Alternating array of tyrosine hydroxylase and heat shock protein 25 immunopositive Purkinje cell stripes in zebrin II-defined transverse zone of the cerebellum of rolling mouse Nagoya

Abstract

The present study examined the spatial organization of tyrosine hydroxylase (TH) immunopositive Purkinje cells in the cerebellum of rolling mouse Nagoya with reference to the distribution pattern of the cerebellar compartmentation antigen, heat shock protein 25 (HSP25). Whole-mount immunostaining revealed a striking pattern of parasagittal stripes of TH staining in the rolling mouse cerebellum but not in the control cerebellum. Although the TH stripes resembled the zebrin II stripes in the rolling cerebellum, these two distributions did not completely overlap. The TH stripes were present in the lobules VI and VII (central zone), the lobule X (nodular zone), and the paraflocculus, where zebrin II immunostaining was uniformly expressed. Double immunostaining revealed that TH stripes were aligned in an alternative fashion with HSP25 stripes within the caudal half of lobule VIb, lobules IXb and X, and paraflocculus. Some, but not all, TH stripes shared boundaries with HSP25 stripes. These results revealed an alternating array of TH immunopositive Purkinje cell subsets with HSP25 immunopositive Purkinje cells in the zebrin II-defined transverse zone of the rolling mouse cerebellum. The constitutive expression of HSP25 may prevent the ectopic expression of TH in zebrin II immunopositive Purkinje cell subsets.

Introduction

Rolling mouse Nagoya is an ataxic mutant mouse characterized by a severe ataxic gait and abnormal hindlimb extension (Oda, 1973). It carries a mutation in a recessive autosomal allele on chromosome 8 that encodes a gene for the P/Q-type Ca²⁺ channel α_1A subunit (Ca_v2.1) (Mori, et al., 2000), as do tottering, leaner (Fletcher et al., 1996), rocker (Zwingman et al., 2001) and wobbly mice (Xie et al., 2007). In humans, defects in this gene are responsible for several neurological disorders such as episodic ataxia type-2 and spinocerebellar ataxia type-6 (Ophoff et al., 1996). Strong Ca_v2.1 channel immunostaining has been demonstrated in Purkinje cells (Sawada et al., 2000, Sawada et al., 2001), and selective reductions in the voltage sensitivity and activity of the P/Q-type Ca²⁺ channels have also been shown in these neurons within the cerebellum of rolling mice (Mori et al., 2000).

The Purkinje cells of the cerebellar cortex form a complex arrangement of parasagittal stripes and transverse zones, which are reflected in the diversity of gene expression patterns (Hawkes, 1997, Herrup and Kuemerle, 1997, Oberdick et al., 1998, Armstrong and Hawkes, 2000). The antigen zebrin II is the most extensively studied expression marker of Purkinje cell phenotypes (Brochu et al., 1990: = aldolase C — Ahn et al., 1994). Currently, four transverse expression domains (“zones”) have been defined in the cerebellum based on the expression pattern of zebrin II: the anterior zone (AZ: lobules I–V), the central zone (CZ; lobules VI to VII), the posterior zone (PZ; lobules VIII to dorsal IX), and the nodular zone (NZ; ventral lobule IX to lobule X) (Ozol et al., 1999, Armstrong and Hawkes, 2000, Sillitoe and Hawkes, 2002). Each transverse zone is further subdivided into a reproducible array of parasagittal stripes — for example in the AZ and PZ by the expression pattern of zebrin II (Brochu et al., 1990, Eisenman and Hawkes, 1993, Ozol et al., 1999, Sillitoe and Hawkes, 2002) and phospholipase Cß4 (Sarna et al., 2006, Marzban et al., 2008), and in the CZ and NZ by the expression pattern of the small heat shock protein HSP25 (Armstrong and Hawkes, 2000) and human natural killer cell antigen 1 (HNK 1: Eisenman and Hawkes, 1993, Marzban et al., 2004). Such Purkinje cell patterns are central to normal cerebellar organization (Sillitoe and Hawkes, 2002), and its disruption triggers severe motor control problems (Howell et al., 1997, Gallagher et al., 1998, Croci et al., 2006).

Ca_v2.1 mutant mice such as rolling, tottering, leaner and pogo mice have shown an ectopic expression of tyrosine hydroxylase (TH) in particular subsets of Purkinje cells (Abbott et al., 1996, Sawada et al., 1999, Jeong et al., 2001, Sawada and Fukui, 2001). Such ectopic TH expression is thought to reflect an increase in the levels of intracellular Ca²⁺, and may be involved in the pathogenesis of motor deficits in Ca_v2.1 mutants (Sawada and Fukui, 2001). The distribution of TH immunopositive Purkinje cells resembles, but is not completely overlapped with, that of zebrin II immunopositive Purkinje cell subsets in the Ca_v2.1 mutant cerebella (Abbott et al., 1996, Jeong et al., 2001). However, the distribution pattern of TH in the mutant cerebella has not been precisely mapped. The present study examined an attempt to map out the precise spatial organization of TH immunopositive Purkinje cells in the cerebellum of rolling mice using whole-mount immunostaining. We also compared this pattern to that of the spatial expression of the Purkinje cell compartmentation antigen, HSP25.