482 Preliminary notes registering from a single fixed ... - Science Direct

482 Preliminary notes registering from a single fixed channel (see above, operation on spectral mode, options). The resulting curve is nearly superposable to the spectrum obtained with more intensely fluorescing NAD(P)H crystals, using a polychromator to disperse the emission on about 300 channels. The implications of multichannel microspectrofluorometry are obvious in terms of revealing the ‘multilocalized’ metabolic response and interrelationships of intracellular compartments, down to the limits of optical resolution. Furthermore, a more detailed analysis of spectral changes can lead to in situ conformational studies in correlation with intracellular enzyme activity. The authors acknowledge gratefully the advice of Dr hl. Zatzick, SSR Instruments Co., Santa Monica, Calif. aud the collaboration of Research Engineer L. ~hlund (Department of Pathology, Karolinska Inst.), as well as the help of Engineer J. Bolmgren (Medical Techniques, Karolinska Institutet). This work was supported by NSF (Washington) Grant (no. GB 30224) and Swedish Medical Research Council Grants (nos.‘K 72-12R-3828 and B 73-12X630-09A).

1. Chance, B & Thorell, B, J biol them 234 (1959) 3044. 2. Duysens, L N M & Amesz, J, Biochim biophys acta 24 (1957) 19. 3. Karasek, F W, Res/dev 23 (1972) 47. 4. Kohen, E, Kohen, C & Thorell, B, Quantitative fluorescence techniques as applied in cell biology. Battelle Memorial Institute, Seattle, March 27-31, 1972, p. 173. Springer, Heidelberg (1973). 5. Kohen, E, Kohen, C & Thorell, B, Mikrochim acta (Wien) 1972, 103. 6. Kohen, E, Kohen, C & Thorell, B, Biochim biophys acta 286 (1972) 189. 7. Kohen, E, Michaelis, M, Kohen, C & Thorell, B, Exptl cell res 77 (1973) 195. 8. Kohen, E, Kohen, C & Thorell, B, Mikrochim acta. In press. 9. Olson, G G, Optical spectra, January 1971. 10. - American laboratory, February 1972. 11. Schott & Gen., Mainz, Interferenzfilter: special filter, No. 3711-1972. 12. SSR Instruments Co., Preliminary model 1205 optical multichannel analyzer instruction manual. SSR Instruments Co, Santa Monica, California, 1970. 13. Westinghouse Engineer, May 1971. Recent deExptl Cell Res 81 (1973)

velopments in low-light-level camera tubes. Westinghouse technical information, reprint 48. 14. Zatzick, M R. Personal communication. Received March 29, 1973 Revised version received May 28, 1973

Elongation of axons in an agar matrix that does not support cell locomotion R. J. STRASSMAN, P. C. LETOURNEAU, and N. K. WESSELLS, Department of Biological Sciences, Stanford University, Stanford, Calif. 94305, USA Summary. Axons elongate from I-day embryonic chick spinal ganglion neurons cultured in a soft agar matrix or on a firm agar substratum. Glial cells from the same ganglia are unable to spread or show locomotory behavior (ruffling) in or on the agar, but do spread and show ruffling activity at the agarplastic interface or when replated from agar onto plastic. Axons elongated into or on agar retract much more slowly when treated with colchicine than do similar axons on plastic dishes.

Contact with a solid substratum appears to to be essential for various tissue cells to carry out gliding cell locomotion and cell division [l-4]. Similarly, from the time of Harrison’s [5] initial demonstration of nerve axon elongation in vitro, solid substrata have been deemed necessary for this aspect of nerve growth (a conclusion that also applies to the supposed axon-like neurites of neuroblastoma cells [6]). Cell-substratum adhesivity may be the most important property of various substrata with respect to these cellular activities. Thus, differences in relative adhesion between cell and substratum affect the rate and perhaps even the direction of cell locomotion [7-111. One substratum that is commonly regarded to be relatively nonadhesive is agar. Many cells appear not to attach, spread, or move on an agar substratum [12, 131.On the other hand, cells suspended in agar can carry out mitosis, indicating that an agar matrix meets requirements for that process [14-161. Yamada et al. noted that nerve axons of chick embryonic spinal ganglia elongate into

Preliminary notes 483 a matrix of nutrient agar [17]. This observation, in combination with other studies that led to the proposal that axonal elongation differs from cell locomotion [18], in certain respects, suggested the experiments reported in this paper. The results demonstrate that axons of single nerve cells elongate into a soft agar matrix, that fails to permit glial cell spreading or gliding locomotory behavior. Methods

agar matrix in modified F12 with 10 % FCS and NGF. After making observations over the following 96 h, the medium and loosely adherent cells were picked up in an oral pipette after being dislodged by flushing with the medium. The medium and cell suspension were then plated in a fresh Falcon culture dish that was reincubated. Colchicine (CalBiochem) at a final cont. of 1 lug/ml was added to cells precultured for 48 or 72 h (i.e., an aliquot of appropriate cont. in HBSS was overlaid on the agar matrix). Alternatively, the same colchicine cont. was added to single cell cultures growing on top of the 1% agar substratum as prepared above. For a control, colchicine was added to cultures of single cells grown on uncoated Falcon plastic culture dishes. All sets were observed over the ensuing 24 h of treatment.

Lumbosacral dorsal root ganglia from g-day-old white leghorn chick embryos were dissected out in warm (37°C) Hanks balanced salt solution (HBSS), and were dissected free of most attached mesenchyme in warm modified F12 medium [19] containing 10% fetal calf serum (FCS). The ganglia were then rinsed for 10 min with calcium and magnesium free Tyrode solution (CMFT) at 37°C at slow speed in a gyratory shaker. The CMFT was aspirated, a 0.15 % trypsin solution in CMFT was added, and the ganglia were agitated at low speed in the shaker, at 37”C, for 30 min. The trypsin solution was then aspirated, culture medium with 10 % serum was added, and the ganglia were flushed repeatedly through a narrow-bore pipette. The resultant cell suspension was pelleted at 350 for 90 set and resuspended in culture medium. This rinsing procedure was repeated twice more. The semi-solid agar-cell system was prepared as follows: Enough nerve growth factor (NGF). (prepared as in Yamada et al. [20] as an homogenate of submandibular glands from highly inbred BALB/ adult male mice) to yield a final cont. of 1 : 300 000 was added to the bottom of a 35 mm Falcon Petri dish. Warm F12 with FCS (4/3 of what was desired in the final agar-medium mixture) was added and gently swirled. The cells, in a solution of F12 with a FCS cont. identical to that in the dish, were added but not swirled. Cells were plated at a population of 2-3 x lo5 cells/dish. Warm (40-42”C) agar solution in HBSS was added in varying cont. The ratio of F12 and agar-HBSS was kept at 2 : 1. The final mixture of 3 ml was then gently but quickly swirled to ensure adequate mixing of fluids and dispersal of cells prior to gelatin of the agar. To aid in visualization and photography, randomly selected small aliquots were removed from these dishes before gelatin occurred and were placed in depression slides. Coverslips were added, and the slides were placed in large, loosely covered Petri dishes above a thin layer of distilled water to avoid desiccation. Incubation was in a humidified 5 % CO, incubator at 37°C. ‘Parent’ Petri dishes were cultured in the same incubator. In order to conduct an indirect test for the presence of glial cells, some cultures were prepared as-follows: One ml of warm 1% agar in HBSS was allowed to harden in a 35 mm cuhure dish. Then 2 x lo6 dissociated spinal ganglion cells were plated on top of the

Results The dissociation procedures employed yield mixtures of fibroblast-like ‘glial’ cells and sensory neurons. These two classes of cells behave differently when suspended in a soft (0.33 %) agar matrix. Many nerve cells with elongating axons are present in the matrix. Such axons elongate more slowly than similar ones extending on gelatin or plastic [27]; thus, lengths of ca 250 pm are seen at 3 days in agar, whereas such lengths are found after 1 day under the latter conditions. Axons elongating in the agar matrix are never straight; that is, they curve in three dimensions (figs l-3), and never appear in a single focal plane with light microscopy. In contrast, the relatively small proportion of nerves located at the agaragar-coverslip or agar-culture dish interface invariably have straight axons (figs 5, 6), just as is seen in single cell culture on gelatin or plastic with normal serum levels (ca 10 %). The only glial cells that can be identified as such are also seen at the two agar-solid substratum interfaces (fig. 4). Such cells are spread upon the substratum and frequently show typical ruffled membrane activity. No such flattened cells are seen at any other level in the agar matrix itself. Instead only spherical cells are present. Such cells are presumed to represent the bulk of the glial Exptl Cell Res 81 (1973)

404 Preliminary notes

population derived from ganglia as well as those neurons that fail to elongate axons. That this is the case was demonstrated as follows. Cell suspensions were plated on top of a hard (1 %) agar substratum and were cultured for 4 days. At 24 h, many loose round cells were sitting unattached on the agar (i.e., some were floating in the medium and others rolled about on the agar if the dish was touched). Other cells appeared to be adherent to the agar, though completely unspread (i.e., such cells failed to be moved if the dish was touched). In addition, many small round aggreagatesof ca 5-10 cells were observed on the agar surface. Axons were observed protruding from such aggregates as well as from some single neurons attached to the substratum. At 48 h, many more axons were observed. At 96 h still more were present in the form of extensive branching networks. All such axons appeared in a single focal plane, indicating growth on the surface of the agar and suggesting that the 1% agar could not be penetrated by axons. At no time were any flattened glial cells observed (as in fig. 4). At 96 h, the cells and cell aggregates were removed from the agar substratum and were plated directly on a plastic culture dish. At 90 min many single cells and aggregates were attached to the plastic. Some of the single cells were spread on the plastic (as in fig. 4). Glial cells were also seen as a sheet surrounding some of the aggregates.

A few short axons were present at this time. By 6 h many more spread glial cells appeared to be migrating outward from the aggregates, and longer axons were present. Therefore, glial cells were present as rounded cells on the original agar substratum and in the aggregates. Upon presenting them with a plastic substratum they rapidly adhered, spread, and exhibited locomotory behavior. Returning now to the three-dimensional agar matrix experiments, the effect of differing agar cont. was tested. At 0.66% about l/2 to l/3 the number of axons was present as at 0.33 % agar. Levels of 1% agar permitted very few axons to penetrate the matrix. Glial flattening at the agar-solid substratum interface was observed at all agar concentrations. Effects of colchicine were assessedon: (a) single cells cultured for 48 or 72 h in the soft matrix; (b) single cells cultured on the 1 % agar substratum; (c) single cells plated on a plastic culture dish. At 90 min on plastic, all axons with ‘free’ growth cones originally on the plastic substratum were retracted. Axons whose tips were attached to glial cells or to other axons were not retracted. In contrast to theseresults, all axons in the matrix or on the agar substratum appeared unaffected by colchicine at 90 min. At 3 h, axons on plastic with their tips attached to other cells appeared extraordinarily thin and possessedmany rounded

Fig. I. A single neuron with an elongating axon in the agar matrix. Insets are photographs of the same cell

taken in different focal planes in order to show more clearly all segments of the axon and growth cone. x 500. Fig. 2. Another neuron growing in the agar matrix and showing typical axonal branching and twisting in the three dimensional matrix. Contrast this arrangement with the axons in a single focal plane of figs 5 and 6. Fig. 3a, b. The same single neuron photographed approx. 10 min apart. Note that the tip (A) has elongated in the interval, and that microspikes can be seen protruding from the growth cones despite presence of the enveloping agar matrix. x 530. Fig. 4. A typical flattened glial cell situated at the agar matrix-coverslip interface. The cell is spread, has a typical ‘trailing’ end, and has ruffle-like thickenings anteriorly. Such flattened cells are never observed elsewhere in the matrix proper. x 430. Fig. 5. A neuron and a glial cell located at the agar matrix-plastic interface. The glial cell is spread and the axon elongates in a single focal plane, reflecting the flat substratum upon which it rests. x 360. Fig. 6. A neuron at the matrix-plastic interface. Contrast with fig. 1. x 360. Exptl Cell Res 81 (1973)

Preliminary notes 485

Exptl Cell Res 81 (1973)

486

Preliminary notes

‘varicosities’ down their length. Axons on or in agar were of normal thickness, though some tip regions appeared rounded and lacked microspikes. At 24 h on plastic, only flattened glial cells and rounded cells were seen-no intact axons were present. At 24 h on the 1% agar substratum, no axons remained. Only rounded single cells and cell aggregateswere visible. At 24 h in the agar matrix, fewer axons than at earlier times were present per dish. However, someaxons remained patent though of narrow diameter and with varicosities. Control dishes with agar but without colchitine contained many more neurons with long axons of normal morphology at this time. Discussion

These results establish that single sensory neurons can elongate axons into an agar matrix, whereas glial cells appear to be unable to spread or carry out gliding cell movement in such a matrix. Such axons tend to have tortuous, irregular shapes, unlike the axons seenin single cell culture on two-dimensional substrata. Axons can also elongate over a 1% agar substratum that does not permit axon penetration or glial cell spreading. Thus either two- or three-dimensional agar provides sufficient interactions to permit axons to elongate but not glia to spread. Perhaps such interactions and the presence of a supportive matrix on all sides accounts for the failure of some axons in agar to retract when treated with colchicine for 24 h. Similarly, such interactions may result in the relatively slow retraction that occurs on the hard agar substratum. Axon retraction does not occur in vivo where the myelin sheath, the presence of branching collaterals, and the adherence of the nerve ending, may all contribute to stability in the absence of axonal microtubules [21-231. Exptl

Cell Res 81 (1973)

Though, to our knowledge, no direct measurements of cell-to-agar adhesivity have been made, it is generally assumed that the low ‘adhesivity’ of cells to agar accounts for failure of cells to attach and locomote. We have proposed that a basic difference distinguishes gliding cell movement from nerve cell elongation. The former movement may entail flattening on a substratum, an extension phase in the ruffled membrane region, attachment to a substratum, and a contractile event to advance the cell soma forward. Axon elongation may involve analogous extension activities, as well as net addition of new axonal surface material near the growth cone [24--261.In the model there is no requirement for firm adhesion between nerve and substratum, since the nerve cell soma need not be advanced over the substratum. The observed elongation of axons into or on a supposedly non-adhesive agar matrix agrees with this model. This work was supported by research grant HD-04708 from USPHS. Thanks are exnressed to Belen Sosa and Joan Wrenn for aid during these studies.

References 1. Harrison, R G, J exptl zoo1 17 (1914) 521. 2. Trinkaus, J P, Cells into organs. Prentice-Hall, Englewood Cliffs, N J (1969). 3. Yaoi, Y & Kanaseki, T, Nature 237 (1972) 283. 4. Yaoi, Y, Onoda, T & Takahashi, H, Nature 237 (1972) 285. 5. Harrison, R G, J exptl zoo1 9 (1910) 787. 6. Schubert, D & Jacob, F, Proc natl acad sci US 67 (1970) 247. 7. Gail, M H & Boone, C W, Exptl cell res 70 (1972) 33. 8. Carter, S B, Nature 208 (1965) 1183. Ibid 213 (1967) 256. 9. 10. Harris, A, Exptl cell res 77 (1973) 285. 11. Curtis, A S G, Symposia Brit sot parasitology (ed A E R Taylor & R Muller) vol. 10. Blackwell, London, 1972. 12. Lieberman, M, Roggeveen, A E, Purdy, J E & Johnson, E A, Science 175 (1972) 909. 13. Abercrombie, M, Nat1 cancer inst monogr 26 (1967) 249. 14. Sanders, F K & Burford, B 0, Nature 201 (1964) 786. 15. MacPherson, I & Montagnier, L, Virology 23 (1964) 291.

Preliminary notes 487 16. Tuffery, A A, J cell sci 10 (1972) 123 17. Yamada, K M, Spooner, B S & Wessells, N K, Proc natl acad sci US 66 (1970) 1206. 18. Luduefia, M A & Wessells, N K, Dev biol 30 (1973) 427. 19. Spooner, B S, J cell physiol 75 (1970) 33. 20. Yamada, K M, Spooner, B S & Wessells, N K, J cell biol 49 (1971) 614. 21. Bunge, R & Bunge, M, Anat ret 160 (1968) 232. 22. Kreutzberg, G W, Proc natl acad sci US 62 (1969) 722. 23. Dahlstriim, A, Symposia int sot cell biol (ed S H Barondes) vol. 8, p. 153. Academic Press, New York, 1969. 24. Bray, D, Proc natl acad sci US 65 (1970) 905. 25. J cell biol 56 (1973) 702. 26. Bunge, M B, J cell biol 56 (1973) 713. 27. Luduefia, M A. Dev biol 33 (1973) 268. Received June 15, 1973 Revised version received July 24, 1973

A simple technique for the measurement of swimming speed of Chlamydomonas G. K. OJAKIAN’ and D. F. KATZ,2” ‘Department of Physiology-Anatomy and VheDeportment of Mechanical Engineering, University of California, Berkeley, Calif. 94720, USA Summary. A simple technique for the measurement of swimming speed of Chlamydomonas reinhardi is described. The technique is based on a rapid statistical counting method which treats the swimming cells as a dilute, kinetic gas and its accuracy has been confirmed by direct photomicrographic measurements. In principle, this technique can be applied to any suspension of organisms satisfying certain statistical criteria.

Determination of the average swimming speed in a suspension of microorganisms can be quite useful, not only as a standard parameter of the organism’s state, but also, in studying physiological aspects of motility. Average swimming speeds can be obtained with some precision utilizing photomicrographic [5, 10, 141and cinephotomicrographic [6, 111 techniques. These methods, however, require considerable time and expense. Alternatively, rapid, statistical counting techniques, such as those introduced by Baker * Present address: Department of Applied Mathematics and Theoretical Physics, University of Cambridge, UK.

et al. [l] and utilized by other workers [2, 8, 91 for measuring the swimming speed of spermatozoa, have been developed. In this report, we present a modification of the counting method of Baker et al. [l] that has been successfully applied to the biflagellar alga Chlamydomonas reinhardi. This method is based on the principle that the number of organisms swimming through a known area in a given time period is related to the average swimming speed. The computational formula is analagous to that previously derived [l], but contains a correction. Direct confirmation of the statistical assumptions implicit in the revised formula, and in the average speeds predicted, has been obtained by use of video tape photomicrography. Materials and Methods Wild-type (strain 137~) cultures of Chlamydomonas reinhardi were kindly provided by Dr R. P. Levine, The Biological Laboratories, Harvard University. The cells were grown at 22-24°C in 500 ml flasks containing 300 ml of minimal media [15] supplemented with 0.2 “,b sodium acetate. The growth light intensity was 4 000 Iux at the level of the cultures and was provided by daylight fluorescent lamps programmed to maintain a cycle of 12 h light, 12 h dark 131.All motility measurements were done on log phase cultures 4-8. h into the light portion of their cycle. Motility measurements were done in a hemaytcometer mounted on the stage of a Zeiss phase-contrast microscope. To prevent pihotactic respbnses, either a red gelatin filter (Kodak no. 92) was placed over the light source [4] or dark-field illumination was utilized. A plane area is defined bv choosing a 0.25 mm line segment on the hemacytometer and-multiplying it by the depth (0.1 mm) of the chamber. Since Chlamydomonas reinhardi is usually no more than 0.01 mm in length, the depth of the hemacytometer is sufficient to permit three-dimensional swimming and still allow visualization of all cells that swim through the plane. By counting the number of cells swimming thiough the defined area in a given time period (usually 60 set), we can calculate the mean swimming speed ising the following formula (cf Appendix):

where V is average swimming speed; N is the number of cells passing through the plane area in a given period of time t; n is cell density; A is the area of the plane; and 4 is a statistical factor (cf Appendix). If the cells are confined to movement in a plane, Exptl Cell Res 81 (1973)