Parasagittal organization of the rat cerebellar cortex: direct comparison of Purkinje cell compartments and the organization of the spinocerebellar projection.

THE JOURNAL OF COMPARATIVE NEUROLOGY 291:79-102 (1990)

Parasagittal Organization of the Rat Cerebellar Cortex: Direct Comparison of Purkiqje Cell Compartments and the Organization of the Spinocerebellar Projection CLAUDE GRAVEL AND RICHARD HAWKES Department of Biochemistry and Laboratory of Neurobiology, Faculty of Medicine, Lava1 University, Ste-Foy, Quebec, Canada

ABSTRACT Retrograde and anterograde transport of tracers, electrophysiological recording, somatotopic mapping, and histochemical and immunological techniques have all revealed a parasagittal parcellation of the cerebellar cortex, including its efferent and many of its afferent connections. In order to establish whether the different compartments share a common organizational plan, a systematic comparative analysis of the patterns of parasagittal zonation in the cerebellar cortex of the rat has been undertaken, by using the parasagittal compartmentation of zebrin I+ and zebrin I Purkinje cells as revealed by monoclonal antibody Q113 as a reference frame. The distribution of mossy fiber terminals originating from the lower thoracic-higher lumbar spinal cord was compared to the distribution of zebrin I bands. Three-dimensional reconstructions from alternate frontal sections processed either for the anterograde transport of tracer or for zebrin I immunoreactivity reveal that the limits of the spinocerebellar terminal fields in the granular layer correlate well with the boundaries of some, but not all, zebrin I compartments in the molecular layer above. This leads to a subdivision of the zehrin I compartments into spinal receiving and spinal nonreceiving portions. In lobules I1 and VIII, the spinocerebellar terminal fields assume different positions relative to the zebrin I compartments in the ventral compared to the dorsal faces. Thus, each longitudinal compartment may be further divided transversely into suhzones, each receiving a specific combination of mossy fiber afferents. The further subdivision of zebrin I compartments by mossy fiber terminal fields increases the resolution of the topography to such a point that anatomical compartment widths become compatible with the width of the microzones and the patches identified by electrophysiological methods. Key words: monoclonal antibody, mossy fibers, parasagittal bands, eerebellum, anterograde transport, immunocytochemistry

The cerebellar cortex is divided functionally into numerous compartments. Initially, the lobular (transverse) subdivisions were emphasized, based either on afferent inputs and phylogenetic development (Larsell, '37) or on retrograde degeneration studies of the olivocerebellar projection (Rrodal, '40). The idea that lobules represent functional subdivisions was further emphasized by Anderson ('43) on the basis of his studies of the spinocerebellar receiving areas. At the same time, it also became clear that rostrocaudal sub-

0 1990 WILEY-LISS, INC.

divisions are present. For example, in their study of the corticonuclear projections, Jansen and Brodal ('40)identified three sagittal zones in each hemicerebellar cortex that project in an orderly way onto the cerebellar nuclei. This per-

Accepted August 17,1989 Address reprint requests to Dr. R. Hawkes, Department of Anatomy, Faculty of Medicine. University of Calgary, Calgary, Alberta T2N 4N1 Canada.

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Fig. 1. A reconstruction of the cerebellum of animal #74 seen at the midsagittal plane. Individual, alternate frontal 50 wm sections are viewed along their edges to yield the classical arbor vitae. Only the molecular layer is shown. The scale is given in millimeters from the rostral extremity, and this convention is followed here throughout. The

rostra1 tip of the cerebellum is located at approximately 10.5 mm interaural arcording to the brain atlas of Paxinos and Watson ('82). No correction has been made for tissue shrinkage. Lobules have heen labelled according to Larsell ('52).

spective was widened by Voogd ('64, '67) with studies of cerebellar myeloarchitecture that identified alternating bundles of Purkinje cell axons and cerebellar afferents. The raphes in the white matter are indicative of a longitudinal parcellation of cerebellar afferents. This has been confirmed by numerous anatomical and electrophysiological studies for the olivocerebellar projection (Oscarsson, '69, '80; Voogd, '69; Van Gilder and O'Leary, '70; Armstrong et al., '74, '82; Courville et al., '74; Courville, '75; Brodal et al., '75; Brodal, '76, '80; Chan-Palay et al., '77; Groenewegen and Voogd, '77; Oscarsson and Sjolund, '77; Beyerl et al., '82; Eisenman, '81, '84; Campbell and Armstrong, '83a,b; Eisenman et al., '83; Sotelo et al., '84), the spinocerebellar projection (Van Rossum, '69; Voogd, '69; Hazlett et al., '71; Ekerot and Larson, '73, '80; Watson et al., '76; Vielvoye and Voogd, '77; Yaginuma and Matsushita, '86; Okado et al., '87a,b; Heckroth and Eisenman, '88), the cuneocerebellar projection (Voogd, '69; Ekerot and Larson, '80; Gerrits et al., '85), and the projection from the lateral reticular nuclei

(Kunzle, '75; Russchen et al., '76; Chan-Palay et al., '77). Finally, there is also evidence of a longitudinal organization within the pontocerebellar projections (Brodal and Walberg, '77). Molecular correlates of the parasagittal organization of the cerebellar cortex have also been described. Scott ('64) and Marani ('82) used 5'-nucleotidase histochemistry to reveal longitudinal alternating bands of high and low enzyme activity in the molecular layers of rats and mice, and others demonstrated similar acetylcholinesterase compartments in the cat (Ramon-Moliner, '72; Marani and Voogd, '77; Marani, '82; Brown and Graybiel, '83),primate (Ingram et al., '85; Hess and Voogd, '86), and rat (Boegman et al., '88). Immunocytochemistry has also revealed unexpected regional heterogeneity in the organization of the cerebellar cortex. Antibodies raised against cysteine sulfinic acid decarboxylase (CSADCase; Chan-Palay et al., '82) revealed parasagittal bands of Purkinje, stellate, and Golgi cells in rat, mouse, and rabbit. A monoclonal anti-synaptophysin

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Fig. 2. Brightfield photomicrograph of a frontal section through the anterior lobe of the rat cerebellum that has been peroxidase stained for zebrin I with 4-chloro-1-naphthol as the chromogen. Immunoreactivity is restricted exclusively to the Purkinje cells, but not all Purkinje cells express zebrin I, and clusters of immunoreactive cells alternate with similar zebrin I clusters. Serial reconstruction reveals that the zebrin I' cells form continuous parasagittal bands. The pattern is symmetrical about the midline, and in each hemicerebellum, there are 7 zebrin I ' compartments. P1' abuts to the midline, P2' and P3' run laterally in the vermia, and P4' is at the interface of vermis and hemisphere. The

P5+, P6+, and P7' are in the hemisphere proper (not labelled). The zebrin 1- (P-) compartments are numbered according to the P' compartment immediately medial. The midline P1' band is, in fact, two contiguous zebrin I' compartments, one in each hemisphere (see Gravel et al., '87b; Leclerc et al., '88). The arrowheads on the dorsal surface of lobule V indicate zebrin I' compartments that serial reconstruction has shown do not extend the full length of the lobule. These have been called satellite bands (Hawkes and Leclerc, '87) and are not numbered. Lobules 11, 111, IV, and V are labelled according to Larsell ('52). Scale bar = 1mm.

antibody (mabQ155) that recognizes all the major classes of synapses in the cerebellum (Hawkes et al., '85; Leclerc et al., '89a) shows alternating patches of high and low activity in the mouse cerebellar cortex (Hawkes e t al., '85). Antibodies raised against synthetic porcine motilin (Chan-Palay et al., '81; Nilaver et al., '82) and glutamic acid decarboxylase (Chan-Palay et al., %l), and monoclonal antibodies to unknown epitopes zebrin I (previously named the mabQ113 antigen; Hawkes et al., '85) and B1 (Ingram et al., '85), all reveal parasagitally organized subsets of Purkinje cells in various mammalian species. The identification of multiple parasagittal representations in the cerebellar cortex raises complex organizational questions. In particular, do the different afferent, efferent, and intrinsic maps all reflect a simple, common ground plan? I t is difficult to answer this question because few direct comparisons have been published, and their results are contentious. For example, in primates, Hess and Voogd ('86) found that acetylcholinesterase patches and the myeloarchitectural compartments coincide, whereas no simple relationship could be identified between CSADCase and other markers (Chan-Palay et al., '82) or between AChE and

B1 (Ingram et al., '85).To relate the distribution of different intrinsic cerebellar markers both to one another and to the afferent termination patterns, we have used the pattern of Purkinje cell rompartments as revealed by using zebrin I immunocytochemistry as our frame of reference. Zebrin I is a polypeptide antigen, apparent molecular mass 120 kD, that is recognized by monoclonal antibody mabQ113 (Hawkes et al., '85; Hawkes and Leclerc, '87). In the cerebellum, zehrin I is confined exclusively to a subset of Purkinje cells arranged into a symmetrical, reproducible pattern of parasagittal compartments (Hawkes et al., '85; Hawkes and Leclerc, '86, '87). This cerebellar map has been compared directly with the distribution of AChE (Boegman et al., '88), 5'-nucleotidase (Eisenman and Hawkes, '89), and cytochrome oxidase (Leclerc et al., '89b), and in all cases, the compartment boundaries align. A direct comparison with the compartmentation of the olivocerebellar projection has also been made (Gravel et al., '87) with the same result-the boundaries of the climbing fiber compartments revealed by using small anterograde tracer injections into the inferior olivary complex coincide with antigenic boundaries between iebrin IT Purkinje cell clusters. Furthermore, the climbing

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fiber bands were never found to subdivide an antigenic compartment (with the apparent exception of the P1+compartment that straddles the midline and is probably composed of two independent, abutting zebrin I + zones). Thus, all our studies to date have suggested that the zebrin I compartmentation is the finest level of structural parasagittal compartmentation present. On the other hand, electrophysiological studies clearly reveal a significantly finer mediolateral parcellation of the olivocerebellar projection (e.g., Andersson and Oscarsson, '78). Moreover, mappings of the cutaneous projections to the granular cell layer have shown a patchy somatotopy in which the different receptive fields are not only segregated mediolaterally, but, also rostrocaudally, with discontinuities occurring between folia and even within a single folium (Joseph et al., '78; Shambes et al., '78a,b; Kassel et al., '84; Welker and Shambes, '85; Welker et al., '88). This complexity is less evident in the afferent organization. In rat, the spinocerebellar projection is reported to be restricted both mediolaterally and longitudinally (e.g., Voogd et al., '85), and a t least one of its components, the dorsal spinocerebellar tract (DSCT), conveys exteroceptive signals (e.g., Ito, '84). In the present study, we have therefore explored the spatial relationship between spinocerebellar mossy fiber terminal fields and the zebrin I+/zebrin I- compartmentation in the adult rat cerebellar cortex.

lMATERLALS AND METHODS WGA-HXP injections into the spinal cord Adult rats (250 g) were anesthetized by using sodium pentobarbital (65 mg/kg), and bilateral laminectomies were performed to expose the lower thoracic-higher lumbar (LTHL) spinal cord. Pressure injections of a 3 % saline solution of WGA-HRP were made via a glass micropipette (internal diameter 75 fim) fitted to a 0.5 MI Hamilton syringe. Sixty nanoliters of tracer solution was injected on each side of the spinal cord, 0.5 mm from the midline and at a depth of 1mm. Following the injections, the wound was sutured shut, and after recovery from the anaesthesia, the animals were returned to their cages. It should be emphasized that the axons and terminals labelled by using this protocol represent only a small fraction of the total spinocerebellar afferents, and differently located injection sites may not yield the same pattern of terminal fields.

Perfusion and sectioning Forty-eight hours following tracer injection, the injected rats were deeply anesthetized by using sodium pentobarbital and fixed by perfusion through the ascending aorta. The

Fig. 3. Photomontages of serial 50 pm frontal sections through lobule I1 of animal #74 taken 0.8 mm caudal to the rostral extreme of the cerebellum and processed either to reveal zebrin I immunoreactivity (A) or anterogradely transported WGA-HRP (B).In both cases, the chro mogen was TMB. Zebrin It compartments (Pl', P2+,P3+)are indicated by arrowheads in A. P1+ and P2' are only one to two cells wide, P3+ is wider but also fainter. In B, labelled axons issue from distinct bundles in the white matter (two are marked by asterisks) and ascend into the granular layer where they terminate as distinct, well-defined terminal fields, separated by regions with few if any labelled terminals. The location of the terminal fields is more medial in the ventral than in the dor sal face of the lobule and is clearly shifted with respect to the white mat ter bundle of origin. Arrows point to sections of the same blood vessels in the two sections. Scale bar = 200 am.

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perfusion started with 100 ml of a 0.9% saline solution, followed by 300 ml of a fixative containing 1TI paraformaldehyde, 1.257; glutaraldehyde in phosphate bufler (PB; 0.1 M, pH 7.4), and finally the fixative was flushed from the circulation by passing 100 ml of PBS (PB + 0.9",# NaC1). The brain and spinal cord were dissected out, and the segmental level of the injections verified by reference to the spinal nerve roots. The cerebellum and its attached brain stem was blocked and sectioned frontally a t 50 pm on a freezing-stage microtome, and the sections stored serially in cold PBS. The spinal cord encompassing the injection site was blocked out and sectioned either horizontally or frontally on a freezingstage microtome. Horizontal sections were cut at a thickness of 75 fim, and all sections were collected in PBS for processing. Frontal sections were cut at 50 pm and two consecutive sections out of every ten collected and stored in buffer.

Histochemical visualization of WGA-HRP T o reveal the presence of anterogradely transported HRP in the cerebellum, alternate frontal sections were processed with tetramethylbenzidine (TMB), dehydrated, and mounted according to the method of Mesulam ('82). No counterstaining was used. To reveal the extent of the injection site, the same protocol was applied to all the spinal cord sections collected, but in this case, alternate sections were counterstained with neutral red.

Zebrin I immunocytochemistry T o compare the distribution of zebrin I to the distribution of LTHL terminals on alternate sections of the cerebellum, we have modified our standard indirect immunoperoxidase protocol (Hawkes et al., '82) by using TMB instead of 4-chloro- 1-naphtholas substrate for the enzymatic reaction. This modification was prompted by the fact that the different incubation media used for TMB and 4-chloro-lnaphthol histochemistry lead to different tissue shrinkages that complicate subsequent serial reconstruction. T o reveal the presence of zebrin I in the cerebellum, alternate sections were first incubated for 3 hours at room temperature in spent culture medium of hybridoma line Q113 (Hawkes et al., '85; Hawkes and Leclerc, '87) diluted 1/500 with a 5% solution of powdered milk in 0.4 MPBS,pH 7.4. After three rinses in 0.4 MPBS (10 minutes each), the sections were incubated for 2 hours a t room temperature in peroxidaseconjugated rabbit-anti-mouse immunoglobulins (Dako, Inc.) diluted 1/200 in a 5% solution of powdered milk in 0.2 MPBS, pH 7.4. The sections were then rinsed three times with 0.1 MPBS,pH 7.4, processed for TMB histochemistry, dehydrated, and mounted on slides all according to the method of Mesulam ('82).

Reconstruction of the distributions of L"HL terminal fields and Purkinje cell compartments A series of camera lucida drawings were produced from all the sections tested for zebrin I. These drawings include the outlines of the molecular layer and of numerous blood vessels. The drawings were then aligned by using blood vessels and general contours as reference points and digitized by using a graphics tablet. The digitized data was processed with a 3-dimensional (3-D) reconstruction software package (PC3D; Jandel Scientific, CA) to generate the foliation pattern of the cerebellar cortex of each individual animal. Midvermal sagittal views served to identify section levels with


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PARASAGITTAL ORGANIZATION OF RAT CEREBELLAR CORTEX respect to lobulation, to compare cerebellar foliation patterns between the experimental animals and to illustrate the regions where the distribution of zebrin I+ Purkinje cells and LTHL terminals were reconstructed. An example is shown in Figure 1. In a second step, the distribution of LTHL terminal fields and zebrin I+ Purkinje cells was reconstructed. First, a set of camera lucida drawings representing the distribution of zebrin 1' Purkinje cells in the cerebellar vermis was obtained that included all the anti-zebrin I-stained sections in a particular lobule. These drawings included outlines of the lobule and of blood vessels, and the position of zebrin I+ Purkinje cells (i.e., the somata and their dendritic arborization) was represented by polygons traced around one or many contiguous zebrin I' Purkinje cells. Second, another set of camera lucida drawings, representing the distribution of HRP-labelled LTHL terminal fields was traced a t the same magnification by using the alternate sections processed with TMB without antibodies. These drawings include outlines of the lobule and of blood vessels, and the position of HRP-positive terminals were represented by tracing polygons around regions of the granular layer containing a cluster of positive terminals. All the drawings were checked a t high magnification, and then the two sets of drawings brought together and aligned by using the blood

Fig. 4. The distribution of spinocerehellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule I1 of animal #74. Part A is a detail from Figure 1 to show the lobulation of part of the anterior lobe vermis. The initial 200 pm of cerebellum is missing. The scale is in millimeters from the rostral extreme of the cerebellum. Curved arrows indicate the unfolding of lobule I1 for the surface projection in B. The region of lobule I1 within the polygon is shown as seen from rostrally ("eye" in C) in part C. B shows the vermis of lobule I1 unfolded. On alternating sections, zebrin I' compartments are shown in dark grey, and HRP-WGA labelled mossy fiber terminal fields, in pale grey. The white areas on the Zebrin I sections are the zebrin I- compartments, and those on the MF sections indicate regions devoid of labelled terminals. The vertical scales is in millimeters from the rostra1 tip of the cerebellum (see A). The horizontal scale is in millimeters from the midline (as judged by the zebrin I P l + compartment). This view tends to exaggerate bandwidths and sometimes makes labelled compartments appear slightly wider than they are. Both the zebrin I+ Purkinje cells and the spinocerebellar mossy fiber terminal fields are grouped to form longitudinal bands. On the dorsal face of lobule 11, there is a close alignment between the P' compartments and the labelled terminal fields beneath. The positions of the P1+,P2+,and P3+ compartments are indicated at the top of the figure. The match between the two is not exact. For example, the first lateral band of mossy fiber terminals is beneath the P1' compartment but extends medially well into P1-. However, the most medial half of P1- tends to be devoid of labelled glomeruli. In contrast, in the ventral aspect of lobule 11, the positions of the labelled terminal fields relative to the zebrin I compartments are quite different. With the exception of a row of terminals beneath P l ' , the terminal fields are located within the P - territories. The first lateral mossy fiber terminal field is confined within P1-hut does not fill it. As for the dorsal aspect, the terminal fields are located preferentially in the lateral half, but unlike dorsally, they do not extend beneath P2'. The second lateral mossy fiber terminal field is within P2- and seems to fill it, but scarcely extends into either P2' or P3'. C shows a frontal view of lobule I1 between 0.8 and 1.35 mm from the rostra1 tip of the cerebellum, as reconstructed from alternating zebrin I-- stained and WGA-HRPlabelled sections. The reconstructed region is identified by the polygon in A (and is seen from the point of view of the eye) and by the square brackets in B (dorsal surface Cd, ventral surface Cv). The P1+,P2+,and P3' compartments are labelled above and the scale is in millimeters. On the dorsal surface (d), mossy fiber terminal fields (light grey) are located immediately beneath the P' Purkinje cell compartments. On the ventral surface, with the exception of the midline terminal field, they lie in the P1- compartments.

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vessels and contours as references points. Neither zebrin I+ or LTHL polygons were used for alignment purposes. The drawings were then digitized by using the graphic tablet and the data processed by using the 3-D reconstruction software. To compare the zehrin I compartmentation to the terminal field distributions over long stretches of cerebellum, we have used projection maps of the unfolded cortex. In these, each frontal section is viewed dorsally and therefore appears as a single 50 Km-thick strip. Seen in this way, the zebrin I+ or LTHL polygons become rectangles whose width represents the maximum mediolateral extent of the polygon. This treatment leads typically to an overestimate of the true width of a compartment. For a rectangular polygon, there is no problem, hut for a trapezoid, the profile seen from above extends from the most medial to the most lateral extent. Thus, these dorsal unfolded views always tend to show the labelled compartments as wider than they really are and to exaggerate any overlaps.

RESULTS General considerations This study was done to compare the zonal organization of spinocerebellar terminals with the zonal organization of two immunologically distinct populations of Purkinje cells in the rat cerebellar cortex, the zebrin I'hebrin I- cells. For this purpose, we have generated interdigitating maps representing the distribution of LTHL terminals and of zebrin I' and zcbrin I- Purkinje cells in the vermis of adult rats. These are based on two comprehensive reconstructions, and four other animals that were used to verify the generality of the conclusions. As the results are similar in all cases, we will concentrate the description on one particular animal, but will refer to other cases to illustrate the variations observed.

Injection sites All the WGA-HRP injections were at the lower thoracichigher lumbar level (LTHL) and were bilateral. This region was chosen for ease of access and the relatively high density of spinocerebellar neurons (Matsushita and Hosoya, '79) and because both the adult distribution and the postnatal development of the projection from this region are well described in rodents (Arsenio-Nunes and Sotelo, '85; Arsenio-Nunes et al., '88). Large, constant volumes (2 x 60 nl) of tracer were injected into each animal to ensure strong and reproducible labelling of LTHL projection zones. Typically, the center of the injection site is situated a t T13-L1, and extends bilaterally between TllLT12 and L2-L3. The use of TMB as substrate in the indirect immunoperoxidase protocol increases the sensitivity by an order vf magnitude when compared to the results obtained with either 4-chloro-1-naphthol or diaminobenzidine. However, it also results in higher nonspecific background staining, and the larger precipitates obscure some of the fine detail. We have partially alleviated the background problem by using lower concentrations of primary antibody and by increasing the molarity of the buffer solutions used to dilute both the first and second antibody. Examples of the results are illustrated in Figures 3, 6, 8, 10, and 13. Zebrin I' Purkinje cell bodies and dendrites are clearly visible in the molecular layer, but the axons are somewhat buried in the background of the granular layer. As we were not able clearly t o identify labelled LTHL rosettes in the granular layer of' sections processed by using this protocol, we have


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Fig. 5. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule I1 of ani ma1 #7R. The labelling is as for Figure 4. A shows a detail from the folial reconstruction made a t the midsagittal plane. In this case, lobule I1 IS subfoliated into sublobules IIa and IIb. B shows the unfolded vermis and the distributions of zebrin I'Purkinje cells (dark grey) and mossy fiber terminal fields (light grey) taken from adjacent sections. The medial LTHL terminal field is generally aligned beneath Pl+throughout the lobule, but often extends laterally into P1-.It often appears to be split into two a t the midline. The first lateral pair of terminal fields occupy

the lateral third of Pl-, and in IIb dorsal, they extend under P2+.In IIb ventral and all of IIa, the terminal fields are displaced medially into P1-. The second lateral pair of terminal fields generally run beneath P3+ in lobule IIb dorsal, but spread extensively into P2-medially. In the rest of lobule 11, they are beneath P2-and avoid P3'. (Note that in IIb/dorsal a fourth pair of zebrin I'compartments are present in the intermediate cortex. These have not been analysed a s the curvature of the lobule is pronounced in this region, and, thus, the relation of granular layer to molecular layer is not straightforward.)

relied strictly on alternate sections to compare the distribution of LTHL terminals to that of zebrin I bands. This study is confined to the vermis. This is where most of the LTHL terminals are found, where the zebrin I - bands are the strongest and cleanest (Fig. a), and also where there

is least mediolateral curvature of the cortex. This last point is important for the interpretation of the graphic reconstructions because the simplest view is that the mossy fiber rosettes are situated immediately below the Purkinje cells they contact via the ascending portion of the granule cell

Fig. 6. Frontal sections through lobule I11 taken 1.4 mm caudal to the rostra1 extreme of the cerebellum, stained either for zebrin I immunoreactivity (A) or WGA-HRP anterograde transport (B). The conventions are as in Figure 5. In A, PI' is about three cells wide; P2+,about five; and P3+, from five to seven. I n B are labelled axons and terminals from sharply defined fields in the granular layer, interposed by label-free zones. The three more medial terminal fields are each about 200 pm wide; the two lateral are significantly wider. Same conventions as as Figure 3. Scale bar = 200 pm.

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Fig. 8. Frontal sections through lobule IV (2.4 mm caudal from the rostra1 extreme of the cerebellum), processed either for zebrin I immunoreactivity (A) or anterogradely transported WGA-HRP (B). The conventions are as in Figure 3. Only the more ventral part of the lobule is shown (see Figs. 1,9).At this level, only P1' and P2+are present: P1' is two to three Purkinje cells wide, P2' is two to five cells wide. The two lateral terminal fields are dense and sharply defined; the medial terminal field is more sparse. Same conventions as Figure 3. Scale bar = 200 m .

Fig. 7. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule 111of case #74. The same conventions are used as in Figure 4. Lobule I11 is divided into sublobules IIIa and IIIb (A). The unfolded vermis at lobule 111 is shown in B. The medial spinocerebellar terminal fields are aligned primarily under P1' but extend out under P1-. The first lateral terminal field is beneath the lateral half of P1- and occasionally encroaches into P2'. The second lateral terminal field is centered under P3' but spills into both P2- and P3-.Frontal reconstructions (C,D) confirm this analysis. Note that mediolateral curvature of the lobule exaggerates the apparent width of the P' compartments when viewed from above (as in B) and tends therefore to suggest overlap between zebrin I and mossy fiber compartments that is probably not real. For example, the first lateral terminal field, in fact, abuts upon the edge of P2' throughout the extent of IIIbidorsal and encroaches into P2+ only in IIIahentral (C) and towards the depth of the sulcus (more than 1.35 mm in C,D).

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axons. However, this may well be an oversimplification, as the natural projections of the granular layer onto the Purkinje cells are not necessarily orthogonal.

Lobule II In lobule 11, zebrin I+ Purkinje cells are arranged into three longitudinal compartments in each hemivermis, P1+P3 ' , with the P- Compartments named according to the P+ immediately medial (Fig. 3A). As is generally the case in the anterior lobe, the zebrin I ' compartments are much narrower than the zebrin I-, so that P1' and P2+ are only one or two Purkinje cells wide on most sections. P3' is broader but i t s immunoreactivity is much weaker, and it is often difficult to clearly define its lateral limit. In both dorsal and ventral aspects of the lobule-labelled LTHL, terminals are clustered to form five fields, one astride the midline and two others laterally on each side (Fig. 3B). Lahelled axons enter the granular layer from discrete bundles in the white matter, but do not necessarily ascend strictly perpendicular to the granular layer. As a result, except for the medial field, terminals on the ventral face are located medial to the bundles from which their axons issue.

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Fig. 9. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule IV of anima1 #74. The same conventions are used as for Figure 4. In a sagittal view (A), lobule IV is seen to have a much larger ventral than a dorsal face. In the surface projection of the unfolded vermis (B), the medial terminal field aligns beneath P1' and extends a little into P I - laterally, the first lateral terminal field is centered beneath the lateral P1- and

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encroaches into PZ', and, dorsally, a more lateral terminal field crosses the PZ'/PZ- boundary. This field extends unilaterally into the ventral aspect of the lobule. In frontal view (C), this is seen to be misleading, and, in fact, the terminal field in individual sections is either within P2' or P2- but never sits astride it. Finally, a still more lateral terminal field is situated beneath P3'. Satellite bands are present within Pl-/dorsal (left side) and Pl-/ventral (right side).

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A comparison of the distribution of LTHL terminals with zebrin I compartments in lobule I1 of animal #74 reveals unexpected complexity (Figs. 3, 4). On the dorsal surface, the mossy fibers terminate beneath PI+,beneath P 2 + ,and beneath P3'. The terminal field beneath P l + extends beyond the limits of the Purkinje cell compartment above. In some sections, it appears split into two at the midline, but this is not always evident. The LTHL terminal field beneath P2 consistently extends medially into the granular layer beneath PI- but seldom laterally into P2-. From a dorsal view (Fig. 4B), the situation beneath P3+ is less clear, but a frontal reconstruction (Fig. 4C) shows that the LTHL terminal field extends somewhat into P2-. One consequence of this is the subdivision of the P- compartments into LTHLreceiving and non-receiving zones. Thus, within P1- dorsally, the medial and lateral edges are innervated by LTHL terminals but the central portion is not, and within P2-, only the lateral edge receives an LTHL input. The ventral aspect of lobule I1 appears different from the dorsal. Only the LTHL terminal distribution a t the midline is the same-with a terminal patch underlying, and spilling beyond, PL'. Otherwise, the two more lateral mossy fiher terminal fields ventrally are restricted almost completely to the granular layer under the P- bands. The first lateral field is entirely within the mediolateral limits of P l - , often approaching hard up against the medial limit of P2' but almost never encroaching beyond. Likewise, the second lateral LTHL terminal field lies within P2- and seems to respect closely both the P2'/P2- and the P2-/P3 ' boundaries. As was the case with the dorsal surface, the ventral P1- compartment is also subdivided into LTHL-receiving and non-receiving patches, but here it is the LTHL-receiving patch that occupies the central position. In animal #74, lobule I1 shows no division into sublobules. However, this is more the exception than the rule-in the rat, lobule I1 shows a certain degree of morphological variability (Larsell, '52) and sublobulation into subfolia IIa and IIb is frequent. In fact, all the other animals analyzed in the present study show some degree of subfoliation in lobule 11. For example, in animal #73, lobule TI is clearly subfoliated into IIa and IIb (Larsell, '52; Fig. 5A). As in animal #74, #73 has five discrete, sagittally oriented terminal fields in the vermis of sublobules IIa and IIb. In the intermediate cortex of the dorsal face of lobule IIb, an additional pair is also present (Fig. 5B). In lobule IIb/dorsal, the medial mossy fiber field is more or less centered under P I + ,is on some sections split a t or near the midline, and encroaches consistently under P1- on each side. Although in some sections the first lateral pair of LTHL terminal fields appears to enter the granular layer beneath P2+,analysis of frontal sections (not illustrated) reveals that it is better described as running under the lateral third to a half of P1-. The second lateral pair occupy the granular layer under P3+and the lateral half of P2-. Proceeding towards the ventral face of the lobule (toward ventral IIa), there is a tendency for the second lateral pair of mossy fiber terminal fields to shift medially under P2-. More labelled glomeruli occupy a medial position beneath P1- ventrally than dorsally, but the majority are still found under the lateral half. Thus, in animal #73 as in #74, there is a tendency for LTHL terminal fields to shift medially going from dorsal to ventral face. However, the difference between the ventral and dorsal sides is not as clear-cut in animal #73-for one thing, the granular layer under the most lateral portion of P1- does not have the LTHL-free zone ventrally that was observed in +

animal G74. Nonetheless, analysis of the pattern obtained in lobule I1 of other animals confirms that LTHL terminals from low thoracic-high lumbar regions tend to occupy more medial positions under P- bands as the terminal field extends from intracentral fissure 1 (between lobules I1 and 111) towards the precentral fissure (between I1 and I). The region of the lobule where this shift begins does not seem to be strictly linked to the subfoliation pattern, as it can occur in either ventral IIb or ventral IIa.

Lobule I l l In all our animals, lobule I11 (which separates into IIIa and IIIb; see Fig. 7A) receives a dense LTHL projection (Fig. 6B). Terminals are found across the entire thickness of the granular layer in Gre sharply delineated mediolateral terminal fields across the vermis and in an additional pair of large terminal fields in the intermediate cortex (in the region of P4+). The organization and position of zebrin I compartments in lobule I11 is similar to that in lobule 11. The distribution of LTHL terminal fields in lobule I11 (Figs. 6, 7 ) resembles that in lobule II/dorsal (see Figs. 3, 4). One mossy fiber terminal field is centered beneath P1' but extends under the medial third of Y1- to each side. The first lateral terminal field runs under the lateral half of P1- and may extend slightly under P2+, but very rarely under P2-. Finally, the most lateral mossy fiber terminal field forms a very broad band beneath P3+ and P3- and frequently reaches P4+ laterally and P2 medially. The organization is the same on the ventral and dorsal faces in both IIIa and IIIb (Fig. 7C,D). This arrangement of terminals subdivides the granular layer under zebrin 1- compartments of lobule 111. The central portion of P1- overlies a region of the granular layer receiving no direct input from the low thoracic-high lumbar spinal cord, but both the medial and lateral thirds of P1 are LTHL receiving areas. For P2-, the boundary is between lateral-receiving and medial non-receiving compartments. Concerning the zebrin I+ Purkinje cell bands, P1' and P3+ overlie LTHL receiving regions, but P2+ is over a non-receiving area for most of its longitudinal extent.

Lobule IV In lobule IV, the pattern of zebrin I immunoreactivity is similar to that in lobules I1 and I11 except for a slight widening of both positive and negative bands (Fig. 8Aj and the frequent presence of "satellite" bands (Hawkes and Leclerc, '87) inside PI- in or close to the dorsal face (Fig. 9B; see also Fig. 2j. The LTHL projection to lobule IV is not as dense as in more anterior lobules, but, in general, territories occupied by labeled LTHL terminals are sharply delimited, especially deep into the fissures (Fig. 8B). Their distribution is in most ways similar to those described for lobules I1 and 111 (Fig. 9B,C). In particular, the central mossy fiber terminal field is centered under P1+ and extends under PI- on each side, the first lateral pair is beneath lateral P1-, and there is a pair of terminal fields under the P3+ territory that may extend slightly under the lateral region of P2- (Figs. 8,9B). Differences are found when the granular layer under the medial half of P2- is considered. We have shown above that in the dorsal face of lobule I1 and throughout lobule 111 no LTHL terminals are found in this region (see Figs. 5, 7). However, at the tip of lobule IV and extending to its dorsal face, there is an extra mossy fiber terminal field under the medial portion of P2- (Fig. 9B,C). Seen in a dorsal unfolded

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PARASAGITTAL ORGANIZATION OF RAT CEREBELLAR CORTEX view, these fields appear to extend under the lateral edge of P2 ' ,but this impression is due to the curvature of the cortex (compare C and D of Fig. 9)-in fact, they occupy the granular layer under P2- and run hard up against the lateral edge of P2+. They are separated from the fields to either side by 50-200 pm-wide terminal free zones, one running beneath P Y , the other beneath P2-/P3+. The region of cortex in which the additional terminal field first appears varies between individuals and is not necessarily symmetrical about the midline. In case #74, a terminal field appears beneath P2- both ventrally and dorsally in the right hemicerebellum (i.e., on the left in Fig. 9B), but only dorsally in the left hemicerebellum. This may represent structural interindividual differences or merely the consequences of subtle shifts in the injection sites.

Lobule V The density of the spinocerebellar projection in lobule V is the lowest of the anterior lobe receiving areas (Fig. 10B). The transition between positive and negative LTHL terminating zones is still quite abrupt at or near the intermediate part of the cortex, but in most of the vermis, labelled rosettes are arranged into low-density clusters. In general, labelled terminals appear less numerous in the dorsal face, but in both dorsal and ventral aspects of the lobule, their distribution is similar to that in lobule IV/dorsal (Fig. 11). The central field of mossy fiber terminals is centered under PI' but includes some medial PI- to each side (Fig. llB,C). The first lateral field runs under lateral Pl-/medial P2+, and the second, under medial P2-. As was found in the other lobules of the anterior lobe, the second lateral field can extend as far laterally as to abut the lateral edge of P2+,but does not cross it (Figs. IOB, 1lC). Finally, the most lateral field is situated under P3+. However, in the dorsal face of lobule V in animal #74, the LTHL terminal field under P2is present only for the first 200-300 ym from the most rostral extent of the lobule, whereas the other fields are still visible deep into the primary fissure (Fig. 11B, see also below). Slight interindividual differences are seen between animals when the pair of terminal field under P2- is considered. For example, in one case, one of these terminal fields stops near the apex of the ventral face, whereas the other continues some distance into the dorsal face. Satellite bands of zebrin I' Purkinje cells are frequent in lobule V. As in lobule IV, they are especially common in the PI- region, a zone in which few LTHL fibers normally terminate (except for the ventral face of lobule 11). Whether the mossy fiber terminal fields are modified with respect to these satellite bands is not easily resolved. In some cases, there is a suggestion that satellite bands may have influenced the LTHL afferents or vice versa (e.g., Fig. 11B). In this animal, the satellite band within P1- territory of the right-hand side has an associated mossy fiber terminal field beneath it that extends from the ventral to the dorsal face of

Fig. 10. Frontal sections through lobule V taken 1.9 mm caudal from the rostra1 extreme of the cerebellum. A is peroxidase stained for zebrin I immunoreactivity; B shows anterograde transport of WGA-HHP. The same conventions are used as for Figure 3. In A, the zebrin 1- Purkinje cell in the P1- compartment ventrally is part of a satellite band. In B, the labelled spinocerebellar terminals are clearly denser and better defined in the lateral terminal fields. In some cases, terminal fields in the dorsal aspect of the lobule have no equivalent ventrally. Same convention as Figure 3. Scale bar = 200 pm.

the lobule. This is also the case for the short satellite bands present in the left P1- territory of the dorsal face; however, in many other examples, the relationship is not so clear (e.g., medial to the left PI- on the ventral face; see also medial to the left P I - in Fig. 9B). The first intraculminate fissure, which separates lobules IV and V, is relatively shallow in the rat (arrow in Fig. 12B). Consequently, an important portion of the anterior lobe between the preculminate and primary fissures, caudal to the depth of the intraculminate fissure 1, cannot be ascribed naturally to one or the other lobule. In this region, the molecular layer that borders the primary fissure is highly convoluted; nonetheless, a frontal reconstruction reveals that both zebrin I compartments and LTHL terminal fields are each closely aligned throughout and that they maintain relative distributions similar to, and in continuity with, those in the deep portions of lobules IV and V proper (Fig. 12A).

Lobule VIlI Lobule VIII is the main target of spinocerebellar neurons from low thoracic-high lumbar segments in the posterior lobe. Most axons terminate in the copula pyramidis proper, but a few are found in the vermis. In frontal sections, the terminals are arranged into loose clusters spaced by terminal-free regions (Fig. 13). As was the case for the anterior lobe, we have concentrated on the vermal portion of the lobule. The zehrin I+Purkinje cell compartments form three pairs (one medial and two lateral) as in the anterior lobe, but here the zebrin I+ bands are wider (Figs. 13,14). In the vermis of lobule VIII, analysis of the longitudinal organization of LTHL terminals is complicated by the low number of labelled structures. In many cases, particularly in t h r dorsal face where they are less numerous, there may be only two to ten rosettes, and thus there are no sharp boundaries (Fig. 13). In lobule VIII/dorsal, there is a general tendency for the terminals to occupy the granular layer under PI' and P3+, with virtually none under P2+ (Fig. 14B). Under P1' , the terminals frequently form two clusters split at the midline and extending into the medial part of P1-. The tendency for LTHL terminals to concentrate under P1' and P3+,as well as the split of the central terminal field, was seen in all animals. However, the long stretches devoid of J,THL terminals under P2- were not, as three of our animals have aggregates of labelled rosettes in this region. A more variable and less symmetrical distribution of LTHL terminal fields in the posterior lobe than in the anterior was also noted by Arsenio-Nunes and Sotelo ('85). The longitudinal organization of LTHL appears different in ventral and dorsal faces. In particular, LTHL terminals are not seen in the granular layer under ventral PI+ and form symmetrical terminal fields running laterally under both positive and negative zebrin I domains. One terminal field is located under the medial half of P2', frequently extends medially beneath the narrow PI-, and at the apex, sometimes encroaches laterally into PI+.Another, more lateral terminal field, is centered under P2- but extends significantly into P2+ and P3+ on each side. Finally, the most lateral terminal field in the vermis is centered beneath lateral P3' and extends beyond. Comparison of zebrin I and mossy fiber terminal distributions near the apex of lobule VIII suggests that the dorsoventral differences are due to shifts in both patterns (Fig. 14B). The differences between the VIII/ dorsal and VIIT/ventral distributions of LTHL terminals, as


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Fig. 11. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule V of anima1 #74. The same conventions are used as for Figure 4. In a sagittal view (A), no sublobulat,ion is present. The surface projection of the unfolded vermis (B) reveals a medial terminal field centered under Pl' that frequently appears to be divided into two by the midline. A second terminal field runs beneath P1- and encroaches a little into P2'. In the

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ventral face, a second lateral terminal field runs beneath P2-, but dorsally, it only extends for the first 500 pm from the apex. The most lateral terminal field runs beneath P3'. SateHite zebrin I+ bands are present on both sides, and both dorsally and ventrally-dorsally, and in right P1ventrally, the satellite bands have underlying terminal fields. C shows P1 and P2 in frontal reconstruction over 450 pm starting 1.9 mm from the rostra1 tip extreme of the cerebellum.

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Fig. 12. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation a t the depth of lobules I V N (culmen) of animal #74. The same conventions are used as for Figure 4. In a sagittal view (A),it is seen that deep in the first intraculminate fissure (arrow in €3) the distinction between lobules I Y and V

is hlurred. The dorsal surface within the primary fissure is highly convoluted, but, nonetheless, the distributions of terminal fields and zebrin I compartments remain remarkably constant (A). With one minor exception, no terminal fields are seen astride the lateral PZ'PZ-edge.

well as the apical shift, can be seen in frontal reconstructions of the basal or apical portions of the lobule (Fig, 14C,D). Near the base of the lobule (Fig. 14D), terminals in the dorsal face are concentrated under PI+ and P3+;in the ventral face, they are under P2+ and P2-. In a more apical portion of the lobule (Fig. 14C),the dorsoventral differences are less evident. The mediolateral shift of mossy fiber aggregates and the location a t which it occurs is not clear in all experimental animals. Only two characteristics appear unique in the organization of LTHL terminals in lobule VIII-a marked dorsoventral difference in distribution and, in contrast to the anterior lobe, the presence of some mossy fiber terminals straddling the lateral edge of P2+.

tion of low thoracic-high lumbar spinocerebellar projections of the rat described in the present manuscript is consistent with previous descriptions from other animals. In the vermis of the anterior lobe, the number of sagittal LTHL terminal fields per hemicerebellum goes from three to four as they extend caudally from lobule TI to lobules 111, IV, and V. One possible explanation is that the most lateral vermal band of lobule I1 splits into two in the region of II/dorsal-III/ventral (see Figs. 6B, 7B, 9B). A very similar situation has been described in ferret and cat (Voogd, '69; Yaginuma and Matsushita, '87). Further, in lobule V of the ferret, the third lateral LTHL field does not extend as far dorsally as do the two more medial ones (Voogd, '69). The same appears to be the case here also in the rat (e.g., Fig. 1lB). In both the anterior and posterior lobes, the medial group of LTHL terminals appears to be divided at the midline, particularly in lobules V and VIII (Figs. 11,14). This may be because it consists of two contiguous longitudinal bands directly adjacent to the midline, as is the case for the olivocerebellar and corticonuclear projections. In the cat, LTHL terminals from the thoracic spinal cord seem concentrated on each side of the midline in most lobules of the anterior lobe (Yaginuma and Matsushita, '87), and in the ferret, the most medial aggregation of LTHL terminals originating from T1 and lower are located at or near the midsagittal plane (Voogd, '69). In X-irradiated rats and homozygous weaver mice, spinocerebellar afferents from lower thoracicupper lumbar segments are also concentrated lateral to the midline in anterior regions of the cerebellum (Arsenio-

DISCUSSION Our description of the distribution of LTHL terminals agrees with previous studies showing that the spinal receiving areas comprise the anterior lobe (mainly lobules 11-V) and lobule VIII of the posterior lobe (Whitlock, '52; Smith, '61; Brodal and Grant, '62; Grant, '62; Hubbard and Oscarsson, '62; Kimmel, '69; Oscarsson, '73; Grottel, '75; Aldskogious et al., '76; Petras, '77; Hirai et al., '78; Matsushita and Hosoya, '79; Wiksten, '79a,b; Matsushita and Ikeda, '80; Grant et al., '82), and that the terminals are clustered into discrete fields that run sagittally in the granular layer of the vermal and intermediate cortex (Voogd, '64, '69; Hazlett et al., '71; Robertson et al., '83; Arsenio-Nunes and Sotelo, '85; Yaginuma and Matsushita, '86). The mediolateral distribu-

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Fig. 13. Frontal sections through lobule VIII taken 6.0 mm caudal to the rostra1 extreme of the cerebellum. A has been peroxidase stained for zebrin I immunoreactivity, and B, processed to reveal the presence of anterogradely transported WGA-HRP. The same conventions are used as in Figure 3. The medial zebrin I ' compartment is much wider in the posterior lobe than in the anterior and slightly wider in the dorsal than

in the ventral face of lobule VIII. P2' is of similar width to P1' and P3' is a little larger. The terminal fields (B) are well defined and dense at the limit of the copula pyramidis laterally, but are only loose, low-density clusters in the vermis proper. Fewer labelled rosettes are seen in the dorsal than the ventral face. Scale bar = 200 wm.

Nunes et al., '88). That the central area of termination for LTHL terminals is split by the midline is also consistent with myeloarchitectonic compartmentation in the mouse (Marani, '82), rat (Voogd et al., '85), and other mammals (Voogd, '69), and also with the organization of corticonuclear projections (e.g., Goodman et al., '63; Armstrong and Schild, "is), olivocerebellar projections (e.g., Campbell and Armstrong, '83a,b), and zebrin I territories (Gravel et al.,

'87; Hawkes and Leclerc, '87). Tn the posterior lobe, the two more medial mossy fiber clusters on each side are clearly separated by a midline gap (Fig. 14), as is the case in the dorsal face of sublobule VIIIa in the cat (Yaginuma and Matsushita, '87). That mossy fiber terminals are confined to discrete longitudinal compartments in the granular layer is a t first sight confusing. Granule cells have the remarkable property that

PARASAGITTAL ORGANIZATION OF RAT CEREBELLAR CORTEX their axons extend mediolaterally over several millimeters and. thus, encounter the dendritic arbors of Purkinje cells from several Compartments. Why confine the mossy fiber terminals to a narrow compartment only immediately to dissipate this precision? One reason may be that the mossy fiber input to Purkinje cells is dominated by the contribution of synapses between the ascending parallel fiber axons and the Purkinje ceII proximal dendrites (Llinas, '82). For example. Bower and Woolston ('83) showed that granule cell stimulation preferentially drives those Purkinje cells immediately above, suggesting that the apparent parallel fiber divergence of the signal is not functionally very significant. However, by this view, it is hard to see why extensive parallel fiber arrays would be generated so consistently throughout the vertebrates if they have only marginal importance. It is usually assumed that parallel fibers form synapses with all the Purkinje cells they traverse, but this need not be the case. It seems possible, instead, that parallel fibers only synapse within certain compartments, perhaps only those driven by common mossy fiber inputs and not with Purkinje cells in inappropriate compartments. This is consistent with calculations suggesting that only 20% of the parallel fibers traversing a Purkinje cell dendritic tree actually make synaptic contact (Palkovits et al., '71; for further discussion, see Ito, '84). A structural consequence of selective synaptogenesis might be differences in synaptic density in the molecular layer that would correspond to the pattern of mossy fiber input to the granular layer beneath. This might be the underlying structural explanation of the patchy distribution revealed in the mouse molecular layer by immunocytochemical staining for the synaptic vesicle antigen synaptophysin (mabQ155) (Hawkes et al., '85).

Comparison of Purkinje cell and mossy fiber compartments A crucial question-and one that is difiicult to decide-is, Do the mossy fiber terminal fields respect the PC compartments? In a general sense, since both PC and mossy fibers are organized longitudinally, there is inevitably some measure of correspondence. However, unlike the olivocerebellar projection, where there is a direct synaptic interaction and precise compartment alignment (Gravel et al., '87), the mossy fiber-Purkinje celI interaction is indirect via the granule cells and may be architecturally less precise. The problem is further compounded by interlobular and intralobular differences and by uncertainty as to the trajectories of the ascending portions of the granule cell axons. The interpretation of the LTHL terminal field distributions depends on the assumption that glomeruli contact the Purkinje cells immediately above them in the molecular layer. If the granule cell ascending axons do not ascend more or less orthogonally, this assumption is incorrect. For this reason, we have chosen to concentrate our analysis on regions of the lobules where the dorsal and ventral molecular layers run more or less parallel to the horizontal plane and where there is minimum sign of distortion. Our assumption is supported by observations of the mossy fiber trajectories that are often shifted with respect to the white matter bundles in one or the other surface of the lobule (e.g., Fig. 2B). With this in mind, three general coriclusions seem justified: 1. Some P + / P boundaries are respected by the terminal fields, whereas others are not. 2. Some terminal fields obey boundaries that are not revealed by using zebrin I (e.g., beneath P l - ) .

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3. Some terminal fields shift position with respect to Lebrin compartments going from the dorsal to the ventral face of a lobule (e.g., lobule 11).

The identification of parasagittal bands of afferents and Purkinje cells serves in part to explain how the ostensibly uniform cerebellar cortex might encode complex somatotopic maps. The further subdivision of zebrin I compartments by mossy fiber terminal fields increases the resolution of the topography to a point such that anatomical compartment widths become compatible with the widths of compartments in the somatotopic map. However, the means by which longitudinal bands are subdivided to give the patchy mosaic characteristic of cerebellar somatotopy remains unclear. The present results may bear on this issue. The obvious means by which longitudinal compartments are subdivided rostrocaudally is the natural cerebellar foliation. Electrophysiological studies of somatosensory projections t o the cerebellum have shown that each folium and folial complex receives inputs from mixtures of peripheral sources and submodal types (Joseph et al., '78; Shambes et al., '78a,b; Kassel et al., '84; Welker and Shambes, '85; Welker e t al., '88). For example, in lobule VIII of Galago, sublobules a, b, and c have receptive fields for the lower neck-upper back region, whereas sublobule d has middleback representations (Welker et al., '88). The cerebellar lobulation provides a natural means by which afferent inputs can be directed to one or other cortical territory. At the base of each sulcus, the white matter tracts must bifurcate, and hence, there is a natural transition at which the granule cells on one side are driven by the mossy fibers ventrally and those on the other side by the mossy fibers located dorsally. However, most somatotopic boundaries are not a t the base of the sulci, and for these, other mechanisms must be invoked. We have shown that the positions of the mossy fiber terminal fields relative to the Purkinje cell compartments in lobules I1 and VIII are different in the dorsal and ventral aspects of the folia. If relative position is an accurate reflection of connectivity, such differences imply that the 1,THL inputs dorsally and ventrally drive Purkinje cell subsets that are sampled by different components of the olivocerebellar projection and project to different cerebellar nuclear targets. Thus, a second class of mediolateral boundary is created a t the tip of the folium, and longitudinal compartments are further subdivided into a quilt of functional modules. There is a quite unexpected variation between lobules in the distribution of terminal fields. One of the more interesting concerns a mossy fiber terminal field that straddles the YZ+/P2- boundary in lobule VIII (Fig. 14), but never does so in the anterior lobe. One explanation may be that the sources of the mossy fiber terminals are different in the anterior and posterior lobes, that they read different cues, and, thus, terminate differently. However, double-labelling studies have shown that many spinal neurons give rise to bifurcating mossy fibers with one collateral branch terminating in the anterior lobe and another in lobule VIII (Heckroth and Eisenman, '88), and so at least a portion of spinocerebellar neurons are common to both anterior and posterior lobes. Alternatively, it may be the Purkinje cells that are different-that is to say, P2+ Purkinje cells in the anterior lobe are not homologous with those in the posterior lobe. It has been pointed out that the rostrocaudal extension of longitudinal corticonuclear zones may not be continuous (e.g., Brodal, '80),and in both rats and mice, there are significant myeloarchitectonic differences between anterior and

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X3&llIo3 llVTI3832I33 LVX d o NOILVZINV3XO TVLLI3VSVXVd

Fig. 15. A cutaway view of the cerebellar vermis to illustrate a possible model by which mossy fiber afferents might differentially innervate dorsal and ventral faces of a lobule. The two kinds of MF are laminated within the white matter. Thus, when they come to identify synaptic

Fig. 14. The distribution of spinocerebellar mossy fiber terminal fields with respect to the zebrin I compartmentation in lobule VIII of animal 874. The same conventions are used as for Figure 4. Themidline sagittal reconstruction (A) shows no sublohulation, although a prominent hump is consistently present on the ventral face, (B) The surface projection of the unfolded lobule shows P1' and P2 ' are of similar Ridth and P3+ is rather wider. On the ventral face, P1+ tapers progressively from the apex and P2' widens. In contrast to the anterior lobe, the Pcompartmentsare narrower than are the P+,and ventrally, PI- and p2may be reduced to a single Purkinje cell in width. The dorsal face terminal fields are found almost exclusively beneath PI' and P3'. Beneath Pl', the terminal field tends to split at the midline to form two adjacent fields that tend to diverge laterally as they extend towards the apex of the lobule. Ventrally, terminal fields are beneath both pt and ppartments. Most of the granular layer beneath PI+ remains devoid of labelled terminals, but otherwise, the terminal fields do not seem to be phasize the difference between dorsal and ventral faces, especially deeper in the lobule (D). In this projection, the widths of the P' compartments are exaggerated.

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partners, the more dorsal MFs naturally innervate the granular layer dorsally, and those more ventral, the granular layer ventrally. The differences in terminal field position arise through chemically or physiologically based decision making during synaptogenesis.

posterior lobe vermis, with zone B not extending into the posterior lobe (Marani, '82; Voogd et al., '85). Thus, P2+ may not be the same thing in the anterior as in the posterior lobe.

Developmental mechanisms Both the Purkinje compartments (Korneliusseny'68; Wassef and Sotelo, '84;Wassef et a1.9 '85) and at least r d i mentary mossy fiber axon compartments (Arsenio-Nunes and Sotelo, '85) are present in the cerebellar white matter at birth, prior to the onsetof granule cell migration and, hence, prior to normal synaptogenesis. ~h~ natural conclusion is that non-synaptic interactions in the axon tracts are responsible for a t least the gross features of axon compartmentation (e.g., Arsenio-Nunes e t al., '88). This is reinforced by warani, '82; voogd et al., '85) showing the of alternating, parasagittal axonal compartments in the mature


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white matter that presumably reflect the migratory histories of the afferent and efferent, growth cones. In this context, how might this differential distribution of LTHL terminals between dorsal and ventral aspects be achieved? There are two aspects to the problem-to distribute different subsets of LTHL afferents to either the dorsal or the ventral half of the lobule, and then to identify appropriate synaptic partners. For the first part, we suggest that a dorsoventral lamination of axons within the white matter leads naturally to a dorsoventral bias in the distribution of their terminals within the granular layer (Fig. 15). The LTHL growth cones are already situated in the white matter a t birth, prior to the arrival of their granule cell targets. As the granule cells descend, during the first three postnatal weeks or so, the mossy fiber growth cones invade the granular layer and form the synaptic glomeruli. With an intrinsic lamination in the white matter, the more dorsal LTHL axons will naturally innervate the dorsal aspect of the lobule and vice versa. Once within the target area, the differences in the terminal field distributions might arise in many ways. For example, molecular differences in the properties of the growth cones, such as differential adhesivity, could bias growth cone migration. However, the great variability in the terminal field locations both within and between lobules would require an remarkable degree of molecular heterogeneity within the LTHL axon population and their targets. More likely, once the general target area has been reached, precise choice of synaptic partners is based on other criteria. For example, the circuitry could be sculpted by competitive interactions. The protracted maturation of the mossy fibergranule cell circuitry during the first postnatal month, and probably beyond, could provide an ideal substrate for experiencdactivity dependent refinement of cerebellar circuitry. Alternatively, it is possible that zebrin I immunocytochemistry underestimates the diversity present among the Purkinje cells and that a more subtle compartmentation of the target cortex is present. For example, the association between satellite zebrin I + bands and LTHL terminal fields (e.g., lobule V), albeit complex, may point to a guidance role for Purkinje cells during mossy fiber development (see also Arsenio-Nunes et al., '88). This view is supported by evidence that the perinatal molecular compartmentation of the cerebellar cortex differs from that in the adult. This is true for zebrin I, where the earliest differential expression, a t postnatal days 6-9, suggests more compartments in the vermis than there are in the adult (Leclerc et al., '88). Similarly, cyclic GMP-dependent protein kinase, vitamin D-dependent calcium binding protein, and Purkinje cell-specific glycoprotein are all expressed transiently by Purkinje cell subsets during the perinatal period, and these compartments are thought neither to be strictly coincident nor to map directly onto the naturally occurring Purkinje cell clusters (b'assef and Sotelo, '84; Wassef et al., '85). Finally, in the adult, several studies, cited above, have claimed that compartrnentation markers are not perfectly coextensive.

ACKNOWLEDGMENTS We thank J. Rafrafi and R. Sasseville for their expert assistance, Marc Colonnier for extensive help with the figures, and Len Eisenman for his comments and advice. We also acknowledge the financial support of the Medical Research Council of Canada (RH) and the FRSQ (CG).

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Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of purkinje cell perikarya.

Cerebellar development: afferent organization and Purkinje cell heterogeneity.

Topographic organization of the cerebellar nucleocortical projection in the albino rat: an autoradiographic orthograde study.

Somatotopic and columnar organization in the corticotectal projection of the rat somatic sensory cortex.

Postnatal development of the cerebellar cortex in the rat. IV. Spatial organization of bipolar cells, parallel fibers and glial palisades.

Cardiac chronotropic organization of the rat insular cortex.

Organization of local axon collaterals of efferent projection neurons in rat visual cortex.

Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. II. Synaptic organization on reinnervated Purkinje cells.

Topographical organization in the origin of serotoninergic projections to different regions of the cat cerebellar cortex.

Spinocerebellar projection in the meander tail mutant mouse: organization in the granular posterior lobe and the agranular anterior lobe.

The organization of plasticity in the cerebellar cortex: from synapses to control.

Abnormal organization of the cerebellar cortex in the mutant Japanese quail, Coturnix coturnix japonica.

Beyond Columnar Organization: Cell Type- and Target Layer-Specific Principles of Horizontal Axon Projection Patterns in Rat Vibrissal Cortex.

Neuropeptide organization of the hypothalamic projection to the parabrachial nucleus in the rat.

The harmonic organization of auditory cortex.

Medial-lateral organization of the orbitofrontal cortex.

The topographical and laminar organization of the presubiculum's projection to the ipsi- and contralateral entorhinal cortex in the guinea pig.

Organization of visceral and limbic connections in the insular cortex of the rat.

Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat.

The laminar organization of certain afferent and efferent fiber systems in the rat somatosensory cortex.

Organization of neurons in the visual cortex, area 17, of the rat.

Organization of corticocortical connections in the parietal cortex of the rat.

Dopamine Modulates the Functional Organization of the Orbitofrontal Cortex.

Organization of cingulo-ponto-cerebellar connections in the cat.