Formation of presynaptic dendrites in the rat cerebellum following neonatal X-irradiation

il’~~~it~c~.

1977.

Vol.2, pp.

275-283.

Pergamon Press

Printed

in Great Britain.

FORMATION OF PRESYNAPTIC DENDRITES IN THE RAT CEREBELLUM FOLLOWING NEONATAL X-IRRADIATION C. SOTELO

Laboratoire de Neuromorpholo~e

(U-106 INSERM) Centre-medico-Chirurgical 92150 Suresnes, France

FOCH,

Abstract-The ultrastructure of the cerebellum was studied after X-irradiation of the whole head of newborn rats. The neuronal elements forming the cerebellar circuitry in rats 30-60 days after irradiation were limited to climbing fibers, mossy fibers, and Purkinje and Golgi cells. Under these conditions the perikarya and dendrites of the Golgi neurons develop presynaptic vesicular grids. These unconventional presynaptic elements establish numerous synaptic contacts with spines and occasionally with shafts of Purkinje cell dendrites. The results indicate that interference with normal cerebellar development, such that granule, basket and stellate cells are absent, generates new types of cellular interactions during synaptogenesis which allow Golgi cells to express their latent potentiality to form presynaptic perikarya and dendrites. It is concluded that this latent potentiality is repressed during development of the normal cerebellum by the presence of the other interneurons.

dritic levels. Furthermore, there is a growing awareness that many of the short-axon neurons may often NUMEROUSmorphologicai and elec~ophysiolo~cal develop such presynaptic dendrites. studies published in the last decade have demonOn the other hand, an outstanding example of a strated that modeling all nerve cells after the spinal neuronal network totally lacking in these unconvenmotoneuron (i.e. consisting of a passive dendritic tree, tional synapses, although it has a plethora of shortan electroresponsive initial segment and a transmitaxon neurons, is the normal cerebellar cortex (LLINAS, ting axon) is no longer acceptable (cf. SCHMITTet af., 1969; MUGNAINI,1972; PALAY& CHAN-PALAY.1974). 1976). In fact, RAMCIN Y CAJAL'S suggestion (1911) that Even in this case, however. unconventional synapses synaptic transmission in axonless neurons (such as have been observed in unusual circumst~ces such retinal amacrine cells and olfactory inner granule as ectopic granule cells in the molecular layer (~PA(SEK cells) could take place through presynaptic dendrites et al., 1973). organotypic cerebellar cultures (KIM, has been validated and enlarged. Thus, it is accepted 1974), and in some neurological mutations affecting today that neurons in many regions of the central the mouse cerebellum (SOTELO, 1975~; MARIANIet al., nervous system, from the spinal cord (SZENTP;GOTHAI 1977). Most commonly, these synapses are established & ARBIB, 1974) to the neocortex (SLOPER,1971) and between granule cell perikarya and Purkinje cell denparticularly in the thalamic nuclei (RALSTON& HERdrites. In addition, dendro-dend~tic synapses between MAN, 1969; FAMIG~IE~I, 1970; LE VAY, 1971; MORgranule cell dendrites and Purkinje cell dendritic EST,1971; WONG-RILEY,1972; PASIKet al., 1973), may spines have also been reported in mutant mice have presynaptic properties at both axonic and den(SOTELO,1975a,; MARIANIer al., 1977). Following previous work (SOTELO, 1975u,b) the conclusion was reached that the intrinsic specificity for afferent-effer1 ‘Heterologous’ synapses are here considered as those ent coupling in the development of the cerebellar cirestablished between two neurons, which under normal concuitry can be overridden, to a certain extent, by local ditions never form synaptic contacts. They are the expresfactors. Further, the most favorable local conditions sion of a certain lack of specificity during synaptogenesis in pathological conditions. for the formation of these unconventional synaptic ‘Ectopic’ synapses are those established between two connections ~heterologous’ and ‘ectopic’ synapses)’ neurons which normally form synaptic connections, but are obtained in the agranular cerebellum, where there the presynaptic partner instead of being the axon, as in are innumerable free postsynaptic receptor sites. Innormal cerebellum, is the dendrite and/or the soma. In deed it is in cerebella devoid of a large proportion these instances the synaptic specificity is preserved. of granule cells that most of the ‘ectopic’ synapses ‘Heterologous-ectopic’ synapses are those established between granule cells and Purkinje cells have been between two neurons, which under normal conditions reported. It seems that in such situations a neuronal never form synaptic contacts. In addition. the presynaptic p~uripotentiality is revealed by the unusually favorelement instead of being the axon, is the dendrite and/or the soma. This type of synapse is the main concern of able local conditions produced by the lack of compethe present paper. tition for the postsynaptic space. INTRODUCTION

FIG. 1. Synapse rn mctrron between a mossy terminal (mf) and a Golgi cell profile (Go P). therefore, identifies the Go P as belonging to the perikaryon. Profiles of Purkinje cell dendrites (Pden) are numerous; one of their spines (s) is directly apposed to the mossy fiber. In the cytoplasm of the Golgi cell, two clusters of spherical synaptic vesicles (arrows) are present; one of them is associated with a synapse on a Purkinje cell dendritic spine (asterisk) x 32,000. Ftc;. 2. Dendro-dendritic synapses between a Golgi dendrite (Go den) and a Purkinje dendrite (P den). Note that the synapse is established on the shaft of the dendrite (asterisk) x 28,000. FK. 3. Synapse en ntarron between a mossy terminal (mf) and the perikaryon of a Golgi cell (Go P). The curved arrows indicate the origins of small dendritic branches leaving the perikaryon. Note that in one of the branches. there is a cluster of spherical synaptic vesicles (arrow) probably synapsing on a Purkinje cell dendritic spine (S). Purkinje cell dendritic profile and free spines are numerous. Profiles containing flattened synaptic vesicles belong to Golgi axon terminals (ax) x 31,000. FK;. 4. Electron micrograph of an irregular profile that in spite of the absence of synapse en man’otr has been considered as belonging to a Golgi cell perikaryon (Go P). In addition to numerous large granulated vesicles dispersed in the cytoplasm, there is a cluster of spherical synaptic vesicles (big arrow) forming a presynaptic vesicular grid contacting a large Purkinje cell dendritic spine (S). The small arrow points to the neck of the spine x 23.000. FK. 5. A large dendritic profile of a Golgi cell (Goden), giving rise to irregularly spaced protrusions (big arrows) and containing a labyrinthine body (lb) establishes a synaptic contact (small arrow) with a Purkinje cell dendritic spine (S). Numerous axon terminals filled with flattened vesicles and probably belonging to Golgi cell axons (ax) are near by the dendritic profile x 30,000. Inset: High magnification of the dendro-dendritic synapse. Note the spherical shape of the Golgi cell synaptic vesicles (arrow) x 87,OCKL

Presynaptic dendrites in the rat cerebellum

281

belonging to Golgi cells. Furthermore. all Purkinje The aim of this paper is to anatyze the ‘heterologousectopic synapses which develop in the rat cere- cell dendrites are studded with spines, most of which are free of innervation. On the other hand, Golgi bellum following X-irradiation damage of the external dendrites have substantially fewer spines which genergranular layer at birth and thus of all the neurons ally are more irregular and longer than those of the which originate from this secondary germinal neuroPurkinje cell. The analysis of the synaptic investment epithelium (granule, stellate and basket cells). Under these conditions, as reported by ALTMAN & ANDER- of both classes of neurons can also be used to clarify their identity. Purkinje cells are contacted generally SON (1972) only the two afferent systems consisting at their spine surfaces and predominantly by climbing of climbing and mossy fibers and Purkinje and Golgi fibers, but occasionally ‘heterologous’ mossy fiber ceils are present in the cerebellar cortex. Since Pursynapses are found. In contrast, Golgi cells are the kinje cells in agranular cerebella develop numerous main postsynapti~ target for mossy fibers, which in free postsynaptic receptor sites (HERNDON et al., 1971; ALTMAN& ANJXRSON, 1972; HIRANO & DEMMBITZER, the absence of granule cells increase their synaptic relationships with Golgi cells (RAKIC& SIDMAN,1973: 1973; LLINAS et al., 1973; RAKIC & SIDMAN, 1973; SOTELO.197.50).Recurrent collaterals of Purkinje cell SOTELO,1973, 197%). these could also offer favorable local conditions for the possible expression of pre- axons form synapses on neurons of both classes. Due to the highly irregular surface of the Golgi synaptic Golgi cell dendrites. cell perikarya (PAL.AY & CHAN-PALAY, 1974), the dendritic or somatic nature of Golgi cell profiles encounMATERIALS AND METHODS tered in single electron micrographs can be very diffiNewborn rats (Sherman rats of a strain from the cult to determine. However, CHAN-PALAY& PALAY C.R.N.S.) were irradiated by Dr N. Delhaye-Bouchaud fol- (1971) indicated that the presence of a special synaptic lowing a standard procedure (CREPELet nl., 1976). Each arrangement, the synapse en marron, between a mossy rat received a total dose of 1100 rads distributed in seven varicosity and a Golgi cell profile can be used to idensuccessive exposures. On the day of birth (day0) they received 200rads and on days 3, 5, 7, 10, 12 and 14 tify the perikaryon. According to the above authors, this structure is characterized by ‘the ridges and furreceived 1.50rads each time. rows of the interface, the ribbon-like synaptic comIrradiated rats, 30 to 60 days of age, were anesthetized plexes in the furrows and the postsynaptic fibrillary by i.p. injection of 3.59; (w/v) chloral hydrate (0.1 ml/lOOg body weight). They were fixed by intracardiac perfusion zone’. In the present study the somatic nature of of 1OOOmlof a warm solution of 1% (w/v) paraformaldeGolgi cell profiles lacking a nucleus will be deterhyde and I a0(w/v) glutaraIdehyde in 0.12 M mono-sodium mined by the presence of synapses en marrm (Figs. di-potassium phosphate buffer (pH 7.3). After fixation, the 1, 3). cerebellum was removed and cut in the sagittal plane. The irradiated cerebellum does not develop the Siices about 1mm thick were post-fixed by immersion for typical cortical layering and Purkinje cells are found 4 h in 2”:, (w/v) osmic acid in 0.12 M phosphate buffer widely dispersed over the cortical zone. Most of the (pH 7.3) containing 7% (w/v) glucose. After routine staining synapses between mossy fibers and Golgi cells are en bloc with uranyl acetate, the slices were dehydrated in situated in the deep region of the cortex, closer to graded solutions of ethanol and embedded an Araldite. One-micron thick sections were cut from the vermis and the white matter where the maximum density of Golgi the hemispheres and stained with toluidine blue. Following cell profiles is located. In a relatively large proportion light microscopic examination of the sections, regions of of these profiles, in addition to numerous large granuthe cortex in which the agranularity was complete were lated vesicles found throughout the cytoplasm (Figs. selected and trimmed. Thin sections were subsequ~tly 1, 3 and 4), there are clusters of synaptic-like vesicles obtained from these regions and mounted on uncoated suspended in the somatic (Figs. 1, 4) or dendritic copper grids. double stained with uranyl acetate and lead (Figs. 2, 3, 5) cytoplasmic matrix. These clusters are citrate. and examined with a Siemens Elmiskop IA. located mainly at the periphery of the neuronal profile in association with the Golgi cell membrane RESULTS where they form typical presynaptic vesicular grids The ultrastructure of the agranular cerebellum (Figs. 1, 4, 5). The postsynaptic elements in these obtained by X-irradiation has been described heterologous-ectopic synapses are almost exclusively (HAMORI,1969; ALTMAN& ANDERSON, 1972). For this dendritic spines (Figs. 1, 3, 4, 5); which are identified reason we will consider only the morphological re- as belonging to Purkinje cells. Furthermore, in favorsponse of Golgi cells to the modified cellular environable sections, as illustrated in Fig. 4, the postsynaptic ment. Discriminating between neuronal profiles spine is connected by a narrow neck with its parent belonging to Golgi and Purkinje cells is a straightfordendrite which can easily be identified as a Purkinje ward process in this agranular cerebellum. Indeed, cell dendrite. Occasionally the postsynaptic elements Purkinje cell profiles conserve all the ultrastructural are the actual shafts of this class of dendrite (Fig. features which allow their identification, in particular 2). In both types of heterologoussectopic synapse the hypolemmal cisterna (Figs. 1, 3 and 4) (PALAY (soma-dendritic and dendro-dendritic) the active zone & CHAN-PALAY,1974). Large cell bodies and dendriformed between the Golgi profile and the Purkinje tic profiles lacking this characteristic are identified as spine resembles that of a type I synapse (Figs. 1 and

S), as is the case in the normal cerebellum between Golgi axons and granule cell dendrites (PALAY &

CHAN-PALAY,1974). However, in the normal cerebellum the synaptic vesicles exhibit a flattened shape, whereas the synaptic vesicles are spherical in the heterologous soma-dendritic (Figs. 1, 4) and dendrodendritic contacts (Figs. 3, 5).

DISCUSSION The results demonstrate that under certain experimental conditions Golgi cells of the rat cerebellar cortex establish soma-dendritic and dendro-dendritic synaptic contacts with Purkinje cells. These classes of synapses are never present in a normal cerebellum. However, the existence of such unconventional synapses appears to be a fundamental feature of neuronal networks in many other regions of the central nervous system, especially in sensory thalamic nuclei (LE VAY, 1971; MORES, 1971; F~IGLIE~I & PETERS, 1972; WOKG-RILEY,1972; LIEBERMAN, 1973; PASIK et al., 1973). Nevertheless, their existence is not universal. This fact raises the question of whether (1) there are two different categories of Golgi II cells, (a) one possessing the autonomous capability of transmitting information not only through their axon terminals, but also via presynaptic perikarya and dendrites, and (b) a second type where only the axon is utilized for synaptic transmission, or whether (2) all Golgi II cells have the intrinsic potential of transmitting through axons and dendrites with this property only being expressed if certain specific celfular interactions occur during the establishment of the neuronal networks. In order to decide which of these two possibilities accounts for the findings in the irradiated cerebellum, it is necessary to establish whether the major determining factor for the occurrence of heterologous synapses is the lack of development of cerebellar interneurons or the direct effect of radiation on the remaining cells. If the former were to be the case, examples of abnormal synaptic connectivity should result from other types of lesions capable of producing either the death or total absence of interneuronal elements of the cerebellar circuit. On the other hand, if radiation alone could produce, due to destruction of surface specificity, this kind of heterologous-ecto-

pit synaptic contact, they should be observed in irradiated cerebella even under conditions where X-irradiation does not prevent the formation of the majority of basket and stellate cells. Since the latter has not been observed (%TELO, unpublished observations), the first possibility appears the most likely. In fact, in other agranular cerebella such as those of U’~UPPI’mice (%TELO, 197%) and the heterotopic regions of the cerebellum in reeler mice (MARIAN] rt al.. 1977), although Golgi cell presynaptic perikarya and dendrites have not yet been observed, granule cells form soma-dendritic and dendro-dendritic synapses with Purkinje cells. This is found, for example, in the cerebellar hemispheres of weauer mice, where only a few granule cells migrate successfuhy to the granule layer, and in the superficial zone of the central cerebellar mass in reeler mice where some rare granule cells and stellate cells are intermingled with numerous Purkinje cells and heavily myelinated fibers. These results indicate that another class of cerebelfar intemeurons, the granule cells, can under ccrtain local conditions, and in the absence of irradiation, form presynaptic grids in their perikarya and dendrites which establish ectopic synapses with Purkinje cells. In conclusion, the present results strongly suggest that the occurrence of heterologous-ectopic synapses demonstrates a basic potentiality of Golgi type II neurons. Thus, it is suggested that the presence or absence of these heterologous synapses might be the result of the cellular milieu in which the cells develop rather than being pr~etermined geneticaI~y. The difference between the genetic or viral-induced agranular cerebellar cortices, in which this kind of heterolo-

goussectopic synapse is not found, and the X-irradiated cortices, I ascribe to the presence in the former of other inhibitory cells besides the Golgi cell. In the latter I believe that the lack of basket or stellate cells is the major factor determining the formation of the heterologous-ectopic synapses. Acknowfedgements-The technical assistance of Mr J. P. Rio. as well as the photographic work of Mr D. LEWREN. are gratefully acknowledged. The work was partially supported by the Institut National de la Sante et de la Recherche Medicale (ATP6-74-27 no. 20) and by the Centre National de la Recherche Scientifique (A.T.P. Genetique et developpement d’un Mammifere).

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(Accepted 2 December 1976)