Download Cerebellar Abnormalities Based on Chemical Neuroanatomy

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]. Such
neurochemical modulation of the cerebellar cortex may well be the key to
understanding the pathogenesis of cerebellar ataxia of Ca2+ channelopathy.
Acknowledgements. The authors wish to thank Dr. S. Oda, of the Graduate
School of Bio-Agricultural Science, Nagoya University, for providing the
rolling mice.
References
[1]
M. Ando, K. Sawada, H. Sakata-Haga, Y. G. Jeong, N. Takeda and Y.
Fukui, Regional difference in corticotropin-releasing factor immunoreactivity in
mossy fiber terminals innervating calretinin immunoreactive unipolar brush cells
in vestibulocerebellum of rolling mouse Nagoya, Brain Research, 1063 (2005),
96-101.
[2]
D. A. Bayliss, Y. W. Li and E. M. Talley, Effects of serotonin on caudal
raphe neurons: inhibition of N- and P/Q-type calcium channels and the
afterhyperpolarization, Journal of Neurophysiology, 77 (1997), 1362-1374.
[3]
G. A. Bishop, Neuromodulatory effects of corticotropin releasing factor on
cerebellar Purkinje cells: an in vivo study in the cat, Neuroscience, 39 (1990),
251-257.
[4]
G. A. Bishop and R. H. Ho, The distribution and origin of serotonin
immunoreactivity in the rat cerebellum, Brain Research, 331 (1985), 195-207.
[5]
G. A. Bishop and J. S. King, Differential modulation of Purkinje cell
activity by enkephalin and corticotropin releasing factor, Neuropeptides, 22
(1992), 167-174.
166
K. Sawada and Y. Fukui
[6]
J. A. Buttner-Ennever, A review of otolith pathways to brainstem and
cerebellum, Ann. New York Academy Science, 871 (1999), 51-64.
[7]
S. Carpenter, G. Karpati, F. Andermann and R. Gold, Giant axonal
neuropathy, Archive of Neurology, 31 (1974), 312-316.
[8]
P. J. Craig, A. D. McAinsh, A. L. McCormack, W. Smith, R. E. Beattie, J.
V. Priestley, J. L. Y. Yip, S. Averill, E. R. Longbottom, and S. G. Volsen,
Distribution of the voltage-dependent calcium channel α1A subunit throughout the
mature rat brain and its relationship to neurotransmitter pathways, Journal of
Comparative Neurology, 397 (1998), 251-267.
[9]
I. Dean, S. J. Robertson, and F. A. Edwards, Serotonin drives a novel
GABAergic synaptic current recorded in rat cerebellar Purkinje cells: a Lugaro
cell to Purkinje cell synapse, Journal of Neuroscience, 23 (2003), 4457-4469.
[10] M. R. Diño, M. G. Nunzi, R. Anelli and E. Mugnaini, Unipolar brush cells
of the vestibulocerebellum: afferents and targets, Progress in Brain Research, 124
(2000), 123-137.
[11] M. R. Diño, R. J. Schuerger, Y. B. Liu, N. T. Slater and E. Mugnaini,
Unipolar brush cell: a potential feedforward excitatory interneuron of the
cerebellum, Neuroscience, 98 (2000), 625-636.
[12] C. F. Fletcher, C. M. Lutz, T. N. O’Sullivan, J. D. Shaughnessy Jr, R.
Hawkes, W. N. Frankel, N. G. Copeland and N. A. Jenkins, Absence epilepsy in
tottering mutant mice is associated with calcium channel defects, Cell, 87 (1996),
607-617.
[13] E. A. Fox and D. L. Gruol, Corticotropin-releasing factor suppresses the
afterhyperpolarization in cerebellar Purkinje neurons, Neuroscience Letters, 149
(1993), 103-107.
[14] F. J. Geurts, E. De Schutter and J. P. Timmermans, Localization of
5-HT2A, 5-HT3, 5-HT5A and 5-HT7 receptor-like immunoreactivity in the rat
cerebellum, Journal of Chemical Neuroanatatomy, 24(2002), 65–74.
[15] G. Giannini, A. Conti, S. Mammarella, M. Scrobogna and V. Sorrentino,
The ryanodine receptor/calcium channel genes are widely and differentially
expressed in murine and peripheral tissues, Journal of Cell Biology, 128 (1995),
893-904.
[16] C. W. Kerr and G. A. Bishop, The physiological effects of serotonin are
mediated by the 5HT1A receptor in the cat’s cerebellar cortex, Brain Research,
591 (1992), 253–260.
[17] E. J. Kilbourne, B. B. Nankova, E. J. Lewis, A. McMahon, H. Osaka, D. B.
Sabban and E. L. Sabban, Regulated expression of the tyrosine hydroxylase gene
by membrane depolarization, Journal of Biological Chemistry, 267 (1992),
7563-7569.
[18] J. S. King, P. Madtes Jr., G. A. Bishop and T. L. Overbeck, The
distribution of corticotropin-releasing factor (CRF), CRF binding sites and CRF1
receptor mRNA in the mouse cerebellum, Progress in Brain Research, 114 (1997),
55-66.
Cerebellar abnormalities in rolling mice
167
[19] G. Kuwajima, A. Futatsugi, M. Niinobe, S. Nakanishi and K. Mikoshiba,
Two types of ryanodine receptors in mouse brain: skeletal muscle type
exclusively in Purkinje cells and cardiac muscle type in various neurons, Neuron,
9 (1992), 1133-1142.
[20] F. C. Lau, L. C. Abbott, I. J. Rhyu, D. S. Kim and H. Chin, Expression of
calcium channel α1A mRNA and protein in the leaner mouse (tgla/tgla) cerebellum,
Molecular Brain Research, 59 (1998), 93-99.
[21] S. Y. Lee, D. Y. Hwang, Y. K. Kim, J. W. Lee, I. C. Shin, K. W. Oh, M. K.
Lee, J. S. Lim, D. Y. Yoon, S. J. Hwang and J. T. Hong, PS2 mutation increases
neuronal cell vulnerability to neurotoxicants through activation of caspase-3 by
enhancing of ryanodine receptor-mediated calcium release, FASEB Journal, 20
(2006), 151–153.
[22] R.J. Miller, The control of neuronal Ca2+ homeostasis, Progress in
Neurobiology, 37 (1991), 255–285.
[23] I. M. Mintz, V. J. Venema, K. M. Swiderek, T. D. Lee, B. P. Bean and M.
E. Adams, P-type calcium channels blocked by the spider toxin ω-Aga-IVA,
Nature, 355 (1992), 837-829.
[24] M. Miyata, D. Okada, K. Hashimoto, M. Kano and M. Ito,
Corticotropin-releasing factor plays a permissive role in cerebellar long-term
depression, Neuron, 22 (1999), 763-775.
[25] F. Mori, M. Fukaya, H. Abe, K. Wakabayashi and M. Watanabe,
Developmental changes in expression of the three ryanodine receptor mRNAs in
the mouse brain, Neuroscience Letters, 285 (2000), 57-60.
[26] Y. Mori, M. Wakamori, S. Oda, C. F. Fletcher, N. Sekiguchi, E. Mori, N.
G. Copeland, N. A. Jenkins, K. Matsushita, Z. Matsuyama and K. Imoto, Reduced
voltage sensitivity and activation of P/Q-type Ca2+ channels is associated with the
ataxic mouse mutation rolling Nagoya (tgrol), Journal of Neuroscience, 20 (2000),
5654-5662.
[27] W. Morishita and B. R. Sastry, Postsynaptic mechanisms underlying
long-term depression of GABAergic transmission in neurons of the deep
cerebellar nuclei, Journal of Neurophysiology, 76 (1996), 59–68.
[28] J. Mouton, I. Marty, M. Villaz, A. Feltz and Y. Maulet, Molecular
interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat
brain, Biochemical Journal, 354 (2001), 597-603.
[29] K. Nagamoto-Combs, K. M. Piech, J. A. Best, B. Sun and A. W. Tank,
Tyrosine hydroxylase gene promoter activity is regulated by both cyclic
AMP-responsive element and AP-1 sites following calcium influx, Journal of
Biological Chemistry, 272 (1997), 6051-6058.
[30] T. Nakamura, M. Honda, S. Kimura, M. Tanabe, S. Oda and H. Ono,
Taltirelin improves motor ataxia independently of monoamine levels in rolling
mouse Nagoya: a model of spinocerebellar atrophy, Biological Pharmaceutical
Bulletin, 28 (2005), 2244-2247.
[31] B. Nankova, B. Hiremagalur, A. Menezes, R. Zeman and E. Sabban,
Promoter elements and second messenger pathways involved in transcriptional
168
K. Sawada and Y. Fukui
activation of tyrosine hydroxylase by ionomycin, Molecular Brain Research, 35
(1996), 164-172.
[32] M. G. Nunzi, S. Birnstiel, B. J. Bhattacharyya, N. T. Slater and E.
Mugnaini, Unipolar brush cells form a glutamatergic projection system within the
mouse cerebellar cortex, Journal of Comparative Neurology, 434 (2001), 329-341.
[33] S. Oda, The observation of rolling mouse Nagoya (rol), a new neurological
mutant, and its maintenance, Experimental Animals, 22 (1973), 281-286.
(Japanese)
[34] K. Ohisugi, K. Adachi and M. Ando, Serotonin metabolism in the CNS in
cerebellar ataxic mice, Experimentia, 42 (1986), 1245-1247.
[35] R. A. Ophoff, G. M. Terwindt, M. N. Vergouwe, R. van Eijk, P. J. Oefner,
S. M. G. Hoffman, J. E. Lamerdin, H. W. Mohrenweiser, D. E. Bulman, M.
Ferrari, J. Haan, D. Lindhout, G. B. van Ommen, M. H. Hofker, M. D. Ferrari and
R. R. Frants, Familial hemiplegic migraine and episodic ataxia type-2 are caused
by mutations in the Ca2+ channel gene CACNL1A4, Cell, 87 (1996), 543-552.
[36] J. J. Plomp, A. M. J. M. van den Maagdenberg and S. Kaja, The ataxic
Cacna1a-mutant mouse rolling Nagoya: An overview of neuromorphological and
electrophysiological findings, Cerebellum, 8 (2009), 222-230.
[37] I. J. Rhyu, L. C. Abbott, D. B. Walker and C. Sotelo, An ultrastructural
study of granule cell/ Purkinje cell synapses in tottering (tg/tg), leaner (tgla/tgla)
and compound heterozygous tottering/leaner (tg/tgla) mice, Neuroscience, 90
(1999), 717-728.
[38] I. J. Rhyu, S. Oda, C. S. Uhm, H. Kim, Y. S. Suh and L. C. Abbott,
Morphological investigation of rolling mouse Nagoya (tgrol/tgrol), cerebellar
Purkinje cells: an ataxic mutant revisited, Neuroscience Letters, 266 (1999),
49-52.
[39] J. R. Sarna, M. Larouche, H. Marzban, R. V. Sillitoe, D. E. Rancourt and
R. Hawkes, Patterned Purkinje cell degeneration in mouse models of
Niemann-Pick type C disease, Journal of Comparative Neurology, 456 (2003),
279-291.
[40] W. A. Sather, T. Tanabe, J. F. Zhang, Y. Mori, M. E. Adams and R. W.
Tsien, Distinctive biophysical and pharmacological properties of class A (BI)
calcium channel alpha 1 subunit, Neuron, 11 (1993), 291-303.
[41] K. Sawada, M. Ando, H. Sakata-Haga, X. Z. Sun, Y. G. Jeong, S. Hisano,
N. Takeda and Y. Fukui, Abnormal expression of tyrosine hydroxylase not
accompanied by phosphorylation at serine 40 in cerebellar Purkinje cells of ataxic
mutant mice, rolling mouse Nagoya and dilute-lethal, Congenital Anomalies, 44
(2004), 46-50.
[42] K. Sawada and Y. Fukui, Expression of tyrosine hydroxylase in cerebellar
Purkinje cells of ataxic mutant mice: its relation to the onset and/or development
of ataxia, Journal of Medical Investigation, 48 (2001), 5-10.
[43] K. Sawada and Y. Fukui, Torpedoes of Purkinje cell axons in deep
cerebellar nuclei of an ataxic mutant, rolling mouse Nagoya, Current
Neurobiology, 1 (2010), 169-172.
Cerebellar abnormalities in rolling mice
169
[44] K. Sawada and Y Fukui, Zebrin II expressing Purkinje cell
phenotype-related and unrelated cerebellar abnormalities in Cav2.1 mutant, rolling
mouse Nagoya, TheScientificWorldJournal, 10 (2010), 2032-2038.
[45] K. Sawada, Y. Fukui and R. Hawkes, Spatial distribution of
corticotropin-releasing factor immunopositive climbing fibers in the mouse
cerebellum: analysis by whole mount immunohistochemistry, Brain Research,
1222 (2008), 106-117.
[46] K. Sawada, H. Haga and Y. Fukui, Ataxic mutant mice with defects in
Ca2+ channel α1A subunit gene: morphological and functional abnormalities in
cerebellar cortical neurons, Congenital Anomalies, 40 (2000), 99-107.
[47] K. Sawada, E. Hosoi, M. Bando, H. Sakata-Haga, N. S. Lee, Y. G. Jeong
and Y. Fukui, Differential alterations in expressions of ryanodine receptor
subtypes in cerebellar cortical neurons of an ataxic mutant, rolling mouse Nagoya,
Neuroscience, 152 (2008), 609-617.
[48] K. Sawada, M. Kawano, H. Tsuji, H. Sakata-Haga, S. Hisano and Y. Fukui,
Over-expression of corticotropin-releasing factor mRNA in inferior olivary
neurons of rolling mouse Nagoya, Molecular Brain Research, 117 (2003),
190-195.
[49] K. Sawada, S. Komatsu, H. Haga, X. Z. Sun, S. Hisano and Y. Fukui,
Abnormal expression of tyrosine hydroxylase immunoreactivity in cerebellar
cortex of ataxic mutant mice, Brain Research, 829 (1999), 107-112.
[50] K. Sawada, S. Komatsu, H. Haga, S. Oda and Y. Fukui, Abnormal
expression of tyrosine hydroxylase immunoreactivity in Purkinje cells precedes
the onset of ataxia in dilute-lethal mice, Brain Research, 844 (1999), 188-191.
[51] K. Sawada, H. Sakata-Haga, M. Ando, N. Takeda and Y. Fukui, An
increased expression of Ca2+ channel α1A subunit immunoreactivity in deep
cerebellar neurons of rolling mouse Nagoya, Neuroscience Letters, 316 (2001),
87-90.
[52] K. Sawada, H. Sakata-Haga and Y. Fukui, 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,
Brain Research, 1343 (2010), 46-53.
[53] K. Sawada, H. Sakata-Haga, S. Hisano and Y. Fukui, Topological
relationship between corticotropin-releasing factor-immunoreactive cerebellar
afferents and tyrosine hydroxylase-immunoreactive Purkinje cells in a hereditary
ataxic mutant, rolling mouse Nagoya, Neuroscience, 102 (2001), 925-935.
[54] N. Schweighofer, K. Doya and S. Kuroda, Cerebellar aminergic
neuromodulation: towards a functional understanding, Brain Research Reviews,
44 (2004), 103-116.
[55] Y. S. Suh, S. Oda, Y. H. Kang, H. Kim and I. J. Rhyu, Apoptotic cell death
of cerebellar granule cells in rolling mouse Nagoya, Neuroscience Letters, 325
(2002), 1-4.
[56] Y. Takeuchi, H. Kimura and Y. Sano, Immunohistochemical
demonstration of serotonin-containing nerve fibers in the cerebellum, Cell and
170
K. Sawada and Y. Fukui
Tissue Research, 226 (1982), 1-12.
[57] Y. Tamaki, S. Oda and Y. Kameyama, Postnatal locomotion development
in a neurobiological mutant of Rolling mouse Nagoya, Developmental
Psychobiology, 19 (1986), 67-77.
[58] T. Teramoto, M. Kuwada, T. Niidome, K. Sawada, Y. Nishizawa, K.
Katayama, A novel peptide from funnel web spider venom, ω-Aga-TK,
selectively blocks P-type calcium channels, Biochemical and Biophysical
Research Communications, 196 (1993), 134-140.
[59] L. C. Triarhou and B. Ghetti, Serotonin-immunoreactivity in the
cerebellum of two neurological mutant mice and the corresponding wild-type
genetic stocks, Journal of Chemical Neuroanatomy, 4 (1991), 321-428.
[60] G. Xie, S. J. Clapcote, B. J. Nieman, T. Tallerico, Y. Huang, I.
Vukobradovic, S. P. Cordes, L. R. Osborne, J. Rossant, J. G. Sled, J. T.
Henderson and J. C. Roder, Forward genetic screen of mouse reveals dominant
missense mutation in the P/Q-type voltage-dependent calcium channel,
CACNA1A, Genes Brain Behavior, 6 (2007), 717-727.
[61] G. Yadid, I. Sotnik-Barkai, C. Tornatore, B, Baker-Cairns, J,
Harvey-White, P,G, Pentchev and E, Goldin, Neurochemical alterations in the
cerebellum of a murine model of Niemann-Pick type C disease, Brain Research,
799 (1998), 250–256.
[62] T. A. Zwingman, P. E. Neumann, J. L. Noebels and K. Herrup, Rocker is a
new variant of the voltage-dependent calcium channel gene Cacna1a, Journal of
Neuroscience, 21 (2001), 1169-1178.
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