Brain Research, 96 (1975) 169-175
169
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Two types of terminal degeneration in the molecular layer of the dentate fascia following lesions of the entorhinal cortex
EVA FIFKOV/~ Department of Psychology, University of Colorado, Boulder, Colo. 80302 (U.S.A.)
(Accepted June 6th, 1975)
One of the hippocampal afferent pathways, known as the perforant path, which originates in the entorhinal area and terminates on the granular cell dendrites in the molecular layer of the fascia dentata, was shown to have potent excitatory properties z-4,11. Being a monosynaptic pathway, as it were, it can serve as a model for the study of changes which might underlie various functional states in this system. For such a type of experiment the pattern of termination of the various entorhinal fibers within the dentate fascia is of primary importance. In preparations impregnated after Nauta, Blackstad 1, Raisman et aL 1~, and Hjort-Simonsen 7 traced terminations from the entorhinal area into the distal third of the dentate molecular layer along the hippocampal fissure. Lesions producing this effect involved mostly the lateral part of the entorhinal area. Degenerated fibers from the medial part of the entorhinal area were traced to the middle third of the dentate molecular layer in Nauta impregnated preparations 8 and were shown to terminate on the spines of granular cell dendrites 13. Comparing the data of Nafstadt 13 and Hjort-Simonsen and Jeune 8 with those of Blackstad ~, Raisman et al. ~5 and Hjort-Simonsen 7, it seems likely that the lateral entorhinal area indeed projects to the distal third of the molecular layer. However, in preparations impregnated after Nauta and examined with the light microscope, the degenerated axon terminal cannot be identified with certainty, because it is difficult to make a definite distinction between degenerated axons and degenerated axon terminals. Since in electron miscroscope preparations this distinction can be readily made, an electron microscope study of the degenerated axon terminals, after lesions of the lateral part of the entorhinal area, seemed to be indicated. Thirteen albino rats were used for this investigation. Under urethane anesthesia a 3 m m trephine opening was drilled in the temporal bone just above the zygomatic arc. A triple electrode, slightly bent, was introduced in such a way that it followed the curvature of the pyriform lobe. The indifferent electrode was positioned in the exposed neck muscles and a 2 m A direct current was passed for 1 min through the uninsulated tips of the electrode. Animals were allowed to survive this procedure for 16, 24, 36, 48, and 72 h. Three unoperated animals were used as controls. After appropriate time periods, the rats were anesthetized with urethane and perfused
170 with a mixture of 4 ~ paraformaldehyde and 0.5~o glutaraldehyde in a phosphate buffer (pH 7.2-7.4). The perfused animals were kept overnight in the refrigerator. The next day the brain was dissected free, and 1 mm thick coronal slabs were taken from the superior hippocampus. From each slab a block from the superior hippocampus including the dentate gyrus was prepared and postfixed in 2~,, OsO4 in phosphate buffer for 1.5 h at 4 °C. Following dehydration in increasing concentrations of acetone and propylene oxide, the blocks were embedded in Epon. During trimming of the block, the 1 # m thick sections were stained with Axur II and methylene blue iv for the purpose of general orientation. The final trim, from which the thin sections were cut, included the molecular layer of the upper blade of the dentate fascia with the hippocampal fissure at one side and with granular cells at the opposite side, thus permitting orientation in the thin section. Silver sections were mounted on formvarcoated slot grids and stained with lead citrate 19. In order to get a survey of the extent of the lesion in the entorhinal area, the pyriform lobe of the operated hemisphere was examined in serial paraffin sections stained after Nissl. Those preparations which revealed lesions in the lateral entorhinal area were used for electron microscope examination. The paraffin Nissl stained sections of the pyri|brm lobe involved also a part of the posterior hippocampus and of the dentate fascia. In animals which survived the lesions for more than 36 h there was a distinctly light stripe in the outer third of the dentate molecular layer, just below the obliterated hippocampal fissure. A similarly positioned light stripe was observed also in the I #m sections obtained from the plastic embedded blocks of the superior part of the dentate fascia. At high magnification (1250 × ) this light stripe revealed a number of vacuoles corresponding in location with the maximum of degeneration observed in electron micrographs. In cases in which the lesion also involved the medial entorhinal area, the light stripe was wider and covered the middle and distal thirds of the dentate molecular layer. This agrees with the finding of degenerations in the middle third of the dentate molecular layer after lesions of the medial entorhinal area 8,13. it therefore can be concluded that fibers from the lateral part of the entorhinal area terminate in the superficial third of the dentate molecular layer throughout the entire extent of the dentate fascia, which is in accord with the observation of Raisman et al. 1'5. The position of the light stripe is schematically demonstrated in Fig. 1. In this region the electron microscope revealed degenerated axon terminals which contacted exclusively dendritic spines. If there were any indications of degeneration 16 h postoperatively, they could not be distinguished from the normal variation of the tissue. The first signs of degeneration became identifiable within 24 h following the lesion and increased markedly in severity following 36 h. Two distinct patterns of degeneration could be observed in axon terminals of the perforant path in the distal third of the dentate molecular layer. The first one, generally referred to as dark, electron-dense degeneration, has been described in a number of brain regions and seems to be a common type of degeneration. A comprehensive review of this topic has been done by Raisman and Matthews 16. The dark type of degeneration of the perforant fiber terminals in the distal third of the dentate molecular layer was similar to that of other areas. The
171
L
L÷M
,
o ~/3 _
_
Fig. 1. A scheme of the position of the triple electrode used for electrolytical lesions of the entorhinal area is in the upper righthand corner. The lesion of the lateral entorhinal area (L) causes terminal degeneration in the distal third (d 1/3) of the dentate molecular layer. A combined lesion of the lateral and medial entorhinal area (L + M) causes degeneration in the distal and medial third of the dentate molecular layer. background matrix of the terminal becomes increasingly dark and the synaptic vesicles, sometimes enlarged, sometimes shrunken, tend to clump together (Fig. 2A and B). The mitochondria were either shrunken or swollen and the outline of the terminal was collapsed. Subsequently, the vesicles and the mitochondria became more difficult to identify. The region of the postsynaptic thickening was the last one to be absorbed, so that the postsynaptic side could have been identified at the latest stage of degeneration investigated (72 h). The second, much less common mode of terminal degeneration has the following morphological features. The synaptic vesicles gain an irregular shape and the mitochondria appear swollen (Fig. 2D). No signs of darkening of the background cytoplasma can be observed (Fig. 2C). Subsequently, the vesicles disintegrate together with the mitochondria, and the terminal becomes considerably swollen (Fig. 2E and F). This type of degeneration was never accompanied by formation of neurofilaments. In some of the already swollen terminals synaptic vesicles could still be recognized clumped together and pushed to one side of the profile. Even at the third day of survival (the longest period of degeneration investigated) the background cytoplasm remained pale and sometimes showed irregular vacuoles. In spite of all the degenerative changes the region of the synaptic thickening remained preserved and the postsynaptic element could be identified. Both dark and pale
172
173 terminals became invaginated into astrocytic processes. The astrocytic reaction to the degeneration first appeared 24 h postoperatively and was characterized by an increase of astrocytic processes, increased content of glycogen and formation o f filaments. It thus appears that the axon terminals of the perforant fibers from the lateral part of the entorhinal area terminate on dendritic spines and have two distinct patterns of degeneration, which occur simultaneously. In an attempt to quantify the degenerating terminals it became obvious that the number can be approximate only. According to Raisman and Matthews16: 'The degree of asynchronism with which degeneration proceeds is such that at any time when terminals are recognizably degenerating but still in contact, a proportion of terminals will not yet show unequivocal signs of degeneration, while others will be completely phagocytized'. In the present material 48 ~o of the total synaptic population showed signs of degeneration (dark and light type) 36 and 48 h postoperatively. That only a part of the population of axon terminals degenerates in the distal third of the dentate molecular layer following lesions in the lateral entorhinal area is consistent with the termination of another system in this r e g i o n - - namely, the association fibers from the CA1 field of the hippocampus 9. The degenerated terminals were about equally distributed between the dark and light types. Since the light type of degeneration is so closely related to the dark type in time and location it is unlikely that the former would be caused by imperfections in tissue preservation. The light, electron-lucent degeneration was for the first time observed as the only mode of degeneration in the ventral cochlear nucleus by De Robertis 5. More recently Gentschev and Sotelo 6 confirmed De Robertis' description, but they also observed the dark type of degeneration in the ventral cochlear nucleus. Westrum z°, Westrum and Black ~1 and O'Neal and Westrum 14 observed the light type of degeneration in the pyriform cortex, spinal trigeminal nucleus and lateral cuneate nucleus, respectively. In these structures the light type of degeneration occurred simultaneously with the dark one. Descriptions of the light type of degeneration resemble very closely the present observation as to the lysis of synaptic vesicles, swelling of the terminal, absence of dark cytoplasm and absence of neurofibrils. Both types of degeneration were also observed in the mossy fibers of the cerebellum is. The light type in the mossy
Fig. 2. Different types of degeneration in the distal third of the molecular layer of the dentate fascia at 48 h survival. A: degenerating terminal - - dark type (DT) contacting a dendritic spine (S). A dendritic profile with an unchanged mitochondrion (M) and unchanged multivesicular body (V) in the upper lefthand corner. B: degenerating terminal - - dark type with a swollen mitochondrion (SM) contacting a spine with a spine apparatus (A). C: degenerating terminal - - light type (LT) contacting a spine (S). In initial state of degeneration, synaptic vesicles are clumped together and the terminal is slightly swollen. D : degenerating terminal - - light type (LT) in an advanced state of degeneration with a swollen mitochondrion (SM), a swollen multivesicular body (SV) and partly lysed vesicles (indicated by an arrow). Degenerating terminal - - dark type (DT). Both degenerating terminals contact dendritic spines (S). In between the degenerating terminals are two glycogen rich astroglial profiles (G). E and F: degenerating terminals - - light type (LT) largely swollen with remnants of lysed vesicles (indicated by arrows) contacting dendritic spines (S). Calibration line 1 pm.
174 fibers did, however, differ from our observation in the occasional appearance of masses of neurofibrils. Although some enlargement of vesicles could be observed in the present material, it was invariably in the terminals which displayed the dark type of degeneration. The vesicle enlargement was never as marked as that described in the avian tectum 4 or rat caudate 9. In the light type of degeneration, in instances where the synaptic vesicles were still present in the swollen terminal, they were irregular in shape rather than enlarged. In most of the studies on terminal degeneration, the survival times were longer (from the third day on) than in the present experiments, which could be one reason why the light type of degeneration has been observed so infrequently. This type of change seems to be an early event in the process of degeneration of some pathways, starting around 20 h postoperatively, and does not occur at any appreciable rate after 3 days of survival 2x. Another reason why these degenerating terminals may not be recognized at later stages of degeneration when they become detached from the postsynaptic side is their electron-lucent appearance which makes them indistinguishable from the surrounding swollen glia. On the other hand, terminals undergoing dark degeneration, even when detached from the postsynaptic side, can still be recognized engulfed in the glial processes. It would seem that the light type may be a more common type of degeneration than has been originally thought. For the period of time investigated, the dark and light types of degeneration occur simultaneously and are therefore likely to be two parallel processes rather than two stages of one process. The question of why terminals of the same pathway undergo different patterns of degeneration cannot, at present, be satisfactorily answered. Biochemical properties of the plasma membrane are the likely factors determining whether the terminal will swell or shrink in the course of degeneration. These properties in turn may be related to the type of transmitter, which the axon terminals carry. Although axon terminals seem to be uniform as to their morphological appearance and content of different organelles, they may differ as to the transmitter substance. In the distal two thirds of the dentate molecular layer acetylcholine and G A B A have been postulated to be the transmitter compounds 12. It could be that the synaptic transmitter is the factor determining the type of degeneration which will occur in a given terminal. This investigation was carried out at the Division of Biology, California Institute of Technology, where it was supported in part by a grant from NSF (NB 07658). The author expresses her gratitude to Dr. A. Van Harreveld for making the financial support as well as laboratory facilities available.
1 BLACKSTAD,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1956) 202-214. 2 BLISS,T. V. P., AND GARDNER-MEDWIN,A. R., Long-lasting potentiation of synaptic transmission
in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 0973) 357-374.
3 BLISS,T. V. P., AND LOMO,T., Long-lasting potentiation of synaptic transmission in the dentate
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9 10
11 12
13 14
15
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area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiok (Lond.), 232 (1973) 331-356. CU~NOD, M., SANDRI,C., AND AKERT, K., Enlarged synaptic vesicles as an early sign of secondary degeneration in the optic nerve terminals of the pigeon, J. Cell ScL, 6 (1970) 605-613. DE ROBERTIS, E., Submicroscopic changes of the synapse after nerve section in the acoustic ganglion of the guinea pig, J. biophys, biochem. Cytol., 2 (1956) 503-512. GENTSCHEV,T., AND SOTELO,C., Degenerative patterns in the ventral cochlear nucleus of the rat after primary deafferentiation, an ultastructural study, Brain Research, 62 (1973) 37-60. HJORT-SIMONSEN,A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neural., 146 (1972) 219-232. HJORT-SIMONSEN,A., AND JEUNE, B., Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation, J. comp. Neurol., 14 (1972) 215-232. KAWANA,E., AKERT, K., AND BRUPPACHER,n., Enlargement of synaptic vesicles as an early sign of terminal degeneration in the rat caudate nucleus, J. comp. Neurol., 142 (1971) 297-308. LAATSCH,R. H., AND COWAN, W. M., Electron microscopic studies of the dentate gyrus of the rat. I. Normal structure with special reference to synaptic organization, J. comp. NeuroL, 128 (1966) 359-396. LOMO, T., Potentiation of monosynaptic EPSPs in the perforant path - - dentate granule cell synapse, Exp. Brain Res., 12 (1971) 46-63. NADLER, V. J., COTMAN, C. W., AND LYNCH, G. S., Subcellular distribution of transmitterrelated enzyme activities in discrete areas of the rat dentate gyrus, Brain Research, 79 (1974) 465-475. NAFSTADT,P. n . J., An electron microscope study on the termination of the perforant path fibers in the hippocampus and the fascia dentata, Z. Zellforsch., 76 (1967) 532-542. O'NEAL, J. T., AND WESTRUM,L. E., The fine structural synaptic organization of the cat lateral cuneate nucleus. A study of sequential alterations in degeneration. Brain Research, 51 (1973) 97-124. RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., The extrinsic afferent, commissural and association fibers of the hippocampus, Brain, 88 (1965) 963-996. RAISMAN,G., AND MATTHEWS,M. R., Degeneration and regeneration of synapses. In G. H. BOURNE (Ed.), The Structure and Function of Nervous Tissue, Vok IV, Academic Press, New York, 1972, pp. 61-104. RICHARDSON,K. C., JARETT, L., AND FINKE, E. M., Embedding in epoxy resins for sectioning in electron microscopy, Stain Technol., 35 (1960) 313-323. SMITH, K. L., JR., HUDGENS, R. W., AND O'LEARY, J. L., An electron microscopic study of degenerative changes in the cat cerebellum after intrinsic and extrinsic lesions, J. comp. NeuroL, 126 (1966) 15-36. VENABLE,J. M., ANDCOGGESHAL,R., A simplified lead citrate stain for use in electron microscopy, J. Cell BioL, 25 0965) 407-408. WESTRUM,L. E., Electron microscopy of degeneration in the lateral olfactory tract and plexiform layer of the prepyriform cortex of the rat, Z. Zellforsch., 98 (1969) 157-187. WESTRUM,L. E., AND BLACK,R. G., Fine structural aspects of the synaptic organization of the spinal trigeminal nucleus (pars interpolaris) of the cat, Brain Research, 25 (1971) 265-287.
169
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Two types of terminal degeneration in the molecular layer of the dentate fascia following lesions of the entorhinal cortex
EVA FIFKOV/~ Department of Psychology, University of Colorado, Boulder, Colo. 80302 (U.S.A.)
(Accepted June 6th, 1975)
One of the hippocampal afferent pathways, known as the perforant path, which originates in the entorhinal area and terminates on the granular cell dendrites in the molecular layer of the fascia dentata, was shown to have potent excitatory properties z-4,11. Being a monosynaptic pathway, as it were, it can serve as a model for the study of changes which might underlie various functional states in this system. For such a type of experiment the pattern of termination of the various entorhinal fibers within the dentate fascia is of primary importance. In preparations impregnated after Nauta, Blackstad 1, Raisman et aL 1~, and Hjort-Simonsen 7 traced terminations from the entorhinal area into the distal third of the dentate molecular layer along the hippocampal fissure. Lesions producing this effect involved mostly the lateral part of the entorhinal area. Degenerated fibers from the medial part of the entorhinal area were traced to the middle third of the dentate molecular layer in Nauta impregnated preparations 8 and were shown to terminate on the spines of granular cell dendrites 13. Comparing the data of Nafstadt 13 and Hjort-Simonsen and Jeune 8 with those of Blackstad ~, Raisman et al. ~5 and Hjort-Simonsen 7, it seems likely that the lateral entorhinal area indeed projects to the distal third of the molecular layer. However, in preparations impregnated after Nauta and examined with the light microscope, the degenerated axon terminal cannot be identified with certainty, because it is difficult to make a definite distinction between degenerated axons and degenerated axon terminals. Since in electron miscroscope preparations this distinction can be readily made, an electron microscope study of the degenerated axon terminals, after lesions of the lateral part of the entorhinal area, seemed to be indicated. Thirteen albino rats were used for this investigation. Under urethane anesthesia a 3 m m trephine opening was drilled in the temporal bone just above the zygomatic arc. A triple electrode, slightly bent, was introduced in such a way that it followed the curvature of the pyriform lobe. The indifferent electrode was positioned in the exposed neck muscles and a 2 m A direct current was passed for 1 min through the uninsulated tips of the electrode. Animals were allowed to survive this procedure for 16, 24, 36, 48, and 72 h. Three unoperated animals were used as controls. After appropriate time periods, the rats were anesthetized with urethane and perfused
170 with a mixture of 4 ~ paraformaldehyde and 0.5~o glutaraldehyde in a phosphate buffer (pH 7.2-7.4). The perfused animals were kept overnight in the refrigerator. The next day the brain was dissected free, and 1 mm thick coronal slabs were taken from the superior hippocampus. From each slab a block from the superior hippocampus including the dentate gyrus was prepared and postfixed in 2~,, OsO4 in phosphate buffer for 1.5 h at 4 °C. Following dehydration in increasing concentrations of acetone and propylene oxide, the blocks were embedded in Epon. During trimming of the block, the 1 # m thick sections were stained with Axur II and methylene blue iv for the purpose of general orientation. The final trim, from which the thin sections were cut, included the molecular layer of the upper blade of the dentate fascia with the hippocampal fissure at one side and with granular cells at the opposite side, thus permitting orientation in the thin section. Silver sections were mounted on formvarcoated slot grids and stained with lead citrate 19. In order to get a survey of the extent of the lesion in the entorhinal area, the pyriform lobe of the operated hemisphere was examined in serial paraffin sections stained after Nissl. Those preparations which revealed lesions in the lateral entorhinal area were used for electron microscope examination. The paraffin Nissl stained sections of the pyri|brm lobe involved also a part of the posterior hippocampus and of the dentate fascia. In animals which survived the lesions for more than 36 h there was a distinctly light stripe in the outer third of the dentate molecular layer, just below the obliterated hippocampal fissure. A similarly positioned light stripe was observed also in the I #m sections obtained from the plastic embedded blocks of the superior part of the dentate fascia. At high magnification (1250 × ) this light stripe revealed a number of vacuoles corresponding in location with the maximum of degeneration observed in electron micrographs. In cases in which the lesion also involved the medial entorhinal area, the light stripe was wider and covered the middle and distal thirds of the dentate molecular layer. This agrees with the finding of degenerations in the middle third of the dentate molecular layer after lesions of the medial entorhinal area 8,13. it therefore can be concluded that fibers from the lateral part of the entorhinal area terminate in the superficial third of the dentate molecular layer throughout the entire extent of the dentate fascia, which is in accord with the observation of Raisman et al. 1'5. The position of the light stripe is schematically demonstrated in Fig. 1. In this region the electron microscope revealed degenerated axon terminals which contacted exclusively dendritic spines. If there were any indications of degeneration 16 h postoperatively, they could not be distinguished from the normal variation of the tissue. The first signs of degeneration became identifiable within 24 h following the lesion and increased markedly in severity following 36 h. Two distinct patterns of degeneration could be observed in axon terminals of the perforant path in the distal third of the dentate molecular layer. The first one, generally referred to as dark, electron-dense degeneration, has been described in a number of brain regions and seems to be a common type of degeneration. A comprehensive review of this topic has been done by Raisman and Matthews 16. The dark type of degeneration of the perforant fiber terminals in the distal third of the dentate molecular layer was similar to that of other areas. The
171
L
L÷M
,
o ~/3 _
_
Fig. 1. A scheme of the position of the triple electrode used for electrolytical lesions of the entorhinal area is in the upper righthand corner. The lesion of the lateral entorhinal area (L) causes terminal degeneration in the distal third (d 1/3) of the dentate molecular layer. A combined lesion of the lateral and medial entorhinal area (L + M) causes degeneration in the distal and medial third of the dentate molecular layer. background matrix of the terminal becomes increasingly dark and the synaptic vesicles, sometimes enlarged, sometimes shrunken, tend to clump together (Fig. 2A and B). The mitochondria were either shrunken or swollen and the outline of the terminal was collapsed. Subsequently, the vesicles and the mitochondria became more difficult to identify. The region of the postsynaptic thickening was the last one to be absorbed, so that the postsynaptic side could have been identified at the latest stage of degeneration investigated (72 h). The second, much less common mode of terminal degeneration has the following morphological features. The synaptic vesicles gain an irregular shape and the mitochondria appear swollen (Fig. 2D). No signs of darkening of the background cytoplasma can be observed (Fig. 2C). Subsequently, the vesicles disintegrate together with the mitochondria, and the terminal becomes considerably swollen (Fig. 2E and F). This type of degeneration was never accompanied by formation of neurofilaments. In some of the already swollen terminals synaptic vesicles could still be recognized clumped together and pushed to one side of the profile. Even at the third day of survival (the longest period of degeneration investigated) the background cytoplasm remained pale and sometimes showed irregular vacuoles. In spite of all the degenerative changes the region of the synaptic thickening remained preserved and the postsynaptic element could be identified. Both dark and pale
172
173 terminals became invaginated into astrocytic processes. The astrocytic reaction to the degeneration first appeared 24 h postoperatively and was characterized by an increase of astrocytic processes, increased content of glycogen and formation o f filaments. It thus appears that the axon terminals of the perforant fibers from the lateral part of the entorhinal area terminate on dendritic spines and have two distinct patterns of degeneration, which occur simultaneously. In an attempt to quantify the degenerating terminals it became obvious that the number can be approximate only. According to Raisman and Matthews16: 'The degree of asynchronism with which degeneration proceeds is such that at any time when terminals are recognizably degenerating but still in contact, a proportion of terminals will not yet show unequivocal signs of degeneration, while others will be completely phagocytized'. In the present material 48 ~o of the total synaptic population showed signs of degeneration (dark and light type) 36 and 48 h postoperatively. That only a part of the population of axon terminals degenerates in the distal third of the dentate molecular layer following lesions in the lateral entorhinal area is consistent with the termination of another system in this r e g i o n - - namely, the association fibers from the CA1 field of the hippocampus 9. The degenerated terminals were about equally distributed between the dark and light types. Since the light type of degeneration is so closely related to the dark type in time and location it is unlikely that the former would be caused by imperfections in tissue preservation. The light, electron-lucent degeneration was for the first time observed as the only mode of degeneration in the ventral cochlear nucleus by De Robertis 5. More recently Gentschev and Sotelo 6 confirmed De Robertis' description, but they also observed the dark type of degeneration in the ventral cochlear nucleus. Westrum z°, Westrum and Black ~1 and O'Neal and Westrum 14 observed the light type of degeneration in the pyriform cortex, spinal trigeminal nucleus and lateral cuneate nucleus, respectively. In these structures the light type of degeneration occurred simultaneously with the dark one. Descriptions of the light type of degeneration resemble very closely the present observation as to the lysis of synaptic vesicles, swelling of the terminal, absence of dark cytoplasm and absence of neurofibrils. Both types of degeneration were also observed in the mossy fibers of the cerebellum is. The light type in the mossy
Fig. 2. Different types of degeneration in the distal third of the molecular layer of the dentate fascia at 48 h survival. A: degenerating terminal - - dark type (DT) contacting a dendritic spine (S). A dendritic profile with an unchanged mitochondrion (M) and unchanged multivesicular body (V) in the upper lefthand corner. B: degenerating terminal - - dark type with a swollen mitochondrion (SM) contacting a spine with a spine apparatus (A). C: degenerating terminal - - light type (LT) contacting a spine (S). In initial state of degeneration, synaptic vesicles are clumped together and the terminal is slightly swollen. D : degenerating terminal - - light type (LT) in an advanced state of degeneration with a swollen mitochondrion (SM), a swollen multivesicular body (SV) and partly lysed vesicles (indicated by an arrow). Degenerating terminal - - dark type (DT). Both degenerating terminals contact dendritic spines (S). In between the degenerating terminals are two glycogen rich astroglial profiles (G). E and F: degenerating terminals - - light type (LT) largely swollen with remnants of lysed vesicles (indicated by arrows) contacting dendritic spines (S). Calibration line 1 pm.
174 fibers did, however, differ from our observation in the occasional appearance of masses of neurofibrils. Although some enlargement of vesicles could be observed in the present material, it was invariably in the terminals which displayed the dark type of degeneration. The vesicle enlargement was never as marked as that described in the avian tectum 4 or rat caudate 9. In the light type of degeneration, in instances where the synaptic vesicles were still present in the swollen terminal, they were irregular in shape rather than enlarged. In most of the studies on terminal degeneration, the survival times were longer (from the third day on) than in the present experiments, which could be one reason why the light type of degeneration has been observed so infrequently. This type of change seems to be an early event in the process of degeneration of some pathways, starting around 20 h postoperatively, and does not occur at any appreciable rate after 3 days of survival 2x. Another reason why these degenerating terminals may not be recognized at later stages of degeneration when they become detached from the postsynaptic side is their electron-lucent appearance which makes them indistinguishable from the surrounding swollen glia. On the other hand, terminals undergoing dark degeneration, even when detached from the postsynaptic side, can still be recognized engulfed in the glial processes. It would seem that the light type may be a more common type of degeneration than has been originally thought. For the period of time investigated, the dark and light types of degeneration occur simultaneously and are therefore likely to be two parallel processes rather than two stages of one process. The question of why terminals of the same pathway undergo different patterns of degeneration cannot, at present, be satisfactorily answered. Biochemical properties of the plasma membrane are the likely factors determining whether the terminal will swell or shrink in the course of degeneration. These properties in turn may be related to the type of transmitter, which the axon terminals carry. Although axon terminals seem to be uniform as to their morphological appearance and content of different organelles, they may differ as to the transmitter substance. In the distal two thirds of the dentate molecular layer acetylcholine and G A B A have been postulated to be the transmitter compounds 12. It could be that the synaptic transmitter is the factor determining the type of degeneration which will occur in a given terminal. This investigation was carried out at the Division of Biology, California Institute of Technology, where it was supported in part by a grant from NSF (NB 07658). The author expresses her gratitude to Dr. A. Van Harreveld for making the financial support as well as laboratory facilities available.
1 BLACKSTAD,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1956) 202-214. 2 BLISS,T. V. P., AND GARDNER-MEDWIN,A. R., Long-lasting potentiation of synaptic transmission
in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 0973) 357-374.
3 BLISS,T. V. P., AND LOMO,T., Long-lasting potentiation of synaptic transmission in the dentate
175
4 5 6 7 8
9 10
11 12
13 14
15
16 17 18
19 20 21
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