DEVELOPMENTAL

BIOLOGY

139,417-426

(1990)

The Development of ACH- and GABA-Activated Currents in Normal and Target-Deprived Embryonic Chick Ciliary Ganglia KATHRIN

L. ENGISCH AND GERALD

D. FISCHBACH~

Department of Anatomy & Neurobiology, Washington University School of Medicine,

660

South Euclid, St. Louis, Missouri

63110

Accepted. January 29, 1990 We have examined the expression of functional ACh and GABA receptors on embryonic chick ciliary ganglion neurons between Stages (St) 29 and 44 (Embryonic Day 6 to Embryonic Day 18). Whole-cell currents activated by ACh or GABA were measured in neurons 3-6 hr after dissociation to estimate the level of functional receptors in viwo. The mean peak IAa increased sevenfold between St 29 (321 PA) and St 44 (2345 PA) in two steps, separated by a plateau between St 35 and St 38 (E9 to E12). Cell size, estimated from measurements of membrane capacitance, increased only threefold over the same interval. Moreover, I AChand cell size were not well correlated at any stage examined. IoaBA increased twofold between St 29 and St 38; the change was gradual and without any indication of two phases. The increase in IAch during development was not dependent on innervation of target cells within the eye. We removed the primordial eye between St 11 and St 13 (E2) and allowed the embryos to mature to various stages. Despite a small (2050%) reduction in IAChat every stage examined, I AChstill increased dramatically (about lo-fold) between St 29 and St 44 in target-deprived neurons. I AChwas not uniquely affected by early target removal; IGAs* and capacitance were also slightly reduced in target-deprived neurons. o 1990 Academic press. IX INTRODUCTION

ACh receptors (Jacob et aL, 1984), and [‘251]Toxin F (neuronal bungarotoxin, K-bungarotoxin; Loring and Zigmond, 1987) are concentrated at areas of presynaptic contact in Embryonic Day 16 (E16) and El7 ganglion neurons suggests a causal relationship between receptor expression and location of the presynaptic terminal. On the other hand, separation of chick ciliary ganglion neurons from their targets through posthatch axotomy induces a dramatic loss in ACh sensitivity (Brenner and Martin, 1976; McEachern et a.& 1989). Binding of [‘“I]mAb35 (Jacob and Berg, 1987) and the amount of neuronal ACh receptor mRNA are also reduced (Boyd et al, 1988). GABA sensitivity is unaffected by axotomy (McEachern et cd, 1989). It is possible that presynaptic and postsynaptic factors influence receptor expression at early stages of development, during the initial phases of synapse formation. Specific binding of [‘251]mAb35 can be detected in homogenates of E8 ganglia, and the number of sites increases during development (Smith et cd., 1985). mRNA that encodes the receptor CY~subunit can be detected by in situ hybridization at E6 and increases with age to at least 1 month posthatch (Boyd et al, 1988). A recent paper reported that ACh-induced currents of acutely isolated embryonic ciliary ganglion neurons increase eightfold between E8 and El6 (Margiotta and Gurantz, 1989). It has been known for some time that neurons cultured from young embryonic ciliary ganglia are sensitive to ACh (Ravdin and Berg, 1979; Bader et uL, 1982; Crean et uL, 1982). The ACh sensitivity of em-

In principle, the expression of ACh’ receptors on autonomic ganglion neurons might be regulated by presynaptic inputs, postsynaptic targets, or by intrinsic mechanisms that are independent of both. The embryonic chick ciliary ganglion has several advantages for studies of these issues (Pilar and Tuttle, 1982). The timing of key developmental events such as the onset of synaptic transmission within the ganglion, the period of nerve cell death, and the innervation of target tissues in the eye by postganglionic axons have been described in detail (Landmesser and Pilar, 1972, 1974a, 1974b; Meriney and Pilar, 1987). Presynaptic inputs form primarily on the neuronal cell bodies or on short “pseudodendrites” that appear transiently during development (Landmesser and Pilar, 1972). The chemical component of synaptic transmission within the ganglion is mediated by nicotinic ACh receptors. GABA receptors are also present on principal neurons within the ganglion, but their function is unknown (McEachern et al, 1985). There is evidence that both inputs and targets influence ACh receptor expression in late embryonic and more mature, posthatch chick ciliary ganglia. The fact that the binding of HRP-conjugated mAb35 (a monoclonal antibody thought to recognize neuronal nicotinic ’ To whom correspondence should be addressed. 2 Abbreviations used: ACh, acetylcholine; GABA, gamma amino butyric acid; mAb, monoclonal antibody; SEM, standard error of the mean. 417

0012-1606/90 $3.00 Copyright All rights

0 1990 by Academic Press. Inc. of reproduction in any form reserved.

418

DEVELOPMENTAL

BIOLOGY

VOLUME

139,199O

Plating medium consisted of Eagle’s Minimal Essential Medium (MEM, Hazelton, Denver, PA) supplemented with 10% heat-inactivated horse serum, 5% chick embryo extract, 2 mlM glutamine, 50 U/ml penicillin, and 50 pg/ml streptomycin (GIBCO Laboratories, Grand Island, NY). Transferrin medium was composed of Eagle’s MEM with 10% heat-inactivated horse serum and 0.04 mg/ml ovotransferrin (Sigma). Some cultures received 5% embryonic eye extract (Nishi and Berg, 1981) or 20% saline brain extract (Jessell et ah, 1979) in transferrin medium. Media were changed on Day 3. Electrophysiology. Coverslip cultures were transferred to a microscope stage and examined at room temperature under phase contrast optics at 400X magnification. The extracellular recording medium was Eagle’s MEM with 11 mM glucose and 600 nM tetrodotoxin but with no glutamine, serum, or embryonic extracts. The mixture was buffered with 2.2 g/l NaHC03, and pH was maintained between 7.2 and 7.4 by the passage of a 10% C02-90% 02-air mixture over the recording chamber. Patch electrodes, fabricated from borosilicate glass (1.2-mm o.d., with filament, World Precision InstruMATERIALS AND METHODS ments, New Haven, CT), were fire polished and filled Fertilized eggs (Spafas, Roanoke, IL) were mainwith intracellular solution containing 140 mM KCl, 11 tained in an incubator at 3’7°C and humidity of 60-‘70%. mM EGTA-K, 10 mM Hepes-K, 2 mM MgCl, and 1 mlM Embryos were staged according to the method of HamCaC12, pH 7.2. Recording electrodes had resistances of burger and Hamilton (1951). In our studies, the term 2-5 MR. “embryonic day” refers to the corresponding stage, reStandard methods (Hamill et al, 1981) were used to gardless of the actual time of incubation. form high-resistance seals (lo-30 GR) on the neuron soma and to achieve the whole-cell recording configuraCell culture. Isolated embryonic chick ciliary ganglia were incubated in 0.05% (w/vol) trypsin (tpck-treated tion. Macroscopic currents filtered at either 3 kHz or 13 bovine type XIII, Sigma Chemical Co., St. Louis, MO) in kHz were recorded with a Dagan 8900 patch clamp amCa’+- and Mg2+-free PBS (phosphate-buffered saline) at plifier (Dagan Corporation, Minneapolis, MN), and 3’7°C for 20 min (St 29/30, St 34, St 35; Nishi and Berg, were digitized and stored on floppy disks with a 1977), 30 min (St 36, St 38, St 40), or 1 hr (St 44). Ganglia MINC-23 computer (Digital, Merrimack, NH). were centrifuged at 1500 rpm for 10 min, resuspended in Resting membrane potential was measured in curplating medium (see below), and triturated through a rent-clamp mode immediately after entering a cell. fire polished Pasteur pipet to form a single-cell suspen- Series resistances calculated from peak capacitative sion. currents evoked by a lo-mV hyperpolarizing voltage Neurons were plated at a density of 1.0-2.0 X 104/35 step (filtered at 13 kHz) were between 6 and 8 MQ and mm dish (each dish contained two 18-mm glass cover- were not electronically compensated. Capacitance was slips) on lysed fibroblasts or multinucleated myotubes. calculated from the time constant of decay of the curFibroblasts were prepared by repeated passage of cells rent transient: C = ((R, + Ri)/(&*Ri)) *T; Ri = input dissociated from El1 pectoral muscle until a confluent resistance. Capacitance was multiplied by the factor 1 layer of nonmuscle cells remained. The fibroblasts were cm2/1 pF, the reciprocal of specific membrane capacilysed in sterile, distilled Hz0 for 1 hr at 37°C. Muscle tance, to estimate cell size. In addition, cell size was cultures were established with cells dissociated from calculated from the formula for an ellipse rotated about El1 pectoral muscles (Role and Fischbach, 1987). Before its major axis, 27rb2 + (2xab(sin-’ t))/f, where a and b seeding with fibroblasts or myoblasts, glass coverslips are the major and minor axes, respectively, and E, the were coated with adult rat tail collagen and dried in a eccentricity = (a2 - b2)1’2/u. The correlation coefficient humid incubator at 37°C for at least 24 hr. Neurons between surface area estimated by capacitance and were maintained in culture for 3 hr to 4 days in a humid surface area calculated using diameters was 0.8 (n 5% CO2 atmosphere at 37°C. = 117). The slope of the linear regression line was 1.0.

bryonic ciliary ganglion neurons grown in vitro appears to depend on the presence of target tissue in the form of muscle cells (Crean et al, 1982) or muscle cell membranes (Tuttle, 1983). We have measured ACh and GABA responses of neurons freshly dissociated from Stage (St) 29130 (E6-E7) through St 44 (E18) ciliary ganglia. The neurons respond to ACh and GABA at the earliest time examined, and the evoked currents increase dramatically during development. GABA responses increase steadily and in proportion to cell size. ACh-activated currents on the other hand increase in two distinct phases. To examine the effect of the target on the early development of chemosensitivity, we removed the eye cup at St 11-13, 24 hr before the arrival of ganglionic axons. In contrast to axotomy of posthatch ciliary ganglia, early eye removal had only a small effect on ACh and GABA sensitivity of neurons examined at later times. We conclude that the increase in functional receptors on chick ciliary ganglion neurons during development does not depend on target-derived factors.

ENGISCH

AND FISCHBACH

Development of ACh- and GABA-Activated

Voltage-activated potassium conductance (Gx) was evoked in voltage-clamp mode with a step of voltage from -80 mV to +50 mV; Gx was calculated from the maximum slope of the current-voltage relationship. Neurons were voltage-clamped to -50 mV. Unless otherwise stated, 250 pM ACh or 100 pM GABA (Sigma) dissolved in recording solution was applied for 200-300 msec by pressure ejection (5 psi) from micropipets (fabricated as described for patch electrodes) positioned 5-10 pm from the neuron soma (Choi and Fischbach, 1981). We confirmed using methylene blue (Fisher Scientific, Springfield, NJ) that solution does not leak from the pipet. However, the dye was diluted within the electrode tip by capillary action up to a distance of 2-4 pm. Therefore, a pulse was given at least one optic field away from the neuron soma prior to test applications to minimize the gradient between drug and bath solution. Under these conditions, the dye cloud rapidly and completely bathed the neuron soma. ACh and GABA were alternately applied to a neuron. In experiments assaying the concentration dependence of ACh-activated responses, ACh application was alternated with application of bath solution. The rinses between applications of ACh or GABA were required in order to evoke successive currents undiminished in peak amplitude. With equimolar chloride in the intracellular solution and in the bath solution, GABA evoked an inward current. Emhqo surgery. Each fertilized egg to be used for surgery was incubated in a horizontal position for 2 days. Embryos were exposed via a lateral window at St 11-13. India ink (Faber Castell, Lewisburg, TN) diluted 1:30 in PBS was injected beneath the embryonic sac to enhance visualization of the young embryo. Using fine forceps, a small hole was opened in the membrane overlying the optic vesicle. The forming eye cup was loosened from surrounding tissue with the opposing action of two fine insect pins (Landmesser and Pilar, 1974a) and was removed with a suction pipet. A few drops of PBS and penicillin (5000 U/ml) plus streptomycin (5000 pg/ml) were released onto the operated embryo, the hole was sealed with cellophane tape and the egg returned to the incubator in a horizontal position. The procedure took approximately 4 min per egg. Of the operated embryos, 30-50% survived to the desired developmental stages.

Cur conclusions about the development of ACh currents in normal and target-deprived ganglia depend on the assumption that responses of acutely dissociated neurons are representative of responses from neurons in intact ganglia. It might be expected that some time in

419

vitro is needed for neurons to recover normal function following exposure to trypsin and tissue disruption; however, we found that embryonic chick ciliary ganglion neurons responded to ACh and GABA as soon as they could be tested after dissociation. Within the first 3-6 hr, St 36 neurons exhibited a peak current evoked by 250 /.tM ACh of 809 f 53 pA (SEM, n = 42) and a peak current evoked by 100 pM GABA of 4294 f 250 pA @EM, n = 30). Remarkably, transmitter-evoked currents were not larger when neurons were dissociated without prior exposure to trypsin. ACh binding sites on myoblasts are virtually eliminated when the cells are dissociated in the presence of 0.1% trypsin (Smilowitz and Fischbach, 1978). Early experiments showed that ACh-activated currents actually declined with time in culture (Table 1). Twenty-four hours after plating St 36 neurons on myotubes in transferrin medium, the peak ACh-induced current (IA&) was only 50% as large as peak currents observed within the first 4 hr. After 4 days in vitro, IACh was reduced by 70%. Peak currents evoked by GABA (IoAaA) were reduced in the same manner. The loss of responses to ACh and GABA was specific; there was no change in resting membrane potential or in potassium conductance of cultured neurons. The loss of transmitter-induced responses was not prevented when the medium was supplemented with whole chick embryo extract (5%) v/v, 1 experiment), eye extract (5%, v/v, two experiments) or brain extract (1:5 dilution of crude brain saline extraction, seven experiments). The reduction in ACh and GABA responses with time in culture was also observed in neurons dissociated from older embryonic ganglia. In contrast, IACh and IoAsA of dissociated St 29 and St 34 neurons did not decline within 24 hr in culture (data not shown). We did not investigate the age-dependent loss in chemosensi-

ACH-

AND GABA-ACTIVATED

TABLE 1 CURRENTS OF ST 36 NEURONS

DECLINE

WITH TIME IN CULTURE Time in culture 4 hr 24 hr

IACh

(PA) 809 + 53 (43) 437 f 60 (10)

RESULTS

Currents

4 day

236 f 60 (7)

IGABA

(PAI

4294 + 250 (30) 1850 rf:186 (10)

732 31119 (7)

(IF) -48k 1 (43) -50+2

(2) 3 (46) 73 f 6

70 f

(10)

(10)

-46+2 (7)

76 + 8 (7)

Note, Neurons were plated on myotubes in transferrin medium and examined at the indicated timepoints. Both IACh and IGABA were reduced as early as 24 hr in culture and did not recover to initial values even after 4 days in vitro. Resting membrane potential and potassium conductance were unaffected by time in culture.

VOLUME 139, 1990

DEVELOPMENTAL BIOLOGY

420

Cyke

,5 -5 10

-4 10 ACh

-3 10

10

CONCENTRATION

-2

10

(M)

-1

v

L-

FIG. 1. Concentration dependence of ACh-activated currents in freshly dissociated St 36 neurons (A). Bars represent *ISEM; the number of neurons tested at each concentration is shown in parentheses. The error bars for currents evoked by 10 fl ACh, f14 pA, are within the dimensions of the plotted symbol. Typical currents evoked by three different concentrations of ACh are shown in (B). (C) Two superimposed responses evoked by 10 mlM ACh applied at an interval of 2 min show that even at high doses, the same peak current could be evoked repeatedly (under our conditions; see Materials and Methods). Calibration bars: 400 pA, 500 msec.

further. Instead, we recorded responses within 3-6 hr of dissociation to estimate the in viva state. No notable decline in IAch or IGAB* was observed during this interval. Concentration deper&nce of I,,. Concentrations bf ACh from 10 pM to 10 mM were applied by pressure ejection to freshly dissociated, St 36 neurons voltageclamped at -50 mV. Responses that peaked in 100 msec or less were accepted for further analyses; responses which peaked after 200 msec were reduced in amplitude compared to faster responses recorded in the same cell, and were discarded. Following the application of 10 pM ACh, there was a small but consistent current (78.8 f 14, n = 6, two experiments). Peak currents reached a maximum at about 1 mM ACh (1605 + 207 pA, n = 13, three experiments) (Fig. 1A). Typical responses are shown in Fig. lB. Under our conditions (see Materials and Methods), stimuli repeated at 2-min intervals evoked essentially identical responses; even concentrations as high as 10 mMdid not produce a long-lasting desensitization (Fig. 1C). However, to avoid problems due to high levels of ACh building up in the bath, 250 JLMwas chosen as the test dose for all subsequent experiments. IAC~ of ?zeuTotzs dissociated from di&rent embryonic stages.The peak ACh-induced current increased sevenfold between St 29/30 and St 44. The mean values plotted in Fig. 2 show that this rise was composed of two separate steps: an early twofold increase, 403 5 47 pA to 752 f 81 PA, between St 34 and St 35 (P < 0.002, Stutivity

dent’s two-tailed t test) and an additional threefold increase (753 f 66 pA to 2330 f 168 pA) between St 38 and St 44. These steps were separated by a 3-day period of no change. Responses of cells from 3-11 separate experiments were pooled for each time point.

Relationship between the mugn&.de of IACAand cell size. There was a clear increase in the diameters of freshly dissociated ciliary ganglion neurons between St 29/30 and St 36 (Fig. 3). Cell size was quantitated by measuring whole cell capacitance; it was assumed that specific membrane capacitance did not change with embryonic stage. The increases in size during development (Fig. 4) do not account for the observed increases in IAch (Fig. 2). First, capacitance increased only threefold, not sevenfold. Second, capacitance increased smoothly from St 29130 to St 38; there was no period of abrupt increase between St 34 and St 35. Third, cell capacitance was unchanged between St 38 and St 44, an interval marked by a threefold increase in I*ch. Finally, ACh-activated currents were not highly correlated with capacitance on an individual cell basis at any embryonic stage (T = 0.43, n = 134) (Fig. 5). The lack of correlation was especially evident at later stages as the range of cell sizes increased (St 44, filled squares; r = 0.39, n = 19).

I GABAOf neu?vns dissociatedfrom daywent emhymic stages.Currents evoked by 100 pM GABA activated and desensitized more slowly than currents evoked by ACh (Fig. 6). We did not explore the GABA dose response relation but 100 PM GABA is reported to evoke a maximum conductance increase in embryonic chick ciliary

ENGISCH 3000

Development of ACh- and GABA-Activated

AND FISCHBACH

Currents

-

421

St 29ao L.

2500

-f (19)

7 A

2000--

6 25 -

1500--

I

St

36

St

44

-ii----

lOOO--

/

(4.2)

((43)

(27)

(17)&-

--i

(27m-----

25 50001 --

27

29 31 6.5 7

33

(24) s

/

35

37 10

EMBRYONIC FIG. 2. The development embryos at the indicated neurons tested are shown pA, 500 msec.

39 12

41 14 i

43 i,s:

45 ST;:;

k

AGE

of ACh-activated currents with time in ouo. Ciliary ganglion neurons were assayed 3-6 hr after dissociation from stages. Each point represents the mean (bars = &EM) of the peak currents evoked by 250 fl ACh. The numbers of in parentheses. Typical currents evoked in St 29130, St 36, and St 44 neurons are shown at right. Calibration bar: 1000

ganglion neurons (McEachern et ab, 1985). GABA-activated currents increased twofold during the interval examined, but in contrast to ACh-activated currents, the change was gradual; the increase paralleled the increase in cell size, and it was more closely related to cell size (capacitance) on an individual cell basis (Fig. 7). The decrease in mean IoAsA at St 44 (Fig. 6) can be explained by the fact that there are two size classes of

Stage

neurons at this stage (see Landmesser and Pilar, 1974b). Cells of less than 10 pF had small GABA currents (~3 nA), while cells greater than 15 pF had large GABA currents (>5 nA). The low mean in this St 44 sample simply reflects the over-sampling of small cells (12119) compared to large ones (3/19). IACh increased in the absence of embryonic target structures. Ciliary ganglion neurons that developed

Stage

29

FIG. 3. Interference contrast micrographs of ciliary ganglion neurons freshly the change in cell size. The neurons are attached to a substratum of embryonic

dissociated myotubes.

from

36

St 29/30

and St 36 chick

embryos,

to illustrate

DEVELOPMENTALBIOLOGY V0~~~~139,1990

422

15--

01 25

27

29 31 6.5 7

33 8

35

37 10

39 12

41 14

43 18

45 STAGE DAY

EMBRYONIC AGE

FIG.4. The increase in parentheses. Mean St 38.

in cell size with time in ouo measured by membrane capacitance of freshly dissociated neurons increased

without eye structures remained sensitive to ACh, although at each stage examined, the mean IAch was reduced compared to age-matched controls (Fig. 8A). The decrease, apparent as early as St 29/30, was not large (20~50%), and was statistically significant only at St 29/30 (P < 0.05), St 36 and St 38 (P < 0.002). Responses of target-deprived cells from two to seven experiments

capacitance. Bars = GEM. The number of neurons tested are shown threefold from St 29/30 to St 38. There was no further increase after

were pooled for each time point. There was an almost threefold increase in IAch between St 29/30 and St 36, but we did not determine if the increase occurred primarily between St 34 and St 35 as in controls. Strikingly, the large increase in IAch between St 38 and St 44 was still evident in neurons deprived of their targets. Altogether ACh-activated currents of target-deprived neurons increased lo-fold from St 29130 to St 44.

IGABAand cell size were also fleded

by early target

removal. The small effect of target removal was not specific to ACh-activated currents. IoAnA was reduced to a similar extent at St 29/30 and St 36 (Fig. 8B). At later stages, GABA responses of control and target-deprived neurons were not statistically different. The capacitances of target-deprived neurons were also only slightly decreased (St 36, 4.1 + 0.4 pF compared to 6.6 + 0.4 pF for control neurons). DISCUSSION

0

5

10

15 capacitance

20

25

30

35

pF

FIG.5. Relationship between magnitude of ACh-activated currents and capacitance. Values (in PA) of peak ACh-activated currents of neurons from St 29/30 to St 44 are plotted as a function of membrane capacitance. ACh-activated currents did not vary linearly with increasing capacitance. Open circles, St 29/30 and St 34; open triangles, St 35; open squares, St 36; open inverse triangles, St 38; open diamonds, St 40. St 44 points are shown with filled squares for emphasis; St 44 neurons as a population were not larger than St 38 and St 40 neurons but had ACh-activated currents that were greater than most responses from these earlier stages.

We examined freshly dissociated neurons from ciliary ganglia of different embryonic stages and found that ACh-activated currents increased sevenfold in two distinct phases between St 29/30 and St 44. The large increase in I Ach IS a probably due to both an increase in receptor number and an increase in receptor function. The number of [‘251]mAb35 binding sites increases several-fold between ES (St 33) and El8 (St 44) (Smith et al., 1985). The increase in [‘251]mAb35 binding does not exactly parallel the change in ACh sensitivity measured in this study: the number of sites increased gradually rather than in two phases and the rise was complete by

ENGISCH

Development of ACh- and GABA-Activated

AND FISCHBACH

423

Currents St

6000

-ii----

T

5000

29/30

St

36

St

44

--ii--

t

L/-----

s 3 $ H

3000 , 2000 4000

2

1

1000 1

04 25

27

29 31 6.5 7

33 6

35 37 39 41 9 10 12 14

EMBRYONIC

AGE

43

16

45

STAGE DAY

I

FIG. 6. The development of GABA-activated currents with time in ouo. Bars = HEM of the peak currents evoked by 100 pM GABA. The numbers of neurons tested are shown in parentheses. Typical currents evoked in St 29/30, St 36, and St 44 neurons are shown to the right. At St 44 there were two readily distinguishable classes of responses, large and small. An example of each is shown. Calibration bar: 2 nA, 509 msec.

St 38 rather than St 44. These disparities may simply reflect the fact that binding of [lZI]mAb35 to cell homogenates was measured, including intracellular as well as surface sites. In the chick ciliary ganglion, surface mAb35 binding sites are only a small fraction of the total (Jacob et aZ., 1986). It has recently been reported that the number of surface [1251]mAb35 sites per neuron increases about 12-fold between ES and El6 (Margiotta and Gurantz, 1989). In addition, the mean

15 1

i 0

5

10

15 capacitance

20

25

30

35

pf

FIG. 7. Relationship between magnitude of GABA-activated currents and capacitance. Values (in nA) of peak GABA-activated currents from St 29/30 to St 44 are plotted as a function of membrane capacitance. Symbols refer to same stages as in Fig. 5. In general, GABA-activated currents increased with increasing capacitance. Values at St 44 are shown with filled squares for emphasis. St 44 GABA responses were within the range of responses from younger neurons.

ACh channel open time increases several-fold over the same developmental period (Margiotta and Gurantz, 1989). There are two reasons to suspect that the fold increase in IAch might be even larger than measured here. First, peak currents were not corrected for series resistance errors. Larger currents would have produced greater voltage drops across R,, and thus the peak would have occurred at a voltage more depolarized than Vhold. Second, in the present study it is possible that a maximal dose was applied at early stages and a submaximal dose at later stages. In a recent study it was found that the maximal dose of ACh increased from 100 PLM at E8,9 to about 500 PIM at E13,14 (Margiotta and Gurantz, 1989). However, we found with our application techniques that the maximal dose at El0 (St 36) is as high as 1 mM. It may be that 250 pM is submaximal at all stages examined. However, an increase in ECW with developmental age would result in a smaller fraction of the total receptors being activated at later stages. The variation in whole-cell ACh-activated currents evident at each stage of development cannot be explained by differences in cell size. In contrast, mean GABA-activated currents increased in parallel with mean cell size, and IoAsA was more closely related to size on an individual cell basis. The lack of correlation between IACi, and capacitance (size) is a strong indication that ACh receptors are not distributed at a fixed density in the membrane throughout development. Receptor clusters have been identified on El6 and El? chick ciliary ganglion neurons and adult frog autonomic neurons, where they are in close apposition to identified

424

DEVELOPMENTALBIOLOGY VOLUME139,199O

.-

A

3000

-*NORMAL l

DEVELOPMENT

NO TARGET

2500

I

25

27

29 6.5

31 7

33 8

35

37

39

10

EMBRYONIC

12

a1

-* NORMAL .

4000--

2

3000--

gz

2000--

.

STAGE DAY

DEVELOPMENT

NO TARGET

-1’

f/

(11, 1000

45 18

AGE

.-

‘;i 2

43

14

-DJ 25

27

29 6.5

i

31 7

33 8

35

EMBRYONIC

37 10

39 12

41 14

43

45 18

STAGE DAY

AGE

FIG. 8. The effect of early target removal on ACh (A) and GABA (B) currents. ACh- and GABA-activated currents were only moderately reduced by early target removal. Bars = +SEM; the numbers of neurons tested are shown in parentheses. The error bars for ACh currents of St 29/30 target-deprived neurons, +38 pA, are within the dimensions of the plotted symbol. The small symbols and dotted lines indicate the normal, unoperated developmental curves (same data as Figs. 2 and 6). Responses of neurons from contralateral control ganglia were not different from normal, age-matched control responses and so all control data were pooled.

presynaptic specializations (Harris et al., 1971; Marshall, 1981; Jacob et al, 1984; Loring and Zigmond, 1987; Sargent and Pang, 1989). It may be that IAch is determined by the number of receptors clustered under presynaptic terminals, and that the number of terminals is independent of cell size. In the adult Xenqpus cardiac ganglion, the number of presynaptic boutons is directly related to cell size (Sargent, 1983), but this might not be the case in developing chick ciliary ganglia. In the chick, after El0 there are two types of presynaptic terminals on ciliary ganglion neurons: large, calciform terminals

on ciliary neurons, and bouton-like terminals on choroid neurons (Landmesser and Pilar, 1972). This heterogeneity may have obscured a relationship between cell size and nerve terminal number (and number of receptor clusters). ACh-activated responses of target-deprived neurons were reduced compared to responses of age-matched controls. Strikingly, the effect of target removal on embryonic neurons was far less than that induced by axotomy of posthatch ciliary ganglia (Brenner and Martin, 1976; Jacob and Berg, 198’7). Furthermore, the reduction

ENGISCH AND FISCHBACH

Development of ACh- and GABA-Activated

was not specific to ACh responses; both IGAB* and cell size were decreased by the same small percentage. However, we do not think that the reduction in IACh is due to the decrease in cell size because cell size does not determine ACh current magnitude in normal development. Following early target removal, over 80% of the ganglionic neurons die between St 33 and St 40, compared to 50% in normal ganglia (Landmesser and Pilar, 1974a). It is unlikely, for several reasons, that the lack of a large effect of target removal on ACh sensitivity was due to the selective loss of relatively insensitive cells. First, ACh-activated responses were reduced at St 29/30, at least 24 hr prior to the onset of cell death. Second, we did not see a unique population of insensitive neurons at early embryonic ages. Third, axotomy of posthatched ganglia also results in a loss of cells compared to controls (37%) (Jacob and Berg, 1987), yet the remaining neurons display a profound reduction in ACh receptors. The relatively large effect of target removal seen in posthatch neurons indicates that ACh receptor expression becomes more dependent on target-derived factors with time after initial contact. Alternatively, axotomy itself, rather than isolation from the target, precipitates the loss in ACh receptors. When the eye is removed on E2, ganglionic axons are not severed (Cowen and Wenger, 1968; Landmesser and Pilar, 1974a). We cannot rule out the possibility that ACh sensitivity is maintained because surviving neurons innervate ectopic structures. It is very likely that ACh receptor-inducing activity is not exclusively contained in eye tissue but is present throughout the environment surrounding the neurons in situ. It is interesting to speculate on what events underlie the two step increases in IACh during development. Neither phase coincides with the initial period of synapse formation in the ganglion. Synaptic transmission, measured as the ratio of ganglion population action potentials following orthodromic and antidromic stimulation, is first detected at St 26; (E5); the proportion of transmitting cells in the ganglion reaches 100% by St 33 (E8) (Landmesser and Pilar, 1972). We measured relatively little change in ACh sensitivity until St 35,24 hr after the establishment of 100% ganglionic transmission. The plateau in the increase in ACh-activated currents between St 35 and St 38 is concurrent with the period of normal cell death. The second and larger increase in IACh begins toward the end of the cell death period and continues until at least St 44. This late rise is undiminished in target-deprived neurons; therefore it cannot depend on initial target contact. As discussed above, receptors observed on late embryonic chick ciliary ganglion neurons are closely associated with presynaptic release sites. It seems likely that the increase

Currents

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between St 38 and St 44 is associated with an IACh elaboration of synaptic contacts on those neurons that have survived the period of cell death (cf. Lichtman, 1977). Changes in the number and distribution of ACh receptors following denervation of adult autonomic ganglia are small and variable (Kuffler et cd., 1971; Dunn and Marshall, 1985; Jacob and Berg, 1987, 1988; Sargent and Pang, 1988; McEachern et cd, 1989). However, the contribution of innervation to the increase in ACh receptors on developing chick ciliary ganglion neurons has not yet been determined. We are currently exploring this question by early removal of the accessory oculomotor nucleus, the source of innervation of the ganglion. in

The authors are indebted to Ms. Cindy Nettrour for advice and assistance with tissue culture techniques. We thank Dr. Jay Yang, who took part in the initial experiments. This work was supported by a grant from NINCDS, NS18458 (G.D.F.), and a Systems and Molecular Neurobiology training grant, GM08151 (K.L.E.). REFERENCES BADER, C. R., BERTRAND, D., and KATO, A. C. (1982). Chick ciliary ganglion in dissociated cell culture. II. Electrophysiological properties. Dez. Biol. 94,131-141. BOYD, R. T., JACOB, M. H., COUTURIER, S., BALLIVET, M., and BERG, D. K. (1988). Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia. Neuron 1,495-502. BRENNER, H. R., and MARTIN, A. R. (1976). Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells. J. P&&d (L.ondon) 260,159-175.

CHOI, D. W., and FISCHBACH, G. D. (1981). GABA conductance of chick spinal cord and dorsal root ganglion neurons in cell culture. J. Newophysd 45,605~643. COWAN, W. M., and WENGER, E. (1968). Degeneration of the nucleus of origin of the preganglionic fibers to the-chick ciliary ganglion following early removal of the optic vesicle. J. I&p. Zool. 168,105-B&t. CREAN, G., PILAR, G., TUTTLE, J. B., and VACA, K. (1982). Enhanced chemosensitivity of chick parasympathetic neurons in co-culture with myotubes. J. PhysioL (London) 331,87-104. DUNN, P. M., and MARSHALL, L. M. (1985). Lack of nieotinic supersensitivity in frog sympathetic neurons following denervation. J. Physiol (London) 363,211-225. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B., and SIGWORTH, F. J. (1981). Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. PJluegers Arch. 391,85-100. HARRIS, A. J., KUFFLER, S. W., and DENNIS, M. J. (1971). Differential chemosensitivity of synaptic and extrasynaptic areas on the neuronal surface membrane in parasympathetic neurons of the frog, tested by microapplication of acetylcholine. Proc. R. Sot. London B 177,541-553. JACOB, M. H., and BERG, D. K. (1987). Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion. J. Cell BioL 105,1847-R%& JACOB, M. H., and BERG, D. K. (1988). The distribution of acetylcholine receptors in chick ciliary ganglion neurons following disruption of ganglionic connections. J. Neurosd 83838-3849.

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The development of ACH- and GABA-activated currents in normal and target-deprived embryonic chick ciliary ganglia.

We have examined the expression of functional ACh and GABA receptors on embryonic chick ciliary ganglion neurons between Stages (St) 29 and 44 (Embryo...
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