immunocytochemical demonstration of topographic ordering of purkinje cell axon terminals in the...

THE JOURNAL OF COMPARATIVE NEUROLOGY 244:481-491 (1986)

Immunocytochemical Demonstration of Topographic Ordering of Purkinje Cell

Axon Terminals in the Fastigial Nuclei of the Rat

RICHARD HAWKES ANI) NICOLE LECLERC Laboratory of Neurobiology and Department of Biochemistry, Lava1 University, Ste-Foy,

Quebec, G1K 7P4

ABSTRACT We have used a monoclonal antibody, mabQ113, which selectively stains

a subset of cerebellar Purkinje cells, to study the topography of the corticonuclear projection of the median vermis to the fastigial nuclei in the rat. The fastigial projection zone contains both mabQ113' and mabQ113- Pur- kinje cells grouped into parasagittal bands. The immunoreactivity extends throughout the Purkinje cell including the axon terminals and thus it is possible to investigate the topographic distribution of mabQ113+ terminals. In the fastigial nuclei the target cells receiving mabQ113+ axon terminals are concentrated in the caudal pole. In the rostra1 pole, cells receive anti- GADt terminals but not mabQ113+ terminals. There is no gradient in anti- GAD staining. Double-labelling experiments with mabQ113, anti-GAD, and an antisynaptic antibody mabQ155 suggest that there is little or no mixing of mabQ113' and mabQ113- Purkinje cell terminals on the same target neuron.

Key words: monoclonal antibody, cerebellum, sagittal zones

Both the afferent and the efferent projections of the mammalian cerebellar cortex are topographically ordered into sagittal zones. With respect to the afferent inputs, the climbing fiber projection fields from discrete regions of the inferior olive have been mapped to precise sagittal bands of cortex both electrophysiologically (Oscarsson, '69, '79; Arm- strong et al., '74; Anderson and Oscarsson, '78a,b; Van Gilder and O'Leary, '70) and by the transport of tracers (Courville, '75; Chan-Palay et al., '77; Groenewegen and Voogd, '77; Walberg, '80; Courville and Faraco-Cantin, '80; Brodal, '80; Beyerl et al., '82; Campbell and Armstrong, '83). Sagittal zonation has also been suggested in the mossy fiber afferents projection fields (Scheibel, '77). In the cerebellar cortex itself, sagittal zones have been demonstrated anatomically (Voogd, '691, histochemically (Scott, '63; Mar- ani and Voogd, '77) and by immunocytochemistry (Chan- Palay et al., '81, '82). Finally, the cerebellar cortical efferents are also arranged into sagittal zones. The sole efferent projection from the mammalian cerebellar cortex is the corticonuclear pathway that is formed exclusively of Pur- kinje cell axons. Each axon terminates as a bushy network of collaterals that innervates a small nuclear area (Ramon- y-Cajal, '11; Palay and Chan-Palay, '74). The projection is

0 1986 ALAN R. LISS, INC.

topographically ordered such that different sagittal zones of the cerebellar cortex project t o individual nuclei (Jansen and Brodal, '40; Goodman et al., '63; Eager, '63, '66; Voogd, '64; Walberg and Jansen, '64; Van Rossum, '69; Sreesai; '74; Haines, '75, '76; Courville and Diakiw, '76; Haines et al., '76; Armstrong and Schild, '78a,b). There is a general tendency for the more lateral cortical zones to project to the more lateral cerebellar nuclei (Voogd, '69; Haines et al., '82). Thus, the most medial nuclei, the fastigial, receive projections from the most medial cortical zones (zone A). Zone B in the lateral vermis projects to the ipsilateral vestibular nucleus. Cortical efferents to the interposed nuclei arise from zones C1, C2, and C3 of the intermediate cortex and the most lateral cortical zones (D1 and D2) project to the lateral cerebellar nuclei.

Recently a monoclonal antibody has been described, mabQ113, that labels a subset of Purkinje cells (Hawkes et al., '85). On Western blots of cerebellar polypeptides mab- Q113 recognizes a single band at apparent molecular weight 120,000. The mabQ113+ and mabQ113- Purkinje cells are

Accepted October 18, 1985.

482

arranged in bands running rostrocaudally through the cerebellar cortex. The mabQ113 labelling of Purkinje cells extends into the axons and their terminals in the cerebellar nuclei and therefore permits us to investigate the overall arrangement of mabQ113' and mabQ113- Purkinje cell efferent fields. We have concentrated our attention on the corticonuclear projection from the medial zone of the vermis where there is a characteristic medial band of mabQ113' Purkinje cells in the rat cerebellar cortex, bordered on either side by mabQ113- cells. Purkinje cells from this region (roughly the equivalent of zone A of Voogd, '69) have been shown to project to the fastigial nuclei and in turn, the fastigial nuclei receive predominantly medial Purkinje cell afferents (Walberg and Jansen, '64; Armstrong and Schild, '78a,b; Haines and Koletar, '79; Oscarsson, '80; Courville and Faraco-Cantin, '80; Haines et al., '82). We have used mabQll3 to study this projection in greater detail and to answer questions concerning the organization of Purkinje cell efferents, specifically: (I) Do both mab- Q113' and mabQ113- cells project to the fastigial nuclei and if so, (2) do target neurons receive pure mabQ113+ or mabQ113- inputs or mixed, and (3) is the mabQ113+/ mabQ113- topographic separation seen between the mabQ113+ and mabQ113 Purkinje cells in the cerebellar cortex maintained in the target nucleus?

MATERIALS AND METHODS The production and characterization of monoclonal anti-

body mabQ113 will be described in detail elsewhere. In brief, adult Balblc mice were immunized intraperitoneally with 3 mghnjection of whole-brain synaptosomal plasma membranes isolated from 13-day-old rats (Jones and Matus, '74). The first immunization was with antigen emulsified in Freund's complete adjuvant, the second, 8 days later, was with Freund's incomplete, and the final injection, in physiological saline only, followed 6 weeks later. The panel of immunized mice were tested for antibody titer using a dot-immunobinding assay (Hawkes et al., '82a) and the two animals with the highest antibody titers were used for fusion. Hybridomas were produced by established tech- niques (Galfre et al., '77; Hawkes et al., '82b,c). Interesting hybridomas were identified by immunocytochemistry by using P13 rat cerebellum and were cloned by limiting dilu- tion at 0.1 cells/welI.

All sections shown here were from tissue fixed by trans- cardiac perfusion with 4% paraformaldehyde, 0.2% glutar- aldehyde followed by overnight fixation in paraformaldehyde alone. Sections were cut a t 40 pm on a freezing microtome. Antibody binding was detected by an indirect immunoperoxidase procedure by using peroxidase-conju- gated rabbit antimouse immunoglobulin (Dako Inc.) and 4- chloro-1-naphthol as substrate. Control sections in which the first antibody was omitted, or replaced by either normal mouse serum or myeloma conditioned medium, gave no staining.

We have used three different antibodies in this study. Two of these are monoclonal-mabQll3, which recognizes a subset of Purkinje cells and their terminals, and mab- Q155, which recognizes all synaptic classes (Hawkes et al., '85). We also used a polyclonal antibody raised in sheep against glutamic acid decarboxylase [anti-GAD] that labels all Purkinje cell terminals in the deep nuclei. Anti-GAD (# 1440-4) was provided through the Laboratory of Clinical Science, NIMH where it was developed under the supervi- sion of Dr. I.J. Kopin with Dr. W. Oertel, D.E. Schmechel,

R. HAWKES AND N. LECLERC

Fig. 1. Irnmunoperoxidase staining of ra t cerebellar cortex with mab- Q113. In A, reaction product is seen in the Purkinje cell somata (P), dendrites, and axons (arrowhead). In B, the immunoreactive Purkinje cell axcins can he seen passing through the granular layer (gl) into the white matter (wrn). Scale bar in A = 50 pm. Scale bar in B = 100 wm.

and M. Tappaz (Oertel et al. '80). Antibodies mabQll3 and mabQ155 were used directly from spent culture medium diluted 1/32 and anti-GAD diluted 1/500 in 10% normal horse serum, 0.1 M phosphate buffer, pH 7.4.

Some sections were double-labelled by using two different antibodies. The first antibody was used as described above except that 4-chloro-1-naphthol was replaced by 33-diami- nobenzidine as substrate. The stained section was then washed for 60 minutes in several changes of phosphate buffer and the staining procedure was repeated with the

TOPOGRAPHY OF CORTICOFASTIGIAL PROJECTION 483

Fig. 2. MabQll3 selectively stains a subset of Purkinje cells in rat cerebellar cortex. Immunoreactive Purkinje cells are arranged in a series of parasagittal bands seen in horizontal section in A. Three-dimensional recon- struction from serial sections of the mabQll3-stained band pattern reveals a midline band (P1') and seven others displayed laterally to either side, three in the vermis (P2+, P3+, P4+) and four in the hemisphere (P5 ' -P8 + )

Hawkes and Leclerc: in preparation). The vermal bands P1 '-P3' are indi- cated. The fastigial nuclei are labelled with arrowheads. Scale bar = 500 pm. In B the stained Purkinje cell axons running into a fastigial nucleus are shown in greater detail from another section. The projection is purely ipsilateral and no immunoreactive fibers are seen crossing the midline. Scale bar = 200 pm.

484 R. HAWKES AND N. LECLERC

second antibody using 4-chloro-1-naphthol as chromogen. In this fashion, the immunoreactivity of the first antibody was revealed in brown, and the second in blue.

RESULTS After immunocytochemical staining with mabQ113, de-

posits of reaction product are found in a subset of Purkinje cells throughout the cerebellar cortex. Stain is deposited throughout the dendritic tree, in the soma, and in the axons and axon collaterals (Fig. lA,B). The stained axons can be followed from the cell soma out though the granular layer and bundles of stained axon tracts are observed throughout in the white matter. It is characteristic of mabQ113 that only about one-third of the Purkinje cells are labelled. All lobules of the cerebellum display short stretches of mab- Q113' Purkinje cells that alternate with similar bands of mabQ113- cells (Fig. 2A,B). The mabQ113+ cells are arranged in a reproducible pattern of parasagittal bands (Hawkes et al., '85). Figure 2A shows a typical horizontal section through the rat cerebellar cortex at the level of the fastigial nucleus (about 4.5 mm interaural; Paxinos and Watson, '82) with the mabQ113 '/mabQ113- bands displayed. For the present purpose, it is necessary to empha- size only the median bands. At the midline there is a narrow mabQ113' band of Purkinje cells (Pl'), ranging from one to ten cells wide, flanked at either side by bands of mabQ113- cells. Depending on the dorsoventral level of section, up to three additional mabQ113+ vermal bands may be found to either side (P2+, P3+, P4+).

The Purkinje cell axons terminate synaptically on neurons of the cerebellar nuclei and the lateral vestibular nuclei. In particular, Purkinje cells located at or near the midline of the vermis project to large relay neurons in the fastigial nuclei (Armstrong and Schild, '78a). Figure 2B shows immunoreactive Purkinje cell axons running through the white matter of the cerebellar cortex from the vermal mabQ113+ bands in the posterior lobe of the cerebellum to the fastigial nucleus. The projection is purely ipsilateral and we see no stained fibers crossing the midline. Sections though the medial nuclei stained with mabQ113 clearly reveal these terminations (Fig. 3). Large (0.5-2.5-pm diam- eter) punctate deposits of reaction product encircle the cell bodies and primary dendrites of large neurons. The target neurons themselves are always unstained. Reaction product is also found in axons en passant.

Not all synaptic boutons in the fastigial nucleus are mabQ113 ' . To demonstrate this point, double-labelling experiments were carried out by using a second monoclonal antibody, mabQ155, that recognizes an antigen associated with synaptic vesicles (Hawkes et al., '85) and that appar- ently stains all synapses in the fastigial nuclei. By treating the section first with one antibody with 3,3'-diaminobenzi- dine as substrate then with the other and by using 4-chloro- 1-naphthol as substrate, we can differentially label mab- Q113,+ and mabQ113,- (mabQ155,+)synapses (Fig. 4) (The s subscript is used to distinguish the staining of the sYn- apses from the staining of the cell bodies themselves.) For example, by first using mabQ155 we label all synapses brown. Subsequent labelling with mabQ113 renders a subset of synapses dark blue. The distribution of immunoreac- mabQ113, t/- mixed (Fig. 4). The relative frequencies of the tivity is the same when mabQll3 is used first. three classes in the fastigial nuclei are given in Table 1. It

By using mabQ113/mabQ155 double-labelling target neu- can be seen that target neurons receiving pure mabQ113, .-

rons can be classified into three groups depending upon the input are more than twice as common as those receiving a input they receive: mabQ113,+ only, mabQ113,- only, and mixed input, roughly in accord with the overall relative

~ l g . 3. MahQ113 staining ofsynaptlc boutons i n the fastigial nuclei. The target neurons are themselves unstained Scale har = 20 pm.


frequencies of mabQ113+ and mabQ113- Purkinje cells in the cortex. Target cells receiving only mabQ113,+ input are very rare. The relative frequencies are independent of which antibody is used first in the double staining. Synaptic boutons are found both on the target cell somata and on prin- ciple dendrites. When both mabQ113,+ and mabQ113,- terminals were seen on the same profile, no obvious segregation of the two types was observed between somata and dendrites.

A polyclonal antibody against glutamate decarboxylase (anti-GAD) was also used to stain terminals of Purkinje cells (Fig. 5). In the cerebellar cortex, the distribution of reaction product corresponds to that described previously (Oertel et al., '81) with basket cell terminals, granule cell somata, and stellate cell terminals all consistently labelled (Fig. 5A). Staining of the Purkinje cell somata is irregular when anti-GAD is used diluted at 1/500, a concentration that gives strong staining of synaptic boutons in the deep cerebellar nuclei: at 1/200 or after colchicine treatment all Purkinje cell bodies were immunoreactive (not shown). Double-labelling experiments with anti-GAD and mabQ113 identify two principal classes of neurons based on the synaptic input they receive on their somata, GAD,+/mab- Q113,+ and GAD,+/mabQ113,- [Table 1, Fig. 61. Profiles that are GAD,-/mabQ113,- would be unstained and thus difficult to observe with confidence but it is probable that they are rare. Cell somata receiving GADs-/mabQ113,+ inputs were never observed. Mixed Purkinje cell inputs to the same cell-that is, a combination of GADs+/mabQ113,- and GAD,+/mabQ113, + terminals on the same soma-were sought for specifically, for these might indicate a target cell receiving input from both mabQ113' and mabQ113- Pur- kinje cells. No convincing example of mixed input was observed. This further suggests that there is no substantial class of GADs+ interneuron terminals on the somata of the large neurons (expected to be mabQ113,-/GADs+/mab- Q155,+). This is supported by the estimate of Chan-Palay ('77) that 86% of axosomatic synapses in the dentate nucleus are Purkinje cell in origin. Similar double-labelling experiments were conducted with anti-GAD and mabQ 155. Both antibodies stained terminals on all target cells. Two types of terminal were noted, GADs+/rnabQ155,+ (presum- ably Purkinje cell derived) and GADS-/mabQ155,+ (pre- sumably derived from non-Purkinje cell sources such as cerebellar afferent axon collaterals or nuclear interneuron terminals). The two classes of terminals occurred together on almost all target profiles (Table 1) with GAD,+/mab- Q 155, + terminals outnumbering GAD, -/mabQ 155, + by about four to one. The frequency of non-GABAergic terminals in the fastigial nuclei corresponds to the frequency of non-Purkinje cell terminals in the dentate nuclei (Chan- Palay, '77). Finally, a labelling experiment was performed with all three antibodies. The first incubation and staining was with anti-GAD and mabQ113 together and the second was with mabQ155. Again 20% of the boutons were labelled by mabQ155 alone and 80% were double-labelled.

The corticonuclear projection to the rat fastigial nuclei arises predominantly from the ipsilateral medial vermis (Armstrong and Schild, '78a,b). The correspondence between zones &marked by mabQ113 and efferent projection fields is ill-defined but from their respective dimensions it is evident that the vermal zone projecting to the fastigial nucleus (zone A) includes both mabQ113+ and mabQ113- bands. Therefore, it is interesting to examine whether the

Fig. 4. Double-immunoperoxidase labelling in the fastigial nuclei with mabQ113 and mabQ155. Some terminals are double-labelled mabQll3,+/ mabQ155,+ others are single-labelled mabQli3,-/mabQ155, I . The double- labelled terminals are marked by arrowheads. The Figure shows three cell profiles, two receiving mixed synaptic inputs (i.e., both mabQll3,+/mab- Q155,+ and mabQ113,-/mabQ155,i, the other exclusively mabQll3, / mabQ155, + terminals. Scale bar = 20 pm.

486 It. HAWKES AND N. LECLERC

TABLE 1. Relative Freuuencies of Target Tvues in the Fastwial Nuclei'

____ Incubation mabQ113' mabQ155+ Anti-GAD No. 1 No. 2 only only only Mixed

mabQ113 mnbQ155 l(1) 69 (167) - 31 (76) 244 mabQ155 mabQl l3 1 (2) 74 (223) - 25 (77) 302

N _ _ _ _ ____-____-

anti-GAD ma bQ 1 1 3 0 (0) - 62 (144) 38 (88) 232 mabQ113 anti-GAD 0 (0) - 70 (161) 30 (69) 230

mabQ155 anti-GAD - 0 (0) 0 (0) 100 (100) 216 anti-GAD niabQ1.55 - 4 (4) 0 (0) 98 (98) 204

'Doubla-lnbellmg Paperiments were performed on horizontal sections through t h e cerebellum selected to inuludc t h c fastipial nuclei Binding 01 t h e first ,+ntihody lincuhation No. 1) was revealed with diammohenzidine, hrnding of the second antihody (Incubation No. 21 with 4 chloro-1-naphthol. Axon te rmina ls were identified as dense, punctate s ta in deposits encircling the penmeter of the somata a n d dendrites of large neurons. To avoid confusion between axon terminal houtons a n d axons cut in cross section, only those structures clearly associated with large neuron profiles were considered. and te rmina ls on dendrites were only included when the dcndnte was contigoous with a cell w n i a in t h e section. All large neuron profiles in a section were considercLd iusually 20-50) and the total numher of profiles sampled IN) w'as obtained from a t least five animals. The frequency ,ofthe various

classei of ta rge t ncwron is given a s ii percentage with t h e total numher found in parenthesis No significant diflhrence in paired 1

tests \\:IS Ibund in t h r frcquencics due to t h e order o f application of t h e antibodies.

cortical bands are reflected in the topography of the axon terminal fields. To explore this question we have examined the distribution within the fastigial nuclei of large neuron profiles receiving either pure mabQ113,- input or mab- Q113, i /mabQ113,- mixed inputs. The fastigial nuclei are not stained uniformly by mabQ113 (Fig. 6A-D). Staining is clearly more intense in the caudal part of the nucleus. The higher caudal concentration of mabQ113 reaction product is found throughout the fastigial nucleus from dorsal to ventral. Most of the mabQ113 immunoreactivity in the rostral region is associated with axonal cross sections. An example of serial sections through the same nucleus is illustrated in Figure 7A-C. The dorsolateral protuberance is always heavily labelled. This difference in intensity arises in two ways. First, the mabQll3+ axons tend to enter the nucleus caudally so that bundles of stained axon profiles are concentrated in the caudal half and second, the axon terminals are segregated such that the mabQ113, timab- Q113,- mixed target neurons are also concentrated caudally with the exclusively mabQ113,- targets found predominantly in the rostral half of the nucleus. This is illustrated from a double-labelling experiment in Figure 8. When target neurons are classified as mabQ113,+imab- Q113, ~ mixed or pure mabQ113,-, it is clear that the mabQll3 + bands in the cerebellar cortex terminate prefer- entially in the caudal half of the nucleus (Fig. 8A). How- ever, there is no gradient apparent in the density of synapses throughout the nucleus after staining with mabQ155. Likewise, and despite the incomplete staining of Purkinje cell bodies, we observe no gradient either rostrocaudal or mediolateral, in anti-GAD immunoreactivity. (Figs. 5B, 8B). Finally, we have examined the distribution of mabQ113 axons. Our observations confirm the previous report of Armstrong and Schild ('78a) that, in the rat, the projection is purely ipsilateral and that axons are not seen crossing the midline (see the axons running from lobe IX to a fastigial nucleus in Fig. 2B).

IIISCUSSION MabQll3 stains a subset of Purkinje cells in the rat

cerebellar cortex, including their axonal projections to the cerebellar nuclei. We can therefore identify the axon terminal field of the mabQ113 ' cells. We have used two other antibodies: the one to identify another subset, perhaps all,

of the Purkinje cell terminals (anti-GAD: Oertel et al., '81) and another to identify all synapses (mabQ155: Hawkes et al., '85). The corticonuclear projection must have extensive convergence of cerebellar efferents since Purkinje cells out- number nuclear target neurons manyfold (Chan-Palay, '73). Despite this convergence it is clear that the cerebellar cortical efferents are not distributed at random. For example, part of the vestibular pathway from the cerebellar cortex to the lateral vestibular nuclei passes via the fastigial nuclei. The anterior cerebellar cortex projects to the rostral poles of the fastigial nuclei and then ipsilaterally to the lateral vestibular nuclei while the posterior cortex projects caudally onto the fastigial nuclei that in turn project to the contralateral lateral vestibular nuclei (Brodal et al., '62). Furthermore, within each pathway, a degree of somatotopy is maintained (e.g., Kuypers, '81).

The analysis of our results assumes that all mabQ113 staining in the fastigial nuclei derives from the hands of mabQll3 ' Purkinje cells in the cerebellar cortex. This is reasonable since neither the climbing fiber nor the mossy fiber synapses in the cerebellar cortex are mabQl13 ' and there are no mabQ113' neurons within the nucleus itself. Furthermore, Chan-Palay ('77) found 86% of axosomatic synapses in the dentate nuclei to be Purkinje cell in origin. When a profile receives mabQ113' boutons, 80%' are mabQ113+ and 20% mabQl13-. The 20% mabQ113 terminals probably do not derive from Purkinje cells since they are also anti-GAD . It could he that they represent the axon terminals from a subset of anti-GAD- Purkinje cells (Chan-Palay et al., '81) but more likely, they are a mixture of excitatory collateral inputs from mossy and climbing fibers, perhaps together with non-GABAergic interneuron synapses. If the anti-GAD' terminals in the caudal part of the nucleus (anti-GAD + imahQll3 imah- Q155' ) all derive from mabQ113+ Purkinje cells, it is reasonable to extrapolate that the anti-GAD l terminals on rostral neurons (anti-GAD+imabQ113 -./mabQ155 ' are derived from the cortical mahQl13 ~ Purkinje cells.

At the level of the individual nuclear large neuron, the present data suggest that little convergence takes place between inputs from different parasagittal cortical zones. Double antibody labelling indicates that there are two pre- dominant classes of target neuron with respect to Purkinje cell input in the fastigial nuclei, those which receive pure


Fig. 5. A. Immunoperoxidase labellinfi of the cerebellar cortex with anti- GAD antiserum. At the concentration used, reaction product is not seen in the Purkinje cells (P) but t he somata are surrounded by dense deposits of reaction product corresponding to the distribution of GABAergic basket cell axon terminals. The characteristic lacework of anti-GAD staining around the grzanule cell somata is seen in the granular layer (gl). Scale bar = 100 pm. B. Anti~GAD staining of the fastigial nuclei is homogeneous and there

is no sign of a rostrocaudal gradient of immunoreactivity. Scale bar = 50pm. C. At higher magnification, the anti-GAD staining in the fastigial nuclei resolves to synaptic boutons surrounding target cell somata and large dendrites. Scale bar = 20 pm. D. A tissue section double-labelled with mabQll3 and anti-GAD. Shown are three target neurons. Anti-Gad+ terminals can be either mabQll3- or mabQll3+ (arrowheads). One profile receives both kinds of input; the other two receive only mabQll3-. Scale as in C.

488 K. HAWKES AN11 N. LECLERC

Fig. 6. MabQllS reveals a rostrocaudal gradient in the rat fastigial nuclei. The plate shows horizontal sections through fastigial nuclei from four individuals each taken at about interaural +4.5 mm (Paxinos and

Watson, '82). Reaction product is concentrated in the caudal pole in each case. Scale bar = 50 pm.


Fig. 8. Distribution of mabQll3,i and mabQll3,- target cells in the fastigial nuclei. A fastigial nucleus has been double-labelled with mabQ113 to study the Purkinje cell projection from the vermal mabQ113 ' bands, and with anti-GAD to reveal all the Purkinje cell terminals (inter alia). Target neuron profiles were identified a t higher magnification as either receiving mixed cabQ113,+/anti-GADs+ and mabQll3, /anti-GAD, 1 terminals or pure mabQ113,- /antiGAD,+ inputs. A map of neuronal profiles receiving mixed (closed circles) or pure (open circles) synaptic inputs is given in A. Figure 8B shows the double-labelled nucleus: no gradlent in immunoreactivity is apparent.

Fig. 7. MabQll3 stains the caudal region of the fastigial nuclei prefer- entially. Three horizontal serial sections through a fastigial nucleus from dorsal to ventral showing the rostrocaudal gradient at all levels. The dorsolateral protuberance (dp) is always uniformly immunoreactive. Scale bar = 200 wm.

490 R. HAWKES AN11 N. LECLEKC

mabQ113,- (anti-GAD +) inputs and those which receive mixed mabQll3,t /mabQ113,-- inputs. Cell profiles receiving only mabQ113,. inputs are very rare and could easily reflect uncharacteristic cross sections through cells in fact receiving mixed input. The two classes of target are highly segregated within the fastigial nuclei: those receiving only mabQ113,- inputs are found in the rostral half, those receiving mixed input in the caudal half. No such segregation is observed in the distribution of anti-GAD' terminals or of terminals stained with mabQ155. Thus, we find it plau- sible that the topographical zonation into mabQ113+ and mabQ113- bands observed in the medial vermis of the cerebellar cortex is preserved in the efferent projection to the fastigial nuclei.

A rostrocaudal bipartitioning of the fastigial nuclei has been revealed in several previous studies both of connectiv- ity (Moolenaar and Rucker, '76; Batton et al., '77; Ruggiero et al., '77) and physiology (Batinin and Pompeiano, '58; Martner, '75). Similar evidence has been presented based on the finding that the seven classes of fastigial neurons identified by Beitz and Chan-Palay ('79a,b) are differentially distributed within the nucleus. The mabQ113' axon terminals are not exclusively associated with any particular class of target neuron.

Previous studies have reported both rostrocaudal and mediolateral topography within the corticonuclear projection to the fastigial nuclei (Courville and Diakiw, '76; Arm- strong and Schild, '78a). For the rostrocaudal axis, this implies that the anterior vermis projects to the rostral pole of the fastigialis and the posterior vermis projects more caudally. There is a difference in the frequency of mab- Q113' Purkinje cells in the vermis between the anterior lobe and posterior lobe with 15-25% of Purkinje cells in the anterior lobe Q113+ and in the posterior lobe 70-90% (Hawkes et al., '85). Thus, the predominance of mabQ113 immunoreactivity in the caudal pole of the fastigial nuclei may be, in part, a consequence of the higher frequency of mabQ113' Purkinje cells in the posterior vermis.

Armstrong and Schild ('78a,b) have addressed the mediolateral topography of the corticofastigial projection in the rat. They concluded that the sagittal segregation of Pur- kinje cells in the vermis was preserved as a mediolateral gradient in the fastigial nuclei. Injections of 3H-leucine into the medial regions of the vermis revealed projections to the medial pole of the fastigjal nuclei. Lateral extension of the injection site resulted in a corresponding lateral enlarge- ment of the terminal field. A mediolateral segregation of inputs has not been detected in the present study.

ACKNOWLEDGMENTS We wish to thank L. Thivierge and R. Sasseville for their

technical assistance. This work was supported by grants from MRC (Canada) and Fonds de Recherche en Paralysie Cerebrale.

LITERATURE CITED Andersson, G., and 0. Oscarsson (1978a) Projections to lateral vestibular

nucleus from cerebellar climbing fiber zones. Exp. Brain Res. 32:549- 564.

Anderson, G., and 0. Oscarsson (1978b) Climbing fiber microzones in cerebellar vermis and their projection to different groups of cells in the lateral vestibular nucleus. Exp. Brain Res. 32565-579.

Armstrong, D.M., R.J. Harvey, and P.B.C. Matthews (1974) Topographical localization in the olivocerebellar projection: An electrophysiological study in the cat. J. Comp. Neurol. 154.287-302.

Armstrong, D.M., and R.F. Schild (1978a) An investigation of the cerebellar cortico-nuclear projections in the rat using an autoradiographic tracing method I. Projections from the vermis. Brain Res. 141:1-19.

Armstrong, D.M., and R.F. Schild (1978b) An investigation of the cerebellar cortico-nuclear projections in the rat using an autoradiographic tracing method. 11. Projections from the hemisphere. Brain Res. 14120-45.

Batini, C., and 0. Pompeiano (1958) Effects of rostro-medial and rostro- lateral fastigial lesions on decerebrate rigidity. Arch. Ital. Biol. 96.315- 329.

Batton, R.R., A. Jayaraman, D. Ruggiero, and M.B. Carpenter (1977) Fasti- gial efferent projections in the monkey. An autoradiographic study. J. Comp. Neurol. 174:281-306.

Beitz, A.J., and V. Chan-Palay (1979a) The medial cerebellar nucleus in the rat: nuclear volume, cell number, densit~y and orientation. Neuroscience 4:31-45.

Beitz, A.J., and V. Chan-Palay (1979b) A Golgi analysis of neuronal wgani- zation in the medial cerebellar nucleus of the rat. Neuroscience 4:47- 63.

Beyerl, B.D., L.F. Borges, B. Swearingen, and R.L. Sidinan (1982) Parasag- ittal organization of the olivocerebellar projection in the mouse. J. Comp. Neurol. 209:339-346.

Brodal, A. (1980) Olivocerebellocortical projection in the cat as determined with the method of retrograde axonal transport of horseradish peroxidase 2. Topographical pattern in relation to the longitudinal subdivision of the cerebellum. In J. Courville, C. de Montigny, and Y. Lamarre (eds): The Inferior Olivary Nucleus: Anatomy and Physioloby, pp. 187-205.

Brodal, A,, 0. Pompeiano, and F. Walberg (1962) The Vestibular Nuclei and their Connections. Anatomy and Functional Correlations. The Hender- son Trust Lectures. Edinburgh: Oliver and Boyd.

Campbell, N.C., and D.M. Armstrong (1983) Topographical localization in the olivocerebellar projection in the rat: An autoradiographic study. Brain Res. 275.235-249.

Chan-Palay, V. (1973) Afferent axons and their relations with the colomns and swirls of neurons in the nucleus lateralis of the cerebellum: A light microscope study. Z. Anat. Entwickl.-(3esch. 14P1-21.

Chan-Palay, V. (1977) Cerebellar Dentate Nucleus. New York: Springer Verlag.

Chan-Palay, V., G. Nilaver, S.L. Palay, M.C. Beinfeld, E.A. Zimmerman, J- Y. Wu, and T.L. ODonahue (1981) Chemical heterogeneity in cerebellar Purkinje cells: Existence and coexistence of glutamic acid decarboxylase-like and motilin-like immunoreaetivities. Proc. Natl. Acad. Sci. U S A . 78:7787-7791.

Chan-Palay, V., S.L. Palay, J.T. Brown, and C. Van Itallie (1977) Sagittal organization of olivocerebellar and reticulocerebellar projections: Auto- radiographic studies with 35S-methionine. Exp. Brain Res. 30:561-576.

Chan-Palay, V., S.L. Palay, and J-Y. Wu (1982) Sagittal cerebellar micro- bands of taurine neurons: Immunocytochemical demonstration by using antibodies against the taurine synthesizing enzyme cysteine sulfinic acid decarboxylase. Proc. Natl. Acad. Sci. U S A . 79:4221-4225.

Courville, J. (1975) Distribution of olivocerebellar fibers demonstrated by a radioautoradiographic tracing method. Brain Res. 95253-263.

Courville, J., and N. Diakiw (1976) Cerebellar corticonuclear projection in the cat. The vermis of anterior and posterior lobes. Brain RILS. 110;1-20.

Courville, J., and F. Faraco-Cantin (1980) Topography of the olivocerebellar projection: An experimental study in the cat with an autoradiographic tracing method. In J. Courville, C. de Montigny, and Y. Lamarre (eds): The Inferior Olivary Nucleus: Anatomy and Physiology. NY: Raven Press, pp. 235-277.

Eager, R.P. (1963) Efferent cortico-nuclear pathways in the cerebellum of the cat. J. Comp. Neurol. 120:81-104.

Eager, R.P. (1966) Patterns and mode of termination of cerebellar corticonuclear pathways in the monkey ( M C Z C ~ Q mulatta). J. Comp. Neurol. 126t551-566.

Galfre, G., S. Howe, C. Milstein, G.W. Butcher, and J.C. Howard (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266~550-552.

Goodman, D.C., R.E. Hallett, and R.B. Welch (1963) Patterns of localization in the cerebellar corticonuclear projections of the albino rat. J. Comp. Neural. 121r51-67.

Groenewegen, H.J., and J. Voogd (1977) The parasagittal zonation within the olivocerebellar projection. I. Climbing fiber distribution in the vermis of cat cerebellum. J. Comp. Neurol. 174:417-488.

Haines, D.E. (1975) Cerebellar cortical efferents of the posterior lobe vermis in a Prosimian primate (Galago) and the tree shrew (Tupaza). J. Comp. Neural. I63:21-40.


Haines, D.E. (1976) Cerebellar cortico-nuclear and corticovestibular fibers of the vermis in a prosimian primate (Galago senegalensis). J. Comp. Neurol. 170:67-96.

Haines, D.E., J.L. Culberson, and G.F. Martin (1976) Laterality and topography of cerebellar cortical efferents in the opossum (Didelphis marsupialis virginiana). Brain Res. 106:152-158.

Haines, D.E., and S.L. Koletar (1979) Topography of cerebellar corticonuclear fibers of the albino rat. Vermis of anterior and posterior lobes. Brain Behav. Evol. I6:271-292.

Haines, D.E., G.W. Patrick, and P. Satrulee (1982) Organization of cerebellar corticonuclear fiber systems. In S.L. Palay and V. Chan-Palay (eds): The Cerebellum-New Vistas. Berlin, Heidelburg, New York Springer- Verlag, pp. 320-367.

Hawkes, R., M. Colonnier, and N. Leclerc (1985) Monoclonal antibodies reveal sagittal banding in the rodent cerebellar cortex. Brain Res. 333,359-365.

Hawkes, R.B., E. Niday, and J. Gordon (1982a) A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. I19:142-147.

Hawkes, R., E. Niday, and A. Matus (1982b) MIT23: A mitochondria1 marker for terminal neuronal differentiation defined by a monoclonal antibody. Cell 28,253258.

Hawkes, R., E. Niday, and A. Matus (1982~) Monoclonal antibodies identify novel neural antigens. Proc. Natl. Acad. Sei. U.S.A. 7952410-2414.

Jansen, J., and A. Brodal(1940) Experimental studies on the intrinsic fibers of the cerebellum 11. The cortico-nuclear projection. J. Comp. Neurol. 73367-321.

Jones, D.H., and A. Matus (1974) Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient cen- trifugation. Biochim. Biophys. Acta 356,276-287.

Kuypers, H.G. J.M. (1981) Anatomy of the descending pathways. In V. Brooks (edj: Handbook of Physiology 1. The Nervous System. Bethesda, MD: Physiol. SOC., pp. 597-666.

Marani, E., and J. Voogd (1977) An acetylcholinesterase band-pattern in the molecular layer of the cat cerebellum. J. Anat. 124t335-345.

Martner, J. (1975) Cerebellar influences on autonomic mechanisms. Acta. Physiol. Scand. Suppl. 4251-42.

Moolenaar, G., and H.K. Rucker (1976) Autoradiographic study of brain- stem projections from fastigial pressor areas. Brain Res. 114t492-496.

Oertel, W.H., D.E. Schmechel, E. Mugnaini, M.L. Tappaz, and I.J. Kopin (1981) Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6:2715-2735.

Oertel, W.H., M.L. Tappaz, I.J. Kopin, D.H. Ransom, and D.E. Schmechel (1980) Production of an antiserum to brain glutamate (GAD)/cysteine sulfinate (CSD) decarboxylase. Brain Res. Bull. 5:713-719.

Oscarsson, 0. (1969) The sagittal organization of the cerebellar anterior lobe as revealed by the projection patterns of the climbing fiber system. In R. Llinas (ed): Neurobiology of Cerebellar Evolution and Develop- ment. Chicago, 11: Am. Med. Assoc., pp. 525-532.

Oscarsson, 0. (1979) Functional units of cerebellum: Sagittal zones and microzones. Trends Neurosci. 2143-145.

Oscarsson, 0. (1980) Functional organization of olivary projection to the cerebellar anterior lobe. In J. Courville, C. de Montigny, and Y. Lamarre (eds): The Inferior Olivary Nucleus: Anatomy and PhysidohT. NY: Raven Press, pp. 279-289.

Palay, S.L., and V. Chan-Palay (1974) Cerebellar Cortex. Berlin, Springer- Verlag.

Paxinos, G., and C. Watson (1982) The Rat Brain in Stereotactic Coordi- nates. New York: Academic Press.

Ramon-y-Cajal, S. (1911) Histologie du Systeme Nerveux de l'homme et des vertebrb. Paris: Maloine.

Ruggiero, D., R.R. Batton, A. Jayaraman, and M.B. Carpenter (1977) Brain stem afferents to the fastigial nucleus in the cat demonstrated by transport of horseradish peroxidase. J. Comp. Neurol. 172:189-210.

Rossum van, J. (1969) Corticonuclear and Corticovestibular Projections of the Cerebellum. Assen: Van Gorcum.

Scheibel, A. (1977) Sagittal organization of mossy fiber terminal systems in the cerebellum of the rat: A Golgi study. Exp. Neurol. 57:1067-1070.

Scott, T.G. (1963) A unique pattern of localization within the cerebellum of the mouse. J. Comp. Neurol. 122:l-8.

Sreesai, M. (1974) Cerebellar cortical projections of the opossum (Didelphis marsupialis virginiana). J. Hirnforsch. 15,529-544.

Van Gilder, J.C., and J.L. OLeary (1970) Topical projection of the olivocerebellar system in the cat: An electrophysiological study. J. Comp. Neurol. 140:69-80.

Voogd, J. (1964) The Cerebellum of the Cat. Assen: Van Gorcum. Voogd, J. (1969) The importance of fiber connections in the comparative

anatomy of the mammalian cerebellum. In R. Llinas (ed): Neurobiology of Cerebellar Evolution and Development. Chicago, IL: Am. Med. As-

Walberg, F. (1980) Olivocerebellocortical projection in the cat as determined with the method of retrograde axonal transport of horseradish peroxidase 1. Topographical pattern. In J. Courville, C. de Montigny, and Y. Lamarre (eds): The Inferior Olivary Nucleus: Anatomy and Physioioby. N Y Raven Press, pp. 169-186.

Walberg, F., and J. Jansen (1964) Cerebellar corticonuclear projection stud- ied experimentally with silver impregnation methods. J. Hirnforsch. 6:338-354.

s ~ . , pp. 493-514.

immunocytochemical demonstration of topographic ordering of purkinje cell axon terminals in the...

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