Download Confocal imaging of glial cells in the intact rat optic nerve

GLIA 10:315-322 (1994)
Confocal Imaging of Glial Cells in the Intact
Rat Optic Nerve
A.M. BUTT,l K. COLQUHOUN,' AND M. BERRY2
'Sherrington School of Physiology and 2Department of Anatomy and Cell Biology, U.M.D.S., Guy's and
St. Thomas's Hospitals, London
KEY WORDS
Oligodendrocyte,Astrocyte, Morphology, Structure
ABSTRACT
Astrocytes and oligodendrocytes in the isolated intact mature rat optic
nerve have been computer imaged in three dimensions by laser scanning confocal microscopy of single cells, dye-filled with lysinated rhodamine dextran (LRD). Our results
illustrate the first application of these techniques to an intact CNS white matter tract
and provide comparative data for previous studies on neonatal rat optic nerve (Butt and
Ransom: Glia 2:470-475,1989;Butt and Ransom: J Comp Neurol338:141-158,1993).
The combined use of intracellular injection of LRD and confocal imaging significantly
improves the resolution of glial cell structure, particularly that of mature astrocytes, for
a number of reasons. 1) Single mature dye-filled glia can be imaged, because LRD does
not pass through gap junctions. 2) The entire process field of astrocytes can be visualized
in a single two-dimensional image. 3) Cell images can be rotated through 360" in all
planes to provide a new perspective of glial cell structure in the intact tissue. 4) Reconstruction of optical sections, within a narrow focal plane, provides a high definition and
resolution of the finer details of glial morphology. Using these techniques, three astrocyte subclasses were distinguished on morphological criteria. It is the conclusion of this
study that the majority of these forms represent a single population of fibrous astrocytes
which are well-suited to perform the multiple functions attributed to astrocytes in the
CNS. The morphology of mature myelin-forming oligodendrocytes was also described.
The results confirm that the structural characteristics of both astrocyte subtypes and
oligodendrocytes described in younger rat optic nerves by Butt and Ransom (Glia 2:470475,1989; J Comp Neurol338:141-158,1993) accurately reflect those of the mature glial
population. 0 1994 Wiley-Liss, Inc.
INTRODUCTION
trocytes were not described in detail. The aim of the
present study is to provide new information on the threeThe investigation of glial morphology in vivo has re- dimensional anatomy of mature astrocytes and oligolied on metal impregnation, electron microscopic, and dendrocytes in the intact rat optic nerve using confocal
immunocytochemicaltechniques (Del Rio Hortega, 1928; microscope imaging of cells iontophoretically injected
Klatzo, 1952; Penfield, 1932; Peters, 1964; Privat and with lysinated rhodamine dextran (LRD). The use of
Rataboul, 1986; Skoff et al., 1986, 1976a,b; Ramon y these novel techniques significantly improves the resoCajal, 1909-1911; Ramon-Moliner, 1958;Vaughn, 1969; lution of mature glial cell structure in situ, which is
Vaughn and Peters, 1967). We previously studied glia essential for fully understanding the correlation of form
in the rat optic nerve by using intracellular dye-injec- with function in the CNS. A further stimulus for this
tion to improve resolution of the three-dimensional morphology of these cells (Butt and Ransom, 1989, 1993;
Received September 23, 1993, accepted December 21,1993.
Ransom et al., 1991). This work focused on the strucAddress reprint requests to Arthur M. Butt, SherringtonSchool of Physiology,
tural changes in glia during development and also on U.M.D.S.,
St. Thomas's Hospital,Lambeth Palace Road, London SE17EH, United
the morphology of mature oligodendrocytes.Mature as- Kingdom
0 1994 Wiley-Liss, Inc.
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BUTT ET AL
investigation is the possible verification that two morphological subtypes of astrocyte may exist in the rat
optic nerve, namely, type-1 and type-2 astrocytes, which
are also distinguished on the basis of their antigenic
phenotype (Miller et al., 1985; Raff et al., 1983,1984).It
is suggested (ffrench-Constand and Raff, 1986; Miller
et al., 1989) that type-2 astrocytes form perinodal processes exclusively and have predominantly longitudinal processes, whereas type-1 astrocytes are specialised
to form the perivascular and subpial glia limitans and
possess predominantly radial processes (but see Suarez
and Raff, 1989).Analysis of dye-filled cells may help to
settle the question of the existence of these morphological astrocyte subtypes in situ. Finally, the results of
this work will ascertain if the results of previous analyses of glia in young optic nerves (Butt and Ransom,
1989, 1993; Ransom et al., 1991) are representative of
the adult glial population.
METHODS
RESULTS
Astrocytes
In our previous studies on the developing optic nerve,
astrocytes were subdivided on the basis of the orientation of their processes into transverse, random, and
longitudinal forms (Butt and Ransom, 1993),all of which
are clearly recognised in the confocal images of mature
astrocytes obtained in the present work (Figs. 1-3).
Random astrocytes
The majority (61%) of mature astrocytes extended
processes which were randomly oriented. Visualization
of whole-cells (n = 19) in a plane parallel to the long
axis of the nerve (Fig. lA,B) and rotated through 90"
(Fig. 1C) into the transverse plane showed processes
extending from a centrally located cell body in all directions within a process field 200-500 pm in diameter.
Processes bifurcated regularly along their length forming branches which extended radially and longitudinally through the nerve (Fig. lA,B). Radial branches
terminated in end-feet at the pial surface (Fig. l C , arrowheads) and on blood vessels, whilst longitudinal
branches ended freely in the nerve (Fig. 1D). The calibre of individual processes of single cells varied (Fig.
1D). The surface texture of processes also varied from
smooth to rough, and others bore short spines or collateral processes which passed longitudinally (Fig. 1D).
Optic nerves of Wistar rats, aged from 30 to 60 days
old, were maintained in a brain slice chamber and superfused with oxygenated saline a t 34°C (containing in
mM: NaC1, 132; KC1, 3; CaCI,, 2.5; MgCl,, 1.1; D-glucose, 10; HEPES, 10). Cells were impaled with glass
microelectrodes back-filled with 2% LRD (Molecular
Probes, Inc., Eugene, OR, USA) in 0.5 M KCl, and dye Transverse astrocytes
was injected using 400 ms pulses of 5 nA depolarizing
These cells made up 30% of dye-filled astrocytes. Concurrent every 1s for 5 min. Following injection of around
five cells, nerves were fixed for 30 min in 4% paraform- focal imaging of these astrocytes (n = 10) shows their
aldehyde, dehydrated in alcohols, and whole-mounted cell bodies and processes to be oriented in the transverse plane, to the long axis of the nerve (Fig. 2A).
in methyl salicylate.
Forty LRD-filled cells were imaged on a confocal la- Processes were predominantly smooth, fine calibre and
ser scanning microscope (Bio-Rad Microscience Divi- branched close to the cell body (Fig.2B). Primary branches
sion, Ltd.). The fluorescent dye is excited by a laser passed radially to the pia or to blood vessels and often
which is focused on a single plane, and the light emitted spanned the nerve diameter (Fig. 2A). Longitudinally
by the cell is collected through a variable aperture. This oriented collateral branches emanated either from the
behaves like a pin-hole camera, eliminating scattered primary processes, or directly from the cell body, and
and reflected light, and also light from out of focus passed for short distances before ending freely in the
planes, thereby providing greater definition and resolu- nerve (Fig. 2B).
tion of the image than is possible using conventional
microscopy. The light is filtered, processed using a series of photomultipliers, and converted into a computer Longitudinal astrocytes
image.
These cells represented 9% of dye-filled astrocytes.
For each cell, low magnification serial optical sections were taken at consecutive focal levels of 1-2 pm, Confocal imaging of this rarely filled cell type (n = 3)
to encompass the entire extent of the process field of the clearly illustrates processes preferentially growing in
cell. Addition of data from all sections enabled the three- the longitudinal plane, paralld to the long axis of the
dimensional morphology of the whole cell to be repre- nerve (Fig. 3A). Their cell bodies were large and irregusented in a single two-dimensional image, and, by rota- lar in outline, from which thick cytoplasmic expansions
tion, the image of the cell could be visualised through radiated in all directions for 10-20 pm, and from these
360" in any plane. A further series of optical sections stemmed three kinds of process; one type was smooth
were taken a t high magnification to provide structural and straight in morphology, growing for approximately
100 pm and ending freely in the nerve (Fig. 3A), andetails of the cell body and processes.
GLIAL MORPHOLOGY
317
Fig. 1. Confocal micrographs of two random astrocytes. Visualization of whole-cells in the sagittal plane (A and B), illustrating the
random extension of processes which bifurcated regularly and passed
in all directions through all planes. (C) Rotation of cell (A) through 90"
shows processes extending radially from a centrally located cell body
and terminating in end-feet at the pial surface (pial surface is indi-
cated by arrowheads). (D) High magnification optical section in the
saggital plane through cell body of cell in (B)illustrating the variable
calibre of processes short spines or collateral processes which emanated from primary processes and passed longitudinally. Bar in B
represents 100 pm in A-C and 20 pm in D.
other formed a dense random network of fine processes
localized to within 30 pm of the cell body (Fig. 3B), and
the other was radially oriented ending at the subpial
glia limitans, a s illustrated in one example after the cell
image was rotated through 90" into the radial plane
(Fig. 3 0 .
long (Fig. 4A,B). It was clear from the confocal imaging
of these cells (n = 8) that longitudinal processes varied
in calibre from around 1 pm (Fig. 4A) to submicron
dimensions (Fig. 4B). Statistically valid quantification
of the small number of cells imaged was not possible,
but our impression was that oligodendrocytes supported
a small number of large calibre longitudinal processes
(Fig. 4A) or a larger number of small calibre longitudinal processes (Fig. 4B). High magnification optical sections in the region of the cell body show that connecting
branches give rise to single or multiple longitudinal
processes (Fig. 4C). Rotation of a n oligodendrocyte
Oligodendrocytes
The three-dimensional cell structure of mature oligodendrocytes is illustrated in Figure 4. Mature oligodendrocytes had 15-20 longitudinal processes, 150-250 pm
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BUTT ET AL.
Fig. 2. Confocal micrographs of transverse astrocyte. (A) In the
saggital plane, these cells were characterised by the preferential extension of their processes perpendicular to the long axis of the nerve.
Primary processes ended in bdlbous end-feet at the pia or on blood
vessels. (B) High magnification optical section in the saggital plane,
illustrating smooth, fine calibre primary processes which branched
close to the cell body, and collateral branches which emanated from
the primary processes or directly from the cell body, and passed longitudinally before ending freely in the nerve. Bar in A represents 100 pm
in A and 20 pm in B.
through 90" into the transverse plane illustrates that
the spatial distribution of axons myelinated by an oligodendrocyte is tightly constrained to a radial field with a
diameter of approximately 30 pm (Fig. 4D).
and Ransom, 1993). 2) Astrocyte whole-cell structure
can be visualized in a single two-dimensional image,
which is not generally possible using other techniques
because astrocyte processes span a depth of field of 200
pm or more. 3) The three-dimensional ramification of
processes can be analyzed in planes not normally accessible in the intact tissue by rotating the cell image through
360°, in any plane. 4) A far greater definition and resolution of microscopic features than is possible using
conventional microscopy can he achieved by imaging
optical sections of the cell within a narrow focal depth,
without interference from out of focus planes. Our confocal microscopic analysis is of' a relatively small sample of cells (n = 40).Nevertheless, our investigation of
mature glial cells recognized three morphological astrtocyte subtypes and oligodendrocytes,which agrees with
previous reports based on the analysis of over 750 dyefilled cells in the intact rat optic nerve during development (Butt and Ransom, 19931, and we are confident
that the cells analyzed in this present report are representative of this large sample of the optic nerve glial
population. Previously (Butt and Ransom, 19931, we
focused on the structural changes in glia during development and astrocytes and oligodendrocytes from mature nerves were not analyzed in detail. The present
study confirms that the morphological characteristics
described in younger nerves accurately reflects those of
the mature glial population.
Astrocytes are attributed with diverse functions including the support and partitioning of axons, the trans-
DISCUSSION
The recent use of the intracellular dye-injection technique has provided new data on the whole-cell morphology of macroglial cells in the rat optic nerve not accessible using standard morphological sections (Butt and
Ransom, 1989,1993; Ransom et al., 1991). In this work,
we have extended previous observations to include mature LRD-filledastrocytes and oligodendrocytesimaged
by confocal microscopy. This is the first use of the confocal microscope to image the whole-cell structure of glial
cells in an intact CNS tissue, although the confocal
microscope has been used to reconstruct serial sections
of immunohistochemically labelled glia in mouse optic
nerve (Morgan et al., 1992). The novel use of confocal
imaging of LRD-filled cells provides a significant improvement on the resolution of the fine structure of
individual cells, particularly in older nerves, for a number of reasons. 1) Single dye-filled cells can be visualised in isolation,because LRD is a high molecular weight
(3,000 Da) fluorescent dye that does not pass through
gap junctions, which are the basis of the extensive dyecoupling in mature astrocytes (Mugnaini, 1986), and
which previously restricted the use of the fluorescent
dye Lucifer yellow to filling cells in young nerves (Butt
GLIAL MORPHOLOGY
Fig. 3. Confocal images of longitudinal astrocyte. (A) In the saggital
plane, these cells were characterised by the preferential extension of
processes parallel to the long axis of the nerve, which ended freely. (B)
High magnification optical section in the saggital plane, illustrating
irregular cell body with thick cytoplasmic expansions from which a
319
dense network of fine processes emanate. (C) 90" rotation of the cell
into the transverse plane, illustrating radially oriented processes in
the nerve which ended at the subpial glia limitans (pial surface is
indicated by arrowheads), Bar in C represents 50 pm in A and C, and
15 p,m in B.
port of solutes, maintaining the subpial glia limitans, Cajal, 1909-191 1; Reichenbach, 1989; Robinson and Dreand the physiology of the blood-brain barrier and nodes her, 1989; Skoff et al., 1986; Susuki and Raisman, 1992).
of Ranvier. Such functions are correlated with the pat- It is possible that the morphological subtypes of astrotern of their process extension and the contacts they cyte described here may represent functionally specialmake (Butt, 1991; Butt and Ransom, 1993; Chan-Ling ised cell types. Astrocytes in the rat optic nerve have
and Stone, 1991; Klatzo, 1952; Penfield, 1932; Ramon y been subdivided into type-1 and type-2 on the basis of
GLIAL MORPHOLOGY
their antigenic phenotype and developmental timetables in vitro (Miller et al., 1985; Raff et al., 1983,19841,
and it was suggested that in vivo type-1 astrocytes form
the perivascular and subpial glia limitans and have a
radial morphology, whilst type-2 astrocytes form perinodal associations, exclusively, and have longitudinal
processes (ffrench-Constant and Raff, 1986; Miller et
al., 1989).In this study, transverse astrocytes are shown
to have radial processes which terminate predominantly
in end-feet which subserve the subpial glia limitans,
whereas longitudinal astrocytes have longitudinal processes which end freely in the nerve and may subserve
nodes (Butt and Ransom, 1989, 19931, as described in
electron microscope studies (Black and Waxman, 1989).
Transverse and longitudinal astrocyte subtypes may
represent functionally specialized astrocyte cell types.
However, this study, like others (Butt and Ransom,
1993; Fulton et al., 1992; Suarez and Raff, 1989), has
not detected a strict morphological and functional divergence of astrocyte forms in the rat optic nerve. We
have found that all astrocytes have both radial and
longitudinal processes, and connect by end-feet with
the subpial and perivascular glia limitans. Moreover,
longitudinal astrocytes are unlikely to be a cell type
exclusively subserving nodes, since they make up only a
small proportion of the total astrocyte population. Accordingly, it is the conclusion of this study that mature
astrocytes form a single population, in which transverse and longitudinal astrocytes represent extreme morphological variants of the random form. These cells are
structurally well suited to perform the multiple functions attributed to astrocytes in the CNS.
Chan-Ling and Stone (1991)came to a similar conclusion that astrocytes in the cat retina are morphological
variations of a single class of cells, the extreme forms
being a direct result of their association with neighbouring structures, such as blood vessels and nerve
fibre bundles, and they described a “bipolar” astrocyte
which may be equivalent to the longitudinal astrocytes
described in the present study. These morphologically
and antigenically distinct astrocyte forms in the retina
(Chan-Lingand Stone, 1991;Robinson and Dreher, 1989)
are considered to be derived from a single optic nerve
astrocyte subtype with a “type-1”lineage (ffrench-Constant et al., 1988; Ling et al., 1989; Watanabe and Raff,
1988). Thus, a single astrocyte cell type can develop in
vivo into a diverse range of forms. Butt and Ransom
Fig. 4. Confocal micrographs of mature myelin-forming oligodendrocytes. (A) In the saggital plane, this oligodendrocyte had ten large
calibre longitudinal processes with a uniform length of approximately
200 pm. (B) An oligodendrocyte, also in the saggital plane, with 20 or
so small calibre longitudinal processes which varied in length from
100 to 200 pm. (C) High magnification optic section through the cell
body of cell in (A), illustrating that connecting branches could give rise
to single or multiple longitudinal processes, or longitudinal processes
may emanate from the cell body, directly. (D) Rotation of the oligodendrocyte in (B) through 90”showing the highly localised radial process
field (cf. astrocyte in Fig. 1B). Bar in D represents 30 (*min A,B, and
D, and 10 pm in C.
321
(1993) concluded that the morphological diversity of
astrocytes in the rat optic nerve may reflect the divergent responses of a single astrocyte cell type to variable
local conditions during development. Transverse astrocytes differentiated prior to birth, at a time when no
cells were far from the pial surface, whilst randomly
oriented forms which extended processes to both the pia
and blood vessels differentiated during the first 2 weeks
after birth, as the nerve became more vascularized, and
finally longitudinal forms developed in the second and
third weeks after birth, as axons and nodes matured
(Butt and Ransom, 1993). The present study demonstrates that after this time astrocytes undergo no further, significant changes in morphology, and confirms
that the morphological characteristics of astrocytes described in these younger nerves accurately reflect those
of the mature glial population. These studies once again
call into question the existence of type-2 astrocytes in
vivo.
Oligodendrocytes have a characteristic morphology
(Bunge, 1968; Butt and Ransom, 1989; Del Rio Hortega,
1928; Hirano, 1968; Penfield, 1924; Suzuki and Raisman, 19921, and mature oligodendrocytesin 60-day-old
rat optic nerves have 10-20 myelin segments with an
internodal length of 150-250 pm. These results confirm
that previous observations on the number and length of
internodal myelin segments, which were based on
younger nerves (Butt and Ransom, 1993), are representative of the mature oligodendrocyte population. Our
previous studies on the rat optic nerve emphasized the
homogeneity of the oligodendrocyte population in comparison with the marked morphological heterogeneity
displayed by astrocytes (Butt and Ransom, 1989,1993;
Ransom et al., 1991). However, subtle morphological
diversity has been reported in oligodendrocytesin other
white matter tracts (Del Rio Hortega, 1928; Hildebrand
et al., 1993) and was also detected in confocal images of
rat optic nerve oligodendrocytes.For example, the number of myelin segments supported by single oligodendrocytes varied between 10 and 20, the calibre of the
longitudinal processes varied from the submicron thickness to around 1 pm, the internodal distance of individual myelin segments varied between 50 and 350 pm,
and myelin segments were connected t o the cell body by
short radial processes, either singly or several were
supported by the same connecting branch (also see Butt
and Ransom, 1993). It is possible these differences are
an indication of oligodendrocyte subtypes in the rat
optic nerve. If the calibre of longitudinal processes is
representative of the thickness of the myelin sheath,
then our qualitative observations support the suggestion that oligodendrocytes may support either many
myelin segments with a small diameter or a few myelin
segments with a large diameter (see Hildebrand, 1993).
However, this apparent inverse relation between number and thickness of the ensheathed axon segments is
based on confocal images of a small number of cells
(n = 8) and further studies, including electron microscope examination of dye-filled oligodendrocytes, will
be required to determine whether this is the case.
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BUTT ET AL.
ACKNOWLEDGMENTS
Morgan, F., Barberese, E., and Carson, J.H. (1992)Visualizing cells in
three dimensions using confocal microscopy, image reconstruction
and isosurface rendering: Application to glial cells in mouse central
This work was supported by the Wellcome Trust and
nervous system. Scanning Microsc., 6:345-357.
Special Trustees of St. Thomas’s Hospital. At different Mugnaini, E. (1986) Cell junctions of’astrocytes, ependyma, and related central nervous system, with tamphasis on the hypothesis of a
periods during the course of this work, Miss Colquhoun
generalized functional syncytium of supporting cells. In: Astrocytes,
was on scholarships from the Worshipful Company of
Vol. 1. S. Federoff and A. Vernadakis, eds. Academic Press, New
York, pp. 329371,
Barber Surgeons, the Nuffield Foundation, and The
Penfield, W. (1924)Oligodendroglia and its relation to classical neuroSpecial Trustees of Guy’s Hospital. We would like to
glia. Brain, 47:430-452.
thank Professor Lumsden for the use of the confocal Penfield, W. (1932)Neuroglia: Normal and Pathological. In: Cytology
and Cellular Pathology of the Nervous System., Vol. 2. Hoeber, New
microscope.
York.
Peters, A.(1964)Observations on the coiineldonsbetweenmyelin sheaths
and glial cells in the optic nerves of young rats. J . Anat., 98:125-134.
Privat, A. and P. Rataboul(1986) Fibro’usand protoplasmic astrocytes.
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