Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
A computer-aided light microscopy system for three-dimensional
reconstruction of axonal projections
Izumi Sugihara and Hirofumi Fujita
Department of Systems Neurophysiology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8519, Japan
Axons allow nerve cells (neurons) to signal each other over long distances. Axonal projection patterns determine the
organization of the central nervous system. Morphological techniques to analyse axonal projections systematically include
tracing the trajectory of labelled axons, mapping the distribution of labelled axonal terminals and nerve cell bodies and
reconstructing their original spatial arrangement. The camera lucida apparatus, in combination with the microscope, has
been used for mapping and tracing labelled microscopic structures such as axons, terminals, and cell bodies for more than
a century. Although two-dimensional mapping and tracing can be done efficiently with the camera lucida apparatus in
single histological sections of the central nervous system, reconstruction of the three-dimensional nature of axonal
projections and microscopic organization of the central nervous system has been too difficult or expensive to be generally
performed using this technique. We have been developing a computer-aided light microscopy system with commercially
available software for personal or industrial use (Adobe Illustrator and Robert McNeel Rhinoceros). In this system, a
computer monitor is connected to the camera lucida apparatus, so that one can directly draw labelled structures viewed
under the microscope with the software. This system enables efficient tracing, mapping and reconstruction of microscopic
structures in three-dimensional space from serial sections. The technical aspects of this system are described in this
chapter.
Keywords light microscopy; serial sections; neuronal reconstruction; camera lucida; Illustrator; Rhinoceros
1. Introduction
Nerve cells (neurons) have unique characteristics. First, they typically have a long process, the axon, which projects to
distant targets (up to one meter away), and conveys signals to these targets via conduction of action potentials. Neurons
also have shorter processes, dendrites, which remain local, and are the primary targets of axons of other neurons.
Second, each neuron has specific functional and morphological properties, and is located in a specific position within
the nervous system. Because of these characteristics, labelling individual neurons has been an important morphological
technique for understanding neurons and the neuronal organization of the brain since Ramón y Cajal [1]. With this
technique, the entire dendritic arbour and axonal tree of individual neurons, and the distribution of cell bodies of
specific neuronal populations can be visualized under light microscopy.
To record and to show morphology of labelled neurons, a drawing is often more efficient than photographs, because
the essential components can be extracted in the drawing process. The camera lucida apparatus has been a powerful
tool for drawing microscopic images. Originally invented for landscape and portrait drawing, the camera lucida
apparatus was subsequently applied to drawing images seen with the microscope. Ramón y Cajal was one of the first to
make extensive use of an early camera lucida apparatus attached to the microscope [2]. With a camera lucida apparatus
one can superimpose a magnified image of the microscopy specimen onto a white sheet of paper. Thus, it allows
accurate drawing of the specimen.
Observing labelled neurons in serial sections significantly enriches the morphological analysis, because the entire
extent of dendritic arbour or axonal tree, or the distribution of a group of neurons is rarely restricted within a single
section of tissue. This analysis requires certain reconstruction procedures to visualize morphological data obtained
from serial sections. In order to show drawings of a labelled process of a neuron that has been cut at the sectioning
plane, a simple reconstruction is to connect two drawings at the cut end. Then, the entire process can be reconstructed.
In order to show the distribution of labelled neurons, drawings obtained in serial individual sections may be
superimposed to show an accumulated distribution pattern [3-5].
The reconstruction of neuronal morphology from serial sections can be facilitated by using a small computer system,
a monitor of which is attached to a light microscope through the camera-lucida apparatus, with commercially available
software for general or industrial use (Adobe Illustrator and Robert McNeel Rhinoceros) installed in the computer. This
system can not only replace previous manual two-dimensional tracing and mapping on a sheet of paper but also enables
one to reconstruct in the three-dimensional space the morphological data obtained by tracing and mapping. Since this
system uses commercially available software for general use, it is much less expensive than other reconstruction
systems specialized for biomedical research.
We have been developing this computer system for more than a decade, and using it for single axonal and other
neuronal reconstructions in our studies of the cerebellar afferent and efferent systems [6-9]. This chapter describes the
technical and practical aspects of this computer system.
©FORMATEX 2010
813
Microscopy: Science, Technology, Applications and Education
A.
Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
2. Histological preparation
Any thin-sectioned histological preparation for standard light microscopy can be analyzed using this computer-aided
reconstruction system. This system is most beneficial to serial sections in which something that has special three
dimensional structure or distribution is labelled across multiple sections. The thickness of the section does not matter,
but it should be determined by efficiencies in cutting, labelling and analysis. If a section is thicker, the number of
sections is smaller and each section is stronger, but a good labelling reaction is more difficult to achieve deep inside of a
thicker section.
To use camera lucida apparatus and superimpose the image of the tissue upon the computer monitor image, intense
labelling with a low background level is preferable. Among standard labelling for light microscopy, labelling with
black or brown colour, as produced with peroxidase reaction with diaminobenzidine as a chromogen, and labelling with
blue or indigo colour, as beta galactosidase reaction with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)
as a chromogen, are acceptable.
3. Equipments
3.1
Microscope
A standard upright light microscope with a camera lucida attachment can be used. It is essential that the illumination
intensity of the microscope should be quickly and precisely controlled manually, since drawing can be done only when
darkness of the labelled structure in the section matches brightness of the computer monitor, i.e. when images of the
microscope preparation and the computer monitor can be seen simultaneously through the eyepieces. For this purpose
the original brightness control knob of most microscopes is not very appropriate, because it is often positioned too far to
reach with the left hand. Therefore, a remote controller for the illumination intensity of the microscope is preferable.
Dim illumination is often preferable since the brightness of the tissue often surpasses the brightness of the CRT monitor
when the microscope image and the monitor image are superimposed upon each other with the camera lucida.
Therefore, it is often necessary to put neutral density (ND) filters in the illumination light pathway.
Fig. 1 Setup of the microscope. To use the
system efficiently, the stage knob, the focus
knob and the light intensity knob are preferably
located closely to each other so that they are
handled quickly by the left hand. An extra
keyboard may also be located close these knobs
so that some frequently-used keys such as
control-Z can be typed blindly with the left
hand.
A so-called 3-D microscope is commercially available (R400 or Perceptra, Edge Scientific Instrument, Santa
Monica, CA, U.S.A.). This type of microscope is particularly useful for tracing multiple axons, because with it, it is
easy to distinguish the axons when they cross each other. There is a certain difference in depth at the crossing point
between the two crossing axons. Difference in the depth is intuitively recognized with a 3-D microscope.
It may be possible to apply camera lucida drawing technique to fluorescent microscopy. However, continuous
illumination for drawing might cause diminishment of fluorescence. Therefore, we have not applied the camera lucida
technique to fluorescent microscopy. Instead, drawing and reconstruction can be done off-line after taking
photomicrographs of the fluorescent material.
During observation and drawing of the labelled structure in the section, one needs to readjust the focus and
brightness of the microscope frequently and efficiently. One’s right hand is used to handle the mouse of the computer
to draw picture in the software. Therefore, the left hand has to readjust both focus and brightness. Installing a remote
control system for setting the light intensity enables one to have a brightness control knob near the focus knob of the
microscope (Fig. 1). Then, one can readjust focus and brightness quickly with one hand. An extra computer keyboard
814
©FORMATEX 2010
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
may be also set near the left hand (below) to facilitate input of frequently-used (combinations of) keys for the left hand
such as control-Z, which means ‘undo’.
Biological microscopes usually allow one to move the slide stage in X (left/right) and Y (front/back) directions. The
knob(s) for moving the stage should be set near the focus knob but not too close to it, since one needs to move them
quickly and efficiently, but unexpected interactions have to be prevented. Rotation of the slide stage of the microscope
is also required, because it allows for compensation of the differences in the orientation of the individual sections as
they are mounted on the slide glass.
3.2
Camera lucida apparatus
A conventional camera lucida apparatus available from microscope manufacturers is designed to protrude to the right
side of the main body of the microscope so as to allow illustrations to be drawn on a sheet of paper spread on top of the
table to the right of the microscope. We modified the direction of the camera lucida apparatus so that it can capture an
image displayed in the computer monitor. Some examples of various possible arrangements of the camera lucida and
the computer monitor are shown in Fig. 2.
Fig. 2 Examples of set-ups of camera-lucida apparatus interfaced with a computer monitor. Orientation of the monitor is indicated
by the word ‘Top’ in each panel.
3.3
Computer monitor
A computer monitor has to satisfy several requirements in this system. Concerning the size of the monitor, the image in
the monitor viewed through the camera lucida has to match the visible field of the light microscope in size (a 17 inch
screen is usually appropriate). Concerning resolution, 1024x768 or a larger number of pixels for a 17 inch monitor are
preferable. Brightness is very important. When the section image seen through the objective and the monitor image
generated by the camera-lucida are superimposed, the section image is usually much brighter than the camera-lucida
image. Since both the images have to be of comparable brightness to work with the system, illumination of the
microscope for the section has to be weakened while the computer monitor has to be enough bright. Brightness of a
computer monitor is often expressed in cd/m2. Our experience suggests brightness equal or higher than 300 cd/m2 is
preferable.
Any type of monitor can be used with a standard biological microscope. However, a liquid crystal display (LCD)
monitor cannot be used with a 3-D microscope (Edge R400 or Perceptra). A 3-D microscope utilizes two orthogonal
components of polarized light for one’s left and right eyes. The left and right eyepieces have polarizing filters to pass
different orthogonal components of the polarized light. However, a LCD monitor transmits a polarized light of single
linear component. So, the light from the LCD monitor is diminished unequally by the polarizing filters in the left and
right eyepieces. This situation hinders work with the system. Therefore, a monitor that transmits non-polarized light
such as a cathode ray display (CRT) monitor has to be used with a 3-D microscope.
While working one may want to see the computer monitor directly, not through the microscope. For this purpose, we
usually have two monitors that show the same image in the system. One monitor is faced to the camera lucida
apparatus and the other is set upright on the right side of the microscope for one to watch directly what he is drawing
with the software. A distributor of monitor signal may be used to split the monitor signal output line that comes from
the computer (Fig. 3).
©FORMATEX 2010
815
Microscopy: Science, Technology, Applications and Education
A.
Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
3.4
Human input devises for the computer
To make drawings with computer software in the system, one has to push some (combinations of) keys of the computer
keyboard, very often in addition to standard clicking and dragging with mouse buttons. However, the right hand is
holding the mouse and the left hand is holding the focus knob of the microscope or the brightness controller knob.
Therefore, there is no extra hand to touch a keyboard. To solve this problem, we use a USB (universal serial bus) foot
switch equipment, which is commercially available (Fig. 3). This equipment has one, two or three foot-switches, to
which one can assign specific keys of the keyboard (USB 3Foot Switch, Scythe, Funabashi, Japan). Another solution is
to install a mouse that has some extra buttons to which one can assign specific (combinations of) keys of the keyboard
(e.g., MX-400BK, Logitech, Apples, Switzerland). In Adobe Illustrator, ‘Control’, ‘Space’, ‘Alternative’ and ‘P
(shortcut key for the pen tool)’ keys are most frequently used together with mouse clicking and drugging. Therefore,
Fig. 3 Human input/output devices of the
computer. The monitor signal is distributed
to the monitor that is attached to the camera
lucida apparatus and to the other upright
monitor for one to look at directly. Beside
the standard keyboard near the upright
monitor, other input devices include a mouse
with many buttons, a foot switch and a
keyboard for the left hand.
we have assigned these keys to the footswitch or to the extra mouse buttons. The extra keyboard for the left hand may
also help solve this problem.
3.5
Computer software
We use Illustrator (Adobe, San Jose, CA, U.S.A.), which enables one to draw various line pictures. The zoom function
of this software is particularly useful, because it can be coordinated with the magnification of the objectives of the
microscope. For example, when the 4x, 10x, 20x and 100x objectives are used, the zoom of Illustrator can be set to 40,
100, 200, 400, and 1000 %. In this way, one can make drawings seamlessly with any objectives in a single file. The
layer function of Illustrator is also useful in making a picture from multi-section preparation. Drawings from a single
section can be placed on a one layer of an Illustrator file, and multiple such layers can be used to recreate the
correspondence of the histological sections. It enables one to makes drawings in a layer of the software while observing
a given section, and in the next layer of the software, while observing the next section, as if sketching on multiple sheets
of transparent paper. This multi-layer drawing is remarkably useful in understanding and restoring the original structure
of the labelled material. Once one is accustomed to using it, curve drawing process can be fast and flexible in
Illustrator. Finally, text messages can be incorporated into an Illustrator file.
Another piece of software that we routinely use with this system is Rhinoceros (Robert McNeel & Associates,
Seattle, WA, U.S.A.), which is a 3-D graphic software. This software is originally designed for modelling industrial
products, such as vehicles and cell phones, but can be also used for neuroanatomical mapping. Although this software
is relatively inexpensive, it fulfils essential functions for our purpose. Drawing is usually done in the XY viewport
(‘top’ viewport) at a fixed Z coordinate which is correlated to the section number. If drawing is done for serial sections
at sequentially shifted Z coordinates, a 3-D picture will be produced. Rhinoceros has a zoom function so its viewport
zoom can be coordinated with the magnification of the objectives. However, since it does not display the zoom
parameter, unlike Illustrator, one has to adjust zoom manually. Layers are available in Rhinoceros as in Illustrator. It
can import from and export to Adobe Illustrator files. Particularly, it has its own macro language based on Visual Basic
Script (Microsoft, Redmond, Washington, U.S.A.) language, with which one can write long data codes in an
alphanumeric text format.
816
©FORMATEX 2010
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
4. Neuronal tracing and mapping
4.1
Reconstruction of single axons with Illustrator
One of the main usages of this system in our laboratory is to reconstruct the complete trajectories of individual labelled
axons in the central nervous system [6]. Reconstruction with the present computer-aided system is illustrated in Figure
4. After moving a given labelled axon to the centre of the visual field of the microscope (Fig. 4A), lines are
superimposed on the axon in a layer of Illustrator (Fig. 4B, C). One may name this layer as ‘section 1’. The next
section is moved to the visual field of the microscope (Fig. 4D). The position and orientation (rotation) of the next
section is carefully aligned to the drawing of the axon of the previous section so that the cut ends of the axon in the
previous section are connected properly to the corresponding cut ends of the same axon in the next section (Fig. 4E).
Then, lines are superimposed on the axon in the next layer of Illustrator, which may be named ‘section 2’ (Fig. 4F, G).
Drawings of different sections should be placed on separate layers. The precise matching of the drawn lines to the
entire labelled element is essential (Fig. 4A-C). One has to follow exactly all branches and swellings of an axon. It is
usually necessary to readjust the focus and brightness. It is also occasionally necessary to temporarily change the
objective. If multiple axons are labelled in the preparation, one has to follow each identified axon very carefully. When
two axons cross each other within the section, the continuity of these axons has to be carefully judged from the
difference in the depths of these axons at the crossing point. The 3-D microscope facilitates this procedure. Finding the
same axon in the next section (Fig. 4E) is also important in the tracing process. To help with this task, we often use
markers other than the given axon, such as the contour of the nucleus, thick blood vessels and other axons. These
markers are also drawn in the Illustrator layer. A calibration bar should be written within the Illustrator file by setting a
micrometer glass slide instead of the preparation glass slide.
Fig. 4 Reconstruction of a single axon with this system. A, Section of the rat cerebellar nucleus in which a part of terminal arbour
of a Purkinje cell axon is labelled. Some parts of the axonal arbour are out of focus. B, Drawing the labelled axon in Illustrator in
progress. Arrowhead indicates the last anchor point in the middle of drawing process. C, Drawing finished in a section. D, The next
section is mounted on the microscope stage, but is not yet aligned with the drawing. E, The section has been aligned to the drawing
with the previous section by moving and slightly rotating the microscope stage. F, Drawing the labelled axon in this section in
progress. Arrowhead indicates the last anchor point. G, As drawing has been finished in the next section, a part of the axonal
terminal arbour has been reconstructed from the two serial sections.
Once the entire axonal trajectory is traced, all the data of the axonal trajectory is stored in the Illustrator file.
Illustrator can produce a text file of these data written in Illustrator language in numerals and text, which is similar to
the postscript language. This file can be read by one’s hand-made program and rendered to any graphic processing if
programs are prepared. We have made programs for several types of analysis, including measuring axonal length,
connecting up of cut ends to make a semi-final output drawing, and converting the original top view of the axonal
trajectory to front and side views. Furthermore, we are now developing another program to convert data of single
reconstructed axons in Adobe illustrator to McNeel Rhinoceros macro language data so that the axonal trajectory traced
in Illustrator is incorporated directly into the 3-D space of Rhinoceros.
4.2
Direct mapping of neuronal distribution in the 3-D space with Rhinoceros
The present system enables one to map directly distributions of axonal terminals and neuronal cell bodies in the 3-D
space by using Rhinoceros. Relative position of the terminals and cell bodies within the specific nucleus or brain area is
generally important in mapping studies. Therefore, we first make a ‘template’ or an ‘atlas’ of the nucleus within which
©FORMATEX 2010
817
Microscopy: Science, Technology, Applications and Education
A.
Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
we map the distribution of axonal terminals and neuronal cell bodies. For this purpose, histological preparations of the
nucleus in serial coronal, parasagittal, and/or horizontal sections have to be made with standard Nissl staining. Then,
outlines of the nucleus and adjacent structures are depicted in every serial section with the present system with Adobe
Illustrator. The Illustrator data of the nucleus outlines are converted to Rhinoceros 3-D data by giving z coordinates
based on the section number. Then, the 3-D structures of the nucleus formed from coronal, sagittal and horizontal
sections are superimposed and compared to each other. Small variations among preparations such as in size, rotation
and tilt (shearing) may be adjusted in Rhinoceros so that all preparations make the best fit to each other. Then, a
surface can be created for the best fit 3-D outline of the nucleus in Rhinoceros. This surface and best fit sets of serial
coronal, sagittal and horizontal sections of the nucleus represent the optimum 3-D image of the nucleus and serve as the
3-D atlas of the nucleus (Fig. 5A).
Fig. 5 Mapping axonal terminal distribution in the 3-D space with Rhinoceros. A, 3-D atlas of the cerebellar nucleus of the rat
prepared in Rhinoceros. B, A coronal section of the cerebellar nucleus (microscope image) and the equivalent coronal section within
the 3-D atlas (green, camera lucida image) are shown. These images were not fit to each other yet. C, The microscope stage has
been moved and rotated and the Rhinoceros viewport zoom has been adjusted to obtain the best fit. D, A curve (blue) has been
drawn to circumscribe the distribution of labelled axonal terminals. E, Magnified photomicrograph of labelled axonal terminals
(arrowheads). F, Mappings have made in serial sections. G, A surface has been produced based on the curves in serial sections.
This surface circumscribes the distribution of axonal terminals in the 3-D space. Axonal terminals in photomicrographs (B, C and D)
are digitally enhanced to improve visualization. Abbreviations: C, caudal; D, dorsal; L, lateral; M, medial; V, ventral.
Fig. 6 Three-dimensional mapping of retrogradely labelled individual neurons with Rhinoceros. A, Several neurons are labelled
(arrowhead) in the medial accessory olivary nucleus in a coronal section (rat). B, A corresponding slice in the 3-D atlas that has been
prepared beforehand is fitted to the section. C, Spheres are plotted on the labelled neuron in Rhinoceros. The depth of the neuron in
the section can be reflected in adjusting the z coordinate of the sphere in the 3-D space. D, Two-dimensional displays of the neuronal
mapping are superimposed to produce a pseudo 3-D impression. E, A 3-D display of the mapped neurons with the contour of the
nucleus produced by Rhinoceros. F, Horizontal trajectory of the mapped neurons and a contour of the nucleus produced by
Rhinoceros. Abbreviations: C, caudal; D, dorsal; L, lateral; M, medial; R, rostral; V, ventral.
To map the distribution of labelled axonal terminals by using the 3-D atlas in Rhinoceros, the relative position within
the nucleus of the given section containing the terminals were determined. The caudal and rostral poles of the nucleus
can be used as markers to calculate the relative rostrocaudal position of a coronal section. Similarly, the dorsal and
ventral poles of the nucleus can be used as markers to calculate the relative dorsoventral position of a horizontal section.
The outline of the nucleus in the 3-D atlas at the determined position is displayed on the computer monitor. The image
818
©FORMATEX 2010
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
of the 3-D atlas outline in Rhinoceros and the image of the tissue through a microscope are superimposed upon each
other in the present system (Fig. 5B, C). If these two images are not very similar, another adjacent position in the 3-D
atlas may be selected to give the best fit to the microscope image. The difference between the relative position of the
section and the actual fitted position in the 3-D atlas is small (less than 8 %) [8]. As the power of the microscope is
increased to see the terminal swellings (Fig. 5E) of the olivocerebellar projections, the display of the 3-D atlas is also
enlarged by the same ratio while carefully maintaining positioning. The area in which axonal terminals were located is
carefully circumscribed by drawing a curve within Rhinoceros (Fig. 5D). The same procedures were repeated for all
sections that contained axonal terminals (Fig. 5F). The ‘loft’ command in Rhinoceros was then used to reconstruct a
surface based on all curves in the 3-D space (Fig. 5G).
The method to map labelled individual neurons (Fig. 6A) is similar. The outline of the nucleus in the 3-D atlas at the
corresponding position is fit to the image in the microscope section (Fig. 6B). Individual labelled neurons can be
directly mapped by plotting small spheres in Rhinoceros (Fig. 6C). While plotting spheres, the depth of the labelled
neuron within the section can be reflected to the z coordinate of the sphere in Rhinoceros. The same procedures were
repeated for all sections that contained labelled neurons (Fig. 6D). The results of mapping can be shown in the 3-D
space (Fig. 6E) or in a two-dimensional trajectory of the 3-D mapping (Fig. 6F).
5. Summary
Technical and practical aspects of our computer-aided light microscopy system have been described in this chapter. We
now routinely use several setups of this system in the laboratory, and are engaged in investigating the axonal projections
and other morphological aspects of the organization of the central nervous system. This system is especially beneficial
to 3-D reconstruction of several aspects of axonal projections. Results obtained by studies with the present computeraided light microscopy system have been published elsewhere [7-9].
Our laboratory has long used the technique of reconstruction of labelled axons with light microscopy equipped with a
camera lucida apparatus [4-6]. In the past we used hundreds of sheets of paper to draw labelled axons and neurons.
However, paper method has several disadvantages. For example, it cannot adapt to changing magnification. Arranging
the data from a long axon is very difficult and time-consuming. As personal computers and graphic software for them
were developed and improved in the last decade of the 20th century, we began developing a computer aided tracing
system that retains good aspects of drawing with sheets of paper such as ease of use and flexibility. Thus, we ended up
with the system that is presented in this chapter.
Nowadays several special computer-aided neuronal tracing systems are commercially available for research use.
Those research-oriented systems are usually expensive, costing tens of thousands of U.S. dollars, whereas the system
described in this article can built for a little more than ten thousand dollars. The software used was not specific to this
system but rather was made for general use (Adobe Illustrator and McNeel Rhinoceros). These commercially available
software programs are not only less expensive, but also have a much friendlier user interface than the software of
research-oriented systems. The present computer-aided light microscopy system makes full use the software for general
use in neuroanatomical research.
Acknowledgements The support by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science
(20300137) is gratefully acknowledged. The authors thank Dr. Eric Lang for comments on the manuscript and correction of English
writing.
References
[1] Ramón y Cajal S. Histologie du Système Nerveux de l'Homme et des Vertébrés, Vol. II. Paris: Maloine; 1911.
[2] DeFelipe J, Jones EG. Santiago Ramón y Cajal and methods in neurohistology. Trend Neurosci. 1992;15:237-246.
[3] Shinoda Y, Yokota J, Futami T. Divergent projection of individual corticospinal axons to motoneurons of multiple muscles in
the monkey. Neurosci Lett. 1981;23:7-12.
[4] Sugihara I, Wu H-S, Shinoda Y. Morphology of single olivocerebellar axons labeled with biotinylated dextran amine in the rat.
J Comp Neurol. J Comp Neurol. 1999; 414:131-148.
[5] Wu HS, Sugihara I, Shinoda Y. Projection patterns of single mossy fibers originating from the lateral reticular nucleus in the rat
cerebellar cortex and nuclei. J Comp Neurol. 1999;411:97-118.
[6] Sugihara I, Fujita H, Na J, Quy PN, Li BY, Ikeda D. Projection of reconstructed single Purkinje cell axons in relation to the
cortical and nuclear aldolase C compartments of the rat cerebellum. J Comp Neurol. 2009;512:282-304.
[7] Sugihara I, Marshall SP, Lang EJ. Relationship of complex spike synchrony bands and climbing fiber projection determined by
reference to aldolase C compartments in crus IIa of the rat cerebellar cortex. J Comp Neurol. 2007;501:13-29.
[8] Sugihara I, Shinoda Y. Molecular, topographic and functional organization of the cerebellar nuclei: analysis by threedimensional mapping of the olivonuclear projection and aldolase C labeling. J Neurosci. 2007;27: 9696-9710.
[9] Fujita H, Oh-Nishi A, Obayashi A, Sugihara I. Organization of the marmoset cerebellum in three-dimensional space: lobulation,
aldolase C compartmentalization and axonal projection. J Comp Neurol. 2010;518:1764-1791.
©FORMATEX 2010
819