Camp. Biochem. Physiol. Vol. SOC, No. I, pp. 53-59, 1985 Printed in Great Britain

0306~4492/85 $3.00 + 0.00 Q 1985 Pergamon Press Ltd

LOCUST NYMPHAL NEURONES IN CULTURE: A NEW TECHNIQUE FOR STUDYING THE PHYSIOLOGY AND PHARMACOLOGY OF INSECT CENTRAL NEURONES

D. P. GILES* and P. N. R. USHERWOOD~

Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Telephone: 0602-506 10 1

(Received 4 June 1984)

Abstract-l. A new technique for studying the physiology and pharmacology of locust central neurones is described. Somata isolated from neurones in the meso and metathoracic ganglia of third instar locusts (Schimcerca gregaria) were maintained for up to 4 weeks in co-culture (monolayer) with embryonic locust neurones.

2. Most of the cultured cells became multipolar but a few were monopolar like their in uiuo counterparts. They had diameters of 40-80pm and “clean” (glial free) surface membranes.

3. Cells 614 days in vitro were depolarized by acetylcholine and usually hyperpolarized by y-aminobutyrate, taurine and glycine. L-Glutamate and L-asparate were inactive but further pharma- cological studies are required to confirm this.

4. Cultured larval neurones should provide excellent opportunities to study the molecular basis for drug-receptor interactions and voltage-sensitive membrane channels using the patch clamp technique.

INTRODUCTION MATERIALS AND METHODS

The morphological characteristics and structural properties of ganglion explants and dissociated neurones taken from the central nervous system of embryonic cockroaches (Periplaneta americana) (Hicks and Beadle, 1980; Usherwood et al., 1980; Skelton, 1980; Hicks et al., 1981; Beadle et al., 1982; Giles et a/., 1983) and locusts (Schistocerca gregaria) (Giles of al., 1978) and maintained in long-term culture have been recently described. Usherwood et (11. (1980) presented a preliminary account of the electrophysiology of dissociated cockroach neurones 7-10 days in vitro which has recently been extended by Lees et al. (1983) who also investigated the acetylcholine sensitivity and r-bungarotoxin binding properties of these cells. The small size and uncertain properties of the embryonic cells led us to investigate whether fully differentiated insect central neurones could be grown in culture.

In this paper the preparation and maintenance of long-term cultures of central neurones taken from third-instar locusts is described, together with a brief introduction to their physiology and pharmacology. These cultures provide neurones with well-developed processes and large cell-bodies which are suitable for a variety of intracellular and extracellular micro- electrode studies and may well prove ideal for patch clamp work of both voltage-sensitive and receptor gated membrane channels. We shall show that the cultured neurones are morphologically distinct from their in zko counterparts, but exhibit qualitatively similar electrophysiological and pharmacological properties to the latter.

All insects used in this study were maintained at 28-29°C. 65-75x relative humidity and with a light/dark cycle of 13.5/10.5 hr. Third instar locust (Schistocerca gregaria) nymphs were surface sterilized by immersion for 5 min in 10% Domestos solution during CO, anaesthesia. The meso and metathoracic ganglia were excised, care being taken to avoid puncturing the gut during dissection. In order to maintain asepsis the excised tissue was immersed for 15 min either in culture medium (Hicks et al., 1981) or in locust saline (Usherwood and Grundfest, 1965) containing a high concentration of penicillin (50 i.u. ml-‘) and streptomycin (5OOpgml-I). Either whole ganglia or specific parts of ganglia were then dissociated by passage through fine bore pipettes (Hicks et al., 1981). The nymphal neurones were difficult to maintain in culture alone for periods > 5 days, but successful growth and maintenance beyond this period was achieved when the cells were plated out onto established monolayer cultures of dissociated embryonic locust neu- rones, 7-14 days in vitro (Fig. ID). Details of culture media and of techniques for preparing and maintaining cultures of embryonic cockroach neurones are described in Hicks et al. (1981). Similar techniques were employed in our studies of locust nymphal neurones in culture. All preparative pro- cedures were undertaken in a microflow cabinet located in a dust-free room. Cultures were examined daily using a Leitz inverted microscope. The techniques used for pre- paring cultures for electron microscopy were also described in Hicks et al. (1981).

*Present address: Schering AG, Frohnau Researdh Station, Postfach 650311, D-1000 Berlin 65, FRG.

tTo whom all correspondence should be addressed.

For electrophysiological studies preparations were placed on the stage of an Olympus inverted microscope and visualized with phase contrast optics. Standard intracellular techniques were used in these studies. Membrane parame- ters were usually determined using two intracellular micro- electrodes, one for recording and the other for passing current, although on occasions a single electrode coupled to a bridge circuit was used to both record and pass current (Yamasaki and Narahashi, 1959). All recordings were made with respect to virtual earth using a current-voltage con- verter. Estimates of changes in input resistance of the nymphal cells during drug application were made by mon-

53

54 D. P. GILES and P. N. R. USHERWOOD

itoring the changes in the amplitude of hyperpolarizations (l&20 mV in absence of drug) induced by current injection with current pulses of 60%800msec duration and 0.1-0.2 Hz. Intracellular recording and current electrodes were filled with 2 M potassium acetate. Drugs were applied either in the bathing medium or by ionophoresis from high resistance (80-100 MR) micropipettes using a constant cur- rent stimulator. Bias currents of O-10 nA were used to counteract background diffusion of drug from the pipettes. Data were collected either on a Grass P7 pen recorder or filmed directly from the screen of an oscilloscope.

The cultures were perfused continuously during experi- ments with saline of the following composition (mM): NaCI, 214; KCl, 3.1; CaCI,, 9.0; Tris-HCI, 1.0; pH, 7.0 (adjusted with 1 M NaOH) (Kerkut et al., 1969). The perfusion rate was 5 ml min’ which was sufficient to change the entire contents of the “bath” within 1.5 min.

RESULTS

Between l&20% of the nymphal neurones grown in co-culture showed outgrowths, some processes growing for IO-14 days to reach lengths up to 500 pm. Initially after dissociation and plating the nymphal neurones were approximately spherical in outline, although they occasionally had axon stumps of up to IOOpm in length. These stumps were ab- sorbed within l-2 days in vitro. Some of the nymphal cells survived for up to 4 weeks in vitro. Cultures of nymphal neurones prepared from either the meso or metathoracic ganglia or from different parts of these ganglia showed no differences in growth. The cul- tured cells flattened as they developed in vitro to produce neurones with cell bodies of 4&80 pm wid- est diameter. Unlike their in rive counterparts, cul- tured cells 6-14 days in vitro were mostly multipolar with extensively branched processes (Fig. lA,B) al- though a few were monopolar (Fig. 1C). In co- cultures, embryonic neurones in the region of a nymphal cell body usually did not survive. However, contacts between the processes of the cultured nymphal neurones and between those of embryonic cells were apparent in the outer reaches of a nymphal cell but it was not established morphologically whether synapses were present in the cultures. Con- tacts between nymphal cells were also apparent in the cultures. Transmission electron microscopy of nymphal neurones 6-14 days in vitro revealed exten- sive damage of some organelles, particularly mito- chondria. However, the surface membrane of these cells was remarkably free of glia material and cell debris.

Physiology

Resting potentials of nymphal cells 614 days in culture were in the range - 20 to - 60 mV (mean 38.9 + 12.8 (SD); N = 16). This mean value is lower than that obtained by Kerkut et al. (1969) for in situ recordings from cell bodies of the adult locust meta- thoracic ganglion and by Usherwood er al. (1980) for freshly isolated neurone somata from adult locusts. Input resistances were in the range lG-40 MD (19.5 + 9.8; N = lo), i.e. similar to those obtained by Usherwood et al. (1980) for freshly isolated neurone somata of adult locusts. About 30”/;; of the cells were spontaneously active, generating all-or-none action potentials which ranged from 10-100 mV amplitude (Fig. 2A,B) and occurred at frequencies of l-20 Hz. In some cells action potential production ceased within Smin of electrode impalement, but in other cells spike activity was maintained throughout the recording period (up to 5 hr). In the latter case spike frequency declined during the first 10 min and then remained at a steady level of l-5 Hz. Crossman et al. (1971) obtained similar data when recording in situ from neurones in the mid-dorsal region of the adult locust (and cockroach) metathoracic ganglion. About half of the “silent” cells generated one or more action potentials in response to current injection. either during the passage of depolarizing current (0.5-3 nA) or after the passage of hyperpolarizing current (7710nA) but the amplitude of these spikes some- times declined with repetition (Fig. 2C,D). The rest of the cells were electrically inexcitable apart from the presence of delayed rectification. There was no cor- relation between the magnitude of the resting poten- tial and the capacity of a cell to spike.

Despite their apparent morphological connectivity, intracellular recordings from the nymphal neurones in vitro gave no evidence for coupling between the cells, either electrical or chemical. Recordings taken simultaneously from adjacent cells, which could also be stimulated by injected current, confirmed the absence of functional connections, There was no evidence from the intracellular recordings for the presence of inhibitory and/or excitatory postsynaptic events.

Pharmacology

Preliminary studies were undertaken to ascertain whether the nymphal cells exhibited sensitivity to putative neurotransmitters after 5 days in vitro. The results of these studies are summarized in Table 1.

Table I. Pharmacology of locust nymphal neurones in culture (&I4 days in ritw)

No. cells No. ceils KSpollSlW

Test compound tested (concentration) Response

Acetylcholine 16 II (IO-‘M) Depolarization chloride

GABA I 7 (IO-‘-IO-‘M) Hyperpolarization 5 Depolarization I

Biphasic I L-Glutamate 4 O(lO-*M) L-Aspartate 3 O(lO_* M) Glycine 4 1(5x10-‘M) Hyperpolarization Taurine 1 I (5 x IO-‘M) Hyperpolarization

All responses were accompanied by a decrease in input resistance.

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56

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D. P. GILFJ and P. N. R. USHERWOOD

Fig. 2. Electrically excitable properties of third instar locust neurones 614 days in oitro. A, B. Action potentials (lower traces) recorded from a spontaneously active cell. The upper traces represent zero potential. Calibration: 25 mV, 0.25 sec. C. Depolarization of a silent cell with injected current (upper trace; 2 nA) elicits an overshooting action potential. Upper trace also represents zero potential. Calibration pulse preceeding response was 50 mV, 25 msec. D. Action potentials following hyperpolarizing current injection (7 nA) into a previously silent cell. Calibration: 25 mV, 125 msec. Note prolonged hyperpolarization

following current injection. Figure slightly retouched.

There were no apparent changes in the properties of the cells from the 6th day onwards.

The majority of cells responded to acetylcholine (ACh) applied in the bathing medium, but only to high concentrations of this drug. For example 10e4 M ACh elicited a depolarization of 5-25 mV (16.4 &- 4.4; N = 5) and decreased the input resistance of the cultured neurone by 3&70%. The response to ACh was fully reversible upon removal of the drug. This sensitivity to ACh compares favourably with that of cockroach neurones in situ (Kerkut et al., 1969) but it is lower than the threshold value of 5 x 10e5 M reported by Callec and Boistel (1967) for such neu- rones. However, when compared with freshly isolated neurone somata taken from the adult locust central nervous system the cultured nymphal cells are cu. 100 x less sensitive to bath applied ACh (Usherwood et al., 1980).

With bath applications of y-aminobutyric acid (GABA) at concentrations < lo-‘M the neurones did not usually respond but with concentrations > 10e3 M the cells were either hyperpolarized (range l&20 mV; 14.1 + 3.3; N = 7) or more rarely depolar- ized. (In one cell a biphasic response to GABA was recorded (Fig. 3A).) The change in membrane poten- tial was accompanied by a decrease in input re- sistance of l&90% (37.2 + 26.8; N = 7) and a cessa- tion of spontaneous and evoked spike generation (Fig. 3A,B). The responses to bath-applied GABA gradually faded in the continued presence of the amino acid. The cell in Fig. 3A was atypically sensitive to concentrations of GABA < 10m4 M. The other six cells on which this amino acid was tested responded only to concentrations > 10e3 M. Iono-

phoretic application of GABA to the somal mem- brane evoked a hyperpolarization, the amplitude of which was dose dependent (Fig. 4). Since membrane potential was the measured parameter and the resting potential was not greatly different from the reversal potential for the GABA response it is not possible to draw any quantitative conclusions from the dose-response data. Significantly no desensitization to either a prolonged pulse of GABA or a series of short pulses was observed. When the ionophoretic electrode was moved away from the soma along one or more of the processes emanating from the latter a gradual decrease in GABA sensitivity was recorded. The cultured cells were cu. 20 x less sensitive to GABA applied ionophoretically than the freshly iso- lated neurone somata of adult locusts.

Only one of four cells tested responded to bath- applied glycine (5 x lo-’ M). This cell was transiently hyperpolarized and exhibited at ca. 75% decrease in input resistance (Fig. 3C). A similar response was obtained with 10m3 M taurine (Fig. 3D). Kerkut et al. (1969) found that glycine inhibited cockroach neu- rones but only at concentrations > 100 x those re- quired with GABA. Application of L-glutamate and L-aspartate at very high concentrations produced negative results (Table l), which contrasts with re- sults for freshly dissociated neurones (Usherwood et al., 1980).

DISCUSSION

When we were first successful in growing locust nymphal neurones in culture we suspected that these cells might be neurosecretory (NS) in origin. Seshan

Cultured locust neurones

(Al

On

(B) 4

Fig. 3. Bath application of (A) 1W4 M and (B) lo-’ M GABA to nymphal neurones 6 and 7 days in vitro respectively evoked transient hyperpolarization, followed by a small depolarization in (A), abolition of action potential production in (B) and a decrease in input resistance (monitored by change in amplitude of hyperpolarizations to injected current (1 nA) (downward deflections). C. 5 x lo-’ M glycine applied to a nymphal neurone 7 days in vitro evoked a transient depolarization, loss of spontaneous action potentials and a fall in input resistance, but these changes quickly waned. D. Hyperpolarization accompanied by a fall in input resistance and loss of spontaneous action potentials following application of 10-l M taurine to a neurone 6 days in oirro. The response was rapidly reversible upon removal of the amino acid when there was a transient increase in spike frequency above the pretreatment control value. In records ED the evoked (B) and spontaneous (C, D) spikes were attenuated by the low frequency

response of the pen-recording system. Calibration: 20 mV, 15 sec.

and Levi-Montalcini (1971) and Aloe and Levi- Montalcini (1972) have successfully grown cultures of cockroach NS cells but they were unable to maintain non-neurosecretory neurones from nymphal cock- roaches in vitro. However, available evidence suggests that the cells grown in our studies are not NS in origin. For example, they were, on average, much larger than locust NS neurones (Chalaye, 1967; see Maddrell, 1974) and did not contain NS granules either immediately after excision from the ganglia or after 6-14 days in vitro. Seshan et a[. (1974) showed that cockroach larval NS cells retain their NS gran- ules when cultured.

Crossman et al. (1971) discovered electrically- excitable neurone somata in the mid-dorsal region of the metathoracic ganglion of Schistocetca gregaria

which produce all-or-none, overshooting action po- tentials. However, they concluded along with Hoyle (1970) that the majority of the cell bodies in the locust thoracic ganglia do not generate action potentials. In the present study most (ca. 70%) of the cultured nymphal cells generated action potentials, although not all of these were spontaneously active, The origins of these cells were not determined precisely, but it is clear that they did not all originate from the mid-dorsal regions of the meso and metathoradc ganglia since cells exhibiting all-or-none action po- tentials were present in cultures prepared from pieces of ganglia which excluded these mid-dorsal cells. Furthermore the adult cell bodies studied by Cross- man et al. (1971) were 40-60 pm in diameter whereas in our studies the larval somata when freshly isolated

58 D. P. GILES and P. N. R. USHERWOOD

-- I

Iontophoretlc dose (nC)

Fig. 4. Dose-response relationship for GABA iono- phoretically applied to the soma of a 3rd instar locust neurone after 7 days in aitro. The resting potential of this cell was - 45 mV. Trace above graph is the response of a larval neurone 7 days in vitro to GABA ionophoresis onto the soma. Calibration: IO mV, 100 msec; resting potential,

- 50 mV.

were 4&8Opm of diameter. When somata of neu- rones of adult locusts are isolated from their axons and maintained for short periods (1-12 hr) in vitro 70% of the surviving cells bodies are capable of spike production (Usherwood et al., 1980). It seems likely, therefore, that axotomy is the causal factor in con- verting so-called electrically-inexcitable somata into spike-generating bodies (see Suter and Usherwood (1985a) for a further discussion of this phenomenon).

There is some evidence that the pharmacology of these cultured nymphal neurones is quantitatively different from that of in situ cells and freshly-isolated somata (Holden et al., 1978; Usherwood et al., 1980; Giles and Usherwood, 1985; Suter and Usherwood, 1985a,b), since they are seemingly less sensitive to both ACh and GABA. Possibly this loss of sensitivity in culture results from a decline in the population densities of extrajunctional receptors consequent upon dedifferentiation of the culture cells. The ab- sence of sensitivity to certain amino acids, e.g. L-glutamate, might also be related to the presence of these compounds in the culture medium (Fischbach and Nelson, 1977), since receptor sensitivity in general tends to decline in the continued presence of its ligand (Luqmani et al., 1979), possibly due to receptor down-regulation. However, further studies will be required to substantiate this.

The object of the studies described in this paper was to establish the feasibility and viability of a new technique for studying the physiology and pharma- cology of insect central neurones. Despite the tech- nical difficulties associated with the culture of non- embryonic insect neurones there is no reason why the methods that we have developed should not be employed to culture identified neurones of larval and adult tissues. The cultured cells clearly require fur- ther, more extensive study, before their pharma-

cological properties are firmly established but the “clean” (glial free) surfaces presented by these cells should provide excellent opportunities for studying the molecular basis of drug-receptor interactions and of receptor gated and voltage sensitive membrane channels using the newly-developed patch clamp technique (Neher et al., 1978).

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