J. Anat. (1977), 124, 1, pp. 83-97 With 9 figures Printed in Great Britain
83
Uptake of horseradish peroxidase by sensory nerve fibres in vitro KLAUS TISCHNER
Institute of Neurology (Edinger Institute) of the Johann Wolfgang Goethe- Universitdt and Neuropathology Department of the Max-Planck-Institute for Brain Research, Frankfurt, Germany
(Accepted 8 June 1976) INTRODUCTION
The expanded fibre tip of a neuron displays a remarkable degree of autonomous activity, as pinocytosis and fibre elongation may continue for hours after transection of the axon (Hughes, 1953; Birks, Mackey & Weldon, 1972). The occurrence of pinocytotic vesicles in living nerve fibres is a well documented phenomenon (Murnaghan, 1941; Lewis, 1945). It has been described how droplets may move inside nerve fibres in a retrograde direction toward the perikarya (Pannese, 1968; Pomerat, Hendelman, Raiborn & Massey, 1967). Also, data exist which demonstrate the incorporation and centripetal spread of 3H-leucine in growth cones of cultured chick sensory ganglia (Tischner & Korr, 1972). The significance of uptake and transport of substances from the surrounding nutrient medium into nerve fibres in vitro remains to be elucidated. Application of horseradish peroxidase has proved a valuable method for investigating the incorporatton of exogenous protein in?to animal cells at both light microscope (Straus, 1957) and electron microscope (Graham & Karnovsky, 1965) levels. Local application of this tracer to hypoglossal nerve terminals leads to its retrograde transport (Kristensson & Olsson, 1971). Moreover, various barrier functions in the central nervous system have been demonstrated with this low molecular weight enzyme tracer (Reese & Karnovsky, 1967; Hirano, Becker & Zimmerman, 1970), and in the peripheral nervous system also (Klemm, 1970). The present study is concerned with the fine structural details of pinocytosis by neurons of explanted sensory ganglia in the presence of peroxidase. Special attention has been given to the growth cones of regenerating neurons. MATERIAL AND METHODS
Explants of dorsal root ganglia from 17-18 day old Wistar rat embryos were sealed into Maximov type double coverslip depression slide assemblies as described by Murray & Peterson (1953) and Bunge, Bunge, Peterson & Murray (1967). The cultures were fed two drops of a mixture containing equal parts of human adult serum, Eagle's minimum essential medium, a balanced salt solution, and embryonic extract from 9 day old chicks, supplemented with 600 mg 00 glucose. After 47T hours in vitro the cultures were exposed to horseradish peroxidase at a concentration of 4 mg!ml of medium for 15 minutes. This procedure was followed by fixation for two hours in a mixture of 3-9 %0 glutaraldehyde and 4 % formaldehyde containing 0 1 M cacodylate buffer. Thereafter the cultures were immersed in 0 1 M cacodylate buffer overnight. A total of 21 cultures was incubated with 10 mg of 3'3-diamino6-2
84 KLAUS TISCHNER benzidine hydrochloride dissolved in 10 ml of 0 I M Tris-HCl buffer at pH 7-6, and 1 % hydrogen peroxide solution (Karnovsky, 1967). Then the cultures were washed in double-distilled water for 20 minutes, and post-fixed in 2 % osmic acid containing 0-1 M phosphate buffer for 1 hour. Dehydration was initiated in 25, 35, 50, 75, and 95 % ethanol (5 minutes each) and completed in two changes of 100 % ethanol (20 minutes each). The cultures were embedded as whole mounts in epoxy resin. Suitable areas of nerve fibres were selected under the phase contrast microscope. After trimming off excess epon, thin sections were taken with a diamond knife and stained with lead citrate (Venable & Coggeshall, 1965). Electron micrographs were taken with a Siemens Elmiskop 102. RESULTS
Light microscopic findings After 48 hours in vitro dorsal root ganglia had formed an intertwining network of regenerating fibres many of which had grown out from the severed nerve stumps (Fig. 1). Phase contrast observations on the living fibres revealed expanded terminal regions from which ruffling membranes and filopodia protruded. The latter engaged in a process of prolongation and retraction as growth of the nerve fibres took place. The region of the growth cone was conspicuous by the presence of pinocytotic vacuoles of up to 2 ,um in diameter (Fig. 3, inset), many of which moved proximally at a rate of 0-5-10 ,tm/min, covering distances of several hundreds of ,cm. On their way toward the perikarya the larger droplets divided into smaller ones (Figs. 2A, B) which soon disappeared from the level of resolution of the conventional microscope. Addition of horseradish peroxidase to living cultures revealed no effects on this specific activity of nerve fibres.
Ultrastructural findings Cultures exposed to horseradish peroxidase showed dense labelling of the external constituents of the plasmalemma of outgrowing nerve processes (Fig. 3, and following figures). The flanges expanding from the growth cones contained 5 nm filaments which usually were distributed in a random network, but were sometimes in sheaves with a predominantly parallel orientation (Fig. 3). Horseradish peroxidase reaction product was located in membrane-bound vacuoles inside such flanges. The microspikes projecting from the area of the growth cone contained identical filaments, but pinocytotic values were absent. Growth cones were filled with aggregates of membrane-bound vesicles of ovoid or elongated shapes, measuring between 0 1 and 0-25 ,um across their largest diameters. In addition, dense-cored vesicles (80-100 nm), cup-shaped profiles, moderate amounts of smooth endoplasmic reticulum, and occasionally a mitochondrion, were encountered (Figs. 4, 5). Neurotubules tended to be restricted to the proximal portions of the growth cones. Numerous vesicles of various sizes were filled with horseradish peroxidase reaction product, whereas dense-cored vesicles rarely contained this marker enzyme. At the plasmalemma, pinocytotic vesicles were common in some growth cones (Fig. 4). Other growth cones were made conspicuous by the presence of prominent vacuoles (1-2 ,tm) in which horseradish peroxidase was lined up along the inner portions of the limiting membranes (Fig. 5). Smaller vesicle&frequently- abutted on
~ ~ .4
Peroxidase uptake by nerve fibres in vitro 85 -11-.-.-
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C_ Fig. 1. Nerve fibres which have grown out of the severed nerve are shown after 48 hours in vitro. Beginning fasciculation of axons can be noted, and spindle-shaped Schwann cells can be seen in the zone of outgrowth. Living dorsal root ganglion culture, untreated. x 400.
86
KLAUS TISCHNER
.re
.1
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Fig. 2. Part of a slender growth cone is depicted at the ultrastructural level. From the cone there extends a broad flange which is filled with a filamentous material. The filaments are in parallel array at the upper right of the figure. The exterior of the plasmalemma is coated with reaction product for horseradish peroxidase. The expanded flange contains a membranebound vacuole in which horseradish peroxidase is present. x 15000. Inset: A corresponding region of the fibre tip of a living neuron is shown at the light microscopic level. Pinocytotic vacuoles in the growth cone are conspicuous. Note the velamentous flange extending from the tip, and several filopodia. x 1000.
Peroxidase uptake by nerve fibres in vitro
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Figs 3A, B. A sequence of micrographs was taken at an interval of 10 minutes. In the living nerve fibre two 'bubbles' have been transferred from the tip, and are present in the intermediate region of the axon. B, Several smaller vacuoles have originated from the two larger ones. x 1200.
the voluminous ones, and sometimes appeared to be fusing with the latter. Horseradish peroxidase was not localized in all large vacuolar compartments. In varicosities of the nerve fibres horseradish peroxidase filled vacuoles were also found. Such distended structures were marked by the presence of numerous mitochondria (Fig. 6). In the more proximal parts of the fibre large droplets were absent. Here, the enzyme label was found in small vesicles, and occasionally in autophagic vacuoles. Pinocytotic phenomena were also encountered in these regions of the fibres
(Fig. 7).
At this stage of development the cell membranes of spinal ganglion cells were still in direct contact with each other over long distances. Intersurface cisternae were frequently found between areas where the membranes were directly apposed (Fig. 8).
88
KLAUS TISCHNER
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Fig. 4. This electron micrograph displays a growth cone which holds vacuoles of varying size, cup-shaped structures, short profiles of tubules, and dense-cored vesicles. A pinocytotic vesicle is seen at the lower left as it forms from the plasmalemma. Microtubules are present at the base of the cone. The cone is surrounded by microspikes which in most cases stem from adjacent neurons. x 40000.
Peroxidase uptake by nerve fibres in vitro
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Fig. 6. The right part of the micrograph depicts a varicosity of the nerve fibre. Note a small, horseradish peroxidase-containing vesicle that abuts on a large vacuole. Mitochondria are numerous. On the left a growth cone of a contiguous neuron contains a wealth of vesicles. Pinocytotic phenomena occur between the adjacent fibres (arrows). x 15000.
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83
Uptake of horseradish peroxidase by sensory nerve fibres in vitro KLAUS TISCHNER
Institute of Neurology (Edinger Institute) of the Johann Wolfgang Goethe- Universitdt and Neuropathology Department of the Max-Planck-Institute for Brain Research, Frankfurt, Germany
(Accepted 8 June 1976) INTRODUCTION
The expanded fibre tip of a neuron displays a remarkable degree of autonomous activity, as pinocytosis and fibre elongation may continue for hours after transection of the axon (Hughes, 1953; Birks, Mackey & Weldon, 1972). The occurrence of pinocytotic vesicles in living nerve fibres is a well documented phenomenon (Murnaghan, 1941; Lewis, 1945). It has been described how droplets may move inside nerve fibres in a retrograde direction toward the perikarya (Pannese, 1968; Pomerat, Hendelman, Raiborn & Massey, 1967). Also, data exist which demonstrate the incorporation and centripetal spread of 3H-leucine in growth cones of cultured chick sensory ganglia (Tischner & Korr, 1972). The significance of uptake and transport of substances from the surrounding nutrient medium into nerve fibres in vitro remains to be elucidated. Application of horseradish peroxidase has proved a valuable method for investigating the incorporatton of exogenous protein in?to animal cells at both light microscope (Straus, 1957) and electron microscope (Graham & Karnovsky, 1965) levels. Local application of this tracer to hypoglossal nerve terminals leads to its retrograde transport (Kristensson & Olsson, 1971). Moreover, various barrier functions in the central nervous system have been demonstrated with this low molecular weight enzyme tracer (Reese & Karnovsky, 1967; Hirano, Becker & Zimmerman, 1970), and in the peripheral nervous system also (Klemm, 1970). The present study is concerned with the fine structural details of pinocytosis by neurons of explanted sensory ganglia in the presence of peroxidase. Special attention has been given to the growth cones of regenerating neurons. MATERIAL AND METHODS
Explants of dorsal root ganglia from 17-18 day old Wistar rat embryos were sealed into Maximov type double coverslip depression slide assemblies as described by Murray & Peterson (1953) and Bunge, Bunge, Peterson & Murray (1967). The cultures were fed two drops of a mixture containing equal parts of human adult serum, Eagle's minimum essential medium, a balanced salt solution, and embryonic extract from 9 day old chicks, supplemented with 600 mg 00 glucose. After 47T hours in vitro the cultures were exposed to horseradish peroxidase at a concentration of 4 mg!ml of medium for 15 minutes. This procedure was followed by fixation for two hours in a mixture of 3-9 %0 glutaraldehyde and 4 % formaldehyde containing 0 1 M cacodylate buffer. Thereafter the cultures were immersed in 0 1 M cacodylate buffer overnight. A total of 21 cultures was incubated with 10 mg of 3'3-diamino6-2
84 KLAUS TISCHNER benzidine hydrochloride dissolved in 10 ml of 0 I M Tris-HCl buffer at pH 7-6, and 1 % hydrogen peroxide solution (Karnovsky, 1967). Then the cultures were washed in double-distilled water for 20 minutes, and post-fixed in 2 % osmic acid containing 0-1 M phosphate buffer for 1 hour. Dehydration was initiated in 25, 35, 50, 75, and 95 % ethanol (5 minutes each) and completed in two changes of 100 % ethanol (20 minutes each). The cultures were embedded as whole mounts in epoxy resin. Suitable areas of nerve fibres were selected under the phase contrast microscope. After trimming off excess epon, thin sections were taken with a diamond knife and stained with lead citrate (Venable & Coggeshall, 1965). Electron micrographs were taken with a Siemens Elmiskop 102. RESULTS
Light microscopic findings After 48 hours in vitro dorsal root ganglia had formed an intertwining network of regenerating fibres many of which had grown out from the severed nerve stumps (Fig. 1). Phase contrast observations on the living fibres revealed expanded terminal regions from which ruffling membranes and filopodia protruded. The latter engaged in a process of prolongation and retraction as growth of the nerve fibres took place. The region of the growth cone was conspicuous by the presence of pinocytotic vacuoles of up to 2 ,um in diameter (Fig. 3, inset), many of which moved proximally at a rate of 0-5-10 ,tm/min, covering distances of several hundreds of ,cm. On their way toward the perikarya the larger droplets divided into smaller ones (Figs. 2A, B) which soon disappeared from the level of resolution of the conventional microscope. Addition of horseradish peroxidase to living cultures revealed no effects on this specific activity of nerve fibres.
Ultrastructural findings Cultures exposed to horseradish peroxidase showed dense labelling of the external constituents of the plasmalemma of outgrowing nerve processes (Fig. 3, and following figures). The flanges expanding from the growth cones contained 5 nm filaments which usually were distributed in a random network, but were sometimes in sheaves with a predominantly parallel orientation (Fig. 3). Horseradish peroxidase reaction product was located in membrane-bound vacuoles inside such flanges. The microspikes projecting from the area of the growth cone contained identical filaments, but pinocytotic values were absent. Growth cones were filled with aggregates of membrane-bound vesicles of ovoid or elongated shapes, measuring between 0 1 and 0-25 ,um across their largest diameters. In addition, dense-cored vesicles (80-100 nm), cup-shaped profiles, moderate amounts of smooth endoplasmic reticulum, and occasionally a mitochondrion, were encountered (Figs. 4, 5). Neurotubules tended to be restricted to the proximal portions of the growth cones. Numerous vesicles of various sizes were filled with horseradish peroxidase reaction product, whereas dense-cored vesicles rarely contained this marker enzyme. At the plasmalemma, pinocytotic vesicles were common in some growth cones (Fig. 4). Other growth cones were made conspicuous by the presence of prominent vacuoles (1-2 ,tm) in which horseradish peroxidase was lined up along the inner portions of the limiting membranes (Fig. 5). Smaller vesicle&frequently- abutted on
~ ~ .4
Peroxidase uptake by nerve fibres in vitro 85 -11-.-.-
-
4' 1R
/ 7
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'a 4'
:1I $1 'C'
C_ Fig. 1. Nerve fibres which have grown out of the severed nerve are shown after 48 hours in vitro. Beginning fasciculation of axons can be noted, and spindle-shaped Schwann cells can be seen in the zone of outgrowth. Living dorsal root ganglion culture, untreated. x 400.
86
KLAUS TISCHNER
.re
.1
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I
S
4¼ -4
C 2. 2
f.
*
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Fig. 2. Part of a slender growth cone is depicted at the ultrastructural level. From the cone there extends a broad flange which is filled with a filamentous material. The filaments are in parallel array at the upper right of the figure. The exterior of the plasmalemma is coated with reaction product for horseradish peroxidase. The expanded flange contains a membranebound vacuole in which horseradish peroxidase is present. x 15000. Inset: A corresponding region of the fibre tip of a living neuron is shown at the light microscopic level. Pinocytotic vacuoles in the growth cone are conspicuous. Note the velamentous flange extending from the tip, and several filopodia. x 1000.
Peroxidase uptake by nerve fibres in vitro
IF
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'.
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Figs 3A, B. A sequence of micrographs was taken at an interval of 10 minutes. In the living nerve fibre two 'bubbles' have been transferred from the tip, and are present in the intermediate region of the axon. B, Several smaller vacuoles have originated from the two larger ones. x 1200.
the voluminous ones, and sometimes appeared to be fusing with the latter. Horseradish peroxidase was not localized in all large vacuolar compartments. In varicosities of the nerve fibres horseradish peroxidase filled vacuoles were also found. Such distended structures were marked by the presence of numerous mitochondria (Fig. 6). In the more proximal parts of the fibre large droplets were absent. Here, the enzyme label was found in small vesicles, and occasionally in autophagic vacuoles. Pinocytotic phenomena were also encountered in these regions of the fibres
(Fig. 7).
At this stage of development the cell membranes of spinal ganglion cells were still in direct contact with each other over long distances. Intersurface cisternae were frequently found between areas where the membranes were directly apposed (Fig. 8).
88
KLAUS TISCHNER
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Fig. 4. This electron micrograph displays a growth cone which holds vacuoles of varying size, cup-shaped structures, short profiles of tubules, and dense-cored vesicles. A pinocytotic vesicle is seen at the lower left as it forms from the plasmalemma. Microtubules are present at the base of the cone. The cone is surrounded by microspikes which in most cases stem from adjacent neurons. x 40000.
Peroxidase uptake by nerve fibres in vitro
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Fig. 6. The right part of the micrograph depicts a varicosity of the nerve fibre. Note a small, horseradish peroxidase-containing vesicle that abuts on a large vacuole. Mitochondria are numerous. On the left a growth cone of a contiguous neuron contains a wealth of vesicles. Pinocytotic phenomena occur between the adjacent fibres (arrows). x 15000.
Peroxidase uptake by nerve fibres in vitro -
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