0306-4522/90 $3.00 + 0.00 Pergamon Press plc Q 1990 IBRO
NeuroscienceVol. 31, No. 2, pp. 523-530, 1990 Printed in Great Britain
NERVE GROWTH FACTOR INDUCES FUNCTIONAL NICOTINIC ACETYLCHOLINE RECEPTORS ON RAT SENSORY NEURONS IN CULTURE A. MANDELZYS,* E. COOPER,*? Q. M. K. VERGES and P. M. R~CHARDSON~ Departments
of *Physiology and SNeurology and Neurosurgery,
McGill University, Montreal, Quebec,
Canada H3G 1Y6 Abstract-Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influen~d the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day I2 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholinesensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.
The expression for
the
system. the
The muscle
best
served
of postsynaptic
development studied
as a model
of nicotinic
receptors
synapses
in
receptor
neurotransmitter to investigate
is essential the
virtually all non-neuronal cells have been eliminated, nodose neurons express nicotinic ACh receptors and cholinergic synapses form among the neurons.13 In contrast, when the neurons are co-cultured with non-neuronal elements from the ganglion (including satellite cells, Schwann cells, and fibroblasts) to mimic their in vivo environment, few neurons express ACh receptorsi These findings suggest that some influence from non-neuronal cells prevents the expression of ACh receptors on these neurons. The factors that induce nodose neurons to express nicotinic ACh receptors in culture in the absence of non-neuronal cells, however, are largely unknown. As nerve growth factor (NGF) was present in all our previous studies, we questioned whether NGF itself may have a role in the induction of nicotinic ACh receptors on these neurons. NGF has been shown to influence the differentiated properties of other neurons: for example, NGF increases neurotransmitter synthesis in sympathetic neurons,4z’ ‘J*,~‘.~‘.~in sensory neurons3t.3a and in cholinergic forebrain neurons,24,26,Uand NGF can affect the expression of voltage-gated ionic channels on PC12 cells’y~~~47~~~55 and on sensory neurons.’ In this paper, we have extended our studies on the development of rat nodose neurons in culture and have investigated the effects of NGF on the expression of nicotinic ACh receptors on nodose neurons in culture.
nervous
is undoubtedly receptor.
It has
the developmental
appearance
of postsynaptic receptors, as well as factors, such as innervation and activity, that influence receptor number and dist~bution on the postsynaptic membrane. a.20.57 The expression of neuronal nicotinic receptors, which are part of a common gene family of ligand-gated channels, is similarly regulated by denervation16~29,33.s3,56 and also by axotomy.7.zg A recent study has shown that this regulation on autonomic neurons can occur, at least in part, at the level of mRNA for the receptor subunits.6 Much less is known about developmental influences that determine why some neurons express particular neurotransmitter receptors. For example, while all autonomic neurons express nicotinic receptors, only a small number of sensory neurons express them,“@ even though most sensory neurons are derived from the same embryological structures as autonomic neurons. 34To learn what factors influence the appearance of nicotinic receptors on some sensory neurons and not on others, we have been studying the development of neonatal rat nodose neurons in dissociated cell culture. Only a few nodose neurons are sensitive to acetylcholine (ACh) in vivo;28 however,
we have
found
that
in conditions
where
tTo whom correspondence should be addressed. ACh, acetylcholine; HEPES, N-Zhydroxyethylpi~razine-~‘-2-ethanesulphonic acid; L- 15 medium, Leibovitz-15 medium; NGF, nerve growth factor.
Abbreoiations:
EXPERIMENTAL PROCEDURES Neuronal cultures
The methods for culturing nodose neurons and sympathetic neurons were similar to those already publjshed.l~.14 Briefly, ganglia were dissected under sterile conditions from
523
524
A. MANDELZYS rt ul.
newborn rats (CD. strain, Charles River, Canada), which
were killed by cervical dislocation. The ganglia were incubated for 15 min at 37°C in a medium contain& collatenase (1 mg/ml, Cooper Biomedical) and a neutral protease (Dispase, Grade II, 2.4 mg/ml, Boehringer Mannheim). They were then transferred to a solution containing only the neutral protease and triturated every IS min for 3-4 h. Y
”
Following the dissociation, the cells were centrifuged through a Percoll (Pharmacia) gradient (35% Percoll) in order to separate ncuronal from non-neuronal cells. The neuron suspension was washed twice and plated in modified culture dishes on collagen-laminin-coated coverslips (0.75cm”) (lOOfig/ml laminin for 12 h) in Leibovitz-15 medium supplemented with sodium bicarbonate, vitamins, co-factors, penicillin--streptomycin, rat serum (5%) and 7s NGF (l~nM~.~9 In some experiments, fi-NGF prepared from submandibular glands of male mice by ion-exchange chromatography’0,4” was used at concentrations of 40 and 400 pM. Cytosine arabinoside (IO HM, Sigma) was added to the cell cultures for the first four days to kill any dividing cells that remained following the Percoll step. The cultures were maintained in a humid atmosphere of 95% air-5% CO, at 37’C and fed every three to four days with fresh growth media. All cultures were initially grown with NGF, However, on day 4, the plating was divided into two groups: those grown in the continual presence of NGF; and those in which NGF had been removed and rabbit antiserum to 2.5 S NGF at a I: 1000 dilution was added. This antiserum at l:lO,OOO, blocks the effect of 10 nM 7 S NGF when added to dissociated rat sympathetic neuron cultures. Neuronal numbers were determined by counting the neurons directly in the living culture using phase optics; cultures typically had 1000-2000 neurons,
ACh-sensitivity was measured electrophysiologically with intracellular recordings; the methods were similar to those previously described. 2.46The cultures were perfused at approximately 0.5 mljmin with media containing: 10% (v/v) L-15, 90% (v/v) Hank’s Balanced Salt Solution. CaC&, (2.8 mM), choline chloride (0.07 mMf, HEPES (IO mM, BDH), glucose (5.6 mM), glutamine (2 mM) and penicillin (100 pg/ml) and streptomycin (0.25 mg/ml) (Gibco): the pH was 7.2.7.4 and temperature was 35537°C. The recording electrodes, filled with 3 M KC1 (resistance; 50-100 MO), were connected to a WPI M707 amplifier to monitor membrane voltage and to pass current, and the signals were displayed on a Tektronic storage oscilloscope (5113). To determine whether a neuron expressed functional ACh receptors, we rapidly exposed as much of the neuron as possible to ACh (10“-lo-* M, Sigma) and observed if this resulted in any membrane depolarization. We found that the best way to apply ACh is via relatively large tipped pipettes (lo-20 pm) positioned at an appropriate distance from the neuron (2&40 pm) so that upon application of pressure we could perfuse a large area. We have verified this approach in several pilot studies with pipettes filled with dyes or suspended oil droplets, and we have ascertained that there is no outflow of drug from the “puffer pipette” into the bath provided that the Ievel of liquid in the pipette is kept low. With this technique, most sensitive neurons depolarized by lo-20mV, whereas insensitive neurons had no detectable change in membrane potential. From whole-cell and singlechannel recordings (Mandelzys and Cooper, unpublished observations), we estimate that sensitive neurons had several hundred ACh receptors open simultaneously at the peak of the response, and that neurons judged as insensitive could not have had more than 20-30 receptors. As there are no visible clues to distinguish ACh-sensitive neurons from ACh-insensitive neurons in the living culture with phase optics, neurons with clearly visible nuclei in the centre of the celJ were selected at random. Only neurons with resting
potentials greater than - 35 mV were included in this study. The proportion of ACh-sensitive neurons in any given culture was determined by recording from 50 randomly chosen neurons.
P-NGF was radioiodinated by the lactoperoxidase method” and separated from free iodine.62 IIZSIINGF with specific activity _of approximately 100 ,utC$pg’ was used within 24 h of preparation. For 4 h before exposure to [‘*‘I]NGF, the cultures were washed with four changes of culture medium without NGF. The cultures were incubated for 1.5 h at 37°C with 40 pM [‘*‘I]NGF in the presence of 0,40, 80, 160, 320, or 4000 pM uniabelled NGF. The incubation solutions were prepared by diluting NGF in 0.1 M phosphate-buffered saline, pH 7.4, with magnesium chloride (0.5 mM), cytochrome C (1 mg/ml), leupeptin (4 p&/ml), and phenylmethylsulfonylfluoride (0.5 mM). After six rinses with cold-buffered saline over 3 min and fixation with cold 4% parafo~aldehyde for 10 min, the cultures were washed with water and dehydrated in a series of alcohols. Coverslips were then dipped in emulsion (NTB, Kodak), exposed in the dark at 4°C for two to five days and developed. Dark-field photomicrography at low magni~cation was accomplished with a scanning condenser. To measure the affinity of NGF binding, low density cultures were used in which neurons were plated at approximately 20.-3O/cm*so that most of the neurites from individual neurons remained separate. After one week, the cultures were incubated with [‘*‘I]NGF (20 PM) and O-2500 pM unlabelled NGF as described above. As previously described image-analysis system5* was slightly modified to measure (percentage of area covered by silver grains minus percentage of area covered by silver grains in background regions of the slide) multiplied by area and divided by neurite length. This measurement, obtained for rectangles over labelled neurites in culture is linearly related to specific grain counts per length of axon.
RESULTS
Development of nodose neurons in culture with and without nerve growth factor
We first determined whether or not rat nodose neurons require NGF for growth and survival when grown in dissociated culture in the absence of nonneuronal cells. All neurons were plated with NGF for the first four days to avoid the possibility of selecting populations of nodose neurons that differ in their
NGF requirements. At day 4, NGF was withdrawn from half the cultures, and in addition, antiserum to NGF was also added at a concentration of 1: 1000. Neurons with and without NGF continued to grow in size and extend processes for several weeks. Phasecontrast photomicrographs of sister cultures grown for three weeks with or without NGF is shown in Fig. 1. At three weeks, the mean cell body diameter of neurons grown without NGF are indistinguishable from those grown with NGF [22.8 k 1.9, II = 150 (mean + S.E.M.) and 23.4 f 1.7, n = 150 (mean f S.E.M.), respectively]. The density of neurite outgrowth at three weeks, although dificult to quantify, generally appeared to be somewhat greater when NGF was present in the culture medium (see Fig. I).
NGF induces ACh receptors on nodose neurons in culture
Fig. 1. Montages of phase-contrast photomicrographs of nodose neurons after three weeks in culture. Both cultures were grown with NGF for the first four days: thereafter, the culture on the left was grown in the continual presence of NGF, while the culture on the right had its NGF removed. Both cultures show spherical cell bodies with clear nuclei containing one or two nucleoli and extensive neurite outgrowth. Scale bar = IO0 pm.
Figure 2 demons~ates that neuronal survival was largely unaffected by NGF. During the first two to three days after NGF withdrawal, there was a 15% decrease in neuronal numbers; the remaining 85%, however, continued to grow for at least three weeks without NGF indicating their lack of dependence on NGF for long-term survival. By comparison, sympathetic neurons from neonatal rat superior cervical ganglion do not survive longer than two days when NGF is withdrawn from the cultures at day 4 (not shown).
Nerve growth factor and the indact~o~ of functional nicotinic acetylcholine receptors Rat nodose neurons express nicotinic ACh recep tom when grown in culture without non-neuronal cells and with NGF.13*14To determine whether NGF was necessary for the expression of these nicotinic recep tors, we measured the proportion of neurons expressing functional ACh receptors when grown either with or without NGF. Neurons sensitive to ACh were
Nerve growth factor receptors on nodose neurons in culture We investigated whether cultured nodose neurons have NGF receptors, even though it was apparent that these neurons do not need NGF for long-term growth and survival. Whether cultured with or without NGF, nodose neurons were consistently shown to bind [lZSI]NGF (Fig. 3). [‘ZSI]NGF (40 PM) binding was visibly diminished by unlabelled NGF at 80 pM and signi~~antly reduced by unlabelled NGF at 4 nM (see Fig. 3). When the grain density per unit neurite length was quantified in low density cultures, labelling by 20pM [‘=I]NGF was reduced to 50% by approximately 50 pM unlabelled NGF (Fig. 4). Therefore, under these conditions, most of the NGF bound with a dissociation equilibrium constant less than 30pM.’ This value is consistent with that for high-affinity NGF receptors on other neurons.z2.59
4
/ / M 8 12 DAYS IN CULTURE
t XI
Fig. 2. Neuronal numbers in culture. This figure shows the number of nodose neurons per culture over a three-week period. The numbers were determined by counting the neurons directly in the living culture under phase optics and expressed relative to the count obtained on day 4. Each point represents a mean of 12 dishes taken from six different platings and the error bars represent the S.E.M. The filled symbois represent cultures grown in the presence of NGF, while open symbols represent cultures that had their NGF removed on day 4.
526
A.
bfANDELZVs
identi~ed by monito~ng the membrane potential as ACh was applied to the cell body. Sensitive neurons, whether grown with or without NGF, usually depolarized by lO-20mV upon ACh application, whereas insensitive neurons had no detectable change in membrane potential {see Fig. 5A). The effect of NGF on the proportion of ACh-sensitive neurons measured in this way is shown in Fig. 5B. Out of 872 neurons grown in culture with NGF for 12-21 days, 64% were ACh-sensitive, whereas only 14% out of 514 neurons had ACh-sensitivity when cultured for the same time period without NGF. There was no signi~~ant difference between resting potentials or the amplitude of the action potentials among neurons grown with or without NGF for two to three weeks: for neurons with NGF, the mean resting potential was 51f 1 mV (mean _t S.E.M., n = 36) and the action potential amplitude was 68 k 2mV (mean If: S.E.M., n = 36), and for neurons grown without NGF after day 4, the mean resting potential was 53 _t 1 mV (mean _t S.E.M., n = 36) and the mean action potential amplitude was 67 + 2 mV (mean _t S.E.M., n = 36). Experiments with 40 and 400 pM fl-NGF gave results that were similar to those with
et al.
7 S NGF indicating that the effects on nicotinic receptor expression can be attributed to interactions with high-affinity NGF receptors.25~59 To determine whether the proportion of AChsensitive neurons grown without NGF remains low over time or whether it simply increases at a rate slower than that for cultures grown with NGF, we measured the proportion of ACh-sensitive neurons at different times after plating. To measure the proportion in a given culture, approximately 50 neurons were chosen at random and tested individually for the presence or absence of ACh receptors. The results from two platings are shown in Fig. 6. When cultured with NGF, the proportion of neurons expressing ACh-sensitivity increased over a two-week period and plateaued near 80%. In contrast, only 10% of neurons grown in sister cultures without NGF had a detectable response to ACh application when tested over the same time period. In addition, at day 4, 28% (99 out of 353) of neurons were sensitive to ACh, whereas less than 10% were ACh-sensitive two to three days after removing NGF; this observation suggests that some neurons may lose functional nicotinic receptors when NGF is withdrawn.
set shows binding of [‘ZSI]NGF(40 PM) to the yower scrj iY”l-. t ne ie,t upp,pn anu 11 The middle set shows binding in the presence of 80 pM unla~ll~ NGF. The right set shows non-specific IOO-fold excess (4 nM) unlabelled NGF. Scale bar = 100 pm. binding in the presev-
w1mout
NGF induces ACh receptors on nodose neurons in culture
Fig. 4. Specific binding of [“‘I]NGF to nodose neurons in culture. Cultures were grown at low density in the presence of NGF for five days and radioautographs were prepared after incubation of sister cultures with 20pM [‘251]NGF and 0-8OpM unlabelled NGF. Binding over labelled neurites (area covered by grains/length) was quantified and expressed as a fraction of the binding with [iZSIJNGF alone (left) and in logit-log form (right). Note that half-maximal displacement occurs at approximately 50 pM unlabelled NGF (m 5 S.E.M.: n = 4247).
A
60
I
40
20
Fig. 5. Influence of NGF on the expression of ACh-sensitivity. (A) The response of two neurons to ACh (10e3 M in the pipette) puffed onto their somas and proximal processes. The upper trace represents the membrane potential, the lower trace indicates the duration of the drug application. For the neuron on the left, a brief (200ms) application of ACh caused a large change in membrane potential which repolarized as the drug diffused away from the neuron. For the neuron on the right, repeated application of ACh caused no change in membrane potential. (3) The mean and standard deviation of the proportion of ACh-sensitive neurons cultured for 12-21 days either in the continual presence of NGF (n = 872) or after having their NGF removed on day 4 in vitro (n = 574).
521
528
A. MANDELZYS et (11
I
*
-I
100
‘“1;
, d
\;/‘-;y’
-NGF
8
1;
16
20
DAYS IN CULTURE
Fig. 6. Developmentai time-course of the proportion of neurons expressing ACh-sens~t~vjty. This figure shows the proportion of ACh-sensitive neurons in cultures from two different platings (A, 0). Each point represents a different culture and shows the proportion of ACh-sensitive neurons determined from a 50.neuron random sample. The filled symbols represent cultures grown continualIy in NGF, the open symbols represent cultures that had NGF removed on day 4. The value on day 4 (28 & 2, mean + SD.) is the mean of 353 neurons from 10 different platings. DISCUSSION
this study differs in that it demonstrates the induction by NGF of a functional neurotransmitter receptor that was not previously expressed by these neurons. Our findings that nodose neurons express nicotinic receptors in culture may be related to reports of nicotinic receptors on sensory neurons in V&W.There is good physiological evidence that some terminals and regenerating endings of sensory axons have nicotinic ACh receptors, although they seem to have little role in the sensory transduction mechanism.“,‘* Recent immunohistochemical studies have shown that presynaptic terminals of sensory axons in the CNS display nicotinic receptors.60 Furthermore, chick dorsal root ganglion neurons have been shown to contain mRNA for the ligand-binding subunit of a neuronal nicotinic ACh receptor.6 The role for these receptors on sensory neurons may become apparent when more is known about the physiology of the various types of neuronal nicotinic ACh receptors~3,5J52345
mRNA for subunits of neuronal nicotinic receptors have been shown to be present in basal forebrain neurons.63 In addition, cholinergic forebrain neurons have been shown to have high-affinity NGF receptors.” Conceivably, NGF may play a role in inducing functional nicotinic receptors on these neurons. Two factors have now been shown to be important in the induction of nicotinic receptors on nodose neurons: the absence of satellite cellsI and the presence of NGF (the present study). The possible interplay between these two trophic influences requires further study and may have some bearing on the general question about factors that influence a developing neuroblast to choose between different phenotypes. Neuroblasts, normally destined to form the nodose ganglion, can migrate to form part of the parasympathetic cardiac ganglion after the removal of the cranial neural crest in developing chicks, and these displaced neurons express a chohnergic phenotype.” Neonatal rat nodose neurons developing in culture without satellite cells can synthesize choline acety1transferase42 and form cholinergic synapses among each other;” such properties are more typical of autonomic neurons in culture.“‘,46.49Conceivably, nodose neurons may be expressing an autonomic neuron phenotype when they develop in culture with NGF and without their ganglionic satellite cells, and the expression of nicotinic receptors may be one manifestation of this change.
In this paper, we have shown that NGF, acting on high-affinity receptors causes neonatal rat nodose neurons to express nicotinic ACh receptors as they develop in tissue culture without other cell types. NGF has little influence on the number of neurons surviving in culture: 85% of the neurons continue to grow in size and extend processes for at least three weeks in the absence of NGF. The present results are consistent with previous observations that antibodies to NGF do not decrease the number of rodent nodose neurons in ci~o,~~~~and with observations that NGF does not support dissociated rat or chick nodose neurons in tissue culture. ‘x~* The 15% decrease in neuronal numbers when NGF is removed from the cultures may indicate that a subpopulation of neurons requires NGF for survival. Recently, embryonic rat nodose ganglia were shown to have low-affinity NGF receptors.64 The present binding studies (Figs 3 and 4) however, identified high-affinity NGF receptors on most nodose neurons in culture, and the experiments with picomolar concentrations of NGF indicate that the effects on nicotinic receptor induction are mediated .~cknowferinements-We thank MS Brigitte Pie for technical by high-a~nity receptors.‘5.59 Studies on adult rat assistance and MS S. James for typingthe manuscript. We nodose neurons indicate that less than one quarter also thank Drs S. Carbonetto for laminin. M. Couahlin for have high-affinity NGF receptors.5’ The difference in anti-NGF, R. Riopelle for [‘251]NGF,W. G. Tatton for the the proportion of neurons with high-affinity receptors image analysis software, and Dr S. Carbonetto, M. W. Cohen and L. Glass for providing comments on the in the adult compared with that in culture suggests manuscript. This work is supported by separate MRC that rat nodose neurons may be plastic in their ability Canada operating grants to EC. and P.M.R. A.M. is to express NGF receptors. supported by a graduate fellowship from FCAR of Although there have been other reports of NGF Quebec and E.C. has a senior scientist award from FRS du Quebec. on the di~erentiation of sensory neurons,9~3’~36~38~4x
NGF induces ACh receptors on nodose neurons in culture
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36. Lindsay R. M. and Harmar A. J. (1989) Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature 337, 3622364. 37. Liuzzi A., Fopper F. H. and Kopin 1. J. (1977) Stimulation and maintenance by nerve growth factor of phenylethanolamine-N-methyltransferase in superior cervical ganglia of adult rats. Brain-Res. 138;309-315. 38. MacLean D. B., Bennett B., Morris M. and Wheeler F. B. (1989) Differential regulation of calcitonin gene-related peptide and substance P in cultured neonatal rat vagal sensory neurons. Brain Res. 478, 349-355. 39. Mains R. E. and Patterson P. M. (1973) Primary cultures of dissociated sympathetic neurons. I. Establishment of long-term growth in culture and studies of differentiated properties. J. CeliBiol. 59, 3299345. 40. Mandel G., Cooperman S. S., Maue R. A., Goodman R. M. and Brehn P. (1988) Selective induction of brain type II Na+ channels by nerve growth factor. Proc. naln. Acud. Sci. U.S.A. 85, 924928. 41. Margiotta J. F., Berg D. K. and Dionne V. E. (1987) The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons. J. Neurosci. 7, 3612-3622. 42. Mathieu C., Moisand A. and Weber M. J. (1984) Acetylcholine metabolism by cultured neurons from rat nodose ganglia: regulation by a macromolecule from muscle-conditioned medium. Neuroscience 13, 1373-1886. 43. Mobley W. C., Schenker A. and Shooter E. M. (1976) Characterization and isolation of proteolytically modified nerve growth factor. Biochemisrry 15, 55433555 1. 44. Mobley W. C., Rutkowski J. L., Tennekoon G. I., Buchanan K. and Johnston M. W. (1985) Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor. Science 229, 284287. 45. Nef P., Oneyser C.. Alliod C., Couturier S. and Ballivet M., (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. Eur. molec. Biol. Org. J. 7, 5955601. 46. O’Lague P. H., Potter D. D. and Furshpan E. J. (1978) Studies on rat sympathetic neurons developing in cell culture. I. Growth characteristics and electrophysiological properties, Devl Biol. 67, 384403. 47. O’Lague P. H., Potter D. D. and Furshpan E. J. (1978) Studies on rat sympathetic neurons developing in cell culture. I. Growth characteristics and electrophysiological properties, Devl Biol. 67, 384403. 48. Otten U., Goedert M., Mayer N. and Lembeck F. (1980) Requirement of nerve growth factor for development of substance P-containing sensory neurons, Nature 287, 158-l 59. 49. Patterson P. H. (1978) Environmental determination of autonomic neurotransmitter functions. A. Rev. Neurosci. 1,
I 17. 50. Pearson J., Johnson E. M. and Brandeis L. (1983) Effects of antibodies to nerve growth factor on intrauterine development of derivatives of cranial neural crest and placode in the guinea pig. Deul Biol. 96, 32-36. 51. Richardson P. M., Verge Issa V. M. K. and Riopelle R. J. (1986) Distribution of neuronal receptors for nerve growth factor in the rat. J. Neurosci. 6, 2312-2321. 52. Richardson P. M., Verge V. M. K. and Riopelle R. J. (1989) Quantitative radioautography for NGF receptors. In Nerve Growth Factors (ed. Rush R. A.), pp. 315-327. John Wiley, New York. 53. Roper S. (1976) The acetylcholine sensitivity of the surface membrane of multiply-innervated parasympathetic ganglion cells in the mudpuppy before and after partial denervation. J. Physiol. 254, 455473. 54. Rudy B., Kirschenbaum B. and Greene L. A. (1982) Nerve growth factor-induced increase in saxitoxin binding to rat PC12 pheochromocytoma cells. J. Neurosci. 2, 1405-141 I. 55. Rudy B., Kirschenbaum B., Rukenstein A. and Greene L. A. (1987) Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells. J. Neurosci. 7, 161331625. 56. Sargent P. B. and Pang D. 2. (1988) Denervation alters the size, number, and distribution of clusters of acetylcholine receptor-like molecules on frog cardiac ganglion neurons. Neuron 1, 877-886. 57. Schuetze S. M. and Role L. W. (1987) Developmental regulation of nicotinic acetylcholine receptors. A. Rev. Neurosci. 10, 403457.
58. Smith M. A,, Margiotta J. F. and Berg D. K. (1983) Differential regulation of acetylcholine sensitivity and r-bungarotoxin binding sites on ciliary ganglion neurons in cell culture. J. Neurosci. 3, 2395-2402. 59. Sutter A., Riopelle R. J., Harris-Warrick R. M. and Shooter E. M. (1979) Nerve growth factor receptors. J. biol. Chem. 254, 597225982.
60. Swanson I,. W., Simmons D. M., Whiting P. J. and Lindstrom J. (1987) Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system. J. Neurosci. 7, 3334-3342. 61 Thoenen H., Angeletti P. V., Levi-Montalcini R. and Kettler R. (1971) Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-/I-hydroxylase in the rat superior cervical ganglion. Proc. natn. Acad. Sci. U.S.A. 68, 159881602.
62 Verge V. M. K., Riopelle R. J. and Richardson P. M. (1989) Nerve growth factor receptors on normal and injured sensory neurons. J. Neurosci. 9, 914-922. 63 Wada E., Wada K., Boulter J., Deneris E., Heinemann S., Patrick J. and Swanson L. W. (1989) The distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptors subunits mRNAs in the central nervous system. A hybridization histochemical study in the rat. J. camp. Neural. 284, 314-335. study of the nerve growth factor receptor in developing 64 Yan Q, and Johnson E. M. Jr (1988) An immunohistochemical rats. J. Neurosci. 8, 3481-3498. (Accepted 13 November 1989)
NeuroscienceVol. 31, No. 2, pp. 523-530, 1990 Printed in Great Britain
NERVE GROWTH FACTOR INDUCES FUNCTIONAL NICOTINIC ACETYLCHOLINE RECEPTORS ON RAT SENSORY NEURONS IN CULTURE A. MANDELZYS,* E. COOPER,*? Q. M. K. VERGES and P. M. R~CHARDSON~ Departments
of *Physiology and SNeurology and Neurosurgery,
McGill University, Montreal, Quebec,
Canada H3G 1Y6 Abstract-Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influen~d the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day I2 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholinesensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.
The expression for
the
system. the
The muscle
best
served
of postsynaptic
development studied
as a model
of nicotinic
receptors
synapses
in
receptor
neurotransmitter to investigate
is essential the
virtually all non-neuronal cells have been eliminated, nodose neurons express nicotinic ACh receptors and cholinergic synapses form among the neurons.13 In contrast, when the neurons are co-cultured with non-neuronal elements from the ganglion (including satellite cells, Schwann cells, and fibroblasts) to mimic their in vivo environment, few neurons express ACh receptorsi These findings suggest that some influence from non-neuronal cells prevents the expression of ACh receptors on these neurons. The factors that induce nodose neurons to express nicotinic ACh receptors in culture in the absence of non-neuronal cells, however, are largely unknown. As nerve growth factor (NGF) was present in all our previous studies, we questioned whether NGF itself may have a role in the induction of nicotinic ACh receptors on these neurons. NGF has been shown to influence the differentiated properties of other neurons: for example, NGF increases neurotransmitter synthesis in sympathetic neurons,4z’ ‘J*,~‘.~‘.~in sensory neurons3t.3a and in cholinergic forebrain neurons,24,26,Uand NGF can affect the expression of voltage-gated ionic channels on PC12 cells’y~~~47~~~55 and on sensory neurons.’ In this paper, we have extended our studies on the development of rat nodose neurons in culture and have investigated the effects of NGF on the expression of nicotinic ACh receptors on nodose neurons in culture.
nervous
is undoubtedly receptor.
It has
the developmental
appearance
of postsynaptic receptors, as well as factors, such as innervation and activity, that influence receptor number and dist~bution on the postsynaptic membrane. a.20.57 The expression of neuronal nicotinic receptors, which are part of a common gene family of ligand-gated channels, is similarly regulated by denervation16~29,33.s3,56 and also by axotomy.7.zg A recent study has shown that this regulation on autonomic neurons can occur, at least in part, at the level of mRNA for the receptor subunits.6 Much less is known about developmental influences that determine why some neurons express particular neurotransmitter receptors. For example, while all autonomic neurons express nicotinic receptors, only a small number of sensory neurons express them,“@ even though most sensory neurons are derived from the same embryological structures as autonomic neurons. 34To learn what factors influence the appearance of nicotinic receptors on some sensory neurons and not on others, we have been studying the development of neonatal rat nodose neurons in dissociated cell culture. Only a few nodose neurons are sensitive to acetylcholine (ACh) in vivo;28 however,
we have
found
that
in conditions
where
tTo whom correspondence should be addressed. ACh, acetylcholine; HEPES, N-Zhydroxyethylpi~razine-~‘-2-ethanesulphonic acid; L- 15 medium, Leibovitz-15 medium; NGF, nerve growth factor.
Abbreoiations:
EXPERIMENTAL PROCEDURES Neuronal cultures
The methods for culturing nodose neurons and sympathetic neurons were similar to those already publjshed.l~.14 Briefly, ganglia were dissected under sterile conditions from
523
524
A. MANDELZYS rt ul.
newborn rats (CD. strain, Charles River, Canada), which
were killed by cervical dislocation. The ganglia were incubated for 15 min at 37°C in a medium contain& collatenase (1 mg/ml, Cooper Biomedical) and a neutral protease (Dispase, Grade II, 2.4 mg/ml, Boehringer Mannheim). They were then transferred to a solution containing only the neutral protease and triturated every IS min for 3-4 h. Y
”
Following the dissociation, the cells were centrifuged through a Percoll (Pharmacia) gradient (35% Percoll) in order to separate ncuronal from non-neuronal cells. The neuron suspension was washed twice and plated in modified culture dishes on collagen-laminin-coated coverslips (0.75cm”) (lOOfig/ml laminin for 12 h) in Leibovitz-15 medium supplemented with sodium bicarbonate, vitamins, co-factors, penicillin--streptomycin, rat serum (5%) and 7s NGF (l~nM~.~9 In some experiments, fi-NGF prepared from submandibular glands of male mice by ion-exchange chromatography’0,4” was used at concentrations of 40 and 400 pM. Cytosine arabinoside (IO HM, Sigma) was added to the cell cultures for the first four days to kill any dividing cells that remained following the Percoll step. The cultures were maintained in a humid atmosphere of 95% air-5% CO, at 37’C and fed every three to four days with fresh growth media. All cultures were initially grown with NGF, However, on day 4, the plating was divided into two groups: those grown in the continual presence of NGF; and those in which NGF had been removed and rabbit antiserum to 2.5 S NGF at a I: 1000 dilution was added. This antiserum at l:lO,OOO, blocks the effect of 10 nM 7 S NGF when added to dissociated rat sympathetic neuron cultures. Neuronal numbers were determined by counting the neurons directly in the living culture using phase optics; cultures typically had 1000-2000 neurons,
ACh-sensitivity was measured electrophysiologically with intracellular recordings; the methods were similar to those previously described. 2.46The cultures were perfused at approximately 0.5 mljmin with media containing: 10% (v/v) L-15, 90% (v/v) Hank’s Balanced Salt Solution. CaC&, (2.8 mM), choline chloride (0.07 mMf, HEPES (IO mM, BDH), glucose (5.6 mM), glutamine (2 mM) and penicillin (100 pg/ml) and streptomycin (0.25 mg/ml) (Gibco): the pH was 7.2.7.4 and temperature was 35537°C. The recording electrodes, filled with 3 M KC1 (resistance; 50-100 MO), were connected to a WPI M707 amplifier to monitor membrane voltage and to pass current, and the signals were displayed on a Tektronic storage oscilloscope (5113). To determine whether a neuron expressed functional ACh receptors, we rapidly exposed as much of the neuron as possible to ACh (10“-lo-* M, Sigma) and observed if this resulted in any membrane depolarization. We found that the best way to apply ACh is via relatively large tipped pipettes (lo-20 pm) positioned at an appropriate distance from the neuron (2&40 pm) so that upon application of pressure we could perfuse a large area. We have verified this approach in several pilot studies with pipettes filled with dyes or suspended oil droplets, and we have ascertained that there is no outflow of drug from the “puffer pipette” into the bath provided that the Ievel of liquid in the pipette is kept low. With this technique, most sensitive neurons depolarized by lo-20mV, whereas insensitive neurons had no detectable change in membrane potential. From whole-cell and singlechannel recordings (Mandelzys and Cooper, unpublished observations), we estimate that sensitive neurons had several hundred ACh receptors open simultaneously at the peak of the response, and that neurons judged as insensitive could not have had more than 20-30 receptors. As there are no visible clues to distinguish ACh-sensitive neurons from ACh-insensitive neurons in the living culture with phase optics, neurons with clearly visible nuclei in the centre of the celJ were selected at random. Only neurons with resting
potentials greater than - 35 mV were included in this study. The proportion of ACh-sensitive neurons in any given culture was determined by recording from 50 randomly chosen neurons.
P-NGF was radioiodinated by the lactoperoxidase method” and separated from free iodine.62 IIZSIINGF with specific activity _of approximately 100 ,utC$pg’ was used within 24 h of preparation. For 4 h before exposure to [‘*‘I]NGF, the cultures were washed with four changes of culture medium without NGF. The cultures were incubated for 1.5 h at 37°C with 40 pM [‘*‘I]NGF in the presence of 0,40, 80, 160, 320, or 4000 pM uniabelled NGF. The incubation solutions were prepared by diluting NGF in 0.1 M phosphate-buffered saline, pH 7.4, with magnesium chloride (0.5 mM), cytochrome C (1 mg/ml), leupeptin (4 p&/ml), and phenylmethylsulfonylfluoride (0.5 mM). After six rinses with cold-buffered saline over 3 min and fixation with cold 4% parafo~aldehyde for 10 min, the cultures were washed with water and dehydrated in a series of alcohols. Coverslips were then dipped in emulsion (NTB, Kodak), exposed in the dark at 4°C for two to five days and developed. Dark-field photomicrography at low magni~cation was accomplished with a scanning condenser. To measure the affinity of NGF binding, low density cultures were used in which neurons were plated at approximately 20.-3O/cm*so that most of the neurites from individual neurons remained separate. After one week, the cultures were incubated with [‘*‘I]NGF (20 PM) and O-2500 pM unlabelled NGF as described above. As previously described image-analysis system5* was slightly modified to measure (percentage of area covered by silver grains minus percentage of area covered by silver grains in background regions of the slide) multiplied by area and divided by neurite length. This measurement, obtained for rectangles over labelled neurites in culture is linearly related to specific grain counts per length of axon.
RESULTS
Development of nodose neurons in culture with and without nerve growth factor
We first determined whether or not rat nodose neurons require NGF for growth and survival when grown in dissociated culture in the absence of nonneuronal cells. All neurons were plated with NGF for the first four days to avoid the possibility of selecting populations of nodose neurons that differ in their
NGF requirements. At day 4, NGF was withdrawn from half the cultures, and in addition, antiserum to NGF was also added at a concentration of 1: 1000. Neurons with and without NGF continued to grow in size and extend processes for several weeks. Phasecontrast photomicrographs of sister cultures grown for three weeks with or without NGF is shown in Fig. 1. At three weeks, the mean cell body diameter of neurons grown without NGF are indistinguishable from those grown with NGF [22.8 k 1.9, II = 150 (mean + S.E.M.) and 23.4 f 1.7, n = 150 (mean f S.E.M.), respectively]. The density of neurite outgrowth at three weeks, although dificult to quantify, generally appeared to be somewhat greater when NGF was present in the culture medium (see Fig. I).
NGF induces ACh receptors on nodose neurons in culture
Fig. 1. Montages of phase-contrast photomicrographs of nodose neurons after three weeks in culture. Both cultures were grown with NGF for the first four days: thereafter, the culture on the left was grown in the continual presence of NGF, while the culture on the right had its NGF removed. Both cultures show spherical cell bodies with clear nuclei containing one or two nucleoli and extensive neurite outgrowth. Scale bar = IO0 pm.
Figure 2 demons~ates that neuronal survival was largely unaffected by NGF. During the first two to three days after NGF withdrawal, there was a 15% decrease in neuronal numbers; the remaining 85%, however, continued to grow for at least three weeks without NGF indicating their lack of dependence on NGF for long-term survival. By comparison, sympathetic neurons from neonatal rat superior cervical ganglion do not survive longer than two days when NGF is withdrawn from the cultures at day 4 (not shown).
Nerve growth factor and the indact~o~ of functional nicotinic acetylcholine receptors Rat nodose neurons express nicotinic ACh recep tom when grown in culture without non-neuronal cells and with NGF.13*14To determine whether NGF was necessary for the expression of these nicotinic recep tors, we measured the proportion of neurons expressing functional ACh receptors when grown either with or without NGF. Neurons sensitive to ACh were
Nerve growth factor receptors on nodose neurons in culture We investigated whether cultured nodose neurons have NGF receptors, even though it was apparent that these neurons do not need NGF for long-term growth and survival. Whether cultured with or without NGF, nodose neurons were consistently shown to bind [lZSI]NGF (Fig. 3). [‘ZSI]NGF (40 PM) binding was visibly diminished by unlabelled NGF at 80 pM and signi~~antly reduced by unlabelled NGF at 4 nM (see Fig. 3). When the grain density per unit neurite length was quantified in low density cultures, labelling by 20pM [‘=I]NGF was reduced to 50% by approximately 50 pM unlabelled NGF (Fig. 4). Therefore, under these conditions, most of the NGF bound with a dissociation equilibrium constant less than 30pM.’ This value is consistent with that for high-affinity NGF receptors on other neurons.z2.59
4
/ / M 8 12 DAYS IN CULTURE
t XI
Fig. 2. Neuronal numbers in culture. This figure shows the number of nodose neurons per culture over a three-week period. The numbers were determined by counting the neurons directly in the living culture under phase optics and expressed relative to the count obtained on day 4. Each point represents a mean of 12 dishes taken from six different platings and the error bars represent the S.E.M. The filled symbois represent cultures grown in the presence of NGF, while open symbols represent cultures that had their NGF removed on day 4.
526
A.
bfANDELZVs
identi~ed by monito~ng the membrane potential as ACh was applied to the cell body. Sensitive neurons, whether grown with or without NGF, usually depolarized by lO-20mV upon ACh application, whereas insensitive neurons had no detectable change in membrane potential {see Fig. 5A). The effect of NGF on the proportion of ACh-sensitive neurons measured in this way is shown in Fig. 5B. Out of 872 neurons grown in culture with NGF for 12-21 days, 64% were ACh-sensitive, whereas only 14% out of 514 neurons had ACh-sensitivity when cultured for the same time period without NGF. There was no signi~~ant difference between resting potentials or the amplitude of the action potentials among neurons grown with or without NGF for two to three weeks: for neurons with NGF, the mean resting potential was 51f 1 mV (mean _t S.E.M., n = 36) and the action potential amplitude was 68 k 2mV (mean If: S.E.M., n = 36), and for neurons grown without NGF after day 4, the mean resting potential was 53 _t 1 mV (mean _t S.E.M., n = 36) and the mean action potential amplitude was 67 + 2 mV (mean _t S.E.M., n = 36). Experiments with 40 and 400 pM fl-NGF gave results that were similar to those with
et al.
7 S NGF indicating that the effects on nicotinic receptor expression can be attributed to interactions with high-affinity NGF receptors.25~59 To determine whether the proportion of AChsensitive neurons grown without NGF remains low over time or whether it simply increases at a rate slower than that for cultures grown with NGF, we measured the proportion of ACh-sensitive neurons at different times after plating. To measure the proportion in a given culture, approximately 50 neurons were chosen at random and tested individually for the presence or absence of ACh receptors. The results from two platings are shown in Fig. 6. When cultured with NGF, the proportion of neurons expressing ACh-sensitivity increased over a two-week period and plateaued near 80%. In contrast, only 10% of neurons grown in sister cultures without NGF had a detectable response to ACh application when tested over the same time period. In addition, at day 4, 28% (99 out of 353) of neurons were sensitive to ACh, whereas less than 10% were ACh-sensitive two to three days after removing NGF; this observation suggests that some neurons may lose functional nicotinic receptors when NGF is withdrawn.
set shows binding of [‘ZSI]NGF(40 PM) to the yower scrj iY”l-. t ne ie,t upp,pn anu 11 The middle set shows binding in the presence of 80 pM unla~ll~ NGF. The right set shows non-specific IOO-fold excess (4 nM) unlabelled NGF. Scale bar = 100 pm. binding in the presev-
w1mout
NGF induces ACh receptors on nodose neurons in culture
Fig. 4. Specific binding of [“‘I]NGF to nodose neurons in culture. Cultures were grown at low density in the presence of NGF for five days and radioautographs were prepared after incubation of sister cultures with 20pM [‘251]NGF and 0-8OpM unlabelled NGF. Binding over labelled neurites (area covered by grains/length) was quantified and expressed as a fraction of the binding with [iZSIJNGF alone (left) and in logit-log form (right). Note that half-maximal displacement occurs at approximately 50 pM unlabelled NGF (m 5 S.E.M.: n = 4247).
A
60
I
40
20
Fig. 5. Influence of NGF on the expression of ACh-sensitivity. (A) The response of two neurons to ACh (10e3 M in the pipette) puffed onto their somas and proximal processes. The upper trace represents the membrane potential, the lower trace indicates the duration of the drug application. For the neuron on the left, a brief (200ms) application of ACh caused a large change in membrane potential which repolarized as the drug diffused away from the neuron. For the neuron on the right, repeated application of ACh caused no change in membrane potential. (3) The mean and standard deviation of the proportion of ACh-sensitive neurons cultured for 12-21 days either in the continual presence of NGF (n = 872) or after having their NGF removed on day 4 in vitro (n = 574).
521
528
A. MANDELZYS et (11
I
*
-I
100
‘“1;
, d
\;/‘-;y’
-NGF
8
1;
16
20
DAYS IN CULTURE
Fig. 6. Developmentai time-course of the proportion of neurons expressing ACh-sens~t~vjty. This figure shows the proportion of ACh-sensitive neurons in cultures from two different platings (A, 0). Each point represents a different culture and shows the proportion of ACh-sensitive neurons determined from a 50.neuron random sample. The filled symbols represent cultures grown continualIy in NGF, the open symbols represent cultures that had NGF removed on day 4. The value on day 4 (28 & 2, mean + SD.) is the mean of 353 neurons from 10 different platings. DISCUSSION
this study differs in that it demonstrates the induction by NGF of a functional neurotransmitter receptor that was not previously expressed by these neurons. Our findings that nodose neurons express nicotinic receptors in culture may be related to reports of nicotinic receptors on sensory neurons in V&W.There is good physiological evidence that some terminals and regenerating endings of sensory axons have nicotinic ACh receptors, although they seem to have little role in the sensory transduction mechanism.“,‘* Recent immunohistochemical studies have shown that presynaptic terminals of sensory axons in the CNS display nicotinic receptors.60 Furthermore, chick dorsal root ganglion neurons have been shown to contain mRNA for the ligand-binding subunit of a neuronal nicotinic ACh receptor.6 The role for these receptors on sensory neurons may become apparent when more is known about the physiology of the various types of neuronal nicotinic ACh receptors~3,5J52345
mRNA for subunits of neuronal nicotinic receptors have been shown to be present in basal forebrain neurons.63 In addition, cholinergic forebrain neurons have been shown to have high-affinity NGF receptors.” Conceivably, NGF may play a role in inducing functional nicotinic receptors on these neurons. Two factors have now been shown to be important in the induction of nicotinic receptors on nodose neurons: the absence of satellite cellsI and the presence of NGF (the present study). The possible interplay between these two trophic influences requires further study and may have some bearing on the general question about factors that influence a developing neuroblast to choose between different phenotypes. Neuroblasts, normally destined to form the nodose ganglion, can migrate to form part of the parasympathetic cardiac ganglion after the removal of the cranial neural crest in developing chicks, and these displaced neurons express a chohnergic phenotype.” Neonatal rat nodose neurons developing in culture without satellite cells can synthesize choline acety1transferase42 and form cholinergic synapses among each other;” such properties are more typical of autonomic neurons in culture.“‘,46.49Conceivably, nodose neurons may be expressing an autonomic neuron phenotype when they develop in culture with NGF and without their ganglionic satellite cells, and the expression of nicotinic receptors may be one manifestation of this change.
In this paper, we have shown that NGF, acting on high-affinity receptors causes neonatal rat nodose neurons to express nicotinic ACh receptors as they develop in tissue culture without other cell types. NGF has little influence on the number of neurons surviving in culture: 85% of the neurons continue to grow in size and extend processes for at least three weeks in the absence of NGF. The present results are consistent with previous observations that antibodies to NGF do not decrease the number of rodent nodose neurons in ci~o,~~~~and with observations that NGF does not support dissociated rat or chick nodose neurons in tissue culture. ‘x~* The 15% decrease in neuronal numbers when NGF is removed from the cultures may indicate that a subpopulation of neurons requires NGF for survival. Recently, embryonic rat nodose ganglia were shown to have low-affinity NGF receptors.64 The present binding studies (Figs 3 and 4) however, identified high-affinity NGF receptors on most nodose neurons in culture, and the experiments with picomolar concentrations of NGF indicate that the effects on nicotinic receptor induction are mediated .~cknowferinements-We thank MS Brigitte Pie for technical by high-a~nity receptors.‘5.59 Studies on adult rat assistance and MS S. James for typingthe manuscript. We nodose neurons indicate that less than one quarter also thank Drs S. Carbonetto for laminin. M. Couahlin for have high-affinity NGF receptors.5’ The difference in anti-NGF, R. Riopelle for [‘251]NGF,W. G. Tatton for the the proportion of neurons with high-affinity receptors image analysis software, and Dr S. Carbonetto, M. W. Cohen and L. Glass for providing comments on the in the adult compared with that in culture suggests manuscript. This work is supported by separate MRC that rat nodose neurons may be plastic in their ability Canada operating grants to EC. and P.M.R. A.M. is to express NGF receptors. supported by a graduate fellowship from FCAR of Although there have been other reports of NGF Quebec and E.C. has a senior scientist award from FRS du Quebec. on the di~erentiation of sensory neurons,9~3’~36~38~4x
NGF induces ACh receptors on nodose neurons in culture
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