Topographical anatomy of the cerebellum in the guinea pig, Cavia porcellus

Brain Research 965 (2003) 159–169 www.elsevier.com / locate / brainres

Research report

Topographical anatomy of the cerebellum in the guinea pig, Cavia porcellus Matt Larouche, Chi Diep, Roy V. Sillitoe, Richard Hawkes* Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2 N 4 N1 Accepted 5 December 2002

Abstract Zebrin II / aldolase C is expressed in a stereotyped array of parasagittal bands and transverse zones in the cerebellum of many animals including birds and mammals. Here, section and whole mount immunohistochemistry has been used to characterize the expression of zebrin II in the cerebellum of the adult guinea pig. Purkinje cells in the adult guinea pig express zebrin II immunoreactivity at three different levels of intensity—high, medium and low. This expression pattern reveals an arrangement of parasagittal bands that are symmetrical about the midline and reproducible between individuals. The expression of zebrin II divides the vermis into four transverse expression domains from rostral to caudal: an anterior zone consisting of one zebrin II-immunoreactive band at the midline and at least three symmetrical bands laterally; a central zone, in which broad zebrin II-positive bands are separated by narrow bands of zebrin II-negative Purkinje cells that disappear caudally to leave no overt compartmentation; a posterior zone consisting of alternating bands of zebrin II-positive and -negative Purkinje cells; and finally, a nodular zone in which nearly all Purkinje cells express zebrin II. In the anterior and posterior hemispheres, zebrin II is also expressed in a banded pattern. These rostrocaudal and mediolateral patterns of zebrin II expression are reminiscent of those in other mammals including rabbit, rat, and mouse, and suggest that there may be a fundamental compartmental organization of the cerebellum that is conserved in mammals.  2002 Elsevier Science B.V. All rights reserved. Theme: Motor systems and sensory motor integration Topic: Cerebellum Keywords: Zebrin II; Compartmentation; Zone; Immunohistochemistry; Whole mount

1. Introduction Histologically, the cerebellum is a simple structure, composed of three major laminae: the molecular, Purkinje cell, and granular layers. Despite the uniform cytoarchitecture, there are elaborate underlying patterns that can be identified on the physiological, biochemical, and molecular levels (reviewed in Refs. [17–19,28]). In the rodent cerebellum in particular, molecular expression domains, mutations, afferent projections, and functional physiological studies all suggest that the cerebellum may be divided into rostrocaudal transverse zones and mediolateral bands (reviewed in Refs. [3,17,18,28,34]). *Corresponding author. Tel.: 11-403-220-5712; fax: 11-403-2108109. E-mail address: [email protected] (R. Hawkes).

Zebrin II is a widely studied marker of cerebellar heterogeneity. Anti-zebrin II is a monoclonal antibody that recognizes a 36-kDa protein [7] that cloning studies suggest is the respiratory isoenzyme, aldolase C [1,19]. In the rodent cerebellum, zebrin II expression is restricted to subsets of Purkinje cells that are arranged in parasagittal bands distributed symmetrically about the midline (rat [7]; mouse [14]). Zebrin II-immunoreactive bands (P 1 ) are separated by parasagittal bands of Purkinje cells that do not express zebrin II (P 2). This parasagittal pattern is highly reproducible between individuals both in number and arrangement [7]. In rodents, zebrin II has proven useful to correlate cerebellar anatomical and functional maps (e.g. Refs. [8,9,16]) Based on differential gene expression and the phenotypes of several strains of mutant mice, the mouse cerebellum appears to be partitioned into four distinct

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(02)04160-4

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transverse zones: anterior zone (AZ: |lobules I–V), central zone (CZ: |lobules VI, VII), posterior zone (PZ: |lobule VIII) and nodular zone (NZ: |lobules IX and X). (e.g. Refs. [4,5,29]). Each zone is further subdivided mediolaterally into a unique set of bands (e.g. AZ, PZ by zebrin II expression; CZ, NZ by expression of the small heat shock protein HSP25 [4,5]). A similar organization is also present in the rabbit cerebellum [31]. Cerebellar compartmentation [21], as revealed using zebrin immunohistochemistry, is phylogenetically conserved. For example, zebrin expression distinguishes two classes of Purkinje cells in various fish [7,23,26], chick [32], opossum [13], rabbit [31], rat [7], mouse [14], hamster (Sanchez and Hawkes, 2002, unpublished data) and various primates (e.g. Ref. [25]) including human (e.g. Ref. [30]). The guinea pig cerebellum has proven to be a useful model for studies of development and is of particular value in investigations using functional whole brain preparations (e.g. Refs. [6,11,22]). However, there have been few investigations that concentrate on cerebellar heterogeneity in the guinea pig. We have therefore extended our comparative studies of cerebellar compartmentation to describe the general topography of the adult guinea pig as revealed by using zebrin II / aldolase C immunohistochemistry, as seen both in sectioned and in whole mount preparations. In particular, we have focused on the zonation of the vermis. The data reveal that the guinea pig cerebellum is patterned in a manner similar to that described previously in other mammals and therefore suggests the presence of a common topographical ground plan for the mammalian cerebellum.

2. Materials and methods All animal procedures have been approved by the University of Calgary Animal Care and Use Committee in accordance with the Guide to the Care and Use of Experimental Animals from the Canadian Council for Animal Care. Adult albino guinea pigs (Cavia porcellus) weighing 250–300 g were obtained from Charles River (St. Constant, Quebec, Canada).

2.1. Perfusion and sectioning A lethal overdose of sodium pentobarbital (Somnotol, MTC Pharmaceuticals, Ontario, Canada; 100 mg / kg body weight) was administered intraperitoneally. Once toe-pinch reflexes were abolished, the animal was perfused transcardially through the left ventricle with 0.9% ice cold saline followed by either freshly prepared ice cold 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 or Bouin’s fixative (VWR, catalogue number 15204-240). Brains were removed from the skulls and immersed in the same fixative and stored at 4 8C for at least 2 days. Cerebella were cryoprotected by immersion in phosphate buffer containing

0.9% NaCl (PBS) and 10% sucrose for 4 h, followed by PBS120% sucrose for 4 h, and finally at least 24 h in PBS130% sucrose. Cerebella were then frozen, embedded in OCT compound (Sakura Finetek, Torrance, CA, USA), and cryostat sectioned at 20 mm in the transverse plane. Sections were mounted on gelatin-coated slides and allowed to dry overnight at 4 8C.

2.2. Immunohistochemistry Anti-zebrin II is a mouse monoclonal antibody produced by immunization with a crude cerebellar homogenate from the weakly electric fish Apteronotus [7]: it was used directly from spent hybridoma culture medium. Sections mounted on slides were dipped in PBS and allowed to dry for at least 2 h. They were incubated in a 1% hydrogen peroxide solution in PBS for 30 min, and subsequently washed in PBS (235 min). Nonspecific binding was blocked by incubating the slides for 1 h at room temperature in PBS containing 0.1% Triton X-100 (PBST) and 10% normal goat serum (Gibco, Burlington, Ontario, Canada). Specimens were then incubated overnight at 4 8C in PBST containing anti-zebrin II at concentrations between 1:100 and 1:400. Sections were washed and subsequently incubated in PBS containing HRP conjugated rabbit anti-mouse IgG (1:200–1:400, Dako Diagnostics, Ontario, Canada) for 3 h at room temperature. Following this incubation the slides were washed in PBS (335 min) and immunolabeling was revealed by incubating in PBS containing 0.6 mg / ml diaminobenzidine (DAB) and 0.005% H 2 O 2 . Sections were dehydrated through an ascending alcohol series, cleared in Histoclear (Diamed, Mississauga, Ontario, Canada), and coverslipped with Entellan mounting medium (BDH, Toronto, Canada).

2.3. Whole mount immunohistochemistry Whole mount immunohistochemistry was performed by using a modification of a protocol designed for mice [33]). Adult guinea pigs were anesthetized with a lethal dose of Somnotol. Once reflexes were completely abolished, the animals were decapitated and the brains were removed from the skulls. The cerebella were then separated from the rest of the brain and immersion fixed in 4% paraformaldehyde in PBS at 4 8C for at least 48 h. During this period the fixative was changed two to three times to remove blood and throughout the protocol, gentle rocking was employed. After allowing the cerebellum to bathe in fixative, it was post-fixed overnight at 4 8C in Dent’s fixative (4 parts absolute methanol (MeOH):1 part dimethysulfoxide (DMSO)). Next the cerebellum was incubated in Dent’s bleach (4 parts MeOH:1 part DMSO:1 part 30% hydrogen peroxide (H 2 O 2 ) for |8 h until completely white, then dehydrated in 2330 min each in MeOH. The cerebellum was passed through 4–5 cycles of chilling to 280 8C and thawing to room temperature in MeOH

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followed by overnight incubation in MeOH at 280 8C. For peroxidase anti-zebrin II immunohistochemistry the cerebellum was rehydrated for 90 min each in 50% MeOH, 15% MeOH, and PBS then enzymatically digested in 10 mg / ml proteinase K (.600 Units / ml: Boehringer-Mannheim, Laval, Quebec, Canada) in PBS for 5 min at room temperature. After rinsing 3310 min in PBS, non-specific antibody binding was attenuated by incubating in 2% non-fat skim milk powder, 0.1% Triton X-100 in PBS (PBSMT) for 6–8 h at room temperature, changing the PBSMT 2–3 times during this period. The cerebellum was incubated for 48 h in primary antibody diluted either 1:150 or 1:300 in PBSMT containing 5% DMSO at 4 8C. Afterwards it was rinsed in PBSMT (332 h at 4 8C) and incubated overnight at 4 8C in rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) diluted 1:200 in PBSMT15% DMSO. Finally, the tissue was rinsed in PBSMT (433 h each at 4 8C followed by a final overnight rinse), incubated in 0.2% bovine serum albumin in PBST for 2 h at room temperature. Antibody binding sites were revealed by incubating with a PBS solution containing 0.05% DAB and 0.015% H 2 O 2 . The reaction was stopped by quenching it in PBST that contained 0.05% sodium azide. Photomicrographs were captured with a SPOT Cooled Color digital camera (Diagnostic Instruments, Sterling Heights, MI, USA) and montages were assembled in Adobe Photoshop 4.0. The images were cropped and corrected for brightness and contrast but not otherwise manipulated.

2.4. Western blotting Adult guinea pig and mouse tissues were isolated, homogenized in 10 volumes (w / v) of ice-cold sample buffer (20% glycerol, 0.004% bromophenol blue, 5% sodium dodecyl sulfate in Tris-buffered saline (0.125 M Tris–HCl, pH 6.8)) and stored at 270 8C until required. Samples were thawed, further diluted (1:5–1:10) in sample buffer containing 10% b-mercaptoethanol, and boiled for 5 min. 20 ml of sample was then loaded onto a polyacrylamide gel (4.5% stacking gel, 10% running gel). Protein molecular weight markers (biotinylated-broad range, 4 mg per lane; BioRad Laboratories, Hercules, CA, USA) were treated in the same way and loaded in parallel. Samples were electrophoresed at 80 V for 20 min and then at 125 V for 45 min. After electrophoresis, proteins were electroblotted onto an Immobilon-P membrane (Millipore, Mississauga, ON, Canada) at 70 V for 3 h. For immunostaining, membranes were rinsed with PBS (pH 7.6) and then blocked for 1 h in 5% skim milk in PBS10.1% Triton X-100 (PBST2). Mouse monoclonal anti-zebrin II was diluted 1:500 in PBST2. Membranes were incubated with primary antibody at 4 8C for 18 h with gentle agitation. Membranes were washed 3320 min in PBS and then incubated for 1 h with biotinylated goat anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted

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1:2000 in PBST2. Blots were washed 6310 min in PBS and binding was detected by using the Vectastain ABC Staining Kit (Vector Laboratories). This was followed by 335-min washes in PBS buffer (pH 7.6) and a 5-min reaction period in 0.6 mg / ml DAB10.005% H 2 O 2 in PBS.

3. Results Early descriptions of the gross anatomy of the guinea pig cerebellum were given by Allen [2]. The lobule nomenclature of the vermis used here is based on the anatomical atlas of the guinea pig of Cooper and Schiller [10]; the hemispheric lobules are named by analogy with rats and mice. Western blots of guinea pig brain reveal a single antizebrin II immunoreactive band that is indistinguishable from the immunoreactive band seen in Western blots of mouse cerebellum (Fig. 1A). The cellular localization of zebrin II resembles that seen in other mammals, including rat [20], mouse [14], and rabbit [31]. Immunocytochemical staining of sections through the guinea pig cerebellum shows immunoreactivity is prominent in the Purkinje cells (Fig. 1B,C). Reaction product is deposited throughout the Purkinje cell cytoplasm, including the somata, axons and dendrites (Fig. 1B,C) but is excluded from the nuclei (Fig. 1C, arrowheads). No other neurons in the cerebellum express zebrin II, but in cases where immunostaining is strong, there is evidence of weak immunoreactivity in glial cells (data not shown: see Ref. [35]). The expression of zebrin II is heterogeneous between Purkinje cells and transverse sections immunoperoxidasestained by using anti-zebrin II reveal an elaborate array of bands. However, in contrast to other rodents (e.g. rat [7]; mouse [14]) where there is a clear-cut distinction between those Purkinje cells that express zebrin II (the P 1 bands) and those that do not (the P 2 bands), the expression pattern in the guinea pig appears more complex. In sectioned material we have defined three levels of zebrin II expression that are reproducible between individuals: high (cells that exhibit immunoreactivity in the somata and dendrites, e.g. Fig. 1C: H), medium (lighter deposits of DAB product in the somata and dendrites, e.g. Fig. 1C: M) and low (little or no immunoreactivity in the dendritic tree, and light or no labeling in the somata, e.g. Fig. 1C: L). Thus, most Purkinje cells appear to express at least low levels of zebrin II, particularly in their somata. It is more difficult to see these quantitative distinctions, with respect to somata, in the whole mount images because they lie so deep within the tissue. Since the cell bodies are not visible and the dendrites dominate the whole mount images, the labeling intensities described here are generally based on dendritic levels of zebrin II immunoreactivity in the molecular layer (e.g. Fig. 2B: high—the P11, P21 and P31 stripes; medium—the P41 stripe; low—the P12 stripe, indicated by the bracket). Despite this complication,

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Fig. 1. Zebrin II is expressed by Purkinje cells in the guinea pig cerebellum. (A) Western blot comparing guinea pig (gp) and mouse (ms). Anti-zebrin II recognizes an identical band in each case. (B) Transverse section through the adult guinea pig cerebellum immunoperoxidase-stained with anti-zebrin II. Peroxidase reaction product is deposited in the Purkinje cell dendrites in the molecular layer (ml), in the somata in the Purkinje cell layer (pcl) and in axons in the white matter (wm). Immunoreactive axons are not prominent in the granular layer (gl). Purkinje cell nuclei are unreactive. No other neurons appear to express zebrin II. (C) A higher magnification view of immunostained Purkinje cells. Three levels of immunostaining intensity are illustrated (bracketed at the bottom of the figure: low (L), medium (M) and high (H)). In the area bracketed by ‘H’ peroxidase reaction product is deposited throughout the cytoplasm of the somata and dendrites but is excluded from the nuclei (arrowheads). Purkinje cells with low levels of zebrin II immunoreactivity, generally have DAB deposits only in the somata, while cells with medium (M) and high (H) immunoreactivity contain DAB reaction product in the dendrites in addition to the soma. Other labels are as in ‘B’ (D) Not all Purkinje cells express zebrin II. Anti-zebrin II immunoperoxidase staining of a transverse section through lobule IIIb of the vermis reveals an array of alternating bands of immunoreactive Purkinje cells (labeled P11–P31) separated by similar zebrin II-negative bands (P12, P22: nomenclature as in the mouse, in accordance with Eisenman and Hawkes [14]). Scale bar in B5100 mm; C550 mm; D5500 mm.

Fig. 2. Zebrin bands in the anterior zone of the guinea pig cerebellum. (A) Cartoon view of the anterior cerebellum showing the distribution of zebrin II expression. Dark grey shading indicates high expression levels, light grey indicates medium levels, and low levels of expression are in white. Lobules in the vermis are labeled IIa–V; the lobulus simplex (LS), and hemispheric lobule V (HV) are labeled in the hemisphere. (B) Whole mount peroxidase immunohistochemistry of the guinea pig cerebellum seen in anterior view. The P11–P41 bands are labeled (as 1–4 for reasons of clarity). Lobules IIa–V in the vermis are also labeled. The bracketed area delineates an area of low zebrin II immunoreactivity. (C) A transverse section through the anterior cerebellum immunoperoxidase stained for zebrin II. The P11, P21, P31, P12 and P22 bands are labeled (‘P’ is omitted for clarity). Lobules II–V in the vermis are also labeled. Scale bar51 mm in A, B and C. (D) Higher magnification view of lobules IIIb–V immunoperoxidase stained in whole mount, to show the possible interpolation of a zebrin II1 band between P11 and P31 (P21). (E) Higher magnification view of lobules IIIb–V immunoperoxidase stained in transverse section. The interpolation of a zebrin II1 band between P11 and P21 is not clearly evident here. (F) High magnification view of the lobulus simplex. Medium intensity zebrin positive bands are labeled, with ‘P’ omitted for clarity. (G) Whole mount immunoperoxidase stained cerebellum. Two sublobules of the lobulus simplex (LS) are visible as well as crus I (CI). Scale bar5500 mm (applies to D–G).

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the antigenic topography of the guinea pig cerebellum is highly reminiscent of that in rat and mouse. Therefore, the same terminology has been adopted: high and medium levels of zebrin II expression are classified as zebrin II-positive (P 1 ) while low-expressing Purkinje cells appear to be equivalent to the zebrin II-negative (P 2) array found in rats and mice. There are ample precedents in mouse to differentiate subsets within both the zebrin II-positive and the zebrin II-negative subsets (e.g. Refs. [4,24,29]), and for at least transient zebrin II expression by the Purkinje cells in the P 2 bands (e.g. Ref. [24]). Since the zebrin II immunoreactive patterns in the guinea pig cerebellum resembles that seen in the rat, the same nomenclature used for naming zebrin II-positive and -negative bands in rat [20] and mouse [14] has been adopted for guinea pig. Accordingly, zebrin II-positive areas in the vermis are labeled P11–P41, and in the hemispheres P4a1, P4b1, P5a1, P5b1, P61, and P71. As in rat and mouse, bands of Purkinje cells with low levels of zebrin II immunoreactivity are named according to the zebrin II-positive band immediately medial to it.

3.1. Vermis The compartmentation of the anterior lobe as revealed using zebrin II immunoreactivity is illustrated in Fig. 2, and summarized in a cartoon in Fig. 2A. In all lobules of the anterior lobe vermis (I–V), Purkinje cells expressing high levels of zebrin II immunoreactivity form a narrow band at the midline (P11), with flanking zebrin II1 bands about 500 mm symmetrically to either side (Fig. 2B,C). In early descriptions of the rat cerebellum, these bands were labeled as P21. However, more detailed analysis in mouse (e.g. Refs. [14,15]), opossum [13] and rabbit [31] suggest they are better named P31, and this is the terminology used here for guinea pig. Lateral to P31 differences are seen in the pattern of zebrin II expression between individual lobules in the anterior lobe. In lobules I and II, a broad band of zebrin II-immunoreactive Purkinje cells, which occupies over half the width of each lobule, flanks P31 laterally on either side. More caudally, in whole mount views of lobules IIIb–V, this broad lateral band resolves into two additional bilateral bands of medium zebrin II1 expression (P41, P51: Fig. 2B). In transverse sections, this is not evident, and the cerebellum lateral to P32 appears uniformly zebrin II-immunoreactive (Fig. 2C). In the caudal portion of lobule IV and throughout lobule V the P31 band widens. In whole mount, it appears that this widening is due to the interpolation of an additional zebrin II-immunoreactive band between P11 and P31 separated by Purkinje cells that express zebrin II at medium intensity (Fig. 2D,E). The interdigitation of a P21 band in between P11 and P31 has previously been described in opossum [13], mouse [14,15] and rabbit [31]. At the boundary in the vermis between lobules V–VIa the zebrin II expression pattern undergoes a transition (Fig. 3). The whole mount views of lobules VIa, VIb and VIIa

reveal a narrow P11 band flanked by a broad P21 band (Fig. 3A,B). Caudally, in lobule VIIb, the P11 band can no longer be discerned (arrow; Fig. 3B). Moving caudally from lobule VIa to lobule VIIa, the P21 band broadens, resulting in a P21 band in lobule VIIa that is roughly twice as wide as in lobule VIa. In the most lateral vermian region, medial to the lobulus simplex in lobule VIa, there are two narrow zebrin II1 bands (Fig. 3B, arrowheads). Caudally in lobule VIIa only a single high-intensity immunoreactive band is apparent in the lateral vermis. The transition from the anterior lobe pattern in lobule VI is clearly seen in transverse sections (Fig. 3C) where alternating bands in the anterior (lobule VIa) supplant a continuous field of zebrin II-immunoreactive Purkinje cells (lobule VIb). In sectioned material a midline P11 band separated from the rest of the vermis by P12 bands cannot be distinguished in the anterior aspect of lobule VIb. The distribution of anti-zebrin II immunoreactivity in lobules VIII–X is summarized as a cartoon (Fig. 4A), and illustrated both in whole mount (Fig. 4B) and transverse section (Fig. 4C). Lobule VIII is subdivided into parasagittal bands of high-zebrin II expressing cells interposed with bands of Purkinje cells exhibiting medium-intensity immunoreactivity (Fig. 4B–D). The bands of zebrin II-positive Purkinje cells in lobule VIII are generally much wider than in the anterior lobe vermis (Fig. 4B–D): here is a midline P11 band flanked by at least two highly immunoreactive bands (P21, P31; Fig. 4A,D). The Purkinje cells in the P 2 bands in lobule VIII express medium levels of zebrin II immunoreactivity. In sublobules IXa and IXb, thin bands of zebrin-negative Purkinje cells at the midline and about 500 mm on either side of the midline divide the molecular layer into four broad parasagittal bands of highly-immunoreactive Purkinje cells (Fig. 4E). More caudally, in lobules IXc and X, almost all Purkinje cells express zebrin II (Fig. 4B,C).

3.2. Hemispheres In the lobulus simplex, bands of medium-intensity immunoreactivity diverge from the rostrocaudal axis at about 458 and are separated by parallel, low-intensity bands: following the rat nomenclature these are P41–P71 (Fig. 2F,G). Purkinje cells in crus I and crus II uniformly exhibit medium-level zebrin II immunoreactivity (Fig. 3B, CI and CII, respectively), and in the paraflocculi almost all Purkinje cells are strongly immunoreactive for zebrin II (data not shown). Some interindividual variability is observed in whole mount preparations of the anterior ventral paraflocculus, with regions of high immunoreactivity juxtaposed by regions of low-immunoreactivity but this is not observed consistently (data not shown). In the paramedian lobules in the posterior hemisphere there multiple bands of highly positive bands of Purkinje cells separated by bands of medium-intensity staining: by analogy with other rodents these are named P4a1, P4b1, P5a1, P5b1 and a fused P61 / 71 (Fig. 4F,G). As for the zebrin II-positive

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Fig. 3. Zebrin bands in the central zone of the guinea pig cerebellum. (A) Cartoon view of the dorsal cerebellum showing the distribution of zebrin II expression in the anterior and central zones. Dark shading indicates high expression levels of immunoreactivity, grey indicates medium levels, and low levels of expression are in white. Lobules in the vermis are labeled V–VIIb; the paramedian (PM), crus I (CI) and II (CII), and simplex (LS) are labeled in the hemisphere. (B) Whole mount peroxidase immunohistochemistry of the guinea pig cerebellum seen in dorsal view. Lobules are labeled as in A. Scale bar51 mm (A, B). (C) Transverse section through lobules VIa,b immunoperoxidase stained for zebrin II. The P11–P31 bands in VIa are labeled. These disappear in VIb to leave a uniform zebrin II-immunoreactive band of Purkinje cells that extends almost 2 mm either side of the midline. Scale bar5500 mm.

bands of the lobulus simplex these bands also diverge from the rostrocaudal axis at a 458 angle. In the copula pyramidis, all Purkinje cells express zebrin II immunoreactivity at a medium intensity and compartmentation is not reliably observed.

4. Discussion Two previous studies have characterized heterogeneous protein expression in the guinea pig cerebellum. First, Neustadt et al., [27] characterized banded muscarinic

receptor heterogeneity in the granular and molecular layers. Secondly, Dino et al. [12] described a differential distribution of calretinin immunoreactivity in the granular layer, associated primarily with the unipolar brush cells. The current study provides further evidence of heterogeneous protein expression in the molecular layer and suggests that despite its relatively homogeneous appearance, the guinea pig cerebellum is composed of smaller heterogeneous units. Zebrin II immunoreactivity reveals at least three subpopulations of Purkinje cells arranged into rostrocaudal parasagittal bands. Two pieces of evidence strongly sug-

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gest that zebrin II is evolutionarily conserved. First, Western blots of guinea pig tissue produce a single immunoreactive band with a molecular weight that is identical to the band revealed in concurrent blots of mouse tissue and resembles that revealed in Western blots using tissue from fish, and other mammals [7,13,23,25,31]. Secondly, like all other animals studied so far, the zebrin II antigen is expressed in only one type of neuron in the guinea pig cerebellum—Purkinje cells. Cloning studies have revealed that in mice the zebrin II antigen is 98% homologous to the metabolic enzyme aldolase C [1]. Thus far the function of zebrin II / aldolase C in a Purkinje cell subset has not been determined. The parasagittal bands of Purkinje cells expressing zebrin II in the guinea pig cerebellum are reminiscent of those in other cerebella previously described—rat [20], opossum [13], mouse [14,29] and rabbit [31]. This suggests that there may a common ground plan used for the organization of the cerebellum in all mammals. However, zebrin II expression in the guinea pig is somewhat different to that described to date in other mammals. In rodents such as rats and mice, zebrin II is detectable in one subset of Purkinje cells while the remaining Purkinje cells express none. In the guinea pig, however, zebrin II immunoreactivity is detected in the somata of nearly all Purkinje cells, but in the dendrites, the protein is expressed at different levels. These differential dendritic expression levels have been exploited as the basis of the compartmental classification proposed here. Previous studies have shown that the vermis of the mouse [29] and rabbit [31] cerebellum is comprised of four transverse zones. The pattern of zebrin II expression in the guinea pig suggests that this cerebellum is also composed of four rostrocaudal expression zones: two striped for zebrin II expression (AZ, PZ), and two uniformly zebrin II-immunoreactive (CZ, NZ). Based on a zebrin II pattern that is reminiscent of the expression in rabbit and mouse, the guinea pig AZ includes all lobules of the classical anterior lobe (I–V) [14,29,31]. In this region, zebrin II is expressed in bands that are symmetrical about the midline. This zone is characterized by the presence of one prominent band of high zebrin expression (P11) flanked on either side by a band of high-level zebrin II expression

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(P21). Additional bands of medium-level zebrin II expression are also found, particularly in lobules III–IV. A transition in expression from alternating high and low bands of zebrin II immunoreactivity to a nearly-uniform field of high-expressing Purkinje cells defines the CZ as lobules VI and VII. This transition is recapitulated in lobules VI and VII of both mouse and rabbit [29,31]. Lobule VIa in the guinea pig consists of P11 and P21 bands that are much thicker than P11 and P21 in lobule V. Moving caudally from lobule VIa to VIIb more Purkinje cells express zebrin II in each successive lobule until caudal lobule VIIb in which nearly all Purkinje neurons are highly immunoreactive. A pattern of zebrin II expression where bands of high-expressing Purkinje neurons are interspersed by equal-width bands of medium-expressing zebrin II cells defines the PZ in mouse and rabbit. A similar striped pattern of expression is also found in lobule VIII of the guinea pig cerebellum, suggesting that this lobule constitutes a PZ in this animal also. These characteristics are consistent with lobule VIII in several other species, including mouse [14,29], rat [7] and rabbit [31]. Finally, in lobule IXa, highly-expressing zebrin II positive Purkinje cells again dominate, although thin bands of low-expressing Purkinje cells are still evident. Moving caudally to lobule X, the bands of low-expressing Purkinje cells gradually disappear. Once again this pattern of expression is reminiscent of NZ zebrin II expression in mouse and rabbit cerebella. Although the zebrin II expression pattern in lobules IX and X suggests that protein expression in the molecular layer of these two lobules is homogeneous, this is not the case. Muscarinic receptors are expressed in alternating high- and low-zones in both lobules of the guinea pig cerebellum [27]. Furthermore the granular layer in both of these lobules reveals a parasagittally organization of unipolar brush cells [12]). Although the topographical relationship between these granular layer stripes and Purkinje cell compartments is not known, it suggests that zebrin II expression does not reveal the full complexity of cerebellar organization in the guinea pig. This conclusion supported by data obtained from mouse demonstrating that Purkinje cells in both the CZ and the NZ express zebrin II homogeneously but express heat shock protein HSP25 [4]

Fig. 4. Zebrin bands in the posterior lobe of the guinea pig cerebellum. (A) Cartoon view of the posterior cerebellum showing the distribution of zebrin II expression. Dark shading indicates high expression levels, pale grey indicates medium levels, and low levels of expression are in white. Lobules VIIa–X are labeled in the vermis and the paramedian (PM), crus I (CI) and II (CII), and copula pyramidis (CP) lobules are labeled in the hemisphere. The paraflocculus (PF) is also labeled. (B) Whole mount peroxidase immunohistochemistry of the guinea pig cerebellum seen in posterior view with lobules labeled as described in A. (C) Transverse section through the posterior cerebellum immunoperoxidase stained for zebrin II. Lobules VIIa–IXb are labeled. Scale bar51 mm (A, B, C). Scale bar51 mm (A–C). (D) Immunoperoxidase stained transverse section through the midline vermis of lobule VIII to show the distribution of zebrin II immunoreactivity. Scale bar5500 mm (applies to ‘D’ and ‘E’). (E) Traverse section through lobule IX, immunoperoxidase stained for zebrin II. The section illustrates gaps in the mostly positive array of Purkinje cells (arrows) in lobule IXa. These gaps are apparent in whole mounts as well as in transverse sections. In lobule IXb, zebrin II staining becomes homogenous and shows no obvious spaces. (F) Transverse section through the posterior hemisphere. At least four bands of high-intensity immunoreactive Purkinje cells are seen in the paramedian lobule (PM). (G) Whole mount immunohistochemistry of the guinea pig cerebellum illustrating zebrin II immunoreactivity in the posterior hemisphere. The paramedian lobule (PM) is labeled. Scale bar5500 mm (applies to ‘F’ and ‘G’).

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and an L7 / pcp-2-lacZ transgene (e.g. Ref. [29]) in parasagittal bands. Finally, the pattern of zebrin II expression appears to be conserved across species (e.g. compare the AZ in wholemounts of mouse [32], rabbit [31] and guinea pig), with the result that a similar array of parasagittal bands can be identified in each. This conservation of both bands and zones suggests that there is a basic cerebellar ground plan that has been conserved through evolution. It also supports the hypothesis that changes in cerebellar size between members of different species is accomplished by the expansion or contraction of a constant set of bands and zones, rather than by the addition of new ones.

Acknowledgements

[13]

[14]

[15]

[16]

[17] [18]

We thank Estrella Gonzales for technical help and Dr Keith Sharkey for guinea pigs. These studies were supported by a grant from the Canadian Institutes of Health Research (RH).

References

[19] [20]

[21] [22]

[1] A.H. Ahn, S. Dziennis, R. Hawkes, K. Herrup, The cloning of zebrin II reveals its identity with aldolase C, Development 120 (1994) 2081–2090. [2] J. Allen, The associative processes of the guinea pig. A study of the psychical development of an animal with a nervous system well medullated at birth, J. Comp. Neurol. 14 (1904) 294–339. [3] C.L. Armstrong, R. Hawkes, Pattern formation in the cerebellar cortex, Biochem. Cell Biol. 78 (2000) 551–562. [4] C.L. Armstrong, A.M. Krueger, R.W. Currie, R. Hawkes, Constitutive expression of the 25 kDa heat shock protein Hsp25 reveals novel parasagittal bands of Purkinje cells in the adult mouse cerebellar cortex, J. Comp. Neurol. 416 (2000) 383–397. [5] C.L. Armstrong, A.M. Krueger, R.W. Currie, R. Hawkes, Expression of heat-shock protein Hsp25 in mouse Purkinje cells during development reveals novel features of cerebellar compartmentation, J. Comp. Neurol. 429 (2001) 7–21. [6] A.L. Babalain, P.P. Vidal, Floccular modulation of vestibuloocular pathways and cerebellum-related plasticity: an in vitro whole brain study, J. Neurophysiol. 84 (2000) 2514–2528. [7] G. Brochu, L. Maler, R. Hawkes, Zebrin II: A polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum, J. Comp. Neurol. 291 (1990) 538–552. [8] G. Chen, C.L. Hanson, T.J. Ebner, Functional parasagittal compartments in the rat cerebellar cortex: an in vivo optical imaging study using neutral red, J. Neurophysiol. 76 (1996) 4169–4174. [9] V. Chockkan, R. Hawkes, Functional and antigenic maps in the rat cerebellum: zebrin compartmentation and vibrissal receptive fields in lobule IXa, J. Comp. Neurol. 345 (1994) 33–45. [10] G. Cooper, A.L. Schiller, Anatomy of the Guinea Pig, Harvard University Press, Cambridge, MA, 1975. [11] D. Coren, Y. Yarom, Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: questioning the role of the parallel fibers, Proc. Natl. Acad. Sci. USA 95 (1998) 15032–15036. [12] M.R. Dino, F.H. Willard, E. Mugnaini, Distribution of unipolar

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

brush cells and other calretinin immunoreactive components in the mammalian cerebellar cortex, J. Neurocytol. 28 (1999) 99–123. ´ C. Jacobson, R. Hawkes, Organization and postnatal L. Dore, development of zebrin II antigenic compartmentation in the cerebellar vermis of the grey opossum, Monodelphis domestica, J, Comp. Neurol. 291 (1990) 431–449. L.M. Eisenman, R. Hawkes, Antigenic compartmentation in the mouse cerebellar cortex: zebrin and HNK-1 reveal a complex, overlapping molecular topography, J. Comp. Neurol. 335 (1993) 586–605. E. Gallagher, B.W. Howell, P. Soriano, J.A. Cooper, R. Hawkes, Cerebellar abnormalities in the disabled (mdab1 -1 ) mouse, J. Comp. Neurol. 402 (1998) 238–251. J.S. Hallem, J.H. Thompson, G. Gundappa-Sulur, R. Hawkes, J.G. Bjaallie, J.M. Bower, Spatial correspondence between tactile projection patterns and the distribution of the antigenic Purkinje cell markers anti-zebrin I and anti-zebrin II in the cerebellar folium crus IIA of the rat, Neuroscience 93 (1999) 1083–1094. R. Hawkes, An anatomical model of cerebellar modules, Prog. Brain Res. 114 (1997) 39–52. R. Hawkes, C. Gravel, The modular cerebellum, Prog. Neurobiol. 36 (1991) 309–327. R. Hawkes, K. Herrup, Aldolase C / zebrin II and the regionalization of the cerebellum, J. Mol. Neurobiol. 6 (1996) 147–158. R. Hawkes, N. Leclerc, Antigenic map of the rat cerebellar cortex: the distribution of parasagittal bands as revealed by monoclonal anti-Purkinje cell antibody mabQ113, J. Comp. Neurol. 256 (1987) 29–41. K. Herrup, B. Kuemerle, The compartmentalization of the cerebellum, Annu. Rev. Neurosci. 20 (1997) 61–90. F. Jursky, N. Nelson, Developmental expression of the neurotransmitter transporter NTT4, J. Neurosci. Res. 55 (1999) 24–35. M.J. Lannoo, L. Ross, L. Maler, R. Hawkes, Development of the cerebellum and its extracerebellar Purkinje cell projection in teleost fishes as determined by zebrin II immunocytochemistry, Prog. Neurobiol. 37 (1991) 329–363. N. Leclerc, C. Gravel, R. Hawkes, Development of parasagittal zonation in the rat cerebellar cortex: MabQ113 antigenic bands are created postnatally by the suppression of antigen expression in a subset of Purkinje cells, J. Comp. Neurol. 273 (1988) 399–420. ´ A. Parent, R. Hawkes, The compartmentalizaN. Leclerc, L. Dore, tion of the monkey and rat cerebellar cortex: zebrin I and cytochrome oxidase, Brain Res. 506 (1990) 70–78. J. Meek, T.G.M. Hafmans, L. Maler, R. Hawkes, The distribution of zebrin II in the gigantocerebellum of the mormyrid fish Gnathonemus petersii compared with other teleosts, J. Comp. Neurol. 316 (1992) 17–31. A. Neustadt, A. Frostholm, A. Rotter, Topographical distribution of muscarinic cholinergic receptors in the cerebellar cortex of the mouse, rat, guinea pig, and rabbit: a species comparison, J. Comp. Neurol. 272 (1988) 317–330. J. Oberdick, S.L. Baader, K. Schilling, From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum, Trends Neurosci. 21 (1998) 383–390. K. Ozol, J.M. Hayden, J. Oberdick, R. Hawkes, Transverse zones in the vermis of the mouse cerebellum, J. Comp. Neurol. 412 (1999) 95–111. A.V. Plioplys, J. Thibault, R. Hawkes, Selective staining of a subset of Purkinje cells in human cerebellum with monoclonal antibody mabQ113, J. Neurol. Sci. 70 (1985) 245–256. M. Sanchez, R.V. Sillitoe, P.J.E. Attwell, M. Ivarsson, S. Rahman, C.H. Yeo, R. Hawkes, Compartmentation of the rabbit cerebellar cortex, J. Comp. Neurol. 444 (2002) 159–173. R. Sillitoe, C.C. Logan, R. Hawkes, En-2 expression in the developing chick cerebellum, Soc. Neurosci. Abstr. 26 (2000) 308. R.V. Sillitoe, R. Hawkes, Whole mount immunohistochemistry: a

M. Larouche et al. / Brain Research 965 (2003) 159–169 high throughput screen for patterning defects in the mouse cerebellum, J. Histochem. Cytochem. 50 (2002) 235–244. [34] J. Voogd, M. Glickstein, The anatomy of the cerebellum, Trends Neurosci. 21 (1998) 370–375. [35] E.U. Walther, M. Dichgans, S.M. Maricich, R.R. Romito, F. Yang, S.

169

Dziennis, S. Zackson, R. Hawkes, K. Herrup, Genomic sequences of aldolase C (zebrin II) direct lacZ expression exclusively in nonneuronal cells of transgenic mice, Proc. Natl. Acad. Sci. USA 95 (1998) 2615–2620.