Brain Research, 105 (1976) 547-550

547

© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

Microtubules and membrane specializations*

LESNICK E. WESTRUM AND E. G. GRAY Departments of Neurological Surgery and of Biological Structure, University of Washington, Seattle, Wash. 98195 (U.S.A.) and Deportment of Anatomy, University College London, London WC1E 6BT (Great Britain)

(Accepted January 5th, 1976)

Gray 5-7 has recently introduced a method of treatment of fresh nervous tissue with albumin solution prior to fixation in osmium tetroxide. The technique shows microtubules to greater advantage than conventional methods of processing. It appears that the protein solution stabilizes microtubules, which may otherwise be more labile, using standard fixation procedures. With this improved preservation of microtubules, Gray has shown at the synapse a clear association of microtubules with synaptic vesicles and with dense projections (presynaptic specializations on the terminal membrane)5, 7. We present here preliminary observations which suggest that microtubules may also be associated with postsynaptic 'thickenings' and other specializations of membranes. Brain from adult and neonatal rats (1-5 days old) was prepared according to the albumin-osmium-glutaraldehyde-uranyl method of GrayL The fixation procedure is based on Kanaseki and Kadota s. The material is primarily taken from the olfactory and neocortex and the neonatal material was included as part of a larger study o f synapse formation in the region 16. Control preparations were processed similarly, but excluding the albumin pretreatment and also processed by conventional methods of fixation in buffered osmium or aldehydes. A goniometer stage was used to obtain various optimal views of sections. Appropriately preserved regions show variable numbers of microtubules in both axonal and dendritic profiles. In the presynaptic terminals the arrangement of the microtubules is similar to that described by Gray 5-7, but it is less common in the neonatal material where terminals have fewer synaptic vesicles than in mature preparations. It is of special interest that postsynaptic dendrites are seen, in both adult and neonatal tissue, to have one or more microtubules in close proximity to the 'thickening' or near a mature or possibly developing contact (Figs. 1, 2 and 3). Sometimes the microtubules appear to be closely related to regions of the postsynaptic membrane * Reprint requests should be sent to: Dr. L. E. Westrum, Department of Neurological Surgery, University of Washington, RI-20, Seattle, Wash. 98195, U.S.A.

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549 where the postsynaptic 'thickening' develops. Fragmented microtubules are more frequently seen in tissue not treated with albumin before fixation (Fig. 3). In the initial segments of axons in either neonatal or adult preparations, microtubules are frequently seen immediately adjacent to, attached to, or embedded in the trilaminar undercoating of the surface membrane (Figs. 4-6). This is particularly obvious in transverse sections (Figs. 5 and 6) viewed with optimal tilt. Occasionally some of the cross-sections of the 'linked' microtubules appear as incomplete semicircles, lacking parts of the cylinder, even when tilted through wide angles (Fig. 6). The observations show a distinct proximity and possibly a meaningful association between microtubules and two sorts of membrane specializations. The relationship of microtubules to postsynaptic 'thickenings' is particularly noteworthy as there is substantial recent biochemical and immunocytochemical evidence that the main postsynaptic junctional protein is tubulin1,10,14,15. Thus, microtubules may play an important role in the formation and maintenance of the postsynaptic 'thickening' and hence in the way pre- and postsynaptic membranes become related to each other in ontogeny. In the past, using conventional methods, there has been no good evidence for the association of microtubles with the postsynaptic 'thickening', Here we are suggesting that the enigmatic dense material of the 'thickening' is, at least in part, derived from the debris of in vivo microtubules (see also Gray4). The axon initial segment characteristically contains numerous microtubulesa, 9, 1~,13,17 and possesses a unique undercoating of the plasma membrane, the features of which have been described in detail by Chan-Palay 2. The association in fish brain initial segments between the nearby microtubules and the undercoating was mentioned briefly by Nakajima 11, but she did not pursue the subject. Our results show clearly a more intimate association between initial segment microtubules and subsurface undercoating than has previously been described and since fragmentation of the microtubules can be observed, it is possible that the initial segment undecoating is composed, at least in part, of microtubule material. In this regard, it would be interesting to apply the antibody technique of Matus et al. ~o to initial segment undercoating.

Fig. 1. Olfactory cortex of a 5-day-old rat. A microtubule abuts against a postsynaptic contact site at the larger arrow. A nearby postsynaptic 'thickening' (t) has a fragment of a microtubule indicated at the small arrow suggestinga transition of microtubules to postsynaptic 'thickening'. Albumin-treated. Scale equals 0.25 pm throughout. Fig. 2. Same region from an adult rat to show one or more microtubules (m) embedded within the postsynaptic 'thickening' at large arrow and possibly fragmented microtubules at small arrow. Albumin-treated. Fig. 3. Similar material as in Fig. 1, but without albumin pretreatment, to show disintegrating microtubules (m) related to the site of an axonal contact (arrow). Fig. 4. Initial segment of an axon in olfactory cortex from a 5-day-old rat, showing the close proximity of microtubules (m) with the undercoating (arrows) in longitudinal section. Albumin-treated. Fig. 5. Initial segment of an axon in olfactory cortex of an adult rat, in transverse section, to demonstrate microtubules attached to or embedded in the undercoating (arrows). Albumin-treated. Fig. 6. Similar preparation to Fig. 5 to show a microtubule embedded in undercoating (large arrow) and three disrupted microtubules (small arrows) nearby.

550 This w o r k was s u p p o r t e d by N a t i o n a l Institutes o f H e a l t h G r a n t s N S 09678 and N S 04053, a w a r d e d by the N a t i o n a l Institute o f N e u r o l o g i c a l an d C o m m u n i c a t i v e Di s o rd er s a n d Stroke, P H S / D H E W a n d a g r an t f r o m the British Med i cal R e s e a r c h Council. Th e au t h o r s gratefully ackowledge this su p p o r t and also wish to t h a n k Mrs. H. S a m s o n fo r valuable technical c o l l a b o r a t i o n and A n n Harris for secretarial help.

1 BANKER,G., CHURCHILL,L., AND COTMAN,C. W., Proteins of the postsynaptic density, J. Cell BioL, 63 (1974) 456-465. 2 CHAN-PALAY,V., The tripartite structure of the undercoat in initial segments of Purkinje cell axons, Z. Anat. EntwickL-Gesch., 139 (1972) 1-10. 3 GRAY, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study, J. Anat. (Lond.), 93 (1959) 420-433. 4 GRAY, E. G., Synaptic fine structure and nuclear, cytoplasmic and extracellular networks, J. NeurocytoL, 4 (1975) 315-339. 5 GRAY,E. G., Presynaptic microtubules and their association with synaptic vesicles, Proc. roy. Soc. B, 190 (1975) 369-372. 6 GRAY, E. G., Microtubules in synapses of the retina, J. Neuroo,tol., in press. 7 GRAY, E. G., Problems in understanding synaptic substructure. In Progr. Brain Res., Elsevier, Amsterdam, in press. 8 KANASEKI,T., AND KADOTA,K., The vesicle in a basket, J. Cell Biol., 42 (1969) 202-220. 9 KOHNO, K., Neurotubules contained within the dendrite and axon of Purkinje cell of the frog, Bull. Tokyo reed. dent. Univ., ll (1965) 411-442. 10 MATUS,A. I., WALTERS,B. B., AND MUGHAL,S., Immunohistochemical demonstration of tubulin associated with microtubules and synaptic junctions in mammalian brain, J. Neurocytol., 4 0975) 733-744. 11 NAKAJIMA,Y., Fine structure of the synaptic endings on the Mauthner cell of the goldfish, J. comp. Neurol., 156 (1974) 375-402. 12 PALAY,S. L., SOTELO,C., PETERS,A., AND ORKAND,P. M., The axon hillock and the initial segment, J. CellBioL, 38 (1968) 193-201. 13 PETERS,A., PALAY, S. L., AND WEBSTER, H. DE F., The Fine Structure q/'the Nervous System. The Cells and their Processes, Hoeber-Harper and Row, New York, 1970. 14 WALTERS,B. B., AND MATUS, A. I., Proteins of the synaptic junction, Biochem. Soc. Trans., 3 (1975) 109-112. 15 WALTERS,B. B., AND MATUS,A. I., Tubulin in postsynaptic junctional lattice, Nature (Lond.), 257 (1975) 496-498. 16 WESTRUM,L. E., Electron microscopy of synaptic structures in olfactory cortex of early postnatal rats, J. NeurocytoL, 4 (1975) 713-732. 17 WESTRUM,L. E., Observations on initial segments of axons in the prepyriform cortex of the rat, J. comp. NeuroL, 139 (1970) 337-356.

Microtubules and membrane specializations.

Brain Research, 105 (1976) 547-550 547 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands Microtubules and membrane sp...
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