Lipid Metabolism During Early Stages of Wallerian Degeneration in the Rat Sciatic Nerve

We examined changes in biosynthetic capacity of sciatic nerve during the early stages of Wallerian degeneration, utilizing a model that permits exclusion of nonresident cells from degenerating nerve. Sciatic nerve segments were placed in either 5-microns pore (allowing infiltration of nonresident cells) or 0.22-microns pore (excluding nonresident cells) Millipore diffusion chambers and then implanted in the peritoneal cavity of the same 32-34-day-old rat. At times up to 7 days postsurgery, nerve segments from the chambers, as well as control segments from the contralateral sciatic nerve, were removed and their capacity to incorporate radioactive precursors into lipids and proteins assayed in vitro. In nerve segments from both the 0.22- and 5-microns pore chambers, incorporation of [14C]acetate into total lipids was decreased relative to control by 50% at 12 h postsurgery and by 85% at day 3. This decreased incorporation of [14C]acetate reflects primarily decreased de novo synthesis of cholesterol and of fatty acyl residues incorporated into glycerolipids and sphingolipids. There was a preferentially decreased synthesis of cerebrosides and cholesterol (components enriched in myelin) relative to other lipids, while cholesterol esters and free fatty acids (products of membrane degradation) accounted for a greater proportion of the greatly reduced levels of total lipid label. In contrast to [14C]acetate, incorporation of [3H]glycerol into lipids was increased up to fourfold, relative to control, 1 day after nerve transection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triacylglycerol Lipase in Developing Rat Sciatic Nerve

Triacylglycerol lipase activity, with a pH optimum of 7.5, was demonstrated in cell-free homogenates of rat sciatic endoneurium. 1,2-Diacylglycerol was the major product of triacylglycerol hydrolysis. A rapid decline in lipase activity was found in rats up to 2 months of age. After this time, the decrease continued, but at a much slower rate. Such developmental changes in triacylglycerol lipase activity may, at least in part, account for the slower metabolic turnover of endoneurial triacylglycerol in adult rat sciatic nerve.     

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Abstract: Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg²⁺ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.     

Changes in Composition of Endoneurial and Perineurial Fatty Acids During Glycerol-Induced Wallerian Degeneration and Regeneration in the Sciatic Nerve of the Adult Rat

Intraneural injection of pure glycerol induces Wallerian degeneration with subsequent regeneration. In agreement with other reports, we observed an increase in endoneurial polyunsaturated fatty acids 8 days after the glycerol injection. Levels then fell until day 30. After a period of 5 months, there was an increase in C18:2(n-6) in the intrafascicular tissue, concomitant with a marked fall in this fatty acid in the remaining extrafascicular perineurium. The rise in C18:2(n-6) in endoneurium correlated with infiltration of this tissue by perineurial cells. Interactions between perineurium and endoneurium during nerve regeneration are discussed.     

Effect of Wallerian Degeneration on Histamine Concentration of the Peripheral Nerve

One sciatic nerve of a White Leghorn hen was severed and the distal portion was allowed to undergo Wallerian degeneration. The change in histamine and DNA concentration and mast cell number was measured at different times following nerve sectioning in the proximal regenerating, distal degenerating, and intact, contralateral nerves. The experimental results revealed a significant accumulation of histamine in the proximal desheathed segment and in the contralateral “functional nerve,” whereas the biogenic amine in the distal desheathed nerve significantly decreased. The pattern of change of histamine in the distal and proximal nerve sheaths was different: it dropped at 2 h and then rose in the later stages of Wallerian degeneration. In the distal desheathed nerves and in both the proximal and distal nerve sheaths DNA increased significantly by 14 days. The number of mast cells appeared to be highest in the 14-day distal nerve and in the 7-day proximal nerve sheaths. These results support a dual localization of histamine in the peripheral nerve, and are consistent with the interpretation that the amine has either some role in neurotransmission or in the process of growth and regeneration.     

Effect of Electrical Stimulation on Phosphoinositide Metabolism in Rat Sciatic Nerve In Vivo

The metabolism of phosphoinositides in rat sciatic nerves in vivo during electrical stimulation was studied. Nerves were prelabeled by injection of [2-3H]-myo-inositol alone for periods of 2 and 20 h or together with [32P]orthophosphate for 2 h and then electrically stimulated (100 Hz) for 5 or 20 min. Contralateral unstimulated nerve served as the control. When tritiated myo-inositol was used alone for prelabeling the nerves, approximately 6% and 14% of the label was incorporated into lipids after 2 h and 20 h, respectively. Both 5 and 20 min of electrical stimulation caused an insignificant change in the percentage of radioactivity recovered in lipids from the nerves prelabeled with either myo-inositol or with a mixture of myo-inositol and phosphate. The proportion of label associated with phosphoinositides of nerves prelabeled with myo-inositol for both 2 h and 20 h showed an increase in phosphatidyl-inositol-4-phosphate at the expense of phosphatidylinositol in stimulated nerves. Similar results were obtained with nerves prelabeled for 2 h with a mixture of [32P]orthophosphate and [2-3H]myo-inositol. No significant changes in the radioactivity associated with water-soluble inositol phosphates were found in stimulated versus control nerves.     

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.     

Membrane-Associated Phospholipase D Activity in Rat Sciatic Nerve

Rat sciatic nerve contains a membrane-bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 micrograms of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg of protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one-half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.     

Regulation of Sulfoglucuronyl Glycolipid Synthesis in the Developing Rat Sciatic Nerve

Sulfoglucuronyl glycolipids (SGGLs) have been considered as target antigens in demyelinating peripheral neuropathies associated with IgM monoclonal gammopathy. The regulation of expression of SGGLs in the rat sciatic nerve during development was studied by assaying the levels of SGGLs and activities of four glycosyltransferases sequentially involved in their synthesis from lactosylceramide. The levels of SGGLs in the sciatic nerve increased with development and reached a maximum at sixty days after birth. The rate of increase in the level of SGGLs between day 5 to 20 was similar to rate of deposition of myelin in the nerve. Analysis of the activities of the glycosyltransferases showed that only lactotriosylceramide galactosyltransferase (LcOse₃Cer-GalTr) increased in parallel with the levels of SGGLs during development. The other three enzymes were not co-relative with the synthesis of SGGLs. The product of LcOse₃Cer-GalTr reaction, nLcOse₄Cer is the key intermediate for all neolactoglycolipids, particularly NeuAc2-3nLcOse₄Cer or nLM1, which is the major ganglioside (60%) of myelin in rat sciatic nerve. The results suggest that in the sciatic nerve SGGLs are mostly associated with Schwann cell myelin and their synthesis is regulated by LcOse₃Cer-GalTr, unlike in the cerebral cortex and cerebellum where SGGLs are associated with the neuronal membranes and their synthesis is regulated by lactosylceramide N-acetylglucosaminyltransferase (LcOse₂Cer-GlcNAcTr).     

Decreased Metabolism of Cerebrosides and Sulfatides in Rat Sciatic Nerve After Intraneural Injection of Colchicine

To obtain an understanding of the importance of the neuronal cytoskeleton in Schwann cell metabolism, an antimicrotubular agent (colchicine) was injected into the rat sciatic nerve 24 or 48 h before incubation of the nerve with labeled precursor: [35S]sulfate, [14C]galactose, or [3H]-galactose. Colchicine inhibited the incorporation of 35S radioactivity into sulfatides and, to a lesser extent, into proteins. With galactose as the radioactive precursor, synthesis of cerebrosides was reduced by colchicine injection, whereas incorporation of radioactivity into phosphatidylserine and phosphatidylcholine increased. Intraneural injection of lumicolchicine had no effect. The effects of colchicine on the metabolism of the Schwann cell are discussed in relation to its action on microtubules.     

Retrograde Axonal Transport of Gangliosides and Glycoproteins in the Motoneurons of Rat Sciatic Nerve

Axonal transport of glycoconjugates was studied in the motoneurons of rat sciatic nerve following injection of [3H]glucosamine into the lumbosacral spinal cord. After varying time intervals, the sciatic nerve was exposed, and two ligatures were tied for collection of materials undergoing anterograde and retrograde transport. Gangliosides and glycoproteins were found to undergo fast anterograde transport, estimated at 284-446 mm/day. Both classes underwent retrograde transport as well, with labeled glycoproteins returning slightly ahead of labeled gangliosides. Only minor quantities of labeled proteoglycans were detected. Purified gangliosides extracted from nerve segments were fractionated according to sialic acid number on diethylaminoethyl-Sephadex; the distributional pattern tended to resemble that of brain gangliosides. The similarity between anterograde and retrograde patterns suggested absence of metabolic changes in gangliosides entering and leaving the axon-nerve terminal structures.     

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.     

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [³H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ～6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (～130 kD, pI ～5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (～50 kD, pI ～5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (～45 kD, pI ～4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (～60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.     

Neurofilament Proteins Are Distributed in a Diminishing Proximodistal Gradient Along Rat Sciatic Nerve

Neurofilament (NF) proteins are distributed in a diminishing proximodistal gradient along rat sciatic nerve when compared with total noncollagen or other proteins in nerve. About a twofold decline of NF proteins can be detected by quantitating nerve proteins that have been separated by gel electrophoresis. A similar decrease of immunoreactivity to each NF subunit is seen in distal nerve segments when noncollagen nerve proteins are immunoblotted. Parallel decreases occur in all three NF proteins, thereby maintaining neurofilament subunit stoichiometry along the neuraxis. The same NF gradient can be detected when the NF contents in nerve branches to the gluteus and gastrocnemius muscles are compared with each other and with those in nerve segments taken from the same proximodistal levels of the parent sciatic nerve. The gradient of NF proteins increases during postnatal development and is readily detected by postnatal day 16. During the same period of development, the heavy NF subunit appears for the first time and is rapidly incorporated throughout the sciatic nerve. Hence, the NF gradient becomes manifest during the development and maturation of the adult form of the axonal cytoskeleton. The basis for the proximodistal gradient of NF proteins in peripheral nerve is presently unknown. The extent of the gradient cannot be accounted for on the basis of diminishing numbers of nerve fibers or increasing amounts of other nerve proteins, e.g., collagen, in distal nerve. An alternative interpretation is that the gradient reflects a low level of NF protein turnover during axonal transport.     

Cold Blockade of Axonal Transport Activates Premitotic Activity of Schwann Cells and Wallerian Degeneration

Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell-associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11 degrees C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked- and control-degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann-cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann-cell premitotic response to axotomy.     

The Sequential Alterations of Endoneurial Cholesterol and Fatty Acid in Wallerian Degeneration and Regeneration

Abstract: The rate of tryptophan hydroxylation in vivo was estimated in discrete rat brain nuclei by measuring L-5-hydroxytryptophan (5-HTP) accumulated after pharmacological blockade of ^L-5-hydroxytryptophan decarboxylase by NSD 1015, using a sensitive radioenzymatic microassay. Endogenous serotonin, a major contaminant in this assay, was quantitatively removed by cationexchange chromatography prior to analysis. In non-treated animals, endogenous 5-HTP could be detected in small but measurable amounts. Following NSD 1015, accumulation occurred linearly for at least 30 min. At this time the recorded figures were two to six times higher when compared to values obtained in the same discrete structure from non-treated animals. This allows an accurate estimation of the rate of tryptophan hydroxylation in vivo in small fragments of grossly dissected brain regions (e.g. cortex) as well as in discrete nuclei containing either serotoninergic (5-HT) cell bodies (brain stem raphe nuclei) or 5-HT-terminals (e.g. catecholaminergic group A l, A2, A6.,. etc). Parachlorophenylalanine drastically reduced the rate of tryptophan hydroxylation in vivo in both terminal regions and raphe nuclei, with similar figures, 3 h or 3 days after injection. Chloral hydrate anaesthesia was attended by a transient decrease which appeared delayed in the raphe nuclei. Finally, pargyline pretreatment led to an 80% decrease in the forebrain, while no significant change appeared in the raphe nuclei. Thus, as illustrated by these few pharmacological manipulations, this method allows the study of the regulation of tryptophan hydroxylation in vivo with an improved anatomical resolution. Investigations can be carried out in the various raphe nuclei and their corresponding terminals in discrete brain areas simultaneously.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.