J. Anat., Lond. (1963), 97, 3, pp. 389-392 389 With 3 plates Printed in Great Britain A note on the dendritic spine apparatus By E. G. GRAY AND R. W. GUILLERY Anatomy Department, University College, London Many of the synapses in the mammalian cerebral cortex are formed by axonal contacts upon dendritic spines. The cytoplasm of these spines often contains a structure that has been called the 'spine apparatus' (Gray, 1959a, b). It consists of a series of membrane bound sacs separated by sheets or plates of dense material, and has been described in the mammalian hippocampus (Hamlyn, 1961, 1962) and in the neocortex of a number of mammalian species (Pappas & Purpura, 1961; Rosenbluth, 1962), including man (M. Kidd, personal communication). Although dendritic spines occur in many parts of the vertebrate nervous system (see Cajal, 1911) past electron microscopical studies of these synaptic regions suggest that the spine apparatus may be unique to the mammalian cortex (Gray, 1961 a, b, 1962 a; Boycott, Gray & Guillery, 1961). In view of its known location and distribution it has been reasonable to regard the apparatus as specifically synaptic in function, and to consider it in particular related to the specialized functions of the mammalian cerebral cortex (see Hamlyn, 1962). It was thus of interest to find structures identical to the spine apparatus in the basal parts of apical dendrites of dog sensori-motor cortex and also within the dendrites of dorsal horn cells of rat and cat spinal cord. The following is a brief report of these observations.

METHODS Small pieces of a dog's sensori-motor cortex, the visual cortex of rats at various intervals after birth, and the dorsal horn of the lumbar region of the rat's and cat's spinal cord were removed under anaesthesia and fixed in 1 % osmium tetroxide at pH 7*4. Ethanol was used for dehydration, followed by staining in 1 % phospho- tungstic acid in absolute ethanol. The pieces were embedded in Araldite for section- ing (see Gray, 1959b, for details). OBSERVATIONS Sensori-motor cortex of adult dog. Electron microscopy of this region showed numerous spine synapses, many with the characteristic spine apparatus in the post- synaptic cytoplasm. In addition, structures resembling the spine apparatus were seen in the thick basal regions of some dendrites. P1. 1, fig. 1, shows the basal region of a dendrite. The cytoplasm contains mitochondria and a general background of dendritic tubules orientated along the main axis, which is here turning out of the plane of the section. Three structures (S1, S2 and S.) lie parallel to the tubules. S, consists of three sacs alternating with two dense plates. S2 and S3 each consists of two sacs flanking a single plate. Two of these structures (S: and S3) differ from the apparatus found in dendritic spines by showing particles, presumably ribosomes (r) close to the sac membranes. However, ribosomes also occur in small groups else- where in the cytoplasm of this dendrite, whereas they are never found in the spine 390 E. G. GRAY AND R. W. GUILLERY cytoplasm. PI. 2, fig. 2, shows a second example. Ribosomes occur in masses along the margin of the dendrite and also in relationship to one of the sacs (x). A mem- brane (y) can be seen apposed to the other side of the dense plate, but there is not a complete sac, possibly because of oblique sectioning. A further example (P1. 3, fig. 4) shows only a few ribosomes close to the sac membrane, and here a few fine filaments are apparently associated with each end of the structure. This apparatus is one of two similar structures, which form an axial chain in the dendrite, and are joined by filaments (P1. 3, fig. 5). Dorsal horn of spinal cord. After extensive examination of the dorsal horn of the rat and cat a few structures (nine to date) with the characteristics of a spine. apparatus have been seen. PI. 3, fig. 3 (S) shows two sacs flanking a single plate. These lie within a process that cannot be definitely identified as a dendrite or axon. In the dorsal horn, axons with tubules can be seen frequently, and a number of synapses show vesicles on both sides of the asymmetrical membrane thickening (cf. Gray, 1962b). Thus it is not possible to say that the spine apparatus is confined to dendrites in the dorsal horn, but it has never been seen in an indubitable axon. PI. 3, fig. 6 shows a similar structure, but here the characteristics of the synaptic region, with synaptic vesicles and pronounced post-synaptic thickening, suggest that the structure lies in a thin dendrite, or its spine. None of the examples that have been found have shown more than three sacs alternating with two plates, in contrast to the more complex forms that are common in the cerebral cortex. The precise location of these structures within the dorsal horn remains uncertain at present, although they probably lie deeper than the substantia gelatinosa, where, so far, they have never been observed.

DISCUSSION Since the structures that have been described, clearly resemble the spine appara- tus, and since they have been found in portions of dendrites that bear no spines, it may be held that the term 'spine apparatus' is no longer appropriate. However, observations to date show that the apparatus occurs with greatest frequency in the dendritic spines, and reaches its greatest complexity there. No useful purpose can be served by renaming the structure until more is known of its function. A study of the development of the spine apparatus in ontogeny has not yet proved very rewarding. In the rat's visual cortex the spine apparatus can be first seen at 16 days after birth, and at 32 days it is seen frequently. Between 16 and 32 days the apparatus often lies in the dendritic trunks, and is simple in form, resembling that found in the dorsal horn (PI. 3, figs. 3, 6). Since the dendritic trunks of adult rats show no spine apparatus it is possible that the apparatus migrates towards the spines during post-natal development, and concurrently becomes more complex, so that in the adult four or more sacs may alternate with three or more plates. It is possible that the structures seen in the dog's sensori-motor cortex were destined to migrate towards the spines and to become more complex. There is no doubt a difference between the sensori-motor cortex of the adult dog and the visual cortex of the adult rat in the distribution of the spine apparatus. Whether this is a species difference or a difference between cortical areas remains to be determined. It is possible that the difference is simply related to cell size. A note on the dendritic spine apparatus 391 The spine apparatus has never been seen in an indubitable axon. This and the fact that it occurs most commonly and in its most complex form in the spine cytoplasm suggests that it plays a special role in the activity of the post-synaptic region.

SUMMARY 1. Structures resembling the spine apparatus but in addition having associated ribosomes occur in the basal regions of dendrites of the sensori-motor cortex of the dog. 2. Structures resembling simple forms of the spine apparatus also occur in small dendritic trunks of the lumbar dorsal horn of the spinal cord of the cat and rat.

We are indebted to Prof. J. Z. Young, F.R.S., for advice, to Mr K. Watkins for technical assistance and Mr J. Hustwaithe for photography.

REFERENCES BoycoTT, B. B., GRAY, E. G. & GUILLERY, R. W. (1961). Synaptic structure and its alteration with environmental temperature: a study by light and electron microscopy of the central ner- vous system of lizards. Proc. Roy. Soc. B, 154, 151-172. CAJAL, S. R. (1911). Histologie du Systkme Nerveux de l'Homme et des Vertebres, Vols. i and ii. Paris: A. Maloirne. GRAY, E. G. (1959a). Electron microscopy of synaptic contacts on spines of dendrites of the cerebral cortex. Nature, Lond., 183, 1592-1593. GRAY, E. G. (1959b). Axosomatic and axodendritic synapses of the cerebral cortex: an electron microscope study. J. Anat., Lond., 93, 420-433. GRAY, E. G. (1961 a). Ultrastructure of synapses of the cerebral cortex and of certain specialisa- tions of neuroglial membranes. In Electron Microscopy in Anatomy (ed. Boyd et al.). London: Edward Arnold. GRAY, E. G. (1961 b). The granule cells, mossy synapses and Purkinje spine synapses of the cere- bellum: light and electron microscope observations. J. Anat., Lond., 95, 345-356. GRAY, E. G. (1962 a). Electron microscopy of synaptic organelles of the central nervous system. In lVth International Congress of Neuropathology, Munchen, 1961 (ed. Jacob). Stuttgart: Georg Thieme. GRAY, E. G. (1962b). A morphological basis for presynaptic inhibition? Nature, Lond., 193,82-83. HAMLYN, L. H. (1961). Electron microscopy of mossy fibre endings in Ammon's Horn. Nature, Lond., 190, 645-646. HAMLYN, L. H. (1962). The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J. Anat., Lond., 96, 112-120. PAPPAS, G. D. & PURPURA, D. P. (1961). Fine structure of dendrites in the superficial neocortical neuropil. Exp. Neurol. 4, 507-530. ROSENBLUTH, J. (1962). Subsurface cisterns and their relationship to the neuronal plasma membrane. J. Cell Biol. 13, 405. 392 E. G. GRAY AND R. W. GIUILLERY

EXPLANATION OF PLATES PLATE 1 Fig. 1. Basal region of dendrite of sensori-motor cortex of adult dog.

PLATE 2 Fig. 2. Basal region of dendrite of sensori-motor cortex of adult dog.

PLATE 3 Fig. 3. Region of dorsal horn of cat's lumbar cord, showing a spine apparatus. Fig. 4. Central portion of a basal region of a dendrite of the sensori-motor cortex of adult dog, containing spine apparatus. Fig. 5. Low-power view showing structure described in fig. 4 in linear relationship with a second spine apparatus. Fig. 6. Synapse of dorsal horn of cat's lumbar cord with postsynaptic spine apparatus.

Key f = filaments (neurofilaments?); m = mitochondrion; r = ribosomes; S = spine apparatus, sv, = synaptic vesicles; x, y, = sacs of spine apparatus; unlabelled arrows = surface membrane of dendrite. Journal of Anatomy, Vol. 97, Part 3 Plate 1

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