TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

PATTERN OF RNA SYNTHESIS IN THE SCIATIC NERVE OF THE HEN DURING WALLERIAN DEGENERATION

The pattern of synthesis of rapidly-labelled RNA of hen sciatic nerve was studied during Wallerian degeneration. At 2,4,8, 16 and 30 days of degeneration the proximal and distal stumps of the severed nerve as well as the intact contralateral sciatic nerve (functional control) were excised and incubated with either [5-³H]uridine or [2-¹⁴C]uridine for 0.5 h. The electrophoretic pattern of RNA from the normal adult sciatic nerve showed that most of the radioactivity was incorporated into RNA species migrating between the 18 S and 4 S components of the bulk RNA. The synthesis of RNA was sensitive to actinomycin-D, an indication that it was directed by a DNA template. The electrophoretic patterns of the rapidly-labelled RNA in the proximal and distal nerve stumps demonstrated a change following nerve section. After 2–4 days of Wallerian degeneration the degenerating distal nerves incorporated more radioactivity in the 4 S region than the corresponding controls, but at 8 and 16-days after degeneration relatively more label appeared in higher molecular weight RNA species. In the intact sciatic nerve of the operated hens progressively more radioactivity was detected in the 4 S region with increasing time after the contralateral nerve section. At each stage of Wallerian degeneration the specific radioactivities of RNA in the control nerves from experimental hens were higher than those of the normal adult sciatic nerve. These results indicated a change of RNA metabolism in increased functional activity and during Wallerian degeneration.     

THE EFFECT OF INTRAOCULAR INJECTION OF CORDYCEPIN ON RETINAL RNA SYNTHESIS AND ON RNA AXONALLY TRANSPORTED DURING REGENERATION OF THE OPTIC NERVES OF GOLDFISH

Abstract— The presence of relatively large amounts of RNA has been demonstrated in regenerating axons of the goldfish optic nerve. Previous experiments have suggested that this R NA may be composed of only small molecular weight 4S RNA. The present experiments were performed in order to see if inhibiting RNA transport by intraocular injections of cordycepin causes a selective depletion of 4S RNA arriving in the contralateral optic tectum, and thus add further evidence that 4S RNA is axonally transported. Optic nerves were crushed in a group of goldfish and 18 days later 10.0 /tg of cordycepin was injected into the right eye followed 3 h later by injections of [³H]uridine into the same eye. Six days later the amount of axonally transported [³H]RNA was decreased by 89% compared with non-cordycepin treated controls. The effect of cordycepin on retinal RNA synthesis was shown by autoradiography to be primarily on retinal ganglion cell RNA synthesis with lesser effects on other cellular elements of the retina. SDS polyacrylamide gel electrophoresis at both 1 and 6 days after intraocular injections of cordycepin and [³H]uridine, showed that cordycepin blocks the retinal synthesis of ribosomal RNAs but appeared to have little effect on the synthesis of 4S RNA. When transported RNA in the tectum was fractionated by gel electrophoresis 6 days after injection, it was found that the amount of ribosomal RNA was decreased by approx 70% as a result of cordycepin pretreatment. This correlated well with the effect of cordycepin on the transport of available RNA precursors (also decreased by approx 70%) and is consistent with the contention that in these experiments ribosomal RNA is synthesized in the tectum itself and is not axonal. The amount of [³H] 4S RNA arriving in the tectum, however, was decreased by greater than 90% suggesting that its presence in the tectum was not entirely dependent on the availability of ³H precursors for local synthesis in the tectum. These results are consistent with data suggesting that 4S RNA is the predominant, if not the only, RNA species axonally transported during regeneration of goldfish optic nerves.     

Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons

The accumulation of [³H]leucine- and [³H]fucose-labelled axonal proteins, acetyl-CoA : choline O-acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was studied proximal to a ligature applied to the hypoglossal nerve of the rabbit at different phases of nerve regeneration. After 1 week of regeneration, the accumulation of rapidly migrating [³H]leucine-labelled proteins, ChAc and AChE was reduced as compared to that of the contralateral nerve. In contrast, the accumulation of [³H]fucose-labelled glycoproteins was markedly increased. After a regeneration period of 4-6 weeks, the accumulation of proteins and glycoproteins in the regenerating nerve was increased whereas the accumulation of ChAc and AChE was almost normal. The results indicate an initial depression of the synthesis and axonal transport of the bulk of rapidly migrating proteins, ChAc and AChE in the chromatolytic hypoglossal neurons whereas the synthesis and transport of rapidly migrating glycoproteins is increased. These initial changes are less pronounced during the subsequent regeneration period.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING PROTEINS IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

—The redistribution of rapidly migrating [³H]leucine-labelled proteins was studied using double ligatures applied to the vagus nerve and single ligatures, applied to the hypoglossal nerves. Rapidly migrating proteins accumulating for 16 h proximal to a distal ligature of the cervical vagus redistributed to give a retrograde accumulation distal to a second ligature. Within 6 h a substantial redistribution occurred indicating a rapid retrograde transport. After 21 h there was a further accumulation with 70 per cent of the labelled material accumulating at the distal end of the isolated nerve segment and 16 per cent accumulating at the proximal end. It was shown that about a half of the retrograde accumulation was dependent on the distal accumulation zone. Rapidly migrating proteins accumulated distal to a ligature applied to the hypoglossal nerve 16 h after labelling of the nerve cell bodies indicating that a rapid retrograde transport of labelled macromolecules occurs from the peripheral parts of the nerve in the tongue. Labelled proteins accumulated proximal to ligatures and transections of both the hypoglossal and vagus nerve when applied 16 h after labelling of the nerve cell bodies, indicating the presence of axonal proteins, migrating at a rate of transport intermediate to that of rapidly and slowly migrating proteins.     

Coexistence of GABA-and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat

Summary Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)-and choline acetyltransferase (ChAT)-like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT-and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT-or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established.Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergie neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

THE EFFECT OF CORDYCEPIN ON THE APPEARANCE OF [3H]RNA IN THE GOLDFISH OPTIC TECTUM FOLLOWING INTRAOCULAR INJECTION OF [3H]URIDINE

Abstract— Although biochemical and electron microscopic evidence has shown that RNA molecules may be found within axons, the origin of this RNA is not known. In order to determine if the RNA found in axons is synthesized in the nerve cell body and axonally transported, we have studied the effect of the RNA inhibitor cordycepin (3′-deoxyadenosine) on the retinal synthesis and axonal migration of radioactive RNA. Ten μg of cordycepin was injected into the right eye of 11 fish and 3 h later [³H]uridine was injected into the same eye. Twelve control fish were injected with [³H]uridine only and all fish were sacrificed 6 days later. Results of RNA extraction of retina and tecta showed that cordycepin decreased retinal RNA synthesis by approx 24%, while inhibiting the amount of [³H]RNA appearing in the contralateral tectum by 74%. Since the transport of RNA precursors was depressed by only 50%, (significantly different from the effect on RNA, P < 0.01) it seems unlikely that the action of cordycepin in decreasing tectal [³H]RNA levels was due solely to a decrease in the availability of labeled precursors for tectal RNA synthesis. For the purpose of blocking tectal RNA synthesis, 200 μg of cordycepin was injected intracranially several days after the intraocular injection of [³H]uridine. This route of cordycepin administration failed to significantly block the appearance of [³H]RNA in the tectum, suggesting that at least some of the [³H]RNA in the tectum was synthesized before arrival in the tectum itself. To be sure that cordycepin itself was not being transported, we injected cordycepin into the right eye of fish and 5 days later, injected fish intracranially with [³H]uridine. Autoradiograms were prepared and grains were counted over the fiber layers of left (experimental) and right (control) tecta. No significant difference was observed in the number of grains of left vs right tecta indicating that cordycepin itself is not axonally transported. These experiments support earlier findings from our laboratory which suggest that RNA may be axonally transported in goldfish optic fibers.     

Coexistence of GABA- and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat

Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)- and choline acetyltransferase (ChAT)- like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT- and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT- or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established. Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergic neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.     

Changes of Phospholipid-Metabolizing and Lysosomal Enzymes in Hypoglossal Nucleus and Ventral Horn Motoneurons During Regeneration of Craniospinal Nerves

In order to study the biochemical changes associated with the cell body response to axonal crush injury, two systems, hypoglossal nucleus and spinal cord ventral horn, were used. The time intervals chosen were 7, 14, and 28 days after unilateral crushing of the right hypoglossal nerve and cervicothoracic nerves of the rabbit. Non-crushed, contralateral nerves were used as controls. Three groups of enzyme activities were tested: (a) phospholipase A2, acyl CoA:2-acyl-sn-glycero-3-phosphocholine acyltransferase, and choline phosphotransferase, as indicators of phospholipid degradation and biosynthesis; (b) seven hydrolases, namely, beta-D-glucuronidase, beta-N-acetyl-D-hexosaminidase, arylsulfatase A, galactosylceramidase, GM1-ganglioside beta-galactosidase, and acid RNase, as indicators of lysosomal activity; and (c) free and inhibitor-bound alkaline RNase, as an index of RNA metabolism. Changes could be grouped into three distinct patterns. Compared to contralateral control, choline phosphotransferase showed a slight increase, whereas phospholipase A2 and most lysosomal hydrolases showed a significant increase of activity, especially evident in the ventral spinal cord neurons 14-28 days after crushing. These changes correlate with known increases of membrane and organelle numbers, including lysosomes, in motor and sensory neurons during peripheral regeneration. In contrast, free and acid alkaline RNase activity significantly decreased in the injured sides compared to the controls. This change can probably be correlated with a stabilization of RNAs needed for increased protein synthesis. No changes in total alkaline RNase and acyltransferase activities in either regeneration model were observed.     

Calcitonin gene-related peptide and α-CGRP mRNA expression in cranial motoneurons after hypoglossal nerve injury during postnatal development

Changes in calcitonin gene-related peptide (CGRP) immunoreactivity and α-CGRP mRNA expression were determined in the hypoglossal nucleus after the nerve was crushed or transected in rats at 10, 14 and 21 days postnatal. α-CGRP mRNA expression was determined in normal, noninjured, hypoglossal nuclei at the three ages and after both injuries in 10 and 21 days postnatal rats. Reinnervation and neuronal survival were assayed. Although the three age groups expressed comparable levels of α-CGRP mRNA and its peptide in intact, hypoglossal nuclei, axonal injury produced age-dependent alterations in α-CGRP mRNA and CGRP. In the 21 days postnatal rats, changes in α-CGRP mRNA and peptide mimicked those reported in adult motoneurons after the same injuries. CGRP was elevated until reinnervation after nerve crush, whereas biphasic elevations occurred after nerve transection. In 21 days postnatal rats, increases in α-CGRP mRNA preceded elevations of the peptide but a greater increase resulted initially after nerve transection. An upregulation of α-CGRP mRNA also developed initially after both injuries in 10 days postnatal rats but subsequent elevations of α-CGRP mRNA did not materialize. In contrast, CGRP immunoreactivity did not increase after either injury in 10 days postnatal rats and, in fact decreased. Levels of CGRP immunoreactivity did not differ from normal amounts after either nerve injury in 14 days postnatal rats. Substantial neuronal cell loss occurred after each injury in 10 and 14 days postnatal rats but was not found in 21 days postnatal rats. Tongue reinnervation by surviving motoneurons was established after all injury paradigms except 10 days postnatal transection. The current findings demonstrate an age-dependent correlation between injury-induced expression of CGRP and hypoglossal motoneuron survival.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [³H]thymidine during their prenatal period of proliferation. The ³H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the ³H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [³H]β self-absorption in the material of the sections. Injection of a high dose of [³H]thymidine into young adult mice did not result in ³H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.     

AXONAL TRANSPORT OF [3H]GLUCOSE RADIOACTIVITY IN THE OPTIC SYSTEM OF SCARDINIUS ERYTHROPHTHALMUS1

Abstract— The optic system of Scardinius erythrophthalmus has been used to study the axonal translocation of radioactivity from [³H]glucose. Intraocularly injected precursors were transported intra-axonally along the optic nerve towards the contralateral optic tectum. In comparison with the well known properties of axonal protein transport there were remarkable differences in the proximo-distal translocation of [³H]glucose. These were: (1) a delay in the labelling of the structures investigated, after tracer application; (2) only a rapid phase of transport; and (3) no accumulation of radioactivity in the region of nerve terminals in the optic tectum connected with the injected eye. The transported material was almost exclusively in the form of TCA-soluble compounds and was mainly glucose itself or its low molecular derivatives, but not glycogen. The rate of transport was decreased by lowered temperatures and was not immediately dependent on retinal protein synthesis. Colchicine blocked the axonal transport of glucose by up to 60–70 per cent.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES

Abstract— The redistribution of rapidly migrating [³H]leucine-labelled proteins and [³H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies, rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve, rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 16 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.     

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.     

INTRANEURAL DISTRIBUTION OF EXOGENOUS NOREPINEPHRINE IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

Catecholaminergic neurons, which take up and retain exogenous norepinephrine labeled with tritium, were studied by means of high resolution radioautography, in the substantia nigra, the substantia grisea periventricularis, and the locus coeruleus of the rat. Under the conditions required for the radioautographic detection of exogenous norepinephrine-³H, it was established that (1) glutaraldehyde was the most suitable fixative for preserving the labeled amine in situ; (2) norepinephrine-³H itself, rather than metabolites, accounted for most of the reactions detected in catecholaminergic neurons. At various time intervals after an intraventricular injection of norepinephrine-³H, the tracer reached a concentration 15–100 times higher, and disappeared at a slower rate, in presynaptic axons (t½:4 hr) than in nerve cell bodies (t½:0.8–1.3 hr). After pretreatment with a monoamine oxidase inhibitor, the radioautographic reactions increased and persisted longer, especially in the preterminal axons. Within neurons, the labeled amine was ubiquitously distributed in the nerve cell body and concentrated in presynaptic axons and synaptic terminals of various morphological types. Although large granular vesicles were usually present in the labeled axonal bulbs, no structural characteristic could be specifically ascribed to catecholaminergic neurons. It is suggested that exogenous norepinephrine bound to macromolecular complexes is present in all parts of catecholaminergic neurons and mainly concentrated within presynaptic axons.     

THE PERIOD OF DNA SYNTHESIS PRIOR TO MEIOSIS IN THE MOUSE

Sixteen pregnant female mice were operated on and H³-thymidine was injected into the amniotic cavities of the uterus. The injection was given between the 6th and 14th days of fetal life. Eighty-eight fetuses received thymidine in this way. Another series of 16 pregnant females was injected intraperitoneally with H³-thymidine between the 5th and 14th days of pregnancy. Two of these females were killed 16 days after the observation of the vaginal plug. The remaining 30 females were allowed to give birth to their progeny. The progeny was killed at birth and the ovaries of the newborn females fixed at once. Labeled oocytes at late pachytene and early diplotene were clearly seen in individuals that received the isotope between the 10th and 12th to 13th days of fetal life, but the period of DNA synthesis preceding meiosis is at the 12th to 13th days of fetal life. Since meiosis is recognized by the 14th day, only the oocyte labeling originating from mothers injected at the 12th and 13th days may be considered as representing the DNA synthesis of the premeiotic replication.     

TRANSPORT AND TURNOVER OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Axonal transport and turnover rate of proteins in the supraoptico-neurohypo-physial tract were studied after injection of ³⁵S cysteine into the region of the supraoptic nucleus. The proximo-distal migration of labelled proteins from the nerve cell bodies to the axon terminals in the neurohypophysis was followed by measuring the radioactivity of neurohypophysial proteins at various time intervals (4 h to 30 days) after isotope injection. A rapidly transported phase of proteins with a minimal transport rate of approximately 60 mm/day was demonstrated. An accumulation of protein-bound radioactivity was also observed in the neural lobe at 9 days after isotope injection, representing slowly transported proteins (0-5 mm/day). In addition, an intermediate phase of axonal transport (1-5 mm/day) was found. Fractionation of neurohypophysial proteins by polyacrylamide gel disc electrophoresis revealed that a predominating portion of the radioactivity was recovered in a single protein component (fraction A) at 4 h as well as at 30 days after isotope injection. This protein component was shown to be a constituent both of the rapid and the slow phase of axonal transport. With time an increasing amount of radioactivity was found in another protein component (fraction B), which reached a maximum at 14 days after injection and then remained fairly constant up to 30 days. When the turnover rates of neurohypophysial proteins were estimated, a half-life of 1-2 days and 8 days was calculated for the rapidly and slowly transported proteins, respectively.