that Figs. 1 and 4 of H e m p e P showed increases in b-wave implicit time in reserpine treated rabbits.
hanced oscillatory potentials in D A depleted retinas which are similar to effects produced by increasing stimulus intensity8,13, 2o,21. These results provide further evidence for such a role for amacrine cells. O u r morphological observations that T H immunoreactive amacrine cells have widely-spreading processes
Although a- and b-wave amplitudes were de-
in the IPL are also consistent with this role. However the exact synaptic relationships that these amacrine
creased, horizontal cell responses remained unchanged. In view of the apparently normal retinal
cells have in this hypothesized amacrine cell feedback system is u n k n o w n .
morphology, lack of change in serotonin-like immunoreactive cells, unchanged serotonin content and
This work was supported by N I H Grants EY04711
normal horizontal cell electrophysiology, we con-
and EY00250 to M.C.C. and EY04067, AM17328 and an Alfred P. Sloan fellowship to N.C.B. We
clude that the changes we observed in the E R G s from depleted retinas are due to D A depletion. Amacrine cells are thought to be involved in feedback circuits responsible for initiating oscillatory potentials j3.20, and also to play a role in light adaptation('. Dopaminergic amacrine cells have been implicated in both of these processes8,20. We report en-
1 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 2 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P. C. Emson (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 85-121. 3 Brecha, N. C., Oyster, C. W. and Takahashi, E. S., Identification and characterization of tyrosine hydroxylase immunoreactive amacrine cells, Invest. Ophthalmol. Vis. Sci., 25 (1984) 66-70. 4 Co, C., Smith, J. E. and Lane, J. D., Use of a single compartment LCEC cell in the determinations of biogenic amine content and turnover, Pharmacol. Biochem. Behav., 16 (1982) 641-646. 5 Cohen, J., Hadjiconstantinou, M. and Neff, N. H., Activation of dopamine-containing amacrine cells of retina: lightinduced increase of acidic dopamine metabolites, Brain Research, 260 (1983) 125-127. 6 Dowling, J. E., The site of visual adaptation, Science, 155 (1967) 273-279. 7 Gutierrez, C. O. and Spiguel, R. D., Oscillatory potentials of the cat retina: effects of adrenergic drugs, Life Sci., 13 (1973) 991-999. 8 Hempel, F. G., Modification of the rabbit electroretinogram by reserpine, Ophthal. Res., 4 (1972/73) 65-75. 9 luvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. 10 Jagadesh, J. M., Lee, H. C. and Salazar-Bookaman, M., Influence of chlorpromazine on the rabbit electroretinogram, Invest. Ophthalmol. Vis. Sci., 19 (1980) 1449-1456. 11 Jagadesh, J. M. and Sanchez, R., Effects of apomorphine on the rabbit electroretinogram, Invest. Ophthalmol. Vis.
thank Linda Schultz and Geoff R u b i n for assisting with the m o n o a m i n e analysis, Drs. A. W. T a n k and N. Weiner for the antisera directed to tyrosine hydroxylase, and Dr. T. E. Ogden for helpful comments on this manuscript.
Sci., 21 (1981)620-625. 12 Kramer, S. G., Dopamine in Retinal Transmission. In S. L. Bonting (Ed.), Transmitters in the Visual Process, Pergamon Press, Oxford, 1976, pp. 165-198. 13 Ogden, T. E., The oscillatory wave of the primate electroretinogram, Vision Res., 13 (1973) 1059-1074. 14 Osborne, N. N., Binding of 3H-ADTN, a dopamine agonist, to membranes of the bovine retina, Cell. Mol. Neurobiol., 1 (1981) 167-174. 15 Osborne, N. N., Nesselhut, T., Nicholas, D. A., Patel, S. and Cuello, A. C., Serotonin-containing neurons in vertebrate retinas, J. Neurophysiol., 39 (1982) 1519-1528. 16 Proll, M. A., Kamp, C. W. and Morgan, W. M., Use of liquid chromatography with electrochemistry to measure effects of varying intensities of white light on DOPA accumulation in rat retinas, Life Sci., 30 (1982) 11-19. 17 Shellenberger, M. K. and Gordon, J. H., A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine, and 5-hydroxytryptamine from discrete brain areas, Anal. Biochem., 39 (1971) 356-372. 18 Spano, P. F., Govoni, S., Hofmann, M., Kumakura, K. and Trabucchi, M., Physiological and pharmacological influences on dopaminergic receptors in the retina. In E. Costa and G. L. Gessa (Eds.), Advances in Biochemical Psychopharmacology, Vol. 16, Raven Press, New York, 1977, pp. 307-310. 19 Straschill, M. and Perwein, J., The inhibition of retinal ganglion cells by catecholamines and gamma-aminobutyric acid, Pfliigers Arch., 312 (1969)45-54. 20 Wachtmeister, L. and DoMing, J. E., The oscillatory potentials of the mudpuppy retina, Invest. Ophthalmol. Vis. Sci., 17 (1978) 1176-1188. 21 Yonemura, D. and Hatta, M., Studies of the minor components of the frog's electroretinogram, Jap. J. Physiol., 16 (1966) 11-22.