brain research 1566 (2014) 31–46 Available online at www.sciencedirect.com www.elsevier.com/locate/brainres Research Report Cerebellar afferents originating from the medullary reticular formation that are different from mossy, climbing or monoaminergic fibers in the rat Yuanjun Luo, Izumi Sugiharan Department of Systems Neurophysiology and Center for Brain Integration Research, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan art i cle i nfo ab st rac t Article history: Integration of cortical Purkinje cell inputs and brain stem inputs is essential in generating Accepted 12 April 2014 cerebellar outputs to the cerebellar nuclei (CN). Currently, collaterals of climbing and Available online 18 April 2014 mossy fiber axons, noradrenergic, serotoninergic and cholinergic axons, and collaterals of rubrospinal axons are known to innervate the CN from the brain stem. We investigated Keywords: whether other afferents to the CN from the medulla exist in the rat. Retrograde labeling Precerebellar nucleus revealed the presence of neurons that project to the CN but not to the cerebellar cortex in Cerebellar mossy fiber the median reticular formation in the rostrodorsal medulla (tentatively named ‘caudal Cerebellar afferent raphe interpositus area’, CRI). Anterograde tracer injection into the CRI labeled abundant Raphe interpositus axonal terminals in the CN, mainly in the ventral parvocellular part of the posterior Biotinylated dextran amine interposed and lateral nucleus. Axonal reconstruction showed that a single CRI axon Single axon reconstruction projected to the CN with 170–1086 varicosities, more broadly and densely than collaterals of a mossy or climbing fiber axon. CRI axons had no or a few collaterals that projected to the granular and Purkinje cell layers of the cerebellar cortex with some small terminals, indicating that these axons are different from mossy fiber axons. CRI axons also had Abbreviations: 4V, AIN, forth ventricle; 5-HT, anterior interposed nucleus; BDA, interpositus area; D, dorsal; DAB, DLP, 3,30 -diaminobenzidine; DLH, dorsolateral protuberance; DMC, GL, granular layer; GRN, IO, inferior olive; IN, interposed nucleus; L, cell; PCG, lateral; L.II–VI, left; LVN, medial vestibular nucleus; nVII, pontine central gray; PCL, caudal; CN, horseradish peroxidase; icp, lobules II–VI; LN, RPo, pyramidal tract; PN, raphe pontis; RPa, nucleus; TMB, pontine nucleus; R, raphe pallidus; Rt, right; scp, 3,30 ,5,50 -tetramethylbenzidine; Tz, http://dx.doi.org/10.1016/j.brainres.2014.04.020 0006-8993/& 2014 Elsevier B.V. All rights reserved. ventral parvocellular molecular layer; MN, rostral; RM, medial nucleus; medial longitudinal nucleus reticularis tegmenti pontis; PC, Phaseolus vulgaris leucoagglutinin; PIN, Purkinje posterior interposed nucleus; prepositus hypoglossi nucleus; PRN, nucleus raphe magnus; RO, vestibular nuclei; WM, pontine raphe obscurus; superior cerebellar peduncle; SO, superior olive; SVN, trapezoid body; V, ventral; VN, Y, nucleus Y n Corresponding author. Fax: þ81 3 5803 5155. E-mail address: isugihara.phy1@tmd.ac.jp (I. Sugihara). genu of the facial nerve; middle subdivision of the medial nucleus; mlf, tract of the facial nerve; NRTP, Purkinje cell layer; PHA-L, facial nucleus; caudal raphe inferior cerebellar peduncle; lateral nucleus; LN-parvo, medial; ML, PIN-parvo, ventral parvocellular subdivision of the posterior interposed nucleus; PrH, reticular nucleus; py, abducens nucleus; VII, cerebellar nuclei; CRI, dorsolateral hump of the anterior interposed nucleus; lateral vestibular nucleus; M, caudomedial subdivision of the medial nucleus; MN-M, fasciculus; MVN, trigeminal nucleus; VI, dorsomedial crest of the anterior interposed nucleus; gVII, gigantocellular reticular nucleus; HRP, subdivision of the lateral nucleus; Lt, MN-CM, 5-hydroxytriptamine; V, biotinylated dextran amine; C, superior vestibular white matter; 32 brain research 1566 (2014) 31–46 collaterals that projected to the medial vestibular nucleus and an ascending branch that was not reconstructed. The location of the CRI, electron microscopic observations, and immunostaining results all indicated that CRI axons are not monoaminergic. We conclude that CRI axons form a type of afferent projection to the CN that is different from mossy, climbing or monoaminergic fibers. & 2014 Elsevier B.V. All rights reserved. 1. Introduction Purkinje cell axons form the most dominant projection to the CN (Chan-Palay, 1973), and the CN, in turn, are the major source of outputs from the cerebellum. However, since Purkinje cell axons are inhibitory, how their activity leads to generation of cerebellar output is not simple (Person and Raman, 2012). Besides Purkinje cell axons, the CN receive afferent projections from the brain stem and spinal cord. Thus, CN activity is likely to reflect the integration of Purkinje cell activity with the activity of these afferents. The sources of brain stem projections to the CN, in particular, have been collectively labeled by retrograde tracers injected into the CN (Eller and Chan-Palay, 1976; Oka et al., 1985; Newman and Ginsberg, 1992). Several brain stem nuclei (nucleus reticularis tegmenti pontis, pontine nuclei, inferior olive, trigeminal nuclear complex, lateral reticular nucleus and some other nuclei in the brain stem) have been identified as possible sources of afferents to the CN. Since these nuclei are the origins of mossy and climbing projections to the cerebellar cortex as well, the possibility of tracer uptake through passing axons that project to the cerebellar cortex, but not to the CN, is hard to exclude. Thus, electrophysiological recording of synaptic responses from CN neurons or anterograde tracing studies of CN projecting axons are required for conclusive demonstration of afferent projections to the CN. Such studies have shown that almost all climbing fiber axons (Kitai et al., 1977; Shinoda et al., 1987; Van der Want et al., 1989; Sugihara et al., 2001) and some mossy fiber axons (Matsushita and Yaginuma, 1995; Wu et al., 1999; Shinoda et al., 1992; Quy et al., 2011) give rise to collaterals that provide excitatory innervation of the CN. However, it seems that the frequency of occurrence of CN collaterals differs among mossy fiber axons originating from different sources. Besides collaterals of mossy and climbing fibers, monoaminergic (serotoninergic and noradrenergic) projections have been reported in the CN (Moore and Bloom, 1979; Takeuchi et al., 1982; Bishop and Ho 1985) as well as in the cerebellar cortex, on the basis of specific labeling by immunostaining. These projections presumably exert a modulatory action on the CN neurons (Murano et al., 2011). It is not clear whether the monoaminergic projections to the CN and the cerebellar cortex originate from the same set of axons. Cholinergic projections in the CN appear as fine varicose axons following immunostaining of choline acetyltransferase (Woolf and Butcher, 1989). The pedunculopontine and laterodorsal tegmental nuclei, the major sources of cholinergic fibers in the brain stem, project to the CN (Woolf and Butcher, 1989); however, the major cholinergic innervation of the cerebellum arises from the medial vestibular nucleus (Barmack et al., 1992), the lateral paragigantocellular nucleus, and the raphe obscurus nucleus (Jaarsma et al., 1997). While the cholinergic projection from the vestibular nucleus takes the form of mossy fibers, other cholinergic projections have a varicose termination pattern in the cortex and/or CN. It is generally not clear whether the cholinergic projections to the CN and to the cerebellar cortex originate from the same axons. Except for collaterals of mossy and climbing axons and monoaminergic and cholinergic axons, information on the brain stem afferents to the CN is relatively limited. However, one retrograde fluorescent labeling study that focused on CN afferents showed that some spinal-projecting neurons of the magnocellular red nucleus supply collaterals to the CN, but not to the cerebellar cortex (Huisman et al., 1983). An input that projects primarily to the CN but not to the cerebellar cortex can directly modulate the cerebellar output without affecting information processing in the cerebellar cortex. In order to look into presence of such an input from other areas, we performed a combination of retrograde and anterograde labeling experiments on afferents to the CN from the medulla. We identified a new cerebellar afferent system that predominantly targets the CN rather than the cerebellar cortex. Single axonal morphology, electron microscopy, and immunostaining evidence are presented to support the idea that this is a distinct type of projection from that of the mossy, climbing, or monoaminergic axons. The functional significance of this newly found projection is discussed. 2. Results 2.1. Screening of CN-projecting neurons by retrograde labeling experiments To examine the possible presence of brain stem neurons that project to the CN but not to the cerebellar cortex, we compared the distribution of neurons in the medulla and caudal pons that were labeled by horseradish peroxidase (HRP) injection into multiple positions in the left CN (five cases) and by HRP injection into multiple points in the left cerebellar cortex (four cases). By carefully comparing the two distributions of retrogradely labeled neurons, we noticed that a group of neurons in the median reticular formation in the rostrodorsal medulla was consistently labeled by CN injections but rarely labeled by cortical injections (Fig. 1). We reconstructed the distribution of labeled neurons in the median and paramedian medulla and caudal pons (within 125 mm to the midline) on the sagittal plane from one CN injection and one cortical injection experiment (#100 and #110, respectively, Fig. 2). The area in which the neurons were brain research 1566 (2014) 31–46 33 Fig. 1 – Screening medullary neuronal populations that project to the CN but not to the cerebellar cortex. A and B, Camera lucida drawing of cerebellar HRP injection sites in two rats in which injection sites were centered in the left CN (A) and in the cerebellar cortex (B), respectively. C and D, Camera lucida drawing of the distribution of retrogradely labeled neurons in a coronal section of the rostral medulla in the rats with injections shown in A and B, respectively. HRP-labeled neurons were visualized with the TMB reaction in these sections. Each dot indicates a labeled neuron. Dotted curve with arrows circumscribes the CRI in which neurons were labeled with HRP injections into the CN but not with HRP injections into the cerebellar cortex. E and F, Photomicrographs of labeled neurons in the CRI in the rat shown in (A) and (C). labeled only by the CN injection was observed in the rostrodorsal medulla (dotted curve with arrows in Fig. 2). The extent of this area was as follows: rostral border, rostral edge of the abducens nucleus; caudal border, 1 mm caudal to the rostral edge of the abducens nucleus; ventral border, 2/5 thickness of the medulla dorsal to the ventral surface in the midline; dorsal border, 1/10 thickness of the medulla ventral to the dorsal surface in the midline; lateral border, 125 mm to the midline. Except for this area, retrogradely labeled neurons were distributed similarly in other areas of the median and paramedian medulla and caudal pons: both CN and cortical injections led to dense labeling of neurons in the nucleus reticularis tegmenti pontis, medial vestibular nucleus, and inferior olive, and to sparse labeling in other parts of the reticular formation (Fig. 2A and B). The area of specific neuronal labeling following a CN injection was located dorsal to the raphe magnus, ventral to the medial longitudinal fasciculus, and caudal to the nucleus raphe interpositus. The raphe interpositus is the median pontine reticular formation, 1–3 mm below the floor of the fourth ventricle, which contains pause neurons of the oculomotor system (Büttner-Ennever et al., 1988; Ohgaki et al., 1987; Hittinger and Horn, 2012). The caudal boundary of the raphe interpositus nucleus is around the level of the abducens nucleus (Ohgaki et al., 1987). Therefore, the distribution area of CN-projecting neurons in the present study was immediately caudal to the raphe interpositus, with some possible overlap. Therefore, we will refer to the area of the distribution of the CN projecting neurons as the ‘caudal raphe interpositus area (CRI)’. We counted the number of retrogradely labeled neurons in the CRI in the five CN injection cases and four cortical injection cases. The number of retrogradely labeled neurons in the raphe interpositus was generally larger for CN 34 brain research 1566 (2014) 31–46 Fig. 2 – Distribution of CN-projecting neurons in the median reticular formation in the rostral medulla that delineated the CRI. A and B, Summary of mapping of retrogradely labeled neurons within 125 lm from the midsagittal plane in experiments shown in Fig. 1A (#100) and B (#119), respectively. Mapping was done in every other section that was stained with TMB reaction. Dotted circle (filled arrows) circumscribes the CRI in which neurons were labeled by HRP injections into the CN (A) but not by HRP injections into the cerebellar cortex (B). C–F, Camera lucida drawing of the distribution of retrogradely labeled neurons in sections in experiment #100 as indicated by open arrowheads in A (caudal sections are to the left). Dotted circle (filled arrows) circumscribes the CRI in C–E. 35 brain research 1566 (2014) 31–46 Table 1 – Number of neurons in the CRI retrogradely labeled by HRP injections in the CN or cerebellar cortex. All injections were made in the left side. Rat no. Injection centers Injection volume (ml) No. of labeled neurons in the CRI #100 #158 #114 #115 #116 #109 #110 #112 #156 Medial, interposed and lateral nuclei Interposed nucleus Interposed nucleus Lateral nucleus Medial nucleus Vermis, hemisphere and paraflocculus Vermis, hemisphere and paraflocculus Vermis, hemisphere and paraflocculus Rostral vermis and hemisphere (lobule II–V) 2.0 1.0 0.5 1.0 1.0 3.0 7.0 2.5 3.0 201 74 40 106 78 47 15 36 33 injections than for cortical injections (Table 1). Case #114 had not as many as retrogradely labeled neurons in the raphe interpositus as in other CN injection cases, which may be due to smaller amount of injection volume of HRP in this case. As a whole, the results suggested that there is a population of neurons that projected predominantly to the CN in the CRI. 2.2. Anterograde tracing experiments To confirm the projection of neurons in the CRI to the CN, we injected anterograde axonal tracer (biotinylated dextran amine, BDA or Phaseolus vulgaris leucoagglutinin, PHA-L) into the CRI or adjacent median areas (injection center, o0.2 mm to the midsagittal plane) in 17 rats. In the experiment (#129) shown in Fig. 3 the injection into the CRI labeled numerous axonal terminals within the CN. The terminals were mainly distributed in the ventral parts of the posterior interposed and lateral nuclei, but were also seen in other areas of the CN and in the vestibular nuclei (Fig. 3A–D). On the other hand, terminals were rarely observed in the cerebellar cortex; a small number of terminals (20 at most) were found in the granular and Purkinje cell layers in a section, and many sections showed no terminals. However, the distribution patterns of terminals were differed significantly between injections; for example, many rosette-type mossy fiber terminals were observed in the cerebellar cortex in some cases, and few terminals were observed either in the cortex or in CN in other cases. Therefore, we tried to relate the different terminal distribution patterns to the location of the injection sites. By counting the number of terminals in the CN and cerebellar cortex in serial sections in each case, the 17 injection cases were classified into one of three groups: (1) cases with labeled terminals predominantly in the CN (41000 terminals in the CN and no or o200 terminals in the cerebellar cortex, 5 cases), (2) cases with labeled terminals predominantly in the cerebellar cortex (41000 terminals in the cerebellar cortex, 7 cases), and (3) cases with few labeled terminals either in the CN or in the cerebellar cortex (o1000 terminals in the CN and o1000 terminals in the cerebellar cortex, 5 cases). Injection sites of only the first group were located within the borders of the CRI (group 1, Fig. 4A). Axons and axonal terminals were densely observed in the CN but were not, or were only infrequently, observed in the cerebellar cortex in these cases. Injections that were located outside of the CRI either produced predominant terminal labeling in the cerebellar cortex (group 2, Fig. 4B), presumably due to labeling of mossy fiber axons, or did not label many terminals in either the CN or the cerebellar cortex (group 3, Fig. 4C), presumably because the injections were small or they did not hit a source of mossy fiber axons. These results confirm the presence of a neuronal population in the CRI that projects to the CN, but not to the cerebellar cortex. The area-dependency of the distribution of CN terminals is summarized for five cases of CRI injection in Table 2. Terminals were densely distributed in the ventral parvocellular parts of the posterior interposed and lateral nuclei in all cases as seen in the mapping of #129 (Fig. 3). Terminals were also observed less densely in most parts of the CN but were not found in the dorsomedial crest or dorsolateral hump. 2.3. Anterograde single axon reconstruction The most numerous afferent projections to the CN from the brain stem are presumably the collaterals of climbing and mossy fibers. These afferent axons have morphologically specialized terminations in the cerebellar cortex. Nuclear collaterals of these axons also have a distinct morphology, which has been revealed by the reconstruction of individual axons (Wu et al., 1999; Sugihara et al., 1999; Sugihara, 2011). In the present study, we reconstructed 12 axons in the five rats, in which the anterograde tracer injection was localized within the CRI (cases shown in Fig. 4A). We first located a well-labeled axon in the CN or in the white matter near the CN, and tried to trace the axon backward to its origin. We then tried to trace all branches in the cerebellum including the CN. Four out of 12 axons were traced down to a cell body. The neurons thus identified were located in the CRI, inside or in the close vicinity of the tracer injection site (Fig. 5A), thereby confirming the origin of the predominantly nuclear projection from the CRI. Three axons out of 12 were completely reconstructed in the cerebellum. The cell body was identified in one of these three. The trajectory of the axon that was completely reconstructed and whose cell body was identified as being in the CRI is shown in Fig. 6. The cell body was relatively large (shown in Fig. 5A, long and short diameter, 45 and 12 mm, respectively) and was located nearly on the midline, 0.5 mm ventral to the medullary floor of the fourth ventricle, and 0.5 mm caudal to the center of the abducens nucleus. The 36 brain research 1566 (2014) 31–46 Fig. 3 – Anterograde mass labeling of the projection from the CRI to the CN. A–D, Camera lucida drawing of labeled axonal terminals in the right CN and adjacent areas in coronal sections at four different rostro-caudal levels of the same experiment. E, Photomicrograph of the BDA injection site (arrows) in the CRI in the coronal section, which was located at 0.2 mm caudal to the center of the abducens nucleus. F, Camera lucida drawing of anterograde tracer injection into the CRI. G, The tracer injection site mapped on the midsagittal plane of the medulla, reconstructed from 50 lm-thick serial coronal sections. Dotted curve with arrows indicates the contour of the CRI that was defined by retrograde labeling. All data in this figure are from one rat (#129). size and shape of the soma of this neuron was similar to that of retrogradely labeled CRI neurons (Fig. 1E and F). The stem axon ran rostro-dorso-laterally at first (Fig. 6A). After giving off its first branch in the pontine reticular formation near the floor of the fourth ventricle, the branch that projected to the cerebellum ran caudo-dorso-laterally. The other branch (ascending branch, Fig. 5B asterisk), which was thicker than the cerebellar branch (Fig. 5B arrowhead), continued further rostrally. We did not trace the ascending branch in the present study. As the cerebellar branch passed through the area ventro-lateral to the locus coeruleus and through the medial vestibular nucleus, it gave rise to two side branches (Fig. 6A and B asterisks). These branches were thinner than the main axon that projected to the cerebellum. Therefore, these branches were likely to innervate the medial vestibular nuclei and neighboring local areas, although axonal tracing was not successful in these branches. Consistent with this possibility, we saw some axonal terminals in the medial vestibular nucleus in other cases. The cerebellar branch of the axon ran caudo-dorsally along the surface of the medulla facing the lateral corner of the fourth ventricle as far as the interposed cerebellar nucleus and entered the nucleus from its ventral border (Fig. 6A and B). This trajectory indicates that the axon passed brain research 1566 (2014) 31–46 Fig. 4 – Mapping the CRI by anterograde tracing. Seventeen cases of injection of PHA-L and BDA into median areas in the rostral medulla and caudal pons were sorted into three classes based on the type of terminal labeling in the cerebellum. The injection sites were then mapped on a drawing of the midsagittal section with median and paramedian structures. A, Injections that labeled nuclear terminals predominantly (group 1). B, Injections that labeled cortical terminals predominantly (group 2). Cortical terminals were mostly mossy fibers. C, Injections that labeled few nuclear or cortical terminals (group 3). Dotted curve with arrows indicates the contour of the CRI that was defined by retrograde labeling. through the superior cerebellar peduncle. Several branches were given off from the parent axon in the interposed nucleus. The parent axon came out of the nucleus and reached the lobular white matter, where it gave rise to several branches projecting to lobules III and IV, 1.2–1.7 mm lateral to the midline. Then, the parent axon returned to the interposed nucleus to produce several further branches, including ones innervating the medial and lateral nuclei. Such a hairpinshaped path of the parent axon in the lobular white matter 37 was observed only in this case. In the CN, some branches were short and ended in small terminal arborizations, whereas others were long and gave rise to many terminal branches (Fig. 6C). The diameter of the axon was 1.1–1.6 mm at the stem axon before the first bifurcation, 0.6–1.1 at the parent axon inside the CN, 0.2–0.4 at the primary collateral in the CN, and about 0.2 mm at the terminal branches in the CN. En-passant and terminal varicosities in the CN were roughly roundshaped and 1.0–2.0 mm (mostly 1–1.5 mm) in diameters. The total number of the varicosities or swellings in this neuron was 1086 in the CN and 64 in the cerebellar cortex. In the CN, terminal branches not only bore many en-passant varicosities but also frequently gave rise to short branchlets bearing one to several varicosities. As a result, varicosities often had a clustered appearance on terminal branches (Fig. 5C–F). Most of the cortical varicosities were in the granular layer (Fig. 5G), but some occur in the Purkinje cell layer (Fig. 5H and I). The terminal arbor in the cerebellar cortex was much smaller than that in the CN, and had a smaller number of varicosities as well (Fig. 6D and E). Terminal branches, and most of the varicosities, were similar in size to those in the CN, although a few varicosities were slightly larger than others (up to 3 mm for their shortest diameter). The morphology of this axon in the cerebellar cortex clearly distinguished it from mossy fiber axons, which have large rosette-type terminals, as well as from climbing fibers, which have a dense ivy-shaped arborization in the molecular layer, and from monoaminergic axons, which have small beaded varicosities (Moore and Bloom, 1979). The morphological details of the other 11 reconstructed fibers that projected to the CN were similar to the case described above. The trajectories of two axons that were completely reconstructed in the cerebellum are shown in Fig. 7A and B. Within the CN, all of the reconstructed fibers had wide-spread branches with densely distributed varicosities, as demonstrated by the two axons shown in Fig. 7C and D. In all cases, the sizes of the varicosities and axons were nearly the same as those of the cases shown in Figs. 5 and 6. Concerning the cerebellar cortical projection, six axons did not provide collaterals to the cortex while the five other axons had cortical branches bearing a small number of small varicosities (o100), similar to the case shown in Fig. 6. Collaterals in the medial vestibular nucleus were seen in all cases. The morphology of the terminal arbor in the medial vestibular nucleus (Fig. 7A) was similar to that in the CN. The path of eight axons was peri-ventricular, as in the cases shown in Figs. 6A, 7A and 7B, but the path of three other axons was more lateral, coursing through the medial vestibular nucleus. The ascending branch was seen in all axons that could be traced up to the soma (n ¼4). 2.4. Electron microscopic observations We made electron microscopic observations of the BDAlabeled terminals in the ventral part of the interposed nucleus to clarify further the presynaptic characteristics of the CRI axons in the CN. Labeled terminals of CRI axons were identified by the presence of electron-dense amorphous reaction product (Fig. 8). Although the DAB reaction product made detailed observation of the presynaptic terminal 38 brain research 1566 (2014) 31–46 Table 2 – Distribution of axonal terminals labeled by anterograde tracer injections into the CRI. Rat no. #94 Left Right #107 Left Right #129 Left Right #131 Left Right #134 Left Right MN-CM MN-M DLP þ þ þ þ þ AIN þþ þþ þ þ þ þ þ þ þ þ þ þþ DMC þ þ þ þ þþ DLH PIN PIN-parvo LN LN-parvo þ þþ þþ þ þ þ þþ þ þ þþ þþ þ þþ þþ þ þþ þþ þþþ þ þþ þþþ þþþþ þ þ þþ þþ þ þþ þþ þ þ þþ þþ þ þ þþ þ Definition of symbols, , þ, þþ, þþþ, and þþþþ, less than 0.5, 0.5–4.0, 4.0–20.0, 20.0–100.0, and 100.0o terminals, respectively, per 20  visual field of the microscope. Average of 20 observations in multiple sections. Terminals were scarce in the cerebellar cortex in all cases. Tracers: #94: PHA-L, and #107–#134: BDA. See Fig. 4A for location of injection sites. The medial nucleus was divided into the MN-CM, MN-M and DLP according to Voogd (2004). Fig. 5 – Photomicrographs of a labeled CRI neuron and its axon and axonal terminals. A, Identified cell body. Arrowheads indicate the axon. B, Branching point near the floor of the fourth ventricle in the pons. Arrowhead indicates the branch that projects to the cerebellum. Single asterisk indicates the ascending branch. Double asterisk indicates the stem axon. C–F, Terminal arborization bearing en-passant and terminal varicosities in the posterior interposed cerebellar nucleus. G, Terminal arborization in the granular layer. H and I, Terminal arborization in the Purkinje cell layer. A–E, G and H are from the neuron and axon shown in Fig. 6. F and I are from other reconstructed axons. Scale bar in I applies to C–I. brain research 1566 (2014) 31–46 39 Fig. 6 – Reconstructed single axon of an identified CRI neuron. A, The axonal trajectory was drawn from a caudal perspective onto the outline of the CN in a coronal section. Reconstruction was made from 61 serial coronal sections. This axon was completely reconstructed from the soma to all of its terminals in the CN, except for an ascending branch (double asterisk) and thin collaterals in the medial vestibular nucleus (single asterisks). Inset: entire fiber path to the cell body shown under low magnification. B, The trajectory in lateral view of the same axon as in A, depicted on a montage of parasagittal sections of the rat brain. C, Parts of terminal arborization in the posterior interposed nucleus in a single section shown under high magnification. Broken lines indicate continuation of the axon to the consecutive sections. D, Cortical collateral and boutons in the lobule IV shown under high magnification. Reconstructed from 11 coronal sections. In C–E, thickness of varicosities and fibers is approximately to scale. Shaded areas in A and B indicate the injection site. This axon had 1086 varicosities in the CN and 64 varicosities in the cerebellar cortex. difficult, we could observe some characteristics of the terminals. Specifically, the axonal terminals contained denselypacked, pleomorphic but generally round, pale synaptic vesicles, and made putative asymmetric synaptic contacts onto the dendrites of nuclear neurons (arrowheads, Fig. 8). No dense-core vesicles indicative of a monoaminergic terminal (Richards and Tranzer, 1970; Muller et al., 2007) were observed in the 20 terminals that were examined. This morphology suggested that these terminals make non-monoaminergic synaptic connections. 2.5. axons Distinction between CRI axons and serotoninergic Serotoninergic fibers and terminals are known to be abundant in the CN (Takeuchi et al., 1982; Bishop and Ho 1985). The 40 brain research 1566 (2014) 31–46 Fig. 7 – Samples of reconstructed CRI axons in the CN. A, axon of a CRI neuron that was completely reconstructed in the CN and medial vestibular nucleus. This axon had 170 varicosities in the CN and 98 varicosities in the medial vestibular nucleus. The axonal trajectory (caudal view) was drawn on the brain outlines of two coronal sections (at the level of medial vestibular nucleus slightly caudal to the locus coeruleus, and at the level of the posterior interposed nucleus). B, axon of another CRI neuron that was completely reconstructed in the CN. This axon had 515 varicosities. The axonal trajectory (caudal view) was also drawn on brain outlines of two coronal sections (at the rostral and caudal levels of the CN). C and D, Local terminal arbor in the CN of other partially reconstructed CRI axons. These axons had 404 (C) and 268 (D) varicosities, respectively. Asterisk indicates the stem of the axon. Scale bar in D applies to A–D. raphe nuclei, located in the median region of the reticular formation, contain serotoninergic neurons. In particular, the raphe magnus is located just ventral to the CRI (Figs. 2–4). Although, serotoninergic neurons are otherwise relatively scarce in the dorsal part of the rostral medulla (Jacobs et al., 1984), spread of the tracer may have caused labeling of some serotoninergic neurons in the raphe magnus. Therefore, to further test whether the cerebellum-projecting CRI axons were serotoninergic or not, we injected PHA-L into the CRI and made serial sections through the CN. Sections were alternately stained brain research 1566 (2014) 31–46 41 Fig. 8 – Electron micrographs of BDA-labeled terminals in the ventral part of the posterior interposed nucleus. Two samples of sections of labeled axonal terminals are shown (A and B). BDA injection was made into the CRI. Labeled axons were visualized with DAB reaction. Arrowheads indicate putative asymmetric synapse formation. for 5-HT immunoreactivity and PHA-L. Camera-lucida drawings of PHA-L labeled axons and 5-HT immunoreactive axons in contiguous sections were accurately superimposed blood vessels as references. At the cut plane between the two sections, no PHA-L-labeled axons aligned with 5-HT-immunoreactive axons (e.g., red and black arrowheads in Fig. 9). This result indicates that the cerebellum-projecting axons originating from the CRI are not serotoninergic. 3. Discussion The present study showed that the cerebellar projection from the CRI terminates mainly in the CN, and that only relative few CRI fibers project to the cerebellar cortex. The morphological characteristics of terminal arbor of the CRI axon in the cerebellar cortex and CN were clearly different from those of mossy fibers, which originate from many precerebellar neurons in the brain stem, climbing fibers, and monoaminergic fibers. Thus, this study presented a detailed morphological demonstration of a distinct type of the cerebellar afferent that arises in the brain stem and predominantly targets the CN. 3.1. A cerebellar afferent system from the brain stem that projects predominantly to the CN The CN are the major output source of the cerebellum, which sends cerebellar output signals to various brainstem nuclei including the thalamus, red nucleus, vestibular nuclei and reticular formation (Voogd, 2004). For the cerebellum to execute its function in motor coordination, the CN would have to change their activity in a timely manner in response to changes in the activity of afferents to the cerebellum. In many behavioral experiments, including conditioning of the eye blink reflex, the CN increases its activity when the animal is required to generate modulation in its behavior. However, how the output signal is generated in the CN neurons by their intrinsic electrophysiological properties and the synaptic inputs to them from Purkinje cells and other afferents is not well understood (Person and Raman, 2012). Although Purkinje cell axonal terminals are the predominant inputs to the CN neurons (Chan-Palay, 1973), how this input is converted into the activity of CN neurons is not straightforward, since the Purkinje cell projection is inhibitory (Ito et al., 1970). Rebound excitation upon the temporal cessation of Purkinje cell activity is the most likely cause, however, excitatory inputs from afferents other than Purkinje cells have to be taken into account. In this sense it is important to clarify direct brain stem inputs to the CN. Climbing fiber axons (or olivocerebellar axons) usually have collaterals that project to the CN (Van der Want et al., 1989; Sugihara et al., 1999). The projection pattern of these collaterals has a precise topographic relationship to their origin (Ruigrok and Voogd, 2000; Pijpers et al., 2005; Sugihara and Shinoda, 2007). Mossy fiber axons constitute the most abundant afferent system in the cerebellar cortex (Voogd, 2004), and thus their collaterals presumably form the most abundant input to the CN from the brain stem. However, not all mossy fiber axons have collaterals that project to the CN. 42 brain research 1566 (2014) 31–46 Fig. 9 – Absence of 5-HT immunoreactivity in CRI axons in the CN. A and B, Camera lucida drawing of CRI axons and 5-HTimmunoreactive axons in consecutive sections of the CN. CRI axons were labeled by PHA-L injected into the CRI. Every other section of the CN was immunostained for PHA-L and 5-HT. Black and red drawings are labeled terminal axons and blood vessels (used as position landmarks) in consecutive sections stained for PHA-L (black) and 5-HT (red) in the ventral part of the posterior interposed nucleus. Cut ends of labeled axons at the boundary between the two sections are indicated by arrows. No cut ends of black and red axons met with each other, indicating that they belong to distinct axonal populations. Note that blood vessels (circles) met with each other nearly completely, showing the precise positional alignment of the drawings of two consecutive sections. For example, mossy fiber axons originating from the pontine nucleus and external cuneate nucleus have CN collaterals infrequently (about 1:3 and 1:15, Shinoda et al., 1992; Quy et al., 2011). On the other hand, mossy fiber axons originating from the lateral reticular nucleus always have CN collaterals (Wu et al., 1999). The topographic relationship between the cortical and nuclear projections is not as precise for mossy fiber systems as it is for the climbing fiber systems. Noradrenergic and serotoninergic projections to the CN and cerebellar cortex have been well documented (Moore and Bloom, 1979; Takeuchi et al., 1982). Whether these monoaminergic projections to the CN arise as collaterals of the monoaminergic projection to the cerebellar cortex or not has not been clarified so far. However, it is possible that these projections originate from common axons since there are a relatively small number of monoaminergic neurons in the brain, and since monoaminergic projections are generally divergent (Moore and Bloom, 1979). The present study has reported the presence of a distinct type of cerebellar afferent originating from the medulla: a population of CRI neurons that project predominantly to the CN and only sparsely to the cerebellar cortex. The innervation of the cortex by these neurons was very weak, consisting of a small number of varicosities or swellings (short diameter, mostly 1–3 mm) that was much smaller than mossy fiber rosettes (short diameter, 4–8 mm). The diameter of CRI axons at the entrance to the cerebellum (about 1 mm) was much smaller than that of mossy fibers (2–4 mm; Wu et al., 1999; Quy et al., 2011). Although the diameter of CRI axons was in the same range as that of climbing fiber axons (0.7–1.4 mm; Sugihara et al., 1999), cortical termination of CRI axons was quite dissimilar to that of climbing fibers. Therefore, the CRI axons were clearly different from mossy or climbing fiber axons. Concerning the noradrenergic projection, locations of noradrenergic neurons (locus coeruleus near the lateral wall of the fourth ventricle in the rostral medulla, A5 cell group in the ventrolateral medulla, and A7 cell group in the lateral pons; Dahlströ m and Fuxe, 1964; Moore and Bloom, 1979) are all far separated from the CRI, which is located in the median reticular formation in the rostral medulla. Concerning the serotoninergic projection, the raphe magnus nucleus, one of the sources of the serotoninergic projection to the cerebellum (Hö kfelt and Fuxe, 1969; Takeuchi et al., 1982; Bishop and Ho 1985), is located just ventral to the CRI. However, the reported distribution of serotoninergic neurons does not extend to the CRI, which is located dorsal to the raphe magnus nucleus (Jacobs et al., 1984; Kitzman and Bishop, 1994; Bishop and Ho, 1985; Harding et al., 2004). Moreover, the morphology of the CRI axons in the cerebellar cortex and CN does not fit well with immunostained noradrenergic or serotoninergic fibers, which are characterized as a varicose fine network (Hö kfelt and Fuxe, 1969; Takeuchi et al., 1982; Bishop and Ho 1985). Furthermore, our immunostaining observations and electron microscopic observations did not support that the CRI axons were serotoninergic. In sum, the present study indicated that CRI axons were not monoaminergic. Concerning cholinergic innervation of the CN, the CRI does not seem to overlap with the reported sources of the projections in the brain stem, such as the pedunculopontine and laterodorsal tegmental nuclei (Woolf and Butcher, 1989), the medial et al., 1992), lateral vestibular nucleus (Barmack brain research 1566 (2014) 31–46 paragigantocellular nucleus, and the raphe obscurus nucleus (Jaarsma et al., 1997). This suggests that the CRI projection is not cholinergic. However, other raphe nucleus can also be a source of cholinergic projection to the cerebellum to a lesser extent (Jaarsma et al., 1997). Moreover, the synaptic terminals of the CRI axons are not dissimilar to the cholinergic terminals in the CN when studied by electron microscopy (Jaarsma et al., 1997). Therefore, whether the CRI projection to the CN is cholinergic needs further investigation. Neurons in the raphe obscurus nucleus were labeled retrogradely from both the CN and cortex (Fig. 1), suggesting the possibility that cholinergic neurons in this nucleus project to both the CN and cortex. Rubrospinal axons, which arise from the magnocellular red nucleus, give off collaterals to the CN, and these fibers constitute another type of afferent to the cerebellum, one which does not supply terminals to the cerebellar cortex (Huisman et al., 1983). The cerebellar projection of CRI axons is similar to this projection in that it is formed by the collaterals of the main projection axons, and in that it mainly supplies to the CN. Since no anterograde labeling study has investigated the CN projection from the red nucleus, the present study is the first demonstration of the axonal morphology of CN projection from the brain stem except for climbing and mossy fiber axons. Whether there are any other brain stem neurons that also send a similar new type of cerebellar afferents is not clear. 3.2. Possible function of the CRI projection The terminal arborization of single CRI axons in the CN was characterized by dense distributions of varicosities. Although the CRI does not occupy a large volume and, consequently, the number of CRI neurons was not as large as the number of neurons in major precerebellar nuclei, each CRI axon had a large number of varicosities that were distributed in a relatively wide area of the CN (  1000, Figs. 5 and 6). In comparison to a CRI axon, a single mossy fiber axon has a much smaller number of varicosities in the CN (36–270 in the lateral reticular nucleus axons in rat, Wu et al., 1999; 0–59 in the dorsal column nucleus axons in rat, Quy et al., 2011). On the other hand, the cortical projection of the CRI neurons was very weak. Therefore, the functional significance of CRI axons would be very different from that of mossy fiber systems. Mossy fiber systems are supposed to supply massive sensory and efference copy signals to the cerebellum, which are used in cortical and nuclear information processing to generate appropriate cerebellar output. In contrast, the CN projection from the CRI may be involved in modulating the cerebellar output directly. The terminal arbor of a single CRI axon was more widely and densely distributed than the collaterals of the climbing and mossy fiber axons in the CN. In this regard, activity of CRI axons may produce a particularly strong and broad effect on CN neurons in the ventral parvocellular parts of the posterior interposed and lateral nucleus. However, identification of the neurotransmitter and synaptic action of the CRI axon will be required to further examine the function of CRI axons in the CN. The function of the CRI is not well understood. As a part of the medial reticular formation, which is generally involved in motor function (Rho et al., 1997), the CRI may also have some 43 function related to motor control. Indeed, eye-movementrelated omnipause neurons were located in the raphe interpositus nucleus, which is in the pontine median reticular formation immediately rostral to the CRI (Ohgaki et al., 1987; Hittinger and Horn, 2012). Omnipause neurons project to the area around the abducens nucleus and other brain stem areas, but not to the cerebellum as demonstrated in an intracellular labeling study in cat (Ohgaki et al., 1987). The axonal trajectory of the CN-projecting CRI neurons in the present study was different from that of omnipause neurons in the raphe interpositus. However, it is possible that CRI neurons may also be involved in eye movement control, since (1) the ventral part of the posterior interposed and lateral nuclei, where the CRI axons were found to project in the present study, is connected with the flocculus, paraflocculus, lateral uvula and crus I (Sugihara and Shinoda, 2004; Sugihara et al., 2004, 2009), lobules that are involved in oculomotor control (Kheradmand and Zee, 2011); (2) the ventral part of the posterior interposed and lateral nuclei has also been shown to be involved in some kinds of eye movements in monkey (Zhang and Gamlin, 1998) and project to the superior colliculus (Teune et al., 2000); (3) injection of HRP into the H field of Forel labeled neurons in the area that is equivalent to the CRI in the cat (Ohgaki et al., 1989); and (4) omnipause neurons send axonal branches to the area that is equivalent to the CRI (Ohgaki et al., 1987). Although most of studies on the oculomotor system in the brain stem were done in cat and monkey, the oculomotor system is also developed in the brain stem in rat (Hittinger and Horn, 2012). CRI axons had an ascending branch that was not traced in the present study. Identifying the target of the ascending branch of CRI axons will be needed to understand nature of CRI neurons including the possibility that they are involved in eye movement control in rat. 4. Experimental procedure 4.1. Surgical procedures and tracer injection Long-Evans male and female adult rats weighing 230–350 g (Kiwa Laboratory Animals, Wakayama, Japan) were used. All animal experiments were carried out in accordance with the guidelines of the animal welfare committee of the Tokyo Medical and Dental University, subsequent to approval by the Ethics Review Committee for Animal Experimentation of Tokyo Medical and Dental University (approval numbers 0020238, 0030129, 0040089, and 0130211A). Rats were anesthetized with intraperitoneal injection of ketamine (130 mg/kg body weight), xylazine (8 mg/kg) and atropine (0.4 mg/kg). Supplemental doses of ketamine (26 mg/kg) and xylazine (1.6 mg/kg) were given every 30 min starting 1 h after the initial dose during surgery (less than three hours). A heating pad was used to keep the rectal temperature between 35 and 37 1C. To inject horseradish peroxidase (HRP) in the cerebellar cortex or CN to label projecting neurons retrogradely, the rat was placed in a stereotaxic apparatus in a prone position, 451 nose down. The bone covering the vermis and left hemisphere was removed. A micropipette filled with HRP (PEO-131, Toyobo, Osaka, Japan; 30% solution in saline) was inserted into various positions in the left cerebellar cortex to inject 44 brain research 1566 (2014) 31–46 HRP solution at depths of 100–500 mm at 20–30 points (vermis, pars intermedia, hemisphere, and paraflocculus–flocculus; total injection amount, 2.5–7 ml) in five rats. A stereotaxic atlas (Paxinos and Watson, 2007) was used to locate the CN. In five other rats, a micropipette filled with HRP was inserted into the left CN (medial, interposed and lateral nuclei, 0.5–4.0 mm lateral to the midline, through lobule VIc, crus I and crus IIa, 4.5–3.5 mm deep; total injection amount, 0.5–2 ml). After a survival period of 48 h, the animals were deeply anesthetized with ketamine (200 mg/kg) and xylazine (12 mg/kg), and transcardially perfused with phosphate-buffered saline followed by fixative containing 2% paraformaldehyde, 0.6% glutaraldehyde and 4% sucrose in 0.05 M sodium phosphate buffer (pH 7.4). The brain was dissected and postfixed overnight in the same fixative and then soaked in 30% sucrose solution (pH 7.2 with 10 mM phosphate buffer). To label projections of neurons in the CRI and nearby areas anterogradely, rats were placed in a stereotaxic apparatus in a supine position. A small incision was made in the ventral neck at the midline. Median neck muscles, the trachea, esophagus, and pharynx were retracted to the left to expose the clivus with a parapharyngeal approach. A hole was drilled in the right clivus under the rostral medulla (center: 2.8 mm rostral to the foramen magnum, 0.2 mm right to the midline), and a small incision was made in the dura. A glass micropipette (tip diameter, 4 mm) filled with P. vulgaris leucoagglutinin (PHA-L, L-1110, Vector Labs, Burlingame, CA, U.S.A., 2.5% in sodium phosphate-buffered saline) or biotinylated dextran amine (BDA, D-1956, 10,000 MW, Molecular Probe, Eugene, Oregon, U.S.A.; 10% solution in saline) was inserted into the CRI (depth, 2.3 mm, direction, 51 tilted in the coronal plane toward the midline) and adjacent areas by changing the rostrocaudal and dorsoventral coordinates. PHA-L was electrophoretically injected (2 mA positive current pulses of 1 s duration at 0.5 Hz for 20 min), whereas BDA was pressure-injected ( 0.05 ml). After a survival period of four or five days, the animals were deeply anesthetized with ketamine (200 mg/kg) and xylazine (12 mg/kg), and transcardially perfused with phosphate-buffered saline followed by fixative containing 2% paraformaldehyde, 0.6% glutaraldehyde and 4% sucrose in 0.05 M sodium phosphate buffer (pH 7.4). The brain was dissected and postfixed overnight in the same fixative and then soaked in 30% sucrose solution (pH 7.4 with 10 mM phosphate buffer). 4.2. Histological procedures For brains where an HRP injection was made, the cerebellum and brain stem were dissected apart and processed separately. The cerebellum was cut into serial sagittal sections whereas the brain stem was cut into serial coronal sections (60 mm thick) on a freezing microtome. After washing for 30 min, all the cerebellar sections and a set of every other brain stem section were treated with 3,30 -diaminobenzidine (DAB, 0.5 mg/ml) plus 0.3% hydrogen peroxide in phosphate buffer for 15 min. Sections were washed with PBS, mounted on glass slides, dried, dehydrated with ethanol and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). The remaining set of brain stem sections was treated with 3,30 ,5,50 -tetramethylbenzidine (TMB), which is more sensitive than DAB (Mesulam, 1982). TMB was dissolved first in ethanol (20 mg/10 ml ethanol) and then mixed with cooled solution of sodium nitroprusside (1 g/l), sucrose (10 g/l) and sodium acetate buffer (50 mM, pH 3.3) in distilled water to obtain final concentration of 40 mg/l. Hydrogen peroxide (0.3%) was added immediately before use. Sections were rinsed with acetate buffered saline (pH 3.3) and then incubated two times in the TMB reaction solution at 4 1C for 30 min. Sections were washed with acetate buffered saline, mounted on glass slides, dried, dehydrated with ethanol and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Retrogradely labeled neurons were counted in sections that underwent TMB treatment, because of the more sensitive HRP visualization by the TMB reaction than the DAB reaction. For the PHA-L injections, each brain was embedded in a gelatin block, which was fixed in a Formalin solution (Formalin 20%, sucrose 25%, 4 1C) for 2–4 days. The gelatin block was cut into serial coronal sections (30 mm thick) on a freezing microtome. After washing for 30 min, sections were incubated with 0.1% sodium azide plus 0.3% hydrogen peroxide for quenching intrinsic peroxidase. Sections were incubated with goat anti-PHA(EþL) antibody (1:2000, AS-2224, Vector) in PBST plus 2% normal goat serum for 72 h, washed, then incubated with rabbit anti-goat IgG (305-005-003, 1:200, Jackson, West Grove, PA, U.S.A.), then goat peroxidase antiperoxidase (PAP, 123-005-024, 1:400, Jackson). PHA-L was then visualized by modification of the cobalt-glucose oxidase method (Itoh et al., 1979; Van der Want et al., 1989). Sections were preincubated for 10 min in 0.5% cobalt acetate in Tris– HCl (50 mM, pH, 7.6). After being rinsed in Tris-HCl, and then in PBS, sections were incubated with 0.05% DAB, beta-Dglucose (Sigma), and 0.4% ammonium chloride, glucose oxidase (0.5 mg/100ml) in PBS for 60 min. Sections were then washed with PBS, mounted on glass slides, dried, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ, U.S.A.). For the BDA injections, each brain was embedded in a gelatin block, which was fixed in a Formalin solution (Formalin 20%, sucrose 25%, 4 1C) for 2–4 days. Frozen coronal or parasagittal sections of 50 mm thickness were made and treated with biotinylated HRP–avidin complex (Elite ABC kit KT-6100, Vector) in a sodium phosphate buffer (0.1 M, pH 7.4) overnight at 4 1C. Next, BDA was visualized by modification of the cobalt-glucose oxidase method as described above. Then, the sections were mounted on chrome alum-gelatinized slides as described above. Two brains with PHA-L injection were embedded in gelatin and cut into serial coronal sections (30 mm). A set of every other section was processed for PHA-L labeling as above. The other set of every other section was processed for 5-hydroxytriptamine (serotonin, 5-HT) immunostaining. After quenching intrinsic peroxidase as above, sections were incubated with antiserotonin rabbit polyclonal antibody (NT102, 1:1000, Eugene, Ridgefield Park, NJ, U.S.A.) with 2% normal rabbit serum for 72 h. This antibody specifically labels serotoninergic neurons (Ishida et al., 1998; Michel et al., 2000). Then sections were incubated with biotinylated anti-rabbit IgG antibody (BA 1000, Vector), followed by biotinylated HRP-avidin complex (Elite ABC kit KT6100, Vector) in a sodium phosphate buffer (0.1 M, pH 7.4) overnight at 4 1C. Next, BDA was visualized by modification of the cobalt-glucose oxidase method as described above. Lastly, brain research 1566 (2014) 31–46 the sections were mounted on chrome alum-gelatinized slides as described above. We mapped labeled neurons and reconstructed single axons under a microscope with a camera lucida apparatus by using a computer-aided system (Sugihara and Fujita, 2010) as described before (Quy et al., 2011). We referred to sample sagittal sections of the rat brain atlas (Paxinos and Watson, 2007) to summarize mapping from serial coronal sections onto a single sagittal plane. The number of varicosities were counted manually under 20  objective while adjusting the focus of the microscope to visualize all levels throughout the thickness of each section. 4.3. Electron microscopy BDA was injected into the median area of the median reticular formation in the rostral medulla in three rats as described above. After seven days the rat was perfused with fixative containing 2% paraformaldehyde and 3% glutaraldehyde in 0.10 M phosphate buffer (pH¼ 7.4). The brain was dissected and postfixed in the same fixative overnight. Eighty micrometer sections of the brainstem and the cerebellum were cut with a microslicer. BDA was visualized with DABlabeling as described above except that no cobalt preincubation was done. After confirming that the injection site was in the intended area and that there were labeled axons in the CN, the ventral part of the posterior interposed nucleus was trimmed from the section and postfixed for 1 h with 1% osmium tetroxide in phosphate buffer, dehydrated with 100% ethanol, infiltrated with propylene oxide and epoxy resin (Epon 812, TAAB, Berks, England), embedded, and polymerized in a 60 1C oven for 48 h. Coronal sections of the ventral part of the posterior interposed nucleus were cut at a thickness of 60–90 nm, mounted on copper–rhodium mesh grids and stained with 3% aqueous uranyl acetate for 10 min. The sections were examined and photographed on a Hitachi H-600 electron microscope (75 kV acceleration). Acknowledgments This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to I.S. (25430032). The authors thank Dr. Sizuko Ichinose for technical assistance on electron microscopy and Dr. Eric J. 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