Primary Cultures of Muscle From Embryonic Locusts (Locusta Migratoria, Schistocerca Gregaria): Developmental, Electrophysiological and Patch-Clamp Studies

It is well-established that L-glutamic acid is the transmitter at excitatory nerve-muscle junctions on adult insect skeletal muscle (Usherwood & Cull-Candy, 1975; Usherwood, 1978; Piek, 1985) and that y-aminobutyric acid transmits at the inhibitory junctions (Usherwood & Grundfest, 1965; Duce & Scott, 1984). Glutamate receptors (D-receptors), with similar pharmacological and physiological characteristics to the glutamate receptors present postjunctionally at excitatory synapses, are also widely distributed extrajunctionally on locust leg muscle (Cull-Candy & Usherwood, 1973; Usherwood & Cull-Candy, 1974). These extrajunctional receptors have been extensively studied during the past few years using patch-clamp techniques, and through single channel analysis a detailed account of their channel gating kinetics is now available (Patlak, Gration & Usherwood, 1979; Usherwood, Clark, Gration & Patlak, 1979; Gration, Lambert, Ramsey & Usherwood, 1980; Gration & Usherwood, 1980; Gration et al. 1981, 1982; Cull-Candy, Miledi & Parker, 1981; Cull-Candy & Parker, 1981; Ashford et al. 1984a,b;,Kerry et al. 1986). Advances made in understanding the properties of the glutamate receptor channel complex have been facilitated by the use of concanavalin A to block desensitization (Mathers & Usherwood, 1976, 1978). However, further progress in understanding the gating kinetics and ligand binding properties of this membrane protein is impeded by the inability to obtain giga-ohm seals routinely between patch-clamp pipettes and adult locust muscle membranes. This may be due to the properties of the thick basal lamina which characterizes the leg muscles of adult locusts (Usherwood, 1967; Rees & Usherwood, 1972) and which may restrict access of the patch pipette to the muscle plasma membrane. Enzymatic treatment of the muscle to remove connective tissue has been undertaken but with unsatisfactory results (Gration, 1982). Although Franke & Dudel (1985) have reported successful single channel recordings from crayfish muscle using giga-ohm seals, these are not possible if the muscle is pretreated with concanavalin A (J. Dudel, personal communication).

Developing muscle is often characterized by a thin basal lamina which may account for the ease with which giga-ohm seal techniques can be employed for patchclamping membrane receptors and other channel proteins of embryonic vertebrate skeletal muscle in culture (e.g. Sakmann & Neher, 1983). This paper describes the methods employed to establish primary cultures of locust muscle tissue and contains a preliminary account of patch-clamp recordings, using giga-ohm seals, from single glutamate receptor channel complexes on the cultured myofibres. An abstract of this work has been published previously (Cook, Ramsey & Usherwood, 1985).

The growth and viability of embryonic locust and embryonic cockroach excitable tissue in vitro depend upon the age of the material from which the cultures are prepared, the temperature and humidity at which they are maintained and the medium in which they are grown. Based on studies extending over the past 8 years (Giles, Joy & Usherwood, 1978; Lynch, 1980; Hicks, Beadle, Giles & Usherwood, 1981; Giles, Usherwood, Hicks & Beadle, 1983), we have developed the following techniques for obtaining primary cultures of excitable tissues from embryonic locust (and cockroach).

Developing eggs were obtained from either 10-day-old (Locusta migratoria) or 11-day-old (Schistocerca gregaria) egg pods and the material from these was divided equally to prepare 4-5 primary cultures. Most pods contained 20–30 eggs. When fewer eggs were present embryos from a number of pods were pooled to produce sufficient material for 4–5 cultures. An important feature of the eggs at this stage is that the cuticle of the embryo is still untanned and this facilitates dissociation of the embryos for culturing. Cultures prepared from eggs older than 10(11) days were rarely successful; those from younger eggs gave lower yields of muscle. After removing the eggs from their pods they were washed with distilled water and then immersed in Methcol for 20 min for surface sterilization. All subsequent operations were performed under aseptic conditions, mostly in a laminar flow cabinet. The eggs were transferred to a Petri dish containing sterile saline (in mmol I⁻¹: NaCl, 180; KC1, 10; CaCl₂, 2; NaH₂PO₄, 6; Na₂HPO₄, 4; pH6·8; Usherwood & Grundfest, 1965) and the embryos were separated from the egg cases (Fig. 1A,B) after rupturing the latter with fine forceps (no. 5). The entire gut was then removed from each embryo (Fig. 1C-E). The remainder of the embryo was transferred to a dish of fresh saline, where it was mechanically dissociated with fine forceps. The dissociated material was centrifuged at 250 g for 5 min, resuspended in 5+4 medium (Chen & Levi-Montalchini, 1969) and dissociated further by trituration using a Pasteur pipette. The dissociated material, suspended in 5+4 medium, was cultured in 50X9 mm Falcon dishes using a modified version of the hanging column technique described by Shields, Dubendorfer & Sang (1975). Two pieces of sterile, broken glass slide, coated in sterile Vaseline, were placed in the bottom of each sterile Petri dish as supports. The dissociated tissue was then plated out between the supports. Finally, sterile coverslips were gently lowered onto the supports and pressed down until the medium containing the dissociated material filled the whole area between the coverslips and the Petri dish (Fig. 1F). The dishes were stored under high humidity at 28°C. Medium changes were made initially after 2 weeks in vitro and thereafter ad lib. Apart from removing debris, changes of medium also initiated further development of the cultures.

Cultures of dissociated material from 10-day-oldLocwsta embryos were also grown successfully in Grace’s insect medium (GIBCO 041-1590) (Grace, 1962) using the above procedures.

Unless otherwise stated similar results were obtained from Schistocerca and Locusta cultures.

Cultures of different ages were equilibrated in saline (in mmol l⁻¹: NaCl, 180; KC1, 10; CaC12, 2; Hepes, 3; pH 6·8) for 30 min before being mounted on the stage of an inverted, phase contrast microscope.

Membrane potentials of the myofibres were determined using intracellular, glass microelectrodes filled with 3 mol l⁻¹ KC1. The sensitivity of the myofibres to L-glutamic acid was tested by applying 10⁻⁶moll⁻¹ sodium L-glutamate, in saline, under pressure from micropipettes (tip diameter about 2μm). For single-channel studies, patch-clamp micropipettes with fine tips (diameter 1–2μm), either polished or unpolished and filled with saline containing sodium L-glutamate (5×l0⁻⁹-10⁻⁶ moll⁻¹), were used. When approaching a myofibre a slight positive pressure was applied to the inside of the pipette to keep its tip free of debris. The pressure was removed when contact with the myofibre was made and a giga-ohm seal then often formed spontaneously, although sometimes it was necessary to apply a small negative pressure to the inside of the pipette to achieve a good seal. During the latter part of these studies patch recordings were made after treatment of the muscle cultures for 30 min with 10⁻⁶moll⁻¹ concanavalin A (Mathers & Usherwood, 1976, 1978) and these were compared with recordings made in the absence of this lectin. Singlechannel currents were recorded on a Racal FM tape recorder at a bandwidth of d.c. to 1–3 kHz. Full details of the procedures employed for analysing the channel data can be found in previous publications from our laboratory (Gration et al. 1982; Kerry et al. 1985).

When first plated out the cultures contained mainly rounded cells, but after only about 30 min in vitro some of these had become spindle-shaped with polar processes and had attached to the base of the culture dish. During the first 7 days in culture there was a lot of cell death, particularly of isolated cells, but by the end of this period many spindle-shaped cells remained, particularly in the vicinity of tissue clumps. Careful observation of their later development showed clearly that these remaining cells were myocytes. Either through extending their polar processes, to make contact with adjacent myocytes (Fig. 2A), or by associating laterally with other myocytes they eventually formed reticular arrays or chains of cells (Fig. 2B), before giving rise to multinucleated myofibres (Fig. 2C). By about 10 days a mixture of branched and unbranched myofibres was present in the cultures, the latter often stretched between tissue clumps (Figs 2D, 3A). After 2 weeks in vitro the myofibres were the most abundant structures and it was at this stage that spontaneous contractile activity appeared in the Locusta cultures. (Myofibres in Schistocerca cultures rarely contracted spontaneously.) Application of 10⁻⁶moll⁻¹ L-glutamate in saline by pressure from a micropipette increased the frequency of these contractions. The cultures also contained neurones, glial cells and tracheolar tissue (Fig. 2D). The neurones were usually aggregated into groups and were readily distinguishable from muscle tissue by their fine processes. The neurone processes were often closely associated with myofibres but it remains to be established whether functional connections develop between nerve and muscle tissue in these cultures.

A change of medium after about 4 weeks in vitro precipitated further release of myocytes from tissue clumps and the development of more myofibres (Fig. 2D). The myofibres were fully grown after about 5 weeks in vitro, when they were up to 400/tm in length and 15/im in diameter (Fig. 3A). Myofibres of this size were usually detached from the base of the culture dish for a greater part of their length. Unlike other tissues in the cultures, the myofibres contained a granular and fibrous cytoplasm, but they were rarely striated (Fig. 3A,B). Nervous tissue could still be readily distinguished from the muscle tissue at this stage by its highly branched, fine, recurrent processes radiating from clumps of loosely-packed, round cell bodies (Fig.3C). …

The myofibres continued to develop for a further 2–3 months during which time any remaining clumps of cells and most non-muscular tissues were lost (Fig. 3D). The myofibres no longer contracted spontaneously but many, particularly those of Locusta cultures, still contracted in response to a challenge with sodium L-glutamate (10⁻⁶mol l⁻¹). Although the development of the cultures was similar for Locusta and Schistocerca tissue, a larger area of growth was usually obtained with the Schistocerca cultures.

Freshly-dissociated material from 10-day-old Locusta embryos was also grown in modified Grace’s insect medium. After about 30 min in vitro the majority of the cells in these cultures had attached to the base of the culture dish. At this stage and for the next few hours, most of the attached cells had a rounded outline although by 4 h some spindle-shaped cells were present (Fig. 4A). By 17 h most of the myocytes had polar processes with many cells aggregated into groups with their processes aligned. By 24 h the myocyte groups had fused to form multinucleate structures, whilst isolated myocytes were now characterized by thickened processes (Fig. 4B). After 7 days myofibres with a fibrous appearance had developed either as mononucleate structures from single myocytes or as multinucleate structures from the groups of muscle cells (Fig. 4C). At this time they were up to 30 μm in length and 13 μm in diameter. After 3 weeks many of the myofibres were distinctly striated (Fig. 4D), even those with only one nucleus. Their length had increased to over 75 μm and their diameter to over 20 μm, and they bore a striking resemblance to myofibres which develop in vivo. They survived for up to 1 month in culture but showed no further signs of growth. They were never observed to contract either spontaneously or in response to a challenge with sodium L-glutamate (10⁻⁶moll⁻¹).

Our studies of the physiology and pharmacology of both Locusta and Schistocerca muscle cultures have so far been mainly restricted to those grown in 5+4 medium, despite the fact that myofibres grown in Grace’s medium looked more like those found in vivo. Unfortunately, myofibres grown in Grace’s medium usually became detached from the base of the culture dish, which effectively precluded routine studies using patch-clamp techniques.

Intracellular recordings using KC1 electrodes from two Locusta cultures and two Schistocerca cultures, all of which had been more than 4 weeks in vitro, gave a mean resting potential of — 33·4 ± 13·5 mV (S.D.; N= 19). Unfortunately, because the myofibres were not attached to the base of the culture dish for much of their length and often contracted when impaled by a microelectrode, they were easily damaged. For this reason only those potentials which were maintained for more than 1 min after impalement are included in the above result. It is accepted that the true resting potential of the myofibre may be somewhat higher than the recorded value, i.e. closer to the —60 mV which characterizes adult locust leg muscle after equilibration in locust saline (Usherwood, 1969).

When 10⁻⁶moll⁻¹ L-glutamate was ejected onto the surface of the myofibre there was a transitory fall in membrane potential, sometimes accompanied by a contraction. Application of saline alone had no effect.

When patch pipettes were used to form mega-ohm seals with the surface membrane of myofibres present in cultures 3–8 weeks old, spontaneous spike-like events were recorded from about 5 % of the myofibres in both Locusta (Fig. 5) and Schistocerca cultures, sometimes associated with contractions. These spike-like events were most frequently recorded from myofibres closely associated with nerve fibres. The effects of L-glutamate on the spike-like events have not yet been investigated.

Giga-ohm recordings of glutamate channel currents have been obtained from patches of myofibre membrane either on intact myofibres or as excised, cell-free, outside-out and inside-out patches. With patch pipettes containing 10⁻³ mol I⁻¹ Ca²⁺ and sodium L-glutamate at a concentration as low as 5×10⁻⁹moll⁻¹, inward channel currents were usually seen for a brief period immediately after formation of a gigaohm seal and then rarely thereafter. These channel currents, which were about 4pA in amplitude in cell-attached patches when the patch pipette potential was held at 0mV, were recorded from cultured myofibres as early as 3 weeks in vitro. The impression gained was of a homogeneous glutamate receptor channel population diffusely distributed over the surface of the myofibre and exhibiting rapid desensitization. This impression was subsequently confirmed when myofibres pretreated with concanavalin A were studied. In the presence of this lectin these large conductance channels were found to be present over the entire surface of a myofibre and channel events occurred throughout the period of maintenance of a giga-ohm seal. The latter observation suggests that concanavalin A blocks desensitization of glutamate receptors on embryonic muscle. The channel currents were never seen when sodium L-glutamate was omitted from the patch pipette.

Most of the single-channel recordings, both on myofibres and from excised patches, including those made with only 5×10⁻⁹moll⁻¹ sodium L-glutamate in the patch pipette, were characterized by channel events exhibiting two equal, open conductance states (Fig. 6A). It seems likely that these represent the openings of two receptor channels but there remains the possibility that they are sub-conductances of a single receptor channel, since sudden transitions from the closed state to the double open state and vice versa were sometimes seen. Studies are currently in progress to clarify this issue. The noise level exhibited by a channel in its open state is usually greater than that seen when the channel is closed (Fig. 6A). During the simultaneous occurrence of more than one open conductance state the open noise level is further increased.

The amplitude and polarity of the glutamate channel current was dependent upon the magnitude and polarity of the pipette potential. A reversal potential of about 0mV was routinely obtained for the single-channel current recorded with cell-attached patches (assuming a resting potential of about —35 mV), which suggests that it may be a cation-selective channel like its counterpart on adult locust muscle (Anwyl & Usherwood, 1974). However, further studies are required before this can be firmly established.

The open-closed kinetics of the glutamate-gated channel are complex and vary according to the recording mode, i.e. whether recordings are made from a patch of membrane on a myofibre, from an excised inside-out patch or from an excised outside-out patch. Examples of the channel dwell-time distributions (open and closed) are presented in Fig. 6C,D for data obtained from an inside-out patch. The histogram of channel open times indicates the presence of at least three open states for the channel. The corresponding histogram for channel closed times is less easy to interpret because of the presence of either more than one channel or, perhaps, subconductance open states, in the recording from which the data were obtained, but it suggests that the glutamate channel is characterized by multiple closed states. A fuller account of the channel kinetics for inside-out patches and other recording modes will be presented in due course.

The conductance of the channel varied with the Ca²⁺ environment of the membrane patch. With 10⁻³ mol l⁻¹ Ca²⁺ in the patch pipette, 2×10⁻³ moll⁻¹ Ca²⁺ in the bathing medium and symmetrical distributions of Na⁺ and K⁺, the current-voltage characteristic for channels recorded from an inside-out patch was linear for pipette potentials between +80 mV and —80mV. The slope conductance under these conditions was about 60 pS (Fig. 7). However, when the pipette Ca²⁺ concentration was reduced to 10⁻⁷ mol I⁻¹ (buffered with EGTA) the slope conductance for inward currents (i.e. those flowing from pipette to bathing medium) was twice (about 120pS) that for outward currents (Fig. 7). This is similar to the conductance of the channel gated by extrajunctional D-receptors on adult locust leg muscle (Patlak et al.1979).

Significant advances in understanding the properties of vertebrate skeletal muscle have arisen from studies of primary cultures of muscle (Lake, 1916; Lewis & Lewis, 1917; Firkett, 1967; Bischoff & Holtzer, 1969; Larson, Jenkinson & Hudgson, 1970; Shimada, 1971; Nelson, 1975; Fambrough, 1978), most of which have been made on embryonic tissues. Primary cultures of insect muscle have been prepared using tissue from butterfly (Kurtti & Brooks, 1970), fruitfly (Seecof, Alleaume, Teplitz & Gerson, 1971; Dubendorfer, Blumer & Deak, 1978) and cockroach (Lynch, 1980) embryos, but their physiology and pharmacology have not been investigated. In this paper we have presented techniques for the establishment of primary cultures of muscle from embryonic locust together with a description of in vitro myogenesis. In addition we have shown that the cultured myofibres express receptors for L-glutamic acid and that recordings of channels gated by these receptors can be obtained using giga-ohm seal, patch-clamp techniques.

The development of locust muscle in culture displays many of the characteristics already described for cultured muscle of other insects (Kurtti & Brooks, 1970; Lynch, 1980) and, to a lesser extent, of vertebrates (Nelson, 1975; Bachmann, 1980). However, there are differences in the time course of myogenesis between insect species (Seecof et al. 1971), and between insect and vertebrate species, which tend to complicate such comparisons. Further complications arise because of methodological differences in terms of media used, the temperatures at which the cultures are maintained and the conditions under which the embryonic tissues are dissociated. The growth medium is obviously an important factor in determining the fate of the cultures, since one major result of this study is the discovery of striking differences in the morphological appearance of muscle tissues grown in 5+4 medium compared with Grace’s insect medium. Cultures grown in 5 + 4 medium express glutamate receptors resembling those found on adult muscle, but the morphology of the in vitro myofibres bears little resemblance to mature muscle tissue found in vivo. Myofibres grown in Grace’s insect medium are morphologically similar to mature muscle in vivo, but it remains to be established whether they exhibit similar physiological and pharmacological characteristics. One main advantage of the techniques we have developed is that they involve rather simple procedures. Also, once a culture has been established, only one change of medium is normally required for ‘full’ development of the muscle tissue. In 5+4 medium, myofibres survived for up to 3 months in vivo under these conditions.

The discoveries that receptors for L-glutamic acid are widely distributed over the surface of myofibres grown for 3 weeks or more in 5+4 medium, that desensitization of these receptors is blocked by concanavalin A, that the receptors can be studied using giga-ohm seal recording techniques and that the channel conductance is large provide ample opportunities for studying the channel gating kinetics of these receptors at high recording resolution.

The influence of Ca²⁺ on the conductance of the channel is interesting because, in adult locust muscle, Ca²⁺ blocks the channel gated by junctional glutamate receptor channels (Cull-Candy, 1982) and extrajunctional glutamate D-receptor channels (Kits & Usherwood, 1984), although this only occurs with concentrations of Ca²⁺ in excess of 10⁻²moll⁻¹. In terms of their interactions with Ca²⁺, there thus appears to be a quantitative difference between the glutamate channel in vivo and in vitro which merits further investigation. This is not the only difference, of course. The sensitivity of the glutamate receptor of cultured locust muscle to L-glutamate seems to be at least a thousand times greater than that of the glutamate D-receptor of adult locust muscle, since the frequency of channel openings is similar for 5×10⁻⁹ mol I⁻¹ L-glutamate in the case of the cultured muscle and 5× 10⁻⁵ mol I⁻¹ L-glutamate in the case of the adult muscle. The apparent dissociation constant (K_d) for L-glutamate interaction with the adult extrajunctional D-receptor is 5xl0⁻⁴moll⁻¹ (Gration et al. 1980, 1981). It remains to be established whether the equivalent K_d of the cultured muscle receptor is three orders of magnitude smaller than this.

We wish to thank Mr T. Smith for his invaluable technical assistance during the formative stages of this study. This work was supported by a grant from SERC.