137
J. Anat. (1978), 125, 1, pp. 137-147 With 16 figures Printed in Great Britain
Electron microscopical study of retrograde axonal transport of horseradish peroxidase in the supraoptico-hypophyseal tract in the rat PETER PRICE AND A. W. F. FISHER
Division of Morphological Science, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4
(Accepted 17 December 1976) INTRODUCTION
In recent years much use has been made of the retrograde axonal transport of horseradish peroxidase (HRP) to trace a wide variety of neuroanatomical connexions. However, few have attempted to identify the experimental factors which affect the process and contribute to the specificity and reproducibility of the technique (Beitz & King, 1976; Bunt et al. 1976; Halperin & LaVail, 1975; Janowska, Rastad & Westman, 1976; Jones, 1975; Kim & Strick, 1976; LaVail & LaVail, 1974; Nauta, Kaiserman-Abramof & Lasek, 1975; Theodosis et al. 1976; Turner & Harris, 1974). Recently we have used the technique to locate the afferent connexions of the pars nervosa (PN) and pars intermedia (PI) of the rat pituitary (Fisher & Price, 1975; Fisher & Price, 1976) but were unable to delineate specific connexions of PI and PN because the injection sites were too large, always involving at least two parts of the pituitary. In addition, we began to look at intracortical and transcallosal connexions in the rat and cat cerebral cortex (unpublished observations) but found that the transport of HRP in these situations was very capricious. Therefore, in order to reduce the size of the injection site and to improve the specificity and reproducibility of the technique, we began to study the factors which may affect the retrograde transport process. This report presents our observations of some ultrastructural features of this transport process in the rat magnocellular neurosecretory system, features which are essential to an understanding of the process. MATERIALS AND METHODS
0-3 ,ul of 20 0 HRP (Sigma type VI) in 3 M-KCI was injected via the right ear canal into the pituitary glands of 36 adult male Sprague-Dawley rats (200-250 g) under Diabutal anaesthesia. Injections were made with glass micropipettes (5080,am ID) inserted through a Hoffman-Reiter hypophysectomy instrument, and connected by oil-filled tubing to a 10 ,ul Hamilton syringe driven by a Sage syringe pump at a rate of 1 al/20 min. The animals were allowed to survive for varying intervals (4, 1, 1j, 3, 4, 5, 6, 12, 24, 48, 96 and 192 hours) to allow time for uptake and transport to occur. The rats were killed by intracardiac perfusion, under Diabutal anaesthesia, of 50 ml 0 9 %/O NaCl followed by 100 ml cold (4 °C) 2 5 %0 glutaraldehyde in 0 1 M phosphate buffer at pH 7-3. Brains and pituitaries were immediately removed, trimmed and immersed in fixative for 18 hours at 4 'C. Then 50-75 ,tm sections,
P. PRICE AND A. W. F. FISHER 138 cut on a Smith-Fahrquarson tissue chopper, were washed for 2 hours in cold phosphate buffer and then treated for 30 minutes at 20 °C with fresh 50 mg % 3-3' diaminobenzidine (DAB) and 0-01 % H202 in phosphate buffer. Following transfer to phosphate buffer, areas of interest were dissected from the sections under the dissecting microscope, washed for 2 hours in phosphate buffer, and immersed for 1 hour at 4 °C in 1%Os04 in 0 1 M phosphate buffer at pH 7-3. The tissue blocks were then washed in distilled water, stained en bloc for 1 hour in 0-5 % aqueous uranyl acetate and embedded in Vestopal W. Unstained thin sections were examined in an AEI Corinth 275 electron microscope. Some of the sections, after reaction with DAB and H202, were embedded in glycol methacrylate, sectioned at 1 ,um on a Sorvall JB4 microtome, stained with H & E and examined in a Zeiss Photoscope.
RESULTS
Most injection sites involved parts of both the pars nervosa (PN) and the pars intermedia (PI), and occasionally also involved the pars anterior (PA). In some cases only the PA was involved, and in these animals no transport to hypothalamic neurons occurred. When the PN was part of the injection site HRP was invariably found in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei as well as numerous other hypothalamic areas (Fisher & Price, 1976) after time for transport had elapsed. By both light and electron microscopy most neurons of the SON and the supero-lateral portion of the PVN contained granular HRP reaction product (Fig. 1). HRP uptake in the pars nervosa HRP reaction product was found in the extracellular space, within the cytoplasm of axon swellings and terminals, and within the cytoplasm of pituicytes and endothelial cells. Within 15 minutes of injection HRP had penetrated widely through the extracellular space, producing a dense reaction product associated with basal laminae and collagen fibres (Fig. 2). By this time HRP had also entered into neurosecretory axon swellings and terminals. Clusters of small clear vesicles (40-70 nm) are normally found in these terminals. The HRP reaction product was seen as circular, uniform densities (40-70 nm) located among these clusters of small vesicles. While most of the vesicles were in the 40-50 nm size range, and most of the HRP densities were in the 60-70 nm range, it seems reasonable to conclude that the HRP reaction product was located in the larger of the small clear vesicles (Fig. 5). In spite
Fig. 1. Dark field light micrograph of 1 jum plastic section of the supraoptic nucleus (SON) 24 hours after injection of HRP into the pituitary. Most cells of the SON (arrows) are filled with bright refractile granules of HRP reaction product. OT, optic tract. x 60. Fig. 2. Injected area of the pars nervosa 15 minutes after HRP injection. HRP reaction product (black) is seen throughout the extracellular space, outlining the pituicytes and axon terminals and 'staining' the basal laminae about vessels and tissue lobules. Small amounts of reaction prdduct are also seen already within axon terminals and pituicytes. x 3800. Fig. 3. Neuron in the SON 3 hours after injection. A few black granules of reaction product (arrows) are clustered close to the nucleus and a large Golgi complex. x 5600. Fig. 4. Neuron in the SON 24 hours after injection. There are many granules of reaction product, mainly of large size, distributed widely throughout the cytoplasm. x 6500.
HRP transport in supraoptic neurons
139
P. PRICE AND A. W. F. FISHER 140 of considerable searching, no pinocytotic vesicles associated with plasma membranes were seen. After 45 minutes the density of reaction product in the extracellular space had increased, as had the number of vesicles containing reaction product inside the axon terminals (Fig. 6). In some terminals (Fig. 8) a few larger, irregular profiles filled with HRP were seen, and in some of the axons tubular profiles containing reaction product could be found (Fig. 9). After 1-5 hours the intensity of reaction product within the extracellular space had diminished, and by 3 hours it had virtually disappeared. Reaction product was present in vesicles, granules and tubules of the axon swellings and terminals at 1-5 and 3 hours, but by 4 hours it was becoming scarce in these locations, and it was progressively more difficult to find at later times (Fig. 7). Reaction product was seen at all times up to 4 hours, in pituicyte and endothelial cell cytoplasm, and by 4 hours this was by far the major location.
Transport of HRP through axons of the infundibulum HRP reaction product was found in axons of the infundibulum as early as 1-5 hours, and up to 24 hours, after injection. The amount of reaction product was greatest in the 4-6 hour time period, decreasing to become rare by 24 hours. It was seen in three types of location only: in long irregular tubular profiles (Fig. 10) that resembled agranular reticulum and also resembled the tubular profiles seen in PN axons (Fig. 9); in granular profiles (Fig. 11) that were usually lined up like a string of beads; and in composite arrangements of tubular and granular profiles (Fig. 12) which suggested that both these profiles were local variations in the geometry of the agranular reticulum.
Accumulation of HRP in neurons of the supraoptic nucleus HRP did not appear in the cells of the SON until 3 hours after injection, at which time reaction product was seen in membrane-bound granules of varying size, most of which were small (Fig. 3). These granules tended to be grouped close to the nucleus and Golgi complexes. In some cases tubular structures containing reaction product, resembling the tubules in the infundibular axons, were seen close to these Golgi complexes (Figs. 13, 14). Some of the granules, at 3 and 4 hours, had irregular profiles, many of which could perhaps be those of agranular reticulum tubules (Fig. 15). Fig. 5. Injected area of PN 15 minutes after injection. Reaction product is seen in the extracellular space 'staining' the basal lamina and the surface coat of the axon terminal. It is also in small granules similar in size to the small clear vesicles seen in clusters among the larger neurosecretory granules. x 33 750. Fig. 6. PN 45 minutes after injection. The density of reaction product on the basal lamina and collagen fibres is increased, as is the number of HRP-containing vesicles in the axon terminal. x 33450. Fig. 7. Axon terminal in the PN 3 hours after injection. Reaction product has disappeared from the extracellular space, although there are HRP-containing vesicles in the axon terminal. x 32350. Fig. 8. Axon terminal in the PN 45 minutes after injection. In addition to a few small vesicles containing HRP, there are several larger, irregular granular or tubular structures filled with reaction product (arrows). These structures are distributed among the neurosecretory granules rather than among clusters of the small vesicles. x 33 300.
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as 15 minutes after injection indicates a very rapid uptake. Theodosis et al. (1976) have demonstrated uptake of intravenous HRP within 1 minute of stimulation of the nerve terminals. LaVail (1975) has suggested that uptake probably begins very early; Nagasawa, Douglas & Schulz (1971) have demonstrated in rat pars nervosa that uptake occurs by pinocytosis within 10 minutes of injection, with reaction product located within the small vesicles. Although no pinocytotic vesicles were seen in the experiments reported here, our observations are generally consistent with those of LaVail (1975) and Nagasawa et al. (1971). Douglas, Nagasawa & Schulz (1971) and Nagasawa et al. (1971) have suggested that this pinocytotic activity, indicated by the clusters of small clear vesicles found in neurosecretory axon terminals of the rat pars nervosa, represents a recycling of membrane subsequent to discharge of neurosecretory material by exocytosis. Although this has not been fully substantiated, the rapidity of the process of HRP uptake observed here is in accord with their concept. Recently Theodosis et al. (1976) have shown that, after stimulating the rat pituitary electrically or by acute haemorrhage, intravenous HRP was taken up by axon terminals of the pars nervosa within 1-5 minutes into large irregular vacuoles. Very little HRP was found in micropinocytotic vesicles. Transport of HRP within axons has been observed in granules of varying size, including multivesicular bodies, and in irregular tubular profiles resembling agranular reticulum (LaVail & LaVail, 1974; Nauta et al. 1975). The observations reported here show that the larger irregular granules appear in axon terminals as early as 30 minutes after injection, and can be found in infundibular axons from 1P5 to 24 hours after injection. In addition, however, these granules were frequently seen to be arranged in linear fashion, resembling a string of beads, or in a linear combination with irregular tubular profiles. These results, while consistent with both previous reports (LaVail & LaVail, 1974; Nauta et al. 1975), strongly suggest that the granules and the tubules seen in these axons are simply different geometrical forms of the same structure, the agranular reticulum. Further, our observations also suggest that the larger irregular profiles seen in axon terminals are the dilated, irregular terminations of the agranular reticulum, which somehow collect HRP in the axon terminal and allow retrograde transport to occur. The process by which the small vesicles transfer the HRP to the agranular reticulum remains an open
question. HRP arrived in the neuron somata of the SON within 3 hours of injection into the pituitary. Initially these granules were mainly small, clustered close together Fig. 13. Detail of the Golgi complex of the SON neuron seen in Fig. 3, 3 hours after injection. A series of four irregular HRP vesicles is seen (arrows) which are similar in size and shape to agranular reticulum tubules and probably represent a curved portion of such a tubule. x 33 600. Fig. 14. Golgi complex of another SON neuron 3 hours after injection. A tubular structure filled with reaction product is seen among the Golgi membranes. At least two portions of these Golgi membranes show increased density which may indicate HRP reaction product (short arrows). In addition, a large lysosome-like granule (long arrow) containing some reaction product has a tubular structure apparently leading off from its surface. NUC, nucleus. x 55 000. Fig. 15. Portion of the cytoplasm of an SON neuron 4 hours after injection showing predominantly large granules densely filled with reaction product. Some of these granules have irregular, tail-like projections (arrows). x 17800. Fig. 16. Cytoplasm of SON neuron 4 days after injection. The density of reaction product within the lysosomes is diminished, and more irregular. x 23400. 10
ANA
125
146
P. PRICE AND A. W. F. FISHER and close to Golgi complexes, and often had tubular structures containing HRP nearby. At later times the granules tended to be larger, were more widely distributed in the cytoplasm, and in some instances had irregular tail-like profiles. These results suggest, as did those of Nauta et al. (1975), that the HRP arrives at the perikaryon in agranular reticulum tubules, and is transferred to larger, lysosome-like granules. The nature of the transfer process remains unclear, although two possibilities emerge from the present experiments: either the HRP is transferred by the Golgi complex directly for packaging into the lysosome granules (suggested by the 3 and 4 hour appearance; Figs. 13, 14), or the lysosomes are produced in the Golgi complex and subsequently acquire HRP directly from the agranular reticulum (suggested by the irregular appendages of some granules; Fig. 15). Both of these possibilities are consistent with the fate of transported HRP in other systems (LaVail & LaVail, 1974; Nauta et al. 1975) and with studies of HRP taken up into cortical neurons in the injection site (Turner & Harris, 1974). Extensive observations of many sections of tissue taken at shorter time intervals about the time of arrival of HRP in the cell somata should help to clarify the mode of transfer. The fate of HRP in neuron perikarya appears certain: degradation within lysosomes. In the rat magnocellular neurosecretory cells, this begins to occur by 4 days and is complete by 8 days. In the chick visual system, it is complete by 3-4 days (LaVail & LaVail, 1974) and in cerebral cortex neurons of rabbit, cat and monkey, it is complete by 4-5 days (Turner & Harris, 1974). It thus appears from this study, and from the ultrastructural studies conducted by others, that the processes of HRP uptake, retrograde axonal transport, accumulation in cell somata and ultimate destruction involve, in mildly stimulated neurons, a sequence of cell organelles common to all the cell types studied. Differences between cell types are confined to differences in the rates at which these processes take place. SUMMARY
The uptake of exogenous horseradish peroxidase (HRP) into axon terminals of rat pars nervosa, and its retrograde axonal transport via infundibular axons to the neurons of the supraoptic nucleus (SON), were examined in the electron microscope. HRP was present in the extracellular space of the pars nervosa for 3 hours after injection. Reaction product was found in small vesicles (40-50 nm) within axon terminals from 15 minutes to 3 hours. Infundibular axons showed reaction product from 1*5 hours to 24 hours in tubules of the agranular reticulum, which often appeared as granular or beaded profiles. Reaction product first appeared in cells of the SON at 3 hours, indicating a transport rate of 2-3 mm/hour, or 50-70 mm/day. Initially HRP was mainly in small lysosome-like granules clustered close to Golgi complexes. Later, larger granules predominated and were more widely distributed throughout the cytoplasm. Some tubular forms containing HRP, and resembling agranular reticulum, were seen close to Golgi complexes. Some granules had irregular tails that could represent direct connexions with agranular reticulum. By 4 days the reaction product was less dense in large granules, and by 8 days had disappeared. Thus, injected HRP is rapidly picked up by axon terminals in the PN by pinocytosis into small vesicles, and is subsequently transferred to agranular reticulum in which it is transported within axons to neuron perikarya of the supraoptic
147
HRP transport in supraoptic neurons
nuclei where, in association with Golgi complexes, it is transferred to lysosomes for degradation of its enzymic activity. REFERENCES
ARVIDSSON, J. (1975). Location of cat trigeminal ganglion cells innervating dental pulp of upper and lower canines studied by retrograde transport of HRP. Brain Research 99, 135-139. BEITZ, A. J. & KING, G. W. (1976). An improved technique for the microinjection of horseradish peroxidase. Brain Research 108, 175-179. BUNT, A. H., HASCHKE, R. H., LUND, R. D. & CALKINS, D. F. (1976). Factors affecting retrograde transport of horseradish peroxidase in the visual system. Brain Research 102, 152-155. DENNIS, B. J. & KERR, D. I. B. (1976). Origins of olfactory bulb centrifugal fibres in the cat. Brain Research 110, 593-600. DOUGLAS, W. W., NAGASAWA, J. & SCHULZ, R. A. (1971). Coated microvesicles in neurosecretory terminals of posterior pituitary glands shed their coats to become 'synaptic' vesicles. Nature 232, 340-341. FISHER, A. W. F. & PRICE, P. G. (1975). Retrograde axonal transport of exogenous horseradish peroxidase from the rat hypophysis. Proceedings of the Canadian Federation of Biological Societies 18, 30. FISHER, A. W. F. & PRICE, P. G. (1976). Interconnecting neurons between the third ventricle and the neuro-intermediate lobe of the rat. Anatomical Record 184, 403. HALPERIN, J. J. & LAVAIL, J. H. (1975). A study of the dynamics of retrograde transport and accumulations of horseradish peroxidase in injured neurons. Brain Research 100, 253-269. JANOWSKI, E., RASTAD, J. & WESTMAN, J. (1976). Intracellular application of horseradish peroxidase and its light and electron microscopical appearance in spinocervical tract cells. Brain Research 105, 557-562. JoNEs, E. G. (1975). Possible determinants of the degree of retrograde neuronal labelling with horseradish peroxidase. Brain Research 85, 249-253. KIM, C. C. & STRICK, P. L. (1976). Critical factors involved in the demonstration of horseradish peroxidase retrograde transport. Brain Research 103, 356-361. LAVAIL, J. H. (1975). The retrograde transport method. Federation Proceedings 34, 1618-1624. LAVAIL, J. H. & LAVAIL, M.M. (1974). The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: A light and electron microscopic study. Journal of Comparative Neurology 157, 303-358. NAGASAWA, J., DOUGLAS, W. W. & SCHULZ, R. A. (1971). Micropinocytotic origin of coated and smooth microvesicles ('synaptic vesicles') in neurosecretory terminals of posterior pituitary glands demonstrated by incorporation of horseradish peroxidase. Nature 232, 341-342. NAUTA, H. J. W., KAISERMAN-ABRAMOF, I. R. & LASEK, R. J. (1975). Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum. Brain Research 85, 375-384. STOCKEL, K., SCHWAB, M. & THOENEN, H. (1975). Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory and adrenergic neurons. Brain Research 99, 1-16. THEODOSIS, D. T., DREIFUSS, J. J., HARRIS, M. C. & ORCI, L. (1976). Secretion-related uptake of horseradish peroxidase in neurohypophyseal axons. Journal of Cell Biology 70, 234-303. TURNER, P. T. & HARRIS, A. B. (1974). Ultrastructure of exogenous peroxidase in cerebral cortex. Brain Research 74, 305-326.
IO-2
J. Anat. (1978), 125, 1, pp. 137-147 With 16 figures Printed in Great Britain
Electron microscopical study of retrograde axonal transport of horseradish peroxidase in the supraoptico-hypophyseal tract in the rat PETER PRICE AND A. W. F. FISHER
Division of Morphological Science, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4
(Accepted 17 December 1976) INTRODUCTION
In recent years much use has been made of the retrograde axonal transport of horseradish peroxidase (HRP) to trace a wide variety of neuroanatomical connexions. However, few have attempted to identify the experimental factors which affect the process and contribute to the specificity and reproducibility of the technique (Beitz & King, 1976; Bunt et al. 1976; Halperin & LaVail, 1975; Janowska, Rastad & Westman, 1976; Jones, 1975; Kim & Strick, 1976; LaVail & LaVail, 1974; Nauta, Kaiserman-Abramof & Lasek, 1975; Theodosis et al. 1976; Turner & Harris, 1974). Recently we have used the technique to locate the afferent connexions of the pars nervosa (PN) and pars intermedia (PI) of the rat pituitary (Fisher & Price, 1975; Fisher & Price, 1976) but were unable to delineate specific connexions of PI and PN because the injection sites were too large, always involving at least two parts of the pituitary. In addition, we began to look at intracortical and transcallosal connexions in the rat and cat cerebral cortex (unpublished observations) but found that the transport of HRP in these situations was very capricious. Therefore, in order to reduce the size of the injection site and to improve the specificity and reproducibility of the technique, we began to study the factors which may affect the retrograde transport process. This report presents our observations of some ultrastructural features of this transport process in the rat magnocellular neurosecretory system, features which are essential to an understanding of the process. MATERIALS AND METHODS
0-3 ,ul of 20 0 HRP (Sigma type VI) in 3 M-KCI was injected via the right ear canal into the pituitary glands of 36 adult male Sprague-Dawley rats (200-250 g) under Diabutal anaesthesia. Injections were made with glass micropipettes (5080,am ID) inserted through a Hoffman-Reiter hypophysectomy instrument, and connected by oil-filled tubing to a 10 ,ul Hamilton syringe driven by a Sage syringe pump at a rate of 1 al/20 min. The animals were allowed to survive for varying intervals (4, 1, 1j, 3, 4, 5, 6, 12, 24, 48, 96 and 192 hours) to allow time for uptake and transport to occur. The rats were killed by intracardiac perfusion, under Diabutal anaesthesia, of 50 ml 0 9 %/O NaCl followed by 100 ml cold (4 °C) 2 5 %0 glutaraldehyde in 0 1 M phosphate buffer at pH 7-3. Brains and pituitaries were immediately removed, trimmed and immersed in fixative for 18 hours at 4 'C. Then 50-75 ,tm sections,
P. PRICE AND A. W. F. FISHER 138 cut on a Smith-Fahrquarson tissue chopper, were washed for 2 hours in cold phosphate buffer and then treated for 30 minutes at 20 °C with fresh 50 mg % 3-3' diaminobenzidine (DAB) and 0-01 % H202 in phosphate buffer. Following transfer to phosphate buffer, areas of interest were dissected from the sections under the dissecting microscope, washed for 2 hours in phosphate buffer, and immersed for 1 hour at 4 °C in 1%Os04 in 0 1 M phosphate buffer at pH 7-3. The tissue blocks were then washed in distilled water, stained en bloc for 1 hour in 0-5 % aqueous uranyl acetate and embedded in Vestopal W. Unstained thin sections were examined in an AEI Corinth 275 electron microscope. Some of the sections, after reaction with DAB and H202, were embedded in glycol methacrylate, sectioned at 1 ,um on a Sorvall JB4 microtome, stained with H & E and examined in a Zeiss Photoscope.
RESULTS
Most injection sites involved parts of both the pars nervosa (PN) and the pars intermedia (PI), and occasionally also involved the pars anterior (PA). In some cases only the PA was involved, and in these animals no transport to hypothalamic neurons occurred. When the PN was part of the injection site HRP was invariably found in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei as well as numerous other hypothalamic areas (Fisher & Price, 1976) after time for transport had elapsed. By both light and electron microscopy most neurons of the SON and the supero-lateral portion of the PVN contained granular HRP reaction product (Fig. 1). HRP uptake in the pars nervosa HRP reaction product was found in the extracellular space, within the cytoplasm of axon swellings and terminals, and within the cytoplasm of pituicytes and endothelial cells. Within 15 minutes of injection HRP had penetrated widely through the extracellular space, producing a dense reaction product associated with basal laminae and collagen fibres (Fig. 2). By this time HRP had also entered into neurosecretory axon swellings and terminals. Clusters of small clear vesicles (40-70 nm) are normally found in these terminals. The HRP reaction product was seen as circular, uniform densities (40-70 nm) located among these clusters of small vesicles. While most of the vesicles were in the 40-50 nm size range, and most of the HRP densities were in the 60-70 nm range, it seems reasonable to conclude that the HRP reaction product was located in the larger of the small clear vesicles (Fig. 5). In spite
Fig. 1. Dark field light micrograph of 1 jum plastic section of the supraoptic nucleus (SON) 24 hours after injection of HRP into the pituitary. Most cells of the SON (arrows) are filled with bright refractile granules of HRP reaction product. OT, optic tract. x 60. Fig. 2. Injected area of the pars nervosa 15 minutes after HRP injection. HRP reaction product (black) is seen throughout the extracellular space, outlining the pituicytes and axon terminals and 'staining' the basal laminae about vessels and tissue lobules. Small amounts of reaction prdduct are also seen already within axon terminals and pituicytes. x 3800. Fig. 3. Neuron in the SON 3 hours after injection. A few black granules of reaction product (arrows) are clustered close to the nucleus and a large Golgi complex. x 5600. Fig. 4. Neuron in the SON 24 hours after injection. There are many granules of reaction product, mainly of large size, distributed widely throughout the cytoplasm. x 6500.
HRP transport in supraoptic neurons
139
P. PRICE AND A. W. F. FISHER 140 of considerable searching, no pinocytotic vesicles associated with plasma membranes were seen. After 45 minutes the density of reaction product in the extracellular space had increased, as had the number of vesicles containing reaction product inside the axon terminals (Fig. 6). In some terminals (Fig. 8) a few larger, irregular profiles filled with HRP were seen, and in some of the axons tubular profiles containing reaction product could be found (Fig. 9). After 1-5 hours the intensity of reaction product within the extracellular space had diminished, and by 3 hours it had virtually disappeared. Reaction product was present in vesicles, granules and tubules of the axon swellings and terminals at 1-5 and 3 hours, but by 4 hours it was becoming scarce in these locations, and it was progressively more difficult to find at later times (Fig. 7). Reaction product was seen at all times up to 4 hours, in pituicyte and endothelial cell cytoplasm, and by 4 hours this was by far the major location.
Transport of HRP through axons of the infundibulum HRP reaction product was found in axons of the infundibulum as early as 1-5 hours, and up to 24 hours, after injection. The amount of reaction product was greatest in the 4-6 hour time period, decreasing to become rare by 24 hours. It was seen in three types of location only: in long irregular tubular profiles (Fig. 10) that resembled agranular reticulum and also resembled the tubular profiles seen in PN axons (Fig. 9); in granular profiles (Fig. 11) that were usually lined up like a string of beads; and in composite arrangements of tubular and granular profiles (Fig. 12) which suggested that both these profiles were local variations in the geometry of the agranular reticulum.
Accumulation of HRP in neurons of the supraoptic nucleus HRP did not appear in the cells of the SON until 3 hours after injection, at which time reaction product was seen in membrane-bound granules of varying size, most of which were small (Fig. 3). These granules tended to be grouped close to the nucleus and Golgi complexes. In some cases tubular structures containing reaction product, resembling the tubules in the infundibular axons, were seen close to these Golgi complexes (Figs. 13, 14). Some of the granules, at 3 and 4 hours, had irregular profiles, many of which could perhaps be those of agranular reticulum tubules (Fig. 15). Fig. 5. Injected area of PN 15 minutes after injection. Reaction product is seen in the extracellular space 'staining' the basal lamina and the surface coat of the axon terminal. It is also in small granules similar in size to the small clear vesicles seen in clusters among the larger neurosecretory granules. x 33 750. Fig. 6. PN 45 minutes after injection. The density of reaction product on the basal lamina and collagen fibres is increased, as is the number of HRP-containing vesicles in the axon terminal. x 33450. Fig. 7. Axon terminal in the PN 3 hours after injection. Reaction product has disappeared from the extracellular space, although there are HRP-containing vesicles in the axon terminal. x 32350. Fig. 8. Axon terminal in the PN 45 minutes after injection. In addition to a few small vesicles containing HRP, there are several larger, irregular granular or tubular structures filled with reaction product (arrows). These structures are distributed among the neurosecretory granules rather than among clusters of the small vesicles. x 33 300.
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HRP transport in supraoptic neurons
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as 15 minutes after injection indicates a very rapid uptake. Theodosis et al. (1976) have demonstrated uptake of intravenous HRP within 1 minute of stimulation of the nerve terminals. LaVail (1975) has suggested that uptake probably begins very early; Nagasawa, Douglas & Schulz (1971) have demonstrated in rat pars nervosa that uptake occurs by pinocytosis within 10 minutes of injection, with reaction product located within the small vesicles. Although no pinocytotic vesicles were seen in the experiments reported here, our observations are generally consistent with those of LaVail (1975) and Nagasawa et al. (1971). Douglas, Nagasawa & Schulz (1971) and Nagasawa et al. (1971) have suggested that this pinocytotic activity, indicated by the clusters of small clear vesicles found in neurosecretory axon terminals of the rat pars nervosa, represents a recycling of membrane subsequent to discharge of neurosecretory material by exocytosis. Although this has not been fully substantiated, the rapidity of the process of HRP uptake observed here is in accord with their concept. Recently Theodosis et al. (1976) have shown that, after stimulating the rat pituitary electrically or by acute haemorrhage, intravenous HRP was taken up by axon terminals of the pars nervosa within 1-5 minutes into large irregular vacuoles. Very little HRP was found in micropinocytotic vesicles. Transport of HRP within axons has been observed in granules of varying size, including multivesicular bodies, and in irregular tubular profiles resembling agranular reticulum (LaVail & LaVail, 1974; Nauta et al. 1975). The observations reported here show that the larger irregular granules appear in axon terminals as early as 30 minutes after injection, and can be found in infundibular axons from 1P5 to 24 hours after injection. In addition, however, these granules were frequently seen to be arranged in linear fashion, resembling a string of beads, or in a linear combination with irregular tubular profiles. These results, while consistent with both previous reports (LaVail & LaVail, 1974; Nauta et al. 1975), strongly suggest that the granules and the tubules seen in these axons are simply different geometrical forms of the same structure, the agranular reticulum. Further, our observations also suggest that the larger irregular profiles seen in axon terminals are the dilated, irregular terminations of the agranular reticulum, which somehow collect HRP in the axon terminal and allow retrograde transport to occur. The process by which the small vesicles transfer the HRP to the agranular reticulum remains an open
question. HRP arrived in the neuron somata of the SON within 3 hours of injection into the pituitary. Initially these granules were mainly small, clustered close together Fig. 13. Detail of the Golgi complex of the SON neuron seen in Fig. 3, 3 hours after injection. A series of four irregular HRP vesicles is seen (arrows) which are similar in size and shape to agranular reticulum tubules and probably represent a curved portion of such a tubule. x 33 600. Fig. 14. Golgi complex of another SON neuron 3 hours after injection. A tubular structure filled with reaction product is seen among the Golgi membranes. At least two portions of these Golgi membranes show increased density which may indicate HRP reaction product (short arrows). In addition, a large lysosome-like granule (long arrow) containing some reaction product has a tubular structure apparently leading off from its surface. NUC, nucleus. x 55 000. Fig. 15. Portion of the cytoplasm of an SON neuron 4 hours after injection showing predominantly large granules densely filled with reaction product. Some of these granules have irregular, tail-like projections (arrows). x 17800. Fig. 16. Cytoplasm of SON neuron 4 days after injection. The density of reaction product within the lysosomes is diminished, and more irregular. x 23400. 10
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P. PRICE AND A. W. F. FISHER and close to Golgi complexes, and often had tubular structures containing HRP nearby. At later times the granules tended to be larger, were more widely distributed in the cytoplasm, and in some instances had irregular tail-like profiles. These results suggest, as did those of Nauta et al. (1975), that the HRP arrives at the perikaryon in agranular reticulum tubules, and is transferred to larger, lysosome-like granules. The nature of the transfer process remains unclear, although two possibilities emerge from the present experiments: either the HRP is transferred by the Golgi complex directly for packaging into the lysosome granules (suggested by the 3 and 4 hour appearance; Figs. 13, 14), or the lysosomes are produced in the Golgi complex and subsequently acquire HRP directly from the agranular reticulum (suggested by the irregular appendages of some granules; Fig. 15). Both of these possibilities are consistent with the fate of transported HRP in other systems (LaVail & LaVail, 1974; Nauta et al. 1975) and with studies of HRP taken up into cortical neurons in the injection site (Turner & Harris, 1974). Extensive observations of many sections of tissue taken at shorter time intervals about the time of arrival of HRP in the cell somata should help to clarify the mode of transfer. The fate of HRP in neuron perikarya appears certain: degradation within lysosomes. In the rat magnocellular neurosecretory cells, this begins to occur by 4 days and is complete by 8 days. In the chick visual system, it is complete by 3-4 days (LaVail & LaVail, 1974) and in cerebral cortex neurons of rabbit, cat and monkey, it is complete by 4-5 days (Turner & Harris, 1974). It thus appears from this study, and from the ultrastructural studies conducted by others, that the processes of HRP uptake, retrograde axonal transport, accumulation in cell somata and ultimate destruction involve, in mildly stimulated neurons, a sequence of cell organelles common to all the cell types studied. Differences between cell types are confined to differences in the rates at which these processes take place. SUMMARY
The uptake of exogenous horseradish peroxidase (HRP) into axon terminals of rat pars nervosa, and its retrograde axonal transport via infundibular axons to the neurons of the supraoptic nucleus (SON), were examined in the electron microscope. HRP was present in the extracellular space of the pars nervosa for 3 hours after injection. Reaction product was found in small vesicles (40-50 nm) within axon terminals from 15 minutes to 3 hours. Infundibular axons showed reaction product from 1*5 hours to 24 hours in tubules of the agranular reticulum, which often appeared as granular or beaded profiles. Reaction product first appeared in cells of the SON at 3 hours, indicating a transport rate of 2-3 mm/hour, or 50-70 mm/day. Initially HRP was mainly in small lysosome-like granules clustered close to Golgi complexes. Later, larger granules predominated and were more widely distributed throughout the cytoplasm. Some tubular forms containing HRP, and resembling agranular reticulum, were seen close to Golgi complexes. Some granules had irregular tails that could represent direct connexions with agranular reticulum. By 4 days the reaction product was less dense in large granules, and by 8 days had disappeared. Thus, injected HRP is rapidly picked up by axon terminals in the PN by pinocytosis into small vesicles, and is subsequently transferred to agranular reticulum in which it is transported within axons to neuron perikarya of the supraoptic
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nuclei where, in association with Golgi complexes, it is transferred to lysosomes for degradation of its enzymic activity. REFERENCES
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