- Email: [email protected]
Astrocytes play a role in regulation of synaptic density
Brain Research, 402 (1987) 139-145
139
Elsevier BRE 21971
Short Communications
Astrocytes play a role in regulation of synaptic density Charles K. M e s h u l 1, Fredrick J. Seil I and R o b e r t M. H e r n d o n 2 1Neurology Research (151N), VeteransAdministration Medical Center, and Department of Neurology, Oregon Health Sciences University, Portland OR 97201 (U.S.A.)and 2Centerfor Brain Research, University of Rochester Medical School, Rochester, NY 14642 (U.S.A.)
(Accepted 2 September 1986) Key words: Astrocyte; Synaptic density; Electron microscopy; Cerebellar explant; Cytosine arabinoside
Exposure of neonatal cerebellar explants to cytosine arabinoside destroys granule cells and arrests surviving glia in an early stage of maturation. Purkinje cells lack astroglial ensheathment and are hyperinnervated by sprouted Purkinje cell recurrent axon collateral terminals. Such granuloprival cultures were transplanted with optic nerve in order to supply mature glial cells. It was observed that not only were Purkinje cells almost completely ensheathed by astroglia, but there was a greater than 60% reduction in the number of somatic synapses compared to the non-transplanted granuloprival cultures. This astroglial ensheathment, which may be neuronally directed, could be the physical element provoking the reduction in the number of synapses.
The interaction of axonal terminals and postsynaptic somatic and dendritic surfaces, which results in the formation of synaptic contacts, has long been thought to be regulated by the neurons themselves. Glial cells are believed to have structural and phagocytic functions 26, to serve as ion sinks during neuronal activity 13, to play a role in the sequestration of released neurotransmitters Is and, in some instances, to release neurotransmitters 6,8,17,26. There is also evidence to suggest that they guide neuronal migration during development 2x. A n indication that astrocytes could influence the maintenance of postsynaptic surfaces was reported in 1968, when it was found that after destruction of cerebellar granule cells in mature animals, Purkinje cell synaptic spines were invested by astrocytic processes t°. In addition, these spines maintained a morphologically complete, possibly enhanced, postsynaptic apparatus, including the cleft material. Later, it became clear that the dendritic spines could develop in the absence of afferent connections 11'14,15and that the postsynaptic apparatus would develop if, and
only if, astrocytic processes were present to replace the missing granule cell terminals on the spines 1'4. It has been shown that exposure of neonatal mouse cerebellar explants to cytosine arabinoside (Ara C) for the first 5 days in vitro ( D I V ) destroyed granule cells and arrested those surviving glia in an early stage of maturation 4,24. Purkinje cells lacked an astroglial ensheathment and underwent marked sprouting of recurrent axon collaterals. Recurrent axon collateral terminals synapsed not only with somata and dendrites of other Purkinje cells, but also with dendritic spines, sites normally occupied by terminals from granule cell axons (parallel fibers). Transplantation of such granuloprival cultures with granule cells and mature glia resulted in Purkinje cell ensheathment by astroglia, and a marked reduction in the number of recurrent axon collaterals and of recurrent collateral terminals synapsing on Purkinje cell somata and dendritic spines 3,23. We report here that the transplantation of granuloprival cultures with glial cells, but without granule cells, produces a dramatic reduction in synaptic contacts on the Put-
Correspondence: C.K. Meshul, Neurology Research (151N), Veterans Administration Medical Center, Portland, OR 97201, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
140 kinje cell soma. These results indicate that astrocytes play an important role in regulating synaptic density. Neonatal mouse cerebellar explants, exposed to Ara C (5/~g/ml of medium) for 5 days, and subsequently maintained in normal nutrient medium 24,
Fig. 1. A: for legend see p. 142.
were transplanted with 7-day-old mouse optic nerve at 13 D I V and maintained in normal medium for 22-35 DIV. Untreated control cultures and nontransplanted granuloprival explants were maintained in normal medium between 20 and 30 DIV. Cultures
141
Fig. 1. B: for legend see p. 142.
142
Fig. 1. Electron microscopy of Purkinje cells under various conditions. Astrocytic processes were identified on enlarged photomonrages, colored to add contrast, and rephotographed. A: control, untreated cerebellar explant (20 DIV) where several synaptic connections onto the Purkinje cell soma can be observed (small, dark, double arrows), along with an astroglial covering (large, open arrows). x11,235. B: cerebellar explant exposed to Ara C (5/~g/ml of medium) for 5 days, then maintained in normal medium until 30 DIV. Note the increase in the number of synapses (small, dark, double arrows) and the dramatic decrease in the astroglial covering of the Purkinje cell compared to the control, x 10,220. C: cerebeUar explant exposed to Ara C (5/zg/ml of medium) for 5 days, maintained in normal medium until 13 DIV, transplanted at this time with 7-day-old optic nerve (a source of glial cells), and then maintained in normal medium until 35 DIV. There is a large decrease in the number of synaptic connections (small, dark, double arrows) and an increase in the degree of astroglial ensheathment (large, open arrows) of the Purkinje cell compared to Fig. lB. x 12,600.
143 from all 3 groups were then fixed and processed for electron microscopy 4. Purkinje cells were identified under the electron microscope 19 and the n u m b e r of synaptic contacts around the soma counted. Synapses were counted only if the nerve terminal showed an accumulation of synaptic vesicles along the presynaptic membrane, and if there was a pre- and postsynaptic density. Control, untreated cerebellar explants developed a relatively normal range and density of synaptic connections on the Purkinje cells, as previously described 2'12'22 (Fig. 1A). The origin of terminals making synapses on Purkinje cells in vitro were from Purkinje cell recurrent collaterals, basket cells and stellate cells, as in vivo 19. A r a C treated cultures had a markedly increased density of synapses on the Purkinje cell somata (Fig. 1B), as reported previously 4. The origin of the majority of these synaptic contacts was from Purkinje cell recurrent axon collaterals. Basket and stellate cells contributed much less to the Purkinje cell input in granuloprival cultures since Ara C exposure resulted in a significant decrease in the numbers of these inhibitory interneurons 4. Unlike normal explants, there were few astrocytic processes directly in contact with the Purkinje cells (compare Figs. 1A and 1B). After addition of optic nerve to granuloprival explants, astrocytic processes could be seen in contact with the Purkinje cell somata. Concomitantly there was a decrease in the number of synapses on the Purkinje cell somata (see Table I and Fig. 1C). It is also evident from Table I
TABLE I Mean number of synapses on Purkinje cells following transplantation of granuloprival cerebellar cultures with optic nerve
Data represents mean number of presynaptic terminals making a synapse with Purkinje cell soma +_S.D. Total number of cells examined was 64 for the control cultures (n = 6), 35 for the Ara C treated cultures (n = 3) and 72 for the transplanted cultures (n = 6). Treatment
Synapses on Purkinje cell
Control
3.7 + 2.1
Ara C alone
5.8 + 1.33
Ara C followed by optic nerve transplantation
2.1 + 0.28*
* Significantly different (P < 0.05) vs the Ara C treatment alone.
that the mean number of synapses surrounding the Purkinje cells following transplantation with optic nerve is even less than in untreated control cultures, reflecting the Ara C induced decrease in the n u m b e r of basket and stellate cells. These results demonstrate that the addition of glial cells can directly induce a change in synaptic organization. There was a greater than 60% reduction in the number of synaptic connections along the Purkinje cell somata in the transplanted granuloprival cultures compared to non-transplanted granuloprival explants. The difference in the number of synapses in the Ara C treated cultures compared to untreated controls is actually greater than Table I suggests. Since Ara C also destroys basket and stellate cells, whose axons contribute to the input of the Purkinje cells, the increase in the number of synapses in granuloprival cultures is due almost solely to sprouted Purkinje cell recurrent axon collateral terminals 4. Several other studies have suggested a role for glial cells in synaptic reorganization. Blinzinger and Kreutzberg 5 showed that following transection of the facial nerve, there was a dramatic decrease in the number of synaptic terminals around those m o t o n e u rons. A qualitative and quantitative study 7 of the effects of nerve transection on chromatolyzed spinal motoneurons confirmed and extended the previous study 5. The detachment of terminals from the motoneurons not only involved a disappearance of both pre- and postsynaptic m e m b r a n e thickenings, but was associated with the presence of reactive astrocytes and microglia. The astrocytes appeared to occupy most of the neuronal fnembrane surface, with microglial processes covering only a small portion of the motoneuron. Following axotomy of the postganglionic superior cervical ganglion 16, the decrease in the number of synapses around the soma was associated with satellite cells surrounding the nerve terminal. This was followed by a disappearance of the postsynaptic thickening, with the maintenance of the presynaptic specialization. Neuronal-glial interactions in the hypothalamoneurohypophysial system were investigated in more recent studies 9'20'25. Changes in the physiological and/or hormonal state of the animals resulted in retraction of astrocytes from either neurosecretory cell bodies in the hypothalamus or from nerve terminals in the pituitary. This allowed for closer interaction
144
the Purkinje cell somata. A s A r a C destroys some glial cells and arrests the
d e e d the case, then optic nerve astrocytes have the capacity for behaving similarly to c e r e b e l l a r Golgi epithelial cells with r e g a r d to e n s h e a t h m e n t of Purkinje cell somata. With no previous o p p o r t u n i t y to interact with a Purkinje cell, what directs these optic nerve astrocytes to ensheath P u r k i n j e cells? Since it has been o b s e r v e d that developing axons a p p e a r to direct w h e r e myelination should t a k e place 27, and since m a n y neurons do not have astrocytic sheaths, it seems r e a s o n a b l e to suggest that the P u r k i n j e cell solicits astrocytic e n s h e a t h m e n t . C o n t r o l of the e n s h e a t h m e n t process m a y be the m e a n s of indirectly regulating the synaptic density a r o u n d the n e u r o n a l soma. O u r study is the first clear d e m o n s t r a t i o n that the addition of astrocytes capable of n e u r o n a l ensheathm e a t can induce a d r a m a t i c d e c r e a s e in the n u m b e r of previously established synaptic contacts surrounding Purkinje cell somata. A s this can be a c c o m p l i s h e d by astrocytes naive to Purkinje cells, it a p p e a r s that the n e u r o n regulates the e n s h e a t h m e n t process and m a y t h e r e b y control the synaptic density a r o u n d its soma.
m a t u r a t i o n of the remaining glia 4, the reactive astrocytes o b s e r v e d in the t r a n s p l a n t e d cultures are most p r o b a b l y derived from the optic nerve. If this is in-
S u p p o r t e d by the V e t e r a n s A d m i n i s t r a t i o n . W e thank K e n n e t h L. T i e k o t t e r for technical assistance.
b e t w e e n neuronal cell bodies and an increase in the n u m b e r of presynaptic terminals contacting two adjacent nerve cells, as in the hypothalamus, or allowing nerve terminals direct access to the perivascular space, as in the pituitary. A t the time p e r i o d investigated in the present study there was no evidence to directly show that the astrocytes insinuated b e t w e e n the nerve terminals and Purkinje cell bodies prior to e n s h e a t h m e n t of the neuron. H o w e v e r , b o u t o n - l i k e structures, with accumulations of synaptic vesicles, but lacking presynaptic specializations, were o b s e r v e d in the vicinity of the cell bodies. These terminals could possibly be the result of astrocytic stripping of synapses previously m a k i n g contact with the Purkinje cells. T h e lack of presynaptic specialization is consistent with the observations r e p o r t e d by Chen 7. H o w e v e r , in no instance were the terminals found to be c o m p l e t e l y surr o u n d e d by an astrocytic process. O n l y a d e t a i l e d time course study can evaluate the sequence of events leading to the r e m o v a l of the terminals from
1 Baloyannis, S.L. and Kim, S.U., Experimental modification of cerebellar development in tissue culture: X-irradiation induces granular degeneration and unattached Purkinje cell dendritic spines, Neurosci. Lett., 12 (1979) 283-288. 2 Blank, N.K. and Seil, F.J., Mature Purkinje cells in cerebellar tissue cultures: an ultrastructural study, J. Comp. Neurol., 208 (1982) 169-176. 3 Blank, N.K. and Seil, F.J., Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. II. Ultrastructural studies, J. Comp. Neurol., 214 (1983) 267-278. 4 Blank, N.K., Seil, F.J. and Herndon, R.M., An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture, Neuroscience, 7 (1982) 1509-1531. 5 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by .microglial cells, Z. Zellforsch. "Mikrosk. Anat., 85 (1968) 145-157. 6 Bowery, N.G. and Brown, D.A., Gamma-aminobutyric acid uptake by sympathetic ganglia, Nature (London) New Biol., 238 (1972) 89-91. 7 Chen, D.H., Qualitative and quantitative study of synaptic displacement in chromatolyzed spinal motoneurons of the cat, J. Comp. Neurol., 177 (1978) 635-664. 8 Dennis, M.J. and Miledi, R., Electrically induced release
of acetylcholine from denervated Schwann cells, J. Physiol. (London), 237 (1974) 431-452. 9 Hatton, G.I., Perlmutter, L.S., Salm, A.K. and Tweedle, C.D., Dynamic neuronal-glial interactions in hypothalamus and pituitary: implications for control of hormone synthesis and release, Peptides, 5 (1984) 121-138. 10 Herndon, R.M., Thiopen-induced granule cell necrosis in the rat cerebellum, Exp. Brain Res., 6 (1968) 49-68. 11 Herndon, R.M., Margolis, G. and Kilham, L., The synaptic organization of the malformed cerebellum induced by perinatal infection with the feline panleukopenia virus (PLV), J. Neuropathol. Exp. Neurol., 30 (1971) 557-570. 12 Herndon, R.M., Seil, F.J. and Seidman, C., Synaptogenesis in mouse cerebellum: a comparative in vivo and tissue culture study, Neuroscience, 6 (1981) 2587-2598. 13 Hertz, L., An intense potassium uptake into astrocytes, its further enhancement by high concentrations of potassium, and its possible involvement in potassium homeostasis at the cellular level, Brain Research, 145 (1978) 202-208. 14 Hirano, A. and Jones, M., Fine structure of cycasin-induced cerebellar alterations, Fed. Proc., 31 (1972) 1517-1519. 15 Jones, M.Z. and Gardner, E., Pathogenesis of methylazoxymethanol-induced lesions in the postnatal mouse cerebellum, J. Neuropathol. Exp. Neurol., 35 (1976) 413-444. 16 Matthews, M.R. and Nelson, V.H., Detachment of struc-
145
17
18
19 20
21
turally intact nerve endings from chromatolytic neurones of rat superior cervical ganglion during the depression of synaptic transmission induced by postganglionic axotomy, J. Physiol. (London), 245 (1975) 91-135. Minchen, M.C.W. and Iversen, L.L., Release of [3H]gamma-aminobutyric acid from glial cells in rat dorsal root ganglia, J. Neurochem., 23 (1974) 533-540. Orkand, P.M. and Kravitz, E.A., Localization of the sites of gamma-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations, J. Cell Biol., 49 (1971) 75-89. Palay, S.L. and Chan-Palay, V., Cerebellar Cortex: Cytology and Organization, Springer, New York, 1974, 348 pp. Perlmutter, L.S., Tweedle, C.D. and Hatton, G.I., Neuronal/glial plasticity in the supraoptic dendritic zone: dendritic bundling and double synapse formation at parturition, Neuroscience, 13 (1984) 769-779. Rakic, P., Neuron-glia relationships during granule cell migration in cerebellar cortex, J. Comp. Neurol., 141 (1971) 283-312.
22 Seil, F.J., Cerebellum in tissue culture. In D.M. Schneider (Ed.), Reviews of Neuroscience, Vol. 4, Raven, New York, 1979, pp 105-177. 23 Seil, F.J., Leiman, A.L. and Blank, N.K., Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. I. Light microscopic and electrophysiological studies, J. Comp. Neurol., 214 (1983) 258-266. 24 Seil, F.J., Leiman, A.L. and Woodward, W.R., Cytosine arabinoside effects on developing cerebellum in tissue culture, Brain Research, 186 (1980) 393-408. 25 Tweedle, C.D. and Hatton, G.I., Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states, Brain Research, 309 (1984) 373-376. 26 Varon, S.S. and Somjen, G.G., Neuron-glia interactions, Neurosci. Res. Prog. Bull., 17 (1979) 1-239. 27 Waxman, S.G. and Foster, R.E., Ionic channel distribution and heterogeneity of the axon membrane in myelinated fibers, Brain Res. Rev., 2 (1980) 205-234.
139
Elsevier BRE 21971
Short Communications
Astrocytes play a role in regulation of synaptic density Charles K. M e s h u l 1, Fredrick J. Seil I and R o b e r t M. H e r n d o n 2 1Neurology Research (151N), VeteransAdministration Medical Center, and Department of Neurology, Oregon Health Sciences University, Portland OR 97201 (U.S.A.)and 2Centerfor Brain Research, University of Rochester Medical School, Rochester, NY 14642 (U.S.A.)
(Accepted 2 September 1986) Key words: Astrocyte; Synaptic density; Electron microscopy; Cerebellar explant; Cytosine arabinoside
Exposure of neonatal cerebellar explants to cytosine arabinoside destroys granule cells and arrests surviving glia in an early stage of maturation. Purkinje cells lack astroglial ensheathment and are hyperinnervated by sprouted Purkinje cell recurrent axon collateral terminals. Such granuloprival cultures were transplanted with optic nerve in order to supply mature glial cells. It was observed that not only were Purkinje cells almost completely ensheathed by astroglia, but there was a greater than 60% reduction in the number of somatic synapses compared to the non-transplanted granuloprival cultures. This astroglial ensheathment, which may be neuronally directed, could be the physical element provoking the reduction in the number of synapses.
The interaction of axonal terminals and postsynaptic somatic and dendritic surfaces, which results in the formation of synaptic contacts, has long been thought to be regulated by the neurons themselves. Glial cells are believed to have structural and phagocytic functions 26, to serve as ion sinks during neuronal activity 13, to play a role in the sequestration of released neurotransmitters Is and, in some instances, to release neurotransmitters 6,8,17,26. There is also evidence to suggest that they guide neuronal migration during development 2x. A n indication that astrocytes could influence the maintenance of postsynaptic surfaces was reported in 1968, when it was found that after destruction of cerebellar granule cells in mature animals, Purkinje cell synaptic spines were invested by astrocytic processes t°. In addition, these spines maintained a morphologically complete, possibly enhanced, postsynaptic apparatus, including the cleft material. Later, it became clear that the dendritic spines could develop in the absence of afferent connections 11'14,15and that the postsynaptic apparatus would develop if, and
only if, astrocytic processes were present to replace the missing granule cell terminals on the spines 1'4. It has been shown that exposure of neonatal mouse cerebellar explants to cytosine arabinoside (Ara C) for the first 5 days in vitro ( D I V ) destroyed granule cells and arrested those surviving glia in an early stage of maturation 4,24. Purkinje cells lacked an astroglial ensheathment and underwent marked sprouting of recurrent axon collaterals. Recurrent axon collateral terminals synapsed not only with somata and dendrites of other Purkinje cells, but also with dendritic spines, sites normally occupied by terminals from granule cell axons (parallel fibers). Transplantation of such granuloprival cultures with granule cells and mature glia resulted in Purkinje cell ensheathment by astroglia, and a marked reduction in the number of recurrent axon collaterals and of recurrent collateral terminals synapsing on Purkinje cell somata and dendritic spines 3,23. We report here that the transplantation of granuloprival cultures with glial cells, but without granule cells, produces a dramatic reduction in synaptic contacts on the Put-
Correspondence: C.K. Meshul, Neurology Research (151N), Veterans Administration Medical Center, Portland, OR 97201, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
140 kinje cell soma. These results indicate that astrocytes play an important role in regulating synaptic density. Neonatal mouse cerebellar explants, exposed to Ara C (5/~g/ml of medium) for 5 days, and subsequently maintained in normal nutrient medium 24,
Fig. 1. A: for legend see p. 142.
were transplanted with 7-day-old mouse optic nerve at 13 D I V and maintained in normal medium for 22-35 DIV. Untreated control cultures and nontransplanted granuloprival explants were maintained in normal medium between 20 and 30 DIV. Cultures
141
Fig. 1. B: for legend see p. 142.
142
Fig. 1. Electron microscopy of Purkinje cells under various conditions. Astrocytic processes were identified on enlarged photomonrages, colored to add contrast, and rephotographed. A: control, untreated cerebellar explant (20 DIV) where several synaptic connections onto the Purkinje cell soma can be observed (small, dark, double arrows), along with an astroglial covering (large, open arrows). x11,235. B: cerebellar explant exposed to Ara C (5/~g/ml of medium) for 5 days, then maintained in normal medium until 30 DIV. Note the increase in the number of synapses (small, dark, double arrows) and the dramatic decrease in the astroglial covering of the Purkinje cell compared to the control, x 10,220. C: cerebeUar explant exposed to Ara C (5/zg/ml of medium) for 5 days, maintained in normal medium until 13 DIV, transplanted at this time with 7-day-old optic nerve (a source of glial cells), and then maintained in normal medium until 35 DIV. There is a large decrease in the number of synaptic connections (small, dark, double arrows) and an increase in the degree of astroglial ensheathment (large, open arrows) of the Purkinje cell compared to Fig. lB. x 12,600.
143 from all 3 groups were then fixed and processed for electron microscopy 4. Purkinje cells were identified under the electron microscope 19 and the n u m b e r of synaptic contacts around the soma counted. Synapses were counted only if the nerve terminal showed an accumulation of synaptic vesicles along the presynaptic membrane, and if there was a pre- and postsynaptic density. Control, untreated cerebellar explants developed a relatively normal range and density of synaptic connections on the Purkinje cells, as previously described 2'12'22 (Fig. 1A). The origin of terminals making synapses on Purkinje cells in vitro were from Purkinje cell recurrent collaterals, basket cells and stellate cells, as in vivo 19. A r a C treated cultures had a markedly increased density of synapses on the Purkinje cell somata (Fig. 1B), as reported previously 4. The origin of the majority of these synaptic contacts was from Purkinje cell recurrent axon collaterals. Basket and stellate cells contributed much less to the Purkinje cell input in granuloprival cultures since Ara C exposure resulted in a significant decrease in the numbers of these inhibitory interneurons 4. Unlike normal explants, there were few astrocytic processes directly in contact with the Purkinje cells (compare Figs. 1A and 1B). After addition of optic nerve to granuloprival explants, astrocytic processes could be seen in contact with the Purkinje cell somata. Concomitantly there was a decrease in the number of synapses on the Purkinje cell somata (see Table I and Fig. 1C). It is also evident from Table I
TABLE I Mean number of synapses on Purkinje cells following transplantation of granuloprival cerebellar cultures with optic nerve
Data represents mean number of presynaptic terminals making a synapse with Purkinje cell soma +_S.D. Total number of cells examined was 64 for the control cultures (n = 6), 35 for the Ara C treated cultures (n = 3) and 72 for the transplanted cultures (n = 6). Treatment
Synapses on Purkinje cell
Control
3.7 + 2.1
Ara C alone
5.8 + 1.33
Ara C followed by optic nerve transplantation
2.1 + 0.28*
* Significantly different (P < 0.05) vs the Ara C treatment alone.
that the mean number of synapses surrounding the Purkinje cells following transplantation with optic nerve is even less than in untreated control cultures, reflecting the Ara C induced decrease in the n u m b e r of basket and stellate cells. These results demonstrate that the addition of glial cells can directly induce a change in synaptic organization. There was a greater than 60% reduction in the number of synaptic connections along the Purkinje cell somata in the transplanted granuloprival cultures compared to non-transplanted granuloprival explants. The difference in the number of synapses in the Ara C treated cultures compared to untreated controls is actually greater than Table I suggests. Since Ara C also destroys basket and stellate cells, whose axons contribute to the input of the Purkinje cells, the increase in the number of synapses in granuloprival cultures is due almost solely to sprouted Purkinje cell recurrent axon collateral terminals 4. Several other studies have suggested a role for glial cells in synaptic reorganization. Blinzinger and Kreutzberg 5 showed that following transection of the facial nerve, there was a dramatic decrease in the number of synaptic terminals around those m o t o n e u rons. A qualitative and quantitative study 7 of the effects of nerve transection on chromatolyzed spinal motoneurons confirmed and extended the previous study 5. The detachment of terminals from the motoneurons not only involved a disappearance of both pre- and postsynaptic m e m b r a n e thickenings, but was associated with the presence of reactive astrocytes and microglia. The astrocytes appeared to occupy most of the neuronal fnembrane surface, with microglial processes covering only a small portion of the motoneuron. Following axotomy of the postganglionic superior cervical ganglion 16, the decrease in the number of synapses around the soma was associated with satellite cells surrounding the nerve terminal. This was followed by a disappearance of the postsynaptic thickening, with the maintenance of the presynaptic specialization. Neuronal-glial interactions in the hypothalamoneurohypophysial system were investigated in more recent studies 9'20'25. Changes in the physiological and/or hormonal state of the animals resulted in retraction of astrocytes from either neurosecretory cell bodies in the hypothalamus or from nerve terminals in the pituitary. This allowed for closer interaction
144
the Purkinje cell somata. A s A r a C destroys some glial cells and arrests the
d e e d the case, then optic nerve astrocytes have the capacity for behaving similarly to c e r e b e l l a r Golgi epithelial cells with r e g a r d to e n s h e a t h m e n t of Purkinje cell somata. With no previous o p p o r t u n i t y to interact with a Purkinje cell, what directs these optic nerve astrocytes to ensheath P u r k i n j e cells? Since it has been o b s e r v e d that developing axons a p p e a r to direct w h e r e myelination should t a k e place 27, and since m a n y neurons do not have astrocytic sheaths, it seems r e a s o n a b l e to suggest that the P u r k i n j e cell solicits astrocytic e n s h e a t h m e n t . C o n t r o l of the e n s h e a t h m e n t process m a y be the m e a n s of indirectly regulating the synaptic density a r o u n d the n e u r o n a l soma. O u r study is the first clear d e m o n s t r a t i o n that the addition of astrocytes capable of n e u r o n a l ensheathm e a t can induce a d r a m a t i c d e c r e a s e in the n u m b e r of previously established synaptic contacts surrounding Purkinje cell somata. A s this can be a c c o m p l i s h e d by astrocytes naive to Purkinje cells, it a p p e a r s that the n e u r o n regulates the e n s h e a t h m e n t process and m a y t h e r e b y control the synaptic density a r o u n d its soma.
m a t u r a t i o n of the remaining glia 4, the reactive astrocytes o b s e r v e d in the t r a n s p l a n t e d cultures are most p r o b a b l y derived from the optic nerve. If this is in-
S u p p o r t e d by the V e t e r a n s A d m i n i s t r a t i o n . W e thank K e n n e t h L. T i e k o t t e r for technical assistance.
b e t w e e n neuronal cell bodies and an increase in the n u m b e r of presynaptic terminals contacting two adjacent nerve cells, as in the hypothalamus, or allowing nerve terminals direct access to the perivascular space, as in the pituitary. A t the time p e r i o d investigated in the present study there was no evidence to directly show that the astrocytes insinuated b e t w e e n the nerve terminals and Purkinje cell bodies prior to e n s h e a t h m e n t of the neuron. H o w e v e r , b o u t o n - l i k e structures, with accumulations of synaptic vesicles, but lacking presynaptic specializations, were o b s e r v e d in the vicinity of the cell bodies. These terminals could possibly be the result of astrocytic stripping of synapses previously m a k i n g contact with the Purkinje cells. T h e lack of presynaptic specialization is consistent with the observations r e p o r t e d by Chen 7. H o w e v e r , in no instance were the terminals found to be c o m p l e t e l y surr o u n d e d by an astrocytic process. O n l y a d e t a i l e d time course study can evaluate the sequence of events leading to the r e m o v a l of the terminals from
1 Baloyannis, S.L. and Kim, S.U., Experimental modification of cerebellar development in tissue culture: X-irradiation induces granular degeneration and unattached Purkinje cell dendritic spines, Neurosci. Lett., 12 (1979) 283-288. 2 Blank, N.K. and Seil, F.J., Mature Purkinje cells in cerebellar tissue cultures: an ultrastructural study, J. Comp. Neurol., 208 (1982) 169-176. 3 Blank, N.K. and Seil, F.J., Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. II. Ultrastructural studies, J. Comp. Neurol., 214 (1983) 267-278. 4 Blank, N.K., Seil, F.J. and Herndon, R.M., An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture, Neuroscience, 7 (1982) 1509-1531. 5 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by .microglial cells, Z. Zellforsch. "Mikrosk. Anat., 85 (1968) 145-157. 6 Bowery, N.G. and Brown, D.A., Gamma-aminobutyric acid uptake by sympathetic ganglia, Nature (London) New Biol., 238 (1972) 89-91. 7 Chen, D.H., Qualitative and quantitative study of synaptic displacement in chromatolyzed spinal motoneurons of the cat, J. Comp. Neurol., 177 (1978) 635-664. 8 Dennis, M.J. and Miledi, R., Electrically induced release
of acetylcholine from denervated Schwann cells, J. Physiol. (London), 237 (1974) 431-452. 9 Hatton, G.I., Perlmutter, L.S., Salm, A.K. and Tweedle, C.D., Dynamic neuronal-glial interactions in hypothalamus and pituitary: implications for control of hormone synthesis and release, Peptides, 5 (1984) 121-138. 10 Herndon, R.M., Thiopen-induced granule cell necrosis in the rat cerebellum, Exp. Brain Res., 6 (1968) 49-68. 11 Herndon, R.M., Margolis, G. and Kilham, L., The synaptic organization of the malformed cerebellum induced by perinatal infection with the feline panleukopenia virus (PLV), J. Neuropathol. Exp. Neurol., 30 (1971) 557-570. 12 Herndon, R.M., Seil, F.J. and Seidman, C., Synaptogenesis in mouse cerebellum: a comparative in vivo and tissue culture study, Neuroscience, 6 (1981) 2587-2598. 13 Hertz, L., An intense potassium uptake into astrocytes, its further enhancement by high concentrations of potassium, and its possible involvement in potassium homeostasis at the cellular level, Brain Research, 145 (1978) 202-208. 14 Hirano, A. and Jones, M., Fine structure of cycasin-induced cerebellar alterations, Fed. Proc., 31 (1972) 1517-1519. 15 Jones, M.Z. and Gardner, E., Pathogenesis of methylazoxymethanol-induced lesions in the postnatal mouse cerebellum, J. Neuropathol. Exp. Neurol., 35 (1976) 413-444. 16 Matthews, M.R. and Nelson, V.H., Detachment of struc-
145
17
18
19 20
21
turally intact nerve endings from chromatolytic neurones of rat superior cervical ganglion during the depression of synaptic transmission induced by postganglionic axotomy, J. Physiol. (London), 245 (1975) 91-135. Minchen, M.C.W. and Iversen, L.L., Release of [3H]gamma-aminobutyric acid from glial cells in rat dorsal root ganglia, J. Neurochem., 23 (1974) 533-540. Orkand, P.M. and Kravitz, E.A., Localization of the sites of gamma-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations, J. Cell Biol., 49 (1971) 75-89. Palay, S.L. and Chan-Palay, V., Cerebellar Cortex: Cytology and Organization, Springer, New York, 1974, 348 pp. Perlmutter, L.S., Tweedle, C.D. and Hatton, G.I., Neuronal/glial plasticity in the supraoptic dendritic zone: dendritic bundling and double synapse formation at parturition, Neuroscience, 13 (1984) 769-779. Rakic, P., Neuron-glia relationships during granule cell migration in cerebellar cortex, J. Comp. Neurol., 141 (1971) 283-312.
22 Seil, F.J., Cerebellum in tissue culture. In D.M. Schneider (Ed.), Reviews of Neuroscience, Vol. 4, Raven, New York, 1979, pp 105-177. 23 Seil, F.J., Leiman, A.L. and Blank, N.K., Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. I. Light microscopic and electrophysiological studies, J. Comp. Neurol., 214 (1983) 258-266. 24 Seil, F.J., Leiman, A.L. and Woodward, W.R., Cytosine arabinoside effects on developing cerebellum in tissue culture, Brain Research, 186 (1980) 393-408. 25 Tweedle, C.D. and Hatton, G.I., Synapse formation and disappearance in adult rat supraoptic nucleus during different hydration states, Brain Research, 309 (1984) 373-376. 26 Varon, S.S. and Somjen, G.G., Neuron-glia interactions, Neurosci. Res. Prog. Bull., 17 (1979) 1-239. 27 Waxman, S.G. and Foster, R.E., Ionic channel distribution and heterogeneity of the axon membrane in myelinated fibers, Brain Res. Rev., 2 (1980) 205-234.