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Grid-Mapped Freeze-Fracture Analysis ofGap Junctions in Gray and White Matter

of Adult Rat Central Nervous System,With Evidence for a ‘‘Panglial Syncytium’’

That Is Not Coupled to Neurons

JOHN E. RASH,1,2,3* HEATHER S. DUFFY,1,2 F. EDWARD DUDEK,1,2,3

BRENT L. BILHARTZ,1 L. RAY WHALEN,1,3 AND THOMAS YASUMURA1

1Department of Anatomy and Neurobiology, Colorado State University,Fort Collins, Colorado 80523

2Program in Molecular, Cellular, and Integrative Neurosciences, Colorado State University,Fort Collins, Colorado 80523

3Program in Cell and Molecular Biology, Colorado State University,Fort Collins, Colorado 80523

ABSTRACTIn white matter regions of the brain and spinal cord of adult mammals, gap junctions

previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes andependymacytes. The resulting ‘‘functional syncytium’’ was proposed to modulate the ion fluxesthat occur during electrical activity of the associated axons. Gap junctions also have beenreported linking neurons with glia, and functional neuronal-glial coupling has been postu-lated. To investigate the glial syncytium and the neuron-to-glia coupling hypotheses, we used‘‘grid-mapped freeze fracture,’’ conventional thin-section electron microscopy, and lightmicroscope immunocytochemistry to examine and characterize neurons and glia in gray andwhite matter of adult rat brain and spinal cord. We have obtained quantitative evidence forthe abundance and widespread distribution of gap junctions interlinking the three primarytypes of macroglia throughout both gray and white matter of the mammalian central nervoussystem (CNS), thereby extending the concept to that of a functional panglial syncytium. Incontrast to previous reports, we show that of more than 400 gap junctions in which bothparticipating cells were identified, none were between neurons and glia. Thus, neuronalcoupling and glial coupling involved separate and distinct pathways. Finally, putative waterchannels (i.e., ‘‘square arrays’’) were confirmed to be abundant and in close association withgap junctions in astrocytes and ependymacytes. Because the astrocyte ‘‘intermediaries’’extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, fromthe ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons toastrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abun-dance of water channels and intercellular ion channels, is optimally positioned and equippedto modulate water and ion fluxes across broad regions of the CNS. J. Comp. Neurol.388:265–292, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: aquaporin; electron microscopy; intercellular junctions; water channels

In their pioneering study, Brightman and Reese (1969)used techniques of conventional thin-section electron mi-croscopy to identify the primary types of intercellularjunctions between glia and between neurons in the verte-brate central nervous system (CNS). Subsequently, freeze-fracture electron microscopy revealed that gap junctionsare present in the plasma membranes of all three primary

Grant sponsor: NIH; Grant number: NS31027; Grant sponsor: CollegeResearch Council.

Brent L. Bilhartz’s current address is Department of Pediatrics, Univer-sity of Minnesota, Minneapolis, MN 55455.

*Correspondence to: John E. Rash, Department of Anatomy and Neurobi-ology, Colorado State University, Fort Collins, CO 80523.E-mail: [email protected]

Received 3 March 1997; Revised 16 June 1997; Accepted 17 June 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 388:265–292 (1997)

r 1997 WILEY-LISS, INC.

classes of macroglial cells1 (i.e., ependymacytes, astro-cytes, and oligodendrocytes; Landis and Reese, 1974b;Larsen, 1977; Hatton and Ellisman, 1981; Landis, 1981;Landis and Weinstein, 1983). However, the patterns andextent of intercellular coupling among the glia remained indoubt (Dermietzel et al., 1978).

To analyze the extent of ultrastructural coupling amongglia in white matter regions of the mammalian CNS,Massa and Mugnaini (1982) examined freeze-fracturereplicas of carefully-dissected regions from brain, spinalcord, and optic nerve as simplified model systems thatcontained no neuronal cell bodies, dendrites, or synapsesthat would complicate cell identification. They showedthat the macroglia in white matter are extensively coupledby both ‘‘homologous’’ and ‘‘heterologous’’ couplings. Ho-mologous coupling (the presence of gap junctions betweenlike kinds of cells [Sontheimer, 1995]) occurred amongastrocytes and among ependymacytes, whereas heterolo-gous coupling (the presence of gap junctions betweenunlike cells) occurred between astrocytes and oligodendro-cytes and between astrocytes and ependymacytes (Mug-naini, 1986). However, in contrast to previous assumptions(Dermietzel et al., 1978), Massa and Mugnaini also re-ported that homologous gap junctions between oligodendro-cytes in CNS white matter did not occur (or were ex-tremely rare) and suggested instead that oligodendrocytesare coupled to other oligodendrocytes solely through astro-cyte ‘‘intermediaries’’ (Massa and Mugnaini, 1982; Mug-naini, 1986). Despite the freeze-fracture data of Massa andMugnaini showing that homologous oligodendrocyte-to-oligodendrocyte coupling is essentially nonexistent in CNSwhite matter, widespread homologous coupling between oligo-dendrocytes continues to be invoked to explain immunocyto-chemical and electrophysiological observations (Kettenmannand Ransom, 1988; Butt and Ransom, 1989; Li et al., 1997).

In addition to homologous coupling between ependymalcells lining the brain ventricles (Larsen, 1977; Hatton andEllisman, 1981), Mugnaini (1986) documented heterolo-gous coupling between ‘‘ependymoglial cells’’ and astro-cytes in brain white matter. From those data, Mugnaini(1986) proposed the existence of a ‘‘generalized functionalsyncytium of supporting cells’’ in white matter regions ofthe adult mammalian CNS and suggested that the glialsyncytium is involved in regulating ionic fluxes that occurduring electrical impulse conduction in the associatedaxons. In the succeeding years, it was not feasible todetermine with confidence the degree to which the threetypes of macroglial cell types are coupled ultrastructurallyor physiologically (Butt and Ransom, 1989; Ransom, 1995;Sontheimer, 1995; but see Robinson et al., 1993; Konietzkaand Muller, 1994). In large part because of the lack ofindependent corroboration for the proposed existence ofgap junctions linking the three primary classes of glialcells, and in part because of the lack of data regardingcoupling of glia in CNS gray matter, the concept of afunctional glial syncytium involving all classes of macrog-lia has remained controversial (Kettenmann and Ransom,1988; Robinson et al., 1993; Sontheimer, 1995).

Gap junctions also have been found linking neurons toneurons in several regions of the CNS of adult mammals

(Sotelo and Taxi, 1970; Sloper, 1972; Sotelo and Korn,1978; Kosaka, 1983a,b, 1985; Matsumoto et al., 1988;Matsumoto et al., 1989; Rash et al., 1996) and, untilrecently, neuronal coupling has been assumed to be strictlyhomologous (but see Walker and Hild, 1969; Morales andDuncan, 1975). However, evidence for coordinated changesin intracellular calcium concentration and the apparentblockade of the slow ‘‘calcium waves’’ (Nedergaard, 1994)by octanol, a blocker of gap junctions, have revived earliersuggestions that neurons may be coupled to glia by heter-ologous gap junctions (Walker and Hild, 1969; Morales andDuncan, 1975). For example, Nadarajah et al. (1996), byusing conventional thin-section electron microscopy andlight microscope immunocytochemistry, have suggestedthat 18% of all gap junctions in adult rat cerebral cortexare heterologous neuron-to-glia couplings. Expression ofconnexin32 (Cx32, an oligodendrocyte connexin) and Cx43(an astrocyte connexin) in neurons also has been reportedby using light microscopic techniques (Micevych and Abel-son, 1991; Nadarajah et al., 1996). However, the proposedexistence of gap junctions between neurons and glia hasnot been confirmed by higher-resolution electron micro-scope immunohistochemical staining techniques or byfreeze-fracture. Moreover, recent electrophysiological andpharmacological evidence directly contradicts the pro-posed involvement of gap junctions in the propagation ofglia-to-neuron cytoplasmic calcium waves (Charles, 1994;Hassinger et al., 1995; Charles et al., 1996). Thus, theproposed existence of heterologous glia-to-neuron gap junc-tions remains controversial.

The freeze-fracture technique, because of its high spatialresolution and its ability to reveal the internal macromo-lecular architecture of membranes, is capable of providingunambiguous evidence for the occurrence and distributionof gap junctions within a tissue. In principal, the freeze-fracture technique also allows one to identify the two cellscontributing to an individual gap junction. However, be-cause of limitations thought to be inherent to the freeze-fracture technique, it previously was not possible to makestrong inferential correlations of freeze-fracture detailswith their presumed histological counterparts (Dermietzelet al., 1978). Moreover, ambiguous criteria used to distin-guish neurons from glia in freeze-fracture replicas mayhave led to misidentification of glia as neurons (Perrachia,1973; Andrew et al., 1981; Schmalbruch and Jahnsen,1981; Dudek and Snow, 1985). To overcome many of thelimitations of conventional freeze fracture, we developed‘‘grid-mapped freeze fracture’’ (Rash et al., 1995; Rash etal., 1996), a technique that eliminates replica fragmenta-tion, provides for correlative confocal light microscopy andfreeze-fracture imaging of the same tissue slice, and allowsunambiguous identification and imaging of replicated cellsby using conventional histological and electron microscopeimmunocytochemical labeling techniques.

In this study, we have used grid-mapped freeze fractureto determine whether the ‘‘generalized glial syncytium’’proposed by Massa and Mugnaini for CNS white matter(Massa and Mugnaini, 1982; Mugnaini, 1986) also occursin CNS gray matter, and if so, whether the glial syncytiumin gray matter links all three types of macroglia. Usinggrid-mapped freeze fracture, we describe criteria for distin-guishing the major classes of macroglia in both gray andwhite matter, as well as criteria for distinguishing neuronsfrom glia in diverse areas of the mammalian CNS. Weconfirm that abundant gap junctions extensively interlink

1For further characterization of ependymacytes (or ‘‘ependymoglia’’) andtheir associated tanycytes (or ‘‘radial glial cells’’) as macroglia, see reviewby Reichenbach and Robinson (1995).

266 J.E. RASH ET AL.

all three major classes of macroglia via astrocyte interme-diaries, thereby expanding the scope of the proposed glialsyncytium to that of a vast panglial network that extendsradially from the spinal canal and brain ventricles, acrossgray and white matter regions, to the glia limitans and tothe capillary endothelium. We also confirm that ‘‘squarearrays’’2 (a class of putative water channels [Verbavatz etal., 1994; King and Agre, 1996]) are abundant near gapjunctions in astrocytes and ependymacytes (Hatton andEllisman, 1981; Landis and Reese, 1981), thereby implicat-ing the three-dimensional network of the macroglial syncy-tium as a pervasive pathway in the CNS that may beinvolved in intercellular movement of water and ions andin the resorption of cerebrospinal fluid (King and Agre,1996). We also confirm that gap junctions link astrocytes tooligodendrocytes at the paranodal loops of myelin (Wax-man and Black, 1984; Black and Waxman, 1988), wherepotassium efflux appears to be buffered by direct intercel-lular transfer from the neuron cytoplasm to the oligoden-drocyte cytoplasm (Wiley and Ellisman, 1980). Finally, weprovide statistical evidence that gap junctions betweenneurons and glia (Nadarajah, et al., 1996) either do notexist or they are so rare as to constitute a negligiblefraction of gap junctions in the CNS. Thus, we propose thatby its widespread distribution, membrane molecular com-position, and extensive intercellular coupling, the panglialsyncytium bears a major role in osmotic and ionic regula-tion of the adult nervous system.

MATERIALS AND METHODS

Adult Sprague-Dawley rats (8 males and 2 females)were anesthetized with ketamine (100 mg/kg) and xyla-zine (20 mg/kg) and fixed by transcardiac whole-bodyperfusion (Hudson et al., 1981) with 2.5% glutaraldehydein 0.15 M Sorensen’s phosphate buffer, pH 7.3–7.4, accord-ing to procedures approved by the Colorado State Univer-sity Animal Care and Use Committee. More than onehundred 100-µm-thick slices from transected spinal cord(Fig. 1A) and more than thirty 100-µm-thick slices fromselected brain regions (hippocampus, suprachiasmaticnucleus, supraoptic nucleus, and cerebellum) were frozen,freeze-fractured at 2105° to 2183°C in a JEOL freeze-etchdevice (RFD 9010C; RMC, Inc., Tucson, AZ), and replicatedwith a calculated 0.7–1.5 nm of platinum and 10 nm ofcarbon. (Due to the slow response time of conventionalquartz-crystal thin-film monitors, the thinner films usedin this study could not be measured directly; consequently,

their thicknesses were estimated on the basis of previouslyestablished deposition rates and deposition times; Rashand Yasumura, 1992.)

For grid-mapped freeze fracture (Rash et al., 1995), eachfractured and replicated tissue slice was bonded at 290°Cto a gold index (Finder) grid by using 1% Lexan (polycarbon-ate) plastic dissolved in ethylene dichloride (Steere andErbe, 1983). After solvent evaporation for 3–18 hours at235°C, the Lexan-stabilized samples were thawed, placedon coverslips, and mapped (Fig. 1B) by using a MolecularDynamics (Sunnyvale, CA) Multiport 2001 inverted confo-cal laser scanning microscope. Tissue fragments wereremoved by digestion for up to 48 hours in 5.25% sodiumhypochlorite solution containing 1–2% Tween 20. Eachgrid-bonded replica was rinsed and dried, and a second5-to 10-nm stabilizing coat of carbon was applied to thecleaned platinum surface (to anneal cracks in the carbonsupport film created by warming the sample from 2180 °Cto 130°C). The supporting Lexan film was removed byimmersing the replica in six 5-minute changes of ethylenedichloride.

For immunolabeling, 75-µm-thick cross sections of form-aldehyde-fixed spinal cord were treated for 24 hours withmouse antibodies to Cx43 and Cx32 (Chemicon, Temecula,CA) and counterlabeled for 1 hour with goat anti-mouseIgG conjugated to Texas Red or Cascade Blue (MolecularProbes, Eugene, OR). Immunolabeled samples were photo-graphed with a Zeiss Axiophot microscope equipped forfluorescence microscopy and for Nomarski interferenceoptics. Images were imported into a Macintosh Power PC7100, stored as Adobe Photoshop files, and printed tominimize background fluorescence.

For conventional thin-section electron microscopy, seg-ments from the thoracic region of adult rat spinal cord(prepared as above) were postfixed for 1 hour in 1%osmium tetroxide in 0.15 M Sorensen’s phosphate buffer,pH 7.4, rinsed in distilled water, and stained for 30 hoursin 0.5% aqueous unbuffered uranyl acetate. The samplewas dehydrated in graded ethanol series, rinsed in ac-etone, embedded in Epon-Araldite-dodecenylsuccinic anhy-dride plastic mixture, and polymerized at 70°C for 18hours. (For detailed methods, see Rash et al., 1969, andRash and Fambrough, 1973.) Thin sections (silver to palegold interference colors) were cut by using a Dupontdiamond knife in a Sorvall MT-2B ultramicrotome andstained for 10 seconds with lead citrate (Venable andCoggeshall, 1965).

Replicas and thin sections were examined at 100 kV in aJEOL 2000 EX-II transmission electron microscopeequipped with a long-focal-length objective lens (SAP-20;lattice resolution of 0.14 nm shown with 50-µm objectiveaperture; working resolution of 0.3–0.4 nm). Over 1,000low- to high-magnification images (350–3200,000) werephotographed, almost all as stereoscopic pairs having anincluded angle of 8°. Stereoscopic imaging has been shownto be of value in deciphering complex, three-dimensionalrelationships between cells and their processes and isrequired for proper interpretation of structural subunits ofmacromolecules (Rash and Yasumura, 1992). High-magni-fication images at slight underfocus (less than 100 nmunderfocus; phase contrast granularity less than 0.4 nm)were selected for structural analysis of connexons andsquare array subunits. In stereoscopic images, these arti-facts of underfocus are resolved as a three-dimensional

2We continue to use the term ‘‘square arrays’’ (Rash et al., 1974a) todenote clusters of IMPs and pits arranged in regular crystalline squarelattice in preference to other suggested designations, such as ‘‘rectilineararrays’’ (Kreutziger, 1968; Ellisman and Rash, 1977), ‘‘rectangular arrays’’(Dermietzel, 1973; Rash et al., 1974b), ‘‘orthogonal arrays’’ (Rash andEllisman, 1974), ‘‘membrane orthogonal particle complexes’’ or ‘‘MOPCs’’(Dermietzel et al., 1978), ‘‘orthogonal assemblies’’ (Gulley et al., 1978),‘‘orthogonal assemblies of intramembrane particles’’ or ‘‘OAPs’’ (Verbavatzet al., 1994), or simply, ‘‘assemblies’’ (Landis and Reese, 1974b), becauseboth ‘‘orthogonal’’ and ‘‘rectangular’’ denote any rectangular lattice regard-less of length or width of subunits, whereas ‘‘rectilinear’’ denotes ‘‘straightlines’’ and does not imply either right angles or equilateral sides. Likewise,‘‘assemblies’’ is insufficiently specific, could be applied to any cluster ofIMPs, and lacks precedence. However, with growing evidence that squarearray proteins are members of the ‘‘aquaporin’’ family of proteins (Verba-vatz et al., 1994), future designations will likely be based on the nomencla-ture of the Human Genome Committee (King and Agre, 1996).

HETEROLOGOUS GAP JUNCTIONS CREATE PANGLIA 267

Fig. 1. Images from sequential steps in grid-mapped freeze-fracture imaging of central nervous system (CNS) tissues. A: Image of100-µm-thick slice from the lumbosacral region of an adult rat spinalcord photographed and mapped before freeze fracturing. B: Hemisec-tion of similar slice from cervical region of rat spinal cord followingfreeze fracturing and bonding in Lexan to a gold ‘‘Finder’’ grid. Arrow‘‘1C’’ points to the central canal, which is shown at higher magnifica-tion as Figure 1C, below. Arrow 8A,B points to the area shown in

Figure 8A,B. C: Overview image of freeze-fracture replica from thearea indicated in Figure 1B. The ependymal layer consists of cellswhose nuclei are in two layers (E, ependyma; S, second or ‘‘subependy-mal’’ layer). Four arrows at top point to cilia. Five numbered boxesdelineate areas shown at higher magnification in succeeding figures,which include areas from the apical margin (2C,D), lateral appositions(3A,B), and the ependyma-astrocyte boundary (3C). (In all figures, thescale bar 5 0.1 µm unless otherwise indicated.)

‘‘mist’’ above and below the molecules being imaged,whereas ‘‘real’’ structural details are imaged in the focalplane of the molecule. In thin sections, oblique images ofmembranes were examined for gap junctions by tilting thespecimens with a 660° tilt, 360° rotation ‘‘goniometer’’stage.

Selected areas of electron micrographs were scannedinto a Macintosh Power PC 8100 computer as 2–8 Mbytegray scale files by using a Leaf Lumina camera (LeafSystems, Westborough, MA). Digital images were manipu-lated by using the ‘‘unsharp masking,’’ brightness andcontrast, and selected-area ‘‘dodging’’ functions of AdobePhotoshop 3.0.5. To document the presence of obliquelysectioned gap junctions, one pair of images that had beenexposed at 158° and 230° tilt (included angle of 88°) wasconverted to a ‘‘pseudostereoscopic’’ image by using the‘‘distort’’ function in Adobe Photoshop to correct for severeimage foreshortening created at the original high-tilttransmission electron microscopic (TEM) viewing angles.Several stereoscopic images were altered to fit the rectan-gular publication format by using the ‘‘scale’’ and ‘‘distort’’functions in Adobe Photoshop. Figures for reviewers wereprinted with a Kodak XLS 8300 digital color printer. Forpublication, digital images were provided as Photoshopfiles on Zip disks.

Freeze-fracture images are presented with black shad-ows, according to the original convention of Steere (1957).The freeze-fracture terminology proposed by Branton et al.(1975) is used in this report.

RESULTS

By using grid-mapped freeze fracture (Rash et al., 1995,1996), we examined slices of rat brain and spinal cord byconfocal microscopy before they were frozen (Fig. 1A) andafter freeze fracturing but before replica cleaning andremoval of histologically relevant biological macromol-ecules (Fig. 1B). In addition to holding the replicas in placefor initial light microscopic examination, the Lexan filmsused in grid-mapped freeze fracture protected the samplesfrom fragmentation, often yielding replicas containinggreater than 3 mm2 (or greater than 3,000,000 µm2) ofsurface area to be examined (Fig. 1B). Following tissuedigestion and TEM examination, light microscopic imagesand low-magnification freeze-fracture images were super-imposed and/or the grid coordinates of selected structureswere compared in the two images, thereby allowing thehistological and gross anatomical mapping of cells withinthe tissue slices (compare Fig. 1B, arrow designated 1C,with Fig. 1C) and providing confidence in identification ofindividual cells. For example, the central canal in a100-µm-thick hemisection from the cervical enlargementof an adult female rat appeared as a bright ring inlow-magnification confocal images (Fig. 1B). In the compan-ion freeze-fracture images from the same tissue slice (Figs.1C, 2B–D, 3A–C), individual cells and their subcellularcomponents were revealed at high resolution.

Tissue components recognizable at lowmagnification in freeze-fracture replicas

In freeze-fracture replicas from CNS tissues, the lumenof the brain ventricles and spinal canal (Fig. 1C) appearedas a relatively flat, featureless void whose margins werepunctuated by cross and longitudinal fractures of cilia(Fig. 1C, arrows; Fig. 2A,B) and by much shorter microvilli

(Figs. 1C, 2B,C). In freeze-fracture replicas of the spinalcord, the ependyma was found to consist of mixed ciliatedstratified cuboidal and pseudostratified columnar epithe-lial cells (Peters et al., 1991) surrounding the centrallumen (compare Figs. 1C, 2B,C, and 4A). Subjacent to theependymal cell layer in brain and spinal cord were numer-ous astrocytic processes, myelinated and unmyelinatedaxons, and small blood vessels (described in later sections).

Ultrastructural features of ependymal cells

Microvilli and cilia. The apical margins of manyependymal cells exhibited densely packed microvilli, whichconsisted of a mixture of intertwined tubular projectionsand short ridges (Figs. 2B,C, 3C). Other ependymal cellshad both microvilli and cilia, the latter of which projectedup to 20 µm into the lumen of the spinal canal (Figs. 1C,4A,C), and occasionally, toward the neuropil (Fig. 4C,lower arrow). In freeze-fracture replicas, the base of eachependymal cilium displayed five to eight distinctive rowsof intramembrane particles (IMPs; Fig. 2A, arrow) thatcollectively are called the ‘‘ciliary necklace’’ (Gilula andSatir, 1972). Cross fractures and cross sections revealedthe ‘‘9 1 2’’ configuration of microtubules (Figs. 2B, 4C)that is characteristic of motile cilia.

Intermediate filaments. Distinctive bundles of inter-mediate filaments were present throughout the cyto-plasms of most ependymal cells (Figs. 3A,B, 4F,H). Al-though similar in appearance to glial fibrillary acidicprotein (GFAP) filaments in astrocytes (see below), numer-ous studies have shown that GFAP is not present in theintermediate filaments of ependymacytes (Basco et al.,1981; Langley et al., 1984; but see Mugnaini, 1986). Thecomposition of ependymacyte intermediate filaments isnot yet established.

Ependymacyte-to-ependymacyte gap junctions. Gapjunctions were found to be abundant between ependyma-cytes. In the small area of glial plasma membrane includedin Figure 1C (approximately 30 µm2), more than 20 gapjunctions were found (Fig. 1C and Figs. 2,3). Abundant gapjunctions also were seen in companion thin-section images(Figs. 4A,B–G). Thus, by measuring areas of plasmamembrane in freeze-fracture and thin-section images, weestimated that each ependymacyte had 50–100 homolo-gous gap junctions on its lateral margins and that it wascoupled to 6–8 of the surrounding ependymacytes, eachhaving 6–15 gap junctions per cell apposition.

It was not possible in all freeze-fracture replicas ofspinal cord to ascertain which regions of the ependymacontained stratified cuboidal vs. pseudostratified colum-nar cells (compare Fig. 1C, from the cervical spinal cord,with Fig. 4A, from the thoracic cord). Consequently, theplasma membranes between ependymal cells were exam-ined at all histological levels—from apical margins nearmicrovilli (Fig. 2B–D), to the lateral plasma membranesbetween cells in the apparent upper (Figs. 3A, 4B) andlower (subependymal) layers (Fig. 1C, ‘‘S’’; Fig. 3B), toappositions at the ependymacyte-astrocyte boundary (Figs.3D, 4D–J).

At the apical borders of the ependymal cells (Figs. 2D),small gap junctions (i.e., those with ,100 connexons) weremore prevalent than large gap junctions. The smallest gapjunctions, consisting of only a few, regularly spaced connex-ons, were at the apical border and were resolvable only athigh magnification (boxed area in Fig. 2C; as Fig. 2D; seeleft arrow). However, within 1–2 µm from the lumenal


Fig. 2. Defining features of ependymal cells. A: Stereoscopic imageof cilia on apical surface of ependymacytes from central canal of ratspinal cord. The base of each cilium has five to eight rows of P-faceintramembrane particles (IMPs, arrow) constituting the ‘‘ciliary neck-lace’’ that is characteristic of motile cilia. A few square arrays arepresent between cilia (arrowhead). B: Stereoscopic image of ependyma-cyte microvilli and a cross-fractured cilium (from box ‘‘2B’’ in Fig. 1C).In the apical cytoplasm is an invaginated (or cuplike) gap junction(lower right). (In thin sections, invaginated gap junctions appear

annular; Larsen, 1977.) The cross-fractured cilium reveals the ‘‘9 1 2’’arrangement of microtubules that is characteristic of motile cilia.C: Stereoscopic image of apical margins of two ependymal cells.Microvilli project into the lumen of the spinal canal. Arrows point tothree gap junctions in the plasma membrane P-face. The inscribedarea is shown at higher magnification at Figure 2D. D: Two small gapjunctions in the E-face image of the apical portion of the apposedependymacytes. The pits are in regular hexagonal array.


Fig. 3. Stereoscopic images illustrating the abundance of gapjunctions between ependymal cells. (See Fig. 1C for exact locations of3A,C.) A: Large gap junctions between apposed ependymal cells in the(apparent) upper layer. E-face images of gap junctions are indicated bythe upper arrows; larger gap junctions showing P- to E-face transi-tions (lower arrows) contain several hundred connexons. IF, intermedi-ate filaments. B: Two large gap junctions (arrows) between cells of the(apparent) upper and lower layers of the ependyma. Arrowheads,caveolae. C: Gap junction at margin between ependymacyte andastrocyte processes. Confirmation of the existence and abundance ofependymacyte-astrocyte gap junctions is shown in conventional thinsections, Figure 4. Arrows point to square arrays on the ependymacyte

P-face. D: Large gap junction from the lateral margins of apposedependymal cells. The gap junction IMPs (left edge) and pits arehexagonally packed in small domains (or ‘‘islands’’), which are sepa-rated by small aisles that are devoid of IMPs or pits. Each E-face pitcontains a 2-nm ‘‘peg’’ thought to represent the aqueous matrixextracted from the tubular IMPs (see Fig. 5). Square arrays (arrow-heads) are present on apical, lateral, and basal plasma membranes ofependymacytes. The E-face pits of square arrays contain 1-nm ‘‘pegs’’(arrowheads), whereas the gap junctions E-face pits contain 1.5- to2-nm pegs (arrows). (For comparison at higher magnification, see Figs.5C–E and 6F,H.)

Fig. 4. Conventional thin section image from thoracic region ofadult rat spinal cord showing the abundance of homologous gapjunctions between ependymacytes and of heterologous gap junctionsbetween ependymacytes and astrocytes. A: Low-magnification imageof entire thickness of ependymal layer. The lumen of the spinal canal isat top right. Lettered boxes correspond to higher magnification imagesbelow. B: Two (of many) gap junctions (arrows) between apposedependymacytes. C: Cilia (arrows) usually extend into the central canal(upper arrows) but occasionally extend into the neuropil (lower arrow).mv arrow, microvilli. D: Gap junction (arrow) between ependymal cell(dark cytoplasm) and process from protoplasmic astrocyte (clearcytoplasm). E: Gap junctions between ependymacyte (dark cell) andastrocyte (clear cytoplasm, upper arrow) and between astrocytes(lower arrow). F: Two gap junctions (arrows) on one astrocyte process,linking to two ependymacyte processes (dark cytoplasm). G: Heterolo-

gous ependymacyte-to-astrocyte gap junction (arrow). H: Gap junction(black arrow) between ependymacyte and (likely) fibrous astrocyte.GFAP, glial fibrillary acidic protein-containing intermediate fila-ments; arrowheads, microtubules. Note desmosome (white arrow andinset at upper right) between astrocyte and ependymacyte. Desmo-somes are discrete intercellular junctions having paired cytoplasmicdensities attached where the patches of plasma membrane abruptlyseparate to approximately 20 nm. I,J: Pseudostereoscopic image oftwo gap junctions (arrows) at TEM viewing angles of 230° and 158°(88° included angle). The extreme tilt angles were required to revealthat the near tangentially sectioned, apposed membranes were gapjunctions. Extreme image compression was corrected using the ‘‘dis-tort’’ function of Adobe Photoshop, thereby allowing the images to beviewed as stereoscopic pairs. The image distortion also resulted inartifactual widening of the gap junction in Figure 5J.

border, abundant large gap junctions (i.e., those containingseveral hundred to over a thousand connexons, Figs. 3,4)were found. Additional gap junctions were found invagi-nated within the apical and basal cytoplasm (Fig. 2B),confirming that gap junctions were present on the studlikeinvaginations seen in thin sections (Peters et al., 1991). Inlarger gap junctions (Figs. 3C,D), the connexons character-istically were arranged in distinctive domains (or ‘‘is-lands’’; Larsen, 1977) of hexagonally packed IMPs and/orpits, with each domain oriented at random to those inadjacent domains and separated from its neighboringdomains by 20- to 30-nm corridors of IMP-free plasmamembrane.

High-magnification images revealed that most gap junc-tion E-faces consisted of 7–8 nm E-face pits, each contain-ing a 2-nm-diameter central ‘‘peg’’ (Fig. 3D), whereas theP-face IMPs were resolved as 6- to 7-nm tubes, each with a1- to 1.5-nm central ‘‘dimple’’ or pore (see Fig. 5C–E,below). (The slight discrepancy in diameter of pegs anddimples is due in part to the thin coat of platinum, whichdeposits within and decreases the apparent diameter ofthe dimples and deposits on and increases the diameter ofthe pegs. The platinum-coating effect also is dependent onlocal shadowing angle; Rash and Yasumura, 1992.) Theability to resolve the 2-nm pegs within E-face pits and the1.5-nm dimples within P-face IMPs was useful in distin-guishing gap junctions from other arrays of IMPs and pits.

Criteria for distinguishing gap junctions from other arraysof IMPs are described in Rash et al. (1996).

Square arrays. Scattered square arrays of P-faceIMPs and E-face pits (Figs. 3C,D) were common, but notabundant, in the apical, lateral, and basal plasma mem-branes of ependymal cells, particularly near gap junctions(Landis and Reese, 1974b; Hatton and Ellisman, 1981).The component IMPs of square arrays consisted of 5-nmP-face IMPs and 5-nm E-face pits (Fig. 2C) arranged inregular square lattices having a periodicity of 6.5–7 nm.(For further details regarding square array ultrastructure,see Astrocytes: substructure of the square arrays, below.)

Heterologous ependymacyte-to-astrocyte gap junc-

tions. In freeze-fracture replicas and conventional thinsections of spinal cord, gap junctions were abundant on thebasal plasma membranes of ependymacytes (Fig. 3C).Because both ependymacytes and astrocytes had similarmembrane and cytoplasmic markers (i.e., square arrays,gap junctions, and intermediate filaments; see Table 1), itseldom was possible in freeze-fracture replicas to obtainpositive identification of the cell process to which the basalplasma membranes of ependymacytes were coupled. How-ever, in thin sections (Fig. 4), the second cell or cell processfrequently was identifiable. Thin-section images of theependyma-to-neuropil border in the gray matter of adultrat spinal cord (Fig. 4) revealed abundant gap junctionslinking ependymacytes with astrocytic processes (Fig.

TABLE 1. Affirmative Criteria and Negative Criteria for Distinguishing Between the Five Major Cell Types Presentin Freeze-Fracture Replicas of the CNS1

Diagnosticcriteria

Cell type

Ependyma Astrocytes Oligodendrocytes Neurons2

Vascular3

endotheliaSoma Dendrites Soma Dendrites Soma Dendrites Myelin Soma Dendrites4

Synapses$4 in contact 2 2 2 2 2 2 2 111 111 22AZ (each occurrence) 2 2 22 22 2 2 22 11 11 22Endo-exocytosis/synapse 2 2 2 2 2 2 2 1 1 25

PSDs (each occurrence) 2 2 2 2 2 2 2 11 11 22Cytoplasm

.5 µm6 2 2 22 222 1 2 222 11 111 222

.3 RER stacks (‘‘Nissl’’) 2 2 22 22 2 22 222 111 111 22$2 Golgi stacks 2 222 2 222 2 22 222 11 11 22,0.5-µm-thick sheet 22 22 22 2 22 22 1 22 22 111.10 Caveolae 1 2 2 2 2 2 2 2 2 1115

‘‘Clear’’ cytoplasm 22 22 22 11 22 22 22 222 22 2IF bundles 111 11 111 11 222 222 222 1 1 2

Plasma membraneSquare arrays 111 111 111 111 222 222 222 222 222 222.2 Cilia 111 22 222 22 222 222 222 222 222 222Gap junctions

Regular hexagonal array 11 ND 1 1 2 2 2 111 111 11Multiple domains 111 11 111 11 1 1 1 22 22 2Irregular array 22 22 1 1 11 11 11 22 22 22E-face necklaces 222 222 222 222 111 111 111 222 222 222

IMP-free patches 1 1 22 22 11 11 111 2 2 2‘‘Reciprocal’’ patches 2 2 22 22 111 111 11 2 2 2Tight junctions 27 27 2 2 111 1 111 2 2 111Grooves/Furrows 1 1 2 2 111 111 1 1 1 1‘‘Moth-eaten’’ PMs 22 22 22 22 111 11 111 22 22 222.15 Caveolae 2 2 2 2 2 2 2 2 2 1115

1To identify a cell or cell process, all structural features that are observed are tested against this ‘‘truth table’’ and a value from 111 to 222 is assigned for each marker. (If themarker is not discernable, a zero value is assigned.) The number of pluses and minuses are summed. A negative score signifies that the cell does not correspond to the testdesignation. A score from 0 to 31 signifies ‘‘undetermined’’ (or insufficient data); a score greater than 41 signifies a positive cell identification. Note: Because of similar properties,ependyma and astrocytes may have a similar score; therefore, histological location is used for final discrimination.2Axons not described because they are easily recognized within myelin; unmyelinated axons not yet characterized. However, unmyelinated axons usually have multiple synapticcontacts and, in cross section, contain neurofilaments and microtubules.3Vascular smooth muscle is not included in Table 1 because smooth muscle is relatively rarely encountered, and vascular smooth muscle is always within the perivascular wall oflarge arterioles.4Includes dendritic spines.5In vascular endothelia, prominent caveolae may be misidentified as endocytotic or exocytotic profiles.6Distance from nucleus to plasma membrane, if nucleus is present.7Tight junctions occur on ependymal tanycytes in rat median eminence (Hatton and Ellisman, 1982).AZ, active zones; IF, intermediate filament; IMP, intramembrane particle; ND, not determined; PM, plasma membrane; PSD, postsynaptic density, either clusters of IMPs or pits;RER, rough endoplasmic reticulum.


4D–I), just as occurs in brain white matter (Mugnaini,1986). (Many areas of obliquely sectioned membrane appo-sition between ependymacytes and astrocytes [Fig. 4H,I]required goniometric tilting to identify gap junctions.) Inthree representative thin sections examined by tilting androtating, 23 gap junctions were present in 110 µm ofependymacyte basal plasma membrane cut in cross section(Fig. 4D–I). On the basis of an estimated 0.1-µm sectionthickness (and an included area of approximately 11 µm2

of plasma membrane), we calculate that there are 1–2ependymacyte-to-astrocyte gap junctions per square mi-crometer of ependymacyte basal plasma membrane, or20–50 heterologous ependymacyte-to-astrocyte gap junc-tions per ependymacyte at the neuropil boundary. Thus,each ependymacyte was linked by 50–100 gap junctions toadjacent ependymacytes (i.e., homologous coupling), andon its basal plasma membrane, was coupled by 20–50 gapjunctions to 5–10 different astrocyte processes (i.e., mul-tiple heterologous coupling).

Tanycyte-like ‘‘ependymoglial’’ processes (Mugnaini,1986) also exhibited abundant gap junction couplings withastrocytes at the ependyma-to-neuropil boundary (Fig.4F). These darkly stained, filament-rich protoplasmic ex-tensions near the ependyma-to-neuropil boundary wereseen to couple with astrocytic processes, which wereidentified by their electron-lucent cytoplasms, and occasion-ally by their content of bundles of intermediate filaments.However, deeper in the neuropil of the spinal cord, identifi-cation of ependymoglial processes was precluded except inthe rare instance where the plane of section (or plane offracture) revealed continuity of the tanycyte process withthe ependymoglial soma (Fig. 4F). Filament-rich ependy-moglial processes were positively distinguished from oligo-dendrocyte processes, which had electron-dense cyto-plasms that were devoid of bundles of filaments, and infreeze-fracture replicas, lacked the square arrays thatcharacterize ependymal cells. Likewise, the smooth, un-branched ependymoglial processes were positively distin-guished from neuronal dendrites, which had numerousdendritic spines and synaptic contacts, and in freeze-fracture replicas, lacked square arrays in their plasmamembranes (Table 1).

Homologous and heterologous glial gap junctions

that resemble proposed neuronal-glial couplings. Inconventional thin-section images of the ependymacyte-astrocyte border, the irregular astrocyte processes wereidentified on the basis of the presence of bundles ofintermediate filaments (presumptive fibrous astrocytes;Fig. 4H, arrows) and/or unusually clear cytoplasm (pre-sumptive protoplasmic astrocytes; Fig. 4D–G; see Table 1).Occasionally, both protoplasmic and fibrous astrocytes hadregions containing microtubules (Fig. 4H, arrowhead) andrough endoplasmic reticulum, markers otherwise consid-ered to represent definitive markers for neurons (Parna-velas et al., 1983; Nadarajah et al., 1996). In addition,astrocyte processes were frequently linked to ependyma-cytes or to other astrocytes by desmosomes (Fig. 4H, whitearrow, and inset, upper right; also see Morales and Dun-can, 1975). When obliquely sectioned, astrocyte desmo-somes resembled the ‘‘asymmetric synapses’’ of proposedneuronal-glial couplings (Nadarajah et al., 1996).

Ultrastructural features of astrocytes

Astrocyte somata. The cell bodies of most astrocytesin both gray and white matter of rat brain and spinal cord

were small (8–10 µm in diameter) and contained smallovoid nuclei surrounded by thin rims of cytoplasm thatcontained sparse, nonstacked rough endoplasmic reticu-lum (Fig. 5A,B). However, the most definitive feature forastrocytes in freeze-fracture replicas (Table 1) was thepresence of abundant square arrays of P-face particles(Figs. 5C, 6C, 7B,C) and square arrays of E-face pits (Fig.5C, 6F, 7C) in virtually all astrocyte plasma membranes(Landis and Reese, 1974b; Hatton and Ellisman, 1981;Landis, 1981; Massa and Mugnaini, 1982). (Square arraysalso were present in ependymacytes [Fig. 2C,D, above],but they were much less abundant than in astrocytes.) Inaddition, the somatic and dendritic cytoplasms of bothprotoplasmic and fibrous astrocytes usually exhibited dis-tinctive bundles of 10-nm intermediate filaments (Figs.5B, 7D) that are composed of immunologically identifiedglial fibrillary acidic protein (De Vitri et al., 1981; Langleyet al., 1984). However, many smaller astrocytic fingerswere devoid of intermediate filaments (see below).

Astrocyte gap junctions. Gap junctions were abun-dant in the somatic (Fig. 6A,B) and dendritic plasmamembranes (Fig. 6C–E) of most astrocytes. (It should benoted, however, that Type 2 astrocytes are reported to bedeficient in gap junctions; Belliveau and Naus, 1994.) Gapjunctions also were abundant between astrocyte perivascu-lar end feet (Fig. 6C), between their distal protoplasmicfingers (see below), and on fingerlike processes that couplewith oligodendrocytes (see next section).

Two morphological types of gap junctions were presenton the somata and dendrites of astrocytes: 1) gap junctionsconsisting of small islands of hexagonally packed IMPs(Figs. 5B–E, 6C), which represent homologous astrocyte-to-astrocyte gap junctions (Massa and Mugnaini, 1982), and2) gap junctions consisting of irregular clusters of IMPs/pits (see Figs. 11C, 12C,D), which represent heterologousastrocyte-to-oligodendrocyte gap junctions (Massa andMugnaini, 1982; Mugnaini, 1986; for details, see below). Asin the replicas of gap junctions in ependymacytes, mostIMPs in most astrocyte gap junctions had a 1- to 1.5-nmcentral depression or dimple (Fig. 5C,E, arrows), whereaseach E-face pit had a corresponding 2-nm central peg (Fig.5C–E). Thus, in replicas made at 2180°C using less than 1nm of platinum, details as small as 1 nm were resolved inboth gap junctions and square arrays.

Astrocyte endfeet. Freeze-fracture replicas from deepwithin the neuropil of gray and white matter of adult ratbrain and spinal cord revealed astrocyte endfeet surround-ing capillaries and smaller blood vessels (Fig. 6A–C). Theastrocyte endfoot layer closest to the blood vessel (Fig.6B,C) was characterized by the presence of abundant,densely packed square arrays. In the area of endfeetoverlap, or where tubular or irregular astrocytic processescontacted the endfeet, the plasma membranes frequentlycontained fewer square arrays, but contained small gapjunctions (Fig. 6C) linking the apposed astrocyte processes(Landis and Reese, 1981; Hatton and Ellisman, 1981).

Square array disorganization where multiple astro-

cyte processes overlap. Astrocytic processes that serveas endfeet (Fig. 7A–C) or that participate in the pia-gliallimitans (Fig. 7D) were characterized by the abundance ofsquare arrays, as well as by the presence of densely packedbundles of GFAP-containing intermediate filaments intheir cytoplasms (Fig. 7D, arrowheads). Where the astro-cyte endfeet were present in multiple layers (Fig. 7A,B),only the layer adjacent to the blood vessel wall (or to the


Fig. 5. Stereoscopic images of features used to identify astrocytes.A: Astrocyte soma, with large gap junction to adjacent astrocyteprocess (boxed area). B: Homologous astrocyte-to-astrocyte gap junc-tion. IMPs and pits are in small domains, each in regular hexagonalarray, with occasional particle-free aisles between domains. Gapjunctions (small arrows) and square arrays (large arrow) are usually

in close proximity. C,D: Gap junction between astrocyte ‘‘fingers.’’Arrowheads, square arrays; arrows, tubular connexon IMPs on P-faces. The E-face pits have 2-nm ‘‘pegs’’ corresponding to the ‘‘dimples’’in the IMPs. E: High-magnification image of astrocyte-astrocyte gapjunctions, with confirmation of 2-nm ‘‘dimples’’ (arrows) in IMPs and2-nm ‘‘pegs’’ in E-face pits.

Fig. 6. Stereoscopic images of defining features in astrocyte end-feet: square arrays. A: Neuropil from dentate gyrus of an adult femalerat. An obliquely-fractured capillary is in the center. The inscribedarea is shown at higher magnification in succeeding figures. B: Marginsof two astrocyte end-feet surrounding the capillary. Densely-packedsquare arrays in membrane P-faces (C,E) and E-faces (F and Fig.7A–C) are definitive markers for astrocytes. C: Homologous gapjunction (small arrow) that links overlapping astrocyte end-feet hasregular hexagonal arrays of P-face IMPs and E-face pits. The largerarrow points to one of the many square arrays that characterize

astrocyte plasma membranes. D: Tight junction between overlappingcapillary endothelial cells. This component of the blood-brain barrierconsists of many interlocking tight junctions strands (arrowhead),forming a web-like network. E,G: High-magnification images ofsquare arrays in an astrocyte P-face. Each IMP has a central pore ordimple (arrow) and is linked to adjacent IMPs by 2-nm ‘‘bridges.’’ F,H:E-face image of square arrays. Each pit contains a 1- to 1.5-nm central‘‘peg’’ (arrows) and is linked to its neighboring pit by a 2-nm groove.E-face ‘‘pegs’’ and ‘‘grooves’’ are complementary to the P-face ‘‘dimples’’and ‘‘bridges.’’

Fig. 7. Stereoscopic images showing variability of square arrays inregions where foot processes overlap. A: Overlap region of twoastrocyte end-feet (white arrow). B: Square arrays are abundant onthe nonoverlapping region (to right of black arrows) but are relativelysparse in the region of overlap, where square arrays are replaced bydensely packed 5-nm pits or partially assembled arrays (white arrow).Note the unusually abundant E-face IMPs that characterize astrocyte

plasma membranes. C: High-magnification E-face image of denselypacked square arrays on an astrocyte end-foot. In addition to ‘‘pegs’’ ineach pit, small grooves link each pit (arrowheads). D: Flattenedastrocyte processes at the pia-glia limitans. Pial collagen fibers lieabove the square array-enriched outer glial membrane (arrow). Bundlesof intermediate filaments (GFAP, arrowheads) are present in thesefibrous astrocyte processes.


pia mater) was enriched in square arrays. In the moredistal endfoot layers, the structural subunits of the squarearrays were present, but they often were not assembledinto distinctive arrays (Fig. 7B). The transition fromsquare arrays to dispersed clusters of 6-nm IMPs occurredabruptly at the step where a second astrocyte process (Fig.7A, arrow) was interposed between the outermost endfootand the capillary epithelial cell (Fig. 7B, opposed blackarrows). A functional significance for the transition fromregular square array to dispersed array is presented inDiscussion.

Substructure of the square arrays. Within squarearrays, each P-face IMP was 4–5 nm in diameter andcontained a 1-nm central pore or dimple (Fig. 6E,G,arrows). Each IMP was linked to two of its neighboringIMPs by two 2 3 2-nm bridges oriented at right angles,thereby defining the perpendicular axes of each array (Fig.6E,G). In the complementary E-face images, each 5-nmE-face pit contained a 0.7- to 1-nm central peg (Figs. 6F,Hand Fig. 7C) and was linked to the neighboring pits by2-nm furrows (Figs. 6F,H and 7C, arrowheads). Moreover,the pegs within the square array E-face pits consistentlywere about half the diameters of the pegs in adjacent gapjunctions (1 vs. 2 nm; Figs. 3D, 5C; also see Figs. 6E–H,7C), suggesting that the two different peg diameters werereplicated with fidelity in platinum films that are made at2180°C and that are less than 1 nm thick. The differingpeg diameters may reflect different transport capabilitiesfor connexons and square array proteins. (A more detaileddescription of square array substructure will be providedin a subsequent report; Rash and Yasumura, in prepara-tion.)

Ultrastructural features of oligodendrocytes

Within the gray matter of spinal cord and brain, thesomata of oligodendrocytes often were present adjacent toneurons (Fig. 8A) or capillaries (Fig. 9). In most cases,their triangular shape (Peters et al., 1991) and few cytoplas-mic processes (Figs. 8, 9, 11) gave oligodendrocytes asuperficial resemblance to neurons. However, oligodendro-cytes lacked the abundant synaptic appositions and recog-nizable pre- and postsynaptic specializations (e.g., activezones, postsynaptic densities) that constitute traits foridentifying neurons (Gulley et al., 1978; Walrond andReese, 1985; Rash et al., 1996), and they lacked the squarearrays and GFAP filaments that characterize astrocytes(Massa and Mugnaini, 1982). Instead, as summarized inTable 1, oligodendrocytes were identified by the presenceof 1) small somata (5–15 µm; see Figs. 8–11); 2) small,eccentric nuclei that occasionally are furrowed (Figs. 8C,9A); and 3) a thin rim of cytoplasm (Fig. 8C) that wasfrequently expanded on one side to 5 µm thickness (Fig.9A).

In addition, the plasma membranes of oligodendrocyteswere characterized by the presence of 1) elongated IMPs(Massa and Mugnaini, 1982) on both P-and E-faces (Fig.10D, small arrowhead), and 2) abundant gap junctions intheir somatic and dendritic plasma membranes (Figs.8–11; also see Hatton and Ellisman, 1981; Massa andMugnaini, 1982; Massa and Mugnaini, 1985; Mugnaini,1986). As noted by Massa and Mugnaini (1982) andMugnaini (1986), the IMPs and pits in these gap junctionswere not in regular hexagonal array, but instead, wereirregularly distributed, with only limited areas displayingthe hexagonal packing (Fig. 12).

New criteria for identifying oligodendrocytes. Onthe basis of the studies cited above and on the systematicapplication of grid-mapped freeze fracture, we have identi-fied more than a dozen unambiguous examples each ofoligodendrocytes, neurons, and astrocytes. From thosearchetypal cells, we have identified five additional criteria(Table 1) that were highly correlated with positive identifi-cation of oligodendrocytes:

1. Numerous furrows and ridges in the somatic anddendritic plasma membranes (Figs. 8–11). (Furrowsand ridges marked the impressions of neuronal andglial dendrites and of myelinated and unmyelinatedaxons. Multiple furrows and ridges were not seen inastrocytes, but occasionally were observed in the plasmamembranes of neuronal dendrites; see below.).

2. ‘‘Moth-eaten’’ patches of plasma membrane, including1) where myelin abutted oligodendrocyte somata ordendrites (Figs. 8C, 10B,C); 2) where myelinated pro-cesses were in contact; and 3) within successive myelinlayers (Figs. 10B,C, 12A,B, 13B). (Moth-eaten patchesare regions where the fracture plane made multipleexcursions between closely apposed stacks of mem-branes of nearly identical biochemical composition andidentical resistance to fracture, thereby creating a lacypattern of perforated membrane faces. Where myelinabutted neurons or astrocytes, moth-eaten patches didnot extend to the astrocyte or neuronal plasma mem-branes.

3. A ‘‘necklace’’ of E-face IMPs surrounding the gap junc-tion E-face pits in oligodendrocyte somata, dendrites,and myelin (Figs. 10B,C, 12C,D). Similar E-face IMPnecklaces were not seen surrounding the gap junctionsof astrocytes, ependymacytes, or neurons. Thus, gapjunction E-face necklaces represent a definitive markerfor recognizing oligodendrocytes.

4. ‘‘Reciprocal patches’’ (Fig. 10C), consisting of mixedlarge- and small-diameter IMPs and pits on both P-andE-faces. In addition, the margins of the furrows andridges usually were delineated by bands of mixed IMPsand pits that resemble reciprocal patches (Fig. 10B;also see Fig. 8B). Reciprocal patches were distinguishedfrom gap junctions (Figs. 10D, 11C) by their lack ofhexagonal array on either membrane face, by theadmixture of small and large (5–15 nm) IMPs and pitson both E-faces (Fig. 10B) and P-faces (Fig. 10D), andby the lack of narrowing of the intercellular spacewithin the perimeter of reciprocal patches (Fig. 10D,large arrow). In contrast, gap junctions have 1) clustersof uniform-diameter (7–8 nm) IMPs on P-faces (Fig. 8B;Fig. 10D, arrow; Fig. 11C) and uniform-diameter (8nm), irregularly spaced pits on E-faces (Figs. 11C, 12D);2) narrowing of the intercellular space within gapjunctions (Fig. 3B; Fig. 10D, above and left of arrow;Fig. 11B,C; Fig. 12B); and 3) complementary 2-nm pegsin the E-face pits and 1.5- to 2-nm dimples in the P-faceIMPs.

5. IMP-free patches of plasma membrane (Figs. 10C,13A), particularly in myelin, where the clear patcheswere stacked in successive layers (Fig. 13A; designatedby ‘‘]’’). These particle-free ‘‘lenses’’ were present inmyelin from spinal cords and brains that had been fixedby perfusion or by immersion, using either glutaralde-hyde or formaldehyde, and fixed at 37° or 4°C. They arealso present in most published figures of oligodendro-cytes and myelin (Schmalbruch and Jahnsen, 1981;


MacVicar and Dudek, 1982; Dudek et al., 1983; Wax-man and Black, 1984; Matsumoto et al., 1988; Nadara-jah et al., 1996). Consequently, we do not consider them(solely) an artifact of fixation, but instead, to reflectunidentified aspects of membrane composition and/orapposition.

6. A single stack of rough endoplasmic reticulum (RER)similar to the more abundant stacks of Nissl substancethat characterize neurons (compare Fig. 9B with Fig.14A). The RER stacks in oligodendrocytes were similarin spacing (1⁄4–1⁄2 µm) to those in neurons, but theyseldom included more than six layers (Fig. 9B), whereas

Fig. 8. Stereoscopic images of markers for recognizing oligodendro-cytes. A: Pericapillary oligodendrocyte within gray matter of cervicalspinal cord (from Fig. 1B). Oligodendrocytes (‘‘cells with few den-drites’’) have small somata, few dendrite-like processes (4 arrows),furrows in their somatic and dendritic plasma membrane (markingthe imprints of myelinated and unmyelinated neuronal processes; seefurrow from cross-fractured myelinated fiber at top center of cell). Theinscribed area is shown at higher magnification as (B). B: Oligoden-drocyte plasma membranes are characterized by abundant gap junc-

tions, six of which are included in the , 3-µm2 area shown here.Oligodendrocyte gap junctions, which consist of irregular clusters of10-nm IMPs, are present in all areas of the plasma membrane exceptwithin grooves (asterisk) where dendrites and myelinated processesabut the plasma membrane. C: Typical oligodendrocyte with a smallsoma, very thin rim of cytoplasm, and a small ovoid or groovednucleus. Furrows marking imprints of myelin (from other oligodendro-cytes) never exhibited gap junctions. N, nucleus.


Nissl substance in neurons often consisted of multiplestacks of RER, many of which contain eight or morelayers (Fig. 14A). Thus, the presence of a single stack ofRER resembling Nissl substance was not an absolutemarker for designating a cell soma or dendrite asneuronal, nor did the absence of Nissl substance definea cell as nonneuronal.

7. Tight junctions between the outer layers of myelin atthe outer mesaxon (Fig. 13B) and between adjacentoligodendrocyte somata (Fig. 13C,D; for details, seebelow).

Gap junctions in oligodendrocytes. Based on iden-tification of oligodendrocytes by the criteria in Table 1, gap

junctions were found in all areas of plasma membranes ofoligodendrocytes (Figs. 8–13B), except between successivelayers of myelin (i.e., no ‘‘reflexive’’ gap junctions) or wheremyelin abutted oligodendrocytes (Figs. 8B, 10B,C), confirm-ing the observations of Massa and Mugnaini (1982) fromCNS white matter. As many as 40 gap junctions wereexposed in the plasma membranes of individual freeze-fractured oligodendrocyte somata (see Fig. 8B for a 2-µm2

area containing 6 gap junctions, and Fig 11B for a 3-µm2

area containing 6 gap junctions). Based on the number ofgap junctions found in the somatic plasma membranes ofmore than a dozen representative oligodendrocytes, thesoma and proximal dendrites of a typical oligodendrocytewere calculated to have more than 100 gap junctions (see

Fig. 9 Stereoscopic images of markers shared by oligodendrocytesand neurons. A: Oligodendrocyte abutting astrocyte end-feet thatsurrounds a capillary. Cap, capillary lumen. B: A stack of roughendoplasmic reticulum (RER) in this oligodendrocyte is indistinguish-able from Nissi substance found in neurons. Grooves in plasma

membrane (asterisk), abundant gap junctions (arrows), and ‘‘elon-gated’’ IMPs (see Fig. 10D) are consistent with identification of this cellas an oligodendrocyte, and the absence of synaptic contacts, activezones, and other neuronal markers is inconsistent with identificationas a neuron. G, Golgi.


Fig. 10. Stereoscopic images of plasma membrane markers inoligodendrocytes. A: Portion of dentate gyrus from adult rat hippocam-pus. Dentate granule cells (their nuclei marked by ‘‘Nu’’) abut oligoden-drocytes, which exhibit numerous ridges in their somatic plasmamembranes (upper portion of micrograph). At the edge of the plasmamembrane E-face, the ridges align with cross-fracture neuronalprocesses (center of right edge). B: At higher magnification, E-faceimage of two oligodendrocyte gap junctions (arrowheads) reveal thecharacteristic irregular clustering of pits and the presence of E-faceIMPs at margins of the glial and neuronal impressions. The asteriskmarks a ‘‘moth-eaten’’ patch of membrane at a site where a myelinated

process has indented the cell surface. C: Furrow in E-face of oligoden-drocyte, with myelinated neuron containing tight junction strands(arrow) between myelin layers. Note the two gap junctions at lower left(arrowheads) and the distinctive IMP ‘‘necklace’’ surrounding each.D: P-face image of a ‘‘reciprocal patch’’ and an adjacent gap junction.Gap junctions consist of uniform 10-nm-diameter P-face IMPs (smallarrow), whereas reciprocal patches consist of mixed 5- to 15-nmparticles and pits and ‘‘elongated’’ IMPs (small arrowhead) on both P-and E-faces. Large arrowhead, square array on E-face of coupledastrocyte. The large arrow points to the intercellular space, whichnarrows as it approaches the gap junction.

below), totaling more than 10,000 connexons. Many addi-tional gap junctions (several hundred per oligodendrocyte)were found on their cytoplasmic processes (Fig. 9B, upperarrow), their outermost layers of myelin (Fig. 12A,B), andon their paranodal loops of myelin at nodes of Ranvier (Fig.13A), thereby providing possible ultrastructural confirma-tion of LM immunohistochemical observations that Cx32is present along myelinated fibers and at nodes of Ranvier(Li et al., 1997). However, ascertaining the identity of cellsto which the oligodendrocytes were coupled required fur-ther analysis.

Homologous vs. heterologous gap junctions in oligo-

dendrocytes. Occasionally, both cells contributing to anoligodendroglial gap junction could be identified. Of 48examples of oligodendrocyte gap junctions where the at-tached cell or cell process was identifiable, all (100%) wereheterologous couplings with astrocytes (Table 2), as con-firmed by the presence of square arrays in the plasmamembrane of the coupled cell or cell process (Fig. 11C),and/or by the presence of bundles of intermediate fila-ments (presumably GFAP). (These observations from spi-nal cord and brain gray matter are similar to those from

Fig. 11. Stereoscopic images confirming the presence of heterolo-gous oligodendrocyte-to-astrocyte gap junctions. A: Small, groovedoligodendrocyte with abundant gap junctions. B: Higher magnifica-tion image containing portions of five gap junctions and one ‘‘reciprocalpatch.’’ C: At high magnification, the gap junction is confirmed to beheterologous. The E-face of the attached plasma membrane contains

two square arrays (1 indicated by an arrowhead), characteristic ofastrocytes. Typically, heterologous oligodendrocyte-astrocyte gap junc-tions have connexons in irregular clusters rather than in regularhexagonal arrays, as are found in homologous gap junctions betweenastrocytes, ependymacytes, or neurons.


Fig. 12. Stereoscopic images of gap junctions in myelin and inoligodendrocyte processes. A: Obliquely fractured myelinated nerve.Small gap junctions (arrow) are frequent on the outermost layer ofmyelin, particularly at the margin of the outer mesaxon. B: Smallheterologous gap junction between outer layer of myelin and small,finger-like astrocyte process. Note the square array (arrow) in theastrocyte E-face and the ‘‘moth-eaten’’ area of myelin (asterisk). The

inset shows the square array and gap junction at higher magnifica-tion. C,D: Five of seven E-face gap junctions (arrows) in a single patchof oligodendrocyte plasma membrane. Each gap junction is sharedwith a tubular or finger-like process of a ‘‘protoplasmic’’ astrocyte,identified by the presence of square arrays in both E- and P-faces(arrowheads), clear cytoplasm (asterisk), abundant P-face IMPs, andpresence of characteristic E-face tubular IMPs (D, small arrowhead).

Fig. 13. Stereoscopic images of gap junctions and tight junctionsassociated with nodes of Ranvier, myelin, and apposed oligodendro-cytes. A: Paranodal region of myelinated axon, with oligodendrocytegap junction on the outer loop of myelin (arrow at upper right) and onthe outer layer of myelin in the internodal region (arrow at upper left).Note the impressions of the paranodal loops (asterisk) in the axonplasma membrane. ‘‘]’’ indicates a lenslike stack of IMP-free myelin.The arrowhead points to a homologous astrocyte-to-astrocyte gapjunction. In higher magnification images (not shown), the oligodendro-cyte plasma membrane was identified based on the presence of

IMP-free patches, distribution of IMPs and pits, and absence ofastrocyte markers. B: Oblique fracture of a myelinated axon, revealingparallel tight junction strands (arrow) between the outermost andsecond layer of myelin. The tight junction strands do not form weblikecross connections. C: Site of contact between two oligodendrocytes.The inscribed area is shown at higher magnification in (D). D: Thebroad area of tight junction contact (zonula occludens) consists of morethan 30 partially interlocking strands (arrow). (D) is tilted approxi-mately 30° from (C).

Fig. 14. Stereoscopic images of markers used to identify neurons.A: Cross-fractured 5-µm-diameter proximal dendrite in dentate gyrusof hippocampus. Approximately 16 synaptic contacts surround thedendrite. Ni, Nissl; sv, synaptic vesicles; arrowhead, exocytosis-endocytosis at active zones. B: Longitudinal fracture of neuronaldendrite in hippocampus, which has a few grooves resembling those in

oligodendrocytes. However, cross-fractured and surface-fractured den-dritic spines (arrows) are found only in neuronal dendrites.C,D: ‘‘Anastomosing’’ gap junctions found between hippocampal neu-rons. SV, synaptic vesicles; arrows mark margins of gap junctions in(C). Arrow in (D) points to ‘‘pegs’’ in gap junction E-face pit.

CNS white matter; Massa and Mugnaini, 1982.) In addi-tion, gap junctions were found linking oligodendrocyteswith tubular astrocyte fingers, which were identified bythe presence of square arrays in their P- or E-faces (Fig.12B, arrow; Fig. 12B, inset; and Fig. 12C,D, arrowheads),by the presence of a disproportionately high number ofscattered 10-nm tubular IMPs in E-faces (Fig. 12D, smallarrowhead), by dense accumulations of 6- to 8-nm IMPs(some representing unassembled components of squarearrays) in their P-faces (Fig. 12C, arrowheads), and by thepresence of clear cytoplasms that were devoid of filamentsor cytoplasmic organelles (Figs. 4D–G, 12C,D; see Table 1).Although the presence of square arrays is a definingfeature for astrocytes (and ependymacyte-tanycyte pro-cesses), square arrays were not always present in thesmaller astrocytic processes, or they were obscured by ahigh local shadowing angle (Fig. 10D, large arrowhead;Fig. 12B, arrow, and 12B, inset). Thus, the second cellcontributing to an oligodendrocyte gap junction frequentlywas not identifiable.

Of 48 oligodendrocyte gap junctions in which both cellscould be identified, all (100%) were oligodendrocyte toastrocyte. Heterologous oligodendrocyte-to-astrocyte cou-plings were found on oligodendrocyte somata (Fig. 11C),their cytoplasmic processes (not shown), and the outer-most layer of internodal myelin (Fig. 12B). In contrast, noexamples (0%) of homologous, autologous, or reflexiveoligodendrocyte-to-oligodendrocyte couplings were found,nor did we find any (0%) heterologous oligodendrocyte-to-neuronal couplings. As evidence that most or all gapjunctions in oligodendrocytes are heterologous, we wereable to identify as many as seven confirmed astrocyte-to-oligodendrocyte gap junctions within a small area ofplasma membrane in a single oligodendrocyte (Fig. 12C,D).Constraints of photographic reproduction and image mag-nification allowed only five of the seven gap junctions to beincluded in the stereoscopic image.) Thus, we confirm thatthe predominant or exclusive form of coupling of oligoden-drocytes in vivo in both white matter (Mugnaini, 1986) andgray matter (this report) is heterologous (i.e., with astro-cytes, only).

Tight junctions (zonulae occludentes). As in CNSwhite matter (Massa and Mugnaini, 1982), oligodendro-cytes in CNS gray matter exhibited two distinctive types oftight junctions. Autologous tight junctions were presentbetween the outer tongue of myelin and the underlyingfirst layer of compact myelin. The tight junctions in myelinconsisted of 3–15 IMP strands arranged in parallel, nonin-terconnected (or seldom-interconnected) rows (Fig. 13B).In CNS white matter, oligodendrocyte somata also oc-curred in chains of cells between axon bundles. Homolo-gous tight junctions frequently occurred between the adja-cent oligodendrocyte somata, where the occluding zonesconsisted of 10–30 or more interconnected strands linkingthe apposed plasma membranes (Fig. 13C,D). Thus, thetight junctions in oligodendrocytes differed from those innearby vascular endothelia (Table 1), which consisted ofonly a few interconnecting, weblike or ladderlike strands(Fig. 6C). Because few other cell types within the gray orwhite matter regions of the CNS have tight junctions(Brightman and Reese, 1969; Hatton and Ellisman, 1981;Peters et al., 1991), tight junctions represent an additionalstrong affirmative criterion for distinguishing oligodendro-cytes from other glia and from neurons (Massa andMugnaini, 1982; Massa and Mugnaini, 1985; Mugnaini,1986). (Cells of the choroid plexus and median eminencealso have tight junctions [Brightman and Reese, 1969;Hatton and Ellisman, 1982; Jarvis and Andrew, 1988], butthese cells are not within the neuropil or within whitematter of the CNS.)

Ultrastructural features of neurons

To develop unambiguous criteria for identifying neu-rons, we initially examined only somata and dendritesthat exhibited multiple (i.e., greater than 4) synapticcontacts, the sine qua non for identifying neurons. Fromthose prototypical neurons and neuronal processes (Fig.14A,B), we identified or confirmed eight additional mark-ers that were highly correlated with neurons (Table 1): 1)multiple synaptic contacts. Structures were identified assynaptic terminals if they contained clusters of more than25 nearly identical synaptic vesicles (Fig. 14A). To identifyprocesses with less than 25 vesicles as ‘‘synaptic’’ contacts,several additional markers were required. 2) ‘‘Active zones,’’which have distinctive presynaptic specializations associ-ated with transmitter release (Gulley et al., 1978; Gulleyand Reese, 1981; Walrond and Reese, 1985). Frequently,active zones had multiple examples of ongoing exocytosisand/or endocytosis (Fig. 14A, arrowhead). 3) Postsynapticdensities (Fig. 5C), usually located opposite active zones.Postsynaptic densities consist of clusters of IMPs or pits oneither P- or E-faces (Landis and Reese, 1974a; Landis etal., 1974; Landis and Reese, 1977; Gulley et al., 1978;Walrond and Reese, 1985).

In their cytoplasms, neurons were characterized by thepresence of: 1) Nissl substance, defined as multiple stacksof rough endoplasmic reticulum consisting of three or moreparallel, widely spaced (i.e., 1⁄4–1⁄2 µm) closed membranesacs (Fig. 14A), and 2) multiple Golgi membrane stacks(not shown) and their associated Golgi vesicles. Clusters ofGolgi vesicles, which are present in all neurons and glia,may be misidentified as synaptic vesicles.

In addition, neurons typically had: 1) one or more largedendrites (2–10 µm in diameter) either projecting from thesoma or free in the neuropil (Fig. 14A). 2) In surfacefracture, dendrites usually had many cross-fractured orsurface-fractured, small (greater than 0.3 µm) dendritic

TABLE 2. Identities of the Two Cell Types Forming Each Gap Junctionand Number of Gap Junctions Between Identified Cell Pairs1

Cell type Neuron AstrocyteOligo-

dendrocyte Ependymacyte Total

Neuron 972 97Astrocyte 0 2003 200Oligodendrocyte 0 484 0 48Ependymacyte 0 225 0 60 82

1Intersections of rows and columns contain the number of gap junctions between eachcell pairing. The percentage of cell pairs with gap junctions are as follows:

Astrocyte-to-Astrocyte 47%Astrocyte-to-Oligodendrocyte 11%4

Astrocyte-to-Ependymacyte 5%Astrocyte-to-Neuron 0%Neuron-to-Neuron 23%2

Neuron-to-Oligodendrocyte 0%Neuron-to-Ependymacyte 0%Ependymacyte-to-Ependymacyte 14%Ependymacyte-to-Oligodendrocyte 0%Oligodendrocyte-to-Oligodendrocyte 0%

2Disproportionately high because of selective search strategy.3Conservative estimate of the number of confirmed astrocyte-to-astrocyte gap junctionsobserved.4Disproportionately low because most patches of astrocytes were too small for positiveidentification.5Includes data from thin sections.


spines (Fig. 14B, arrows). Multiple spinous processes werenot present on the smooth cytoplasmic processes of oligo-dendrocytes (Figs. 8A, 9A, 11A).

Finally, additional criteria for identifying a cell as aneuron included: 1) the presence of a large-diameter soma(greater than 20 µm). However, it should be noted that inmany regions of the CNS, such as the dentate gyrus of thehippocampus (Fig. 10A), neurons may be no larger than8–10 µm in diameter. 2) Large, smooth, hemisphericalnucleus (greater than 8 µm).

Based on these criteria (summarized in Table 1), neuro-nal somata, dendrites, axons, and synaptic terminals, wereidentified in Laminae I–IX of the spinal cord (Fig. 15); in CA1,CA3, and the dentate gyrus of the hippocampus (Fig. 10A);and in the suprachiasmatic nucleus, supraoptic nucleus, andcerebellum (not shown). Neurons in those areas of the CNShave such diverse functions and ultrastructural propertiesthat a comprehensive description was beyond the scope of thisreport. Nevertheless, in most instances, neurons weredistinguishable from glia based on comparison and summa-tion of multiple affirmative criteria and negative criteria,as summarized in Table 1.

Gap junctions. Gap junctions are extremely rare infreeze-fracture replicas and conventional TEM images ofmost neurons (Sotelo and Korn, 1978). In the hippocam-pus, for example, unusual anastomosing gap junctionswere present between hippocampal neurons (Fig. 14C,D),

but the limited area of plasma membrane associated withthe few hippocampal gap junctions found to date (total 5 5)did not permit determination of whether the neuron-to-neuron synapses in hippocampus were predominantlyelectrical or ‘‘mixed.’’ (A detailed study of gap junctions inthe hippocampus is in progress.)

In contrast to neurons of the hippocampus, neurons ofthe spinal cord were relatively enriched in gap junctions(Fig. 15). In previous reports (Rash et al., 1995, 1996), wedescribed an average of several hundred ‘‘mixed synapses’’(combined chemical and electrical synapses) per neuron inlumbosacral, thoracic, and cervical regions of the spinalcord (Rash et al., 1995, 1996). Of the 97 neuronal gapjunctions found to date in spinal cord (previous andpresent reports), all (100%) represented clusters of homolo-gous neuron-to-neuron gap junctions. None (0%) repre-sented heterologous couplings to any type of glial cell.Moreover, the gap junctions in spinal cord neurons wereunusually small (median 5 47 connexons), thereby show-ing that even the smallest gap junctions (6–9 connexons)could be found in grid-mapped freeze-fracture replicas.

Of more than a thousand gap junctions examined in thisstudy (Table 2), approximately 350 contained sufficientareas in the apposed E- and P-faces to permit identifica-tion of both cells. None of those (0%) were neuronal-to-glialgap junctions. Instead, approximately 47% were astrocyte-to-astrocyte couplings (Figs. 6, 7); 11% were oligodendro-

Fig. 15. Stereoscopic images of gap junctions from ‘‘mixed’’ (chemi-cal and electrical) synapses in adult rat spinal cord. A: Gap junction(arrow) adjacent to sites of transmitter release in mixed synapse fromthe cervical enlargement of rat spinal cord. IMPs and pits are in

regular hexagonal array. SV, synaptic vesicles. B: High-magnificationimage of gap junction from lumbosacral region of the spinal cord. At1-nm resolution, the 1.5- to 2-nm ‘‘dimples’’ (arrow) are resolved inmost P-face IMPs.


cyte-to-astrocyte couplings (Figs. 11C, 12B,D), 5% wereastrocyte-to-ependymacyte couplings, 14% were ependyma-cyte-to-ependymacyte couplings, and 23% were neuron-to-neuron gap junctions. (The last number is disproportion-ately high because: 1) we directed our early searchesprimarily to the analysis of neuronal plasma membranes(Rash et al., 1995, 1996) and 2) neuronal gap junctions inspinal cord occurred in clusters. Conversely, the estimateof oligodendrocyte-to-astrocyte coupling may be low be-cause most of the gap junctions on oligodendrocyte E- andP-faces were not assignable because the geometry ofcoupling (i.e., large oligodendrocyte to a small astrocyte finger)minimizes the chance of finding a square array in the smallarea of astrocyte plasma membrane (Figs. 10D, 12B).

Cx32 and Cx43 immunostaining along neuronal

plasma membranes. To investigate the relative proxim-ity of glial gap junctions to neurons, as well as to investi-gate the possibility that neurons may be coupled to glia(Nadarajah et al., 1996), we immunostained formaldehyde-fixed spinal cord gray matter by using antibodies to Cx43(the primary astrocyte connexin; Dermietzel and Spray,1993). Punctate immunofluorescence consistently wasfound surrounding neuron cell bodies (Fig. 16), similar tothat shown by Nadarajah et al. (1996). In our companionfreeze-fracture images of neurons in spinal cord and brain,we also found numerous gap junctions along the neuronalperiphery, but rather than neuronal-to-glial gap junctions,the gap junctions were homologous couplings between twovery small diameter (1⁄4–1⁄2 µm), fingerlike processes ofastrocytes (Fig. 17A,B). Because the smaller astrocyteprocesses are below the limits of resolution of light micros-

copy (0.2 vs. greater than 0.4 µm for immunofluorescenceusing red light), and thus, would not be distinguishablefrom the adjacent neuronal somata, they likely provide thesource of the observed intense immunocytochemical label-ing seen by light microscopy. Therefore, our evidence fromfreeze-fracture images of similar regions in normal ratbrain and spinal cord, as well as from quantitative analy-sis of freeze-fracture replicas from several areas of brainand spinal cord revealed that at least some neurons werelinked by gap junctions with other neurons, but no imageswere found that were consistent with the hypothesis thatneurons are linked by gap junctions to any type of glial cell.

DISCUSSION

In the CNS of adult mammals, neurons and theirprocesses constitute only approximately 10% of the cellsand up to 30% of the tissue volume. In contrast, macroglialcells3 and their elaborate cytoplasmic processes are thepredominant structural components, constituting 80–90%of the cells by number and, depending on the area of the

3Within the glia of nondiseased brain and spinal cord, the macrogliapredominate. However, the remaining wedge-shaped and hemisphericalmicroglia (‘‘triangular’’ and ‘‘round’’ cells in TEM images) constitute from 4to 16% of the cells within many areas of the CNS (Mori and Leblond, 1969;Peters et al., 1991). The lack of identified freeze-fracture markers formicroglia continues to confound freeze-fracture analyses of CNS tissues(Mugnaini, 1986). However, because most microglia are mobile macro-phages of mesodermal origin, they do not make or maintain gap junctions(Mugnaini, 1986; Peters et al., 1991), and thus, they are not consideredfurther in this analysis of homologous vs. heterologous coupling among glia.

Fig. 16. Light microscope immunocytochemical staining of sectionfrom lumbosacral region of rat spinal cord. Mouse anti-connexin 43antibodies were counter-labeled with goat anti-mouse antibodiesconjugated with Texas Red. Punctate immunofluorescence (smallarrow) delineates the edges of two neurons (dark oval areas marked byasterisk). See Figure 17 for freeze-fracture images from a similar area.Large arrow points to astrocyte soma.

Fig. 17. Stereoscopic images of homologous astrocyte gap junctionsat margin of neuron. A: Edge of cross-fractured neuron soma aftershallow etching. A small portion of the neuronal soma is at the upperleft, a synaptic terminal is at lower left (SV, synaptic vesicles), andmyelinated process are at lower right. The area included in (A) isapproximately the same as the area covered by the small arrowhead inFigure 16. B: At higher magnification, two astrocyte fingers are seen tocontact at a gap junction (arrow, square array). The astrocyte pro-cesses are 0.2 µm in diameter and thus are below the limits ofresolution of the light microscope.


CNS, from 30 to 90% by volume (Kandel et al., 1992). Cellsof the circulatory system (capillaries, veins, arteries, andtheir surrounding vascular smooth muscle) correspond tothe remaining 10–20% of the cells and 5–10% of the tissuevolume. Thus, despite their disproportionate representa-tion, the functions of the macroglial cells in adult mamma-lian CNS are not well understood.

In this study, we have used grid-mapped freeze fractureto reinvestigate the ‘‘glial syncytium’’ hypothesis of Massaand Mugnaini (Massa and Mugnaini, 1982; Mugnaini,1986) and have extended that analysis of glial coupling toinclude selected gray matter regions of the brain andspinal cord. In addition, we have examined the pattern ofdistribution of square arrays (putative water channels, seebelow) with respect to the distribution of gap junctions inthe astrocyte ‘‘intermediaries’’ and in the ependymal layer.By analyzing unambiguous examples of ependymacytes,astrocytes, oligodendrocytes, and of several classes ofneurons in selected regions of brain and spinal cord, wewere able to identify or expand 24 criteria (approximately6–8 affirmative criteria and 16–18 negative criteria percell type, Table 1) for identifying and distinguishing evensmall patches of plasma membrane from the somata anddendrites of neurons, ependymacytes, astrocytes, and oligo-dendrocytes.

Using those images from selected regions of brain andspinal cord gray matter, we have shown that oligodendro-cyte somata, their cytoplasmic processes, their outermostlayer of myelin, and the outermost paranodal loop ofmyelin at nodes of Ranvier contain abundant gap junc-tions. However, even though we examined more than athousand gap junctions, we could find no evidence forhomologous or autologous gap junctions within the oligo-dendrocyte populations in gray or white matter. Despitediligent searches, we found no gap junctions betweensuccessive myelin layers, between paranodal loops ofmyelin, or between outer myelin layers and adjacentoligodendrocyte somata. Nor did we find gap junctionsbetween oligodendrocyte somata and dendrites. Instead,in all external plasma membranes, oligodendrocyte gapjunctions were heterologous, i.e., they coupled oligodendro-cytes exclusively to astrocyte processes, primarily at thetips of astrocyte fingers. Thus, our data confirm Massa andMugnaini’s suggestion that the physiological and dyecoupling of oligodendrocyte to oligodendrocyte detected byothers (Kettenmann and Ransom, 1988; Butt and Ransom,1989; Robinson et al., 1993; Konietzka and Muller, 1994)likely occurs primarily or exclusively through astrocyteintermediaries and not directly via homologous coupling.(Note: Since his 1986 report suggesting that oligodendro-cytes do not form homologous couplings, Mugnaini [unpub-lished observation] has found several examples of appar-ent oligodendrocyte-to-oligodendrocyte gap junctions.)

On the basis of the criteria in Table 1, we also found thatependymacytes (and related ependymoglia and tanycytes)were ultrastructurally coupled via abundant homologousgap junctions to other ependymacytes and were coupled byabundant heterologous gap junctions to many fingerlikeprocesses from subjacent astrocytes. Our finding of abun-dant heterologous gap junctions between ependymacytesand astrocytes and between ependymoglial processes andastrocyte processes deeper in the neuropil confirms previ-ous suggestions that ependymacytes are strongly coupledto the astrocyte syncytium, not only in white matter(Mugnaini, 1986) but also in gray matter regions of theCNS.

It is well established that astrocytes in gray matter ofthe spinal cord and brain share abundant homologous gapjunctions with other astrocytes (Brightman and Reese,1969; Konietzka and Muller, 1994; Sontheimer, 1995;Ransom, 1995). The branching fingerlike processes from asingle astrocyte do not converge on an individual oligoden-drocyte, but instead, branch widely to couple with manyoligodendrocytes (Robinson et al., 1993) and many otherastrocytes. In addition, most astrocytes have branchingprocesses that extend from the ependymal layer to the glialimitans (Butt and Ransom, 1989). Thus, we conclude thatthe ‘‘functional syncytium’’ (Massa and Mugnaini, 1985;Mugnaini, 1986) extends throughout gray and white mat-ter—from the ependymal layer, across the gray and whitematter, to the glia limitans, and from the sheathes ofmyelinated and unmyelinated axons to the astrocyte end-feet surrounding vascular endothelia. Moreover, injectionof neurobiotin into astrocytes for periods longer than 10minutes revealed that ‘‘there is virtually no border’’ toastrocyte coupling between anatomical subdivisions of thehippocampus (Konietzka and Muller, 1994). All of thesedata suggest that the glial syncytium is truly ‘‘panglial.’’

With such abundant gap junctions between all classes ofmacroglia, it may be asked, ‘‘Why do most glial cells otherthan astrocytes appear to be only weakly dye coupled?’’Whether the apparent low level of oligodendrocyte-to-astrocyte coupling (Kettenmann and Ransom, 1988; Buttand Ransom, 1989; Ransom, 1995; Sontheimer, 1995)reflects dilution of dye in the vast astrocyte syncytium(Konietzka and Muller, 1994), unidirectional dye transport(Robinson et al., 1993), or restriction of intercellularcommunication to the free flow of water, ions, and verysmall molecules (i.e., molecules smaller than the dyesconventionally employed), our data document the presenceof abundant heterologous gap junctions interlinking allclasses of macroglial cells in the adult rat CNS.

Gap junctions do not link neurons to glia. Our datafrom grid-mapped freeze fracture directly contradict datafrom conventional thin-section EM, which have been inter-preted as suggesting that a substantial fraction (i.e.,17.9%) of all gap junctions in the cerebral cortex graymatter of adult rats represent neuronal-to-glial couplings(Nadarajah et al., 1996). Although we searched extensivelyfor gap junctions between neurons and any type of glialcell, we could find no evidence for neuronal-to-glial gapjunctions in any of several hundred cell pairs that weobserved were linked by gap junctions. Instead, we ob-tained anatomical and statistical evidence that in normaladult rats, gap junctions link neurons solely to otherneurons (97/97) and not to oligodendrocytes (0/48) or toastrocytes (0/200). The discrepancy (0% vs. 18% neuron-to-glia coupling) is much too large to be explained by chancelack of observation of a common ultrastructural feature.We consider it unlikely that the grid-mapped freeze-fracture process selectively exposes all types of heterolo-gous couplings except the abundant neuronal-to-glial cou-plings that were reported by others (Morales and Duncan,1975; Nadarajah et al., 1996). Likewise, we consider itunlikely that abundant large-diameter neuronal-to-glialcouplings would be visible in thin sections but would not berecognizable in freeze-fracture replicas. We note that gapjunctions smaller than 0.07 µm2 (or approximately 64connexons) were readily discerned in our freeze-fracturereplicas, but gap junctions of that size would be difficult todetect in conventional thin sections because their diam-eters would be less than the thickness of a conventional


thin section, and as a consequence, they would appear asindistinct blurs representing the superposition of contigu-ous regions of closely spaced and more widely spacedmembrane profiles. Because we routinely identified gapjunctions with as few as 6–12 IMPs or pits in glia (Fig. 2D)and neurons (Rash et al., 1996), we consider it unlikelythat we were unable to recognize the much larger neuronal-to-glial gap junctions claimed by others to be abundant inconventional thin sections (Nadarajah et al., 1996). Thus,we conclude that neuronal-to-glial couplings are extremelyrare or nonexistent (i.e., statistically >0%) and that neu-rons and glia employ separate and distinct gap junctioncoupling pathways.

Neuron-to-glia couplings likely misidentified in pre-

vious reports. In an attempt to reconcile the observa-tions of Morales and Duncan (1975) and Nadarajah et al.(1996) with that of Massa and Mugnaini (1982), Mugnaini(1986), and those of this report, we applied the affirmativecriteria in Table 1 to their published images. We concurwith all cell identifications provided in the freeze-fracturereports by Massa and Mugnaini (1982, 1985), Mugnaini(1986), Waxman and Black (1984), Landis and Reese(1977, 1981), Landis et al. (1983), and Hatton and Ellis-man (1981, 1982). On the basis of the data in Table 1,however, we conclude that many other published thin-section and freeze-fracture images purporting to show gapjunctions in the plasma membranes of neurons (includingsome images published by members of this group), were infact, either: 1) images of gap junctions on glial cells(primarily oligodendrocytes [Schmalbruch and Jahnsen,1981; Matsumoto et al., 1988; Dudek et al., 1983] or astrocytes[Andrew et al., 1981; Matsumoto et al., 1988]); 2) the cells werenot identifiable because the images contained insufficientmarkers (Fig. 9C in MacVicar and Dudek, 1982; Fig. 10 inNadarajah et al., 1996); or 3) the images of close membraneappositions and of IMP arrays were not reliably identified asgap junctions (Fig. 9C,D in MacVicar and Dudek, 1982;and Fig. 9D in Nadarajah et al., 1996). We attribute thesedifferences in cell identification, in part, to improvementsin freeze-fracture technology during the intervening 15–20years (Rash and Yasumura, 1992) and in part to the use ofgrid-mapped freeze fracture for cell identification andmapping (Rash et al., 1995).

Light microscope immunocytochemical localization

of connexins. Although we confirmed the presence ofimmunocytochemical staining of gap junction Cx43 alongthe plasma membranes of neurons (Fig. 16), our freeze-fracture replicas of similar regions (Fig. 17) revealed onlyhomologous gap junctions between astrocyte fingers, manyof which were below the limits of resolution of the lightmicroscope. Thus, our freeze-fracture images and lightmicroscopic immunocytochemical observations are consis-tent with our hypothesis that images from light micro-scope immunocytochemistry have been misinterpreted assupporting the proposed presence of heterologous gapjunctions between neurons and astrocytes. Similarly, weconclude that light microscope immunocytochemical visu-alization of Cx32 along oligodendrocyte plasma mem-branes and myelin sheaths (Li et al., 1997) does not implyhomologous oligodendrocyte-to-oligodendrocyte coupling.Rather, we suggest that each of those areas is especiallyenriched in heterologous astrocyte-to-oligodendrocyte cou-plings and further suggest that only thin-section or freeze-fracture immunocytochemistry has the resolution re-quired to ascribe labels to specific cells, their cytoplasmicprocesses, or, for that matter, to gap junctions.

Are the gap junctions between astrocyte and oligoden-

drocytes heterotypic or homotypic? The primary con-nexin isoform in oligodendrocytes is Cx32 (Dermietzel andSpray, 1993). However, oligodendrocytes form gap junc-tions primarily or exclusively with astrocytes, which con-tain Cx43 but not Cx32 (Dermietzel and Spray, 1993).Because the heterologous couplings represent essentiallyall of the gap junctions on oligodendrocytes, as well as asignificant fraction of the gap junctions on astrocytes, weconclude that heterologous astrocyte-to-oligodendrocytecouplings must be heterotypic. Possibilities include Cx43coupling with Cx32 (Duffy et al., 1997) or to an as yetundescribed connexin in oligodendrocytes.

Possible roles for square arrays in the panglial

syncytium. In this study, we confirmed that squarearrays were abundant near gap junctions in astrocytes andependymacytes (Hatton and Ellisman, 1981; Landis andReese, 1981; Hatton and Ellisman, 1982). Although theidentities of the ions and/or molecules permeating the glialgap junctions are not established, it is important to notethat antibodies made against the muscle square arrayprotein, CHIP28 (a member of the aquaporin family ofproteins; King and Agre, 1996), also yielded strong immu-nostaining of astrocyte endfeet (Verbavatz et al., 1994),where square arrays are the dominant membrane struc-tural component. Aquaporin IV (likely the same moleculeas CHIP28; King and Agre, 1996) has been involved inresorption of cerebrospinal fluid (reviewed in King andAgre, 1996). The presence of gap junctions in close proxim-ity to square arrays (presumably Aquaporin IV) in epen-dyma and astrocytes and the presence of an extensiveconduit system of cytoplasmic fingers appear to providethe three structural components required for cooperativetransport of water between the several glial compartmentsthat are involved in resorption of cerebrospinal fluid (Kingand Agre, 1996), in transport of K1 (Newman, 1986), andin ‘‘spatial buffering’’ of K1 (Orkand et al., 1966). Thus, wecall attention to the observation that the density of squarearrays is maximal in astrocyte endfeet immediately adja-cent to capillaries, but that the square arrays are dis-rupted (or the components are not assembled) in regionswhere a second endfoot process is interposed. At thistransition zone (where square array components are pre-sent but dispersed), water and ion transport betweenastrocyte endfoot and capillary would be reduced due tothe stacking of multiple diffusion barriers.

It is also noteworthy that astrocyte fingers are coupledto the outermost paranodal loop of myelin at nodes ofRanvier (Waxman and Black, 1984; and Fig. 12C, thisstudy), where potassium conductance during normal im-pulse propagation may occur directly into the paranodalloops of myelin (Wiley and Ellisman, 1980). The abundantgap junctions linking the glial syncytium may provide therequisite intercellular channels to facilitate concentration-dependent intercellular movement of ions and water at arate comparable with or greater than that found for dyemovements (Konietzka and Muller, 1994).

Unresolved questions. Although we assembled datato suggest that heterologous oligodendrocyte-to-astrocytegap junctions may consist of CX32 coupled to CX43, weemphasize that no freeze-fracture or thin-section electronmicroscopic immunocytochemical data are yet availableregarding the connexin types involved in the several typesof heterologous couplings observed between the threemajor classes of macroglial cells (but see Duffy et al., 1997).Nor are data available regarding changes in connexin


isoforms or in gap junction connectivity that may occurduring CNS development, during normal aging, followingneurological trauma, during spreading depression, or dur-ing progression of neurological disease, such as epilepsy.Finally, we note that only limited data are availableregarding the interaction of water channels and gapjunctions in producing osmotic and ionic homeostasis(Mulders et al., 1995; Zampighi et al., 1995). To beginassessing the roles of the several types of glial gapjunctions and of square arrays in health and disease, amore complete understanding of the biochemical identi-ties, functions, and anatomical distributions of glial gapjunctions and their associated square arrays is required.The combination of high-resolution, postreplication immu-nocytochemical labeling procedures (Fujimoto, 1995; alsosee Pinto da Silva et al., 1981; Dinchuk et al., 1987; Severs,1989; Rash et al., 1990) and grid-mapped freeze fracture(this report) may be of value in that effort.

Note added in proof: In support of this contention, we have recentlydemonstrated direct immunogold labeling of aquaporin-4 in square arraysof astrocytes and ependymacytes (Rash et al., 1997).

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