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Histochemical detection of orthograde degeneration in the central nervous system of the rat
Brain Research, 54 (1973) 65-73
65
© ElsevierScientificPublishing Company, Amsterdam- Printed in The Netherlands
H I S T O C H E M I C A L D E T E C T I O N OF O R T H O G R A D E D E G E N E R A T I O N IN T H E C E N T R A L NERVOUS SYSTEM OF T H E RAT
OSWALD STEWARD, GARY LYNCH AND CARL COTMAN
Department of Psychobiology, University of California, Irvine, Calif. 92644 (U.S.A.) (Accepted October 25th, 1972)
SUMMARY
Following interruption of an afferent fiber projection in the rat brain, sites of terminal and possibly pre-terminal axonal degeneration accumulate formazan from iodonitrotetrazolium violet (INT). Increased formazan deposition first appears in sites of terminal degeneration several hours after the causative lesion, and persists for as long as 1 month. The effect has been observed in 7 different fiber systems, and thus appears to be a common characteristic of brain regions containing degenerating axon terminals. The method can serve as an adjunct to the classical silver techniques, and moreover has been found particularly useful for the localization of extremely minute brain lesions, such as the tracks by microelectrodes.
INTRODUCTION
If the axon of a neuron is severed, the axon and its synaptic terminals are isolated from the cell body, and consequently degenerate. This orthograde degenerative process, which ultimately results in the disappearance of the axon and its terminal ramifications, is both interesting and useful as a neuroanatomical tool. After experimental interruption of a fiber system, the route of the fibers and their site of termination may be defined through the use of histological stains that more or less selectively demonstrate degenerating axons and their synaptic terminals. The Nauta 9, Nauta-Gygax 10, and Fink-Heimer 8 techniques are examples of such stains. We report here a new histochemical marker for degenerating nerve terminals that is easier and less time consuming than any technique described to date for the localization of terminal degeneration. The histochemical procedure results in massive deposition of a blue mono-tetrazolium salt at sites spatially comparable with terminal degeneration indicated by Fink-Heimer staining.
66
o. STEWARDe t a / .
MATERIALS A N D METHODS
Experimental animals were 200-400 g male Sprague-Dawley rats, obtained from Simonson Labs. Several fiber systems were studied, including the temporo-ammonic system from the entorhinal cortex to the hippocampal formation2,5,1~, the septo-hippocampal system described by Raisman 11, the hippocampal commissural system described by Blackstad 1,2, the hippocampo-septal system described by Raisman et al. la, the cortico-fugal projection from the posterior cortex to the dorsal thalamus, and the afferent optic system. Stereotaxic lesions were placed electrolytically by means ot 0.01 in. thick stainless steel wires, insulated except at the tip. The entorhinal cortex was approached through the cerebral hemisphere, frequently resulting in simultaneous lesions of the visual cortex. Septal lesions were produced electrolytically or by section of the fimbriafornix, and the afferent optic system was destroyed by enucleation of one eye. All lesions were histologically verified by post-fixation of the lesion area in 10 ~ formalin, and subsequent staining with cresyl violet. For iodonitrotetrazolium violet (INT) histochemistry, animals were sacrificed after post-operative intervals ranging from 4 h to 30 days. The brains were rapidly removed, and immediately sectioned on a freezing microtome. The unfixed 50-75/~m sections were placed directly in the histochemical medium described below. The histochemical medium was composed of 0.1 M Tris buffer, pH 7.4, with 1 m M iodonitrotetrazolium violet (INT) and 75 m M sodium succinate. This medium is a modification of one used by Melgren and Blackstad 8 to measure succinate dehydrogenase activity histochemically. In some experiments 1.5 m M reduced nicotinamide dinucleotide ( N A D H ) or 7.8 m M tryptamine was used as the substrate for oxidation, or the I N T was reduced artificially with ammonium sulfide. After the histochemical reaction had proceeded for 5-10 min, the sections were mounted, allowed to dry, and covered with Kaiser's glycerol jelly 6 and a coverslip. For the purpose of comparison, terminal degeneration from lesions of the type
Fig. 1. The appearance of the hippocampal formation after lesion of a major afferent system, the entorhinal cortex, first in a slide from a Fink-Heimer preparation (A), and (B) following incubation in a histochemical medium in which INT is reduced by SDH. The obliterated hippocampal fissure (F) divides the stratum moleculare of the dentate gyrus from the stratum lacunosum-moleculare of the hippocampus, (P) defines the stratum pyramidale, and (G) the stratum granulosum. Terminal degeneration in a Fink-Heimer preparation, appearing as darker staining in the neuropil (A), and increased INT deposition are obvious in the outer two-thirds of the stratum moleculare of the dentate and in the stratum lacunosum-moleculare of the hippocampus immediately adjacent to the hippocampal fissure. In this INT section, the cell body layers are distinguishable as clear bands (P and G). Fink-Heimer stainability and increased INT deposition is also obvious in the hippocampal formation contralateral to the entorhinal lesion in the stratum lacunosum-moleculare of the CA1 region (C) immediately adjacent to the hippocampal fissure. This corresponds to the terminal field from the crossed temporo-ammonic tract, described by Caja114and by Raisman et al.iL The dark staining in the dorsal thalamus represents the thalamic cortico-fugal projection from posterior cortex. In freshly prepared sections (see Figs. 2 and 3), the cell bodies are more clearly visible. The post-lesion survival time for these examples was 4 days for the Fink-Heimer preparation, and 8 days for the INT.
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
67
described above was also mapped with the Fink-Heimer 3 procedure. In these cases, the rats were perfused transcardially 3-4 days after surgery with 10 ~o formalin, and the brains were post-fixed for 10 days before sectioning.
B
C
P
68
o. STEWARD ~'I ~1/. A
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,
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Fig. 2. Formazan deposition (A) and Fink-Heimer staining (B) in the molecular layer of the dentate gyrus ipsilateral to a lesion of the entire entorhinal cortex. The molecular layer extends from the granule cell body layer (G) to the hippocampal fissure (F), and in both A and B above, the stain is deposited in the outer two-thirds of this layer. With the 1NT procedure, utilizing succinate dehydrogenase (SDH) as the reducing enzyme, the cell body layer is indicated by the deposition of a fine red precipitate, while the areas of degeneration appear bright blue. This section was incubated for a shorter time in the histochemical medium than was the example shown in Fig. l, resulting in a less massive deposition of INT. The blue color from the sites of degeneration emanates from the large granules indicated by the vertical arrows. Distributed among the large formazan deposits are smaller grains (1-2 /~m) corresponding at least in size to the Fink-Heimer degeneration products. With increased incubation times, the smaller grains are obscured by the accumulation of formazan, and the cell bodies are more clearly visible. The post-lesion survival time for these examples was 4 days for the Fink-Heimer preparation, and 3 days for the INT.
RESULTS
As early as 4-6 h after lesion of a fiber system, sites of terminal degeneration can be detected by increased accumulation of INT. This increased formazan deposition is maximal 4-8 days after the lesion, but is obvious as early as 1 day and as late as 1 month postoperatively. For example, Figs. 1 and 2 show the deposition in the hippocampus and dentate gyrus 8 days after destruction of the entire ipsilateral entorhinat cortex, and consequent degeneration of the major non-commissural projection to the hippocampal formation2,5,1L Massive formazan deposits appear in the outer twothirds of the stratum moleculare of the dentate gyrus, and in the stratum lacunosummoleculare of the hippocampus proper (terminology according to Lorente de N67). These are regions showing massive terminal degeneration with the Fink-Heimer technique, both in earlier studiesS, 12, and in our own preparations (Figs. 1 and 2). The increased deposition of INT formazan is not peculiar to the degeneration resulting from an entorhinal cortical lesion. For example, Fig. 3 shows the INT depo-
HISTOCHEMICALDETECTIONOF NEURAL DEGENERATION
69
r
I
Fig. 3. The spatial correspondence between Fink-Heimer terminal degeneration (A) and INT deposition (B) is shown after a lesion of the contralateral hippocampus. In this preparation, clear degeneration may be seen in the stratum radiatum and stratum oriens in the hippocampus, corresponding to the published reports of other authorsl,~, lz. In the dentate gyrus, degeneration may be seen in the stratum moleculare of the dentate adjacent to the stratum granulosum. The degeneration in the CA2CA4 regions of the hippocampus tends to be lighter than in CAI, and this is quite consistent with the INT deposition. Post-lesion survival was 3 days for the Fink-Heimer preparation and 1 day for the INT.
sition in the h i p p o c a m p a l f o r m a t i o n after a b l a t i o n of the contralateral hippocampus. In this case, massive I N T deposition occurs in the proximal apical a n d basal dendritic regions of the h i p p o c a m p a l p y r a m i d a l cells, particularly in the CA1 region, a n d in the
¸
70
o. STEWARDet al.
TABLE I Summary describing the appearance of INT formazan in areas of terminal degeneration following various lesions. Column A describes the anatomical system tested; column B simply describes the lesioning method; and column C describes the appearance and distribution of the 1NT formazan granules in areas of afferent fiber termination. All systems were examined 4-6 days following the lesion to the site of origin of afferent fiber systems. Other examples are described in detail in Figs. 1 and 3. Anatomical projection system
Lesioning method
Appearance of formazan
Septo-hippocampal projections
Section of the fimbria- Formazan granules appear in the basal fornix or electrolytic dendritic area of the hippocampal formation, lesions of the medial and in the proximal apical dendritic tree. septal nucleus. Some diffuse granularity also is present in the hilus and in the molecular layer of the dentate immediately adjacent to the stratum granulosum. This corresponds spatially to the degeneration described in Raisman11.
Hippocampo-septal projections
Unilateral aspiration of Ipsilateral to the lesion formazan granules the hippocampal for- are evident in the dorsal and lateral septum, while contralaterally, only in the dorsal mation. septum. This is in agreement with Raisman et al.l:L
Posterior cortico-fugal projections to the dorsal thalamus
Lesioned coincidental- Large formazan granules are obvious in the ly with entorhinal cor- thalamus dorso-medial to the lateral geniculate nucleus, sometimes includingthe most tex. dorsal aspects of the lateral geniculate. This can be seen clearly as a dark area in Fig. 1.
Retino-geniculate projections Enucleation of one eye. Extremely small formazan granules can be found diffusely in the lateral geniculate nucleus. The formazan increase in this system is not as profound as that seen in the above examples.
proximal dendritic area of the granule cells of the dentate gyrus. As in the case of e n t o r h i n a l cortex lesion, the location of the deposits coincides with terminal degeneration revealed by the F i n k - H e i m e r m e t h o d (Fig. 3) and by earlier studies of N a u t a stainingl,Z, u . As Table I indicates, we have f o u n d a similar correspondence between I N T deposition a n d terminal degeneration following lesion of the septo-hippocampal system, the cortico-fugal projections from the posterior cortex to the thalamus, the projection of the septum to the midline thalamus, a n d in a t t e n u a t e d form in the projection from the retina to the lateral geniculate nucleus. In all these systems, areas of increased I N T deposition are bright blue against a pale blue or slightly red background. I n systems where degenerating fiber tracts were observable, there was some increase in I N T deposition along the degenerating fiber tract, but this was not as dramatic as the deposition at sites of terminal degeneration. This increase in f o r m a z a n deposition in I N T histochemistry is extremely useful in localizing small lesions in the central nervous system of the rat a n d in tracing the
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
71
Fig. 4. The perforation of a micropipette recording electrode, outlined by INT formazan deposition. Two other faint electrode tracks are visible on each side of the major track (unlabeled arrows), but these are out of the plane of section. Since the INT halo around the electrode perforation is over 100 #m wide, localization of tracks out of the plane of section is quite feasible. It is of interest to note the discontinuity of the INT path. At that level of the hippocampal formation where neuropil becomes a cell body layer (at the border between the stratum moleculare and stratum granulosum of the dentate), the INT path evidently ceases, only to reappear when the cell body layer has been transversed. Evidently, INT only 'reacts' with some kind of degeneration occurring in the neuropil, and does not react with degeneration products from cell bodies.
resulting terminal degeneration. For example, after insertion of the electrode into the entorhinal cortex, the termination of the perforant path may be easily detected in the hippocampal formation even if no lesioning current is passed. If the lesion produces massive degeneration (for example, after complete destruction of the ipsilateral entorhinal cortex or contralateral hippocampus) the area of INT deposition is distinguishable even without the aid of a magnifying device. An indication of the unusual sensitivity of this method in identifying minute lesions is shown in Fig. 4. Here a saline-filled micropipette for electrophysiological studies was lowered into the hippocampus. The path of the electrode is outlined sharply by a zone of INT granules several hundred micra wide. It is occasionally possible to localize the track of microelectrodes with the Fink-Heimer stain, again by the deposition of an indicator for terminal degeneration, but the utility of this method is limited by the relatively long survival times required (2-3 days) and the time consuming nature of the Fink-Heimer procedure. We have found that microelectrode tracks may be traced easily with the INT method even with less than 1 day survival. These observations emphasize the
72
O. S T E W A R D
eta].
sensitivity of the technique for the localization of minute amounts of degenerating nervous tissue. DISCUSSION
Deposition of INT formazan at sites of terminal degeneration offers an extremely useful means of localizing such degeneration. The histochemical procedure is simple, fast and requires only one treatment viz., incubation in the histochemical medium. For this incubation, we have found the succinate dehydrogenase (SDH) linked reduction of the 1NT to be the most reliable. With this method, there is a wide flexibility in post-lesion survival time, since the effect appears a few hours after the lesion and persists for at least one month. We have found that the largest increase in formazan deposition over background occurs 4-8 days after the lesion. Even during the early post-lesion periods, the stain is extremely sensitive; for example, the minute track left by a micropipette measuring only a few micra at the tip is surrounded by INT formazan granules. These characteristics make the stain useful first as a rapid means to identify areas of dense terminal degeneration, second, as an adjunct to the more arduous and often capricious silver stains and, third, as an efficient method to localize minute electrode tracks after electrophysiological experiments. This last benefit of the method is particularly valuable, since the localization of microelectrode tracks by conventional histological methods is usually difficult and unreliable. The method also has disadvantages. First, the INT granules are relatively large and diffuse (50-100 #m) and it is therefore impossible that one 1NT granule could represent a single degenerating axon terminal. Within areas of heavy INT deposition there is however a suggestion of granularity within the INT deposits approximating the size of a degenerating synaptic terminal (or Fink-Heimer granule of 1.0/~m) (see Fig. 2). The positive identification of these granules must await superior histological preparations. A second disadvantage is that the stain only works in unfixed sections aS, thus limiting the time which may elapse between removal of the brain and the histochemical process. In addition, histological preservation of the sections is difficult without formalin fixation; but since it is possible to formalin-fix the sections after the short incubation in the histochemical medium 15, this is a less significant disadvantage. Several areas of the normal rat brain also accumulate INT formazan. These areas include several brain stem nuclei, notably the nucleus of the hypoglossal nerve and the inferior olive, and to some extent also the pyramidal cell layer of the cortex and the interpeduncular nucleus. Normal areas of unusually high INT accumulating ability must be noted if this stain is to be diagnostic for degeneration. A final limitation which this stain may share with silver stains is some capriciousness with regard to the type of degeneration which is stainable. For example, the afferent optic projection is difficult to stain in INT procedures (see Table I) or with silver techniques 4. Since we have in general made no attempt to define optimal survival time following lesion for each fiber system, it is possible that our lack of success with the optic projections reflects this. It is also possible that the INT accumulation maximally occurs only with certain types of orthograde degeneration.
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
73
A s is the case with the classical silver stains, it has been impossible thus far to d e t e r m i n e the m e c h a n i s m o f the I N T a c c u m u l a t i o n by degenerating tissue, a l t h o u g h some possibilities have been eliminated. F o r e x a m p l e the a c c u m u l a t i o n is clearly i n d e p e n d e n t o f the activity o f oxidizing e n z y m e s l L Beyond this, I N T a c c u m u l a t i o n could be a reflection o f some process involved in glial phagocytosis, a l t h o u g h the f o r m a z a n granules are n o t associated with a n y methylene-blue positive cell bodies in the n e u r o p i l o f the h i p p o c a m p u s . Alternatively, if the response is neural in origin, it could either be a degenerative change associated with the disintegrating synaptic terminal, or it c o u l d be some highly selective response o f some p o s t s y n a p t i c element to denervation. W h a t e v e r the m e c h a n i s m o f the a c c u m u l a t i o n , it is clear t h a t the increased I N T f o r m a z a n d e p o s i t i o n is spatially associated with degenerating nerve t e r m i n a l s in all systems studied. F o r this reason, it can serve as a valuable m e a n s o f m a r k i n g areas o f t e r m i n a l degeneration. ACKNOWLEDGEMENTS
S u p p o r t e d b y Research G r a n t s N S F G B 35315X a n d N I M H M H 19793 to G . L . ; N I M H 19691 to C.C. ; a n d U.S. Public H e a l t h Service a n d R e s e a r c h Fellowship 1FO1 M H 53558-01 to O.S. REFERENCES 1 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-521. 2 BLACKSTAD,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1958) 202-214. 3 FINK, R. P., ANDHEIMER,L., Two methods for selective silver impregnation of degeneration axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 4 GUILLERY,R. W., Light and electron microscopical studies of normal and degenerating axons. In W. J. H. NAUTAAND S. O. E. EaBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970. 5 HJORTH-SIMONSEN,A., ANDJEUNE,l. , Origin and termination oftbe hippocampal perforant path in the rat, studied by silver impregnation, J. comp. Neurol., 144 (1971) 215-232. 6 HUMASON,G. L., Animal Tissue Techniques, Freeman, San Francisco, 1967, p. 130. 7 LORENTEDE N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 113-177. 8 MELGREN,S. I., ANDBLACKSTAD,T. W., Oxidative enzymes (tetrazolium reductases) in the hippocampal region of the rat, distribution and relation to architectonics, Z. Zellforsch., 78 (1967) 167-207. 9 NAUTA,W. J. H., Ober die sogenannte terminale Degeneration im Zentralnervensystem und ihre Darstellung dutch Silberimpr/ignation, Schweiz. Arch. Neurol. Psychiat., 66 (1950) 353-376. 10 NAUTA,W. J. H., AND GYGAX,P. A., Silver impregnation of degenerating axons in the central nervous system. A modified technic, Stain Technol., 29 (1954) 91-93. 11 RAISMAN,G., The connexions of the septum, Brain, 89 (1966) 317-348. 12 RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., The extrinsic afferent commissural and association fibers of the hippocampus, Brain, 88 (1966) 963-996. 13 RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., An experimental analysis of the efferent projection of the hippocampus, Brain, 89 (1968) 83-108. 14 RAM6NYCAJAL,S., Histologie du Systdme Nerveux de l'Homme et des Vertdbrds, Vol. 2, Maloine,
Paris, 1911. 15 STEWARD,O., COTMAN,C., ANOLYNCh, G., The histochemical nature of altered formazan deposition in brain of sites undergoing orthograde degenerative alterations, In preparation.
65
© ElsevierScientificPublishing Company, Amsterdam- Printed in The Netherlands
H I S T O C H E M I C A L D E T E C T I O N OF O R T H O G R A D E D E G E N E R A T I O N IN T H E C E N T R A L NERVOUS SYSTEM OF T H E RAT
OSWALD STEWARD, GARY LYNCH AND CARL COTMAN
Department of Psychobiology, University of California, Irvine, Calif. 92644 (U.S.A.) (Accepted October 25th, 1972)
SUMMARY
Following interruption of an afferent fiber projection in the rat brain, sites of terminal and possibly pre-terminal axonal degeneration accumulate formazan from iodonitrotetrazolium violet (INT). Increased formazan deposition first appears in sites of terminal degeneration several hours after the causative lesion, and persists for as long as 1 month. The effect has been observed in 7 different fiber systems, and thus appears to be a common characteristic of brain regions containing degenerating axon terminals. The method can serve as an adjunct to the classical silver techniques, and moreover has been found particularly useful for the localization of extremely minute brain lesions, such as the tracks by microelectrodes.
INTRODUCTION
If the axon of a neuron is severed, the axon and its synaptic terminals are isolated from the cell body, and consequently degenerate. This orthograde degenerative process, which ultimately results in the disappearance of the axon and its terminal ramifications, is both interesting and useful as a neuroanatomical tool. After experimental interruption of a fiber system, the route of the fibers and their site of termination may be defined through the use of histological stains that more or less selectively demonstrate degenerating axons and their synaptic terminals. The Nauta 9, Nauta-Gygax 10, and Fink-Heimer 8 techniques are examples of such stains. We report here a new histochemical marker for degenerating nerve terminals that is easier and less time consuming than any technique described to date for the localization of terminal degeneration. The histochemical procedure results in massive deposition of a blue mono-tetrazolium salt at sites spatially comparable with terminal degeneration indicated by Fink-Heimer staining.
66
o. STEWARDe t a / .
MATERIALS A N D METHODS
Experimental animals were 200-400 g male Sprague-Dawley rats, obtained from Simonson Labs. Several fiber systems were studied, including the temporo-ammonic system from the entorhinal cortex to the hippocampal formation2,5,1~, the septo-hippocampal system described by Raisman 11, the hippocampal commissural system described by Blackstad 1,2, the hippocampo-septal system described by Raisman et al. la, the cortico-fugal projection from the posterior cortex to the dorsal thalamus, and the afferent optic system. Stereotaxic lesions were placed electrolytically by means ot 0.01 in. thick stainless steel wires, insulated except at the tip. The entorhinal cortex was approached through the cerebral hemisphere, frequently resulting in simultaneous lesions of the visual cortex. Septal lesions were produced electrolytically or by section of the fimbriafornix, and the afferent optic system was destroyed by enucleation of one eye. All lesions were histologically verified by post-fixation of the lesion area in 10 ~ formalin, and subsequent staining with cresyl violet. For iodonitrotetrazolium violet (INT) histochemistry, animals were sacrificed after post-operative intervals ranging from 4 h to 30 days. The brains were rapidly removed, and immediately sectioned on a freezing microtome. The unfixed 50-75/~m sections were placed directly in the histochemical medium described below. The histochemical medium was composed of 0.1 M Tris buffer, pH 7.4, with 1 m M iodonitrotetrazolium violet (INT) and 75 m M sodium succinate. This medium is a modification of one used by Melgren and Blackstad 8 to measure succinate dehydrogenase activity histochemically. In some experiments 1.5 m M reduced nicotinamide dinucleotide ( N A D H ) or 7.8 m M tryptamine was used as the substrate for oxidation, or the I N T was reduced artificially with ammonium sulfide. After the histochemical reaction had proceeded for 5-10 min, the sections were mounted, allowed to dry, and covered with Kaiser's glycerol jelly 6 and a coverslip. For the purpose of comparison, terminal degeneration from lesions of the type
Fig. 1. The appearance of the hippocampal formation after lesion of a major afferent system, the entorhinal cortex, first in a slide from a Fink-Heimer preparation (A), and (B) following incubation in a histochemical medium in which INT is reduced by SDH. The obliterated hippocampal fissure (F) divides the stratum moleculare of the dentate gyrus from the stratum lacunosum-moleculare of the hippocampus, (P) defines the stratum pyramidale, and (G) the stratum granulosum. Terminal degeneration in a Fink-Heimer preparation, appearing as darker staining in the neuropil (A), and increased INT deposition are obvious in the outer two-thirds of the stratum moleculare of the dentate and in the stratum lacunosum-moleculare of the hippocampus immediately adjacent to the hippocampal fissure. In this INT section, the cell body layers are distinguishable as clear bands (P and G). Fink-Heimer stainability and increased INT deposition is also obvious in the hippocampal formation contralateral to the entorhinal lesion in the stratum lacunosum-moleculare of the CA1 region (C) immediately adjacent to the hippocampal fissure. This corresponds to the terminal field from the crossed temporo-ammonic tract, described by Caja114and by Raisman et al.iL The dark staining in the dorsal thalamus represents the thalamic cortico-fugal projection from posterior cortex. In freshly prepared sections (see Figs. 2 and 3), the cell bodies are more clearly visible. The post-lesion survival time for these examples was 4 days for the Fink-Heimer preparation, and 8 days for the INT.
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
67
described above was also mapped with the Fink-Heimer 3 procedure. In these cases, the rats were perfused transcardially 3-4 days after surgery with 10 ~o formalin, and the brains were post-fixed for 10 days before sectioning.
B
C
P
68
o. STEWARD ~'I ~1/. A
•
ia
,
IG
Fig. 2. Formazan deposition (A) and Fink-Heimer staining (B) in the molecular layer of the dentate gyrus ipsilateral to a lesion of the entire entorhinal cortex. The molecular layer extends from the granule cell body layer (G) to the hippocampal fissure (F), and in both A and B above, the stain is deposited in the outer two-thirds of this layer. With the 1NT procedure, utilizing succinate dehydrogenase (SDH) as the reducing enzyme, the cell body layer is indicated by the deposition of a fine red precipitate, while the areas of degeneration appear bright blue. This section was incubated for a shorter time in the histochemical medium than was the example shown in Fig. l, resulting in a less massive deposition of INT. The blue color from the sites of degeneration emanates from the large granules indicated by the vertical arrows. Distributed among the large formazan deposits are smaller grains (1-2 /~m) corresponding at least in size to the Fink-Heimer degeneration products. With increased incubation times, the smaller grains are obscured by the accumulation of formazan, and the cell bodies are more clearly visible. The post-lesion survival time for these examples was 4 days for the Fink-Heimer preparation, and 3 days for the INT.
RESULTS
As early as 4-6 h after lesion of a fiber system, sites of terminal degeneration can be detected by increased accumulation of INT. This increased formazan deposition is maximal 4-8 days after the lesion, but is obvious as early as 1 day and as late as 1 month postoperatively. For example, Figs. 1 and 2 show the deposition in the hippocampus and dentate gyrus 8 days after destruction of the entire ipsilateral entorhinat cortex, and consequent degeneration of the major non-commissural projection to the hippocampal formation2,5,1L Massive formazan deposits appear in the outer twothirds of the stratum moleculare of the dentate gyrus, and in the stratum lacunosummoleculare of the hippocampus proper (terminology according to Lorente de N67). These are regions showing massive terminal degeneration with the Fink-Heimer technique, both in earlier studiesS, 12, and in our own preparations (Figs. 1 and 2). The increased deposition of INT formazan is not peculiar to the degeneration resulting from an entorhinal cortical lesion. For example, Fig. 3 shows the INT depo-
HISTOCHEMICALDETECTIONOF NEURAL DEGENERATION
69
r
I
Fig. 3. The spatial correspondence between Fink-Heimer terminal degeneration (A) and INT deposition (B) is shown after a lesion of the contralateral hippocampus. In this preparation, clear degeneration may be seen in the stratum radiatum and stratum oriens in the hippocampus, corresponding to the published reports of other authorsl,~, lz. In the dentate gyrus, degeneration may be seen in the stratum moleculare of the dentate adjacent to the stratum granulosum. The degeneration in the CA2CA4 regions of the hippocampus tends to be lighter than in CAI, and this is quite consistent with the INT deposition. Post-lesion survival was 3 days for the Fink-Heimer preparation and 1 day for the INT.
sition in the h i p p o c a m p a l f o r m a t i o n after a b l a t i o n of the contralateral hippocampus. In this case, massive I N T deposition occurs in the proximal apical a n d basal dendritic regions of the h i p p o c a m p a l p y r a m i d a l cells, particularly in the CA1 region, a n d in the
¸
70
o. STEWARDet al.
TABLE I Summary describing the appearance of INT formazan in areas of terminal degeneration following various lesions. Column A describes the anatomical system tested; column B simply describes the lesioning method; and column C describes the appearance and distribution of the 1NT formazan granules in areas of afferent fiber termination. All systems were examined 4-6 days following the lesion to the site of origin of afferent fiber systems. Other examples are described in detail in Figs. 1 and 3. Anatomical projection system
Lesioning method
Appearance of formazan
Septo-hippocampal projections
Section of the fimbria- Formazan granules appear in the basal fornix or electrolytic dendritic area of the hippocampal formation, lesions of the medial and in the proximal apical dendritic tree. septal nucleus. Some diffuse granularity also is present in the hilus and in the molecular layer of the dentate immediately adjacent to the stratum granulosum. This corresponds spatially to the degeneration described in Raisman11.
Hippocampo-septal projections
Unilateral aspiration of Ipsilateral to the lesion formazan granules the hippocampal for- are evident in the dorsal and lateral septum, while contralaterally, only in the dorsal mation. septum. This is in agreement with Raisman et al.l:L
Posterior cortico-fugal projections to the dorsal thalamus
Lesioned coincidental- Large formazan granules are obvious in the ly with entorhinal cor- thalamus dorso-medial to the lateral geniculate nucleus, sometimes includingthe most tex. dorsal aspects of the lateral geniculate. This can be seen clearly as a dark area in Fig. 1.
Retino-geniculate projections Enucleation of one eye. Extremely small formazan granules can be found diffusely in the lateral geniculate nucleus. The formazan increase in this system is not as profound as that seen in the above examples.
proximal dendritic area of the granule cells of the dentate gyrus. As in the case of e n t o r h i n a l cortex lesion, the location of the deposits coincides with terminal degeneration revealed by the F i n k - H e i m e r m e t h o d (Fig. 3) and by earlier studies of N a u t a stainingl,Z, u . As Table I indicates, we have f o u n d a similar correspondence between I N T deposition a n d terminal degeneration following lesion of the septo-hippocampal system, the cortico-fugal projections from the posterior cortex to the thalamus, the projection of the septum to the midline thalamus, a n d in a t t e n u a t e d form in the projection from the retina to the lateral geniculate nucleus. In all these systems, areas of increased I N T deposition are bright blue against a pale blue or slightly red background. I n systems where degenerating fiber tracts were observable, there was some increase in I N T deposition along the degenerating fiber tract, but this was not as dramatic as the deposition at sites of terminal degeneration. This increase in f o r m a z a n deposition in I N T histochemistry is extremely useful in localizing small lesions in the central nervous system of the rat a n d in tracing the
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
71
Fig. 4. The perforation of a micropipette recording electrode, outlined by INT formazan deposition. Two other faint electrode tracks are visible on each side of the major track (unlabeled arrows), but these are out of the plane of section. Since the INT halo around the electrode perforation is over 100 #m wide, localization of tracks out of the plane of section is quite feasible. It is of interest to note the discontinuity of the INT path. At that level of the hippocampal formation where neuropil becomes a cell body layer (at the border between the stratum moleculare and stratum granulosum of the dentate), the INT path evidently ceases, only to reappear when the cell body layer has been transversed. Evidently, INT only 'reacts' with some kind of degeneration occurring in the neuropil, and does not react with degeneration products from cell bodies.
resulting terminal degeneration. For example, after insertion of the electrode into the entorhinal cortex, the termination of the perforant path may be easily detected in the hippocampal formation even if no lesioning current is passed. If the lesion produces massive degeneration (for example, after complete destruction of the ipsilateral entorhinal cortex or contralateral hippocampus) the area of INT deposition is distinguishable even without the aid of a magnifying device. An indication of the unusual sensitivity of this method in identifying minute lesions is shown in Fig. 4. Here a saline-filled micropipette for electrophysiological studies was lowered into the hippocampus. The path of the electrode is outlined sharply by a zone of INT granules several hundred micra wide. It is occasionally possible to localize the track of microelectrodes with the Fink-Heimer stain, again by the deposition of an indicator for terminal degeneration, but the utility of this method is limited by the relatively long survival times required (2-3 days) and the time consuming nature of the Fink-Heimer procedure. We have found that microelectrode tracks may be traced easily with the INT method even with less than 1 day survival. These observations emphasize the
72
O. S T E W A R D
eta].
sensitivity of the technique for the localization of minute amounts of degenerating nervous tissue. DISCUSSION
Deposition of INT formazan at sites of terminal degeneration offers an extremely useful means of localizing such degeneration. The histochemical procedure is simple, fast and requires only one treatment viz., incubation in the histochemical medium. For this incubation, we have found the succinate dehydrogenase (SDH) linked reduction of the 1NT to be the most reliable. With this method, there is a wide flexibility in post-lesion survival time, since the effect appears a few hours after the lesion and persists for at least one month. We have found that the largest increase in formazan deposition over background occurs 4-8 days after the lesion. Even during the early post-lesion periods, the stain is extremely sensitive; for example, the minute track left by a micropipette measuring only a few micra at the tip is surrounded by INT formazan granules. These characteristics make the stain useful first as a rapid means to identify areas of dense terminal degeneration, second, as an adjunct to the more arduous and often capricious silver stains and, third, as an efficient method to localize minute electrode tracks after electrophysiological experiments. This last benefit of the method is particularly valuable, since the localization of microelectrode tracks by conventional histological methods is usually difficult and unreliable. The method also has disadvantages. First, the INT granules are relatively large and diffuse (50-100 #m) and it is therefore impossible that one 1NT granule could represent a single degenerating axon terminal. Within areas of heavy INT deposition there is however a suggestion of granularity within the INT deposits approximating the size of a degenerating synaptic terminal (or Fink-Heimer granule of 1.0/~m) (see Fig. 2). The positive identification of these granules must await superior histological preparations. A second disadvantage is that the stain only works in unfixed sections aS, thus limiting the time which may elapse between removal of the brain and the histochemical process. In addition, histological preservation of the sections is difficult without formalin fixation; but since it is possible to formalin-fix the sections after the short incubation in the histochemical medium 15, this is a less significant disadvantage. Several areas of the normal rat brain also accumulate INT formazan. These areas include several brain stem nuclei, notably the nucleus of the hypoglossal nerve and the inferior olive, and to some extent also the pyramidal cell layer of the cortex and the interpeduncular nucleus. Normal areas of unusually high INT accumulating ability must be noted if this stain is to be diagnostic for degeneration. A final limitation which this stain may share with silver stains is some capriciousness with regard to the type of degeneration which is stainable. For example, the afferent optic projection is difficult to stain in INT procedures (see Table I) or with silver techniques 4. Since we have in general made no attempt to define optimal survival time following lesion for each fiber system, it is possible that our lack of success with the optic projections reflects this. It is also possible that the INT accumulation maximally occurs only with certain types of orthograde degeneration.
HISTOCHEMICAL DETECTION OF NEURAL DEGENERATION
73
A s is the case with the classical silver stains, it has been impossible thus far to d e t e r m i n e the m e c h a n i s m o f the I N T a c c u m u l a t i o n by degenerating tissue, a l t h o u g h some possibilities have been eliminated. F o r e x a m p l e the a c c u m u l a t i o n is clearly i n d e p e n d e n t o f the activity o f oxidizing e n z y m e s l L Beyond this, I N T a c c u m u l a t i o n could be a reflection o f some process involved in glial phagocytosis, a l t h o u g h the f o r m a z a n granules are n o t associated with a n y methylene-blue positive cell bodies in the n e u r o p i l o f the h i p p o c a m p u s . Alternatively, if the response is neural in origin, it could either be a degenerative change associated with the disintegrating synaptic terminal, or it c o u l d be some highly selective response o f some p o s t s y n a p t i c element to denervation. W h a t e v e r the m e c h a n i s m o f the a c c u m u l a t i o n , it is clear t h a t the increased I N T f o r m a z a n d e p o s i t i o n is spatially associated with degenerating nerve t e r m i n a l s in all systems studied. F o r this reason, it can serve as a valuable m e a n s o f m a r k i n g areas o f t e r m i n a l degeneration. ACKNOWLEDGEMENTS
S u p p o r t e d b y Research G r a n t s N S F G B 35315X a n d N I M H M H 19793 to G . L . ; N I M H 19691 to C.C. ; a n d U.S. Public H e a l t h Service a n d R e s e a r c h Fellowship 1FO1 M H 53558-01 to O.S. REFERENCES 1 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-521. 2 BLACKSTAD,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1958) 202-214. 3 FINK, R. P., ANDHEIMER,L., Two methods for selective silver impregnation of degeneration axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 4 GUILLERY,R. W., Light and electron microscopical studies of normal and degenerating axons. In W. J. H. NAUTAAND S. O. E. EaBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970. 5 HJORTH-SIMONSEN,A., ANDJEUNE,l. , Origin and termination oftbe hippocampal perforant path in the rat, studied by silver impregnation, J. comp. Neurol., 144 (1971) 215-232. 6 HUMASON,G. L., Animal Tissue Techniques, Freeman, San Francisco, 1967, p. 130. 7 LORENTEDE N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 113-177. 8 MELGREN,S. I., ANDBLACKSTAD,T. W., Oxidative enzymes (tetrazolium reductases) in the hippocampal region of the rat, distribution and relation to architectonics, Z. Zellforsch., 78 (1967) 167-207. 9 NAUTA,W. J. H., Ober die sogenannte terminale Degeneration im Zentralnervensystem und ihre Darstellung dutch Silberimpr/ignation, Schweiz. Arch. Neurol. Psychiat., 66 (1950) 353-376. 10 NAUTA,W. J. H., AND GYGAX,P. A., Silver impregnation of degenerating axons in the central nervous system. A modified technic, Stain Technol., 29 (1954) 91-93. 11 RAISMAN,G., The connexions of the septum, Brain, 89 (1966) 317-348. 12 RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., The extrinsic afferent commissural and association fibers of the hippocampus, Brain, 88 (1966) 963-996. 13 RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., An experimental analysis of the efferent projection of the hippocampus, Brain, 89 (1968) 83-108. 14 RAM6NYCAJAL,S., Histologie du Systdme Nerveux de l'Homme et des Vertdbrds, Vol. 2, Maloine,
Paris, 1911. 15 STEWARD,O., COTMAN,C., ANOLYNCh, G., The histochemical nature of altered formazan deposition in brain of sites undergoing orthograde degenerative alterations, In preparation.