PAPER (Pham Nguyen Quy) Projection Patterns of Single Mossy Fiber Axons...

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



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.


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



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.


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.



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.


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.



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.


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.]



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.


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.



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.


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



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.


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-



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.]



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.


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.]



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.


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



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.

LITERATURE CITEDAkintunde A, Eisenman LM. 1994. External cuneocerebellar

projection and Purkinje cell zebrin II bands: a direct com-parison of parasagittal banding in the mouse cerebellum.J Chem Neuroanat 7:75–86.

Alisky JM, Tolbert DL. 1997. Quantitative analysis of converg-ing spinal and cuneate mossy fibre afferent projections tothe rat cerebellar anterior lobe. Neuroscience 80:373–388.

Atkins MJ, Apps R. 1997. Somatotopical organisation withinthe climbing fibre projection to the paramedian lobule andcopula pyramidis of the rat cerebellum. J Comp Neurol389:249–263.

Barmack NH, Yakhnitsa V. 2008. Functions of interneurons inmouse cerebellum. J Neurosci 28:1140–1152.

Berretta S, Perciavalle V, Poppele RE. 1991. Origin of cuneateprojections to the anterior and posterior lobes of the ratcerebellum. Brain Res 556:297–302.

Brochu G, Maler L, Hawkes R. 1990. Zebrin II: a polypeptideantigen expressed selectively by Purkinje cells revealscompartments in rat and fish cerebellum. J Comp Neurol291:538–552.

Brodal A. 1981. Neurological anatomy in relation to clinicalmedicine, 3rd ed. New York: Oxford University Press.

Brodal A, Pompeiano O. 1957. The vestibular nuclei in cat.J Anat 91:438–454.

Buisseret-Delmas C, Angaut P. 1993. The cerebellar olivocorti-conuclear connections in the rat. Prog Neurobiol 40:63–87.

Buttner-Ennever JA. 1999. A review of otolith pathways tobrainstem and cerebellum. Ann N Y Acad Sci 871:51–64.

Campbell SK, Parker TD, Welker W. 1974. Somatotopic orga-nization of the external cuneate nucleus in albino rats.Brain Res 77:1–23.

Cerminara NL, Makarabhirom K, Rawson JA. 2003. Somato-sensory properties of cuneocerebellar neurones in themain cuneate nucleus of the rat. Cerebellum 2:131–145.

Gerrits NM, Voogd J, Nas WS. 1985. Cerebellar and olivaryprojections of the external and rostral internal cuneatenuclei in the cat. Exp Brain Res 57:239–255.

Groenewegen HJ, Voogd J. 1977. The parasagittal zonationwithin the olivocerebellar projection. I. Climbing fiber distri-bution in the vermis of the cat cerebellum. J Comp Neurol174:417–488.

Gundappa-Sulur G, De Schutter E, Bower JM. 1999. Ascendinggranule cell axon: an important component of cerebellarcortical circuitry. J Comp Neurol 408:580–596.

Hallem JS, Thompson JH, Gundappa-Sulur G, Hawkes R, Bjaa-lie JG, Bower JM. 1999. Spatial correspondence betweentactile projection patterns and the distribution of the anti-

genic Purkinje cell markers anti-zebrin I and anti-zebrin IIin the cerebellar folium crus IIA of the rat. Neuroscience93:1083–1094.

Hawkes R, Leclerc N. 1987. Antigenic map of the rat cerebellarcortex: the distribution of parasagittal bands as revealedby monoclonal anti-Purkinje cell antibody mobA113. J CompNeurol 256:29–41.

Ito M. 1984. The cerebellum and neural control. New York:Raven Press.

Itoh K, Konishi A, Nomura S, Mizuno N, Nakamura Y, SugimotoT. 1979. Application of coupled oxidation reaction to elec-tron microscopic demonstration of horseradish peroxidase:cobalt-glucose oxidase method. Brain Res 175:341–346.

Ji Z, Hawkes R. 1994. Topography of Purkinje cell compart-ments and mossy fiber terminal fields in lobules II and IIIof the rat cerebellar cortex: spinocerebellar and cuneocer-ebellar projections. Neuroscience 61:935–954.

Jorntell H, Ekerot CF. 2006. Properties of somatosensory synap-tic integration in cerebellar granule cells in vivo. J Neurosci26:11786–11797.

Kawauchi D, Taniguchi H, Watanabe H, Saito T, Murakami F.2006. Direct visualization of nucleogenesis by precerebel-lar neurons: involvement of ventricle-directed, radial fibre-associated migration. Development 133:1113–1123.

Larsell O. 1952. The morphogenesis and adult pattern of thelobules and fissures of the cerebellum of the white rat.J Comp Neurol 97:281–356.

Low JS, Mantle-St John LA, Tracey DJ. 1986. Nucleus z in therat: spinal afferents from collaterals of dorsal spinocerebel-lar tract neurons. J Comp Neurol 243:510–526.

Mackel R, Miyashita E. 1993 Nucleus Z: a somatosensoryrelay to motor thalamus. J Neurophysiol 69:1607–1620.

Matsushita M, Yaginuma H. 1995. Projections from the cen-tral cervical nucleus to the cerebellar nuclei in the rat,studied by anterograde axonal tracing. J Comp Neurol 353:234–246.

Matsushita M, Ragnarson B, Grant G. 1991. Topographic rela-tionship between sagittal Purkinje cell bands revealed bymonoclonal antibody to zebrin I and spinocerebellar pro-jections arising from the central cervical nucleus in therat. Exp Brain Res 84:133–141.

Pijpers A, Ruigrok TJH. 2006. Organization of pontocerebellarprojections to identified climbing fiber zones in the rat.J Comp Neurol 496:513–528.

Pijpers A, Apps R, Pardoe J, Voogd J, Ruigrok TJ. 2006. Pre-cise spatial relationships between mossy fibers and climb-ing fibers in rat cerebellar cortical zones. J Neurosci 26:12067–12080.

Ruigrok TJH, Cella F. 1995. Precerebellar nuclei and red nu-cleus. In: Paxinos G, editor. The rat nervous system. SanDiego: Academic Press. p 277–308.

Snider RS. 1950. Recent contributions to the anatomy andphysiology of the cerebellum. Arch Neurol Psychiatry 64:196–219.

Somana R, Walberg F. 1980. A re-examination of the cerebel-lar projections from the gracile, main and external cuneatenuclei in the cat. Brain Res 186:33–42.

Sugihara I. 2005. Microzonal projection and climbing fiberremodeling in single olivocerebellar axons of newborn ratsat postnatal days 4–7. J Comp Neurol 487:93–106.

Sugihara I, Shinoda Y. 2004. Molecular, topographic, andfunctional organization of the cerebellar cortex: a studywith combined aldolase C and olivocerebellar labeling.J Neurosci 24:8771–8785.

Sugihara I, Shinoda Y. 2007. Molecular, topographic, andfunctional organization of the cerebellar nuclei: analysis bythree-dimensional mapping of the olivonuclear projectionand aldolase C labeling. J Neurosci 27:9696–9710.

Quy et al.


Sugihara I, Wu H-S, Shinoda Y. 1999. Morphology of singleolivocerebellar axons labeled with biotinylated dextranamine in the rat. J Comp Neurol 414:131–148.

Sugihara I, Wu H-S, Shinoda Y. 2001. The entire trajectoriesof single olivocerebellar axons in the cerebellar cortex andtheir contribution to cerebellar compartmentalization.J Neurosci 21:7715–7723.

Tolbert DL, Gutting JC. 1997. Quantitative analysis of cuneo-cerebellar projections in rats: differential topography in theanterior and posterior lobes. Neuroscience 80:359–371.

Tracey D. 2004. Somatosensory system. In: Paxinos G, editor.The rat nervous system. Amsterdam: Elsevier AcademicPress. p 797–815.

Voogd J, Bigare F. 1980. Topographical distribution of olivaryand cortico nuclear fibers in the cerebellum: a review. In:Courville J, de Montigny C, Lamarre Y, editors. The inferiorolivary nucleus: anatomy and physiology. New York: RavenPress. p 207–234.

Voogd J, Ruigrok TJH, 1997. Transverse and longitudinal pat-terns in the mammalian cerebellum. In: De Zeeuw CI,

Strata P, Voogd J, editor. The cerebellum: from structureto control. Prog Brain Res Vol. 114. Amsterdam: Elsevier.p 21–37.

Voogd J, Pardoe J, Ruigrok TJH, Apps R. 2003. The distributionof climbing and mossy fiber collateral branches from thecopula pyramidis and the paramedian lobule: congruence ofclimbing fiber cortical zones and the pattern of zebrin band-ing within the rat cerebellum. J Neurosci 23:4645–4656.

Waespe W, Cohen B, Raphan T. 1985. Dynamic modificationof the vestibulo-ocular reflex by the nodulus and uvula.Science 228:199–202.

WelkerW. 1987. Spatial organization of somatosensory projec-tions to granule cell cerebellar cortex: functional and con-nectional implications of fractured somatotopy (summary ofWisconsin studies). In: King JS, editor. New concepts in cer-ebellar neurobiology. New York: Liss. p 239–280.

Wu H-S, Sugihara I, Shinoda Y. 1999. Projection patterns ofsingle mossy fibers originating from the lateral reticular nu-cleus in the rat cerebellar cortex and nuclei. J Comp Neu-rol 411:97–118.



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