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Synaptic rearrangement in the ventrobasal complex of the mouse following partial cortical deafferentation
Brain Research, 125 (1977) 351-355 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
351
Synaptic rearrangement in the ventrobasal complex of the mouse following partial cortical deafferentation
J. P. DONOGHUE* and J. WELLS Department of Anatomy, University of Vermont, Burlington, Vt. 02154 (U.S.A.)
(Accepted January 7th, 1977)
Morphological rearrangement has been shown in certain areas of the adult mammalian central nervous system in response to afferent denervation. Lynch et al. 6 and Steward et al. 1°,11 have demonstrated newly formed entorhinal fibers in the hippocampus of the adult rat in response to ipsilateral deafferentation. Raisman v has found synaptic reorganization in the lateral septal nucleus after removal of inputs from either the hippocampus or the medial forebrain bundle. Comparable changes also have been demonstrated in other areas of the CNS including the spinal cord 5 and parts of the visual system 3. The present study indicates that synapses can reform in ventrobasal thalamus (VB) after partial loss of the principal somatic sensory (Sm I) cortical input. To identify the pattern of cortical termination in VB, lesions were placed in Sm I of the right cortical hemisphere of C57B1/6J mice, and the subsequent degeneration was traced to VB using the F i n k - H e i m e r stain for degenerating axon terminals. The somatotopic distribution of the cortical input was similar to that demonstrated by Cowan et al. 1. A small area in the lateral part of VB was chosen as a sample area for ultrastructural study of the effect of deafferentation. This area was chosen for study since the rather distinct lateral border of VB could be used as a consistent landmark for uniform sampling. For deafferentation of the sample area, a small (1 m m × 1 m m × depth of cortex) lesion was placed stereotaxically in the medial part of Sm I cortex. The results of the degeneration study (above) confirmed that these lesions produced a well-defined patch of degenerating terminals in the lateral part of VB. The sample area was within the borders of this degeneration field. F r o m electron micrographs of the sample area in 4 normal animals, 4 categories of vesicle-containing profiles were established on the basis of consistent differences in their morphology, and the number of synaptic contacts made by each of these types as well as the total were computed per 100 sq. # m of neuropil (excluding blood vessels and cell bodies). For quantitation a synapse was defined as any profile containing three or more vesicles which were adjacent to an * Current address: Section of Neuroscience, Division of Biology and Medicine, Brown University, Providence, R.I. 02912, U.S.A.
352 area of m e m b r a n e differentiation. The density of synaptic contacts for each category was c o m p u t e d in electron micrographs from the sample area in 5 mice that survived cortical lesion by 4 days a n d 4 mice that survived 60 days, to determine any compensatory changes in the n u m b e r of synaptic contacts after long term survival. Synapse counts were carried out o n mixed a n d coded electron micrographs from each of the three groups. I n n o r m a l VB, small profiles 1/~in or less in diameter c o n t a i n e d r o u n d vesicles a n d made asymmetric contacts o n dendrites of approximately the same size. Synaptic contacts made by such terminals were categorized as type S (Fig. 1A). These terminals were the most c o m m o n in the neuropil, f o r m i n g 19.02 contacts/100 sq. #m. Two types of large terminals 2-4 # m across were present. The first, type G, con-
Fig. 1. A: a type G terminal from VB of a normal mouse. At least one of the 4 dendritic excrescences (e) has a synaptic contact on it (arrow). A type S terminal is also present in the field. B: in this micrograph from normal VB a spine-like dendritic excrescence arising from a large dendrite is surrounded by a type G terminal. The glial capsule can be clearly seen (arrow). The appearance of the neuropil in A and B is similar to that seen 60 days after the Sm I cortical lesion. C: two degenerating synapses (arrows) present 4 days after an Sm I cortical lesion. Each bar equals 1/~m.
353 tained several mitochondria, round vesicles and made two or more asymmetric contacts on large dendrites and spine-like dendritic invaginations into the terminal (Fig. 1A and B). The entire G terminal-dendritic contact area was at least partially enclosed by a thin glial process. No other vesicle-containing profiles were enclosed within this glial layer. There were 1.08 G-type synapses/100 sq. #m. The second type of large terminal, type L, contained several mitochondria and either flat or small round synaptic vesicles but did not contain dendritic invaginations. These terminals made 0.58 contacts/100 sq. ~m. Vesicle-containing profiles contacting perikarya (type AS) were rare, forming 0.30 contacts/100 sq./zm. The synaptic organization of mouse VB is like that of other major thalamic relay nuclei, such as the dorsal lateral geniculate 2, or VB of other species 4,s, except for one difference. In other principle thalamic nuclei the relay afferents (e.g. from the dorsal column nuclei or the retina) terminate in synaptic glomerulP 2 consisting of a glial encapsulated complex of several presynaptic elements and at least one dendrite. These structures are not evident in mouse VB. We have identified the type G profiles as the terminals of axons from the medial lemniscus since these terminals degenerate after lesions of the dorsal column nuclei (unpublished observations). Therefore, for at least this part of VB the synaptic glomerulus is replaced by a different synaptic arrangement consisting of a single type G presynaptic element which is invaginated by dendritic processes. Similar findings have been reported in the rat VB by ~pa~ek and Lieberman 9. Four days after the small cortical lesion, numerous small electron dense axon terminals were seen that formed asymmetric synapses in VB (Fig. 1C). Quantitative analysis revealed a significant decrease (P=0.001) in the number of normal S contacts to 14.25, a decrease of about 25 ~o. The density of contacts made by G terminals increased by 64 ~ (P=O.05) after 4 days, while type L and AS were unchanged from controls. At 60 days the neuropil appeared similar to normal VB. There was a significant increase in the S terminal population from 14.25 (after 4 days) to 16.76 contacts/100 sq. # m (an increase of 17.7~, P=O.05). However, this was still less than controls (P=O.05). The number of G contacts at 60 days was decreased to control levels. Two changes, then, were observed following the removal of the cortical input to VB: (1) a loss of type S synaptic contacts shortly after cortical deafferentation, followed by a partial return to control values 60 days later; (2) a transient rise in the number of glomerular contacts/100 sq. # m which decreased to control levels 60 days post lesion (see Fig. 2). The conclusion that contacts are formed as a result of partial cortical deafferentation must be made with some reservation. Combinations of shrinkage and swelling by glial reaction and retrograde degeneration complicate the interpretation of synaptic changes on a density basis. For example, a rise in the number of contacts could reflect either shrinkage of the neuropil or formation of new synaptic contacts. Two points argue against the idea of non-specific change in the density of the neuropil. First, at the very short (4 days) and long (60 days) survival times little glial or retrograde change was observed; and second, neither the L- or AS-type synaptic density
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Fig. 2. Graphic summary of VB synaptic alterations following partial cortical deafferentation. (Each point represents the mean number of synapses for each terminal type/1000 sq. #m ± S.E.M.). For each group (control, 4-day, 60-day survival) 4000-5000 sq.~m were sampled. The total number of synaptic contacts (T, solid line) decreased significantly at 4 days survival, but did not show any long term change. Analysis of separate subpopulations did show changes. The number of contacts made by the small (S) terminals showed a significant decrease at 4 days and then a significant increase at 60 days to a level still below normal (P - 0.05). Contacts made by glomerular terminals (G) increased at 4 days but were not different from controls at 60 days (P - 0.05). The number of contacts made by large terminals (L) or terminals making axosomatic contacts (AS) remained unchanged from control at either survival time. showed a n y change at either time after the lesion. A t present the significance of the transient rise in G contacts is unclear. A t 60 days survival the n u m b e r of S synapses approached control levels, suggesting that there had been a partial return towards n o r m a l synaptic density, perhaps by the f o r m a t i o n of new type S synapses. These could arise from adjacent intact corticothalamic axons, although no data are available to show this to be true. We feel that these experiments indicate that there is a limited but real potential for synaptic plasticity in the mouse ventrobasal complex after incomplete removal o f the Sm I cortical input.
355 1 Cowan, W. M., Gottlieb, D. I., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-32. 2 Famiglietti, E. V. and Peters, A., The synaptic glomerulus and intrinsic neuron in the dorsal lateral geniculate of the cat, J. comp. Neurol., 144 (1972) 285-334. 3 Goodman, D. C. and Horel, J. A., Sprouting of optic projections in the brain stem of the rat, J. comp. Neurol., 127 (1966) 71-88. 4 Jones, E. G. and Powell, T. P. S., Electron microscopy of synaptic glomeruli in the thalamic relay nuclei of the cat, Proc. roy. Soc. B, 172 (1969) 153-171. 5 Liu, C. N. and Chambers, W. W., Intraspinal sprouting of dorsal root axons, Arch. Neurol. (Chic.), 79 (1958) 46-61. 6 Lynch, G., Deadwyler, S. and Cotman, C. W., Postlesion axonal growth produces permanent functional connections, Science, 180 (1973) 1364-1366. 7 Raisman, G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. 8 Ralston, H. J. and Herman, M. M., The fine structure of neurons in the ventrobasal thalamus of the cat, Brain Research, 14 (1969) 77-97. 9 ~pa~ek, J. and Lieberman, A. R., Ultrastructure and three-dimensional organization of synaptic glomeruli in the rat somatosensory thalamus, 3. Anat. (Lond.), 117 (1974) 487-516. 10 Steward, O., Cotman, C. W. and Lynch, G., Re-establishment of electrophysiologically functional entorhinal cortical input to the dentate gyrus deafferented by ipsilateral entorhinal lesions: innervation by contralateral entorhinal cortex, Exp. Brain Res., 18 (1973) 396-414. 11 Steward, O., Cotman, C. W. and Lynch, G., Growth of a new fiber projection in the brain of adult rats: re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions, Exp. Brain Res., 20 (1974) 45-66. 12 Szentfigothai, J., Glomerular synapses, complex synaptic arrangements, and their operational significance. In F. O. Schmitt (Ed.), The Neurosciences Second Study Program, Rockefeller University Press, New York, 1970, pp. 427-442.
351
Synaptic rearrangement in the ventrobasal complex of the mouse following partial cortical deafferentation
J. P. DONOGHUE* and J. WELLS Department of Anatomy, University of Vermont, Burlington, Vt. 02154 (U.S.A.)
(Accepted January 7th, 1977)
Morphological rearrangement has been shown in certain areas of the adult mammalian central nervous system in response to afferent denervation. Lynch et al. 6 and Steward et al. 1°,11 have demonstrated newly formed entorhinal fibers in the hippocampus of the adult rat in response to ipsilateral deafferentation. Raisman v has found synaptic reorganization in the lateral septal nucleus after removal of inputs from either the hippocampus or the medial forebrain bundle. Comparable changes also have been demonstrated in other areas of the CNS including the spinal cord 5 and parts of the visual system 3. The present study indicates that synapses can reform in ventrobasal thalamus (VB) after partial loss of the principal somatic sensory (Sm I) cortical input. To identify the pattern of cortical termination in VB, lesions were placed in Sm I of the right cortical hemisphere of C57B1/6J mice, and the subsequent degeneration was traced to VB using the F i n k - H e i m e r stain for degenerating axon terminals. The somatotopic distribution of the cortical input was similar to that demonstrated by Cowan et al. 1. A small area in the lateral part of VB was chosen as a sample area for ultrastructural study of the effect of deafferentation. This area was chosen for study since the rather distinct lateral border of VB could be used as a consistent landmark for uniform sampling. For deafferentation of the sample area, a small (1 m m × 1 m m × depth of cortex) lesion was placed stereotaxically in the medial part of Sm I cortex. The results of the degeneration study (above) confirmed that these lesions produced a well-defined patch of degenerating terminals in the lateral part of VB. The sample area was within the borders of this degeneration field. F r o m electron micrographs of the sample area in 4 normal animals, 4 categories of vesicle-containing profiles were established on the basis of consistent differences in their morphology, and the number of synaptic contacts made by each of these types as well as the total were computed per 100 sq. # m of neuropil (excluding blood vessels and cell bodies). For quantitation a synapse was defined as any profile containing three or more vesicles which were adjacent to an * Current address: Section of Neuroscience, Division of Biology and Medicine, Brown University, Providence, R.I. 02912, U.S.A.
352 area of m e m b r a n e differentiation. The density of synaptic contacts for each category was c o m p u t e d in electron micrographs from the sample area in 5 mice that survived cortical lesion by 4 days a n d 4 mice that survived 60 days, to determine any compensatory changes in the n u m b e r of synaptic contacts after long term survival. Synapse counts were carried out o n mixed a n d coded electron micrographs from each of the three groups. I n n o r m a l VB, small profiles 1/~in or less in diameter c o n t a i n e d r o u n d vesicles a n d made asymmetric contacts o n dendrites of approximately the same size. Synaptic contacts made by such terminals were categorized as type S (Fig. 1A). These terminals were the most c o m m o n in the neuropil, f o r m i n g 19.02 contacts/100 sq. #m. Two types of large terminals 2-4 # m across were present. The first, type G, con-
Fig. 1. A: a type G terminal from VB of a normal mouse. At least one of the 4 dendritic excrescences (e) has a synaptic contact on it (arrow). A type S terminal is also present in the field. B: in this micrograph from normal VB a spine-like dendritic excrescence arising from a large dendrite is surrounded by a type G terminal. The glial capsule can be clearly seen (arrow). The appearance of the neuropil in A and B is similar to that seen 60 days after the Sm I cortical lesion. C: two degenerating synapses (arrows) present 4 days after an Sm I cortical lesion. Each bar equals 1/~m.
353 tained several mitochondria, round vesicles and made two or more asymmetric contacts on large dendrites and spine-like dendritic invaginations into the terminal (Fig. 1A and B). The entire G terminal-dendritic contact area was at least partially enclosed by a thin glial process. No other vesicle-containing profiles were enclosed within this glial layer. There were 1.08 G-type synapses/100 sq. #m. The second type of large terminal, type L, contained several mitochondria and either flat or small round synaptic vesicles but did not contain dendritic invaginations. These terminals made 0.58 contacts/100 sq. ~m. Vesicle-containing profiles contacting perikarya (type AS) were rare, forming 0.30 contacts/100 sq./zm. The synaptic organization of mouse VB is like that of other major thalamic relay nuclei, such as the dorsal lateral geniculate 2, or VB of other species 4,s, except for one difference. In other principle thalamic nuclei the relay afferents (e.g. from the dorsal column nuclei or the retina) terminate in synaptic glomerulP 2 consisting of a glial encapsulated complex of several presynaptic elements and at least one dendrite. These structures are not evident in mouse VB. We have identified the type G profiles as the terminals of axons from the medial lemniscus since these terminals degenerate after lesions of the dorsal column nuclei (unpublished observations). Therefore, for at least this part of VB the synaptic glomerulus is replaced by a different synaptic arrangement consisting of a single type G presynaptic element which is invaginated by dendritic processes. Similar findings have been reported in the rat VB by ~pa~ek and Lieberman 9. Four days after the small cortical lesion, numerous small electron dense axon terminals were seen that formed asymmetric synapses in VB (Fig. 1C). Quantitative analysis revealed a significant decrease (P=0.001) in the number of normal S contacts to 14.25, a decrease of about 25 ~o. The density of contacts made by G terminals increased by 64 ~ (P=O.05) after 4 days, while type L and AS were unchanged from controls. At 60 days the neuropil appeared similar to normal VB. There was a significant increase in the S terminal population from 14.25 (after 4 days) to 16.76 contacts/100 sq. # m (an increase of 17.7~, P=O.05). However, this was still less than controls (P=O.05). The number of G contacts at 60 days was decreased to control levels. Two changes, then, were observed following the removal of the cortical input to VB: (1) a loss of type S synaptic contacts shortly after cortical deafferentation, followed by a partial return to control values 60 days later; (2) a transient rise in the number of glomerular contacts/100 sq. # m which decreased to control levels 60 days post lesion (see Fig. 2). The conclusion that contacts are formed as a result of partial cortical deafferentation must be made with some reservation. Combinations of shrinkage and swelling by glial reaction and retrograde degeneration complicate the interpretation of synaptic changes on a density basis. For example, a rise in the number of contacts could reflect either shrinkage of the neuropil or formation of new synaptic contacts. Two points argue against the idea of non-specific change in the density of the neuropil. First, at the very short (4 days) and long (60 days) survival times little glial or retrograde change was observed; and second, neither the L- or AS-type synaptic density
354
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I
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4 POST-LESION
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i
6O SURVIVAL TIME ( d a y s )
Fig. 2. Graphic summary of VB synaptic alterations following partial cortical deafferentation. (Each point represents the mean number of synapses for each terminal type/1000 sq. #m ± S.E.M.). For each group (control, 4-day, 60-day survival) 4000-5000 sq.~m were sampled. The total number of synaptic contacts (T, solid line) decreased significantly at 4 days survival, but did not show any long term change. Analysis of separate subpopulations did show changes. The number of contacts made by the small (S) terminals showed a significant decrease at 4 days and then a significant increase at 60 days to a level still below normal (P - 0.05). Contacts made by glomerular terminals (G) increased at 4 days but were not different from controls at 60 days (P - 0.05). The number of contacts made by large terminals (L) or terminals making axosomatic contacts (AS) remained unchanged from control at either survival time. showed a n y change at either time after the lesion. A t present the significance of the transient rise in G contacts is unclear. A t 60 days survival the n u m b e r of S synapses approached control levels, suggesting that there had been a partial return towards n o r m a l synaptic density, perhaps by the f o r m a t i o n of new type S synapses. These could arise from adjacent intact corticothalamic axons, although no data are available to show this to be true. We feel that these experiments indicate that there is a limited but real potential for synaptic plasticity in the mouse ventrobasal complex after incomplete removal o f the Sm I cortical input.
355 1 Cowan, W. M., Gottlieb, D. I., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-32. 2 Famiglietti, E. V. and Peters, A., The synaptic glomerulus and intrinsic neuron in the dorsal lateral geniculate of the cat, J. comp. Neurol., 144 (1972) 285-334. 3 Goodman, D. C. and Horel, J. A., Sprouting of optic projections in the brain stem of the rat, J. comp. Neurol., 127 (1966) 71-88. 4 Jones, E. G. and Powell, T. P. S., Electron microscopy of synaptic glomeruli in the thalamic relay nuclei of the cat, Proc. roy. Soc. B, 172 (1969) 153-171. 5 Liu, C. N. and Chambers, W. W., Intraspinal sprouting of dorsal root axons, Arch. Neurol. (Chic.), 79 (1958) 46-61. 6 Lynch, G., Deadwyler, S. and Cotman, C. W., Postlesion axonal growth produces permanent functional connections, Science, 180 (1973) 1364-1366. 7 Raisman, G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. 8 Ralston, H. J. and Herman, M. M., The fine structure of neurons in the ventrobasal thalamus of the cat, Brain Research, 14 (1969) 77-97. 9 ~pa~ek, J. and Lieberman, A. R., Ultrastructure and three-dimensional organization of synaptic glomeruli in the rat somatosensory thalamus, 3. Anat. (Lond.), 117 (1974) 487-516. 10 Steward, O., Cotman, C. W. and Lynch, G., Re-establishment of electrophysiologically functional entorhinal cortical input to the dentate gyrus deafferented by ipsilateral entorhinal lesions: innervation by contralateral entorhinal cortex, Exp. Brain Res., 18 (1973) 396-414. 11 Steward, O., Cotman, C. W. and Lynch, G., Growth of a new fiber projection in the brain of adult rats: re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions, Exp. Brain Res., 20 (1974) 45-66. 12 Szentfigothai, J., Glomerular synapses, complex synaptic arrangements, and their operational significance. In F. O. Schmitt (Ed.), The Neurosciences Second Study Program, Rockefeller University Press, New York, 1970, pp. 427-442.