LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM ERIC HOLTZMAN, ALEX B. NOVIKOFF, and HUMBERTO VILLAVERDE From the Department of Pathology, Albert Einstein College of Medicine of Yeshiva University, D o New York wn lo a d e d fro m ABSTRACT h ttp The rat ganglion nodosum was used to study chromatolysis following axon section. After ://ru p fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as re markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL ss.o rg (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to /jcb develop) (31-33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; /a thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance rticle -p (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the df/3 mitochondrial enzyme were studied by electron microscopy as well as light microscopy. 3/2 In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in /41 9 the center of the cell and segregation of the remainder to the cell periphery. Golgi ap- /13 8 paratus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. 37 1 6 GERL is prominent in both normal and operated perikarya. Electron microscopic images /4 1 9 suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several .p d ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from f b focal areas dilated with granular or membranous material; )c( "multivesicular bodies" y gu e in which vesicles and other material are sequestered; (d) autophagic vacuoles containing st o n endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and 0 7 mitochondria. The number of autophagic vacuoles increases following operation. Fe b ru a INTRODUCTION ry 20 2 3 Chromatolysis, the process that ensues in the In the present study, marker enzyme activities perikaryon following interruption of its axon, long and electron microscopy have been used to study has been of interest to cytologists and cell patholo- lysosomes and other cytoplasmic organelles in gists (see reviews in references 1-6). Although the normal and chromatolytic neurons. The results process has been analyzed by many techniques draw attention to the variety of lysosomes in including electron microscopy (7-12), it seemed perikarya and emphasize the likely role of Golgi- likely that a study of the intracellular digestive associated smooth endoplasmic reticulum (GERL) system, lysosomes and associated structures, (14- in the formation of lysosomes. They suggest further 15) would lead, in time, to a fuller understanding that changes in chromatolytic neurons shed light of cytological processes within neurons. on ribonucleoprotein turnover in normal neurons. 419 MATERIALS AND METHODS After 20-30 min the right nodosal ganglion was re- moved and fixation was continued by immersion in The right vagus nerves of ether-anesthetized female glutaraldehyde at 5°C. All fixation was carried out Sprague-Dawlcy rats (180-280 g) were sectioned in a 5°C coldroom. about 0.5-1.0 cm below the nodosal ganglion. No Unincubated tissues for fine structure studies attempt was made to approximate or suture the generally were fixed by immersion for 11/~-3 fir, but severed ends. This operative procedure leaves intact occasionally longer periods up to about 20 hr were a small laryngeal branch oft he vagus nerve. Probably used. They then were rinsed for a few minutes in the as a result of this, some 10-20v/v of neurons in the appropriate buffer without glutaraldehyde but with ganglion of the operated animal appear normal. added 0.2 ~ sucrose, and postfixed for 2-3 hr in 1% These cells serve as "internal controls" in addition to osmium tetroxide buffered either with 0.I M phos- the controls mentioned below with which changes in phate containing glucose (19), for material fixed with chromatolytic neurons can be compared. phosphate-buffered glutaraldehyde, or with Veronal- The animals were sacrificed from 6 hr 46 days acetate-sucrose (20, 21), for tissue fixed with cacodyl- after vagotomy. They were anesthetized with Nem- ate-buffered glutaraldehyde. After post-fixation the butal and perfused through the ascending aorta, material was dehydrated in ethanols and embedded in first with warm, then with cold (5°C) 3% glutaralde- Araldite 506 (Ciba) by the method of Luft (13). hyde (see reference 66) buffered at pH 7.4 with either Cacodylate-buffered glutaraldehyde fixation was D 0.1 M cacodylic acid with 0.25% calcium chloride used for enzyme cytochemistry. The time of im- ow n (16, 17) or 0.1 M phosphate (18). The descending mersion fixation was 30-45 rain. The tissues then loa d aorta was clamped shortly after perfusion was started. were rinsed for 45 rain or longer in cacodylate-buffer ed fro m h ttp ://ru p re ss.o rg /jcb /a rticle -p d f/3 3 /2 /4 1 9 /1 3 8 3 7 1 6 /4 1 9 .p d f b Unless otherwise noted, all tissues were fixed in cacodylate-buffered glutaraldehyde. Electron micro- y g graphs are from sections stained with both uranyl and lead. Figs. 1-12 are light micrographs from frozen ue sections. For acid phosphatase preparations, CMP was the substrate for all preparations but that in st o n Fig. 45, for which ~-glycerophosphate was used. The incubation times are indicated in parentheses. 07 F e b FIGURE 1 Neuron from an unoperated ganglion, stained with methyl green-pyronin. The pyronin ru stains the Nissl material (arrows) and the nucleoli (NU). Methyl green staining of the chromatin is ary 2 too light to show in the photograph. X 1,000. 02 3 FIouR~. 2 Neuron from a ganglion 7 days after operation. In the cytoplasm most of the pyronin-stained material is seen as a peripheral band (arrow). The nucleus is eccentric and shows a prominent nucleolus (NU). X 1,100. FmtmE 3 Neuron from an unoperated ganglion, incubated for NADH-TNBT reductase activity (10 min). The numerous mitochondria, one of which is indicated by the arrow, are colored by the formazan. The nucleus is seen at N. X 1,200. FmuaE 4 Neuron from a ganglion 7 days after operation, incubated for NADH-TNBT reductase activity (10 min). The mitochondria (M) are segregated to the center of the cell. A few individual mito- chondria may be seen (arrow) in the thin layer of cytoplasm over the eccentric nucleus (N). X 1,200. 420 THE JOURNAL FO CELL YGOLOIB • VOLUME 33, 1967 D o w n lo a d e d fro m h ttp ://ru FmuaE 5 Neuron from an unoperated ganglion, incubated for TPPase activity (60 rain). Much re- p re action product is seen in the Golgi apparatus (G) and moderate amounts in the nucleoli (NU). Along- ss.o side the perikarya reaction product is seen in an mlmyelinated nerve fiber (arrow) (see reference 39). rg X 1,100. /jcb /a ERUGIF 6 Neuron from a ganglion 7 days after operation, incubated for TPPase activity (60 rain). rticle -p The Golgi apparatus (G) is segregated to the center of the cell. The nucleolus (NU) is lightly stained. df/3 X 1,000. 3/2 /4 1 ERUGIF 7 Neuron from an unoperated ganglion, incubated for acid phosphatase activity (45 mill). 9/1 3 Reaction product is present in numerous granules, the lysosomes, and in GERL (arrows). The upper 8 3 7 cell shows a characteristic tendency of the GERL of the smallest neurons to be concentrated in a narrow 1 6 zone around the nucleus. Larger cells, like the lower one in this figure, often show a somewhat more /41 9 dispersed distribution. The nucleus is seen at N. X 1,100. .pd f b FlalmE 8 Neuron from a ganglion 4 days after operation, incubated for acid phosphatase activity (45 y g u e rain). The lysosomes and GERL have become segregated to the center of the cell (arrow) while the st o nucleus (h0 is seen at the periphery. X 1,100. n 0 7 F e b with 0.2 M sucrose. Frozen sections were cut at 10/~ copy, 5% sucrose was added to the media): acid rua for light microscopy and 40/z for electron microscopy. phosphatase, with the Gomori medium with /3- ry 2 0 Some material also was fixed with cold formalde- glycerophosphate (23) or CMP (24) as substrate; 2 3 hyde-calcium for enzyme cytochemistry (22). The inosine diphosphatase and thiamine pyrophosphatase, initial perfusion lasted 30 rain. It was followed by with the Novikoff-Goldfischer medium (25); acetyl- overnight immersion at 5°C. cholinesterase, using Karnovsky's medium (26); The frozen sections were incubated for demonstra- NADH-TNBT reductase, substituting tetraNitro for tion of the following enzymes 1 (for electron micros- Nitro BT in the medium of Novikoff (27). The reductase activity was used only in frozen 1 The following abbreviations are used: CMP, 5'- sections for light microscopy, while the other incuba- cytidylic acid; IDPase, inosine diphosphatase; tions were done for both light microscopy and electron TPPase, thiamine pyrophosphatase; NADH-TNBT microscopy. reductase, reduced nicotinamide adenine dinucleo- Methyl green-pyronin staining was done according tide-tetranitro blue tetrazolium reductase. to the procedure of Taft (28). :E. ,~IAMZTLOH A. B. ,PFOKlVON DNA H. EDREVALLIV Neuronal semososyL dna GERL 421 D o w n lo a d e d fro m ERUGIF 9 Neurons from an unoperated ganglion, fixed in formaldehyde-calcium and incubated for http IDPase activity (40 rain). Reaction product in the perikarya is present in the Golgi apparatus (G) and ://ru in the Nissl substance (arrows). N, nucleus. )< 1,100. pre ss.o FIGURE 10 Neurons from a ganglion 4 days after operation, fixed in formaldehyde-calcium and in- rg cubated for IDPase activity (60 min). The Golgi apparatus has become segregated to the center of the /jcb /a cell( see (G) Fig. and 9) thise present Nissl substance at the sheath to the eell-pepriekrairpyhaelr y (arrows). interface Reaction but the product possibility at the of border diffusion of the from ad- neuron rticle-p jacent unmyelinated fibers and blood vessels has not been eliminated (see reference 39). The eccentric df/3 nucleus is seen at N. )< 1,000. 3/2 /4 1 9 FIGURE 11 Neurons from an unoperated ganglion, incubated for aeetylcholinesterase activity (1~0 /1 3 min). Reaction product is present in the Nissl substance (arrows) and in the nuclear envelope (N). Re- 83 7 action product, due to unspecific cholinesterase (see text), is present at the sheath eell-perikaryal in- 16 /4 terface (S). X 1,100. 19 .p d FIGURE 1~ Neuron from a ganglion 7 days after operation, incubated for aeetyleholinesterase activity f b (1~0 min). No reaction product occurs in the perikarya. However, the sheath cell-perikaryal interface y gu e (S) and unmyelinated fibers (arrow) (see references 39, 8~) continue to show activity of the unspecific st o cholinesterase. The eccentric nucleus is seen at N. <) 1,000. n 0 7 F All enzyme reactions were carried out at 37°C, days; 13-26 days following surgery. Ganglia from un- eb with the exception of the acetylcholinesterase which operated animals and the contralateral ganglia from rua was done at 0°C. operated rats served as controls for both electron ry 2 0 The 10-/z sections and sample 40-/x sections were microscopy and light microscopy. It should be noted 23 mounted in glyeerogel for examination in the light that the cells from both operated and unoperated microscope, after brief treatment with ammonium ganglia are from animals anaesthetized with Nembu- sulfide, for the phosphatases. The remaining 40-/~ tal and that 20-60 seconds elapsed between opening sections were postfixed for 30 min in Veronal-ace- of the chest cavity and initiation of the perfusion of tate--sucrose-buffered 1~ osmium tetroxide, then fixative. embedded in Araldite as above. As enzyme controls, the frozen sections that were Material from animals 6 and 12 hr, ,1 2, and 4 days, examined in the light microscope and electron micro- and ,1 2, 3, 4 and 6 wks after operation was studied scope were incubated as follows. For the phosphatases, by light microscopy. Five or more ganglia were the media used were complete except for the sub- studied in the electron microscope for each of the strates (23). For acid phosphatase, incubation also following four groups: 12 hr-1 day; 2-4 days; 5-7 was performed in the full medium to which 0.01 M 422 THE LANRUOJ FO CELL YGOLOIB • EMULOV 33, 1967 NaF was added (23). 01 -5 M BW 62C47 (Burroughs- been sectioned, the distribution of organelles is Wellcome & Co., Inc., Tuckahoe, N.Y.) was added changed drastically. Initial changes are visible to the medium to inhibit acetylcholinesterase without within 24 hr, and by 4-7 days the alterations inhibiting unspecific cholinesterase. To inhibit un- have reached a peak. The altered distribution of specific cholinesterase, RO 20683 (Hoffman- organelles persists, at least in part, for the longest LaRoche, Inc., Nutley, N.J.) was used. intervals we have studied, 4-6 wk. Thin sections were cut with glass knives on a In chromatolytic neurons, the cell nuclei Porter-Blum MT-1 microtome and were mounted on (Figs. 2, 4, 6, 8, 10, and 12) and most of the naked grids. They were stained either with a satu- rated solution of uranyl acetate in 50% ethanol (29), pyronin-stained material (Fig. 2) are seen at the lead citrate (30) or, most often, with uranyl followed periphery of the cells. In contrast, mitochondria by lead. Micrographs were taken with an RCA (see references 7, 43) (Fig. 4), lysosomes (see EMU B3 or 3H microscope at 100 kv and initial references 6, 44, 45), and GERL (Fig. 8) are magnifications of 2000-19,000. located toward the center. The Golgi apparatus also appears at the center of the cell (Fig. 6) RESULTS rather than segregating to the periphery as has been reported for some other neurons (46-48). Light Microscopy IDPase reaction product is seen (Fig. 10) in D o UNOPERATED GANGLIA: Light microscopy the Golgi apparatus in the center of the cell and wnlo of unoperated ganglia shows a more or less sym- in the Nissl substance at the periphery. Although ad e metrical distribution around the nucleus of the traces of activity remain in some neurons, in d fro major organelles of the neurons although some most perikarya little or no acetylcholinesterase m h variation from cell to cell does occur (Figs. 5, 7). activity is demonstrable (Fig. 12) (see references ttp The symmetrical distribution is shown for the 49, 50). ://rup NFiisgs.l 3, substance and for in the Fig. Golgi ,1 for apparatutshe mitochondria in Fig. i5n, deposition In both of normal precipitate and chraotm atolytic all sites within neurons, the ress.org as seen in methyl green-pyronin, NADH- perikarya is abolished by omission of substrates /jcb /a TspeNctiBveTly. reductase Bodies andc ontaining TPPase demonstrablper eparations, acid re- fhirboimt ors. the Howeivenrc, ubation as media expected, or by the inclusion acetylcholin- of in- rticle-p d phosphatase are distributed similarly, as may be esterase inhibitor (BW 62C47) does not prevent f/3 3 seen in Fig. .7 These include lysosomes and larger staining of the sheath cell-perikaryal interface /2/4 1 bodies similar to the GERL described previously (Figs. I ,1 12); this indicates that the enzyme here 9/1 in dorsal root ganglia (31-33) as a special region is an unspecific cholinesterase (see reference 39). 38 3 7 of smooth endoplasmic reticulum. As is true in the As in dorsal root ganglia (39), the sheath cell- 16 /4 dorsal root perikarya, the areas occupied by perikaryal interface but not the perikaryon also 19 GERL in the nodosal ganglia neurons are ellip- has demonstrable activity (abolished by RO .pd f b soidal or roughly spherical and appear to fit within 20683) when butyrylthiocholine is used as sub- y g u Fitgh.e mes5 hwork and Fig. of 7). the Electron Golgi microscopy, apparatus d(esccormipbaerde strate in place of acetylthiocholine. est on Electron Microscopy 0 below, confirms this impression. 7 F IDPase activity is demonstrated in the Golgi Electron microscopy confirms the light micro- ebru astpupdairead tus (34, and, 35), unlikien the otNhiseslr substnanecuer ons we (Fig. have 9). socvoeper -all observations changes in as organelle to enzyme distribution. localizations Other and ary 202 3 Activity in the Nissl substance is demonstrated authors have described the changed distribution best in formaldehyde-calcium-fixed tissues, but of mitochondria and the disruption of the rough some activity also is present after glutaraldehyde endoplasmic reticulum of the Nissl substance in fixation. Acetylcholinesterase reaction product is the center of the cell (7-I I). Most of our attention seen in the Nissl substance, the nuclear envelope, will be devoted to the lysosomes, GERL, and and the sheath cell-perlkaryal interface (Fig. 11) Golgi apparatus. (see references 36-40). UNOPERATED GANGLIA: In unoperated OPERATED GANGLIA: After the axons ~ have neurons, GERL is seen as a region of agranular 2 See Bodian (41) for a discussion of terminology re- membranes associated closely with the Golgi ap- lating to axons and dendrites. paratus and showing reaction product in acid E. ,N~MZTLOH A. B. NOVIKOFF, AND H. EDREVALLIV Neuronal searososyL dna GERL 423 phosphatase-incubated preparations (Fig. 13). stance (Fig. 17). None is seen in the nuclear While we refer to it as smooth endoplasmic reticu- envelope. In acetylcholinesterase preparations lure, it may be continuous with the Golgi appara- reaction product is observed in the endoplasmic tus. Numerous vesicles of the type called "coated" reticulum of both nuclear envelope and Nissl sub- or "alveolate" (52) are attached to the GERL (Fig. stance (Fig. 18). 19) or lie nearby, apparently unattached. The OPERATED GANGLIA: Chromatolytic neu- coat of the delimiting membrane shows radial rons were identified in the electron microscope projections. Some of the coated vesicles show by the peripheral position of the nucleus and the reaction product in ganglia incubated for acid altered distribution of the other organelles. Only phosphatase activity (Fig. 20). The coated vesicles those cells were studied in which sufficient por- fulfill the cytochemical criteria for lysosomes: tions of nucleus and cytoplasm had been included membrane-delimited cytoplasmic bodies with in the plane of section to indicate that the nuclei acid hydrolase activity (see reference 53). were peripheral. Many other lysosomes are present in tmoper- GERL continues to be prominent for the first ated neurons, often in association with GERL. few days after operation (Figs. 16, 27). As in These include large numbers of dense bodies con- unoperated preparations (see reference 31), con- taining grains (Fig. 43) and "crystalline" or nections between rough and smooth endoplasmic Do w membranous material. "Multivesicular bodies" reticulum are seen often in the GERL area (Figs. nlo a also are observed (Figs. 23, 40); and, on occasion, 15, 27), and clusters of ribosomes occasionally are de d autophagic vacuoles (14, 24, 55-57) are encoun- found within the meshwork of smooth endoplas- fro m tered. The latter are identifiable by the organelles mic reticulum (Fig. 16). Lysosomes of the types h tbhuety more contain: commonly sometimes ribosomes mitochondria and fragmenting (Fig. 21) daesssoccriiabteedd for with unoperated GERL in gcahnrgolmiaat olytic also are neurons. found ttp://rup re endoplasmic reticulum (Fig. 23). Their delimiting Of special interest are the autophagic vacuoles ss.o membrane appears thickened and irregular. Fur- which are seen relatively infrequently in neurons rg ther discussion of these various lysosomes will be from unoperated preparations. Their number /jcb/a deferred until consideration of the operated gan- increases considerably, especially during the first rticle glia. day or two after operation (Figs. ,22 24-26). -pd Electron microscopy shows reaction product for Mitochondria within autophagic vacuoles are f/33 /2 IDPase activity in the Golgi apparatus and in the much more common in chromatolytic than in /4 1 9 endoplasmic reticulum of GERL and Nissl sub- normal neurons. As in neurons from unoperated /1 3 8 3 7 1 6 /4 1 9 .p d FIGUR~ 31 Portion of a neuron from an unoperated ganglion, incubated for acid phos- f by g phatase (30 rain). Reaction product is present in the dense bodies (D) and in GERL u e (arrows; see Figs. ,61 ,91 27). None is seen in the Golgi saccules (G). X 33,500. st o n 0 ERUGIF 41 Portion of the Golgi area from a neuron of an unoperated ganglion, incu- 7 F e bated for IDPase activity 55( min). Reaction product is seen in two saccules of the Golgi bru apparatus )G( and in GERL (arrow). None is seen in the dense body (D).)K 38,000. ary 2 0 2 ERUGIF 51 Portion of a neuron from the same ganglion shown in Fig. .61 Connections 3 between rough endoplasmic reticulum and smooth endoplasmic reticulum (GE) are in- dicated by the arrows. X 51,000. ERUGIF 61 Portion of a neuron from a ganglion 1 day after operation. The Golgi ap- paratus )G( borders the fenestrated smooth endoplasmic reticulum (arrow) of GERL. Coated vesicles (C) and dense bodies (D) are seen in this area, as is frequently the case. Note the clusters of ribosomes (RI) within the GERL meshwork. A mitochondrion is seen at M, and rough endoplasmic reticulum and free ribosomes of the Nissl substance at RE. At V, Golgi vesicles are seen next to Golgi saccules (G) possibly dilated as a prepara- tive artifact. The relations between GERL and Golgi saccules, with which it may prove to be continuous, are discussed in reference 15. X 38,000. 424 THE JOURNAL FO CELL BIOLOGY • VOLUME 33, 1967 D o w n lo a d e d fro m h ttp ://ru p re ss.o rg /jcb /a rticle -p d f/3 3 /2 /4 1 9 /1 3 8 3 7 1 6 /4 1 9 .p d f b y g u e st o n 0 7 F e b ru a ry 2 0 2 3 524 D o w n lo a d e d fro m h ttp ://ru p re ss.o rg /jcb /a rticle -p d f/3 3 /2 FIOURE 17 Portion of a neuron from an unoperated ganglion, fixed in formaldehyde-calcium and in- /41 9 cubated for IDPase activity (60 rain). Reaction product is present in the rough endoplasmic reticulum /1 3 8 of the Nissl substance (arrow). None is seen in the nuclear envelope (N). × 14,500. 3 7 1 6 /4 FIOUaE 18 Portion of a neuron from an unoperated ganglion, incubated for aeetylcholinesterase ac- 1 9 tivity (105 min). Reaction product is present in the rough endoplasmic reticulum (arrows) and the nu- .pd clear envelope (N). The thin section was stained only with uranium. X 14,500. f by g u e FIGURE 19 Part of the GERL of a neuron from an unoperated ganglion. A coated vesicle (arrow) st o with a number of projections radiating from its surface is continuous with smooth endoplasmic reticu- n 0 lure. The projections are separated, at their tips, by ~0~0 m#. Another coated vesicle is seen at ,7( and 7 F e portions of Golgi saeeules at G. (See also Fig. 16.) )< 45,000. bru a FIGURE 20 Three coated vesicles in the GERL region of a neuron from an unoperated ganglion, incu- ry 20 2 bated for acid phosphatase activity (30 min). Reaction product is present within the vesicles. Note 3 the projections of the coats of the vesicles indicated by the arrows (cf. Fig. 19). X 69,000. See also Fig. 58 in reference 15. FIGva~. ~1 Portion of a neuron from an unoperated ganglion. In the GERL region an autophagie vacuole contains a mitochondrion (M), endoplasmic reticulum (E), and ribosomes (arrow). A dense body is seen at D, and portions of Golgi saccules at G. <) 60,000. FIGURE ~ Portion of the GERL region of a neuron from a ganglion two days after operation. Fixed in phosphate-buffered glutaraldehyde. An autophagie vacuole containing ribosomes is seen at AV. Part of a mitoehondrion is seen at M. (> 55,500. 426 THE LANRUOJ FO CELL YGOLOIB " EMULOV 33, 1967 D o w n lo a d e d fro m ~3 FIGURE Portion of a neuron from an unoperated ganglion. Fixed in phosphate-buffered glutaralde- http hyde. Part of the Golgi apparatus is seen at ;G the rest of the field is occupied by GERL. The arrow ://ru indicates an autophagic vacuole containing ribosomes present in the GERL region. Its delimiting mem- pre brane in some places is double (arrow), suggesting that it arises by the close apposition or "compac- ss.o tion" of two or more membranes (see Fig. ~o, 4~ and reference 58). A multivesicular body is seen at rg MV, and two dense bodies at D. X .005,54 /jcb /a FIGURE 4~ Portion of a neuron from a ganglion 7 days after operation, A mitochondrion (M) is present rticle -p within an autophagic vacuole. Endoplasmic reticulum (E) is seen close to the surface of this vacuole. df/3 X 87,500. 3/2 /4 1 ERVGIF .5~ Portion of a neuron from a ganglion 5 days after operation. Fixed in phosphate-buffered 9/1 3 glutaraldehyde. This may be an autophagic vacuole in the process of formation. Thickened membranes 83 7 (see Fig. g8 and reference 58) continuous with smooth endoplasmic retlculum (arrow) appear to be sur- 16 rounding a cisterna of rough endoplasmic reticulum (E) and a mitochondrion (M). X 88,000. /41 9 .p d g ERUGIFlutaraldehyde. ~6 Portion This may of a be neuron from an autophagic a ganglion vacuole ~ in days the process after operation. of formation. Fixed A mienm brane phosphate-buffered continuous f by gu e with smooth endoplasmic reticulum (arrow) appears to be sequestering a mitochondrion (M). X 47,000. st o n 0 7 F e ganglia, autophagic vacuoles also contain ribo- micrographs because of the large numbers present bru a somes and endoplasmic reticulum. Their de- initially. However, information was obtained ry 2 limiting membrane is thickened and often is com- relating to the modes of origin of these bodies. 02 3 posed of two or more distinct membranes (Fig. The numerous multivesicular bodies present in 23). Connections between these membranes and the neurons before and after operation contain smooth endoplasmic reticulum sometimes are small vesicular and tubular elements of varying observed (Figs. 25, 26; see reference 58). morphology (Figs. 27, 28, 32, 34, 36). The de- While they cannot be related specifically to limiting membrane often is seen attached to chromatolysis, a number of other observations smooth surfaced tubular elements of the endo- made on chromatolytic neurons should be noted. plasmic reticulum of GERL (Figs. 27, 34, 37) Increase or decrease in the size, number, or dis- (see reference 34). As reported by others (59-62), tribution of dense bodies, multivesicular bodies, portions of the surface of the multivesicular bodies and coated vesicles is difficult to assess in electron frequently show a coating (Figs. ~82 38). In our .~] ~NAMZTLOH A. B. ,FFOKIVON DNA H. EDREVALLIV Neuronal Lysosomes dna GERL 427 D o w n lo a d e d fro m h ttp ://ru p re ss.o rg /jcb /a rticle -p d f/3 3 /2 /4 1 9 /1 3 8 3 7 1 6 /4 1 9 .p d f b y g u e st o n 0 7 F FIGURE 27 Portion of a neuron from a ganglion 1 day after operation. Most of the field is occupied eb by GERL. Note the fenestrations of the endoplasmic reticulum (GE). The arrow indicates a region of rua this endoplasmic reticulum that shows a surface coating with short projections. At MV a multivesicular ry 2 0 body is seen attached to smooth endoplasmic reticulum (E). A coated vesicle is present at C, dense 23 bodies at D, continuity of rough and smooth endoplasmic reticulum at R, and a microtubule at T. G, Golgi saccules. )< 59,500. FIGURE ~8 Portion of a neuron from a ganglion one day after operation. A multivesicular body is seen in the center of the field. A portion of its surface (arrow) shows a coating with short projections spaced ~0-30 mlx apart. This coating is similar to that seen on the endoplasmic reticulum (see Figs. 27, 29-31, 35). As is often the case, the delimiting membrane of the coated region (arrow) consists of two or more distinct layers. × 44,000. FIGURE 29 Portion of a neuron from a ganglion 2 days after operation. Fixed in phosphate-buffered glutaraldehydc. Coated agranular membranes appear (arrow) as if surrounding a group of vesicles or tubules (V) probably derived from endoplasmic reticulum. Golgi saccules are indicated by G. )< 62,000. FIGURE 30 Portion of a neuron from a ganglion 5 days after operation. Fixed in phosphate-buffered glutaraldehyde. Coated endoplasmic reticulum (arrow) appears as if surrounding small vesicles and tubules (see Fig. 29). )< 51,000. 428