Morphological alterations of Purkinje cell axons and ... - Springer Link

Acta Neuropathologica

Acta Neuropathol (Berl) (1981) 54:301 - 310

9 Springer-Verlag 1981

Morphological Alterations of Purkinje Cell Axons and Presynaptic Terminals in Organotypic Cerebellar Cultures Exposed to Ionizing Irradiation St. J. B a l o y a n n i s x'3, a n d S. U. K i m 2 Aristotelian University, Thessaloniki, Greece 2 University of Pensylvania, Philadelphia, PA, USA

Summary. I o n i z i n g i r r a d i a t i o n i n d u c e d m a r k e d morphological alterations in the P u r k i n j e cell axons a n d axonic terminals in vitro. The m y e l i n a t e d segments d e m o n s t r a t e d a very p o o r d e v e l o p m e n t of the myelin sheath. The a x o p l a s m d e m o n s t r a t e d various alterations of the m i t o c h o n d r i a , a n d the s m o o t h endoplasmic r e t i c u l u m a n d the n u m e r o u s e l o n g a t e d presynaptic terminals c o n t a i n e d large aggregates of tubulovesicular structures and neurofilaments a r r a n g e d in parallel a n d reticular array.

Key words: D y s t r o p h i c axons - I o n i z i n g i r r a d i a t i o n Tissue culture - Presynaptic terminals

Introduction I o n i z i n g i r r a d i a t i o n induces r e m a r k a b l e alterations in the n e w b o r n mice cerebellar cortex cultured in vitro. A l t h o u g h the m o s t impressive one is the d r a m a t i c r e d u c t i o n o f the granule cell p o p u l a t i o n , resulting in a practically a g r a n u l a r cerebellum [2]. I n a d d i t i o n to this several other m o r p h o l o g i c a l changes take place, concerning the n e u r o n s which are quite resistant to the ionizing irradiation. The P u r k i n j e cells exhibit m a r k e d anomalies o f their dendritic a r b o r i z a t i o n a n d develop n u m e r o u s u n a t t a c h e d dendritic spines [3]. These alterations could be a t t r i b u t e d either to the direct effect o f the i r r a d i a t i o n u p o n the P u r k i n j e cells, or to extensive d e o r g a n i z a t i o n of the n e u r o n a l circuits in the deafferented a g r a n u l a r cerebellar cortex. I n the p r e s e n t study we describe some m o r p h o l o g i cal alterations in the P u r k i n j e cell axons, seen in cerebellar cortex cultured in vitro, exposed to Xirradiation, which became practically a g r a n u l a r , 3 Present address: Aristotelian University, School of Medicine, Department of Neurology, Thessaloniki, Greece Offprint requests to." Seung U. Kim, MD, PhD, Ass. Prof., Division of Neuropathology, Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Material and Methods Cerebella were removed from 16 day Swiss albino mice fetuses and each was cut parasagittally into six sections. One part of these sections was exposed to ionizing irradiation, been given a total irradiation of 1,000 tad by a CS-I37 irradiator (Gammacell 40, Atomic Energy of Canada). The other part was used as a normal control. Immediately after the irradiation, the explants were placed on collagen covered rectangular coverslips 11 x 22 ram, incorporated into test tubes and allowed to stand in a static position for 24 h. Then they were placed in a roller drum and revolved at 12 rev/h. The nutrient medium consisted of 20 % fetal calf serum, 80 ~ Eagle's minimal essential medium and 0.6 % glucose. It was renewed once a week. After 14 days explants of each group, controls and irradiated, were fixed in 2.5 ~ glutaraldehyde and 1% paraformaldehyde in 0.2M cacodylate buffer (pH7.4) and postfixed in 2% osmium tetroxide in the same buffer. After dehydration in graded alcohols and propylen oxide they were embedded in Araldite. Semithin sections were prepared with a Porter Blum microtome and stained with a LKB Ultramicrotome, stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop-I, electron microscope.

Fig. 1.1,000 rad irradiated fetal mouse cerebellum cultured 2 weeks in vitro. The cerebellar cortex became practically agranular. The Purkinje cells were disarrayed and clustered. Semi thin section. Toluidine blue strain • 250

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Acta Neuropathol (Berl) 54 (1981)

Fig.2. Neuraxon (ax) and axonic terminal (at) of Purkinje cell in tissue culture exposed to 1,000 rad 7 irradiation. Large lysosomes and marked mitochondrial alterations are seen in the axon. The axonic terminal demonstrates remarkable tubnlovesicular deformation ( x 38,620)

St. J. Baloyannis and S. U. Kim: Purkinje Cell Axons in Organotypic Cerebellar Cultures

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Fig.3. Axonic terminal of Purkinje cell axon, in organotypic tissue culture exposed to 1,000 rad 7 irradiation. Numerous tubulovesicular profiles (tv) occupy most of the terminal. A marked polymorphism is seen in the disarrayed synaptic vesicles. ( x 30,000)

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Fig.4. Axonic terminal of Purkinje cell, in organotypic tissue culture exposedto 1,000 rad ~irradiation, demonstrating marked alterations of the mitochondria, elongated cisternae containing osmiophilic material and numerous polymorphic vesicles. ( x 71,490)

Results In the light microscope the control explants demonstrated normal development of the cerebellar cortex. The granule cells formed a well populated layer. The

Purkinje cells were intact and the stellate and basket cells formed a well defined layer. In the electron microscope the fine structure of the granule cells the Purkinje cells, the stellate cells, the basket cells and the glial cells was unremarkable. The


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Fig.5. Axosomatic synapse between an axonic collateral and the Purkinje cell body, in organotypic tissue culture exposed to 1,000 tad ~/ irradiation. Large number of polymorphic vesicles and multivesicular body are seen in the axonic terminal. ( x 56,000)

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Fig.6. Myelinatedaxon in organotypiccultureof cerebellarcortexexposedto y irradiation.The myelinsheath is composedof fewlamellae,which exhibit an atypical periodicity. ( x 45,650)

parallel fibers were seen forming synaptic contacts with the dendritic spines of the Purkinje and granule cells were normal. The myelin sheaths around the axons were well developed. In the 1,000 rads irradiated explants light microscopy revealed complete destruction of the granule cell population and clustering of the Purkinje cells all over

the explants. The interneurons and the glial cells remained intact (Fig. 1). The electron microscope verified the absence o f the granule cells, parallel fibers were seen embedded in large numbers in the perikaryon of the astrocytes, while several others remained entirely free (Fig. 7).


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Fig.7. Unattached Purkinje cell dendritic spine in organotypic culture of cerebellar cortex exposed to 1,000 rads 7 irradiation. The unattached spine is embedded in the cellular process of an astrocyte. ( x 28,800)

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Fig.8. Purkinjecellaxon in organotypicculturc of cerebellarcortexexposedto ~irradiation. Few lamellaeof atypicalmyelinsurround the axon ( x 28,800)

The axons of the Purkinje cells demonstrated marked morphological alterations. Many of them were enlarged and contained a large number of lysosomes, vesicular or tubular structures of various sizes and shapes numerous round or elongated vesicles and some lysosome like structures surrounded by halos (Fig. 2). Numerous elongated presynaptic axonic terminals included aggregates of tubulovesicular structures and neuroflaments arranged in parallel and reticular array (Fig. 3). At the periphery of the axonic terminals large number of synaptic vesicles were arranged irregularly or were locally concentrated near the active synaptic contacts (Fig. 3). In the large majority o f the axonic terminals the number o f mitochondria was reduced and they were smaller and darker than usual, although occasionally the mitochondria were very large elongated, and had a bizzare appearance (Fig. 4). The tubulovesicular material was almost exclusively seen in the unmyelinated segments of the axons with a

higher preponderance in the presynaptic terminals. The membranes forming the tubulovesicular structures were 5 - 7 nm thick. The myelinated segments demonstrated a very poor development of the myelin sheath. This consisted only of three to four lamellae, which exhibited an atypical periodicity (Figs.6,8), The axoplasm included numerous neurofilaments, microtubules, dilated vesicles and small dark mitochondria with irregular cristae and fbrillary inclusions in the matrix (Fig. 6). Almost all the presynaptic terminals contained numerous coated vesicles arranged mostly near the periphery. Apparently there was no continuity between the tubulovesicular elements and the coated vesicles (Fig.9). Discussion

The tubulovesicular profiles were the most impressive f n d i n g in the nonmyelinated axonal segments and in the presynaptic terminals in the irradiated cerebeUar


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Fig.9. Axodendritic synapse in organotypic culture of cerebellar cortex exposed to 7 irradiation. In the presynaptic terminal large number of pleomorphic vasicles, some of them very dilated, are roughly distributed. ( x 54,780)

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explants. Similar structures have been described in dilated axons and axonic terminals in various other axonal dystrophy processes, such as in Alzheimer disease in the human [6], in degeneration of the cerebellum after intrinsic or extrinsic lesions [15], adjacent to brain tumours [14], in early WaUerian degeneration [11], in cases of infantile neuroaxonal dystrophy [8,91, in case of mental retardation, convulsions and cortical blindness [5]. Tubulovesicular profiles have been described also in axons of the cat brain, in cases of degeneration within the serotonin pathways [1], in the lateral vestibular nuclei of apparently normal adult rats [16] and in the axon growth cones in the developing rat cerebellum [4]. In experimental conditions dystrophic axons with tubulovesicular aggregates have been noticed in the nucleus cuneatus of vitamin deficient rats [12] and in experimental spongiform encephalopathy in chimpanzees [13]. These tubulovesicular profiles presumably arose by an atypical proliferation of the smooth endoplasmic reticulum, which partially failed in the final step of synaptic vesicie recycling [7]. The ionizing irradiation seemed to interfere either in the mechanism of the conversion of the smooth endoplasmic reticular membranes into synaptic vesicles, or in the normal liberation of the neurotransmitter, due to deorganization of the synaptic formation in the agranular cerebellum, leading to a distruction of the local neuronal circuits. The defective liberation of the neurotransmitter, therefore, leads to a conversion of many synaptic vesicles into tubulovesicular profiles.

Acta Neuropathol (Bed) 54 (1981)

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References 1. Aghajanian GK, Bloom FE, Sheard MH (1966) Electron microscopy of degeneration within the serotonin pathway on rat brain. Brain Res 13:266-273 2. Altman J, Anderson W (1972) Experimental reorganization of the cerebellar cortex. I. Morphological efforts of elimination of

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all microneurons with prolonged X-irradiation started in birth. J Comp Neurol 146:355-406 Baloyannis S, Kim SU (1979) Experimental modification of cerebellar development in tissue culture: X-irradiation induces granular degeneration and unattached Purkinje cell dendritic spines. Neurosci Lett 12:283-288 Del Cerro MP, Snider RS (1968) Studies on the developing cerebellum ultrastructure of the growth cones. J Comp Neurol 133:341-361 Gonatas NK, Goldensohn ES (1965) Unusual neocortical presynaptic terminals m a patient with convulsions, mental retardation and cortical blindness: an electron microscopic study. J Neuropath Exp Neurol 24:539-562 Gonatas NK, Gambetti P (1970) The pathology of the synapse in Alzheimer disease. In: Walstenholme GEW, O'Connor M (eds) Ciba Foundation Symposium on Alzheimer disease and related conditions. Summit, New Jersey, pp 169-183 Gonatas NK, Moss A (1975) Pathologic axons and synapses in human neuropsychiatric disorders. Hum Pathol 6:511 - 582 Hedley-Whyte ET, Gilles FH, Uzman BG (1968) Infantile neuroaxonal dystrophy. A disease characterized by altered terminal axons and synapfic endings. Neurology 18 : 891 - 9 0 6 Herman MM, Huttenlocher PR, Bench KG (1969) Electron microscopic observations in infantile neuroaxonal dystrophy. Arch Neurol (Chicago) 2 0 : 1 9 - 3 9 Kim SU (1974) Granule cell with somatodendritic synapse in organotypic cuRures of mouse cerebellum. Exp Neuro[ 4 5 : 6 5 9 662 Laatsch RH (1969) Aggregates ofirregular axoplasmic tubules in early Wallerian degeneration of guinea pig optic nerve. Brain Res 14: 745 - 748 Lambert P, Blumberg JM, Pentschew A (1964) An electron microscopic study of dystrophy axons in the gracile and cuneate nuclei of vitamin E deficient rats. J Neuropath Exp Neuro123 : 60 Lambert PW, Cajdusek DC, Gibbs CJ Jr (1971) Experimental spongiform encephalopathy (Creutzfeld-Jacob desease) in chimpanzees. Electron microscopic studies. J Neuropath Exp NeuroI 30: 20 - 32 Ramsey HJ (1967) Altered synaptic terminals in cortex near tumor. Am J Path 51 : 1093 - 1109 Smith KR Jr, Hugens RW, O'Leary JL (1965) An elect~on microscopic study of degenerative changes in the eat after intrinsic and extrinsic lesions. J Comp Neurol 126:15 - 36 Sotelo C, Palay SL (1971) Altered axons and axon terminals in the lateral vestibular nucleus of the rat. Lab Invest 2 5 : 6 5 3 - 671

Received October 17, 1980/Accepted January 5, 1981