J o m u l of Nrurochernisfrg, 1977. Vol. 28, pp. 159-163. Pergarnon Press. Printed in Great Britain
DISTRIBUTION OF FOUR POTENTIAL TRANSMITTER AMINO ACIDS IN MONKEY RETINA SOSAMMA J. BERGER, M. L. MCDANIEL, JOYCEG. CARTER and 0. H. LOWRY Department of Pharmacology and the Beaumont-May Institute of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA. (Received 24 March 1976. Accepted 20 Jury 1976)
Abstract-Discrete layers from frozen dried sections of Rhesus monkey retina were analyzed for each of four amino acids. Peak levels of glycine were found near the border of the inner nuclear and inner reticular layers, and were high throughout these two layers. The levels were less than 50% of the peak in the adjacent ganglion cells and outer reticular layers and fell to very low levels elsewhere. GABA was much more sharply restricted to the inner reticular layer and fell off on both sides to levels of 10% or less of the peak in the fiber and photoreceptor cell layers. Glutamate and aspartate were highest in the ganglion cell layer. On a lipid-free dry weight basis the peak aspartate level was about twice that of brain. Moderately high levels of both aspartate and glutamate were found in the inner reticular and fiber layers. Elsewhere the levels ranged from 20 to 50% of the peak, and both amino acids were relatively low in optic nerve. The amino acid distributions are compatible with a transmitter function for GABA in amacrine cells and for glycine in horizontal and amacrine cells. Glutamate and aspartate may be especially high in Miiller fibers, ganglion cells or both.
THISIS a record of the distribution of glycine, glutamate, aspartate and GABA among the layers of retina et a/. (1968) found in the Rhesus monkey. KURIYAMA GABA and glutamic decarboxylase (EC 4.1.1.15) in rabbit retina to be highest in the ‘ganglion cell layer’ which probably included at least part of the inner (1972) found GABA and glureticular layer. GRAHAM tamic decarboxylase in frog retina to be highest in the inner reticular layer, both fell to low levels in the outer layers (outer nuclear and outer segment (1972) found in the rat that deslayers). MACAIONE truction of the inner retinal layers (by perinatal glutamate excess) caused an 80%loss of GABA and almost complete disappearance of glutamic decarboxylase and GABA transaminase (EC 2.6.1.19).COHENet a/. (1973) confirmed the GABA loss with glutamate treated mice. The uptake of exogenous GABA as seen in radioautographic studies of a variety of species is greatest in inner retinal layers, especially in the inner reticular layer and in the neighborhood of amacrine cells (EHINGER,1970; LAM & STEINMAN,1971; VOADEN et al., 1974; BRUNN& EHINGER, 1974). However, NEAL& IVERSEN (1972) reported uptake to be correlated with the distribution of Muller fibers. BARBER& SAITO (1976) with immunochemical radioautography found glutamic decarboxylase sharply limited to the inner reticular layer. WOODet a!. (1976) with an electron microscopic version of the same technique reported that the enzyme is concentrated at synapses typical for amacrine cells. There is less information concerning glycine distribution. COHENet a/. (1973) presented evidence that in the mouse the inner layers are about 4 times richer in glycine than the outer layers. (Destruction of inner
layers with glutamate reduced the concentration 50%; absence of outer layers due to a genetic defect resulted in a doubling of the concentration.) Radioautographic graphic studies have shown exogenous glycine uptake to be greatest in approximately the same area as for GABA, i.e. in the inner reticular layer and in the inner & FALCK, aspect of the inner nuclear layer (EHINGER 1971; BRUNN& EHINGER, 1974; VOADENet al. (1974). In an early study, glutamate in monkey retina was found t o be fairly evenly distributed, except for high levels in the ganglion cell and fibre layers (LOWRY et al., 1956). GRAHAM et al. (1970) found little difference in glutamate levels among 3 subdivisions of frog retina.
MATERIAL AND METHODS Preparation of samples
Rhesus monkeys were sedated with Phencyclidine (Sernyl) and anesthetized with pentobarbital. The eyes were removed and frozen within 5 or 10 s of the time the blood supply was cut off. Rapid freezing was accomplished by immersing in liquid nitrogen that had been evacuated in a Dewar flask until part of the nitrogen had frozen. This accelerates freezing, because no gas bubbles form on contact with the tissue. The detailed technique for preparing samples for analysis has been described (LOWRYet al., 1961; LOWRY & PASSONNEAU, 1972). Frozen sections, 5 pm in thickness, were cut at - 25” to - 30°C (from a region near the fovea) and dried under vacuum at -40°C. Samples weighing 2G80 ng were dissected from each layer, weighed and transferred into aqueous droplets in ‘oil wells’ (MATSCHINSKY et al., 1968) for the first analytical steps.
159
160
S. J. BERGER,M. L. MCDANIEL, J. G. CARTERand 0. H. LOWRY SHEET FOR TABLEI . FLOW
Step 1
ANALYSES
Step 2
Step 3
Step 4
Step 5
Enzyme destruction
Specific reagents
Destruction of excess pyridine nucleotide
Enzymatic cycling
Indicator step
Glutamate
0.1 p1 20 mM-NaOH
0.5pI 30min RT
0.2 pl 0.5 N-NaOH
0.5 pl to 50 pI cycl. rgt.?
I ml indic. rgt.
Aspartate
0.1 p1 20 mM-NaOH
0.1 pl 30min RT
0.5 pl 0.1 N-HCI
Samet
Same
0.05pI 10 mM-HC1
0.05 p1 40min RT
2 PI 50 mM-NaOH
6~ t l Cycl. rgt.1
6 PI to I ml indic. rgt.
0.05pl
0.05 pl A* 18 h RT 0.1 pI B* 40 min RT
0.1 pl 0.25 N-HCI
64 Cycl. rgt.3
Same
GABA Glycine
10 mM-NaOH
-
Steps 1-3 were carried out in oil wells. In the case of GABA and glycine, Step 4 was also in the oil well. The last step, or steps, was in the fluorometer tube. At Step 1 in all cases, and also at Step 3 for glutamate and GABA, the oil well racks were heated 20min at 80°C. Room temperature (RT) was 20-25°C. * Reagent A contained D-aminO acid oxidase; reagent B contained glyoxylate reductase and NADH. The samples were not heated before adding reagent B. t This step was in the fluorometer tube and cycling was stopped by heating 3 min in a 95°C water bath. $This step was in the oil well and cycling was stopped with 2p1 of 0 . 2 5 ~ - N a O Hfollowed by heating 20min in an oven at 80°C.
Analytical details
The analytical methods are nearly the same as those described in a companion paper for somewhat larger samples (0.24.3 pg dry weight) from brain and spinal cord (BERGERet a/., 1976) except for adjustments to increase sensitivity and reduce blank values. Only the diflerences will be described. A flow sheet (Table 1) describes the logistical details. Glutamate and aspartate. Volumes for the first two steps were cut in half. The reagents were identical to those given previously except that the enzyme concentrations in the NAD cycling reagent were tripled to give greater 'amplification' (300 pg/ml of alcohol dehydrogenase and 30 pg/ml of malic dehydrogenase). Glycine and GABA. Blank problems have been greater with glycine and GABA than with glutamate and aspartate, therefore volumes for the first steps were reduced by a factor of 4 or 5. Cycling was carried out in the oil wells instead of in the final fluorometer tubes. The same NAD cycling reagent (required for the glycine method) was used as above. For NADP (GABA method) the same cycling reagent was used as before, but greater amplification (about 40,000 fold) was achieved by incubating for 4 h at 37°C or overnight at 4°C plus 2 h at 37°C. Source of materials. GABase and D-amino acid oxidase were from Sigma Chem. Co., St. Louis, MO; the other enzymes were from Boehringer Mannheim Corp., New York, NY. Most of the special chemicals were from Sigma. RESULTS The most detailed data are for a single retina (Figs. 1 and 2). Less complete data were obtained from retinas from two other monkeys (Table 2). GABA. This is almost entirely restricted t o the inner reticular layer and the deepest third of the inner
nuclear layer (Fig. 1, Table 2). The peak GABA levels in the inner reticular layer are at least 5 times the brain average. The levels in the ganglion cell layer were more variable than in the other layers. GABA concentrations in the inner reticular layer are similar t o those found by GRAHAM (1972) in the frog, but elsewhere in the retina the levels are much lower than he found. In frog the outer portion of the inner nuclear layer was 50% of the peak, in monkey only
6-30%.
a Epi 0 5
IS
ON
p OR
b IN
c , ,a
b IR
c,
GC Fib
FIG.1. Glycine and GABA in retina of monkey A. The abbreviations for the layers are pigmented epithelium, Epi; outer segments, 0 s ; inner segments, IS; outer nuclear, ON; outer reticular, OR; inner nuclear, IN; inner reticular, IR; ganglion cell, GC; fiber, Fib. The schematic representation of cells in the inner nuclear layer are horizontal cells, H; bipolar cells, B; and amacrine cells, A. The band widths rcprcscnt 2 S.E.M.for an avcrage of 6 or 7 samplcs from each layer or sublayer.
Potential transmitter amino acids in retina
161
TABLE2. FOURAMINO ACIDS IN MONKEY RETINA Glycine
GABA B
Monkey Pig. epi. Outer seg. Inner seg. Outer nucl. Outer retic. a Outer retic. b
0.8
k 1.4 Inner nucl. a
7.2
k 1.3 Inner nucl. b Inner nucl. c
Inner retic. a Inner retic. b Inner retic. c Gangl. cell Fiber
.
5.6 f2.0 18.0 f 1.2 36.4 k 2.4 41.1 f 1.3 32.5 2.5 12.0 f 1.4 3.1 5 1.4
C 2.6 f0.3 3.8 k0.5 2.0 f 0.2 5.4 f0.5 3.8 0.5 4.8 k0.6 6.3 k0.6 10.0 kl.0 15.6 1.2
B
Glutamate C (mmol/kg dry weight)
+
*
21.4.
k 1.9
+
18.1
k 1.0
12.1 k1.4 18.0 fl.1 22.2 k1.4 21.1 f 2.2 25.0 k 1.6 20.4 k2.1 17.0 k 1.7 3.6 f0.1 5.8 f1.6
C
7.9 f1.2 4.3 50.5 4.6 k0.6 3.7 f0.4 12.7 f 1.0 18.5 k0.8 26.0 k1.5 30.3 k1.7 39.9 f1.6
33.3* k1.7
B
28.6 k3.2 39.4
k3.1 28.1 f1.7 30.3 k1.7 49.2 k5.0 53.3 f3.5 48.9 f2.1 83.7 f3.3 40.7 k4.2
Aspartate B
C
lipid
t
%
17.2 *16.0 1.2
26.6 f 1.3 7.5
k0.8 36.9 k0.8 37.5 1.9 36.8 1.6 40.1 k2.0 46.0 & 1.8 45.1 3.2 39.8 k2.7
f0.5 8.4
22 25 24
k0.6
8.5 k1.4 5.8 k0.8 7.1 f1.0 8.0
k1.3
13.0 L-0.5 9.0 k0.5 9.0 k0.8 8.9 k0.7 9.1 f0.6 7.8 k0.7
12 18*
17*
14.3
k0.8 56.9. k3.7 90.3 k4.0 86.4 k 5.2
9.0 k1.7 9.6 2.3 19.5 k2.3 10.2 k0.4
13.2* k1.6
37*
27.4 52.4 17.3 f1.3
20 34
With a few exceptions, each value is the average for 5 or mores samples f S.E.M. The optic nerve of monkey C (sampled where it passes through the sclera) had respective glutamate and aspartate levels ( ~ s . E . M . ) of 34.6 f 0.8 and 5.2 & 0.15 mmol/kg lipid-free dry weight. (The lipid was 51.5 k 0.7% of the total dry weight.) For comparison, on a lipid free basis the peak levels shown in the fiber layer of the same monkey are 130 and 26 mmol/kg. * Values for the whole layer. t Percent of total dry weight, from LOWRYet al. (1956). The lipid content of brain is about 50% of the dry weight. Glycine. This is much less sharply restricted than GABA, however, the range of values (30-fold) is about the same (Fig. 1, Table 2). The broad peak centers at about the boundary between inner nuclear and inner reticular layers where the level is 4-6 times that in average brain. Highest levels are limited to these two layers, although in the ganglion cell and outer reticular layers the levels ranged from 10% to 45% of the peak. Throughout the photoreceptor cells, (except for one value from part a of the outer reticular layer, monkey C Table 2 ) the glycine level is much lower than in average brain. Glutamate (Fig. 2, Table 2). From the inner segments to the inner nuclear layer, inclusive, glutamate levels are about the same, calculated on the basis of dry weight, as in average brain (THORNet al., 1958). Because most of the retina is much lower in lipid than brain (Table 2), glutamate in these areas, on a lipid-free dry weight basis, is only about two thirds as high 9 in average brain. Outer segments are much lower, whereas further inward the concentration rises to a peak in the ganglion cell layer where it is slightly higher than the average for brain on a lipid-free dry weight basis. The level in the fiber layer was substantially lower than in the ganglion cell layer in two
out of the three cases. High levels in the ganglion cell and fiber layers were also seen earlier (LOWRY et al., 1956). In the optic nerve of monkey C (Table 2 ) the glutamate level on a lipid-free basis was only 27% as high as in the retinal fiber layer (legend to Table 2). Aspartate (Fig. 2, Table 2). The level of aspartate is slightly below the brain average (about 10mmol/kg total dry wt) in the outer layers, with an irregular increase in the inward direction rising to a peak in the ganglion cell layer, where aspartate on a lipid-free dry weight basis, is about twice the average brain level. In the optic nerve of monkey C aspartate was only about 20% as high as in the retinal fiber layer (legend to Table 2). DISCUSSION Peak amino acid levels in the retina are as high or nearly as high as any found so far in the nervous system. OTSUKA et al. (1971) reported a G A B A level of 6.6mmol/kg wet weight in Purkinje cell bodies. O n a wet weight basis the peak levels in the inner reticular layer were over 8 mmol/kg. The distribution coincides very closely to the distribution of glutamic
S.
-
100
BERGER, M. L. MCDANIEL, J. G. CARTER and 0. H. LOWRY
ter function on the basis of distribution alone. Nevertheless, it is a first step to seek for cells especially rich in these amino acids. 80The distribution pattern in the retina suggests that glutamate and aspartate may be especially high in f 60ganglion cells. However, the levels of glutamate and aspartate in optic nerve were very low, only 27% and U 1 0 20% as high, respectively, on a lipid free dry 40weight basis as in the retinal fiber layer of the same a monkey, i.e. much too low to be accounted for by E dilution with myelin sheaths. Also, the optic nerve 20 z levels are substantially lower than in several other myelinated fiber layers in the brain and cord (BERGER 0et al., 1976). Therefore, either the ganglion cells are b c , & o b, .a .a b c, Epi 0 5 IS ON R> IN not especially rich in glutamate and aspartate or these IR GC Fib amino acids are sequestered in the ganglion cell FIG. 2. Glutamate and aspartate in retina of monkey A. bodies and processes within the retina. If these amino The data are for the same retina as Fig. 1, and abbreviations and significance of the band widths are the same. acids are ganglion cell transmitters it is hard to An average of 6 samples were analyzed from each layer see why they would be high in the somata but low or sublayer. (The lipid free dry weight in the ganglion cell in the axons leading to the central terminals where layer is about 80% of total dry weight Table 2). Note the transmitters would need to be released. An alterthe aspartate values in the figure have been multiplied by 2. nate possibility is that the high levels in the ganglion cell layer are attributable to Muller fibers. Radiographic studies of the uptake of exogenous glutamate decarboxylase as it appears with immunochemical and aspartate led EHINCER& FALCK(1971) to conradioautography (BARBER & SAITO,1976). The highest clude they were being taken up predominantly by (1972) found in a quantitative glycine level in rabbit spinal cord is about 15 mmol/l Miiller cells. RASMUSSEN of H 2 0 (BERGER et a/., 1976), whereas the peak retinal study of rat retina that Miiller cell cytoplasm is distrilevel is calculated to be 8-12mmol/l of H20.(The buted in a manner roughly parallel to the distribution inner nuclear layer of monkey retina was reported of glutamate and aspartate shown here, except that to contain 3 liters of H,O/kg dry weight (LOWRYrt the Miiller fibers rose in a steep gradient from the d.,1956)) Glutumutr levels in the ganglion cell layer outer border of the ganglion cell layer to the inner are somewhat higher than for average brain. To our limiting membrane. Thus the average proportion of knowledge the highest reported aspartate concen- Miiller cytoplasm was much higher in the fiber layer tration is about 9mmol/l of water in the extreme tip than in the ganglion cell layer, whereas aspartate is of ventral horn of the spinal cord (BERGERet at., much lower in the fiber layer in all three retinas, and 1976). This is comparable to the levels found in the glutamate was lower in two out of three. Since a ganglion cell layer which calculate to be quantitative study of Miiller cytoplasm has not been 6.5-1 1 mmol/l of H 2 0 . reported for monkey, the question must remain open. Thus on the basis of concentration alone, a good case could be made for at least three of these amino Acknowledgements-Supported in part by grants from the acids to be transmitters in retina. The case for GABA American Cancer Society (BC4Q), and National Institutes and glycine is already strong (AMES& POLLEN, 1969; of Health (NS-08862). CURTIS & JOHNSTON, 1974). The difference in distribution of these two amino acids concurs with functional evidence that they are inhibitory transmitters for different cell types. (BURKHARDT, 1972; WYATT& DAW, REFERENCES 1976). Both could be amacrine cell transmitters, although probably not in the same population. In A m s A., 111 & POLLEND. A. (1969) J . Neurophysiol. 32, monkey at least, the low level of GABA in the outer 424. R. & SAITOK. (1976) in GABA in Nervous System part of the inner nuclear layer probably rules it out BARBER Function (ROBERTS E., CHASET. M. & TOWERD. B., as a general transmitter in horizontal cells, although eds) p. 113. Raven Press, New York. uptake studies have made it a good candidate in the BERGER S . J., CARTERJ. G. & LOWY 0. H. (1976) J . 1971) and frog (VOADEN goldfish (LAM& STEINMAN, Newrochem. 27, 149-1 58. et a/., 1974). The distribution of glycine, however, BRUNNA. & EHINGER B. (1974) Expl. Eye. Res. 19, 435. makes it an excellent candidate as an inhibitory trans- BURKHARDT D. A. (1972) Brain Res. 43, 246. mitter in horizontal as well as amacrine cells. COHENA. I., MCDANIEL M. L. & ORRH. T. (1973) Invest. The important metabolic roles of glutamate and Ophthalmol. 12, 686. aspartate, and their generally high levels throughout CURTISD. R. & JOHNSTON G. A. R. (1974) Ergebnisse der the body, make it unsatisfactory to attribute transmitPhysiologie 69, 97. % L
.
."I
Potential transmitter amino acids in retina EHINCER B. (1970) Experientia, Basel 26, 1063. EHINCERB. & FALCK B. (1971) Brain Res. 33, 157. GRAHAM L. T., JR. (1972) Brain Res. 36, 476. GRAHAML. T., JR., BAXTER C. F. & LOLLEYR. W. (1970) Brain Res. 20, 379. KURIYAMA K., SISKENB., HABERB. & ROBERTS E. (1968) Brain Res. 9, 165. LAMD. M. K. & STEINMAN L. (1971) Proc. natn. Acad Sci., U.S.A. 68, 2177. LOWRY0. H. & PASSONNEAU J. V. (1972) A FlexibIe System of Enzymatic Analysis. Academic Press, New York. LOWRY0. H., ROBERTS N. R. & LEWISC. (1956) J . biol. Chern. 220, 879. LOWY 0. H., ROBERTS N. R., SCHULZD. W. & CLOW J. E. (1961) J . biol. Chem. 236, 2813. MACAIONE S. (1972) J . Neurochem. 19, 1397.
163
MATSCHINKSY F. M., PASSONNEAU J. V. & LOWRY0. H. (1968) J . Histochem. Cytochem. 16, 29. NEALM. J. & IVERSENL. L. (1972) Nature, New Biol. 235, 217.
OTSUKAM., OBATAK., MIYATAY. & TANAKA Y. (1971) J . Neurochem. 18, 281. RASMUSSEN K-E. (1972) J. Ultrastructure Res. 39, 413. THORN W., SCHOLLH., PFLEIDERER G. & MUELDENER B. (1958) J. Neurochem. 2, 150. VOADENM. J., MARSHALL J. & MURANIN. (1974) Brain Res. 67, 115. WOODJ. G., MCLAUGHLIN B. J. & VAUGHNJ. E. (1976) In GABA in Nervous System Function (ROBERTSE., CHASET. M. & TOWERD. B., eds) p. 133. Raven Press, New York. WYATTH. J. & DAWN. W. (1976) Science 191, 204.
DISTRIBUTION OF FOUR POTENTIAL TRANSMITTER AMINO ACIDS IN MONKEY RETINA SOSAMMA J. BERGER, M. L. MCDANIEL, JOYCEG. CARTER and 0. H. LOWRY Department of Pharmacology and the Beaumont-May Institute of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA. (Received 24 March 1976. Accepted 20 Jury 1976)
Abstract-Discrete layers from frozen dried sections of Rhesus monkey retina were analyzed for each of four amino acids. Peak levels of glycine were found near the border of the inner nuclear and inner reticular layers, and were high throughout these two layers. The levels were less than 50% of the peak in the adjacent ganglion cells and outer reticular layers and fell to very low levels elsewhere. GABA was much more sharply restricted to the inner reticular layer and fell off on both sides to levels of 10% or less of the peak in the fiber and photoreceptor cell layers. Glutamate and aspartate were highest in the ganglion cell layer. On a lipid-free dry weight basis the peak aspartate level was about twice that of brain. Moderately high levels of both aspartate and glutamate were found in the inner reticular and fiber layers. Elsewhere the levels ranged from 20 to 50% of the peak, and both amino acids were relatively low in optic nerve. The amino acid distributions are compatible with a transmitter function for GABA in amacrine cells and for glycine in horizontal and amacrine cells. Glutamate and aspartate may be especially high in Miiller fibers, ganglion cells or both.
THISIS a record of the distribution of glycine, glutamate, aspartate and GABA among the layers of retina et a/. (1968) found in the Rhesus monkey. KURIYAMA GABA and glutamic decarboxylase (EC 4.1.1.15) in rabbit retina to be highest in the ‘ganglion cell layer’ which probably included at least part of the inner (1972) found GABA and glureticular layer. GRAHAM tamic decarboxylase in frog retina to be highest in the inner reticular layer, both fell to low levels in the outer layers (outer nuclear and outer segment (1972) found in the rat that deslayers). MACAIONE truction of the inner retinal layers (by perinatal glutamate excess) caused an 80%loss of GABA and almost complete disappearance of glutamic decarboxylase and GABA transaminase (EC 2.6.1.19).COHENet a/. (1973) confirmed the GABA loss with glutamate treated mice. The uptake of exogenous GABA as seen in radioautographic studies of a variety of species is greatest in inner retinal layers, especially in the inner reticular layer and in the neighborhood of amacrine cells (EHINGER,1970; LAM & STEINMAN,1971; VOADEN et al., 1974; BRUNN& EHINGER, 1974). However, NEAL& IVERSEN (1972) reported uptake to be correlated with the distribution of Muller fibers. BARBER& SAITO (1976) with immunochemical radioautography found glutamic decarboxylase sharply limited to the inner reticular layer. WOODet a!. (1976) with an electron microscopic version of the same technique reported that the enzyme is concentrated at synapses typical for amacrine cells. There is less information concerning glycine distribution. COHENet a/. (1973) presented evidence that in the mouse the inner layers are about 4 times richer in glycine than the outer layers. (Destruction of inner
layers with glutamate reduced the concentration 50%; absence of outer layers due to a genetic defect resulted in a doubling of the concentration.) Radioautographic graphic studies have shown exogenous glycine uptake to be greatest in approximately the same area as for GABA, i.e. in the inner reticular layer and in the inner & FALCK, aspect of the inner nuclear layer (EHINGER 1971; BRUNN& EHINGER, 1974; VOADENet al. (1974). In an early study, glutamate in monkey retina was found t o be fairly evenly distributed, except for high levels in the ganglion cell and fibre layers (LOWRY et al., 1956). GRAHAM et al. (1970) found little difference in glutamate levels among 3 subdivisions of frog retina.
MATERIAL AND METHODS Preparation of samples
Rhesus monkeys were sedated with Phencyclidine (Sernyl) and anesthetized with pentobarbital. The eyes were removed and frozen within 5 or 10 s of the time the blood supply was cut off. Rapid freezing was accomplished by immersing in liquid nitrogen that had been evacuated in a Dewar flask until part of the nitrogen had frozen. This accelerates freezing, because no gas bubbles form on contact with the tissue. The detailed technique for preparing samples for analysis has been described (LOWRYet al., 1961; LOWRY & PASSONNEAU, 1972). Frozen sections, 5 pm in thickness, were cut at - 25” to - 30°C (from a region near the fovea) and dried under vacuum at -40°C. Samples weighing 2G80 ng were dissected from each layer, weighed and transferred into aqueous droplets in ‘oil wells’ (MATSCHINSKY et al., 1968) for the first analytical steps.
159
160
S. J. BERGER,M. L. MCDANIEL, J. G. CARTERand 0. H. LOWRY SHEET FOR TABLEI . FLOW
Step 1
ANALYSES
Step 2
Step 3
Step 4
Step 5
Enzyme destruction
Specific reagents
Destruction of excess pyridine nucleotide
Enzymatic cycling
Indicator step
Glutamate
0.1 p1 20 mM-NaOH
0.5pI 30min RT
0.2 pl 0.5 N-NaOH
0.5 pl to 50 pI cycl. rgt.?
I ml indic. rgt.
Aspartate
0.1 p1 20 mM-NaOH
0.1 pl 30min RT
0.5 pl 0.1 N-HCI
Samet
Same
0.05pI 10 mM-HC1
0.05 p1 40min RT
2 PI 50 mM-NaOH
6~ t l Cycl. rgt.1
6 PI to I ml indic. rgt.
0.05pl
0.05 pl A* 18 h RT 0.1 pI B* 40 min RT
0.1 pl 0.25 N-HCI
64 Cycl. rgt.3
Same
GABA Glycine
10 mM-NaOH
-
Steps 1-3 were carried out in oil wells. In the case of GABA and glycine, Step 4 was also in the oil well. The last step, or steps, was in the fluorometer tube. At Step 1 in all cases, and also at Step 3 for glutamate and GABA, the oil well racks were heated 20min at 80°C. Room temperature (RT) was 20-25°C. * Reagent A contained D-aminO acid oxidase; reagent B contained glyoxylate reductase and NADH. The samples were not heated before adding reagent B. t This step was in the fluorometer tube and cycling was stopped by heating 3 min in a 95°C water bath. $This step was in the oil well and cycling was stopped with 2p1 of 0 . 2 5 ~ - N a O Hfollowed by heating 20min in an oven at 80°C.
Analytical details
The analytical methods are nearly the same as those described in a companion paper for somewhat larger samples (0.24.3 pg dry weight) from brain and spinal cord (BERGERet a/., 1976) except for adjustments to increase sensitivity and reduce blank values. Only the diflerences will be described. A flow sheet (Table 1) describes the logistical details. Glutamate and aspartate. Volumes for the first two steps were cut in half. The reagents were identical to those given previously except that the enzyme concentrations in the NAD cycling reagent were tripled to give greater 'amplification' (300 pg/ml of alcohol dehydrogenase and 30 pg/ml of malic dehydrogenase). Glycine and GABA. Blank problems have been greater with glycine and GABA than with glutamate and aspartate, therefore volumes for the first steps were reduced by a factor of 4 or 5. Cycling was carried out in the oil wells instead of in the final fluorometer tubes. The same NAD cycling reagent (required for the glycine method) was used as above. For NADP (GABA method) the same cycling reagent was used as before, but greater amplification (about 40,000 fold) was achieved by incubating for 4 h at 37°C or overnight at 4°C plus 2 h at 37°C. Source of materials. GABase and D-amino acid oxidase were from Sigma Chem. Co., St. Louis, MO; the other enzymes were from Boehringer Mannheim Corp., New York, NY. Most of the special chemicals were from Sigma. RESULTS The most detailed data are for a single retina (Figs. 1 and 2). Less complete data were obtained from retinas from two other monkeys (Table 2). GABA. This is almost entirely restricted t o the inner reticular layer and the deepest third of the inner
nuclear layer (Fig. 1, Table 2). The peak GABA levels in the inner reticular layer are at least 5 times the brain average. The levels in the ganglion cell layer were more variable than in the other layers. GABA concentrations in the inner reticular layer are similar t o those found by GRAHAM (1972) in the frog, but elsewhere in the retina the levels are much lower than he found. In frog the outer portion of the inner nuclear layer was 50% of the peak, in monkey only
6-30%.
a Epi 0 5
IS
ON
p OR
b IN
c , ,a
b IR
c,
GC Fib
FIG.1. Glycine and GABA in retina of monkey A. The abbreviations for the layers are pigmented epithelium, Epi; outer segments, 0 s ; inner segments, IS; outer nuclear, ON; outer reticular, OR; inner nuclear, IN; inner reticular, IR; ganglion cell, GC; fiber, Fib. The schematic representation of cells in the inner nuclear layer are horizontal cells, H; bipolar cells, B; and amacrine cells, A. The band widths rcprcscnt 2 S.E.M.for an avcrage of 6 or 7 samplcs from each layer or sublayer.
Potential transmitter amino acids in retina
161
TABLE2. FOURAMINO ACIDS IN MONKEY RETINA Glycine
GABA B
Monkey Pig. epi. Outer seg. Inner seg. Outer nucl. Outer retic. a Outer retic. b
0.8
k 1.4 Inner nucl. a
7.2
k 1.3 Inner nucl. b Inner nucl. c
Inner retic. a Inner retic. b Inner retic. c Gangl. cell Fiber
.
5.6 f2.0 18.0 f 1.2 36.4 k 2.4 41.1 f 1.3 32.5 2.5 12.0 f 1.4 3.1 5 1.4
C 2.6 f0.3 3.8 k0.5 2.0 f 0.2 5.4 f0.5 3.8 0.5 4.8 k0.6 6.3 k0.6 10.0 kl.0 15.6 1.2
B
Glutamate C (mmol/kg dry weight)
+
*
21.4.
k 1.9
+
18.1
k 1.0
12.1 k1.4 18.0 fl.1 22.2 k1.4 21.1 f 2.2 25.0 k 1.6 20.4 k2.1 17.0 k 1.7 3.6 f0.1 5.8 f1.6
C
7.9 f1.2 4.3 50.5 4.6 k0.6 3.7 f0.4 12.7 f 1.0 18.5 k0.8 26.0 k1.5 30.3 k1.7 39.9 f1.6
33.3* k1.7
B
28.6 k3.2 39.4
k3.1 28.1 f1.7 30.3 k1.7 49.2 k5.0 53.3 f3.5 48.9 f2.1 83.7 f3.3 40.7 k4.2
Aspartate B
C
lipid
t
%
17.2 *16.0 1.2
26.6 f 1.3 7.5
k0.8 36.9 k0.8 37.5 1.9 36.8 1.6 40.1 k2.0 46.0 & 1.8 45.1 3.2 39.8 k2.7
f0.5 8.4
22 25 24
k0.6
8.5 k1.4 5.8 k0.8 7.1 f1.0 8.0
k1.3
13.0 L-0.5 9.0 k0.5 9.0 k0.8 8.9 k0.7 9.1 f0.6 7.8 k0.7
12 18*
17*
14.3
k0.8 56.9. k3.7 90.3 k4.0 86.4 k 5.2
9.0 k1.7 9.6 2.3 19.5 k2.3 10.2 k0.4
13.2* k1.6
37*
27.4 52.4 17.3 f1.3
20 34
With a few exceptions, each value is the average for 5 or mores samples f S.E.M. The optic nerve of monkey C (sampled where it passes through the sclera) had respective glutamate and aspartate levels ( ~ s . E . M . ) of 34.6 f 0.8 and 5.2 & 0.15 mmol/kg lipid-free dry weight. (The lipid was 51.5 k 0.7% of the total dry weight.) For comparison, on a lipid free basis the peak levels shown in the fiber layer of the same monkey are 130 and 26 mmol/kg. * Values for the whole layer. t Percent of total dry weight, from LOWRYet al. (1956). The lipid content of brain is about 50% of the dry weight. Glycine. This is much less sharply restricted than GABA, however, the range of values (30-fold) is about the same (Fig. 1, Table 2). The broad peak centers at about the boundary between inner nuclear and inner reticular layers where the level is 4-6 times that in average brain. Highest levels are limited to these two layers, although in the ganglion cell and outer reticular layers the levels ranged from 10% to 45% of the peak. Throughout the photoreceptor cells, (except for one value from part a of the outer reticular layer, monkey C Table 2 ) the glycine level is much lower than in average brain. Glutamate (Fig. 2, Table 2). From the inner segments to the inner nuclear layer, inclusive, glutamate levels are about the same, calculated on the basis of dry weight, as in average brain (THORNet al., 1958). Because most of the retina is much lower in lipid than brain (Table 2), glutamate in these areas, on a lipid-free dry weight basis, is only about two thirds as high 9 in average brain. Outer segments are much lower, whereas further inward the concentration rises to a peak in the ganglion cell layer where it is slightly higher than the average for brain on a lipid-free dry weight basis. The level in the fiber layer was substantially lower than in the ganglion cell layer in two
out of the three cases. High levels in the ganglion cell and fiber layers were also seen earlier (LOWRY et al., 1956). In the optic nerve of monkey C (Table 2 ) the glutamate level on a lipid-free basis was only 27% as high as in the retinal fiber layer (legend to Table 2). Aspartate (Fig. 2, Table 2). The level of aspartate is slightly below the brain average (about 10mmol/kg total dry wt) in the outer layers, with an irregular increase in the inward direction rising to a peak in the ganglion cell layer, where aspartate on a lipid-free dry weight basis, is about twice the average brain level. In the optic nerve of monkey C aspartate was only about 20% as high as in the retinal fiber layer (legend to Table 2). DISCUSSION Peak amino acid levels in the retina are as high or nearly as high as any found so far in the nervous system. OTSUKA et al. (1971) reported a G A B A level of 6.6mmol/kg wet weight in Purkinje cell bodies. O n a wet weight basis the peak levels in the inner reticular layer were over 8 mmol/kg. The distribution coincides very closely to the distribution of glutamic
S.
-
100
BERGER, M. L. MCDANIEL, J. G. CARTER and 0. H. LOWRY
ter function on the basis of distribution alone. Nevertheless, it is a first step to seek for cells especially rich in these amino acids. 80The distribution pattern in the retina suggests that glutamate and aspartate may be especially high in f 60ganglion cells. However, the levels of glutamate and aspartate in optic nerve were very low, only 27% and U 1 0 20% as high, respectively, on a lipid free dry 40weight basis as in the retinal fiber layer of the same a monkey, i.e. much too low to be accounted for by E dilution with myelin sheaths. Also, the optic nerve 20 z levels are substantially lower than in several other myelinated fiber layers in the brain and cord (BERGER 0et al., 1976). Therefore, either the ganglion cells are b c , & o b, .a .a b c, Epi 0 5 IS ON R> IN not especially rich in glutamate and aspartate or these IR GC Fib amino acids are sequestered in the ganglion cell FIG. 2. Glutamate and aspartate in retina of monkey A. bodies and processes within the retina. If these amino The data are for the same retina as Fig. 1, and abbreviations and significance of the band widths are the same. acids are ganglion cell transmitters it is hard to An average of 6 samples were analyzed from each layer see why they would be high in the somata but low or sublayer. (The lipid free dry weight in the ganglion cell in the axons leading to the central terminals where layer is about 80% of total dry weight Table 2). Note the transmitters would need to be released. An alterthe aspartate values in the figure have been multiplied by 2. nate possibility is that the high levels in the ganglion cell layer are attributable to Muller fibers. Radiographic studies of the uptake of exogenous glutamate decarboxylase as it appears with immunochemical and aspartate led EHINCER& FALCK(1971) to conradioautography (BARBER & SAITO,1976). The highest clude they were being taken up predominantly by (1972) found in a quantitative glycine level in rabbit spinal cord is about 15 mmol/l Miiller cells. RASMUSSEN of H 2 0 (BERGER et a/., 1976), whereas the peak retinal study of rat retina that Miiller cell cytoplasm is distrilevel is calculated to be 8-12mmol/l of H20.(The buted in a manner roughly parallel to the distribution inner nuclear layer of monkey retina was reported of glutamate and aspartate shown here, except that to contain 3 liters of H,O/kg dry weight (LOWRYrt the Miiller fibers rose in a steep gradient from the d.,1956)) Glutumutr levels in the ganglion cell layer outer border of the ganglion cell layer to the inner are somewhat higher than for average brain. To our limiting membrane. Thus the average proportion of knowledge the highest reported aspartate concen- Miiller cytoplasm was much higher in the fiber layer tration is about 9mmol/l of water in the extreme tip than in the ganglion cell layer, whereas aspartate is of ventral horn of the spinal cord (BERGERet at., much lower in the fiber layer in all three retinas, and 1976). This is comparable to the levels found in the glutamate was lower in two out of three. Since a ganglion cell layer which calculate to be quantitative study of Miiller cytoplasm has not been 6.5-1 1 mmol/l of H 2 0 . reported for monkey, the question must remain open. Thus on the basis of concentration alone, a good case could be made for at least three of these amino Acknowledgements-Supported in part by grants from the acids to be transmitters in retina. The case for GABA American Cancer Society (BC4Q), and National Institutes and glycine is already strong (AMES& POLLEN, 1969; of Health (NS-08862). CURTIS & JOHNSTON, 1974). The difference in distribution of these two amino acids concurs with functional evidence that they are inhibitory transmitters for different cell types. (BURKHARDT, 1972; WYATT& DAW, REFERENCES 1976). Both could be amacrine cell transmitters, although probably not in the same population. In A m s A., 111 & POLLEND. A. (1969) J . Neurophysiol. 32, monkey at least, the low level of GABA in the outer 424. R. & SAITOK. (1976) in GABA in Nervous System part of the inner nuclear layer probably rules it out BARBER Function (ROBERTS E., CHASET. M. & TOWERD. B., as a general transmitter in horizontal cells, although eds) p. 113. Raven Press, New York. uptake studies have made it a good candidate in the BERGER S . J., CARTERJ. G. & LOWY 0. H. (1976) J . 1971) and frog (VOADEN goldfish (LAM& STEINMAN, Newrochem. 27, 149-1 58. et a/., 1974). The distribution of glycine, however, BRUNNA. & EHINGER B. (1974) Expl. Eye. Res. 19, 435. makes it an excellent candidate as an inhibitory trans- BURKHARDT D. A. (1972) Brain Res. 43, 246. mitter in horizontal as well as amacrine cells. COHENA. I., MCDANIEL M. L. & ORRH. T. (1973) Invest. The important metabolic roles of glutamate and Ophthalmol. 12, 686. aspartate, and their generally high levels throughout CURTISD. R. & JOHNSTON G. A. R. (1974) Ergebnisse der the body, make it unsatisfactory to attribute transmitPhysiologie 69, 97. % L
.
."I
Potential transmitter amino acids in retina EHINCER B. (1970) Experientia, Basel 26, 1063. EHINCERB. & FALCK B. (1971) Brain Res. 33, 157. GRAHAM L. T., JR. (1972) Brain Res. 36, 476. GRAHAML. T., JR., BAXTER C. F. & LOLLEYR. W. (1970) Brain Res. 20, 379. KURIYAMA K., SISKENB., HABERB. & ROBERTS E. (1968) Brain Res. 9, 165. LAMD. M. K. & STEINMAN L. (1971) Proc. natn. Acad Sci., U.S.A. 68, 2177. LOWRY0. H. & PASSONNEAU J. V. (1972) A FlexibIe System of Enzymatic Analysis. Academic Press, New York. LOWRY0. H., ROBERTS N. R. & LEWISC. (1956) J . biol. Chern. 220, 879. LOWY 0. H., ROBERTS N. R., SCHULZD. W. & CLOW J. E. (1961) J . biol. Chem. 236, 2813. MACAIONE S. (1972) J . Neurochem. 19, 1397.
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