Chromatolysis in a pair of identifiable metathoracic neurons in the cockroach, Diploptera punctata

TISSUE 8 CELL 1970 2 (2) 255-279 Published by Oliver & Boyd, Edinburgh. Printed in Great Britain

MARGARET READ BYERS*

C H R O M A T O L Y S I S IN A PAIR OF IDENTIFIABLE M E T A T H O R A C I C N E U R O N S IN THE C O C K R O A C H , DIPL OP TERA P U N C TA TA ABSTRACT. The effect of axonal transection upon a specific pair of identifiable nerve cell bodies in the metathoracic ganglion of the cockroach, Diploptera punctota, was studied by electron microscopy. The response of these cells resembles vertebrate chromatolysis in most respects and occurs in two phases : initially, the Golgi bodies decrease in size and the Nissl bodies become more concentrated next to the nucleus and less concentrated in the periphery of the perikaryon; the morphology then changes to one that is more directly concerned with active regeneration of the axon. Autoradiographic experiments showed that the initial changes in basophilia depend primarily upon movement of the Nissl bodies toward the nucleus rather than upon synthesis of nev~ RNA.

Introduction

Much has been learned about the response of the nerve cell body to axonal injury since the first studies by Franz Nissl (1892). The early investigations by light microscopy revealed that the alterations in cytoplasmic basophilia varied in intensity and pattern depending upon the type of neuron, the location and severity of injury, and the age and species of the animal (Nissl, 1894a; Lugaro, 1896; Marinesco, 1909; Ramon y Cajal, 1909, 1928; Young, 1932). Such variability was also encountered in other morphological changes including displacement of the nucleus to the edge of the cell body, enlargement of the nucleus and nucleolus, disruption of the neurofibrillar network, and alterations in size and location of the Golgi apparatus. In neurons that survive the initial effects of injury, there is a long period of recovery which in many cases leads to restora-

* Department of Anatomy, Harvard Medical School, Boston, Mass., U.S.Ao Received 6 November1969.

tion of the normal perikaryal morphology. This entire morphological response to axonal injury has come to be known as 'chromatolysis' or the 'chromatolytic cycle' even though the term originally was used merely to indicate the initial breakdown of the Nissl bodies (Marinesco, 1897). Recent studies of chromatolytic neurons have shown that the loss of basophilia in certain regions of the perikaryon depends upon dilution of the cytoplasmic RNA, ribosomes, or rough endoplasmic reticulum as a result of cellular swelling, displacement of ribosomes, or destruction of ribosomes (Hyddn, 1943; Brattgard et al., 1957; Andres, 1961; Evans and Gray, 1961; Smith, 1961; Pannese, 1963; Porter and Bowers, 1963; Bodian, 1964; Holtzman et al., 1967). In addition, the rates of protein and RNA synthesis increase during chromatolysis (Fischer et al., 1958; Brattgard et al., 1958; Porter and Bowers, 1963; Watson, 1965; Gutmann, 1968) as does the rate of phospholipid synthesis (Miani, 1964). Some enzymatic activities are also altered during chromatolysis (Bodian and Mellors, 1945; Friede, 1959);

255

256 for example, acetylcholinesterase activity disappears from the cell body soon after axonal transection and only reappears when reinnervation has been achieved (Schwarzacher, 1958). Furthermore, some of the electrical properties of the neuron change following axonal injury. At the height of chromatolysis in motor neurons there is a decreased effect of excitatory synapses upon generation of the action potential even though patches of the soma-dendritic membrane have become more excitable (Eccles et al., 1958); in chromatolytic sympathetic neurons there is a reduced sensitivity to acetylcholine and the ceils are less excitable (Brown and Pascoe, 1954). ]n spite of this knowledge, much is still unclear concerning the effects of axonal injury upon the nerve cell body. One of the difficulties in studying this problem is that the normal interactions between the nerve cell body and its axon and synapses are only partially understood. Another difficulty is that previous studies have examined large groups of neurons in which it is impossible to identify any particular cell. Since there is wide variation in the response of different neurons to axonal injury, it has been difficult to establish the exact sequence of events or to have adequate control cells for experimentation. Even when one limits the investigation to a relatively uniform group of neurons--such as those in the facial nucleus studied by Nissl (1892)--there is wide variation in the rate and intensity of the response. Recently, Cohen and Jacklet (1965) observed chromatolytic changes in injured cock~ roach nerve cells. These neurons regenerate especiallywell (Bodenstein, 1957; Jacklet and Cohen, 1967ab), and many of them can be specifically identified (Cohen, 1967; Cohen and Jacklet, 1967)./t is therefore possible to study chromatolysis in a specific neuron that is capable of complete regeneration. F o r this reason, an investigation was undertaken on the effect of axonal transection upon the fine structure of a bilateral pair of identical nerve ceils in the metathoracic ganglion of the cockroach, Diploptera punctata Eschscholtz; in all experiments, one member of the pair was i~jured while the other served as a control.

BY ERS Autoradiographic experiments performed on these cells showed that the Nissl bodies move toward the nucleus soon after injury, thereby causing some of the observed changes in perikaryal basophilia. The response of these two cockroach neurons to axonal injury is compared with vertebrate chromatolysis, and the significance of the Nissl body movement is discussed. Materials and Methods

A breeding colony of the cockroach, Diploptera punctata Eschscholtz, was obtained from Dr L. M. Roth, U.S. Army Quartermaster Research and Engineering Center, Natick, Mass. The roaches were maintained on Purina Lab Chow Pellets and water, and their development under these conditions was similar to that described by Willis et aL (1958). Adults and last-instar larvae were collected soon after molting, and transection of the right or left metathoracic nerve 3B was performed 1-5 days later as follows: the roach was lightly anesthetized with ether, and the nerve was cut at the point where it passes along the inner surface of the soft, transparent cuticle located between the sternum and coxa on the ventral surface of either metathoracic leg (Pipa and Cook, 1959). When the incision was made carefully, the roach did not lose any hemolymph, and the wound healed rapidly. The metathoracic ganglion was fixed for 89 hour in a solution (modified from Karnovsky, 1965) of t% formaldehyde and 4% glutaraldehyde in 0-07M cacodylate buffer. After being washed in buffer and postosmicated for 1-2 hours, the tissue was embedded in Epon (Luft, 1961). Because the ganglion is very sensitive to mechanical damage, part of the abdominal cord was removed along with the ganglion for use as a handle in all manipulations. Fixation was also improved by immersing the entire roach in fixative during the dissection and by removing the ganglion from the animal as rapidly as possible. For light microscopy, thick sections (89 TISSUE 8 CELL 1970 2 (2)

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C H R O M A T O L Y S I S IN N E U R O N S were stained on an 80~ hot plate for 10M5 seconds with 0-1% toluidine blue dissolved in 1"0% sodium borate. For electron microscopy, silver sections were placed on grids coated with Formvar and carbon, and they were stained either with 3% aqueous uranyl acetate (Watson, 1958) followed by 0.4% lead citrate (Reynolds, 1963; Yenable and CoggeshM1, 11965) or with 4% uranyl acetate in 50% methanol followed by 04% aqueous lead citrate. The sections were examined in an RCA-3F or RCA-3G electron microscope at 50 kV. I n the autoradiographic experiments, either uridine-aH (generally labelled, l#C//xl, 11 9 C/raM) or uridine-5-aH (I/xC//zl, 26-9C/mM) (obtained from New England Nuclear Corp.) was injected into the base of either the right or left mesothoracic leg in dosages of 20-80#C./gm. This injection route not only eliminated the possibility of piercing the gut or any metathoracic nerves but also achieved an even distribution of the injected fluid throughout the hemocoel within 30 seconds, as determined by injections of buffered Neutral Red. After the desired intervals of time, the labelled ganglia were removed, fixed, and embedded as described above, and 1/z or 1/x sections were mounted on gelatincoated slides. The sections were coated with l/ford-L4 emulsion (diluted 1 : 2 with water) using the methods of Caro and van Ttibergen (1962) and stored for 3 weeks to 4 months at 18~:C. They were then developed in Kodak Dektol for 5 minutes and stained on an 80~ hot plate for 10-15 seconds with 0"1% toluidine blue in 1-0% sodium borate. The rate at which the concentration of radioactive uridine in the hemolymph decreased following injection of the isotope was determined as follows: samples of hemolymph were collected at intervals of I minute, 7 minutes, 1 t~our, 6 t~ours, 1 day, 2 days, and 5 days after the injection of uridine-5-aH (dosage: 60/~C/gm). The samples were mixed with cold 10% trichloroacetic acid to precipitate high molecular weight nucleic acids (Schmidt, 1957). The precipitates and the cells of the hemolymph were pelleted by centrifugation for 10 minutes at 2500 rpm. Aliquots TJSSUE 8- CELL 1970 2. (2)

of the supernatants were then added to Bray's solution (dioxane) for scintillation counting. It was found that the concentration of radioactive, low molecular weight nucleotides per microliter of hemolymph decreased 90% between 1 minute and 1 hour after injection. From 1 hour to 1 day, the radioactivity slowly fell to 5% of the concentration at 1 minute, and by 5 days it had become insignificant. The uridine fraction was isolated from the supernatant of each sample by thin layer chromatography (StahI, I965) (solvents: ethyl acetate and formic acid in a proportion of 13 : 1) (Merck AG Darmstadt TLC plates; silica gel F254). The radioactivity of the uridine was measured by scintillation counting. This procedure indicated that uridine-5-'~H (which made up 97% of the injected radioactivity) was 70% of the total radioactivity in the hemoIymph at 1 hour after injection, and 40% of that in the hemolymph at 1 day after injection. The concentration of radioactive uridine in the hemolymph therefore falls 93% during the first hour after injection, and 98% during the first day. This analysis suggests that an effective pulse label was given relative to the time course studied here.

Results A. NORMAL MORPHOLOGY

Metathoracic neurons 3L and 3R a are a bilateral pair of nerve cells located between the anterior connective and the second peripheral root on either side of the metathoracic ganglion (Fig. 1). Their cell bodies are over twice the size of others in that part of the ganglion and can therefore be identified with certainty

The terms 3L ai~d dR. h~dicate the left and right members of the third bilateral pair of metathoracic neurons according to the classification by Cohen and Jacklet (1967) (Cohen, 1967). Their map conceres the metathoracic ganglion in the cockroach, Periplaneta americana, which undoubtedly has a somewhat different structure from the metathoracic ganglion of Diploptera punctata. However, their terminology was used here because this particular pair of unusually large l~eurons is present in both cockroach species in the same location.

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Fig, 1, This section through the metathoraclc ganglion shows the cell bodies of neurons 3L and 3R (arrows) located on either side between the anterior connective (AC) and the second peripheral root (11). In this plane of section, the centrally-located neuropil fills almost the entire space and only a few nerve cell bodies are present, The peripheral connectives (PC) lie at the bottom of the field. • 175,

ita a n y a n i m a l . T h e a x o n f r o m e a c h o f t h e s e m o n o p o l a r cells first gives o f f m a n y b r a n c h e s in t h e n e u r o p i l a n d t h e n l e a v e s t h e g a n g l i o n in t h e i p s i l a t e r a l n e r v e 3B z. A l t h o u g h t h e exact functions of these two neurons are not known, ~ there are good reasons for consideri n g t h e m to b e a b i l a t e r a l p a i r o f i d e n t i c a l n e u r o n s : (1) t h e y a r e t h e o n l y n e u r o n s o f t h a t size in t h a t p a r t o f t h e g a n g l i o n ; (2) t h e y h a v e a n i d e n t i c a l p e r i k a r y a l m o r p h o l o g y ; (3) t h e y s h o w a s i m i l a r r e s p o n s e to t r a n s e c t i o n o f t h e i p s i l a t e r a l m e t a t h o r a c i c n e r v e 3B.

2 The classification of metathoracic nerves by Pipa and Cook (1959) is for the species Periplaneta americana. Although there are some anatomical differences in the metathoracic segment of Diploptera punctata, there are the same number of peripheral nerves as in Periplaneta americana, and nerve 3B has a pathway similar to that given by Nijenhuis and Dresden (1955), Pipa and Cook (1959), and Jacklet and Cohen (1967b). a Metathoracic nerve 3B in Periplanem americana contains many sensory axons from chordotonal organs, hair plates, and setae in the coxa and trochanter of the metatfioracic leg; it also contains a few motor axons (Pipa and Cook, 1959). That neurons 3L and 3R are probably motor neurons is indicated by their position and large size.

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Fig. 2. This section through the pear-shaped cell body of metathoracic neuron 3L shows the centrally located nucleus, the small Nissl bodies (NB) scattered throughout the perikaryon, the dense lipofuscin granules (L), and the trophospongial interdigitations (arrows). The axon, which is in a ventromedial orientation, enters the neuropil in the upper left-hand corner of the field. • 1330.

The morphology of the cell bodies of neurons 3L and 3R resembles that of many other insect and invertebrate neurons (Zawarzin, 1924; Hess, 1958; Trujillo-Cendz, 1962; Smith and Treherne, 1963; Coggeshall and Fawcett, 1964; Bullock and Horridge, 1965; Lane, 1968). There is a large dilute nucleus in the center of the monopolar cell body, clusters of organelles are scattered fairly evenly throughout the perikaryon, and the cell membrane interdigitates with the surrounding glia to form the trophospongium TISSUE 8- CELL 1970 2 (2)

(Figs. 2, 3). Scattered throughout the cytoplasm are many small, irregularly shaped spaces which contain relatively few organelles. These spaces are of approximately the same size in all regions of the perikaryon, and many of them are interconnected. In three-dimensions they form a system of channels which probably contain rapidly flowing cytoplasm similar to that described by Pomerat et al. (1967). The clusters of organelles would thus be located in more stationary zones of the cell body.

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In neuron 3L or 3R, the large nucleus contains dilute chromatin, one or two nucleoli, and a variety of dense, basophilic aggregates (Figs. 2-4). The number of nucleoli within any nucleus is variable, but the total nucleolar volume is not. Reconstruction of serially sectioned cells demonstrated that in any animal either neuron 3L or 3R can have one or two nucleoli, and when there is only one n u d e olus, its volume is approximately equal to the combinedvolumes of the nucleoli in cells that have two. The Nissl bodies of neurons 3L and 3R appear in the light microscope as small, irregularly shaped clumps of basophilia (Figs. 2, 4). The main aggregates of basophilia are 1-4/~ wide whereas those in the trophospongial region are somewhat smaller (Fig. 3). Some authors have stated that cockroach neurons normally lack Niss~ bodies (Hess, 1958; Ashhurst, 1961; Cohen and Jacklet, 1965; Cohen, 1967). This statement, however, is based upon studies of thick paraffin sections in which these small Nissl bodies would overlap, thus obscuring their actual profiles. In the thinner sections studied here, the outlines of individual basophilic aggregates are clearly visible and can be considered to be Nissl bodies (Nissl, 1894b; Palay and Palade, 1955; Deitch and Murray, 1956; Deitch and Moses, 1957). In the electron microscope, the Nissl bodies are found to consist of free ribosomes, polysomes, tubular rough endoplasmic reticulum, and a fine fibrous matrix (Figs. 3, 8, 9, 19, 20). They do not contain ordered stacks of cisternae of the rough endoplasmic reticulum as are found in some other neurons, but instead have a highly irregular form of the endoplasmic reticulum. This form is very similar to that of

the N issl bodies of certain vertebrate neurons, such as the Purkinje cell (Palay and Palade, 1955). The Nissl bodies in the trophospongial region are slightly different from the others in that they encircle the penetrating glial processes and contain rough and smooth subsurface cisternae (Fig. 19). The mitochondria are closely associated with the Nissl bodies and occur either singly or in apparent clusters (Figs. 8, 19). Serial sections indicated that many of the clustered profiles are interconnected parts of large, contorted mitochondria. The fine structure of both the isolated and contorted mitochondria is similar: there are several tubular cristae in each profile, and between the cristae there is a loose, fibrous matrix and a number of dense granules 100-150 /~ in diameter. The Golgi bodies of neurons 3L and 3R are located mainly in the more peripheral regions of the perikaryon, although they are occasionally found next to the nucleus. They have a roughly hemispheric shape similar to the Golgi bodies--or dictyosomes--of other cockroach neurons (Smith and Treherne, 1963), as well as the other following characteristics: the cisterna on the convex side is dilated, there are 4-7 flat cisternae in the middle, and the cisterna on the concave surface is partially vesiculated (Figs. 8, 19, 23, 24). A variety of vesicles occur on all sides, and they are either smooth or coated, and either empty or dense-cored. The Golgi bodies in the trophospongial zone are all oriented such that their outer, dilated cisterna faces adjacent rough or smooth subsurface cisternae (Fig. 19). The frequent occurrence of vesicles between the Golgi bodies and the subsurface cisternae suggests that there may

Fig, 3. This low power electron micrograph shows a portion of the perikaryon of neuron 3R. Part of the nucleus, containing a nucleolus (NU) and dilute chromatin, can be seen at the bottom of the field. Clumps of ribosomes and rough endoplasmic reticulum, which make up the Nissl bodies (*), are scattered throughout the cytoplasm ; those in the trophospongial area (arrows) are smaller than the others. Numerous Golgi bodies (G), mitochondria (M), lipofuscin granules, and lysosomes are present. It should be noted that the spaces between the clusters of organelles are roughly the same size in all regions of the cell body. The glial-neuronal interdigitations of the trophospongium lie at the top of the field; some of the overlapping gliar folds (GL) surrounding the perikaryon can also be seen. • 7000. TISSUE 8 CELL 1970 2 (2)

~!i~!~8!i4

LEE

226 structure of the excretory system in adult N.

brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods

Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results

Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend

backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum

Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to

the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.

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263

C H R O M A T O L Y S I S 1N N E U R O N S be a functional relationship between these apposed organelles. There is a similar morphological relationship between the other Golgi bodies and cisternae of rough endoplasmic reticulum in the rest of the cell. The morphology of neurons 3L and 3R changes in three important respects as the adult roach ages: both the nucleus and nucleoli decrease in size and the lipofuscin granules become more numerous (Figs. 4, 6). Measurement of nuclear and nucleolar volumes by reconstruction of serially sectioned cells revealed that both of them decrease during the first month after the adult molt, the nuclear volume by 25-40% and the nucleolar volume by 30-60%. These changes are probably related to the reductions in juvenile hormone and ecdysone that occur in the adult insect (Gilbert, 1964; Wigglesworth, 1964). Regardless of the cause, these changes suggest that during adult life, which lasts at least 5 months, neurons 3L and 3R require less RNA and ribosome production than they do in the younger stages. B. THE RESPONSEOF THE CELL BODYTO AXONAL INJURY

The hdtial Response MorphologicaI changes are first observed in the cell body of neuron 3L or 3R by 24 to 36

hours after transection of the axon. At this time, a basophilic aggregate begins to form in the cytoplasm around the nucleus, and the concentration of basophilia in the more :peripheral zones of the cell body begins to decrease. By 3 to 5 days after injury, these changes are well developed (Figs. 4, 5). This perikaryal response is similar to that of the injured neurons in Periplatwta ameHca~la (Cohen and Jacklet, 1965) but it develops more slowly. The perinuclear basophilic aggregate has been called a nuclear 'ring' by Cohen and Jacklet; this term, however, will not be used here because the basophilia actually forms a complete shell aroundthe nucleus rather than a ring. Twenty-five pairs of experimental and control cells were examined in the electron microscope 1 to 5 days after injury to either neuron 3L or 3R. In all cases, there was no change in nuclear morphology in the injured cell. By three days after injury, however, two important alterations in cytoplasmic morphology are apparent. First, the Nissl bodies in the periphery of the cell body are smaller and more widely spaced, while those :near the nucleus are larger and more closely spaced than in this region of the control cell (Figs. 8-11); this change is demonstrated in Fig. 12 in which the Nissl bodies of Figs. 8-11 have been painted black to emphasize their size

Fig. 4. Neuron 3L. This control cell for Fig. 5 has the morphology typical of neurons in young adults. Note the even distribution of N issl bodies, the centrally located nucleus, the large nucleolus, and the infrequent lipofuscin granules, • 950. Fig. 5. Neuron 3R. This cell was injured three days before fixation, The Nissl bodies have become more concentrated in the perinuclear cytoplasm, and less concentrated in the more peripheral regions. • 950. Fig. 6. Neuron 3L. This control cell for Fig, 7 has the morphology typically found one month after the adult molt. Comparison with the younger neuron in Fig. 4 indicates the decrease in nuclear and nucleolar size (this section passes through the centre of both oganelles and there is only one nucleolus), The lipefuscin granules are also more numerous than in the younger cell, • 950. Fig. 7, Neuron 3R. This cell was injured 4 weeks before fixation. The nucleus, which is larger and more dilute than that of the control cell (Fig, 6), is displaced from its central position toward the axon hillock. There is still a greater concentration of Nissl bodies around the nucleus than in the peripheral areas, and the perinuclear Nissl bodies are larger than those of the healthy neurons (Figs. 4, 6), or the recently-injured neuron (Fig. 5). • 950, TISSUE 8- CELL 1970 2 (2)

ii?!~:i'i,'i~i~ :' :i~

LEE

226 structure of the excretory system in adult N.

brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods

Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results

Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend

backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum

Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to

the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.

TISSUE 8- CELL 1970 2 (2)

266 and distribution. It should be noted that the Nissl bodies in the perinuclear aggregate have a similar fine structure to that of the Nissl bodies in the control cell, and that they are still associated with mitochondria and other organelles. In addition, those in the periphery are much smaller than the comparable ones in the control cell; however, they still contain the subsurface cisternae. The second alteration in fine structure is a reduction in the size of the Golgi bodies (Figs. 8, 10, 23, 25). This is caused largely by a decrease in the number and length of the cisternae; there are usually 4 to 7 flat cisternae in the Golgi bodies of the healthy cell, but by 3 days after injury, there are only 1 to 5 of them in the Golgi bodies of the injured cell. The location of the Golgi bodies is not different at this time. Moreover, those in the trophospongium remain associated with the subsurface cisternae and undergo the same morphological changes as the other Golgi bodies.

Nissl Body Movement The alteration in the pattern of perikaryal basophilia occurring during the initial response may be the result of perinuclear production of Nissl bodies coupled with destruction of those in the periphery; alternatively, it may result from the movement of Nissl bodies toward the nucleus; or both processes

BYERS could occur. Cohen (1967) has given some preliminary evidence from autoradiographic experiments that suggests that increased R N A synthesis is a major factor in the production of the perinuclear basophilic aggregate, This problem was examined in the present study by two different autoradiographic experiments, and the alternative conclusion was reached. One set of experimental animals was given uridine-aH or uridine-5-3H at either 18, 24, 36, or 48 hours after axonal transection, and the metathoracic ganglia were fixed at either 6, 24, 48, or 72 hours after injection. If the perinuclear aggregate were composed of newly synthesized RNA, one should observe a large proportion of the radioactivity in that zone of the injured cell, In the second experiment, the animals were injected with radioactive uridine 1 to 6 weeks prior to injury, and their ganglia were fixed at 1, 2, or 389days after injury. In autoradiograms of these ganglia, the silver grains represent the loci of RNA molecules synthesized long before axonal injury, and most of them are located over the Nissl bodies. /f the perinuclear aggregate resulted from the centripetal movemerit of Nissl bodies, there should be a heavy concentration of radioactivity in the perinuclear region of the injured cell in these ganglia. If, on the other hand, there is much

Figs. 8 and 9. Neuron 3 R. These views of the control cell for Figures 10 and 11 show the normal cytoplasmic morphology of the juxtanuclear and peripheral regions. The spacing between the Nissl bodies (NB) is approximately the same throughout the c e l l and each Nissl body is composed of disorganized tubular rough endoplasmic reticulum, free ribosomes and polysomes, and a fine fibrous matrix. They are closely associated w i t h mitochondria (M), which occur either singly or in clusters. The Golgi bodies (G) are more frequently found in the peripheral cytoplasm and contain 4 - 7 flat, stacked cisternae. Numerous rough or smooth subsurface cisternae (arrows) are present, especially at the tips of the indentations made by penetrating glial processes. • 18,000. Figs. 10 and 11. Neuron 3L. This cell was injured three days before fixation, The N issl bodies in the juxtanuclear zone (Fig. 11 ) are more closely spaced than those in the control cell (Fig. 9). They have the same fine structure and association with mitochondria as the Nissl bodies in the control cell. In the peripheral cytoplasm (Fig. 1 0), the Nissl bodies are smaller and more widely spaced than in the similar region of the control cell (Fig. 8). Many subsurface cisternae (arrows) are still present in the injured cell. The Golgi bodies have only 1 - 5 flat, stacked cisternae, and they are smaller and more vesiculated than those of the healthy cell. • 18,000. TISSUE ~ CELL 1970 2 (2)

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Fig. 1 2. The Nissl bodies in Figures 8-11 are painted black here to emphasize the changes in their size and distribution that occur during the intitial response, x 6000.

synthesis of new R N A , then the injured neuron could only have a labelled perinuclear aggregate if the old R N A were broken down and its nucleotides reutilized; in this case the nucleus of the injured cell should be more heavily labelled than that in the control cell. In the first experiment, when the radioactive uridine was injected after axonal transection, very little difference in the distribution of label was found between the experimental and control cell in any of the animals. The pair of ceils in Figs. 13 and 14 illustrates the greatest labelling of the perinuclear aggregate that was found; in this case, the injured cell has only 10-20% more silver grains near the nucleus than the control cell. This is not a large enough difference to suggest that the synthesis of new R N A is an important factor in the initial stages of the formation of the perinuclear aggregate. In TISSUE 8- CELL 1970 2 (2)

addition, no difference in nuclear label was found; if R N A synthesis were rapidly stimulated byaxonal transection, one would expect to find more radioactivity in the nucleus of the injured cell. In the second experiment, there was a significantly greater amount of label over the perinuclear aggregate in the injured ceils than in the same area in the controls, and the size of the zone of increased radioactivity was proportional to the size of the basophilic aggregate. The cells in Figs. 15 and 16 come from an animal that was injected with radioactive uridine 6 weeks prior to fixation; neuron 3R was injured 48 hours before fixation. The perinuclear aggregate is only partly formed in this case, but it is accompanied by increased radioactivity. At this stage in the formation of the basophilic aggregate, there is no difference in the amount of label over the nucleus.

Figs. 13 and 14. Uridine-5-SH was injected 48 hours before fixation : neuron 3R (Fig, 14) was injured 83 hours before fixation; neuron 3L (Fig. 13), uninjured. Although a prominent perinuclear aggregate of Nissl bodies is evident in neuron 3R, it is not heavily labelled as compared with the perinuclear zone of neuron 3 L The nuclear label is similar in both cells. • 600. Figs. 15 and 16. Uridine-'~H was injected 6 weeks before fixation: neuron 3R (Fig. 16) was injured 48 hours before fixation; neuron 3L (Fig, 15), uninjured. The perinuclear aggregate is only partly formed in neuron 3R, but there is a significantly greater number of silver grains over the aggregated Nissl bodies than in the similar zone of neuron 3L. The nuclear label is similar in both cells. • 600. Figs. 17 and 18. Uridine-5-3H was injected 11 days before fixation: neuron 3R (Fig. 18) was injured 389 days before fixation; neuron 3L (Fig. 17), uninjured. There is a heavy concentration of radioactivity and basophilia in the perinuclear region of neuron 3R, while neuron 3L has evenly distributed Nissl bodies and silver grains. Nuclear label is approximately 50% greater in the injured cell than the control. The background was low in these experiments as evidenced by the infrequent silver grains over zones outside the tissue (Fig, 18) or over large axons in the neuropil (Fig. 17). x 600.

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269

The cells in Figs. I7 and 18 come from an animal that was injected with radioactive uridine 11 days prior to fixation; neuron 3R was injured 3} days before fixation. In the injured cell, the initial response is well developed and there is a pronounced concentration of radioactivity over the aggregated Nissl bodies. By 389 days after injury, there is also more radioactive RNA in the nucleus of the injured cell. This result indicates either that the labelled RNA returns to the nucleus or, more probably, that it is broken down and its nucleotides reutilized. This return of label to the nucleus was not observed during the earlier stages of the initial response (Figs. t l let cl u l

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in labelling the aggregate until the later stages of its formation. The comparison of these two experiments suggests that movement of Nissl bodies toward the nucleus occurs during the initial response to axonal transection. This conclusion is also supported by the fine structure of the injured cell (Figs. 8-11). If the basophilic aggregate were the result of increased R N A and ribosome production, as suggested by Cohen (1967), one would expect to find a disproportionate number of ribosomes in the juxtanuclear region as well as alterations in the morphology of the nucleoli; neither of these was observed in either neuron 3L or 3R during the initial response. Instead, at all stages of the formation of the perinuclear aggregate, the Nissl bodies near the nucleus have the same fine structure as those of the control cell. It should be noted that the Nissl bodies in the peripheral zones of the cell body decrease in size during the initial :response (Figs. 8, 10). The loss of basophilia in this region may therefore involve partial destruction of the Nissl bodies as well as movement toward the nucleus.

The Morphology of Regeneration The morphology of neuron 3L or 3R during the initial response is gradually replaced by a very different one which is well developed by three weeks after injury. This morphology is assumed to be present during the active regeneration of the axon for several reasons. TISSUE 8- CELL 1970 2 (2)

First, most cockroach nerves regenerate well and can re-establish functional connections (Roeder and Weiant, 1950; Bodenstein, 1955, 1957; Guthrie, 1962; Jacklet and Cohen, 1967ab). In the injured Diploptera punctata studied here, a tuft of fibers could be seen growing into the coxa from the central stump of the injured nerve 3B by four weeks after injury. Another argument in favor of this assumption is that Jacklet and Cohen (1967b) have demonstrated that rapid regeneration of metathoracic nerve 3B in Periplaneta americana begins several weeks after transection and is accompanied by the development of a special perikaryal morphology: a,,u ,,u~.,~.~,,us injured neurons become hypertrophied, the nucleus assumes an eccentric position, and the basophilic pattern of the cytoplasm returns to normal. In the injured neurons of Diploptera punctata, the nucleus and nucleolus were enlarged by three weeks after injury, the nucleus was slightly displaced from its central position, and the zone of aggregated basophilia around the nucleus had become broader and usually less dense (Figs. 6, 7). It might be suggested that this morphology represents a preliminary stage in the eventual degeneration of the cell. This suggestion, however, can be discounted, for in several cases a specific morphology of degeneration appeared by 3 to 4 weeks after injury: the nucleus was pyknotic, and the cytoplasmic structure severely disrupted. It seems that the fate of these cells has been determined by 3 or 4 weeks after injury: either they are regenerating or they are moribund. The morphology of regeneration persists for several weeks; during this time the zone of aggregated basophilia gradually expands and becomes less concentrated. By two months after injury, the cytoplasm has regained a normal appearance, although the nucleus and nucleolus remain enlarged. The regenerating neuron and the control cell from six animals that ihad been injured 389 to 5 weeks earlier were examined in the electron microscope. The fine structure of the regenerating neuron (Figs. 21-22) is strikingly different from either that of the

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226 structure of the excretory system in adult N.

brasiliensis and to try to ascribe a function to the various parts of the system. Materials and Methods

Infections of N. brasiliensis were maintained in laboratory rats. The adult nematodes were removed from the intestine 8 to 10 days after infection and fixed in Carnoy's fixative or in 5% formal saline for general histological study. Paraffin sections were stained with haematoxylin and eosin. For work on the detection of enzymes frozen sections of fresh nematodes, or of nematodes fixed in 5% normal saline or 2.5 ~o glutaraldehyde at 4~ for 2 to 24 h were used. The histochemical methods used for the detection of enzymes were as given by Pearse (1960). Control sections were heated to destroy enzyme activity or incubated in appropriate inhibitors. For electron microscopy the nematodes were chopped and fixed in 2.5% glutaraldehyde in cacodylate buffer at 4~ for 24 h: washed in sucrose buffer (Gordon, Miller and Bensch, 1963); postfixed in 1% osmium tetroxide in a balanced salt solution (Rosenbluth, 1965); dehydrated in ethanol, followed by propylene oxide and embedded in Araldite. Sections were cut on a LKB III ultratome, stained with uranyl acetate and lead citrate and examined in an AE1 E M 6B or a Philips EM 300 electron microscope at 60 kV or 80 kV. Results

Morphology. The sub-ventral glands of N. brasiliensis consist of a pair of elongate structures which lie in the pseudocoele and extend

backwards from their junction with the excretory duct towards the tail and occupy about half the length of the nematode. The two lateral canals are embedded in the lateral cords and extend towards the mouth and towards the tail from the transverse canal which links the two canals in the region o f the base of the oesophagus. The sub-ventral glands also open into this transverse canal. An excretory duct, which is lined with cuticle, passes from the transverse canal to the ventral excretory pore. Histochemistry.The sub-ventral glands gave a strong positive result for non-specific esterase with the bromo-indoxyl acetate method. Heated control sections gave a negative result but 10 4 M E600 did not markedly inhibit the enzyme. A strong positive result was also obtained with the acetylthiocholine iodide method for cholinesterases and 10 a M E600 did inhibit this enzyme. The glands gave a strong positive result with the 'leucine' aminopeptidase method. The excretory canals gave negative results with the above methods. UItrastructure. Each lateral canal of the excretory system consists of an elongate cell which has a convoluted intracellular lumen along its length (Fig. 1). This central lumen is lined by a plasma membrane and has a very irregular outline. Numerous membranebound vesicles or canaliculi are congregated around the lumen of the canal (Fig. 1) and apparently open into the lumen. There are very few mitochondria in the cytoplasm of the cell but there are many more mitochondria in the cytoplasm of the hypodermis adjacent to the lateral canal. A few short strands of granular endoplasmic reticulum

Fig. 1. Electron micrograph of a longitudinal section through a lateral cord of N . brasiliensis to show the structure of the lateral canal and its relationship to

the hypodermis, x 22,000. Fig. 2. Electron micrograph of a section through a sub-ventral gland to show the t w o types of granule, the extensive rough endoplasmic reticulum and the formation of the granules, x 21,000. c wall of lateral canal; ca canaliculi; er rough endopiasmic reticulum; g Golgi complex; h hypodermis; j junction of endoplasmic reticulum with granule ; / lumen of lateral canal ; rn mitochondrion ; s.g t w o types of secretory granule.

TISSUE 8- CELL 1970 2 (2)

272 healthy cell (Figs. 19-20) or that of the cell undergoing the initial response. First of all, the Nissl bodies near the nucleus of the regenerating cell are larger than any in the control cell, and they contain more ribosomes. The peripheral Nissl bodies are still small, but their ribosomes are more numerous than those of the recently injured neuron (Fig. 10). In addition, a larger proportion of the ribosomes are free rather than attached to the endoplasmic reticulurn; examination of many electron rnicrographs indicated that normally one-fifth of the ribosomes are bound to the endoplasmic reticulum and that this proportion decreases to one-seventh in the regenerating neuron. Secondly, the Golgi bodies of the regenerating neuron are hypertrophied, and some of them are located nearer to the nucleus. Many of the Golgi bodies, however, are still adjacent to subsurface cisternae, and these are ~ot as hypertrophied as the other Golgi bodies. The cisternae of the hypertrophied Golgi bodies are longer and more curved than those of the control cell (Figs. 24, 26). They also have more complex membrane systems and are surrounded by a greater variety of vesicles. Thirdly, the lysosomes in the regenerating neuron have become more numerous (Figs. 19--22); this is especially true of the small lysosomes and the autophagic vacuoles. No

BYERS specific organelles could be distinguished within the autophagic vacuoles so that the objects of their activity in the regenerating neuron remain unclear. In the fourth place, the mitochondria during axon regeneration not only become enlarged, but also gain a denser matrix. Moreover, the clustered profiles of the highly contorted mitochondria become less tightly packed together (Figs. 19, 21). Finally, the volumes of the nucleus and nucleolus are up to 50% greater than those of young healthy cells (Figs. 4, 6, 7). This increase occurs in the regenerating neurons from both young and old animals, even though the control cells of the older adults are undergoing a decrease in nuclear and nucleolar size. The requirements of axonal regeneration therefore must be able to overcome the factors which would ordinarily cause a shrinkage of these organelles in the old animals. This morphology of regeneration is not exactly the same as that described by Jacklet and Cohen (1967b) for the neurons of Periplaneta americana: the nucleus does not move all the way to the edge of the cell body, and the perinuclear aggregation of basophilia persists for a longer time. It is probable that these differences are caused more by variation in fixation techniques and the site of injury than by the species difference.

Figs. 1 9 and 20. Neuron 3L. These views of the control cell for Figures 2 1 - 2 2 show the typical cytoplasmic morphology in the peripheral and juxtanuclear regions. The association of peripheral Golgi bodies (G), and Nissl bodies (NB) with subsurface cisternae is indicated (arrows). Two clusters of mitochondrial profiles (M) are present, and the glial processes of the trophospongium which penetrate into the neuronal cell body are indicated (*). The Nissl bodies in Figure 20 have an especially representative fine structure : they contain tubular rough endoplasmic reticulum, free ribosomes and polysomes, and a fine fibrous matrix, x 18,000. Figs. 21 and 22. Neuron 3R. This cell was injured 4 weeks before fixation. The hypertrophied Golgi bodies (G), mitochondria ( M ) , and numerous small lysosomes (L) are indicated. Many small autophagic vacuoles are also present (AV). The Nissl bodies ( N 8 ) near the nucleus (Fig. 22) are larger than those in the comparable zone of the control cell (Fig. 20) and contain more ribosomes; there is also less space between the Nissl bodies that surround the nucleus in the regenerating neuron. The Nissl bodies in the periphery of the regenerating cell (Fig. 21) are still smaller than those in the similar region of the control cell (Fig. 19). • 18,000. TISSUE 8" CELL 1970 2 (2)

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Figs. 23-26. These micrographs show the m o r p h o l o g y o f representative Golgi bodies in the healthy cell (Figs. 23 and 24), the neuron injured for 3 days (Fig. 25), and the neuron injured for 389 weeks (Fig. 26). The Golgi bodies of the healthy cell contain 4 - 7 flat cisternae ; by three days after axon transection, there are never more than 5 such cisternae, and there are usually only 1 or 2. In the regenerating neuron the cisternae are longer, more curved, and surrounded by a greater variety of vesicles; there are usually only 3 or 4 flat cisternae and these do not extend all the way across the Golgi body. • 21,000.

Discussion

The response of this cockroach neuron to a• transection has certain characteristics in common with that of other nerve cells. First of all, during the early phase of chromatolysis, Nissl bodies aggregate in one region of the perikaryon and disperse in another zone. The same general process occurs in other invertebrate (Young, 1932) and vertebrate nerve cell bodies following axonal injury. Vertebrate neurons such as the monopolar sensory cell (Andres, 1961 ; Smith, 1961 ; Holtzman et al., 1967) or the motor neuron TISSUE 8" CELL 1970 2 (2)

(Porter and Bowers, 1963; Bodian, 1964) have a reverse pattern of basophilic change from that in the cockroach neuron: the Nissl bodies aggregate around the periphery of the cell body and disperse in the more central regions. The Purkinje cell, however, shows the same pattern of peripheral dispersion and perinuclear aggregation (Llinfls et al., 1967). The basophilic change following axonal injury should therefore be considered similar in the cockroach neuron to that in other nerve cells and not a fundamentally different process as suggested by Cohen (1967). Another similarity between the response of

274 the cockroach neuron and that of other nerve cells is that the Golgi bodies undergo an initial decrease in size (Ramon y Cajal, 1914; Penfield, 1920; Young, 1932; Novikoff and Essner, 1962). These changes also seem to involve a reduction in the length of the cisternae and the number of flat cisternae (Porter and Bowers, 1963; Bowers et al., unpublished observations). Finally, the initial morphological changes in the cockroach neuron and other injured nerve cells are followed by: a shift in nuclear position; hypertrophy of the nucleus, nucleolus, and Golgi apparatus; an increased concentration of cytoplasmic basophilia and ribosomes; and an increased number of lysosomes (Nissl, 1894a; Bodian and Mellors, 1945; Porter and Bowers, 1963; Jacklet and Cohen, 1967b). These morphological features all seem to characterize the cell body during the period of rapid axonal outgrowth. In some cases lysosomes reach a peak concentration during the earlier phase of chromatolysis (Holtzman et al., 1967), but in other cases, their numbers are greatest during the regenerative phase. In regenerating frog motor horn cells, for instance, lysosomes are most numerous at six weeks after injury when most of the perikaryal basophilia has been restored (Bowers et al., unpublished observations). In addition, Bodian and Mellors (1945) found that the peak increase in acid phosphatase activity extended well into the regenerative period. The hypertrophy of the mitochondria during regeneration is the only morphological change observed here that is not usually reported in chromatolytic vertebrate neurons. However, altered mitochondrial form was observed in injured sensory ganglion cells in raIs (Smith, 1961) and in the newt Triturus (Lentz, 1967); mitochondria were also found to increase in number following axonal transection of hypoglossal neurons in rats (Hartmann, 1954). It should be noted, in this regard, that succinic dehydrogenase and triphosphopyridine nucleotide dehydrogenase activities decrease during chromatolysis (Friede, 1959) in motor neurons, while no changes in mitochondrial form are found in

BYERS this type of cell (Bodian, 1964; Bowers et al., unpublished observations). Mitochondria therefore seem to undergo changes in function that are only morphologically apparent in some cases. This study of a specific cockroach neuron has suggested certain general features of the chromatolytic process. First, autoradiographic experiments demonstrated that movement of Nissl bodies is an important part of the initial perikaryal response. Such movernent has previously been suggested by some of the morphological studies of vertebrate neurons (Smith, 1961; Porter and Bowers, 1963; Holtzman et al., 1967); this paper provides stronger evidence for this phenomenon, at least in an invertebrate neuron. Two other explanations of the shifts in distribution of basophilia are often given: either there has been destruction of Nissl bodies and ribosomes (Bodian and Mellors, 1945; Holtzman et al., 1967) or there has been dilution of cytoplasmic R N A or ribosomes as a result of cellular swelling (Hyd6n, 1943; Brattgard et al., 1957; Porter and Bowers, 1963). Both of these explanations for the chromatolytic changes are undoubtedly valid for many neurons, but movement of Nissl bodies should also be considered as a possible factor. The degree to which movement, dilution, and destruction of Nissl bodies are important in the basophilic changes probably depends upon the cell and the kind of injury. In the cockroach neuron studied here, Nissl body movement is the most important factor, although some destruction of the Nissl bodies seems to occur in the peripheral regions of the cell body. In the second place, this study has found that the response of the cockroach neuron occurs in two phases. Initially, the Nissl bodies move toward the nucleus and the Golgi bodies decrease in size. These changes do not seem to be directly related to the regeneration of the axon for they occur before axonal outgrowth is established, and they are replaced by a different morphology when regeneration begins. Jacklet and Cohen (1967b) have suggested that the initial response is a constructive stage in which the cytoplasmic TISSUE 8- CELL 1970 2 (2)

C H R O M A T O L Y S I S IN N E U R O N S structure is reorganized in preparation for regeneration of the axon. This idea is supported by the observations of increased synthesis of protein (Brattgard et aL, 1958; Fischer et al., 1958; Gutmann, 1968), RNA (Porter and Bowers, 1963; Watson, 1965), and phospholipids (Miani, 1964) in some neurons during the early phase of chromatolysis. It is possible, however, that most of the events of the initial response are disruptive ones which must be overcome in order for fine cell to regenerate its axon (Bodian and Mellors, 1945). Both the movement of the Nissl bodies (rather than synthesis of new ones) and the shrinkage of the Golgi bodies suggest that the response in this cockroach neuron during the first few days after injury is not a constructive one. It is not until three days after injury, when the changes mentioned above have already occurred, that an apparent increase in synthesis of RNA is detected (Figs.

17 18). A third point concerning chromatolysis is that the regeneration of the axon involves a somewhat different activity in the perikaryon than is found in the neuroblast. Some investigators have suggested that the nerve cell body reverts to embryonic functions following axonal injury, for both the regenerating neuron and the neuroblast have many morphological features in common (Pannese, 1963). The regenerating neuron is different, however, in that it contains numerous lysosomes; these are rarely present in micrographs of embryonic neurons (Lyser, 1964). Although their exact role is not known, the lysosomes seem to play a specific role in the :regenerative process. This role does not seem to be concerned with the losses of basophilia, as suggested by Holtzman et al. (1967), since the peak lysosomal activity is often at a time when the cytoplasmic basophilia is being restored (Bodian and Mellors, 1945; Bowers et al., unpublished observations). The phenomenon of Nissl body movement, which has been studied here, could alternatively be viewed as a change in the size of the spaces between the Nissl bodies: those near the nucleus decrease in size while the more peripheral spaces increase. Studies by TISSUE 8- CELL 1970 2 (2)

275 Pomerat et al. (1967) have shown that the spaces between the Nissl bodies are channels for rapidly moving cytoplasm, while the Niss/ bodies are rather immobile islands. In the cockroach neuron, it seems plausible that a shrinkage in the volume of these channels near the nucleus and an expansion of the channel volume in the periphery could be the mechanisms producing the altered location of the Nissl bodies. These changes in channel volume may be explained by the fact that axonal transect ion necessarily interrupts axoplasmic flow both the slow, centrifugal axoplasmic movement (Weiss and Hiscoe, 1948; Droz and Leblond, 1963) and the rapid, highly variable, bidirectional streaming (Lubinska, 1964; Dahlstr6m and Hfiggendal, 1966; Lasek, 1968; McEwen and Grafstein, 1968; Ochs et al., 1968). The products of the perikaryon would consequently accumulate in the proximal segment and possibly return to the cell body; similarly, materials from the distant axon and synapses could no longer reach the perikaryon. These changes could cause specific alterations in the movement of cytoplasm within the cell body with the observed changes in perikarya/morphology. It is an interesting possibility that alterations in cytoplasmic flow may be a signal by which the perikaryon learns of the axonal injury (Lasek, 1967). The suggestion that the distributional changes in basophilia are caused by alterations in cytoplasmic flow is supported by some other observations. First, chromatolysis is more severe and begins sooner when the injury occurs close to the cell body than when it is far away (Marinesco, 1897). This is what one would predict from the above model, for when injury is close to the cell body, the decrease in return flow would occur earlier, and the perikaryal outflow would become congested sooner. It is interesting, in this regard, that basophilic changes begin near the axon hillock in most injured neurons; it is this zone which would first be affected by altered axoplasmic flow. In the second place, the intensity of chromatolysis depends upon the normal rates of cytoplasmic flow within the axon,

BYERS

276 L a s e k (1968) has s h o w n t h a t faster rates of r a p i d flow occur in the peripheral a x o n of feline sensory n e u r o n s t h a n in the central one, a n d L u g a r o (1896) f o u n d t h a t t r a n s e c t i o n of the f o r m e r causes a greater c h r o m a t o l y t i c r e s p o n s e t h a n t h a t of the latter. Similarly, axoplasmic flow is faster in y o u n g n e u r o n s t h a n in old ones ( D r o z a n d Leblond, 1963; G u t m a n n , 1968), a n d chromatolysis is frequently m o r e i n t e n s e in the y o u n g e r n e u r o n s ( R a m o n y Cajal, 1928). Thus, t r a n s e c t i o n of a n axon possessing m o r e r a p i d c y t o p l a s m i c flow causes a m o r e violent d i s r u p t i o n o f the p e r i k a r y a l structure. In conclusion, t h e similarities between the post-injury r e s p o n s e of this c o c k r o a c h n e u r o n a n d t h a t of o t h e r n e r v e cells suggests t h a t a n y particular p a t t e r n of chromatolysis is only one expression of a general process w h i c h has as m a n y specific variations as there are k i n d s o f n e r v e cells. R e s t r i c t i o n of the s t u d y to a

single cell is a n i m p o r t a n t way to c o n t r o l for this v a r i a t i o n in the c h r o m a t o l y t i c r e s p o n s e a n d allows a m o r e precise d e t e r m i n a t i o n of the sequence of changes a n d their relationship to axonal regeneration. T h e i n v e r t e b r a t e n e r v o u s systems offer m a n y such identifiable n e u r o n s with which it s h o u l d be possible to learn m u c h m o r e a b o u t t h e p h e n o m e n o n of n e r v e regeneration.

Acknowledgements I w o u l d like to t h a n k D r S a n f o r d L, P a l a y for his extensive advice t h r o u g h o u t this p r o ject. I w o u l d also like to t h a n k D r s S. J. Adelstein, R, E. Coggeshall, D. W. Fawcett, S. Ito, a n d J. P. Revel for helpful discussions. T h e w o r k reported here was p a r t of a Ph.D. thesis a n d was s u p p o r t e d by N.1.H. T r a i n i n g G r a n t No. T 01 NB-05591 to D r S. L. Palay a n d No, G M 0 0 4 0 6 T G to D r D. W. Fawcett.

References ANDRES, K.H. 1961. Untersuchungen 0ber morphologische Ver~nderungen in Spinalganglien wfihrend der retrograden Degeneration. Z. Zellforsch. mikrosk. Anat., 55, 49-79. AS~JHURST, D. E. 1961. The cytology and histochemistry of the neurones of Periplaneta americana. Q.J. microsc. Sci., 102, 399-405. BODENSTE1N, D. 1955. Contributions to the problem of regeneration in insects. J. exp. Zool., 129, 209-224. BODENSTEIN,D. 1957. Studies on nerve regeneration in Periplaneta americana. J. exp. Zool., 136, 89115. BODIAN, D. 1964. An electron microscopic study of the monkey spinal cord. Bull. Johns Hopkins Hosp., 114~ 13-120. BODIAN, D. and MELLORS, R. C. 1945. The regenerative cycle of motoneurons, with special reference to phosphatase activity. J. exp. Meal., 81, 469-487. BowzRs, M. B., FEDER, N., and PORTER, K. R. Unpublished observations. BRATTOARD,S. O., EDSTR()M,J. E., and H•Df:N, H. 1957. Chemical changes in regenerating neurons. Y. Nenrochem., 1, 316-325. BRA'ITGARD, S. O., HYDI~N, H., and SJOSTRAND, J. 1958. Incorporation of orotic acid-l~C and lysine-l~C in regenerating single nerve cells. Nature, Loud., 182, 801-802. BROWN, G. L. and PASCOE, J. E. 1954. The effect of degenerating section of ganglionic axons on transmission through the ganglion. Y. Physiol., Lond., 123, 565-573. BULLOCK, T. H. and HORRIDGE, G. A. 1965. Structure and Function in the Nervous Systems o f Invertebrates. W. H. Freeman, San Francisco. CARO, L. G. and VAN TOBERGEN, R~ P. 1962. High resolution radioautography. I. Methods. J. Cell Biol., 15, 173-188. COGOESHALL, R. E. and FAWCETT,D. W. 1964. The fine structure of the central nervous system of the leech, Hirudo medichlalis. Y. Neurophysiol., 27, 229-289. COHEN, M. J. 1967. Correlations between structure, function, and R N A metabolism in central neurons TISSUE 8 CELL 1970 2 (2)

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