Neurochemical Research (3) 479- 492 (1978)
A X O N A L TRANSPORT OF LIPID IN GOLDFISH OPTIC AXONS J. R . C U R R I E , 1 B . G R A F S T E I N ,
M.
H.
WHITNALL,
AND
R.
ALPERT
Department of Physiology, Cornell University Medical College New York, New York 10021
Accepted January 20, 1978
After injection of labeled glycerol, choline, or serine into the eye of goldfish, labeled lipids were axonally transported along the optic nerve to the optic tectum. Although the different precursors were presumably incorporated into somewhat different lipid populations, all three were approximately equally effective in labeling the lipids transported to the tectum, but the amount of transported material remaining in the nerve was different, being highest with choline and lowest with serine. The labeled lipids appeared in the tectum within 6 hr of the injection, indicating a fast rate of transport, but continued to accumulate over a period of 1-2 weeks, which presumably reflects the time course of their release from the cell body. Since there was a gradual increase in the proportion of labeled lipid in the rectum during this period, some other process in addition to fast axonal transport may have affected the distribution of the lipids along the optic axons, When [3Hlcholine was used as precursor, the transported material included a small amount of TCA-soluble material, which was probably mainly phosphorylcholine, with labeled acetylcholine appearing in only insignificant amounts. With serine, which gave rise to a large amount of axonally transported protein in addition to lipid, a late increase in the amount of labeled lipid in the tectum was seen, accompanied by a decrease in labeling of the protein fraction.
INTRODUCTION After injection of a radioactively labeled amino acid into the goldfish eye, labeled protein is axonally transported in the retinal ganglion cell i Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, New York.
479 0364-3190/78/08~0-0479505.00/0t~ 1978PlenumPublishingCorporation
480
CURRIE ET AL.
axons to their terminals in the contralateral optic tectum. There are both a fast component and a slow component of protein transport, proceeding at rates of about 100 mm/day and 0.5 mm/day, respectively (11). Axonal transport of phospholipid in this system has been studied using [3H]glycerol as the injected precursor (12). In this case, the amount of transported labeled phosphoglyceride appearing in the tectum was found to increase over a period of about a week after the injection. Analysis of the characteristics of the transport indicated that the rate of transport of the lipid was equivalent to the rate of the fast component of protein transport, the relatively slow rate of accretion of labeled lipid in the tectum being determined by the rate of release of labeled material from the cell body into the axon, rather than by its rate of movement along the axon. Axonal transport of glycolipids labeled with either [3H]N-acetylmannosamine or [ZH]glucosamine also appears to have the same characteristics (6; M. Whitnall, unpublished results). There is general agreement that various lipids are associated with the fast component of transport (1, 8, 13, 17, 18, 21, 31). However, it has been suggested that some lipids are also involved in slow axonal transport (13, 21, 23), although our findings with glycerol provided no evidence of this. The present study, therefore, was intended as a brief survey of the transport characteristics of lipids labeled with serine or choline in comparison to those labeled with glycerol. Serine has been previously used for studying axonal transport of lipids in rabbit visual system (18, 31). Like glycerol, serine and choline are incorporated into a wide variety of cellular lipids, but there are some differences in their distribution within the cell (19). For example, glycerol is excluded from sphingolipids, whereas serine is, to a large extent, converted to these compounds, which are important constituents of the plasma membrane and of myelin. Choline also enters some sphingolipids, but most of it (33) goes into choline phosphoglyceride (lecithin), the major lipid constituent of cell membranes. Compared to the plasma membrane, intracellular membranes such as the endoplasmic reticulum have a higher concentration of choline phosphoglyceride and a lower concentration of sphingolipids and serine phosphoglyceride (22). Hence choline is more likely than serine to label intracellular membranes. Another characteristic of choline is that it may exchange readily from one cellular compartment to another (24, 25, 30), in contrast to glycerol which is less likely to be recycled within the cell (2). The use of choline as a precursor also enabled us to test for the presence of acetylcholine (ACh), which is reputed to be the synaptic transmitter in some retinal ganglion cells (10, 15, 26), and which has been demonstrated to be rapidly transported in an ACh-synthesizing cell (16).
AXONAL TRANSPORT OF LIPID
EXPERIMENTAL
481
PROCEDURE
In goldfish (Carassius auratus) of 3-4 in. overall length, the left eye was injected with 2/xl of normal saline containing 13/~Ci of [methyl-3H]choline chloride (specific activity 4.2 Ci/mmol) or 1.4/xCi of [3H]L-serine (specific activity 3.12 Ci/mmol) or 3 ~1 of a combination of 0.24 /zCi of [1,2-14C]choline chloride (specific activity 7.8 mCi/mmol) and 2.3 /~Ci of [2-3H]glycerol (specific activity 6.48 Ci/mmol). All radiochemicals were obtained from New England Nuclear Corporation, Boston, Massachusetts. Six fish were used for each determination. At an appropriate interval after the injection, each fish was decapitated and a sample of the nerve from the injected eye as well as both optic tecta were dissected out. The nerve sample, which was 1-2 mm long, began about 1 mm behind the eye. Each tissue sample was placed in 0.5 ml cold 10% trichloroacetic acid (TCA) to extract the small-molecular-weight (TCA-soluble) materials and precipitate the larger molecules. After the tissues had remained in TCA at 4~ for 24 hr, the lengths of the nerve samples were measured to the nearest 0.1 mm and each sample was placed in 1 ml chloroform-methanol (2 : l v/v) at 4~ for 24 hr. Then each sample was removed and air dried, and the tecta were weighed. The TCA-soluble material, the chloroform-methanolsoluble material, and the residual material (protein fraction) obtained by this technique have been found to correspond closely to, respectively, the "dialyzable" fraction of the Folch upper phase, the lipid fraction (including gangliosides), and the "residue" obtained by conventional lipid-separation techniques (6, 8, 12). The relatively low proportion of methanol used for lipid extraction may have given a somewhat incomplete extraction of polar lipids, but ensured that contamination of the lipid fraction by protein was kept to a minimum. Radioactivity in each of the three fractions obtained from each sample was measured by liquid scintillation counting under the following conditions: the TCA extract (0.5 ml) was combined with Aquasol scintillation medium (New England Nuclear Corporation); the chloroform-methanol extract (1 ml) was air dried, the residue was redissolved in 0.5 ml methanol, and a toluene-based scintillation liquid was added; the dried lipidextracted tissue containing the labeled protein fraction was solubilized in Soluene-100 (Packard Instrument Co.) and then disperse d in a toluene-based scintillation liquid formulated to minimize chemiluminescence. Counting efficiency was 25-35% for 3H and 55-65 % for 14C determined by internal radioactivity standards. For the experiments involving high-voltage electrophoresis, pooled tissues from three fish were used for each experimental group. The fresh tissues were homogenized in ice cold acetone-1 M formic acid (85:15). The supernatant fraction (which contained about 98% of the total radioactivity) was dried in a stream of N2 and then subjected to electrophoresis on Whatman 3 MM paper at pH 4.7 (see reference 16). The areas on the electrophoresis paper containing various labeled constituents were cut out, and the radioactivity was eluted from the paper with distilled water for liquid scintillation counting in Aquasol. About 25% of the applied radioactivity was recovered, most of the lipids having been leached out by the solvent in the electrophoresis tank. A large proportion of the radioactive material injected into the eye escapes into the circulation and becomes distributed throughout the body, producing a background of radioactivity in the brain. This background appears equally on both sides of the brain, whereas the radioactivity that is axonally transported from the injected eye appears only on the side to which the eye projects, i.e., in the ipsilateral optic nerve and the contralateral optic rectum. In this study, the transported radioactivity reaching the optic tectum was always calculated from the difference between the right and left tecta, but the nerve values were not corrected for background, because the background was very low (-
A X O N A L TRANSPORT OF LIPID IN GOLDFISH OPTIC AXONS J. R . C U R R I E , 1 B . G R A F S T E I N ,
M.
H.
WHITNALL,
AND
R.
ALPERT
Department of Physiology, Cornell University Medical College New York, New York 10021
Accepted January 20, 1978
After injection of labeled glycerol, choline, or serine into the eye of goldfish, labeled lipids were axonally transported along the optic nerve to the optic tectum. Although the different precursors were presumably incorporated into somewhat different lipid populations, all three were approximately equally effective in labeling the lipids transported to the tectum, but the amount of transported material remaining in the nerve was different, being highest with choline and lowest with serine. The labeled lipids appeared in the tectum within 6 hr of the injection, indicating a fast rate of transport, but continued to accumulate over a period of 1-2 weeks, which presumably reflects the time course of their release from the cell body. Since there was a gradual increase in the proportion of labeled lipid in the rectum during this period, some other process in addition to fast axonal transport may have affected the distribution of the lipids along the optic axons, When [3Hlcholine was used as precursor, the transported material included a small amount of TCA-soluble material, which was probably mainly phosphorylcholine, with labeled acetylcholine appearing in only insignificant amounts. With serine, which gave rise to a large amount of axonally transported protein in addition to lipid, a late increase in the amount of labeled lipid in the tectum was seen, accompanied by a decrease in labeling of the protein fraction.
INTRODUCTION After injection of a radioactively labeled amino acid into the goldfish eye, labeled protein is axonally transported in the retinal ganglion cell i Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, New York.
479 0364-3190/78/08~0-0479505.00/0t~ 1978PlenumPublishingCorporation
480
CURRIE ET AL.
axons to their terminals in the contralateral optic tectum. There are both a fast component and a slow component of protein transport, proceeding at rates of about 100 mm/day and 0.5 mm/day, respectively (11). Axonal transport of phospholipid in this system has been studied using [3H]glycerol as the injected precursor (12). In this case, the amount of transported labeled phosphoglyceride appearing in the tectum was found to increase over a period of about a week after the injection. Analysis of the characteristics of the transport indicated that the rate of transport of the lipid was equivalent to the rate of the fast component of protein transport, the relatively slow rate of accretion of labeled lipid in the tectum being determined by the rate of release of labeled material from the cell body into the axon, rather than by its rate of movement along the axon. Axonal transport of glycolipids labeled with either [3H]N-acetylmannosamine or [ZH]glucosamine also appears to have the same characteristics (6; M. Whitnall, unpublished results). There is general agreement that various lipids are associated with the fast component of transport (1, 8, 13, 17, 18, 21, 31). However, it has been suggested that some lipids are also involved in slow axonal transport (13, 21, 23), although our findings with glycerol provided no evidence of this. The present study, therefore, was intended as a brief survey of the transport characteristics of lipids labeled with serine or choline in comparison to those labeled with glycerol. Serine has been previously used for studying axonal transport of lipids in rabbit visual system (18, 31). Like glycerol, serine and choline are incorporated into a wide variety of cellular lipids, but there are some differences in their distribution within the cell (19). For example, glycerol is excluded from sphingolipids, whereas serine is, to a large extent, converted to these compounds, which are important constituents of the plasma membrane and of myelin. Choline also enters some sphingolipids, but most of it (33) goes into choline phosphoglyceride (lecithin), the major lipid constituent of cell membranes. Compared to the plasma membrane, intracellular membranes such as the endoplasmic reticulum have a higher concentration of choline phosphoglyceride and a lower concentration of sphingolipids and serine phosphoglyceride (22). Hence choline is more likely than serine to label intracellular membranes. Another characteristic of choline is that it may exchange readily from one cellular compartment to another (24, 25, 30), in contrast to glycerol which is less likely to be recycled within the cell (2). The use of choline as a precursor also enabled us to test for the presence of acetylcholine (ACh), which is reputed to be the synaptic transmitter in some retinal ganglion cells (10, 15, 26), and which has been demonstrated to be rapidly transported in an ACh-synthesizing cell (16).
AXONAL TRANSPORT OF LIPID
EXPERIMENTAL
481
PROCEDURE
In goldfish (Carassius auratus) of 3-4 in. overall length, the left eye was injected with 2/xl of normal saline containing 13/~Ci of [methyl-3H]choline chloride (specific activity 4.2 Ci/mmol) or 1.4/xCi of [3H]L-serine (specific activity 3.12 Ci/mmol) or 3 ~1 of a combination of 0.24 /zCi of [1,2-14C]choline chloride (specific activity 7.8 mCi/mmol) and 2.3 /~Ci of [2-3H]glycerol (specific activity 6.48 Ci/mmol). All radiochemicals were obtained from New England Nuclear Corporation, Boston, Massachusetts. Six fish were used for each determination. At an appropriate interval after the injection, each fish was decapitated and a sample of the nerve from the injected eye as well as both optic tecta were dissected out. The nerve sample, which was 1-2 mm long, began about 1 mm behind the eye. Each tissue sample was placed in 0.5 ml cold 10% trichloroacetic acid (TCA) to extract the small-molecular-weight (TCA-soluble) materials and precipitate the larger molecules. After the tissues had remained in TCA at 4~ for 24 hr, the lengths of the nerve samples were measured to the nearest 0.1 mm and each sample was placed in 1 ml chloroform-methanol (2 : l v/v) at 4~ for 24 hr. Then each sample was removed and air dried, and the tecta were weighed. The TCA-soluble material, the chloroform-methanolsoluble material, and the residual material (protein fraction) obtained by this technique have been found to correspond closely to, respectively, the "dialyzable" fraction of the Folch upper phase, the lipid fraction (including gangliosides), and the "residue" obtained by conventional lipid-separation techniques (6, 8, 12). The relatively low proportion of methanol used for lipid extraction may have given a somewhat incomplete extraction of polar lipids, but ensured that contamination of the lipid fraction by protein was kept to a minimum. Radioactivity in each of the three fractions obtained from each sample was measured by liquid scintillation counting under the following conditions: the TCA extract (0.5 ml) was combined with Aquasol scintillation medium (New England Nuclear Corporation); the chloroform-methanol extract (1 ml) was air dried, the residue was redissolved in 0.5 ml methanol, and a toluene-based scintillation liquid was added; the dried lipidextracted tissue containing the labeled protein fraction was solubilized in Soluene-100 (Packard Instrument Co.) and then disperse d in a toluene-based scintillation liquid formulated to minimize chemiluminescence. Counting efficiency was 25-35% for 3H and 55-65 % for 14C determined by internal radioactivity standards. For the experiments involving high-voltage electrophoresis, pooled tissues from three fish were used for each experimental group. The fresh tissues were homogenized in ice cold acetone-1 M formic acid (85:15). The supernatant fraction (which contained about 98% of the total radioactivity) was dried in a stream of N2 and then subjected to electrophoresis on Whatman 3 MM paper at pH 4.7 (see reference 16). The areas on the electrophoresis paper containing various labeled constituents were cut out, and the radioactivity was eluted from the paper with distilled water for liquid scintillation counting in Aquasol. About 25% of the applied radioactivity was recovered, most of the lipids having been leached out by the solvent in the electrophoresis tank. A large proportion of the radioactive material injected into the eye escapes into the circulation and becomes distributed throughout the body, producing a background of radioactivity in the brain. This background appears equally on both sides of the brain, whereas the radioactivity that is axonally transported from the injected eye appears only on the side to which the eye projects, i.e., in the ipsilateral optic nerve and the contralateral optic rectum. In this study, the transported radioactivity reaching the optic tectum was always calculated from the difference between the right and left tecta, but the nerve values were not corrected for background, because the background was very low (-
