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An ultrastructural study of ipsilateral corticospinal terminations in the rat
Brain Research, 89 (1975) 327-330
327
Elsevier Sclenttfic Pubhshlng Company, Amsterdam - Printed m The Netherlands
Short Communications
An ultrastructural study of ipsilateral corticospinal terminations in the rat
J. ROSS McCLUNG* AND ANTHONY J CASTRO Depat tment o] Anutotto', Loutstana State Untvet slty Me&cal Center, New O~lean~, La 70112 ( U S A )
(Accepted February 18th, 1975)
Using both hght and electron microscopic techmques, Brown a has reported that the rodent cortmospmal tract ~s entirely crossed. [t was found to terminate exclusively in the dorsal horn of the spinal cord contralateral to the side of its cortical origin. However, recent mvestlgatlons~, 5,6 have demonstrated, at the light microscopic level, the presence of ,psllateral cortmospmal fibers These ipsllateral fibers were observed m adult rats that had sustained unilateral, fi'ontal cortical lesions as neonates They originated from frontal cortical areas on the side opposite to the neonatal lesion The rodent cortmospmal tract exhibits considerable postnatal development m that it does not reach its medullary decussatlon until after birth a, Accordingly, ,t has been hypothesized by Leong and Lurid 6 that crossing normally occurs due to an interaction between the two comcospmal tracts as they grow through the medulla. This interaction is thought to be disrupted by a neonatal cortical lesJon which destroys one of the pathways and thereby causes an incomplete decussatlon of the remammg tract. There ,s no direct evidence in support of the functional significance, if any, of these ipsilateral projections. And even more fundamentally, there is no ultrastructural ewdence of synaptm endings associated with this aberrant pathway. Therefore, the object of this report is to present anatomical evidence of functional synapses corresponding to the aberrant ipsllateral comcosplnal tract Five 1-day-old Long-Evans male rats received unilateral frontal cortical ablations by asplratmn under hypothermia. Two additional neonates that recmved only scalp incisions served as hypothermic and surgical controls. Three to 6 months later, ammals that sustained neonatal cortmal ablatmn received a les~on of the opposite (previously unoperated) frontal cortex while under sodmm pentobarbital anesthesia. The two control animals were also anesthetized at this time. At 3 or 6 days after the second surgery, ammals were perfused through the aorta with 0 1 M cacodylate * Present address Department of Anatomy, Medmal College of Vwgmla, MCV Station, P O Box 906, Rmhmond, Va. 23298, U S A
328
Fig. 1. Dark degenerating axon (da) in the dorsal horn of the cervical spinal cord 3 days after ipsilateral cortical ablation. × 25,500. buffer (pH 7.3) followed by 500 ml of a 4 ~ paraformaldehyde and 0.5 5/ooglutaraldehyde solution in the same buffer. Cervical spinal cord cross-sections were cut freehand at approximately 0.5 mm thickness, trimmed to include only the gray matter and dorsal funiculi, osmicated, and embedded in Epon. Toluidine blue-stained sections (1/~m) were used for orientation of corresponding 80-90 nm (gold and silver) thin sections which were stained with uranyl acetate and lead citrate and viewed with a Philips EM 300. In the spinal cord on the side ipsilateral to the second (adult) lesion, many degenerated myelinated axons were observed in the atrophied corticospinal tract and the adjoining portion of the dorsal horn. These degenerated axons (Fig. 1) displayed typical dark degeneration changes ~. Similar degenerated axons were found in the medial aspect of the dorsal horn within zones equivalent to laminae II-V of Rexe& ; however, progressively fewer were observed either dorsally or laterally in the dorsal horn. Degenerated, unmyelinated terminal axons were most commonly observed just ventral to the substantia gelatinosa in the area corresponding to lamina III, and recognizable synaptic contacts (Fig. 2) were frequently found between these axon terminals and small dendrites in the region. At 3 days postoperative, these synaptic boutons (Fig. 2) displayed a dark axoplasmic matrix which partially masked the decomposed plasma membranes, mitochondria and synaptic vesicle remnants. However, the shrinkage which resulted from this degeneration occasionally enhanced visualization of the synaptic contacts because of their tight adhesion to the postsynaptic membrane
329
Fig. 2 Dark, degenerating bouton (db) in the dorsal horn (lamina 111) of the cervical spmal cord 3 days after lpsllateral cortical ablation The reset illustrates, at hJgher magmfication, the synaptlC contact of th~s bouton \ 24,500, reset, , 67,500
(insert Fig. 2). By 6 days postoperative, axonal degeneration was still present, but most unmyehnated degenerated structures were no longer recogmzable as defimtJve synaptlc boutons because of advanced degeneration and glial envelopment. The presence of darkened, ghal-enveloped, unmyelinated profiles rather than clearly recogmzable darkened boutons further indicates that the dense boutons observed at 3 days were in fact degenerating corticospmal terminals. These degenerating boutons are believed to represent the terminations of degenerating axons which appeared in and emerged from the dorsal funiculus on the side ipsilateral to the second les~on. They are not considered to be residual degenerated elements caused by the initial neonatal cortical les~on These ammals survived 3 6 months after neonatal ablation, and th~s amount of time ~s considerably sufficient for degeneration debris to be removed. Furthermore, light microscopic analysis, using silver staining methods for degenerated axons and their synaptlc endings, revealed no evidence of cortlcospinal degeneration ~n animals that were killed only 2 months after neonatal frontal cortical ablation 2. It ~s concluded that the degenerating boutons observed m the present study represent anatomical evidence for functional synapses being established by aberrant lpsllateral corticospmal fibers which resulted from neonatal cortical injury. The pattern of termination of these fibers appears to correspond with the normal contralateral distribution of cortlcospmal fibers as reported by Brown 1 and this is being more precisely examined with continuing experimentation.
330 T h i s r e b e a r c h vva~ s u p p o r t e d by a [~OUlSldn;.t S t a t e U m ~ e r b J t \ N~.ht)t~J ol Medw~J~c G e n e r a l R e s e a r c h S u p p o r t G r a n t zw~arded t o J R M a n d a G r a n l N S 1 1 2 0 0 - 0 2 IF,,n~ the National Institute of NeurologJc Drseascs and Stroke to A J ¢ T h e p h o t o g r a p h i c a s s i s t a n c e t~l M r
I BROWN,
G a r b l b K e H l m a n is e c k n o ~ l e d g e d
L 1" , JR ~ ProJections and termmatmn of the cortmospmaI tract n3 rodents, /zvp B, atn
Rea, 13 [1971) 432 450
2 CASTRO, A J , lpsllateral corttcospJnal projectmns m neonatal rats after large cerebral hem~,,phelc lesions, E.V~. Ncltro[, 46 (1975) 1 8. 3 DEMp,'r:R, W , Ontogene~,s of the rat cortlcospmal tract, Arch Neurol. ( Ctm ,~, I c) (1967) 203 211 4 GUILLER~, R W , Light and electron m~croscopJcal studies of normal and degenerating axons In W J H NAUrA arnD S O E EBBIZSSO',,(Eds), Contempotat)' Research ~,lethod, m ,Vemo,anatomy, Springer, New York, 1970, pp 77 105 5 HICKS, S P, anl~ D'AnlA1o, C J , Motor sensory and wsual behavior aftra helmspherectom5 m newborn and mature rats, E.vp A'et#ol, 29 (1970) 416-438. 6 LEONe, S K , aNI~ Lt.JNl), R D , Anomalous bilateral cort~cofugal pathways m albino lain after neonatal lesions, Brain Research, 62 ( 1973 ~ 218-221 7 REXED, B , The cytoarch~tectomc orgamzatmn of the spinal cold m the cat, ,I ~omp N e r o , / , 96 (1952) 415 496
327
Elsevier Sclenttfic Pubhshlng Company, Amsterdam - Printed m The Netherlands
Short Communications
An ultrastructural study of ipsilateral corticospinal terminations in the rat
J. ROSS McCLUNG* AND ANTHONY J CASTRO Depat tment o] Anutotto', Loutstana State Untvet slty Me&cal Center, New O~lean~, La 70112 ( U S A )
(Accepted February 18th, 1975)
Using both hght and electron microscopic techmques, Brown a has reported that the rodent cortmospmal tract ~s entirely crossed. [t was found to terminate exclusively in the dorsal horn of the spinal cord contralateral to the side of its cortical origin. However, recent mvestlgatlons~, 5,6 have demonstrated, at the light microscopic level, the presence of ,psllateral cortmospmal fibers These ipsllateral fibers were observed m adult rats that had sustained unilateral, fi'ontal cortical lesions as neonates They originated from frontal cortical areas on the side opposite to the neonatal lesion The rodent cortmospmal tract exhibits considerable postnatal development m that it does not reach its medullary decussatlon until after birth a, Accordingly, ,t has been hypothesized by Leong and Lurid 6 that crossing normally occurs due to an interaction between the two comcospmal tracts as they grow through the medulla. This interaction is thought to be disrupted by a neonatal cortical lesJon which destroys one of the pathways and thereby causes an incomplete decussatlon of the remammg tract. There ,s no direct evidence in support of the functional significance, if any, of these ipsilateral projections. And even more fundamentally, there is no ultrastructural ewdence of synaptm endings associated with this aberrant pathway. Therefore, the object of this report is to present anatomical evidence of functional synapses corresponding to the aberrant ipsllateral comcosplnal tract Five 1-day-old Long-Evans male rats received unilateral frontal cortical ablations by asplratmn under hypothermia. Two additional neonates that recmved only scalp incisions served as hypothermic and surgical controls. Three to 6 months later, ammals that sustained neonatal cortmal ablatmn received a les~on of the opposite (previously unoperated) frontal cortex while under sodmm pentobarbital anesthesia. The two control animals were also anesthetized at this time. At 3 or 6 days after the second surgery, ammals were perfused through the aorta with 0 1 M cacodylate * Present address Department of Anatomy, Medmal College of Vwgmla, MCV Station, P O Box 906, Rmhmond, Va. 23298, U S A
328
Fig. 1. Dark degenerating axon (da) in the dorsal horn of the cervical spinal cord 3 days after ipsilateral cortical ablation. × 25,500. buffer (pH 7.3) followed by 500 ml of a 4 ~ paraformaldehyde and 0.5 5/ooglutaraldehyde solution in the same buffer. Cervical spinal cord cross-sections were cut freehand at approximately 0.5 mm thickness, trimmed to include only the gray matter and dorsal funiculi, osmicated, and embedded in Epon. Toluidine blue-stained sections (1/~m) were used for orientation of corresponding 80-90 nm (gold and silver) thin sections which were stained with uranyl acetate and lead citrate and viewed with a Philips EM 300. In the spinal cord on the side ipsilateral to the second (adult) lesion, many degenerated myelinated axons were observed in the atrophied corticospinal tract and the adjoining portion of the dorsal horn. These degenerated axons (Fig. 1) displayed typical dark degeneration changes ~. Similar degenerated axons were found in the medial aspect of the dorsal horn within zones equivalent to laminae II-V of Rexe& ; however, progressively fewer were observed either dorsally or laterally in the dorsal horn. Degenerated, unmyelinated terminal axons were most commonly observed just ventral to the substantia gelatinosa in the area corresponding to lamina III, and recognizable synaptic contacts (Fig. 2) were frequently found between these axon terminals and small dendrites in the region. At 3 days postoperative, these synaptic boutons (Fig. 2) displayed a dark axoplasmic matrix which partially masked the decomposed plasma membranes, mitochondria and synaptic vesicle remnants. However, the shrinkage which resulted from this degeneration occasionally enhanced visualization of the synaptic contacts because of their tight adhesion to the postsynaptic membrane
329
Fig. 2 Dark, degenerating bouton (db) in the dorsal horn (lamina 111) of the cervical spmal cord 3 days after lpsllateral cortical ablation The reset illustrates, at hJgher magmfication, the synaptlC contact of th~s bouton \ 24,500, reset, , 67,500
(insert Fig. 2). By 6 days postoperative, axonal degeneration was still present, but most unmyehnated degenerated structures were no longer recogmzable as defimtJve synaptlc boutons because of advanced degeneration and glial envelopment. The presence of darkened, ghal-enveloped, unmyelinated profiles rather than clearly recogmzable darkened boutons further indicates that the dense boutons observed at 3 days were in fact degenerating corticospmal terminals. These degenerating boutons are believed to represent the terminations of degenerating axons which appeared in and emerged from the dorsal funiculus on the side ipsilateral to the second les~on. They are not considered to be residual degenerated elements caused by the initial neonatal cortical les~on These ammals survived 3 6 months after neonatal ablation, and th~s amount of time ~s considerably sufficient for degeneration debris to be removed. Furthermore, light microscopic analysis, using silver staining methods for degenerated axons and their synaptlc endings, revealed no evidence of cortlcospinal degeneration ~n animals that were killed only 2 months after neonatal frontal cortical ablation 2. It ~s concluded that the degenerating boutons observed m the present study represent anatomical evidence for functional synapses being established by aberrant lpsllateral corticospmal fibers which resulted from neonatal cortical injury. The pattern of termination of these fibers appears to correspond with the normal contralateral distribution of cortlcospmal fibers as reported by Brown 1 and this is being more precisely examined with continuing experimentation.
330 T h i s r e b e a r c h vva~ s u p p o r t e d by a [~OUlSldn;.t S t a t e U m ~ e r b J t \ N~.ht)t~J ol Medw~J~c G e n e r a l R e s e a r c h S u p p o r t G r a n t zw~arded t o J R M a n d a G r a n l N S 1 1 2 0 0 - 0 2 IF,,n~ the National Institute of NeurologJc Drseascs and Stroke to A J ¢ T h e p h o t o g r a p h i c a s s i s t a n c e t~l M r
I BROWN,
G a r b l b K e H l m a n is e c k n o ~ l e d g e d
L 1" , JR ~ ProJections and termmatmn of the cortmospmaI tract n3 rodents, /zvp B, atn
Rea, 13 [1971) 432 450
2 CASTRO, A J , lpsllateral corttcospJnal projectmns m neonatal rats after large cerebral hem~,,phelc lesions, E.V~. Ncltro[, 46 (1975) 1 8. 3 DEMp,'r:R, W , Ontogene~,s of the rat cortlcospmal tract, Arch Neurol. ( Ctm ,~, I c) (1967) 203 211 4 GUILLER~, R W , Light and electron m~croscopJcal studies of normal and degenerating axons In W J H NAUrA arnD S O E EBBIZSSO',,(Eds), Contempotat)' Research ~,lethod, m ,Vemo,anatomy, Springer, New York, 1970, pp 77 105 5 HICKS, S P, anl~ D'AnlA1o, C J , Motor sensory and wsual behavior aftra helmspherectom5 m newborn and mature rats, E.vp A'et#ol, 29 (1970) 416-438. 6 LEONe, S K , aNI~ Lt.JNl), R D , Anomalous bilateral cort~cofugal pathways m albino lain after neonatal lesions, Brain Research, 62 ( 1973 ~ 218-221 7 REXED, B , The cytoarch~tectomc orgamzatmn of the spinal cold m the cat, ,I ~omp N e r o , / , 96 (1952) 415 496