COMPARATIVE ELECTRON MICROSCOPY OF SYNAPSES IN THE … · 2005. 8. 19. · The white matter shows the usual compact mass of ascending and descending myelinated fibres. A bundle comparable

J. Cell Sci. i, 67-80 (1966) 67

Printed in Great Britain

COMPARATIVE ELECTRON MICROSCOPY OF

SYNAPSES IN THE VERTEBRATE SPINAL CORD

B. T. CHARLTON AND E. G. GRAYDepartment of Anatomy, University College London, Gower Street, London, W.C.i

SUMMARY

Synapses with a cleft with ' thickened' membranes and presynaptic vesicles and mito-chondria occur commonly throughout the grey matter of the spinal cord of goldfish, frog andvarious mammals studied. Such synapses are generally thought to have a chemical mode oftransmission. The absence or rare occurrence of presynaptic neurofilaments in fish and frogaccounts for the failure to detect boutons by silver methods, and there is no need to postulatemorphologically unspecialized synaptic contacts in the lower vertebrates as some light micro-scopists did. Both fish and frog show axo-somatic or axo-dendritic tight junctions, which couldbe sites of electrical synaptic transmission. No neuronal tight junctions have yet been seen inthe mammalian spinal cord. Axo-axo-dendritic synapses have been seen in the frog and mam-malian cord, but not so far in the fish. Such serial synapses may be responsible for presynapticinhibition. Neuroglia offish, frog and mammals have tight junctions at their apposed surfaces.These differ structurally from neuronal tight junctions. Neuroglia in fish cord have, in addition,desmosomes at their apposed surfaces.

INTRODUCTION

Nieuwenhuys (1964) has made a comprehensive review of the comparative anatomyof the spinal cord of protochordates and of the several groups, cyclostomes, fishes,amphibia, reptiles, birds and mammals, usually classed together as the vertebrates.Observations have been derived almost entirely from light microscopy and there isan urgent need for the use of the electron microscope (EM) in this field. EM observa-tions have been made on the cyclostome spinal cord by Bertolini (1964), on theteleost cord by Bennett, Aljure, Nakajima & Pappas (1963) and on the mammaliancord by Wyckoff & Young (1956), Luse (1956), Gray (1962a, b, 1963, 1964), Bodian(1964), Gray & Guillery (1961, 1963, 1965), Ralston (1965) and Bunge, Bunge &Peterson (1965).

The present paper is an effort to start to close the gap with a comparative EM studyof synaptic morphology of certain regions of the fish and frog spinal cord. Answersto the following questions are sought. Is it true that, in the cord of lower vertebrates,specialized synaptic contacts are rare (frog) or absent (fish) and neuronal interactiontakes place largely by undifferentiated contacts (see Nieuwenhuys, 1964)? How dothe junctions compare in morphological complexity and distribution with those ofmammals ? Do the junctions show well-defined synaptic clefts with associated mem-brane thickenings and presynaptic vesicles (features usually associated with a chemicalmode of transmission), or are neuro-neuronal tight junctions present which could beindicative of an electrical mode of transmission, as described, for example, by Robert-

5-2

68 B. T. Charlton and E. G. Gray

son, Bodenheimer & Stage (1963) and Stefanelli (1964) in the Mauthner cell of thefish brain? Are there axo-axonal synapses comparable to those of the mammaliancord, which are thought to be responsible for presynaptic inhibition (see Gray, 1962 a,1963; Eccles, 1964)?

METHODS

Observations were made on (a) the lumbosacral enlargement of the cord of maturefrogs (Rana temporaria) weighing 15 to 20 g, and (b) the cord of mature goldfish(Carassius auratus) weighing 8 to 12 g, between the levels of the 3rd and the 10thvertebrae.

Frog cord. The brain was destroyed with a fine needle and the vascular system perfusedwith Ringer's solution (2-4 °C) to wash out the blood so as to facilitate penetration of thefixative. The vertebral column containing the lumbosacral region was dissected outin a cold room at 4 °C, freed of investing musculature and cut away from the uppervertebral column and pelvic girdle. The vertebral column, still containing the cord,was then placed in a groove of comparable size made in a small block of Plasticine,which was then squeezed firmly round the preparation. In this way a firm coat ofPlasticine and vertebrae gave support, so that a series of cross-sections of the cordcould be cut with a razor blade. The whole was then placed in fixative and thesections of the cord freed with needles. These were then transfered to fresh fixative.

Fish cord. A goldfish was killed by decapitation and the required region of thevertebral column was cut free and the musculature cut or scraped away in the coldroom. The preparation was placed on a wax block and cut into a series of sections witha razor blade, which usually passed between the vertebrae. The pieces were placed infixative and the cord sections carefully removed from the vertebrae with fine needles.They were then transferred to fresh fixative.

In this way complete cross-sections were obtained of frog and fish cord so thatappropriate regions to be sectioned could be easily located by phase-contrast micro-scopy of thick sections from the Araldite block before sectioning for the EM.

The fixative was 1 % OsO4 dissolved in Ringer, buffered with veronal acetate atpH 7-4 and maintained at 4 °C. Fixation lasted for 3 h and was followed by rinsing in20% ethanol and dehydration in graded ethanol solutions. Staining was carried outwith 1 % phosphotungstic acid (PTA) in absolute ethanol and Araldite was used forembedding (see Gray, 1964). PTA is especially suitable for delineating the membranecomplexes of tight junctions and 'thickenings' bounding the synaptic cleft (seebelow).

For light microscopy Nissl, reduced silver and Golgi preparations were made andexamined in the usual way. Much information was obtained by phase-contrast micro-scopy of Araldite sections.

Mammalian cord. Observations on the lumbar region of the cat and rat spinal cordhave been published elsewhere (Gray, 1962a, b, 1963, 1964; Gray & Guillery, 1961,1963, 1965). The material was fixed with OsO4 and stained with PTA, as in the frogand fish material. Reference will be made to this work from time to time and these

Spinal cord synapses 69

papers should be consulted for illustrations and full details. Ralston's (1965) descrip-tion of the substantia gelatinosa and deeper laminae of the dorsal horn of the cat cordwill also be referred to for comparison.

OBSERVATIONS

Frog

General organization of lumbosacral cord. Only a few general remarks are made here,and the review of Nieuwenhuys (1964) should be referred to for details and references.The grey matter clearly shows dorsal and ventral horns (Fig. 1 a), and the ependymallayer of the central canal is ciliated. The numerous small neurons of the substantiagelatinosa form a well-defined apical zone and small neurons are also scattered through-out the intermediate grey zone and grey matter in general. The perikarya of the largemotor neurons supplying the hind limbs may reach 50 /i or more in diameter and lielaterally in the ventral horns. Their dendrites run medially, sometimes crossing themidline, and laterally, penetrating the white matter, often in parallel bundles. Thedendritic branches terminate in a submeningeal marginal plexus, which is up to200 /t thick. Axons also enter this peripheral zone from the small cells of the greymatter. The marginal zone has generally been considered a synaptic region becauseof this profuse intermingling of axons and dendrites, but boutons have not beendemonstrated with the usual reduced-silver methods. As mentioned above, fewboutons can be demonstrated throughout the grey matter in general (see Discussion).A second group of large motor cells supplying the trunk musculature lies mediallyto the leg ventral horn cells, in the more anterior regions of cord, but such motorcells are absent from the lumbosacral region. Cajal (1911) has described recurrentcollaterals of ventral horn cells in the toad (Bufo). It has not yet been fullyestablished whether incoming branches of dorsal root fibres make direct contactwith ventral horn cells. A few large neurons occur in the dorsal regions of the greymatter.

The white matter shows the usual compact mass of ascending and descendingmyelinated fibres. A bundle comparable to Lissauer's tract can be distinguished atthe apex of the dorsal horn near the entry of the dorsal root fibres. The giant myelinatedaxons of Mauthner are absent from adult frogs (anurans), but are present in the larvaeand in adult urodele amphibia, in bony fishes, and also in cyclostomes (Miiller's fibres),where, however, they are uncrossed and unmyelinated, as are all the fibres in thecyclostome nervous system (see Nieuwenhuys, 1964).

EM observations. The fine structure of the frog neuronal perikarya and dendriticramifications has not been studied in detail but the main organelles are directlycomparable to those of mammalian neurons (see Palay & Palade, 1955). Granularendoplasmic reticulum, free ribosomes in clusters and rosettes, Golgi apparatus,lipid droplets, mitochondria, lysosome-like bodies (see Gray, 1964), multivesicularbodies and neurofilaments can all be distinguished in the perikaryon. Curious whorledorientations of granular reticulum also occur (see Palay & Palade, 1955) and some-times surround a clear zone of cytoplasm containing bundles of neurofilaments. The

7o

Marginalplexus

B. T. Charlton and E. G. Gray

Dorsal horn

Central canal

Anterior commissure White matter

Marginalplexus

smmimDorsal horn

(substantia gelatinosa)

Mauthner axon White matter

(b)

Fig. i a, b. Diagrams based on Nissl and Golgi preparations, phase-contrast microscopyof Araldite sections, and electron microscopy, (a) Cross-section of lumbosacral regionof frog cord. Dotted rectangle shows, on one side, the region where neuronal tightjunctions occur, (b) Cross-section of goldfish cord between 3rd and ioth vertebrae.Only some of the large ventral horn neuron cell bodies have been included in thediagrams.


dendrites contain both neurofilaments and tubules; the latter predominate in thethinner distal branches. Some dendrites also contain glycogen granules.

Throughout the grey matter, including the marginal plexus (Fig. 1a) synapticcontacts can be recognized with all the morphological complexity found in synapsesof the mammalian cord. Contacts may be on cell bodies or dendrites or their spines.Both myelinated and unmyelinated axons contain tubules and/or neurofilaments.Transverse sections of myelinated sheaths sometimes show radial striations (seePeters, 1961). The bare areas of nodes of Ranvier sometimes reach 4 [i in length (seeHess & Young, 1952). It is not uncommon to see the terminal myelinated region of anaxon expanding directly into a vesicle-containing presynaptic bag in the plane ofsection (see below).

The neuroglial cells resemble those of mammals. The numerous astrocyte-like,highly branched cells often contain glycogen granules and bundles of glial fibrilsembedded in pale cytoplasm. Tight junctions between glial profiles are common (seebelow). Oligocyte-like glia resemble those of mammals in the dense granular natureof the cytoplasm, which forms a rather narrow zone around the nucleus. The nucleusis more irregular in the amphibian than in the mammalian oligocyte. Tubules mayoccur in both the cell body and processes of frog oligocytes, and the processes mayeasily be confused with dendrites.

Two axo-dendritic synapses from the lateral intermediate region of the frog cordare shown in Fig. 2. The presynaptic knobs (a and b) contain groups of mitochondriaand glycogen granules in the distal zone and aggregates of synaptic vesicles 300-600 Ain diameter, near the 'thickened' regions (t) of the apposed axo-dendritic membranes.The synaptic clefts are about 200 A wide. Granular vesicles (gv) are present in (a) andalso in a third presynaptic knob (c), which in this plane of section shows no membranethickenings at the apposition region with the dendrite. The granulated vesicles rangefrom 700 to 1300 A across. A complex, coated vesicle (cv) is seen in continuation withthe membrane of bag (a) (see Gray, 1961a, 19626,1964; Andres, 1964; Westrum, 1965).

The synaptic clefts and the associated dense material are directly comparable instructure to those of the mammalian cord. The cytoplasmic surface of the pre-synaptic membrane shows dense projections (dp) and marked postsynaptic thickenings(Fig. 2 and insets). In tangential sections the dense projections show clearly in thefrog, as in the mammal, a regular hexagonal spacing. The interpretation of thesestructures has been considered in detail elsewhere (see Gray, 1963, 1964; Gray &Guillery, 1965). The synaptic complex described above will be referred to as a cleftjunction.

Rarely, as in the presynaptic knob (a in Fig. 2; Fig. 3) bundles of neurofilaments(nf) can be seen with ring-shaped orientation. In Fig. 2 they surround a group ofmitochondria and in Fig. 3 they surround both mitochondria and synaptic vesicles.The filaments lie in clear zones of cytoplasm free from synaptic vesicles. It must beemphasized that most presynaptic knobs, however, contain no orientated bundles ofneurofilaments.

Axo-somatic synapses occur commonly on the large ventral horn cells (Fig. 1 a)and on smaller neurones scattered throughout the grey matter except in the marginal


plexus, where neuronal perikarya have not been observed. Fig. 5 shows such a contacton a small ventral horn neuron with a perikaryon approximately 15/i in diameter.Fig. 4 shows an axo-somatic contact with a subsynaptic sac (ss) with tightly apposedmembranes. Part of the cleft region is shown at higher magnification in Fig. 6. Thepresence of a subsynaptic sac (membranes x) is correlated with the absence of'thickening' material on the postsynaptic membrane (y). Such an arrangement isdirectly comparable to that seen at some axosomatic synapses in the mammaliancord (Gray, 19626).

Axo-axonal synapses. Using the criteria for the identification of a synapse (asabove) axo-axonal synapses have been identified in the ventral horn and intermediateregions of the frog cord (see Fig. 1 a) but they have not been seen so far in the sub-stantia gelatinosa and dorsal horn or in the marginal plexus. Such contacts aredirectly comparable to those described in the mammalian cord by Gray (1962 a,1963) and Ralston (1965).

An example from frog cord is shown in Fig. 7, where the terminal region of themyelin sheath can be seen related to a presynaptic knob (a). This bag makes a synapticcontact (s) with a dendrite which has a second contact (b). The knob (a) is itself con-tacted by a presynaptic knob (c). The synaptic knob in Fig. 3 is also contacted by apresynaptic knob (c).

Axo-dendritic tight junctions. Occasionally in the intermediate region of the cord(see Fig. 1 a, rectangle) tight junctions can be observed between axonal knobs anddendrites. The knobs are identified as axonal 'presynaptic' expansions, first becauseof the characteristic accumulations of synaptic vesicles, complex vesicles and mito-chondria and secondly because the knob can sometimes be traced in the plane ofsection in continuity with a myelinated terminal region of an axon (see below). Theseknobs are often relatively large, reaching 5 ju, in diameter.

Fig. 8 shows an example of an axo-dendritic tight junction (ntj). It is shown athigher magnification in the inset. At this region the axonal and dendritic membranescome into very close apposition, seen in section as a three-lined complex with an overallthickness of about 150 A. The tight junction has fine fibrils (/) of indefinite lengthassociated with and running at various angles to it. Some of the fibrils appear topenetrate through the tight junction, and high-resolution work is in progress to tryand settle this point. Often a mitochondrion (m) lies very close to the tight junctionon the axonal side. Synaptic vesicles are not invariably aggregated close to the axonalside of the tight junction.

So far it has not been possible to detect an axo-dendritic contact where both a tightjunction and a cleft junction occur between the same axon and dendrite. However,axonal knobs with tight junctions may also have cleft junctions but these are madewith other dendritic profiles. The presynaptic knob in Fig. 13, for example, makesa tight junction (ntj) with a dendrite below and a cleft junction (cj) with anotherdendritic profile to the right. Whether these dendrites arise from the same neuron orare invariably of different neurons has not been determined. Only rarely is it possibleto see both sorts of contact in the plane of section, but a similar arrangement has beentraced in serial sections.


Fig. 9 shows an axonal knob in continuity with the terminal myelinated region(my) of an axon. It makes an axo-dendritic tight junction (ntj), shown at highermagnification in the inset. The tight junction opens into a cleft (za) lined on bothaxonal and dendritic surfaces with dense material. Since this thickened zone shows nomarked asymmetry in contrast to that of the cleft junctions described above (pre-synaptic dense projections, vesicle aggregates and marked post-synaptic thickening)it is here considered comparable with the zonula adhaerens of Farquhar & Palade

(I963)-So far all the neuronal tight junctions encountered in the frog have been found in

the intermediate zone of the cord. None has been seen in the vicinity of the ventralhorn cells, on the dendrites as they pass out through the white matter to the marginalplexus, in the marginal plexus itself, or in the extremities of the dorsal horns, in-cluding the substantia gelatinosa. Also dendro-dendritic tight junctions have not beenobserved, although dendrites commonly run parallel to each other with the usual200 A cleft between their apposed surfaces.

Neuroglial tight junctions. Tight junctions occur between apposed neuroglial pro-cesses in the brain and spinal cord of mammals (Gray, 19616, 1964; Peters, 1962).Neuroglial tight junctions also occur commonly between astrocytic neuroglia in thefrog cord. Fig. 10 shows four such tight junctions (gtj) in the marginal plexus. Anaxo-dendritic cleft synapse (cj) is included for comparison. A second example, alsofrom the marginal plexus, is shown in Fig. 11 (gtj). It will be noted that the neuroglialtight junctions have no webs of fine fibrils associated with them, in contrast to theneuronal tight junctions described above. All the material has been stained with PTAand its affinity for fine fibrous material (possibly protein) emphasizes the structuraldifference between the two.

Note on the marginal plexus of the frog. Several comments have been made aboveon this region (see Fig. 1 a). It is a zone rich in axo-dendritic synaptic contacts(Fig. 12). The presynaptic knobs in section have diameters up to 2/1, and twelve ormore profiles may occur in a micrograph showing an area of 50 /i2. There is a subpialpalisade layer of astrocytic processes (ap) with the usual tight junctions (gtj) betweenthem and separating the synaptic zone from the pia mater. A basement membrane(bm) lines the outer surface of the glial palisade. Synapses (arrow) occur immediatelybelow the palisade, some being only 1 or 2 fi from the pia. Many of the dendrites haveclub-shaped terminal regions up to 3 fi in diameter, which correspond with the shapesdescribed by Cajal (1911) in Golgi preparations. Synapses occur on the dendritictrunks and their terminal clubs. Many of these dendrites are the terminal ramificationsof ventral horn cells whose perikarya may be situated as far as 500 /* away. As men-tioned previously, in this zone the presynaptic knobs have no neurofilamentous ringsor nets; axo-axonic contacts have not been observed nor have neuronal tight junctionsbeen seen.

Fish

General organization of the spinal cord. Only a few general remarks are made hereabout the organization of the cord of the bony fish, and again Nieuwenhuys (1964)


should be consulted for details and references. A diagram of a section of the goldfishcord (between vertebrae 4 and 10) is shown in Fig. ib. The dorsal horns of the greymatter form a compact mass above the central canal and consist largely of small cellsof the substantia gelatinosa intermixed with a few large cells. The ventral hornsextend laterally. The large cells of the ventral grey matter situated around the sidesof the central canal are said to innervate the trunk musculature, while the moreventrolateral motor cells supply the pectoral fins (see Ariens Kappers, Huber & Crosby,1936, and Beccari, 1943). The perikaryaof the largest neurons reach 50 ft, in diameter.The white matter forms an extensive area of myelinated axons and the dendritesextend into it. In the goldfish the terminal ramifications are not as complex as in thefrog so that there is no extensive marginal plexus. The plexus in fact consists of smallsubpial pockets of axodendritic synapses. The two giant Mauthner axons lie ventro-laterally to the canal, and their diameters may exceed 50 fi, including the myelinsheath. These giant fibres show several interesting features by light microscopy(Leghissa, 1956). Observations on these fibres with the electron microscope are inprogress and will be published elsewhere. Light microscopy shows that collateralsfrom the Mauthner axons synapse with the axons of the motor neurons just distalto the axon hillock (see Discussion).

Electron microscopy of goldfish cord. Examination of the grey matter indicates thatthe general structure and cytoplasmic organelles of the neuronal perikarya, axons anddendrites show no essential difference from those described above in the frog andmammalian cord. The neuroglia also show basic similarities and tight junctions occurcommonly between apposed glial surface membranes. One marked difference, how-ever, is the common occurrence of desmosomes between the contacting astrocyticprocesses in the white and grey matter of the fish cord, which are not present in thefrog or mammalian cord (excluding ependymal cells). The astrocytic fibrils are associ-ated with the dense plaques in a manner similar to that of the tonofibrils of the desmo-somes of other tissues. Dilly (1961) has described a similar desmosomal organizationin the neuroglia of the goldfish brain.

Synaptic organization of fish cord. Throughout the grey matter typical synapsesoccur in abundance, showing all the features mentioned above for such synapses infrog and mammal. Synaptic vesicles, complex vesicles, occasional granular vesiclesand mitochondria are the usual features of the presynaptic knob and the membranethickenings lining the cleft show the usual asymmetry. Axo-somatic and axo-dendriticsynapses (either on a trunk or dendritic spine) are common. However, unlike the frogand mammal, no neurofilamentous rings have yet been observed in the presynapticknobs, nor have axo-somatic synapses been identified with subsynaptic sacs. Clear(possibly glial) processes contacting neuronal perikarya, with submembrane sacs, are,however, common in the goldfish. Further, no axo-axonal synapses forming an axo-axo-dendritic serial arrangement (described above in the frog and also found in themammalian cord) have so far been encountered.

Of especial interest in the fish cord is the presence of tight junctions at axo-somaticsynapses on the surfaces of some of the large neurons of the ventral cord near thecentral canal (see Fig. ib). An example is shown in Fig. 16. The tight junction (ntj)


has a mitochondrion just above it and the same presynaptic knob forms a cleftjunction (cj) about 1000 A to the right. Such an arrangement is commonly found.Sometimes only a tight junction is seen, as in Fig. 17 (again, note the proximity ofa mitochondrion). Other axosomatic synapses show only a cleft junction. The greatmajority of synapses in the fish cord are, as in the frog, of the cleft type.

Whether these contacts with tight junctions also always have cleft junctions remainsto be determined. In sections the presynaptic knobs range from about 0-3 to 2-3 /i indiameter. Sometimes, in the plane of section, they can be seen to arise as directdilatations from the terminal regions of myelinated axons 1-2 /.t across including themyelin sheath (Figs. 14, 15). A similar arrangement for an axo-dendritic tight junctionwas described above in the frog. Sometimes synaptic knobs have been seen contactingsmall spinous projections of the cell bodies (Figs. 15, 18) and small perikaryal spinousprojections can be seen in Golgi preparations by light microscopy (Fig. 19). Fig. 20shows a structure presumed to be a section of a perikaryal spine. It is contacted bythree presynaptic knobs. The one above and the one on the right have cleft junctions(cj) and the one below makes a cleft and a tight junction (njt). Occasionally axo-somatictight junctions show continuity with a zonula adhaerens as described in the frog.

Marginal plexus of the fish. In the region of the goldfish studied there is no extensivemarginal synaptic zone, in contrast to the frog, and the myelinated fibres of the whitematter abut directly on to the subpial glial limiting layer. However, there are pocketsof axo-dendritic contacts in this zone (Fig. 1 b). The synapses are of the common clefttype and neither neuronal tight junctions nor axo-axonal contacts have been seen inthese pockets. The presynaptic knobs lack rings or nets of neurofilaments.

DISCUSSION

In the past, light microscopists, using reduced-silver methods, have demonstratedvery few boutons terminaux in the amphibian cord and none in the cords of fishes(but see below) or cyclostomes. From these observations, Nieuwenhuys (1964) hassuggested that in the course of evolution the interneuronic connexions in the spinalcord have changed from unspecialized contacts into the more localized, morpho-logically differentiated synapses of higher vertebrates, especially mammals. Ourobservations with the electron microscope do not support this view. Typical axo-somatic and axo-dendritic synapses can be seen in abundance in the spinal cord of thefrog and fish, with all the morphological complexity of synapses of the mammalian cord.The features include presynaptic vesicles, including granulated and complex varieties,mitochondria and a well-defined synaptic cleft with asymmetrical and polarizeddistribution of associated dense material. The demonstration of only a few boutons inthe frog and none in the fish is simply due to the scarcity or absence of argyrophilicneurofilaments, orientated either as a ring or reticulated mass, within the cytoplasmof the presynaptic knob. The evidence and arguments for this view are given in detailby Gray & Guillery (1965). In the present work presynaptic rings of neurofilamentswere observed only very rarely in the frog and not at all in the fish.

Bertolini (1964) has demonstrated morphologically specialized synapses in the


cyclostome (lamprey) cord. They have characteristic synaptic vesicles and a synapticcleft with associated dense material.

The discovery of neuronal tight junctions in the frog has already been described ina brief report (Charlton & Gray, 1965). They have so far been seen only in the inter-mediate grey region and since they constitute less than 1 % of the neuronal junctions(the majority being of the cleft-type synapse) they are not easily found. It sometimestook two or more hours scanning the intermediate region with the electron microscopeat a magnification of x 20000 before a neuronal tight junction could be found. Thethree-lined structure of the tight junction indicates two unit membranes either veryclosely aligned or completely fused (see Robertson et al. 1963). PTA staining showsthat fine fibrous material is associated with neuronal tight junctions of frog and fish,but is absent from the tight junctions which occur commonly between adjacentneuroglial processes (Gray, 19616). This may reflect a functional difference. Thedense material is similar to that described in teleost dendro-dendritic tight junctionsby Bennett et al. (1963).

There is now strong evidence to suggest that neuronal tight junctions are sites ofelectrical synaptic transmission (see, for example, Bennet et al. 1963; Robertson et al.1963; Eccles, 1964; Evans & Evans, 1964; Furshpan, 1964; Stefanelli, 1964; Barr,Dewey & Berger, 1965; Gray & Guillery, 1965; Takahashi & Hama, 1965). In thefrog cord there is evidence for electrical transmission (A. D. Grinnell & R. Miledi,personal communication) between adjacent ventral horn cells, possibly by direct inter-action of their apposed dendrites. In the present work no tight junctions were seenbetween adjacent dendrites, although dendrites are commonly seen with their surfacemembranes apposed across a 200 A extracellular zone. In the frog cord all the tightjunctions observed so far have been between axon knobs and dendrites in the inter-mediate region. These dendrites could be branches of the large ventral horn motorcells, for they spread into this region, but this has not been established. Electricalinteraction between adjacent ventral horn cells could then be accounted for by as-suming that the presynaptic knobs with the tight junctions are the terminals ofrecurrent axon collaterals of adjacent ventral horn cells.

On only one occasion so far has it been possible to trace a dendrite, with an axonaltight junction, to its perikaryon in the plane of section. The dendrite was 2 fi indiameter and the junction was situated about 10/i from the perikaryon. The peri-karyon was about 16 /* in diameter. This neuron from the intermediate region is muchsmaller than one of the ventral horn motor cells and its precise nature is uncertain.

The occurrence of synaptic knobs with both cleft and tight junctions suggests adual function of both chemical and electrical transmission. In the frog presynapticknobs may show a cleft synapse with one dendrite and a tight junction with a differentprofile. Whether these dendrites are branches of one parent dendrite or dendritesfrom different neurons is not yet known. In the fish both the cleft and tight junctionsoccur along the apposed membranes of an axo-somatic synapse. Such contacts occuron the large cells situated laterally and ventrally to the central canal. They are pre-sumably motor cells supplying the somites. Such a dual arrangement at one synapticcontact was described in the medulla and spinal cord of two gymnotid fish by M. L. V.


Bennett and G. D. Pappas at a Neuroscience Research Program in Boston in 1964,before this present work was started. Bennett & Pappas (personal communication)state in addition that such endings are found in the electric catfish {Malapterurus) onthe two giant electromotoneurons. According to Bennett, there is evidence only forelectrical, and not for chemical, transmission at these junctions.

In both frog and fish, observations on a dual synapse show that vesicles are invari-ably aggregated near the presynaptic side of the cleft junction but only sometimesaggregated near the tight junction. This observation supports the theory that synapticvesicles play a special role in chemical transmission, presumed to take place at thecleft synapse. As described above, a mitochondrion is frequently situated near theaxonal surface of a neuronal tight junction, perhaps indicating a special metabolicrelationship. On the other hand, however, mitochondria occur commonly in pre-synaptic knobs and are always situated away from the synaptic cleft and synapticvesicles. Since at the dual synapse the tight junction is a region 'away from the cleft',and often free of synaptic vesicles, it could be argued that the region contains a mito-chondrion simply as a result of packing restrictions on the presynaptic knob.

Work is in progress to try to obtain more information about the origin of the axonsof the dual contacts on the large cells of the goldfish cord. Possibly they are theterminals of branches of the contralateral Mauthner axon, which is known to exert apowerful contralateral inhibition on the effect of ipsilateral Mauthner axon excitation(J. Diamond, personal communication). The excitatory ipsilateral connexions of thebranches from the Mauthner axon with the motor cells also need elucidating. Light-microscopical observations of silver preparations show collaterals from the Mauthneraxon terminating as expansions on the initial segment of the motor cell axon (Leghissa,1956). This, incidentally, is the exception, mentioned above, to the general rule thatboutons cannot be demonstrated in fish cord (see Nieuwenhuys, 1964). Electron micro-scopy has so far failed to reveal any especially large terminals on the axon hillock. Theproblem is complicated in that the Mauthner collaterals were only clearly demonstratedin young and not in adult fishes (Leghissa, 1956).

Axo-axonal synapses similar to those described in the cat cord (Gray, 1962a, 1963)occur in the frog cord and may be responsible for the dorsal-root potential observedin the frog (Katz & Miledi, 1963).

We are grateful to Professor J. Z. Young, F.R.S., for his interest and advice, and to Mr S.Waterman for skilful photography.

REFERENCES

ANDRES, K. H. (1964). Mikropinozytose im Zentralnervensystem. Z. Zellforsch. mikrosk. Anat.64. 63-73.

ARIENS KAPPERS, C. U., HUBER, G. C. & CROSBY, E. C. (1936). The Comparative Anatomy ofthe Nervous System of Vertebrates including Man, vol. 1. New York: Macmillan.

BARR, L., DEWEY, M. M. & BERGER, W. (1965). Propagation of action potentials and thestructure of the nexus in cardiac muscle. J. gen. Physiol. 48, 797-823.

BECCARI, N. (1943). Neurologia comparata. Firenze: Sansoni Edizione.BENNETT, M. V. L., ALJURE, E., NAKAJIMA, Y. & PAPPAS, G. D. (1963). Electrotonic junctions

between teleost spinal neurons: electrophysiology and ultrastructure. Science, N.Y. 141,262-264.


BERTOLINI, B. (1964). Ultrastructure of the spinal cord of the lamprey. J. Ultrastruct. Res. 11,1-24.

BODIAN, D. (1964). An electron-microscope study of the monkey spinal cord. Bull. JohnsHopkins Hosp. 114, 13-119.

BUNGE, R. P., BUNGE, M. B. & PETERSON, E. R. (1965). An electron microscope study ofcultured rat spinal cord. J. Cell Biol. 24, 163-191.

CAJAL, S. R. (191 I ) . Histologie du systeme nerveux de Vhomme et des verte'bre's, vol. I. Paris:Maloine.

CHARLTON, B. T. & GRAY, E. G. (1965). Electron microscopy of specialized synaptic contactssuggesting possible electrical transmission in frog spinal cord. J. Physiol., Lond. 179,2-4P-

DILLY, N. (1961). A comparison of the cytoplasmic organisation of glial and neuronal con-nections in the goldfish C.N.S. In Cytology of Nervous Tissue, pp. 92-94. London: Taylor &Francis.

ECCLES, J. C. (1964). The Physiology of Synapses. Berlin: Springer.EVANS, D. H. L. & EVANS, E. M. (1964). The membrane relationships of smooth muscles: an

electron microscope study. J. Anat. 98, 37-46.FARQUHAR, M. G. & PALADE, G. E. (1963). Junctional complexes in various epithelia. J. Cell

Biol. 17, 375-412.FURSHPAN, E. J. (1964). 'Electrical transmission' at an excitatory synapse in a vertebrate brain.

Science, N. Y. 144, 878-880.GRAY, E. G. (1961a). The granule cells, mossy synapses and Purkinje spine synapses of the

cerebellum: light and electron microscope observations. J. Anat. 95, 345-356.GRAY, E. G. (1961 b). Ultrastructure of synapses of the cerebral cortex and of certain specialisa-

tions of neuroglial membranes. In Electron Microscopy in Anatomy (ed. J. D. Boyd et al.),PP- 54~73- London: Arnold.

GRAY, E. G. (1962a). A morphological basis for presynaptic inhibition? Nature, Lond. 193,82-83.

GRAY, E. G. (19626). Electron microscopy of synaptic organelles of the central nervoussystem. In 4th Int. Congr. Neuropath. Vol. 11, 1961 (ed. H.Jacob), pp. 57-61. Stuttgart:Thieme.

GRAY, E. G. (1963). Electron microscopy of presynaptic organelles of the spinal cord. J. Anat.97, 101-106.

GRAY, E. G. (1964). Tissue of the central nervous system. In Electron Microscopic Anatomy(ed. S. M. Kurtz), pp. 369-417. New York: Academic Press.

GRAY, E. G. & GUILLERY, R. W. (1961). The basis for silver staining of synapses of themammalian spinal cord: a light and electron microscope study. J. Physiol., Lond. 157,581-588.

GRAY, E. G. & GUILLERY, R. W. (1963). A note on the dendritic spine apparatus. J. Anat.97. 389-392.

GRAY, E. G. & GUILLERY, R. W. (1965). Synaptic morphology in the normal and degeneratingnervous system. Int. Rev. Cytol. 19, 111-182.

HESS, A. & YOUNG, J. Z. (1952). The nodes of Ranvier. Proc. R. Soc. B 140, 301-320.KATZ, B. & MILEDI, R. (1963). A study of spontaneous miniature potentials in spinal moto-

neurones. J. Physiol., Lond. 168, 389-422.LEGHISSA, S. (1956). Contribution ulte>ieure h une meilleure connaissance de l'appareil de

Mauthner chez les poissons et observations sur la morphologie de la fibre. In Progress inNeurobiology (ed. J. Ariens Kappers), pp. 45-62. Amsterdam: Elsevier.

LUSE, S. A. (1956). Electron microscope observations of the central nervous system. J. biophys.biochem. Cytol. 2, 531-542.

NIEUWENHUYS, R. (1964). Comparative anatomy of the spinal cord. In Progress in BrainResearch, vol. xi, pp. 1-57. Amsterdam: Elsevier.

PALAY, S. L. & PALADE, G. E. (1955). The fine structure of neurons. J. biophys. biochem. Cytol.1, 69-99.

PETERS, A. (1961). A radial component of central myelin sheaths. J. Cell Biol. 11, 733-735.PETERS, A. (1962). Plasma membrane contacts in the central nervous system. J. Anat. 96,

237-248.


RALSTON, H. J. (1965). The organisation of the substantia gelatinosa in the cat lumbosacralspinal cord. Z. Zellforsch. mikrosk. Anat. 67, 1-23.

ROBERTSON, J. D., BODENHEIMER, T. S. & STAGE, D. E. (1963). The ultrastructure of Mauthnercell synapses and nodes in goldfish brains. J. Cell Biol. 19, 159-199.

STEFANELLI, A. (1964). II significato morfologico delle sinapsi interneuroniche desunto daricerche di microscopia electronica sui neuroni di Mauthner dei Pesci. Atti Accad. naz.Lincei Re. 36, 31—36.

TAKAHASHI, K. & HAMA, K. (1965). Some observations on the fine structure of the synapticarea in the ciliary ganglion of the chick. Z. Zellforsch. mikrosk. Anat. 67, 174-184.

WESTRUM, L. E. (1965). On the origin of synaptic vesicles in cerebral cortex. J. Physiol., Lond.179, 4-6P.

WYCKOFF, R. W. G. & YOUNG, J. Z. (1956). The motorneuron surface. Proc. R. Soc. B 144,440-450.

(Received 1 October 1965)

8o B. T. Charlton and E. G. Gray

ABBREVIATIONS

aapbbmccjcvcytdendp

fggfgtjgvm

presynaptic knobastrocytic processpresynaptic knobbasement membranepresynaptic knobsynapse with cleft junctioncomplex vesiclecytoplasm of glial celldendritepresynaptic dense ' projection'fibrils of neuronal tight junctionglycogen granulesneuroglial fibrilsneuroglial tight junctiongranular vesiclemitochondrion

mynfntjnucPpcssospsssvtX

yza

myehn sheathneurofilamentsneuronal tight junctionnucleuspial cellpial collagensynaptic contactsoma of neuronspinesubsynaptic sacsynaptic vesiclessynaptic 'thickening'membranes of subsynaptic sacpostsynaptic membranezonula adhaerens

Journal of Cell Science, Vol. i, No. i

Fig. 2. Axo-dendritic synapses of the frog cord. (Insets) Examples of the membranespecializations of axo-dendritic contacts of frog cord.

B. T. CHARLTON AND E. G. GRAY (Facing p. 8o)

Fig. 3. Synaptic knob containing a ring of neurofilaments. Frog cord.Fig. 4. Axo-somatic synapse with subsynaptic sac. Frog cord.Fig. 5. Axo-somatic synapse. Frog cord.Fig. 6. Enlarged part of Fig. 4, showing synaptic cleft and subsynaptic sac. Frog cord.


B. T. CHARLTON AND E. G. GRAY

fcd

HO

>

O

zDW

OO

Fig. 7. A large synaptic knob seen in continuity with a myelinated axon. It synapses with a dendrite. The knob is itselfcontacted by a presynaptic knob (c). Arrows indicate direction of transmission. Frog cord.




.Fig. 9. Axo-dendntic tight junction contiguous with a zonula adhaercns (both enlargedin inset). The presynaptic knob is seen as a continuation of a myelinated axon.Frog cord.



*cyt'i\

0-4//i i

g«j

\

Fig. 10. A cleft synapse (right) and four neuroglial tight junctions (left). Frog cord.Fig. I I . An astrocyte with a tight junction at its surface. Frog cord.


Fig. 12. Pia and submeningeal marginal plexus. Arrow indicates an axo-dendriticsynapse. Frog cord.



Fig. 13. Presynaptic knob making an axo-dendritic tight junction (below) and an axo-dendriticcleft junction (right). Frog cord.Fig. 14. Myelinated axon with presynaptic knob making an axo-somatic tight junction.Goldfish.Fig. 15. Myelinated axon with presynaptic knob synapsing with a perikaryal spine. Goldfish.



Fig. 16. Axo-somatic synapse with both a tight and a cleft junction. Goldfish.Fig. 17. Axo-somatic synapse with a tight junction only. Goldfish.Fig. 18. Axo-somatic synapse on a perikaryal spine. Goldfish.Fig. 19. Golgi (rapid) preparation showing a perikaryal spine. Light microscopy. Goldfish.Fig. 20. Presumed perikaryal spine with two synaptic knobs (above) with cleft junctionsand one (below) with both a cleft and a tight junction. Goldfish.



Documents

COMPARATIVE ELECTRON MICROSCOPY OF SYNAPSES IN THE … · 2005. 8. 19. · The white matter shows the usual compact mass of ascending and descending myelinated fibres. A bundle comparable