Purkinje cells zebrins

Hallem JS, Thompson JH, Gundappa-Sulur G, Hawkes R, Bjaalie JG, Bower JM (1999) 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 Ila of the rat. Neuroscience 95 1083-1094. 

Fig. 14. Phaseolus vulgaris lectin-labelled climbing fibers of rat cerebellum. A, Sagittal section. B. Coronal section. Abbreviations G = granular layer M = molecular layer P = Zebrin- labelled Purkinje cells. Bar = 100 jum. Courtesy of Dr. T.J.H. Ruigrok.

IX of the caudal vermis and low activity in the hemisphere, was described by Mufson et al. (1991) for primates and man. The administration of colchicine results in the expression of NGF-R in most cerebellar Purkinje cells (Pioro and Cuello, 1988, 1990 Pioro et al, 1991). Koh et al. (1989) and Fusco et al. (1991) found NGF-R mRNA expression and NGF-R immunoreactivity in adult rats to be present in alternating Purkinje cell zones of strong and weak activity (Fig. 38C,D). This zonal pattern was also observed by Pioro and Cuello (1990). Its correspondence to the pattern of mabQ113 (Zebrin) immunoreactive zones (Hawkes and Leclerc, 1987) was noticed by Sotelo and Wassef (1991) and verified by Dusart et al. (1994) in adult rats. Lesions of the white matter, or knife cuts isolating the dorsal portion of the vermis of the rat cerebellum induces NGF-R immunoreactivity in previously unstained Purkinje cells (Martinez-Murillo et al., 1993 Dusart et al., 1994). 

N in the molecular layer of mouse cerebellum is distributed in positive and negative parasagittal bands (Scott, 1963). The distribution of cerebellar 5 -N has been reviewed by Marani (1986). Its zonal distribution in mice is very similar to the distribution of the mabQ113 (Zebrin)-positive dendrites of Purkinje cells in the molecular layer (Marani, 1986 Eisenman and Hawkes, 1989) (Figs 58A, 130, 131, 135) (Section 6.1.4.). The... 

Zebrin-immunoreactive and non-immunoreactive Purkinje cells are distributed in parallel longitudinal bands in the cortex of rat cerebellum (Hawkes et al., 1985) and distribute their axons to different parts of the cerebellar nuclei (Hawkes and Leclerc, 1986). Light microscopical observations showed that all Zebrin positive boutons on the soma and dendrites of large central nuclear cells contained GAD, and that most GAD-positive boutons on individual cells either were Zebrin-positive or -negative. The two populations of Purkinje cells, therefore, terminate on different central nuclear cells. Zebrin-positive Purkinje cells of the vermis projected to the caudal part and a majority of Zebrin-negative Purkinje cells to the rostral part of the fastigial nucleus of the rat. 

The possibility that zonally distributed differenees in size and connections of the Purkinje cells are correlated with specific chemical properties of these cells was first raised by Marani (Marani and Voogd, 1977 Marani, 1981, 1982a Marani, 1986) on the basis of the distribution of 5 -nucleotidase and acetyleholinesterase in the molecular layer and by Chan-Palay (1984) who reported a restricted distribution of certain peptides in subsets of Purkinje cells. More recently a complete pattern of alternating zones of immunoreactive and non-immunoreactive Purkinje cells was described by Hawkes and Leclerc (1986, 1987) with a Purkinje cell-specific antibody (anti Zebrin-I) in the rat and by Brochu et al. (1990) with anti-Zebrin II in the rat and other species (see Section... 

The distribution of the Zebrin-positive Purkinje cells was very similar to the distribution of the enzyme 5 -nucleotidase in the molecular layer of certain rodents (Eisenman and Hawkes, 1989). 

The distribution of 5 -nucleotidase (5 -N) (Section 3.5.) in alternate longitudinal bands of high and low enzyme activity in the molecular layer of the cerebellar cortex of the mouse (Scott, 1963,1964,1965,1967) was the first evidence for the biochemical compart-mentalization of the cerebellar cortex. The pattern of 5 -N-positive and -negative zones is complete in the sense that it is present in all the lobules of vermis and hemisphere and unequivocal, because, in the mouse at least, the bands are clearly delineated (Marani, 1986). The 5 -N band pattern is very similar, if not identical, to the more recently described distribution of Purkinje cells in the rat, reacting with Purkinje cell-specific monoclonal antibodies to Zebrin-I (mabQl 13) (Eisenman and Hawkes, 1989). 

The epitopes recognized by Hawkes family of monoclonal antibodies known as the anti-Zebrins are localized on Purkinje cells (see Section 3.1.8.). Zonal patterns that are identical or very similar to Zebrin I and II have been described for the distribution of 5 -nucleotidase (see above), the p75 low affinity nerve growth factor receptor protein in the rat (Section 3.1.10., Fig. 38), protein kinase C delta (Fig. 133) (see Section 3.1.5.) and the B30 antibody of Stainier and Gilbert (1989) (see Section 3.1.8.). Immunoreactiv-ity in mouse Purkinje cells for an antibody against HNK is partially congruent with the Zebrin negative Purkinje cells, but Zebrin+/HNK-l- Purkinje cells also exist (Hawkes, 1992 Eisenman and Hawkes, 1993). The similarity between the Zebrin pattern and the transient zonal patterns in the development of the Purkinje cell specific marker L7 is discussed in Section 6.2. 

Fig. 133. Distribution of protein kinase C delta-immunoreactive Purkinje cells in transverse section through the posterior lobe of rat cerebellum. This pattern is similar to the distribution of Zebrin. Bar = 0,5 mm. Chen and Hillman (1993a).

Differences between both patterns involve the distribution of the marker within the bands. Both for 5 -N and Zebrin I the intensity of the staining falls off in the more laterally located bands, but the sharp lateral borders and the differences in reactivity between the 5 -N-positive bands of the caudal vermis are not as clear with staining for Zebrin I. Moreover Zebrin I immunoreactivity extends into the Purkinje cell axons and compartments of Zebrin I-positive and -negative axons are present in the white matter that reflect the zonal distribution of the corresponding Purkinje cells. 

Zebrin I in rat cerebellum was compared to the distribution of AChE in the cerebellum of the rat (Boegman et al., 1988). These authors stressed the congruence of Zebrin-positive Purkinje cells with the accumulations of AChE in patches in the underlying granular layer. AChE in these patches is present in glomeruli, in certain Golgi cells and in other, unidentified components of the neuropil. 

Hawkes and Leclerc (1987) grouped the Zebrin I-positive Purkinje cells in a midline band (P1+) and seven symmetrically disposed parasagittal bands (P2-I--P8+). Hawkes and Leclerc s (1987) numbering system for the Zebrin-positive and -negative bands in rat is indicated in Fig. 136. The P- bands ofZebrin-negative Purkinje cells bear the same number as the next medial P-i- band. The pattern of Zebrin I-immunoreactive Purkinje cells is virtually identical in rat and mouse (Figs 139 and 140) (Eisenman and Hawkes,... 

VIII. P2-I- was identified in the anterior and posterior lobes, but its continuity cannot be established because it fuses with other P+ bands in lobule VII. P3+ is weakly immunoreactive in the anterior lobe, and looses its identity among the Zebrin-positive Purkinje cells of lobule VII. It reappears in caudal lobule VII and can be traced as a distinct band in lobule VIII. The apparent continuity of P3+ between the lobules VIII and IX may be false, a fusion of P3+ with P4+ into the P3+ band of lobule IX should... 

Short strips of Zebrin-positive Purkinje cells have been noticed between the P1+, P2+ and P3+ bands in the anterior lobe and the simple lobule. Two narrow strips of Zebrin-positive Purkinje cells were identified in the ansiform lobule, between P4-t and P5+. They were considered as bifurcations of these bands, that were indicated as P4+ and P5b-t with the additional strips as P4b+ and P5a+. The narrow stretches of Zebrin-positive Purkinje cells in the dorsal vermis of the anterior lobe and the simple lobule, were considered as satellite bands . They are inconstant and not necessarily bilaterally symmetrical. 

Fig. 136. The reconstruction of parasagittal bands of Zebrin I (mabQl 13-immunoreactive) Purkinje cells in the adult rat cerebellar cortex as seen from the anterior (a) and posterior (b). The band pattern is based upon the serial reconstruction of nine complete and five partial cerebella from sections cut in the horizontal plane and four complete reconstructions from sections cut coronally. Bands PI + through P7+ are labelled. Hawkes and Leclerc (1987).

Zebrin I compartmentalization in Saimiri sciurus was studied by Leclerc et al. (1990). Both in the vermis and the hemispheres clusters of Zebrin I-immunoreactive Purkinje cells were separated by weakly stained Purkinje cell somata or unstained cells. Zebrin-negative bands, therefore, are less distinct than in rodents. P1+, P2+ and P3+ bands are continuous from lobule to lobule and become narrower in the anterior lobe. P4+-P7+ bands were tentatively identified in the hemisphere, but not analysed in detail. A complementary histochemical zonation was detected for cytochrome oxidase, that was present in patches in the granular layer corresponding to the P- bands both in squirrel monkey and rat cerebellum. It is obvious from a comparison of the illustrations from the paper of Dore et al. (1990), showing the distribution of Zebrin I immunoreactivity in Purkinje cells and their axons and the zonation of AChE in monkey cerebellum, that the P2+ immunoreactivity in the anterior vermis corresponds to the X zone, and P2-... 

Fig. 138. Three different interpretations of the adult Purkinje cell compartmentalization for Zebrin in the cerebellum of adult Monodelphis, illustrated in Fig. 136, are possible. A. The P2+ (dark grey) and P3 + (medium grey) bands are continuous and unbranched from lobule I to X. B. P2+ is continuous and unbranched, but a novel Zebrin 11+ band is inserted between PI and P2 in lobules VI to X (unshaded). C. P2+ bifurcates within lobule V to give two branches in lobules VI. Dore et al. (1990).

Fig. 139. Drawings of three surface views of the mouse cerebellum, anterior, dorsal and posterior, showing the locations of the Zebrin+ bands of Purkinje cells. The Purkinje cell bands PI +-P7 + are labelled in the dorsal view (for clarity, only the numerals have been used). Note that in the vermis of the posterior lobe the immunoreactive Purkinje cells form five to seven bands (posterior and dorsal views), whereas in lobules VII and VI all vermal Purkinje cells are immunoreactive (posterior and dorsal view). This pattern gradually changes in the anterior lobe to result in three to five very narrow immunoreactive bands (anterior view). In the hemispheres there are three major immunoreactive bands of Purkinje cells on either side (P5b+, P6+, P7+) plus two sub-bands in the para vermal area of the paramedian and ansiform lobules (P4b-t, P5a+). Note too that the Purkinje cells are all Zebrin+ in the nodulus (lobule X, illustrated as indicated by arrows reflected out from the ventral surface of the cerebellum), the paraflocculus, and the flocculus. From Eisenman and Hawkes (1993).

Bands of P-path immunoreactive Purkinje cells alternate with zebrin II immuno-reactive neurons in the cerebellum of the mouse (Leclerc et al., 1992) (Fig. 134). In the P3+ band in the anterior vermis, lobule VII, VIII and dorsal IX, the P4+ band in the lobules V and VIII and the P2+ band in dorsal lobule IX the two epitopes are colocalized. The B1 monoclonal antibody of Ingram et al. (1985) also detects a subset of Purkinje cells in monkey cerebellum, but their distribution did not correspond to the distribution of Zebrin I in the squirrel monkey (Leclerc et al., 1990) or to AChE as reported by Hess and Voogd (1986). 

Fig. 140. Computer drawing of the reconstruction of the Zebrin Purkinje cells bands in the unfolded adult C57/B6 mouse cerebellum. The drawing was from immunostained 40 fim thick coronal frozen sections. The continuity of the bands has been determined as best as possible. On the left and bottom are the scales in millimeters. The two axes have different magnifications. On the right are marked the approximate boundaries of the vermal lobules. The flocculus and paraflocculus are not illustrated. One place where the data are ambiguous is within lobule V-VI, where a large number of short bands more caudally are dramatically reduced to just three at the rostral limit. It is not clear whether the P2 + or P3 + bands extend through the anterior lobe vermis (see also Fig. 139). The reconstruction data from coronal sections were not suitable to resolve the issue, so the cerebellum has also been reconstructed from horizontal sections. The upper inset panel shows the data from such a reconstruction, equivalent to the region indicated by a rectangle on the main drawing (scale in millimeters). The preferred interpretation is that the P2+ compartment does not extend far into the anterior lobe vermis, and that the first lateral Zebrin + band in lobules I-IV is continuous with P3+ (as indicated by continuous lines in the upper inset panel and as shown in the main drawing). The alternative hypothesis, that the first lateral Zebrin + band in lobules I-IV is continuous with P2+, is shown schematically in the lower inset panel. Eisenman and Hawkes (1993).

Fig. 143. Comparison between Purkinje cells of the A and B zone (open circles) retrogradely labelled from the vestibular nuclei (left side) and retrograde labelling of Purkinje cells of the X zone after an injection of WGA-HRP in the transitional region of the fastigial and posterior interposed nucleus the rat (right side). Purkinje cells of the X zone occupy the Zebrin-positive P2+ zone labelled Purkinje cells of the lateral A zone and the B zone are located in the Zebrin-negative PI- and P2- zones. Graphical reconstructions of transverse sections double labelled for HRP reaction product and Zebrin 1 immunocytochemistry. COP = copula pyra-midis CrI and II = crus I and II of the paramedian lobule PMD = paramedian lobule SI = simple lobule I-X = lobules I-X. Voogd et al. (1991b).

Fig. 144. Localization of retrogradely labelled Purkinje cells (open circles) from an injection of WGA-HRP in the dorsolateral protuberance of the medial cerebellar nucleus (left) and of anterograde transport of Phaseolus vulgaris leucaglutinin (PhaL) in climbing fibers (stripes) from the medial portion of the MAO (tectorecipient area), with respect to bands of Zebrin-I labelled Purkinje cells in the rat. Reconstructions from sections double-stained for Zebrin and WGA-HRP or PhaL. Numbering of Zebrin-immuno-reactive Purkinje cell zones according to Hawkes and Leclerc (1987). COP = copula pyramidis Crl(II) = crus I(II) of the ansiform lobule PMD =paramedian lobule SI = simple lobule I-X = lobules I-X. Voogd et al. (1991b).

The zonal disposition of Zebrin-immunoreactive and non-immunoreactive Purkinje cells has not been compared in any detail to the olivocerebellar projection. According to Gravel et al. (1987) some of the borders between Zebrin-positive and negative zones coincide with borders of certain climbing fiber strips, but these strips were not further identified. Judging from the reported identity of some of the Zebrin-positive and -negative zones with certain corticonuclear and cortico-vestibular projection zones it seems likely, that the correspondence between the zonal organization in the olivocerebellar projection and the Zebrin pattern will be close (Fig. 181). 

Zonal projections of the inferior olive to the nodulus (lobule X) and the flocculus have been substantiated in different species. Purkinje cells of these lobules are uniformly Zebrin-positive in rat, mouse, opossum, squirrel monkey (see Section 6.1.3.) and rabbit (Jaarsma unpublished observations). A compartmental subdivision of the white matter of the nodulus and the flocculus was demonstrated with AChE-histochemistry in the rabbit and the monkey (Section 6.1.5.) and correlated with the olivocerebellar projection to these lobules (Voogd et al., 1987a,b Tan et al., 1995a,b). 

Correlations between the terminations of mossy fibers and cytochemical maps are rare. Gerrits et al. (1985b) mapped the localization of cuneocerebellar fibers with respect to the borders of white matter compartments in adjacent, Haggqvist-stained sections (Fig. 202). The projections of low thoracic-lumbar cord to the cerebellum of the rat were compared to the localizations of Zebrin 1-immunoreactive Purkinje cells in surface maps of all relevant cerebellar lobules by Gravel and Hawkes (1990). These authors noticed... 

P2- (i.e. the B zone) extending into P3+ (Fig. 207). Matsushita et al. (1991), who mapped fibers from the cervical cord in Zebrin-I stained sections of rat cerebellum found less correspondence of the concentrations of rosettes with the borders of the im-munoreactive Purkinje cell zones. Ji and Hawkes (1994) showed that cuneocerebellar mossy fiber terminals are located between the concentrations of lumbar spinocerebellar mossy fiber rosettes in P1+, PI - and P2- of lobules II and III of the rat cerebellum (Fig. 207). A close correspondence between multiple patches of mossy fibers with vibrissal receptive fields and the Zebrin-negative P1-, P2- and P3- zones of lobule IX of the rat cerebellum, was observed by Chockkan and Hawkes (1994). 

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