Abdominal nerves of Rhodnius prolixus were studied with the light microscope under high-power Nomarski optics with a minimum of surgical interference. The preparation was perfused with bathing solutions which could be changed during time-lapse cinematography.
The structure of the nerve trunks was studied by light and electron microscopy.
The movements of intracellular organelles are described and discussed.
Saltatory movements of organelles, probably mitochondria, were followed at different temperatures. Rate of saltation varied linearly with temperature.
Axonal flow (bulk movement of cytoplasm) did not occur in healthy abdominal nerves.
Most of the methods used to study axonal transport suffer from the disadvantage that a single measurement of the rate of transport depends on observations extending over several hours (Heslop, 1975). For example, it may be necessary to allow time for the incorporation of a labelled precursor into a macromolecule and then to follow its translocation to a different site or to estimate the slow accumulation of transported material at a ligature along a nerve or in an end organ. The fastest rates of axonal transport in mammals rarely exceed 5 μm/s (18 mm/h) and to study such movements over short periods some form of microscopical technique is essential. Much information is still being obtained by autoradiography (for example, Droz, Rambourg & Koenig, 1975) but the saltatory movements of particles within cells form the basis of at least part of the axonal transport system and can be studied by established techniques of time-lapse cine-micrography. Work has been reported even on human sural nerve in situ (Kirkpatrick & Stern, 1973) but it has usually been necessary to carry out painstaking dissection of individual fibres from larger trunks (e.g. Cooper & Smith, 1974; Kirkpatrick, Bray & Palmer, 1972) in order to obtain acceptable optical conditions.
We found that a Zeiss/Nomarski microscope made possible observations of individual axons in whole nerve trunks of some insects. The nerves from the fused Pesothoracic ganglion to the abdominal wall of Rhodnius offer an attractive preparation. They are small enough to have good optical properties and run for several millimetres across the haemocoel completely free from any connective or other tissues outside the neural lamella. There are no tracheae associated with them and a preparation can be made in which the cell bodies and dendrites are undamaged, being deep within the ganglionic mass, and any injury to the axon terminals is remote from the point of observation.
This paper describes the structure of two abdominal nerves of Rhodnius and the saltatory movements of organelles within them at different temperatures. The phenomena reported are compared with those previously described in vertebrates and in isolated cells in culture.
MATERIALS AND METHODS
Rhodnius prolixus Stal (Insecta, Hemiptera) were reared in the laboratory at 28 °C on rabbit blood, and fifth instar nymphs were taken for experiment 1–2 weeks after they had been fed. They were dissected from the dorsal side.
Some cuticle, the fat body, Malpighian tubules and alimentary tract were removed and a single strand of silk was tied tightly between the pro- and mesothoracic ganglia. The head, thorax and appendages and part of the abdominal wall were cut away and the mesothoracic ganglion gently folded over the remaining portion of the ventral abdominal wall to give it support. The preparation was then transferred to the perfusion chamber. After transfer the silk strand was trapped between the top coverglass and the chamber, passing through the wax seal. It served to hold the ganglion in position so that the nerves were under slight tension. Lateral movement of the nerves in the stream of perfusing fluid was negligible since both the silk strand holding the ganglion and the piece of abdominal wall were held firmly (Fig. 1). The perfusion chamber was a slightly modified version of that described by McGee-Russell & Allen (1971). Pieces of thick broken coverglass were placed beneath the nerves under observation to prevent them dropping out of focus. Ringer solution (NaCl 129 mM, glucose 34·4 mM, NaHCO3 10·2 mM, KC1 9·6 mM, MgCl2 8·5 mM,NaH2PO4 4·3 mM, CaCl2 2·0 mM, pH 6·9 after Maddrell, 1969) was shaken with air and allowed to flow by gravity at 0·2–1·0 ml/min. A two-way tap was provided to permit changes of perfusing fluid without losing the microscopic field of view.
The optical system was a Zeiss/Nomarski WL microscope using a 7#x00D7; 100 1·25 N.A. oil immersion objective and 1·4 N.A. substage condenser oiled to the underside of the perfusion chamber (Allen, David & Nomarski, 1969). Illumination was by a 100 W quartz-halogen lamp with an additional heat filter and two Wratten IA filters (inserted to prevent photolysis of colchicine and possible cell damage due to near ultraviolet light) in the light path. Time lapse exposures, usually at 1/s, were made with a Vinten scientific mark III camera on 16 mm Ilford type V film. Film was developed for maximum contrast in Kodak D19 and the negatives examined on a frame analyser (Vanguard Instrument Corp. Melville, L.I., New York, U.S.A.). The images of individual particles were plotted frame by frame and rates of movement while in saltation, termed ‘instantaneous rates’, were calculated.
For structural studies preparations were fixed in the perfusion chamber after they had been filmed. About 150 ml of glutaraldehyde (3% solution in 0·05 M sodium cacodylate pH 7·4 with 1·71 % sucrose) was passed over the preparation in 1 h. It was then removed from the perfusion chamber, rinsed in several changes of 12% sucrose in buffer and post-fixed for a further hour in 1 % 0s04 with 10·3% sucrose in 0-05 M cacodylate pH 7·2. Some preparations were stained for 1 h with 2% uranyl acetate in sodium maleate buffer pH 6·2, and all were dehydrated and embedded in Araldite. In two cases this was done in 800 μm wafers of Araldite between two fluorocarbon-coated coverglasses (McGee-Russell & Allen, 1971). Such preparations could be examined under the Nomarski differential interference contrast microscope and the area previously filmed could be identified. It was then mounted for serial sectioning. Sections were cut on an LKB Ultratome III, stained with lead citrate (Venable & Coggeshall, 1965) or double-stained with aqueous uranyl acetate and lead citrate (Reynolds, 1963) before examination on a Philips EM 300 electron microscope.
Both the electron microscopical and time-lapse cinematograph studies were confined to the two paired nerves which pass back from the mesothoracic ganglion to abdominal segments 2 and 3. The nerve trunks are up to 26 μm in diameter where they pass free and unsupported across the body cavity. About 2 mm behind the ganglion there is often a branch of variable size up to 15 μm in diameter. When it was desired to study by electron microscopy an area which had already been filmed up to e time of fixation, a nerve with a side branch was chosen to provide a convenient point of reference.
A transverse section through a nerve trunk is shown in Fig. 2. The whole trunk is ensheathed in a fibrous acellular neural lamella 0·15 –0·5μm thick. Immediately beneath this is a layer of specialized glial cells of varying thickness, the perineurium, which completely envelops the mixed population of axons and neuroglia within. The large and prominent nuclei of the perineurial glial cells appear as elongated oval images up to 1·5 μm across and 7 μm long on light micrographs. Saltating particles within axons were seen to curve around the edges of glial cells at the level of their nuclei. Perineurial cells are morphologically distinct from the glia deeper within the trunk, having many fewer microtubules and more granular bodies 0·2–0·3 μm long. Both contain prominent rosettes of glycogen granules, clear vesicular profiles and mitochondria. Saltatory movements of subcellular particles were also seen at the very edge of nerve trunks, probably in perineurial glia.
Just inside the perineurium there are many neurosecretory axons. These contain numerous vesicles of two main types. Larger vesicles 0·15–0·2 μm in diameter with dense cores or more diffuse granular contents are found together with small heavily staining dense core vesicles about 0·05 μm in diameter. Neurosecretory axons are flattened and in transverse section can be seen to be pressed against the edge of the nerve bundle. They are up to 2·5 μm wide but less than 0·5 μ;m thick. A few were seen to pass through the perineurium to lie just beneath thinner (0·25 μm) areas of neural lamella (Fig. 3 a). The swollen nerve endings at these points act as hormone release sites and granules are seen lying outside the nerve cells and in intermediate stages of exocytosis (Fig. 3 a) indicating that there is no substantial diffusion barrier between them and the haemolymph (Lane, Leslie & Swales, 1975).
The centre of the nerve trunk is occupied by 160-380 mostly non-neurosecretory axons and their supporting glia. In these nerves, axons range from 0·16 to 2μ;m across and the number to be seen at any point depends on the branching pattern and varies from one individual to another. Groups of one to about ten axons are surrounded by glial extensions 0·08–0·34μm wide in transverse section (Fig. 3). Glial cells are very long, extending along the length of the trunk for 60 μm or more. They contain mitochondria and densely staining granular material but their most striking feature is the presence of tightly packed arrays of presumably structural microtubules (Figs. 3,4). Glial cells in the central parts of these nerves contain 63 microtubules per square μm compared with 25 microtubules μm−2 in axons. The alternation of axon bundles and glia gives thin longitudinal sections a striated appearance (Fig. 4) which is clearly seen in the ‘optical sections’ produced by the light microscope although the latter at up to 1 μm are at least ten times as thick. The resolution so obtained (both vertical and lateral) does not permit a categorical statement that saltatory movements occur in glia as well as in axons but the frequent observation of long saltations at the edge of trunks where glial cells predominate suggest that this is the case. Estimations of particle rate have been taken only from optical sections through the middle of nerve trunks in the hope that all observations will represent intra-axonal movements.
Three sorts of body were seen to saltate. The commonest gave small round images 0·6 μm in diameter (Fig. 5). Stationary images of the same size were seen and the proportion saltating at a given time varied from 2 to 14%. No information as to the size and shape of the organelles responsible for 0·6 μm round images can be obtained from the light microscope since the size (Airy’s disc) depends on the lens system and the wavelength of light used, and its contrast also upon the difference in refractive index between the organelle and its surrounds. Smaller particles would give fainter images. Less common were images 0·6 μm in diameter but up to 1·8 μm long. These were usually noticed only when they moved and were always aligned with the longitudinal axis of the nerve trunk. Occasionally (five times in 6000 ft of film) a larger type of image was seen to saltate. Under Nomarski optics it was oval (or square with rounded corners) and 1–2 μm across with no gradient of refractive index in the centre. Small nuclei have this appearance but although the glial nuclei in these nerves of Rhodnius were of similar diameter they were usually much longer. Mitochondria were 0·127#x2013;0·25 μm in diameter with a very few up to 0·5 μm, and 0·25–1·5 μm long with some up to 3 μm. There were sufficient mitochondria in the nerve trunks to account for all the small particles and elongated images seen although the granular bodies of perineurial glia and the large vesicles of neurosecretory cells may also have contributed to the saltatory movements at the edges of nerve trunks. In electron micrographs there are clear vesicular profiles which, if they represent real structures in vivo, could produce light microscope images like the large ones mentioned above.
Three nerves were selected because a fine branch part way along them provided a convenient reference point. They were filmed at 1 frame/s up to the moment of fixation, fixed in the perfusion chamber and embedded while still in position on the lower coverglass. The identified area was cut out and in one case sectioned for electron microscopy at eleven different levels. Montages of micrographs were built up at each level so that the filmed area could be studied at high resolution. Despite close examination no other candidate structure than mitochondria and small clear vesicular profiles was found to account for the images seen, and the authors conclude that one or both of these must account for the majority of the saltations studied. There were no very large images in these preparations.
Particles typically behaved in one of two ways. Most remained more or less stationary. They often moved irregularly backwards for distances which might be as much as 1 μm but always along the nerve trunk. Movements of this amplitude in random directions were seen in dying preparations. This was taken as a sign of damage and such preparations were discarded.
About 8 % of particles in healthy preparations seemed to move along the length of fibres (but the percentage is biased against identification of stationary ones). Some followed curved pathways passing out into side branches. Provided they stayed in focus they could usually be followed right across the field of view of the optical system, because their overall directional progress was maintained despite changes in rate, halts, or even occasional reversals along the same track. Their motion was always discontinuous. Particles often stopped for periods varying from less than a second to several minutes (Fig. 6 a). Whilst stopped they sometimes oscillated backwards and forwards (Fig. 6b) and occasionally one would reverse on its track for up to 5 μm (Fig. 6 c), before continuing in the original direction. Four such particles reversed for over 4 μm (enough to permit an accurate estimate of their speed in the reverse direction). All moved at the same rate backwards as forwards. The distance travelled between halts varied from 1 to 18·5 μm. There was no sign of a favoured saltation length in the data that could be collected but the accidental factors of position relative to the edge of the field and particles passing out of the focal plane have as much influence as physiological parameters on such measurements.
It was most noticeable that a pathway in which a saltation had been seen was likely to be traversed later by others. The busier pathways seemed to carry a constant stream of traffic while others were nearly deserted. Attempts were made to find evidence that the positions of halts between saltations in busy ‘streets’ had a structural basis. None was found. This directly contradicts the observation that reversing particles often oscillated many times between fixed points before moving on. In one case a second particle then proceeded to oscillate along exactly the same 2 μm pathway that had just been vacated by another. More reversing particles were seen in preparations showing some evidence of Brownian movement. It is suggested that reversals themselves may be pathological rather than normal phenomena.
Different particles in a single pathway often moved at different speeds and individual particles could accelerate or decelerate without any intermediate halt that could be detected on 1 frame/s time-lapse film. To confirm this point, particles, such as that in Fig. 6a, were selected from preparations in which there was no other moving organelle visible within 3 μm of the selected one so that the observer’s attention could not have been diverted from one to another. The criterion of change of velocity was that the particle should have deviated at least one image diameter from the expected position estimated from its progress in the preceding 5 s.
Experiments were conducted in controlled rooms at different temperatures on animals that had been reared at 28 °C but kept for 8 days at the experimental temperature before use. The median rate of actual saltation was found to be linearly dependent on temperature over the range studied (if it were exponentially dependent the Q10 would equal 2) (Fig. 7). Where the velocity of a particle changed during saltation the speed plotted was the total displacement divided by time.
The possibility that there might be a bulk movement of cytoplasm in these nerves - corresponding to ‘axonal flow’ (Weiss, 1972)-was tested by filming at one frame every 12 or 20 s. The criterion by which bulk flow was recognized was that adjacent organelles could be seen to move relative to the nerve trunk but maintained a more or less constant position relative to one another. In preparations which already showed signs of deterioration it had been noticed that bulk flow did sometimes occur at rates as high as 0·2 μm/s but that this phenomenon was followed by death. Bulk movements of cytoplasm were never seen in preparations which showed normal saltations and it was concluded that axonal flow does not occur in these nerves.
Even when the flow of perfusing fluid is interrupted, apparently normal saltations continue in the Rhodnius abdominal nerve preparation for at least 16–20 h. This great stability is obtained in a simple salt solution designed for other purposes, and must reflect a well-maintained homeostasis within the nerve trunk. The existence and nature of the ‘blood-brain barrier’ in insects has been discussed by Treherne (1974) and Treherne & Pichon (1972) and its site in this species has been shown to lie in the perineurium (Lane et al. 1975 and Figs. 3, 4). The consequences for this preparation are twofold. The diffusion barrier of the perineurium gives enough protection from ie external environment to permit lengthy experiments. On the other hand acute effects of drugs introduced into the perfusing fluid may be masked.
The alternating bands of glia and bundles of axons in these nerves are prominent on electron micrographs and on the cine film. Saltations were seen in both light and dark bands on the same preparation (i.e. without movement of the upper Wollaston prism which would have altered their appearance under differential interference contrast optics) and also from time to time at the very edges of trunks, presumably in perineurial glia. This suggests that saltatory movements may be common to neurones and glia. Since many glial cells are at least 60 μm long the presence in them of a well- developed intracellular transport system would not be surprising. The implication for this study is that some at least of the saltations that were measured probably took place in glial tissue. There is no doubt that saltatory movements do occur in neurones since they were seen in teased preparations in fine branches and in neurones from embryonic cockroach CNS grown in tissue culture (unpublished observations).
The cell bodies of neurones studied could have been either in the mesothoracia ganglion or at the periphery. Since there were similar numbers of intracellular movemente towards and away from the ganglion and no difference in speed or character between them could be discerned, all were treated equally.
Some of the organelles which were seen to saltate gave images 0·6 μm wide and 1·0–1·8 μm long under the light microscope. These are the size that one would expect from the mitochondria in this tissue. Similar movements of mitochondria have been reported by other authors (e.g. Cooper & Smith, 1974 in toad sciatic nerve and Pomerat et al. 1967 in cultured cells). Close study of electron micrographs revealed that there were enough mitochondria present to account for the long images mentioned above and for all the small round particles too. No other candidate structure was seen except for clear vesicles or ‘profiles of endoplasmic reticulum’. These present a problem. We have no evidence that they exist as true in vivo structures (they could be fixation artifacts) or that if they did exist they would have a sufficiently different refractive index from the cytoplasm to be visible.
The third class of images which moved were faint and fast. Both authors are convinced of their reality and that they moved faster than 1 μm per s. It is tempting to suggest that they might be caused by the dense-core vesicles, probably neurosecretory granules, which are a prominent feature of some axons and are known to be transported down these nerves from cell bodies high in the mesothoracic ganglion (Maddrell, 1966). Similar fast images have been seen in cultured vertebrate cells by Breuer et al. (1975), but their exact nature is uncertain. Their presence serves to remind us of the limitation on visible particle size that is imposed by the optical system.
Particles in motion moved in an irregular manner. Not only did they start and stop, covering very variable distances in successive saltations, but they also frequently changed rate in mid-saltation. Changes of rate between rest and saltation and during saltations were all immediate. There was no graded acceleration or deceleration that could be observed on 1 frame/s time-lapse film.
Wide differences were also noticed between the speeds of individual particles within a single nerve trunk. All of these characteristics are similar to those earlier described in a variety of animal cells (Rebhun, 1972).
Among the mechanisms that have been proposed to explain axonal transport are two - the micro-stream hypothesis of Gross (1975) and the movement of particles within the axolemma itself (Marchisio, Gremo & Sjöstrand, 1975) - which cannot reasonably account for saltatory movements of large organelles such as mitochondria. The most plausible mechanism for the translocation of large organelles depends upon sliding filaments analogous to those of skeletal muscle and it has been proposed (Ochs, 1971) that a variety of organelles could be carried by a single type of structure, a ‘transport filament’ .
Heslop (1974) pointed out that there is no need to postulate that a discrete organelle serves as the transport filament. A specific ‘transport vector protein’ which can engage with and disengage from deformable filaments serving the function of myosin molecules in muscle is required. Provided this protein was capable of non-specific binding to intracellular particles, both natural and extraneous in origin, all the known properties of axonal transport can be explained. There is strong evidence for the involvement of formed microtubules, or at least tubulin molecules, in axonal transport, and in the present study all but five out of 100 mitochondria seen in transverse section (e.g. Fig. 2 a) were within 30 nm of one or more microtubules. Many mitochondria were surrounded by rings of microtubules, and it seems likely that they are involved in the saltatory movements seen. The movements of macromolecules and possibly synaptic vesicles in association with a subaxolemmal network of smooth endoplasmic reticulum has been studied in chick and rat ciliary ganglia by Droz et al. (1975). The possibility that a similar network exists in Rhodnius and that it acts as the stator in this animal cannot be ruled out on the evidence available but it must be regarded as a less likely candidate structure than microtubules on grounds of the distribution of mitochondria within the nerve. Saltatory movements have been seen in artificial axopodia (which lack microtubules) produced by passing a fine glass rod through the body of the protozoan Echinosphaerium so as to push out the body wall on the other side into a fine extension (Edds, 1975). In this case the body wall probably acted as stator and the movements continued in the presence of colchicine, a microtubule disaggregation agent. Not all saltations in cultured cells are prevented by colchicine (Freed & Lebowitz, 1970) again suggesting the possibility that there might be more than one stator in a given nerve and that structures other than microtubules may carry out this function in saltatory movements.
Previous reports (Edström & Hanson, 1973; Heslop & Howes, 1972) have shown that in poikilothermic animals, at least after acclimatization to low temperature, there can be compensating mechanisms which raise the rate of axonal transport at lower temperatures. No such effect was found upon saltations in Rhodnius nerves but it proved hard to find saltations at all at 13 °C. Those which were found were at the expected speed.
Since the instantaneous velocity of saltation did not vary as the logarithmic function of temperature, calculation of apparent Arrhenius activation energies as by Cosens, Thacker & Brimijoin (1976) would not be justified. The saltation velocities found in Rhodnius nerves were slower at corresponding temperatures than those found in amphibians by Cooper & Smith (1974), Hammond & Smith (1977) and Foreman, Padjen & Siggins (1975) but owing to the small size of the insect this would occasion no functional disadvantage.
The authors wish to thank Dr R. S. Smith for kindly reading the manuscript and for stimulating and informative discussion.