The Olivocerebellar Projection in the Cat Studied with the Method of Retrograde Axonal Transport of Horseradish Peroxidase VII. THE PROJECTION TO LOBULUS SIMPLEX, CRUS I AND II N. KOTCHABHAKDI,' F. WALBERG AND A. BRODAL Anatomical Institute, Uniuersity of Oslo, Oslo 1, Norway
The olivocerebellar projection to lobulus simplex, crus I and I1 in the cat was investigated by means of retrograde axonal transport of horseradish peroxidase (HRP). The distribution of labeled cells in the inferior olive following HRP injections in lobulus simplex, crus I and I1 confirmed the findings by Brodal ('40b) t h a t the rostral half of the principal olive projects to these areas of the cerebellar hemisphere. However, concerning details there are some differences in so far as the heaviest contribution to crus I comes from the medial parts of the ventral and dorsal lamella, that to crus I1 from its lateral part, especially the ventral bend. The present findings show that in addition the rostral part of the medial and the rostromedial part of the dorsal accessory olive project to these areas of the cerebellar cortex. Further details in the projection are shown in figure 8B. The findings agree fairly well with the electrophysiological results of Armstrong et al. ('74) and the experimental anatomical data of Groenewegen and Voogd ('77a,b). An attempt is made to correlate the findings with the pattern of longitudinal zonal subdivision of the cerebellum. There is evidence for a topical organization within the projection to crus I and I1 and parts of their projection areas in the principal olive. The distribution of the labeled cells which project to lobulus simplex, crus I and I1 is discussed in relation to afferent pathways to the inferior olive.
ABSTRACT
The olivocerebellar projection has for many years been the subject of experimental investigations in this laboratory. Thus, Brodal ('40b) using the modified Gudden method (Brodal, '40a) showed that crus I and I1 in the cat receive olivary afferents from two different but adjoining regions in the rostral part of the principal olive. The projection t o crus I and lobulus simplex was shown to come from the dorsal lamella, that to crus I1 from the ventral lamella. Recently i t has been possible to study the topographical organization of the olivocerebellar projection anatomically with techniques where anterograde transport of radioactive tracer substances has been used or by means of the retrograde axonal transport of horseradish peroxidase. In addition, electrophysiological studies have given important information (Armstrong et al., '74). Although the results of studies with anterograde tracer techniques and electrophysiological investigations concerning the projections to crus I and J. COMP. NEUR. (1978)182: 293-314.
I1 in general agree with the descriptions in the early work of Brodal ('40b), it appears from these studies that the olivary projections to the crus I and I1 are more complexly organized than concluded by Brodal ('40b). It would be of interest, therefore, to reinvestigate the olivocerebellar projection to the cerebellar hemispheres with t h e technique of retrograde transport of horseradish peroxidase, since previous studies with this method on other parts of the olivocerebellar projection have given much new information. The present study is part of a systematic experimental mapping of the entire olivocerebellar projection in the cat with the use of horseradish peroxidase as a tracer. So far the projections to the paramedian lobule (Brodal et al., '751, the uvula (Brodal, '761, the vermal visual area (Hoddevik et ' On leave from the Laboratory of Neurobiology and Department of Anatomy, Faculty of Science, Mahrdol University, Bangkok, Thai. land, under the Fellowahip Program of the Norwegian Agency for International Development (NORAD).
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N. KOTCHABHAKDI. F. WALBERG AND A. BRODAL
al., '761, the anterior lobe (Brodal and Walberg, '77a1, the flocculonodular lobe and the paraflocculus (Walberg et al., '78) and the projection onto longitudinal zones of the paramedian lobule (Brodal and Walberg, '77b) have been described. The present study attempts to provide answers to the following questions: (a) Do the projections to crus I and I1 come from subdivisions of the inferior olive other than those shown by Brodal ('40b)? (b) Is there any topographical organization within this projection? (c) Can findings made with horseradish peroxidase as a tracer be correlated with recent findings concerning the longitudinal subdivision of crus I and I1 (Groenewegen and Voogd, '77b)? Finally, (d) can the findings be correlated with recent experimental physiological studies? MATERIALS AND METHODS
The experimental material consists of brains from 17 c a t s 2 Two normal brains and several brains from cats with injections in other parts of the brain than the cerebellum, and treated as the experimental material, were used as controls for the presence of endogenous peroxidatic activity in neurons of the inferior 01ive.~Details of surgical techniques of injection procedure for horseradish peroxidase (HRP), fixation and sectioning of the brains and processing of the histological sections have been given previously (Walberg e t al., '76; Brodal and Walberg, '77a,b). Of every five serial consecutive sections of the brain stem, two were mounted. In addition, sections from the levels where labeled cells were observed, were later processed in order to obtain complete information on the distribution of labeled cells in the different subdivisions of the inferior olive. The presence and distribution of HRP labeled cells in the inferior olive were studied by bright-, dark-field, and interference contrast (Normarski) microscopy in two sets of sections, one set stained weakly with cresylviolet, the other unstained. The localization of the labeled cells was carefully mapped in drawings of transverse sections of the inferior olive made in a projection apparatus. In order to facilitate comparison between cases, t h e distributions of labeled cells were transferred to a standard diagram of the olive as seen in 15 equally spaced transverse sections, and to a diagram (fig. 1) of a reconstruction of the olivary complex as imagined unfolded (Brodal,
'40b). The distribution of labeled cells in the olive is indicated as dots in the drawings of transverse sections and in the reconstruction diagram. The varying densities of the dotting indicate relative densities of labeled cells, but each dot does not represent one cell. The extent of the HRP staining in the cerebellum was mapped from sagittal sections and was transferred to a diagram of the cerebellar surface imagined unfolded, taken from Larsell ('70). In cases where a microinjection of HRP (nanoliters) was made in the cerebellum, instant photographs of the needle tip on the cerebellar surface were made to facilitate the localization of t h e injection site on the fixed cerebellum. Table 1 summarizes the animals' weight, the amount, concentration and type of HRP suspension injected, and the survival time in each case. RESULTS
While all injections were made in either crus I or crus I1 of the ansiform lobule (corresponding to Larsell's lobule HVIIA, see fig. 11, in some cases the injected HRP fluid had diffused to the hemispheral part of the lobulus simplex (lobulus HVI of Larsell, '70). The border between the vermal and hemispheral parts of the lobulus simplex is arbitrarily set a t the caudal prolongation of the paravermal sulcus in the anterior lobe. We have no cases with injections restricted to the narrow lobule HVI. However, no clearcut difference in the distribution of labeled cells could be discerned between cases where the HRP fluid injected into the crura, had or had not, spread to the lobulus simplex. The cases with spreading to this lobule will therefore be considered in connection with those of injections into the crus I. It should be noted that we have not succeeded in placing injections isolated to the cortex of the most lateral part of crus I. In the cases where this was attempted, there was always spreading either to the dentate nucleus and/or to the dorsal paraflocculus. All the cases used in the present study were selected from those in which the HRP injection did not spread to the deep cerebellar nuclei. Following HRP injections in various parts The three series C.CO.L.197, 199 and 202 were prepared by Dr. P. Brodal. We acknowledge his permission to use them in the present study. As indicated previously (Kotchabhakdi e t al., '781, no labeling of neurons in the cerebellum and brain stem of the cat by endogenous peroxidatic activities was observed under the conditions and method used in this laboratory.
295
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES TABLE 1 Peroxidase injected Case Weight
Concentration x WIV
B.St.L.667 B.St.L.749 B.St.L.751 B.St.L.747 B.St.L.789
3.4 2.6 1.8 2.5 4.0
50 50 50 50 25
Sigma VIP Sigma VIP Sigma VIP Serva Serva
B.St.L.773 B.St.L.779 B.St.L.792 B.St.L.666 C.Co.L.201 C.Co.L.199 C.Co.L.202 B.St.L.791 B.St.L.784 B.St.L.785
3.5 2.5 1.7 3.4 3.6 2.0 3.4 2.2 3.3 3.1
25 25 25 50 50 50 50 25 25 25
Serva Serva Serva Sigma VIP Sigma VIP Sigma VIP Sigma VIP Serva Serva ' Serva '
B.St.L.776
2.5
25
Serva
C.Co.L.197
3.8
50
Sigma VIP
Type
Amount
'
Survival time in days
0.4 pl 0.2 p1 0.1 pl 0.3 p1 R-0.2 p1 L-0.1 pl 75 nl 70 nl 0.1 p1 0.5 pl 0.2 pl 0.1 pl 0.2 p1 0.15 pl 200 nl R-200 nl L-200 nl R- 70 nl L - 70 nl 0.25 p1
1 2 1 2 2
1 2 1 2 2 2 2 2 1 2
3
2
' In these cases, where nanoliters of HRP were injected into the cerebellar cortex, the HRP suspensron also contained 2%of Dimethyl sulfoxide (DMSOI which has been claimed to increase the uptake of HRP by nerve terminals (Keefer et al., '76). ' In this case, in spite of a relatively large volume of HRP fluid used In the injection, there was only B very small spot of s t a i n ~ ing in the molecular layer comparable to the other cases with microinjections. Preferably this is due to leakage of the fluid injected into the cerebrospinal fluid. of crus I and 11, labeled cells are always found only in t h e contralateral inferior olive. These labeled cells are easily recognized, particularly under dark-field and interference contrast (Normarski) microscopy. Their appearance and characteristics are similar t o those described in previous studies (Walberg e t al., '76; Brodal, '76; Hoddevik et al., '76). As will be described in detail below, following injections in t h e crura H R P labeled cells in t h e inferior olive have been found most consistently in t h e rostral part of t h e dorsal and ventral lamellae of t h e principal olive. In addition, labeled cells have been observed in t h e rostral parts of t h e medial and dorsal accessory olives. Although t h e intensity and density of labeled cells in various subdivisions of t h e inferior olive varied among cases according to the site and extent of t h e H R P injection in t h e crura, the positive cells in t h e ventral lamella of t h e principal olive consistently exhibited a somewhat greater variation in intensity of t h e HRP labeling t h a n those present in t h e olivary subdivisions. In cases where small amounts of H R P (nanoliters) were injected in t h e cerebellar folia within the crura, a small number of closely spaced labeled cells were consistently
found in t h e regions of t h e inferior olive mentioned above. Successful labeling of these few cells in t h e inferior olive appeared to depend on staining of t h e molecular and t h e Purkinje cell layers in t h e cerebellar cortex, in agreement with what was found in a previous study with small injections (Brodal and Walberg, '77b). However, with large injections including several folia, there was usually spreading of t h e HRP fluid to t h e granular layer and, to a varying degree, to t h e white matter underlying t h e folia. Consistent with our previous studies, special attention was paid to t h e extent of t h e HRP staining in the molecular versus t h e granular layers (indicated by different symbols in t h e diagrams in t h e figures). The projection to crus I
The information on t h e olivocerebellar projection to crus I is based on t h e findings in eight cases. A case with a n extensive cortical staining covering most of t h e lateral part of crus I will be presented first. In cat l3.St.L. 667 (fig. 1) t h e tip of the needle was stereotactically placed in t h e central part of crus I. The HRP fluid had spread to all folia of crus I. The staining included a small part of t h e adjacent posterior folium of t h e lobus simplex and had
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N. KOTCHABHAKDI, F. WALBERG AND A. BRODAL
encroached upon the medial part of the anteriormost folium of crus 11. There was considerable staining of the granular layer and white matter beneath the cortex. In the contralateral principal olive a great number of HRP labeled cells were found in the rostral parts of the dorsal and ventral lamellae. Many labeled cells were also present in a circumscribed area in the rostral part of the medial accessory olive. Almost similar findings were made in the olive in another case, cat B.St.L. 749 (fig. 2A). The only difference was that the number of labeled cells was somewhat less, particularly in the medial accessory olive. The cortical staining in this case covers mainly the lateral part of crus I with a trifling encroachment upon the anteriormost part of the first folium of crus 11. In a third case, cat B.St.L. 751 (fig. 2B) only the lateralmost parts of the posterior folia of crus I had been stained. The HRP fluid had diffused somewhat to the anteriormost folium of crus 11. HRP labeled cells were found only in two circumscribed regions in the rostral parts of the ventral and dorsal lamellae of the principal olive. A more extensive distribution of labeled cells in the inferior olive is found in cat B.St.L.
747 (fig. 3A) where the cortical staining includes all folia of crus I and of the lobulus simplex and in addition involves the posteriormost folium of lobule V. In this case there was a considerable staining of the granular layer and white matter beneath the surface. A great number of HRP labeled cells were found in the rostral halves of the dorsal and ventral lamellae of the principal olive. Contrary to what was found in the cases described above, the labeling in the dorsal lamella extended to its caudal region which is continuous with the ventrolateral outgrowth. Many labeled cells were present in the rostromedial part of the dorsal accessory olive. In the rostral part of the medial accessory olive, there was a rather small patch of labeled cells. In another case, cat B.St.L. 789 R 4 (fig. 3B), the cortical staining covers the medial part of the two most posterior folia of crus I with considerable staining of the granular layer and the white matter beneath these folia. A large number of labeled cells were present in the rostrodorsal part of the ventral lamella near ' In cat B.St.L. 789. two separate HRP injections (referred to as 789 R and 789 Ll were made, one in crus I of the right and one in crus I1 of the left cerebellar hemisphere. The description of labeled cells in the inferior olive in the two cases is based on the findings in the olive contralateral to the side of injection.
Abbreviations A, B, C,, C2. C,, D, cerebellar zones of Voogd ANSI, ansiform lobule Cr. I, crus I Cr. 11, crus I1 D, dorsal accessory olive d.cap, dorsal cap d.L, dorsal lamella dm.c.col., dorsomedial cell column FLOC, flocculus Flocc., flocculus H I-VI and H IX, hemispheral lobules I-VI, IX H VIIA cr. I a,p and cr. I1 a,p, anterior and posterior folia of crus I and crus I1 of the ansiform lobule H VIIB and VIIIA,B, sublobules A and B of hemispheral lobules VII and VIII I-VI. vermian lobules I-VI
VIIA, B and VIIIA, B, anterior and posterior sublobules of lobules VI and VIII IX, uvula X, nodulus L.pm., paramedian lobule L.simpl., lobulus simplex l., lateral M, medial accessory olive m., medial nucl. p, nucleus 6 0, nucleus 0 PFLD, dorsal paraflocculus P.fl.d., dorsal paraflocculus PFLV, ventral paraflocculus P.fl.v., ventral paraflocculus PMD, paramedian lobule SI, lobulus simplex v.I.o., ventrolateral outgrowth v.L, ventral lamella
Fig. 1 Presentation of the findings in cat B.St.L. 667 with HRP injection in crus I. In the diagram of the cerebellar surface imagined unfolded (from Larsell, '70) t h e extent of the cortical staining is indicated. Black denotes staining of the molecular layer. In t h e middle, a standard diagram of a series of 15 equally spaced transverse sections through the inferior olive (taken from Brodal, '40b). The various subdivisions of t h e olivary complex are indicated (see list of Abbreviations). Dots show the occurrence of labeled cells, transferred from drawings of particular sections to t h e corresponding levels of the diagram. Density of dots indicates relative density of labeling of olivary neurons (each dot does not correspond to one labeled cell). Below, a diagram of t h e inferior olive imagined unfolded in one plane (from Brodal, The thin broken lines in t h e middle and lower dia'40b) by pulling it apart as indicated by arrows in the inset (1,2,3). grams refer to borders between projection areas as determined by Brodal ('40b). For abbreviations see list.
B. St.L.667
MEDIAL ACC OLIVE
DORSAL ACC O L I V E
rostrol
caudal
Figure 1
298
N
KOTCHABHAKDI, F WALBEHG AND A BHODAL
B St L. 749
A MEDIAL ACC OLIVE
PRINCIPAL OLIVE
DORSAL ACC O L I V E
MEDIAL ACC O L I V E
PRINCIPAL OLIVE
DORSAL ACC O L I V E
rostral
caudal
6.St.L.75I
rostral
caudal
Fig. 2 Diagrams of the findings in two cases (cats B.St L. 749 and 751) with HRP-injections in crus I. For each case is shown a diagram of part of the cerebellar surface indicating t h e extent of t h e cortical staining, some representative transverse sections through t h e olive, and a diagram of t h e unfolded olivary complex. Symbols and abbreviations as in figure 1.
OLIVOCEREBELLAR PROJECTION TO H E M I S P H E R E S
B.St.L.747
MEDIAL ACC OLIVE
P R lNClPAL OLIVE
DORSAL ACC O L I V E
PRINCIPAL OLIVE
DORSAL ACC OLIVE
rostral
caudal
6.St.L789 R
XN
B MEDIAL ACC OLIVE
Fig. 3 Diagrams of the findings in two cases (cats B.St.L. 747 and 789 R) presented as in figure 2. S y m bols and abbreviations as in figure 1.
299
300
N KOTCHABHAKDI F WALBERG AND A BRODAL
XIII
7 7 3 -.
MEDIAL ACC OLIVE
B St L 773,779
PRINCIPAL OLIVE
779
DORSAL ACC O L I V E
777
rostra1
caudo
Fig. 4 Diagrams of t h e findings in two cases (cats B.St.L. 773 and 779) with microinjections in crus I. The extent and localization of t h e staining in the molecular layer a r e indicated by a patch of dots in t h e diagram of part of t h e cerebellar surface. The location of labeled cells (dottingsi in each case is shown in selected transverse sections and in t h e diagram of t h e unfolded olivary complex. The number of dots does not rep re^ sent t h e number of labeled olivary neurons
t h e junction with the rostral part of t h e dorsal accessory olive. There were also many labeled cells in t h e rostral part of the medial accessory olive, and a patch of labeled cells in t h e middle part of t h e dorsal lamella approaching the rostralmost part of t h e ventrolateral outgrowth of the principal olive. Interesting distributions of labeled cells were found in two cases with microinjections in the same folium of crus I, cats B.St.L. 7 7 3 and 779 (fig. 4). In these cases, 75 and 70 nanoliters, respectively, of HRP fluid were deposited in t h e molecular layer in t h e middle part of one of t h e middle folia. The HRP staining of t h e molecular layer in cat B.St.L. 7 7 3 was located slightly more posterior and caudal to t h a t in cat B.St.L. 779. In t h e former case (7731, there was no evidence of labeling in t h e principal olive. However, several labeled cells were found laterally in t h e rostral part of t h e medial accessory olive. In the latter case (779). labeled cells were found only ventrally in t h e middle part of the dorsal lamella. Additional information for t h e control of distribution of labeled cells comes from cat l3.St.L. 792 (not illustrated). In this case, t h e
cortical staining covers the medial part of the posteriormost folium of lobulus simplex and t h e adjacent anterolateral folia of lobule VIIA. Unlike all other cases with injection in t h e ansiform lobule t h e labeled cells in this case were found only in the middle part of the medial accessory olive, just lateral to t h e nucleus B. This region of the inferior olive has been shown t o project to the posterior lobe vermis (Hoddevik et al., '76).
T h e projection t o crus I I Six cases with large injections and four cases with small injections (cat B.St.L. 776 received a bilateral injection) provided information on the olivocerebellar projection to crus 11. In cat l3.St.L. 666 (fig. 5A), t h e cortical staining of HRP covers almost the entire mediolateral extent of three adjacent folia in crus 11. There was no involvement of the dorsal paraflocculus but the HRP fluid had spread into the white matter beneath these folia. There was, however, no staining of the deep cerebellar nuclei. Many labeled cells were found in the rostral part of t h e principal olive, especially near t h e ventral fold joining
301
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES
B.St.L.666
MEDIAL ACC OLIVE
PRINCIPAL OLIVE
DORSAL ACC O L I V E
MEDIAL ACC OLIVE
PRINCIPAL OLIVE
DORSAL ACC O L I V E
roslrol
coudol
C.Co.L.20 I
rostrol
coudol
Fig. 5 Diagrams of the findings in two cases (cats B.St.L. 666 and C.Co.L. 201) with large HRP injections in crus I1 Symbols and abbreviations as in figure 1.
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N. KOTCHABHAKDI, F. WALBERG AND A. BRODAL
the two lamellae. A distinct patch of labeled cells also occurs in the ventrolateral outgrowth, and in addition, there are many labeled cells in the entire rostral part of the medial accessory olive. In another case, cat C. Co. L. 201 (fig. 5B) the injected fluid had been placed more rostrally, with a moderate staining of t h e neighboring p a r t of t h e dorsal paraflocculus. In t h e inferior olive a somewhat lower number of labeled cells than in the previous case (B.St.L. 666) were found in the same regions of the olivary complex, but with a more caudal extension of distribution of labeled cells in the principal olive (probably a result of the involvement of the dorsal paraflocculus, see DIsCUSSION). An almost identical distribution of labeled cells was also found in another case, cat CC0.L. 199 (not illustrated), where the injection in crus I1 was placed more caudally, and where the HRP fluid had spread laterally and encroached upon the neighboring caudal folia of the dorsal paraflocculus. In cat CC0.L. 202 (fig. 6A), the injected HRP fluid stained chiefly medial parts of the caudal folia in crus I1 and encroached slightly upon the lateral part of the rostral folia of the paramedian lobule and the medialmost part of the caudal folia of crus I. Some labeled cells were found in the middle part of the ventral lamella near the junction towards the dorsal accessory olive (at levels XII-XIII), centrally in the rostral part of the medial accessory olive and in the rostromedial part of the dorsal accessory olive. A similar distribution of labeled cells in the principal and medial accessor y olives was also observed in two other cats, B.St.L. 789 L (fig. 6B) and B.St.L. 791 (fig. 6C),in which the HRP fluid covered the medialmost part of the folia of crus 11. In the former case (789 L), where the HRP fluid reached the medial part of the caudal folia of crus I, there was a patch of labeled cells in the rostrolateral part of the dorsal accessory olive. Following microinjections in different parts of crus 11, small patches of labeled cells were found in the contralateral inferior olive. Their site varies with the site of injection. In cat B.St.L. 784 (fig. 7 ) a small amount of HRP was deposited in the cerebellar cortex of t h e second caudal folium of the right crus 11, with no spreading into the dorsal paraflocculus. Unlike the preceding cases with involvement of the dorsal paraflocculus, there was no evidence of labeling of cells in the caudal part of the dorsal lamella joining the ventrolateral outgrowth.
In three other cats, B.St.L. 776R,5 B.St.L. 785 and C.Co.L. 197 (fig. 71, a small amount of HRP was deposited in the middle folia of crus 11. In the two former cases (B.St.L. 776 R and 785) the injections were made in the same folium on the right side, one slightly more medial than the other. In cat C.Co.L. 197 the injection was situated a little caudal to t h a t in cat B3t.L. 785. In all these cases labeled cells were found in the same area of the principal olive around the vertex region (the junction between the ventral and dorsal lamellae) a t level XIV. In cat B.St.L. 776 L the microinjection is rather medially placed. In this cat the only group of labeled cells present was located in the rostral part of the medial accessory olive. No labeling was seen in the principal olive. Figure 8B shows a summarizing diagram of our findings.
Interpretation of findings The interpretation and evaluation of the presence and distribution of labeled cells in the inferior olive following HRP injection in t h e cerebellum have been discussed previously (Walberg et al., '76; Brodal and Walberg, '77a,b). Therefore, the following interpretation will be restricted to specific findings in the present study. As indicated earlier, one of the objectives of our work is to correlate our findings made with the HRP method with the recent electrophysiological (Armstrong et al., '74) and anterograde autoradiographic (Groenewegen and Voogd, '77a,b) studies. This correlation will be considered in the DISCUSSION. A summary of the findings made in the present study is shown in figure 8B, and for comparison the findings of Armstrong et al. and Groenewegen and Voogd are shown in figures 8A and C. It is seen from the description of our results that following injections in either crus I or crus I1 in most of our cases labeled cells are found in the principal olive. When added the labeled areas in the principal olive cover the rostral two-thirds approximately, of the ventral as well as the dorsal lamella, as shown in In cst B.St.L. 7 7 6 , the injections were made on both sides of crus I1 (referred to as 7 7 6 R and 7 7 6 L). In figure 7 the injection site in the left hemisphere is transferred to the right side for the conven. ience of comparison.
Fig. 6 Diagrams of the findings in three cases (cats C.G.L. 202, B.St.L. 789 L, and B.St.L. 791) with HRP injections in crus 11. Symbols and abbreviations as in figure 1.
C.Co.L.202
PRINCIPAL OLIVE
DORSAL ACC OLIVE
PRINCIPAL OLIVE
DORSAL ACC OLIVE
PRINCIPAL OLIVE
DORSAL ACC OLIVE
iostr01
caudal
6.St. L.789L
MEDIAL ACCOLIVE
rostrol
caudol
B.St.L.791
rostra1
coudol
Figure 6
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N. KOTCHABHAKDI, F. WALBERG AND A. BRODAL
B S t . L ,784, 776R, 776L, 785J.Co.L.197 784
776 R
776L
776
197 J
MEDIAL ACC.OLIVE
784
PRINCIPAL OLIVE
DORSAL
ACC.OLIVE
rostrol
coudol Fig. 7 Diagrams of the findings in cases of microinjections in crus I1 (cats B.St.L. 784, 776 R, 776 L, 785, and C.Co.L. 197).The locations of the injections on the cerebellar hemisphere are indicated by black dots on the diagram of part of the cerebellar surface in the middle of t h e upper part. (The location of t h e injection and the findings in t h e olive in case B.St.L. 776 L. are transferred to t h e opposite side for the convenience of comparison). On each side of the upper part are shown representative transverse sections of the contralateral inferior olive indicating the location of the labeled olivary neurons. In the diagram of the unfolded olivary complex (below) these locations are represented by the encircled areas.
Fig. 8 Diagrams of the olivocerebellar projection onto t h e ansiform lobule and the lobulus simplex in the cat. A The projection as found by Armstrong et al. ('74) in their electrophysiological study. To the left their drawing of the cerebellum, in the middle transverse sections of the inferior olive, showing the olivary areas projecting to t h e different longitudinal zones indicated in the diagram of the cerebellum. The Roman numerals indicate the approximate levels of the olive (as marked in the present study) to which t h e levels in Armstrong et al.'s diagram correspond. On the basis of this, their findings are transferred to our diagram of the unfolded olive (to the right). Note t h a t areas left white were not examined. B Diagram of the projection as found in the present study, presented according to the same principles as in A. The projection of the principal olive (dots) goes exclusively to t h e lateral area of lobulus simplex. Crus I and 11, and their total terminal region appears to correspond to Voogd's zone D (see below). The crus I (including the lobulus simplex) receives fibers from other parts than the crus 11, but the two olivary areas overlap. Arrows indicate approximate topical relations between the crura and their projection areas in the olive. The projections from the medial and dorsal accessory olives (horizontal and vertical hatchings, respectively) go to the medial parts of the hemisphere. The distinction between t h e projections to zone C, from the medial accessory olive and to zones C, -Cs from the dorsal accessory olive, is tentative and is based on the pattern emerging from t h e studies of Voogd and his collaborators. C Diagram of t h e total olivocerebellar projection as constructed by Groenewegen and Voogd ('77a,b) on the basis of tracing of fibers following lesions of, or injections of tritiated amino acids in the inferior olive. The connections with the cerebellar nuclei are deleted from the figure. In order to facilitate comparisons with figures A and B, t h e diagrams of the unfolded parts of the olive have been turned 180' and rearranged.
305
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES
\
b
m
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N . KOTCHABHAKDI, F. WALBERG AND A. BRODAL
figure 8B. However, the total area found to project to each of the two crura is not identical. When the injections are placed in t h e cortical regions of crus I, labeled cells in the principal olive are observed in the dorsal as well as in the ventral lamella. Despite a n extensive staining of the cerebellar cortex of crus I in many cases, there is consistently no labeling of neurons in the ventrolateralmost region of the rostral part of the principal olive (the ventral folding). This area, separating the two patches of cells projecting from the dorsal and ventral lamellae to crus I, projects to crus 11. With large injections in the crus I1 (cats B.St.L. 666 and C.Co.L. 201: figs. 5A,B; and C.Co.L. 202: fig. 6A), there is consistently labeling of neurons in this olivary area, a s well as in the adjoining parts of the dorsal and ventral lamellae. A high density of labeled cells within the projecting areas of the principal olive was observed when the cortical staining involved the lateralmost parts of either crus (figs. 1-3,5).It is worthy of notice that when the cortical staining is limited to the lateral part (as in cats B.St.L. 751: fig. 2B; 779: fig. 4; 776 R, 785 and C.Co.L. 197: fig. 71, labeled cells in the inferior olive were observed in the principal olive only. Furthermore, the extension of the areas in the principal olive containing labeled cells varies approximately proportionally to the size of the cortical area in the cerebellar hemisphere stained by the HRP fluid. In two cases (C.Co.L. 201: fig. 5B; C.Co.L. 199, not illustrated) there is also labeling in the caudal region of the principal olive in the bend region. In these cases there was slight spreading of HRP to the dorsal paraflocculus. This presumably explains the occurrence of labeled cells in this region since in the cat this part of the paraflocculus receives a projection from that olivary region (Brodal, '40b, and unpublished observations). As is seen from the figures, in two cases the area in the dorsal lamella containing labeled cells following injections of HRP in crus I (cat B.St.L. 747: fig. 3) or crus I1 (cat B.St.L. 666: fig. 5) extends caudally into the ventrolateral outgrowth, or is found as a patch here, but these areas are not included among those projecting onto the crura in our summarizing diagram (fig. 8B). The caudal extension of the labeled area in cat B.St.L. 747 is presumably due to the concomitant spreading of HRP to the lateralmost part of lobule V of t h e anterior lobe. which has been found to receive afferents
from this part of the principal olive (Brodal and Walberg, '77a). We are unable to explain the occurrence of t h e patch of labeled cells in the ventrolateral outgrowth in cat B.St.L. 666. This part was not labeled in other cases where the injections covered parts of the same area as in t h a t case.6 We conclude from these findings that in the principal olive the total projection area of crus I covers most of the ventral and the dorsal lamellae a t these levels. The projection area of crus I1 fills in the intervening space and covers particularly the lateral bend of the principal olive, but i t overlaps with the areas of crus I, in both lamellae, particularly in the ventral lamella. From a comparison of cases with differently placed injections it appears t h a t there is a certain degree of topical relation within the projection areas of the principal olive and the folial sequence of crus I, as indicated by the thin arrows in figure 8B. Our findings suggest that the caudolateral folia of crus I (crus I p of HVIIA of Larsell) receive an olivary projection from neurons located in more caudal parts of the total projecting area. A corresponding relation may be present within the projection to crus I1 (thick arrows). (The projections found from the principal olive to crus I and I1 appear to belong to Voogd's zone D, see DISCUSSION).
In several of our cases labeled cells were found in the rostral half of the medial accessory oliue. In most of these cases there were labeled cells in the principal olive as well. However, this was not always so. The microinjections in crus I in cat B.St.L. 773 (fig. 4) and in crus I1 in cat B.St.L. 776 L (fig. 7) gave rise to labeling only in the medial accessory olive. It is further seen that the number of labeled cells in the medial accessory olive increases the further medially the injected area extends in the crura; compare, for example cats B.St.L. 666 and C.Co.L. 201 (figs. 5A,B; see also cat B.St.L. 667: fig. 1).We conclude from these findings that the projection found in our studies from the rostral part of the medial accessory olive to crus I and I1 supplies medial areas of the two lobules. (As will be considered in the DISCUSSION, these parts of crus I and I1 probably correspond to Voogd's zone C2.) Cons Since the ventrolateral outgrowth proJects to the flocculus (Hoddevik and Brodal. '77; Groenewegen and Voogd, '77b; Walberg et al., '78;fig. 0C here), it is possible that the labeling here was due to uptake by fibers to the flocculus on 8CCOUnt of spreading of the HRP fluld to the white matter.
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES
vincing evidence for a difference between parts of this olivary area with regard to projections to crus I and I1 has not been brought forward, even if i t is likely to be present. As concerns t h e projection f r o m the dorsal accessory olive (its rostromedial part) found following injections of crus I (cat B.St.L. 747: fig. 3) as well as of crus I1 (cats C.Co.L. 202 and B.St.L. 789 L: fig. 6) our material is not decisive, except for t h e fact t h a t in all these cases the injection has covered t h e molecular layer in rather medial parts of t h e crura. As will be considered below, we are inclined to interpret these findings as reflecting a projection to zones C, and C3 of Voogd which in other parts of t h e cerebellum, e.g., in t h e anterior lobe, are supplied by fibers from the rostromedial part of t h e dorsal accessory olive.
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rograde cell loss was not observed in the dorsal and medial accessory olives, this may be due to t h e collateralization of axons. These olivary regions supply other cerebellar regions as well as crus I and 11, and injury to one of the axonal branches may not be sufficient to evoke convincing retrograde cellular changes. A collateralization of branches of axons from cells in t h e principal olive to supply both crura may likewise, at least in part, explain the differences found in t h e projections from t h e dorsal and ventral lamellae. With respect to a topical organization within t h e projecting areas of t h e inferior olive, Brodal ('40b) indicated in t h e rabbit a topical representation of cerebellar folia in each of the crura in the dorsal and ventral lamellae of the principal olive, while i t could not be ascertained in t h e cat. DISCUSSION As mentioned above, a n analysis of our In t h e present study t h e distribution of la- cases strongly suggests t h a t there is in the cat beled cells in t h e inferior olive was studied fol- a topical relation within t h e projection from lowing HRP injections in different parts of the principal olive to crus I (indicated by thin crus I and 11. Our findings confirm t h e classi- arrows in fig. 8B). There appears to be a simical description of the olivocerebellar projec- lar organization within t h e projection to crus tion by Brodal ('40b) in as far as t h e crura re- I1 (thick arrows). ceive projections from t h e rostral part of t h e Concerning t h e medial accessory olive, t h e principal olive. However, several findings projection to crus I as well as to crus I1 comes show t h a t t h e projections are less simply orga- from olivary cells situated in a relatively exnized than concluded by Brodal ('40b). First, it tensive area in its rostral half (fig. 8B). An apis clear t h a t crus I and 11, in addition to t h e parently more modest projection to crus I1 and projection from t h e principal olive, also re- presumably crus I (see below), comes from t h e ceive fibers from cells in t h e rostral half of t h e rostral and medialmost part of t h e dorsal acmedial accessory olive and a rostromedial area cessory olive. in t h e dorsal accessory olive. Second, with reA comparison of our results with the elecspect to t h e projection from t h e principal trophysiological study of Armstrong et al. olive, our results do not show separate projec- ('74) and t h e studies of autoradiographic tractions from t h e dorsal lamella to crus I and ing of t h e olivocerebellar fibers of Groenefrom t h e ventral lamella to crus 11,respective- wegen and Voogd ('77a,b) shows many points ly, as indicated by Brodal ('40b). With large as of agreement. Below we will consider first t h e well as small injections in crus I labeled cells electrophysiological findings and then a t a r e found in t h e rostral part of the dorsal as tempt a correlation with t h e data on the zonal well as t h e ventral lamella, thus indicating subdivision of t h e crus I and I1 emerging from t h a t crus I receives a projection from both the studies of Voogd ('64, '67, '69) and his colthese regions of t h e principal olive. The find- laborators. This will be done with reference to ings furthermore show that t h e projection to figure 8A, 8B and 8C (see legend to this crus I1 from the principal olive comes pre- figure). dominantly from t h e rostral part of t h e venFrom figure 8A it is seen t h a t t h e two latertral folding. There is considerable overlapping almost zones of crus I and I1 (indicated by between the areas projecting to crus I and filled triangles and fine short irregular lines crus 11. in t h e diagram of Armstrong et al., '741, were The discrepancy between t h e findings ob- found by these authors to receive afferents tained i n studies of retrograde cellular from a n extensive area of the principal olive, changes (Brodal, '40b) and with t h e use of t h e particularly its lateral portions. It is somemethod of retrograde axonal transport is prob- what more restricted in t h e medial parts of ably due t o several factors. When clearcut ret- ventral and dorsal lamellae than the area
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found by us (fig. 8B) to project to t h e crura. (Note, however, t h a t t h e areas left white i n t h e diagram of Armstrong et al. were not explored.) The medial ends of both lamellae, found by us to project to crus I, are indicated by Armstrong et al. ('74) to project to more medial parts (zones) of crus I and 11. The lateral part of t h e zone filled with rings with dots may belong to areas from which we found labeling in t h e principal olive. On this point t h e discrepancy may be only apparent. However, t h e responses recorded in t h e principal olive following stimulation of the medial three zones in Armstrong et al.'s diagram do not agree with our findings, nor with autoradiographic findings (see below). They may be t h e results of weaknesses of the method (spreading of current, problems with the exact determination of t h e recording sites in t h e olive, particularly difficult as concerns the thin ventral lamella and its close vicinity to t h e medial accessory olive). If this is not t h e explanation for this discrepancy, t h e electrophysiological findings would indicate spreading of fibers from a restricted part of t h e olive to areas of t h e cerebellum very far removed from each other in t h e mediolateral direction. It should be recalled t h a t the zones indicated by Armstrong et al. ('74) in t h e hemisphere in figure 8A are present in other lobules as well, and t h a t t h e labelings in t h e olive (see their original complete diagram) indicated with corresponding symbols represent the total area of the olive projecting to a particular zone. We will return to this question below. The area outlined by Armstrong e t al. in t h e rostral half of the medial accessory olive as projecting to crus I and I1 extends somewhat more caudally t h a n found by us, but is otherwise similarly placed. However, this region is indicated by these authors as projecting not only to medial zones of t h e crura but to lateral parts as well, even t h e lateralmost zone. This discrepancy raises t h e same questions as discussed above. The area in t h e dorsal accessory olive found by Armstrong et al. (fig. 8A) to project to crus I and I1 is likewise more extensive than t h a t found by us, but as concerns its position the results agree fairly well, likewise (for particulars, see below) t h e indication t h a t this area projects to medialmost parts of t h e crura. The regions of both accessory olives projecting to crus I and I1 were thus found t o be somewhat more extensive when studied with anti-
dromic stimulation t h a n with t h e retrograde axonal transport of HRP. This may mean t h a t with t h e latter method parts of these olivary areas remained unlabeled because our injections did not cover virtually all parts of t h e crus I and I1 which receive fibers from these parts of t h e olive. This assumption is compatible with the findings t h a t in several olivary areas there is some degree of topical organization in t h e projection from parts of t h e area to its cerebellar projection field (for example, as found for crus I in t h e present study; for t h e paramedian lobule, Brodal et al., '75; t h e anterior lobe, Brodal and Walberg, '77a; and verma1 lobules VI-VIII, Hoddevik e t al., '76). In figure 8C is reproduced t h e diagram of t h e total olivocerebellar projection as determined by Groenewegen and Voogd ('77b) on t h e basis of tracing t h e afferents from t h e olive following lesions of this or following injections of tritiated amino acids. We are here concerned only with t h e projection to crus I and 11, and as referred to briefly in t h e description, to t h e lobulus simplex (labeled ANSI and SI, respectively in fig. 8C). According to these authors t h e entire ventral and dorsal lamellae supply t h e lateralmost zone D. The zone C, (dots) receives i t s fibers from t h e rostral half of t h e medial accessory olive, while zones C, and C3 both a r e supplied from t h e rostromedial part of t h e dorsal accessory olive. The three olivary areas indicated in their diagram as projecting to t h e crus I and I1 a r e similarly placed as those found by us, but are more extensive (cf. figs. 8B and 8C).This is probably due to the fact t h a t distinctions between projections to different regions within a longitudinal zone are not made and topical differences thus a r e obscured (for example the caudal part of t h e dorsal lamella projects to zone D of t h e anterior lobe, Brodal and Walberg, '77a). When this is taken into account there is a very good correspondence with the areas of t h e olive shown in figures 8B and 8C to project to crus I and 11. In the diagram of figure 8B a n attempt is made to interpret our findings in t h e light of those of Groenewegen and Voogd ('77b). I t is implicit in t h e idea of a longitudinal zonal pattern within t h e olivocerebellar projection t h a t each cerebellar zone receives its olivary afferents from a particular subdivision of the olive as indeed found by Groenewegen and Voogd ('77a,b).In general this is confirmed in HRP studies as well. (For example, t h e medial zone A of t h e vermis is supplied throughout its ex-
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES
tent from the caudal half of the olive, even if different parts of this project to the anterior lobe vermis, Brodal and Walberg, '77a; and to lobules VI-VIII, Hoddevik et al., '76). It is in complete agreement with the data of Groenewegen and Voogd ('77b) that the most lateral part of the hemisphere, belonging to zone D receives its olivary afferents from the principal olive, and from this only. When our more medially situated injections were found to result in labeling in the accessory olives this may likewise be correlated with their data, as shown in the diagram of the cerebellum in figure 8B. The rostral half of the medial accessory olive supplies zone C, in other cerebellar subdivisions according to Groenewegen and Voogd ('77b). In HRP studies labeling is found in this olivary region when injections involve the intermediate part of the anterior lobe (Brodal and Walberg, '77a) and the middle part of the paramedian lobule (Brodal and Walberg, '77b), in regions which correspond well with the zone referred to as C2 in Voogd's diagrams. It is not possible from our present findings to indicate precisely the borders of the cerebellar territory receiving fibers from the rostral half of the medial accessory olive, but it may definitely be said to be situated medial to the area receiving fibers from the principal olive and identified with zone D. It may well be broader than suggested in the diagram of figure 8B. In crus I and I1 the zones C, and C3,on either side of Cz, according to Groenewegen and Voogd ('77b1, like other parts of these zones, receive their olivary afferents from the rostromedial part of the dorsal accessory olive. In HRP studies this olivary region has been shown to project to the intermediate part of the anterior lobe (Brodal and Walberg, '77a) and to a medial zone in the paramedian lobule (Brodal and Walberg, '77b), while definite correlations with zones C, and C, could not be made. In the present material labeling has been found in the rostromedial part of the dorsal accessory olive only in a few cases of injections covering the medial part of crus I and 11, particularly those extending farthest medially, i.e., they probably cover areas which belong to Voogd's zone C,. In the diagram of figure 8B we have, on the basis of the pattern of zonal subdivision, taken the projection found from the dorsal accessory olive to reflect a projection to zones C, and C3. These may well be more narrow than indicated in figure 8B. When labeling in the olivary area projecting
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to zones C, and C3 was not found in all cases in our material where t h e HRP injections covered the medial parts of crus I and 11, this may be due to the zones being narrow. The HRP fluid may not have covered sufficiently large parts of these zones to result in labeling of cells in the olive. In addition it may be that zones C, and C3 have to be labeled together if sufficient uptake of HRP from their terminals is to be achieved. (Since a common olivary region gives rise to projections to each of these zones it appears likely that collaterals of a single cell are distributed to both zones). When interpreted as above our findings are seen to correspond in principle with those of Groenewegen and Voogd ('77b). They support the conception that medial parts of the hemispheres are supplied from the accessory olives, lateral parts from the principal olive. In particular, no evidence can be found for the view that lateral parts of the hemisphere receive olivary afferents from the medial accessory olive, as indicated in the diagram in figure 8A of Armstrong et al. ('74). Interesting details concerning topical relations between parts of an olivary area and parts of its cerebellar longitudinal projecting zones can be recognized when the findings in t h e present study are correlated with HRP studies of the olivary projections to other parts of the cerebellum. For example, in the medial accessory olive the area projecting to the intermediate part (zone C,) of crus I and I1 appears to overlap with, but to extend further rostrally, than that projecting jointly to the intermediate part of the anterior lobe (Brodal and Walberg, '77a) and the paramedian lobule, its middle region (Brodal et al., '75; Brodal and Walberg, '77b). The lateral part (zone D) of the anterior lobe receives its afferents from an area in the dorsal lamella which in part overlaps with, but extends further caudally than the area which supplies crus I (Brodal and Walberg, '77a). The topical relations will not be considered further here. It is of importance, however, to remember them when differences are found in experimental studies between projections to particular parts of a cerebellar zone. The projections to the crus I and I1 in the cat found in the present study are purely contralateral, in agreement with almost all previous investigations. However, it may be mentioned that in a recent study in the cat with the HRP method Rosina and Marini ('77) following unilateral injections of the cerebellum describe labeled cells bilaterally in the prin-
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cipal olive (with a contralateral preponderance). Details of t h e injection site and extent of the HRP staining are not given. This prevents a n evaluation of their conclusions. Chan-Palay et al. ('77) described a bilateral olivocerebellar projection to t h e molecular layer and cerebellar nuclei in t h e rat after unilateral injections of 35S-methionine in t h e inferior olive. In this study i t is possible t h a t t h e injected amino acid could have spread into t h e reticular formation and t h e raphe. Moreover, if interolivary commissural connections exist, transneuronal transfer of radioactively labeled amino acid from one olive to t h e other might explain the bilaterality observed. Functional correlations Via the inferior olive afferent impulses from many different sources a r e relayed to t h e cerebellum. Each part of t h e olive appears to have its particular pattern of afferents. The afferent inputs will be of major importance in determining t h e functional role of t h e particular olivary area in its influence on t h e cerebellum. Some points of relevance to the subject of t h e present study will be mentioned. As concerns t h e rostral p a r t of the medial accessory olive projecting onto t h e intermedia t e parts of crus I and I1 (zone C,?) this receives only very few fibers from t h e cerebral cortex (particularly its motor regions). These fibers, however, appear to end in a restricted area below and medial to t h e region projecting to t h e ansiform lobule (Sousa-Pinto and Brodal, '69) as do some fibers from t h e cuneate nucleus (Boesten and Voogd, '75). Fibers from t h e caudate nucleus were found in t h e cat by Walberg ('56) to end in t h e rostral half of t h e medial accessory olive. While some other s t u dents (Martin e t al., '75, in t h e opossum; Brown et al., '77, in t h e rat; Nauta and Mehler, '66, in t h e monkey) did not find these fibers in other species, t h e physiological studies of Sedgwick and Williams ('67) are in agreement with Walberg's ('56) findings. Several authors have described fibers to t h e inferior olive from regions in t h e mesencephalon, but whether they supply t h e rostral part of t h e medial accessory olive is not always clear. According to Walberg ('74) this part of t h e olive receives fibers from t h e periaqueductal gray, from t h e nucleus of Darkschewitsch and from t h e mesencephalic reticular formation. The latter projection was not found in the autoradiographic study of Edwards ('72, in t h e cat; see also Mizuno et al.,
'73, in t h e rabbit). Fibers from t h e nucleus interstitialis of Cajal have been described (for example by Carpenter e t al., '70) but again i t is not clear whether they supply t h e rostral part of t h e medial accessory olive. Following H R P injections i n the olive in the opossum and t h e c a t (Henkel e t al., '75; Brown et al., '77) labeled cells have been found in t h e ventral periaqueductal gray, t h e nucleus of Darkschewitsch and t h e interstitial nucleus, but information of t h e terminal areas of these projections cannot be obtained from these studies. An important contingent of afferents comes from t h e cerebellum, chiefly from n u cleus interpositus posterior (Tolbert et al., '76). It appears from t h e above t h a t t h e rostral part of t h e medial accessory olive is concerned in mediating influences on zone C, in crus I and I1 (as well as t h e paramedian lobule and t h e intermediate part of t h e anterior lobe) mainly from t h e caudate nucleus and some areas in t h e mesencephalon. In addition i t influences t h e nucleus interpositus posterior since it gives off efferent fibers to this nucleus as well (Courville e t al., '77; Kitai et al., '77). The rostromedial part of t h e dorsal accessory olive, projecting to t h e medial part of crus I and I1 (probably zones C, and C,) receives afferents from t h e cerebral cortex, particularly from t h e motor region and from area 6 in t h e cat (Walberg, '56; Sousa-Pinto, '69; SousaPinto and Brodal, '69) as well as in t h e opossum (Martin e t al., '75). The somatotopical pattern found within this corticoolivary projection (Sousa-Pinto and Brodal, '69) indicates t h a t cortical forelimb as well as hindlimb impulses reach t h e area projecting to zones C, and C,. In contrast to t h e other olivary areas projecting to crus I and I1 t h e area in t h e dorsal accessory olive receives a n input from t h e spinal cord from i t s cervical levels, via the cuneate nucleus (Boesten and Voogd, '75; Groenewegen e t al., '75; Berkley and Hand, '76). Most medially, in t h e area shown here to project to t h e crura, anatomical (Kawamura, '71; Berkley and Hand, '76) as well as physiological (Cook and Wiesendanger, '76) studies have demonstrated a trigeminal input. The afferent fibers appear to come from t h e spinal trigeminal nucleus. Fibers traced from t h e pretectum by Itoh ('77) appear not, or only to a little extent, to end in the medial part of t h e dorsal accessory olive. Some other minor afferent contingents may exist a s well, for example from t h e raphe nuclei (Bobillier e t al.,
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES
'76) and probably the reticular formation. Finally an input from the nucleus interpositus anterior deserves attention (Tolbert e t al., '76). The rostromedial part of the dorsal accessory olive thus appears to be a link in a pathway which is engaged first and foremost in the transmission of integrated input from sensory afferents from face and upper spinal segments with cortical impulses from the motor cortex to zones CI-C3,including their area in the medial parts of crus I and 11. In addition this olivary area, like the rostral part of the medial accessory olive, projects back t o the nuclei interpositi, particularly the anterior (Kitai et al., '77; Courville et al., '77). The rostral two-thirds of the principal olive which project onto crus I and I1 (their lateral parts, corresponding to zone D) is not uniform with regard t o its afferents. There are marked differences in this respect between the dorsal and ventral lamella, indicating fundamental differences. The ventral lamella receives cerebral cortical afferents from the sensorimotor (Walberg, '56). chiefly the motor (Sousa-Pinto and Brodal, '69) and supplementary motor (SousaPinto, '69) cortical areas.' Cortico-olivary fibers have been described by other authors as well, but their precise sites of endings are often not indicated. A somatotopical pattern appears t o be present in the projections to the rostral part of the ventral lamella (Sousa-Pint o and Brodal, '69). Physiological data of Armstrong and Harvey ('66; for particulars, see Sousa-Pinto and Brodal, '69: p. 381) appear t o be in agreement. The ventral lamella receives afferents from the mesencephalon, particularly, it appears, from the nucleus of Darkschewitsch and the adjoining reticular formation (Walberg, '74). In the medial and rostralmost part of the ventral lamella there is a small area receiving trigeminal afferents (Berkley and Hand, '76; Cook and Wiesendanger, '761, but spinal inputs do not appear to reach the principal olive. The dorsal lamella is free from cortical afferents, but receives a substantial fiber contingent from the ipsilateral red nucleus described by a number of authors in the cat (Walberg, '56; Hinman and Carpenter, '59; Edwards, '72) as well as in the monkey (Kuypers et al., '62; Poirier and Bouvier, '66; Miller and Strominger, '73; Courville and Otabe, '74). It is of interest that according to the autora-
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diographic study by Edwards ('72) in the cat the rubral fibers appear t o end particularly in the medial part of the dorsal lamella whose main efferent projection goes to crus I. (In the monkey the rubral afferents appear to cover a more extensive area including parts of the ventral lamella.) The cells of origin are assumed to be chiefly small cells in the rostral (so-called parvicellular) part of the red nucleus, as has been confirmed with the HRP method by Bishop et al. ('76). The dorsal lamella, like the ventral, has been found t o receive fibers from the mesencephalon, derived from the same structures which give off fibers t o the ventral lamella. Walberg ('56) described afferents from the caudate nucleus and the pallidum. Some other afferents, e.g., from the raphe nuclei and the pontomedullary reticular formation may be present. Finally, it should be recalled that the ventral as well as the dorsal lamella receive a topographically organized projection from the dentate nucleus (Beitz, '76; Tolbert et al., '76). Thus the ventral lamella receives fibers from the ventral part of the dentate, the dorsal lamella from its dorsal part. Furthermore, there appears to be a correspondence between medial and lateral parts of the dentate and medial and lateral parts of the lamellae, respectively. Further studies of the precise sites of termination of the (purely descending) afferents t o the principal olive are necessary before final conclusions can be reached concerning possible functional differences between the dorsal and ventral lamella and their projections t o crus I and 11. The overlapping between the olivary areas projecting t o each of them makes evaluations particularly difficult. Some facts appear, however, to be settled. In the first place those parts of the principal olive which project onto the lobulus simplex, crus I and crus I1 do not receive spinal impulses (at least not via direct pathways). Their main afferent input comes from rostrally situated structures, particularly the cerebral cortex, the red nucleus and different structures in the mesencephalon. It appears that the red nucleus is likely to exert its influences on the cerebellum mainly on crus I1 via the dorsal lamella. Cerebral cortical influences via fibers to the ventral lamella may be quantitatively, at least, more important for crus I than for crus 'These origins of cortico-olivary fibers have recently been confirmed in studies following injections of HRP in the ohve (Bishop et al., '76; see also Brown et al., '77).
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N. KOTCHABHAKDI, F. WALBERG AND A. BRODAL
II. However, since t h e same cortical areas which project onto t h e ventral lamella give off fibers (or collaterals) to t h e red nucleus, t h e dorsal lamella might be influenced via t h e cortex as well. This is consistent with t h e results of physiological studies of responses recorded in t h e inferior olive following cortical stimulation (for references, see Allen and Tsukahara, '74 and Armstrong, '74). A considerable literature has grown up on t h e climbing fiber responses occurring in various parts of t h e cerebellum following stimulation of different afferent sources to t h e olive. Most of them deal with the responses obtained on stimulation of peripheral nerves and of t h e cerebral cortex, with particular emphasis on t h e possible role played by t h e olive in the performance of voluntary movements (for a recent review, see Armstrong, '74). Many hypotheses have been set forth. The present, like many other anatomical studies from recent years, makes i t clear t h a t t h e inferior olive is a far more complexly organized nuclear region t h a n previously assumed and t h a t many anatomical details still remain to be clarified. These may hopefully be studied with t h e new anatomical techniques now available. In physiological studies i t will be essential t o indicate precisely t h e olivary region stimulated or recorded from if fruitful correlations between morphological and functional observations are to be expected. LITERATURE CITED Allen, G . I., and N. Tsukahara 1974 Cerebrocerebellar communication systems. Physiol. Rev., 54: 957-1006. Armstrong, D. M. 1974 Functional significance of t h e connections of the inferior olive. Physiol. Rev., 54: 358-417. Armstrong, D. M., and R. J. Harvey 1966 Responses in the inferior olive to stimulation of the cerebellar and cerebral cortices in the cat. J. Physiol. (London), 187: 553.574. Armstrong, D. M., R. J . Harvey and R. F. Schild 1974 Topographical localization in the olivo-cerebellar projection. An electrophysiological study in the cat. J. Comp. Neur., 154: 287-302. Beitz, A. J. 1976 The topographical organization of the olivo-dentate and dentate-olivary pathways in the cat. Brain Res., 115: 311-317. Berkley, K. J., and P. J. Hand 1976 Projections to the inferior olive from t h e gracile, cuneate and trigeminal nuclei in t h e 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. Bobillier, P. S.,F. Petitjean, D. Solvest, M. Fouret and M. Jouvet 1976 The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography. Brain Res., 113: 449-486. Boesten, A. J. P., and J. Voogd 1975 Projections of the dor-
sal column nuclei and t h e spinal cord on t h e inferior olive in the cat. J. Comp. Neur., 161: 215-238. Brodal, A. 1940a Modification of Gudden method for study of cerebellar localization. Arch. Neurol. Psychiat. (Chicago), 43: 46-58. 1940b Experimentelle Untersuchungen uber die olivo-cerebellare Lokalisation. Z. ges. Neurol. Psychiat., 169: 1-153. 1976 The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. 11.The projection to the uvula. J. Comp. Neur., 166: 417-426. Brodal, A., and F. Walberg 1977a The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. IV. The projection to the anterior lobe. J. Comp. Neur., 172: 85-108. 1977b The olivocerebellar projection in t h e cat studied with t h e method of retrograde axonal transport of horseradish peroxidase. VI. The projection onto longitudinal zones of t h e paramedian lobule. J . Comp. Neur., 176: 281-294. Brodal, A,, F. Walberg and G. H. Hoddevik 1975 The olivocerebellar projection in t h e cat studied with t h e method of retrograde axonal transport of horseradish peroxidase. I. The projection to the paramedian lobule. J . Comp. Neur., 164: 449-470. Brown, J. T., V. Chan-Palay and S. L. Palay 1977 A study of afferent input to t h e inferior olivary complex in cat by retrograde axonal transport of horseradish peroxidase. J. Comp. Neur., 176: 1-22. Carpenter, M. B., J. W. Harbison and P. Peter 1970 Accessory oculomotor nuclei in the monkey: projections and effects of discrete lesions. J . Comp. Neur., 140: 131-154. Chan-Palay, V., S. L. Palay, J. T. Brown and C. Van Itallie 1977 Sagittal organization of olivocerehellar and reticulo-cerebellar projections; autoradiographic studies with '%-methionine. Exp. Brain Res., 30: 561-576. Cook, J. R., and M. Wiesendanger 1976 Input from trigeminal cutaneous afferents t o neurons of t h e inferior olive in rats. Exp. Brain Res., 26: 193-202. Courville, J., J. R. Augustine and P. Martel 1977 Projections from t h e inferior olive to t h e cerebellar nuclei in the cat demonstrated by retrograde transport of horseradish peroxidase. Brain Res., 130: 405-419. Courville, J., and S.Otabe 1974 The rubro-olivary projection in the macaque: an experimental study with silver impregnation methods. J. Comp. Neur., 158: 479-494. Edwards, S. 1972 The ascending and descending projections of the red nucleus in the cat: an experimental study using a n autoradiographic method. Brain Res., 48: 45-64. Groenewegen, H. J., A. J. P. Boesten and J. Voogd 1975 The dorsal column nuclear projection 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 zonation within the olivocerebellar projection. I. Climbing fiber distribution in t h e vermis of cat cerebellum. J . Comp. Neur., 174: 417-488. 1977b The parasagittal zonation within the olivocerebellar projection. 11. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum. J. Comp. Neur., in press. Henkel, C. K., M. Linauts and G. F. Martin 1975 The origin of the annulo-olivary tract with notes on other mesencephalo-olivary pathways. A study by the horseradish peroxidase method. Brain Res., 100: 145-150. Hinman, A,, and M. B. Carpenter 1959 Efferent fiber projections of the red nucleus in the cat. J. Comp. Neur., 113: 61-82.
OLIVOCEREBELLAR PROJECTION TO HEMISPHERES Hoddevik, G. H., and A. Brodal 1977 The olivocerehellar projection studied with the method of retrograde axonal transport of horseradish peroxidase. V. The projection to the flocculonodular lobe and t h e paraflocculus in the rabbit. J. Comp Neur., 176: 269-280. Hoddevik, G . H., A. Brodal and F. Walberg 1976 The olivocerebellar projection in t h e cat 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. Itoh, K. 1977 Efferent projections of the pretectum in the cat. Exp. Brain Res., 30: 89-105. Kawamura, K. 1971 Efferent projections of the nucleus caudalis of the spinal trigeminal complex in t h e cat. Okajimas Folia anat. jap., 47: 377-405. Keefer, D. A., W. B. Spatz and U. Misgeld 1976 Golgi-like staining of neocortical neurons using retrograde transport of horseradish peroxidase. Neurosci. Letter, 3: 233- 237. Kitai, S. T., R. A. McCrea, R. J. Preston and G. A. Bishop 1977 Electrophysiological and horseradish peroxidase studies of precerehellar afferents to the nucleus interpositus anterior. I. Climbing fiber system. Brain Res., 122: 197-214. Kotchabhakdi, N., G. H. Hoddevik and F. Walberg 1978 Cerebellar afferent projections from the perihypoglossal nuclei: An experimental study by the method of retrograde axonal transport of horseradish peroxidase. Exp. Brain Res., 31: 13-29. Kuypers, H. G. J. M., W. R. Fleming and J. W. Farinholt 1962 Subcortical projections in the rhesus monkey. J. Comp. Neur., 118: 107-137. Larsell, 0. 1970 The Comparative Anatomy and Histology of t h e Cerebellum from Monotremes through Apes. J. Jansen, ed. University of Minnesota Press, Minneapolis. Martin, G . F., R. Dom, J. S. King, M. Robards and C. R. R. Watson 1975 The inferior olivary nucleus of the opossum, its organization and connections. J. Comp. Neur., 160: 507-534. Miller, R. A., and N. L. Strominger 1973 Efferent connections of the red nucleus in the brainstem and spinal cord of the rhesus monkey. J. Comp. Neur., 152: 327-346. Mizuno, N., K. Nochizuki, C. Akimoto and R. Matsuhina 1973 Pretectal projections to the inferior olive in the rabbit. Exp. Neurol., 39: 498-506. Nauta, W. J. H., and W. R. Mehler 1966 Projections of the lentiform nucleus in the monkey. Brain Res., 1: 3-42. Poirier, L. J., and G. Bouvier 1966 The red nucleus and its
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efferent nervous pathways in the monkey. J. Comp. Neur., J28: 223-244. Rosina, A., and G. Marini 1977 Afferents to the cerebellar hemispheres in the cat. An analysis utilizing horseradish peroxidase as a tracer. Proc. Int. Union physiol. Sci., 13; 638. Sedgwick, E. M., and T. D. Williams 1967 Responses of single units in t h e inferior olive to stimulation of the limb nerves, peripheral skin receptors, cerebellum, caudate nucleus, and motor cortex. J. Physiol. (London), 189: 261-279. Sousa-Pinto, A. 1969 Experimental anatomical demonstration of a cortico-olivary projection from area 6 (supplementarymotor area?) in the cat. Brain Res., 16: 73-83. Sousa-Pinto, A., and A. Brodal 1969 Demonstration of a somatotopical pattern in the cortico-olivary projection in the cat. An experimental study. Exp. Brain Res., 8: 364-386. Tolbert, D. L., L. C. Massopust, M. G. Murphy and P. A. Young 1976 The anatomical organization of the cerebello-olivary projection in the cat. J . Comp. Neur., 170: 525-544. Voogd, J. 1964 Cerebellum of the Cat. Structure and Fibre Connexions. Van Gorcum and Co., N.V., Assen. 1967 Comparative aspects of the structure and fibre connexions of the mammalian cerebellum. Progr. Brain Res., 25: 1-94. 1969 The importance of fiber connections in the comparative anatomy of the mammalian cerebellum. In: Neurobiology of Cerebellar Evolution and Development. R . Llinas, ed. Amer. med. Ass., Chicago, Illinois, pp. 493-514. Walberg, F. 1956 Descending connections to the inferior olive. J. Comp. Neur., 104: 77-173. 1974 Descending connections from t h e mesencephalon to t h e inferior olive: An experimental study in t h e cat. Exp. Brain Res., 21: 145-156. Walberg, F., A. Brodal and G. H. Hoddevik 1976 Notes on the method of retrograde transport of horseradish peroxidase as a tool in studies of afferent cerebellar connections, particularly those from the inferior olive, with comments on the orthograde transport of horseradish peroxidase. Exp. Brain Res., 24: 383-401. Walberg, F., N. Kotchabhakdi andG. H. Hoddevik 1978 The olivocerehellar projections to the flocculus and paraflocculus in t h e cat, compared to those in the rabbit. A study using horseradish peroxidase as a tracer. Brain Res., in press.