Postnatal development of the inferior olivary complex in the rat. I. An electron microscopic study of the medial accessory olive

291

Developmental Brain Research, 8 (1983) 291 310 Elsevier

P o s t n a t a l D e v e l o p m e n t o f the I n f e r i o r O l i v a r y C o m p l e x in the Rat. I. A n E l e c t r o n M i c r o s c o p i c S t u d y o f the M e d i a l A c c e s s o r y O l i v e

F R A N C K B O U R R A T and CONSTANTINO SOTELO*

Laboratoire de Neuromorphologie, U-106 1NSERM, Centre Mbdico Chirurgical Foch, 42, Rue Desbassavns de Richemont, 92150 Suresnes (France) (Accepted December 7th, 1982)

Key words: neuronal development - synaptogenesis - medial accessory olive - olivocerebellar system rat - electron microscopy

The postnatal development of the medial accessory olive (MAO) was studied in the rat from birth to adulthood. In newborn rats, the inferior olivary complex exhibited an adult cytoarchitectonic pattern, facilitating the precise delimitation of the MAO. Computer-assisted measurements of neuronal perikarya in 1 p~m thick plastic sections revealed a 40% increase in perikaryal diameters from day of birth (PO) to the twenty-first postnatal day (P21). This growth takes place mainly during the first postnatal week, the phase of perikaryal maturation, whereas it is almost non-existent during the second week, the phase of sudden neuropil expansion. The ultrastructural study gave the following results: at PI-P5, only the neuronal perikarya have attained a certain degree of maturity. The neuropil is composed of profiles of unknown origin, among which growing dendrites are numerous, but mature synapses are scarce. By P7-PI0, the cytological characteristics of the perikarya reached an adult stage. The dendrites begin to acquire their adult features by their emission of racemose protrusions and by their organization into protoglomerular formations. The most important step in the structural differentiation of the MAO was found to occur between PI0 and P I5. It is at this later age that the neuropil exhibits a complex neuronal organization similar to the adult, characterized by the presence ofolivary glomeruli and of neuro-neuronal gap junctions. The fact that these electrotonic junctions appear a long time after the appearance of chemical synapses, indicates that the ontogeny of the MAO chemical transmission precedes electrical transmission. On PI 5 and thereafter, the maturation of the MAO proceeds mainly by increasing the number of synaptic connections and by glial differentiation. These structural developmental stages of the MAO were related to the different steps of functional development of the olivocerebellar system. INTRODUCTION

The inferior olivary complex (IOC) is the sole known source of climbing fibers projecting to the cerebellum4,~1.15.16.31.In order to better understand the progressive and sequential processes underlying the formation and maturation of the synapses between climbing fibers (CF) and Purkinje cells (PC), the study of the development of this major precerebellar nucleus becomes therefore essential. We have started a long-term project aimed at analyzing the developmental stages of IOC neurons which parallel the establishment of olivocerebellar projections, and more specifically the synaptogenesis between CFs and PCs. The animal selected for this study * To whom correspondence and reprint requests should be sent. 0165-3806/83/$03.00 (~) 1983 Elsevier Science Publishers B.V.

was the rat, because this is the species which has generated most of the electrophysiological data on CF-PC synaptogenesis5-9.-'7.'-9.3~.Moreover, in the rat these synaptogenic events occur postnatally, a situation which greatly facilitates not only their analysis under normal conditions, but also the experimental manipulation of the system. Electrophysiological analyses of CF-PC synaptic activity have provided evidence that immature CF responses can be obtained in some PCs as early as postnatal day 2 (P2), whereas by P3 and P4 the recorded responses were already very similar to adult oness.6.24. Furthermore, from P3 up to PI3 P159, these CF responses are graded by steps with the increased intensity of

292 the stimulation, instead of being all-or-none in character like in adult animals j3. These results6,9.24provide evidence that in the rat, the maturation of CF-PC synapses needs the long period of 10 days. A transient redundancy of the connectivity, corresponding to a developmental stage in which PCs are multiply innervated by CFs, occurs before reaching the final mature stage. Our aim is, therefore, to disclose all kinds of developmental changes taking place in the IOC of the rat from birth up to adulthood, which could be correlated with the protracted process ofCF-PC synaptogenesis. The first paper of this series summarizes the results of a qualitative electron microscope study, aimed at analyzing the developmental phases through which the immature olivary neuropil of the newborn rat must pass until the acquisition of its characteristic adult pattern 3'~7-1s'33. Since our still unpublished quantitative analysis (Bourrat and Sotelo, in preparation) on the developing IOC of the rat indicates that the growing rate (represented by the volumetric increase of tigsue) of the different subnuclei is not synchronous, it is therefore necessary to investigate each of them separately. The present study will be only concerned with the medial accessory olive (MAO). This choice is based upon the fact that the CFs innervating the vermis originate mainly from MAO neurons, and the vermis has been the cerebellar region used for electrophysiological studies on CF-PC synaptogenesisS,9,27->. MATERIAL AND METHODS

Female albino rats of the Wistar strain were placed in the same cage with males for 4--5 days. Those later observed to be pregnant were placed in separate cages and checked every day till parturition. Day of delivery was considered as postnatal day 0 (P0). Pups aged P1, P3, P5, P7, P10 and P15 were anesthetized with ether vapors and sacrificed by intracardiac perfusion of a solution containing 1% paraformaldehyde, 1% glutaraldehyde in 0.12 M phosphate buffer (pH 7.3), maintained for about 30 min. The brain in the skull was immersed overnight in the same

double-aldehyde fixative at 4 %1. After removal of the skull, the region of the brainstem containing the IOC was cut into frontal slices approximately 1 mm thick. These slices were postfixed by immersion in a solution of 25~ osmium tetroxide and 0.12 M phosphate buffer (pH 7.3), with 7% glucose added, for 3 4 h. After en bloc staining with uranyl acetate, the slices were dehydrated in graded solutions of ethanol and embedded in Araldite. In addition, 3 young adult rats, 3 0 4 0 days old, were also perfused with the double-aldehyde fixative, Blocks containing the IOC were prepared for their ultrastructural study with the same histoprocessing method indicated above for the rat pups. Sections 1 pm thick were cut from the blocks and stained with toluidine blue. The limits of the MAO were drawn with a camera tucida. The blocks were consequently trimmed to a restricted area containing the MAO of both sides. Thin sections were obtained from the trimmed blocks, picked up on bare cooper grids and double stained with uranyl acetate and lead citrate. They were examined with a Philips 400 electron microscope. Additional material, already available in the laboratory for a further morphometric analysis of the postnatal development of the rat IOC, was used here to estimate the mean diameters of neuronal cell bodies lying in the MAO. This material consists of the brainstems of 10 female rats aged 0, 3, 8, 12, and 21 days (2 animals for each age). The fixation procedure and the plastic embedding were similar to those described above. Each IOC was serially cut into 1 t~m thick sections. Only one section out of every 100 was mounted and stained with toluidine blue. From this collection, 40 MAO neurons containing distinct nucleoli and located at different levels of the subnucleus were selected at each age. The areas of the selected neurons were measured using a camera lucida projecting image onto a HP 9111A graphics tablet interfaced with an HP 9826A microcomputer. Since the somatic profiles were found to be roughly circular, the diameters were calculated as being those of circles of the same areas.

293 RESULTS

P1-P5 rats: Stage of perikaryal maturation and of intense synaptogenesis.

In newborn rats, the IOC is already organized into the same subnuclei that are present in adulthood, making the identification of the MAO for ultrastructural study very easy. The main difference, at the light microscope level, is the lack of myelinated axons in the pyramidal tract, the hypoglossal fibers and the IOC itself. Most of the MAO neurons have a homogeneous size and shape. They appear as rounded profiles with a characteristic dispersion pattern throughout the subnucleus. In order to obtain a maturation parameter of these neurons, their diameters were estimated (see Material and Methods). The youngest studied neurons, taken from newborn rats (P0) have an average diameter of 12.2 ± 1.5 ~tm. They increase in size mainly during the first postnatal week. At P8, they have reached almost their adult diameter, with an increase of about 31% (16.0 ± 1.2/~m). In the oldest rats analyzed the mean neuronal diameter is 17.4 ± 1.4 ~m, indicating a slight 9% increase during the second and third weeks (Fig. A). Since obvious qualitative changes are only disclosed between P5 and P7 rats and between P10 and P15 animals, in the following description the results will be organized into 4 groups: (i) rats aged 1 5 days; (ii) rats aged 7-10 days; (iii) 15-day-old rats; and (iv) young adult rats.

As in older animals, the neuronal perikarya are not evenly distributed, but aggregated into separate clusters. In each single cluster, the cell bodies are either separated by a small amount of neuropil or directly apposed (Fig. 1). Whereas most of the neuronal organelles appear fully mature, some signs of immaturity, mainly concerning the rough endoplasmic reticulum, are still obvious. Nissl bodies are almost non-existent and, as in other immature neurons, the cytoplasmic matrix contains numerous free polyribosomes and scattered cisterns of the rough endoplasmic reticulum (Figs. 1 and 2). Specific inclusion bodies, similar to nematosomes ~4, have been occasionally observed (Fig. 2) in olivary neurons of all the studied P1-P5 rats. Another important difference with more mature neurons is the smooth contour of their perikarya. If the cell bodies have already attained an advanced stage of maturation, this is certainly not the case for the neuropil, which, for the most part, exhibits a totally immature appearance. In most instances it is formed by numerous small profiles of unknown origin, measuring 0.2-0.5 t~m in diameter. They contain microtubules, some neurofilaments and the largest ones, a mitochondrion (Fig. 3). Among these small processes, a few are much bigger; they look swollen with irregular contours, a flocculent content (Fig. 3), and occasionally free polyribosomes. These bigger profiles are therefore considered to be growing dendrites (Fig. 3). Intermingled with these extensive areas of immature neuropil, there are islands in which the neuronal processes exhibit a more advanced stage of maturity (Fig. 4), allowing the identification of their axonic or dendritic origin by their intrinsic features. Most of the axons resemble axon terminals and contain synaptic vesicles. The dendrites can be identified by the presence of a few rosettes of free polyribosomes and their occasional postsynaptic position (Fig. 4). A peculiar observation in these immature animals is the high occurrence of large granulated vesicles (LGV), present not only in axonic processes but in dendritic ones as well (Fig. 4).

Dl|metcr (miorDnel

~'0

++

)~

÷

10

i 3

I B

I

12

I 21

Rgo

Fig. A. Rate of growth of neuronal perikarya from P0 to P21 rats. The perikaryal size is expressed in diameter length. Each value is the mean _ S.E.M. of 40 MAO neurons.

i

I

295 Synaptogenesis must have started in the prenatal period, because in spite of the immaturity of the MAO neuropil in newborn rats, a few synaptic connections are already visible. The majority of these scarce contacts look like mature synapses, since the pre- and postsynaptic differentiations are fully developed (Fig. 5). Occasionally, immature synaptic contacts can be encountered. In these cases, the presynaptic elements are almost devoid of synaptic vesicles and of their corresponding vesicular grids; however, the dense material constituting the postsynaptic webs is fully differentiated (Figs. 6 and 7). The various kinds of axosomatic and axodendritic synapses, characteristics of the adult MAO, are present in the newborn. Their number and their reciprocal proportions are, however, very different from those of adulthood. A few axon terminals synapse directly on the perikaryal surface (Figs. 2 and 8); the majority, however, are axodendritic lying within the neuropil, especially in the islands (Fig. 4). By far, the bulk of these early synaptic contacts are Gray's type 1 (Figs. 4, 5, 6 and 8) and the presynaptic axons contain rounded vesicles (Figs. 4, 5 and 8). Some rare Gray's type 2 synaptic complexes have been observed between axon terminals and dendrites. In these instances, the axon terminal may contain pleomorphic vesicles (Fig. 9).

P7-PIO rats: stage of dendritic specialization and ofprotoglomeruli formation The neuronal cell bodies maintain their clustered pattern of distribution described above. They can still be directly apposed to each other, but their contours are no longer smooth since short and stubby protrusions can emerge from the neuronal surface (Fig. 12). At this age, the cellular organelles have acquired a fully mature appearance. Nissl bodies are present in the cyto-

plasm, extending toward the base of the dendrites. As in other neurons, this basophilic material avoids the axon hillock region (Fig. 10) where only some free polyribosomes are present. Inclusion bodies similar to nematosomes (Fig. 11) are also encountered in some of these neurons. There are fundamental changes in the neuropil, although it still maintains for a large part an immature appearance. As in P1-P5 rats, large masses of neuropil are formed by closely packed profiles of small size and of an undetermined origin. However, the frequency of the islands containing more mature neuropil is increased, and within these islands, important qualitative changes appear. The most obvious change is the presence of dendritic protrusions that, like the somatic ones, are not merely spines to be contacted by axon terminals. Differently to spines, these protrusions may contain, with the exception of microtubules, the usual dendritic organelles, mainly free polyribosomes (Figs. 15 and 16). Moreover, they are directly apposed to small dendritic profiles (Fig. 16) and more often to other similar protrusions. They correspond to immature racemose formations, commonly seen in adult olivary neurons 3-33, which will evolve into the dendritic central core of the complex synaptic arrangements characteristic of the mature olivary neuropil, called olivary glomeruli26.33. Occasionally, 2 or 3 profiles of these racemose protrusions can be associated with one or more surrounding axon terminals in a protoglomerular arrangement (Fig. 18). These immature glomeruli differ from mature ones since, although their dendritic protrusions are directly apposed, they are not linked together with junctional complexes. Furthermore, the peripheral ring of axon terminals is incomplete, and the astrocytic envelope has not yet been formed (Fig. 18).

Fig. 1. Electron micrograph ofa perikaryal cluster in PI rat. Portions of the cytoplasm of 3 neurons (N l-N3) are illustrated in this micrograph. The arrows indicate zones of directly apposed plasma membranes. Note the large amount of free polyribosomes suspended in the cytoplasmic matrix × 22,000. Fig. 2. Immature perikaryon in the MAO ofa PI rat. Note the absence of Nissl bodies and the frequency of free polyribosomes. A nematosome lies close to the nucleus (n). The arrow points to a mature axosomatic synapse, x 17,000. Fig. 3. Electron micrograph illustrating the neuropil between two neuronal perikarya (Ni and N2). This neuropil is composed of undifferentiated profiles, among which dilated elements (asterisks), corresponding to growing dendrites, are present. The arrows point to axon terminals. Some of the latter establish synaptic contacts on dendritic profiles. × 32,000.

297 The synaptogenesis must still be very active in this developmental period, due to the frequency of i m m a t u r e synapses (Figs. 13 and 14). The latter, as those described in P1-P5 rats, are characterized by the advanced development of the postsynaptic differentiations, which contrasts with the immaturity (Fig. 13) or even the absence (Fig. 14) of active zones in the presynaptic terminals. Fully mature synaptic complexes are, however, more c o m m o n than at earlier stages. They can occasionally establish axosomatic contacts, but they are much more frequent in the neuropil, contacting either dendritic shafts (Figs. 12 and 15), dendritic spines (Fig. 12) or dendritic protrusions (Fig. 18). The majority of these synapses are of the G r a y type 1, and are filled with spherical vesicles. However, a few axon terminals containing a pleomorphic vesicular population can also be encountered. As is the case with P1 P5 rats, L G V are c o m m o n l y present in pre- and postsynaptic structures (Figs. 12, 13, 15, 17and 18). Some exceptional axon terminals, not engaged in synaptic junction, contain numerous LGV, as well as minute tubular vesicles or microcanaliculi intermingled with tubular and vesicular profiles of the smooth endoplasmic reticulum (Fig. 17). These terminals exhibit, therefore, the same cytological features as those which selectively take up tritiated serotonin in the IOC of the adult rat 35. Their presence suggests that at least at this stage of development, serotoninergic innervation is not exclusively confined to the lateral portion of the dorsal accessory olive, but extends to the M A O as well. Although the degree of m a t u r a t i o n of the M A O in P7 and P10 rats is very much alike, an

important difference can be observed in the neuronal organization of the M A O at both ages. At P7, no neuronal gap junction has been encountered, whereas at P10, punctiform junctions, similar to minute gap junctions, have been occasionally observed. In these few instances, the junctions are established between neuronal perikarya and thick dendritic branches (Fig. 19). The plasma m e m b r a n e s of these adjoining profiles are directly apposed and they approach each other focally at one or two points in which the extracellular space is almost obliterated (Fig. 20). The apposed m e m b r a n e s at the focal points, as well as in between them, are undercoated with a layer of semidense material (Figs. 19 and 20). The presence of this cytoplasmic material allows one to exclude the possible artefactual nature of the 'kissing junctions' and favors the idea that they are immature gap junctions, which will rapidly evolve into the typical gap junctional maculae observed in more mature animals. In conclusion, the main developmental events occurring at P7-P10 are the differentiation of dendritic appendages to form protoglomeruli and the great increase in the a m o u n t of mature synaptic contacts. In addition, at P10 takes place the formation of 'kissing junctions', considered here as immature forms of gap junctions.

P15 rats: stage of complete maturation of the neuropil by the acquisition of olivary glomeruli and electrotonicjunctions The most dramatic change in the history of the postnatal development of the M A O occurs between P10 and PI5. In PI5 rats, the neuropil has acquired most of its adult features, mainly

Fig. 4. Neuropil island mainly composed of differentiated dendritic and axonal profiles in P5 rat. The arrow points to a mature axodendritic synapse. Note the abundance of large granulated vesicleslying in axonal as well as in dendritic profiles. Most of the synaptic vesiclesare rounded. × 27,000. Fig. 5. High magnification of a fully developed axodendritic synaptic contact in a P5 rat. The 3 components of this synapse, the presynaptic vesicular grid, the postsynaptic web and the cleft material, allow it to be classifiedas belonging to the Gray type 1. x 110,000.

Fig. 6. Immature axosomatic synapse in PI rat. The postsynaptic web is fully differentiated, whereas the presynaptic element only contains some few synaptic vesicles. × 53,000. Fig. 7. Immature axodendritic synapse in P5 rat. In contrast with the well-developed postsynaptic web, the presynaptic axon is devoid of vesicular grid. x 69,000. Fig. 8. Mature axosomatic synapse in a P5 rat. This contact exhibits features of a Gray type 1synapse, x 42,000. Fig. 9. Mature axodendritic synapse in P5 rat. This contact exhibits features of a Gray type 2 synapse. × 36.000.

299 the olivary glomeruli and the electrotonic junctions. The neuronal perikarya exhibit a fully mature appearance, with small masses of Nissl substance dispersed throughout the cytoplasm (Fig. 21). Nematosome-like bodies are no longer present, but laminated inclusions of rough endoplasmic reticular origin, characteristic of olivary cells in adult animals 33 can be observed within the Nissl bodies. The neuronal cell bodies maintain their clustered pattern. Direct appositions between somatic plasma membranes are common, although most of the neurons within a cluster are separated by a small neuropil region up to 6/~m consisting mostly of axon terminals and dendritic profiles (Fig. 21). In only one case of somatal direct apposition, is a gap junction bridging the two neuronal cell bodies. It consists of a small plaque (0.15 /zm long) in which, in material stained en bloc with uranyl acetate, the two opposing plasma membranes converge to form the typical 7-layered structure which characterizes the gap junctions (Fig. 22). Similarly to all described gap junctions between neuronal elements ~2, a cytoplasmic semidense material undercoats the whole length of the inner surfaces of the junctional plasma membranes (Fig. 22). With the exception of the glial investment, which is still incomplete, the immediate environment of the MAO neurons in P15 rats is similar to that encountered in adult animals. It is mainly composed of axonal and dendritic profiles and some rare axon terminals synapsing on the somatic surface (Fig. 21). Direct appositions between perikarya and dendritic profiles, without any ultrastructural specialization of the opposing membranes, are frequent. They are also c o m m o n in adult IOC, where they were called by Sotelo et al. 33 'casual appositions'.

The most distinctive feature of the neuropil, which clearly demarcates the MAO of P15 rats from that of younger ones, is the presence of complex synaptic arrangements, called olivary glomeruli. They are composed of a central core of dendritic elements, surrounded by a peripheral ring of axon terminals, and wrapped in an astrocytic sheath, which, contrary to mature glomeruli, is only partially formed at this age of development (Fig. 23). As in adult MAO, the central dendritic core comprises 4 10 profiles of about 0.5 ~tm in cross-section diameter (Figs. 23 27). Each profile contains a flocculent matrix in which vesicular and tubular organelles of the smooth endoplasmic reticulum, together with occasional multivesicular bodies, mitochondria and free polyribosomes, are suspended (Figs. 23-27). The origin of the elements forming the central core of the glomeruli has been identified in younger rats (in the protoglomeruli of P7- P 10 rats) as being the racemose protrusions emerging from main dendritic stems a n d / o r perikarya. The preciseness of this origin has been confirmed in the MAO of Pl5 rats because it is still frequent in these more mature glomeruli, and shows the continuity between one of these central profiles and its parent neuronal element. Thus, Fig. 25 illustrates one case in which a central element originates from a thick dendritic branch, whereas, in Figs. 26 and 27, one of these profiles directly emerges from a neuronal cell body. Two types of specialized junctional zones occur between the plasma membranes of central core elements: (i) small attachment plates (Figs. 25 and 30); and (ii) gap junctions (Figs. 28 and 29). The latter will be considered below. The peripheral ring of synaptic terminals in a glomerulus is mainly formed by small-sized boutons.

Fig. 10. Electron micrograph of a MAO neuron in PI0 rat. This perikaryon exhibits mature cytological features: a Nissl body in perinuclear position, a process showing dendritic characteristics (D), and a second one, the axon hillock (AH), gives rise to the initial segment (IS). x 12,000. Fig. 11. Nematosome in a neuron o f a PI0 rat. × 28,000. Fig. 12. Electron micrograph illustrating a somatic protrusion (arrow) in a P7 rat. Note the abundance of mature axodendritic synapses (arrowheads). x 24,000. Fig. 13. Electron micrograph of the MAO neuropil in a P7 rat. Two immature axodendritic synapses are visible. In both of them the postsynaptic web is well differentiated, whereas the presynaptic vesicular grid is either absent (arrowhead) or contains only some few vesicles (arrow). x 54,000. Fig. 14. Immature axodendritic synapse in a PI0 rat. In this case, the presynaptic element is devoid of paramembranous differentiations, x 75,000.

1

301 The large majority of them contain spherical vesicles (Figs. 23-27) and only occasionally some of the axon terminals are filled with pleomorphic vesicles. Similarly, most of the synaptic complexes are of the Gray type 1, and only contact one single postsynaptic profile. Another outstanding feature, indicating that the MAO of P 15 rats has qualitatively reached its adult stage, is the presence of gap junctions between neuronal elements. With the exception of one gap junction encountered between two adjacent perikarya (Fig. 21) and reported above, all the numerous gap junctions present in the MAO of P15 rats are, as in adult animals 17,18,32,33,dendrodendritic, They exhibit similar ultrastructural features: 7-layered membrane complexes in which the outer leaflets of the opposing membranes are separated by a minute gap of about 2 nm (Figs. 29, 32 and 34). The semidense differentiations, attached to the inner leaflets of the junctional membranes occupy large areas of the adjacent cytoplasm (Figs. 31 and 33). The neuronal elements engaged in the gap junctions can be: (i) small dendritic profiles composing the central core of the glomeruli (Figs. 28 and 29); (ii) one of these central profiles and a parent dendrite of another glomerular protrusion (Figs. 31 and 32); and (iii) extraglomerular dendrites (Figs. 33 and 34). Contrary to adult IOC, extraglomerular dendrodendritic gap junctions are more numerous than glomerular ones. It seems, therefore, that the complex neuronal organization of the adult MAO is already achieved in P15 rats.

Young adult rats The ultrastructural description of the IOC of adult animals has been the subject of numerous publications3.~s.26.33.34; therefore, we shall report here only a few features concerning the mature olivary glomeruli and mainly the distribution of gap junctions in the MAO of adult rats, in order to compare them with those described above in younger rats. This comparison will allow us to determine the nature of the late maturation changes. As stated above, the differences between the olivary glomeruli in P 15 rats and in adult rats are only quantitative. The adult glomeruli are better isolated from the surrounding neuropil since the glial envelope is fully developed. Although without a serial reconstruction of the glomeruli it is not possible to determine if these structures are totally covered by astrocytic processes, it is clear that the glial investment of adult glomeruli is further extended than in PI5 rats (Fig. 35). The peripheral ring of axon terminals has also evolved. In adult glomeruli, some boutons are of large size and establish synaptic contacts with several elements of the central core (Fig. 35). Moreover, the number of boutons containing pleomorphic vesicles is greatly increased (Fig. 35), although it is always inferior to the number of boutons with spherical vesicles. In contrast to immature olives, there is no constant association between vesicular shape and pattern of synaptic complexes. Thus, boutons with pleomorphic vesicles can establish Gray type 1 synapses with their postsynaptic partners (Fig. 35).

Fig. 15. Electron micrograph of a thick dendritic branch, probably belonging to a main dendrite, in a P7 rat. Two thinner processes, comparable to adult racemose protrusions, emerge from the main dendritic trunk (arrows). Note the presence of free polyribosomes within these protrusions. The arrowhead points to a mature axodendritic synapse, x 24,000. Fig. 16. Another example of racemose protrusion in a P 10 rat. In this micrograph, the protrusion (arrow) emerges from a medium-sized dendritic branch, and contains polyribosomes, x 29,000. Fig. 17. Electron micrograph of the neuropil in a P 10 rat, illustrating various kinds of axon terminals. One of them (S) contains numerous large granulated vesicles as well as microcanaliculi intermingled with tubules of the smooth endoplasmic reticulum. This terminal is identified as serotoninergic. Another axon terminal (P) contains a pleomorphic population of synaptic vesicles. x 32,000. Fig. 18. Protoglomerular formation in a P7 rat. Three dendritic profiles (DI D3) mainly containing smooth endoplasmic reticulum are directly apposed. Axon terminals (T) are localized at the periphery of the dendritic cluster; one of them (arrows) synapses on two different dendritic profiles, x 30,000. Fig. 19. Junctional zone between a neuronal perikaryon and a dendritic profile. A small portion of the nucleus (n) allows the identification of the cell body. The plasma membranes of the apposed element approach each other at two points, leaving between them an almost normal extracellular space (arrow). This 'kissing junction' is considered as an immature gap junction, x 90,000, Fig. 20. High magnification of the 'kissing junction' illustrated in Fig. 19. The arrows point to the two focal apparent obliterations of the extracellular space. Note the cytoplasmic dense material decorating the apposed membranes. × 220,000.

L

i i

303 Neuronal gap junctions are, in these young adult rats, more often encountered in a glomerular than in an extraglomerular location. In the glomeruli they connect together two of the central dendritic profiles (Figs. 35 and 36). In some fortuitous sections, the two dendritic profiles bridged by the gap junction are postsynaptic to the same axon terminal (Fig. 14 in ref. 32). Extraglomerular gap junctions can engage either isolated dendrites (Figs. 37 and 38), which due to their size, must belong to secondary branches, or smaller dendritic profiles. In these instances, they generally emerge from larger dendritic branches (Figs. 39 and 40), indicating that these gap junctions are closer to the neuronal perikarya than to the distal portion of the dendritic field. DISCUSSION

The important morphological changes in dendritic complexity and neuronal connectivity described in this study clearly indicate that most of the dendritic maturation and of the synaptogenesis in the rat MAO are postnatal events. Although in newborn rats the cytoarchitectonic distribution of olivary neurons is already mature ~, these neurons appear almost undifferentiated. In the MAO only the cell bodies have attained a certain degree of maturity; the neuropil .is mainly composed of undifferentiated profiles where growing dendrites are numerous, but mature synapses scarce. Signs of dendritic maturity start in P7 P10 rats, in which racemose protrusions and protoglomerular formations are for the first time observed. Concomitantly, the number of mature synapses is greatly increased. However, the most important step in the postnatal olivary differentiation occurs between PI0

and P15. It is at this latter age that the neuropil of the MAO exhibits the same complex organization as in adulthood which is characterized by the presence of olivary glomeruli and electrotonic junctions. On P15 and thereafter, the maturation of the MAO proceeds mainly by increasing the number of synaptic connections and by glial development. Although the main developmental stages which the inferior olivary neurons need to cover to attain their maturity are quite similar in the rat and in the opossum2.22, the time schedule of these postnatal events differs greatly. In the opossum, the olivary neurons do not reach their adult cytoarchitectonic distribution until P18, whereas in the rat this distribution is already achieved at birth. Important qualitative changes mainly concerning dendritic maturation, like the formation of protrusions and protoglomeruli, take place in P40 opossums, but in P7-PI0 rats. Finally, in the opossum, the transformation of protoglomeruli into glomeruli and the acquisition of electrotonic junctions is a long process which lasts between P40 and P70. On the contrary, in the rat, this important developmental stage occurs suddenly: by P 10 only protoglomeruli and immature gap junctions are present, whereas, by P15 the neuropil has already attained its adult appearance. The rate of growth of the neuronal cell bodies seems also to differ in these two species. According to Maley and King22, the biggest olivary neurons have a diameter of about 5/xm in P3 opossums, 12 txm in P21 and 30 lam in adulthood, indicating a progressive growth up to 6 times the initial diameter. In the rat, there is an overall increase in diameter of only 40% between newborn and young adult MAO neurons. About 75% of this growth takes place during the first postnatal week, a period of

Fig. 21. Electron micrograph of 2 nearby neuronal perikarya (N 1 and N2) in a PI5 rat. The cytoplasm is, as in P7-P10 rats, fully mature. The two neurons are separated from each other by a narrow band of neuropiL where axon terminals (T) are abundant. The arrow points to an axosomatic synapse, x 20,000 Fig. 22. High magnification of a somatosomatic gap junction in a P I5 rat. The arrows point to the cytoplasmic differentiations which undercoat both internal leaflets of the junctional membranes. × 161,000. Fig. 23. Electron micrograph illustrating the advanced maturity of the neuropil in a PI5 rat. The center of the micrograph is occupied by an olivary glomerulus consisting of a central dendritic core (asterisks) and a peripheral ring of axon terminals (T). The latter are small in size, contain rounded vesicles and mainly establish synaptic contacts on one single dendritic profile. Note that the astroglia (g) does not completely enwrap the glomerulus, x 29,000.

i

!

F

305 slow maturation of the neuropil, but of complete differentiation of the neuronal perikarya. However, during the period of sudden neuropil expansion, the increase in perikaryal diameter is almost non-existent. From the numerous regions of the mammalian brain known to be provided with electrotonic synapses (see refs. in ref. 32), the IOC is the first one in which the chronology of their development has been studied. Contrary to the hypothetical concept that electrotonic transmission is a phylogenetic remnant which would precede chemical transmissionzS, the acquisition of this kind of neuronal communication between MAO neurons is a late event, which occurs between P10 and P15, a long time after the onset of chemical synapses. It could be argued that since electrotonic junctions in adult IOC mainly bridge racemose protrusions within the olivary glomeruli, such junctions cannot be formed until an advanced stage of differentiation, in which the dendrites have reached their maturity. However, this argument does not fit with the fact that in P10 rats, the age at which focal 'kissing junctions' appear for the first time, these immature gap junctions are bridging together neuronal perikarya and proximal dendritic branches. Furthermore, in P15 rats, mature gap junctions are more numerous in extraglomerular locations, mainly between large dendritic branches than within glomeruli. Thus, dendritic maturation does not seem to be the limiting factor for the onset of neuro-neuronal gap junctions. It is also worth noting that in spite of the quantitative differences encountered between developing and adult MAO neurons, these junctions couple in both instances partners which belong to the same category, since/txodendritic gap junctions

were never observed. Thus, the concept of synaptic specificity elaborated for chemically transmitting synapses might also be applied to electrical synapses. As stated in the introduction, the main interest of this study is to obtain a time-table of the developmental stages of postnatal MAO neurons to allow a comparative analysis of the events taking place in the olive with those already known concerning the maturation of CFPC synapses. The first important observation is the asynchrony between the differentiation of the axons of the MAO neurons and the maturation of their dendrites. Retrograde and anterograde tracing experiments (Sotelo, Bourrat and Triller, in preparation) performed in newborn rats have demonstrated that inferior olivary axons are already in the cerebellar cortex of P1 rats, and that the pattern of distribution of these early olivocerebeUar projections mimics that of adult rats. Moreover, the electrophysiological studies on the ontogenesis of olivocerebellar relationships5,6-9,24.27.29,demonstrate that as early as 3 days after birth, CF responses on PCs already resemble adult responses, with the exception that they are not all-or-none in nature. Even an anlage of CF response can be observed in most PCs in P2 rats. The anatomical and physiological studies converge to demonstrate that the axons of MAO neurons are not only in the close vicinity of PCs in newborn rats, but that they make functional synapses. However, in P 1 P5 rats, the dendrites of MAO neurons are still very undifferentiated and they receive few mature synaptic contacts. It is, therefore, evident that MAO neurons are provided with axons which establish functional and specific synapses on their proper targets, while their dendrites are

Figs. 24-27. Olivary gl~rneruli in PI5 rats. Fig. 24, The central core of this glomerulus is composed of 5 dendritic profiles. Each one contains vesicular elements of the smooth endoplasmic reticulum suspended in a floccular matrix. The axon terminals (T) forming the peripheral ring contain rounded vesicles. The arrow indicates a junctional zone, probably an attachment plaque, linking 2 of the dendritic profiles, x 51,000. Fig. 25. This electron micrograph illustrates the dendritic origin of one profile at the central core of a glomerulus. A mediumsized dendritic branch (D) gives offa process (arrow) that enters a glomerulus. Attachment plaques (arrowheads) are present between the central profiles, x 57,000. Figs. 26 and 27. These two electron micrographs illustrate two examples of glomeruli in which one of the profiles of the central core emerge directly (arrow) from a neuronal perikarya (N). Note that most of the axon terminals (T) synapsing on the dendritic core contain rounded vesicles. Fig, 26. × 35,000: Fig. 27, x 41,000.

307 still not mature and far from receiving the complete contingent of their afferent inputs. Although the early CF responses on PCs resemble adult ones, during the first 2 postnatal weeks these responses are graded in a stepwise manner as the intensity of the stimulus is increased9, indicating that immature PCs are multiply innervated by CF. The numerical one-toone relationship, characteristic of adult animals, is not completely achieved until P 13-P 159. It is at this age that the MAO neuropil has already acquired its adult pattern and that the olivary neurons are electrotonically coupled. It becomes therefore tempting to correlate the regression of the multiple innervation with the occurrence of electrotonic junctions. Uncoupled olivary neurons could share the same postsynaptic space in their search for specific targets in order to ensure the innervation of all PCs. It may be speculated that once the IOC neurons develop gap junctions and, therefore, become electrotonically coupled, only the axon collateral which, for instance, has succeeded in establishing the highest amount of synaptic contacts, will remain, whereas, the supranumerary ones emerging from axons of coupled neurons will withdraw, due to a repelling influence generated by the electrotonic coupling. However, the formation of gap junctions between de~,eloping MAO neurons cannot be the unique mechanism for the regression of the multiple innervation. As has been repeatedly said, these junctions are formed between P10 and P15, whereas it is at P%PI0 that there is an abrupt decrease in the number ofCFs synapsing on single PCs, as well as in the percentage of PCs muptiply innervated by CFs TM. Moreover, in the

various mutant mice, in which the granule cells fail to develop synaptic contacts with PCs, as in weaver and staggerer mice (see refs. in ref. 30), the regression of this multiple innervation is not fully achieved7.8,23, whereas the IOC neurons in these mutants are linked together by gap junctions (Sotelo, unpublished observations), as in control mice. Thus, the coincidence of different factors seems necessary for the normal maturation of the olivocerebellar system. The IOC neurons have complex electrophysiological properties mainly dependent on their oscillatory behavior and on the fact that they are electrotonically coupled ~°,~9-2~. According to Llimis and Yarom 2t , the autorhythmicity is predominantly Ca-dependent and is basically a dendritic property. The study of the spontaneous activity o f l O C neurons and CF-mediated activity of PCs in developing rats25 suggests that the dendritic mechanisms necessary for the oscillatory behavior of olivary neurons are established at P%P10. Therefore, there is a perfect matching between the structural differentiation of MAO dendrites (it is at this period that they begin to acquire their adult organization), and the development of the dendritic ionic conductances (indicated by the appearance of their autorhythmicity). On the other hand, the electrophysiological study of Dupont and Crepe112 on the correlations among CF responses of nearby PCs, also in developing rats, suggests that the neuronal organization underlying the synchronization of IOC neurons must be established at P7-P9. Surprisingly, at this early period we have not succeeded in finding gap junctions between olivary neurons; these low resistance

Figs. 28-34. Electron micrographs illustrating the various locations in which neuro-neuronal gap junctions have been observed in PI5 rats. Fig. 28. Gap junction (arrow) in a glomerular location. This junction is present between two of the central dendritic appendages. x 50,000. Fig. 29. High magnification of the gap junction illustrated in Fig. 28. × 258,000. Fig. 30. Gap junction (arrow) in a extraglomerular location. This junction is bridging together a large dendritic branch (D) and a small dendritic appendage (asterisk). x 45,000. Fig. 31. Gap junction (arrow) in the vicinity ofa glomerulus. This junction is present between a dendritic appendage, forming the central core of a glomerulus (asterisk), and a large dendritic branch (D) from which emerges (arrowhead) another dendritic appendage of the same glomerulus. X 60,000. Fig. 32. High magnification of the gap junction illustrated in Fig. 3 I. x 145,000. Fig. 33. Gap junction (arrow) between two medium-sized dendritic branches in an extraglomerular location, x 60,000. Fig. 34. High magnification of the gap junction illustrated in Fig. 33. × 145,000.

i~~ i

i!~'I

I

309 junctions develop later at P10-PI5. The time lag between functional synchronization and appearance of gap junctions suggests that the underlying mechanisms must differ in immature and adult animals. Electrotonic coupling through gap junctions could be only evoked in rats at least 10 days old, but not before.

ACKNOWLEDGEMENTS

REFERENCES

11 Desclin, J., Histological evidence supporting the inferior olive as the major source ofcerebellar climbing fibers in the rat, Brain Res., 77 (1974)365-384. 12 Dupont, J. L. and Crepel. F., Correlations among climbing fibers responses of nearby cerebellar Purkinje cells in the immature rat, Exp. Brain Res., 37 (1979) 525-535. 13 Eccles, J. C., Llin~is, R. and Sasaki, K., The excitatory synaptic action of climbing fibers on the Purkinje cells of the cerebellum, J. Ph vsioL (Lond.), 182 (1966) 268- 296. 14 Grillo, M., Cytoplasmic inclusions resembling nucleoli in sympathetic neurons of adult rats, J. Cell BioL. 45 (1970) 100- 117. 15 Groenewegen, H. J. and Voogd, J.. The parasagittal zonation within the olivocerebel|ar projection. I. Climbing fibers distribution in the vermis of cat cerebellum, Y. comp. Neurol.. 174 (1977) 417-488. 16 Groenewegen, H. J., Voogd, J. and Freedman, S. m The parasagittal zonation within the olivocerebellar projection. II. Climbing fibers distribution in the intermediate and hemispheric parts of cat cerebellum, J. comp, NeuroL, 183(1979)551 602. 17 Gwyn, D. G., Nicholson, G. P. and Flumerfelt, B. A,, The inferior olivary nucleus of the rat: a light and electron microscopy study, J. comp. Neurol.. 174 (1977) 489520. 18 King. J. S,, The synaptic cluster (glomerulus) in the inferior olivary nucleus, J. comp. NeuroL. 165 (1976) 387 400. 19 Llin~is, R., Baker, R. and Sotelo, C., Electrotonic coupling between neurons in cat inferior olive, J. Neuropl~vsiol.. 37 (1974) 560-571. 20 Llinfis, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurons in vitro. Different types of voltage-dependent ionic conductances, J. Physiol. (Lond.), 315 (1981) 549-567. 21 Llin~is, R, and Yarom, Y., Properties and distribution of

1 Altman, J. and Bayer, S. A., Prenatal development of the cerebellar system in the rat. II. Cytogenesis and histogenesis of the inferior olive, pontine gray and the precerebellar reticular nuclei, J. comp. Neurol., 32 (1978) 49- 76. 2 Baner-Moffet, C. and King, J. S., The development of the inferior olivary complex in preweanling opossums, A nat. Embrvol., 162 ( 1981 ) 249- 280. 3 Bowman, M. H. and King, J. S., The conformation, cytology and synaptology of the opossum inferior olivary nucleus, J. comp. NeuroL, 148 (1973) 491- 524. 4 Courville, J. and Faraco-Cantin, F., On the origin of the climbing fibers of the cerebellum. An experimental study in the cat with an autoradiographic tracing method, Neuroscience, 3 (1978) 797-809. 5 Crepel, F., Maturation of climbing fibers responses inthe rat, Brain Res., 35 ( 1971) 272 276. 6 Crepel, F., Delhaye-Bouchaud, N. and Dupont, J. L., Fate of the multiple innervation of cerebellar Purkinje cells by climbing fibers in immature control, X-irradiated and hypothyroid rats, Develop. Brain Res., 1 (1981) 5°. 71. 7 Crepel, F., Delhaye-Bouchaud, N., Guastavino, J. M, and Sampaio, I., Multiple innervation of cerebel|ar Purkinje cells by climbing fibers in staggerer mutant mouse, Nature (Lond.), 283 (1980) 483-484. 8 Crepel, F. and Mariani, J., Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the weaver mutant mouse, J. NeurobioL. 7 (1976) 579- 582. 9 Crepel, F., Mariani, J. and Delhaye-Bouchaud, N., Evidence for a multiple innervation of Purkinje cells by climbing fibers in the immature rat cerebellum, J. Neurobiol., 7(1976) 567 578. 10 Crill, W. E., Unitary multiple-spiked responses in cat inferior olive nucleus, J. Neurophysiol., 33 (1970) 199- 209.

Mr. J. P. Rio and Mr. D. Le Cren are gratefully acknowledged for technical and photographic assitance, and Mrs. C. Verven for typing the manuscript. Research was supported by INSERM and by a grant from the CNRS (ATP 'Petits Mammif~res').

Figs. 35-40. Electron micrographs illustrating the various locations of neuro-neuronal gap junctions in young adult rats. Fig. 35. Gap junction (arrow) in a glomerular location. The axon terminals (T) in this adult glomerulus are bigger than in P15 rats. They mainly contain pleomorphic vesicles and each one establishes synaptic contacts on various dendritic profiles. Note that the gila (g) enwraps the glomerulus completely, x 40,000. Fig. 36. High magnification of the gap junction illustrated in Fig. 35. × 250,000. Fig. 37. Gap junction (arrow) in an extraglomerular location. This junction bridges two medium-sized dendritic branches. × 23,000. Fig. 38. High magnification of the gap junction illustrated in Fig. 37. x 176,000. Fig. 39. Gap junction (arrow) in an extraglomerular position. This junction is present between a dendritic appendage (asterisk) and a small protrusion emerging (curved arrow) from a medium-sized dendritic branch (D). × 28,000. Fig. 40. High magnification of the gap junction illustrated in Fig. 39. x 220,000.

310

22

23

24

25

26

27

28

ionic conductances generating electroresponsiveness of mammalian inferior olivary neurons in vim), J. Phv.vio/. (Lond.),315(1981)569 584. Maley, B, E. and King. J. S., Early development of the inferior olivary complex in pouch young opossums. 11. An electron microscopic study, J. comp. Neurol., 194 (1980) 741 760. Mariani, J. and Changeux, J. P., Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the adult staggerer mutant mouse, J. NeurobioL. 11 (1980) 4t 50. Mariani. J. and Changeux, J. P., Ontogenesis of olivocerebellar relationships. I. Studies by intracellular recordings of the multiple innervation of Purkinje cells bv climbing fibers in the developing rat cerebellum, J. Neurosci., 1 ( 1981 ) 696 702. Mariani, J. and Changeux, J. P., Ontogenesis ofolivocerebellar relationships. II. Spontaneous activity of inferior olivary neurons and climbing fibers mediated activity of cerebellar Purkinje cells in developing rats, J. Neurosci., I ( 1981 ) 703 709. Nemecek, St. and Wolff, J., Light and electron microscopic evidence of complex synapses (glomeruli) in olivo inferior (cat). Lrperientia. 25 (1969) 634-635. Puro, D. G. and Woodward, D. J., Maturation of evoked climbing fiber input to rat cerebellar Purkinje cells, Exp. Brain Res., 28 (1977) 85 100. Shapovalov, A. 1.. lnterneuronal synapses with electrical, dual, and chemical mode of transmission in vertebrates, Neuroscience, 5 (1980) 1113 1124.

29 Shimano. T., Nosaka, S. and Sasaki. K., l-lc~lrophysioh)gical stud} on the postnatal development ~t netlrolla[ mechanisms in thc rat cerebellar cortex. Bre~i, Res. !0,~ (19761279 294. 30 Sotelo, C., Mutant mice and the formation ,~f ccrcbellar circuitry, Trend, Neuro,~ci., 3(1980)33 35. 31 Sotelo, C., Hillman, D. E., Zamora, A. ,1. and Llin:is, R,, Climbing fiber deafferentation: its action on Purkinje cell dendritic spines, Brain Res.. 98 (1975) 574 581. 32 Sotelo, C. and Korn. H., Morphological correlates of electrical and other interactions through low-resistance pathways between neurons of the vertebrate central nervous system, Int. Rev. CvtoL. 55 (1978) 67 I{)7 33 Sotelo, C., l,limis, R. and Baker, R.. Structural ~tudy of inferior olivarv~ nucleus of the cat: morphological correlates of electrotonic coupling, J, Neurophvsiol. 37 (1974) 541 559, 34 Walberg, F., An electron microscopic study' of the inferioroliveofthecat, J. comp. Neurol., 120(1963) I 17 35 Wiklund. L., Descarries. L. and Mollgi~rd, K., Serotoninergic axons terminals in the rat dorsal accessory olive: normal ultrastructure and light microscopic demonstration of regeneration after 5,6-dihydroxytrypmmme lesioning, J. Neurocytol., 10 ( 1981 ) 1009 1027. 36 Woodward, D. J., Holler, B. J,, Siggins, G. R. and Bloom, F. E., The ontogenetic development of synaptic junctions, synatic activation and responsiveness to neurotransmitter substances in ral cerebeltar Purkinje cells, Brain Res., 34 ( 1971 ) 73 97.