Bidirectional axonal transport of free glycine in identified neurons R3--R14 of Aplysia.

The axonal transport of 3H-amino acids was studied in the axons of identified neurons R3--R14 in the parietovisceral ganglion (PVG) of the mollusc Aplysia. The PVG was incubated (3--24 hr) in media containing physiological concentrations of single 3H-amino acids while the isolated nerve was superfused with plain or chemically altered media. The nerve was then sliced into sequential segments for biochemical analyses or fixed for autoradiography. 3H-glucine was transported at 70 mm/day in 6X greater quantities than other amino acids which were transported at less than 40 mm/day. In the 3H-glycine experiments, greater than 80% of the label transported into the nerve remained as free glycine, comigrating with glycine in thin-layer chromatographs. In autoradiographs of sections 4 mm from the ganglion-nerve barrier, greater than 50% of the silver grains were over R3--R14 axons which occupy less than 10% of the nerve cross-sectional area. EM autoradiographs confirmed that grains were within R3--R14 and not in surrounding glia. The selective transport of glycine was inhibited by Hg2+, by vinblastine and Nocodazole, and by low Ca2+ media. Autoradiographs of vinblastine-treated nerves showed a drastic reduction in label over R3--R14 and other axons. Label was also transported retrogradely; this transport rate was similar to the orthograde rate, but 5--10 times less label moved retrogradely. Autoradiographs showed that the retrograde label was localized to R3--R14 axons. This report clearly demonstrates the rapid, selective, and bidirectional transport of a free amino acid and provides further evidence that glycine may be used as a neurochemical messenter by neurons R3--R14.     

Axonal Transport of Choline Lipids in Normal and Regenerating Rat Sciatic Nerve

Abstract: Biochemical methods were used to study the time course of transport of choline phospholipids (labeled by the injection of [³H]choline into the ventral horn of the lumbar spinal cord) in rat sciatic nerve. Autoradiographic methods were used to localize the transported lipid within motor axons. Transported phospholipid, primarily phosphatidylcholine, present in the nerve at 6 h, continued to accumulate over the following 12 days. No discrete waves of transported lipid were observed (a small wave of radioactive phospholipid moving at the high rate would have been missed); the amounts of radioactive lipid increased uniformly along the entire sciatic nerve. In light-microscope autoradiographs, a class of large-caliber axons, presumably motor axons, retained the labeled lipid. Some lipid, even at 6 h, was seen within the myelin sheaths. Later, the labeling of the myelin relative to axon increased. The continued accumulation of choline phospholipids in the axons probably signifies their prolonged release from cell bodies and their retention in various axonal membranes, including the axolemma. The build-up of these phospholipids in myelin probably represents their transfer from the axons to the myelin sheaths surrounding them. When nerves are crushed and allowed to regenerate for 6 or 12 days, choline phospholipids transported during these times enter the regenerating nerve. In light and electron microscope autoradiographs, transported lipid was seen to be localized primarily in the regenerating axons. However, grains overlay the adjacent Schwann cell cytoplasm, indicating transported lipids were transferred from the regenerating axons to the associated Schwann cells. In addition, some cells not associated with growing axons were labeled, suggesting that phosphatidylcholine and possibly acetylcholine, carried to the regenerating axons by axonal transport, were actively metabolized in the terminal, with released choline label being used by other cells. These results demonstrate that axonal transport supplies mature and growing axons and their glial cells with choline phospholipids.     

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.     

Depression of fast axonal transport in axons demyelinated by intraneural injection of a neurotoxin fromK. humboldtiana

Tullidinol, a neurotoxin extracted from the Karwinskia humboldtiana fruit, dissolved in peanut oil was injected into the right sciatic nerve of adult cats. The contralateral sciatic nerve received an equivalent volume of peanut oil alone. The fast axonal transport of labeled ([³H]Leucine) protein was studied in sensory and motor axons of both sciatic nerves. The radioactive label was pressure injected either into the L7 dorsal root ganglion or the ventral region of the same spinal cord segment. Several days after the toxin injection, the cat limped and the Achilles tendon reflex was nearly absent in the right hind limb. The amount of transported label was decreased distal to the site of toxin injection. Proximal to this site, the transported material was dammed. Sensory and motor axons showed similar changes. In addition, the toxin produced demyelination and axonal degeneration. Axonal transport and the structure of the axons were normal in the contralateral nerve. Both, Schwann cells and axons of the right sciatic nerve showed globular inclusions, presumably oil droplets containing the toxin. We conclude that Schwann cells and axons as well are tullidinol targets.Departamento de Química. Centro de Investigación y de Estudios Avanzados del IPN.Special issue dedicated to Dr. Sidney Ochs.     

Reversal of axonal transport at a nerve crush.

Abstract— —We have compared retrograde axonal transport of ³H-labeled protein in normal rat motor and sensory axons, and axons which were injured by a distal ligation of the sciatic nerve. After injection of L-[³H]leucine into the vicinity of the neuron cell bodies, labeled protein was transported into the axons. A premature return of protein towards the cell bodies occurred in the injured axons, which we interpret as a reversal of axonal transport occurring at the site of injury. We estimate that reversal of transport occurred within 1.9–2.4 h of the arrival of labeled protein at the injury, and that the minimum velocity of the subsequent retrograde transport was 112–133 mm day^?1. The ability of the injured axons to reverse transport developed about 0.8 h after making the injury. A large fraction of the orthograde transported protein was returned towards the cell body: it is estimated that by 28 h after labeled protein in sensory axons reached the injury, 46% of the³H-labeled protein originally transported to the injury site had been returned. In intact sensory nerves at this time only 15% of the transported protein had returned. It is suggested that axonal injury produces a sudden increase in the return of newly synthesized protein to the cell body, and that this might serve as a signal for chromatolysis.     

AXONAL TRANSPORT OF SULFATED GLYCOPROTEINS AND MUCOPOLYSACCHARIDES IN THE GARFISH OLFACTORY NERVE

—Application of ³⁵SO₄ to the olfactory mucosa of the long-nosed garfish is found to label sulfated macromolecules which are transported down the olfactory nerve. The transported molecules pass along the nerve as a discrete peak whose leading edge has a transport velocity of 206 ± 6 mm/day. A large portion of the radioactivity from the peak is deposited along the axon. At 2 days after isotope application 83% of the total nerve radioactivity is in the axons and the remaining 17% has accumulated at the terminals in the olfactory bulb. Characterization of sulfated material in the migrating peak indicates that both sulfated glycoproteins (isolated as glycopeptides) and mucopolysaccharides, including chondroitin sulfate and heparan sulfate, are undergoing transport.     

Components of fast and slow axonal transport in the goldfish optic nerve

The composition of the fast and slow components of axonal transport in the goldfish optic nerve was investigated, using specific radioactive precursors injected into the eye. Tritiated glucosamine and fucose label macromolecules, presumably glycoproteins, which are rapidly transported from the eye to the optic tectum. Material labeled with these precursors is not evident in the slowly transported component. Glucosamine and fucose incorporation are blocked when a protein synthesis inhibitor, acetoxycycloheximide, is injected into the eye concurrently with the precursors. As well as labeling macromolecules, ³H-glucosamine and ³H-N-acetylmannosamine ( a precursor of sialic acids) also label rapidly-transported chloroform-methanol-extractable material which may contain transported glycolipids. Two procedures were used to show that the slow component of axonal transport contains tubulin, a protein characteristic of the microtubules:

• (a) Tracer doses of tritiated colchicine injected into the eye label a wave of radioactivity which moves 0.5 mm/day, the rate of slow axonal transport in the goldfish optic nerve. We believe this wave represents the movement of colchicine which is bound to colchicine-binding protein moving in the slow component of axonal transport.
• (b) Tritiated proline labels a slowly transported protein which is precipitated by vinblastine and has a mobility on polyacrylamide gels comparable to authentic tubulin. These results indicate that the fast and slow components of axonal transport each provide specific chemical substances to the nerve endings.

     

RAPID AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG OF FUCOSE-, GLUCOSAMINE- AND SULPHATE-CONTAINING MATERIAL

—An in vitro system from the frog has been used to study fast axonal transport of glycoproteins. The migration of [³H]fucose-, [³H]glucosamine- and [³⁵S]sulphate-labelled material was followed from the dorsal ganglia, along the sciatic nerve towards the gastrocnemius muscle. The distribution in different subcellular fractions, effect of cycloheximide and transport kinetics did not differ very much between fucose- and glucosamine-incorporation into the nerve. Cycloheximide blocked the synthesis of TCA-insoluble radioactivity, which was transported at a rate of 60–90 mm per day at 18°C, more effectively than the synthesis of stationary proteins in the ganglia. About 10 per cent of the TCA-insoluble and transported radioactivity was extracted by chloroform-methanol (2:1, v/v) and might be glycolipids and the rest glycoproteins. Results suggest that TCA-soluble activity, which was recovered in the nerve, originated in part from labelled macromolecules consumed along the axons. The rapidly transported TCA-insoluble radioactivity was 85 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. [³⁵S]Sulphate-labelled TCA-insoluble material was resistant towards chloroform-methanol (2:1, v/v) extraction and rapidly transported from the ganglia into the nerve. The synthesis was inhibited by cycloheximide. The material, probably proteoglycans, represented a quantitatively minor part of transported glycoproteins.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood–retinal barrier (inner BRB). [¹⁴C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [¹⁴C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.     

Studies on regional blood flow of the mouse using whole-body autoradiography of 14C-iodoantipyrine

Summary In order to visualize regional blood flow in various tissues of the mouse at the same time, the distribution of radioactive carbon from ¹⁴C-iodoantipyrine was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-hexane at 1, 10, 30 min, and 1 h and 3 h after intravenous injection of ¹⁴C-iodoantipyrine. Whole-sagittal sections of the frozen mouse, obtained by using a cryostat microtome, were dried in a cryostat and subjected to autoradiography. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO₄, dried at room temperature and again subjected to autoradiography. Autoradiographs thus obtained are referred to as treated autoradiographs. It was found that the method could be suitable for the estimation of regional blood flow of the renal cortex, spleen, lung, skeletal muscle, bone marrow, thymus, testes, and brain.     

Retrograde Axonal Transport of Phospholipid in Rat Sciatic Nerve

Abstract: Retrograde axonal transport of phospholipid was studied in rat sciatic motoneuron axons by placing collection crushes on the nerve at intervals after injection of [methyl-³H]choline into the lumbosacral spinal cord, and allowing labelled material undergoing anterograde or retrograde movement to accumulate adjacent to the collection crushes. Control experiments showed that the accumulations of label were not a result of local uptake of circulating precursor. The majority of the ³H label was associated with phosphatidylcholine. Accumulation of label at the distal collection crush, representing retrograde transport, was observed subsequent to the anterograde transport of phospholipid. In comparison with previous study on retrograde transport of protein, the following points were noted: (1) onset of retrograde transport occurred at approximately the same time after precursor injection (10–20 h) for both protein and phospholipid; (2) retrograde transport of lipids was more prolonged: maximum retrograde transport occurred later for phospholipid (30 h) than for protein (15–20 h), and declined to half-maximum between 49 and 99 h, compared to a corresponding value of 24–28 h for protein; (3) the proportion of total anterograde-transported activity subsequently undergoing retrograde transport was less in the case of phospholipid, at least over the time interval studied (up to 99 h after precursor injection). The similar times of onset of retrograde transport of phospholipid and protein support the concept of retrograde transport as a recycling mechanism returning to the cell body membrane fragments that were earlier transported into the axon. Coordinated retrograde transport of labelled protein and phospholipid components of the recycled membranes would be predicted. Differences between protein and phospholipid in the subsequent time course and amount of retrograde transport may reflect differences in axonal handling of protein and lipid. Both the more prolonged outflow of labelled lipids from cell body into axon and exchange with a distal pool of unlabelled phospholipid may account for the prolonged time course of retrograde transport of labelled lipid.     

Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo

Brain dynein is a microtubule-activated ATPase considered to be a candidate to function as a molecular motor to transport membranous organelles retrogradely in the axon. To determine whether brain dynein really binds to retrogradely transported organelles in vivo and how it is transported to the nerve terminals, we studied the localization of brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using affinity-purified anti-brain dynein antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Interestingly, brain dynein accumulated both at the regions proximal and distal to the ligation sites and localized not only on retrogradely transported membranous organelles but also on anterogradely transported ones. This is the first evidence to show that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activates brain dynein only for retrograde transport.     

Axonal transport of glycerophospholipids following intracerebral injection of glycerol into substantia nigra or lateral geniculate body

[2-³H]Glycerol was injected into one substantia nigra of adult rats. Incorporation of radioactivity into lipids at the injection site was maximal by 2 hr, after which it declined. Rapidly transported³H-labeled lipids were just beginning to accumulate in the primary projection site, the ipsilateral corpus striatum by 2 hr, as evidenced by 20-fold higher levels of lipid radioactivity in the projection site relative to control regions. However, the bulk of labeled lipid arrived between 6 hr and 3 days postinjection, suggesting either a prolonged period of release of rapidly transported lipids from the nerve cell bodies or a slow rate of transport for the later arriving lipids. Colchicine applied locally to the fibers of this tract blocked the axonal transport of lipids to the striatum almost completely. Choline and ethanolamine phosphoglycerides were the major transported lipids, accounting for approximately 60% and 25%, respectively, of the total. Similar results were obtained in studies of [2-³H]glycerol-labeled lipids synthesized in the lateral geniculate body and transported to the visual cortex. The rapid axonal transport of lipids labeled with [³²P]phosphate (injected simultaneously with [2-³H]glycerol) could also be demonstrated in both tracts. However, in contrast to [2-³H]glycerol, considerable amounts of³²P soluble label were present in the projection sites, and colchicine only partially blocked the accumulation of³²P-labeled lipid. These results demonstrate the relative utility of [2-³H]glycerol as a lipid precursor for examination of axonal transport in intrabrain tracts. Characteristics of lipid axonal transport in these two intrabrain tracts are similar to each other and are also similar to those previously described for retinal ganglion cells, indicating a common requirement for the axonal transport of these membrane constituents to axons and nerve endings in widely divergent CNS tracts.Presented in part at the 11th meeting of the American Society for Neurochemistry, Houston, Texas, March 1980.     

Significance of transported glycine in the conjugation of sodium benzoate in spf mutant mice with ornithine transcarbamylase deficiency

3H-glycine and 14C-serine were injected intraperitoneally, during treatment of spf mutant mice with 2% sodium benzoate in drinking water. Urinary hippurate was separated by thin layer chromatography and counted for 3H and 14C labels representing transported and newly synthesized glycine, respectively. The specific activity of 3H-hippurate increased significantly in mutant and normal groups, while the increase of 14C was seen only in mutants. The ratio of specific activity 3H:14C showed significant increases in normal (0.99 to 1.93; p less than 0.01) and mutant (1.53 to 3.05; p less than 0.05) groups, which shows that glycine transported from body pools played a significantly greater role in the conjugation of benzoate, compared to glycine synthesized de novo from serine. In spf mice, benzoate treatment also resulted in a decrease in orotate excretion, indicating amelioration of the hyperammonemic state. It is postulated that the elimination of glycine transported from body pools may be the primary mechanism for the reduction of ammoniagenicity in benzoate therapy, and that the de novo synthesis of glycine may have a secondary effect.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo-[3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol-soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Spinal subarachnoid perfusion in the rat: glycine transport from spinal fluid

Abstract— A method was developed for perfusion of the spinal subarachnoid space in the rat. Bidirectional steady-state fluxes of [¹⁴C]glycine between spinal fluid and plasma were measured. [¹⁴C]glycine clearance from spinal fluid was 5-fold greater than its clearance from plasma. Glycine was transported out of spinal fluid by a saturable process, and the rate of transport was unaffected by the other depressant amino acids, GABA, β-alanine, and taurine. Perfused [¹⁴C]glycine and [³H]GABA distributed in an intracellular compartment in spinal cord. The preparation should be useful for study of the release of these inhibitory amino acids from the intact spinal cord.     

Polar transport related to mobilization of plant constituents

Indole-3-acetic acid (IAA), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), sucrose, and mannitol, were tested for polar transport through 5-mm hypocotyl segments of bean (Phaseolus vulgaris var. Pinto) seedlings. All 4 compounds were transported in a polar direction. Autoradiographs of segments through which ¹⁴C-labeled compounds were transported indicated areas of ¹⁴C concentration near the morphological base of segments. There was a direct correlation of IAA transport with the ability to initiate roots as well as with the degree of ¹⁴C accumulation in the morphological base of segments. Unlabeled IAA in lanolin applied to segments appeared to reduce transport of IAA-¹⁴C through those segments when measured by the accumulation of ¹⁴C in a receiver-block of agar, caused an increase in ¹⁴C accumulation in the region to which the unlabeled IAA was applied, and also caused a decrease in accumulation of ¹⁴C at the morphological base of upright segments. Histological studies showed that IAA accelerated cell division and the formation of root primordia particularly at the basal ends, and that these responses sometimes occurred at the expense of cell proliferation at the apical ends of segments. The data presented support the hypothesis that polar movement of IAA, and other endogenous and exogenous substances, in isolated stem segments was controlled by mobilization and utilization of plant constituents at the growth centers.