SYNAPSES ON THE AXON HILLOCKS AND INITIAL SEGMENTS OF PYRAMIDAL CELL AXONS IN THE CEREBRAL CORTEX

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1 J. Cell Sci. 5, (1969) 495 Printed in Great Britain SYNAPSES ON THE AXON HILLOCKS AND INITIAL SEGMENTS OF PYRAMIDAL CELL AXONS IN THE CEREBRAL CORTEX E. G. JONES AND T. P. S. POWELL Department of Human Anatomy, Oxford, England SUMMARY The axon hillocks and initial segments of pyramidal cell axons can be clearly recognized in electron micrographs of the somatic sensory cortex. The initial segment is characterized by three features: bundles of neurotubules linked together by electron-dense bands, a layer of dense material attached to the inner surface of the plasma membrane, and small membranebound dense bodies. All of these elements and the few ribosomes usually present disappear at the commencement of the myelin sheath. The initial segment of the axon often contains a cluster of cisternae similar to the spine apparatus, and this part of the axon sometimes gives off small branches. Axon terminals end on both the axon hillock and the initial segment, and there is an increase in number on the latter as the distance from the hillock increases. All of these terminals are relatively large, contain a high proportion of small flattened or pleomorphic synaptic vesicles and terminate in symmetrical synaptic contacts. These morphological features suggest that the synapses may be inhibitory in function. INTRODUCTION The close similarity between the internal structure of an axon and that of a dendrite has made the electron-microscopic identification of the initial axon segment difficult unless seen in continuity with its parent soma (Laatsch & Cowan, 1966). Recently, however, Palay, Sotelo, Peters & Orkand (1968) have shown that the initial segments of axons possess two unique features: a layer of electron-dense material beneath the plasma membrane and clusters of neurotubules linked by bands of electron-dense material. In a detailed description of the axon hillock and initial segment of the axon of the pyramidal neuron of the cerebral cortex Peters, Proskauer & Kaiserman- Abramof (1968) have extended these findings, and have provided information on the synapses on this part of the neuron. During the course of a study on the somatic sensory area of the cerebral cortex, many examples of axon hillocks have been encountered, and in addition to confirming the observations of Palay et al. (1968) and of Peters et al. (1968), this material has provided further evidence on the synaptic relations of the hillock and initial segment. MATERIAL AND METHODS This material was collected from the cerebral cortex of 25 cats, both normal and experimental, which have been used mainly for studies of the connexions of the somatic sensory cortex (Jones & Powell, 19696). All animals were perfused under hypothermia with a o-i M

2 496 E. G. Jones and T. P. S. Powell phosphate-buffered mixture of 4% paraformaldehyde and 1% glutaraldehyde at ph 7-4. Blocks from the somatic sensory cortex were post-fixed in osmium tetroxide and embedded in Araldite; thin sections were stained with lead citrate (Reynolds, 1963) or with lead citrate and uranyl acetate (Watson, 1958). RESULTS The axon hillock and initial segment The perikarya of the pyramidal cells of layers II-IV are readily recognized in lowmagnification electron micrographs by their characteristic triangular shape (Fig. 1). The majority of the axons arise from the basal aspects of these cells or less commonly from a basal dendrite; they have a characteristic appearance and a mode of origin which is more definite than that of the apical and basal dendrites, but as the present observations are in accord with the descriptions of Palay et al. (1968) and of Peters et al. (1968), the features of these parts of the pyramidal neuron will not be described in detail. The axons are much narrower than these dendrites, and the angle which they make with the perikaryon is far more acute than that made by any dendrite at its origin. At the site of origin of the axon, a distinct, semicircular region which is relatively free of most cytoplasmic organelles and in which free and attached ribosomes are particularly reduced in numbers extends for a variable distance into the perinuclear cytoplasm; the region is the equivalent of the axon hillock of light microscopy, and its size is usually directly proportional to the diameter of the initial axon segment, and this in turn to the size of the perikaryon. Neurotubules are the most conspicuous constituent of the hillock and they have a very characteristic orientation, as though being funnelled into the initial axon segment. A number of features distinguish this part of the axon. The neurotubules converging on it from the axon hillock become aggregated into bundles (Figs. 2-5) of three or more, and within these bundles individual neurotubules appear more electron-dense and are separated from their neighbours by strands of electron-dense material. In transverse section it is apparent that the neurotubules themselves are not greatly increased in electron density, the density being due to the intervening dense material which joins adjacent tubules to one another. A thin layer of amorphous, electron-dense material A thick is applied to the inner surface of the plasma membrane of the initial segment; this material may commence at the perikaryon or a short distance from it, and can sometimes be traced to the commencement of the myelin sheath, where it disappears. A variable number of small, oval or bilobed membrane-bound dense bodies are found, both in the axon and the hillock. These are identical to similar dense bodies which are also present in normal neuronal perikarya, but they are a constant feature of the initial axon segment, usually continuing as far as the commencement of the myelin sheath. The bundles of neurotubules, the dense material beneath the plasma membrane and the small dense bodies make it possible to identify the initial segment of an axon in the cortex, even when it is not attached to its parent cell soma. Another feature of the initial segment of the axon, which has been described in detail by Peters et al. (1968), is the presence of a group of cisternae which are more similar to the spine apparatus than to the subsurface cisternae of the cell soma.

3 Axon hillocks and initial axon segments 497 Whether this organelle is constantly present is not known, but up to three such clusters have been found within an individual axon (Figs. 4, 5). It usually consists of a few flattened cisternae arranged parallel to each other and in the long axis of the axon,. and separated by dense granular material. The cisternae may be situated close to the plasma membrane, and in relation to a synapse, or more deeply within the axon. These organelles differ from the subsurface cisternae found in the cell soma principally in not being so closely applied to the plasma membrane and in having dense material between the individual cisternae. Also present within the initial segment are varying numbers of neurofilaments and mitochondria and a few scattered rosettes of free ribosomes, which are conspicuous at the site of origin of small branches (Fig. 8). Ribosomes may extend as far as the beginning of the myelin sheath, but thereafter they are absent except occasionally at a branch (Fig. 7). At the node of Ranvier the bare portion of the axon usually contains beneath its plasma membrane dense material similar to that in the initial segment (Fig. 7). Synapses on the axon hillock and initial segment Axon terminals are commonly seen to make synaptic contact on the axon hillock or on immediately adjoining parts of the perikaryon (Figs. 2-6). These terminals are relatively large, and, unlike the majority of axon terminals in the cortex, have a clear background cytoplasm; sometimes they are bilobed or irregular in shape. The number of synaptic vesicles in each terminal is relatively low when compared with the number in the smaller, dense terminals ending on dendritic spines in the vicinity. In this aldehyde-fixed material the vesicles of the clear terminals on the axon hillock tend to be flattened or irregular in shape, while those of neighbouring axo-spinous terminals are spherical and often slightly larger. The flattening of the vesicles is tosome extent variable: in some brains all terminals on the axon hillock contain a majority of flattened or pleomorphic forms; in others a small proportion only may be flattened, while in a few cases most vesicles are spherical though smaller than those in adjoining axo-spinous terminals. The terminals have one or more short synaptic thickenings associated with the plasma membrane of the axon hillock region. These are invariably symmetrical thickenings with small, but equal, accumulations of electron-dense material on the pre- and postsynaptic sides. Although it is probably equivalent to Gray's type 2 synapse, it does not correspond precisely (cf. Colonnier v 1968), and a little dense material may fill the synaptic cleft in this aldehyde-fixed material. The number of synaptic vesicles in the immediate vicinity of the presynaptic thickening is usually small. Similar axon terminals are also seen on the initial axon segment and become increasingly common on moving away from the axon hillock (Fig. 6). All make symmetrical synaptic contacts and usually possess a high proportion of flattened or pleomorphic synaptic vesicles. At the point of synaptic thickening (usually single) the electron-dense material typical of the initial segment as a whole disappears from beneath the plasma membrane, and there is commonly a local aggregation of free ribosomes (Fig. 9). The initial segment, where not contacted by axon terminals, may be surrounded by a variety of other neuronal and glial profiles;, it is not wholly surrounded by glia. None of the adjoining profiles are in intimate 32 Cell Sci. 5

4 498 E. G. Jones and T. P. S. Powell contact with the initial segment, and the impression is gained that, although variable, the extracellular space separating them from the initial segment is frequently wider than that separating other profiles throughout the cortex. Axon hillocks and initial segments have also been encountered on round or oval perikarya, particularly in layers II, IV and VI of the cortex. The initial segment contains similar linked bundles of neurotubules and dense bodies and has dense material beneath the plasma membrane. The hillock, however, is smaller and less obvious than on the pyramidal cells, although it receives clear axon terminals containing a proportion of flattened vesicles and ending in symmetrical contact zones. Similar terminals are also encountered on the initial segments of these cells, but with slightly lower frequency than on the pyramidal cells. DISCUSSION In addition to the aggregations of neurotubules linked by electron-dense bands and the 'undercoating' of dense material beneath the plasma membrane of the initial segments which have been described in detail by Palay et al. (1968) and Peters et al. (1968), the initial segments and axon hillocks consistently contain a number of small membrane-bound dense bodies. The exact significance of these structures is uncertain. They are frequently also found in neuronal perikarya in the cortex and other sites, although never so consistently as in the initial segment. They are smaller than lipofuscin particles (Samorajski, Ordy & Keefe, 1965), and unlike such particles are not granular or vacuolated so they probably represent some other neuronal inclusion. A further feature is the cisternal organelle; although this has often been seen, both in the present study and by Peters et al. (1968), it remains to be determined whether it occurs constantly. Peters et al. (1968) have commented upon the similarity of this organelle to the spine apparatus and have speculated upon its possible relation to synaptic transmission at these two sites on a neuron. It should be noted, however, that by far the commonest type of synapse upon spines is the asymmetrical (or type 1 of Gray), whereas on the axon hillock the synapses are invariably symmetrical (or type 2 of Gray). Furthermore, the spine apparatus may occasionally be found in dendrites, and there also it is associated with an asymmetrical synapse. As has been emphasized previously (Laatsch & Cowan, 1966; Palay et al. 1968; Peters et al. 1968), the axon hillock is only relatively deficient in ribonucleoprotein, because a small number of free and attached ribosomes are invariably present here. Some of these extend into the initial segment itself and often accumulate beneath synaptic contacts and at points of branching, both from the initial segment and from nodes of Ranvier. The axon hillocks of small pyramidal neurons and of other neurons in the cortex, in being smaller, are far less distinctive than those of large pyramidal neurons. Similarly, in the initial segments of axons arising from these smaller hillocks, fewer linked clusters of neurotubules are present, although the same dense material appears beneath the plasma membrane. Palay et al. (1968) have suggested that the cross-linked neurotubules may be contractile and serve to pump material down the axon. As the size of a cell soma is generally proportional to the amount of axoplasm, the number of

5 Axon hillocks and initial axon segments 499 bundles of linked neurotubules in the initial segment may be a function of the length and degree of branching of the axon. This raises the possibility that many interneurons and other short axon cells may lack such bundles of neurotubules. Some of the initial segments observed in the present study gave off small side branches close to the axon hillock. None of these could be traced for long distances, but there seems little doubt that these are the collaterals which have been known for many years from light-microscopic studies (Ramon y Cajal, 1911). Their origin so close to the parent soma appears unusual, but is not unique to the cortex for some collaterals have been observed arising from similar positions of neurons in thalamic nuclei (Jones & Powell, 1969a). Perhaps the most significant finding of the present study is that large numbers of axon terminals may make synaptic contact upon the initial segment and axon hillock. Some of these may even surround the initial segment at its point of emergence from the axon hillock. The functional significance of synapses upon this region, from which impulses arise, is of considerable interest, and there is evidence that synapses on the initial segments of Purkinje cells are inhibitory (Eccles, Ito & Szentagothai, 1967). In the present aldehyde-fixed material most of the terminals ending on the axon hillock and initial segment contain a high proportion of synaptic vesicles which are flattened or pleomorphic. Moreover, they invariably end in symmetrical synaptic complexes. Similar observations have been made by E. G. Gray (personal communication) in the spinal cord of the goldfish. The association of flattened vesicles and symmetrical synaptic thickenings with known inhibitory synapses in the cerebellum (Uchizono, 1967; Larramendi, Fickenscher & Lemkey-Johnston, 1967) has raised the possibility that these two features may be the morphological basis for inhibitory synapses in other parts of the central nervous system (Bodian, 1966; Colonnier, 1968); there are, however, reasons for caution in too readily accepting this relationship (Walberg, 1968). Peters et al. (1968) also found synapses to be quite common upon the axon hillock and initial segment but, while their description and illustrations of the membrane thickenings of these synapses would conform with what has been termed symmetrical, these authors found the vesicles within the terminals to be spherical. As little is known about the factors affecting the shape of synaptic vesicles one can only speculate about the possible reasons for this difference. The most probable explanation is the different aldehyde mixtures used in the two studies, and particularly the relatively greater concentration of paraformaldehyde in the present investigation. Another factor may be the age of the animals, as Larramendi et al. (1967) have found that in the cerebellum the proportion of flattened vesicles increases with age; the present material was taken from adult cats whereas Peters et al. (1968) used young rats. The proportion of flattened vesicles found in these endings varied from brain to brain, but the association of such vesicles with symmetrical membrane thickenings, which was first recognized by Colonnier (1968) in the visual cortex of the cat, has been confirmed in this study of the somatic sensory area. The origin of the fibres terminating upon the axon hillock and initial segment is not known, but from other experimental studies there is indirect evidence to suggest that they are intrinsic to the cortex. In experiments involving selective interruption of 32-2

6 500 E. G. Jones and T. P. S. Powell the main extrinsic afferent fibres to the somatic sensory cortex (Jones & Powell, 19696) it was found that these fibres all terminate on dendritic spines or shafts and in asymmetrical synaptic thickenings. Even after undercutting a topographic subdivision of the first somatic sensory area no degenerating endings were found on axon hillocks, so that from the available evidence it is probable that the fibres ending on the hillock and initial segment have a local origin. No axon terminals were observed on the preterminal unmyelinated segments of myelinated axons in the cerebral cortex despite the common occurrence of quite long segments of this type. Westrum (1966) noted that in the prepyriform cortex of the rat bare segments of myelinated axons receive axon terminals containing flattened or pleomorphic vesicles and ending in symmetrical thickenings. He was unable to determine, however, whether the recipient axons were initial or preterminal segments. The present evidence would suggest that all were initial segments. The somatic sensory cortex appears to be the only part of the somatic sensory pathway from which axo-axonic synaptic contacts upon axon terminals or preterminal segments are absent, as synapses of this type are common at the spinal (Gray, 1961; Ralston, 1968; Heimer & Wall, 1968; Walberg, 1965) and thalamic (Jones & Powell, 1969a) relay sites, in both of which presynaptic inhibition has been demonstrated (Eccles, 1964). This work was supported by grants from the Medical and Science Research Councils, and was done during the tenure of a Nuffield Dominions Demonstratorship by E.G.J. on leave from the University of Otago, New Zealand. REFERENCES BODIAN, D. (1966). Electron microscopy: two major synaptic types on spinal motoneurons. Science, N.Y. 151, COLONNIER, M. (1968). Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res. 9, ECCLES, J. C. (1964). The Physiology of Synapses. Berlin: Springer-Verlag. ECCLES, J. C, ITO, M. & SZENTAGOTHAI, J. (1967). The Cerebellum as a Neuronal Machine. Berlin: Springer-Verlag. GRAY, E. G. (1961). A morphological basis for presynaptic inhibition? Nature, Lond. 193, HEIMER, L. & WALL, P. D. (1968). The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat. Expl Brain Res. 6, JONES, E. G. & POWELL, T. P. S. (19690). Electron microscopy of synaptic glomeruli in the thalamic relay nuclei of the cat. Proc. R. Soc. B 172, JONES, E. G. & POWELL, T. P. S. (19696). An electron microscopic study of the laminar pattern and mode of termination of afferent fibre pathways to the somatic sensory cortex of the cat Phil. Trans. R. Soc. B (in the Press). LAATSCH, R. H. & COWAN, W. M. (1966). Electron microscopic studies of the dentate gyrus of the rat. I. Normal structure with special reference to synaptic organization.^, comp. Neurol. 128, LARRAMENDI, L. M. H., FICKENSCHER, L. & LEMKEY-JOHNSTON, N. (1967). Synaptic vesicles of inhibitory and excitatory terminals in the cerebellum. Science, N. Y. 156, PALAY, S. L., SOTELO, C, PETERS, A. & ORKAND, R. N. (1968). The axon hillock and initial segment.,7. Cell Biol. 38, PETERS, A., PROSKAUER, C. C. & KAISERMAN-ABRAMOF, I. R. (1968). The small pyramidal neuron of the rat cerebral cortex. The axon hillock and initial segment. J. Cell Biol. 39,

7 Axon hillocks and initial axon segments 501 RALSTON, H. J. (1968). The fine structure of neurons in the dorsal horn of the cat spinal cord. J. comp. Nenrol. 132, RAMON Y CAJAL, S. ( ). Histologie du systbne nervenx de VHomme et des Vertibris. Paris: Maloine. REYNOLDS, E. S. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, SAMORAJSKI, T., ORDY, J. M. & KEEFE, J. R. (1965). The fine structure of lipofuscin age pigment in the nervous system of aged mice. J. Cell Biol. 26, UCHIZONO, K. (1967). Synaptic organization of the Purldnje cells in the cerebellum of the cat. Expl Brain Res. 4, WALBERG, F. (1965). Axoaxonal contacts in the cuneate nucleus, a probable basis for presynaptic depolarization. Expl Neurol. 13, WALBERG, F. (1968). Morphological correlates of postsynaptic inhibitory processes. In Structure and Function of Inhibitory Neuronal Medianisms (ed. C. von Euler, S. Skoglund & U. Soderberg), pp Oxford: Pergamon. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biocliem. Cytol. 4, WESTRUM, L. E. (1966). Synaptic contacts on axons in the cerebral cortex. Nature, Lond. 210, (Received 15 January 1969)

8 502 E. G. Jones and T. P. S. Powell All the electron micrographs are from material stained with lead citrate or with lead citrate and uranyl acetate. Fig. i. The perikaryon of a large pyramidal neuron in layer V of the somatic sensory cortex showing the initial segment (a) of its axon arising from a typical axon hillock (h) at the basal aspect of the cell. The arrows indicate axon terminals which make synaptic contact with the hillock and initial segment, x Fig. 2. The initial segment of the axon illustrated in Fig. 1, showing the linked neurotubules (small arrows) typical of this structure and an axon terminal (large arrow) ending on it. Note also the small dense bodies in the axon and the dense material beneath the plasma membrane, x

9 Axon hillocks and initial axon segments

10 504 E. G. Jones and T. P. S. Powell Figs Three electron micrographs which overlap at the short vertical lines, showing the hillock and initial axon segment of a pyramidal neuron. Both the hillock and initial segment receive axon terminals which contain flattened or pleomorphic vesicles and terminate in symmetrical synaptic contacts (arrow heads and inset). As well as containing the typical linked neurotubules and dense bodies, the initial segment contains 3 clusters of cisternae and dense material resembling a spine apparatus (small arrows and inset). Figs. 3-5, x 15000; inset, x

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12 5o6 E. G. Jones and T. P. S. Powell Fig. 6. A typical initial axon segment receiving 6 axon terminals all of which contain a high proportion of flattened synaptic vesicles and terminate symmetrically (arrows), x Fig. 7. A small branch arising from the node of Ranvier of a myelinated axon in the somatic sensory cortex. Note the dense material (small arrows) beneath the bare portion of the plasma membrane and the small collection of ribosomes (large arrows) at the point of branching, (g, astroglial processes; m, common myelin sheath of the parent axon and its branch.) x Fig. 8. A short side branch (arrows) of an initial segment beneath the origin of which is a small cluster of ribosomes (arrow head), x Fig. 9. A little distal to the point on the initial segment shown in Fig. 8, an axon terminal containing flattened vesicles terminates in a symmetrical synaptic contact upon the initial segment (arrow head). Beneath the site of synaptic contact, the dense material beneath the plasma membrane is deficient (small arrows) and there is a further small aggregation of ribosomes (double arrows), x

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