- Email: [email protected]
Altered synaptophysin-immunoreactive pattern in human olivary hypertrophy
ELSEVIER
Neuroscience Letters 176 (1994) 178 180
. . . . . .
Altered synaptophysin-immunoreactive pattern in human olivary hypertrophy Toru K a w a n a m i "'b'*, Takeo Kato c, Josephina F. Llena a, Asao Hirano ", Hideo Sasaki c "Division of Neuropathology, Department of Pathology, Montefiore Medical Center, New York, USA bDepartment of Internal Medicine, Sanyu-do Medical Center, Yonezawa, Japan ~Third Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan Received 16 May 1994: Revised version received 9 June 1994; Accepted 10 June 1994
Abstract
We examined the inferior olivary nucleus from four cases of olivary hypertrophy and six control cases immunohistochemicatty with an anti-synaptophysin (SYP) antibody. Our study revealed the altered SYP-immunoreactivepattern in the hypertrophied olives, consisting of an increased number of SYP-immunoreactive dots on neuronal cell bodies in spite of its decreased immunoreactivity in the neuropil. Because SYP is a marker protein for presynaptic vesicles, presynaptic terminals in the inferior olivary nucleus seem to change their distribution during the course of olivary hypertrophy. Key words: Synaptophysin; Inferior olivary nucleus; Olivary hypertrophy; Immunohistochemistry; Synapse; Presynaptic terminal; Synaptic reorganization
Olivary hypertrophy, or hypertrophied inferior olive, is frequently associated with palatal myoclonus and has several neuropathological features which were demonstrated by histopathological [7,12], histochemical [8] and ultrastructural [1,5] studies. It is attributed to the interruption of the dentato-olivary projection [9], resulting in transsynaptic degeneration of the inferior olivary nucleus [3]. Since ultrastructural evaluation of synapses in autopsied materials is difficult due to postmortem changes, we have examined hypertrophied olives immunohistochemically with anti-synaptophysin (SYP) antibody [14]. The antibody had been shown to recognize presynaptic vesicles in various regions of the brain [4,14]. Although the previous SYP immunohistochemical study [2] demonstrated some SYP-immunoreactive neurons in the hypertrophied olive, few information is available concerning the synaptic changes in the hypertrophied olives [1]. In this communication, we report an altered
* Corresponding author. Address: Third Department of Internal Medicine, Yamagata University Schoolof Medicine,Yamagata, Japan. Fax: (81) (236) 25-2044. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 4 6 0 - R
presynaptic terminal distribution in the hypertrophied olives using SYP immunohistochemistry. Four autopsied cases of neuropathologically diagnosed olivary hypertrophy (one man and three women, 69 79 years at the time of death t were used in the present study. Additional six autopsied cases of various nonneurological disorders (three men and three women. 4578 years at the time of death) were used as control cases. Tissue from the medulla oblongata was fixed in 10% buffered formalin and submitted for 6-/,tm-thick paraffin sections. Some of these sections were examined with hematoxylin and eosin, luxol fast blue-PAS and modified Bielshowsky stains. In addition, we have immunostained sections from each case with the anti-SYP monoclonal antibody (BioGenex, CA) by the ABC method [6]. Several sections were treated with non-immunonized mouse sera instead of the anti-SYP antibody or by omission of the primary antibody. The immunostained sections were faintly counterstained with hematoxylin Sections from the hypertrophied olives examined with routine neuropathological stains showed elongation and widening of the olivary folds (Fig. 1) in addition to pathological changes, such as hypertrophic and/or vacu-
179
77 Kawanami et al./Neuroscienee Letters 176 (1994) 178 180
olated neurons and glomeruloids, consistent with the previous reports [7,12]. The inferior olivary nucleus from the control cases did not show any significant histopathological changes. SYP-immunostained sections from the control cases showed numerous SYP-immunoreactive dots in the neuropil while the cell bodies of the olivary neurons were surrounded by a few immunoreactive dots (Fig. 2). There were no neurons with SYP-immunoreactive cytoplasm. In the hypertrophied olives, SYP-immunoreactive dots were diffusely decreased in the neuropil (Figs. 3, 4). On the other hand, there were many SYP-immunoreactive dots on the cell bodies of many olivary neurons, including hypertrophic or vacuolated neurons (Figs. 3, 4). Some neurons had diffuse SYP-immunoreactive cytoplasm. Also noted were SYP-immunoreactive glomeruloids (Fig. 3) and a few immunoreactive granules within the neuronal perikarya (Fig. 4), In two cases of unilateral olivary hypertrophy, the unaffected side of the inferior olivary nucleus showed the immunoreactive pattern similar to the control cases. Omission of the primary antibody or use of the non-immunized mouse sera instead of the primary antibody abolished all immunostaining above. The present study revealed altered SYP-immunoreacrive pattern in the hypertrophied olives, consisting of numerous immunoreactive dots on the neuronal cell bodies in spite of diffusely decreased immunoreactivity in the neuropil. In addition, we demonstrated SYP-immunoreactivity in glomeruloids and intraneuronal granules in the hypertrophied olives, consistent with the previous ultrastructural study by Barron et al. [1], who have reported presynaptic vesicles in glomeruloids and presynaptic buttons deeply invaginated into the neuronal cytoplasm. The altered SYP-immunoreactive pattern suggests changes of the presynaptic terminal distribution
__..) Fig. 1. Hypertrophied inferior olive (right) and inferior olivary nucleus on unaffected side (left). Hypertropied olive shows elongation and widening of olivary folds (bar, 3 mm; luxol fast blue-PAS stain). Fig. 2. Inferior olivary nucleus from a control case immunostained with anti-synaptophysin antibody. There are numerous immunoreactive dots in neuropil while olivary neurons have only a few immunoreactive dots on cell bodies (bar, 50/am; counterstained with hematoxylin). Fig. 3. Hypertrophied olivary nucleus immunostained with anti-synaptophysin antibody. Olivary neurons are densely decorated by numerous synaptophysin immunoreactive dots in spite of greatly reduced immunoreactivity in neuropil. Arrow indicates a synaptophysin-immunoreactive glomeruloid (bar, 50/am; counterstained with hematoxylin). Fig. 4. Hypertrophied olivary nucleus immunostained with anti-synaptophysin antibody. Four olivary neurons have numerous immunoreactive dots on the cell body, one of which (upper) has hypertrophic and vacuolated cytoplasm. Arrow indicates intraneuronal SYP-immunoreactlvc granules (bar, 50 pm: counterstained with hematoxylin).
because SYP is a marker protein for presynaptic termirials [4,14]. The reduced immunoreactivity in the neuropil would correlate with loss of the dentate-olivary projection [9] which synapses with olivary neurons.
....... '.....
180
T Kawanami et aL/Neuroscience Letters 176 (1994) 178-180
However, this c a n n o t a c c o u n t for n u m e r o u s SYP-immunoreactive dots on the neuronal cell bodies in the hypertrophied olives. One possible explanation is that the presynaptic terminals could be rearranged o n t o the neuronal cell bodies during the course o f olivary hy-
pertrophy. A n o t e w o r t h y finding o f the present study is that im-
munoreactivity of presynaptic terminals changed their distribution as one o f the pathological responses in 01ivary h y p e r t r o p h y . A similar p h e n o m e n o n , 'synaptic reorganization' [10,11], has been reported in the cat red nucleus n e u r o n s after partial denervation caused by a lesion o f the interpositorubral projection: the red nucleus neurons h a d newly f o r m e d synapses on the p r o x y m a l
thick dendrites and the cell bodies, demonstrated electrophysiologically [13] and m o r p h o l o g i c a l l y [10,11]. O u r study suggests that the synaptic reorganization also occurs in h u m a n inferior olivary neurons after the interrup-
tion of the dentato-olivary projection. [1] Barron, K.D., Dentinger, M.P. and Koeppen, A.H., Fine structure of neurons of the hypertrophied human inferior olive, J. Neuropathol. Exp. Neurol., 41 (1982) 186-203. [2] Barron, K.D., Najjar, S. and Koeppen, A.H., Transneuronat degeneration (TND) in the human inferior olive: an immunohistochemical study, Neurology, 42, Suppl., 3 (1992) 153. [3] Cowan, W.M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W.J.H. Nauta and S.O,E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, NY, 1970, pp. 217-251. [4] Goto, S.. Hirano, A. and Pearson J., Calcineurin and synapto-
physininthehumanspinalcordofnormalindividualsandpatients with familial dysautonomia, Acta Neuropathol., 79 (1990)644652. [5] Horoupian, D.S. and Wisniewski, H., Neurofilamentous hyperplasia in inferior olivary hypertrophy, J. Neuropathol. Exp. Neurol.. 30 (1971)571-582. [6] Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin complex (ABC) in immuno-peroxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. [7] Jellinger, K., Hypertrophy of the inferior olives. Report on 29 cases, z. Neurol., 205 (1973) 153-174. [8] Koeppen, A.H., Barron, K.D. and Dentinger M.P., Olivary hypertrophy: histochemical demonstration of hydrolytic enzymes, Neurology, 30 (1980) 471--480. [91 Lapresle, J., Rhythmic palatal myoclonus and the dentate-olivary pathway, J. Neurol., 220 (1979) 223-230. [10] Murakami, F., Katsumaru, H., Saito, K. and Tsukahara N., A quantitative study of synaptic reorgan;zation in red nucleus neurons after lesion of the nucleus interpositus of the cat: an electron microscopic study involving intracellular injection of horseradish peroxidase, Brain Res., 242 (1982) 41. [11] Nakamura, Y., Mizuno, N., Konishi, A. and Sato, M., Synaptic reorganization of the red nucleus after chronic deafferentation from cerebellorubral fibers: an electron microscope study in the cat, Brain Res., 82 (1974) 298-301. [12] Sohn, D. and Levine, S., The hypertrophy of the olives. A report of 43 cases. In H.M. Zimmermann (Ed.), Progress in Neuropathology, Grune and Stratton, New York, NY, 1971, pp. 202-217. [13] Tsukahara, N., Hultborn, H., Murakami, F. and Fujito, Y., Electrophysiological study of formation of new synapses and collateral sprouting in red nucleus neurons after partial denervation, J. Neurophysiol., 38 (1975)1359-1372. [14] Wiedenman, B. and Franke, W.W., Identification of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles, Cell, 41 (1985) 1017-1028.
Neuroscience Letters 176 (1994) 178 180
. . . . . .
Altered synaptophysin-immunoreactive pattern in human olivary hypertrophy Toru K a w a n a m i "'b'*, Takeo Kato c, Josephina F. Llena a, Asao Hirano ", Hideo Sasaki c "Division of Neuropathology, Department of Pathology, Montefiore Medical Center, New York, USA bDepartment of Internal Medicine, Sanyu-do Medical Center, Yonezawa, Japan ~Third Department of Internal Medicine, Yamagata University School of Medicine, Yamagata, Japan Received 16 May 1994: Revised version received 9 June 1994; Accepted 10 June 1994
Abstract
We examined the inferior olivary nucleus from four cases of olivary hypertrophy and six control cases immunohistochemicatty with an anti-synaptophysin (SYP) antibody. Our study revealed the altered SYP-immunoreactivepattern in the hypertrophied olives, consisting of an increased number of SYP-immunoreactive dots on neuronal cell bodies in spite of its decreased immunoreactivity in the neuropil. Because SYP is a marker protein for presynaptic vesicles, presynaptic terminals in the inferior olivary nucleus seem to change their distribution during the course of olivary hypertrophy. Key words: Synaptophysin; Inferior olivary nucleus; Olivary hypertrophy; Immunohistochemistry; Synapse; Presynaptic terminal; Synaptic reorganization
Olivary hypertrophy, or hypertrophied inferior olive, is frequently associated with palatal myoclonus and has several neuropathological features which were demonstrated by histopathological [7,12], histochemical [8] and ultrastructural [1,5] studies. It is attributed to the interruption of the dentato-olivary projection [9], resulting in transsynaptic degeneration of the inferior olivary nucleus [3]. Since ultrastructural evaluation of synapses in autopsied materials is difficult due to postmortem changes, we have examined hypertrophied olives immunohistochemically with anti-synaptophysin (SYP) antibody [14]. The antibody had been shown to recognize presynaptic vesicles in various regions of the brain [4,14]. Although the previous SYP immunohistochemical study [2] demonstrated some SYP-immunoreactive neurons in the hypertrophied olive, few information is available concerning the synaptic changes in the hypertrophied olives [1]. In this communication, we report an altered
* Corresponding author. Address: Third Department of Internal Medicine, Yamagata University Schoolof Medicine,Yamagata, Japan. Fax: (81) (236) 25-2044. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 4 6 0 - R
presynaptic terminal distribution in the hypertrophied olives using SYP immunohistochemistry. Four autopsied cases of neuropathologically diagnosed olivary hypertrophy (one man and three women, 69 79 years at the time of death t were used in the present study. Additional six autopsied cases of various nonneurological disorders (three men and three women. 4578 years at the time of death) were used as control cases. Tissue from the medulla oblongata was fixed in 10% buffered formalin and submitted for 6-/,tm-thick paraffin sections. Some of these sections were examined with hematoxylin and eosin, luxol fast blue-PAS and modified Bielshowsky stains. In addition, we have immunostained sections from each case with the anti-SYP monoclonal antibody (BioGenex, CA) by the ABC method [6]. Several sections were treated with non-immunonized mouse sera instead of the anti-SYP antibody or by omission of the primary antibody. The immunostained sections were faintly counterstained with hematoxylin Sections from the hypertrophied olives examined with routine neuropathological stains showed elongation and widening of the olivary folds (Fig. 1) in addition to pathological changes, such as hypertrophic and/or vacu-
179
77 Kawanami et al./Neuroscienee Letters 176 (1994) 178 180
olated neurons and glomeruloids, consistent with the previous reports [7,12]. The inferior olivary nucleus from the control cases did not show any significant histopathological changes. SYP-immunostained sections from the control cases showed numerous SYP-immunoreactive dots in the neuropil while the cell bodies of the olivary neurons were surrounded by a few immunoreactive dots (Fig. 2). There were no neurons with SYP-immunoreactive cytoplasm. In the hypertrophied olives, SYP-immunoreactive dots were diffusely decreased in the neuropil (Figs. 3, 4). On the other hand, there were many SYP-immunoreactive dots on the cell bodies of many olivary neurons, including hypertrophic or vacuolated neurons (Figs. 3, 4). Some neurons had diffuse SYP-immunoreactive cytoplasm. Also noted were SYP-immunoreactive glomeruloids (Fig. 3) and a few immunoreactive granules within the neuronal perikarya (Fig. 4), In two cases of unilateral olivary hypertrophy, the unaffected side of the inferior olivary nucleus showed the immunoreactive pattern similar to the control cases. Omission of the primary antibody or use of the non-immunized mouse sera instead of the primary antibody abolished all immunostaining above. The present study revealed altered SYP-immunoreacrive pattern in the hypertrophied olives, consisting of numerous immunoreactive dots on the neuronal cell bodies in spite of diffusely decreased immunoreactivity in the neuropil. In addition, we demonstrated SYP-immunoreactivity in glomeruloids and intraneuronal granules in the hypertrophied olives, consistent with the previous ultrastructural study by Barron et al. [1], who have reported presynaptic vesicles in glomeruloids and presynaptic buttons deeply invaginated into the neuronal cytoplasm. The altered SYP-immunoreactive pattern suggests changes of the presynaptic terminal distribution
__..) Fig. 1. Hypertrophied inferior olive (right) and inferior olivary nucleus on unaffected side (left). Hypertropied olive shows elongation and widening of olivary folds (bar, 3 mm; luxol fast blue-PAS stain). Fig. 2. Inferior olivary nucleus from a control case immunostained with anti-synaptophysin antibody. There are numerous immunoreactive dots in neuropil while olivary neurons have only a few immunoreactive dots on cell bodies (bar, 50/am; counterstained with hematoxylin). Fig. 3. Hypertrophied olivary nucleus immunostained with anti-synaptophysin antibody. Olivary neurons are densely decorated by numerous synaptophysin immunoreactive dots in spite of greatly reduced immunoreactivity in neuropil. Arrow indicates a synaptophysin-immunoreactive glomeruloid (bar, 50/am; counterstained with hematoxylin). Fig. 4. Hypertrophied olivary nucleus immunostained with anti-synaptophysin antibody. Four olivary neurons have numerous immunoreactive dots on the cell body, one of which (upper) has hypertrophic and vacuolated cytoplasm. Arrow indicates intraneuronal SYP-immunoreactlvc granules (bar, 50 pm: counterstained with hematoxylin).
because SYP is a marker protein for presynaptic termirials [4,14]. The reduced immunoreactivity in the neuropil would correlate with loss of the dentate-olivary projection [9] which synapses with olivary neurons.
....... '.....
180
T Kawanami et aL/Neuroscience Letters 176 (1994) 178-180
However, this c a n n o t a c c o u n t for n u m e r o u s SYP-immunoreactive dots on the neuronal cell bodies in the hypertrophied olives. One possible explanation is that the presynaptic terminals could be rearranged o n t o the neuronal cell bodies during the course o f olivary hy-
pertrophy. A n o t e w o r t h y finding o f the present study is that im-
munoreactivity of presynaptic terminals changed their distribution as one o f the pathological responses in 01ivary h y p e r t r o p h y . A similar p h e n o m e n o n , 'synaptic reorganization' [10,11], has been reported in the cat red nucleus n e u r o n s after partial denervation caused by a lesion o f the interpositorubral projection: the red nucleus neurons h a d newly f o r m e d synapses on the p r o x y m a l
thick dendrites and the cell bodies, demonstrated electrophysiologically [13] and m o r p h o l o g i c a l l y [10,11]. O u r study suggests that the synaptic reorganization also occurs in h u m a n inferior olivary neurons after the interrup-
tion of the dentato-olivary projection. [1] Barron, K.D., Dentinger, M.P. and Koeppen, A.H., Fine structure of neurons of the hypertrophied human inferior olive, J. Neuropathol. Exp. Neurol., 41 (1982) 186-203. [2] Barron, K.D., Najjar, S. and Koeppen, A.H., Transneuronat degeneration (TND) in the human inferior olive: an immunohistochemical study, Neurology, 42, Suppl., 3 (1992) 153. [3] Cowan, W.M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W.J.H. Nauta and S.O,E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, NY, 1970, pp. 217-251. [4] Goto, S.. Hirano, A. and Pearson J., Calcineurin and synapto-
physininthehumanspinalcordofnormalindividualsandpatients with familial dysautonomia, Acta Neuropathol., 79 (1990)644652. [5] Horoupian, D.S. and Wisniewski, H., Neurofilamentous hyperplasia in inferior olivary hypertrophy, J. Neuropathol. Exp. Neurol.. 30 (1971)571-582. [6] Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin complex (ABC) in immuno-peroxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. [7] Jellinger, K., Hypertrophy of the inferior olives. Report on 29 cases, z. Neurol., 205 (1973) 153-174. [8] Koeppen, A.H., Barron, K.D. and Dentinger M.P., Olivary hypertrophy: histochemical demonstration of hydrolytic enzymes, Neurology, 30 (1980) 471--480. [91 Lapresle, J., Rhythmic palatal myoclonus and the dentate-olivary pathway, J. Neurol., 220 (1979) 223-230. [10] Murakami, F., Katsumaru, H., Saito, K. and Tsukahara N., A quantitative study of synaptic reorgan;zation in red nucleus neurons after lesion of the nucleus interpositus of the cat: an electron microscopic study involving intracellular injection of horseradish peroxidase, Brain Res., 242 (1982) 41. [11] Nakamura, Y., Mizuno, N., Konishi, A. and Sato, M., Synaptic reorganization of the red nucleus after chronic deafferentation from cerebellorubral fibers: an electron microscope study in the cat, Brain Res., 82 (1974) 298-301. [12] Sohn, D. and Levine, S., The hypertrophy of the olives. A report of 43 cases. In H.M. Zimmermann (Ed.), Progress in Neuropathology, Grune and Stratton, New York, NY, 1971, pp. 202-217. [13] Tsukahara, N., Hultborn, H., Murakami, F. and Fujito, Y., Electrophysiological study of formation of new synapses and collateral sprouting in red nucleus neurons after partial denervation, J. Neurophysiol., 38 (1975)1359-1372. [14] Wiedenman, B. and Franke, W.W., Identification of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles, Cell, 41 (1985) 1017-1028.