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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. 316 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 318 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. 322 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. 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