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The Journal of Neuroscience, October 1, 2001, 21(19):7715-7723
The Entire Trajectories of Single Olivocerebellar Axons in the
Cerebellar Cortex and their Contribution to Cerebellar
Compartmentalization
I.
Sugihara1,
H.-S.
Wu1, 2, and
Y.
Shinoda1, 2
1 Department of Systems Neurophysiology, Tokyo Medical
and Dental University Graduate School of Medicine, Bunkyo-ku , Tokyo 113-8519, Japan, and 2 The Core Research for
Evolutional Science and Technology Program, Kawaguchi, 332-0012, Japan
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ABSTRACT |
The functional partitioning of the cerebellar cortex depends on the
projection patterns of its afferent and efferent neurons. However, the
entire morphology of individual projection neurons has been
demonstrated in only a few classes of neurons in the vertebrate
CNS. To investigate the contribution of the projection pattern
of individual olivocerebellar axons to the cerebellar functional
compartmentalization, we labeled individual olivocerebellar axons,
which terminate in the cerebellar cortex as climbing fibers, with
biotinylated dextran amine injected into the inferior olive in the rat,
and completely reconstructed the entire trajectories of 34 olivocerebellar axons from serial sections of the cerebellum and
medulla. Single axons had seven climbing fibers on average, which
terminated at similar distances from the midline in a single or in
multiple lobules. Cortical projection areas of adjacent olivary neurons
were clustered as narrow but separate longitudinal segments and often
innervated by collaterals of single neurons. Comparison of the
cerebellar distribution of olivocerebellar axons arising from different
sites within a single olivary subnucleus indicated that slightly
distant neurons projected to complementary sets of such segments in a
single longitudinal band. Several of these longitudinal bands formed a
so-called parasagittal zone innervated by a subnucleus of the inferior
olive. Single olivocerebellar axons projected rostrocaudally to
segments within a single band but did not project mediolaterally to
multiple bands. These results suggest fine substructural organization
in the cerebellar compartmentalization that may represent functional units.
Key words:
climbing fibers; biotinylated dextran amine; inferior
olive; rats; cerebellar cortex; cerebellum; afferent neurons; neural
pathways; neuroanatomy; brain mapping
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INTRODUCTION |
The uniformity of the local circuit
of the cerebellar cortex (Ramón y Cajal, 1911 ; Eccles et al.,
1967; Palay and Chan-Palay, 1974; Ito, 1984) implies that functional
partitioning of the cerebellar cortex depends on the patterns of
afferent and efferent connections to each cerebellar cortical region.
Indeed, anatomical studies have shown that the mammalian cerebellar
cortex can be divided into several parasagittal zonal compartments
(designated A, B, C1-3, and D1-2) on the basis of the topography of
the olivocerebellar projection (Groenewegen and Voogd, 1977; Azizi and
Woodward, 1987; Buisseret-Delmas and Angaut, 1993; Voogd, 1995).
However, electrophysiological studies involving sensory mapping have
suggested an even finer compartmentalization in which the A-D zones
are subdivided into narrower "microzones" (Oscarsson and
Sjölund, 1977; Ito, 1984; Ekerot et al., 1991; Jörntell et
al., 2000). Multiple electrode and optical recording studies have also
identified such narrow longitudinal microzones in the cerebellar cortex
based on correlations in Purkinje cell complex spike activity
(Llinás and Sasaki, 1989; Hansen et al., 2000). Thus, the
olivocerebellar projection appears to be organized into many more
functional zones than have been identified anatomically. Several fine
zones have been anatomically demonstrated in the flocculus and nodule
(Ruigrok et al., 1992; Tan et al., 1995); however, in general,
morphological correlates for electrophysiologically identified narrow
zones in the olivocerebellar projection remain elusive.
One way to attempt to identify a finer anatomical organization to the
olivocerebellar system is to analyze the projection patterns of
individual olivary axons. Recently, the complete trajectories of single
axons of inferior olive neurons have been reconstructed (Sugihara et
al., 1999). Individual olivary neurons project to rather localized
areas with several climbing fibers by ramifying mostly in the
cerebellar white matter and occasionally in the cerebellar granular
layer (Sugihara et al., 1999). This conforms to the idea that the
projection pattern of olivocerebellar axons is tightly related to a
compartmentalization of the cerebellar cortex and suggests that the
analysis of individual axonal projections is useful for understanding
the morphological correlates underlying the functional
compartmentalization of the cerebellar cortex. Thus, the present study
was undertaken to investigate the basic structural organization of the
olivocerebellar projection by analyzing the entire axonal trajectories
of individual olivary neurons.
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MATERIALS AND METHODS |
Tracer injection and histological procedure.
Thirty-six adult Long-Evans rats were used. Methods of anesthesia,
surgery, and histological procedures and axonal reconstruction were the
same as described previously (Sugihara et al., 1999). The experimental animals were treated ethically, and the experimental protocols were
approved by the Institutional Animal Care and Use Committee in Tokyo
Medical and Dental University. Biotinylated dextran amine was
pressure-injected into the inferior olive (0.1 to <0.01 µl of 10%
solution in saline). The rats in the present study had a small
injection with a spread of dye in the injection site limited to <0.2
mm, unless otherwise stated. Fifty-micrometer-thick serial frozen
sections were cut from the entire cerebellum and medulla parasagittally
in most cases and in others coronally. The protocols for histological
labeling of biotinylated dextran amine were the same as described
previously (Sugihara et al., 1999). Some photomicrographs were obtained
with a computer-aided dynamic focusing system (MCID image analysis
system; Imaging Research, St. Catharines, Ontario, Canada).
Reconstruction of individual axons. Axonal trajectories of
single labeled olivocerebellar axons were reconstructed from serial parasagittal sections using a three-dimensional 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 properly to the corresponding
cut ends of the same axon on the successive section (Shinoda et al.,
1981; Sugihara et al., 1999). Only axons that were well labeled,
isolated from other axons, and could be traced from the injection site
to every climbing fiber terminal were regarded as completely
reconstructed, whereas axons that could not be traced at any point on
their pathway because of poor labeling or intermingling with other
axons were regarded as "not fully reconstructed."
Mapping climbing fiber locations onto the unfolded cerebellar
cortex. In the diagram of the unfolded cerebellar cortex, as in
Figures 3b, 5, and 6, the distances from the midline to each climbing fiber and the cerebellar outline were to scale (measured by
the number of the parasagittal sections). A small difference in the
lateral dimension of the cerebellum between animals (<10%) was
adjusted in the figures by normalizing the maximum width measured at
Crus Ib to the average value (11.4 mm) obtained from seven rats in
which the sections were cut coronally. The rostrocaudal dimension of
each cerebellar lobule was determined by measuring the length of the
Purkinje cell layer in each lobule in parasagittal sections at the
midline and 1.5 and 3 mm lateral from the midline. The primary fissure
was made straight arbitrarily. The rostrocaudal distances in the
paraflocculus and flocculus are not to scale (enlarged for clarity).
Broken outlines in the paraflocculus and flocculus indicate the
continuation of the Purkinje cell layer. When mapping from parasagittal
sections, the position in the rostrocaudal axis for each climbing fiber
was determined by measuring its relative distance from the borders of
the lobule in a parasagittal section, and the mediolateral distance was
determined from the number of the sections. When mapping from coronal
sections, the parasagittal outline of the tissue at several
mediolateral distances was reconstructed first. Climbing fibers were
mapped onto these parasagittal outlines and then transferred onto the
unfolded cerebellar cortex diagram.
To map climbing fibers in the unfolded hemisphere along a tilted
parasagittal plane, as in Figure 4b, the three-dimensional coordinates of each climbing fiber were measured at the point where it
contacted a target Purkinje cell. The equation for the plane was
calculated by minimizing the sum of the squares of the distance from
the plane to the individual points. The distance from this plane for
each point was used as the mediolateral position in the unfolded
hemisphere. A homemade computer program was used for these
calculations. The rostrocaudal position of each climbing fiber was
measured along the Purkinje cell layer on this plane.
The diagram of the inferior olive represents a dorsal view that was
reconstructed from the preparation in which the majority of olivary
neurons were labeled retrogradely by a large injection of biotinylated
dextran amine into the inferior cerebellar peduncle.
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RESULTS |
Branches of individual olivocerebellar axons are confined to a
single rostrocaudally oriented plane
Olivocerebellar axons were labeled with biotinylated dextran
(Sugihara et al., 1999), an anterograde neuronal tracer. Injections were made into the inferior olive (Fig.
1c), and the complete trajectories of 34 axons were reconstructed from parasagittal or
coronal serial sections in 14 rats. Individual olivocerebellar axons
ramified many times, mainly within the deep cerebellar white matter,
into thick branches that terminated as climbing fibers in the
cerebellar cortex and thin collaterals that terminated mainly in the
cerebellar nuclei and the granular layer (Fig. 1a) (Sugihara
et al., 1999). The number of climbing fibers per axon ranged from 2 to
17 (6.6 ± 3.7; mean ± SD; n = 34). This
number was consistent with the average number of climbing fibers per axon (approximately seven) inferred by counting the total numbers of
Purkinje cells and olivary neurons in the rat (Schild, 1970), supporting complete labeling of reconstructed axons. Axons could be
classified into two types based on their climbing fiber distribution pattern. For most axons (n = 22 of 34) all of their
climbing fibers terminated within a single rostrocaudally oriented area
that was limited to one lobule or multiple contiguous lobules (Fig.
1b). However, a significant percentage of axons
(n = 12 of 34) had a distinct termination pattern in
which climbing fibers were distributed to multiple non-contiguous
lobules (Fig. 2). In both cases, all climbing fibers originating from a single neuron were located at almost
equal distances from the midline and therefore were contained within a
single parasagittal plane (Fig. 1d). The width of these
planes was measured as the maximum mediolateral spread of climbing
fibers from an individual olivocerebellar axon (the distance between
the nearest and farthest climbing fibers from the midline). For
reconstructed axons in the vermis the average width was 217 ± 173 µm (n = 16). The maximum mediolateral spread of
branches was larger (1114 ± 441 µm; n = 18) for
axons terminating in the pars intermedia and hemisphere; however, this
was because the plane that contained climbing fibers of individual
axons was tilted from the parasagittal plane (Fig. 2b,c).
When measured perpendicularly to this tilted plane, the widths of the
planes that contained the branches of individual axons were as narrow as in the vermis (0.2-0.3 mm).
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Figure 1.
Projection patterns of single olivary axons in the
cerebellum. a, Intracerebellar trajectory of a single
olivocerebellar axon labeled after a small injection of biotinylated
dextran amine (BDA) into the mediocaudal portion of the MAO
(reconstructed from 77 serial parasagittal sections). This axon gave
rise to nine climbing fibers terminating in uvula (lobule IXb-c) and
nodule (lobule Xa), and thin collaterals terminating in the cerebellar
nucleus and the granular layer. b, Entire trajectory of
the same axon shown in a with all the other climbing
fibers (n = 28, red) labeled
by the tracer injection. All climbing fibers were distributed from
caudal lobule VIII to lobule X. c, Photomicrograph of
the injection site in the inferior olive shown in b.
d, Photomicrograph of labeled climbing fibers in a
parasagittal section. Arrowheads indicate the three
labeled climbing fibers belonging to a single inferior olive neuron.
Digital focusing and enhancement were used. Abbreviations in this and
subsequent figures and legends: I-X, lobules I-X;
a-d, sublobules a-d; CP, copula
pyramidis; Cr I, crus I ansiform lobule; Cr
II, crus II ansiform lobule; DN, dentate
nucleus; DPFL, dorsal paraflocculus; FL,
flocculus; FN, fastigial nucleus; IN,
interposed nucleus; IO, inferior olive;
MAO, medial accessory olive; Par,
paramedian lobule; PO-DL, dorsal lamella of the
principal olive; Sim, simple lobule;
VPFL, ventral paraflocculus.
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Figure 2.
Narrow longitudinal band distribution of climbing
fibers originating from single olivary neurons. Results in three
animals are shown in individual columns a-c, in which
climbing fibers were labeled in the vermis (a),
pars intermedia (b), and hemisphere
(c) by a small tracer injection in the caudal MAO
(a), rostral MAO (b), and
dorsomedial subnucleus of the ventral leaf of the principal olive
(c), respectively. Coronal sections in each
column show climbing fibers of a single reconstructed
olivary axon (open rectangles indicated by open
arrowheads) and other labeled climbing fibers (black
bars) that were observed in neighboring four to six sections. A
maximum mediolateral spread of labeled climbing fibers was ~0.3 mm.
Injection sites are indicated in coronal sections of the inferior
olive. The bottom drawing shows a lateral view of a
trajectory of reconstructed olivary axon with other labeled climbing
fibers (open circles). The climbing fiber distribution
areas in a-c belonged to A, C2, and D0 zones
(Buisseret-Delmas and Angaut, 1993; Voogd, 1995), respectively.
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Distribution of axonal branches from neighboring cells
The activity of adjacent olivary neurons is functionally
correlated because of the electrotonic coupling among olivary neurons (Llinás et al., 1974) and because of focal afferent projection patterns to the inferior olive (Apps, 1998; McCurdy et al., 1998). To
determine the translation of such correlated olivary activities into
patterns of activity across the cerebellar cortex, it is necessary to
know the relationship of axonal projection patterns of adjacent olivary
neurons. To address this issue, we reconstructed all labeled axons from
neighboring olivary cells in each animal. Figure
3a shows an example in which
six individual olivocerebellar axons were completely reconstructed from
a single injection site. Forty-two climbing fibers originating from
these six axons (Fig. 3a, colored fibers) and 36 climbing
fibers originating from other axons (not fully reconstructed,
gray fibers) were all located in vermal lobules VIa-c and
VII. Within this region there was significant overlap of the projection
areas, with individual axons projecting to lobules VIa, VIb, and VII
(light blue), to lobules VIa, VIb, and VIc
(violet), to lobules VIa and VIb (green),
to lobules VIb and VIc (orange and magenta), and
to lobule VII (brown). These results indicated that olivary
neurons located close to each other shared projection areas. To clarify
the mediolateral and rostrocaudal extents of the cortical projection
areas shared by adjacent neurons, henceforth referred to as
"segments," the labeled climbing fibers shown in Figure
3a were mapped onto an unfolded representation of the
cerebellar cortex (Fig. 3b). All labeled climbing fibers
were distributed as clustered segments within a narrow rostrocaudally
oriented area, which was almost in parallel with the midline. These
segments were more or less continuous in a rostrocaudal direction and
formed a clearly delineated narrow longitudinal band. The mediolateral
width of this longitudinal band was ~0.2-0.4 mm when a slightly
curved boundary was fitted (broken line) and was only
3.5-7% of the entire width of the left vermis. In contrast, the
rostrocaudal dimension of this band was almost 40 times as long as the
mediolateral dimension.
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Figure 3.
Climbing fibers originating from small areas in
the inferior olive distribute within narrow longitudinal bands in the
cerebellar cortex. a, Lateral view of six
olivocerebellar axons (colored) reconstructed from 81 serial parasagittal sections. Forty-two climbing fibers arising from
these six axons, and the other 36 labeled climbing fibers
(gray) are included. Inset shows
lateral view under low magnification of the entire axonal trajectories
from the injection site in the centromedial portion of the MAO (single
injection of 0.01 µl of BDA). b, The distribution of
climbing fibers plotted on the unfolded vermal cortex from the midline
to the left by 1.3 mm. Colors used for the climbing fibers in
b correspond to those used for individual axons in
a. Light and dark gray
areas in the unfolded scheme represent the cerebellar cortex
exposed in the cerebellar surface and hidden in the sulci,
respectively. Dotted line indicates the contour of the
distribution area. Inset shows the area for the unfolded
display.
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To examine further whether this narrow longitudinal band structure
exists in the cerebellar cortex outside the vermis, a similar analysis
was made of axons terminating in the hemisphere (Fig. 4). Axonal trajectories of three well
labeled axons were completely reconstructed from a small injection in
the principal olive. Climbing fibers originating from these axons
(colored) and five other labeled climbing fibers
(gray) were located in crus Ia and Ib (Fig.
4a). All climbing fibers were mostly positioned in a single
plane that was tilted laterally (Fig. 4b, inset). This
tilted plane was numerically obtained (see Materials and Methods), and
the cerebellar cortex in the hemisphere was unfolded along this plane.
The plot of climbing fibers on the unfolded cortex shows that they were
arranged in a single narrow longitudinal band (Fig. 4b).
Although this band was slightly twisted presumably along the foliation
of the lobules, the maximum width of the band was 0.25 mm, similar to
the width of a band in the vermis. A similar longitudinal band with
extensive overlap of axonal projection areas of neighboring neurons in
the dorsal cap of the inferior olive was observed in the flocculus (six
axons) in another rat.
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Figure 4.
Climbing fiber distribution in a narrow
longitudinal area in the hemisphere. a, Lateral view of
trajectories of three olivocerebellar axons (colored)
reconstructed on 31 serial coronal sections. A tracer injection was
made in the rostral portion of the dorsal lamella of the principal
subnucleus of the inferior olive. Of 23 labeled climbing fibers, 18 climbing fibers arose from these three axons, and the other five
labeled climbing fibers (gray) from other olivary
neurons. The outline of the cerebellum is deformed to show all climbing
fibers. b, The distribution of climbing fibers was
plotted on the unfolded hemisphere along a tilted parasagittal plane.
Inset shows the tilted parasagittal plane in which
climbing fibers were aligned. Colors used for the climbing fibers in
b correspond to those used for individual axons in
a. Light and dark gray
areas in the unfolded scheme represent the cerebellar cortex exposed in
the cerebellar surface and hidden in the sulci, respectively.
Dotted line indicates the contour of the distribution
area.
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To obtain a general view of the olivocerebellar organization in the
entire cerebellar cortex, tracer injections were made into small areas
within the various subnuclei of the inferior olive in 30 rats; the
caudal portion of the medial accessory olive (MAO) (n = 12 animals), the rostral portion of the MAO (n = 4), the dorsal accessory olive (DAO) (n = 6), the principal
olive (PO) (n = 6), and the dorsal cap
(n = 2). These injections resulted in labeled climbing
fibers in the vermis, pars intermedia, pars intermedia, hemisphere, and
flocculus, respectively, consistent with the broad topography of the
olivocerebellar system described previously (Groenewegen and Voogd,
1977; Azizi and Woodward, 1987; Buisseret-Delmas and Angaut, 1993;
Voogd, 1995). The number of labeled climbing fibers in each rat ranged
from 16 to 161 (mean = 53, SD = 41). Labeled climbing fibers
clustered in a few longitudinal segments, each of which ranged from a
part of a lobule to some consecutive lobules in length, and from 0.2 to
0.5 mm in width, indicating that such basic organization of the
olivocerebellar projection as shown in Figures 3b and
4b is consistent throughout the cerebellar cortex and
represents the basic organization of the olivocerebellar projection.
Neighboring cells but not branches of a single axon may project to
different bands
Investigation of the arrangement of segments in the above 30 cases
showed three distinct projection patterns: labeled climbing fibers
could be clustered within (1) a single segment (n = 8 of 30) (Fig. 1b), (2) multiple separate segments in a single
longitudinal band (n = 17 of 30) (Fig. 2), or (3)
multiple segments in separate longitudinal bands located at different
distances from the midline (n = 5 of 30). The
relationship was examined between projection areas of a single axon and
the multiple projection segments in the above cases (2 and 3). Cortical
segments that were separate in the rostrocaudal direction but within a
single longitudinal band were often innervated by a single neuron, as
shown in Figure 2 [11 of 14 axons reconstructed in five rats in the
above case (2)]. These 11 axons terminated in two segments that were
separated rostrocaudally but contained all labeled climbing fibers in
these five rats. This indicated that a specific combination of
rostrocaudally separate segments was coupled by axon collaterals of
single olivocerebellar neurons, and these segments might be
functionally interrelated. On the other hand, cortical segments that
were separate mediolaterally were not simultaneously innervated by
single olivocerebellar axons, suggesting that different populations of
adjacent olivary neurons might be labeled by chance.
Only few climbing fibers were seen in the vermis and pars intermedia
ipsilateral to the injection site in the inferior olive in 3 of the
above 30 rats. These ipsilateral climbing fibers were located
symmetrical to the midline with the contralateral main climbing fibers
(Fig. 5a, red). The
ipsilateral climbing fibers are formed by branches of olivocerebellar
axons crossing the midline in the cerebellum, and the majorities of
climbing fibers belonging to the same axons are located in the
contralateral cerebellum (Sugihara et al., 1999). We regarded such
ipsilateral projections as exceptional and did not include them in the
above analysis.
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Figure 5.
Projection patterns of climbing fibers originating
from adjacent areas in the inferior olive showing complementary
segments within the same longitudinal band. Climbing fibers labeled by
small injections in two adjacent sites in the lateral and caudal
portions of the MAO (green and
red) (a), three sites in the
medial and rostral portions of the MAO (orange, dark
green, and light blue)
(b), and three sites in the lateral portion of
the dorsal lamella of the principal olive (PO-DL)
(yellow-green, pink, and
blue) c, Results of eight experiments are
presented in three groups based on olivary injection sites and their
related cerebellar projections (a, vermis;
b, medial hemisphere or pars intermedia;
c, lateral hemisphere). Inset shows the
dorsal view of the injection sites (colored spots) in
the inferior olive. Dotted lines drawn along labeled
climbing fibers in the cerebellar cortex indicate putative single
longitudinal bands. Each colored dot, which is often
fused to others, indicates a labeled climbing fiber.
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Complementary projection patterns of adjacent regions within single
inferior olivary subnuclei produce longitudinal bands in the cerebellar
cortex
To understand the finer organization of individual longitudinal
bands, small injections of tracer were made into adjacent parts of a
specific olivary subnucleus (Fig. 5). Injections were made into the
caudal MAO nucleus (n = 2 animals), the rostral MAO
nucleus (n = 3 animals), and into the lateral portions
in the dorsal lamella of the principal olive nucleus (n = 3 animals). Climbing fibers originating from neurons in the caudal
MAO, rostral MAO, and the principal olive projected to the vermis, the
pars intermedia, and the hemisphere, respectively (Fig. 5a-c,
three dotted longitudinal lines). These relationships between the
subnuclei of the inferior olive and the locations of the bands in the
cerebellar cortex were consistent with the gross topographical
relationships of the olivocerebellar projection (A, C2, and D1 zones)
(Groenewegen and Voogd, 1977; Azizi and Woodward, 1987;
Buisseret-Delmas and Angaut, 1993; Voogd, 1995), respectively. In the
two cases of the projection to the vermis, climbing fibers were mostly
distributed in small longitudinal segments in lobules I to V and apical
lobule VIII in one case (red) and in lobules VIb-c and
rostral lobule VIII in the other (green). These
projection segments were arranged in the same longitudinal band in a
roughly complementary manner. Similarly, in the other cases, climbing
fiber distributions were roughly complementary and formed longitudinal
bands (orange, dark green, and light blue in the
pars intermedia, and yellow-green, pink, and
blue in the hemisphere) (Fig. 5). These results indicated that slightly separate groups of olivary neurons in a single subnucleus projected to distinct sets of segments in the same longitudinal band in
a complementary manner.
To examine further the organization of substructures and
suprastructures of single longitudinal bands in the olivocerebellar projection, different volumes of tracer were injected to vary the sizes
of labeling sites in the inferior olive, and the projection patterns of
labeled climbing fibers were compared with each other (Fig.
6). The caudal portion of the MAO was
used in this analysis, because it is relatively separated from the
other subnuclei, and the probability of tracer spread into other
subnuclei was low. With a small injection site (diameter, 0.1 mm),
there was a single longitudinal band with two small projection segments
occupying lobules I-IV and VIII, and two of three reconstructed
olivary axons innervated both segments by their ramification (Fig.
6a). With a slightly larger injection site (diameter, 0.3 mm), the projection areas in the single longitudinal band extended in
the rostrocaudal direction, covering most of the lobules except for some portions of lobules VIc, VII, IXa-b, and Xa-b with only slight extension in the lateral direction (Fig. 6b). A much larger
injection site (diameter, 0.8 mm) produced two additional longitudinal
bands in the pars intermedia, but the original band near the midline now extended further in the rostrocaudal direction to cover nearly all
of the lobules in the vermis without significant lateral extension (Fig. 6c). Similar changes in the projection patterns in
relation to the sizes of the injection sites were observed in four
other rats. All bands except those in the flocculus in Figure
6c belonged to the so-called A zone that is innervated by
the caudal portion of the MAO (Groenewegen and Voogd, 1977; Azizi and
Woodward, 1987; Buisseret-Delmas and Angaut, 1993; Voogd, 1995). These
results support the above conclusion that adjacent olivary neurons
project to the same set of segments that are all arranged in a single longitudinal band, and slightly distant neurons project to different sets of segments in the same longitudinal band. Furthermore, more widely separated neurons project to different longitudinal bands. These
findings on the substructure of the A-zone also suggest that individual
cerebellar zones, which are defined according to the topographical
innervation of the cortex by entire olivary subnuclei (Groenewegen and
Voogd, 1977; Azizi and Woodward, 1987; Buisseret-Delmas and Angaut,
1993; Voogd, 1995), consist of several of the longitudinal bands.
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Figure 6.
The distribution patterns of climbing
fibers labeled by injections of different volumes of tracer into the
inferior olive indicating substructures in the zonal organization of
the olivocerebellar projection. a, Two longitudinal
segments in lobules I-IV and VIII in the vermis along the midline
produced by a small injection in the caudal portion of the MAO
(right inset). Red dots indicate climbing
fibers of a single reconstructed olivocerebellar axon terminating in
both areas. b, Longitudinal band-shaped pattern
occupying most of the lobules in the vermis along the midline produced
by a medium-sized injection including the site in a.
c, Striped pattern produced by an
injection larger than in b and covering the sites in
a and b in the MAO. Curvatures of some
lateral bands are attributable to the tilt of the longitudinal plane
and the foliation of the cortex. Each dot, which is
often fused to others, indicates a labeled climbing fiber.
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DISCUSSION |
The present study revealed that the traditionally defined
anatomical zones defined on the basis of projections from the olivary subnuclei to the cerebellum have a substructure consisting of 0.2- to
0.3-mm-wide longitudinal bands. These bands may provide a morphological
substrate for physiologically defined narrow longitudinal zones
(Oscarsson and Sjölund, 1977; Ito, 1984; Llinás and Sasaki, 1989; Ekerot et al., 1991; Jörntell et al., 2000; Hansen et al., 2000; Fukuda et al., 2001). The width of the band observed in the
present morphological study is in accordance with the width determined
by high synchrony of climbing fiber responses of multiple Purkinje
cells (250-500 µm; Fukuda et al., 2001). Furthermore, the
present finding that adjacent olivary neurons project to the same band
may explain the physiologically observed high-synchrony band (Fukuda et
al., 2001), assuming that electrotonic coupling within nearby olivary
neurons underlies the synchrony. However, physiological studies so far
have not much clarified substructures of the longitudinal band. In the
present morphological study, each longitudinal band was not evenly
organized along its length, but consisted of several sets of segments
that were arranged in a complementary manner within the overall
confines of the band. We suggest that each segment represents the
morphological substructure for the functional unit of the
olivocerebellar projection. It is not clear whether neighboring
segments may have clear borders or instead can be intermingled with
each other at their border. The organization of such segments followed
more or less the lobular division of the cerebellar cortex.
The segmental organization, revealed here, significantly increases the
number of compartments in the cerebellar cortex in terms of the
olivocerebellar projection. A very fine and complex topography may
underlie the innervation of each segment by a small group of olivary
neurons. The present labeling of a very localized population of olivary
neurons has made it possible to reveal this fine topography. It has
been hypothesized that origin-target matching with specific molecules
underlies the formation of the topographical olivocerebellar projection
(Chédotal et al., 1996). The fine topography revealed in this
study suggests a sophisticated matching mechanism involving several of
such molecules. Although mediolateral borders of olivocerebellar bands
may coincide with the borders of molecular marker labelings (Wassef et
al., 1992; Zagrebelsky et al., 1996), it is a remaining question
whether molecular marker labelings (Hawkes et al., 1985) have any
rostrocaudal organization compatible with the segmentation of each
longitudinal band. Branches of a single olivocerebellar axon projecting
to the same segments were sometimes completely separated in the deep
cerebellar white matter (Figs. 1b, 3a,
4a), suggesting independent growth of these branches
affected by the same target specificity.
Each small segment where adjacent olivary neurons project may be
related to specific activity of climbing fiber input to the cerebellar
cortex. Some segments reported here coincide well with physiologically
defined zones in terms of specific receptive fields. For example, the
segment in lobule I-IV in Figure 5 roughly coincides with the "a1
zone", which is responsive to somatosensory inputs from the proximal
area in the rat (Jörntell et al., 2000). Lobules VIa-c and VII,
in which the segments shown in Figure 3 are located, are related to eye
movements (Godschalk et al., 1994). Some single olivary neurons
innervated separate segments in the same band. This finding provides
clear morphological evidence of interlobular branching of single
olivocerebellar axons previously demonstrated electrophysiologically or
by retrograde labeling studies (Armstrong et al., 1973; Brodal et al.,
1980; Rosina and Provini, 1987; Apps, 2000). There seems to be some
general principle organizing the location of the segments innervated by
common axons across lobules. For example, individual axons often
innervated segments in simple lobule and crus II-paramedian lobule
(Fig. 2b,c; some in Fig. 5), or segments in lobules I-V and
lobule VIII (Fig. 2a; some in Fig. 5). Simple lobule and
crus II-paramedian lobule may be functionally related to some extent,
because both areas receive trigeminal input via mossy fibers in rats
(Welker, 1987). Although little is known about the relationship of
activities in lobules I-V and lobule VIII, the present results suggest
that one would be found. None of the single olivary neurons thus far
examined projected to segments located in mediolaterally separated
longitudinal bands. This finding does not fit the electrophysiological
finding that axon collaterals of single olivocerebellar axons often
project to transversely separate microzones in cats (Ekerot and Larson, 1982). More detailed information on the organization of the
corticonuclear and olivonuclear projections will be required to further
understand the functional roles of the cerebellar compartmentalization
in control of movement.
| |
FOOTNOTES |
Received May 14, 2001; revised July 2, 2001; accepted July 9, 2001.
This work was supported by Core Research for Evolutional Science and
Technology of Japan Science and Technology Corporation (Y.S.) and
Grants-in-Aid for Scientific Research from the Ministry for Education,
Science, and Culture of Japan (I. S., Y. S.). We thank Dr.
E. J. Lang for reading this manuscript and Dr. K. Miura of
FujiFilm for his support of the computer-aided dynamic focusing system.
Correspondence should be addressed to Dr. Yoshikazu Shinoda, Department
of Systems Neurophysiology, Tokyo Medical and Dental University,
Graduate School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 Japan. E-mail: yshinoda.phy1{at}tmd.ac.jp.
| |
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