The Olivocerebellar Projection in the Cat Studied with the Method of Retrograde Axonal Transport of Horseradish Peroxidase VI. THE PROJECTION ONTO LONGITUDINAL ZONES OF THE PARAMEDIAN LOBULE A. BRODAL A N D F. WALBERG Anatomical Institute, University ofoslo, Oslo I , Norway

ABSTRACT Microinjections (30-50 nl) of a horseradish peroxidase (HRP) suspension of 25% (wt./vol.) were made in different folia of the paramedian lobule of cats, and the sites of occurrence of labeled cells in t h e inferior olive were precisely determined. In each case only a small number of cells are labeled, aggregated in a minute area. The labeled cells a r e found within three only of the four olivary areas previously determined (Brodal e t al., '75) to project onto the paramedian lobule (fig. 1): one area in the rostral half of t h e medial accessory olive, another in the dorsal accessory olive (except its caudalmost part), and a third in part of t h e caudal half of the dorsal lamella of the principal olive. Labeled cells were never found in t h e fourth area, t h e ventral lamella. A distinct zonal pattern in t h e projection is demonstrated (figs. 3, 5B): a middle longitudinal zone of t h e paramedian lobule receives olivary afferents from the area in the medial accessory olive, a medial zone from part of the projection area in t h e dorsal accessory olive, a lateral zone from part of t h e projection area in t h e dorsal lamella. This zonal projection appears to extend throughout t h e length of the paramedian lobule (the two caudalmost folia could not be studied). The somatotopical pattern in t h e projections from t h e accessory olives described previously (Brodal et al., '75) is confirmed. The pattern of a zonal projection obtained with t h e HRP-method (fig. 5B) is simpler t h a n t h a t deduced by Armstrong et al. ('74) from recordings of antidromic potentials in the olive (fig. 5A). Concerning main points there is satisfactory agreement. The phenomenon t h a t following microinjections of HRP in superficial parts of t h e folia labeled cells occur within parts only of the regions of the olive which contain labeled cells following large HRP-injections in the paramedian lobule is discussed. In a preceding study t h e total projection from the inferior olive to t h e paramedian lobule was determined by means of t h e method of retrograde axonal transport of horseradish peroxidase (HRP) (Brodal e t al., '75). The rostral two-thirds of t h e paramedian lobule were found to receive afferents from four different areas of the inferior olivary complex, situated in t h e medial and in the dorsal accessory olives and in t h e dorsal and ventral lamella of t h e principal olive. The caudal one-third receives afferents from t h e J. COMP. NEUR., 176: 281-294

former three subdivisions, with some overlapping, but not from the latter (fig. 1). There is anatomical (Korneliussen, '69; Voogd, '69; Courville, '75) and physiological (Szabo and Albe-Fessard, '54; Cooke e t al., '72; Armstrong e t al., '71, '74; Oscarsson and Sjolund, '77b) evidence t h a t t h e longitudinal zonal subdivision of t h e cerebellum is valid for t h e paramedian lobule. As concerns t h e olivocerebellar projection the study of Armstrong e t al. ('74) indicates t h a t the medial, middle and lateral longitudinal zones of t h e parame-

281

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A. BRODAL AND F WALBERG

dian lobule receive their olivary fibers from the dorsal accessory olive, from t h e medial accessory and ventral lamella, and from the dorsal lamella and parts of the ventral lamella and t h e medial accessory olive, respectively (fig. 5A). Because of the spread of HRP along the folia of t h e paramedian lobule, the zonal pattern in t h e projection could not be determined in our previous study, except for suggestive evidence for a projection from the dorsal accessory olive to the medial zone. The present study was undertaken in t h e hope t h a t by using microinjections of HRP, and if t h e injections could be limited to one of t h e zones, it might be possible to demonstrate a zonal pattern in this projection anatomically. As will be seen this has turned out to be t h e case. To a large extent t h e findings agree with those made by Armstrong et al. ('74) by recording antidromic potentials in the olive, but on some points there are discrepancies. The present study has further revealed details which have so far not been recognized. MATERIAL A N D METHODS

Of altogether 20 cats 15 could be used in the present study. Under Mebumal anesthesia the bone covering the posterior fossa was removed and the underlying dura cleaned. Especially in small cats, i t is usually possible to identify under the dissecting microscope the borders of t h e paramedian lobule. Following a small incision in the dura overlying t h e part of the folium of t h e paramedian lobule to be

injected, the arachnoid was gently opened with a small forceps to expose the cortex and facilitate t h e penetration of the tip of t h e needle. The injections of horseradish peroxidase (HRP) were made through a glass micropi) to pette (tip diameter about 1 0 0 ~ attached l syringe. Most of the the tip of a l - ~Hamilton cats received a single injection of 30 nl of a HRP suspension of 25% (wt/vol) in buffered saline, in a few cats 50 or 100 nl were injected. Since the olivary projection to the paramedian lobule is completely crossed, in some animals bilateral injections were made (for details see table 1). The injection time was t e n m i n u t e s . Prior t o t h e injection t h e plunger of t h e syringe was advanced so t h a t a tiny drop of HRP was visible a t the tip of the micropipette. The drop was immediately removed by flushing the tip with saline. Care was taken not to insert the pipette until no more HRP fluid appeared on the tip. After completion of the injection the micropipette was withdrawn immediately. In order to control, a s far as possible, t h e depth of the penetration, the micropipette was inserted on the top of a folium. On account of t h e removal of the arachnoid i t was usually possible to stop t h e pipette immediately after i t had pierced the surface. This is especially essential with injections in the caudalmost folia visible, where t h e needle has to be inserted through t h e cerebellomedullary cistern. Most of the injections turned out to be limited to the molecular layer and a small part of the

TABLE I Peroxidase injected Cat B S t L

Weight kg

Concrntration

TSP

Amount in nl

Survival time days

Iwt/vol J

726

0.75

25

727 728 729 730 733 737 739 740 74 1 743 744 745 746

0 75

25 25 25 25 25 25 25 25 25 25 25 25 25

0.6 0 85 0.95 1.6 1.6 1.5 1.4 1.4 2.6 1.6 1.2 1.2

Sigma VI Sigma Sigma Serva Serva Serva Serva Serva Serva Serva Serva Serva Serva Serva

100 L

1

l0OL 100 L 50 L 30 L 30 L 30 L 30 L 30 L 30 L 30R+L 30R+L 30R+L 30R+L

2 2 1

1 2 2 2 2 2 2 3 3 3

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION

underlying granular layer, and on microscopical examination appeared as shown in figure 2A. Our equipment for microinjection was not calibrated as done by Eide et al. ('76). We cannot exclude, therefore, that a little more or less than the intended volume was injected. For our purpose, however, it was not critical whether exactly 30, 50 or 100 ml were injected. The main point is that the procedure used permits the production of very small, localized, areas of injected HRP in the superficial layers of the cerebellar cortex. The weight of the animals, the survival times and the amount of HRP suspension injected are given in table 1. The animals were killed under Mebumal anesthesia by intracardiac perfusion of a mixture containing 0.4% formaldehyde and 1.25%glutaraldehyde in phosphate buffer. After careful removal from the skull, the cerebellum and the brain stem were isolated and further fixed for one day, and then moved to phosphate buffer added 30%sucrose. On the following day 50-pthick frozen serial sections were cut of the cerebellum (in the sagittal plane) and of the brain stem (in the transverse plane). The sections were then activated as described by Graham and Karnovsky ('66). From groups of five consecutive sections two were selected and mounted, one unstained, the other weakly stained with thionin or cresyl violet. The sections were drawn in a projection apparatus and the distribution of cortical staining and the occurrence of labeled cells as studied under the microscope, were entered in the drawings. In some cases supplementary sections were mounted from areas of the olive containing labeled cells. All neurons in the olive containing granules visible with bright and/or dark field microscopy (fig. 2B) were recorded. For the identification of the various subdivisions of the inferior olive, the diagrams of Brodal ('40) were used. To facilitate comparisons, in each case the spatial distribution of labeled cells was transferred to a standard diagram of the olive as seen in transverse sections and to a diagram of the unfolded inferior olive taken from Brodal ('40, fig. 1 here). This map is based upon sections from two to three weeks old kittens, but the differences from adult cats are so small (Sousa-Pinto and Brodal, '69) that the diagram can be used also for adult animals. The work of Larsell ('70) served as a guide

283

in the identification of the cerebellar lobules. The injection sites as seen in the sections were transferred to a diagram of the cerebellar cortex taken from Larsell ('70). RESULTS

Following successful injections of minute amounts of HRP in the cortex of the paramedian lobule a small number of labeled cells is consistently found in the contralateral inferior olive. In this as in our previous studies it is apparent that in general HRP-staining of the molecular layer is required for labeling of olivary neurons. In some of the cases there is some staining of the granular layer, but only occasionally slight staining of the white matter underneath. The stained area of the cortex in the positive cases is often barely visible with the naked eye and present only in one, two or three neighboring mounted sections (separated by intervals of 200 p , see MATERIAL A N D METHODS). Figure 2A shows a n example of a minute stained patch in the cerebellar cortex. The degree of HRP labeling of the olivary neurons varies somewhat between cells in the same case and between different animals. All labeled neurons are found within three of the four olivary regions determined previously (Brodal e t al., '75) as projecting onto the paramedian lobule, i.e., parts of the medial and dorsal accessory olives and of the dorsal lamella of the principal olive (fig. 1).In the ventral lamella, which likewise projects onto the paramedian lobule, labeled neurons were, however, never found in the cases with single injections (see DISCUSSION). The labeled olivary neurons are always found aggregated as small groups, consisting of from 2 to about 20 cells (fig. 2C). Their location within the paramedian-lobule projecting olivary areas depends on the site of the injection, particularly upon its medio-lateral placement in the folia. In the presentation of the findings below, the cases will be grouped in a diagram according to the olivary region containing labeled cells in the particular case (fig. 3). In the medial accessory olive (fig. 3A) labeled cells are found in its rostra1 half approximately, mainly medially, and always within the previously determined total area of this projecting onto the paramedian lobule (interrupted line in fig. 3A, see also fig. 1). Labeled cells in this olivary area are found only follow-

284

A. BRODAL AND F. WALBERG Abbreviations

8. nucleus 8 of inferior olive D. dorsal accessory olive d. cap, dorsal cap d.1.. dorsal lamella of principal olive dm.c.col., dorsomedial cell column I.. lateral L. left

M, medial accessory olive m., medial nucl. p , nucleus 8 of inferior olive R, right v.L, ventral lamella of principal olive v.I.o., ventrolateral outgrowth

I.

,OStlOI

MEDIAL ACC OLIVE

PRINCIPAL OLIVE

DORSAL ACC OLIVE

Fig. 1 Diagram summarizing the gross pattern in the projection of the inferior olive onto the paramedian lobule in the cat as determined with the method of retrograde axonal transport of HRP. Above the surface of the left paramedian lobule. In the middle a series of transverse sections through the olive from caudal (I) to rostral (XV) with areas projecting to the paramedian lobule indicated. Below a diagram of the contralateral inferior olive imagined unfolded by pulling the medial and accessory olives apart as indicated in the lowermost drawings 1. 2 and 3. The olivary projection areas of the rostral two-thirds and the caudal one-third are indicated by horizontal and vertical hatchings, respectively. Arrows indicate sequence of representation of folia of paramedian lobule from rostral to caudal. From Brodal e t al. (’75).Abbreviations: see list above.

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION

ing injections situated in a middle longitudinal zone of the paramedian lobule. For t h e rostral two-thirds of the lobule a n approximately rostromedial to caudolateral correspondence between the site of t h e injection and t h e position of the labeled cells in t h e olive can be discerned (in agreement with previous observations, Brodal et al., '75). I t is particularly

285

striking t h a t t h e caudalmost injections (cat B.St.L. 745L, see also cat B.St.L. 746L, x, fig. 4C) result in labeling of cells lying most caudally and laterally of all groups, i.e., within the olivary area determined previously (Brodal e t al., ' 7 5 ) a s projecting onto the caudal one-third of the paramedian lobule. The medialmost part of the projection area to t h e

Fig. 2 Photomicrographs illustrating t h e distribution of H R P a t t h e injection s i t e and labeled cells in t h e olive. A The injection site in t h e paramedian lobule in c a t B.St.L. 740 (cp. fig. 3C). The H R P stainingof the m o ~ lecular layer is restricted to a small a r e a of one folium (between arrows). There is some staining of the granular layer a n d some bleeding making the granular layer appear too d a r k i n t h e photograph. X 25 B One of the labeled cells shown in C with labeled dendrites (arrows) Interference contrast photograph. X 1.035. C Low power view of part of t h e dorsal accessory olive icp. inset) in c a t B.St.L. 746R (fig. 3C). showing four closely spaced labeled cells (arrows). Dark-field photograph. X 330. Abbreviations: see list on p. 284.

286

A. BRODAL AND F . WALBERG

C

A I 17291

Zi730) 317261

MEDIAL ACC OLIVE

PRINCIPAL OLIVE

DORSAL

ACC O L I V E

rostra1

caudal

D Fig. 3 Diagrams summarizing t h e findings following microinjections of HRP in the middle IAI. the lateral IB) and the medial IC) longitudinal zone of the paramedian lobule, respectively. I n each of these figures: above the left paramedian lobule with indications (black) of sites of injection. Findings made following injections in the right paramedian lobule are transferred to the left lobule to facilitate comparisons between cases. In the middle diagrams of different parts of the olivary complex imagined unfolded (cf. fig. 1).The areas determined previously as projecting onto the paramedian lobule lcf. fig. 11are outlined by interrupted lines. The small areas containing labeled cells are encircled and numbered (case numbers shown above). Below (D) the distribution of labeled cells in all cases shown is indicated (dotted) in drawings of transverse sections of the olive (levels indicated in the diagram of the unfolded olive). Note t h a t the projection from the medial accessory olive passes only to a middle longitudinal zone of the paramedian lobule (A), those from t h e dorsal lamella of the principal olive (B) and the dorsal accessory olive (C) to a lateral and medial zone, respectively. A roughly rostromedial to caudolateral topographical correspondence (cp. arrows in fig. 1) between the olive and t h e paramedian lobule can be seen in the projections from the accessory olives. For particulars see text. Abbreviations: see list on p. 284.

rostral two-thirds contains labeled cells only in one case (cat B.St.L. 739, fig. 3A). In this case the tip of t h e injection needle was found to have been in the cortex of a folium in a sulcus, a t some distance from t h e surface. Figure 3B shows t h e findings in three cases where labeled cells are found only in t h e dorsal lamella of the principal olive. I n these a s well as i n a fourth case (cat B.St.L. 746L, xx, fig. 4C, to be considered below) the more or less overlapping groups of labeled cells a r e found in the caudal part of the total paramedian projecting area (interrupted lines in fig. 3B, see also fig. l), while its rostral part is always free. I n these cases the injection has been in t h e lateralmost end of the rostral three folia and the caudalmost visible folium (figs. 3B, 4C). No clear topographical correspondence between t h e folia of the parame-

dian lobule and the labeled area in the dorsal lamella can be ascertained. I n the dorsal accessory olive labeled cells are again found within the area of this determined previously to project onto the paramedian lobule (interrupted line in fig. 3C, see also fig. 1). Two small groups of cells (groups 1 and 2 in fig. 3C) are found caudally in the medial part of t h e dorsal accessory olive (a third one in cat B.St.L. 737, see fig. 4A), and three heavily overlapping groups are situated rather far caudally in the lateral part of the dorsal accessory olive (groups 3-5 in fig. 3C). All these groups of labeled cells a r e found in cases with injections of the medial ends of paramedian folia (except for cat B.St.L. 737, fig. 4A, see below). As will be seen there is some correspondence between injection sites and t h e olivary location of labeled cells in par-

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION

A

B St L 737

B

BStL744R

C

287

B St L 746 L

DORSAL ACC OLIVE

Fig. 4 Diagrams illustrating the findings in three particular cases, in two of them with labeling of cells in two olivary subdivisions. The distribution of labeled olivary neurons is indicated by dottings In the drawings. On the basis of the findings in figure 3 the two olivary areas marked x and xx in C are indicated as projecting to correspondingly marked injection points in the paramedian lobule. Cp. in text. Abbreviations: see list on p. 284.

tial agreement with the pattern determined earlier (see arrow in dorsal accessory olive in fig. 1). It is particularly apparent that injections in the caudalmost visible folium result in labeling within the area projecting to the caudal one-third of the paramedian lobule. It is striking that in none of our cases are labeled cells to be found in the larger rostra1 part of the paramedian projecting areas of the dorsal accessory olive (fig. 1). It is seen from the above that the olivary projection to the paramedian lobule is remarkably precise. Each of the olivary regions considered projects to one of three longitudinal zones of the paramedian lobule. Furthermore, the small cell groups labeled in the accessory olives following injection of minute parts of the cortex are arranged in an approximately rostromedial to caudolateral sequence (see arrows in fig. 1) corresponding to the rostrocaudal sequence of folia of the paramedian lobule. Four further cases illustrate the precision in the projection and give some supplementary information. In cat B.St.L. 737 (fig. 4A) an injection of the rostralmost folium of the paramedian lobule involves approximately its middle onethird, while the medial and lateral ends of the folium are free. There is in addition a trifling staining of the caudalmost folium of lobule

VI. In the medial accessory olive a few labeled cells are found, some more are present in the medial part of the dorsal accessory olive.' In cat B.St.L. 744R (fig. 4B) an injection in the second folium from above covers its middle part. I t extends a little further medially than laterally. (Of the 13 sagittal sections passing through this particular folium altogether three show staining, leaving 4 free in the medial direction, 6 in the lateral direction.) In the olive labeled cells are found only in the medial part of the dorsal accessory olive, a t levels IX-X. There are none in the medial accessory olive. A corresponding distribution of labeled cells is found in cat B.St.L. 733 (not illustrated), having a small injection in the first folium from above, situated in the midline, but extending further in the lateral than the medial direction. A comparison of the distribution of labeled cells and the extent of the injected area of cortex in these cases indicates that the medial zone of the paramedian lobule, receiving fibers from the dorsal accessory olive, is somewhat broader rostrally than more caudally. Furthermore, it is seen from a comparison of these cases with those shown in figures 3A ' T h e trifling involvement of lobule VI h a s probably not resulted In cell labeling. since n o labeled cells were found in t h e projection area of t h i s lobule a s determined previously (Hoddevik e t al , '761

288

A. BRODAL A N D F. WALBERG

and C t h a t the findings largely agree with the topographical relation between the paramedian lobule and the accessory olives mentioned above (arrows in fig. 1 ) . In a third case (cat B.St.L. 746L, fig. 4C) t h e injection in t h e left paramedian lobule is of interest (the findings on the right side are shown in fig. 3 0 . In the lowermost visible folium two minute patches (fig. 4C) of stained molecular layer have been produced, separated by a n area where the granular layer and some white matter have a faint brown color. One patch (marked xx) is situated a t the extreme lateral end of the folium, the other (marked x) a little lateral to its midline. A few labeled cells occur in the contralateral medial accessory olive, rather far laterally (marked x), a t levels VIII-IX, another small group (marked xx) is found in t h e dorsal lamella medially, at levels VIII-X. The location of t h e former cell group corresponds almost completely to that found in c a t B.St.L. 745L (fig. 3A'l with a n injection in t h e same place as t h e median one in the present case. The cell group in the dorsal lamella is found a little more medially than those in three cases with injections in t h e lateral end of the uppermost folia (fig. 3B). In addition to illustrating t h e preciseness of the projection, this case shows t h a t the zonal subdivision in the paramedian lobule extends a t least as far caudally as its third folium from below.

cells in the different cases, but the variations a r e only small. On account of its characteristic picture in transverse sections a t different levels (fig. 1) the inferior olive permits a n extraordinarily precise identification of its minor regions. Nevertheless, in some instances there may be a slight uncertainty concerning the level of a particular section, but it will not exceed more than one level of the diagram. Of greater importance as a source of error is probably t h e allocation of a n injection to a particular folium, since the pattern of foliation of t h e paramedian lobule shows considerable individual variations. The exact rostrocaudal placement of an injection, therefore, cannot always be transferred with certainty to a diagram (while the latero-medial coordinates can be well established). Presumably this uncertainty explains that the sequential topographical correspondence between injection sites and labeled cell groups is not evident in all instances (cp. for example cats B.St.L. 741 and 7431, in fig. 3C; cat B.St.L. 739 in fig. 3A). I n the latter case, the injection was made in t h e cortex somewhat deeper than in the other cases. The open spaces between t h e spots containing labeled cells within the olivary region in different cases, presumably indicate t h a t these areas project to folia which have not been injected (see for example fig. 3C). The pattern shown in the summarizing diagram (fig. 5B) is constructed on this assumption.

DISCUSSION

While fairly large injections of HRP demonstrate t h e total olivary projection to a particular cerebellar lobule a s the paramedian (see Brodal et al., '75, and fig. 1 here) i t is seen from the present study that the use of microinjections is a procedure suited to determine details in the pattern. Following microinjections only a few labeled cells are found in the olive, and they are always aggregated in a very small territory of this. This as well as t h e fact that injections in a particular part of a folium most often results in labeling of cells in exactly the same olivary spot (see for example, cases B.St.L. 744L and 743R in fig. 3A; 745L in fig. 3A and 746R, x in fig. 4C; 740, 745R and 746R in fig. 3C) a r e convincing illustrations of the sharpness and precision in t h e olivocerebellar projection. I n some instances, following injections of apparently identical marked spots i n t h e paramedian lobule, there are, however, some variations between locations of the labeled

The olivary projection to longitudinal zones of the paramedian lobule as determined in the present study As seen from figures 3A, B and C, our findings clearly demonstrate t h e presence of a longitudinal zonal pattern in the olivary projection to the paramedian lobule. I t should again be emphasized t h a t all labeled cells in t h e present experiments occurred within olivary areas shown previously to project to the paramedian lobule as a whole (Brodal e t al., '75, and fig. 1 here). While three longitudinal zones of the paramedian lobule have been distinguished in the present study there is evidence (see description and fig. 5) that the medialmost zone is considerably broader in the rostralmost folia t h a n more caudally. The two caudalmost folia are not accessible for direct microinjections in t h e cortex, and no evidence concerning a zonal subdivision of these has, therefore, been obtained. However, i t appears from our findings t h a t t h e zonal subdivi-

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION I 2 3 iw-Vml

A

I0

0 rxr-w,

3 5

B

MEDIAL ACC O L I V E

PRINCIPAL OLIVE

DORSAL ACC O L I V E

MEDIAL ACC OLIVE

PRINCIPAL OLIVE

DORSAL ACC OLIVE

imi

w I

289

0

7

Fig. 5 A comparison between (A) the electrophysiologically determined pattern of the zonal olivary projections to the paramedian lobule of Armstrong e t al. ('75),and (B) as deduced in the present study from anatomical findings following microinjections of HRP (hased on cases shown in figs. 3 and 4). In each figure to the left a diagram of the paramedian lobule, with three longitudinal zones indicated. I n the middle a series of diagrams of transverse sections through the olive, indicating the areas projecting to the different zones. The order of the sections in A has been reversed from Armstrong et al.'s figure 5 in order to correspond to t h a t in B, and the approximate corresponding levels are given in parentheses. To the right t h e findings of both studies are transferred t o a diagram of the unfolded olivary complex (cf. fig. 1).The heavy interrupted lines in B indicate the entire areas of t h e olive projecting to the paramedian lobule (fig. 1). Cp. text. Abbreviations: see list on p. 284.

sion extends a t least as far caudally as to the third folium from below (figs. 3A,C, 4C). In addition to providing an anatomical demonstration of a longitudinal zonal projection from the olive to the paramedian lobule, the present study has confirmed the conclusion drawn in the previous study, that there is a topical correspondence between the paramedian lobule and its projection area in the medial accessory olive, arranged in the direction approximately of the arrow through the medial accessory olive in figure 1. With the exception of one animal (cat B.St.L. 739) the pattern is revealed as clearly as one can expect, the sources of errors taken into consideration. In the dorsal accessory olive a similar correspondence is seen, but the direction of

the sequential order is more transversally oriented than indicated in figure 1 (fig. 6). In the projection area in the dorsal lamella the pattern is obscure. It is striking that with the exception of the medial accessory olive (figs. 3A, 5B) labeled cells were not found in all parts of the areas shown to project onto the rostral two-thirds of the paramedian lobule when injections of larger amounts of HRP were used (Brodal et al., '75; see fig. 1 here). Labeled cells were never encountered in the ventral lamella, nor in the rostral parts of the total paramedian lobule projecting areas in the dorsal accessory olive and the dorsal lamella (see interrupted lines in the diagram of the unfolded olive in fig. 5B). How is this to be explained?

290

A. BRODAL AND F. WALBERG

Deep parts I n the present study the zonal pattern in of fallo? the olivary projection to t h e paramedian I lobule has been demonstrated for those parts of the folia only which are visible on the surface. Since the zonal longitudinal pattern in the cerebellum appears to be valid not only for t h e surfaces of the folia (Voogd, '64, '69; Cour-Hmdlimb ville, '75; Gronewegen and Voogd, '77a,b), i t is Caudal a likely assumption t h a t the rostral parts of port of the paramedian lobule projecting areas in t h e I Dm dorsal accessory olive and the dorsal lamella send fibers to the deeper parts of t h e folia Fig. 6 A diagram showing patterns of organization (medial and lateral zone, respectively). t h e projection of the dorsal accessory olive onto the As concerns the area in t h e dorsal accessory within medial zone of t h e paramedian lobule a s deduced from our olive a comparison with our cases with injec- HRP-studies. There is a somatotopical pattern approxtions of relatively large amounts of HRP (Bro- imately from rostromedial to caudolateral (hindlimb repredal et al., '75) is of interest. In cases (for sented most laterally) and a n approximately rostrocaudal according to t h e depth of the cortex of t h e folia ( s u example, cats B.St.L. 619, 620, 636 in t h a t pattern perficial parts of folia lowermost). Cp. text. study) where good labeling was found in the rostral part of t h e dorsal accessory olive, there was always staining of t h e cortex (including gested above for the dorsal accessory olive, the molecular layer of the medial zone) rather but our data are not incompatible with this deep in many folia (fig. 2 in Walberg e t al., assumption. In t h e medial accessory olive the labeled '76). When relatively little of the deeper parts (of the medial zone) of t h e folia had been ex- spots in the present study occur rather scatposed to HRP, or the molecular layer here was tered throughout the total paramedian proslightly brown only (for example, cat B.St.L. jection area (fig. 3A) and thus give no evi6471, there was only little labeling (the level dence for different projections to deep and suin t h e olive depends on the folia injected). Of perficial parts of t h e folia (the middle zone). particular interest is cat B.St.L. 634, where (Note, however, t h e rather medially placed the injection had covered mainly deep parts of spot of labeled cells in cat B.St.L. 739 with a some folia (Walberg et al., fig. 21, and the rela- not quite superficial injection.) The somatotively few labeled cells in t h e dorsal accessory topical pattern agrees with our previous olive occurred in its rostral part (Brodal e t al., findings (fig. 1). fig. 4A). These data lend support to t h e above The total absence of labeling in t h e ventral assumption, and suggest that in the dorsal lamella in our cases with single injections is accessory olive there is not only a somatotopical surprising. I n the evaluation of this negative pattern (from rostromedial to caudolateral), finding it should be kept in mind t h a t clearcut according to the rostrocaudal sequence of the cell loss is seen only in t h e ventral lamella folparamedian folia (their medial zone), but in lowing lesions of t h e paramedian lobule (Broaddition an approximately rostrocaudal pat- dal, '401, and t h a t the ventral lamella is contern, reflecting the sequence of deep towards su- sistently labeled following large injections of perficial parts of this zone of the paramedian HRP in the rostral two-thirds of the paramelobule. The diagram of figure 6 serves to illus- dian lobule (Brodal e t al., '75). (In three only t r a t e this. The somatotopical pattern agrees of the cases in t h a t study, B.St.L. 650,662 and with the demonstration t h a t within t h e dorsal 663, was there no labeling in the ventral laaccessory olive fibers from the trigeminal nu- mella, but these injections were all rather sucleus end most medially, then follow fibers perficial.) There can thus scarcely be any from the cuneate nucleus, and most laterally doubt (see also Armstrong e t al., '74) t h a t the terminate fibers from the gracile nucleus (see ventral lamella has a projection onto the paramedian lobule. From Groenewegen and below). On account of the relatively small size of Voogd's ('77b) study i t appears t h a t this goes the projection area in the dorsal lamella t h e to the lateralmost part of the lobule, Voogd's available data do not permit conclusions zone D (cf. below). whether there is a similar difference in t h e I t does not appear likely (see above, and projection from this onto deep and superficial Groenewegen and Voogd, '77b) t h a t climbing parts of the folia (the lateral zone) as sug- fibers from the ventral lamella supply only

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION

the deeper, but not the superficial, parts of the folia.2However, other explanations are possible. 1. If the ventral lamella projects only to a minor part of a paramedian zone our injections may not have covered this. 2. The ventral lamella is a very thin and rather cell poor sheet of cells. It might be conceived that each of its climbing fibers have to supply a wider longitudinal area than climbing fibers from the other olivary areas projecting onto the paramedian lobule. Injections restricted to one folium, might, therefore, be insufficient to give labeling of the cells of origin. In order to test this possibility, multiple superficial injections in four adjoining rostra1 folia were made in two cats. In one of them (cat B.St.L. 760, the medialmost and lateralmost parts of the folia were not stained, and there was some HRP-staining of the granular layer) a few labeled cells were found in the ventral lamella on both sides. The other case (cat B.St.L. 761) was negative. The positive findings lend some support to the above suggestion. 3. A further possible explanation deserves mention: Can the observations be explained by assuming that the ventral lamella projects to the paramedian lobule by way of mossy fibers? In spite of numerous anatomical studies i t can as yet not be excluded that some olivocerebellar fibers end as mossy fibers (for a review, see Armstrong, '741, and "electrophysiological investigations cannot show directly whether the olivocerebellar fibers terminate as mossy or as climbing fibers" (Armstrong, '74: p. 366). If the inferior olive gives off mossy fibers, these may well be imagined to come from a particular part of it only, for example, the ventral lamella. Some data make this assumption less heretic than might be thought. The ventral lamella appears in many respects to be a particular region of the inferior olive. Thus cells with long unramified axons, abundant in the accessory olives, are absent in the ventral (and dorsal) lamella (Scheibel et al., '56). In silver impregnated sections (method of Glees, '46) the caudal half of the ventral lamella stands out from all other parts of the olive by containing a n abundance of argyrophilic rings and by being very poor in the feltwork of fine fibers (Blackstad et al., '51; see also Walberg, '71). Further, while the three other olivary areas projecting onto the paramedian lobule in addition project to the anterior lobe, the ventral lamella lacks this projection (Brodal and Walberg,

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'77). I t is of particular relevance t h a t VanGilder et al. ('67) following electrical stimulation of the olive found widespread climbing fiber responses from the cerebellar cortex, with the exception of the paramedian lobule where responses of much longer duration were consistently found. It is compatible with a mossy fiber projection from the ventral lamella that there is no labeling of its cells following the small injections used here where only minute areas of the granular layer are exposed to HRP, while large injections of one or more folia, which result in HRP-diffusion to considerable parts of the granular layer, give rise to labeling in the ventral lamella (see fig. 2 in Walberg et al., '76). It appears from the above that the caudal half of the ventral lamella differs in some respects from the other olivary areas projecting onto the paramedian lobule, and that its projection onto the paramedian lobule has a particular ~ r g a n i z a t i o n . Further ~ studies are needed to decide which of the explanations suggested above (or others) for the lack of labeled cells in the ventral lamella in the present study is correct.

Comparison with data in the literature on the zonal subdiuision of the paramedian lobule There are some differences in the diagrams of the cerebellar longitudinal subdivision as concerns the paramedian lobule. On the basis of physiological studies Szabo and AlbeFessard ('541, Cooke et al. ('72) and Armstrong et al. ('74) distinguish three zones. Anatomically Voogd ('691, based on studies of normal and experimental material, indicates four zones. Three of them (his zones C1, C2, and D,) are present along the entire length of the paramedian lobule, while zone C3 is found only in its rostralmost folia (see also Groenewegen and Voogd, '77b). In a recent physiological study Oscarsson and Sjolund ('77b) in The situation would not be truly corresponding to that suggested above for the dorsal accessory olive and the dorsal lamella, since in this case a particular part of the olive. the ventral lamella, must he assumed to supply onlydeeper parts of the folia. and only in the TOStral two-thirds of the paramedian lobule (fig. 1 ) . It is worth recalling in this connection that the longitudinally arranged projection of olivary climbing fibers to the cerebellum is certainly not as schematic a s generally assumed For example, there is no convrncing evidence for the presence of longitudinal zones in the middle vermis (lobules VI-VIIII, and each of these lobules receives its afferents from separate areas of the medial accessory olive (Hcddevik et a l . ,'761 surrounding a central area projecting to the middle vermal zone (Voogds zone A) of the anterior lobe ( B r d a l and Walberg. '771 Other data likewise suggest that many small parts of the olive may have their peculiar anatomical and functional organizational pattern.

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addition describe a small caudal area as belonging to zone CB. I t is of interest to compare our results with Armstrong et al.'s ('74) study of the zonal subdivision of t h e paramedian lobule (recording of antidromic responses in the olive following stimulation of the cerebellum) with reference to its afferents from t h e inferior olive. I t should be emphasized t h a t neither in their physiological nor in our HRP-study has it been possible to define sharp borders between the zones, and that small differences in t h e size of olivary projection areas may be due to the different techniques employed. I t should further be noted t h a t with regard to identifying a particular site in t h e olive, the HRPmethod allows greater precision t h a n t h e method used by Armstrong et al. As is seen from figures 5A and B there is general agreement concerning main points. There is one clear difference between our results: In contrast to our findings, in Armstrong et al.'s diagram each zone is indicated as receiving in addition to afferents from its main olivary projection area afferents from other olivary regions a s well (fig. 5A). Two facts may be adduced as possible causes of these differences. It might be surmised t h a t a cell whose axon terminals have not taken up sufficient HRP and accordingly will not be identified anatomically, might, nevertheless, be influenced on stimulation of i t s cortical terminal field. On the other hand, i t is conceivable t h a t spreading of the stimulating current (in addition to problems of a n exact identification of the spot recorded from) may be responsible for the recording of units outside t h e main fields determined in our study where each zone is found to receive afferents from one subdivision of t h e olive only. The most marked discrepancies concern t h e projections from t h e ventral lamella and t h e dorsal accessory olive. While superficial injections of HRPdid not result in labeling in the ventral lamella or in the rostral part of the dorsal accessory olive, on superficial stimulation antidromic potentials could be recorded. If i t is assumed t h a t terminals of olivocerebellar fibers are stimulated in a more extensive area of t h e cortex than t h a t covered by a microinjection, the discrepancies between Armstrong et al.'s ('74) a n d our present findings concerning t h e ventral lamella may be explained according to any of the three alternatives mentioned in t h e preceding section. If the fibers from t h e rostral part of the dorsal accessory olive supply

t h e deeper parts of the folia only, a s assumed here, spreading of the current to deeper parts of the folia (not necessarily to the bottom of t h e sulci) might explain the discrepancy on this point. Our findings concerning olivary projections to p a r t i c u l a r longitudinal zones of t h e paramedian lobule are largely in complete agreement with Groenewegen and Voogd's ('77b) results of t h e tracing of fibers following lesions or tritiated leucine injections of the olive. Thus in both studies t h e middle zone (hatchings in fig. 5B), which appears to correspond to Voogd's zone C, receives fibers from t h e rostral half of the medial accessory olive. Our medial paramedian zone (open rings in fig. 5B) appears to correspond to Voogd's zone C,, and according to Groenewegen and Voogd ('77b) it receives fibers from the major rostral part of the dorsal accessory olive. The caudal part of this area is found here to project to the superficial folia of zone C1, but on the basis of the findings of Groenewegen and Voogd, the rostral parts of the projection area as determined by t h e HRP method (Brodal e t al., '75; fig. 1 here) likewise project to zone C,. The lateralmost zone outlined here (squares in fig. 5B) appears to correspond to Voogd's zone D as given by Groenewegen and Voogd ('77b). These authors suggest that the dorsal lamella projects to the most lateral part of it, zone DZ. This is compatible with our results. Zone D, they feel probably receives fibers from the ventral lamella. To some extent the findings of Armstrong e t al. ('74) a r e compatible with t h a t view (fig. 5A). As discussed above, we have no evidence concerning this question.

Functional correlations The finding t h a t each of the three main longitudinal zones of the paramedian lobule receives its olivary afferents from a particular region of this nucleus (figs. 3, 5B) strongly supports other data showing that the zones a r e not functionally equivalent. A consideration of the afferent connections to the three olivary areas illustrates this. All of them receive afferents from more than one source, and a particular afferent contingent may supply more t h a n one of them. However, each of these olivary areas appears to have its particular pattern of afferents. In a study of t h e olivary projection onto the anterior lobe (Brodal and Walberg, '77) a review was given of the main afferent connec-

ZONAL OLIVOCEREBELLAR PARAMEDIAN LOBULE PROJECTION

tions of the three olivary areas which project to the anterior lobe as well as to t h e paramedian lobule (rostral part of the medial accessory olive, the major part of t h e dorsal accessory olive and part of t h e dorsal lamella, see fig. 1 and interrupted lines in fig. 5B). For references the reader is referred to t h a t paper (mainly papers which have appeared later are mentioned below). Oscarsson's and his collaborators' studies of t h e cerebellar distribution in the anterior lobe of climbing fiber responses mediated via the inferior olive have demonstrated a number of functionally differently organized spino-olivocerebellar pathways. Only t h e findings concerning pathways through olivary regions which receive afferent fibers from the ventral spino-olivary tract (VF-SOCPs, ventral funiculus-spino-olivocerebellarpaths) or from the dorsal column nuclei (DF-SOCPs) can at prese n t be correlated with anatomical data (see Brodal and Walberg, '77; Oscarsson and Sjolund, '77a). The zones lateral to the vermis (Voogds zones C1, Cz, C3 and D) extend into t h e paramedian lobule. However, t h e zonal pattern of this lobule, based on climbing fiber responses, is insufficiently known. I n recent studies of t h e VF-SOCPs (Oscarsson and Sjolund, '74, '77b) the C, zone was identified medially in t h e caudalmost p a r t of t h e paramedian lobule (for t h e hindlimb). This is compatible with the findings t h a t this part of zone C, receives i t s afferents from t h e dorsal accessory olive, more particularly its lateral half (figs. 1, 5A,B, 6), and t h a t this olivary area (but not t h e medial part of the dorsal accessory olive) receives direct spinal afferents from the hindlimb segments of t h e cord (for references, see Brodal and Walberg, '77). The absence of a representation of VF-SOCPs in the forelimb area of zone C, is likewise in agreement with t h e anatomical data. I t appears, however, from t h e short report of Cooke et al. ('72) that t h e rostral part of zone C, is activated by DF-SOCP fibers, mediating impulses from t h e forelimb. This fits in with the finding t h a t t h e rostral parts of t h e paramedian lobule, more particularly the medial zone, receives its afferents from the medial half, approximately, of t h e major rostral part of t h e dorsal accessory olive (figs. 1, 5A,B, 6) which receives spinal influences chiefly via t h e c u n e a t e nucleus (for references, see Groenewegen et al., '75; Brodal and Walberg, '77). In contrast to t h e lateral part of t h e dorsal

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accessory olive, which in addition to i t s dominating direct spinal afferents receives one major afferent contingent only, coming from the nucleus interpositus anterior (Tolbert e t al., '761, t h e medial part receives a more varied afferent input. I n addition to fibers from the cuneate nucleus, most medially i t receives some from t h e trigeminal nucleus (Berkley and Hand, '76; see also the physiological study of Cook and Wiesendanger, '761, in agreement with the somatotopical pattern in the dorsal accessory olive (fig. 61, further fibers from t h e primary and supplementary motor cortex and from the nucleus interpositus anterior (Tolbert e t al., '76). I t appears from the above that the zone C, can scarcely be functionally identical as concerns i t s relation to fore- and hindlimb. Zone C,, receiving its afferents from a large medial area in t h e rostral half of the medial accessory olive (figs. 1, 3A, 5A,B) has not been found to receive climbing fiber input from t h e cord via either VF-SOCP or DF-SOCP routes. This agrees in general with t h e absence of direct spinal afferents and afferents from the dorsal column nuclei to this part of the 01ive.~ The main afferents to the rostral part of t h e medial accessory olive appear to come from t h e caudate nucleus, some from t h e sensorimotor cerebral cortex (see also Bishop e t al., '76) and not least from the cerebellar nuclei, presumably only the interpositus posterior (Tolbert e t al., '76). Zone C3, according to Voogd ('69, see also Groenewegen and Voogd, '77b1, present only in the rostralmost few folia of t h e paramedian lobule, rather far laterally, was found by them to receive its afferents from t h e medial part of t h e dorsal accessory olive (its forelimb region, cp. above). Oscarsson and Sjolund, ('74, '77b) found a "hindlimb part" of i t in the caudalmost folia of the lobule to receive climbing fibers via VF-SOCP route. The olivary area concerned, as discussed above, supplies in addition the Zone C,. The restricted distribution of zone C3 may explain t h a t we have not been able to identify a particular projection to i t in the paramedian lobule (nor in the anterior lobe, Brodal and Walberg, '77). On account of the lateral position of zone C3, injections as those in figure 3B might have been expected to show i t s projection, but this was not t h e However, fibers from the cuneate nucleus were found hy Boesten and Voogd ('75: see, however, Groenewegen et al.. '751 to supply a small area corresponding in site approximately to the labeled area in cat B.St.L. 728 (no. 4 in fig. 3A1, which projects to what appears to belong to the forelimb projection area in the pararnedran lobule.

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case. However, t h e findings in cat B.St.L. 737 (fig. 4A) are suggestive. They a r e interpreted by us as showing t h a t zone C, is broader most rostrally, but may a s well indicate a projection from the dorsal accessory olive to a small zone CB. Zone D has not been found to show climbing fiber activation via VF-SOCP or DF-SOCPs. This is in agreement with t h e absence of spinal afferents to the dorsal (and ventral) lamella. LITERATURE CITED Armstrong, D. M. 1974 Functional significance of connections of t he inferior olive. Physiol. Rev., 54: 358-417. Armstrong, D. M., R. J . Harvey and R. F. Schild 1971 Climbing fibre pathways from t h e forelimb to the paramedian lobule of t he cerebellum. Brain Res., 25: 199-202. 1974 Topographical localization in t h e olivocerebellar projection: An electrophysiological study in th e cat. J . Comp. Neur., 154: 287-302. Berkley. K. J., and P J . Hand 1976 Projections to t h e inferior olive from the gracile, cuneate and trigeminal nuclei in t he cat. Anat. Rec., 184: 359. Bishop, G. A., R. A. McCrea and S. T. Kitai 1976 A horseradish peroxidase study of the cortico-olivary projection in the cat. Brain Res.. 116. 306-311. Blackstad, T., A. Brodal and F. Walberg 1951 Some observations on normal and degenerating terminal boutons in t h e inferior olive of t he cat. Acta Anat., 11: 461-477. Boesten. A. J . P., and J . Voogd 1975 Projections of the dorsal column nuclei and t h e spinal cord on the inferior olive in t he cat. J . Comp. Neur., 161: 215-238. Brodal, A. 1940 Experimentelle Untersuchungen uber die olivocerebellare Lokalisation. Z. ges. Neurol. Psychiat., 169: 1-153. Brodal. A., and F. Walberg 1977 The olivocerebellar projection in t h e cat studied with t h e method of retrograde axonal transport of horseradish peroxidase. IV. The Projection to t he Anterior Lobe. J . Comp. Neur., 172: 85.108. Brodal, A,. F. Walberg and G. H. Hoddevik 1975 The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. I The projection to t h e paramedian lobule. J . Comp. Neur.. 164: 449-470. Cook, J . R., and M. Wiesendanger 1976 Input from trigeminal cutaneous afferents to neurones of the inferior olive in rats. Exp. Brain Res.. 26: 193-202. Cmke, J . D., 0. Oscarsson and B. Sjolund 1972 Termination areas of climbing fibre paths in paramedian lobule Acta physiol. scand., 84: 37A-38A. Courville, J. 1975 Distribution of olivocerebellar fibers demonstrated by a radioautographic tracing method. Brain Res.. 95: 253-263. Eide, E.. M. Illert and R. Tanaka 1976 Injection of horseradish peroxidase solution in calibrated volumes (nonlitre) into t he spinal cord. Neurosci. Letters, 2: 51-56. Glees, P. 1946 Terminal degeneration within the central nervous system as studied by a new silver method. J . Neuropath. exp. Neurol., 5: 54-59. Graham, R. C., and M. J . Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in t h e proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J . Histochem. Cytochem., 14: 291-302. Groenewegen, H. J., A. J . P. Boesten a nd J . Voogd 1975 The dorsal column nuclear projections to t h e nucleus

ventralis posterior lateralis thalami and the inferior olive in the cat: An autoradiographic study. J Comp. Neur., 162; 505-518. Groenewegen, H . J., and J. Voogd 1977a The parasagittal zonal organization within t h e olivocerebellar projection I. Climbing fiber distribution in t h e vermis of the cat cerebellum. J . Comp. Neur.. 174: 417-488. Groenewegen. H. J., and J . Voogd (1977b. in preparation) The parasagittal zonal organization within t h e olivocerebellar projection I1 Climbingfiber distribution in t h e intermediate and hemispheric parts of cat cerebellum. Hoddevik. G H., A. Brodal and F. Walberg 1976 The olivocerebellar projection in t h e c a t studied with the method of retrograde axonal transport of horseradish peroxidase. 111. The projection to the vermal visual area. J . Comp. Neur.. 169:155-170. Korneliussen, H. K. 1969 Cerebellar organization in the light of cerebellar nuclear morphology and cerebellar corticogenesis. I n : Neurobiology of Cerebellar Evolution and Development. R. Llinas. ed. Education & Research Foundation, Chicago, Illinois, pp. 515-523. Larsell, 0. 1970 The Comparative Anatomy and Histology of t h e Cerebellum from Monotremes through Apes. J Jansen. ed. The University of Minnesota Press, Minneapolis, 269 pp. Oscarsson, O., and B. Sjolund 1974 Identifiication of 5 spino-olivocerebellar p a t h s ascending through t h e ventral funiculus of t h e cord. Brain Res.. 69. 331-335. (Short communication) 1977a The ventral spino-olivocerebellar system in t h e cat. I. Identification of five paths and their termination in t h e cerebellar anterior lobe. Exp. Brain Res.. in press. 1977b The ventral spino-olivocerebellar system in t h e cat. 11. Termination zones in the cerebellar poste. rior lobe. Exp. Brain Res., in press. Scheibel, M., A. Scheibel, F. Walberg and A. Brodal 1956 Areal distribution of axonal and dendritic patterns in inferior olive. J . Comp. Neur.. 106: 21-49. S z a b , Th. e t D. Albe-Fessard 1954 Repartition e t characteres des affeiences somesthesiques e t d’origine corticale sur le lobe p a r a m d i a n du cervelet du chat. J . de Physiol.. 46: 528-531. Tolbert, D. L., L. C. Massopust, M. G. Murphy and P. A Young 1976 The anatomical organization of the cerebel. lo-olivary projection in the cat. J. Comp. Neur.. 170: 525-544. VanGilder, J. C.. J. L. O’Leary and J . P. Ferguson 1967 Steady potential of cerebellar cortex. Results of direct, olivo- a n d ponto-cerebellar a c t i v a t i o n . El e c t r o e n cephalog. Clin. Neurophysiol., 22.-401-413. Voogd, J . 1964 The Cerebellum of t h e Cat. Structure and Fibre Connexions. Thesis. Assen. Van Gorcum, 215 pp. 1969 The importance of fiber connections in the comparative anatomy of the mammalian cerebellum. In: Neurobiology of Cerebellar Evolution and Development. R. LlinBs, ed. Education and Research Foundation, Chicago, Illinois, pp. 493-514. Walberg, F. 1971 Does silver impregnate normal and degenerating boutons? A study based on light and electron microscopical observations of the inferior olive. Brain Res., 31: 47-65. Walberg, F.. A. Brodal a n d G. H. Hoddevik 1976 Notes on t h e method of retrograde transport of horseradish peroxidase a s a tool in studies of afferent cerebellar connections, particularly those from the inferior olive, with comments on t h e orthograde transport of horseradish peroxidase. Exp. Brain Res., 24: 383-401.