Ultrastructure of dissociated nerve cells of Periplaneta americana (L.) (Dictyoptera : Blattidae) growing in culture

Int. J. Insect Morphol. & Embryol., Vol. 10, No. 3, pp. 225 Io 233.1981. Primed in Great Brilain.

0020-7322/811030225- 0 9 $02.00/0 © 1981 Pergamon Press Lid.

ULTRASTRUCTURE OF DISSOCIATED NERVE CELLS OF P E R I P L A N E T A A M E R I C A N A (L.) (DICTYOPTERA : BLATTIDAE) GROWING IN CULTURE D. HIC]~S,* D. J. BEADLE,* D. P. GILESt~ and P. N. R. USHERWOODI" (Accepted 18 November 1980) *School of Biological Sciences, Thames Polytechnic, London SEI8 6PP, U.K. "pDepartment of Zoology, University of Nottingham, Nottingham NG7 2RD, U.K. Abstract--Cells from dissociated nervous systems of 16-21-day-old Periplanetaamericana were grown in vitro and their ultrastructural properties characterised as part of a long-term study of the insect central nervous system (CNS) in order to determine the value of these cultures in pharmacological studies. Most cells had a spherical cell body, a prominent nucleus and nucleolus, and cytoplasm containing numerous mitochondria and lysosomes. Endomembranous elements were rare. These cells are considered to be neurones. After 48 hr in culture, the neurones produced long axonal processes containing mitochondria and microtubules and terminating in flattened growth cones. Although the processes formed an extensive neuronal network, no synapses were seen. Someglial cells were also present in the cultures. Index descriptors (in addition to those in title): Neurones, axons, growth cones.

INTRODUCTION EXPLANT CULTURES have been widely used in investigations of the structure and physiology of the insect nervous system. Regenerating leg explants from Leucophaea maderae (Marks and Reinecke, 1965) and brain and ganglia of Periplaneta americana (Levi-Montalcini and Chen, 1969) have provided valuable insights into the growth and development of nerve and glial elements (Chen and Levi-Montalcini, 1969; LeviMontalcini and Chen, 1971), the effects of endocrine glands on nerve growth (Marks and Reinecke, 1965; Levi-Montalcini and Seshan, 1973) the interrelationship of visceral muscle and nerve cells (Aloe and Levi-Montalcini, 1972) and the electrophysiological characteristics of ganglia in vitro (Provine et al., 1973). However, the use of explant cultures in studies of many aspects of the insect nervous system is limited because the explants tend to retain the complexity of the tissue of origin (Giles 1980), few nerve ceils migrate from the explant (Marks and Reinecke, 1965; Usherwood et al., 1980) and nerve fibres in the migratory zone around explants are still closely associated with glial elements (Levi-Montalcini and Chen, 1969). These difficulties have been overcome in studiesof the vertebrate nervous system by the use of dissociated cell cultures (Peacock et al., 1973; Fischbach and Dichter, 1974; Fishbach and Nelson, 1977) and attempts have been made to develop a similar system for insect nerve cells. Although dissociated insect neurones from Drosophila gastrulae have been grown in culture and successfully used to follow the development of neurones from neuroblasts and the formation of neuromuscular junctions (Seecof and Teplitz, 1971; Seecof et al., 1971; Seecof and Donady, 1972) this system is perhaps not ideal since the neurones are grown in cultures containing a range of other cell types. Chen and Levi-Montalcini (1970a) used embryos of Periplaneta americana to produce dissociated neuronal cultures of central ~:Present address: The Boots Co. Lid., Lenton Research Station, Nottingham NG7 2QD, U.K. 225

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neurones by mechanically disrupting central ganglia. They found that this technique destroyed all cells, except neurones. These cultures were maintained in a chemically defined medium for several months. They responded to the trophic effects of other cockroach tissues, such as gut, and formed synapses with effector cells (Levi-Montalcini et al., 1973). Cultures of this type would appear to provide a defined, simple system for use in studies of cockroach neurones. Our laboratories are currently attempting to use these cultures in a number of structural, biochemical and pharmacological investigations (Giles et al., 1978). We report here on an initial investigation of the uitrastructure of dissociated neurones obtained from the central nervous system of 16-21-day-old cockroach embryos and maintained in vitro for 2 - 2 8 days. MATERIALS

AND

METHODS

Oothecae were collected from adult, female P. americana and incubated at 29°C for 1 6 - 2 1 days before use. If left, hatching occurred at 31 days. Entire nerve cords were removed from embryos under sterile conditions, and transferred to a plastic tube containing culture medium that consisted of 5 parts Schneider's insect culture medium and 4 parts Eagle's basal medium in Hepes buffer containing 100 i . U . / m l penicillin and 100 lag/ml streptomycin (Chen and Levi-Montalcini, 1970a). Approximately 2 nerve chains were used per culture, and these were dissociated by sucking the tissue through pasteur pipettes of progressively smaller openings and by forcing out the tissue with some pressure onto the walls of the plastic tube. After repeating this treatment several times, the cell suspension was plated out (2 drops of culture medium per dish) on the bases of 500 m m x 12 m m Falcon, tightseal, petri dishes. Explants of embryonic cockroach foregut were added to some of the dissociated cell cultures (Giles, 1980; Skelton, 1980). A modification of the 'Hanging C o l u m n ' technique (Shields et al., 1975) was employed to maintain the cultures (Giles, 1980). The culture dishes were left to stand until the cells had adhered (l hr) and were then inverted to prevent the debris contaminating the growing surface. Observations of living cultures were made under phase optics on either an Olympus OM - 2 or a Leitz inverted microscope. Specimens were prepared for transmission electron microscopy by initial fixation in 2.5o10 glutaraldehyde in 0.1 M Na cacodylate buffer at pH 7.2 with 0.1 M sucrose at room temperature for 3 hr, followed by washing in buffer and further fixation in 1010osmium tetroxide in 0. I M cacodylate buffer for I hr. The tissue was subsequently dehydrated through a series of ethanols to absolute and embedded in Epon resin. Thin sections were cut on an LKB ultratome Ill and were stained with lead citrate and uranyl acetate. For scanning electron microscopy, cultures on Falcon dishes were fixed in 2.5010 glutaraldehyde in 0.1 M cacodylate buffer with 0.1 M sucrose at room temperature for 3 hr, followed by dehydration through a series of alcohols to absolute. The tissue was then critical-point dried. Specimens were then coated with carbon and gold in a Polaron sputterer. OBSERVATIONS

The growth and longevity of the cultured neurones depended on the degree of dissociation of the nervous tissue rather than the presence or absence of other cell types or tissues. When dissociation resulted in isolated, single neurones these did not survive for more than 3 - 4 weeks even in the presence of foregut explants, although some neurones exhibited outgrowths. However, a mixture of isolated neurones and cell clumps (containing perhaps a dozen cells) could be maintained for at least 2 months and fibre outgrowth from neurones in such cultures was prolific. The major cell type present in the cultures had a spherical or ovoid soma, 6 - 10 lam in diameter, and a number of long, cellular processes (Fig. 1). These processes were quite thick near the cell body but, farther out, some processes branched profusely to produce numerous thin fibres that formed a neuronal network over the floor of the culture dish (Fig. 2). Occasionally, monopolar neurones were encountered (Fig. 3). Cultures were always contaminated with debris (Fig. 3). Scanning and transmission electron microscope observations confirmed that there was substantial fibre growth from and cellular inter-connections between neurones after only 48 hr in culture (Fig. 4). The neurone cell bodies were usually quite spherical with smooth surfaces, although occasionally a "fried egg" shaped cell was seen with a prominent,

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d~ FiG. 1. Dissociated neurones after 7 days in vitro. Cell bodies have produced large outgrowths and fine processes arise from these, x 250. F]G. 2. Established culture of neurones. Cell bodies have produced numerous outgrowths that form a complex network apparently connecting cell bodies, x 400. FIc. 3. Monopolar nerve cells after 7 days growth. Small spherical structures are probably nuclei of disintegrated glial cells (arrow). x 400. FIG. 4. Two nerve cell bodies connected by outgrowths after only 48 hr in culture. Scanning E.M. x 3300.

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central nuclear region and a surrounding skirt of cytoplasm. The main area of the cell body was occupied by a large nucleus containing dense heterochromatic regions attached to the nuclear envelope and often one or more prominent nucleoli (Fig. 5). The cytoplasm was very granular and the most obvious organelles were mitochondria, lysosomes and vacuoles. Mitochondria were very numerous being tubular in shape and only 0.15 p.m in cross-section. Lysosomes, 1 p.m in diameter and containing dense, osmiophilic material and membrane profiles were also present. Dictyosomes and endoplasmic reticulum were seen only rarely (Fig. 6). Contacts between neurones either at the cell body or fibre level were quite common, and where these occurred, membrane thickenings were evident at the point of contact (Fig. 7). As the cells aged in isolated cultures, they first became filled with numerous lipid droplets (Fig. 8) and then disintegrated. The cultured neurones produced 2 types of fibre outgrowth. One type was cylindrical and straight, branched infrequently and connected cell bodies of adjacent neurones (Fig. 9). The other type was present in large numbers and followed a more tortuous path branching profusely to form numerous, thin processes, 0.1 p.m in diameter (Fig. 4). Type 2 fibres were varicose and at points along their length were attached to the culture vessel by flattened, expanded regions from which new processes emerged (Fig. 10). When examined in the electron microscope there was no obvious separation of the fibres into 2 types. All contained long, straight micro-tubules and narrow, elongated mitochondria (Fig. 11). Some variations in cytoplasmic density and numbers of organelles were apparent from fibre to fibre but these variations were not correlated with the fibre types identified under the light microscope. The tip of advancing fibres was flattened and expanded to form growth regions approx. 5 I~m long and 2 pm wide that were characterised by cytoplasmic depressions and mounds, and membranous folds (Fig. 12). A number of minute filipodia extended distally from this structure. These expanded areas contained a complex network of microtubules. Between these skeletal structures there were large numbers of small vesicles and lysosomes and numerous narrow, tubular mitochondria with indistinct cristae (Fig. 13). Some of the growth cones contained large, osmiophilic, lysosome-like structures (Fig. 14). Where fibres came into contact they formed complex junctions, but there was no evidence of synaptic structures and no synaptic vesicles were seen. Some glial cells had survived the dissociation process. Isolated glial cells did not survive for more than a few days, although during this time some produced quite long processes (Fig. 15). However, when glial cells were present along with neurones in a clump of cells, they survived for as long as the culture was maintained. Glial cells in this situation produced extremely long (100 p.m) processes that could be easily recognised by their flattened, expanded form (Fig. 16). DISCUSSION The cells grown in dissociated culture in this investigation produced extensive fibre growth and developed numerous intercellular connections after only 48 hr. In agreement with Schalpfer et al., (1972), but in contrast to Chen and Levi-Montalcini (1970a,b), we found that the presence of foregut explants had no effect on the maintenance and development of dissociated neurone cultures. We obtained no evidence of synapses in our cultures and, using a sensitive histochemical technique (Beadle et al., 1979), we were unable to demonstrate cholinesterase activity in the cultured neurones, although explants of embryonic

Ultrastructure of Dissociated Nerve Cells of Periplaneta americana

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FIG. 5. Electron micrograph of nerve cell body with prominent nucleus and nucleolus (Nu), numerous mitochondria (M) and lysosomes (L) x 5500. FiG. 6. Electron micrograph of nerve cell body with densely stained mitochondria (arrows), Golgi body (G) and lysosome (L). x 5500. FiG. 7. Electron micrograph of cellular outgrowths containing mitochondria (M) and microtubules (MI). Membrane thickenings occur where outgrowths contact cell bodies or other fibres (arrows). x 10,000. FiG. 8. Electron micrograph of neurones 23 days in culture containing lipid droplets (Li). x 8250.

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FtG. 9. Electron micrograph of cell bodies connected by neuronal network. Some fibres are smooth and branch rarely (F). × 6500. FIG. 10. Electron micrograph of cellular outgrowth with flattened region (F) attached to culture vessel from which many processes have developed. × 13,000. FIG. I1. Electron micrograph of axonal processes from 28-day-old culture with elongated milochondria (M) and microlubules (arrows). × 16,500. FiG. 12. Electron micrograph of growth cone at advancing tip of neurone outgrowth. Scanning E.M. × 14,625.

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FiG. 13. Electron micrograph of region of growth cone with numerous mitochondria, iysosomes (L) and several microtubules (arrow), and small vesicles (white arrow), x 16,500. FIG. 14. Electron micrograph of growth cone containing large numbers of dense lysosomes (L). Points of contact with adjacent processes are characterised by membrane thickenings (arrows) × 16,500. FxG. 15. Electron micrograph of flattened glial cell process (G) emanating from glial cell that has survived dissociation process, x 6500. FiG. 16. Electron micrograph of neuronal network on floor of culture vessel with well-developed, flattened glial process (G) growing amongst neuronal outgrowths (N). x 13,000.

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c o c k r o a c h ganglia exhibit high activity (Schlapfer et al., 1972; W h i t e and Beadle, unpublished observation). A l t h o u g h the cells in o u r cultures exhibited m a n y o f the u l t r a - s t r u c t u r a l characteristics o f n e u r o n e s they d i f f e r e d in s o m e respects f r o m n e u r o n e s identified in explant cultures (Hicks and Beadle, 1980). In particular, the c o m p l e x Golgi system and associated vesicular structures that are characteristic o f n e u r o n e s in explants are absent f r o m dissociated neurones. In view o f the o c c u r r e n c e o f dissociated cultures o f both m u l t i p o l a r and m o n o p o l a r n eu r o n e s , it is t e m p t i n g to assume that the cultures c o n t a i n b o t h m o t o n e u r o n e s and i n t e r n e u r o n e s . H o w e v e r , large n e u r o n e s f r o m t h o r a c i c ganglia o f locust n y m p h s , p r e s u m a b l y m o t o n e u r o n e s , usually b e c o m e m u l t i p o l a r when dissociated and cultured (Giles et al., 1978). T h e g r o w t h cones which c h a r a c t e r i z e the a d v a n c i n g n e u r o n e process resemble those o f v e r t e b r a t e n e u r o n e s in vitro (Bunge, 1973; Rees, 1978), a l t h o u g h the a r r a n g e m e n t and type o f organelles present in this region o f the n e u r o n e a p p e a r to be d i f f e r e n t in insects and vertebrates. In particular, the a g r a n u l a r e n d o p l a s m i c re t i c u l u m that is so p r o m i n e n t in the v e r t e b r a t e g r o w t h cone ap p ear s to be absent in the insect c o n e wh er e the m o s t p r o m i n e n t feature is the presence o f t u b u l a r m i t o c h o n d r i a . H o w e v e r , the skeletal a r c h i t e c t u r e o f b o t h types o f g r o w t h cone is essentially similar. O n e peculiarity o f o u r cultures was the survival o f s o m e glial cells. C h e n an d LeviM o n t a l c i n i (1970b) f o u n d no evidence o f these cells in their cultures. H o w e v e r , the e l o n g a t e d , flattened cell bodies a n d cellular processes seen in o u r cultures are w i t h o u t d o u b t glial in c h a r a c t e r . A l t h o u g h these cells did not survive f o r long in isolation, they did survive when associated with n e u r o n e s in cell clumps. It is possible that the s u r r o u n d i n g n e u r o n e s p ro t ect ed t h e m f r o m the m e c h a n i c a l d i s r u p t i o n o f the g a n g l i o n that occurs d u r i n g the p r e p a r a t i o n o f these cultures. Acknowledgements--We thank R. Lamb and J. Beasley for photographic assistance and scanning electron

microscopy, T. Smith for technical assistance and J. L Skelton for useful discussion. D. Hicks was supported by S.R.C. and Wellcome Research Laboratories. Part of this work was supported by a grant from the British Agricultural Research Council to P.N.R.U. REFERENCES ALOE. L. and R. LEVI-MONTALCINI.1972. In vitro analysis of the frontal and ingluvial ganglion from nymphal specimens of the cockroach, P. americana. Brain Res. 44:147 - 163. BEADLE, D. J., C. D. LIVINGSTONEand S. READ. 1979. UItrastructural localisation of acid phosphatase, non-specific esterase and 13-glucuronidase in the mid-gut-epithelium of Tenebrio molitor, Schistocerca gregaria and Carausius morosus. Histochemie 28:243 -49. BUNGE,M. B. 1973. Fine structure of nerve fibres and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56:713 -35. CHEN, J. S. and R. LEvI-MONTAECINI.1969. Axonal outgrowth and cell migration in vitro from the nervous system of cockroach embroys. Science (Wash., D.C.) 166: 631-32. CHEN, J. S. and R. LEVI-MONTALCINI.1970a. Axonal growth from insect neurons in glia-free cultures. Proc. Nat. Acad. Sci. (Wash.) 66:32 - 9. CHEN, J. S. and R. LEVI-MONTALCINI.1970b. Long term cultures of dissociated nerve cells from the embryonic nervous system of the cockroach, Periplaneta americana. Arch. Ital. Biol. 108:503 - 37. FISCHBACH,G. D. and M. A. DICHTER. 1974. Electrophysiologic and morphologic properties of neurones in dissociated chick spinal cord cell cultures. Dev. Biol. 37: 100- 16 FISCHBACH,G. D. and P. G. NELSON. 1977. Cell culture in neurobiology, pp. 719-74. In E. R. Kandel (ed.) Handbook o f Physiology. Ann. Physiol. Soc. Bethesda, Maryland. GILES, D. P. 1980. Studies on the insect CNS in vitro. Ph.D. Thesis, University of Nottingham. GLEES. D. P., R. T. JoY and P. N. R. USHERWOOD.1978. Growth of isolated locust neurones in culture. J. Physiol. 276:74 p. HICKS. D. and D. J. BEADLE.1980. An investigation of the ultrastructure of neuronal cultures of P. americana, pp. 193 - 200. In Insect Neurobiology and Pesticide Action, Society of Chemical Industries, London.

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