Development of Purkinje cells in absence of climbing fibers

Brain Research, 111 (1976) 389 395

389

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Short Communications

Development of Purkinje cells in absence of climbing fibers*

C. SOTELO and M. L. ARSENIO-NUNES** Laboratoire de Neuromorphologie (U-I06 INSERM), Hdpital de Port Royal, 75014 Paris (France)

(Accepted April 13th, 1976)

From the classical studies of Ramdn y Cajal s, it is known that the first contacts established between climbing fibers (CFs) and Purkinje cells (PCs) occur as pericellular nerve terminations, referred to as 'nests'. The postsynaptic elements of these immature synapses are the long, abundant filopodia arising from the PC somata. R a m 6 n y Cajal s described three different phases that the C F - P C synapses must pass through to reach maturity. During these phases, there are parallel growths of the PC dendrites and of the CFs, taking place simultaneously with resorption of the somatic expansions. These events suggest that the CF induces the development of the PC dendritic tree; a hypothesis which has been advanced recently by Kornguth and Scott 5. A close correlation between CF function and PC integrity has been postulated by H~imori 3 who assumes that the CF presence is necessary for the formation and the maintenance of PC dendritic spines. The functional significance of such a hypothesis, including the role of CFs in the maintenance of PC dendritic spines, has been discussed in a previous paper 11. In this communication we present preliminary results on the development of PCs following deafferentation of olivary input in immature rats. In this study we used 12 pregnant rats. At birth, litters were culled to 6 pups which were operated on 48 h postnatally. The operation consisted ofthermocoagulating the left inferior cerebellar peduncle. The majority of the operated pups died within the first week following the operation. Ten surviving animals were obtained and maintained on a normal diet for two months. These 10 rats were sacrificed by intracardiac perfusion of a double aldehyde fixative consisting of 500 ml solution of 1 ',~oparaformaldehyde and 1 ~ glutaraldehyde in 0.12 M phosphate buffer (pH 7.3) at room temperature. After fixation the cerebellum was detached from the brain stem * Part of this work was presented to the "7th International Neurobiology Meeting", G6ttingen, Sept. 15-19, 1975 (ref. 9). ** Charg6e de Recherche it I'I.N.S.E.R.M. (Laboratoire de Neuropathologie, H6pital Saint Vincent de Paul~ 74 av. Denfert Rochereau, 75014 Paris, France.)

390 and divided by mid-sagittal section. From each half of the cerebellum 2 mm thick slices were prepared in the sagittal plane. A slice from each of the hemispheres and from the left and right vermis was impregnated using the Golgi-Rio Hortega method. The remainder of the slices were dissected in sagittal blocks of folia. "These blocks were postfixed by immersion in solution of 21!; osmic acid and 0.12 M phosphate buffer (pH 7.4), stained "en bloc' with uranyl acetate, dehydrated in graded soluiions of ethanol and embedded in Araldite. The brain stem was embedded in paraltin, serially sectioned and stained with Luxol fast blue and cresyl violet. The light microscopic study of the inferior olivary complex and the inferior peduncles of the 10 fixed rats has allowed us to identify the animals in which the operation was completely successful. That is to say, the left restiform body has disappeared, but the other peduncles (middle and superior) are untouched and in addition the right inferior olivary nuclei no longer exist (Fig. 4). In only three animals were all these requisites fulfilled. Therefore, the qualitative results reported in this communication are concerned only with the observations from the left hemispheies of the three rats in which the operation was successful. In thick plastic sections stained with toluidine blue the cortex of the deafferented left hemisphere exhibits the typical layered arrangement. The molecular layer mainrains its normal dimension, and contains numerous PC dendritic profiles running in all directions, but predominantly they tend to run in the vertical direction perpendicular to the pial surface. In Golgi-Rio Hortega impregnated material, the dendritic arborization of the deafferented PCs spreads out in the same vertical plane and with the same three-dimensional form observed in PCs with inferior olivary input. In addition primary, secondary, and tertiary branches as well as spiny branchlets are present in the dendritic lrees (Fig. I). On the other hand, a closer analysis of the dendritic arborizations of' some deafferented PCs shows some important differences: (a) the amount of branching of the main dendritic trunks in deafferented PCs is lower than in similar normal cells. This decrease gives rise to an increase in the length of dendritic segments, mainly at the superficial 2/3 of the arborization (Figs. 1 and 2); (b) some of the distal dendritic segments run in a straight, vertical direction at right angles to the longitudinal axis of the folium (Figs. l and 2); (c) at all levels of the molecular layer, the thick dendritic trunks are studded with spines (Figs. 2 and 3). Thus, they resemble the superficial 2/3 of the PCs dendritic trees following removal of CFs in adulthood l l. At the ultrastructural level PC perikarya contain the usual organelles and exhibit a smooth contour. Basket terminals surround the axonal pole of the PC bodies and constitute the complex nest formation around the initial segment of the PC axon which is characterized by the synaptic arrangement between the tip of the brush of basket fibers and the initial segment of the PC axon. The primary dendrite at the basal portion is generally smooth and contacted synaptically by ascending collaterals of the basket fibers. In confirmation of results obtained with the Golgi impregnation, there is a considerable increase in the number of spines emerging from tertiary, secondary and upper primary dendritic branches (Figs. 5, 6 and 7). The large majorit5 of these ectopic spines are innervated by axon terminals, and only occasionally ar~

Fig. 1. Photomontage of a Golgi impregnated Purkinje cell dendritic tree from a hemisphere devoid of climbing fibers. Note the presence of numerous spiny branchlets (small arlows) and the existence of tertiary unbranched segments (large arrows) running in a vertical direction at the superficial molecular layer, x 700. Fig. 2. Large magnification of the right upper inset in Fig. 1. Note the abundance of spines emerging from this straight unbranched segment. × 2400. Fig. 3. Large magnification of the left lower inset in Fig. 1. The secondary dendritic trunk branches in several thick segments, each one being studded with spines, x 2400.

Fig. 4. Light micrograph of the inferior olive (IO) at the level of the hypoglossal nerve (large arrows) in one of the operated rats. The left olivary complex, of which the bounderies are limited by small arrows, keeps a normal size and appearance. The right olivary complex does no longer exist. × 50. Fig. 5. Electron micrograph of the molecular layer of a left cerebellar hemisphere devoid of climbing fibers. A thick Purkinje celt dendritic profile (PCD) is cut at a tangential plane. Axon terminals belonging to parallel fibers (PF) are in synaptic contact with abundant spines which, in all likelihood, emerge from the thick dendritic profile. The dendrite and the axon terminaWs are enwrapped on the same glial envelope (GI) and have a straight vertical orientation, x 15,000.

393 dendritic spines bearing a postsynaptic differentiation free of innervation and directly covered by glial processes. Spiny branchlets keep their normal morphology and synaptic relationships 6. Most of the ectopic spines emerging from thick dendritic profiles are in synaptic contact with varicosities of parallel fibers (Fig. 6). In tangential sections to large and straight PC dendritic branches, such as illustrated in Fig. 5, the dendritic profile is draped with numerous synaptic varicosities from the parallel fiber, which are enwrapped in the same glial sheath covering the PC dendrite. Each parallel fiber varicosity is in synaptic contact with 2-4 spines within the plane of the section. Therefore, there appears to be an increase in the number of multiple synaptic contacts as also observed by Raisman and Field v in central axonal sprouting. Axon terminals belonging to stellate cells (Fig. 7), including ascending collaterals of basket fibers, can also innervate the ectopic spines. The present results indicate that the interruption of the olivocerebellar fibers prior to the establishment of functional synapses 2 between CFs and PCs does not interfere with the growth and spine formation of PC dendrites. Therefore, these results are not in support of the theory postulated by Hfimori 3 that CFs are necessary for the formation of PC dendritic spines (i.e., hypothesis of the indirect or heterotopic induction of spines). On the other hand, the present results in part confirm and enlarge on those of previous workers concerning the pattern of arborization of PC dendrites developed in absence of CFs. Kawaguchi et al. 4 recently reported that subtotal unilateral lesion of the inferior olive in 2-day-old kittens produces a "scantiness of branching of the PC d e n d r i t e s . . , many of the primary d e n d r i t e s . . , were seen to run through the molecular layer without branching . . . . . " These results were obtained with Bodian's impregnation method, which probably is not the most appropriate one for the study of dendritic patterns. In our Golgi impregnated rat material, we have disclosed a slight reduction in PC branching, with some secondary and mainly tertiary trunks coursing without branching in a vertical direction. Bradley and Berry 1 have described PCs in the rat devoid of CFs to have a reduced dendritic tree due to a 'decrease in the total number of dendritic segments and not to any decrement in segment length', that is to say, due to a reduction in branching. In their Golgi study, Bradley and Berry also reported the presence of spiny branchlets and the existence of large spines on the main dendritic trunks of the deafferented PCs; but they did not describe the presence of large tertiary segments running in a vertical direction through the molecular layer. The present ultrastructural study affords new data on the synaptic relationships of PCs devoid of CFs. The soma and initial segment of the PC axon receive a normal contingent of basket fibers. In addition, PC perikarya developed in absence of CFs reach maturity, because somatic filopodia have not been observed in the 2-month-old rats. In respect to PC dendrites, those branches in normal cerebella not contacted by CFs, namely the spiny branchlets, maintain their synaptic connections with parallel fibers. The thicker dendritic branches, which normally form the postsynaptic territory for CFs, are of course devoid of CF varicosities, but they retain their other synaptic

Fig. 6. Electron micrograph of a thick dendritic trunk of a Purkmje cell (PCD). [ rom lhe 5 ect,.:,p~c spines emerging from this dendritic profile (arrows), three are contacted by parallel Jibers (PF) wiflm-~ the plane o f / h e section. ;:. 24,000. Fig. 7. Electron m i c r o g r a p h of a sccondary dendritic branch of a Purkinje cell (PC I)). Eour ectop~c spines emerge from the dendrite (arrows). Some of t h e m are in synaptic contact ~ith parallel fiber~ (PF), while others are contacted by stellate a x o n s (SAt. 17,000.

395 relationships with stellate axons and ascending collaterals of the basket fibers. In addition, numerous ectopic spines emerge f r o m these thicker dendritic branches and a large majority o f them are contacted by parallel fibers. The morphological appearance o f some o f the axon terminals innervating these ectopic spines resembles that o f the few remaining parallel fiber varicosities in agranular cerebella (compare Fig. 5 o f this paper with Fig. 15 o f a previous paper1°). In fact, in both cases, there is an increase in the n u m b e r of multiple synaptic contacts 1° due to terminal sprouting of parallel fibers induced by an excess in postsynaptic targets. In conclusion, the effect o f C F deprivation u p o n immature PCs is only reflected in a reduced branching pattern of the dendritic arborizations. Resorption of somatic filopodia, as well as three-dimensional arrangement and spine formation o f the dendritic tree occur in absence o f CFs. The present results clearly demonstrate that CFs are not necessary for the development o f PC dendritic spines but that, on the contrary, their absence induces spine formation. In addition, these results indicate that in the normal ontogenesis CFs and parallel fibers would appear to compete for postsynaptic space. The authors are grateful to J. P. Rio for technical assistance, and to D. ke Cren for p h o t o g r a p h i c assistance. This work was partially supported by the lnstitut National de la Sant6 et de la Recherche Mddicale (ATP 6-74-27 No. 20) and by the Centre National de la Recherche Scientifique ( A T P : G6n6tique et ddveloppement d'un M a m m i Fire).

1 Bradley, P. and Berry, M., The effect of specific deprivation of either the climbing or the parallel fibre input on the development of the dendritic tree of Purkinje cells in the cerebellum of the rat, J. Anat. (Lond.), 120 (1975) 407-408. 2 Crepel, F., Maturation of climbing fiber responses in the rat, Brain Research, 35 (1971) 272-276. 3 HS_mori, J., Developmental morphology of dendritic postsynaptic specialization. In Recent Developments of Neurobiology in Hungary. Results in Neuroanatomy, Neuroendocrinology Neurophysiology and Behaviour, Neuropathology, Vol. IV, Akad6miai Kiad6, Budapest, 1973.

pp. 9-32. 4 Kawaguchi, S., Yamamoto, T., Mizuno, N. and lwahori, N., The role of climbing fibers in the development of Purkinje cell dendrites, Neurosci. Lett., 1 (1975) 301-304. 5 Kornguth, S. E. and Scott, G., The role of climbing fibers in the formation of Purkinje cell dendrites, J. comp. Neurol., 146 (1972) 61 82. 6 Palay, S. L. and Chan-Palay, V., Cerebellar Cortex, Cytology and Organization, Springer, Berlin, 1974. 7 Raisman, G. and Field, M. M., A quantitative investigation of the development of collateral reinnervation after partial deafferentation of the septal nuclei, Brain Research, 50 (1973) 241 264, 8 Ram6n y Cajal, S., Histologie du SystOme Nerveux de l'Homme et des Vertdbrds, Vol. II, Maloine, Paris, 1911, pp. 100-103. 9 Sotelo, C., Formation and maintenance of Purkinje spines in the cerebellum of "mutants" and experimental animals. In Afferent and Intrinsic Organization of Laminated Structures in the Brain, 7th International Neurobiology Meeting, Max-Planck-lnstitute for Biophysical Chemistry, G6ttingen, 1975. 10 Sotelo, C., Synaptic remodeling in mutants and experimental animals. In F. Vital-Durand and M. Jeannerod (Eds.), Aspects of Neural Plasticity, Vol. 43, Colloque 1NSERM, Paris, 1975, pp. 167-190. 11 Sotelo, C., Hillman, D. E., Zamora, A. J. and Llin~.s, R., Climbing fiber deafferentation: its action on Purkinje cell dendritic spines, Brain Research, 98 (1975) 574-581.