Axoplasmic transport of free amino acids

Brain Research, 56 (1973) 271-284

271

© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

AXOPLASMIC T R A N S P O R T OF F R E E A M I N O ACIDS

VILMOS CSANYI*, J U D I T GERVAI AND ABEL LAJTHA

New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y. 10035 (U.S.A.) (Accepted December 6th, 1972)

SUMMARY

(1) Following the injection of L-[14C]proline into one eye in carp, the label appeared in the contralateral optic nerve via axoplasmic flow. As reported previously, protein-bound activity showed faster and slower axoplasmic transport. Labeled free proline appeared in nerve before labeled protein and thus did not originate from labeled protein. (2) When [14C]a-aminoisobutyric acid, L-proline, or D-glutamic acid was injected into the eye, the label in the nerve decreased in a distal direction. The radioactivity gave two peaks of maximal activity with time in the whole nerve and in nerve sections, indicating a faster and a slower component of the axoplasmic transport of amino acids. There were no peaks of activity in the nerve on the control side. Estimated transport rates for the fast components were 90-110 mm/day; values for the slow component varied between 5 and 20 mm/day. The acid-soluble activity did not reach the end of the optic tact or the optic lobe in significant quantities. (3) The injected tracer amino acid levels rapidly declined in the eye; the rate of exit was different for the various compounds. The amino acid content in the eye or in the circulation did not seem to influence the measured flow significantly. The amino acids were not restricted to the external portion of the nerve but along with their axonal flow left the nerve via exchange or exit. (4) Axonal transport in the goldfish optic system occurred at rates similar to those in carp. In goldfish a sharp temperature gradient ofaxonal transport could be measured with a maximum at 20 °C. Axonal amino acid transport in chick and rabbit nerve was about 3 times as fast as in fish, possibly because of higher body temperature. (5) The composition of the free amino acid pool is characteristic for the various peripheral nerves and is different from that in brain. Some of its components have a proximo-distal concentration gradient. Axonal transport of amino acids may play a * Present address: Institute of Medical Chemistry, SemmelweisUniversity Medical School, Budapest, Hungary.

272

v. CSANVl et al.

part in maintaining such gradients or in regulating amino acid metabolism in nerve.

INTRODUCTION

The flow of particulates and macromolecules from the neuronal cell body along the axon has been well established in numerous observations (for recent reviews, see refs. 2, 18, 21, 23). Other studies have demonstrated the axoplasmic flow of a number of metabolites of small molecular weight. These include the transport of orotic acid in the chick sciatic nerve l, glucosamine in goldfish optic nerve s, fucose in chick optic nerve 3, and putrescine in zebrafish optic nerve 7. Although these studies indicated that compounds of both high and low molecular weight are transported along the axon, results on the axoplasmic flow of free amino acids were less clear cut. Flow of free amino acids could be shown in a number of systems, including D-glutamate in snail ganglion 1'~, and leucine in cat ventral roots 14, goldfish optic nerve 5, and rabbit optic nerve 12. In many of these experiments the possibility could not be excluded that at least part of the free amino acid in the axon originated from local proteolysis rather than from flow of the free amino acid from the cell body. Experiments in which no axoplasmic transport of the tested amino acids could be detected included those with leucine in chick sciatic nerve 1, pigeon optic nerve 4, and goldfish optic nerveS; proline in chick optic nerve3; and cycloleucine in goldfish optic nerve 17. Since free amino acids have an important role in cerebral metabolism, and some participate directly or indirectly in neurotransmission, it seemed important to establish whether or not flow of free amino acids from the cell body along the axon occurs. The present paper reports the transport of a number of amino acids along the optic nerve of various species. Some of the results were reported in a preliminary communication 9. METHODS

White Leghorn chicks (1-6 days old), goldfish (10-12 cm long), albino rabbits (2.5-2.8 kg), and carp (2-3 kg) were used. Unless otherwise noted, goldfish were kept in a 20 °C aquarium, carp in 12-14 °C running water. Labeled amino acid was injected in a volume of 2-20/zl into the vitreous body of one or both eyes with a Hamilton microsyringe. Rabbits and chicks were lightly anesthetized with ether for the injection. After decapitation, the optic system was dissected; the optic nerve and the optic tract were cut to several specific lengths and frozen in dry ice. For the determination of the free amino acid pool, freshly excised, frozen, and weighed tissue was homogenized in 1 ml of 1% (w/v) perchloric acid, and the amino acids were separated and analyzed in a Technicon Autoanalyzer 16. With non-metabolizable amino acids, the tissue sections were dissolved in 1 ml of 1 N NaOH and the radioactivity was determined. With the other amino acids, the tissue sections were homogenized in 10% perchloric acid at 0 °C, then heated for 20 min at 85 °C and centrifuged; from an aliquot of the supernatant, the acid-soluble radioactivity was determined. The precipitate was wash-

AXONAL FLOW OF AMINO ACIDS

273

ed twice with 5 ~o perchloric acid and once with alcohol-ether (2:3, v/v). After drying, the precipitate was dissolved in 1 N NaOH, and portions of this solution were used to measure radioactivity and protein content ~5. Radioactivity was measured in an Intertechnique scintillation counter; quench correction was by an external standard. The scintillation mixture contained 6.35 g PPO (2,5-diphenyloxazole), 115 mg dimethyl POPOP (1,4-bis-5-phenyloxazolyl-2-benzene), 538 ml toluene, and 462 ml Triton X-100 per liter. To 10 ml of this mixture, 2-4 ml portions of the solution to be measured were added~°. L-[U-14C]proline (233 mCi/mmole) and [3H]2-aminoisobutyric acid (AIB) (125 mCi/mmole) were from New England Nuclear, L-[14C]2-aminoisobutyric acid (58 mCi/mmole) was from Amersham/Searle and D-[14C]glutamic acid (8 mCi/ mmole) was from Calatomic. RESULTS

Transport of free and protein-bound [14C]proline in optic nerve of carp In goldfish optic nerve the axoplasmic flow of proteins can be easily observed if proline is the labeled amino acid used ~. We administered this amino acid to a species closely related to goldfish, the carp, which conveniently has an optic nerve and tract about 4 cm long. The acid-soluble and acid-insoluble radioactivity in the optic system was measured at various times after the administration of labeled proline (Fig. 1). As had been observed in previous studies, the protein-bound radioactivity showed, 6 h after administration of the amino acid, 3 peaks of protein radioactivity which corresponded to protein movement by rapid and slow flow along the axon. In contrast to protein the amino acid radioactivity did not show these maxima along the nerve. The radioactivity was always highest in the end of the nerve closest to the injection and rapidly decreased distally, showing only traces in the terminus of the optic tract. At 2 h significant radioactivity could be detected in the acid-soluble fraction, with no label in proteins, indicating that the labeled free amino acid in the nerve did not originate from the local breakdown of labeled proteins. In contrast with the differences found with protein, in the optic lobes no difference between control and experimental tissue was found in acid-soluble radioactivity during the first 20 h, and label appeared only in the 30-h experiment, at which time it was higher than in proteins.

Transport of 2-aminoisobutyric acid in the optic nerve of carp We also tested the axonal transport of a non-metabolizable amino acid that is not incorporated into proteins. Following the injection of [14C]aminoisobutyric acid, two well-defined peaks could be observed in the experimental nerve and tract, the first about 10 h and the second about 70 h after the injection (Fig. 2). Calculating from the average total length of the nerve and tract (35-40 mm), the flow rate of the first peak would be about 90 mm/day, and the second peak, 10-15 mm/day. In the nerve connected with the non-injected eye there was negligible radioactivity, which showed no peaks and gave a saturation-type curve. The label appearing in the control nerve, presumably through the circulation, was therefore much less than the transported

274

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distance from eyeball (mm) Fig. 1. Free and protein-bound [14C]prolinealong the optic nerve in carp. Carp were given an injection of 5 #Ci L-[14C]prolinein the right eye and were sacrificed at the times marked on the figure. The optic nerve and tract were dissected in one piece and cut into 5 mm long sections; free and proteinbound radioactivity was measured in each. The values given are the differences between the corresponding experimental and control sections. ©, free amino acids; 0, protein. Each time point is the average of two experiments. label in the experimental nerve. There was no difference in the label appearing in the optic lobe between the experimental and the control sides in these experiments. At each time point investigated the portion of the nerve closest to the injection site contained the highest amount of radioactivity, but with time label in the proximal portion decreased and in distal portions increased (Fig. 3). The relative distribution of radioactivity in the different parts of the nerve showed maxima (peaks), which occurred at different time points in the different sections (Fig. 4). The calculated rate of flow was 5-10 mm/day from the peak of the first 5 mm piece and 90-110 mm/day from the peak of the second 5 mm piece. These values measured from sections correspond well with the values of 10-15 and 90 mm/day respectively calculated from the whole nerve (Fig. 3).

Transport of free proline and D-glutamic acid in sections of nerve The change in distribution of labeled proline and glutamate with time in the first and second 5 mm section (Fig. 5) was very similar to that shown with aminoisobutyric acid (Fig. 4). The maximum of the first section would be utilized for calcula-

275

AXONAL FLOW OF AMINO ACIDS

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Fig. 2. Content of radioactivity in carp optic nerve after [~4C]aminoisobutyric acid injection. Carp were given an injection of 10 #Ci [14C]AIB in the right eye; the optic nerve and tract were dissected and the radioactivity was measured. The solid line represents the total radioactivity of the experimental side, the dotted line that oftbe control side. Values are the averages of 4-8 experiments; the standard error is shown on the figure. • • , experimental; • . . . . . . • , control.

ting the lower rate o f t r a n s p o r t , a n d t h a t o f the second section, the highest rate o f t r a n s p o r t o f these a m i n o acids a l o n g the nerve. T h e values for the faster flow were a p p r o x i m a t e l y 90-110 m m / d a y with p r o l i n e a n d g l u t a m i c acid; the slower flow w a s 13 m m / d a y with p r o l i n e a n d 20 m m / d a y with glutamate. I n these experiments, r a d i o activity a p p e a r e d also in the optic t r a c t with time, b u t in t o o small a n a m o u n t to reliably estimate a n y rates o f m o v e m e n t .

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Fig. 3. Changes in the relative distribution of [14C]aminoisobutyrate along the nerve with time. Carp were given an injection of 10 #Ci [14C]AIB in the right eye and were sacrificed at 6, 48, and 100 h. The optic nerve and tract were dissected in one piece and were cut into 5 mm sections; radioactivity was measured for each. The numbers (averages of 4-8 experiments) represent the difference between the experimental and control side, and are expressed as per cent in section of the total in the nerve. • , 6 h; v , 48 h; l , 100 h.

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276

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Fig. 4. Relative distribution curve of [14C]aminoisobutyrate in 3 sections of the optic nerve. The numbers represent the radioactivity of the given piece, experimental minus control side, as per cent of the total. Values were calculated from the experiments shown in Fig. 3.

Composition of the free amino acid pool of the optic system in carp Differences in migration rate among the various amino acids may be related to their concentration in the nerve. The composition of the free amino acid in optic nerve was different from that in brain (Table I). There were no great differences between cerebellum and the optic lobe; but in optic nerve and tract cystathionine levels were very high, serine and lysine levels were more than twice as high as in brain, and gluta-

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Fig. 5. Relative distribution of [14C]proline and D-[14C]glutamic acid in the first two sections of the optic nerve. Carp were given injections of either 10/,Ci L-[14C]proline or 10/,Ci D-[14C]glutamate and sacrificed at different intervals. Optic nerve and tract were dissected in one piece and cut into 5 mm sections; acid-soluble radioactivity was measured for those injected with proline; total radioactivity was measured witl~ glutamate. Differences between experimental and control sides as per cent of total radioactivity in the sections were plotted against time. Averages of 2-3 experiments are given. A, L-proline; B, D-glutamic acid.

277

AXONAL FLOW OF AMINO ACIDS

TABLE I THE FREE AMINO ACID POOL IN THE OPTIC SYSTEM OF CARP

Tissues were rapidly dissected, frozen, weighed, and homogenized in approximately 3 voi. of 1 perchloric acid. 1 ml extract was analyzed in a Technicon autoanalyzer. Averages of 3 experiments (4 with nerve and tract) are given.

#moles amino acid/gfresh tissue

P-serine Taurine P-ethanolamine Aspartic acid Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Cystathionine Isoleucine Leucine Tyrosine Phenylalanine Ethanolamine Ornithine Lysine Histidine Arginine

Optic nerve Optictract Opticlobe

Cerebellum Retina

Vitreous body

0.67 -4-0.33 4.12 4- 0.09 0.23 ± 0.03 2.52 4- 0.90 0.30 4- 0.10 0.80 ± 0.08 0.94 i 0 . 1 5 1.98 4- 0.05 0.65 ± 0.45 0.39 4, 0.18 0.86 4- 0.19 0.26 4- 0.04 9.55 4, 0.13 0.17 4- 0.02 0.32 4- 0.04 0.10 4- 0.03 0.14 4- 0.02 0.27 4- 0.07 0.20 4- 0.06 1.01 4. 0.50 0.21 4- 0.03 0.314,0dl

0.15 ! 0.01 4.84 ± 0.92 0.62 4- 0.07 3.54 4- 0.53 0.36 4- 0.09 0.33 4- 0.09 5.49 4- 0.30 7.73 4- 0.33 0.12 5:0.04 0.62 4- 0.11 0.66 4- 0.07 0.15 4- 0.04 0.32 4- 0.08 0.13 4- 0.03 0.22 4, 0.04 0.09 -4-0.02 0.13 4. 0.02 0.42 4- 0.04 0.07 4- 0.02 0.34 4, 0.03 0.36 4- 0.07 0.144-0.03

0.04 + 0.02 1.26 -4-0.12 0.09 4- 0.03 0.55 -4- 0.14 0.07 4- 0.01 0.06 4- 0.01 0.13 4- 0.02 0.60 4- 0.04 0 0.06 4- 0.02 0.11 4- 0.02 0.03 4- 0.01 0.08 4- 0.03 0.05 4- 0.01 0.13 4, 0.01 0.05 4- 0.01 0.06 4- 0.01 0.06 4, 0.01 0.02 4- 0.01 0.10 4- 0.02 0.05 4- 0.01 0.054,0.01

0.27 4- 0.07 4.12 4- 0.81 0.50 4- 0.20 3.05 i 0.09 0.36 i 0.12 0.64 i 0.18 1.97 i 0.81 5.43 4- 1.59 0.22 i 0.21 0.50 4, 0.22 0.59 4, 0.18 0.20 4, 0.04 4.44 4- 0.71 0.13 4- 0.06 0.21 4, 0.05 0.09 4- 0.02 0.13 4- 0.05 0.30 4- 0.14 0.15 4, 0.02 0.67 4, 0.15 0.26 4- 0.03 0.244-0.07

0.15 + 0.02 4.65 ± 0.74 0.54 ± 0.06 3.77 4- 1.01 0.32 4- 0.05 0.29 4- 0.03 3.65 4- 0.18 6.35 4- 0.32 0.17 4- 0.05 0.60 4- 0.06 0.58 4- 0.07 0.13 4- 0.02 0.39 4- 0.01 0.10 4- 0.02 0.18 4- 0.02 0.08 -4-0.02 0.10 4- 0.01 0.55 4- 0.07 0.10 4- 0.01 0.39 4. 0.05 0.56 4- 0.04 0.154,0.02

0.15 ± 0.04 9.93 i 0.30 0.37 4- 0.05 3.64 4- 0.82 0.34 4- 0.03 0.37 4- 0.11 0.83 + 0.05 1.04 4- 0.12 0.30 4- 0.08 0.61 4- 0.15 0.91 4- 0.08 0.34 ± 0.02 1.05 4- 0.28 0.23 4- 0.02 0.41 4- 0.02 0.14 4- 0.03 0.18 4, 0.02 0.32 4- 0.09 0.09 4, 0.03 0.42 4- 0.13 0.17 4- 0.07 0.214-0.08

mic acid a n d g l u t a m i n e levels were lower. Differences between b r a i n a n d nerve were greater in the optic nerve t h a n in the optic tract in that cystathionine, serine, a n d lysine were higher, glutamic acid a n d g l u t a m i n e lower, in the optic nerve t h a n in the optic tract. There was a gradient in the nerve, increasing with some a n d decreasing with other a m i n o acids in the distal direction. Proline levels decreased distally. The c o m p o sition of the pool in carp nerve was very different from that f o u n d for the a m i n o acid pools in other nerves; lobster a n d crab nerve contains very high levels o f aspartic acid, alanine, a n d glycine a n d rat sciatic nerve had rather low levels o f most c o m p o u n d s 16.

Exit o f the free amino acids from the injected eye I n order to evaluate experiments of longer d u r a t i o n , we measured the rate o f decrease o f label in the injected eye. L-Proline, o - g l u t a m i c acid, a n d a - a m i n o i s o b u t y r i c acid showed a rapid decrease of radioactivity in the eye (Fig. 6). More experimental points were o b t a i n e d with proline a n d a m i n o i s o b u t y r a t e ; o f these two, the exit o f proline seemed tO be more rapid. After a b o u t 20 h, 50 ~ o f a m i n o i s o b u t y r a t e a n d 25 ~o o f proline were still present in the eye. This rapid exit of label from the eye raised the possibility that some of this label present in the p l a s m a c o n t a m i n a t e d nerve samples,

v. CSANYI et aL

278

2O

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0

20

40

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time after injection (hours) Fig. 6. Exit of labeled amino acids from the eye. Carp were given injections of either l 0 # C i [14C]AIB, L-proline, or D-glutamate and sacrificed at different times; the total radioactivity in the injected eye was measured. O, 2-aminoisobutyric acid; D, D-glutamic acid; O, L-proline.

especially since a vein coming from the eye is present in the first 2 mm section of the optic nerve in the carp and then branches off from it. In a separate set of experiments, after the injection of [14C]aminoisobutyric acid this vein was dissected separately, and its radioactivity was measured at various times. Fig. 7 shows that the label was initially significant in the vein, but it rapidly decreased and within a few hours was too low to significantly affect values for the nerve. In order to exclude the possibility that the flow of amino acids is accounted for by the changes in circulation of the nerve sheath, outer and inner portions of the nerve were separated and analyzed separately (Table II). All parts contained more radioactivity in the inner portion of the part measured, and the ratio of inner:outer part

100

0

I

20 40 60 80 time after injection (hours)

100

Fig. 7. Possible label in nerve contributed by blood. Carp were given injections with 10 #Ci [14C]AIB and were sacrificed at different times; the optic nerve was taken out and the first 10 mm o f the central vein was dissected. The vein was dissolved in 1 N sodium hydroxide, and its total radioactivity was measured. Values are averages of two experiments.

AXONAL FLOW OF AMINO ACIDS

279

TABLE II D I S T R I B U T I O N OF

[14ClAIB BETWEEN

INNER A N D O U T E R P A R T OF THE NERVE

Carp were given injections of 10 #Ci [14C]AIB and sacrificed 6 h later; the optic nerve was dissected, frozen in dry ice, and cut into 3 mm sections. The outer part of each section was separated from the inner portion as illustrated, and the total radioactivity was measured in each part. Averages of two experiments are given. Distance from eye (ram)

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3.7

6-- 9

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15-18

1.7

18-21

1.9

r a d i o a c t i v i t y was highest in the p r o x i m a l p a r t o f the nerve; thus there was no significant c o n t a m i n a t i o n f r o m the circulation. F u r t h e r tests for excluding c o n t r i b u t i o n s f r o m the circulation to m e a s u r e d values o f a x o p l a s m i c t r a n s p o r t c o m p a r e d the d i s t r i b u t i o n o f two a m i n o acids injected together into the eye. A solution c o n t a i n i n g [ a H ] a m i n o i s o b u t y r a t e a n d D-[14C]glutamate was administered, a n d the level o f the two a m i n o acids was m e a s u r e d in the optic system at v a r i o u s times. A m i n o i s o b u t y r a t e was lost f r o m the eye at a higher rate t h a n D-glutamate a n d the r a t i o o f 3H :14C decreased f r o m 3 (present in the injecting solution) to a b o u t 0.5 at the e n d o f the experiment. The m o r e r a p i d release o f a m i n o -

TABLE III C H A N G E S IN THE RATIO OF C O N T E N T OF AMINOISOBUTYRATE TO G L U T A M A T E W I T H TIME

Carp were given injections of a solution containing 50 ~Ci [aH]a-aminoisobutyrate and O-[14C]glutamate in a molar ratio of 3:1. After different times the animals were sacrificed, the optic pathway was dissected, and aH and 14C content measured in each part. Averages of two experiments are given. Hours after injection

1.5 6 18 30 100

Concentration ratio (ttmoles) aminoisobutyrate to glutamate Eye

2.7 2.1 1.7 1.8 0.48

Optic lobe Contralateral

lpsilateral

0.9 6.6 6.6 11 11

0.9 6.3 6.7 I1 11

First 5 mm of ipsilateral nerve

2.0 2.7 7.0 8.5 8.1

v. CSANYI et u/.

280

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temperature (°C) Fig. 8. Temperature dependence of the content of aminoisobutyrate in the eye and in the optic nerve

of goldfish. Goldfish ordinarily kept at 20 °C were adapted to different temperatures for 2 days. An injection of I/~Ci [1 4 C]AIB was given into the right eye of each goldfish. After 1 h they were sacrificed, and the optic pathways and the eyes were dissected. A" remaining radioactivity of the eye, 2 × 106 disint./min injected. B: total radioactivity of the optic nerve and tract of the experimental side minus control side. Values are the averages of 6--8 experiments; standard errors are given by the vertical

lines. isobutyrate from the site of injection probably resulted in higher levels of labeled aminoisobutyrate than D-glutamate in the circulation. This was reflected in the increasing ratio of AIB to glutamate in the optic lobe with time. There were no significant differences in this ratio between the contralateral and ipsilateral portions of the lobe (Table III). The ratio also changes in the first 5 mm portion of the experimental nerve. It increased in time, but the values in nerve were different from those in the optic lobe, indicating that the label in the nerve did not originate from the circulation.

Temperature dependence of the amino acid transport Transport of proteins along the axon was shown to be strongly temperature dependent 6,1°,19. Temperature dependence of free amino acid transport was tested in goldfish, because carp do not tolerate extreme temperature changes. In a set of experiments we found that distribution of a-aminoisobutyrate in goldfish is similar to that in carp (as shown in Fig. 3). Goldfish were kept for 2 days at the various experimental temperatures (3-36 °C) in order to adapt them. The goldfish tolerated these changes in temperature without loss of any animals; the fish kept at 15-36 °C readily accepted food, but those kept at 10 °C or below would not eat. When the radioactivity of the optic nerve was measured 1 h after the injection of labeled aminoisobutyrate, strong

281

AXONAL FLOW OF AMINO ACIDS TABLE IV THE DISTRIBUTION OF AMINOISOBUTYRATEIN TIIE OPTIC SYSTEMOF VARIOUSSPECIES

Carp and rabbits were given injections of 10/tCi, chicks 1 #Ci [14C]AIB; after different times the animals were sacrificed, the optic pathways dissected, and the total radioactivity of each part was measured. The numbers represent the difference between the experimental and control sides and are the averages of 2-3 experiments.

Disint./min/part, experimental minus control lh

2.5h

6h

24h

2750 990

3580 80

12300 120

6520 50

419 0

742 47

327 5

23 0

50 0

3200 0

250 0

40 0

Carp Optic nerve Optic tract

Chick Optic nerve Optic tract

Rabbit Optic nerve Optic tract

dependence on temperature was found (Fig. 8), with maximal label in the nerve at 20 °C (the temperature at which the animals were kept before being adapted to the various temperatures). In contrast to results with the nerve, the radioactivity remaining in the eye after 1 h did not show strong temperature dependence (Fig. 8A). The sharp peak of label in the nerve at 20 °C (Fig. 8B) indicated little contribution from passive diffusion to the level of label in the nerve.

Aminoisobutyric acid transport in various species Since some of the contradictory results in previous reports on the existence or nonexistence of flow of free amino acids in the axon may have been due to the use of different species in different laboratories, the appearance of labeled AIB in the optic nerve was measured in chicks and rabbits as well as in carp (Table IV). Labeled amino: isobutyric acid could be found in the optic nerve of all 3 species, although the labeling in chicks and rabbits was lower, and maximal labeling was at an earlier time point than in carp. The calculated rate of transport was approximately 3 times as high in rabbits and chicks as in carp. The optic lobe in chicks and carp and the lateral geniculate body in rabbits under these experimental conditions did not contain a significant amount of radioactivity. The injected label left the eye more rapidly in chicks and rabbits than in carp; the label remaining in carp, chicks and rabbits was 75, 47 and 32 % respectively after 2.5 h; 60, 31 and 26% after 6 h; and 20, 12 and 8% after 24 h. DISCUSSION

Measurement of axonal amino acid transport We interpret the results of our experiments as showing that compounds of low

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molecular weight, in our case amino acids and nonmetabolizable amino acid analogs, are transported along the axon in the optic nerve when the amino acids are injected into the eye of several species. The fact that there is movement of amino acids does not necessarily indicate that a process specific for amino acids exists in the axoplasm. It is quite clear from numerous studies that particles and macromolecules such as proteins and nucleic acids participate in the axoplasmic flow and that for proteins there are at least two flow rates 2,18,2a. Compounds of low molecular weight may be transported protein- or particulate-bound. Since the axoplasmic flow of particulates which most likely contain amino acids is well established, it is somewhat surprising that the available experiments are contradictory on the axonal transport of low-molecularweight metabolites. There may be several reasons for the difficulty in detection of axoplasmic transport of amino acids. The concentration of free amino acids in most cases is 1 ~ or less of their protein-bound form; amino acids may be metabolized and may also be rapidly incorporated into macromolecules. (In one study ~8, it was estimated that within 20 min most of the amino acid administered was already incorporated into proteins.) Experiments with compounds less active metabolically, such as galactosamine, fucose, and putrescine, did indicate the presence of axoplasmic transport for these materials a,7,s. An additional complication with amino acids may be that some could originate from the breakdown of proteins traveling along the axon. The fact that in our experiments there was little difference between the transport of amino acids that are incorporated into proteins and that of analogs that are not, indicates that local breakdown does not greatly contribute to the appearance of axonal flow of the amino acids. With amino acids, it is difficult to calculate the flow rates with great precision, partly because exit from the axon into the circulation or the surrounding extra-axonal space is superimposed. Of equal importance, the true specific activity of the amino acid traveling down is not known since amino acid uptake into neurons may be not uniform throughout the retina. The rates of flow of the free amino acids that we estimated seem to be very close or identical to those of proteins, both in the slow and in the fast flow. Rates of fast flow of proteins vary from 50 m m to 2000 mm/day; the variations between the different reports may be partly due to differences in the methods of estimating this flow. In warm-blooded animals, Ochs estimated the fast flow for proteins to be about 400 mm/day in cat, rabbit, monkey, rat, and dog nerves 18. Our rates of approximately 100 mm/day in fish optic nerve are similar to the rate of flow of proteins in fish nerve of about 70-100 mm/day 6, and the faster flow that we found in chicks and rabbits, about 3 times as much, is close to rates reported for the sciatic nerve in warm-blooded animals. Since the flow (Fig.8) is temperature dependent, the difference between fish and cat may be, at least in part, due to differences in temperature. These values are also similar to the fast flow of o-glucosamine s, which was estimated at approximately 50 mm/day, but lower than the value for D-glutamic acid flow in snail ganglia, estimated at 720 mm/day 13.

Mechanism of flow The mechanism of axoplasmic transport of the various molecules is not clear. It

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is thought 2a that the low rate of flow of macromolecules is due to the peristaltic movement of axoplasm, which would not be a selective mechanism, but would result in the movement of most components present in the axoplasm - - particles, macromolecules, and also metabolites of low molecular weight. The fast flow mechanism could be more specific where the microtubular proteins play an important role. It is difficult to perceive how such a mechanism could be involved in the selective transport of metabolites of low molecular weight, although this possibility could not be excluded. Recently the intra-axonal flow of free nucleosides and nucleotides was shown in goldfish nerve it. It is possible that the amino acids are adsorbed to proteins or are present in particulates and are, therefore, transported in a bound form. The exchange ~f free amino acids along the axon may be a reason for differences in distribution of label along the nerve between amino acids and proteins, the movement of free amino acid across nerve membranes altering labeling by a counter-current type of mechanism. This exchange, which may be in itself temperature dependent, may be one of the reasons that in warm-blooded animals the axonal transport of amino acids could not be observed in a number of experiments. The exit or exchange along the nerve may in addition serve to establish increasing or decreasing proximo-distal gradients in amino acid levels; we observed such gradients not only in carp nerve (Table I) but also in other nerve, including lobster and crab leg nerve and rat sciatic nerve t6. Differences in the composition of the free amino acid pool between various species are much greater in the peripheral than in the central nervous system. In order to test the movement of amino acids between the axoplasm and its environment, in a preliminary set of experiments we tried to measure the exit of amino acids from the optic system in vitro. In these experiments, the system was dissected in one piece, the eye was suspended in a plastic cup and the attached nerve was suspended in the buffered medium, and the movement of the amino acid (injected into the eye) from the nerve to the medium was measured. These experiments indicated a very slow exit of the administered amino acid from the nerve to the surrounding fluid, but this may have been due to the changes of permeability in the in vitro preparation. If the proteins transported proximo-distally along the axon were broken down at nerve terminals and thus supplied ample quantities of amino acids, a separate transport process supplying additional amounts would be superfluous. Further work has to clarify the role, if any, of axonal transport of amino acids in the function of the nervous system. ACKNOWLEDGEMENTS

The authors are indebted to Mr. Jeno Toth for the amino acid analyses. This investigation was supported in part by Public Health Service Grants NS03226 and RR05707.

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