A combined golgi-electron microscopic study of the synapses made by the proximal axon and recurrent collaterals of a pyramidal cell in the somatic sensory cortex of the monkey

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A COMBINED GOLGI-ELECTRON MICROSCOPIC STUDY OF THE SYNAPSES MADE BY THE PROXIMAL AXON AND RECURRENT COLLATERALS OF A PYRAMIDAL CELL IN THE SOMATIC SENSORY CORTEX OF THE MONKEY D. A. WINFIELD, R. N. L. BROOK& J. J. SLOPER and T. P. S. POWELL Department of Human Anatomy, South Parks Road, Oxford OX1 3QX, U.K. Abstract-The synapses made within the cortex by the proximal axon and recurrent collateral branches of a pyramidal cell, the soma of which was at the boundary of layers II and III of the somatic sensory cortex of the monkey, have been studied by the combined Golgi-electron microscopic technique. In a large number of serial sections 62 synapses were found, all of which were asymmetric, 49 being formed by the main axon and 13 by the collaterals. Sixty per cent of the synapses were upon the shafts of dendrites, approximately half of which were identified as being of the large or aspinous stellate cell, and the remainder were upon dendritic spines. If the large stellate cell is inhibitory, then these findings provide a morphological basis for recurrent collateral inhibition.

ELECTRON microscopic study of Golgi impregnated material is proving to be a powerful technique for the analysis of the intrinsic connections of the cerebral cortex, and it has provided valuable information particularly about the stellate interneurons (LE VAY, 1973; PARNAVELAS, SULLIVAN, LIEBERMAN & WEBSTER, 1977; PETERS & FAIR~N, 1978; PETERS & PROSKAUER, 1980; SOMOGYI, 1978; WHITE, 1978). The synapses made by the axon and collateral branches of the pyramidal cell, which is the most conspicuous and frequent cell type in the cortex, have, however, received relatively little attention (LEVAY, 1973; PARNAVELASet ui., 1977; SOMOGYI, 1978) although the functional importance of the collaterals has been recognised for many years (PHILLIPS, 1956, 1959; STEFANIS & JASPER, 1964a,b; BROOKS & ASANUMA, 1965). In a section of Golgi impregnated material from the somatic sensory cortex of the monkey a pyr~idai cell was found in the superficial layers which had an axon passing vertically through most of the depth of the cortex and which gave off a number of collateral branches. This cell has now been studied systematically with the electron microscope in a large series of serial sections, and a quantitative estimate has been made of the synapses formed with other profiles in the cortex. EXPERIMENTAL

PROCEDURES

RESULTS

Blocks from area 3b of the somatic-sensory cortex in the posterior wall of the central sulcus of an adult rhesus monkey perfused under hypothermia with a mixture of I”/:,giutaraldehyde and 4% paraformaldehyde (dissolved in 0.9% NaCI, pH 7.2 phosphate buffer) were processed for combined Golgi-electron microscopy according to the method of FAIRBN,PETERS& SALDHANA,1977. The blocks were cut YS(‘. 0 7 fl

at 100 pm thickness with a Sorvall tissue chopper. We have found it necessary to increase the concentration of oxalic acid to 0.2% because 0.05”/, failed to give us a consistent gold precipitate. An isolated pyramidal cell with a well impregnated axon and collaterals whose soma was at the junction between layers II and III was selected for detailed study. Before gold-toning, the cell was photographed (Figs l-4) and drawn with the camera lucida. Becuse of the length of the impregnated axon it was necessary to process the cell for electron microscopy as two blocks, one containing the cell body, dendrites and proximal third of the axon with the major collaterals (Fig. 5), and the other containing the long descending axon (Fig. 6). No other similar impregnated processes were present in this second block. After embedding, the sections were re-examined in the light microscope viewed with transmitted light, and mesas were prepared from each block. Ultra-thin sections (approximately 0.5 x 0.2mm) were cut, in series of about 80 at a time, with a glass knife and mounted on Formvarcoated grids with a single 1 x 2mm hole. They were stained with alkaline lead citrate (REYNOLDS,1963) and uranyl acetate (a 5% solution in 50% ethanol). Altogether about 600 serial sections were required from each block to include all the impregnated branches of the axon and all the sections were studied on the electron microscope. About two to three sections were lost between each series of SO, but the effect of this loss in tracing the continuity of processes between sections was negligible.

Light microscopy

The cell body of the neuron studied here was at the boundary of layers II and III and was clearly pyramidal (Figs l-6). Its apical dendrite ascended perpendicular to the pial surface through layer II, giving rise to four main branches near the soma, and bifurcating

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about midway through layer I. Four basal dendrites emerged from the soma and arhorized in layers II and III. The axon arose from one of the basal dendrites and could be traced for a distance of about 700 pm, to the upper part of layer V. It followed a relatively straight course perpendicular to the pial surface and gave rise to several collaterals. Three recurving collaterals arose at distances of about SO, 55. and 80 jirn from the cell body, and could be followed as they turned up to pass through layer II and enter layer I. They gave rise to several short side-branches. The territory of the three collaterals was contained within the dendritic arborization of the pyramid, but they were not observed to come into close proximity with any of the dendrites. The main descending axon also gave rise to a group of three side branches (about 2.5 kirn in length), approximately 350pm from the cell soma. and to a single horizontal branch, a further 1OOym away in layer IV, which could be traced for a distance of about 45pm (Figs 4 and 6). Numerous short (2-10pm) side branches resembling spines with a long narrow stalk and bulbous ending arose at irregular intervals along the main axon and collaterals (Figs 2--6). These were counted and their average separations measured from the camera lucida drawing; there were 83 such branches at an average IO pm apart on the main axon and 16 pm apart on the collaterals.

The appearance of the Golgi impregnated neuron under the electron microscope (Fig. 7) resembled that of the pyramidal cells in the visual cortex of the rat and in the somatic sensory cortex of the mouse studied after a similar gold-toning procedure (FAIR~N et al., 1977; PETERS & PROSKAUER, 1980; WHITE. 1978). Small electron-dense particles were scattered throughout the cytoplasm of the cell soma, most being con-

t’l trl.

tained by the plasma membrane of the cell, but a few were found outside the membrane and some overlay the nucleus. The distribution of particles varied in different parts of the cell: they were larger and more densely packed in processes of fine diameter, such as dendritic spines and the axon and its collaterals, compared with the perikaryon. The overall intensity of the staining was slightly greater in this cell compared with those of FAIR~N et trl. (1977) and of WHITE (1978). The cell soma was typically triangular in sections which passed through the apical dendrite as it emerged from the cell body. but in other sections it was more oval. The transverse diameter was 11pm on sections passing through the nucleolus. The appearance of the cell corresponded well with previous descriptions of this cell type for sensory cortex (JONES& PDWELL. 1970; PETERS& KAISERMA~-ABRAM~)F,I970; SLOPER, HI~RNS & POWELL. 19791, and the soma and

proximal dendrites received a small number of exclusively symmetric synaptic contacts. The main axon was directed on a relatively straight course through the cortical depth, but in the electron microscope the small irregularities in the course of the axon seen with the light-microscope were very large so that only small fragments of the axon were visible in individual sections as it continually passed in and out of the plane of the section (Figs 8 and 9). Serial sections were required to trace the fragments in continuity through neighboljring sections but a description of the axon and its collaterals will be given which is a composite view based upon that seen in individual sections. The main axon could be traced from its origin from one of the basal dendrites throughout its course in the cortex. It was ilnmyelinated and accompanied a bundle of myelinated axons through the deeper aspect of the cortex (Figs 8 and 9). Occasionally. it was closely apposed to other cell somata as it curved around those lying along its course. but it

FIG. 1. The Golgi impregnated pyramidal cell studied (arrow) at the boundary between layers II and III in area 3b of the primate somatic sensory cortex. The main descending axon and three recurrent collaterals are visible (arrowheads). x 250. FIG. 24. Montage of the Golgi impregnated pyramidal cell studied at higher magnification. The axon (a) gives off three recurrent collaterals close to the cell (large arrowheads) and a further horizontal collateral in layer IV (small arrow). Several examples of small stalked appendages may be seen (small arrowheads). Single and double bars correspond in the three figures. x 650. FIG. 5. Camera

lucida

drawing

FIG. 6. Camera lucida drawing

of the cell soma and dendrites of the pyramidal proximal part of the axon. Bar = 50iim.

cell studied

showing

of the pyramidal

the full extent of the axon and collaterals studied. Bar = 50 pm.

with the cell

RG. 7. The appearance of the cell soma of the deimpregnated cell under the electron microscope. Most of the electron-dense deposit has been removed from the cell somn but more remains in the origin of one of the dendrites (arrow). x 9500. FtGs 8 and 9. The axon of the Golgi impregnated pyramid passing through the lower part of layer III and into the upper part of layer IV, which is characterised by the presence of numerous small myelinated axons. Figs 8 and 9 are continuous at the bars. The impregnated axon is unmyelinated but in this and other sections formed a bundle with several vertical myelinated axons (a). Parts of two small side branches are seen (arrows); the one in layer IV makes a synapse on to a dendrite (d). Both x 7500.

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II

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III

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Golgi-electron microscopy of synapses by proximal axon of pyramidal cell never made synaptic contacts at such sites. Numerous short ~dun~ulated branches were found in continuity with the main axon and its collaterals. These had a narrow neck and expanded spherical head approximately 1 pm in diameter. These boutons tended to be heavily impregnated with gold particles but spherical synaptic vesicles and mitochondria could be seen. The boutons were the sites at which the majority of the synapses were formed by the cell as most synapses were on terminal boutons rather than en passant along the length of the axon. Only one varicosity was found to make synaptic contacts with a dendritic spine and the smooth shaft of a dendrite. Synaptic vesicles were rarely seen in other varicosities, but it cannot be excluded that others may have formed synaptic contacts either in front or behind the varicosity. Altogether, 60 boutons were traced in continuity with either the main axon or its collateral branches, and these made a total of 62 synapses. Synaptic contacts could not be identified on several other boutons or side branches although they were traced through their extent in serial sections, and it is also probable that some side branches were not fully impregnated and so were not followed to their terminations, an example being the long horizontal collateral in layer IV. All of the synapses were symmetric. The majority of the 62 synapses found (~%) were upon the smooth shafts of dendrites (Figs 10-1.5; 20 and 21) and the rest were upon dendritic spines (Figs 16-19). Approximately one half (46%) of the dendritic shafts received a large number of other normal synapses, both asymmetric and symmetric, contained abundant mitochondria and ribosomes, and were slightly varicose in outline, sometimes running in straight lines for considerable lengths (Figs 10-13). These features identify them as arising from large or sparsely-spined stellate cells (PETERS& FAIRI?N,1978; SLOPERet al., 1979). One such dendrite received two synaptic contacts from different impregnated boutons at separate sites in serial sections. The other dendrites were of small or medium diameter, rarely received other synapses in the same section, were not varicose and could not be identified as belonging to a particular class of neuron. All except two boutons made a single synaptic contact when followed in serial sections; two boutons made two synapses, and in both cases one synapse was with a dendritic spine, the other with a smooth dendritic shaft. The main axon established more synaptic contacts than the three recurving collaterals (49 from the axon, 13 from the collaterals), but the distribution of the synapses was similar for both: most were upon dendritic shafts, with similar proportions on to the dendrites of large stellate cells. DISCUSSION The cell that has been studied here was clearly pyramidal with the cell body at the junction of layers II

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and HI, and it bore a striking resemblance to the pyramidal cell at this level in the cortex described and illustrated by RA&N Y CAJAL(1911, pp. 535 and 544). The collateral arborization pattern corresponds well with previous descriptions (e.g. RAMONY CAJAL,1911; VALVERDE, 1971) and the number of recurrent collaterals falls within the normal range of 3-10 for Golgi impregnated cells given by SCHEIBEL& SCHEIBEL (1970). Although the axon of this cell was similar to a number previously iilustrated, several observations suggest that it may not have been completely impregnated. The drawing of three neighbouring pyramidal cells in the cat cerebral cortex by SCHEIBEL& SCHEIBEL (1970) shows that collaterals may branch profusely and extend for 3 mm within the cortex which was far more than those of the cell studied here; but it is not clear, however, whether all pyramidal cells have a similar degree of arborization nor whether collaterals are similar in different species and different layers. Pyramidal cells in the visual and motor cortex of the cat injected with horseradish peroxidase showed even richer branching of collaterals than had previously been seen with the Golgi method (GILBERT & WIESEL, 1979; LANDRY,LABELLE& DESCH~NES, 1980) so it is probable that even apparently well impregnated neurons with the Golgi method are not fully impregnate, the horseradish peroxidase method being more sensitive. It should be noted also that the impregnation of the axon did not extend far into layer V where many collaterals from pyramidal cells in layer III may be given off (LORENTEDE No, 1949; MARTINEZ-MILLAN& HOLL;~NDER,1975; LUND & BOOTHE,1975; HENRY, LUND & HARVEY,1978; GILBERT& WIESEL,1979). For these reasons it is probable that although the axon and collaterals were traced as completely as possible in serial sections they were not followed through their entire course. The problem of the completeness of the impregnation has been discussed in some detail because of its relevance to the interpretation of the observations, and particularly the quantitative estimates. Sixty-two synapses were found of which most (60) were on terminal boutons of the short side branches of the main axon and collaterals with only two being on an en passant bouton. The high proportion of synapses formed by terminal boutons is surprising but correlates well with the number of short side branches (83) seen with the light-microscope. Even when followed through in serial sections, a number of boutons could not be seen to form synapses; the most probable reason for the failure to find synaptic contacts was that the membrane thickening may have been obscured by the metallic deposit being too heavy. Perhaps the most significant finding has been the pattern of distribution of the synapses made by axon and collaterals, 60% of the synapses being directly upon the shafts of dendrites and the remainder upon spines. It was not possible to identify the type of cell from which the spines and some of the dendrites arose, but approximately half of the dendrites had all

D. A. WINFIELDer ul

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the features characteristic of those of the large stellate (SLOPER et al., 1979) or sparsely-spined stellate cell (PETERS& FAIR~N, 1978; WHITE, 1978). In earlier combined light- and electron microscopic studies the axon and collaterals of pyramidal neurons were not studied systematically but the observations which have been made in the visual cortex of several species agree with the present interpretations. LEVAY (1973) and PARNAVELASet al. (1977) state briefly that synapses were formed with dendritic spines and shafts. and of the 16 synapses found by SOMOGYI (1978) half were upon spines and half upon dendrites which ‘invariably had other non-stained asymmetric synapses on their surface’. In conventionally-fixed material of the motor cortex one axon which was traced in serial sections formed a synapse upon a dendrite of a large stellate cell (SLOPER & POWELL. 1979a). Possible functional

implicutions

An individual pyramidal cell may exert an influence upon cells within a considerable extent of the adjoining cortex; vertically, through the recurving collaterals and its descending axon it may have an effect through most of the depth of the cortex, and horizontally over a few millimetres by the spread of its collaterals. The large or aspinous stellate in the somatic sensory cortex of the monkey in turn has horizontally disposed branches of its axon that may extend for 1 mm (JONES, 1975). All the synapses formed by the pyramidal cell axon and collaterals are asymmetric and presumably excitatory, but the large stellate cell is known to make symmetrical synapses upon the somata and proximal dendrites of pyramidal cells (PETERS & PROSKAUER, 1980) and to contain gluta-

mate decarboxylase, the enzyme involved in the synthesis of the inhibitory transmitter y-aminobutyric acid (RIBAK. 1978). It is not known whether the axons and collaterals of pyramidal cells in other layers form synapses within the cortex upon similar processes to those found here, but if one can assume that it is so for pyramidal cells in layer V. the interpretation of earlier physiological studies for the important role of the collaterals and an inhibitory interneuron in cortical physiology would be confirmed. Electrical stimulation of corticospinal axons in the medulla causes an excitatory-inhibitory sequence of activation of pyramidal cells and excitation of nonpyramidal tract neurons in area 4 (PHILLIPS, 1956. 1959; STEFANIS & JASPER. 1964~. h: BROOKS& ASANUMA,1965). and it was considered that the most likely interpretation of these effects was that they were due to the activation of recurrent axon collaterals within the cortex, the early excitatory effect through synapses upon the dendrites of pyramidal cells and the more marked and longer lasting inhibition through an inhibitory interneuron whose axon terminates upon pyramidal cells. The present findings of the axon and collaterals forming synapses, presumably excitatory, upon the dendrites of a stellate interneuron that in turn presumably makes inhibitory synapses, at which y-aminobutyric acid is the transmitter, upon pyramidal cells provides an anatomical basis for the inhibitory effect, The cell type which gives rise to the other dendrites and spines upon which the axon and collaterals make synapses was not identified, but if some of these were from pyramidal cells there would be a basis for the direct excitation of them after stimulation of the corticospinal axons.

FIG. 10. Higher magnification of the upper Impregnated bouton of Fig. 8 on the same section showing it making an asymmetric synapse on to a dendrite (d). A few round synaptic vesicles are visible in the impregnated

axon terminal.

x 31.000.

FIG. 11. The same impregnated bouton and synapse on to a dendrite as Figs 8 and 10 in a serial section 3 sections away. This confirms the asymmetric nature of the synaptic membrane thickening.

FIG. 12. The same impregnated bouton as Figs LO and 11(arrowhead), 4 sections the opposite direction, showing its relationship to the main axon (a) and showing the vertically running postsynaptic dendrite (d). x 16,000. FIG. 13. The same impregnated appearance of the postsynaptic

away from Fig. 10 in the varicose nature of

bouton 14 sections from Fig. 10 in the opposite direction to Fig. 12. The dendrite (d) in Figs 12 and 13 is characteristic of the dendrite of a large stellate cell. x 2O.OC0.

Fms 14 and 15. Two adjacent

serial sectlons showing another impregnated bouton arising from the main axon in layer III and making an asymmetric synapse on to a small dendrite (d). Both x 31,000.

FIGS 16 and

17. Adjacent serial sections of an impregnated bouton from the main asymmetric synapse on to a spine (sp) in layer IV. Both x 31,000.

FIG. 18. An impregnated FE.

19. Another

axon terminal

impregnated

FIGS 20 and 21. Adjacent

terminal

axon

making

an

making

an asymmetric x 31.000.

synapse

on to a spine (sp) in layer

V.

making

an asymmetric x 31.000.

synapse

on to a spine (sp) m layer V.

serial sections showing at higher magnification the impregnated axon terminal at the top of Fig. 8 making an asymmetric synapse on to a dendrite (d). Both x 31.000.

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Golgi-electron

microscopy

of synapses

If the axon of the cell studied here enters the white it will almost certainly pass to another area of cortex where most of the terminals make synapses upon spines and only a few upon the dendrites of the same large stellate type that receives synapses from the proximal axon and its collaterals (SLOPER & POWELL, 19796). This pattern of postsynaptic profiles is quite different from those that receive synapses from the beginning of the axon and collaterals, where dendrites predominate. Approximately half of these dendrites are those of large stellate cells so it would matter

by proximal

axon of pyramidal

cell

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seem that this cell may be more strongly influenced by the proximal axon and collaterals than by the termination of the axon, supporting earlier interpretations in which the important role of an interneuron in recurrent collateral inhibition was emphasised (PHILLIPS,1956, 1959; STEFANIS& JASPER,19644 b). Acknowledgements-This work was supported by a grant from the Medical Research Council. D. A. WINFIELD is the Horace Le Marquand and Dudley Bigg Research Fellow of the Royal Society.

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SOMOGYIP. (1978) The study of Golgi stained ceils and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain. Neuroscience 3, 167-180. STEFANIS C. & JASPERH. (1964~) Intracellular microelectrode studies of antidromic responses in cortical pyramidal tract neurones. J. Neuruphpsiol. 27, 828-854. STEPANIS C. & JASPERH. (1964b) Recurrent collateral inhibition in pyramidal tract neurones. J. Neurophysiol. 27, 8555877. VAI_VERDE F. (1971) Short axon neuronal subsystems in the visual cortex of the monkey. Int. J. Nrurosci 1, 181-197. WHITEE. L. (1978) Identified neurons in mouse Sml cortex which are postsynaptic to thalamocortical axon terminals: a combined Golgi-electron microscopic and degeneration study. J. camp. Neural. 181, 627 -662.