Structure and function in synapses: emerging correlations

TINS- June 1986

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peripheral process extending along the lateral line, innervating neuromasts. Metcalfe has studied the development of the posterior lateral line in zebra fish, confirmed that it resembles the am- Recent observations on membrane structure at neuromuscular junctions of phibian case, and has uncovered an vertebrates and arthropods suggest that a class of prominent membrane particles at elegantly simple plan of development. the active zone, tentatively idenafied as calcium channels, can be reliably discerned. He used scanning EM and light These particles are packed more densely at synapses which are thought to release microscopy with HRP for tracing axons, more transmitter, and in some instances occur in greater numbers at such synapses. A and confined his observations to the 'structure-function' hypothesis derived from these observations is that the period 18--48 hours post-fertilization. probability o f release of a quantal unit of transmitter (synaptic vesicle?) is enhanced The migratory primordium was visible by having more membrane calcium channels close to the site at which transmitter is on the animal's flank as a bump that released. Variation in performance among different synapses could be determined b~ progressed from the head to the tip of part by the pattern of distribution of presynaptic calcium channels. the tail, a distance of about 2 mm, in 22 hours. The growth cones of the Behavioural acts are regulated by Peripheral synapses (especially presumptive ganglion cells chased the synapses, both central and peripheral. neuromuscular junctions) are easiest to migrating cells; indeed, one of the The characteristics of many responses study in this correlative fashion. They growth cones was often in among the can be attributed to properties of provide a wide range of synaptic migratingcells, with others strung along synapses involved in the neural net- performance, are accessible for detailed behind. Thus, from the beginning, the works governing the response. Thus, a ultrastructural investigation, and are neuritic link from ganglion cell to research goal of continuinginterest is to important in locomotion and behavpresumptive neuromast is present. The elucidate the factors that determine iour, being the final necessary synaptic component of the motor system. neurites do not have to find their target; synaptic performance. One approach to the problem is to Neuromuscular junctionsof arthropods instead, they travel with it and lay down the cable that later neurites will follow. examine synaptic ultrastrncture and to are very diverse in performance, and It is possible that this may prove to be see whether function can be correlated offer good models for the less accessible the general pattern for the development with it. To be successful and convincing, central synapses. Vertebrate neuroof placodally derived structures in- this approach demands that ultrastruc- muscular junctions, though generally tural information be obtained from the specialized for high output of transnervated by ganglion cells. These three diverse topics: restriction same synapses used to provide measure- mitter, also show considerable diversity of cell fates, axonal pathfinding by ments of physiological efficacy. Few in transmitter-releasingproperties, and motor neurons, and development of the investigations have met this test at a provide good material for freezelateral line, have all been impressively truly rigorous level. However, recent fracture investigations of membrane clarified by these careful studies on the studies of both vertebrate 1 and in- structure. In their studies of neuromuscular zebra fish. It seems likely that the vertebrate 2 synapses have provided favorable preparation and the elegant new information on membrane struc- junctions of lizard (Anolis) intercostal techniques will continue to increase our ture which could lead to a more muscles, Walrond and Reese 1 comunderstanding of old problems and our complete understanding of how syn- pared junctions on tonic muscle fibers with those on twitch (phasic) muscle aptic function depends on structure. awareness of new ones. fibers. Neuromuscular transmission in twitch fibers involves release of enough • g ••Be• transmitter to drive the membrane Selected references potential past the threshold for generation of a propagating muscle action 1 Kimmel, C. B. and Warga, R. M. (1986) potential. In tonic fibers of amphibians Science 231,365-368 2 Eisen, J. S., Myers, P. Z. and Westerfield, M. and reptiles surnmatingjunction poten20nm (1986) Nature 320,269-271 tials, rather than action potentials, 3 Myers, P. Z., Eisen, J. S. and Westerfield, M. control contraction, and the junctional J. Neurosci. (in press) output of transmitter for a single 4 Westerfield, M., McMurray, J. V. and Eisen, presynaptic action potential is about 10 J. S. J. Neurosci. (in press) times less3. There is evidence for 5 Lance-Jones, C. and Landmesser, L. (1981) " " ' structural features which correlate with Proc. R. Soc. London Ser. B. 214, 1-18 the difference in transmitter output. 6 Hollyday, M. (1980) Curt. Top. Dev. Biol. 15, At vertebrate neuromuscular junc181-215 Fig. 1. Arrangement of prominent membrane tions, ultrastructural studies have given 7 Raper, J., Bastiani, M . J . and Goodman, particles (putative calcium channels) at active C. S. (1983) J. Neurosci. 3, 3 1 4 1 rise to the concept of the active zone, zones of lizard tonic (A) and phasic 01), frog phusic characterized by a double row of 8 Metcalfe, W. (1985) J. Comp. Neurol. 238, (C), and rat (D) neuromuscular junctions (after 218-224 prominent membrane particles on the Walrond and Reesel ). Active zone particles (small solid circles) and associated synaptic vesicles (large transmitter-releasing surface of the hatched circles) are drawn to scale. The double nerve terminal, together with associarray of particles at lizard phasic (B) and rat phusic ated intracellular synaptic vesicles and STEPHEN S. EASTER , JR. (D) neuromuscular junctions could provide more presynaptic 'dense bodies' or 'dense intense calcium entry at the site of a synaptic vesicle, Division of Biological Sciences, University of andconsequentlyagreaterprobabilityofreleaseof projections'4. If one accepts the hypothesis, supported by a large body of Michigan, Ann Arbor, MI 48109, USA. the vesicle. Scale bar is 20ran.

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TINS-June 1986 work at the neuromuscular junction (e.g. Ref. 5), that nerve-evoked transmitter release is mediated by exocytosis of synaptic vesicles6 (though this concept remains in dispute7), there is good reason to believe that the membrane particles at the active zone are calcium channels, and that the clustered synaptic vesicles are the morphological counterpart of the readily releasable pool of transmitter. Evidence for the identity of' active zone' membrane particles as calcium channels comes from quantitative comparison at the squid giant synapses . In this work, it was found that the number of prominent membrane particles at release zones (putative calcium channels) matched the number of calcium channels calculated from electrophysiological measurements of calcium currents. Entry of calcium is necessary for phasic release, and the short time between calcium entry and release of transmitter dictates that only vesicles within about 50 nm of the site of calcium entry can be involved in immediate release s'9. Active zones at 'tonic' neuromuscular junctions in the lizard have single arrays of paired membrane particles. In contrast, 'phasic' neuromuscular junctions have double arrays (Fig. 1). The latter feature is found also in mammalian neuromuscular junctions, while the more modest single array is characteristic of frog neuromuscular junctions. Quantitative analysis of the number of release sites and approximate values for synaptic vesicle release at these neuromuscular junctions showed t h a t for a single nerve impulse, a higher proportion of releasable synaptic vesicles would be expected to participate in transmission at the 'phasic' junction (6%) than at the tonic junction (0.4%). The numbers of active zones per terminal, and the total numbers of active zone particles, are somewhat larger for the 'phasic' junction, but only by a factor of about 1.5. In itself, this cannot account for the 10-fold difference in transmitter output or the 15fold difference in probability of release of a synaptic vesicle, even if the traditional fourth-power relationship between incoming calcium and transmitter release is invoked 1°. The greater density of active zone particles near releasable vesicles provided by the double array configuration (Fig. 1) could, however, provide a higher density of calcium near the vesicle during a nerve impulse, and this could underlie the higher probability of

249 transmitter release at phasic neuromuscular junctions. Recent models of transmission have been developed by Simon et al. H, and by Fogelson and Zucker 12, in which overlapping 'calcium domains' around individual calcium channels~3 are postulated to account for increased transmitter release. Such a model can account for a higher density of calcium ions at release zones of the phasic neuromuscular j unction. If this hypothesis can be more firmly substantiated, an important structural feature governingtransmitter release would be the precise arrangement of calcium channels at the active zone. Although this hypothesis is attractive, it must be borne in mind that the evidence is still not as conclusive as it could be. There has not been a direct measurement of transmitter release (quantal content) at identified lizard neuromuscular junctions to provide a direct correlation with the morphological studies. Figures for quantal content were taken from other preparations. Some attempts at a more direct correlation have been made at frog neuromuscular junctions, which differ considerably in the amount of transmitter released by a single impulse, even among apparently similar twitch muscle fibers 14. Quantal content of transmission (measured at low external Ca 2÷) varies in a roughly linear fashion with length of active zone along the junctional terminal1s'16. These results, though preliminary, are in the direction expected from the structure-function hypothesis. An assumption in this work is that the physiological measurements made in subnormal calcium can be applied to the normal operating conditions of the junction. Neuromuscular junctions on fastacting (phasic) and slow-acting (tonic) muscles of an insect (the moth Manduca sexta) have been analysed at the ultrastructural level in an attempt to explain large differences in transmitter release 2. The excitatory j unction potentials of tonic muscle fibers are only 20 to 30% of the amplitude of those in phasic muscle fibers. This reflects a difference in amount of transmitter released per nerve impulse. (The exact values for quantal content are not yet available, and the numbers of junctionsper muscle fiber are not specified.) Each nerve terminal innervating a muscle fiber bears a number of discrete synapses, each typically with a single relatively small active zone. The well-

organized linear arrays of particles characteristic of vertebrate active zones are not apparent; instead, less organized clusters (roughly linear in shape) are seen. Neuromuscular junctions on tonic muscle fibers have about one third the number of synapses found on terminals innervating phasic muscle fibers, and about half the number of particles per active zone. Thus, over the junction as a whole, there are about six times as many active zone particles for phasic junctions. This result lends additional support to the hypothesis that number and density of active zone particles may be one determinant of transmitter release; but here again, the available evidence does not yet permit a tight correlation. Crustacean neuromuscular junctions are well known for diversity of physiological response 17. Individual synapses of terminals that release relatively large amounts of transmitter per impulse are larger, and have larger active zones, than those of low-output terminalsts. The observed correlation is in qualitative, but not quantitative agreement with the structure-function hypothesis 19.However, the distribution of active zone particles has not yet been determined for crustacean synapses of known quantal content. The evidence for a strong correlation between transmitter output for a single nerve impulse and membrane ultrastructural features has yet to be obtained. An interesting feature of crustacean high-output and low-output terminals is that there are substantially more individual synapses on the latter 19. Also, although transmitter output for a single nerve impulse is much less for a low-output terminal, it equals or exceeds that of the high-output terminal when a high frequency of nerve impulses arrives2°. The powers of facilitation are evidently much greater for low-output terminals. A tentative explanation is that most synapses are ineffective at releasing transmitter for a single impulse (perhaps due to a lower density of active zone particles), but become capable of release as Ca 2+ accumulates within the terminal at a high frequency of nerve impulses. Both insect and crustacean motor terminals are markedly varicose on tonic muscle fibers, and non-varicoseon phasic muscle fibers2'21. Synapses, mitochondria, and synaptic vesicles are preferentially associated with the varicosities on tonic fibers. Recent evidence21 suggests that the varicose structure, and perhaps also the distri-

250 bution of calcium channels, could be determined by the level of activity experienced by the motor neuron. Increasing the activity of a crustacean phasic motor neuron produces an increase in the motor terminal's mitochondrial volume. The phasic motor terminals become more varicose, with the synapses located mainly on the varicosities. Physiologically, the initial level of transmitter release declines, but ability to sustain release becomes much improved. Membrane particle distributions have not yet been determined for transformed and naive terminals. The likelihood that synapses on a single terminal differ in effectiveness, at least for crustacean neuromuscular junctions, raises the question of the possible non-equivalence of individual active zone particles. Do they all represent equally potent calcium channels? Could some be latent or inactive? Before a complete model can be put forward, this question must be addressed. The structure-function hypothesis remains elusive, but the recent observations on active zone ultrastructure point the way to a more complete resolution, which may eventually provide some insight into the meaning of structural changes of uncertain functional significance seen in the central nervous system under different conditions of activity and experience 22.

Selected references 1 Walrond, J. P. and Reese, T . S . (1985) J. Neurosci. 5, 1118-1131 2 Rheuben, M. B. (1985) J. Neurosci. 5, 17041716 3 Proske, V. and Vaughn, P. (1968) J. Physiol. (London) 199, 495-509 4 Couteaux, R. and Pecot-Dechavassine, M. (1974) C. R Acad. Sci. Ser. X. 280,299-301

5 Cohen, I. S. and Van der Kloot, W. (1983) J. Physiol. (London) 336, 335-344 6 Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, L. and Evans, L. (1979)J. CellBiol. 81, 275-300 7 Tauc, L. (1982) Physiol. Rev. 62, 857-893 8 Pumplin, D. W., Reese, T. S. and Llinas, R. (1981) Proc. Natl Acad. Sci. USA 78, 72107214 9 Llinas, R., Steinberg, I. Z. and Walton, K. (1981) Biophys. J. 33,323-352 10 Dodge, F. A. and Rahamimoff, R. (1967) J. Physiol. (London) 193,419--433 11 Simon,S., Sugimori,M. and Llinas, R. (1984) Biophys. J. 45,264a 12 Fogelson, A. L. and Zucker. R.S. Biophys. J. (in press) 13 Chad, J. E. and Eckert, R. (1984) Biophys. J. 45~ 993-999 14 Pawson, P. A. and Grinnel, A. D. (1984) Brain Res. 323,311-315

TINS - June 1986 15 Herrera, A. A., Grinnell, A.D. and Wolowske, B. (1985) J. Neurocytot. 14, 193202 16 Propst, J. W. and Ko, C. P. (1985) Soc. Neurosci. Abstr. 11,303 17 Atwood, H. L. (1982) The Biology of Crustacea Vol. 3. (Neurobiology: Structure and Function). (Atwood, H. L. and Sandeman, D. C., eds), pp. 105-150, Academic Press 18 Govind, C. K. and Chiang, R. G. (1979) Brain. Res. 161,377-388

19 Atwood, H. L. and Marin, L. (1983) ('ell Tissue Res. 231, 103-115 20 Bittner,G. D. (1968)J. Gen. Physiol. 51,731-

758 21 Atwood, H. L., Lnenicka, G. A. and Marin, L. (1985) J. Physiol. (London) 365, 26P 22 Greenough, W. T. (1984) Trend~NeuroSci. 7. 229-233 H. L. ATWOOD AND G. A. LNENICKA

Department of Physiology, University of Toronto. Toronto, Ontario, Canada, M5S 1A8.

The astroc e as a component of the node of Ranvler A number of recent studies have demonstrated the presence of astrocytic processes that contact the axon at nodes o f Ranvier in the CNS. These studies suggest that, in addition to the axon and the myelin-forming oligodendrocyte, the perinodal astrocyte constitutes a third cell type participating in the formation of CNS nodes of Ranvier. However, the earlier studies left open the question of how the astrocyte is attached to the node. Recent immunocytochemical studies I have extended the observations on the perinodal astrocyte, and demonstrate that the J1 glycoprotein, a recently described 160 kDa molecule that has been postulated to play a role in cell-cell adhesion in the nervous system, is concentrated specifically at the interface between the perinodal astrocyte processes and the axons. These findings not only provide evidence for a biochemical specialization of the perinodal astrocyte processes, but also suggest a molecular mechanism by which the astrocyte interacts with the axon and its associated oligodendrocytes at the node. While it is well established that the axon and the oligodendrocyte participate in the formation of nodes of Ranvier in the CNS, there is also evidence that astrocytes have a specific relationship to nodes. In early electron microscopic studies, Hildebrand 2"3 noted the presence of perinodal astrocytic processes at nodes of Ranvier in feline spinal cord. These processes extend through the nodal gap substances to the region of the node, where they come into close contact with the nodal axon membrane, The ubiquity of these astrocytic processes prompted Hildebrand to suggest that they might play a role in the metabolism of the node, the regulation of the extracellular ion concentrations in the perinodal area, or the production of the nodal gap substance. More recently, a number of other studies have confirmed and extended the observations on the association of astrocytic processes with CNS nodes of Ranvier. Raine 4 found perinodal astrocytic processes to be a consistent feature of nodes of Ranvier in guinea pig spinal cord. Waxman and Black 5 used freezefracture analysis to study the rat optic nerve. The freeze-fracture method exposes relatively large membrane surfaces not necessarily constrained to a single sectional level, and thus permits a clear demonstration of the intimate relationship between astrocytes and

CNS nodes. The astrocytic membranes exhibit orthogonal particle arrays 6'7 that are characteristic of this cell type; observations of these orthogonal arrays in freeze-fractured material thus permit definitive identification of the plasmalemma of astrocytes. As viewed in freeze-fractured tissue, perinodal astrocytic processes are closely associated with the node, and often tend to cover the nodal axon in a shield-like configuration. Gap junctions are also observed between the perinodal astrocytes and the outermost loop of oligodendroglial cytoplasm adjacent to the node of Ranvier, suggesting a coupling of these two cell types in the perinodal region. Notably, astrocyte processes often extend through the neuropil for distances of more than several microns, arcing toward the node in a highly specific manner (Fig. 1). The association between astrocytes and specialized regions of the axon membrane apparently does not require the presence of myelin. Island-like patches of node-like membrane are present along some non-myelinated axons, e.g. the axons in the intraretinal nerve fiber layer of ganglion cells, which acquire myelin only when they enter the optic nerve more distally along their course. Notably, despite the absence of myelin, these patches of node-like membrane are surrounded by radially

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