Brain Researeh, 152 (1978) 151 156 ~

15l

Elsevier/North-Holland Biomedical Press

An in vitro method for studying the retrograde intra-axonal transport of horseradish peroxidase in sympathetic neurons

P. N. ANDERSON, J. M. MITCHELL and D. MAYOR

Human Morphology, Southampton University, Medical and Biological Sciences Building, Southampton ( Great Britaht) (Accepted March 9th, 1978)

The use of horseradish peroxidase (HRP) to study retrograde axonal transport in the central nervous system is now well established. Since injected HRP is taken up by nerve terminals and transported to their perikarya this technique has been widely used for the demonstration of specific fibre pathwaysT, s. More recently it has also been used in studies of the autonomic nervous system, but with variable results 2,3,9,1°. However, little attention has been given to the probability that injected HRP may leak into the surrounding tissue and be taken up by neuronal systems other than those under investigation. This criticism is particularly valid in studies on abdominal autonomic ganglia which involve opening the peritoneal cavity in order to apply the HRP to a nerve. Under these conditions leakage of HRP into the peritoneal cavity is unavoidable. The present communication describes preliminary results obtained using an in vitro system which has been developed to study the mechanism of the uptake and transport of HRP and the fibre connections to and from the guinea pig inferior mesenteric ganglion (I MG). An in vitro system has many advantages with regard to investigations of the mechanism of retrograde transport and also allows adequate control measures to be taken which should eliminate false positive results caused by the possible diffusion of HRP. However, other workers have failed to demonstrate the uptake and retrograde transport of HRP within frog sciatic nerve trunks in vitro '~. Male Hartley guinea pigs weighing 280-350 g were anaesthetized with intraperitoneal Nembutal (40 mg/kg: Abbott). The hypogastric nerves were located in the mesentery of the colon and ligated with a fine (5/0) silk ligature approximately 2.5 cm distal to the IMG. The inferior mesenteric artery was ligated distal to the ganglion. Tile ligated nerve/IMG preparation was dissected free from the surrounding tissues. This dissection involved cutting the preganglionic nerves and the inferior mesenteric artery proximal to the ganglia. By means of the thread ligatures, which had been left long, the preparation was suspended in the incubation chamber. A double chamber similar in design to that used by Banks et al. 1, but with smaller compartments, was employed. Twenty five mg of Sigma type II HRP was dissolved in one drop of oxygenated tissue culture medium (Eagles MEM; Gibco Bio-Cult).

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Fig. I. Phase contrast photomicrograph from a 2 pm unstained araldite section of guinea pig IMG showing black particles representing HRP reaction product (arrowed) within the cytoplasm of a neuronal perikaryon. N nucleus.Calibration bar 10 .m. This concentrated H R P solution was applied around the ligation on the hypogastric nerve. The top of the chamber was assembled and tissue culture medium added to the ganglion side of the compartment. Ten minutes later 5 ml of tissue culture medium was added to the ligated nerve compartment. The chamber was placed in a w a t e r b a t h maintained at 37 °C for 24 h. The tissue culture medium within each compartment of the chamber was gassed with 95 O/yo oxygen/5 y,, carbon dioxide throughout the 24 h period of incubation. The preparation was then removed and fixed for 5 h at room temperature in a solution containing: I o/~ paraformaldehyde, 3.5 ~o glutaraldehyde, 7~£i sucrose and 0.1 M phosphate buffer at p H 7.4. Following fixation the preparation was washed overnight in phosphate buffer/sucrose mixture at 4 ~C. The I M G was sectioned at 50 #m on a Sorvall tissue chopper. These sections and 0.5 m m lengths of the hypogastric nerves were rinsed in distilled water and reacted for peroxidase activity using the technique of G r a h a m and Karnovsky 4, The tissues were then osmicated, dehydrated in ethanol, passed through epoxy propane and embedded in araldite. Unstained sections 2 #m thick were examined using phase contrast light microscopy. Uttrathin sections, both stained and unstained, were studied electron microscopically. Frequently the guinea pig I M G consists of one lobe of tissue o n either

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Fig. 2. Electron micrograph of part of the cytoplasm of a neuronal cell body from the guinea pig I MG showing HRP reaction product within membrane-bound vesicles, some of which resemble dense bodies (DB) with others being much smaller (arrowed). Calibration bar .... 1 /~m.

side of the inferior mesenteric artery. In some experiments the two parts were separated prior to sectioning to ascertain whether or not both parts had contributed axons to the hypogastric nerves. The H R P reaction product appeared as discrete brown granules within the cytoplasm of some neuronal perikarya (Fig. 1). These HRP-positive neurones were often grouped together, the great majority being found within the larger caudal lobe of the ganglion. Those neurones which contained reaction product when viewed with the light microscope also showed reaction product localized within membrane-bound organelles similar to dense bodies (lysosomes) at the ultrastructural level (Fig. 2). The HRP-positive organelles were more difficult to identify in sections stained with uranyl acetate and lead citrate because the contrast between them and normal dense bodies was lost. The localization of H R P within dense bodies is in agreement with previous studies of other neuronal systems in vivo 6,7. In order to establish that the H R P within the neuronal perikarya had arrived there by retrograde axonal transport, control experiments were carried out using

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Fig. 3. Schematic diagram of the guinea pig IMG/ligated hypogastric nerve preparation maintained in a two compartment incubation chamber. Concentrated H R P applied in region P1, immediately proximal to distal ligation. Top: arrangement used to demonstrate retrograde intra-axonal transport from P1 to neuronal perikarya in 1MG, Bottom: the position of the second proximal ligation in ganglion compartment used to demonstrate obstruction to retrograde axonal transport,

Fig. 4. Electron micrograph taken from segment Dip (see Fig. 3) of the guinea pig hypogastric nerve showing the contents of a swollen non-myelinated axon. Note the numerous organelles laden with H R P reaction product (arrowed). Calibration bar -- 0.5 ~m.

155 similar ganglion/ligated nerve preparations from 3 further guinea pigs. In addition to the first distal ligation, a second proximal ligation was applied to the hypogastric nerve close to the ganglion. The preparation was positioned in the chamber such that the second proximal ligation was in the same compartment as the ganglion (Fig. 3). The HRP was applied to the distal ligation as described above, and the preparation subsequently treated in an identical manner. No HRP-positive neurones were found within the IMG of these preparations. HRP reaction product was present in the Pl segment (Fig. 3) of the nerve and was localized within Schwann ceils, fibroblasts, interstitial spaces and within both myelinated and non-myelinated axons. The HRP within the axons was present both in a diffuse form and within membrane-bound organelles. No HRP was found in the interstitial spaces of the hypogastric nerves at or beyond 2 mm proximal to the distal ligation. However, in the D~p segments (Figs. 3 and 4) HRP reaction product, localized within membrane bound organelles, was present in swollen axons. It seems probable, therefore, that HRP does not penetrate the perineurium of the normal hypogastric nerve, nor does it spread through the interstitial spaces of the nerves for a distance of more than 2 mm in 24 h. In view of these observations the most likely explanation of the presence of intra-axonal HRP reaction product in the Dip segment is that it represents enzyme taken up by the damaged axons at the site of the distal ligation which is then transported retrogradely towards the proximal ligation. It is apparent therefore that the retrograde transport of HRP occurs in vitro, even in axons isolated from the perikarya. Some previous studies 0,1° have failed to demonstrate the ability of sympathetic neurones to take up HRP at their terminals and transport it back to their perikarya, but this may be due to diftbrences in technique, e.g. the dose of HRP employed and the method used for detecting the HRP within the neuronal perikarya. However Ellison and Clark a found evidence for the retrograde transport of HRP from the anterior chamber of the eye to the superior cervical ganglion in guinea pigs. The present experiments clearly demonstrate that HRP can be taken up by damaged sympathetic axons in the guinea pig hypogastric nerve and transported retrogradely to their perikarya within the IMG. It should be noted that the small size of the HRP-positive granules within the perikarya would probably make the granules very difficult to demonstrate in frozen sections, which have been widely used by previous workers. The 2/~m araldite sections used in the present study allow much greater resolution and better preservation of the tissue. The present in vitro system and method of demonstrating HRP offer further scope for the investigation of the phenomenon of retrograde axonal transport.

156 1 Banks, P., Mayor, D.. Mitchell. M. and Tomlinson, D., Studies on the translocation o f noradrenaline-containing vesicles in post-ganglionic sympathetic neurones in vitro. Inhibition of movement by colchicine and vinblastine and evidence for the involvement of axonal microtubules. J. Physiol. (Lond.), 216 (1971) 625-639. 2 Elfvin, L.-G. and Dalsgaard. C. J.. Retrograde axonal transport of horseradish peroxidase in afferent fibres of the inferior mesenteric ganglion of the guinea pig. Identification of the cells of origin in dorsal root ganglia, Brain Research. 126 (1977) 149-153. 3 Ellison, P. J. and Clark, G. M.. Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves, J. comp. Neurol.. 161 (1975) 103-114. 4 Graham, R. C. and Karnovsky, M. J.. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney: ultrastruetural correlates by a new technique, J. Histochem. Cytochem., 14 (1966) 291--299. 5 Hanson, M., Tonge, D. and Edstrom, A., Retrograde axonal transport of exogenous protein in frog nerves, Brah~ Research, 100 (1975) 458-461. 6 Kristensson, K. and Olsson, Y.. Diffusion pathways and retrograde axonal transport of protein tracers in peripheral nerves, Progr. Neurobiol.. 1 (1973) 85-109. 7 LaVail, M. M. and LaVail, J. H.. Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick, Brain Research. 85 (1975) 273 280. 8 Rinvik, E., Demonstration of nigrothalamic connections in the cat by retrograde axonat transport of horseradish peroxidase, Brain Research, 90 (1975) 313-318. 9 Schwab, M. E., Ultrastructural lccalization of a nerve growth factor-horseradish lzeroxidase (NGFHRP) coupling preduct after r e t r ~ rade a×onal transport in adrenergic neurons, Brcht Research, 130 (1977) 190-196. 10 St~ckel, K., Faravicini, U. and q-hoenen, H., Sl:ecificiiy of the retrograde axonal transport of nerve growth facte, r, Brt, in Research, 76 (1974) 413-421.

An in vitro method for studying the retrograde intra-axonal transport of horseradish peroxidase in sympathetic neurons.

Brain Researeh, 152 (1978) 151 156 ~ 15l Elsevier/North-Holland Biomedical Press An in vitro method for studying the retrograde intra-axonal transp...
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