- Email: [email protected]
A parallel change of taurine and the ERG in the developing rat retina
030~4492:82’030109-03803.00/o C 1982Pergamon Press Ltd
Camp. Biochem. Phpiol. Vo!. 72C. No. 1. pp. 109-l I I, 1982 Prmted in Great Britain
A PARALLEL CHANGE OF TAURINE AND THE ERG IN THE DEVELOPING RAT RETINA RONALD PABMER.’ KHALID H. SHEIKH,~ WILLIAM W. DAWSON’ and PHILIP P. TOSKES* ‘Departments
of Ophthalmology and Physiology; and ‘Department of Medicine. College of Medicine. University of Florida. Gainesville. FL 32610. U.S.A. (Receioed 25 August 1981)
Abstract-l. Electroretinogram (ERG) amplitude and retinal taurine concentration were determined in rats of various ages. 2. ERG b-wave amplitude and retinal taurine concentration show a parallel change in rats of 30-100 days of age. 3. ERG b-wave amplitude and retinal taurine concentration decreased in lOO-day-old as compared to 30-day-old rats. 4. ERG a- and b-wave implicit time decreased in lOO-day-old as compared to 30-day-old rats.
INTRODUCTION Taurine is present in high concentration in the retinas of many species (Orr er al., 1976; Pasantes-Morales et al., 1972a). It is located in each of the layers of the retina in varying concentration (Voaden et al., 1977; Kennedy et al., 1977). Taurine has been implicated in the process of neural communication, possibly as an inhibitory transmitter (Pasantes-Morales et al., 1972b; Bonaventure et al., 1976). Others are less sure of its function (Huxtable, 1980). Taurine has been shown to be necessary for structural integrity of the retina and normal electrical responsiveness (ERG) of the retina (Schmidt et al., 1976; Berson et al., 1976). Ma&one et al. (1974) and Baskin et al. (1977) report variations of taurine level with age in the rat retina which is expected if taurine is a factor in retinal function. We have measured the electroretinogram (ERG) and the retinal taurine content of pigmented rats at various ages. We find that the ERG and retinal taurine content exhibit a parallel change over the time period reported. METHODS Male Long-Evans hooded rats (Rattus noruegicus) 30-100 days of age constituted the population sample. Animals were drawn periodically from the sample to measure cross-sectionally the developmental variation of taurine concentration and the ERG. Following ERG measurements, the eyes were removed and taurine was measured in the retinas which produced the ERGS. Light level in the animals home cage was maintained at 0.4 log fL with a 12:12 1ight:dark cycle. Subjects were fed standard Purina rat chow. Prior to ERG testing, rats were anesthetized with sodium pentobarbital (35-40 mg/kg). Pupils were dilated with Mydriacyl (Tropicamide 1%) and Neo-Synephrine (Phenylephrine HCl 10%). A plastic contact lens with a platinum electrode was fitted to each eye. A Sn Ag-AgCI reference electrode was placed on the scalp between the eyes, and a ground electrode was attached to the animal’s tail. These procedures were carried out under white tungsten light of l.Olog !L and required ca. 15 min for completion.
The rat was placed in a 160; ganzfeld and was maintamed at an adaptation level of 2.2 log fL (tungsten source) for 15 min prior to recording and during the subsequent presentation of stimuli. Stimuli were a series of flashes from a xenon source (Grass PS2) which diffusely illuminated the geld. Xenon flash stimuli of lO-psec duration were presented binocularly (20-set interflash period) at peak power settings from 0.015 to 3,0mW/cm* (visible band). ERGS were recorded differentially (passband 0.8-l kHz) through a 1000 x amplifier. Eight responses were recorded at each stimulus intensity. The signals were averaged and digitally stored by computer. Rats were anesthetized with an overdose of sodium pentobarbital. Eyes were enucleated immediately after death, anterior and posterior chambers were separated by dissection posterior to the ora serrata, and retinas were detached in normal saline. Retinas were stored at -7O’C or were prepared for immediate analysis. Pooled samples of both retinas from a single animal were used for the analysis. Retinal taurine levels were determined by a modification of a chromatographic assay described by Pentz et al. (1957). The assay is based on the fact that taurine does not adhere to the acid form of Dowex 50 ion exchange resin and that taurine, when coupled with dinitrofluorobenzene (DNFB), forms the colored dinitrophenol derivative, exhibiting maximum absorbance at 355 nm. The retinas were homogenized in 2.5cm3 ice-cold distilled H,O in a chilled Thomas glass homogenizer at 1500 rev/min for 2 min at 0°C. The homogenate was centrifuged at 2000 g x 15 min. The supernatant was assayed for protein content (Bio-Rad method) and was then deproteinized (Bradford, 1976). The supernatant was diluted with potassium acid phthalate buffer to a volume of 10 cm3 and applied to the taurine assay. Taurine content of retina was expressed as fig taurine/mg protein.
Figure
1 presents
RESULTS a comparison
(for 30 and
100
days of age) of the ERG response at each stimulus intensity level (0.015-3.0 mW/cm’). We see a decrease in amplitude of a- and b-waves of 100-day-old relative to 30-day-old rats at each of the intensity levels tested. There is also a decrease in implicit time of both the aand b-waves in the lOO-day-old animals as compared to the 30-day-old 109
animals.
RONALD PARMER et al.
110 r
INTENSITY (mrfcm*)
0.015
I
0.047
a15
1.5
3.0
Fig. 1. ERG waveforms at 30 and 100 days of age for each stimulus intensity. Each waveform represents the digitally combined signals from both eyes of 10 animals.
Examination of the ERG b-wave response amplitudes to the 3.0 mW/cn? stimulus (Fig. 2) indicates an orderly amplitude change during the 30-lOO-day period. ERG data are plotted as mean amplitudes (+ 1 SEM) for 10 animals (both eyes) at each age. Figure 2 demonstrates a parallel change in retinal taurine concentrations which is plotted as means (f 1 SEM) for the same animals (both eyes) at each age. Similar findings were obtained for other stimulus levels. With advancing age, there is a significant decline in both the ERG b-wave [F = 7.2 (3,76 d.f.), P c O.OOl] and in the retinal taurine content [F = 21.7 (3,36 d.f.), P < O.OOl]. Across all age groups, a strong correlation is found between the ERG amplitude and retinal taurine content [r = 0.97, P = 0.001, coefficient of determination (r’) = 0.941. There was a less impressive correlation between the decline in b-wave amplitude and advancing age (r = -0.81, P < 0.05, r2 = 0.66). The correlation of retinal taurine concentration with age was insignificant (r = -0.64, P > 0.10, rz = 0.40).
publications. However, our data on pigmented rats show a much greater decrease in retinal taurine past 30 days of age than did Baskin et al. and Macione er al. with albinos. The adverse effects of high or continuous ambient light on the rat retina, especially the albino, are well established in the recent literature (Lanum, 1978). We used pigmented rats maintained under low-level cyclic lighting to avoid this problem area. It is also possible that the pigmented rat retina ages differently. A causal relation between taurine concentration and retinal function as measured by ERG b-wave cannot be established from our results since the normal level of taurine concentration may be secondary to other age-sensitive factors. It may be for this reason that the inhibitory transmitter role proposed for taurine (Bonaventure er a/., 1976; Pasantes-Morales et al., 1973) is not supported by our findings. Pasantes-Morales et al. (1973) observed a decrease in the ERG b-wave of the chicken, after an intravitreal injection of taurine. Bonaventure et al. (1976) also observed that intravitreal injection of taurine caused a decrease in the ERG b-wave which could be reversed by the subsequent injection of strychnine. The decline of the rat ERG with increasing age is not surprising since this effect is firmly established in humans (Weleber. 1981). The decrease in implicit time, however, is not an expected result. Normally, there is an inverse relation between stimulus intensity and b-wave amplitude so that as the b-wave amplitude decreases, the implicit time increases. This inverse relation between stimulus intensity and response latency is a well-established principle of visual system physiology which has been reviewed by Arm-ington (1974). Therefore, one would expect the implicit time of the lOO-day-old rat ERG to be greater than the implicit time of the 30-day-old rats. At this time,
.
-
70
t
r
.
-60
g
DlSCIJSSlON
We have presented data which compare the normal ERG a- and b-waves and normal retinal taurine levels in rats of varying ages (30-100 days). Previous reports have compared the effects of age on taurine levels in the retina. Macaione et al. (1974) reported an increase in retinal taurine between 5 and 30 days of age, followed by a decrease and stabilization between 30 and 90 days of age. Another report offers general agreement with the work reported by Macaione er al. Baskin et al. (1977) show a slight, although probably not significant, decrease in retinal taurine levels between 60 and 90 days of age. Figure 2 is consistent with both
_
-40 I
I
1
30
50
75
100
AS* (dd
Fig. 2. Mean (f 1 SEM) ERG b-wave amplitude and retinal taurine concentration for each age group. B-wave amplitude is measured from the trough of the first negative component to the peak of the following positive going component of the waveform.
Taurine and ERG changes we cannot explain why the implicit time of both the aand b-waves of the MO-day-old animals has decreased in comparison to the 30-day-old animals while at the same time, the amplitude for the lOO-day-old animals decreased. Weleber (1981) did not find a change in implicit time of the human a- and b-wave with increasing age. Lanum (1981) reviews several studies which have dealt with b-wave amplitude changes in the albino rat. These studies, however, do not examine changes in ERG b-wave implicit time for normal rats. Acknowledgemenr-This grant 5 ROl EY02831.
research was supported by NIH
REFERENCES ARMINGTON J. C. (1974) The Electroretinogram, pp. 28. 255. Academic Press, New York. BAXIX S. I., COHN E. M. & KOCSISJ. J. (1977) The effect of age on taurine levels in eye tissues. Expl Eye Res. 24, 315-319. BERSONE. L., HAYESK. C., RABINA. R., SCHMIDTS. Y. & WATSON G. (1976) Retinal degeneration in cats fed casein. II. Supplementation with methionine, cysteine or taurine. Invest. Ophthalm. 15, 52-58. BONA~ENTUREN., WIOLAND N. & BEZAUT M. (1976) Recent data on two putative inhibitory transmitters in the retina: taurine and GABA. Dot. Ophthalm. Proc. Ser. 10, 213-219. BRADFORDM. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyl. Biothem. 72, 248-254.
111
HUXTABLER. J. (1980) Does taurine have a function? Fedn Proc. Fedn Am. Sots exe. Biol. 9. 2678-2679. KENNEDY A. J., NEAL M.’ J. & L~LLEY R. N. (1977) The
distribution
of amino acids within the rat retina. J.
Neurochem. 29, 157-159.
LANUM J. (1978) The damaging effects of light on the retina. Empirical findings, theoretical and practical implications. Sar. Ophthalm. 22, 221-249. MACAIONES., RUGGERIP., DELLJCAF. & TUCCI G. (1974) Free amino acids in developing rat retina. J. Neurochem. 22, 887-891. ORR H. T., COHENA. I. & LOWRY 0. H. (1976) The distribution of taurine in the vertebrate retina. J. Neurochem. 26, 609-611. PASANTES-MORALES H.. BONAVENTURE N.. WIOLANDN. & MANDELP. (1973) Effect of intravitreal injections of taurine and GABA on chicken electroretinogram. Inr. J. Neurosci. 5, 235-241. PASANTES-MORALES H.. KLETHIJ., LEDIG M. & MANDELP. (1972a) Free amino acids of chicken and rat retina. Brain Res. 41, 494-497. PASANTES-MORALES H., KLETHIJ., URBANP. F. & MANDEL P. (1972b) The physiological role of taurine in retina: uptake and effect on electroretinogram. PhysioL Chem. Phys. 4, 339-348.
PENTZE., DAVENPORT C.. GLOVERW. & SMITHD. (1957) A test for the determination of taurine in urine. J. biol. Chem. 228, 433-444.
SCHMIDTS. Y., BER~ONE. L. & HAYESK. C. (1976) Retinal degeneration in cats fed casein. I. Taurine deficiency. Invest. Ophthalm. 15, 47-52. VOADEHM. J., LAKE N., MARSHALL J. & MORJARIA B. (1977) Studies on the distribution of taurine and other neuroactive amino acids in the retina. Exn[. . Eve . Res. 25, 249-257.
WELEBERR. G. (1981) The effect of age on human cone and rod ganzfeld electroretinograms. Invest. Ophthalm. Vis. Sci. 20, 392-399.
Camp. Biochem. Phpiol. Vo!. 72C. No. 1. pp. 109-l I I, 1982 Prmted in Great Britain
A PARALLEL CHANGE OF TAURINE AND THE ERG IN THE DEVELOPING RAT RETINA RONALD PABMER.’ KHALID H. SHEIKH,~ WILLIAM W. DAWSON’ and PHILIP P. TOSKES* ‘Departments
of Ophthalmology and Physiology; and ‘Department of Medicine. College of Medicine. University of Florida. Gainesville. FL 32610. U.S.A. (Receioed 25 August 1981)
Abstract-l. Electroretinogram (ERG) amplitude and retinal taurine concentration were determined in rats of various ages. 2. ERG b-wave amplitude and retinal taurine concentration show a parallel change in rats of 30-100 days of age. 3. ERG b-wave amplitude and retinal taurine concentration decreased in lOO-day-old as compared to 30-day-old rats. 4. ERG a- and b-wave implicit time decreased in lOO-day-old as compared to 30-day-old rats.
INTRODUCTION Taurine is present in high concentration in the retinas of many species (Orr er al., 1976; Pasantes-Morales et al., 1972a). It is located in each of the layers of the retina in varying concentration (Voaden et al., 1977; Kennedy et al., 1977). Taurine has been implicated in the process of neural communication, possibly as an inhibitory transmitter (Pasantes-Morales et al., 1972b; Bonaventure et al., 1976). Others are less sure of its function (Huxtable, 1980). Taurine has been shown to be necessary for structural integrity of the retina and normal electrical responsiveness (ERG) of the retina (Schmidt et al., 1976; Berson et al., 1976). Ma&one et al. (1974) and Baskin et al. (1977) report variations of taurine level with age in the rat retina which is expected if taurine is a factor in retinal function. We have measured the electroretinogram (ERG) and the retinal taurine content of pigmented rats at various ages. We find that the ERG and retinal taurine content exhibit a parallel change over the time period reported. METHODS Male Long-Evans hooded rats (Rattus noruegicus) 30-100 days of age constituted the population sample. Animals were drawn periodically from the sample to measure cross-sectionally the developmental variation of taurine concentration and the ERG. Following ERG measurements, the eyes were removed and taurine was measured in the retinas which produced the ERGS. Light level in the animals home cage was maintained at 0.4 log fL with a 12:12 1ight:dark cycle. Subjects were fed standard Purina rat chow. Prior to ERG testing, rats were anesthetized with sodium pentobarbital (35-40 mg/kg). Pupils were dilated with Mydriacyl (Tropicamide 1%) and Neo-Synephrine (Phenylephrine HCl 10%). A plastic contact lens with a platinum electrode was fitted to each eye. A Sn Ag-AgCI reference electrode was placed on the scalp between the eyes, and a ground electrode was attached to the animal’s tail. These procedures were carried out under white tungsten light of l.Olog !L and required ca. 15 min for completion.
The rat was placed in a 160; ganzfeld and was maintamed at an adaptation level of 2.2 log fL (tungsten source) for 15 min prior to recording and during the subsequent presentation of stimuli. Stimuli were a series of flashes from a xenon source (Grass PS2) which diffusely illuminated the geld. Xenon flash stimuli of lO-psec duration were presented binocularly (20-set interflash period) at peak power settings from 0.015 to 3,0mW/cm* (visible band). ERGS were recorded differentially (passband 0.8-l kHz) through a 1000 x amplifier. Eight responses were recorded at each stimulus intensity. The signals were averaged and digitally stored by computer. Rats were anesthetized with an overdose of sodium pentobarbital. Eyes were enucleated immediately after death, anterior and posterior chambers were separated by dissection posterior to the ora serrata, and retinas were detached in normal saline. Retinas were stored at -7O’C or were prepared for immediate analysis. Pooled samples of both retinas from a single animal were used for the analysis. Retinal taurine levels were determined by a modification of a chromatographic assay described by Pentz et al. (1957). The assay is based on the fact that taurine does not adhere to the acid form of Dowex 50 ion exchange resin and that taurine, when coupled with dinitrofluorobenzene (DNFB), forms the colored dinitrophenol derivative, exhibiting maximum absorbance at 355 nm. The retinas were homogenized in 2.5cm3 ice-cold distilled H,O in a chilled Thomas glass homogenizer at 1500 rev/min for 2 min at 0°C. The homogenate was centrifuged at 2000 g x 15 min. The supernatant was assayed for protein content (Bio-Rad method) and was then deproteinized (Bradford, 1976). The supernatant was diluted with potassium acid phthalate buffer to a volume of 10 cm3 and applied to the taurine assay. Taurine content of retina was expressed as fig taurine/mg protein.
Figure
1 presents
RESULTS a comparison
(for 30 and
100
days of age) of the ERG response at each stimulus intensity level (0.015-3.0 mW/cm’). We see a decrease in amplitude of a- and b-waves of 100-day-old relative to 30-day-old rats at each of the intensity levels tested. There is also a decrease in implicit time of both the aand b-waves in the lOO-day-old animals as compared to the 30-day-old 109
animals.
RONALD PARMER et al.
110 r
INTENSITY (mrfcm*)
0.015
I
0.047
a15
1.5
3.0
Fig. 1. ERG waveforms at 30 and 100 days of age for each stimulus intensity. Each waveform represents the digitally combined signals from both eyes of 10 animals.
Examination of the ERG b-wave response amplitudes to the 3.0 mW/cn? stimulus (Fig. 2) indicates an orderly amplitude change during the 30-lOO-day period. ERG data are plotted as mean amplitudes (+ 1 SEM) for 10 animals (both eyes) at each age. Figure 2 demonstrates a parallel change in retinal taurine concentrations which is plotted as means (f 1 SEM) for the same animals (both eyes) at each age. Similar findings were obtained for other stimulus levels. With advancing age, there is a significant decline in both the ERG b-wave [F = 7.2 (3,76 d.f.), P c O.OOl] and in the retinal taurine content [F = 21.7 (3,36 d.f.), P < O.OOl]. Across all age groups, a strong correlation is found between the ERG amplitude and retinal taurine content [r = 0.97, P = 0.001, coefficient of determination (r’) = 0.941. There was a less impressive correlation between the decline in b-wave amplitude and advancing age (r = -0.81, P < 0.05, r2 = 0.66). The correlation of retinal taurine concentration with age was insignificant (r = -0.64, P > 0.10, rz = 0.40).
publications. However, our data on pigmented rats show a much greater decrease in retinal taurine past 30 days of age than did Baskin et al. and Macione er al. with albinos. The adverse effects of high or continuous ambient light on the rat retina, especially the albino, are well established in the recent literature (Lanum, 1978). We used pigmented rats maintained under low-level cyclic lighting to avoid this problem area. It is also possible that the pigmented rat retina ages differently. A causal relation between taurine concentration and retinal function as measured by ERG b-wave cannot be established from our results since the normal level of taurine concentration may be secondary to other age-sensitive factors. It may be for this reason that the inhibitory transmitter role proposed for taurine (Bonaventure er a/., 1976; Pasantes-Morales et al., 1973) is not supported by our findings. Pasantes-Morales et al. (1973) observed a decrease in the ERG b-wave of the chicken, after an intravitreal injection of taurine. Bonaventure et al. (1976) also observed that intravitreal injection of taurine caused a decrease in the ERG b-wave which could be reversed by the subsequent injection of strychnine. The decline of the rat ERG with increasing age is not surprising since this effect is firmly established in humans (Weleber. 1981). The decrease in implicit time, however, is not an expected result. Normally, there is an inverse relation between stimulus intensity and b-wave amplitude so that as the b-wave amplitude decreases, the implicit time increases. This inverse relation between stimulus intensity and response latency is a well-established principle of visual system physiology which has been reviewed by Arm-ington (1974). Therefore, one would expect the implicit time of the lOO-day-old rat ERG to be greater than the implicit time of the 30-day-old rats. At this time,
.
-
70
t
r
.
-60
g
DlSCIJSSlON
We have presented data which compare the normal ERG a- and b-waves and normal retinal taurine levels in rats of varying ages (30-100 days). Previous reports have compared the effects of age on taurine levels in the retina. Macaione et al. (1974) reported an increase in retinal taurine between 5 and 30 days of age, followed by a decrease and stabilization between 30 and 90 days of age. Another report offers general agreement with the work reported by Macaione er al. Baskin et al. (1977) show a slight, although probably not significant, decrease in retinal taurine levels between 60 and 90 days of age. Figure 2 is consistent with both
_
-40 I
I
1
30
50
75
100
AS* (dd
Fig. 2. Mean (f 1 SEM) ERG b-wave amplitude and retinal taurine concentration for each age group. B-wave amplitude is measured from the trough of the first negative component to the peak of the following positive going component of the waveform.
Taurine and ERG changes we cannot explain why the implicit time of both the aand b-waves of the MO-day-old animals has decreased in comparison to the 30-day-old animals while at the same time, the amplitude for the lOO-day-old animals decreased. Weleber (1981) did not find a change in implicit time of the human a- and b-wave with increasing age. Lanum (1981) reviews several studies which have dealt with b-wave amplitude changes in the albino rat. These studies, however, do not examine changes in ERG b-wave implicit time for normal rats. Acknowledgemenr-This grant 5 ROl EY02831.
research was supported by NIH
REFERENCES ARMINGTON J. C. (1974) The Electroretinogram, pp. 28. 255. Academic Press, New York. BAXIX S. I., COHN E. M. & KOCSISJ. J. (1977) The effect of age on taurine levels in eye tissues. Expl Eye Res. 24, 315-319. BERSONE. L., HAYESK. C., RABINA. R., SCHMIDTS. Y. & WATSON G. (1976) Retinal degeneration in cats fed casein. II. Supplementation with methionine, cysteine or taurine. Invest. Ophthalm. 15, 52-58. BONA~ENTUREN., WIOLAND N. & BEZAUT M. (1976) Recent data on two putative inhibitory transmitters in the retina: taurine and GABA. Dot. Ophthalm. Proc. Ser. 10, 213-219. BRADFORDM. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyl. Biothem. 72, 248-254.
111
HUXTABLER. J. (1980) Does taurine have a function? Fedn Proc. Fedn Am. Sots exe. Biol. 9. 2678-2679. KENNEDY A. J., NEAL M.’ J. & L~LLEY R. N. (1977) The
distribution
of amino acids within the rat retina. J.
Neurochem. 29, 157-159.
LANUM J. (1978) The damaging effects of light on the retina. Empirical findings, theoretical and practical implications. Sar. Ophthalm. 22, 221-249. MACAIONES., RUGGERIP., DELLJCAF. & TUCCI G. (1974) Free amino acids in developing rat retina. J. Neurochem. 22, 887-891. ORR H. T., COHENA. I. & LOWRY 0. H. (1976) The distribution of taurine in the vertebrate retina. J. Neurochem. 26, 609-611. PASANTES-MORALES H.. BONAVENTURE N.. WIOLANDN. & MANDELP. (1973) Effect of intravitreal injections of taurine and GABA on chicken electroretinogram. Inr. J. Neurosci. 5, 235-241. PASANTES-MORALES H.. KLETHIJ., LEDIG M. & MANDELP. (1972a) Free amino acids of chicken and rat retina. Brain Res. 41, 494-497. PASANTES-MORALES H., KLETHIJ., URBANP. F. & MANDEL P. (1972b) The physiological role of taurine in retina: uptake and effect on electroretinogram. PhysioL Chem. Phys. 4, 339-348.
PENTZE., DAVENPORT C.. GLOVERW. & SMITHD. (1957) A test for the determination of taurine in urine. J. biol. Chem. 228, 433-444.
SCHMIDTS. Y., BER~ONE. L. & HAYESK. C. (1976) Retinal degeneration in cats fed casein. I. Taurine deficiency. Invest. Ophthalm. 15, 47-52. VOADEHM. J., LAKE N., MARSHALL J. & MORJARIA B. (1977) Studies on the distribution of taurine and other neuroactive amino acids in the retina. Exn[. . Eve . Res. 25, 249-257.
WELEBERR. G. (1981) The effect of age on human cone and rod ganzfeld electroretinograms. Invest. Ophthalm. Vis. Sci. 20, 392-399.