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Advanced Studies in Biology, Vol. 2, 2010, no. 4, 159 - 178 Cerebellar Abnormalities Based on Chemical Neuroanatomy in Cav2.1 Mutant, Rolling Mouse Nagoya K. Sawada Laboratory of Anatomy Department of Physical Therapy Faculty of Medical and Health Sciences Tsukuba International University Tsuchiura, Ibaraki 300-0051, Japan [email protected] Y. Fukui Department of Anatomy and Developmental Neurobiology University of Tokushima Graduate School Institute of Health Biosciences Tokushima, 770-8503, Japan Abbreviations: CI, crus I of the ansiform lobule; CII, crus II of the ansiform lobule; CRF, corticotropin-releasing factor; F, flocculus; Gr. granule layer; HSP25, small heat shock protein 25; mol, molecular layer; P, Purkinje cell layer; PF, paraflocculus; sim, lobulus simplex; TH, tyrosine hydroxylase 160 K. Sawada and Y. Fukui Abstract This review summarizes cerebellar abnormalities based on chemical neuroanatomy in the ataxic mutant, rolling mouse Nagoya. This mutant mouse carries a mutation in the gene encoding for the α1A subunit of the voltage-gated P/Q-type Ca2+ channel (Cav2.1), as do tottering, leaner, rocker and wobbly mice, and is a useful model for human neurological diseases such as episodic ataxia type-2 and familial hemiplegic migraine. Whereas no obvious cerebellar deformations are detected in rolling mice, the altered functions of cerebellar cortical neurons can be revealed as expressional changes in molecules related to the regulation of intracellular Ca2+. The non-enzymatically active form of tyrosine hydroxylase (TH) was ectopically expressed in zebrin II-positive/heat shock protein 25-negative Purkinje cell population. Although types 1, 2 and 3 ryanodine receptors (RyR1, RyR2 and RyR3) were uniformly expressed in cerebellar Purkinje and/or granular cells in wild-type mice, the rolling cerebellum uniformly exhibited a reduced RyR1 expression in Purkinje cells and a slight enhancement of RyR3 expression in granular cells. An increased Cav2.1 channel expression was also revealed in the deep cerebellar nuclei of rolling mice. On the other hand, increased levels of neurotransmitters/neuromodulators in cerebellar afferents were noted. Corticotropin-releasing factor (CRF) immunostaining in climbing and mossy fiber subsets was more intense in the rolling cerebellum than in the wild-type mouse cerebellum without changes in the overall distribution. Serotonin immunostaining was also increased in serotonergic fiber terminals in the vermis of rolling mice. Thus, cerebellar abnormalities have been identified in rolling mice by chemical neuroanatomical techniques. Such neurochemical modulation of the cerebellar cortex may well be the key to understanding the pathogenesis of cerebellar ataxia of Ca2+ channelopathy. Keywords: Ca2+ channelopathy, zebrin, tyrosine hydroxylase, ryanodine receptor, CRF, serotonin Cerebellar abnormalities in rolling mice 161 1. Introduction Voltage-dependent Ca2+ channels play a crucial role in Ca2+-regulated neuronal functions including membrane excitability and neurotransmitter release [22]. The α1A subunit (Cav2.1) is one gene family of the pore-forming subunits of the Ca2+ channel, and it play roles in ion selectivity and voltage sensitivity [40]. The Cav2.1 channel classifies as the P/Q-type Ca2+ channel by its electrophysiological and pharmacological characteristics [23,58]. Rolling mouse Nagoya is one of the Cav2.1 channel mutant mice first described by Oda (1973) [33], and exhibits motor deficits characterized by their frequent lurching and abnormal cyclic movements of the hindlimbs when walking, yet not exhibiting epilepsy as seen in tottering and leaner [36,57]. The gene mutation is present at the nucleotide position 3784 of the Cav2.1 channel gene by the substitution of C to G, changing a charge-neutralizing amino acid from arginine to glycine at position 1262 in the Cav2.1 channel protein (Figure 1) [26]. Several different autosomal mutations in the Cav2.1 channel gene have been reported in the mouse such as tottering, leaner [12], rocker [62] and wobbly mice [60]. These mutant mice have provided important information on the underlying pathogenesis in the human Ca2+ channelopathy, such as episodic ataxia type-2, and familial hemiplegic migraine (Figure 1) [35]. Cav2.1 channel dysfunctions are thought to contribute fundamentally to the motor deficits of rolling mice and other Cav2.1 mutant mice [46]. However, the underlying pathogenesis of this mutant is not easily understood. This review summarizes our and other studies about cerebellar abnormalities in rolling mice based on chemical neuroanatomy. 2. Expression of mutated Cav2.1 channels In the cerebellum, the Cav2.1 channel immunostaining appeared in somata and primary dendrites of Purkinje cells, granule cell somata, and neuropils of the molecular layer (parallel fibers). Such Cav2.1 channel staining was uniform throughout the cerebellar cortex, and the expression levels of this channel were not altered in Cav2.1 mutant mice [12,20]. However, the altered function of the mutated Cav2.1 channel may be involved in abnormal Purkinje cell morphology. The rolling mouse cerebellum has shown a number of abnormal swellings (or "torpedoes”) of Purkinje cell axons [44,46] and Purkinje cell axonal terminals [43]. These predict impairments of axonal transport and neurotransmission to the deep cerebellar neurons [7]. In the molecular layer of rolling mice [38] and other Cav2.1 mutants [37], multiple synapses of the dendritic spines of Purkinje cells at single parallel fiber varicosity have been revealed by electronmicroscopic observations. The presence of Cav2.1 channel immunostaining in the neuropil of the molecular layer of rolling cerebellum [51] suggests the multiple synapses of the Purkinje cell dendritic 162 K. Sawada and Y. Fukui spines is related to the altered function of mutated Cav2.1 channel. Cav2.1 channel immunostaining was also found in a few neurons in the deep cerebellar nuclei [46]. Unlikely in the cerebellar cortical neurons, an enhanced Cav2.1 channel immunostaining in deep cerebellar neurons was revealed by an increase in the number of Cav2.1 channel immunostained neurons in rolling mice [51]. While the role of the Cav2.1 channel in deep cerebellar neurons has not been fully evident, an increased postsynaptic Ca2+ influx is known to induce the long-term depression (LTD) of GABAA receptor-mediated synaptic transmission in deep cerebellar neurons [27]. Therefore, an increased expression of the Cav2.1 channel may compensate for the altered function of this channel and thereby induce the LTD in the deep cerebellar neurons of rolling mice. 3. Expression of ryanodine receptors (RyRs) Ryanodine receptors (RyRs) are Ca2+ release channels on the membrane of the endoplasmic reticulum (ER), and play a role in Ca2+ mobilization from the ER [22]. Three types of RyRs (RyR1, RyR2 and RyR3) have been identified, and all of them are known to be expressed in the mouse cerebellum [15,19,25]. In the rolling mouse cerebellum, RyR1 expression in Purkinje cells was uniformly reduced through all cerebellar lobules [47]. Such expressional change in RyR1 may be linked with the expression of the mutated Cav2.1 channel because of the co-presence of RyR1 and Cav2.1 channel immunostaining in the rolling Purkinje cells [47]. RyR1 is known to have a direct conformational linkage with the L-type Cav1.3 channel (known as the dihydropyridine receptor; DHPR) in the neuronal plasma membrane of the brain [28]. Co-presence of RyR1 and the Cav1.3 channel in the rolling Purkinje cells [47] suggests that DHPR-RyR1 complexes are reduced by a diminished RyR1 expression. This may reduce Ca2+ release from the ER through RyR1 (DHPR-induced Ca2+ release) in Purkinje cells of rolling mice. A small increase in RyR3 expression but no change in RyR2 expression has been revealed in cerebellar granular cells of rolling mice [47]. RyR3 is known to mediate an increased neuronal vulnerability to L-glutamate [21]. Apoptotic granular cells increased in number in the rolling cerebellum [55]. Therefore, a slight increase of RyR3 expression may enhance the vulnerability to excitotoxicity of cerebellar granular cells in rolling mice. 4. Tyrosine hydroxylase (TH) expression TH is the first-step enzyme for catecholamine synthesis, and is mainly expressed in catecholaminergic neurons in the brain. Our immunohistochemical study revealed that TH expression in the cerebellar Purkinje cells was observed in wild-type mice at low levels, but was ectopically enhanced in rolling mice (Figure 2A-D) [49]. Ectopic TH expression was not specific to the Cav2.1 mutants, because it is observed in other allelic mutant mice such as dilute-lethal [42,49,50] Cerebellar abnormalities in rolling mice 163 and Neimann-Pick type C1 [39]. The role of TH in the Purkinje cells has not been fully understood, while it is thought to vary among allelic groups. In Niemann-Pick type C mice, L-DOPA levels in the cerebellum of (NPC) increased in correlation with the number of TH-immunopositive Purkinje cells [61], suggesting a role played by TH in L-DOPA production in the Purkinje cells. Conversely, the enzymatically active, phosphorylated form of TH is not detected immunohistcohemically in the cerebellum of rolling and dilute-lethal mice [41]. This may mean that TH in the Purkinje cells of these mutants does not catalyze conversion of tyrosine to L-DOPA, and is not related to catecholamine synthesis. In vitro studies reveal that the Ca2+ response element is present in the TH promoter, and non-catecholaminergic neurons express the TH transcripts following Ca2+ influx [17,29,31]. Therefore, ectopic TH expression in Purkinje cells of rolling and dilute-lethal mice may reflect an increase of intracellular Ca2+ concentrations. Ectopic TH expression is seen in particular subsets of Purkinje cells, which form a zebrin II-like stripe array (Figure 2A, B) [42,49,52]. However, the distribution of TH-immunopositive Purkinje cells did not completely overlapped with that of the zebrin II-immunopositive Purkinje cells. TH-immunopositive Purkinje stripes were present in the zebrin II-defined transverse zones, i.e., lobules VI to VII (central zone) and ventral IX and lobule X (nodular zone) (Figure 2E, F)[52]. TH-immunopositive Purkinje cell stripes in these regions make an alternating array to heat-shock protein HSP25 (HSP25)-immunopositive Purkinje cell stripes (Figure 2F.G) [52]. Since the mutated Cav2.1 channel was expressed uniformly through all Purkinje cells of the rolling cerebellum [51], the ectopic TH expression may not be linked with the expression of the mutated Cav2.1 channel. The ectopic TH expression in the rolling Purkinje cells may be related to the corticotropin-releasing factor (CRF) immunopositive-climbing fiber projections (see next section). 5. Innervation of CRF-Immunopositive climbing fibers CRF serves as a neuromodulator in normal cerebellar circuits to enhance the glutamate sensitivity and reduce the GABA sensitivity of Purkinje cells [3,5] by suppressing the afterhyperpolarization [13]. It is also known that the CRF from climbing fibers serves a critical role in the induction of long-term depression (LTD) at the parallel fiber synapses of Purkinje cells [24]. In normal mice, CRF-immunopositive climbing fibers are organized into parasagittal stripes in the cerebellum that resemble the zebrin II-positive Purkinje cell stripes [18,45]. Our previous studies revealed an enhanced CRF immunostaining in the climbing fiber subsets without changes in the overall distribution (Figure 3)[53], and an increased expression of CRF mRNA in the inferior olivary origin of these CRF-positive climbing fibers in rolling mice [48]. Therefore, increased levels of CRF in the climbing fibers of the rolling mouse cerebellum may alter the excitability and/or LTD induction of the Purkinje cells. 164 K. Sawada and Y. Fukui Our previous immunohistochemical study also revealed that the ectopic TH expression in the rolling Purkinje cells appeared corresponding to the terminal fields of CRF-immunopositive climbing fibers (Figure 3) [53]. Therefore, the ectopic TH expression in the rolling Purkinje cells may not be linked with the mutated Cav 2.1 expression, and may rather involve increased levels of CRF in a particular component of olivocerebellar tracts. 6. Innervation of CRF-immunopositive mossy fibers CRF is also contained in the particular subsets of cerebellar mossy fibers, which are distributed prominently in the vermis, lobulus simplex, crus I of the ansiform lobule, copula pyramidis, flocculus and ventral paraflocculus, and rarely in the crus II of the amsiform lobule, paramedian lobule and dorsal paraflocculus in wild-type mice [53]. Our immunohistochemical study revealed that CRF-immunopositive mossy fiber terminals were denser in the rolling cerebellum than in the wild-type mouse cerebellum (Figure 4A-D) [53]. In particular, CRF-immunopositive mossy fiber terminals innervating calretinin-immunopositive unipolar brush cells (UBCs) selectively increased in number in the lobule X of rolling mice (Figure 4A-D) [1]. UBCs are a class of glutamatergic interneurons in the vestibulocerebellum, and play an important role in cerebellar control of equilibrium by amplifying excitatory inputs from vestibulocerebellar mossy fibers (Figure 4E) [10,11,32]. Therefore, CRF may alter the UBC-mediated excitatory pathway selectively in the lobule X via mossy fibers in the cerebellum of rolling mice. This may disturb functions of lobule X, such as the cerebellar adaptation for linear motion of the head [6]. 7. Innervation of serotonergic (5-HTergic) fibers 5-HTergic fibers have been demonstrated immnohistochemically in the cerebellar cortex [4,56], and originated prominently from the lateral paragigantocellular reticular nucleus of medulla oblongata [4]. Cerebellar cortical neurons have been shown to express various types of 5-HTergic receptors such as 5-HT1A , 5-HT2A, 5-HT3, 5-HT5A, and 5-HT7 [14,16]. Serotonin (5-HT) decreases the Purkinje cell activity either directly or via Lugaro cells in the cerebellar circuits [9,54]. In rolling mice, 5-HT-immunopositive fibers in the cerebellar vermis and hemisphere were much denser in wild-type mice (Figure 5) [44]. Our results show a possible linkage between the disturbance of cerebellar 5-HTergic inputs with cerebellar ataxia, together with previous studies on other hereditary ataxic mutant mice that have shown enhancements of 5-HT metabolism and the density of 5-HTergic fibers in the cerebellum [34,59]. Our immunohistochemical study further revealed an increase of 5-HT immunostaining in the origins of cerebellar 5-HTergic fibers (the lateral Cerebellar abnormalities in rolling mice 165 paragigantocellular reticular nucleus of the medulla oblongata) in rolling mice [44]. Consistently, the 5-HT concentration increased in both the cerebellum and brainstem of rolling mice [30]. The Cav2.1 channel is expressed in medullary raphe nuclei [8], and a blockade of this channel is known to enhance the firing rate of medullary raphe neurons by reducing afterhyperpolarization [2]. Therefore, the Cav2.1 channel dysfunction in rolling mice may increase the excitability of medullary raphe neurons, resulting in 5-HT biosynthesis. 8. Conclusions Although the rolling mouse exhibits motor deficits characterized by frequent lurching and abnormal cyclic movements of the hindlimbs when walking [36,57], no obvious cerebellar deformations have been reported in this mutant. Our studies of rolling mice have revealed several cerebellar abnormalities based on chemical neuroanatomy; a reduced expression of RyR1 in all Purkinje cells [47]; an ectopic expression of TH in zebrin II positive/HSP25 negative Purkinje cells [52]; and an increased number and/or density of CRF-immunopositive climbing fibers, CRF-immunopositive mossy fibers, and 5-HT-immunopositive fibers [1,53]. 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Cerebellar abnormalities in rolling mice 171 Figure 1 Membrane topology of the Cav2.1 channel. Arrows indicate positions of amino acid substitutions in mice (tgrol, rolling; tg, tottering; tgla, leaner; tgrkr, rocker; and tgwb, wobbly) and humans (familial hemiplegic migraine; and episodic ataxia type-2). The drawing was made with reference to Sawada et al, 2000 [46]. 172 K. Sawada and Y. Fukui Figure 2 Tyrosine hydroxylase (TH) immunostaining in cerebellum. Dorsal views of the TH whole mount immunostained cerebellum in rolling mouse (A) and wild-type mouse (B). In the rolling mouse cerebellum, an array of TH-immunopositive stripes was obvious throughout the vermis and hemispheres, and these TH stripes were delineated by TH-immunopositive transverse zones in lobules VII and X. On the other hand, no TH-immunopositive stripes were detected by whole mount immunostaining in the wild-type mouse cerebellum. Section TH immunostaining in the lobule X of rolling mouse (C) and wild-type mouse (D). TH immunostaining was heterogeneous with alternating rows of TH-immunopositive/immunonegative Purkinje cell subsets in the rolling mouse cerebellum. In wild-type mouse, a few TH-immunopositive Purkinje cells (indicated by open arrowheads) were sparsely present. Comparison of whole-mount staining patterns among anti-zebrin II (E), anti-TH (F) and anti-HSP25 (G) in the rolling mouse cerebellum. TH-immunopositive stripes and HSP25-immunopositive stripes were alternately arranged in the lobule VIb, whereas zebrin II immunostaining was uniformly expressed in this region. Bar = 2 mm in (A) [applied to (B)]. Bar =200 μm in (C) [applied to (D)]. Bar = 1 mm in (E) [applied to (E), (F) and (G)]. (Adapted from Ref. 52). Cerebellar abnormalities in rolling mice 173 Figure 3 Topological relationship between corticotrophin-releasing factor (CRF)-immunopositive cerebellar afferents and tyrosine hydroxylase (TH)-immunopositive Purkinje cells in cerebellum. Double-section immunostaining for CRF (blue) and TH (brown) in the lobule II of vermis of rolling mouse (A) and wild-type mouse (B). CRF-immunopositive mossy fiber terminals were numerously present in the granular layer of rolling mice, but few (arrowheads) in the granular layer of wild-type mice. In rolling mice, CRF-immunopositive climbing fiber terminals were also present in the molecular layer, and were arranged into parasagittal stripes indicated by half brackets. TH-immunopositive Purkinje cells were distributed within terminal fields of 174 K. Sawada and Y. Fukui CRF-immunopositive climbing fibers. Drawings of representative transverse sections of the cerebellum of rolling (C-E) and wild-type mice (F-H) that illustrate distributions of CRF-immunopositive climbing fiber terminals (lines), CRF-immunopositive mossy fiber terminals (small dots), and TH-immunopositive Purkinje cells (red dots). In the rolling cerebellum, the distribution of TH-immunopositive Purkinje cells corresponded to terminal fields of CRF-immunopositive climbing fibers, but not CRF-positive mossy fiber terminals. In wild-type mice, CRF-immunopositive climbing fibers were seen, while few TH-positive Purkinje cells were distributed within CRF-immunopositive climbing fiber terminals in lobules IX and X of the vermis and the flocculus. Bar =200 μm in (A) [applied to (B)]. (Adapted from Ref. 53) Cerebellar abnormalities in rolling mice 175 Figure 4 Corticotrophin-releasing factor (CRF)-immunoreactive mossy fiber terminals in the vestibulocerebellum. Double-immunofluorescence for CRF (Cy3; red color) and calretinin (FITC; green) in the lobule X of the vermis of rolling (A) and wild-type mice (B). CRF-immunopositive mossy fiber terminals were denser in rolling mice than in wild-type mice, and some of those fibers were closely apposed to calretinin-immunopositive UBCs. Open arrowheads indicate CRF-immunopositive mossy fiber terminals. Closed arrowheads indicate CRF-immunopositive mossy fiber terminals adjoining calretinin-immunopositive UBCs. (C). The number of CRF-immunopositive mossy fiber terminals in the granular layer of the lobule X and flocculus. Two-way analysis of variance (ANOVA) revealed significant effects on group (F1,28 = 18.67, P < 0.001), region 176 K. Sawada and Y. Fukui (F1,28 = 13.22, P < 0.002), and group x region interaction (F1,28 = 5.15, P < 0.05). The number of CRF-immunopositive mossy fibers in the lobule X but not in the flocculus were significantly higher in rolling mice than in wild-type mice (*P < 0.001; contrast tests based on two-way ANOVA). (D). The number of CRF-immunopositive mossy fiber terminals adjoining calretinin-immunopositive UBCs in the granular layer of the lobule X and flocculus. Two-way ANOVA revealed significant effects on group (F1,28 = 9.16, P < 0.01), region (F1,28 = 10.71, P < 0.005) and group x region interaction (F1,28 = 7.14, P < 0.05). The number of CRF-immunopositive mossy fibers adjoining calretinin-immunopositive UBCs in the lobule X but not the flocculus were significantly higher in rolling mice than in wild-type mice (*P < 0.001; contrast tests based on two-way ANOVA). A schematic diagram of the unipolar brush cell (UBC)-mediated microcircuit in the mouse vestibulocerebellum (E). UBCs (green) receive extrinsic mossy fibers of the primary and secondary vestibulocerebellar tracts (red), and give rise to axons (intrinsic mossy fibers), which innervate granular cells (blue) and other UBCs. Thus, UBCs make excitatory feed-forward pathways within the basic cerebellar circuits. Bar = 50 μm in (A) [applied to (B)]. (Adapted from Ref. 1) Cerebellar abnormalities in rolling mice 177 Figure 5 Serotonin (5-HT) immunostaining in the cerebellar cortex of vermis (lobule III) (A, B) and hemisphere (paramedian lobule) (C, D). 5-HT-immunopositive fibers (indicated by arrowheads) were denser in either cerebellar region of rolling mouse (A, C) than that of wild-type mouse (B, D). Drawings of representative transverse sections of the cerebellum of rolling (E-I) and wild-type mice (F-J) that illustrate distributions of 5-HT-immunopositive fibers terminals. In rolling mice, 5-HT positive fibers in the cerebellar vermis and hemisphere were much denser in wild-type mice. Bar = 50 μm in (A) [applied to (B), (C) and (D)]. 178 Received: September, 2010 K. Sawada and Y. Fukui