Nvuroscience Vol.4.pp.615to624 PergamonPress Ltd. 1979.Printed in GreatBritain

ULTRASTRUCTURAL PLASTICITY IN STIMULATED NERVE TERMINALS: PSEUDOPODIAL INVASIONS OF ABUTTED TERMINALS IN TORPEDINE RAY ELECTRIC ORGAN A. F. BOYNEand Department of Pharmacology, Northwestern

SUSAFJ

MCLEOD

Medical and Dental Schools, Chicago, IL 60611, U.S.A.

Abstract-In a previous morphometric analysis of fatigued Torpedine ray electric organ, it was shown that loss of vesicles from nerve terminals was correlated with growth of plasma membrane in the form of double-wales structures containing vesicles, which were seen inside the nerve terminals. In this paper, we show that the nerve terminals form a fen&rated sheet on the ventral eleetrocyte surface. Detailed three-dimensional reconstructions show that the processes within stimulated nerve terminals are pseudopodia arising from adjacent, abutted terminal branches. Apparently disconnected pseudopodia were also encountered. The possible relevance to ultrastructural plasticity in stimulated central nervous systems is discussed.

STIMULATION can change the shape of nerve terminals in the frog neuromuscular junction (HEUSER& RBE%$ 1973), the cat superior cervical ganglion (PYSH & WILEY, 1974), the electric organ of Torpedine rays (ZIMMERMANN & WHITTAKER,1974; BOY% BOHAN& WILLIAM 197% and at the Mauthner cell of the hatchet fish (MODEL,HIGH~TEIN& BENNET& 1975). Such changes apparently occur when the rate of addition of synaptic vesicles to the plasma membrane exceeds the rate of membrane retrieval. ZIMMERMANN & WHITTAKER(1974) first reported that st~ulation at 5 Hz of Torpedo ~ur~orato electric organ resulted in about a 5wA loss of synaptic vesicles after about 13 min. They reported that the terminals appeared to have become segmented, resulting in smaller, more frequent cross sections per micron length of electrocyte surface. A quantitative morphometric analysis of membrane changes in stimulated nerve terminals of the related ray, Nurcine brasiliensis, was subsequently carried out by BOYNE et al. (1975). In 5 fish, it was found that similar stimulation to that used in 7: marmorata produced a similar loss in the total vesicle population (49%) and that small v~~cle-containing structures were then frequently found embedded within terminal cytoplasm. These structures were encircled by two separate sets of unit membrane and it was suggested that they might represent interdigitating pseudopodia from adjacent terminals. Since the implication of exocytotitally driven shape change in nerve terminals is not yet clear, we decided to make r~o~tru~io~ of serial sections of control and stimulated N. brasiliensis electric organ in order to determine more precisely the nature of the change in this system.

EXPER~ME~AL

lated but non-fatigued tissue, an unanesthetized specimen of N. brusi1ien.G (obtained from Turtle Cove Labs, Port Aransas, Texas 78373 and maintained in laboratory aquaria for 1 week after delivery) was subjected to tactile stimulation. It was then anesthetized (BOYNE et nl.,1975) and a small piece of tissue was removed for fixation. Because of the report that discharge of the organ in air leads to an exaggerated loss of synaptic vesicles (DUNANT, ISRAEL, LESBAT~ & MANARANCHE, 1976) care was taken that the fish was never out of water. The fixation procedure has been described previously (BCWNEet al., 1974); the media containing 90 mM Mg and had a salt and buffer osmolarity of 840 ms2. In order to check that the fish had not been fatigued, it was electrically stimulated at 5 Hz and was found to be able to maintain a slowly falling discharge for several min. Discharges were recorded on a Gould Brush Recorder (Model N. 220).

PROCEDURES

The analyses of fatigued and control electric organ were carried out with tissue samples prepared for a previous study (BOYNE et al.,1975). For the preparation of stimu615

Reconstruction

ofserial sections

Serial silver sections were cut with a diamond knife on a Huxley Mk. 2 ultr~icrotome. The sections were picked up on l-hole copper grids coated with formvar and carbon. They were stained with uranyl acetate (6 min) and lead citrate (45 s) and were examined in a Hitachi HS-9 electron microscope. ~ve~iew of the inn~v~ion pattern Undulations in the ventral, innervated surface of the electrocytes make it difficult to determine the pattern of spread of nerves in single horizontal sections. In addition, the regularity of the layers of electrocytes is not evident in this plane, so that location of corresponding regions in a series of such sections is difficult. The horizontal pattern of nerve terminals was therefore built up on graph paper from measurements of terminal positions and ramifications as seen in successive cross sections in the vertical plane. The details of the procedure were as follows. Overlapping negatives of about 20 pm lengths of the innervated surface were photographed at a magnification of 5000 in 60 to 80 consecutive sections. The negatives from every fourth section were printed to give a final magnification of 15,000. The prints from a given section were trimmed and overlapped to give a continuous montage of the neural processes. The width and position of each process was

A. F.

616

BOYNE

and SUSANMCLEOD

marked by dropping perpendiculars down to an X axis. The resulting me~urements were then plotted on graph paper. It was assumed that the silver sections were 70mm thick and that successive montages therefore represented 4 x 70 = 280mn steps along a horizontal axis. The measurements made on successive montages were therefore plotted in increments of 280 x 15,ooO = 4.2mm along the Y axis of the graph paper. A flat representation of an undulating field of neural processes was thereby built up. The outlines of the cross sections in the first montage were then added to the bottom of the graph to convey an impression of the third dimension. Analysisof regions containingencircled neural

Cytophn

Examples of such regions were sought in the middle of a series of about 80 consecutive sections. The example were photographed at a magnification of 10,000 and their locations were noted on a map of the section face. The periodic structure of the cross-sectioned tissue simplifies both map-drawing and relocation of structures in adjacent sections. The negatives from a given grid were developed and used as an additional aid to identification of the structureS being traced from one grid to the next. The analysis was continued in one direction until the end of the structure was found or until the iast of the sections. The analysis then returned to the middle of the series and those processes for which one end had been located were pursued in the opposite direction. ~eco~tr~f~n. The negatives were projected onto sheets of wood to give a final magn~cation of 45,300. The sheet thickness (3.17mm) was then equivalent to that of a 70nm section. The outlines of the appropriate terminals were then drawn with a pencil onto the wood, together with the position of the encircling unit membranes within the terminals. The pencil outlines were then cut out of the sheets with a small band saw. When an interaction between distinctly separate terminal branches was being reconstructed, different woods were used for the 2 branches (e.g. bass and mahogany). When this distinction was not evident, basswood sheets were used and wherever an outline of an encirclement had been sawn, the cut edges were stained black. The encircled area was reassembled into the outer terminal. The successive wooden replicas were then glued together, creating a solid modet. The ventral aspect of the model (i.e. the surface lying away from the electroWe) was then sanded away until the contrasting woods or the blackened saw cuts in the original sheets revealed the progress of the inner structure in the third dimension, The plane of sanding was adjusted so that the origin and major ramifications of the structure were apparent; this sometimes required a curved face. The process is illustrated in Figs 1 and 2. The sanded face was then photographed and a transparency was projected onto paper so that a predominatly 2 dimensional diagram could be traced from it. RESULTS

Gross

pattern

of

innervation

before and

aper

stimu-

lation The genera1 patterns of nerve tracks over the ventral electrocyte surface of a paired controf and fatigued tissue are shown in Figs 3 and 4. As can be seen in the control (Fig. 3), the terminals arise from nerve fibers which run in the space between elec-

trocytes and which extend ramifying processes onto the cell surface. It is noteworthy that, while the termitt& divide and spread over the surface as might

be expected, they also reunite, thereby forming a fenestrated sheet. In the study which prompted the present reconstructions (BOYNE et al., 1979 approximately 80 control and SOfatigued nerve terminals were examined in cross section in each of 5 fish. A 26.8-fold increase in membrane involved in ‘invaginations’ was measured in fatigued tissues. Examination of Figs 3 and 4 may assist the reader in appreciating the frequency of the ‘invaginations’ (hatched areas of Fig. 4) in fatigued nerve beds. In the cross sections used to reconstruct this field, 6275 of the terminal profiles contained the doubIe-walled ‘invaginations’. Origins and structure of the small processes

Seven sets of serial sections of inva~natio~ in fatigued tissues were collected, one of which suggested that the structure was an invading pseudopodium from a nearby terminal The other 6 were complex and no clear i~ication of their origin could be found. Since it seemed possible that fatigue had contributed to the complexity of the structures, stimulated but non-fatigued tissue was prepared (see Experimental Procedures). Serial sets of rni~ro~ap~ were obtained in 9 cases. The origins were clearly apparent in 5. Figures 5-10 provide an illustration. A blind ending appears as a shadow (Fig, 5) and, in the next section, it resolved into an encircled process (Fig. 6). Further along the process, it can be seen to arise from an adjacent terminal branch (Figs 7-9) and to become encircled again as it continued within the penetrated branch (Fig. 10). Four of the stimulated tissue reconstructions revealed unconnected sacs. Figure lid illustrates this. It should be realized that every cross-section was inspected for evidence of attachment of these sacs to external sources but none were found. The illustrations of Fig. 11 were prepared from different reconstructions as described in Experimental Procedures and they serve to summarize the significant features in each case. Hatched areas in the diagrams correspond to regions of pseudopodia or sacs which were completely encircled in the individual cross sections. Examples a-d of Fig. 11 are arranged in what appears to be a simple hypothetical sequence to illustrate how pseudopodia could invade adjacent terminals and become disconnected. (It should be realized, however, that all these configurations could be observed in a single piece of stimulated tissue; we have not correlated the number of stimulation pulses with the average extent of pseudopodial growth, invasion and disconnection.) (a) In this example, a minimal invasion has occurred in which only the upper and Iower tips of a fold in one terminal are encircled by an adjacent branch. (b) An extensive invasion of 1 branch by an abutted branch is shown. (c) An extensive invasion which is apparently close to being disconnected from

FIGS l-2. Wooden reconstructions of abutted terminal branches. The surface of the right-hand branch has been smoothed and painted. The left-hand branch contains an encircled process which is not visible in Fig. 1, but which can be seen after sanding it open (Fig. 2). Magnification, x 14,000. The diagram derived from this model is shown in Fig. llb.

617

FIGS 1-4. Innervation pattern of the ventral face of electrocytes in paired, unstimulated control and fatigued electric organ of N. brasilienses. Magnification, x 10,000. The drawings were made from graphical plots of membrane positions in serial sections, as described under Experimental Procedures. Hatched areas in the fatigued drawing indicate the position of processes within the terminal branches.

618

FIGS 5-10. Electron micrographs of stimulated but non-fatigued electric organ illustrating how the small processes were traced to their source. Magnification, x 32,700. FIG. 5. The blind end of a process is seen as a shadow where the membranes of the structure are predominantly parallel to the plane of section. FIG. 6. In the adjacent section the two encircling membranes can just be resolved. FIG. 7. In the fifth section, the 2 encircling membranes are clearly resolved and both cytoplasmic compartments can be seen to contain vesicular forms and glycogen (?) particles. The upper (dorsal) aspect of the process appears to be turning out of the surrounding branch. FIGS 8 and 9. In the sixth and seventh sections, the origin of the process from a separate terminal branch has become apparent. FIG 10. In the ninth section the process continues in the opposite direction and is once more enclosed.

619

621

Nerve terminal plasticity its source is evident. (d) This illustrates 2 examples of completely enclosed sacs presumably representing disconnected peudopodia. Complex processes, protrusions and Schwann cells In the original description of this phenomenon in random cross sections, the occurrence of small processes within other small processes was noted (see Fig. 4 of BOYNEet al., 1974). Figures lid and e show how these configurations can arise. In Fig. lld a sealed sac has a concavity containing cytoplasm from the outer terminal. In Fig. lle, the cytoplasm of a ~udo~dium had itself been counter-invaded along its side by a pseudopodium from the receiving terminal. Protrusions of pseudopodia from terminals which they had penetrated were also encountered in three cases; this configuration is illustrated in Fig. llf. In the fatigued tissue, 2 examples of Schwann cell processes embedded in the nerve terminals were found and 1 had become pinched off. Interdigitating Schwann cell processes were not seen in the nonfatigued tissue. DISCUSSION

Fatigued and control tissues were always fixed and processed in matched pairs. Profiles of pseudopodia were very rare in control tissues, very common in stimulated tissues and frequently greater than 2 I.rrn in length. Furthermore, this appearance correlated with the loss of synaptic vesicles (measured ultrastructurally) and the loss of vesicle-bound chemicals (acetylcholine and adenosine S-triphosphate). We are led to the conclusion that the vesicle loss had already occurred in the living, stimulated tissue before fixation and that the pseudopodia represent the physiological fate of the lost vesicles. The possible physiological reality of the pinchingoff of these processes must be regarded with more caution. From the reconstruction shown in Fig. lld and from 2 others not shown, it was quite clear that there were no external attachments of the enclosed sacs, nor were there obvious nearby terminals from #which they may have become readily detached. Whether the detachment had occurred in the living tissue or during fixation remains a crucial question, particularly in view of the possible physiological implication of the finding (see below). We have therefore abandoned wet fixation and are attempting to use

Anatomy of the terminal processes BENNETT & GRUNDFE~T(1961) originally described the innervation and electrophysiology of the polygonal electrocytes of N. brasiliensis. Nerve branches arise near the angles of the polygons and subdivide in a non~verlapping fashion on the electrically inexcitable, ventral electrocyte surface. SHERIDAN (1965) described the fine structure of the nerve terminals as seen in cross sections. The present work reveals how the ultimate nerve branches drop fenestrating terminal ramifications onto the electrocyte surface. This ~angement allows for a large propo~ion of the post-junctional membrane to be chemically depolarized by released acetylcholine. The morphology shown in Figs 3 and 4 leaves open the question of whether the terminal network supports action potentials or depolarizes passively. The physiological behaviour would depend, not only on the anatomy, but also on the space constants and the d~tribution of ionic gates in the terminal area. Two techniques have recently been described whereby purely cholinergic synaptosomes can be prepared from minced adult (MOREL, ISRAEL,MANARANCHEdt M~TouR-F~~~N, 1977) or homogenized 1977) ray elecjuvenile (I~~~DALL & Z ~A~, tric organ. In both cases, low recoveries of about IVk were noted. The innervation patterns shown in Figs 3 and 4 suggest that production of closed nerve terminal sacs from this system requires tearing and resealing of multiple ‘necks’. This configuration can be contrasted to the situation in brain tissue where boutons have 1, or at most 2 ‘necks’, and it may account for the relatively low recoveries of synaptosomes from electric organ.

11

Ftc;. 11. Diagrams prepared from sanded reconstructions of nerve terminal branches showing various forms of the ~udo~~l invasions and blind sacs. ~agn~cxtio~ x 12$X Blackened areas indicate sites of attachment to the neural net. Hatched areas indicate regions that were totally enriched by the receiving, branch. Stippling in e’distinguishes the members of an interwoven pair of terminals.

622

A. F. BOYNE and SUSANMCLEOD

extra~IluI~ tracers and fast-freezing physical fixation to test whether closed sacs of neural cytoplasm can be produced in living tissue. Interpretation of cross-sections BOYNE et al. (1975) previously speculated that interdigitati~ pseudopodia develop between adjacent nerve terminal branches when N. br~ilie~~ electric organ is stimulated. The present results clearly confirm this. In the study of ‘I tnarmorata preparation by ZDAMERMANN & WHITTAKER(1974), it was suggested that the terminals had become segmented to as to give smaller cross-section profiles. While it is entirely possible that the 2 electric organs accommodate added vesicle membrane in different ways, it is also possible that we have emphasized different aspects of a complex response which occurs in both preparations. Thus, the reconstruction in Fig. lla is generally consistent with the se~entation of a terminal branch in the form of a fold, only the upper and lower tips of which are penetrating adjacent neural cytoplasm. On the other hand, Fig. 7b of the ZIMMERMANN & WHITTAKER(1974) paper could be interpreted either as showing segmentation of a terminal or as a large terminal with a partially enclosed ~eudo~ium. Is it possible to assess the relative importance of these phenomena in accounting for lost vesicle membrane? Segmentation should be reflected in an increase in nerve terminal perimeter (per ,um of electrocyte). Such an increase was reported in the N. brasiliensis system but the v~iab~ity (which increased with stimulation) was such that the effect did not reach statistical significance (BOYNEet al. 1975). Nevertheless, it is possible to compare how much of the lost vesicle membrane could be accounted for by these 2 mechanisms :

Lost vesicle surface area = 0.096 ~~/~ - I. Increased terminal membrane = 0.022 ~rn*/~rn-‘. Increased encircled membrane = 0.052 ~‘/~rn-‘. Segmentation could thus account for 23% and interdigitation for 55% of the lost vesicle membrane. It therefore seems quite plausible that both processes go on and contribute to membrane growth. Because of the inherent variability in terminal cross section sizes and the unusual appearance of interdigitations, it is relatively easier to establish the statistically significant contribution of the latter phenomenon, Driving forces

It seems likely that stimulation-induced exocytosis of synaptic vesicles causes the growth of the plasma membrane. In electric organ, this occurs simultaneously in adjacent terminals. which often touch each other with no intervening glial barrier. Under these conditions, it appears that a mutually beneficial interaction occurs whereby one membrane foids outward in a pseudopodium, perhaps persuading the other membrane to fold inward in an invagination.

Both terminal branches thereby accommodate added surface area. Although Fig. 11 emphasized interdigitations between short lengths of adjacent terminal branches, it seems likely that, when a pseudopodium develops in a given branch, an invagination or a shrinkage occurs at some point further along its length so as to maintain the overall volume constant. The occasional engulging of Schwann cell cytoplasm may also occur by an invaginating tendency in the underlying nerve terminal. Other workers have described phenomena which may have similar driving forces. By using elevated calcium levels, SMITH,CLARK & KUSTER(1977) were abIe to limit the effect of black widow spider venom in emptying the frog neuromuscular junction of synaptic vesicles. In these partially vesicle-depleted preparations, they observed branched infoldings of the nerve terminal plasma membrane, in addition to Schwann cell invasions. GENNARO? NASTUK & R~~R~RD (1978) have obtained similar but reversible vesicle depletion and terminal infoldings in frog neuromuscular junctions by applying 115 mM K* Ringer’s solution for 30 min. BURWEN& SATIR(1977) recently proposed that the lengthening of surface membrane folds in secreting mast cells is also caused by the addition of secreting granule membrane to the plasma membrane. Reasons for the pinching-off of the pseudopodia are less evident. ZELIGS& WOLLMAN(1977) have shown that cytoplasmic blebs on hyperplastic thyroid epithelial cells may be phagocytosed by pseudopodia extendjng from ~ighboring cells; they were able to show that the pinching-off was associated with a ring of possibly contractiie micro-filaments within the pseudopodia. No such ring was evident in the present material.

ZIMME&ANN & WHITTAKER(1974) reported that recovery from fatigue occurred in 2 stages. After 24 h of rest, the numbers of vesicles and the nerve terminal profiles appeared normal. However, reloading of vesicles with a~etyIcholine and aden~ine-~-t~ph~phate took about 3 days. We have not attempted a threedimensional analysis of the recovery prows, but several relevant considerations are presented below. In theory, the infolding at sites of invaginations could always be reversed by reformation of vesicles from the excess membrane. Similarly, pseudopodia which remain connected to the nerve net couid also be ‘recalled’ by vesicle reformation. Once an invading process becomes pinched off, however, it seems probable that it would be irretrievable. If pinched-off pseudopodia perfoiated the terminals in which they lie (e.g. Fig. lie), they may be extruded in conjunction with the elimination of the su~ou~i~ invagination. Disconnected sacs of nerve terminal cytoplasm would then come to lie in the extracellular space. This would be equivalent to an in viuo production of synapto-

Nerve terminal plasticity somes (MORELer al., 1977) or T sacs (DOWDALL8r

Z~MMERMANN, 1977). ifesicular exocytosis as a driving force for structural change in the central nervous system? A correlation between nerve terminal firing and structural rearrangement has been shown in a few, peripheral locations, as noted in the Introduction. In each case, vesicular exocytosis appears to drive the diverse shape changes (reviewed in BOYNE, 1978). Since the occurrence and/or the rate of net vesicle transfer to the terminal membrane are a function of stimulation frequency (CECCARELLI,HURLBUT & MAURO,1973; F%H & WILEY, 1975; ZIMMERMANN & DENSTON,1977), it seems clear that similar plastic change in the CNS could only occur if a terminal fires rapidly enough and long enough. The quanta1 content of evoked potentials at CNS synapses is thought to be lower than at peripheral junctions. However, this appears to correlate with the smaller size of bouton terminaux as opposed to neuromuscular nerve endings (see KUNO, 1971) and therefore it cannot be regarded as a protection mechanism against exocytotically driven structural change (i.e. a small number of vesicles undergoing exocytosis in a small nerve terminal may also bring about a

623

significant structural alteration). Nevertheless, it seems highly unlikely that changes such as those described herein could occur during normal physiological functioning of the CNS. A further argument is that directly abutted nerve terminals are relatively infrequent in mammalian CNS. On the other hand, the clinical use of electroconvulsive therapy to induce repeated seizures (JUTCHINSON& SMIEDBERG, 1963; ABRAMS& FINK, 1972) is exactly the manipulation needed to drive vesicle exocytosis beyond normal physiological limits. Furthermore, although sites of presynaptic inhibition seem to be relatively infrequent, they are important regulatory elements of neuronal circuits and they fulfil the requirement of nerve terminal abutment. It has therefore been suggested that a search for similar effects in electroconvulsed mammalian CNS may throw light on the mechanism by which electroconvulsive therapy relieves endogenous mood depression (BOYNE,1978). Acknowledgements-A.F.B. would like to thank Dr L. T. WEKCXER, Jr. and the faculty of the Department of Pharmacology for providing an electron microscope. This work was supported by National Institute of Neurological and Communicative Disorders and Stroke grant no. NS 13043.

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1978)