Projection Patterns of Single Mossy Fiber AxonsOriginating from the Dorsal Column Nuclei Mappedon the Aldolase C Compartments in the RatCerebellar Cortex
Pham Nguyen Quy*,1 Hirofumi Fujita,1 Yukiyo Sakamoto,1 Jie Na,1,2 and Izumi Sugihara1*1Department of Systems Neurophysiology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan2Laboratory of Brain and Cognitive Science, Shenyang Normal University, Shenyang 110034, China
ABSTRACTAlthough cerebellar mossy fibers are the most abundant
cerebellar afferents and are deeply involved in cerebel-
lar function, the organization of their projection has
remained obscure, particularly in relation to cerebellar
compartmentalization. The dorsal column nuclei (DCN)
are a major source of cerebellar mossy fibers and pos-
sess distinct somatotopic representations of specific
somatosensory submodalities. We reconstructed individ-
ual dextran-labeled DCN axons completely from serial
sections and mapped their terminals on the longitudinal
cerebellar compartments that were visualized by aldol-
ase C immunostaining to clarify their projection pattern.
Individual axons branched and formed about 100 ro-
sette terminals in the cerebellar cortex, but infrequently
projected to the cerebellar nuclei (1 out of 15 axons).
Cortical terminals were clustered in multiple areas in
the vermis and pars intermedia mostly, but not exclu-
sively, ipsilateral to the origin of the axon. The gracile,
cuneate, and external cuneate nuclei (ECuN) mainly
projected to the copula pyramidis and lobule V, para-
median and simple lobules, and lobules I–V and VIII–IX,
respectively, although there was some overlap. The ma-
jority of terminals were located within aldolase C nega-
tive or lightly positive compartments. However, terminals
of a single axon can be located on aldolase C-negative as
well as on aldolase C-positive compartments. In particular,
the rostral ECuN, which is responsive to shoulder move-
ments, projected consistently to lobule IX, which were
mostly aldolase C-positive. In sum, DCN-cerebellar axons
project to multiple compartments with terminals clustered
mainly in the conventional spinocerebellar region with a
coarse topography, which shows some relationship to the
cortical compartments defined by aldolase C. J. Comp.
Neurol. 519:874–899, 2011.
VC 2010 Wiley-Liss, Inc.
INDEXING TERMS: external cuneate; cuneate; gracilis; somatosensory; biotinylated dextran; zebrin
Anatomically, the cerebellar cortex is subdivided trans-
versely by its lobular folding (Larsell, 1952) and longitudi-
nally by so-called A–D zones based on the corticonuclear
Purkinje cell projection pattern and the olivocerebellar pro-
jection pattern (Groenewegen and Voogd, 1977; Voogd
and Bigare, 1980; Buisseret-Delmas and Angaut, 1993).
Longitudinal compartments have also been defined by the
expression patterns of specific molecules, such as aldol-
ase C (¼zebrin II), in Purkinje cells (Hawkes and Leclerc,
1987; Brochu et al., 1990). Determination of the olivocere-
bellar projection pattern to each aldolase C compartment
indicated that these compartments are either congruent
with, or contained within, one of the A–D zones (Voogd
et al., 2003; Sugihara and Shinoda, 2004). Furthermore,
there is evidence that aldolase C-positive compartments
receive cerebral, tectal, vestibular, and visual inputs via
the olivocerebellar system, whereas aldolase C-negative
areas receive somatosensory inputs (Sugihara and Shi-
noda, 2004). Thus, these anatomical longitudinal subdivi-
sions appear to have functional significance with regard to
the olivocerebellar climbing fiber system.
*Pham Nguyen Quy’s present address: Department of Physiology andCell Biology, Tokyo Medical and Dental University Graduate School, Tokyo,Japan.
Grant sponsor: Japan Society for the Promotion of Science; Grantnumber: Grant-in-Aid for Scientific Research 20300137 (to I.S.).
*CORRESPONDENCE TO: Dr. Izumi Sugihara, Dept. of SystemsNeurophysiology, Tokyo Medical and Dental University Graduate School,1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.E-mail: [email protected]
VC 2010 Wiley-Liss, Inc.
Received August 23, 2010; Revised October 27, 2010; AcceptedNovember 12, 2010
DOI 10.1002/cne.22555
Published online November 30, 2010 in Wiley Online Library(wileyonlinelibrary.com)
874 The Journal of Comparative Neurology |Research in Systems Neuroscience 519:874–899 (2011)
RESEARCH ARTICLE
The situation is much less clear with regard to the
mossy fiber systems. Different cerebellar lobules or areas
tend to receive mossy fibers from distinct sources (Bro-
dal, 1981; Ito, 1984), and some relationship to the aldol-
ase C compartments has been reported for mossy fiber
projections from the basilar pontine nucleus (Pijpers
et al., 2006; Pijpers and Ruigrok, 2006), lateral reticular
nucleus (Ruigrok and Cella, 1995; Pijpers et al., 2006),
external cuneate nucleus (ECuN), spinal cord (Matsushita
et al., 1991; Ji and Hawkes, 1994; Akintunde and Eisen-
man, 1994), and trigeminal system (Hallem et al., 1999).
However, these studies do not supply information on the
actual density or arrangements of mossy fiber rosettes,
since they were done with retrograde labeling, mass an-
terograde labeling, or receptive field mapping.
On the other hand, recordings of granule cell activity
suggest that somatosensory mossy fiber receptive fields
form a patchy fractured somatotopic map across the cor-
tex (Welker, 1987), which suggests that the mossy fiber
pathway topography does not directly reflect a longitudi-
nal zonation pattern. However, most of the previous mor-
phological studies that traced many neurons cannot be
directly related to the physiologically determined maps.
An analysis of the cerebellar projection pattern of single
mossy fiber axons, as performed for lateral reticular nu-
cleus (LRN) axons (Wu et al., 1999), would be important,
since it can show the complete morphology of individual
identified axons, including the branching pattern and the
location and the number of terminals. Such information
would be directly related to physiology, and could also
become a basis for systematically understanding the or-
ganization of the mossy fiber projection.
The dorsal column nuclei (DCN) are a relay station in
the dorsal column-medial lemniscal somatosensory path-
way, and one of the major sources of cerebellar somato-
sensory mossy fibers (Cerminara et al., 2003) besides the
spinal cord (including Clarke’s column), LRN, and trigemi-
nal nucleus. The DCN are generally subdivided into four
parts, the cuneate nucleus (CuN), gracile nucleus (GN),
external cuneate nucleus (ECuN), and the small nucleus
Z, in accordance with their somatotopic organization and
distinct region-dependent representations of somatosen-
sory submodality (Tracey, 2004). The ECuN is sometimes
regarded as a precerebellar nucleus rather than a mem-
ber of the DCN (Brodal, 1981; Ji and Hawkes, 1994;
Kawauchi et al., 2006). However, in the present study the
four nuclei were all considered a part of the DCN that
also contained precerebellar components. Thus, DCN-
cerebellar projection may provide a clue to understanding
the essential organization of mossy fiber systems in
terms of somatotopy and somatosensory submodalities.
To analyze this projection systematically and precisely,
single axons that originate from various subdivisions of
the DCN in the rat cerebellum were completely recon-
structed. Immunostaining was also performed to localize
the axonal terminals with respect to aldolase C compart-
ments. The reconstructed axons showed multiple soma-
totopic localizations in the rat cerebellar cortex that have
a complex relationship with aldolase C compartments.
MATERIALS AND METHODS
Anterograde labeling of DCN axonsLong-Evans male and female adult rats (Kiwa Labora-
tory Animals, Wakayama, Japan) were used. All of the ex-
perimental animals in this study were treated according
to the guiding principles for the care and use of animals
in the field of physiological sciences of the Physiological
Society of Japan (2001, 2002), and the experimental pro-
tocols were approved by the Institutional Animal Care
and Use Committee of Tokyo Medical and Dental Univer-
sity (No. 0060121). The methods used for anesthesia, sur-
gery, and histological procedures were similar to those
described previously (Sugihara et al., 2001; Sugihara and
Shinoda, 2004). In brief, the animals were anesthetized
with an intraperitoneal injection of ketamine (130 mg/kg
body weight) and xylazine (8 mg/kg). Atropine (0.4 mg/kg)
was also given intraperitoneally. Supplemental doses of
ketamine (13 mg/kg) and xylazine (1 mg/kg) were given
every 30 minutes starting 1 hour after the initial dose, as
Abbreviations
1þ, 1�, etc. Compartment 1þ, 1�, etc.I-X Lobules I-Xa-d Sublobules a-d (as in VId-a)AIN Anterior interposed nucleusBDA Biotinylated dextran amineC CaudalCuN Cuneate nucleusCop Copula pyramidisCr I Crus I of the ansiform lobuleCr II Crus II of the ansiform lobuleD DorsalDAB Diaminobenzidine tetrahydrochlorideDCN Dorsal column nucleidPFl Dorsal paraflocculusECuN External cuneate nucleusFL ForelimbFl FlocculusGL Granular layerGN Gracile nucleusHL HindlimbL LateralM MedialML Molecular layerPar Paramedian lobulePBS Phosphate-buffered salinePBST Phosphate-buffered saline containing 0.15% Triton X-100PCL Purkinje cell layerpf Primary fissureR RostralSim a Simple lobule sublobule aSim b Simple lobule sublobule bV VentralvPFl Ventral paraflocculusWM White matter
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 875
required. A heating pad was used to keep the rectal tem-
perature between 35 and 37�C.Small biotinylated dextran amine (BDA) injections
were made into the DCN in 26 rats. Rats were placed in
a stereotaxic apparatus in a prone position with the head
rotated 45–55� nose-down. An incision was made
through the skin and muscle at the midline of the dorsal
side of the neck. The caudal part of the right occipital
bone and the membrane between the occipital bone and
the first vertebra were removed to expose the dorsal me-
dulla. A glass microelectrode (tip diameter, 5 lm) filled
with saline was inserted to the DCN to record fields
evoked by somatosensory stimuli. The receptive field of a
particular location in the DCN was defined using light
touching at various body areas with a fine sable-hair
paintbrush and by manually moving the limbs and the
neck. After the response properties for the injection
point were characterized, a glass micropipette (tip diame-
ter, 5 lm) filled with BDA solution in saline (10%, BDA; D-
1956, molecular weight 10,000; Molecular Probes,
Eugene, OR) was inserted to the same position and
depth. In later experiments BDA with molecular weight
3,000 (D-7135) was used without any clear disadvant-
age. The evoked response was characterized again using
the injection pipette as an electrode (Fig. 1G,H). To eject
a drop of BDA solution (5–10 nl) from the pipette, a pneu-
matic pressure pulse was applied using an electronic
valve device (Picopump PV820, WPI, Sarasota, FL) con-
nected to a nitrogen tank. A change in the evoked
response was noted as a sign of a successful injection.
After the injection was made the micropipette was with-
drawn and the skin was sutured.
After a survival period of 7 days the rats were anesthe-
tized with an overdose of ketamine (195 mg/kg) and xyla-
zine (12 mg/kg). They were then perfused intracardially
with phosphate-buffered saline (PBS) followed by fixative
containing 5% paraformaldehyde, 2% sucrose, and phos-
phate buffer 50 mM (pH: 7.4). After the animal was per-
fused the cerebellum and medulla were dissected free,
stored in the same fixative for 1–2 days, and then embed-
ded in gelatin.
Retrograde labeling of DCN neuronsLarge injections of BDA (molecular weight 3,000) into a
single cerebellar lobule were made in 16 other rats. Anes-
thesia, survival, and dissection for this procedure were
the same as for the anterograde labeling (above). Rats
were placed in a stereotaxic apparatus in a prone position
with the head rotated between �10 and þ80 degrees
nose-down. Depending on the specific lobule to be
injected, parts of the left occipital, temporal, and/or pari-
etal bones were removed to allow access. To access the
rostral lobules (lobules III–V and simple lobule), caudal
parts of the cerebrum and the inferior colliculus were
removed and the transverse sinus was coagulated so that
it could be transected. A micropipette was then used to
inject BDA solution at depths of 100–500 lm at 20–40
penetration points within a single lobule. A total of 1–2
lL was injected. After the injection was made the micro-
pipette was withdrawn and the skin was sutured.
Although BDA is more efficient for anterograde tracing
than retrograde tracing, it can be used for retrograde
labeling with large volume injections (Sugihara et al.,
1999). Its benefits are that it does not spread much and
that it barely deteriorates the brain tissue in which it has
been injected. To support its efficiency many inferior olive
neurons were labeled in each experiment.
Figure 1. Schematic composition of the rat DCN and method illustrations. A: Outlines of three major nuclei of the DCN were mapped on
a photo of the dorsal surface of the medulla. B: Three-dimensional scheme of the DCN. This scheme is based on camera lucida drawings
of the DCN in thionine-stained serial coronal sections of a rat and adjusted slightly according to coronal and parasagittal sections of sev-
eral other rats. The entire ECuN and the rostral parts of the CuN and GN were included in the scheme since the cerebellar projection ori-
ginated from the rostral parts of the CuN and GN. C: Camera lucida drawings of the DCN in coronal sections at three different levels as
indicated in A (a–c, corresponding to top to bottom). D: Horizontal trajectories of the contours of the ECuN, CuN, and GN. They were sep-
arated by being shifted mediolaterally for visualization. These horizontal contours are used in this article to map neurons and BDA injection
sites in the DCN. Dotted line indicates the level for the parasagittal section in F. E: Functional subdivisions of the GN. Responses were
recorded in various sites in the GN. Recording sites were then located by injecting BDA and mapped on the horizontal scheme of the GN.
White circles, trunk cutaneous responses (lateral GN); gray circles, hindlimb cutaneous responses (centromedial GN); black circles, hind-
limb proprioceptive responses (rostral pole of the GN). Dotted curves indicate putative boundaries of different response areas. F: Photomi-
crograph of a BDA injection site in the DCN in a parasagittal section. G,H: Responses from the DCN. The electrode was located in the
ECuN and the best responses were evoked by elbow extension in this case. Responses are shown with slower (G) and faster (H) sweep
speeds. I–K: Schemes that show how to map a mossy fiber terminal in the unfolded scheme of the cerebellar cortex. In the coronal sec-
tion the relative mediolateral distances of the terminal within an aldolase C compartment were measured by extending the boundary of
compartments from the molecular layer to the granular layer (I, a and b). The relative rostrocaudal distances of the terminal within the
given lobule, in which the terminal was located, were measured by counting numbers of sections in which that aldolase C compartment
was seen within the given lobule (J, c, d, and e). The terminal was plotted on the unfolded scheme by using these measured distances (K,
a0/b0 ¼ a/b, c0/(d0þe0) ¼ c/(dþe)).
Quy et al.
876 The Journal of Comparative Neurology |Research in Systems Neuroscience
Histological procedures for BDA andaldolase C labeling
Histological procedures for visualizing BDA and aldol-
ase C were described previously (Sugihara and Shinoda,
2004, 2007). Serial frozen coronal sections (80-lm
thick) were cut from the entire cerebellum and medulla
and from the most rostral levels of the spinal cord. In
some of the experiments involving DCN injections the
cerebellum and the brainstem were separated before
being embedded in gelatin to allow the former to be cut
Figure 1
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 877
coronally and the latter parasagittally. Before sectioning,
the right side of the gelatin block was partially painted
with alcian blue (015-13805; Wako, Tokyo, Japan) to
help identify the orientation of the sections. Sections
were first incubated with biotinylated peroxidase-avidin
complex (PK6100 Elite ABC kit; Vector Laboratories,
Burlingame, CA), and then BDA was visualized by incu-
bating the sections for 30–60 minutes in a solution con-
taining diaminobenzidine (0.5 mg/mL), glucose oxidase
(Itoh et al., 1979; 0.01 mg/mL), nickel ammonium sul-
fate (2 mg/mL), ammonium chloride (4 mg/mL), and
beta-D(þ)-glucose (2 mg/mL) in Tris buffer (50 mM, pH
7.4) followed by washes with Tris buffer and then with
PBS. For DCN injections, the sections that contained
the cerebellum were then incubated with biotin-conju-
gated anti-aldolase C antibody (320 ng/mL, #69076;
Sugihara and Shinoda, 2004) in PBS with Triton X-100
(PBST, 0.15%) and normal rabbit serum (2%) for 48
hours. This antibody was raised in the laboratory by
immunizing a rabbit with a synthetic peptide represent-
ing amino acids 322–344 from rat aldolase C (Sugihara
and Shinoda, 2004). This antibody stains a single band
on western blot and immunostaining is abolished by
adding immunizing peptide to the primary antibody solu-
tion (Sugihara and Shinoda, 2004). The sections were
then washed, incubated with biotinylated peroxidase-avi-
din complex (PK6100 Elite ABC kit; Vector Laboratories)
for 4–8 hours, washed, and finally incubated with diami-
nobenzidine (0.5 mg/mL), glucose oxidase (0.01 mg/
mL; type II, G-6125; Sigma, St. Louis, MO), ammonium
chloride (4 mg/mL), and beta-D(þ)-glucose (2 mg/mL)
in PBS for 30–60 minutes to stain the diaminobenzidine
reaction product brown. Sections were washed with
PBS, mounted on glass slides, dried, counterstained
with thionine, and coverslipped with Permount (Fisher
Scientific, Fair Lawn, NJ).
Reconstruction of individual axonsAxonal trajectories of selected fibers were recon-
structed from serial sections using a conventional bright-
field microscope (BX50; Olympus, Tokyo, Japan) and a 3D
imaging microscope (Edge R400; SNT Microscopes, Los
Angeles, CA) equipped with a camera lucida apparatus
with objectives of 10�, 20�, 40�, 60�, and 100�. Cut
ends of an axon on one section were connected to the
corresponding cut ends of the same axon on the neigh-
boring sections (Sugihara et al., 1999; Wu et al., 1999).
To ensure the correct matching of labeled axons in the
cerebellum and brainstem, reconstruction was performed
for those cases in which the injection labeled only a small
number of cerebellum-projecting axons (fewer than
eight).
Mapping mossy fiber terminals on the unfoldedcerebellar cortex or in 3D space
A previous standardized unfolded representation of the
Purkinje cell layer of the whole cerebellar cortex with an
aldolase C labeling pattern (Sugihara and Shinoda, 2004)
was used as a template for mapping mossy fiber termi-
nals. We identified the aldolase C compartments in which
the terminals of a labeled mossy fiber were distributed.
The relative mediolateral positions of the labeled termi-
nals in each aldolase C compartment and the relative ros-
trocaudal position of the fiber within the folium were then
measured (Fig. 1I–J). Although aldolase C compartments
are not labeled in the granular layer, the bounds of aldol-
ase C-positive and -negative compartments in the granu-
lar layer were extrapolated from those in the molecular
layer. These measurements were used to plot the termi-
nals on the unfolded representation of the cerebellar cor-
tex (Fig. 1K).
To map the mossy fiber terminals in 3D space, outlines
of cortical layers as well as labeled axons and terminals
were depicted in every section with a camera lucida.
These drawings were digitized using 2D graphics software
(Illustrator, Adobe, San Jose, CA). The outlines in serial
sections were drawn in different ‘‘layers’’ of the software.
The positions and tilts of the drawings of serial sections
were adjusted so that all sections, including segments of
single axons, fit each other when superimposed. The Illus-
trator graphic files were then imported into 3D graphics
software (Rhinoceros, Robert McNeel & Associates, Seat-
tle, WA). Outlines in different sections were shifted in the
direction of the z-axis by a distance equivalent to the
thickness of the section to reconstruct the cortical layers
in the 3D space of the software. The position of individual
terminals was further adjusted in the z-direction accord-
ing to the depth from the cut surface of the section.
Mapping BDA injection sites and retrogradelylabeled neurons in the DCN
In one rat, the contour of the DCN and other landmark
structures including the surface of the medulla, the cen-
tral canal, hypoglossal nucleus, and dorsal cochlear nu-
cleus were depicted with the camera lucida in every serial
coronal section of the caudal medulla and the most ros-
tral levels of the spinal cord. A 3D atlas of the DCN was
then built in Rhinoceros. Drawings of the same structures
in parasagittal sections obtained in other rats were used
to adjust the 3D atlas.
To map the retrogradely labeled neurons and BDA
injection sites within the 3D image of the DCN in Rhinoc-
eros, the relative position of each coronal section con-
taining labeled neurons or an injection site was deter-
mined. The caudal poles of the cochlear nucleus and the
ECuN and the opening of the central canal were used as
Quy et al.
878 The Journal of Comparative Neurology |Research in Systems Neuroscience
landmarks for rostrocaudal positioning. The midline and
lateral poles of the ECuN, CuN, and GN were used to align
mediolateral positions for parasagittal sections. The out-
lines of the 3D atlas at that position were displayed on
the computer monitor. The computer monitor and the
view of the tissue through the microscope were superim-
posed on each other. A small sphere was plotted at each
position where a retrogradely labeled neuron was
located. Circle-like closed curves were drawn to outline
the injection sites in serial sections. These curves were
then transformed to a sphere by using the Loft command
in Rhinoceros (Sugihara and Shinoda, 2007). The mapped
neurons and injection sites, as well as the contour of the
DCN, were then projected onto a horizontal plane for use
in the present figures.
Photomicrographs were taken by using a digital camera
(DP-50; Olympus, Tokyo, Japan) attached to a microscope
(BX41; Olympus). Photographs were assembled using Pho-
toshop (Adobe, San Jose, CA) and Illustrator software. The
software was used to adjust contrast and brightness, but
no other digital enhancements were applied.
RESULTS
Tracer injection into the DCNThe CuN, ECuN, GN, and nucleus Z occupy roughly the
caudolateral, rostrolateral, caudomedial, and rostrome-
dial parts of the DCN, and are generally involved in proc-
essing forelimb cutaneous, forelimb proprioceptive, hind-
limb cutaneous, and hindlimb proprioceptive information,
respectively (Tracey, 2004), although the sensory modal-
ities are not strictly separated (Cerminara et al., 2003).
To map tracer injection sites and retrogradely labeled
neurons, a 3D scheme of these nuclei was constructed
based on tracings of the cytoarchitectural contours of the
ECuN, CuN, and GN in serial sections (Fig. 1B). Horizontal
trajectories of these contours were separated by being
shifted mediolaterally and were used to map neurons and
BDA injection sites (Fig. 1D). The nucleus Z, the smallest
of these subdivisions, has been identified morphologically
and physiologically at a position slightly rostrolateral to
the rostral pole of the GN in the cat (Brodal and Pom-
peiano, 1957; Mackel and Miyashita, 1993). One study
has identified the nucleus Z in the same position morpho-
logically in the rat (Low et al., 1986). However, the nu-
cleus Z was not distinguishable by cytoarchitectural crite-
ria in the present study. Therefore, its location was
defined using physiological methods (see below).
Although the nucleus Z is not a major relay nucleus of
hindlimb proprioceptive information to the cerebellum in
comparison to the Clarke’s column nucleus, we studied it
as a part of the DCN in the present study. The caudal
parts of the CuN and GN in the cervical spinal cord were
not included in our 3D scheme since these parts did not
contain cerebellum-projecting neurons (see later sec-
tion). The rostral ends of these nuclei formed a protuber-
ance in the dorsal surface of the medulla (Fig. 1A,C),
which was a landmark for these structures during electro-
physiological recording.
We made 26 BDA injections in various areas in the
DCN, where field potential or multiple unit responses to
touch and passive movement of the forelimb, trunk, or
hindlimb were recorded prior to injection (Fig. 1G,H).
Field responses were sometimes evoked by wide range of
stimuli, i.e., both by proprioceptive and cutaneous stimuli
(Cerminara et al., 2003). However, it was not difficult to
identify the stimulus that evoked the largest field
response in the recording site. We did not systematically
map the DCN electrophysiologically. Although we could
not identify the response property of each reconstructed
neuron, we presume it would be similar to the field poten-
tial recorded prior to the tracer injection in this study
because the injection was localized. Subsequent histolog-
ical examination showed that the forelimb cutaneous and
proprioceptive response sites were located in the CuN (n
¼ 6) and ECuN (n ¼ 11), respectively, consistent with the
above general organization of the DCN. Different
responses were recorded in the area that was regarded
as the GN in the present study (Fig. 1E). Injection sites
where trunk and hindlimb cutaneous responses were
recorded were located in the lateral (n ¼ 3) and centro-
medial (n ¼ 3) parts of the GN (Fig. 1E, gray and white
circles, respectively). Sites that showed hindlimb proprio-
ceptive responses, which presumably correspond to the
nucleus Z, were most difficult to find. These hindlimb pro-
prioceptive sites (n ¼ 3) were mapped to the rostral pole
of the GN in our 3D scheme of the DCN (Fig. 1E, black
circles). Therefore, the rostral pole of the GN in this study
may be equivalent to the nucleus Z described in the rat
and cat (Low et al., 1986; Mackel and Miyashita, 1993).
Injections, which were recognized as black spots with
a diameter of 200–300 lm after histological visualization
(Fig. 1F), were found to be located in the ECuN, rostral CuN,
or rostral GN in 21 of the 26 cases. Each subnucleus of the
DCN has somatotopic organization. For example, the whole
ECuN is separated into six divisions of different somatotopy
(neck, thorax, shoulder, arm, forearm, and hand, from the
rostrolateral to caudomedial poles; Campbell et al., 1974).
The spread of the tracer in an injection in the present study
seemed localized in one or two adjacent somatotopic divi-
sions based on the size. In each of these cases, labeled
axons projected to the cerebellum. In contrast, no such
axons were found in the remaining five injections, which
were located in the caudal CuN and GN, suggesting that cer-
ebellar projecting neurons are rare in the caudal CuN and
GN. This conclusion is also supported by retrograde labeling
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 879
results (see later section). We reconstructed single DCN-
cerebellar axons in 13 of these injections in which one or a
small number of axons were well labeled.
Axonal path and general morphologyof DCN axons
We reconstructed 15 axons that were labeled by DCN
injections in 13 animals. Individual axons were recon-
structed by starting from the inferior cerebellar peduncle
and following their trajectory both up to their cerebellar
terminations and down to the injection site within the
DCN. All branches at each ramification point were recon-
structed completely. Ten axons from nine of the injec-
tions could be traced down to their soma of origin, which
was located within the DCN, adjacent to the injection
center. These cell bodies were generally large and polygo-
nal-shaped (Fig. 2D). Four other axons could be traced
down to the injection site, but their somata were not iden-
tifiable because of the dense staining present. One other
axon could be traced down to a point in the inferior cere-
bellar peduncle 2.5 mm rostral to the injection sites. We
considered that these five axons also originated from the
DCN, even though their somata of origin were not identi-
fied, since the pathways and branching patterns of these
neurons within the medulla and deep cerebellar white
matter were essentially identical to those of the ten axons
whose somata were identified.
We occasionally encountered other kinds of axons.
When traced backward, these axons did not enter the
injection site, but instead passed through the medullary
reticular formation ventral to the DCN and could be fol-
lowed down into the spinal cord. We concluded that they
were spinocerebellar axons that were labeled by retro-
grade tracer uptake from their branches terminating in
the DCN, since BDA works as a retrograde tracer,
although much less efficiently than it does as an antero-
grade tracer (Sugihara et al., 1999). All of these spinocer-
ebellar axons gave off multiple collaterals in the reticular
formation in the medulla and in the cerebellar nuclei and
they also terminated as mossy fiber terminals in the cere-
bellar cortex in the present study. Abundant collaterals in
the cerebellar nuclei of spinocerebellar mossy fiber axons
have also been reported by Matsushita and Yaginuma
(1995). Since nuclear collaterals were relatively unusual
in DCN-cerebellar axons (see later section), the branching
pattern of spinocerebellar axons was clearly different
from that of DCN-cerebellar axons. This difference in the
branching pattern between spinocerebellar and identified
DCN-cerebellar axons supports the earlier decision to
categorize as DCN axons those axons whose somata
were not identified, since the latter two groups of axons
showed the same branching pattern.
The trajectory of a completely reconstructed axon is
shown in Figure 2. This axon originated from a neuron
located near the injection center in the central ECuN (Fig.
2E). The field potential recorded through the micropipette
prior to the injection showed proprioceptive responses
tuned most strongly to abduction in the ipsilateral
shoulder. The axon ran rostrolaterally in the dorsolateral
superficial white matter of the medulla to enter the ipsi-
lateral inferior cerebellar peduncle (Fig. 2A). It gave rise
to no collaterals in the medulla. After entering the cere-
bellum, the stem axon gave rise to several collaterals
while running medially in the ipsilateral deep white matter
(Fig. 2A). These collaterals entered the folial white matter
of several lobules and further branched several times
before entering the granule cell layer. Each branch
formed a cluster of mossy fiber rosettes in a patch- or
zone-shaped small area in a lobule. Rosette terminals
(long diameter, usually 13–23 lm and occasionally 6–12
lm) could be located at the ends or middles of the axon
collaterals and at branching points.
All reconstructed DCN-cerebellar axons showed trajec-
tories and ramification patterns that were essentially sim-
ilar to those just described, although the specific cortical
termination areas varied. Almost all reconstructed axons
projected only in the ipsilateral cerebellum. This agreed
with the previous reports that the DCN-cerebellar projec-
tion is predominantly ipsilateral (Somana and Walberg,
1980; Gerrits et al., 1985). However, we encountered an
ECuN axon that projected bilaterally by transcommissural
elongation of the main axonal trunk in the deep cerebellar
white matter (Fig. 3). This axon was traced back to the
soma in the ECuN. Because of the long main axonal trunk,
the basic branching pattern of the ECuN axon is clearly
recognized in this case: several lobular branches were
given off perpendicularly from the transverse main axonal
trunk. This branching pattern is similar to that of LRN
axons (Wu et al., 1999).
The number of rosette terminals per axon ranged from
57 to 202, with an average of 123.3 6 42.3 (SD, n ¼ 15).
One or a few short collaterals (length, 2–10 lm) with a smallsatellite terminal (diameter, �1 lm) extended from about
one-fifth of the rosette terminals. In thionine-counterstained
sections, we did not observe apparent contact between a
rosette or satellite terminal and a Golgi cell soma.
Since the brain sections were double-labeled for BDA
and aldolase C, locations of axonal terminals could be
identified in terms of aldolase C compartments. Aldolase
C is a molecule expressed in a compartmentalized popu-
lation of Purkinje cells (Hawkes and Leclerc, 1987; Bro-
chu et al., 1990). About 20 longitudinal compartments, in
which PCs have positive, negative, or lightly positive
expression of aldolase C, have been defined in the rat cer-
ebellar cortex. A scheme that shows the aldolase C
Quy et al.
880 The Journal of Comparative Neurology |Research in Systems Neuroscience
immunostaining pattern on the unfolded whole cerebellar
cortex has been developed (Fig. 2C; Sugihara and Shi-
noda, 2004). In this scheme, gray and white longitudinal
stripes represent aldolase C-positive and -negative com-
partments, respectively. A specific name, such as 1þ,
1�, and so on (usually a numeral or a letter followed by a
Figure 2. Morphology of a reconstructed single mossy fiber axon of an ECuN neuron. The axon was traced from the soma, which was
located near the injection center, to the terminations of all branches. A: Frontal view of the axonal trajectory, drawn on the montage of
rostral and caudal cerebellar sections and a caudal medullar section. B: Lateral view of the trajectory of the same axon, drawn on the
montage of parasagittal sections of different mediolateral levels. C: Distribution of all the rosette terminals of this axon mapped on the
unfolded scheme of the cerebellar cortex. D: Photomicrograph of the DCN neuron from which the axon (arrowhead) originated. E: Injection
site mapped on the horizontal scheme of the DCN. It responded best to shoulder abduction.
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 881
sign indicating positive or negative), is given to each com-
partment (Fig. 2C). Tracing studies have identified the
specific olivocerebellar projection to each compartment
and consequently clarified the correspondence between
the aldolase C compartments and the conventional A–D
zones (Voogd et al., 2003; Sugihara and Shinoda, 2004).
Furthermore, it has been suggested that aldolase C-nega-
tive and -positive areas mainly receive somatosensory
and other (cerebral, tectal, vestibular, and visual) inputs,
respectively, through the olivocerebellar projection (Sugi-
hara and Shinoda, 2004). Thus, aldolase C compartments
may delineate distinct functional areas of the cerebellar
cortex. Therefore, the aldolase C immunostaining pattern
can be regarded as a molecular as well as a functional
map of the cerebellar cortex.
All terminals of the reconstructed axon shown in Figure
2A,B were mapped on this scheme (Fig. 2C). Terminals
were distributed in lobules II and III (in medial compart-
ment 2�), lobules IV, V, and simple lobule (in compart-
ment 4�) and IXa (in compartments 2þ and 3þ).
Nuclear projection of DCN axonsAmong 15 completely reconstructed DCN axons, only
one had a collateral that terminated in the cerebellar
nuclei (Fig. 4A,B,E), indicating that DCN axons possess
nuclear collaterals infrequently. None of the 15 axons
had a collateral that projected to the vestibular complex.
The axon shown in Figure 4 could be traced to a soma
located in the BDA injection site in the lateral GN (Fig.
4D), which responded to touching of the abdominal skin
Figure 3. Morphology of a reconstructed single mossy fiber axon of an ECuN neuron, which projected to the bilateral cerebellar cortex. A:
Frontal view of the axonal trajectory, drawn on the montage of rostral and central cerebellar sections and a caudal medullar section. B:
Distribution of all the rosette terminals of this axon mapped on the unfolded scheme of the cerebellar cortex. C: Injection site mapped on
the horizontal scheme of the DCN. It responded best to elbow extension.
Quy et al.
882 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 4. Nuclear projection of a reconstructed DCN axon. A: Frontal view of the axonal trajectory, drawn on the montage of rostral and
caudal cerebellar sections and a caudal medullar section. This axon was traced to the soma. B: Lateral view of the axonal trajectory,
drawn on the montage of parasagittal sections of different mediolateral levels. Arrowheads in A and B indicate the nuclear collateral.
C: Distribution of all the rosette terminals of this axon mapped on the unfolded scheme of the cerebellar cortex. Terminals of this axon
were distributed mainly in various aldolase C compartments near the apex of the copula pyramidis. D: Injection site in the lateral GN
mapped on the horizontal scheme of the DCN. It responded best to touching of the abdominal skin at the level of the liver. E: Magnified
view of the trajectory of terminal arborization of the nuclear collateral, which was reconstructed from three coronal sections. F: Photomi-
crograph of terminal branching of the nuclear collateral.
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 883
at the level of the liver. None of the other reconstructed
axons (n ¼ 14), which included those originating from the
abdomen cutaneous area in the lateral GN (n ¼ 2), had
nuclear collaterals. It was not clear whether occurrence
of nuclear collaterals is related to certain subpopulational
distinctions among DCN neurons.
The lone observed nuclear collateral branched several
times to form a loose terminal arbor, which bore 59 en-
passant and terminal swellings and occupied a small area
in the rostromedial part of the anterior interposed nu-
cleus (Fig. 4E,F). The diameters of the collateral and swel-
lings were �0.4 lm and 1.5–2.0 lm, which were similar
to those of LRN axons (Wu et al., 1999). The terminal
arbor of this nuclear collateral was more compact than
those of LRN axons, which were usually elongated to
more than 500 lm in length (Wu et al., 1999).
In agreement with the infrequency of the nuclear collat-
erals of individual DCN-cerebellar axons, in all cases of
BDA injections to the DCN labeled axonal terminals were
rarely observed in sections of the cerebellar nuclei. These
nuclear terminals may belong to either DCN-cerebellar
axons or to spinocerebellar axons that were concomi-
tantly labeled retrogradely (see earlier section).
Variation of axonal projection of adjacentDCN neurons
We must clarify the variation in the axonal projection
pattern before examining the topography of the DCN-cer-
ebellar projection, since a precise topography requires
that axons originating from adjacent neurons project to
mostly similar areas, as is the case with olivocerebellar
axons (Sugihara et al., 2001). Therefore, the distribution
of all labeled terminals was compared with that of the ter-
minals of individual reconstructed axons for some ECuN
injections (Fig. 5).
Four injections were made in slightly different areas in
the ECuN and all labeled mossy fiber terminals were
mapped. While the two caudal injection sites responded
best to elbow extension (Fig. 5A,B), the rostral injection
sites responded best to shoulder abduction (Fig. 5C) and
shoulder adduction (Fig. 5D). This arrangement agreed
with the reported somatotopic organization of the ECuN
in that proximal muscles are represented in its more ros-
trolateral parts (Campbell et al., 1974). Mossy fibers that
were labeled by these four injections projected mainly to
medial 2� in lobule I–V, negative compartments in pars
intermedia in lobule V, and sublobule a of the simple
lobule in the rostral cerebellum. They also projected to
caudal lobule VII and paramedian lobule, lobule VIII and
copula pyramidis, and lobule IX in the caudal cerebellum
with variable degrees. These projection patterns seem
common for the ECuN in general, and can be compared
with the projection patterns of other parts of the DCN.
Concerning topography, elbow areas in the caudal
ECuN projected more to the paramedian lobule and cop-
ula pyramidis (Fig. 5A,B), while shoulder areas in the ros-
tral ECuN projected more to lobule IX (Fig. 5C,D). Thus,
some topography was indicated within the ECuN-cerebel-
lar projection, which was not as precise as that of the oli-
vocerebellar projection (Sugihara et al., 2001).
Mappings in Figure 5 compare the distribution of termi-
nals from a single axon (red or cyan dots) to that of all ter-
minals (black and colored dots) labeled by an injection
into a specific region of the ECuN. The distribution of ter-
minals from an individual axon was always smaller than
the distribution of all terminals, which indicates that there
is a significant variation in the distribution of terminals in
axons originating from neurons located within each small
region of the ECuN. Indeed, in the case of Figure 5B, in
which all labeled terminals belonged to two reconstructed
axons, the distribution of terminals of the two axons were
mostly located in different areas with a small overlap in
lobule III and copula pyramidis. The neuronal somata of
these axons were located in the tracer injection site, only
90 lm apart from each other. The termination patterns of
two reconstructed axons in Figure 5C (red and cyan dots)
was also different, with only one of the axons terminating
in lobules II–V. These variations of the projection patterns
of adjacent neurons support the idea that topography of
the projection was not precise.
Topography of DCN-cerebellar projectionTo further examine the topography of DCN-cerebellar
projection, the axonal morphology and terminal distribu-
tion of axons originating from different nuclei of the DCN
were compared. Axonal trajectories and terminal distribu-
tions for three completely reconstructed axons are shown
in Figure 6 in a format similar to that in Figure 2. The injec-
tion sites of these axons belonged to different nuclei of the
DCN (axon in Fig. 6A–C: ECuN, responsive to shoulder
adduction; Fig. 6D–F, CuN, responsive to touching of the
dorsal arm; Fig. 6G–I, lateral GN, responsive to touching of
the side of the trunk over and caudal to the lower ribs).
The ECuN axon (Fig. 6A–C) terminated mainly in compart-
ments 2þ and 3þ in caudal lobule VIII and lobules IXa and
IXc. The CuN axon (Fig. 6D,E) terminated mainly in several
compartments in the pars intermedia of sublobule a of the
simple lobule, paramedian lobule, and rostral copula pyra-
midis. The lateral GN axon (Fig. 6G–I) terminated in several
aldolase C compartments of the pars intermedia and hemi-
spheric portions of the apical copula pyramidis. The projec-
tion areas were clearly different among these axons.
Figure 7 shows the axonal trajectories and terminal dis-
tributions for an axon labeled by an injection to a site in
the centromedial GN that responded to touching of the
heel and an axon originating from a site at the rostral pole
Quy et al.
884 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 5. Distribution of mossy fiber terminals labeled by BDA injections to various areas in the ECuN. All labeled terminals were mapped for four
injections to the ECuN (A–D). The terminals that belong to single reconstructed axons are shown in distinct colors (red or cyan) in A, C, and D. Insets
in the top right corner of each panel indicate the injection sites in the horizontal ECuN contour. Response characteristics of the injection sites, (A)
elbow extension; (B) elbow extension; (C) shoulder abduction; (D) shoulder adduction. A small number of terminals were mapped in the contralateral
cerebellar cortex in A–D. It was not clear whether they belonged to DCN axons or to spinocerebellar axons labeled by retrograde tracer uptake.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 885
of the GN (putatively equivalent to the nucleus Z) that
responded to knee extension. The former terminated
mainly in the apical copula pyramidis (Fig. 7A–D), while
the latter terminated mainly in the caudal (ventral) copula
pyramidis (Fig. 7E–H). The terminal distributions of these
axons were more similar to that of the axon from the lat-
eral GN shown in Figure 6G–I and clearly different from
those of ECuN or CuN axons (Fig. 6A–C,D–F).
To further examine the topography of DCN-cerebellar
projection systematically, mossy fiber terminals of all
reconstructed axons (n ¼ 15 in 13 injections) were plot-
ted on an unfolded map of the cerebellar cortex, color-
coded according to their origin in the DCN (Fig. 8). Taken
together, the terminals of these reconstructed axons orig-
inating from the ECuN, CuN, and GN were distributed in
the vermal and intermedial regions in the rostral and
Figure 6. Different projections of reconstructed single mossy fiber axons originating from the ECuN (A–C), CuN (D–F), and GN (G–I). Fron-
tal view of the axonal trajectories, drawn on the montage of rostral and caudal cerebellar sections and a caudal medullar section (A,D,G),
distribution of all the rosette terminals of the same axon mapped on an unfolded scheme of the cerebellar cortex (B,E,H), and injection
sites mapped on a horizontal scheme of the DCN (C,F,I) are shown for each axon. Response characteristics of the injection sites, A–C,
shoulder adduction; D–F, touching of the dorsal skin of the arm; G–I, touching of the skin of the side of the trunk over and caudal to the
lower ribs. Somata were identified for axons in A–C and D–F.
Quy et al.
886 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 7. Projection of reconstructed single mossy fiber axons originating from hindlimb cutaneous (A–D) and proprioceptive (E–H) areas in
the GN. Frontal view of the axonal trajectories, drawn on the montage of rostral and caudal cerebellar sections and a caudal medullar section.
(A,E) Lateral view of the axonal trajectories, drawn on the montage of parasagittal sections of different mediolateral levels (B,F), distribution of
all the rosette terminals of the axons mapped on an unfolded scheme of the cerebellar cortex (C,G), and injection sites mapped on a horizontal
scheme of the DCN (D,H) are shown for each axon. Response characteristics of the injection sites, A–D, touching of the heel; E–H, knee exten-
sion. Somata were not identified for these axons. The axon in A–D could not be traced beyond the middle of the inferior cerebellar peduncle in
the proximal side, but was reconstructed completely in distal parts.
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 887
caudal cerebellar cortex, but were nearly absent in the
central lobules (lobules VIc–VII, crus I–II) and lateral hem-
ispheric regions of other lobules, and in the paraflocculus,
flocculus, and nodulus (lobule X) (Fig. 8). Although the
injection sites did not systematically cover the whole
nuclei, and despite the wide scattering of terminals, a
general topographic organization in the distribution of ter-
minals originating from different nuclei was discernable
(Fig. 8). In the rostral vermis, ECuN terminals were mostly
distributed in a single longitudinal compartment (medial
2� in lobules I–V). In the rostral pars intermedia, CuN ter-
minals were generally distributed more caudally (sublobule
a of the simple lobule) than ECuN terminals (lobule V and
rostral sublobule a of the simple lobule), and GN terminals
were sparsely distributed further rostrally (lobules III–V). In
the caudal vermis, ECuN terminals were densely distrib-
uted in lobules VIII–IXa and IXc in multiple compartments
(1þ to 3þ), while CuN and GN terminals were sparsely dis-
tributed mainly in lobule IXb. The rostral ECuN injections
(purple in Fig. 8; responsive to shoulder movement) pro-
jected more to lobule IX, while the caudal ECuN injections
(blue in Fig. 8; responsive to elbow movement) projected
more to lobules VII–VIII. In the caudal pars intermedia,
CuN terminals and caudal ECuN terminals were distributed
in multiple compartments in the apical and caudal parts of
the paramedian lobule and in the rostral copula pyramidis,
with the distribution in the former lobule being more
medial than that in the latter. The GN terminals were
densely distributed in the apical and caudal pars interme-
dia in multiple compartments. The lateral (trunk cutane-
ous, yellow green) and centromedial (hindlimb cutaneous,
yellow) GN mainly projected to the apical copula pyrami-
dis, while the rostral pole of the GN (hindlimb propriocep-
tive, cyan) projected to the caudal copula pyramidis.
The results showed that terminals of axons originating
from different nuclei of the DCN, which are involved in
sensation in different body regions or different sensory
modalities, were generally distributed in distinct areas in
the cerebellar cortex. The distribution roughly showed a
mirrored pattern about the ‘‘rostrocaudal boundary’’ of
the cerebellum, which is located in lobule VIc and crus I,
and is defined by the pattern of aldolase C compartments
(transverse dotted curve in Fig. 8; Sugihara and Shinoda,
2004). ECuN terminals were distributed most medially in
the rostral and caudal vermis. In the rostral and caudal
Figure 8. Topography of the DCN-cerebellar projection. The dis-
tributions of mossy fiber terminals of the 11 reconstructed axons
in 10 injections are summarized (top). Terminals were color-coded
according to the somatosensory response of the injection sites,
which were mapped in the horizontal scheme of the DCN (bot-
tom). Blue, distal forelimb proprioceptive, Caudal ECuN; purple,
proximal forelimb proprioceptive, rostral ECuN; red, forelimb cuta-
neous, CuN; cyan, hindlimb proprioceptive, rostral pole of the GN;
yellow-green, trunk cutaneous, lateral GN; orange, hindlimb cuta-
neous, medial GN. Vertical dotted curves indicate tentative boun-
daries between the vermis, pars intermedia and hemisphere.
Transverse dotted curve indicate the rostrocaudal boundary of
the cerebellar cortex (Sugihara and Shinoda, 2004). [Color figure
can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Quy et al.
888 The Journal of Comparative Neurology |Research in Systems Neuroscience
pars intermedia, CuN terminals were distributed closer to
the rostrocaudal boundary, whereas GN terminals were
distributed farther from the rostrocaudal boundary.
Retrograde labeling of DCN neurons by largeBDA injections into individualcerebellar lobules
To further examine the differences in the projections of
the DCN subdivisions, large injections of BDA were made
into individual cerebellar lobules to retrogradely label neu-
rons in the DCN. Retrogradely labeled neurons were of a
similar size and shape to that shown in Figure 2D. Neu-
rons located inside of and around the apparent contour of
the nuclei were counted. The number of labeled neurons
in the inferior olive nucleus was also counted to judge the
extent of the injection. Injections were made into vermal
lobules VIa, VIb, VII, VIII, IXa–b, IXc, and Xa; hemispheric
sublobule a and b of the simple lobule, crus I, crus IIa, par-
amedian lobule, copula pyramidis, dorsal paraflocculus,
ventral paraflocculus; and vermal and hemispheric lobules
III and IV–V. Among these injections, those made into
hemispheric simple lobule, paramedian lobule, copula pyr-
amidis, III, IV–V, vermal lobule VIII, IXa–b, IXc (dark gray
areas in Fig. 9I) labeled more than 50 neurons in the ipsi-
lateral DCN, which are mapped in Figure 9A–H. The num-
ber of labeled neurons in the ipsilateral DCN was 0.13–
0.99 of the number of labeled neurons in the inferior olive.
Injections in other lobules (light gray areas in Fig. 9I) la-
beled much smaller number of neurons in the DCN, only 0
to 5 neurons (mapping not shown), although they labeled
171–615 neurons in the inferior olive.
In the CuN and GN, all labeled neurons were located in
their rostral parts (within about 2 mm from the rostral
pole of the CuN and about 1 mm from the rostral pole of
the GN, Fig. 9J,K). Although neurons located around the
rostral pole of the GN were carefully mapped, no separate
group of neurons that might correspond to ‘‘nucleus Z’’
were distinguishable. We summed all of the injections
shown in Figure 9A–H, and found that the total numbers
of labeled neurons in the ipsilateral ECuN, CuN, and GN
were 924, 440, and 38, indicating that the ECuN gener-
ally provides a larger number of mossy fiber axons to the
cerebellum than the CuN or GN. As seen in the number of
labeled neurons in the IO (144–1,094), the extent of the
injection varied among experiments. Therefore, we com-
pared the numbers of labeled neurons in different nuclei
of the DCN or in different subareas of a nucleus within
each case. The numbers of labeled neurons in the CuN
were similar to or greater than those in the ECuN with
injections into the hemispheric simple lobule (Fig. 9A),
paramedian lobule (Fig. 9B), and copula pyramidis (Fig.
9C). In contrast, many more labeled neurons were found
in the ECuN than in the CuN for injections into vermal-
hemispheric lobules III, IV–V and vermal lobules VIII, IXa–
b, and IXc (Fig. 9D–H). These results generally agreed
with those of anterograde mapping (Fig. 8) and support
the conclusion that the projection fields of the ECuN and
CuN are relatively distinct, but do have some overlap.
While injections into lobule IX labeled more neurons in
the rostral ECuN, injections into the copula pyramidis and
paramedian lobule labeled more neurons in the caudal
ECuN than in the rostral ECuN (Fig. 9B,C,F,G,H) and injec-
tions in lobules III and IV–V labeled similar number of neu-
rons in the caudal and rostral ECuN (Fig. 9C,D). This indi-
cated that distinct topographic projections from the
caudal and rostral ECuN exist, as suggested in antero-
grade tracing experiments (Fig. 5).
The relatively small number of labeled neurons in the
GN indicated that there were many fewer cerebellar pro-
jecting neurons in the GN than in the CuN and ECuN,
which was presumably because most GN and nucleus Z
neurons project to the thalamus (Low et al., 1986; Mackel
and Miyashita, 1993; Tracey, 2004). For example, only
injections in copula pyramidis labeled more than 10 neu-
rons in the GN. This agrees with the anterograde labeling
result that GN-cerebellar axons project mainly to the cop-
ula pyramidis (Fig. 8). Most labeled neurons were seen in
and near the rostral pole of the GN and others were dis-
tributed more caudally. Since electrophysiological map-
ping and anterograde injections suggested that the hind-
limb proprioceptive neurons were located in the rostral
pole of the GN (see previous section), the results of retro-
grade mapping indicates that a population of propriocep-
tive axons as well as cutaneous-sensation axons projects
to the cerebellum from the GN.
A small number of labeled neurons was seen in the con-
tralateral DCN (indicated within the parentheses in Fig.
9A–H). They may be explained by the spread of the
injected tracer to the contralateral cerebellum in the cases
of vermal injections. However, they may also indicate the
presence of transcommissural contralateral projection in
a few DCN neurons, as found in the anterograde labeling
(Fig. 2), in the cases of hemispheric injections (Fig. 9A–C).
Relationship between the overall projectionof a single DCN axon and aldolaseC compartmentalization in thecerebellar cortex
Projection patterns of olivocerebellar climbing fiber
axons are closely related to aldolase C compartmentaliza-
tion. Olivocerebellar axons often project to the rostral
and caudal cerebellum simultaneously by branching
(Sugihara et al., 2001). Aldolase C compartments in the
rostral and caudal cerebellum innervated by such an
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 889
Figure 9. DCN projection to individual cerebellar lobules revealed by mapping retrogradely labeled neurons in the ipsilateral DCN (ECuN,
CuN, and GN). A–H: BDA injection into single cerebellar lobules mapped in a coronal section (top) and the distribution of neurons (bottom) in
the horizontal contour of the DCN. First, neurons were mapped in our 3D atlas of the DCN used as a template. Next, the ECuN, CuN, and GN
were separated by shifting them mediolaterally to improve the view, as in Figure 1D. The numeral beside each nucleus of the DCN indicates
the number of labeled neurons. The numbers of labeled neurons in the contralateral DCN (in parentheses) and in the inferior olive are also
indicated. I: Injection areas mapped on the unfolded scheme of the cerebellar cortex. Dark gray areas indicate injections in which a number of
neurons were labeled in the DCN (A–G) and are plotted in this figure. Light gray areas indicate injections that labeled fewer than five neurons
in the DCN. J,K: Photomicrographs of retrogradely labeled DCN neurons in coronal sections of the medulla (J, case B; K, case C). [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Quy et al.
890 The Journal of Comparative Neurology |Research in Systems Neuroscience
olivocerebellar axon usually belong to a specific linked
pair of compartments (Sugihara and Shinoda, 2004). Con-
cerning mossy fiber projections, no comparable system-
atic or detailed results about the relationship between al-
dolase C compartments and axonal projection have been
obtained so far. Therefore, we examined the relationship
between mossy fiber terminals and aldolase C compart-
mentalization for DCN axons.
We counted the number of terminals in each lobule
and in each compartment for every reconstructed axon
and summed the number of all axons for each compart-
ment (Table 1). The terminals were distributed in many
compartments mainly in the vermis and pars intermedia.
Aldolase C compartments have been classified into five
groups on the basis of the intensity of immunoreactivity
and the pattern of the olivocerebellar projection to the
compartments (Sugihara and Shinoda, 2004). The termi-
nals were most frequently (927/1844 ¼ 50.3%) dis-
tributed in the group IV compartments (Table 1), which
consists of aldolase C-negative and -lightly positive com-
partments in the pars intermedia of the rostral and caudal
cerebellum (compartments 2�, 3þ, 3�, 3bþ, 3b�, and
4� in the rostral cerebellum and 4�, fþ, f�, e1þ, e1�,
e2þ in the caudal cerebellum) and of aldolase C-negative
compartments in the hemisphere. It has been speculated
that the group IV compartments are involved in somato-
sensory-related function (see Discussion). Terminals of
DCN axons were also distributed in aldolase C compart-
ments belonging to other groups, including groups I (186/
1844 ¼ 10.1%) and II (391/1844 ¼ 21.2%), which consist
of aldolase C-positive compartments, and group III (340/
1844 ¼ 18.4%), which consist of aldolase C-negative com-
partments (Table 1). Therefore, we carefully looked at the
terminal distribution of individual axons to understand the
general relationship between the DCN-cerebellar projec-
tion and aldolase C compartmentalization.
The axon shown in Figure 10 arose from a BDA injec-
tion site in the caudal ECuN that responded to elbow
extension (Fig. 10D). This axon projected to lobules II, III,
V, VIII, IXa, and the copula pyramidis, the lateral exten-
sion of lobule VIII (Fig. 10A,B). In lobules II, III, and V the
terminals were consistently located in the medial half of
compartment 2� (Fig. 10C,E), which agreed with the
report by Ji and Hawkes (1994). Other branches of this
axon projected to compartment 3þ in lobules VIII–IXa
(Fig. 10C,F) and e1þ in the copula pyramidis. In lobule
IXa, other labeled axons projected to compartments 2þ,
2�, 3þ, 3�, and 4þ (Fig. 10F), while this reconstructed
axon projected only to compartment 3þ. Whereas com-
partment e1þ is a lightly positive compartment, which is
rather akin to a negative compartment in terms of the
climbing fiber projection pattern (Sugihara and Shinoda,
2004), compartment 3þ in lobules VIII and IXa is a
strongly positive compartment, which has a distinct
climbing fiber projection. Similar to this axon, the ECuN
axon shown in Figure 2 innervated negative compart-
ments in lobules II–V (2� and 4�) and positive compart-
ments in lobule IXa (2þ and 3þ).
Another axon shown in Figure 11 originated from an
injection site in the lateral GN that responded to touch of
the abdominal skin at the level of the liver (Fig. 11C). This
axon projected mainly to compartments 5� of lobule III,
4� of lobule V, and e1þ, e2þ, e2�, 5þ of the copula
pyramidis (Fig. 11A,B). This axon also projected to the
dorsal paraflocculus, which is almost completely aldolase
C-positive (Sugihara and Shinoda, 2004), and to 1� and
2� of lobule IXa. Although compartments 1� and 2� are
relatively narrow in lobule IXa, most of the terminals were
located within these negative compartments (Fig. 11D).
In summary, the general relationship between the
DCN-cerebellar projection and aldolase C compartmen-
talization was more complex than that of the olivocerebel-
lar projection. A single DCN axon often projected to both
aldolase C-positive and -negative (including -lightly posi-
tive) compartments in contrast to individual olivocerebellar
axons, whose terminations are usually limited to a single
type of compartment. Furthermore, the DCN-cerebellar
projections to the rostral and caudal cerebellum did not
generally link pairs of compartments as does the olivocere-
bellar projection.
Local relationship between a cluster ofterminals of DCN axons and analdolase C compartment
The relationship between the entire axonal termination
area in the cerebellum from each region of the DCN and the
aldolase C compartmentalization was not straightforward
(preceding section). We next examined the spatial relation-
ship between the aldolase C compartments of a single
lobule and the distribution of a cluster of mossy fiber termi-
nals that belong to one or a few branches of an axon.
The spatial organization of a cluster of terminals was
analyzed by mapping all terminals in a cluster in 3D space
(Fig. 12). The axon used in this analysis originated from
an injection site in the CuN (Fig. 12D), which responded
to touching of the dorsal skin of the ipsilateral fingers.
This axon projected mainly to compartments d� and 4þof sublobule a of the simple lobule, compartments 3�,
4þ, and 4� of lobules VII–VIII, 5a� of the paramedian
lobule, e2� of the copula pyramidis, and 3þ of lobule
IXa (Fig. 12A–C). The terminals in e2� of the copula pyra-
midis were so abundant (n ¼ 120) that they were appro-
priate for analyzing the spatial conformation of their
cluster. In the dorsal view of the mapping (filled arrow in
Fig. 12E), terminals are shown together with a half-
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 891
TABLE1.
Numberanddistributionofterm
inals
ofevery
reconstructedDCNaxon
Axon
Origin
Proximalend
ofaxonal
reconstruction
Best
response
Term
inals
in
thecerebellar
nuclei
Numberofcorticalterm
inals
sortedfirstbylobulesandsecondbycompartments
Total
IIIII
IVV
21
22
42
52
21
22
32
52
21
22
b1
3b2
42
21
22
31
32
3b1
3b2
41
42
51
502(Fig.7G)
rostralGN
injectionsite
kneeextension
093
24
13
491(Fig.6H)
lateralGN
injectionsite
abdomentouch
099
490(Fig.4)
lateralGN
soma
abdomentouch
59
112
23
35
448(Fig.11)
lateralGN
soma
abdomentouch
0122
17
44
511(Fig.7C)
medialGN
dorsalmedulla
heeltouch
075
504(Fig.12)
CuN
soma
fingers
touch
(dorsalside)
0167
1
450(Fig.6E)
CuN
soma
upperarm
touch
(dorsalside)
0126
526(Fig.6B)
rostralECuN
soma
shoulderadduction
0125
471A(Fig.2)
rostralECuN
soma
shoulderabduction
0127
27
912
12
215
2
471D(Fig.5C)
rostralECuN
injectionsite
shoulderabduction
057
595
caudalECuN
soma
elbowextension
0167
216
312
79
29
8
489A(Fig.5B)
caudalECuN
soma
elbowextension
0186
526
12
10
415
416
489C(Fig.5B)
caudalECLIN
soma
elbowextension
0114
28
10
488(Fig.3)
caudalECuN
soma
elbowextension
0202
(26)1)
466(Fig.10)
caudalECuN
injectionsite
elbowextension
072
14
13
29
Sum
ofallreconstructedaxons
Total
1844
749
26
19
81
27
315
72
12
982
518
45
10
43
2
Sortedbythegroupsof
compartments
defined
bythepattern
ofthe
olivocerebellarprojection2)
groupI
186
79
39
10
2
groupII
391
groupIII
340
groupIV
927
49
26
181
27
15
72
12
82
518
45
43
Numberofcorticalterm
inals
sortedfirstbylobulesandsecondbycompartments
(continued)
VId-a/Sim
a
VIb/
Sim
bVIc/Crl
VII-Par
VIII-Cop
IX
dPFI
12
a1
a2
21
22
2b1
c1c2
d1
d2
41
42
d1
41
c232
41
4a2
5a2
51
21
31
32
41
42
f1f2
e11
e12
e21
e22
51
61
12
21
22
31
32
14
20
29
13
11
23
116
74
26
59
219
12
31
516
528
79
4(1)
224
521
15
12
313
34
16
14
10
65
42
120
11
3
711
20
13
22
30
13
87
110
10
1
524
840
18
28
2
42
10
14
28
722
68
79
525
713
1
110
43
22
610
27
92
10
216
735
6
620(16)
19(3)
8(63)
(8)
(8)
28
4(5)
(6)
66
4
636
23
76
18
87
13
28
31
12
42
52
68
110
61
15
35
13
49
17
12
42
81
86
209
165
75
25
84
55
90
521
12
135
75
221
36
76
87
28
52
15
49
84
90
623
13
31
68
10
13
165
555
5
18
42
61
17
12
42
81
86
209
1) Figuresin
theparenthesesindicate
term
inalsin
thecontralateralside.
2) Sugihara
andShinoda,2004.
Figure 10. Projection to aldolase C-positive and -negative compartments of an ECuN axon. A: Frontal view of the axonal trajectory, drawn
on the montage of rostral and caudal cerebellar sections and a caudal medullar section. This axon originated from an injection site that
responded to elbow extension. B: Lateral view of the axonal trajectory, drawn on the montage of parasagittal sections of different medio-
lateral levels. C: Distribution of all the rosette terminals of the axon mapped on the unfolded scheme of the cerebellar cortex. D: Injection
site mapped on the horizontal scheme of the DCN. E,F: Photomicrograph of terminals of this axon in immunostained coronal sections of
lobules III (E) and IXa (F). Terminals of this axon were distributed in medial 2� in lobule III and in 3þ in lobule IXa (arrowheads in E,F). All
terminals in E belonged to this reconstructed axon, while some terminals in F belonged to other labeled axons. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 893
vtransparent sheet representing the Purkinje cell layer on
which the aldolase C immunostaining was mapped (Fig.
12F). In this view the distribution of terminals was nearly
exactly aligned within compartment e2� (Fig. 12F), indi-
cating a close relationship between the distribution of a
cluster of terminals and an aldolase C compartment. Simi-
larly, the clusters of terminals that belonged to a single
axon shown in Figures 11D and 8E were tightly located in
2� of lobule IXb and in medial 2� of lobule III, respec-
tively. These results indicated that the local distributions of
the cluster of terminals of individual DCN axons were often
closely related to the aldolase C compartmentalization.
An electrophysiological study has demonstrated that
mossy fiber inputs terminate in a depth-specific manner
in the granular layer (Jorntell and Ekerot, 2006). There-
fore, the terminals in the cluster shown in Figure 12E
were viewed from the side in the direction tangential to the
folial surface (open arrow in Fig. 12E,F) to measure their
depths in the granular layer (Fig. 12G,H). Since the thick-
ness of the granular layer varied, the cluster was divided
into three parts (rostral, central, and caudal) and the short-
est distance to the Purkinje cell layer was measured for
individual terminals. In the histograms for the three parts
(Fig. 12H, left to right), most of the terminals were distrib-
uted in intermediate depths (60–120 lm from the Purkinje
cell layer). This supported the notion of depth-specific ter-
mination of mossy fibers in the granular layer. In another
cluster of terminals shown in Figure 11D, terminals were
mostly distributed in depths between the center and bot-
tom of the granular layer, again indicating depth-specific
termination. However, the cluster in Figure 11D was
located deeper than that in Figure 12G,H.
Figure 11. Localization of a cluster of terminals of an GN axon in a single aldolase C compartment. A: Frontal view of the axonal trajec-
tory, drawn on the montage of rostral and caudal cerebellar sections and a caudal medullar section. This axon was traced to the soma.
B: Distribution of all the rosette terminals of the axon mapped on the scheme of the unfolded cerebellar cortex. Terminals of this axon were
distributed in compartment 5� in lobules II and III, 1� and 2� in lobule IXb, e1�, e2þ, e2� and 5þ in the copula pyramidis, and in the
dorsal paraflocculus. C: Injection site in the lateral GN mapped on the horizontal scheme of the DCN. It responded best to touching of the
abdominal skin at the level of the liver. D: Photomicrograph of terminals of this axon in immunostained coronal sections of lobules IXb.
Arrowhead indicates that the cluster of terminals was localized mostly in 2�. All terminals shown here belonged to this reconstructed axon.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Quy et al.
894 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 12. Three-dimensional analysis of the distribution of a cluster of terminals of a CuN axon in an aldolase C compartment. A: Frontal
view of the axonal trajectory, drawn on the montage of rostral and caudal cerebellar sections and a caudal medullar section. This axon
was traced to the soma. B: Lateral view of the axonal trajectory, drawn on the montage of parasagittal sections of different mediolateral
levels. C: Distribution of all the rosette terminals of the axon mapped on the unfolded scheme of the cerebellar cortex. Terminals of this
axon were distributed mainly in compartment d� and 4þ in sublobule a of the simple lobule, 3� and 5a� in paramedian lobule, e2� in
the copula pyramidis, and 3þ in lobule IXa. D: Injection site mapped on the horizontal scheme of the DCN. It responded best to touching
of the dorsal skin of the fingers. E: Photomicrograph of terminals (arrowheads) of this axon clustered in e2� in copula pyramidis. F,G: 3D
plots of the terminals (n ¼ 121 of a total of 167 terminals of this axon) in this cluster mostly in e2� of the copula pyramidis. In F, they
are viewed perpendicularly from the dorsolateral direction (filled arrow in E) and plotted underneath the Purkinje cell layer that is repre-
sented by a semitransparent sheet. Reconstructed aldolase C compartments are illustrated on this sheet. Almost all of these terminals
belonged to e2� and the cluster was arranged in a narrow longitudinal band-shaped patch. In G, the terminals are viewed from the side
in the direction tangential to the folial surface (open arrow in E,F) and plotted with the boundaries of cortical layers at the center of com-
partment e2� represented by curves. H: Distribution of terminals in terms of depth in the granular layer. The depth was measured as the
shortest distance to the Purkinje cell layer for individual terminals in 3D space. The cluster of terminals in G was divided into three parts
(rostral, central, and caudal, from left to right separated by dotted lines in G). The distribution of the terminal depths was plotted for each
part in the left, center, or right histogram. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 895
DISCUSSION
We reconstructed 15 DCN axons completely and
mapped their terminals on the aldolase C compartments
in the cerebellar cortex. These axons projected as mossy
fibers in the ipsilateral rostral and caudal cerebellar cor-
tex topographically, whereas they rarely provide a collat-
eral branch to the cerebellar nuclei. The majority of axo-
nal terminals were distributed in aldolase C-negative
bands but some were distributed in positive bands.
Single mossy fiber morphologyGenerally, very limited information is available on the
complete arborization of individual cerebellar mossy
fibers, or even of any class of axons, in the vertebrate
central nervous system. Previous anterograde and retro-
grade mass labeling studies have indicated that mossy
fibers from the DCN branch and project to multiple longi-
tudinal zones in the rostral and caudal cerebellum (Ber-
retta et al., 1991; Ji and Hawkes, 1994; Tolbert and Gut-
ting, 1997; Alisky and Tolbert, 1997) and lack nuclear
collaterals (Gerrits et al., 1985), but could not reveal the
exact extent of single axonal projection. We systemati-
cally studied the mossy fiber projection of DCN neurons
from the viewpoint of single axons, which allows a new
interpretation of earlier studies. The sole previous report
on the complete mossy fiber morphology considered
axons originating from the LRN, which is also related to
somatosensory processing (Wu et al., 1999). Thus, the
morphology of DCN axons in the present study also
allows a comparison of two classes of mossy fibers.
Axons arising from both the DCN and the LRN give off
several cortical branches while running transversely in
the deep cerebellar white matter rostrodorsal to the cere-
bellar nuclei. While LRN axons often cross the midline in
the deep cerebellar white matter to project bilaterally,
DCN axons did not cross the midline. While all recon-
structed LRN axons have nuclear collaterals, which usu-
ally branched from the transverse stem axon, DCN axons
have nuclear collaterals only infrequently. Neither of
these axons gives rise to collaterals in the medulla. Corti-
cal branches of both DCN and LRN axons terminate as a
cluster of terminals in a specific region of the cerebellar
cortex. While the LRN axons terminate mostly in lobules
IV, V, and VIa (Wu et al., 1999), DCN axons terminate in
other combinations of lobules in the caudal (paramedian
lobule, copula pyramidis, lobules VIII–IX) and rostral cere-
bellum (lobules II–V, and simple lobule). Thus, nuclear,
lobular and transverse projection patterns are essential
in characterizing these mossy fiber systems.
The infrequency of nuclear collaterals from DCN axons
is noteworthy regarding the formation of the cerebellar
output signal. In a classical scheme of the cerebellar
microcomplex, nuclear collaterals of mossy fibers are
essential for forming the direct excitation of the nuclear
output neuron, while the Purkinje cell projection provides
inhibitory signals when cerebellar modulation is required
(Ito, 1984). However, the present results suggest that this
scheme cannot be directly applied to the DCN system.
DCN as a precerebellar nucleusThe output axons of the DCN project to the thalamus
and cerebellum (Tracey, 2004). The present study sug-
gested that the neurons that project to the cerebellum
and those that project to the thalamus are generally dis-
tinct populations, since axons of the former had no collat-
erals that project to the thalamus. This conclusion seems
to be related to the different origins of these neurons.
Neurons that project to the cerebellum migrate to the
DCN from the caudal rhombic lip (Kawauchi et al., 2006).
Concerning the cerebellar projection from the subdivi-
sions of the DCN, forelimb areas (ECuN and CuN) were
much more predominant than the hindlimb areas (GN),
and the mainly proprioceptive forelimb area (ECuN) was
more predominant than the mainly cutaneous forelimb
area (CuN) in the present study. The involvement of DCN-
cerebellar projection has not been clarified for hindlimb
inputs, since hindlimb inputs are conveyed to the cerebel-
lum mainly through spinocerebellar projections including
those from the Clarke’s column (Brodal, 1981; Ito, 1984)
and because the GN and nucleus Z neurons mainly pro-
ject to the thalamus (Tracey, 2004). However, this study
indicated the presence of DCN-cerebellar projections for
hindlimb cutaneous and proprioceptive inputs originating
from the GN and its rostral pole, which was putatively
equivalent to the nucleus Z. Essential morphological char-
acteristics, including the arborization pattern, of cerebel-
lum-projecting axons that originated from any subdivision
of the DCN were similar in the present study. Although
only the ECuN is usually considered a precerebellar nu-
cleus among the DCN (Kawauchi et al., 2006), our results
suggested that the population of cerebellum-projecting
neurons in the whole DCN may be considered a relatively
homogeneous source of cerebellar mossy fibers.
Organization of DCN-cerebellar projectionPhysiological studies have long demonstrated a soma-
totopic organization in the cerebellar cortex (Snider, 1950;
Brodal, 1981). Previous anatomical studies on DCN-cerebel-
lar or spinocerebellar projections have roughly supported
such an organization (Somana and Walberg, 1980; Tolbert
and Gutting, 1997). The present study demonstrated that
the DCN-cerebellar projection is distinctively organized
according not only to a somatotopy but also to a sensory
submodality at the level of single axons. Thus, these results
should help us to interpret the input organization of the
Quy et al.
896 The Journal of Comparative Neurology |Research in Systems Neuroscience
conventional spinocerebellum region (Brodal, 1981),
which generally covers the rostral and caudal vermal and
intermediate regions that receive the DCN-cerebellar pro-
jection. The present finding that single DCN axons often
project to the rostral and caudal cerebellum by branch-
ing, which has been also suggested in other mossy fiber
systems (Voogd et al., 2003; Pijpers et al., 2006), is con-
sistent with the conventional double somatotopic repre-
sentation in the spinocerebellum region. However, to con-
sider the functional aspects of this projection, and to
compare them to those of other mossy fiber systems
(e.g., the LRN-cerebellar projection), we would have to
know the functional significance of each lobule and each
aldolase C compartment targeted by these various mossy
fiber systems, which, in turn, would require studying their
output connections through the cerebellar nuclei to other
areas in the CNS.
The present study showed that hindlimb-proprioceptive
and hindlimb-cutaneous axons mainly terminate in the
copula pyramidis and that forelimb-proprioceptive and
forelimb-cutaneous axons generally terminate in different
areas, although there was partial overlap. If we suppose
that the copula pyramidis in the rat is equivalent to a
group of ventral folia of the paramedian lobule in the cat
(Larsell, 1952), the present results generally agree with
the results in the cat examined by mass labeling from
subdivisions of the DCN (Somana and Walberg, 1980).
The present study identified aldolase C compartments
in which DCN mossy fiber terminals were distributed in
each lobule. About half of the DCN axon terminals were
distributed in the negative and lightly positive aldolase C
compartments that were classified into group IV in the
previous study (compartments 2�, bþ, b�, 3þ, 3�,
3dþ, and 3d� in the rostral cerebellum, and fþ, f�,
e1þ, e1�, e2þ in the caudal cerebellum; Sugihara and
Shinoda, 2004). Since the inferior olivary regions that pro-
ject to these compartments (dorsal accessory olive)
receive mainly somatosensory inputs from the spinal cord
and the DCN (Gerrits et al., 1985), it has been hypothe-
sized that group IV compartments have a somatosensory-
related function (Sugihara and Shinoda, 2004). These
indicate that DCN signals or spinal somatosensory signals
conveyed by the mossy and climbing fiber systems par-
tially converge in the group IV compartments of the cere-
bellar cortex. Indeed, the responses of climbing fibers to
forelimb and hindlimb sensation have been recorded in
the paramedian lobule and copula pyramidis, respectively
(Atkins and Apps, 1997), where the projection of mossy
fibers conveying forelimb and hindlimb inputs were identi-
fied in the present study. The local interaction of related
signals may be significant for cerebellar function.
The present study revealed that some branches of
DCN axons innervate aldolase C compartments other
than those that belong to group IV. In particular, ECuN
axons strongly innervated lobules IX, as reported in the
cat (Somana and Walberg, 1980) and mouse (Akintunde
and Eisenman, 1994). The present study demonstrated
that the projection to lobule IX originated mainly from the
rostral ECuN, which was mainly involved in the proprio-
ceptive sensation of the neck and shoulder (Campbell
et al., 1974). Lobule IX is mostly occupied by the positive
compartments (2þ, 3þ and 4þ; group II) that receive
mainly vestibular inputs through the climbing fibers origi-
nating from the beta and adjacent subnuclei of the infe-
rior olive (Sugihara and Shinoda, 2004). Lobule IX also
receives trigeminal somatosensory and vestibular infor-
mation through mossy fibers and is involved in the spatial
transformation of the head and body (Waespe et al.,
1985; Welker, 1987; Voogd and Ruigrok, 1997; Buttner-
Ennever, 1999). Lobule IX is the fastest in cortical matu-
ration in the early postnatal weeks and, therefore, has
been hypothesized to be related to suckling behavior of
the pup (Sugihara, 2005). The present results suggested
a strong influence of shoulder and neck proprioceptive
sensation relayed by the rostral ECuN (Campbell et al.,
1974) on the function of lobule IX, which would be rea-
sonable for the hypothesized function of lobule IX.
Relationship between aldolase Ccompartments and projectionof DCN-cerebellar axons
The present study demonstrated that the entire projec-
tion of a single DCN axon was not as precisely related to
aldolase C compartmentalization as that of climbing fiber
axons. Branches of a single DCN axon often projected to
both aldolase C-positive and -negative compartments, as
seen in many examples of reconstructed axons in the
present study (Table 1, Figs. 2, 3, 4, 6, 7, 10, 11, 12).
Thus, the DCN projection did not show a simple straight-
forward mapping onto the aldolase C compartmentaliza-
tion. However, some relationship has been observed. For
example, it may be inferred that mossy fibers from the
pontine nucleus and the LRN preferentially project to al-
dolase C-positive and -negative compartments, respec-
tively, from data in a retrograde labeling study (Pijpers
et al., 2006). Therefore, further systematic studies are
needed to clarify the general relationship between mossy
fiber projections and aldolase C compartmentalization.
However, locally, the distribution of terminals of a DCN
axon was closely related to aldolase C compartmentaliza-
tion, as shown in Figure 12. Therefore, the local distribu-
tion of a cluster of DCN mossy fiber terminals looked like
a longitudinal strip, or a patch when it was small. The
functional significance of the clustering of mossy fiber
terminals of a single axon is not clear. They would
Single cerebellar mossy fiber axons
The Journal of Comparative Neurology |Research in Systems Neuroscience 897
synapse onto a local population of granule cells, which
then send parallel fibers to a more or less similar popula-
tion of Purkinje cells. Another possibility is that these
granule cells may strongly affect a local population of Pur-
kinje cells through synapses in their ascending axons
(Gundappa-Sulur et al., 1999). It is also possible that
these granule cells may receive inhibition from a Golgi
cell located in the same compartment, since a Golgi cell
axonal arbor is organized longitudinally (Barmack and
Yakhnitsa, 2008).
ACKNOWLEDGMENT
We thank Dr. E.J. Lang for reading the article.
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