Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Neuron.

Vol. 3, 597-607,

November,

1989, Copyright

0 1989 by Cell Press

Functional Properties and Developmental Regulation of Nicotinic Acetylcholine Receptors on Embryonic Chicken Sympathetic Neurons Brenda 1. Moss, Stephen M. and lorna W. Role Department of Anatomy and Center for Neurobiology and Columbia University College and Surgeons New York, New York 10032

Schuetze, Cell Biology Behavior of Physicians

Summary Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity. Introduction The establishment of transmitter sensitivity is a crucial aspect of synaptic development. Studies of the neuromuscular synapse suggest that innervation plays an important role in this process, in part by regulating transmitter receptor number and distribution (reviewed in Poo, 1985; Salpeter and Loring, 1986; Merlie and Sanes, 1986; Steinbach and Bloch, 1986; Laufer et al., 1989). Innervation of embryonic muscle induces an increase in acetylcholine (ACh) sensitivity at the site of nerve-muscle contact. In addition, the response to transmitter is further modified by subsequent changes in the channel properties of the acetylcholine receptor (AChR): the single-channel conductance increases while the apparent mean channel open time decreases (reviewed in Schuetze and Role, 1987; Brehm and Henderson, 1988). As with muscle, the ACh sensitivity of sympathetic neurons is enhanced by innervation both in vitro (Role, 1988) and in vivo (L. M. Marshall, personal communication; D. C. Valenta and L. W. Role, unpublished data). The observed increase in ACh sensitivity following innervation may be the result of an increased number offunctional nicotinic AChRs on the cell surface. Alternatively, there may be changes in AChR channel properties, leading to a net increase in ACh-induced conductance. Clearly these mechanisms are not mutually exclusive.

We have investigated the possibility that changes in channel properties accompany the enhanced ACh sensitivity observed following innervation of sympathetic neurons. To address this question, we have compared ACh-activated single-channel currents in neurons isolated from chicken lumbar sympathetic chain ganglia on embryonic day (ED) 10, before innervation in vivo, with currents in neurons removed on ED17. By this age, there is a substantial increase in the synaptic input as indicated by the number of synaptic boutons, both in an electron microscopic study (Hruschak et al., 1982) and in experiments with dil injection into the preganglionic nucleus (B. L. Moss and L. W. Role, unpublished data). Results The results presented here are based on 156 patches from individual neurons in 40 separate platings. ACh Activates Three Distinct Channel Amplitudes in ED10 Neurons In neurons isolated from ED10 chickens, ACh activates three distinct amplitude classes of channels as shown in Figure 1A. This figure shows nonconsecutive traces from one cell-attached patch recording (1 @I ACh, patch membrane potential = -50 mV relative to rest potential). The record illustrates channel openings of all three amplitude classes, which we denote ST0 (for the smallest amplitude class at EDlO), MI0 (for the mediumsized amplitude class at EDlO), and LIO (for the largest amplitude class at EDlO). Figure 1B is an amplitude histogram from the same recording. The values of all points in the sampled portion of the record are plotted to provide an unbiased estimate of the single-channel amplitudes. The smallest amplitude component with a mean of 0 pA represents the baseline noise. The three remaining peaks indicate three amplitude classes. Superimposed on the histogram is the sum of three Gaussian curves with means it SD of 2.1 k 0.2 pA, 3.8 f 0.3 pA, and 5.2 f 0.2 PA. These three amplitude classes most likely represent three different AChR subtypes. Each class has been observed in isolation; that is, we have recorded from cellattached patches containing just SIO, Ml,,, or L,,, openings. It is unlikely that the three amplitudes are different conductance states of a single-channel type, since in patches with more than one amplitude, there were no transitions from one amplitude to another. It is also unlikely that the larger amplitude events are due to simultaneous openings and closings of smaller amplitude channels, since the rate of channel activation is so low (summed frequency of opening for all ACh-activated channels = 1 opening per s, 2.5 uM ACh). Thus the probability of two channels simultaneously opening and closing is negligible. In fact, overlapping events (doublets) are infrequently observed in patches from ED10 neurons.

Neuron 598

Figure 1. ED10 Neurons nel Amplitude Classes

ho

-80

-40

-60

-20 -1

-2 -3 -4 -5 0

2

4

--6

6

I(pA)

Have

Three

Chan-

(A) Nonconsecutive traces of a cell-attached patch recording from an ED10 neuron showing the three amplitude classes, Slu, Mro, and L,o. The traces were digitally low-passfiltered at 2 kHz. ACh concentration = 1 pM. Patch potential = -50 mV relative to rest. Bars = 7.5 PA, 5 ms. (B) Amplitude histogram of the record in (A) showing that the three classes give rise to three peaks in the distribution. Superimposed on the histogram is the sum of three Gaussian curves with means (* SD) of 2.1 * 0.2 pA, 3.8 f 0.3 PA, and 5.2 f 0.2 pA. K) Mean current amplitude plotted against potential (patch potential relative to rest potential) for a different cell-attached patch. In this example, SIO, MID, and LIO had slope conductances of 15 pS, 34 pS, and 48 pS, respectively.

1Cp.V

Several lines of evidence indicate that each of the three amplitude classes is ACh-activated. First, using the outside-out patch clamp technique, channel openings were never observed before the application of ACh. The subsequent application of 1 uhl ACh to the patch with a pressure ejection pipette resulted in channel openings corresponding to each amplitude class. The results of one such experiment are shown in Figure 2. The top three traces are segments from a 2 min recording ob-

l@fACh

SlO

Ml0

ho --I

Figure 2. The Three Activated

Amplitude

Classes in ED10 Neurons

Are ACh-

(Top) Nonconsecutive traces from a 2 min outside-out patch recording before the application of ACh. No channel openings were observed. (Bottom) Nonconsecutive traces from a 5 min recording from the same patch during the continuous application of ACh with a pressure ejection pipette. Single-channel currents were observed immediately following the start of ACh application, and channel activity persisted for the duration of the recording. This patch contained three amplitude classes with means of approximately 2.1 PA, 3.1 PA, and 4.2 pA;corresponding to S,,,, MIo, and LIO. The opening frequencies of the three classes are as follows: S,,,, 0.1 openings per s; Mro, 0.1 openings per s; and LlO, 0.05 openings per s. Membrane potential = -90 mV. ACh concentration = 1 $vt. The records were digitally low-pass-filtered at 2 kHz. Bars = 7.5 PA, 5 ms.

tained before the application of ACh; no channel openings were observed. The bottom traces are nonconsecutive segments of a 5 min recording obtained during the application of 1 ptvI ACh, demonstrating that each class is seen in this configuration. The record shows three amplitude classes of channels that closely match those observed in cell-attached patches. For example, in the patch shown in Figure 2, the three classes had amplitudes averaging 2.1 PA, 3.1 pA, and 4.2 PA, respectively, at a membrane potential of -90 mV. These correspond to the mean current amplitudes for SIO, Mlo, and L,,, seen in cell-attached patches. Additional observations suggest that all three amplitude classes represent AChactivated channels. First, in cell-attached patches, these amplitude classes are observed only when ACh is included in the patch pipette. Second, each amplitude class desensitizes at high ACh concentrations (>5 NM). Channel activity persists for the duration of the recording in both outside-out (S-10 min recordings) and cellattached patch configurations with lower ACh concentrations (l-5 PM). Third, we observed all three classes in both patch configurations when calcium was omitted from the external ACh-containing solution, indicating that none of the observed amplitude classes is activated by calcium entry into the cell. Finally, preliminary results indicate that all three amplitude classes are blocked by hexamethonium (100 PM), a specific nicotinic antagonist. The same three classes of ACh-elicited channels are also seen on neurons dispersed without enzymatic treatment and assayed l-5 hr later (data not shown). Thus, these conductance classes are quite likely normally expressed in vivo. In many cell-attached patches, we observe a class of channel openings that does not require ACh for activation. However, these events are readily distinguished from the ACh-activated channels since the single-channel current amplitude is >2-fold larger than any of the ACh-elicited channels. Furthermore, this channel is not observed when calcium is omitted from the patch pi-

Developmental 599

Changes

pette, nor in either patches.

in Neuronal

Nicotinic

outside-out

AChRs

or inside-out

excised

The ACh-Activated Channel Classes Can Be Distinguished by Slope Conductance Representative steady-state current-voltage plots from one patch are shown in Figure 1C. Varying the patch potential from -30 mV to -70 mV relative to rest potential resulted in ohmic changes in the single-channel current amplitude for each class. Linear regression of these data yielded slope conductance values of 15 pS, 34 pS, and 48 pS. To determine the mean slope conductance for each class, we constructed a histogram of all conductance values obtained from 16 patches (see Figure SA). Mean conductances were calculated by averaging values from 9-20 pS, 17-36 pS, and 41-60 pS for SIO, Mro, and l-10, respectively. This yielded mean slope conductance values (* SE) of 14 f 1 pS, 27 f 1 pS, and 49 f 1 pS (Table 1). We also estimated the reversal potential for each class by extrapolating to the potential corresponding to zero current and then adding the mean rest potential (-55 mV). We found that both MI0 and LIO have extrapolated reversal potentials of approximately 0 mV. The extrapolated reversal potential for SIO is considerably more positive (approximately +70 mV, if the current-voltage relationship is truly ohmic). However, the current-voltage curve for S10 may rectify at more positive potentials not examined in these experiments, StO, kilo, and LIO Differ in Their Frequency of Occurrence To determine the relative frequency of occurrence of the three channel classes, we scored each patch forthe presence of S,,,, Mlo, and L10 (see Experimental Procedures). It is important to note that this analysis reveals the relative frequency of appearance of a given channel class over all the patches examined rather than differences in the frequency of channel opening between the classes. In fact, the opening frequencies of the three classes are comparable (each class =: 0.05-1.0 opening per s at 2.5 PM ACh). Analysis of the relative frequency of occurrence of each channel class indicates that MI0 is the most common conductance class, occurring in about 83% (25/30) of the patches. SIO, which is found in 63% (19/30) of the patches, is slightly more common than LIO, which is observed in 50% (15/30) of the patches. These differences

Table 1. Comparison and ED17 Neurons

of Mean

Slope

Conductances

Mean Slope Conductance Amplitude 5 M L

Class

f

in ED10

SE (pS)

ED10

ED17

14 * 1 27 + 1 49 f 1

23 f 38 f 50 f

1 1 1

in the relative frequency of occurrence may be due to differences in the number of channels of each class present on the cell surface and/or differences in ACh sensitivity. It should be noted, however, that each channel class makes a robust contribution to the observed channel activity, in contrast to previous reports on other neuronal AChRs (see Discussion). The Three Conductance Classes Differ in Mean Burst Duration Figure 3 shows representative burst duration distributions for each conductance class at EDlO. The distributions for both SIO and MI0 are best fit by double exponentials, with time constants of 1.5 and 7.6 ms for SIO, and 1.5 and 12.0 ms for MI0 (23”C, patch potential = -50 mV relative to rest). The faster components account for 35% and 63% of the area of the burst duration distribution for SIO and MIo, respectively. In contrast, the burst duration distribution of L,o is fit well by a single exponential with a time constant of 3.0 ms. Table 2 gives average mean burst duration values for each conductance class. It should be noted that, in each distribution, it is likely that there are still faster components below our limit of resolution (minimum resolvable open time = 0.5 ms). Developmental Changes in Channel Properties Prior results indicate that the ACh sensitivity of sympathetic neurons increases following innervation. To see whether changes in channel properties contribute to the enhanced ACh sensitivity, we compared the AChRs in neurons isolated on EDlO, prior to innervation, with those in neurons removed on ED17, during the first major wave of synapse formation in chick lumbar ganglia. We found several changes during this period in AChR channel properties consistent with the observed increase in transmitter sensitivity. An Additional Amplitude Class Is Observed in Neurons at a later Stage of Development In neurons isolated on ED17, in addition to the three ACh-activated amplitude classes (now denoted SIT, Mr7, LIT), about 8% of the patches contain a fourth, even larger amplitude class (denoted XL). Figure 4A shows nonconsecutive traces from a single cell-attached patch recording from an ED17 neuron, illustrating all four amplitude classes (2.5 PM ACh, patch potential = -50 mV relative to rest). Figure 48 is the amplitude histogram from the same patch, showing that XL gives rise to a fourth peak in the amplitude distribution. Superimposed on the histogram is the sum of four Gaussian curves with means (* SD) of 1.7 + 0.2 pA, 2.9 + 0.3 PA, 4.4 f 0.3 PA, and 7.0 f 0.3 pA. The XL amplitude class may represent a new AChR subtype. However, unlike S, M, and L, each of which has been observed in isolation, XL has always been observed accompanied by one or more of the other classes. Therefore, it is theoretically possible that XL represents the simultaneous opening and closing of smaller amplitude channels. For example, simultaneous openings of

Neuron 600

MI7 and L,, would give rise to openings with approximately the same amplitude as XL. However, the probability

S10 Burst duration

0

6

12

18

(mscc)

24

30

M 10 (msec)

Burst duration

0

6

12 18 24 Burst duration (msec)

30

Burst duration (msec) 3. ED10 Burst

Duration

Distributions

2. Comparison

Amplitude Class

S" M L XL

of Burst Short

Burst

Durations Duration

between

ED10

and

ED17

Long ED17

1.5 (35%) 1.7 * 0.2 (72% 3.4 * 0.5 Not present

1.1 1.7 3.2 2.5

f 7%)

Values represent the mean * SE. Figures in parentheses a For SlO and 517, the values are based on one burst

(40%) f 0.1 f 0.4 f 0.0

independent

channels

Neurons

(ms)

ED10

for

is small

The Slope Conductances of the Smaller and Medium Channel Classes increase A second change in channel properties during development is an increase in the slope conductances of the smaller and medium channel classes. This can be seen by comparing the ED10 and ED17 conductance histograms (compare Figures 5A and 58). As described ear-

Representative burst duration distributions. The distributions for SIO and Ml0 are fit by double exponentials. In contrast, the distribution for Llu is fit well by a single exponential. Each inset shows the initial part of the distribution on a faster time scale. Dashed bins include events greater than the maximum burst duration indicated. Patch potential - -50 mV relative to rest potential. Time constants (and percentage areas) are as follows: S,O, 1.4 ms (35%) and 7.6 ms (65%); Mlo, 1.5 ms (63%) and 12.0 ms (37%); LlO, 3.0 ms.

Table

occurring

XL Differs in Slope Conductance and Burst Structure XL can also be distinguished from the other classes based on slope conductance, as shown in the current-voltage plot for all four classes of ACh-activated channels in ED17 neurons (Figure 4C). In this example, XL has a slope conductance of 64 pS. The mean slope conductance value (+ SE) for XL is 69 f 6 pS (Table 1). Combined data from three patches yielded an average apparent mean open time (it SE) of 2.5 * 0.8 ms (23OC, patch potential = -50 mV relative to rest; see Experimental Procedures for details). XL openings have a distinctive burst structure; at a filter frequency of 2 kHz, we usually do not observe brief closures or gaps during openings of the other three channel types. In contrast, XL openings are typically interrupted by brief closures (Figure 4A).

1 lo

Figure

of this

because of, the low frequency of channel opening. For example, in one patch containing XL, the frequencies of channel opening for MI7 and L17 were only ~0.2 openings per s and ~1.4 openings per s, respectively, which predicts a negligible probability for their simultaneous opening and closing. Furthermore, in this patch, the frequency of XL openings matched that of MI,. Also, if XL comprised simultaneous openings of smaller channels, one would expect the openings or closings of XL events to have step transitions; however, XL events typically opened and closed smoothly. Several lines of evidence suggest that the XL class is ACh-activated. First, in outside-out patches, XL has been observed only during the application of ACh (data not shown). Secdnd, in cell-attached patches, XL was seen only when ACh is included in the patch pipette. Finally, XL has been obgerved when calcium was omitted from the external ACh-containing solution, suggesting that XL is not actiiated by calcium entry into the cell.

(33% (61%

* 13%) f 11%)

indicate percentage duration distribution

Burst

Duration

(ms)

ED10

ED1 7

7.6 13.0 * 1.0 No long burst Not present

7.0 10.8 * 0.4 16.6 * 3.1 No long burst

areas of the fast components. at each age.

Developmental 601

Changes

in Neuronal

Nicotinic

AChRs

Figure 4. An AdditIonal Amplitude Observed in ED17 Neurons

-80

-60

-40

The Relative Frequency of Occurrence of the Three Conductance Classes Changes In ED10 neurons, as described earlier, we found that SIO, MIo, and LIO are found in 63%, 83%, and 50% of the patches, respectively. When ED17 patches were scored for the presence of S,,, M17, and L1, (based on current amplitude at a patch potential of -50 mV relative to rest potential), we found that the two larger classes, MI7 and L,,, predominate. MI7 occurs in about 57% (17/30) of the patches, and L17 occurs in about 67% (20/30) of the patches. S17 is the least common class; it is seen in about 30% (10/30) of the patches. The Larger Conductances Become More Predominant in ED17 Neurons A consequence of the increase in slope conductances of the two smaller classes and the change in relative fre-

1s

(A) Nonconsecutive traces of a cell-attached patch recording from an ED17 neuron, illustrating SIT, MI,, L,,, and the new XL class. The traces were digitally filtered at 1.5 kHz. ACh concentration = 2.5 PM. Patch potential = -50 mV relative to rest potential. Bars = 7.5 pA, 5 ms. (B) Amplitude histogram of the record in (A) showing XL as a fourth peak in the distribution. Superimposed on the histogram is the sum of four Gaussian curves with means (f SD) of 1.7 f 0.2 pA, 2.9 f 0.3 PA, 4.4 + 0.3 PA, and 7.0 f 0.3 PA. (C) Mean current amplitude plotted against potential (patch potential relative to rest potential). In this example, S17, M,:, L,,, and XL had slope conductances of 23 pS, 40 pS, 50 pS, and 64 pS, respectively. Each currentvoltage curve was taken from a separate cellattached patch since only approximately 5% of the patches on ED17 neurons contain all four conductance classes.

-20

7.,

lier, the mean slope conductance of SIO is 14 f I pS. In contrast, the mean slope conductance of the smallest class of channels seen at ED17 (called S17), which was calculated by averaging all values from 13-32 pS, is 23 f 1 pS. This represents an increase of about 64%. Similarly, the mean slope conductance for MI0 is 27 * 1 pS, whereas the mean conductance for MI,, which was taken as the average of all values from 29-44 pS, is 38 f 1 pS (an increase of approximately 41%). The mean slope conductances of L10 and L,, are not significantly different. The mean conductance for L10 is about 49 f 1 pS, and the mean conductance for L1,, which was calculated by averaging values from 45-56 pS, is 50 f 1 pS. These differences in slope conductance are probably not due to changes in the rest potential and/or input impedance of cells at the two different stages, since there is little change in either (V, =: -50 to -55 mV; Ri” =: 0.3-0.5 GSZ at ED10 and ED17).

Class

quency of occurrence of the three classes, is that the relative contribution of the larger conductance events increases with development. Figure 6 is a histogram in which the distribution of conductance values for each age are compared. From ED10 to ED17, the distribution shifts such that the percentage of conductances falling in the 10-25 pS range decreases from 46% to 26%, whereas the percentage of conductances in the 50-65 pS range increases from 14% to 32%.Thus, later in development, following innervation, the larger conductances become more predominant. The Burst Duration Distribution of L17 Contains a Second, Slow Component A fourth change in AChR channel properties is that the L conductance class acquires a second, slow component in its burst duration distribution. The burst distribution of LIo, as described earlier, is well fit by a single exponential with a mean burst duration of 3.4 ms. In contrast, the burst duration distribution of L17 is best fit by a double exponential. This is shown in Figure 7, which includes a representative burst duration distribution for L,,. The superimposed exponential fit has time constants of 3.8 and 22.3 ms; the slow component accounts for 24% of the area (23”C, patch potential = -50 mV relative to rest). Combined data from three experiments indicate that the mean time constants (* SE) for L,, are 3.2 f 0.4 ms and 16.6 f 3.1 ms (Table 2). The mean percentage of the slow component is 39% f 11%. It is unlikely that a significant contribution of a slower component escaped detection in records from ED10 neurons since comparison of burst duration distributions with the same number of events at both developmental stages yields the same result. In each case, the ED10 distribution is fit well by a single exponential, whereas

ponential

the

ED17

distribution

fit to the data.

requires

a double

ex-

Neuron

602

B I 74

ED 10 8 ,

.

s- 40-

I

fj . -ii 302 . 3 3k 202 3 loE-" & OIO-25 Conductance (pS)

Figure during

6. The Larger Development

30-45 SO-65 Conductance (pS)

Conductances

Become

More

Predominant

For EDlO, 37 conductance values were obtained from 17 patches. For ED17, 31 conductance values were obtained from 22 patches. The histogram includes only patches in which a slope conductance could be determined for all channel classes present.

B 6 Lo a w 2 4 s _m

ii 2

0

Figure 5. The Mean Slope Conductances dium Channel Classes Increase

of the Smaller

and Me-

(A) Histogram of 39 slope conductance values obtained from 16 patches. The mean conductances (arrows) for Sre, M,e, and L,e are 14 * 1 pS, 27 k 1 pS, and 49 f 1 pS, respectively. These means were calculated by averaging all values from 9-20 pS for So, 17-36 pS for M,e, and 41-60 pS for Lre. (B) Histogram of 42 slope conductance values obtained from 25 patches. The mean conductances (arrows) for S,,, MI,, and Lt7 are 23 f 1 pS, 38 f 1 pS, and 50 f 1 pS, respectively. These means were determined by averaging all values from 13-32 pS for S,,, 29-44 pS for MI,, and 45-56 pS for L,,. Only slope conductance determinations based on a minimum of three holding potentials were included in each histogram. Some conductance values were obtained from patches in which a slope conductance could not be determined for all channel classes present. Therefore, these histograms do not reflect the relative proportions of the conductance classes.

The Percentage of Patches With Just One Amplitude Class Increases Nearly 90% of the cell-attached patches on ED10 neurons contain more than one amplitude class. In these

patches, a given channel class has an equal probability of being associated with either of the other two classes. In contrast, examination of patches from ED17 neurons revealed a 4-fold increase in the number of patches with only one amplitude class. It should be noted that the duration of recordings used for this analysis would have reliably revealed the presence of a channel with an opening frequency as low as 0.01 opening per s, which is TO-fold lower than the lowest non-zero channel opening frequency detected. As shown in Figure 8, 10% (4/44) of the patches from ED10 neurons have just one channel amplitude class, whereas approximately 40% (18/46) of the patches from ED17 neurons have a single amplitude class. Of these “single-class” patches, about 95% contained exclusively MI7 or L,r, with each constituting about one-half of these patches. Discussion This study provides the first description of ACh-activated channels in embryonic sympathetic neurons as well as developmental changes in AChR channel properties coincident with innervation. The array of channel types and developmental changes in channel function seen are particularly striking in view of recent molecular studies demonstrating extensive diversity in neuronal ACh receptor subunit clones. Properties of ACh-Activated Channels on Neurons before Innervation In Viva Early in development of the sympathetic neurons (before significant synaptic input in vivo; Hruschack et al., 1982), ACh activates three distinct channel classes based on their relative mean slope conductances. This is in contrast to that found in most other avian and mam-

Developmental 603

Changes

in Neuronal

Nicotinic

AChRs

$lZO-

8A 90! 60s 3

Burst duration

(msec)

$ 30& 0

6 Burst

12 18 24 duration (mm)

30 --

70

ED 10

8% p ?xs sy 14 &O 9,. u. 0 4

56

M l7

42 28

T !i 8

12

Burst duration

YiL 0

8

16

20

(msec)

24 32 16 Burst duration (msec)

40

ED 17

Figure 8. The Percentage of Patches Increases During Development

erties ported 6

12

18

Burst duration

0

8

24

30

(msec)

16 24 32 Burst duration (mm)

40

Figure 7. While the Time Constants for the Sand M Classes Do Not Change during Development, the Burst Duration Distribution of the L Class Acquires a Second, Slow Component Representative burst duration distributions. The superimposed line on each distribution shows the fit of two exponentials. Each inset shows the initial part of the distribution on a faster time scale. Dashed bins include events greater than the maximum burst duration indicated. Patch potential = -50 mV relative to rest potential. Time constants (and percentage areas) are as follows: $7, 1.1 ms (40%) and 7.0 ms (60%); M,r, 1.7 ms (45%) and 10.4 ms (54%); L,,, 3.8 ms (76%) and 22.3 ms (24%).

malian neurons; in each case, a single conductance class predominates. For example, ACh activates primarily one conductance class in chicken ciliary ganglion (Ogden et al., 1984; Margiotta et al., 1987) and in rat thoracic and superior cervical sympathetic neurons (CullCandy and Mathie, 1986; Mathie et al., 1987; Derkach et al., 1987). Similarly, in the CNS, ACh activates primar-

Class

ily one conductance class in hippocampal, brain stem, and medial habenula neurons (Arcava et al., 1987; C. Mulle and J. P Changeux, personal communication). In most of these preparations, a second amplitude class was noted; however, these channels were relatively rare

lfune and Steinbach, 1989, The diversity in neuronal

r$ ’ 0

Amplitude

Percent of patches that have a single amplitude class versus embryonic age. In ED10 neurons, only about 10% (4/44) of the patches have just one amplitude class. In ED17 neurons, however, almost 40% (18/46) of the patches contain a single amplitude class.

and not clearly ACh-activated. PC12 cells, has been reported class of AChR (Bormann and

117

with Just One

implies several by the results

receptor of recent

Only one other cell type. to have more than one Matthaei, 1983; but see

Biophys. J., Abstract). AChR single-channel subtypes. cloning

This idea experiments.

propis supUn-

like the muscle AChR, which is a pentamer composed of four distinct subunits in the stoichiometry a2, /3, y (01 E), 6 (reviewed in Karlin, 1980; Changeux and Giraudat, 1987: Schuetze and Role, 1987), the neuronal receptor appears to comprise only two subunits, a and 8 (reviewed in Berg and Halvorsen, 1988; Steinbach and Ifune, 1989). The stoichiometry of these subunits is not known. However, in contrast to muscle, which apparently expresses only one type of a subunit (al), to date four different neuronal a subunit clones have been identified (a2, a3, a4, and a5) in rat (Boulter et al., 1986; Goldman et al., 1986, 1987; Wada et al., 1988; McKinnon et al., 1988, Sot. Neurosci., abstract). Furthermore, at least three distinct rat 8 subunit clones (82, 83, and 84) have been reported (Deneris et al., 1988, 1989; S. Heinemann, personal communication). A similar diversity of clones has been reported for the chicken a and 8 (referred to as non-a subunits (Nef et al., 1988; Schoepfer et al., 1988; M. Ballivet, personal communication). Additional evidence that these a and 8 clones code for neuronal AChR subunits has come from experiments in which they have been functionally expressed in Xenopus oocytes. Combined injection of a and 8 mRNA results in an ACh-induced macroscopic response (Boulter et al., 1987; Wada et al., 1988). More recent experiments

Neuron 604

indicate that different combinations of subunits give rise to AChRs with distinct single-channel properties (Papke et al., 1989; Ballivet et al., 1988). In particular, injection of a4 and B2 chicken cDNA results in a single class of ACh-activated channels with a conductance of 20 pS (Ballivet et al., 1988). The similarity between this channel and the smaller conductance classes we see in the sympathetic neurons suggests that these AChRs may have the same subunit composition. It is clearly important to determine the precise relationship between subunit composition and functional properties of AChRs expressed in sympathetic neurons. Developmental Changes in ACh-Activated Channels The observed changes in neuronal AChR properties during development may contribute to their net increase in ACh sensitivity following innervation in vivo and in vitro (Role, 1988; L. M. Marshall, personal communication). These changes include the following: the appearance of a fourth, larger conductance class, XL; an increase in the slope conductances of the two smaller channel classes; a shift in the relative contribution of the smallest and larger conductance classes such that L predominates; the addition of a slow component to the burst duration distribution of the L class. In addition, AChRs may also be segregated and clustered with development, since we detect an increase in the percentage of patches with only one conductance class. The appearance of XL suggests that a new ACh-activated channel may beexpressed in sympathetic neurons following innervation. This observation has precedence in the extensive studies of developmental changes in the properties and subunit composition of muscle AChRs (Sakmann and Brenner, 1978; Fischbach and Schuetze, 1980; Brehm et al., 1982; Kullberg and Kasprzak, 1985; Mishina et al., 1986). In this system, innervation and denervation regulate the expression of the adult form of the AChR (i.e., higher y, smaller r; Brenner and Sakmann, 1983; Schuetze and Vicini, 1984; Brenner et al., 1987) as well as the expression of the E subunit (Witzemann et al., 1989). Examination of developmental changes in the expression of individual AChR subunits in sympathetic neurons may reveal a molecular basis for the appearance of the XL class. From ED10 to ED17, the distribution of slope conductance determinations changes from predominantly low (z-10-45 P’S) to predominantly high (~30-65 pS) values. This overall shift in conductance is due to two underlying developmental changes in channel properties: the increase in slope conductance of the two smaller classes, and the change in the relative contribution of the S, M, and L classes. These changes in channel behavior may represent posttranslational modifications of preexisting channels, the expression of different subunits or different combinations of existent subunit(s). The observation that both SIO and S17 have burst duration distributions with identical time constants (both about 1 and 7 ms), as do MI0 and MI7 (both about 2 and 12 ms), suggests that the increase in conductance may be due to posttranslational modification. This is in contrast to muscle,

where the increase in conductance results from replacing they subunit with E (Mishina et al., 1986). The change in relative contribution of the different classes, however, may be due to differential expression of the channel types. Another developmental change in AChR channel properties is that the largest amplitude class acquires a second, relatively long-lived open state, while its conductance remains unchanged. It is intriguing that the mean burst duration of L,o and the time constant for the fast component of L17 are both about 3 ms. This observation, combined with the fact that LIO and L17 have identical slope conductances (50 pS) suggests that a more conservative mechanism (i.e., modification of a preexisting channel) may be involved. A similar phenomenon occurs in Xenopus muscle cells in vitro: the low conductance AChR undergoes a 4-fold reduction in mean burst duration, with no accompanying change in conductance (Leonard et al., 1988). Furthermore, the authors have shown that this change is not blocked by treatment with tunicamycin (an inhibitor of glycosylation), which blocks insertion of newly synthesized AChRs into the membrane (Carlson and Leonard, 1989; but see Brehm et al., 1987). This suggests that the reduction in burst duration of the low-conductance AChR may be due to posttranslational modification. If the L channel class is synaptic, then the addition of a slow component to the burst duration distribution would likely prolong synaptic current decay. Marshall and Kojima have reported similar results for bullfrog B-type sympathetic neurons developing in vivo; in larval frogs, the EPSC decays as a single exponential with a time constant of 4.0 ms, compared with about 5.2 ms in the adult (Marshall and Kojima, 1986, Sot. Neurosci., abstract). These findings are in contrast to muscle, in which a developmental decrease in mean burst duration results in a faster rate of synaptic current decay (reviewed in Schuetze and Role, 1987; Brehm and Henderson, 1988). Obviously there are many classification schemes to account for the increase in functional channel classes other than the one outlined here. For example, one might propose that the channel behavior at ED17 could be due to the disappearance of the S class, the addition of two new classes with conductances of ~40 pS and 60 pS, a shift in the conductance of the MI0 class from 27 pS to 23 pS and a change in the r, for this class. We have adopted the indicated nomenclature because it is the most parsimonious with regard to the number of changes in channel properties that need be proposed. That is, since the open-time kinetics between the S and M classes of channels at each stage are identical, only a shift in their conductance and the addition of one new class are required to account for the observations. Other possibilities (such as the one proposed above) are equally possible, though somewhat more complex. Additional changes with development and innervation of sympathetic neurons implicate both segregation and clustering of the AChR channels. Segregation of channel types is suggested by the observed 4-fold in-

Developmental 605

Changes

in Neuronal

Nicotinic

AChRs

crease in the percentage of patches that have a single conductance class from ED10 to ED17. These data suggest homogeneous domains of individual channel types. Other preliminary data suggest a developmental increase in AChR clustering; that is, an increase in the local density of AChR channels. First, the percentage of patches with doublets (i.e., multiple openings that temporally overlap) increases about 2-fold. In some ED17 patches, even triplets were seen, which were never observed in ED10 patches. In addition the percentage of patches with no channel activity increases between ED10 and ED17, implying areas of membrane devoid of AChRs (B. L. Moss and L. W. Role, unpublished data). Thus, it seems likely AChRs are clustered with development. Segregation of different types of neuronal AChRs has also been reported. The distribution of bungarotoxin binding sites on ciliary ganglion neurons indicates that AChRs binding neuronal bungarotoxin are present both in synaptic and extrasynaptic regions, whereas a-bungarotoxin binding sites are excluded from sites of presynaptic apposition (Jacob and Berg, 1983; Loring et al., 1985). Although the function of a-bungarotoxin sites remains unclear, their separation from conducting AChRs indicates that this level of segregation has precedence. Segregation of classes of functional AChRs has also been suggested previously. Work by Marshall and colleagues in frog sympathetic neurons shows that selective expression of specific channel types depends on whether they are innervated by B- or C-type fibers (Marshall, 1985, 1986). Furthermore, when neurons are innervated by both types of presynaptic input (i.e., both B and C fibers to a single neuron), the postsynaptic responses evoked suggests that B- and C-type AChR channels are restricted to the sites of their respective presynaptic inputs (Marshall, 1989, Sot. Neurosci., abstract). There is ample precedent for clustering of AChRs with innervation at the neuromuscular junction where increased AChR density at the synaptic site is among the earliest events in synaptogenesis (reviewed in Salpeter and Loring, 1986; Schuetze and Role, 1987; Laufer et al., 1989). Likewise, at established interneural synapses in autonomic ganglia, AChRs are clearly clustered at the synapse (Marshall, 1981; Jacob et al., 1984; Loring and Zigmond, 1987; Sargent and Pang, 1989).

Contribution of Changes Increased ACh Sensitivity

in Channel

Properties

to

Several changes in AChR channel properties described above may contribute to the 4-fold increase in ACh sensitivity from ED10 to ED17 (measured by peak whole-cell currents elicited by applied ACh; D. C. Valenta and L. W. Role, unpublished data). In a preliminary analysis to evaluate the contribution of changes in channel function, we determined the mean ACh current by integrating single-channel recordings from ED10 to ED17 neurons. Preliminary results indicate that the mean current in cell-attached patches increases about 2-fold, from 0.008 f 0.001 pA in ED10 neurons to 0.015 * 0.004 pA in ED17 neurons (2.5 uM ACh, patch potential = -50

mV relative to rest; B. L. Moss and L. W. Role, unpublished data). Since the mean current is a function of n, P,, and i, (where n is the number of channels in the patch; P,, the probability that a channel is open; and i, the single-channel current amplitude), and we do not know how n varies with development, its contribution cannot be separated from other changes in channel properties. However, this analysis allows us to put an upper limit on the potential contribution of changes in channel properties to the observed increase in ACh sensitivity.

Possible Functional Significance Changes in Channel Properties Neuronal AChR

of Developmental of the

In addition to contributing to increased ACh sensitivity, developmental changes in channel properties of the neuronal AChR may alter synaptic transmission. Recent work on the muscle AChR has demonstrated that the kinetic changes in AChR channels observed in developing rat muscle have direct functional consequences: the embryonic form of the receptor, with its relatively long apparent mean open time (~6 ms at room temperature), promotes spontaneous contractions necessary for normal muscle fiber differentiation (Jaramillo et al., 1988). Given that all the changes in the neuronal AChR channel properties favor an increase in the postsynaptic response, these changes would likely enhance the efficacy of nicotinic synaptic transmission. Experimental

Procedures

Single-Channel Recording Cultured sympathetic neurons were obtained by dissecting the lumbar chain ganglia from ED10 and ED17 chicks, dispersing the ganglia to individual cells, and plating under conditions that suppress proliferation of nonneuronal cells. As an additional measure to prevent the growth of nonneuronal cells in ED17 cultures, the neurons were y-irradiated (=5000 rads) lust prior to plating. Details of the cell culture protocol have been published previously (Role, 1984). Most cultures were used for physiological recording 4-10 days after plating. In some experiments we used acutely dispersed neurons from both embryonic ages. We recorded from these cells l-5 hr after dispersal without enzyme treatment. Single-channel recording was done using conventional cellattached and outside-out patch-clamp techniques (Hamill et al., 1981). In each experiment, the plating medium was replaced with L-15 tissue culture medium (GIBCO) supplemented with 4.5 mM calcium. The culture dish was placed on the stage of a Nikon Diaphot inverted microscope equipped wtth phase contrast optics. Cells were viewed at 500x with a 40x objective. The bath temperature was monitored with a telethermometer. All experiments were carried out at room temperature (22”C24”C). Patch pipettes were pulled from Boralex 100 pl capillaries (Rochester Scientific) on a Kopf vertical puller and coated with either Sylgard (Dow Corning) or Sigmacote (Sigma) to reduce capacitance. The recording pipette was connected to a List EPC-7 patch-clamp amplifier. Pipette resistances were 5-10 MD. For cell-attached patch recording, the pipette contained ACh (l-5 PM; Sigma) dissolved tn an extracellular recording solution consisting of 150 mM NaCI, 3 mM KCI, 1 mM MgCI1, 1 mM CaCI,, and 10 mM HEPES titrated to pH 7.2 with 1 M NaOH. In \ome experiments, calcium was omitted and 1 mM EGTA was added to buffer free calcium. For outside-out patch recording, the patch pipette was filled with an Intracellular solution consisting of 150 mM KCI, 3 mM NaCI, 1 mM MgCI,, 1 mM EGTA, and 10 mM HEPES buffered to pH 7 2

NWKJll

606

with KOH. The pressure ejection pipette used to apply agonist contained ACh (l-2.5 pM) dissolved in the extracellular solution. In some experiments, calcium was omitted from the ACh solution and 1 mM EGTA was added. For each outside-out patch, we first recorded for 2 min prior to ACh-application to test for channels that are not ACh-activated. We then pressure-applied ACh (~15 psi) using a Picospritzer II (General Valve Corporation). The current output signal from the amplifier was low-pass filtered at 2-3 kHz (&pole Bessel, Frequency Devices, Inc., Haverhill, Massachusetts) and connected to the analog-to-digital converter of a Digital Equipment Corp. LSI 11/73 computer. The signal was sampied at 10 or 15 kHz (5 times the corner frequency of the analog filter) and stored on the computer’s hard disk. The digitized records were then stored on magnetic tape using a Kennedy 9600 recording system (Kennedy, Monrovia, California).

and brain stem of the rat characterized’by FEBS Lett. 222, 63-70.

Single-Channel Analysis The single-channel records were analyzed using BASIC 23 software developed by S. M. Schuetze. Details of the procedure, which is based on half-amplitude threshold analysis, has been described previously (Jaramillo and Schuetze, 1988). All analysis was identical to that described in Jaramillo and Schuetze (1988) with the following exceptions. In the generation of burst duration distributions, ~b was always set to 5 ms. We found that the precise value of Tb was not critical because of the very low frequency of channel opening. Also, all burst durations distributions were fit with one or two exponential components, using the method based on the log scale histogram display.

Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S., and Patrick, J. (1987). Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Natl. Acad. Sci. USA 84, 7763-7767.

Relative Frequency of Occurrence of the Channel Classes To determine the relative frequency of occurrence of the channel classes, we identified the classes based on current amplitude at a patch potential of -50 mV relative to rest (2.5 pM ACh) and scored the patches accordingly for the presence or absence of each individual channel class. To be certain that we would detect the contribution of channel classes at low levels of activity, we analyzed records that were at least 5 min in duration. This is sufficient to detect channels with a mean opening frequency as low as 0.01 opening per s (-3 openings per 5 min), which is about 5-to lo-fold less than the actual minimum opening frequency observed at ED10 and ED17, respectively (opening frequency for individual channel classes ranged from ~0.05 to 1.0 opening per s at EDlO, and from ~0.1 to 2.3 openings per s at ED17; 2.5 JIM ACh). Mean Burst Duration from the Arithmetic Mean We were unable to obtain records that contained enough channel openings of the XL conductance class to estimate reliably the mean burst duration from an exponential fit to the distribution. Therefore, we estimated the mean burst duration by taking the arithmetic mean of XL openings across 3 different patches (patch potential = -50 mV relative to rest potential). This value was then corrected for events that were below our limit of resolution, by subtracting the minimum resolvable open time (0.5 ms) from the arithmetic mean. Acknowledgments B. L. M. is affiliated with the Department of Biological Sciences, Columbia University. We dedicate this work to the memory of our friend and colleague, Steve Schuetze. We also thank K. Dunlap, J. Koester, S. Siegelbaum, and D. C. Valenta for critically reading an earlier version of the manuscript. Support was provided by the National Institutes of Health (L. W. R. - NS 22061; S. M. 5. NS 17774), awards from the Klingenstein and McKnight Foundations (L. W. R.) and EBR (L. W. R.) Received

June

30, 1989;

revised

August

30, 1989

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Added

B., Criado, M., Stein, E., and Sakmann, B. regulation of five subunit specific mRNAs receptor subtypes in rat muscle. FEBS Lett.

in Proof

Margiotta and Curantz have recently reported developmental changes in AChR channel properties in chicken ciliary ganglion neurons (Margiotta, J. F., and Curantz, D., 1989. Changes in the number, function, and regulation of nicotinic acetylcholine receptors during neuronal development. Dev. Biol. 735, 326-339).