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Axonal migration of taurine in the goldfish visual system
Brain Research, 115 (1976) 535-539
535
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Axonal migration of taurine in the goldfish visual system
NICHOLAS A. INGOGLIA*, JOHN A. STURMAN, THOMAS D. LINDQUIST and GERALD E. GAULL Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, New Jersey 07103, Department of Pediatric Research, Institute for Basic Research in Mental Retardation, Staten Island, New York 10314 and Department of Pediatrics, Mount Sinai School of Medicine of the City University of New York, New York, N. Y. 10029 (U.S.A.)
(Accepted July 9th, 1976)
Taurine is an ubiquitous amino sulfonic acid present in high concentrations in neural and muscle tissue. Except for its role in the conjugation of bile acids, its biological significance is uncertain 5,7. However, there is considerable recent evidence which suggests that it may function as a neurotransmitter or neuromodulator in nervous tissue and in retina 1, and that it might be involved in brain development 1~-16. The present experiments were performed in order to test the possibility that taurine might be axonally transported in the optic nerve of the goldfish, a system which has been used extensively to study the axonal transport of macromolecules 4,6,1°. We also have used this system to investigate the axonal migration of cysteine, a precursor of taurine, and of?-aminobutyric acid (GABA), an amino acid analog of taurine and also a putative neurotransmitter. [85S]Taurine (64 mCi/mmole, obtained from Amersham/Searle Corp.), [~5S] cysteine (22 mCi/mmole, obtained from Amersham/Searle Corp.), or [14C]GABA (49 mCi/mmole, obtained from New England Nuclear Corp.) were dissolved in 0.9 saline. Four microliters of each solution (containing 0.5-1.0/~Ci) were injected in separate experiments into the vitreous of the right eye of goldfish (4-5 in. in length, obtained from Ozark Fisheries, Stoutland, Mo.). Fish were killed at various times after injection and their right retinas and both optic tecta were removed and homogenized in deionized H20. Trichloroacetic acid (TCA), 30 ~o w/v, was added to the homogenate to give a final concentration of 1 0 ~ TCA. Determination of TCAinsoluble and TCA-soluble radioactivity was performed as described previously 6. Since visual fibers of the goldfish cross completely at the optic chiasm, radioactivity in the tectum ipsilateral to the injected eye (right tectum in this case) represents material which has arrived by general circulation, whereas radioactivity in the left tectum represents material which has arrived by axonal transport or axonal diffusion
* Correspondence and reprint requests should be addressed to: Dr. N. A. Ingoglia, Department of Physiology, New Jersey Medical School, 100 Bergen Street, Newark, N.J. 07103, U.S.A.
536
~
3000-
_o-
2
~
~ooo
o:
IO00-
I
0.25
I
6
14
DAYS AFTER INJECTION OF3SS-TAURINE
Fig. 1. [z~S]Taurine was injected into the right eye and the fish were killed at various times after injection. TCA-soluble and insoluble radioactivity was extracted from left and right tecta. Each point is the mean ± S.E.M. of left minus right tecta for TCA-soluble radioactivity, n -- 6.
as well as by the general circulation. Thus, the a m o u n t o f r a d i o a c t i v i t y actually m i g r a t i n g via the optic nerve is c a l c u l a t e d by s u b t r a c t i n g the r a d i o a c t i v i t y f o u n d in the right tectum f r o m t h a t f o u n d in the left tectum. I n t r a o c u l a r injection o f [35S]taurine resulted in large differences in the a m o u n t s o f r a d i o a c t i v i t y arriving in left vs. right optic tecta within 24 h (Fig. l). This r a d i o activity was all present in the T C A - s o l u b l e fraction. Analysis o f T C A - soluble extracts o f 30 p o o l e d tecta a n d retinas using an a u t o m a t i c a m i n o acid analyzer in c o n j u n c t i o n with a flow cell scintillation s p e c t r o m e t e r 14, showed f u r t h e r that all r a d i o a c t i v i t y in b o t h the retina a n d tecta r e m a i n e d in the f o r m o f taurine. Experiments p e r f o r m e d with [85S]cysteine, a p r e c u r s o r o f taurine, also resulted in the a p p e a r a n c e o f r a d i o a c t i v i t y in the c o n t r a l a t e r a l optic t e c t u m (Fig. 2). In this
600 >-
_> 450 0
o 300"
(3_ CO Z
e:
t
tt
150-
0 TCA SOLUBLE TCA INSOLUBLE
F-
DAYS A F T E R
INJECTION OF 35S-CYSTEINE
Fig. 2. [3~S]Cysteine was injected into the right eye and the fish were killed at various times after injection. TCA-soluble and insoluble radioactivity was extracted from left and right tecta. Each value is the mean ± S.E.M. of left minus right tecta for 4-6 fish/point. ©, TCA-soluble, @, TCA-insoluble.
537 case, however, radioactivity was found not only in the TCA-soluble fraction, but also in the TCA-insolub!e fraction, presumably as [aSS]cysteine incorporated into proteins. Analysis of the TCA-soluble radioactivity in the tectum showed the presence of only [asS]taurine and [asS]inorganic sulfate with no evidence of [aSS]cysteine. [asS]Taurine and [asS]inorganic sulfate also were present in the retina, in approximately the same ratio as in the tectum. Also found in the retina were other radioactive compounds, presumptively identified (by elution volume) as reduced and oxidized glutathionine. No [aSS]cysteine remained in the retina one day after injection. Since amino acids are not axonally transported in appreciable amounts2, a, the most likely explanation for these findings is as follows: when cysteine is injected into the vitreous, it enters retinal cells, where some of it is rapidly incorporated into protein, a portion of which is then axonally transported and appears in the contralateral optic tectum as TCA-insoluble radioactivity (Fig. 2). The portion of [a5S]cysteine which is not incorporated into protein is converted to TCA-soluble metabolic products, of which taurine and inorganic sulfate are transferred to the optic tectum, while glutathione remains in retinal cells. GABA is a putative inhibitory neurotransmitter which resembles taurine structurally (it is an amino carboxylic acid rather than an amino sulfonic acid) and, like taurine, is not incorporated into protein. Because of these similarities, we decided to compare GABA and taurine in order to determine whether the axonal migration of taurine is limited to that compound, or is a more generalized phenomenon characteristic of a class of compounds. GABA has been shown to be taken up by neuronal cell
I01 35S-TAURINE:
IOs
ul
o
35S-CYSTEINE
10 4
14C-GABA
10 3
•
I
3
~
6
DAYS AFTER INJECTION
Fig. 3. TCA-soluble radioactivity in the retina at various times after the intraocular injection of [ass]taurine (A, values are taken from experiments shown in Fig. 1); [asS]cysteine (©, values are taken from experiments shown in Fig. 2); and [14C]GABA ( 0 , see text for description of experiments). Values are mean 4- S.E.M. for 4-6 fish/point. N.B. ordinate is a 3 cycle log scale.
538 populations, including the goldfish retina 9, and has been reported to be axonally transported in the chick visual systemL When we injected [14C]GABA into the goldfish eye, we found that little or no radioactivity ( < 50 disint/min//~Ci of injected material) arrived in the contralateral optic tectum at times up to 21 days after injection. Thus it appears that the axonal migration of taurine in this system is relatively specific. However, the axonal migration of any radioactive material injected into the vitreous depends initally on the uptake of that material by retinal ganglion cells. Our results (Fig. 3) demonstrate clearly that taurine is taken up by the retina to a much greater extent (approx. 50-fold) than GABA. This may be the result of GABA being taken up only by horizontal cells 9, whereas taurine appears to be taken up by all retinal cells although predominantly by photoreceptor cellss,11. Thus, a limiting factor in the axonal migration of GABA may be its initial uptake by retinal ganglion cells. Radioactive uptake in retina following [35S] cysteine injection into the vitreous was found to be intermediate between that of taurine and that of GABA (Fig. 3). These results demonstrate that taurine is unique in its axonal migration when compared with similar compounds studied in the goldfish visual system. All other amino acids studied, thus far (including GABA and cysteine in the present experiments), do not migrate along the axon in appreciable amounts 2,3. [85S]Taurine, however, appears in large amounts in the optic tectum following its intraocular injection. The earliest arrival of radioactivity, 6 h after injection, suggests a rate of migration similar to fast axonal protein transport reported in the same system 4. These experiments do not completely rule out the possibility that taurine is migrating within the axon by simple diffusion. However, this seems unlikely since molecules of similar size and characteristics (amino acids) have been shown not to migrate along the optic nerve, even under circumstances when the rapid incorporation of amino acids into protein has been prevented by inhibition of protein synthesis. Thus, it seems likely that following intraocular injection of [35S]taurine, relatively large amounts of labeled taurine are axonally transported unchanged to the optic tectum. These findings suggest that taurine may have some specialized function in the goldfish optic system. Whether this function is involved in neurotransmitter activity or in some other physiological activity of goldfish optic axons remains to be demonstrated. These studies were supported by a Neurological Diseases and Stroke Grant NS 11259 from N I H and the N.Y. State Department of Mental Hygiene. We thank Mr. Paul Hartunian and Ms. Judith Fagan for expert technical assistance.
1 Barbeau, A., Inoue, N., Tsukada, Y. and Butterworth, R. F., The neuropharmacology of taurine, Life ScL, 17 (1975) 669-678. 2 Bondy, S. C., Axonal transport of macromolecules. I. Protein migration in the central nervous system, Exp. Brain Res., 13 (1971) 127-134.
539 3 Elam, J. S. and Agranoff, B. W., Rapid transport of protein in the optic system of the goldfish, J. Neurochem., 18 (1971) 375-387. 4 Grafstein, B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198. 5 Huxtable, R. and Barbeau, A., Taurine, Raven Press, New York, 1976. 6 Ingoglia, N. A., Grafstein, B., McEwen, B. S. and McQuarrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled R N A precursors, J. Neurochem., 20 (1973) 1605-1615. 7 Jacobsen, J. G. and Smith, L. H., Biochemistry and physiology of taurine and taurine derivatives, Physiol. Rev., 48 (1968) 424-511. 8 Lake, N., Marshall, J. and Voaden, M. J., Studies on the uptake of taurine by the isolated neural retina and pigment epithelium of the frog, Biochem. Soc. Trans., 3 (1975) 524-525. 9 Lain, D. M. K. and Steinman, L., The uptake of [aH]y-aminobutyric acid in the goldfish retina, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2777-2781. l0 Neale, J. H., Neale, E. A. and Agranoff, B. W., Radioautography of the optic tectum of the goldfish after intraocular injection of aH-proline, Science, 176 (1972) 407-410. 11 Orr, H. T., Cohen, A. I. and Lowery, O. H., The distribution of taurine in the vertebrate retina, J. Neurochem., 26 (1976) 609~11. 12 Rassin, D. K., Sturman, J. A. and Gaull, G. E., Taurine: Subcellular distribution in brain of the developing rat, Trans. Amer. Soc. Neurochem., 7 (1976) 163 (abstract). 13 Sturman, J. A., Taurine pool sizes in the rat: Effects of vitamin B-6 deficiency and high taurine diet, J. Nutr., 102 (1973) 1566-1580. 14 Sturman, J. A. and Gaull, E. G., Taurine in the brain and liver of the developing human and monkey, J. Neurochem., 25 0975) 831-835. 15 Sturman, J. A., Rassin, D. K. and Gaull, G. E., Taurine: Maternal-fetal transfer to developing rat brain, Trans. Amer. Soc. Neurochem., 7 (1976) 164 (abstract). 16 Sturman, J. A., Rassin, D. K. and Gaull, G. E., Taurine in development: Is it essential in the neonate? Pediat. Res., 10 (1976) (abstract).
535
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Axonal migration of taurine in the goldfish visual system
NICHOLAS A. INGOGLIA*, JOHN A. STURMAN, THOMAS D. LINDQUIST and GERALD E. GAULL Departments of Physiology and Neuroscience, New Jersey Medical School, Newark, New Jersey 07103, Department of Pediatric Research, Institute for Basic Research in Mental Retardation, Staten Island, New York 10314 and Department of Pediatrics, Mount Sinai School of Medicine of the City University of New York, New York, N. Y. 10029 (U.S.A.)
(Accepted July 9th, 1976)
Taurine is an ubiquitous amino sulfonic acid present in high concentrations in neural and muscle tissue. Except for its role in the conjugation of bile acids, its biological significance is uncertain 5,7. However, there is considerable recent evidence which suggests that it may function as a neurotransmitter or neuromodulator in nervous tissue and in retina 1, and that it might be involved in brain development 1~-16. The present experiments were performed in order to test the possibility that taurine might be axonally transported in the optic nerve of the goldfish, a system which has been used extensively to study the axonal transport of macromolecules 4,6,1°. We also have used this system to investigate the axonal migration of cysteine, a precursor of taurine, and of?-aminobutyric acid (GABA), an amino acid analog of taurine and also a putative neurotransmitter. [85S]Taurine (64 mCi/mmole, obtained from Amersham/Searle Corp.), [~5S] cysteine (22 mCi/mmole, obtained from Amersham/Searle Corp.), or [14C]GABA (49 mCi/mmole, obtained from New England Nuclear Corp.) were dissolved in 0.9 saline. Four microliters of each solution (containing 0.5-1.0/~Ci) were injected in separate experiments into the vitreous of the right eye of goldfish (4-5 in. in length, obtained from Ozark Fisheries, Stoutland, Mo.). Fish were killed at various times after injection and their right retinas and both optic tecta were removed and homogenized in deionized H20. Trichloroacetic acid (TCA), 30 ~o w/v, was added to the homogenate to give a final concentration of 1 0 ~ TCA. Determination of TCAinsoluble and TCA-soluble radioactivity was performed as described previously 6. Since visual fibers of the goldfish cross completely at the optic chiasm, radioactivity in the tectum ipsilateral to the injected eye (right tectum in this case) represents material which has arrived by general circulation, whereas radioactivity in the left tectum represents material which has arrived by axonal transport or axonal diffusion
* Correspondence and reprint requests should be addressed to: Dr. N. A. Ingoglia, Department of Physiology, New Jersey Medical School, 100 Bergen Street, Newark, N.J. 07103, U.S.A.
536
~
3000-
_o-
2
~
~ooo
o:
IO00-
I
0.25
I
6
14
DAYS AFTER INJECTION OF3SS-TAURINE
Fig. 1. [z~S]Taurine was injected into the right eye and the fish were killed at various times after injection. TCA-soluble and insoluble radioactivity was extracted from left and right tecta. Each point is the mean ± S.E.M. of left minus right tecta for TCA-soluble radioactivity, n -- 6.
as well as by the general circulation. Thus, the a m o u n t o f r a d i o a c t i v i t y actually m i g r a t i n g via the optic nerve is c a l c u l a t e d by s u b t r a c t i n g the r a d i o a c t i v i t y f o u n d in the right tectum f r o m t h a t f o u n d in the left tectum. I n t r a o c u l a r injection o f [35S]taurine resulted in large differences in the a m o u n t s o f r a d i o a c t i v i t y arriving in left vs. right optic tecta within 24 h (Fig. l). This r a d i o activity was all present in the T C A - s o l u b l e fraction. Analysis o f T C A - soluble extracts o f 30 p o o l e d tecta a n d retinas using an a u t o m a t i c a m i n o acid analyzer in c o n j u n c t i o n with a flow cell scintillation s p e c t r o m e t e r 14, showed f u r t h e r that all r a d i o a c t i v i t y in b o t h the retina a n d tecta r e m a i n e d in the f o r m o f taurine. Experiments p e r f o r m e d with [85S]cysteine, a p r e c u r s o r o f taurine, also resulted in the a p p e a r a n c e o f r a d i o a c t i v i t y in the c o n t r a l a t e r a l optic t e c t u m (Fig. 2). In this
600 >-
_> 450 0
o 300"
(3_ CO Z
e:
t
tt
150-
0 TCA SOLUBLE TCA INSOLUBLE
F-
DAYS A F T E R
INJECTION OF 35S-CYSTEINE
Fig. 2. [3~S]Cysteine was injected into the right eye and the fish were killed at various times after injection. TCA-soluble and insoluble radioactivity was extracted from left and right tecta. Each value is the mean ± S.E.M. of left minus right tecta for 4-6 fish/point. ©, TCA-soluble, @, TCA-insoluble.
537 case, however, radioactivity was found not only in the TCA-soluble fraction, but also in the TCA-insolub!e fraction, presumably as [aSS]cysteine incorporated into proteins. Analysis of the TCA-soluble radioactivity in the tectum showed the presence of only [asS]taurine and [asS]inorganic sulfate with no evidence of [aSS]cysteine. [asS]Taurine and [asS]inorganic sulfate also were present in the retina, in approximately the same ratio as in the tectum. Also found in the retina were other radioactive compounds, presumptively identified (by elution volume) as reduced and oxidized glutathionine. No [aSS]cysteine remained in the retina one day after injection. Since amino acids are not axonally transported in appreciable amounts2, a, the most likely explanation for these findings is as follows: when cysteine is injected into the vitreous, it enters retinal cells, where some of it is rapidly incorporated into protein, a portion of which is then axonally transported and appears in the contralateral optic tectum as TCA-insoluble radioactivity (Fig. 2). The portion of [a5S]cysteine which is not incorporated into protein is converted to TCA-soluble metabolic products, of which taurine and inorganic sulfate are transferred to the optic tectum, while glutathione remains in retinal cells. GABA is a putative inhibitory neurotransmitter which resembles taurine structurally (it is an amino carboxylic acid rather than an amino sulfonic acid) and, like taurine, is not incorporated into protein. Because of these similarities, we decided to compare GABA and taurine in order to determine whether the axonal migration of taurine is limited to that compound, or is a more generalized phenomenon characteristic of a class of compounds. GABA has been shown to be taken up by neuronal cell
I01 35S-TAURINE:
IOs
ul
o
35S-CYSTEINE
10 4
14C-GABA
10 3
•
I
3
~
6
DAYS AFTER INJECTION
Fig. 3. TCA-soluble radioactivity in the retina at various times after the intraocular injection of [ass]taurine (A, values are taken from experiments shown in Fig. 1); [asS]cysteine (©, values are taken from experiments shown in Fig. 2); and [14C]GABA ( 0 , see text for description of experiments). Values are mean 4- S.E.M. for 4-6 fish/point. N.B. ordinate is a 3 cycle log scale.
538 populations, including the goldfish retina 9, and has been reported to be axonally transported in the chick visual systemL When we injected [14C]GABA into the goldfish eye, we found that little or no radioactivity ( < 50 disint/min//~Ci of injected material) arrived in the contralateral optic tectum at times up to 21 days after injection. Thus it appears that the axonal migration of taurine in this system is relatively specific. However, the axonal migration of any radioactive material injected into the vitreous depends initally on the uptake of that material by retinal ganglion cells. Our results (Fig. 3) demonstrate clearly that taurine is taken up by the retina to a much greater extent (approx. 50-fold) than GABA. This may be the result of GABA being taken up only by horizontal cells 9, whereas taurine appears to be taken up by all retinal cells although predominantly by photoreceptor cellss,11. Thus, a limiting factor in the axonal migration of GABA may be its initial uptake by retinal ganglion cells. Radioactive uptake in retina following [35S] cysteine injection into the vitreous was found to be intermediate between that of taurine and that of GABA (Fig. 3). These results demonstrate that taurine is unique in its axonal migration when compared with similar compounds studied in the goldfish visual system. All other amino acids studied, thus far (including GABA and cysteine in the present experiments), do not migrate along the axon in appreciable amounts 2,3. [85S]Taurine, however, appears in large amounts in the optic tectum following its intraocular injection. The earliest arrival of radioactivity, 6 h after injection, suggests a rate of migration similar to fast axonal protein transport reported in the same system 4. These experiments do not completely rule out the possibility that taurine is migrating within the axon by simple diffusion. However, this seems unlikely since molecules of similar size and characteristics (amino acids) have been shown not to migrate along the optic nerve, even under circumstances when the rapid incorporation of amino acids into protein has been prevented by inhibition of protein synthesis. Thus, it seems likely that following intraocular injection of [35S]taurine, relatively large amounts of labeled taurine are axonally transported unchanged to the optic tectum. These findings suggest that taurine may have some specialized function in the goldfish optic system. Whether this function is involved in neurotransmitter activity or in some other physiological activity of goldfish optic axons remains to be demonstrated. These studies were supported by a Neurological Diseases and Stroke Grant NS 11259 from N I H and the N.Y. State Department of Mental Hygiene. We thank Mr. Paul Hartunian and Ms. Judith Fagan for expert technical assistance.
1 Barbeau, A., Inoue, N., Tsukada, Y. and Butterworth, R. F., The neuropharmacology of taurine, Life ScL, 17 (1975) 669-678. 2 Bondy, S. C., Axonal transport of macromolecules. I. Protein migration in the central nervous system, Exp. Brain Res., 13 (1971) 127-134.
539 3 Elam, J. S. and Agranoff, B. W., Rapid transport of protein in the optic system of the goldfish, J. Neurochem., 18 (1971) 375-387. 4 Grafstein, B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198. 5 Huxtable, R. and Barbeau, A., Taurine, Raven Press, New York, 1976. 6 Ingoglia, N. A., Grafstein, B., McEwen, B. S. and McQuarrie, I. G., Axonal transport of radioactivity in the goldfish optic system following intraocular injection of labeled R N A precursors, J. Neurochem., 20 (1973) 1605-1615. 7 Jacobsen, J. G. and Smith, L. H., Biochemistry and physiology of taurine and taurine derivatives, Physiol. Rev., 48 (1968) 424-511. 8 Lake, N., Marshall, J. and Voaden, M. J., Studies on the uptake of taurine by the isolated neural retina and pigment epithelium of the frog, Biochem. Soc. Trans., 3 (1975) 524-525. 9 Lain, D. M. K. and Steinman, L., The uptake of [aH]y-aminobutyric acid in the goldfish retina, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2777-2781. l0 Neale, J. H., Neale, E. A. and Agranoff, B. W., Radioautography of the optic tectum of the goldfish after intraocular injection of aH-proline, Science, 176 (1972) 407-410. 11 Orr, H. T., Cohen, A. I. and Lowery, O. H., The distribution of taurine in the vertebrate retina, J. Neurochem., 26 (1976) 609~11. 12 Rassin, D. K., Sturman, J. A. and Gaull, G. E., Taurine: Subcellular distribution in brain of the developing rat, Trans. Amer. Soc. Neurochem., 7 (1976) 163 (abstract). 13 Sturman, J. A., Taurine pool sizes in the rat: Effects of vitamin B-6 deficiency and high taurine diet, J. Nutr., 102 (1973) 1566-1580. 14 Sturman, J. A. and Gaull, E. G., Taurine in the brain and liver of the developing human and monkey, J. Neurochem., 25 0975) 831-835. 15 Sturman, J. A., Rassin, D. K. and Gaull, G. E., Taurine: Maternal-fetal transfer to developing rat brain, Trans. Amer. Soc. Neurochem., 7 (1976) 164 (abstract). 16 Sturman, J. A., Rassin, D. K. and Gaull, G. E., Taurine in development: Is it essential in the neonate? Pediat. Res., 10 (1976) (abstract).