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The olivocerebellar projections to the flocculus and paraflocculus in the cat, compared to those in the rabbit. A study using horseradish peroxidase as a tracer
Brain Research, 161 (1979) 389-398
389
© Elsevier/North-HollandBiomedicalPress
THE OLIVOCEREBELLAR PROJECTIONS TO THE FLOCCULUS AND PARAFLOCCULUS IN THE CAT, COMPARED TO THOSE IN THE RABBIT. A STUDY USING HORSERADISH PEROXIDASE AS A TRACER
F. WALBERG,N. KOTCHABHAKDI*and G. H. HODDEVIK Anatomical Institute, University of Oslo, Oslo (Norway) and Laboratory of Neurobiology and Department of Anatomy, Faculty of Science, Mahidol University, Bangkok (Thailand)
(Accepted May 25th, 1978)
SUMMARY The projections from the inferior olive to the flocculus and paraflocculus in the cat have been mapped by means of the method of retrograde axonal transport of horseradish peroxidase. The findings show that the afferents to the flocculus are derived from the dorsal cap, ventrolateral outgrowth, the principal olive (the caudal parts of the ventral and dorsal lamella) and from the rostral part of the medial accessory olive. The fibres to the paraflocculus come from the caudal part of the principal and from the rostral part of the medial accessory olive. Details in the projections are seen from Figs. 1 and 2. Concerning some points the findings are at variance with those made in the rabbit by Hoddevik and Brodal 9, and suggest that there are species differences hitherto not known.
INTRODUCTION In a preceding study from this laboratory 9, the projections from the inferior olive to the flocculus and paraflocculus in the rabbit were mapped by means of the method of tracing the retrograde axonal transport of horseradish peroxidase (HRP). This study was undertaken as part of an examination of the entire olivocerebellar projection. The rabbit was used in that study as the experimental animal, while cats were used for mapping of the olivary connections to the other cerebellar regions, the paramedian lobuleS, 8, the anterior lobe 4, the uvula a, the vermal lobules VI-VII110, and the ansiform lobule 11. During these studies some injections were accidentally placed in * On leavefrom the Laboratory of Neurobiologyand Department of Anatomy, Faculty of Science, Mahidol University,Bangkok,Thailand, under the FellowshipProgramof the NorwegianAgencyfor International Development(NORAD).
390 TABLE [ Weight
Conc.
Amount
(kg)
( % w/v)
(/d)
Survival time in days
50 50 25 50 50 50 50 50
0.3 0.2 0.2 0.2 0.2 0.3 0.2 0.1
2 2 1 2 2 1 2 2
Cat B.St.L. 763 2.5 762 2.5 782 2.7 755 2.6 754 2.4 748 3.2 757 2.5 Rabbit R63
Sigma VIP Sigma VIP Serva SigmaVIP Sigma VIP Sigma VIP SigmaVIP Serva
the flocculus and/or paraflocculus. A scrutiny of the distribution of labelled cells in these cases suggested that there might be some differences between the cat and the rabbit as concerns the olivary areas supplying the flocculus and paraflocculus. Additional experiments with intentional injections of HRP into these lobules were therefore performed. Although the species differences found are relatively modest, they seem important enough to merit attention, and will be briefly reported below. MATERIAL A N D METHODS
Altogether, 7 cats and one rabbit were used. The injections of HRP were made stereotactically under Nembutal or mebumal anaesthesia and the cat material further processed as described in earlier publications from this laboratory (see, e.g., ref. 6). The sections from the rabbit were treated for the blue reaction product as described by Mesulam 13. The distribution of HRP-labelled cells was entered in drawings of transverse sections of the olive and transferred to a diagram of the olive as imagined unfolded (taken from Brodall). Table I gives the weight, amount and concentration of the HRP fluid injected, and the survival time in the various cases. Below, some cases will be described in detail; others illustrating main points will be mentioned briefly. RESULTS
Flocculus
In cat B.St.L. 763 (Fig. 1A) the injection was made in the lateral part of the left flocculus. The injection was restricted to the molecular and granular layers and the adjacent part of the white matter. No staining was present in the dentate nucleus. In the inferior olive labelled cells were present only contralaterally. Heavily labelled cells are found in the dorsal cap (from level IV) including the adjoining part of the ventrolateral outgrowth and the ventral bend of the principal olive (to level X). Faintly labelled cells are present further rostrally in the ventral bend (levels X-XII) and in a patch in the central part of the ventral lamella (levels XI-XII). In addition, labelled
391
B.St.L. 7 6 3
pfl.
A
~
left B.St.L.762
right
r0str01 ~
MC EDI PR AC .OA LIVLE OINLC IVIP EAL dmcco v ~ - - I~ -dl" - - - - [ ....... _-- . . . . "-
B.St. L. 782 R
B ~B St.L.755 ~B.Sf.L.754
MC E.O DLIAIVLE PR AC OINLC IVIP EAL ccol vl ~ - - - ~ rostrol)O,l--~--~---[-2...... "~7-~z'~7"<-~'~dl-~ '........
~v [
Tl--4nuc]~-L
-_11-~r.o./-_~T%_---F-
Fig. 1. A-B: diagram of two cases (cats B.St.L. 763 and 782R) with injections in the lateral part of the left flocculus and medial part of the left dorsal paraflocculus, respectively. Insets show cases described in the text. Abbreviations: d.cap, dorsal cap; d.l., dorsal lamella of principal olive; dm.c.col., dorsomedial cell column; floc., flocculus; 1., lateral; m., medial; nucl. fl, nucleus fl; pfl.d., paraflocculus dorsalis; pfl.v., paraflocculus ventralis; v.l., ventral lamella of principal olive; v.l.o., ventrolateral outgrowth.
392 cells are found in the rostralmost portion of the medial accessory olive (levels XI-XI i I). The cells are concentrated in two patches, one lateral and one medial. The laterally located cells are faintly labelled, the medial ones heavily labelled. In another case the injection was placed in the two lateralmost folia of the left flocculus (cat B.St.L. 762, Fig. 1A, inset). The injection was also in this animal very localized and restricted to the granular and molecular layers and the adjacent white matter. No spreading of fluid to the dentate nucleus occurred. The distribution of the labelled cells in this animal was almost similar to that in the previous case. The only difference was that the labelled cells were fewer and located more caudally in the ventral bend of the principal olive (only up to level XI).
Dorsal paraflocculus In cat B.St.L. 782R* (Fig. 1B) a small amount of HRP had been deposited in the sixth folium (counted from the paramedian lobule). The brown colour was restricted to the molecular and granular layers of the folium and to the immediately adjacent part of the white matter. In the contralateral inferior olive labelled cells are present at levels X-XI in two patches in the principal olive, one in the ventral and one in the dorsal lamella (overlapping into the ventral bend), and in the rostral part of the medial accessory olive in a lateromedially extended band at levels XI-XII. The medialmost part of the medial accessory olive (adjacent to the dorsomedial cell column) is free of labelled cells. In another cat (B.St.L.755) in which the needle was placed more laterally in the dorsal paraflocculus (Fig. 1B, inset) and where the injection likewise was restricted to the cortex and adjacent part of the white matter, labelled cells were found in the principal as well as in the medial accessory olive, but somewhat differently distributed in the principal olive. Here there was a patch of weakly labelled cells only in the rostrolateral part of the ventral lamella (levels XIII-XV). All cells in the medial accessory olive were faintly labelled, those in the rostralmost part very faintly. In a third cat (B.St.L. 754) in which the injection had been placed in the lateralmost folia of the dorsal paraflocculus (Fig. 1B, inset) and restricted to the cortex and the immediately adjacent part of the white matter, labelled cells were present only rostrally in the ventral bend and the adjacent part of the dorsal lamella (levels X-XV). The rostrally located cells were heavily labelled.
Ventral paraflocculus In cat B.St.L. 748 (Fig. 2A) the needle was inserted through the posterior folium ofcrus I on the left side. The larger part of the H R P fluid was deposited in the fourth to sixth folium (counted from the paramedian lobule) of the ventral paraflocculus. An additional patch of staining was, however, also present in the posteriormost folium of crus I and the adjacent part of the anteriormost folium of crus II. No staining was present in the cerebellar nuclei. Labelled cells were present in the ventral bend of the * IncatB.St.L. 782theinjectionwasmadeintheright dorsalparaflocculus. InFig. 1B the injection site in the right hemisphere is transferred to the left side for the convenience of comparsion.
393
B. St.L. 748
left
right MEDIAL ACC.OLIVE
A
rostrol ) 3 1 - - ~ -
PF?INCIPAL OLIVE
~ d ~ c col.
vL _ ~ _ _ ~ J
....
B.St.L. 757
B
MEDIAL ACC.OLIVE rostral XV . . . . . . . .
PRINCIPAL OLIVE
d m_.c=col. ___ _ v . I . _ ~ / . ~ _
_ _7_-_
.
2 ~
co,o, ~
.
.
.
.
d.l= . . . . . . . . .
.................
555-_
:
:i
Fig. 2A-B. Diagram of two cases(cats B.St.L. "148and 757) with injections in the left ventral parafloccu]us and media] part of the ]eft paraflocculus. A detailed description of the cases is given in the text. Abbreviations as in Fig. 1.
394 contralateral principal olive and in the immediately adjoining part of the ventrolateral outgrowth (levels VI-XII), and in the rostral part of the medial accessory olive, its lateral region from levels IX-XIV. A few scattered labelled cells are found in the dorsal cap.
Dorsal and ventral paraflocculus In cat B.St.L 757 (Fig. 2B) the injection had been placed medially in the paraftocculus so that some folia of the dorsal as well as the ventral part of this lobule were stained. In the principal olive labelled cells are found caudally in the ventral bend (level VIII), but extend rostrally within the adjoining parts of the ventral and dorsal lamella (levels IX-XIII). The labelled cells in the medial accessory olive are concentrated laterally at levels XI-XII. The best labelled cells are in all areas found at level XI. DISCUSSION From their findings, Hoddevik and Brodal 9 concluded that in the rabbit the olivary projection to the flocculus arises from the dorsal cap and the adjoining part of the ventrolateral outgrowth, and to a slight extent from the rostral pole of the medial accessory olive. Our findings concerning the distribution of cells from the dorsal cap, ventrolateral outgrowth and from the rostral part of the medial accessory olive to the flocculus in the cat agree with the observations by Hoddevik and Brodal ° in the rabbit. These authors found that the floccular afferent fibres originated from the same olivary areas. From a description of their cases it is, however, clear that in several of the animals (rabbits R 26, 17 and 12) there were also a number of faintly labelled cells in the lateral bend of the principal olive, mainly the ventral lamella, but the labelling of these cells was considered to be a result of spreading of the HRP fluid to the lateral part of the dentate nucleus and to the adjacent parafloccular cortex. Since in the cases described here (cats B.St.L 762 and 763) the HRP fluid has been restricted to the flocculus, it must be concluded that in the cat there is a projection to the flocculus from the main principal olive. As is seen from the description of the cases, this projection comes from the lateral bend of the principal olive and from the ventral lameUa. Although our findings are not decisive on this point, it appears furthermore that there is some kind of topical distribution from the principal olive to the flocculus. Thus, the lateralmost part of this lobe receives fibres only from the caudal portion of the ventral lamella (cat B.St.L. 762), whereas the area of the lobe immediately medial to this (cat B.St.L. 763) receives fibres from the ventral bend up to level XII. However, since we have no injections in the medialmost part of the flocculus, we cannot comment further upon this distribution. The cat appears to differ from the rabbit also in the projection from the olive to the paraftocculus. Whereas the dorsal paraflocculus in the rabbit was found to receive its olivary afferents only from the lateral part of the rostrat half of the medial accessory olive °, in the cat this part and the principal olive are regions of origin for the afferent
395 fibres to the dorsal paraflocculus. Although the situation is not so clear for the ventral paraflocculus, there appears to be a similar situation for this part of the cerebellar cortex. Thus, in cat B.St.L. 748, with an injection covering part of the ventral parafiocculus with an additional patch of HRP in the border zone between the left crus I and II (Fig. 2A), labelled cells are found in the medial accessory olive as well as in the principal olive. In a previous study we have shown that the border zone between crus I and II receives afferents only from two circumscribed regions in the ventral and dorsal lamella, but not from the ventral bend (cat B.St.L. 751, Fig. 2b in ref. 11). In cat B.St.L. 748 a few labelled cells are present lateralmost in the part of the ventral lamella sending its fibres to the border zone between crus I and II, but in this case the majority of the labelled cells lie in the ventral bend of the principal olive and in the rostral part of the medial accessory olive, areas which in cat B.St.L. 751 (Fig. 2b in ref. 11) showed no labelled cells. From a comparison of the various cases it is therefore permissible to conclude that the ventral as well as the dorsal paraflocculus in the cat receive afferents from the principal as well as from the medial accessory olive, and that the fibres to the ventral paraflocculus take their origin from more caudolateral areas of the principal olive than do those to the dorsal parafiocculus. This conclusion is furthermore supported by the findings in cat B.St.L. 757 with an injection including the medial part of the dorsal as well as the ventral paraflocculus (Fig. 2B). In this cat the labelled cells are found in the ventral bend and adjacent parts of the ventral and dorsal lamella, and in the rostral part of the medial accessory olive*. A comparison of our cases with injections in the dorsal paraflocculus indicates that, for this region as for the fiocculus, there is a certain topical arrangement in the projection from the principal olive. Thus, when the injections are located medially in the dorsal paraflocculus, the labelled cells in the principal olive are located somewhat more caudally than when the injection is placed more laterally (compare cases B.St.L. 782R and 754). Comparative anatomical investigations as well as experimental studies have demonstrated a precise pattern of narrow sagittally oriented zones in the cerebellar cortex (for references, see, e.g., ref. 9). As concerns the paraflocculus, the autoradiographic studies by Groenewegen and Voogd s and Kawamura and Hashikawa (personal communication) indicate that Voogd's zones C2 and D are present in both divisions of the parafiocculus. Although details are not clear, it appears that zone C2 is present ventrally in the dorsal and dorsally in the ventral paraflocculus, while the rest of the lobule belongs to zone D. Furthermore, the studies show that in the cat the input to zone D comes from the caudal part of the principal olive, its bend region, and that the input to zone Cz comes from the rostrolateral part of the medial accessory olive. These observations fit well with our findings that labelled cells are present in the principal as well as in the medial accessory olive when our injections cover zones C2 and D (cats B.St.L. 782, 755, 748 and 757), but that labelled cells are found only in the principal olive when the injection lies within zone D (cat B.St.L. 754). * In this connection it should be recalled that Brodal2in his studies with the modified Gudden method t describes a projection from the ventral bend to the entire paraflocculus in the cat.
396 As concerns the flocculus, the autoradiographic findings are less clear. One of Kawamura and Hashikawa's (personal communication) cases suggests that zone C2 is situated medially in the flocculus, an observation obviously supported by the observations made in one of the cases by Groenewegen and Voogd 8 (see their Fig. 18). This zone Ca appears to receive its fibres from the rostral part of the medial accessory olive, while the rest of the flocculus receives its afferents from the ventrolateral outgrowth and the dorsal cap. Our injections in the ftocculus are not limited to the abovementioned medial and lateral parts, and we can therefore not comment upon a possible zonation within the flocculus, the more so since the autoradiopgraphic studies have not confirmed our projection from the main principal olive to the flocculus in the cat. As is evident from the description of our cases, heavily labelled as well as weakly labelled cells are found in most of the animals. This is especially well seen in cat B.St.L. 763 (Fig. IA), which received an injection in the flocculus, and where weakly labelled cells are present in two patches in the principal olive, and laterally in the rostral part of the medial accessory olive. It is well known that a certain concentration of terminals is necessary for a visualization of cells when H R P is used as a tracer (for a discussion of this point, see especially ref. 12). The weak labelling of cells in the abovementioned regions of the olive after injections in the flocculus probably indicates that only (a few) collaterals from cells in the two patches in the principal, and the laterally located cells in the rostral part of the medial accessory olive, terminate in the flocculus. Our previous study 11 has shown that the cells in the areas of the olive here considered also project to crus I and II of the cerebellar hemisphere. Since after injection of HRP in crus I and II these cells are heavily labelled, it is probable that their main axons supply the cerebellar hemispheres. Hoddevik and Brodat 9 found labelled cells only in the medial accessory olive following injections of H R P in the dorsal paraflocculus in the rabbit. After injections in this part of the cerebellum in the cat, labelled cells are present also in the principal olive. The reason for this negativity in the rabbit could obviously be that in this animal only a few collaterals of axons of cells in the principal olive terminate in the dorsal paraflocculus, and that the main branch of the fibres terminates elsewhere in the cerebellum, for example in the ventral paraflocculus. Because of this we have included one rabbit in our material, in which an H R P injection was placed in the dorsal paraflocculus, and where the sections were processed for the blue reaction product described by Mesulam 13.. The visibility of this blue reaction product is better than that of the commonly used brown reaction product 7. This rabbit had, however, similar to the animals described by Hoddevik and Brodal 9, labelled cells only in the rostral part of the medial accessory, and not in the principal olive. This observation could indicate that there are virtually species differences in the terminal branching of climbing fibres within certain parts of the cerebellar cortex. An alternative explanation
* The HRP injection in rabbit R63 was restricted to the dorsal paraflocculusand almost simitar tothat in rabbit R t9 of Fig. 3D in the study of Hoddevik and BrodalL
397 rostrol
pfl,v. coudol
left MEDIAL kCC.OLIVE
PRINCIPAL OL)VE . . . . . . .
7-_'555555 ~
f . . . . . . . . . . Yf-T
coudo~ ~ _~_V_'_-_TL.T
right
DORSAL ACC.OLIVE . . . . . . . . . . . . . .
. . . . . . .
&--_-
-~5
...... 7 - - - 7 -
.T]L-L-.-_V,T.-.-_VLL
Fig. 3. Diagram showing our findings of the olivocerebellar projection onto the flocculus and paraflocculus in the cat. Abbreviations as in Fig. 1.
could be that zone D in the dorsal paraflocculus is absent in the rabbit. Fig. 3 gives a summary of our findings in the cat. ACKNOWLEDGEMENTS
Supported by the Norwegian Agency for International Development (NORAD). REFERENCES 1 Brodal, A., Modification of Gudden method for study of cerebral localization, Arch. Neurol. Psychiat. (Chic.), 43 (1940) 46--58. 2 Brodal, A., Experimentelle Untersuchungenfiber die olivocerebellare Lokatisation, Z. ges. Neurol. Psychiat., 169 (1940) 1-153. 3 Brodal, A., The olivocerebellar projection in the cat as studied with the method ofretrograde axonal transport of horseradish peroxidase. II. The projection to the uvula, J. comp. NeuroL, 166 (1976) 417-426. 4 Brodal, A. and Walberg, F., 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. Neurot., 172 (1977) 85-108. 5 Brodal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. VL The projection onto longitudinalzones of the paramedian lobule, Jr. comp. Neurol., I76 (1977) 281-294. 6 Brodal, A., Walberg,F, and Hoddevik, G. H.,The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase, J. comp. Neurol., 164 (1975) 449-470.
398 7 Graham, R. C., Jr. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 8 Groenewegen, H. J. and Voogd, J., The parasagittal zonation within the olivocerebellar projections. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum, J. comp. Neurol., in press. 9 Hoddevik, G. H. and Brodal, A., The olivocerebellar projection studied with the method of retrograde axonal transport of horseradish peroxidase. V. The projections to the flocculonodular lobe and the paraflocculus in the rabbit, J. comp. Neurol., 176 (1977) 269-280. 10 Hoddevik, G. H., Brodal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. Ill. The projection to the vermal visual area, J. comp. Neurol., 169 (1976) 155-170. 11 Kotchabhakdi, N., Walberg, F. and Brodal, A., 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, J. comp. Neurol., in press. 12 LaVail, J. H., Retrograde cell degeneration and retrograde transport techniques. In W. M. Cowan and M. Cuenod (Eds.), The use o f Axonal Transport for Studies o f Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 219-248. 13 Mesulam, M.-M., The blue reaction product in horseradish peroxidase. Neurohistochemistry: incubation parameters and visibility, J. Histochem. Cytochem., 24 (1976) 1273-1280.
389
© Elsevier/North-HollandBiomedicalPress
THE OLIVOCEREBELLAR PROJECTIONS TO THE FLOCCULUS AND PARAFLOCCULUS IN THE CAT, COMPARED TO THOSE IN THE RABBIT. A STUDY USING HORSERADISH PEROXIDASE AS A TRACER
F. WALBERG,N. KOTCHABHAKDI*and G. H. HODDEVIK Anatomical Institute, University of Oslo, Oslo (Norway) and Laboratory of Neurobiology and Department of Anatomy, Faculty of Science, Mahidol University, Bangkok (Thailand)
(Accepted May 25th, 1978)
SUMMARY The projections from the inferior olive to the flocculus and paraflocculus in the cat have been mapped by means of the method of retrograde axonal transport of horseradish peroxidase. The findings show that the afferents to the flocculus are derived from the dorsal cap, ventrolateral outgrowth, the principal olive (the caudal parts of the ventral and dorsal lamella) and from the rostral part of the medial accessory olive. The fibres to the paraflocculus come from the caudal part of the principal and from the rostral part of the medial accessory olive. Details in the projections are seen from Figs. 1 and 2. Concerning some points the findings are at variance with those made in the rabbit by Hoddevik and Brodal 9, and suggest that there are species differences hitherto not known.
INTRODUCTION In a preceding study from this laboratory 9, the projections from the inferior olive to the flocculus and paraflocculus in the rabbit were mapped by means of the method of tracing the retrograde axonal transport of horseradish peroxidase (HRP). This study was undertaken as part of an examination of the entire olivocerebellar projection. The rabbit was used in that study as the experimental animal, while cats were used for mapping of the olivary connections to the other cerebellar regions, the paramedian lobuleS, 8, the anterior lobe 4, the uvula a, the vermal lobules VI-VII110, and the ansiform lobule 11. During these studies some injections were accidentally placed in * On leavefrom the Laboratory of Neurobiologyand Department of Anatomy, Faculty of Science, Mahidol University,Bangkok,Thailand, under the FellowshipProgramof the NorwegianAgencyfor International Development(NORAD).
390 TABLE [ Weight
Conc.
Amount
(kg)
( % w/v)
(/d)
Survival time in days
50 50 25 50 50 50 50 50
0.3 0.2 0.2 0.2 0.2 0.3 0.2 0.1
2 2 1 2 2 1 2 2
Cat B.St.L. 763 2.5 762 2.5 782 2.7 755 2.6 754 2.4 748 3.2 757 2.5 Rabbit R63
Sigma VIP Sigma VIP Serva SigmaVIP Sigma VIP Sigma VIP SigmaVIP Serva
the flocculus and/or paraflocculus. A scrutiny of the distribution of labelled cells in these cases suggested that there might be some differences between the cat and the rabbit as concerns the olivary areas supplying the flocculus and paraflocculus. Additional experiments with intentional injections of HRP into these lobules were therefore performed. Although the species differences found are relatively modest, they seem important enough to merit attention, and will be briefly reported below. MATERIAL A N D METHODS
Altogether, 7 cats and one rabbit were used. The injections of HRP were made stereotactically under Nembutal or mebumal anaesthesia and the cat material further processed as described in earlier publications from this laboratory (see, e.g., ref. 6). The sections from the rabbit were treated for the blue reaction product as described by Mesulam 13. The distribution of HRP-labelled cells was entered in drawings of transverse sections of the olive and transferred to a diagram of the olive as imagined unfolded (taken from Brodall). Table I gives the weight, amount and concentration of the HRP fluid injected, and the survival time in the various cases. Below, some cases will be described in detail; others illustrating main points will be mentioned briefly. RESULTS
Flocculus
In cat B.St.L. 763 (Fig. 1A) the injection was made in the lateral part of the left flocculus. The injection was restricted to the molecular and granular layers and the adjacent part of the white matter. No staining was present in the dentate nucleus. In the inferior olive labelled cells were present only contralaterally. Heavily labelled cells are found in the dorsal cap (from level IV) including the adjoining part of the ventrolateral outgrowth and the ventral bend of the principal olive (to level X). Faintly labelled cells are present further rostrally in the ventral bend (levels X-XII) and in a patch in the central part of the ventral lamella (levels XI-XII). In addition, labelled
391
B.St.L. 7 6 3
pfl.
A
~
left B.St.L.762
right
r0str01 ~
MC EDI PR AC .OA LIVLE OINLC IVIP EAL dmcco v ~ - - I~ -dl" - - - - [ ....... _-- . . . . "-
B.St. L. 782 R
B ~B St.L.755 ~B.Sf.L.754
MC E.O DLIAIVLE PR AC OINLC IVIP EAL ccol vl ~ - - - ~ rostrol)O,l--~--~---[-2...... "~7-~z'~7"<-~'~dl-~ '........
~v [
Tl--4nuc]~-L
-_11-~r.o./-_~T%_---F-
Fig. 1. A-B: diagram of two cases (cats B.St.L. 763 and 782R) with injections in the lateral part of the left flocculus and medial part of the left dorsal paraflocculus, respectively. Insets show cases described in the text. Abbreviations: d.cap, dorsal cap; d.l., dorsal lamella of principal olive; dm.c.col., dorsomedial cell column; floc., flocculus; 1., lateral; m., medial; nucl. fl, nucleus fl; pfl.d., paraflocculus dorsalis; pfl.v., paraflocculus ventralis; v.l., ventral lamella of principal olive; v.l.o., ventrolateral outgrowth.
392 cells are found in the rostralmost portion of the medial accessory olive (levels XI-XI i I). The cells are concentrated in two patches, one lateral and one medial. The laterally located cells are faintly labelled, the medial ones heavily labelled. In another case the injection was placed in the two lateralmost folia of the left flocculus (cat B.St.L. 762, Fig. 1A, inset). The injection was also in this animal very localized and restricted to the granular and molecular layers and the adjacent white matter. No spreading of fluid to the dentate nucleus occurred. The distribution of the labelled cells in this animal was almost similar to that in the previous case. The only difference was that the labelled cells were fewer and located more caudally in the ventral bend of the principal olive (only up to level XI).
Dorsal paraflocculus In cat B.St.L. 782R* (Fig. 1B) a small amount of HRP had been deposited in the sixth folium (counted from the paramedian lobule). The brown colour was restricted to the molecular and granular layers of the folium and to the immediately adjacent part of the white matter. In the contralateral inferior olive labelled cells are present at levels X-XI in two patches in the principal olive, one in the ventral and one in the dorsal lamella (overlapping into the ventral bend), and in the rostral part of the medial accessory olive in a lateromedially extended band at levels XI-XII. The medialmost part of the medial accessory olive (adjacent to the dorsomedial cell column) is free of labelled cells. In another cat (B.St.L.755) in which the needle was placed more laterally in the dorsal paraflocculus (Fig. 1B, inset) and where the injection likewise was restricted to the cortex and adjacent part of the white matter, labelled cells were found in the principal as well as in the medial accessory olive, but somewhat differently distributed in the principal olive. Here there was a patch of weakly labelled cells only in the rostrolateral part of the ventral lamella (levels XIII-XV). All cells in the medial accessory olive were faintly labelled, those in the rostralmost part very faintly. In a third cat (B.St.L. 754) in which the injection had been placed in the lateralmost folia of the dorsal paraflocculus (Fig. 1B, inset) and restricted to the cortex and the immediately adjacent part of the white matter, labelled cells were present only rostrally in the ventral bend and the adjacent part of the dorsal lamella (levels X-XV). The rostrally located cells were heavily labelled.
Ventral paraflocculus In cat B.St.L. 748 (Fig. 2A) the needle was inserted through the posterior folium ofcrus I on the left side. The larger part of the H R P fluid was deposited in the fourth to sixth folium (counted from the paramedian lobule) of the ventral paraflocculus. An additional patch of staining was, however, also present in the posteriormost folium of crus I and the adjacent part of the anteriormost folium of crus II. No staining was present in the cerebellar nuclei. Labelled cells were present in the ventral bend of the * IncatB.St.L. 782theinjectionwasmadeintheright dorsalparaflocculus. InFig. 1B the injection site in the right hemisphere is transferred to the left side for the convenience of comparsion.
393
B. St.L. 748
left
right MEDIAL ACC.OLIVE
A
rostrol ) 3 1 - - ~ -
PF?INCIPAL OLIVE
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PRINCIPAL OLIVE
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Fig. 2A-B. Diagram of two cases(cats B.St.L. "148and 757) with injections in the left ventral parafloccu]us and media] part of the ]eft paraflocculus. A detailed description of the cases is given in the text. Abbreviations as in Fig. 1.
394 contralateral principal olive and in the immediately adjoining part of the ventrolateral outgrowth (levels VI-XII), and in the rostral part of the medial accessory olive, its lateral region from levels IX-XIV. A few scattered labelled cells are found in the dorsal cap.
Dorsal and ventral paraflocculus In cat B.St.L 757 (Fig. 2B) the injection had been placed medially in the paraftocculus so that some folia of the dorsal as well as the ventral part of this lobule were stained. In the principal olive labelled cells are found caudally in the ventral bend (level VIII), but extend rostrally within the adjoining parts of the ventral and dorsal lamella (levels IX-XIII). The labelled cells in the medial accessory olive are concentrated laterally at levels XI-XII. The best labelled cells are in all areas found at level XI. DISCUSSION From their findings, Hoddevik and Brodal 9 concluded that in the rabbit the olivary projection to the flocculus arises from the dorsal cap and the adjoining part of the ventrolateral outgrowth, and to a slight extent from the rostral pole of the medial accessory olive. Our findings concerning the distribution of cells from the dorsal cap, ventrolateral outgrowth and from the rostral part of the medial accessory olive to the flocculus in the cat agree with the observations by Hoddevik and Brodal ° in the rabbit. These authors found that the floccular afferent fibres originated from the same olivary areas. From a description of their cases it is, however, clear that in several of the animals (rabbits R 26, 17 and 12) there were also a number of faintly labelled cells in the lateral bend of the principal olive, mainly the ventral lamella, but the labelling of these cells was considered to be a result of spreading of the HRP fluid to the lateral part of the dentate nucleus and to the adjacent parafloccular cortex. Since in the cases described here (cats B.St.L 762 and 763) the HRP fluid has been restricted to the flocculus, it must be concluded that in the cat there is a projection to the flocculus from the main principal olive. As is seen from the description of the cases, this projection comes from the lateral bend of the principal olive and from the ventral lameUa. Although our findings are not decisive on this point, it appears furthermore that there is some kind of topical distribution from the principal olive to the flocculus. Thus, the lateralmost part of this lobe receives fibres only from the caudal portion of the ventral lamella (cat B.St.L. 762), whereas the area of the lobe immediately medial to this (cat B.St.L. 763) receives fibres from the ventral bend up to level XII. However, since we have no injections in the medialmost part of the flocculus, we cannot comment further upon this distribution. The cat appears to differ from the rabbit also in the projection from the olive to the paraftocculus. Whereas the dorsal paraflocculus in the rabbit was found to receive its olivary afferents only from the lateral part of the rostrat half of the medial accessory olive °, in the cat this part and the principal olive are regions of origin for the afferent
395 fibres to the dorsal paraflocculus. Although the situation is not so clear for the ventral paraflocculus, there appears to be a similar situation for this part of the cerebellar cortex. Thus, in cat B.St.L. 748, with an injection covering part of the ventral parafiocculus with an additional patch of HRP in the border zone between the left crus I and II (Fig. 2A), labelled cells are found in the medial accessory olive as well as in the principal olive. In a previous study we have shown that the border zone between crus I and II receives afferents only from two circumscribed regions in the ventral and dorsal lamella, but not from the ventral bend (cat B.St.L. 751, Fig. 2b in ref. 11). In cat B.St.L. 748 a few labelled cells are present lateralmost in the part of the ventral lamella sending its fibres to the border zone between crus I and II, but in this case the majority of the labelled cells lie in the ventral bend of the principal olive and in the rostral part of the medial accessory olive, areas which in cat B.St.L. 751 (Fig. 2b in ref. 11) showed no labelled cells. From a comparison of the various cases it is therefore permissible to conclude that the ventral as well as the dorsal paraflocculus in the cat receive afferents from the principal as well as from the medial accessory olive, and that the fibres to the ventral paraflocculus take their origin from more caudolateral areas of the principal olive than do those to the dorsal parafiocculus. This conclusion is furthermore supported by the findings in cat B.St.L. 757 with an injection including the medial part of the dorsal as well as the ventral paraflocculus (Fig. 2B). In this cat the labelled cells are found in the ventral bend and adjacent parts of the ventral and dorsal lamella, and in the rostral part of the medial accessory olive*. A comparison of our cases with injections in the dorsal paraflocculus indicates that, for this region as for the fiocculus, there is a certain topical arrangement in the projection from the principal olive. Thus, when the injections are located medially in the dorsal paraflocculus, the labelled cells in the principal olive are located somewhat more caudally than when the injection is placed more laterally (compare cases B.St.L. 782R and 754). Comparative anatomical investigations as well as experimental studies have demonstrated a precise pattern of narrow sagittally oriented zones in the cerebellar cortex (for references, see, e.g., ref. 9). As concerns the paraflocculus, the autoradiographic studies by Groenewegen and Voogd s and Kawamura and Hashikawa (personal communication) indicate that Voogd's zones C2 and D are present in both divisions of the parafiocculus. Although details are not clear, it appears that zone C2 is present ventrally in the dorsal and dorsally in the ventral paraflocculus, while the rest of the lobule belongs to zone D. Furthermore, the studies show that in the cat the input to zone D comes from the caudal part of the principal olive, its bend region, and that the input to zone Cz comes from the rostrolateral part of the medial accessory olive. These observations fit well with our findings that labelled cells are present in the principal as well as in the medial accessory olive when our injections cover zones C2 and D (cats B.St.L. 782, 755, 748 and 757), but that labelled cells are found only in the principal olive when the injection lies within zone D (cat B.St.L. 754). * In this connection it should be recalled that Brodal2in his studies with the modified Gudden method t describes a projection from the ventral bend to the entire paraflocculus in the cat.
396 As concerns the flocculus, the autoradiographic findings are less clear. One of Kawamura and Hashikawa's (personal communication) cases suggests that zone C2 is situated medially in the flocculus, an observation obviously supported by the observations made in one of the cases by Groenewegen and Voogd 8 (see their Fig. 18). This zone Ca appears to receive its fibres from the rostral part of the medial accessory olive, while the rest of the flocculus receives its afferents from the ventrolateral outgrowth and the dorsal cap. Our injections in the ftocculus are not limited to the abovementioned medial and lateral parts, and we can therefore not comment upon a possible zonation within the flocculus, the more so since the autoradiopgraphic studies have not confirmed our projection from the main principal olive to the flocculus in the cat. As is evident from the description of our cases, heavily labelled as well as weakly labelled cells are found in most of the animals. This is especially well seen in cat B.St.L. 763 (Fig. IA), which received an injection in the flocculus, and where weakly labelled cells are present in two patches in the principal olive, and laterally in the rostral part of the medial accessory olive. It is well known that a certain concentration of terminals is necessary for a visualization of cells when H R P is used as a tracer (for a discussion of this point, see especially ref. 12). The weak labelling of cells in the abovementioned regions of the olive after injections in the flocculus probably indicates that only (a few) collaterals from cells in the two patches in the principal, and the laterally located cells in the rostral part of the medial accessory olive, terminate in the flocculus. Our previous study 11 has shown that the cells in the areas of the olive here considered also project to crus I and II of the cerebellar hemisphere. Since after injection of HRP in crus I and II these cells are heavily labelled, it is probable that their main axons supply the cerebellar hemispheres. Hoddevik and Brodat 9 found labelled cells only in the medial accessory olive following injections of H R P in the dorsal paraflocculus in the rabbit. After injections in this part of the cerebellum in the cat, labelled cells are present also in the principal olive. The reason for this negativity in the rabbit could obviously be that in this animal only a few collaterals of axons of cells in the principal olive terminate in the dorsal paraflocculus, and that the main branch of the fibres terminates elsewhere in the cerebellum, for example in the ventral paraflocculus. Because of this we have included one rabbit in our material, in which an H R P injection was placed in the dorsal paraflocculus, and where the sections were processed for the blue reaction product described by Mesulam 13.. The visibility of this blue reaction product is better than that of the commonly used brown reaction product 7. This rabbit had, however, similar to the animals described by Hoddevik and Brodal 9, labelled cells only in the rostral part of the medial accessory, and not in the principal olive. This observation could indicate that there are virtually species differences in the terminal branching of climbing fibres within certain parts of the cerebellar cortex. An alternative explanation
* The HRP injection in rabbit R63 was restricted to the dorsal paraflocculusand almost simitar tothat in rabbit R t9 of Fig. 3D in the study of Hoddevik and BrodalL
397 rostrol
pfl,v. coudol
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Fig. 3. Diagram showing our findings of the olivocerebellar projection onto the flocculus and paraflocculus in the cat. Abbreviations as in Fig. 1.
could be that zone D in the dorsal paraflocculus is absent in the rabbit. Fig. 3 gives a summary of our findings in the cat. ACKNOWLEDGEMENTS
Supported by the Norwegian Agency for International Development (NORAD). REFERENCES 1 Brodal, A., Modification of Gudden method for study of cerebral localization, Arch. Neurol. Psychiat. (Chic.), 43 (1940) 46--58. 2 Brodal, A., Experimentelle Untersuchungenfiber die olivocerebellare Lokatisation, Z. ges. Neurol. Psychiat., 169 (1940) 1-153. 3 Brodal, A., The olivocerebellar projection in the cat as studied with the method ofretrograde axonal transport of horseradish peroxidase. II. The projection to the uvula, J. comp. NeuroL, 166 (1976) 417-426. 4 Brodal, A. and Walberg, F., 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. Neurot., 172 (1977) 85-108. 5 Brodal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. VL The projection onto longitudinalzones of the paramedian lobule, Jr. comp. Neurol., I76 (1977) 281-294. 6 Brodal, A., Walberg,F, and Hoddevik, G. H.,The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase, J. comp. Neurol., 164 (1975) 449-470.
398 7 Graham, R. C., Jr. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 8 Groenewegen, H. J. and Voogd, J., The parasagittal zonation within the olivocerebellar projections. II. Climbing fiber distribution in the intermediate and hemispheric parts of cat cerebellum, J. comp. Neurol., in press. 9 Hoddevik, G. H. and Brodal, A., The olivocerebellar projection studied with the method of retrograde axonal transport of horseradish peroxidase. V. The projections to the flocculonodular lobe and the paraflocculus in the rabbit, J. comp. Neurol., 176 (1977) 269-280. 10 Hoddevik, G. H., Brodal, A. and Walberg, F., The olivocerebellar projection in the cat studied with the method of retrograde axonal transport of horseradish peroxidase. Ill. The projection to the vermal visual area, J. comp. Neurol., 169 (1976) 155-170. 11 Kotchabhakdi, N., Walberg, F. and Brodal, A., 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, J. comp. Neurol., in press. 12 LaVail, J. H., Retrograde cell degeneration and retrograde transport techniques. In W. M. Cowan and M. Cuenod (Eds.), The use o f Axonal Transport for Studies o f Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 219-248. 13 Mesulam, M.-M., The blue reaction product in horseradish peroxidase. Neurohistochemistry: incubation parameters and visibility, J. Histochem. Cytochem., 24 (1976) 1273-1280.