- Email: [email protected]
An investigation of the cerebellar corticonuclear projections in the rat using an autoradiographic tracing method. II. Projections from the hemisphere
Brain Research, 14l (1978)235-249 ~:~') Elsevier/North-Holland Biomedical Press
235
AN INVESTIGATION OF THE CEREBELLAR C O R T I C O N U C L E A R PROJECTIONS IN THE RAT USING AN A U T O R A D I O G R A P H I C T R A C I N G M ETHOD. I1. PROJECTIONS FROM T H E H E M I S P H E R E
D. M. ARMSTRONG and R. F. SCH[LD Department o]" Physiology, The Medical School, University o[ Bristol, Bristol BS8 ITD (Great Britahl)
(Accepted May 25th, 1977)
SUMMARY Autoradiographic tracing was used to study the distribution within the intracerebellar nuclei of axon terminals from Purkinje cells in the cerebellar hemisphere of the rat. Only selected areas of hemisphere were studied but terminal labelling was found in most of nucleus lateralis and nucleus interpositus indicating extensive convergence with projections from uninvestigated cortex. The projections were entirely i psilateral. Injections into rostral crus II and nearby caudal curs I showed that these regions project largely to different portions of the nuclear complex (terminations from crus 11 predominantly dorsal to those from crus I). Each region projected to all three deep nuclei and there was an obvious mediolateral localisation in the connexions. There was a substantial projection from the medial part of curs II to the dorsolateral protuberance of nucleus fastigius (Fdlp) and, although the labelled projection to fastigius from medial crus I was small, a larger projection from crus 1 may in fact exist because medial portions of lobulus simplex and paramedian lobule also projected substantially to Fdlp. These observations suggest that, in the rat, the lateral part of fastigius derives its main input from a rostrocaudal strip of cortex in the medial part of the hemisphere (cf. Goodman et al.9). The ventral paraflocculus projected to the caudoventral portion of lateralis and the adjoining small-celled portion of interpositus posterior. Finally, major projections from two somatosensory areas of cerebellar cortex (the lateral part of the anterior lobe and the copula pyramidis plus paramedian lobule) converged onto the medial part of interpositus anterior. This convergence presumably has significance for cerebellar control of the rubrospinal tract.
236 INTRODUCTION
In the present paper we have used the autoradiographic tracing technique to study projections from hemispheral areas of the rat cerebellar cortex to the intracerebellar nuclei (excluding the vestibular complex). This report is therefore complementary to the preceding paper '~ in which vermal projections were studied. We have not attempted a complete 'map' of the hemispheral projections but instead we have focussed on specific points which we believe to be of special interest. Nevertheless, our observations do provide a partial picture of the topography of corticonuclear relations in the hemisphere. In the previous paper we confirmed that a degree of topographical locatisation exists (particularly in the mediolateral plane) in the connections of the vermis with nucleus fastigius. We have attempted here to determine whether a similar degree of mediolateral localisation could be demonstrated for the projections of Purkinje cells from the hemisphere. To this end, three injections of tritiated leucine were spaced tin different experiments) along the length of a single folium m each crus and the distributions of the resultant terminal labelling in the deep nuclei were compared. We have chosen to inject the most caudal folium of crus I and the most rostrat of crus II because the degree of overlap of the terminations from these two neighbouring region s might be expected to reveal the maximum extent to which the projections of the two crura overlap, and thus the minimum degree of rostrocaudal localisation existing in the hemisphere. In addition, two rejections were made into the paraflocculus for comparison with the crural results. Finally, one injection was made into the copula pyramidis and paramedian lobule, which have been shown in the rat to constitute an Important somatosensory receiving area (cf. ref. 7 and Adlam. Armstrong and Clarke, unpublished observations) and another into the lateral part of the anterior lobe which is known to be another important somatosensory area in mammals (e.g. ref. 11). METHODS
Ten male albino rats, ranging in weight from 300 to 400 g, were used for these experiments. Under sodium pentobarbit0ne anaesthesia (40-50 mg/kg, Lp.) a small (0.1-0.7 #1) volume of [3H]leucine solution (pre-concentrated to 7-25 #Ci/#l) was injected into a folium of the left cerebellar hemisphere. Full details of the isotope concentration and injection techniques and of all other experimental and recording procedures may be found in the Methods section of the preceding paperL Postoperative survival times ranged from 3.5 to 68 h. Five brains were sectioned frontally and 5 sagittally. Two series of 10 #m sections (usual interval 50 /~m) for each experiment were dipped in autoradiographic emulsion (Kodak N T B 2) and poststained with cresyl fast violet, one series having in addition been prestained with Luxol fast blue. Slides were examined using: a Zeiss Ultraphot microscope equipped for bright- and dark-field illumination and some sections were drawn with a microprojector. Background counts varied from t.7 to 4.9 grains/1000 # m 2 (mean 2.3/1000
237 #m2). The distribution of labelled material transported from each injection site in the cerebellar cortex to the intracerebellar nuclei was registered, as in the previous paper, on the numbered outline diagrams of the rat intracerebellar nuclei as published by Korneliussen 1°. Correlation of the sections with the diagrams proved to be straightforward. The deep nuclei and their subdivisions are fully labelled in Figs. 2 and 5 which respectively demonstrate the morphology of the nuclear region in frontal and in sagittal sections. RESULTS The projections revealed in these experiments were exclusively ipsilateral. A typical injection site in shown in Fig. la.
Mediolateral Iocalisation in the projections from the crura Crus L Three injections (cases 7, 1 and 6) were made at different distances from the cerebellar midline into the most caudal folium of crus I. The findings from cases 7 and 1, which were sectioned frontally, are presented in Fig. 2. In case 7 the injection
Fig. 1. a: dark-field photograph of a typical injection site (case 5) sectioned frontally. Calibration represents 1 ram. c and d: respectively bright- and dark-field micrographs of a sagittal section through the intracerebellar nuclei in case 17. Section corresponds to level K61, Fourth ventricle is at bottom right. For further explanation see text and Fig. 3. Calibration 500/tin. b: detail from the field shown in c and d. Note dense accumulation of terminal labelling in subnucleus Fdlp. Calibration 500/~m.
CN-7 LEFT
CN-7 "~"~
_
CAUDAL
0.'.5 m m
~~
CN-1 LEFT
ROSTRAL
59
53
38
o~
239 was placed well laterally in the folium and the injection site was confined to crus 1. Labelled fibres descended to form a compact rostroventrally directed stream alongside the lateral surface of nucleus lateralis. Fibres leaving this stream terminated mainly in nucleus lateralis: at caudal levels (corresponding to Korneliussen 18 38) heavy terminal labelling was present throughout the nucleus but further rostrally labelling was confined mainly to the ventral portion and its density progressively decreased. Modest to sparse terminations were also provided to the most lateral portions of both the anterior and posterior interpositus nuclei, the largest contribution (at levels K I8 and 23) being in the ventrally placed, small-celled portion of interpositus posterior
(Ips). In case 1 an injection into the same folium produced light labelling of some Purkinje cells in crus II, but labelled axons occurred only in crus I. The injection site was centred approximately one millimetre nearer the midline than in case 7 and the termination field was also displaced towards the midline. Thus between levels K28 and 43 there was labelling in nucleus lateralis, but it was mainly confined to the smallcelled portion (Ls) and the heaviest labelling was located in the lateral half of interpositus posterior (levels K13-23). Further rostrally (K38 59) a second termination field, substantial in size and moderate in density, was present in the lateral part of interpositus anterior. In addition, although the injection site quite definitely included no vermal cortex, its medial portion gave rise to a minor bundle of fibres which terminated in the caudolateral part of nucleus fastigius - - principally in the medial subnucleus (Fro), but also in the adjoining part of the dorsolateral subnucleus (Fdlp). In case 16 (Fig. 3; but see Fig. 5 for labelling conventions) an injection was placed between those of cases 7 and 1. This produced substantial transport from crus I and a very few crus II axons were labelled. They mingled with those from crus I in the white matter dorsocaudal to the deep nuclei, and could not be followed further. The cerebellum was sectioned sagittally and labelled terminals were present in sections corresponding to K levels 88-138. Labelled fibres streamed ventrally from the injection site, and the majority swung caudally to penetrate between the caudal pole of nucleus lateralis and the adjoining (lateral) border of nucleus interpositus. These fibres provided some terminations to the interpositus subnucleus termed dorsolateral h u m p
Fig. 2. Results from frontally sectioned cases 7 and I. Postoperative survivals 5 and 3.5 h, respectively. Semi-diagrammatic frontal sections through the intracerebellar nuclei are numbered according to Korneliussen l°. Distance of each section from the caudal pole of the nuclear complex is obtained by multiplying the K number by 20/~m. Fcm, Fm and Fdlp respectively denote caudomedial, medial and dorsolateral protuberance subnuc[ei of fastigius; la, Ip denote anterior and posterior portions of interpositus (note that the junction between la and Ip cannot be precisely identified in frontal sections); interrupted line divides Ip into dorsal, large-celled (Ipl) and ventral, small-celled (Ips) subdivisions; dlh and dmc are respectively the dorsolateral hump and the dorsomedial cap of 1, LI and Ls respectively denote large and small-celled subdivisions of lateralis. Vestibular complex not shown. Stippling indicates distribution and density of terminal labelling within the intracerebellar nuclei. Arrows show direction and span of labelled fibres entering the nuclear complex. Central diagrams indicate distributions of all Purkinje cells in sections through the injection sites. Distance between sections shown in ram. Regions with labelled Purkinje cells are indicated by dotted lines. Lobules labelled after Larsell.
240
~
CN-16
MEDIAL
rusZ
MEDIAL
cruslI
°'
cr~I
O,40mm
CN-17
c ~~ s i [
~ qmm
040mm~ [')~'-~
0.40 m m ~ LATERAL
MEDIAL L E F T
LAI"ERAL
MEDIA,
LEFT
05mm 77
8
77
104
104
113
113
117
@
38 ~
117
t
~ 127
127
138
~
148 ~
LATERAL
Fig. 3. Results from sagittally sectioned cases 16 and 17. Survivals 4 and 68 h respectively. Semidiagrammatic sagittal sections through whole nuclear complex (case 17) and lateral half of complex (case 16) are numbered according to Korneliussen so that distance of each section from medial pole of nuclear complex is obtained by multiplying the K number by 20 #m. (For identification of subnuclei as seen in this plane of section see Fig. 5.) Other conventions as in Fig. 2. The upper three diagrams illustrate the injection site. The distributions of unlabelled (heavy filled lines) and labelled (dotted lines) Purkinje cells are shown in sagittal sections separated by the distances shown.
b~
ce
~ . / , ) " -
~____ CAUDAL
•~
- "~
3
CN-5
i 0.5ram
k,J
LEFT
ROSTRAL
4
CAUDAl.
.
CN-3
CN-5
lmm
CAUDAL
~
'~mm 0,5
CN-3
ROSTRAL
~o gp
LEFT
t,gl
242 by G o o d m a n et al. 9 (dlh: see Fig. 3) but the majority were divided approximately equally between the medial half of lateralis and the lateral portion of lps. A small number of fibres descended from the source without swinging caudally and these terminated in the lateral half of interpositus anterior after entering through the rostrodorsal surface. The term,nation pattern in this case was, therefore, in the main. a hybrid of the distributions found for cases 7 and 1 including the two main fields found for I and part of the field for case 7. This finding seems entirely compatible with the location of the injection site. which partially overlapped the site in each of the other two cases. Collectively, the three cases establish clearly thai there ~s a distinct mediolateral Iocalisation in the projections from the injected folium. Crus H. In three cases (5, 3 and 17) injections were made into the most rostral folium of crus !1. Cases 5 and 3 were sectioned frontally and the results are displayed in Fig. 4. In case 5, the injection was made into the most lateral exposed portion of the folium, and the injection site is shown in Fig. la. In addition to the abundant labelled fibres from crus II, a minute number from crus I were labelled. They joined the main contingent in the deep white matter and could not be followed further. The labelled fibres passed medially from the source to form a rostrally directed capsule (alongside nucleus tateralis) which became progressively depleted as axons entered k through its dorsolateral surface. Terminal labelling was confined to the dorsal half of the largecelled portion of the nucleus iLl) and, though sparse at caudal levels, it was heavy in the rostral half of this subnucleus. Heavy labelling was also present in the interpositus subnucleus dlh, especially in its lateral half adjoining nucleus lateralis, in case 3. the bulk of the transport originated from Purkinje cells m c r u s !1 and in the adjoining paramedian lobule. The labelled axons from these two regions came together in the deep white matter and thereafter could not be separated. They were joined by a small contingent from crus I, which likewise could not be traced separately beyond the point of junction. The pattern of terminal labelling was somewhat unexpected because. although there was sparse termination in the mid-portion of interpositus posterior, the large majority of the terminations were in nucleus fastigius. Most were present in Fdlp, which was labelled throughout its extent, but the distribution field atso included modest ventral extensions into the adjoining portion of Fm. In case 17, which was sectioned sagittally, the injection was placed close to that of case 3, but slightly further laterally. Transport originated only from the rostral portion of crus II and, as in case 3. the main termination field included the whole of Fdlp together with some extension into adjacent parts of Fro. Photomicrographs demonstrating the projections to Fdlp are shown in Fig. t. Fig. Ic is a low power bright-field micrograph of a section corresponding to level K61, and showing Fdlp together with the anterior and posterior subdivisions of interpositus. Fig. t d is a darkfield micrograph of the same section m which an approximately spherical accumulation of terminal labelling can be seen to fill the whole extent of Fdlp. This termination field is shown again at higher magnification in Fig. lb. In case 17 there were also smaller projections to more lateral parts of the nuclear complex: the lateral part of the injection site gave rise to a bundle of fibres which coursed rostroventrally toterminate in the lateral part of lp and the adjoining area of lateralis and minor contributions were also made to Ia and to the dorsolateral hump (dlh).
243 Thus in crus II, as in crus I, the three cases demonstrate unequivocally the existence of a mediolateral localisation in the projections from a single folium.
Comparison of the projection fields for crus I and crus H Each of the two folia studied provided projections to both large- and smallcelled portions of lateralis, to both the anterior and posterior subdivisions of interpositus and also to the lateral part of fastigius. However, despite the widespread nature of the projections revealed, comparison of the cases reveals scarcely any overlap between the terminations from the two folia. In all three deep nuclei, the termination fields for crus II were centred dorsally to those from crus I. Pro/ections from the paraflocculus Injections were made into the paraflocculus in two cases (14 and 19) and both were sectioned sagittally. Reconstruction revealed that in each brain, although the ventral limb of the paraflocculus was heavily labelled, the labelling did not extend into the dorsal limb, so that a complete account of the parafloccular projections cannot be given. In these cases both the injection site and the termination fields were almost identical (see Fig. 5). From the injection site, fibres ran dorsomedially to form a capsule in the white matter just outside the surface of nucleus lateralis. The capsule extended medially beyond the junction with nucleus interpositus, but during its course fibres were continually given off which penetrated the caudal surface oflateralis before terminating in the caudoventral part of the nucleus, in both the large- and small-celled portions (L1 and Ls). The termination in interpositus was dense but confined almost entirely to the lateral two-thirds of the subnucleus which Korneliussen 1° identifies as lps. Injections into 'somatosensory' areas in case 2 (Fig. 6) the injection pipette was inserted into lobulus VI and advanced well beneath the cerebellar surface in order to deliver the leucine into the lateral portion of lobules IV and V in the anterior lobe. The injection site failed to reach the cerebellar midline and, by contrast with cases in which injections were placed near the midline of the anterior lobe, there was no terminal labelling whatsoever in fastigius. There was, however, heavy terminal labelling in the vestibular complex, and large numbers of labelled fibres passed through the raphe which separates the fastigial and interpositus nuclei. A smaller number of corticovestibular fibres passed through the medial portion of interpositus. In the intracerebellar nuclei proper, heavy accumulations of label were present in the medial one-third of la throughout its rostrocaudal extent and labelling was also present in the medial part of lp though fading out at level KI3. In case 18 (sectioned sagittally) an injection made into the copula pyramidis resulted in an injection site which included parts of both the copula and the paramedian lobule (see Fig. 6). Large numbers of labelled fibres were intercepted in longitudinal section as they arched rostrally from the injection site to the intracerebellar nuclei. The great bulk of the terminal labelling was in Ia, but there was also
244 MEDIAL
~
CN-14 LEFT
Fcm
CN-19 LEFT
4EDIAL
Ia~<~Ipl
FdIp/~T~Fcm~.m__.~28 m ~
~/~,LI DIh N ~
~,'~Ips
117
Fdlp
0 Ie ~--~{~ IDI
~
~
88
LI_~
LAT ~ ERA1 ~ ~ 4 8~48
~5mm -ATERAL
Fig. 5. Results in sagittal cases 14 and 19. Survivals 5 and 22 h, respectively. Diagrams on the left identify the left intracerebellar nuclei as seen in sagittal sections. The vestibular complex is not ShOwn. Abbreviations and other conventions as in Fig. 2.
245
.......
C N-18
.......
"" 045 m m _ ~
C N-2
oE~Y~_~_~,,
~
v
LATERAL
V
lmm
LEFT
28 ~
104 @
38 ~
113 @
°'~'~'~
t
CAUOAL
LEFT
117
61 68
148
33
ROSTRAL 59
Fig. 6. Results obtained in sagittal case 18 and frontal case 2. Survivals 24 and 29 h. respectively. Conventions as in Figs. 2 and 5. Arrows descending beyond the intracerebellar nuclei denote corticovestibular axons. Interrupted vertical line indicates that lateral part of right hemispheral cortex was trimmed off before sectioning. a small amount in Ip (levels K88 and 104) and in Fdlp (levels K48 and 61). There was no labelling in the vestibular complex. Thus, in cases 18 and 2, in which widely separate portions of the cortical sheet were injected, there was very marked convergence (into interpositus anterior) between the major corticonuclear projections. DISCUSSION The present series of experiments in which 10 injections of the tracer were made at different locations in the hemisphere revealed only ipsilateral corticonuclear
246 projections (cf. refs. 12 and 13). Considered together with the data from the previous paper z in which vermal projections were studied, this finding suggests very strongly that the corticonuclear connexion in the rat is entirely uncrossed. Although the hemispheral injections were collectively far from involving the whole of the cerebellar hemisphere, they nevertheless led to heavy terminal labelling throughout most of the lateral and interpositus nuclei. Clearly, therefore, considerable convergence must occur between the projections demonstrated here and those from cortical areas which were not labelled. It should be noted there was very little overlap between the present terminal fields and those which were found in the preceding paper after injections into the vermis. Thus, the one part of fastigius which was heavily labelled in the present series of experiments (i.e. Fdlp) is precisely that which was scarcely labelled in the vermal series. The medial border of interpositus was the only area involved from both vermal and hemispheral injection sites. However, this paucity of overlap does not necessarily imply a sharp discontinuity in the corticonuclear projection. Thus, only in two of the hemispheral cases (2 and 18) were the injection sites close to the vermis, and in the preceding study the majority of the injection sites were centred near the cerebellar midline. The facts that cases 2 and 18 led to heavy labelling of the most medial part of la, that some hemispheral injections led to labelling of lateral F, and that some vermal cases produced modest labelling in lp together suggest that injections near the paravermal vessels would almost certainly demonstrate a greater degree of overlap between the vermal and hemispheral projections than is shown by our existing cases. Nevertheless, it is obvious that, in general, the hemispheral termination fields are more laterally placed in the nuclear complex. Furthermore. within the crura as within the vermls, there is undoubtedly a mediolateral localisation of projection as previously claimed on the basis of degeneration experiments by Goodman et al. 9. It is similar in degree to that existing within the vermis, since both the injection sites and the termination fields for individual experiments were comparable in size to those in the vermal experiments. In a recent preliminary report by Courville and Faraco-Cantin 5, corticonuclear connexions in the cat have been traced in the retrograde direction using horseradish peroxidase. This study has indicated that in crus II the superficial and deep parts of the folia may project to different parts of the nuclear complex. Our injection sites have been too extensive, relative to the small folia of the rat cerebellum, to allow any comparison with this finding. However, application of the autoradiographic technique to larger species should provide an alternative method of investigating this interesting phenomenon. As regards any rostrocaudal localisation in the crural projections, our results do not provide a complete picture because so much cortex remained unexplored. Nevertheless, we were surprised at the extent to which the nuclear territories labelled after injections into the caudal part of crus I were complementary to those labelled from the rostral portion of crus I1. This finding seems to conflict directly with the conclusion reached in their degeneration study by Goodman et al.~. These workers concluded that the hemisphere in the rat could be divided into two rostrocaudal
247 'functional-zones' (lateral and intermediate) running across both crura and that small lesions placed anywhere within one zone led to very similar distributions of terminal degeneration within the nuclear complex. The paucity of overlap in our experiments suggests that each crus may have its own distinct projection field within the nuclei. However, this can only be proved by a more comprehensive study of the crural projections. It should be noted that if such private termination fields do exist, some difference in rostrocaudal organization is implied between the hemispheral and the vermal projections, since in the latter the terminations from different lobules overlapped heavily 2. The extent of the projection from crus 11 to Fdlp (cases 3 and 17) came as a surprise, but in fact it confirms findings made by Goodman et al., who indeed suggest that Fdlp should be included with the neocerebellum (hemisphere) rather than the paleocerebellum. These authors also suggest that the hemispheral projection to Fdlp derives from the whole rostrocaudal extent of the paravermal or 'intermediate' cortex and that the same termination field is found irrespective of the particular location of the lesion within this zone. Our series provides only limited evidence regarding this point, but in case 3 the whole subnucleus was densely labelled except for the rostral pole where terminations were fewer. Again, in case 17 heavy labelling was present throughout, excepting only the medial border which was lightly labelled. These findings are compatible with the views of Goodman et al., but it should be noted that, in case 4 of the preceding paper, labelling of the left lobulus simplex led to dense terminal labelling in only the rostral half of Fdlp. It seems, therefore, that at least one area in the hemisphere projects to only a part of Fdlp. Furthermore, other injections described in the preceding paper labelled small portions of the subnucleus and in case 1 of the present series a (small) projection to fastigius was directed to the region where Fm and Fdlp merge. Our finding that the (ventral) paraflocculus projects to the ventrocaudal part of nucleus lateralis is in good agreement with the results of Goodman et al. and with results obtained by Dow 6 using the Marchi method. Like Dow, but unlike Goodman et al., we found, in addition, that the projection continued medially to provide abundant terminations in lps. From comparison with Korneliussen we are convinced that we have correctly identified this subnucleus, but it should be noted that we and Dow used sagittal sections, whilst Goodman et al. employed frontal sections. The difference in results might therefore be a difference of interpretation regarding the relevant nuclear boundaries. If the discrepancy is to be resolved by any future study, the use of horizontal sections would perhaps be advisable, since these are said to provide the best display of boundaries TM. In the cat, corticonuclear projections originating from crus II have been studied in great detail by Brodal and Courville 3 using the Nauta technique. It was concluded, as in the present study, that this lobule projects to more than one deep nucleus. Two separate termination zones were distinguished, one in nucleus lateralis and continuing into the neighbouring interpositus anterior, the other in interpositus posterior. Even very small lesions produced degeneration in both zones. The study also considered the possibility of a mediolateral localisation within the projection from crus lI, and it was
248 concluded that, although lateral areas projected somewhat more laterally and ventrally in each projection zone, the terminal fields for medial and lateral parts of the crus nevertheless overlapped heavily so that there was no marked localisation. This conclusion is apparently at odds with that reached here, but it must be remembered that the terms lateral and medial were applied in the cat study to two separate groups of folia, whilst we have applied them to different portions of the same folia. The major difference between that study and our own was the absence in the cat of any projection from crus II to nucleus fastigius. Courville et al. 4 have also investigated projections from the paramedian lobule in the cat. This very detailed study concluded that the lobule projected to both lateralis and interpositus. We have found no projection to lateralis, but the lateral part of t he lobule was labelled by neither of our injections (3 and 18) which involved this lobule. In one of these cases (case 3) fibres from the lobule travelled together with others from the medial part of crus I1 to terminate mainly in Fdlp, and few terminals were provided to interpositus. In case 18, however, fibres from the paramedian lobule and copula pyramidis terminated mainly in interpositus anterior. Unfortunately, one case was sectioned sagittally and the other frontally so that it was difficult to compare accurately the distributions of labelled Purkinje cells in the lobule. However, it seems likely that one part of the lobule projects to Fdlp and an adjoining portion to nucleus interpositus. If the lateral part of the lobule projects to nucleus lateralis as claimed for the rat by G o o d m a n et al (and for the cat by Courville et al. 4) then the paramedian lobule would display the same kind of mediolateral localisation as the portions of the two crura we have studied. However, further experiments are needed to verify this speculation. It is worth noting that Eager 8 has reported projections to all three ipsilateral nuclei in the cat. Finally, the injection site involving the lateral portion of vermal lobules 1V and V (case 2) projected mainly to the medial part of interpositus anterior. Studies in the cat have reported a similar connexion~,S,lZ, ~4. it is noteworthy that the injection site involving the copula pyramidis and paramedian lobule (case 18) also projected mainly to the same portion of interpositus. In the cat, both the pars intermedia of the anterior lobe and the paramedian lobute are well documented as the main somatosensory receiving areas of the cerebellar cortex1,11 and in the rat also lobulus V (the Culmen) and the region of the copula pyramidis and paramedian lobule are loci from which short latency evoked potentials are readily recorded following stimulation of afferems from the limbs (cf. ref. 7 and Adlam, Armstrong and Clarke, unpublished observations). It is likely therefore that there is extensive convergence within interposltus anterior between outputs from the two main somatosensory areas, even though these are widely separated in the rostrocaudal plane. Since interpositus anterior projects mainly to the magnocellular red nucleus, which in turn gives origin to the rubrospinal tract, this convergence is presumably of considerable functional significance in relation to cerebellar control of the limbs.
249 A C K N O W L E D G E M ENTS W e t h a n k Mrs. S. R u c k l i d g e for e x p e r t t e c h n i c a l assistance, Mrs. A. B a c o n and B. C o l f e r for the p h o t o g r a p h y , Mrs. B. R i d d i c k for the t y p i n g a n d Dr. R. J. H a r v e y for c o n s t r u c t i v e c o m m e n t o d the m a n u s c r i p t . T h i s r e s e a r c h was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h C o u n c i l o f G r e a t Britain.
REFERENCES I Armstrong, D. M., Functional significance of connections of the inferior olive, Physiol. Rev., 54 (1974) 358-417. 2 Armstrong, D. M. and Schild, R. F., An investigation of the cerebellar cortico-nuclear projections in the rat using an autoradiographic tracing method. I. Projections from the vermis, Brain Research, 14l (1978) 1-19. 3 Brodal, A. and Courville, J., Cerebellar corticonuclear projection in the cat. Crus II. An experimental study with silver methods, Brain Research, 50 (1973) 1 23. 4 Courville, J., Diakiw, N. and Brodal, A., Cerebellar corticonuclear projections in the cat. The paramedian lobule. An experimental study with silver methods, Brain Research, 50 (1973) 24-45. 5 Courville, J. and Faraco-Cantin, F., Cerebellar corticonuclear projection demonstrated by the horseradish peroxidase method, Soc. Neurosci. Mtg., Toronto, 1976, Vol. H, p. 108 (Abstract no. 153). 6 Dow, R. S., The fiber connections of the posterior parts of the cerebellum in the rat and cat, J. comp. Neurol., 63 (1936) 527 548. 7 Dow, R. S. and Anderson, R., Cerebellar action potentials in response to stimulation of proprioceptors and exteroceptors in the rat, J. Neurophysiol., 5 (1942) 363 371. 8 Eager, R. P., Efferent cortico-nuclear pathways in the cerebellum of the cat, J. comp. Neurol., 120 (1963) 81 104. 9 Goodman, D. C., Hallett, R. E. and Welch, R. B., Patterns of localization in the cerebellar cortico-nuclear projections of the albino rat, J. comp. Neurol., 121 (1963) 51-67. 10 Korneliussen, H. K., On the morphology and subdivision of the cerebellar nuclei of the rat, J. Hirnforsch., 10 (1968) 109-122. 1l Oscarsson, O., Functional organization of spinocerebellar paths. In H. Antrum et al. (Eds.), Handbook of Sensory Physiology, Vol. 11, Somatosensory Systems, Springer, Berlin, 1973, pp. 339-380. 12 Rossum van, J., Corticonuclear and Corticovestibular Projections {~f the Cerebellum, Van Gorcum, Assen, 1969. 13 Voogd, J., The Cerebellum of the Cat, Van Gorcum, Assen, 1964. 14 Walberg, F. and Jansen, J., Cerebellar corticonuclear projection studied experimentally with silver impregnation methods, J. HirnJbrsch., 6 (1964) 338-354.
235
AN INVESTIGATION OF THE CEREBELLAR C O R T I C O N U C L E A R PROJECTIONS IN THE RAT USING AN A U T O R A D I O G R A P H I C T R A C I N G M ETHOD. I1. PROJECTIONS FROM T H E H E M I S P H E R E
D. M. ARMSTRONG and R. F. SCH[LD Department o]" Physiology, The Medical School, University o[ Bristol, Bristol BS8 ITD (Great Britahl)
(Accepted May 25th, 1977)
SUMMARY Autoradiographic tracing was used to study the distribution within the intracerebellar nuclei of axon terminals from Purkinje cells in the cerebellar hemisphere of the rat. Only selected areas of hemisphere were studied but terminal labelling was found in most of nucleus lateralis and nucleus interpositus indicating extensive convergence with projections from uninvestigated cortex. The projections were entirely i psilateral. Injections into rostral crus II and nearby caudal curs I showed that these regions project largely to different portions of the nuclear complex (terminations from crus 11 predominantly dorsal to those from crus I). Each region projected to all three deep nuclei and there was an obvious mediolateral localisation in the connexions. There was a substantial projection from the medial part of curs II to the dorsolateral protuberance of nucleus fastigius (Fdlp) and, although the labelled projection to fastigius from medial crus I was small, a larger projection from crus 1 may in fact exist because medial portions of lobulus simplex and paramedian lobule also projected substantially to Fdlp. These observations suggest that, in the rat, the lateral part of fastigius derives its main input from a rostrocaudal strip of cortex in the medial part of the hemisphere (cf. Goodman et al.9). The ventral paraflocculus projected to the caudoventral portion of lateralis and the adjoining small-celled portion of interpositus posterior. Finally, major projections from two somatosensory areas of cerebellar cortex (the lateral part of the anterior lobe and the copula pyramidis plus paramedian lobule) converged onto the medial part of interpositus anterior. This convergence presumably has significance for cerebellar control of the rubrospinal tract.
236 INTRODUCTION
In the present paper we have used the autoradiographic tracing technique to study projections from hemispheral areas of the rat cerebellar cortex to the intracerebellar nuclei (excluding the vestibular complex). This report is therefore complementary to the preceding paper '~ in which vermal projections were studied. We have not attempted a complete 'map' of the hemispheral projections but instead we have focussed on specific points which we believe to be of special interest. Nevertheless, our observations do provide a partial picture of the topography of corticonuclear relations in the hemisphere. In the previous paper we confirmed that a degree of topographical locatisation exists (particularly in the mediolateral plane) in the connections of the vermis with nucleus fastigius. We have attempted here to determine whether a similar degree of mediolateral localisation could be demonstrated for the projections of Purkinje cells from the hemisphere. To this end, three injections of tritiated leucine were spaced tin different experiments) along the length of a single folium m each crus and the distributions of the resultant terminal labelling in the deep nuclei were compared. We have chosen to inject the most caudal folium of crus I and the most rostrat of crus II because the degree of overlap of the terminations from these two neighbouring region s might be expected to reveal the maximum extent to which the projections of the two crura overlap, and thus the minimum degree of rostrocaudal localisation existing in the hemisphere. In addition, two rejections were made into the paraflocculus for comparison with the crural results. Finally, one injection was made into the copula pyramidis and paramedian lobule, which have been shown in the rat to constitute an Important somatosensory receiving area (cf. ref. 7 and Adlam. Armstrong and Clarke, unpublished observations) and another into the lateral part of the anterior lobe which is known to be another important somatosensory area in mammals (e.g. ref. 11). METHODS
Ten male albino rats, ranging in weight from 300 to 400 g, were used for these experiments. Under sodium pentobarbit0ne anaesthesia (40-50 mg/kg, Lp.) a small (0.1-0.7 #1) volume of [3H]leucine solution (pre-concentrated to 7-25 #Ci/#l) was injected into a folium of the left cerebellar hemisphere. Full details of the isotope concentration and injection techniques and of all other experimental and recording procedures may be found in the Methods section of the preceding paperL Postoperative survival times ranged from 3.5 to 68 h. Five brains were sectioned frontally and 5 sagittally. Two series of 10 #m sections (usual interval 50 /~m) for each experiment were dipped in autoradiographic emulsion (Kodak N T B 2) and poststained with cresyl fast violet, one series having in addition been prestained with Luxol fast blue. Slides were examined using: a Zeiss Ultraphot microscope equipped for bright- and dark-field illumination and some sections were drawn with a microprojector. Background counts varied from t.7 to 4.9 grains/1000 # m 2 (mean 2.3/1000
237 #m2). The distribution of labelled material transported from each injection site in the cerebellar cortex to the intracerebellar nuclei was registered, as in the previous paper, on the numbered outline diagrams of the rat intracerebellar nuclei as published by Korneliussen 1°. Correlation of the sections with the diagrams proved to be straightforward. The deep nuclei and their subdivisions are fully labelled in Figs. 2 and 5 which respectively demonstrate the morphology of the nuclear region in frontal and in sagittal sections. RESULTS The projections revealed in these experiments were exclusively ipsilateral. A typical injection site in shown in Fig. la.
Mediolateral Iocalisation in the projections from the crura Crus L Three injections (cases 7, 1 and 6) were made at different distances from the cerebellar midline into the most caudal folium of crus I. The findings from cases 7 and 1, which were sectioned frontally, are presented in Fig. 2. In case 7 the injection
Fig. 1. a: dark-field photograph of a typical injection site (case 5) sectioned frontally. Calibration represents 1 ram. c and d: respectively bright- and dark-field micrographs of a sagittal section through the intracerebellar nuclei in case 17. Section corresponds to level K61, Fourth ventricle is at bottom right. For further explanation see text and Fig. 3. Calibration 500/tin. b: detail from the field shown in c and d. Note dense accumulation of terminal labelling in subnucleus Fdlp. Calibration 500/~m.
CN-7 LEFT
CN-7 "~"~
_
CAUDAL
0.'.5 m m
~~
CN-1 LEFT
ROSTRAL
59
53
38
o~
239 was placed well laterally in the folium and the injection site was confined to crus 1. Labelled fibres descended to form a compact rostroventrally directed stream alongside the lateral surface of nucleus lateralis. Fibres leaving this stream terminated mainly in nucleus lateralis: at caudal levels (corresponding to Korneliussen 18 38) heavy terminal labelling was present throughout the nucleus but further rostrally labelling was confined mainly to the ventral portion and its density progressively decreased. Modest to sparse terminations were also provided to the most lateral portions of both the anterior and posterior interpositus nuclei, the largest contribution (at levels K I8 and 23) being in the ventrally placed, small-celled portion of interpositus posterior
(Ips). In case 1 an injection into the same folium produced light labelling of some Purkinje cells in crus II, but labelled axons occurred only in crus I. The injection site was centred approximately one millimetre nearer the midline than in case 7 and the termination field was also displaced towards the midline. Thus between levels K28 and 43 there was labelling in nucleus lateralis, but it was mainly confined to the smallcelled portion (Ls) and the heaviest labelling was located in the lateral half of interpositus posterior (levels K13-23). Further rostrally (K38 59) a second termination field, substantial in size and moderate in density, was present in the lateral part of interpositus anterior. In addition, although the injection site quite definitely included no vermal cortex, its medial portion gave rise to a minor bundle of fibres which terminated in the caudolateral part of nucleus fastigius - - principally in the medial subnucleus (Fro), but also in the adjoining part of the dorsolateral subnucleus (Fdlp). In case 16 (Fig. 3; but see Fig. 5 for labelling conventions) an injection was placed between those of cases 7 and 1. This produced substantial transport from crus I and a very few crus II axons were labelled. They mingled with those from crus I in the white matter dorsocaudal to the deep nuclei, and could not be followed further. The cerebellum was sectioned sagittally and labelled terminals were present in sections corresponding to K levels 88-138. Labelled fibres streamed ventrally from the injection site, and the majority swung caudally to penetrate between the caudal pole of nucleus lateralis and the adjoining (lateral) border of nucleus interpositus. These fibres provided some terminations to the interpositus subnucleus termed dorsolateral h u m p
Fig. 2. Results from frontally sectioned cases 7 and I. Postoperative survivals 5 and 3.5 h, respectively. Semi-diagrammatic frontal sections through the intracerebellar nuclei are numbered according to Korneliussen l°. Distance of each section from the caudal pole of the nuclear complex is obtained by multiplying the K number by 20/~m. Fcm, Fm and Fdlp respectively denote caudomedial, medial and dorsolateral protuberance subnuc[ei of fastigius; la, Ip denote anterior and posterior portions of interpositus (note that the junction between la and Ip cannot be precisely identified in frontal sections); interrupted line divides Ip into dorsal, large-celled (Ipl) and ventral, small-celled (Ips) subdivisions; dlh and dmc are respectively the dorsolateral hump and the dorsomedial cap of 1, LI and Ls respectively denote large and small-celled subdivisions of lateralis. Vestibular complex not shown. Stippling indicates distribution and density of terminal labelling within the intracerebellar nuclei. Arrows show direction and span of labelled fibres entering the nuclear complex. Central diagrams indicate distributions of all Purkinje cells in sections through the injection sites. Distance between sections shown in ram. Regions with labelled Purkinje cells are indicated by dotted lines. Lobules labelled after Larsell.
240
~
CN-16
MEDIAL
rusZ
MEDIAL
cruslI
°'
cr~I
O,40mm
CN-17
c ~~ s i [
~ qmm
040mm~ [')~'-~
0.40 m m ~ LATERAL
MEDIAL L E F T
LAI"ERAL
MEDIA,
LEFT
05mm 77
8
77
104
104
113
113
117
@
38 ~
117
t
~ 127
127
138
~
148 ~
LATERAL
Fig. 3. Results from sagittally sectioned cases 16 and 17. Survivals 4 and 68 h respectively. Semidiagrammatic sagittal sections through whole nuclear complex (case 17) and lateral half of complex (case 16) are numbered according to Korneliussen so that distance of each section from medial pole of nuclear complex is obtained by multiplying the K number by 20 #m. (For identification of subnuclei as seen in this plane of section see Fig. 5.) Other conventions as in Fig. 2. The upper three diagrams illustrate the injection site. The distributions of unlabelled (heavy filled lines) and labelled (dotted lines) Purkinje cells are shown in sagittal sections separated by the distances shown.
b~
ce
~ . / , ) " -
~____ CAUDAL
•~
- "~
3
CN-5
i 0.5ram
k,J
LEFT
ROSTRAL
4
CAUDAl.
.
CN-3
CN-5
lmm
CAUDAL
~
'~mm 0,5
CN-3
ROSTRAL
~o gp
LEFT
t,gl
242 by G o o d m a n et al. 9 (dlh: see Fig. 3) but the majority were divided approximately equally between the medial half of lateralis and the lateral portion of lps. A small number of fibres descended from the source without swinging caudally and these terminated in the lateral half of interpositus anterior after entering through the rostrodorsal surface. The term,nation pattern in this case was, therefore, in the main. a hybrid of the distributions found for cases 7 and 1 including the two main fields found for I and part of the field for case 7. This finding seems entirely compatible with the location of the injection site. which partially overlapped the site in each of the other two cases. Collectively, the three cases establish clearly thai there ~s a distinct mediolateral Iocalisation in the projections from the injected folium. Crus H. In three cases (5, 3 and 17) injections were made into the most rostral folium of crus !1. Cases 5 and 3 were sectioned frontally and the results are displayed in Fig. 4. In case 5, the injection was made into the most lateral exposed portion of the folium, and the injection site is shown in Fig. la. In addition to the abundant labelled fibres from crus II, a minute number from crus I were labelled. They joined the main contingent in the deep white matter and could not be followed further. The labelled fibres passed medially from the source to form a rostrally directed capsule (alongside nucleus tateralis) which became progressively depleted as axons entered k through its dorsolateral surface. Terminal labelling was confined to the dorsal half of the largecelled portion of the nucleus iLl) and, though sparse at caudal levels, it was heavy in the rostral half of this subnucleus. Heavy labelling was also present in the interpositus subnucleus dlh, especially in its lateral half adjoining nucleus lateralis, in case 3. the bulk of the transport originated from Purkinje cells m c r u s !1 and in the adjoining paramedian lobule. The labelled axons from these two regions came together in the deep white matter and thereafter could not be separated. They were joined by a small contingent from crus I, which likewise could not be traced separately beyond the point of junction. The pattern of terminal labelling was somewhat unexpected because. although there was sparse termination in the mid-portion of interpositus posterior, the large majority of the terminations were in nucleus fastigius. Most were present in Fdlp, which was labelled throughout its extent, but the distribution field atso included modest ventral extensions into the adjoining portion of Fm. In case 17, which was sectioned sagittally, the injection was placed close to that of case 3, but slightly further laterally. Transport originated only from the rostral portion of crus II and, as in case 3. the main termination field included the whole of Fdlp together with some extension into adjacent parts of Fro. Photomicrographs demonstrating the projections to Fdlp are shown in Fig. t. Fig. Ic is a low power bright-field micrograph of a section corresponding to level K61, and showing Fdlp together with the anterior and posterior subdivisions of interpositus. Fig. t d is a darkfield micrograph of the same section m which an approximately spherical accumulation of terminal labelling can be seen to fill the whole extent of Fdlp. This termination field is shown again at higher magnification in Fig. lb. In case 17 there were also smaller projections to more lateral parts of the nuclear complex: the lateral part of the injection site gave rise to a bundle of fibres which coursed rostroventrally toterminate in the lateral part of lp and the adjoining area of lateralis and minor contributions were also made to Ia and to the dorsolateral hump (dlh).
243 Thus in crus II, as in crus I, the three cases demonstrate unequivocally the existence of a mediolateral localisation in the projections from a single folium.
Comparison of the projection fields for crus I and crus H Each of the two folia studied provided projections to both large- and smallcelled portions of lateralis, to both the anterior and posterior subdivisions of interpositus and also to the lateral part of fastigius. However, despite the widespread nature of the projections revealed, comparison of the cases reveals scarcely any overlap between the terminations from the two folia. In all three deep nuclei, the termination fields for crus II were centred dorsally to those from crus I. Pro/ections from the paraflocculus Injections were made into the paraflocculus in two cases (14 and 19) and both were sectioned sagittally. Reconstruction revealed that in each brain, although the ventral limb of the paraflocculus was heavily labelled, the labelling did not extend into the dorsal limb, so that a complete account of the parafloccular projections cannot be given. In these cases both the injection site and the termination fields were almost identical (see Fig. 5). From the injection site, fibres ran dorsomedially to form a capsule in the white matter just outside the surface of nucleus lateralis. The capsule extended medially beyond the junction with nucleus interpositus, but during its course fibres were continually given off which penetrated the caudal surface oflateralis before terminating in the caudoventral part of the nucleus, in both the large- and small-celled portions (L1 and Ls). The termination in interpositus was dense but confined almost entirely to the lateral two-thirds of the subnucleus which Korneliussen 1° identifies as lps. Injections into 'somatosensory' areas in case 2 (Fig. 6) the injection pipette was inserted into lobulus VI and advanced well beneath the cerebellar surface in order to deliver the leucine into the lateral portion of lobules IV and V in the anterior lobe. The injection site failed to reach the cerebellar midline and, by contrast with cases in which injections were placed near the midline of the anterior lobe, there was no terminal labelling whatsoever in fastigius. There was, however, heavy terminal labelling in the vestibular complex, and large numbers of labelled fibres passed through the raphe which separates the fastigial and interpositus nuclei. A smaller number of corticovestibular fibres passed through the medial portion of interpositus. In the intracerebellar nuclei proper, heavy accumulations of label were present in the medial one-third of la throughout its rostrocaudal extent and labelling was also present in the medial part of lp though fading out at level KI3. In case 18 (sectioned sagittally) an injection made into the copula pyramidis resulted in an injection site which included parts of both the copula and the paramedian lobule (see Fig. 6). Large numbers of labelled fibres were intercepted in longitudinal section as they arched rostrally from the injection site to the intracerebellar nuclei. The great bulk of the terminal labelling was in Ia, but there was also
244 MEDIAL
~
CN-14 LEFT
Fcm
CN-19 LEFT
4EDIAL
Ia~<~Ipl
FdIp/~T~Fcm~.m__.~28 m ~
~/~,LI DIh N ~
~,'~Ips
117
Fdlp
0 Ie ~--~{~ IDI
~
~
88
LI_~
LAT ~ ERA1 ~ ~ 4 8~48
~5mm -ATERAL
Fig. 5. Results in sagittal cases 14 and 19. Survivals 5 and 22 h, respectively. Diagrams on the left identify the left intracerebellar nuclei as seen in sagittal sections. The vestibular complex is not ShOwn. Abbreviations and other conventions as in Fig. 2.
245
.......
C N-18
.......
"" 045 m m _ ~
C N-2
oE~Y~_~_~,,
~
v
LATERAL
V
lmm
LEFT
28 ~
104 @
38 ~
113 @
°'~'~'~
t
CAUOAL
LEFT
117
61 68
148
33
ROSTRAL 59
Fig. 6. Results obtained in sagittal case 18 and frontal case 2. Survivals 24 and 29 h. respectively. Conventions as in Figs. 2 and 5. Arrows descending beyond the intracerebellar nuclei denote corticovestibular axons. Interrupted vertical line indicates that lateral part of right hemispheral cortex was trimmed off before sectioning. a small amount in Ip (levels K88 and 104) and in Fdlp (levels K48 and 61). There was no labelling in the vestibular complex. Thus, in cases 18 and 2, in which widely separate portions of the cortical sheet were injected, there was very marked convergence (into interpositus anterior) between the major corticonuclear projections. DISCUSSION The present series of experiments in which 10 injections of the tracer were made at different locations in the hemisphere revealed only ipsilateral corticonuclear
246 projections (cf. refs. 12 and 13). Considered together with the data from the previous paper z in which vermal projections were studied, this finding suggests very strongly that the corticonuclear connexion in the rat is entirely uncrossed. Although the hemispheral injections were collectively far from involving the whole of the cerebellar hemisphere, they nevertheless led to heavy terminal labelling throughout most of the lateral and interpositus nuclei. Clearly, therefore, considerable convergence must occur between the projections demonstrated here and those from cortical areas which were not labelled. It should be noted there was very little overlap between the present terminal fields and those which were found in the preceding paper after injections into the vermis. Thus, the one part of fastigius which was heavily labelled in the present series of experiments (i.e. Fdlp) is precisely that which was scarcely labelled in the vermal series. The medial border of interpositus was the only area involved from both vermal and hemispheral injection sites. However, this paucity of overlap does not necessarily imply a sharp discontinuity in the corticonuclear projection. Thus, only in two of the hemispheral cases (2 and 18) were the injection sites close to the vermis, and in the preceding study the majority of the injection sites were centred near the cerebellar midline. The facts that cases 2 and 18 led to heavy labelling of the most medial part of la, that some hemispheral injections led to labelling of lateral F, and that some vermal cases produced modest labelling in lp together suggest that injections near the paravermal vessels would almost certainly demonstrate a greater degree of overlap between the vermal and hemispheral projections than is shown by our existing cases. Nevertheless, it is obvious that, in general, the hemispheral termination fields are more laterally placed in the nuclear complex. Furthermore. within the crura as within the vermls, there is undoubtedly a mediolateral localisation of projection as previously claimed on the basis of degeneration experiments by Goodman et al. 9. It is similar in degree to that existing within the vermis, since both the injection sites and the termination fields for individual experiments were comparable in size to those in the vermal experiments. In a recent preliminary report by Courville and Faraco-Cantin 5, corticonuclear connexions in the cat have been traced in the retrograde direction using horseradish peroxidase. This study has indicated that in crus II the superficial and deep parts of the folia may project to different parts of the nuclear complex. Our injection sites have been too extensive, relative to the small folia of the rat cerebellum, to allow any comparison with this finding. However, application of the autoradiographic technique to larger species should provide an alternative method of investigating this interesting phenomenon. As regards any rostrocaudal localisation in the crural projections, our results do not provide a complete picture because so much cortex remained unexplored. Nevertheless, we were surprised at the extent to which the nuclear territories labelled after injections into the caudal part of crus I were complementary to those labelled from the rostral portion of crus I1. This finding seems to conflict directly with the conclusion reached in their degeneration study by Goodman et al.~. These workers concluded that the hemisphere in the rat could be divided into two rostrocaudal
247 'functional-zones' (lateral and intermediate) running across both crura and that small lesions placed anywhere within one zone led to very similar distributions of terminal degeneration within the nuclear complex. The paucity of overlap in our experiments suggests that each crus may have its own distinct projection field within the nuclei. However, this can only be proved by a more comprehensive study of the crural projections. It should be noted that if such private termination fields do exist, some difference in rostrocaudal organization is implied between the hemispheral and the vermal projections, since in the latter the terminations from different lobules overlapped heavily 2. The extent of the projection from crus 11 to Fdlp (cases 3 and 17) came as a surprise, but in fact it confirms findings made by Goodman et al., who indeed suggest that Fdlp should be included with the neocerebellum (hemisphere) rather than the paleocerebellum. These authors also suggest that the hemispheral projection to Fdlp derives from the whole rostrocaudal extent of the paravermal or 'intermediate' cortex and that the same termination field is found irrespective of the particular location of the lesion within this zone. Our series provides only limited evidence regarding this point, but in case 3 the whole subnucleus was densely labelled except for the rostral pole where terminations were fewer. Again, in case 17 heavy labelling was present throughout, excepting only the medial border which was lightly labelled. These findings are compatible with the views of Goodman et al., but it should be noted that, in case 4 of the preceding paper, labelling of the left lobulus simplex led to dense terminal labelling in only the rostral half of Fdlp. It seems, therefore, that at least one area in the hemisphere projects to only a part of Fdlp. Furthermore, other injections described in the preceding paper labelled small portions of the subnucleus and in case 1 of the present series a (small) projection to fastigius was directed to the region where Fm and Fdlp merge. Our finding that the (ventral) paraflocculus projects to the ventrocaudal part of nucleus lateralis is in good agreement with the results of Goodman et al. and with results obtained by Dow 6 using the Marchi method. Like Dow, but unlike Goodman et al., we found, in addition, that the projection continued medially to provide abundant terminations in lps. From comparison with Korneliussen we are convinced that we have correctly identified this subnucleus, but it should be noted that we and Dow used sagittal sections, whilst Goodman et al. employed frontal sections. The difference in results might therefore be a difference of interpretation regarding the relevant nuclear boundaries. If the discrepancy is to be resolved by any future study, the use of horizontal sections would perhaps be advisable, since these are said to provide the best display of boundaries TM. In the cat, corticonuclear projections originating from crus II have been studied in great detail by Brodal and Courville 3 using the Nauta technique. It was concluded, as in the present study, that this lobule projects to more than one deep nucleus. Two separate termination zones were distinguished, one in nucleus lateralis and continuing into the neighbouring interpositus anterior, the other in interpositus posterior. Even very small lesions produced degeneration in both zones. The study also considered the possibility of a mediolateral localisation within the projection from crus lI, and it was
248 concluded that, although lateral areas projected somewhat more laterally and ventrally in each projection zone, the terminal fields for medial and lateral parts of the crus nevertheless overlapped heavily so that there was no marked localisation. This conclusion is apparently at odds with that reached here, but it must be remembered that the terms lateral and medial were applied in the cat study to two separate groups of folia, whilst we have applied them to different portions of the same folia. The major difference between that study and our own was the absence in the cat of any projection from crus II to nucleus fastigius. Courville et al. 4 have also investigated projections from the paramedian lobule in the cat. This very detailed study concluded that the lobule projected to both lateralis and interpositus. We have found no projection to lateralis, but the lateral part of t he lobule was labelled by neither of our injections (3 and 18) which involved this lobule. In one of these cases (case 3) fibres from the lobule travelled together with others from the medial part of crus I1 to terminate mainly in Fdlp, and few terminals were provided to interpositus. In case 18, however, fibres from the paramedian lobule and copula pyramidis terminated mainly in interpositus anterior. Unfortunately, one case was sectioned sagittally and the other frontally so that it was difficult to compare accurately the distributions of labelled Purkinje cells in the lobule. However, it seems likely that one part of the lobule projects to Fdlp and an adjoining portion to nucleus interpositus. If the lateral part of the lobule projects to nucleus lateralis as claimed for the rat by G o o d m a n et al (and for the cat by Courville et al. 4) then the paramedian lobule would display the same kind of mediolateral localisation as the portions of the two crura we have studied. However, further experiments are needed to verify this speculation. It is worth noting that Eager 8 has reported projections to all three ipsilateral nuclei in the cat. Finally, the injection site involving the lateral portion of vermal lobules 1V and V (case 2) projected mainly to the medial part of interpositus anterior. Studies in the cat have reported a similar connexion~,S,lZ, ~4. it is noteworthy that the injection site involving the copula pyramidis and paramedian lobule (case 18) also projected mainly to the same portion of interpositus. In the cat, both the pars intermedia of the anterior lobe and the paramedian lobute are well documented as the main somatosensory receiving areas of the cerebellar cortex1,11 and in the rat also lobulus V (the Culmen) and the region of the copula pyramidis and paramedian lobule are loci from which short latency evoked potentials are readily recorded following stimulation of afferems from the limbs (cf. ref. 7 and Adlam, Armstrong and Clarke, unpublished observations). It is likely therefore that there is extensive convergence within interposltus anterior between outputs from the two main somatosensory areas, even though these are widely separated in the rostrocaudal plane. Since interpositus anterior projects mainly to the magnocellular red nucleus, which in turn gives origin to the rubrospinal tract, this convergence is presumably of considerable functional significance in relation to cerebellar control of the limbs.
249 A C K N O W L E D G E M ENTS W e t h a n k Mrs. S. R u c k l i d g e for e x p e r t t e c h n i c a l assistance, Mrs. A. B a c o n and B. C o l f e r for the p h o t o g r a p h y , Mrs. B. R i d d i c k for the t y p i n g a n d Dr. R. J. H a r v e y for c o n s t r u c t i v e c o m m e n t o d the m a n u s c r i p t . T h i s r e s e a r c h was s u p p o r t e d by a g r a n t f r o m the M e d i c a l R e s e a r c h C o u n c i l o f G r e a t Britain.
REFERENCES I Armstrong, D. M., Functional significance of connections of the inferior olive, Physiol. Rev., 54 (1974) 358-417. 2 Armstrong, D. M. and Schild, R. F., An investigation of the cerebellar cortico-nuclear projections in the rat using an autoradiographic tracing method. I. Projections from the vermis, Brain Research, 14l (1978) 1-19. 3 Brodal, A. and Courville, J., Cerebellar corticonuclear projection in the cat. Crus II. An experimental study with silver methods, Brain Research, 50 (1973) 1 23. 4 Courville, J., Diakiw, N. and Brodal, A., Cerebellar corticonuclear projections in the cat. The paramedian lobule. An experimental study with silver methods, Brain Research, 50 (1973) 24-45. 5 Courville, J. and Faraco-Cantin, F., Cerebellar corticonuclear projection demonstrated by the horseradish peroxidase method, Soc. Neurosci. Mtg., Toronto, 1976, Vol. H, p. 108 (Abstract no. 153). 6 Dow, R. S., The fiber connections of the posterior parts of the cerebellum in the rat and cat, J. comp. Neurol., 63 (1936) 527 548. 7 Dow, R. S. and Anderson, R., Cerebellar action potentials in response to stimulation of proprioceptors and exteroceptors in the rat, J. Neurophysiol., 5 (1942) 363 371. 8 Eager, R. P., Efferent cortico-nuclear pathways in the cerebellum of the cat, J. comp. Neurol., 120 (1963) 81 104. 9 Goodman, D. C., Hallett, R. E. and Welch, R. B., Patterns of localization in the cerebellar cortico-nuclear projections of the albino rat, J. comp. Neurol., 121 (1963) 51-67. 10 Korneliussen, H. K., On the morphology and subdivision of the cerebellar nuclei of the rat, J. Hirnforsch., 10 (1968) 109-122. 1l Oscarsson, O., Functional organization of spinocerebellar paths. In H. Antrum et al. (Eds.), Handbook of Sensory Physiology, Vol. 11, Somatosensory Systems, Springer, Berlin, 1973, pp. 339-380. 12 Rossum van, J., Corticonuclear and Corticovestibular Projections {~f the Cerebellum, Van Gorcum, Assen, 1969. 13 Voogd, J., The Cerebellum of the Cat, Van Gorcum, Assen, 1964. 14 Walberg, F. and Jansen, J., Cerebellar corticonuclear projection studied experimentally with silver impregnation methods, J. HirnJbrsch., 6 (1964) 338-354.