J. Anat., Lond. (1964), 98, 1, pp. 47-53 47With 4 plates

Printed in Great Britain

Experimental degeneration in the cerebral cortex

BY MARC COLONNIER*Department of Anatomy, University College London

INTRODUCTION

Ever since the establishment of the neuron doctrine (Cajal, 1911), the identifica-tion of synapses has been a major problem in neurohistology. Nerve endings havebeen related by light microscopists to ring- or club-shaped structures seen on sectionsstained by silver methods for neurofibrils, the classical 'boutons terminaux' (Cajal,1911; Bartelmez & Hoerr, 1933; Bodian, 1937) and to agglomerations of mitochon-dria along the soma and dendrites of neurons (Armstrong, Richardson & Young,1956). They have been described by electron microscopists as cytoplasmic bags withthickened membranes, containing numerous vesicles and mitochondria (De Robertis& Bennett, 1955; Palay, 1956; Gray, 1959).The classical neurofibrillar boutons are only present in some parts of the normal

central nervous system; they are numerous, for example, around the large anteriormotor horn cells of the spinal cord, although it is obvious (with other stains) thateven here they represent but a fraction of the total number of synapses (Wyckoff &Young, 1956). In other areas they are absent in normal material but may appear asa degenerative phenomenon when stained by the Glees (1946) method, as in theavian optic tectum after section of the optic nerve (Evans & Hamlyn, 1956). Instill other regions, notably the cerebral cortex, they are not only virtually absent innormal, well-fixed material (Glees, 1946; Brodal & Walberg, 1952; Evans &Hamlyn, 1956; Smythies & Inman, 1960), but few, if any, appear during Walleriandegeneration (Brodal & Walberg, 1952; Evans & Hamlyn, 1956). In contrastnumerous synapses can be seen in the cortex with mitochondrial stains (Armstrong& Young, 1957).

In correlation, electron microscopy has shown that neurofilaments, 100A indiameter, sometimes orientated in the form of rings, are often present in boutons inthe normal spinal cord (Boycott, Gray & Guillery, 1961; Gray & Guillery, 1961),that they are absent from those of the normal avian optic tectum but appear aftersection of the optic nerve (Gray & Hamlyn, 1962), and finally that they are alsoabsent from normal cortical boutons (Boycott, Gray & Guillery, 1960). Thus thetheory that the neurofibrils of light microscopy, at least in some situations, corres-pond to the neurofilaments of electron microscopy (Schmitt & Geren, 1950; Palay& Palade, 1955) is largely confirmed.

Since neurofibrils do not appear in the boutons during Wallerian degeneration inthe cerebral cortex, Gray & Hamlyn (1962) have suggested that the synapticterminals in this region do not produce neurofilaments as a Wallerian change. Thepresent work was undertaken to study the fate of degenerating terminals in this

* Medical Research Fellow, Medical Research Council of Canada. Present Address: Departmentof Anatomy, University of Ottawa, Ontario, Canada.

4-2

48 MARC COLONNIERsituation. Some of the observations have already been described in a brief report(Colonnier & Gray, 1962).

MATERIAL AND METHODS

Slabs of rat visual cortex were isolated from the rest of the central nervous system,in anaesthetized animals, by undercutting with a hooked wire the tip of whichskimmed the undersurface of the pia (Burns, 1950). The blood supply remainedundisturbed in the central part of the isolated cortex. The animals were allowed tosurvive 1, 3, 5, 7, 9, 11 and 15 days, when they were anaesthetized and the slabsremoved, fixed in 1% buffered osmic acid, counterstained in 1 % alcoholic P.T.A.and embedded in Araldite (see Gray, 1959 for details).

RESULTS

After 24 hr. some endings become electron dense, granular and their vesicles nolonger appear as discrete structures. The mitochondria become extremely electronopaque. No neurofilaments are seen (P1. 1, fig. 1). In a few endings, the only sign ofdegeneration is the presence of large, crowded vesicles (PI. 1, fig. 3, inset). On thethird and fifth day, typically altered endings are common in the neuropil (PI. 1,figs. 2-4). Throughout this period most terminals (P1. 3, fig. 12) and the vastmajority of cell bodies and dendrites remain normal (a few abnormal cells are pro-bably undergoing retrograde degeneration as a result of the undercutting). There areno massive autolytic changes. The alterations seen in the endings are thereforeprobably Wallerian in nature. By the seventh day degenerating endings havealmost completely disappeared from the neuropil.Some of the degenerating endings found in the neuropil are applied in the normal

way to the post-synaptic membranes and are surrounded by several distinct glialand neuronal processes (PI. 1, figs. 1, 2). The one illustrated in PI. 1, fig. 2 has acup shape not seen in the normal cortex (although the presynaptic membrane isoften concave with respect to the post-synaptic thickening). It is likely that thisshape represents a distortion of a large pre-synaptic bag into a shrunken massstill anchored at the site of synaptic contact and that adjoining glial processeshave swollen to occupy the space left by the shrinking terminal.

Other endings though remaining attached to the post-synaptic membrane, losetheir normal relationships with other elements of the neuropil. They seem to becompletely invaginated within large, clear processes (P1. 1, figs. 3, 4; PI. 2, figs. 5-7)or within glial cell bodies (PI. 3, fig. 9). The post-synaptic membrane is often seenadhering to the ending within the glial cytoplasm (PI. 2, figs. 5-7; PI. 3, fig. 9) andserves as a convenient label.Dense masses are also present within the glial cytoplasm. Because they appear in

this situation, together with the degenerating endings, at a time when there is noevidence that the other parts of the neurons have been phagocytosed, they have beeninterpreted as terminals undergoing further degenerative changes and losing theiridentifying characteristics. P1. 2, fig. 7, shows two dense masses (d.m.s.) within glialcytoplasm which also contains numerous fibrils and a still recognizable degeneratingending. The dense bodies both have a clear region (a) possibly corresponding to thepost-synaptic cytoplasm, and a darker element, the terminal itself. Dense debris is

Experimental degeneration in the cerebral cortexalso present in the glial process illustrated in PI. 2, fig. 8. The process completelysurrounds a capillary which contains a red blood cell. The inset shows a high-powerview of the debris: one mass can still be interpreted as a synapse (see m.t.); theother is an irregular mass (d.m.s.).Some of the inclusions acquire 'whorly' lamellated patterns before disintegrating

into amorphous vacuoles. PI. 3, fig. 10, shows a terminal with its attached post-synaptic membrane within a glial cell body. Lamellation can be seen at one side ofits periphery (lam). Other inclusions within this and other glial cells form lamel-lated structures (P1. 3, fig. 10, inset). PI. 3, fig. 11, shows less distinctly lamellatedbodies (l.b.) and amorphous figures (am.f.) within another glial cell. They arebelieved to represent the final stages of degeneration. The whole sequence ofdegeneration can be seen in one glial cell (PI. 4, fig. 13, g.l.c.) closely apposed as asatellite to a neuron (nc.). The cell has a relatively small round nucleus and granu-lar cytoplasm. It contains the easily identifiable terminal (pre.) illustrated in fig. 10,completely lamellated structures (l.b.) and amorphous figures (am.f.).The lamellated structures are not degenerating myelin for in this material, as in

the optic tectum of the chick (Gray & Hamlyn, 1962), the electron microscopicappearance of myelin sheaths is not grossly altered until the fifth day. No doubtbiochemical changes and breaks in the axon and myelin occur before this. Degenerat-ing myelin within phagocytic cells is only seen from about the seventh day, at whichtime few degenerating endings are present in the neuropil. PI. 4, fig. 15, showsmyelin, ingested within glial cytoplasm at 7 days. The lamellae are only beginningto separate from one another. A low power view of a phagocyte, 9 days afterthe lesion, contains myelin sheaths at different stages of degeneration (PI. 4, fig. 16).

DISCUSSION

The absence of neurofilaments in the endings and their rapid phagocytic destruc-tion by glial cells account for the refractoriness of the mammalian cerebral cortexto the Glees method for the staining of degenerating neurofibrillar endings. Theseobservations add negative evidence in favour of the theory that the neurofibrils oflight microscopy correspond to the neurofilaments of electron microscopy.The changes undergone by the degenerating endings in the cerebral cortex differ

considerably from those occurring in other areas of the central nervous system.Not only do they not produce neurofilaments in contrast to the endings in theoptic tectum of the chick, but their cytoplasm and mitochondria become moreopaque than in the normal after osmium tetroxide fixation. Evidently there hasbeen a drastic change in the molecular structure of the lipids and proteins.The mechanism of phagocytosis seems to be essentially that postulated by Palade

(1956) and Bennett (1956) for the incorporation of substances into the cytoplasmof a cell, i.e. invagination of the membrane and formation of vacuoles containingthe ingested material. Lysis of the glial cytoplasmic membrane presumably occurs,since the dense inclusions are in direct continuity with the cytoplasm, without theintervention of a double membrane. One would therefore expect to find intermediatestages of the phagocytic process in the form of partial or single membranes, and thismay be represented by the apparently single membrane (s.m.) found to one side ofthe terminal illustrated in PI. 2, fig. 6.

4-3

49

It is intriguing that the post-synaptic contacts of the degenerating terminals arealso phagocytosed. It may be that the post-synaptic membranes which are phago-cytosed are in fact those of cells undergoing retrograde degeneration as a result ofthe undercutting. However, many of the phagocytosed post-synaptic elementsappear quite normal, in spite of the fact that elsewhere, lamellated configurations,similar to those of degenerating terminals, are occasionally seen in dendritic trunks,and that a similar reaction associated with an increase in electron opacity is also seenin a few dendritic spines, presumably undergoing retrograde degeneration as a resultof the undercutting. It is of course well known that normal synaptic contacts are noteasily broken in the central nervous system. Carpenter (1911) describes how preter-minals remain attached to the cells of the ciliary ganglion of the chick in teasedpreparations. In an electron microscopic study, Gray (1959) describes post-synapticthickenings adhering to terminals in damaged tissue at the periphery of corticalblocks. They also remain attached, and serve as a label for synaptic terminals, incentrifuged preparations of the cerebral cortex (Gray & Whittaker, 1960). The phago-cytosis of post synaptic membranes along with degenerating endings implies thatsynaptic contacts, at least in the cerebral cortex, are sites of inseparable membraneadhesion which results in the removal of the specialized post-synaptic region,containing the receptor and enzymic deactivator of the transmitter substance,together with the presynaptic process.The lamellated configuration of the degenerating material is not surprising;

Revel & Fawcett (1958) have demonstrated that such figures are obtained fromhydrated lipids. Similar whorls have been seen by a number of workers duringdegeneration in different situations as, for example, by Sobin (1962) in degeneratingcytoplasmic inclusions in cells of lymphosarcoma, where the lamellated pattern alsobegins at the periphery of the inclusion. In the central nervous system such patternsmay be terminal stages in the degeneration of endings and of myelin sheaths. Theymay even represent a common pattern of degeneration for all parts of the neuron.The nucleus and cytoplasm of a neuron, probably undergoing retrograde degenera-tion as a result of the undercutting, and identifiable by an axosomatic contact, canbe seen in P1. 4, fig. 14. The chromatin material is accumulated at the periphery ofthe nucleus. The cytoplasm exhibits the typical 'whorly' pattern of degeneration.(The danger of interpreting this, in sections cut in other planes, as a degeneratingaxon (a) with its degenerating myelin sheath (b), is quite evident.)

Microglia transformed into Gitter cells are usually held responsible for phagocyticactivity in the central nervous system (Penfield, 1932; del Rio-Hortega, 1932). Thepresence of terminals within large, swollen, clear processes, sometimes containingglial fibrils and occasionally related to blood vessels, as well as within satellite cellswith granular cytoplasm suggests that astrocytes and oligodendroglia (Schultz,Maynard & Pease, 1957; Farquhar & Hartman, 1957) are also phagocytic. Swollenastrocytes have been described by light microscopists during Wallerian degeneration(Adams, 1958).The identification of degenerating endings by electron microscopy may be a valu-

able adjunct for the determination of the precise ending of fibre tracts inthe central nervous system, especially when the Nauta or Glees methods areunsuitable.

50 MARC COLONNIER

Experimental degeneration in the cerebral cortex 51In short, therefore, it may be said that axon terminals in the cerebral cortex do

not develop neurofilaments during degeneration, and are hence negative to neuro-fibrillar stains. There is therefore the intriguing situation in the central nervoussystem where some terminals, as in the spinal cord, normally have neurofilaments,some where they only develop during degeneration, as in the chick optic tectum, andothers where they are present neither in the normal nor in the degenerating state,notably the cerebral cortex.

S UMMARY

1. As early as 24 hr after undercutting the cerebral cortex, degenerating endingsappear. They are electron dense and granular. The vesicles no longer appear as dis-crete structures. Mitochondria become extremely electron opaque. No neuro-filaments are formed.

2. Three to five days after the lesion, the endings together with their attachedpost-synaptic thickenings are invaginated and apparently incorporated within theneighbouring glial cells. They are believed to acquire a lamellated configuration andfinally to disintegrate into amorphous figures.

3. By the seventh to ninth day, the tissue is quite free of degenerating endings.Phagocytosis of the degenerating myelinated axons is at this time only beginning.

4. All types of neuroglial cells seem to be involved in phagocytic activity.

I wish to thank Dr E. G. Gray, for his help and advice, and Prof. J. Z. Young, forhis encouragement and criticism during the course of this work.

REFERENCES

ADAMS, R. D. (1958). Implication of the biology of neuroglia and microglia cells for clinical neuro-pathology. In Biology of Neuroglia, pp. 245-263, Ed. W. F. Windle. Thomas: Springfield, Ill.

ARMSTRONG, J., RICHARDSON, K. C. & YOUNG, J. Z. (1956). Staining neural end feet and mito-chondria after postchroming and carbowax embedding. Stain Tech. 31, 263-270.

ARMSTRONG, J. & YOUNG, J. Z. (1957). End feet in the cerebral cortex. J. Physiol. 137, 10P.BARTELMEZ, G. W. & HOERR, N. R. (1933). The vestibular club endings in Ameiurus. J. comp.

Neurol. 57, 401-428.BENNETT, H. (1956). The concept of membrane flow and membrane vesiculation as mechanisms for

active transport and ion pumping. J. biophys. biochem. Cytol. suppl. 2, 99-103.BODIAN, D. (1937). The structure of the vertebrate synapse. A study of axon endings in Mauthner's

cell and neighbouring centres in the goldfish. J. comp. Neurol. 68, 117-160.BOYCOTT, B. B., GRAY, E. G. & GUILLERY, R. W. (1960). A theory to account for the absence ofboutons in silver preparations of the cerebral cortex, based on a study of axon terminals bylight and electron microscopy. J. Physiol. 152, 3-5P.

BOYCOTT, B. B., GRAY, E. G. & GUILLERY, R. W. (1961). Synaptic structure and its alterationwith environmental temperature: a study by light and electron microscopy of the centralnervous system of lizards. Proc. roy. Soc. B, 154, 151-172.

BRODAL, A. & WkLBERG, F. (1952). Ascending fibres in pyramidal tract of cat. 4rch. Neurol.Psychiat., Chicago, 68, 755-775.

BURNS, B. D. (1950). Some properties of the cat's isolated cortex. J. Physiol. 111, 50-68.CAJAL, S. R. Y (1911). Histologie du systeme nerveux de l'homme et des vertebras. Vol. 1. Maloine:

Paris.CARPENTER, F. W. (1911). The ciliary ganglion of birds. Folia neuro-biol., Lpz., 5, 738-754.COLONNIER, M. and GRAY, E. G. (1962). Degeneration in cerebral cortex. Electron Microscopy.

Fifth International Congress for Electron Microscopy, Vol. 2, U3, Ed. S. S. Breese, Jr. AcademicPress: New York.

52 MARC COLONNIERDE ROBERTIS, E. D. P. & BENNETT, H. S. (1955). Some features of submicroscopic morphology of

synapses of frog and earthworm. J. biophys. biochem. Cytol. 1, 47-58.EvANs, D. H. L. & HAMLYN, L. H. (1956). A study of silver degeneration methods in the central

nervous system. J. Anat., Lond., 90, 193-202.FARQUHAR, M. G. & HARTMAN, J. F. (1957). Neuroglial structure and relationships as revealed by

electron microscopy. J. Neuropath. exp. Neurol. 16, 18-39.GLEES, P. (1946). Terminal degeneration within the central nervous system as studied by a new

silver method. J. Neuropath. exp. Neurol. 5, 54-59.GRAY, E. G. (1959). Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron

microscope picture. J. Anat., Lond., 93, 420-483.GRAY, E. G. & GUILLERY, R. W. (1961). The basis for silver staining of synapses of the mammalian

spinal cord: a light and electron microscope study. J. Physiol. 157, 581-588.GRAY, E. G. & HAmLYN, L. H. (1962). Electron microscopy of experimental degeneration in the

avian optic tectum. J. Anat., Lond., 96, 309-316.GRAY, E. G. & WHITTAKER, V. P. (1960). The isolation of synaptic vesicles from the central

nervous system. J. Physiol. 153, 35-47P.PALADE, G. E. (1956). The endoplasmic reticulum. J. biophys. biochem. Cytol. Suppl. 2, 85-98.PALAY, S. L. (1956). Synapses in the central nervous system. J. biophys. biochem. Cytol. Supply. 2,

193-206.PALAY, S. L. & PALADE, G. E. (1955). The fine structure of neurons. J. biophys. biochem. Cytol. 1,

69-99.PENFIELD, W. (1932). Neuroglia: normal and pathological. In Cytology and Cellular Pathology of

the Nervous System, Vol. 2, pp. 423-479. Ed. W. Penfield. New York: Hoeber.REVEL, J. P. & FAwCETT, D. W. (1958). Electron micrographs of myelin figures of phospholipids

simulating intracellular membranes. J. biophys. biochem. Cytol., 4, 495-496.DEL RIO-HORTEGA, P. (1932). Microglia. In Cytology and Cellular Pathology of the Nervous System,

Vol. 2, pp. 483-534, Ed. W. Penfield. New York: Hoeber.SCHMITT, F. 0. & GEREN, B. B. (1950). The fibrous structure of the nerve axon in relation to

localization of neurotubules. J. exp. Med. 91, 499-504.SCHULTZ, R. L., MAYNARD, E. A. & PEASE, D. C. (1957). Electron microscopy of neurons and

neuroglia of cerebral cortex and corpus callosum. Amer. J. Anat. 100, 369-388.SOBIN, L. H. (1962). Cytoplasmic inclusions in cells of lymphosarcoma 6C3HED. II. Electron

microscopic observations. Exp. Cell Res. 26, 280-289.SMYTHIES, J. R. & INMAN, O. P. (1960). The effect of post mortem autolysis on synaptic terminals

in cerebral cortex of dog. J. Anat., Lond., 94, 241-243.WYCKOFF, R. W. & YOUNG, J. Z. (1956). The motor neuron surface. Proc. roy. Soc. B, 144,440-450.

KEY TO LETTERING

am.f. amorphous figure m. mitochondrionax.s. axo-somatic synapse m.t. membrane thickeningcap. capillary nc. neuronc.m. cytoplasmic membrane post. post-synaptic component of synapsed.m. double membrane pre. presynaptic component of synapsed.m.&. deiise masses r.b.c. red blood celld.my. degenerating myelin s.m. single membranegl. glial process v. synaptic vesiclesgl.c. glial cell body N.B. structures marked a and b: for explana-lam. lamellation tion, see text.I.b. lamellated body

EXPLANATION OF PLATES

PLATE 1

Fig. 1. Degenerating ending, 1 day after undercutting.Fig. 2. Degenerating ending, 8 days after undercutting.Fig. 3. Degenerating ending invaginated within a clear glial process, 8 days after undercutting.Fig. 4. Degenerating ending surrounded by clear glia, 8 days after undercutting.

Plate 1

', - -..

I r

Lf t 1'

p I

-m.

- pre.

'-14444.

MARC COLONNIER

Journal of Anatomy, Vol. 98, Part 1

#. ?.

i *4

.4

..4

Vt'44.

A-

.4 .

9W

.s -e ... 4

t

i.tI.ip *1 "'l. 'e

A

I'4 .f

,

gl.

m.

t... .. Y

t. . h. t..~l

.. ..

NI ,0.

lli gL

2.1w'F4 '4."-11

* t~~~I#

,fit.

0.5p.~ ~~~~~....

4.

N

:,.!3 4.-, ". 4-;-.tlim&-V..,I

., A6:,O- % S. 7

2

!

g ,,,,4?>*s .4 1

I ri

(Facing p. 52)

IP t.r.

Journal of Anatomy, Vol. 98, Part 1

pre. _'

g.;Y::- .;M:

a1 '"

31i* ~:.ryn. t. *.A

6i

;...4

r pre. jjiF S:; -ee;t '.

:. § "' '. a6;7

'..::''dR.;.; '# '' '. _! t'

1ltw | F-aS;> > '.

.'Q' '.ibpe'XW i X '.4 ',%J-.v '" l s ..

X$ rS *;J 9ii I ,,++.llte..,ySci I_ ' .. _: t.s

.s...;jS 1 81'$''.3 i. 8 8 | i. | | | ! [..,A

_ ,,a: 'S ! v-1s 3w * *w-qms_| r eg;.b2. .,:

;;e t.@X-_ X,1;a-;

||W > j!

d.n1,s. /.. ^.§. 2 s

X..X.

*. >s Fya

.N,

7

MARC COLONNIER

Plate 2

k ew .- j - vs,, ,4

< twSt S .

mllz. tjw"sg . *

Mr

...p

El

Journal of Anatomy, Vol. 98, Part 1 Plate 3 v . Bybs :W.t '.iZi..!

,.St'':. '?' .-' i' Ad| Too,, do'..w;e We; Am............. .... ..........

I [..-EI'.::nr s* W-i,,.1.,.

Axis, .},,¢t..X.. use.'>4w..;- E

X w EE

OR,,.

MARC COLONNIER

Journal of Anatomy, Vol. 98, Part 1

MARC COLONNIER

Plate 4

Experimental degeneration in the cerebral cortex 53

PLATE 2

Fig. 5. Degenerating ending within glial process, 3 days after undercutting.Fig. 6. Degenerating ending within glial process, 5 days after undercutting.Fig. 7. Degenerating ending within glial process, 3 days after undercutting.Fig. 8. Debris in swollen process surrounding a blood vessel, 5 days after undercutting.

PLATE 3

Fig. 9. Degenerating ending within glial cell body, 3 days after undercutting.Fig. 10. Degenerating ending within glial cell body, 3 days after undercutting.Fig. 11. Lamellated bodies and amorphous figures within glial cell body, 3 days after undercutting.Fig. 12. Normal synapse, 5 days after undercutting.

PLATE 4

Fig. 13. Degenerating endings within satellite cell, 3 days after undercutting.Fig. 14. Degenerating nerve cell body, 3 days after undercutting.Fig. 15. Myelin sheath within glial cell, 7 days after undercutting.Fig. 16. Degenerating myelin within glial cell, 9 days after undercutting.