Development of receptors for α-bungarotoxin in chick embryo sympathetic ganglion neurons in vitro

Life Sciences, Vol . 22, pp . 407-414 Printed in the U .S .A .

Pergamon Press

DEVELOPMENT OF RECEPTORS FOR a-BUNGAROTOXIN IN CHICK EMBRYO SYMPATHETIC GANGLION NEURONS IN VITRO Diane Dvorak, Effie Gipps, John Leah and Chev Kidson Department of Biochemistry University of Queensland Brisbane 4067 Australia

(Received in final form November 18, 1977)

The development of receptors for a-bungarotoxin was examined in neurons in dissociated cultures of cells derived from chick embryo sympathetic ganglia . Neurons from 12 day embryos showed a marked increase in receptor numbers per cell over 3-4 days in culture . The increase was less marked in neurons from 14 day embryos and absent in 19 day embryos . The incidence of cholinergic synapses in cultures from 12 day and 19 day embryos was also examined . Evidence for synapse formation was found only in cultures from older embryos . The a-bungarotoxin (a-BT) receptors of chick embryo sympathetic ganglion neurons increase progressively in number per ganglion during the later stages of development in ovo and in the period from hatching to maturity, as measured y ending of [ 125 1]a-BT to ganglion membrane fragments (1) . The development of these receptors is correlated with synapse formation involving the principal neurons (days 12 to 20 in ovo) and with neuronal cytological and biochemical differentation (2) . Nicotinic but not muscarinic ligands are potent inhibitors of L%-BT binding at the high affinity site, suggesting that the toxin binding site has some properties of the nicotinic acetylcholine (ACh) receptor (1) . However, the identity of the a-BT and ACh receptors in sympathetic ganglion neurons has not yet been established (3, 4) . This question and the relationship of receptor development to synapse formation can potentially be examined more easily in vitro than in vivo . We report here patterns of in vitro development of a-BT receptors of sympathetic ganglion neurons cultured from chick embryos at various stages of development in ovo. MATERIALS AND METHODS Paravertebral sympathetic ganglion chains were dissected from White Leghorn chick embryos (Rochedale Hatchery), aged according to Hamburger and Hamilton (5) . Cultures were prepared from 12, 14 or 19 day embryos following the procedure of Greene et al (6) but using different enzyme treatments to dissociate the ganglia . The cells were grown in 33 mm tissue culture dishes (Kayline Plastics, South Australia) without a collaqen substrate in medium F15 (Gibco) + 58 fetal calf serum. 7S nerve growth factor (NGF) was purified from the submaxillary glands of young male mice as 407

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described by Varon et al (7) and assayed for biological activity by the method of Greene (8) . NGF was added to the medium at a concentration of 10 units/ml of medium . a-Bungarotoxin was purified from the venom of Bungarus multicinctus (K & K Laboratories) according to Bosmann (9) but using 0 .05 M ammonium acetate in lieu of 0 .005 M, and radiolabelled with 125, according to published procedures (10) . Binding of [ 125 I]monoiodo-a-bungarotoxin (90Ci/mmole) or [ 125 1]diiodo-a-bungarotoxin (180Ci/mmole) to cultured sympathetic neurons was examined as described below . Incubations contained [ 125 10 nM I]a-BT in 1 ml Puck's saline G containing 2 mg/ml bovine serum albumin (BSA, Sigma) . To correct for non-specific binding, control plates were preincubated for 15 mins with 0 .2 mM nicotine . After incubation with labelled toxin for 30-45 min at 37°C, the cultures were washed with 3 x 3 ml portions of Puck's saline G containing BSA and 3 x 3 ml portions of Puck's saline G . Bound radioactivity was determined by incubating the plates with 1 ml portions of trypsin (10 mg/ml, pH7, Worthington Biochemicals) . The cell suspension was collected, and the process repeated twice . The combined suspensions were counted in a Beckman Biogamma counter . Neuronal numbers were counted during the last wash with Puck's saline G, by counting twenty 1 mm 2 areas of the plate at random . Assays of [ 125 I]a-BT binding to dissociated ganglia were performed as follows . Ganglia were dissociated by repeated passage through a fine bore pasteur pipet, without prior treatment (mechanical dissociation) or after one of the following three enzyme treatments : (a) 0 .258 trypsin, (b) 0 .025% trypsin, (c) 0 .1% collagenase (Sigma type 1) + 0 .058 hyaluronidase (Calbiochem., B grade) . All enzyme incubations were carried out at 37 ° C for 30 min. The dissociated ganglia were washed with Puck's saline G containing BSA and the cells added to small silicoised glass tubes (Kimax, 6 x 50 mm) with approximately 5 x 10-1 cells/tube, in a volume of 100 pl . In control tubes nicotine was present at a final concentration of 0 .2 mM . Tubes were preincubated for 15 minutes at room temperature, then 100 pl of [125, ]a-BT was added to a final concentration of 10 nM . After 30-45 min the cells were centrifuged, the supernatant was removed, and the cells were washed with 0 .5 ml Puck's saline G containing BSA . After resuspension in a further 0 .5 ml Puck's saline G containing BSA, the cells were filtered through a 0 .45 p millipore filter held in a Sartorius filtration manifold under vacuum . Each filter was washed with 15 ml Puck's saline G containing BSA and radioactivity counted . Estimations were performed in duplicate . Nonspecific binding was usually 10-20% of total radioactivity . For electrophysiological studies cultures were held at 25 °C and the plates were fixed to the movable stage of an inverted phase contrast microscope . The pH of the medium was maintained close to 7 .4 by passing a water saturated mixture of 5% C02/95% 02 over the surface . Electrical activity was monitored using glass micropipet electrodes pulled from borosilicate glass tubing containing an inner filament (Clark Electromedical) . These had resistances of 70-120 Mn and were filled with 2M potassium citrate . Electrodes having very gentle tapers over the terminal 50 um gave the best impalements . Stimulating currents and membrane potential changes were monitored simultaneously using a

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Iontophoresis micropipets (pulled current-clamp bridge circuit. from Theta tubing) and having tip diameters approximately 0 .5 um were filled with 1 .0 M acetylcholine chloride (pH4) . Retaining currents were less than 2 .0 nA . RESULTS AND DISCUSSION The effects of different enzyme treatments on the number of receptors per neuron for cells isolated from 12 day embryos are shown in Table 1 . The degree of dissociation obtained with collagenase and hyaluronidase was not as good as that obtained with trypsin. Treatment with 0 .025% trypsin caused a minimal decrease in receptor numbers while giving good dissociation of This concentration was therefore cells suitable for culturing. used in all subsequent experiments . TABLE 1

The effect of enzymes on a-BT receptors Enzyme treatment

Receptors/cell

None (mechanical dissociation)

2 x 104

0 .258 trypsin Collagenase + hyaluronidase

2 x 10 3

0 .025% trypsin

1 x 10 4

7 x 10 3

The pattern of a-BT receptor development in vitro is shown in Fig . 1 . In cultures of neurons isolated from2~ embryos, the number of receptors showed an initial rapid increase and reached a plateau by 3-4 days in vitro . The receptor numbers at this time were much higher than expected for the corresponding period in vivo . Autoradiography showed that virtually all principal ganglion neurons isolated from 12 day embryos were positive for a-BT receptors at one day in culture . The increase in receptor numbers represents, therefore, an increase in the number of receptors per cell, not an increase in the number of cells possessing receptors . Electrophysiological studies were carried out on 6 cultures of In all cultures over 90% of 12 day embryos, 1-6 days in vitro . cells successfully impaled as fudged by membrane time constants) generated action potentials in response to depolarizing current pulses (0 .5 - 2 .0 nA, 70msec) and were responsive to iontophoretically applied acetylcholine . Some cells required hyperpolarization to -60 mV before active responses were seen . Membrane potentials ranged from -20 to -40 mV (Fig . 2a, c) .

Neurons isolated from 14 day embryos showed a smaller increase in receptor number with time in vitro, while neurons from 19 day embryos showed no increase at a F g. 1) even though they had not yet attained the maximal receptor numbers seen in the adult (ti6 x 10 5 cell, calculated from the data of Greene (1)) .

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FIG.

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1

The development of a-BT receptors in vitro as determined by [ I]a-bungarotoxin binding to culture sympathetic neurons from 12 day ( 0), 14 day ( 0) and 19 day (A ) embryos .

-E-

The initial point of each curve represents the number of receptors in ovo . Each point is the mean of several different experiments, the standard error of the measurements being approx . 10% . The dotted line shows the development of a-BT receptors in ovo , and was calculated from the data of Greene (1) .

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b

7

~~rR-rfT'("f

FIG .

L

2

Intracellular recordings from cultured chick embryo sympathetic ganglion neurons . (a)

(b)

Action potential elicited from a neuron (12 day embryo) 6 days in vitro . (Stimulating current 0 .3

W.

Train of action potentials from a neuron (19 day embryo) 3 days in vitro .

(c)

Response of a neuron (12 day embryo) 5 days in vitro to iontophoretically applied acetylcho3ine 7 -7TnA, 70 msec) . The pulse was suprathreshold and elicited an action potential. Note transient increase in membrane conductance .

(d)

Demonstration of a synapse between two neurons (19 day embryo) 3 days in vitro . An action potential elicited in one cell lower trace) initiated an action potential in a second cell (upper trace) .

The upper trace in (a), (b), (c) represents 0 mV and in (a) shows the stimulating current. Vertical bar is 20 mV for (a), (c) and (d), 15 mV for (b) . Horizontal bar is 5 msec for (a) and (d), 35 msec for (b), and 500 msec for (c) .

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FIG.

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3

Phase contrast photomicrographs of living sympathetic ganglion cultures from 12 day (a) and 19 day (b) embryos . Magnification x 200 . Neurons from the older embryos were no more difficult to maintain in culture than those from the younger embryos, although the former were more rapidly overgrown by non-neuronal cells . The older neurons were morphologically intact (Fig . 3) and electrically active, having membrane potentials from -30 to -80MV . In response to stimulating currents many fired multiple action potentials (Fig . 2b) . Thus their failure to show an increase in a-BT receptor number cannot be explained by decreased viability or functional capacity . In view of the failure of other workers to observe any effect of a-BT on electrophysiological responses to iontophoretically applied acetylcholine in rat superior cervical ganglion neurons (3, 4) and spinal cord (11, 12) we performed similar experiments with the cultured chick embryo sympathetic ganglion neurons . We could not observe any blockade of the response to acetylcholine, even after the cells had been incubated for over one hour at 37 °C with 700 nM a-BT prior to electrophysiological measurements . On the other hand 100 UM d-tubocurarine hydrochloride completely blocked the idntophoretic response . The pharmacological characteristics of the ganglion a-BT receptors (1, 13) resemble those of the receptors in brain (14, 15, 16) in that the dissociation constant of the toxin receptor complex in both cases is higher than for a-BT complexes with muscle ACh receptors, and hexamethonium, a potent ganglionic blocking agent is not an efficient inhibitor of a-BT binding in ganglia or brain . Thus the identity of the a-BT binding sites in ganglia and brain with ACh receptors has not been established .

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In contrast to a-BT, structurally related toxins from the elapid snake Dendr0as is viridis are capable of producing blockade of spinal cord cho inergic synapses (11) . By virtue of its slightly different structure, a-BT may bind at the same site as the dendrotoxins yet produce no blockade of the response . It has recently been reported that purified solubilised acetylcholine receptors from Torpedo californica (17) and Electrophorus electricus (18) have twice as many binding sites for the elapid a-toxins as for acetylcholine and other small cholinergic ligands . Also there appear to be two classes of toxin binding sites and two classes of ligand binding sites (17) . These may represent discrete nonidentical subunits, different sites on the same subunit (17) or an interconversion of two different states of the same subunit (18, 19) . If a similar situation occurs in sympathetic ganglia the ACh receptors may have evolved such that a-BT is able to bind only to the low affinity site without affecting the binding of acetylcholine to the high affinity site . However it remains to be determined whether the ligand binding properties of the native membrane-bound receptor resemble those of the solubilised form . Direct evidence for the specificity of the a-BT binding in ganglia must await isolation of the acetylcholine receptor from this source . The formation of cholinergic synapses between principal neurons in cultures of superior cervical ganglion cells from the newborn rat has been reported (20,21, 22) . The incidence of functional synapses in cultures from 12 day embryos, 6 days in vitro, was explored . One cell in each of a pair of cell clusters, connected by thick axonal tracts, was impaled . If both cells had resting potentials of -30 mV or greater, an action potential was elicited in one and responses in the second cell noted . In 18 such pairs no evidence of functional cell interaction, chemical or electrical, was observed . In contrast, in cultures of cells from 19 day embryos, examination of 10 pairs of cells demonstrated functional synapses in 2, at 3 days in vitro (Fig . 2d) . There is thus no direct correlation between synaormation and the development of a-BT receptors . It would appear that transferring ganglion cells from 12 day embryos to an in vitro situation releases them from some developmental constraint, resulting in a rapid increase in receptor numbers, while cells removed from 19 day embryo lack the required signals for further increase . In view of our demonstration of functional synapses in cultures from 19 day but not from 12 day embryos, it may be possible to examine further some of the factors governing interneuronal synapse formation using these cultures, although the nature of these synapses remains to be determined . ACKNOWLEDGEMENT

This work was supported by a grant from the Australian Research Grants Committee . REFERENCES

1.

L . A. GREENE, Brain Research 111, 135-145 (1976) .

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