Developmental Changes in the Nicotinic Responses of Ciliary Ganglion Neurons

Edward M. Blumenthal, Richard D. Shoop, and Darwin K. Berg

Department of Biology, 0357, University of California, San Diego, La Jolla, California 92093

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Blumenthal, Edward M., Richard D. Shoop, and Darwin K. Berg. Developmental changes in the nicotinic responses of ciliary ganglion neurons. J. Neurophysiol. 81: 111-120, 1999. The accumulation of functional neurotransmitter receptors by neurons during development is an essential part of synapse formation. Chick ciliary ganglion neurons express two kinds of nicotinic receptors. One is abundant, contains the alpha 7 gene product, rapidly desensitizes, and binds alpha -bungarotoxin. The other is less abundant, contains multiple gene products (alpha 3, beta 4, alpha 5, and beta 2 subunits), slowly desensitizes, and binds the monoclonal antibody mAb 35. Rapid application of agonist to freshly dissociated neurons elicits responses from both classes of receptors. Between embryonic days 8 and 15, the whole cell response of alpha 3-containing receptors increases fivefold in peak amplitude and, normalized for cell growth, 1.7-fold in current density. In addition, the response decays more slowly in older neurons, suggesting a developmental decrease in the rate of desensitization. The whole cell response of alpha 7-containing receptors increases 10-fold in peak amplitude over the same period and 3-fold in current density. No change in the rate of desensitization was apparent for alpha 7-containing receptors with developmental age, but analysis was limited by overlap in responses from the two kinds of receptors. Indirect immunofluorescence measurements on dissociated neurons showed that the relative levels of alpha 7-containing receptors on the soma increased during development to the same extent as the whole cell response attributed to them. In contrast, the relative levels of alpha 3-containing receptors increased more during the same time period than did the whole cell response they generated. The immunofluorescence analysis also showed that both classes of receptors become distributed in prominent clusters on the cell surface as a function of developmental age. The results indicate that during this period of synaptic consolidation on the neurons, the two major classes of functional nicotinic receptors undergo substantial upregulation; alpha 3-containing receptors as a class may undergo changes in receptor properties as well.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Producing functional neurotransmitter receptors and concentrating them at appropriate locations on the cell surface are important features of synapse formation. These events are best understood at the vertebrate neuromuscular junction where much has been learned about the assembly, membrane localization, and functional regulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane (Sanes 1997). Much less is known about receptor regulation on neurons where often multiple receptor subtypes must be made and distributed to a complex array of synaptic locations.

Like vertebrate muscle, autonomic neurons rely on AChRs to mediate primary synaptic input from preganglionic neurons in the CNS. A variety of studies indicate that cell-cell interactions regulate the number and properties of AChRs expressed by the neurons (Arenella et al. 1993; Boyd et al. 1988; Devay et al. 1994; Gardette et al. 1991; Jacob and Berg 1987; Levey and Jacob 1996; Levey et al. 1995; Margiotta and Gurantz 1989; Moss and Role 1993; Moss et al. 1989). Recently it has been shown that an isoform of neuregulin can enhance the number of functional AChRs expressed by sympathetic neurons in culture (Yang et al. 1998).

One of the most interesting and abundant neuronal AChRs both in the peripheral and central nervous systems is a species that binds alpha -bungarotoxin (alpha Bgt) with high affinity and is thought to be a homopentamer containing alpha 7 subunits (Anand et al. 1993; Chen and Patrick 1997; Couturier et al. 1990a; Conroy and Berg 1998; Schoepfer et al. 1990). The receptor (alpha 7-AChR) has a high relative permeability for calcium and rapidly desensitizes (Alkondon and Albuquerque 1993; Bertrand et al. 1993; Seguela et al. 1993; Zhang et al. 1994). It modulates transmitter release from presynaptic sites both in the central and peripheral nervous systems (Coggan et al. 1997; Gray et al. 1996; McGehee et al. 1995) and can influence developmental events at the growth cone (Fu and Liu 1997; Pugh and Berg 1994). The developmental regulation of functional alpha 7-AChRs has not been examined.

Chick ciliary ganglion (CG) neurons express large numbers of alpha 7-AChRs (Chiappinelli and Giacobini 1978; Vernallis et al. 1993) and concentrate them at extrasynaptic or perisynaptic sites (Jacob and Berg 1983; Loring et al. 1985; Wilson Horch and Sargent 1995). The neurons express a smaller number of AChRs that bind the monoclonal antibody mAb 35 and contain alpha 3, beta 4, alpha 5, and, sometimes, beta 2 subunits (Conroy and Berg 1995; Vernallis et al. 1993). These latter receptors (alpha 3*-AChRs) are concentrated at postsynaptic densities as well as extra/perisynaptic sites on the neurons (Jacob et al. 1984; Loring and Zigmond 1987; Wilson Horch and Sargent 1995). Both alpha 7-AChRs and alpha 3*-AChRs contribute importantly to synaptic transmission elicited by stimulating the preganglionic nerve (Ullian et al. 1997; Zhang et al. 1996).

All CG neurons receive functional nicotinic innervation by embryonic day (E)7 (Landmesser and Pilar 1974). Between E8 and E15, the number of alpha 3*-AChRs in the ganglion, quantified by 125I-mAb 35 binding, increases ~10-fold (Corriveau and Berg 1994; Smith et al. 1985) while the number of alpha 7-AChRs, quantified by 125I-alpha Bgt binding, increases 6-fold (Chiappinelli and Giacobini 1978; Corriveau and Berg 1994). These increases occur despite a 50% decline in the number of ganglionic neurons through naturally occurring cell death (Landmesser and Pilar 1974). Previous analysis of freshly dissociated CG neurons with patch-clamp techniques yielded whole cell nicotinic responses that increased 14-fold between E8 and E15 (Margiotta and Gurantz 1989). The responses were attributed to alpha 3*-AChRs because alpha Bgt had no effect. Surprisingly, single-channel analysis, together with whole cell current recordings and 125I-mAb 35 binding measurements, suggested that only a small fraction of the alpha 3*-AChRs at late developmental stages was functionally competent (Margiotta and Gurantz 1989; Margiotta et al. 1987a).

The present studies were undertaken for two reasons. First, the availability of techniques for rapidly applying agonists to dissociated CG neurons (Zhang et al. 1994) made possible for the first time an analysis of alpha 7-AChR responses as a function of development. Second, the larger and more rapid whole cell responses elicited by the new method of agonist application motivated a reexamination of the inference that many AChRs on the neurons may not be functionally active.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell preparation

Dissociated CG neurons were prepared from E6 to E15 chick ciliary ganglia using a modification of methods previously described (Margiotta and Gurantz 1989). The age of the embryo was confirmed before dissection using the staging method of Hamburger and Hamilton (1951). The ganglia were dissected from the embryo, hemisected (except for ganglia from the youngest embryos), and incubated with collagenase (Boehringer Mannheim, Indianapolis, IN) for 20 min at 37°C. The concentration of collagenase varied with the age of the embryo: 0.1 mg/ml, E6; 0.2, E8; 0.25, E9; 0.3, E10; 0.6, E11; 0.75, E12; 1.0, E13-E14; 1.25, E15. The dissociation medium contained (in mM) 140 NaCl, 3 KCl, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 (with NaOH). After collagenase treatment, the ganglia were transferred to culture medium made up of Eagle's minimal essential medium (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated horse serum (Gemini, Cannon Falls, MN) and 3% (vol/vol) embryonic eye extract (Nishi and Berg 1981). The cells were dispersed by trituration with a fire-polished Pasteur pipette and plated on tissue culture dishes (Falcon) that had been coated with poly-D-lysine (1 mg/ml). Dissociated cells were used within 1-5 h of plating and were kept in a tissue culture incubator at 37°C until use.

Electrophysiology

Currents were recorded using the whole cell patch-clamp configuration controlled by an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) as previously described (Hamill et al. 1981; Zhang and Berg 1995). All experiments were carried out at room temperature. The extracellular solution contained (in mM) 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 (with NaOH). The pipette solution contained (in mM) 140 CsCl, 1 MgCl2, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 glucose, and 10 HEPES, pH 7.2 (with CsOH). Electrodes were pulled from 1.5-mm-OD borosilicate glass (Drummond, type N51, Broomali, PA) and had resistances of ~3 MOmega . Series resistance was always <6 MOmega and was 80% compensated. Cells were clamped at -60 mV, and agonist was rapidly applied as previously described (Zhang and Berg 1995). Solutions were delivered from a linear array of glass tubing (370 µm ID, 470 µm OD; Polymicro Technologies, Phoenix, AZ) mounted on a piezobimorph element (Morgan-Matroc, Bedford, OH). Solution flow was gravity fed and controlled by a set of solenoid valves (General Valve, Fairfield, NJ). The valves and the bimorph were controlled by a Master-8 programmable stimulator (A.M.P.I., Jerusalem, Israel). Using this system, the time of solution exchange at an open pipette was 0.5-3 ms as measured by the change in junction potential. Data were filtered at 1 kHz and digitized at either 0.7 or 1.5 kHz with the pCLAMP software (Axon Instruments). Currents were analyzed using Axograph software (Axon Instruments).

For recordings from alpha Bgt-treated cells, the toxin (Biotoxins Inc.) was applied at 60 nM for >1 h at 37°C, and 18 nM alpha Bgt also was included in the recording solution. When ACh was used as the agonist, 100 nM atropine was included in the bath and agonist solution. Tetrodotoxin was omitted from the bath because previous experiments showed it to be unnecessary when recording nicotinic responses in the neurons with voltage clamp techniques (Zhang et al. 1994).

Immunofluorescence

Freshly dissociated cells were labeled by incubating with either biotinylated alpha Bgt (Molecular Probes, Eugene, OR; 1:500 dilution) or mAb 35 (1:1,000 dilution) in 10 mM HEPES-buffered medium with 3% eye extract for 1-2 h at 4°C. The neurons then were rinsed three times with HEPES-buffered medium and fixed in 4% paraformaldehyde in 0.15 M sodium phosphate, pH 7.4, for 30 min. After rinsing with 0.10 M sodium phosphate (SP), pH 7.5, toxin-labeled neurons were incubated in a 1:1,000 dilution of Cy3-conjugated streptavidin (Jackson Laboratories, Bar Harbor, ME) in SP for 1 h at 4°C. Likewise, mAb-35-labeled cells were incubated for 1 h in a 1:200 dilution of Cy3-conjugated donkey anti-rat antibody (Jackson Laboratories) in SP with 5% normal donkey serum (Sigma, St. Louis, MO). The specificities of these probes for immunofluorescent detection of AChRs on ciliary ganglion neurons have been demonstrated previously (Jacob et al. 1984; Wilson Horch and Sargent 1995). In some experiments, alpha 7-AChRs were labeled with indirect immunofluorescence by fixing the cells as described above, permeabilizing with 0.1% (wt/vol) Triton X-100 in SP, and then labeling with anti-alpha 7-AChR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). In a few experiments, alpha 3*-AChRs were labeled by first fixing the cells and then labeling as described in the preceding text.

Images were visualized either with a Noran ODYSSEY confocal laser scanning microscope or with a Sensys CCD camera (Photometrics, Tucson, AZ) and Zeiss Axioskop. The confocal images represent reconstructions of the neuron surface achieved by assembling 80-120 contiguous individual optical sections taken through an individual cell. An indication that the optical reconstruction did not distort the patterns observed is that computer rotation of the reconstructions in three-dimensional space indicated that fluorescently labeled receptor clusters on the sides of the cells were not significantly different in shape or labeling intensity from those on the top or bottom of the cells, though the former were generated by reconstruction from multiple optical sections while the latter often were contained principally within one or two optical sections. Confocal images of E8 and E15 cells were adjusted individually for brightness and contrast to optimize visualization.

For quantitative comparisons of fluorescence signals, the CCD camera was used. The linearity of fluorescence detection by the camera over the operating range used here was confirmed by calibration experiments. For this purpose, dissociated E8 and E15 ciliary ganglion neurons were incubated with biotinylated alpha Bgt diluted with known concentrations of underivatized alpha Bgt and then carried through the labeling procedure as described. The specific fluorescence signal associated with a cell was determined by measuring the total fluorescence emanating from a circle enclosing the cell and then subtracting the background fluorescence from an equivalent circle lacking a cell in the same field of view. Background values determined in this way were not significantly different from those determined by including an excess of underivatized alpha Bgt or nicotine in the initial binding reaction to block specific binding of the biotinylated toxin. Measurements of specific fluorescence then were used to construct calibration curves that showed the expected dependence on toxin dilution both for E8 and for E15 neurons, corroborating the manufacturer's calibration of linearity supplied with the camera.

Because the optical image captured by the CCD camera represented a slice through the cell rather than the whole cell, a correction for cell size was necessary when comparing relative fluorescence signals for E8 and E15 neurons. E15 neurons were found on average to be 35% larger in diameter than E8 neurons. Accordingly, when calculating the relative levels of whole cell specific fluorescence associated with E15 versus E8 neurons, a 35% positive correction was applied to the calculated E15:E8 ratio. The fact that identical E15:E8 fluorescence ratios were obtained when measurements for an experiment were made with either a ×40 or a ×63 oil immersion objective indicates that the results were independent of the thickness of the optical section over the range employed.

Materials

White Leghorn chick embryos were obtained locally and maintained at 37°C in a humidified incubator. alpha Bgt was purchased from Biotoxins (St. Cloud, FL). All other reagents were purchased from Sigma unless otherwise indicated.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

alpha 3*-AChR responses

Developmental changes in nicotinic responses between E6 and E15 were examined in freshly dissociated CG neurons using whole cell patch-clamp recording techniques. Initial studies focused on alpha 3*-AChRs because their responses could be studied in isolation. This was achieved by using alpha Bgt to block the alpha 7-AChRs. Rapid application of 1 mM ACh to the neurons in the continued presence of alpha Bgt (and atropine to block muscarinic receptors) elicited currents wholly attributable to alpha 3*-AChRs.

Figure 1 illustrates representative responses obtained from E6 (A and C) and E15 (B and D) neurons. The induced currents increase over several milliseconds on agonist delivery and then diminish in the continued presence of agonist. Both the amplitude of the peak response and the kinetics of decay change during development. The decay phases are shown fit with the sums of three exponentials (Fig. 1, C and D), which change during development (see below, this section). Between E6 and E15, the peak current amplitude increases ~15-fold (Fig. 2A). Much of this is due to neuronal growth: normalizing for cell capacitance (Fig. 2B) yields a value for current density that increases just over threefold between E6 and E15 (Fig. 2C). The peak amplitude and the calculated current density both increase smoothly during the 10 days of development examined.


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FIG. 1. Whole cell currents activated by 1 mM acetylcholine (ACh) in the presence of alpha Bgt from embryonic day 6 (E6; A and C) and E15 (B and D) ciliary ganglion (CG) neurons. Agonist was applied to the cells for the time indicated by the horizontal bar. C and D are replicates of A and B, respectively, with superimposed dashed lines that indicate the sum of 3 exponentials used to fit the decay phase of the response in each case.


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FIG. 2. Developmental change in response of CG neurons to 1 mM ACh in the presence of alpha Bgt. A: peak current amplitudes (pA); B: cell capacitance (pF); C: current density (pA/pF). Values represent the means ± SE of 16-172 cells per point.

The observed changes in peak amplitude did not result from a developmental shift in agonist affinity. The concentration of ACh used (1 mM) was equally effective at both early and late developmental times. This can be seen by comparing the responses of individual cells to 1 and 0.1 mM ACh: the ratio of the peak amplitudes elicited by the two concentrations was 2.1 ± 0.7 at E8/9 (27 cells; mean ± SD) and 1.9 ± 0.3 at E14/15 (30 cells). Moreover, normalizing peak amplitudes to those induced by 0.1 mM ACh showed that 0.5 and 1 mM ACh elicited equivalent responses both at E9 (ratios of 1.6 ± 0.2 and 1.7 ± 0.3 for 0.5 and 1 mM vs. 0.1 mM ACh, respectively) and at E15 (ratios of 1.8 ± 0.4 and 1.8 ± 0.2, respectively).

To characterize the developmental change in decay kinetics and accompanying receptor desensitization, we first measured the integral of each current trace over the first second of the recording and normalized the integral to the peak amplitude. The resulting value (desensitization index) gives a rough measure of desensitization, ranging from zero for a current that desensitizes instantaneously to 1.0 for a nondesensitizing current. Almost all of the change in the desensitization index occurred between E8 and E10 and represented a shift from a more rapidly desensitizing early response to a more slowly desensitizing later response (Fig. 3A).


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FIG. 3. Developmental change in the desensitization kinetics of alpha 3*-AChRs. A: change in the integral of the current response for the first second of the recording, normalized for the peak current to create an index of relative desensitization (desensitization index) as a function of developmental age. B: sum of 3 exponentials was required to fit adequately the decay phase of the response. Shown here is the developmental change in the fraction of the peak current contributed by the slowest component. Fractional currents were calculated by extrapolating back to the beginning of the rising phase. Both the desensitization index and the contribution of the slowest component increase most dramatically between E8 and E10. Values represent the means ± SE of 14-64 cells per point.

Analysis of the falling phase of the responses indicated that three exponentials were necessary to fit adequately the vast majority of the records. Time constants of 30 ± 2 and 176 ± 20 ms (n = 14 cells) were calculated for the fast and intermediate components, respectively, at E6; these increased to 44 ± 1 and 238 ± 11 ms (n = 64 cells) by E15. The increases were statistically significant (P <=  0.001). The time constant of the slow component exceeded the length of the recordings (2 s) and could not be measured accurately. Nonetheless, the amplitude of the slow component could be measured and was found to account for most of the change in the desensitization index between E8 and E10 because it contributed an increased proportion of the total response at the later time (Fig. 3B). The desensitization index, as calculated, arithmetically emphasizes slow components. Together, the three exponentials provided a good fit of the falling phase of the response (Fig. 1, C and D).

We considered whether some of the slowdown in desensitization seen with increasing cell age might result from an increase in mean cell size between E6 and E15. Larger cells presumably impede access of transmitter to the far side of the cell more than smaller cells do and thereby would increase the time over which their receptors are activated and desensitize. Support for this comes from the finding of a positive correlation between cell capacitance and desensitization index among cells of different sizes in the population of E12-E15 neurons (R2 = 0.17 for linear regression; n = 139 cells; P <=  0.001). Although the two measures are correlated significantly, the correlation coefficient indicates that most of the variability in the desensitization index is not caused by differences in cell size. Moreover, mean cell size changes gradually between E6 and E15 but most of the change in the desensitization index occurs between the narrow window of E8 and E10. In addition, much of the shift in the desensitization index, as pointed out above, is due to the relative increase in the amplitude of the slowly decaying component, and it is difficult to see how changes in cell size could account for such a shift.

alpha 7-AChR responses

Nicotine was the agonist of choice for activating alpha 7-AChRs because it produced significant temporal separation of the alpha 7-AChR and alpha 3*-AChR peak responses. This was deduced from preliminary experiments, which showed that nicotine activates alpha 7-AChRs much more rapidly than it does alpha 3*-AChRs; ACh produces a much smaller time differential between the two kinds of responses. In fact, all of the rapidly desensitizing response elicited by 20 µM nicotine in both E8 and E14 neurons can be attributed to alpha Bgt-sensitive alpha 7-AChRs (Fig. 4, A-D). Only slowly inactivating currents, indicative of alpha 3*-AChR responses, are observed when nicotine is applied to neurons treated with alpha Bgt (Fig. 4, E and F).


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FIG. 4. Examples of whole cell currents elicited in E8 (A, C, and E) and E14 (B, D, and F) CG neurons by 20 µM nicotine in the absence (A-D) and presence (E and F) of alpha Bgt. Horizontal bar applies to all traces and indicates the time of agonist application to the cells. Traces are from 6 different cells and illustrate by difference (A and C vs. E; B and D vs. F) the time course and magnitude of the alpha Bgt-sensitive component of the responses.

The peak amplitude of the alpha Bgt-sensitive nicotinic response increased ~10-fold between E8 and E15 (Fig. 5A). It was not possible to quantify the alpha Bgt-sensitive component of the nicotinic responses in younger neurons (E6) because it could not be reliably distinguished from alpha Bgt-resistant components, and so those data have been excluded. After correcting for cell size by normalizing the peak response for capacitance, the net increase in current density between E8 and E15 for the alpha Bgt-sensitive response was found to be ~3.5-fold (Fig. 5B). This is greater than the increase in current density seen for the alpha 3*-AChR response over the entire developmental window of E6-E15 and is twice as great as that for the equivalent period of E8-E15 (Fig. 2C).


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FIG. 5. Developmental change in the response of CG neurons to 20 µM nicotine. A: peak current amplitudes (pA); B: current density (pA/pF). Values represent the means ± SE of 16-127 cells per point.

The developmental increase in peak amplitude of alpha 7-AChR responses could not be ascribed to a shift in the dose-response curve of the receptors for nicotine. Although 20 µM nicotine does not elicit a maximal response, the ratio of peak responses evoked by 100 and 20 µM nicotine was unchanged over development: 2.1 ± 0.7 at E9 (n = 16 cells); 1.9 ± 0.7 at E14 (16 cells).

No developmental change was apparent in the desensitization kinetics of alpha 7-AChRs. Exponential fits of the rapidly decaying nicotine-induced current were not possible because the slow alpha Bgt-resistant current usually contaminated much of the falling phase of the alpha Bgt-sensitive response (Fig. 4, C and D). The extent of desensitization during the initial few milliseconds of the recordings was comparable for neurons from young and late developmental stages (data not shown), but only large differences would have been detected in this way.

Relative increases in alpha 7- and alpha 3*-AChRs during development

In view of past results suggesting that a large number of nonfunctional alpha 3*-AChRs appear at late developmental times (Margiotta and Gurantz 1989), it seemed important to compare changes in the number of alpha 3*-AChRs on the neurons at early and late times with those seen in the whole cell alpha 3*-AChR response under the same conditions. Previous quantification of CG AChRs during development usually measured total alpha 3*- or alpha 7-AChRs in ganglion extracts (Chiappinelli and Giacobini 1978; Corriveau and Berg 1994; Smith et al. 1985). It is clear now that such measurements would have included a substantial number of intracellular AChRs and AChRs on neuronal processes (Corriveau and Berg 1994; Jacob and Berg 1988; Stollberg and Berg 1987; R. Shoop and D. Berg, unpublished studies). In addition, at least some of the alpha 7-AChRs present in ganglion extracts are likely to have been preganglionic in origin where they acted presynaptically to modulate neurotransmitter release (Coggan et al. 1997; McGehee et al. 1995).

Indirect immunofluorescence was used in the present experiments to compare the numbers of receptors at early and late developmental times. This approach allowed us to focus exclusively on cell surface AChRs confined to the cell soma and enabled us to prepare the cells in the same manner as those used for electrophysiological analysis. To visualize alpha 7-AChRs, dissociated neurons were incubated first with biotinylated alpha Bgt and then Cy3-conjugated streptavidin before viewing with confocal laser microscopy. Serial reconstruction of labeled optical sections was performed to generate a composite image of the whole neuron. When viewed in this way, E8 neurons had low levels of labeling distributed among small clusters while E15 neurons had large and prominently labeled receptor clusters (Fig. 6, A and B). Such clusters have been seen previously on neurons in situ late in development and have been described as being perisynaptic because they often are adjacent to but never overlapping with presumed presynaptic sites of transmitter release defined immunocytochemically (Wilson Horch and Sargent 1995). The alpha 7-AChR labeling was judged specific because it was eliminated by using native alpha Bgt to block the binding of biotinylated alpha Bgt to the cells (Fig. 6C). Similar labeling patterns were obtained if the cells first were fixed and permeabilized and then labeled with anti-alpha 7 antibodies (data not shown). CCD images of labeled cells with conventional microscopy was used to more accurately depict the relative levels of fluorescence on E8 versus E15 neurons (Fig. 6, D and E; see following paragraphs).


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FIG. 6. Distributions of alpha 7-AChRs and alpha 3*-AChRs on CG neurons during development. Dissociated CG neurons were labeled either with biotinylated alpha Bgt followed by Cy3-conjugated streptavidin (A-E) for alpha 7-AChRs or with mAb 35 followed by Cy3-conjugated donkey anti-rat antibodies (F-I) for alpha 3*-AChRs and visualized either with confocal laser microscopy (A-C, F, and G) or with a CCD camera and conventional fluorescence microscopy (D, E, H, and I). E8 neurons: A, D, F, and H; E15 neurons: B, C, E, G, and I. Cell in C received a large excess of underivatized alpha Bgt to block binding of biotinylated alpha Bgt and serve as a control for nonspecific labeling. Both kinds of imaging used ×63 oil immersion objectives. Scale bar: 10 µm.

Using a similar strategy to image alpha 3*-AChRs, dissociated neurons were incubated first with mAb 35 and then with Cy3-conjugated secondary antibody. Reconstructed confocal images revealed low levels of labeling at E8, and much more pronounced levels at E15 when receptor clusters were again readily apparent (Fig. 6, F and G). Omission of the primary mAb in the binding reaction eliminated the fluorescence signal. The antibody incubations were routinely carried out at 4°C to prevent antibody-induced redistribution of receptors. The adequacy of the low-temperature approach was confirmed by fixing cells before incubation with antibody and finding that similar patterns of labeling were obtained, albeit with higher levels of background staining (data not shown). CCD images of labeled cells with conventional microscopy again were used to obtain better estimates of the relative levels of labeling for E8 versus E15 neurons (Fig. 6, H and I).

The CCD images were used for quantitative comparisons of fluorescence labeling at early and late developmental stages. For alpha 7-AChRs, E15 neurons were found to have ~10-fold more whole cell specific fluorescence than did E8 neurons (Fig. 7A). This value is indistinguishable from the 10-fold increase between E8 and E15 in whole cell nicotinic response attributed to alpha 7-AChRs (Fig. 5). For alpha 3*-AChRs, E15 neurons were found to have ~12-fold greater whole cell fluorescence than did E8 neurons (Fig. 7B). This is significantly greater than the approximately fivefold increase between E8 and E15 in whole cell ACh-induced response attributed alpha 3*-AChRs (Fig. 2A).


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FIG. 7. Relative levels of alpha 7-AChRs and alpha 3*-AChRs on CG neurons at early and late developmental stages. Dissociated CG neurons labeled as described in Fig. 6 for alpha 7-AChRs (A) and alpha 3*-AChRs (B) were visualized with a CCD camera and conventional fluorescence microscopy to measure relative fluorescence levels as described in METHODS. Scale was adjusted to give optimum sensitivity in the linear range of the camera; arbitrary units are shown. Values represent the means ± SE for a total of 31-32 cells in A and a total of 44-46 cells in B compiled from 3 separate experiments in each case.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We examined the nicotinic responses of CG neurons during the embryological period E6-E15, which encompasses the major milestones of CG development. These include innervation of the ganglion by neurons from the accessory oculomotor nucleus, innervation of postsynaptic target muscle in the iris, ciliary body, and choroid layer by CG neurons, and programmed cell death in the ganglion. Patch-clamp recordings from freshly dissociated neurons reported here show large increases in the whole cell response produced both by alpha 7-AChRs and by alpha 3*-AChRs over this time frame and demonstrate further that a substantial portion of the increase in each case represents a developmental change in the current density generated by the receptors. For alpha 7-AChRs immunofluorescence measurements show that the increases in whole cell responses are matched by increases in receptor number on the cells; for alpha 3*-AChRs, the increases in receptor number exceed the increases in whole cell response. The distribution of receptors also changes during this time frame and increasingly takes on the appearance of discrete receptor clusters reminiscent of the distribution on adult neurons in situ.

Previous developmental studies of functional nicotinic receptors did not include alpha 7-AChRs apparently because the methods used for agonist application did not permit resolution of their responses. Only alpha Bgt-resistant currents, attributable to alpha 3*-AChRs, were recorded from freshly dissociated CG neurons in response to puffer-applied ACh (Margiotta and Gurantz 1989; Margiotta et al. 1987a). In those studies, values of 14- and 4.5-fold were obtained for the increases in peak amplitude and current density, respectively, between E8 and E15. Values of 5- and 1.7-fold were obtained in the present studies for the same parameters of the alpha Bgt-resistant response. The differences result from the larger responses obtained from young neurons in the present studies when agonist was applied rapidly. The developmental shift reported here in the desensitization kinetics of alpha 3*-AChR responses toward longer times at later developmental stages is the primary explanation: a greater proportion of the signal would have been lost through receptor desensitization in young neurons when agonist was applied by puffer pipette.

A similar explanation may account for the previous report of a developmental shift in agonist affinity (Margiotta and Gurantz 1989). If desensitization at high agonist concentrations caused a truncation of the dose-response curve when using the puffer pipette and was most pronounced at early developmental times, it could have produced an apparent shift with development in the measured EC50 values. No evidence for a developmental shift in agonist affinities was found in the present studies, but it was not examined directly. Dose-response experiments were designed here to test whether the concentrations being used were sufficient to generate maximal responses.

The slower desensitization of the alpha 3*-AChR response in older neurons is the opposite of what one might have expected from muscle AChRs where the mature form of the receptor (alpha 1beta 1epsilon delta ) produces more rapidly decaying responses than does the embryonic form (alpha 1beta 1gamma delta ) (Naranjo and Brehm 1993). It is unlikely that the slower desensitization observed here results primarily from the effects of size differences between E8 and E15 neurons. Although an increase in cell diameter would extend the time required for agonist to access receptors on the far side of the cell (thereby extending the apparent duration of the response at the cost of peak amplitude), the actual amounts of time required for access are, at most, a few milliseconds (Zhang et al. 1994). Further, most of the slowdown in desensitization occurs between E8 and E10 though cell size increases almost monotonically between E6 and E15. Instead, much of the change in the desensitization index can be attributed to a developmental shift in the proportion of the alpha 3*-AChR response generated by the most slowly desensitizing component at the expense of the most rapidly desensitizing component, and this is unlikely to have been influenced by changes in cell size.

Previous studies came to the conclusion that only a small fraction of the alpha 3*-AChRs present on CG neurons at late developmental stages was functionally competent (Margiotta and Gurantz 1989). The determination made use of single-channel analysis combined with measurements of the whole cell response for comparison with levels of 125I-mAb 35 binding on freshly dissociated cells. The total number of alpha 3*-AChRs on dissociated neurons at E9 (assuming 2 mAb 35 binding sites per receptor) exceeded the number of functional AChRs by a factor of two. At E15, however, the total number exceeded the functional receptors by a factor of eight. The levels of mAb 35 binding increased in parallel with the whole cell response over this time period, but developmental changes in the ensemble of single channel events led to the increasing disparity as a function of age (Margiotta and Gurantz 1989). The disparity was interpreted as a developmentally increasing population of functionally silent receptors.

Using the new data on current amplitudes obtained here and the single channel data and open time probabilities reported previously (Margiotta and Gurantz 1989) produces a value of ~5,500 for the total number of functional alpha 3*-AChRs on E9 neurons. This compares closely with the ~5,000 mAb 35 binding receptors inferred to be on the cells previously, again assuming two mAb sites per receptor (Margiotta and Gurantz 1989). The results clearly argue against nonfunctional alpha 3*-AChRs at early stages. By E14, however, the present whole cell responses, together with previous single channel data, yield a value of 6100 for the number of functional alpha 3*-AChRs on the neurons. This is to be compared with 25,000 for the total number of alpha 3*-AChRs calculated from previous mAb 35 binding data (Margiotta and Gurantz 1989).

The binding assay used by Margiotta and Gurantz (1989) had the advantages of measuring only AChRs on the cell surface and providing readily quantifiable values. A potential disadvantage was that the binding results might have included receptors present on membrane debris from lysed cells or axon fragments. The fluorescence assay used here measured only surface receptors on intact cells. Reassuringly, the two approaches yielded similar results. Thus the original binding results indicated an increase of ~11-fold in mAb 35 binding levels between E8 and E15 while the fluorescence measurements reported here showed an increase of ~12-fold. Notably the developmental increase between E8 and E15 in whole cell response attributed to alpha 3*-AChRs was only fivefold. This, together with the previously reported changes in single channel properties, supports the contention that a population of functionally silent alpha 3*-AChRs may appear late in development and constitute as much as three-fourths of the total alpha 3*-AChRs.

What is the meaning of functionally silent alpha 3*-AChRs? Least interesting is the possibility that the receptors represent assembly errors or receptors rendered dysfunctional by the methods used to prepare the dissociated cells. This seems unlikely because the nonfunctional receptors can be found on neurons after 1 wk in culture (Margiotta et al. 1987a) and in vivo appear to be regulated developmentally, appearing at late times when neuronal development is well advanced. A different possibility is that the receptors only appear nonfunctional in the assay because of more prominent receptor desensitization at late developmental stages. This would seem at odds with the fact that the whole cell response decays more slowly with development rather than more quickly, but the slowdown may be deceptive. The weak correlation between cell size and desensitization index suggests that larger cells impede transmitter access to some extent and, as a result, may experience a greater asynchrony in receptor activation. The consequence would be a diminished peak response in which some receptors already were desensitized while others had not yet been activated. Calculations based on the peak response then would suggest silent receptors. Because the rate of agonist application, however, appears to be so much faster than the rate of alpha 3*-AChR activation (Zhang et al. 1994), this explanation seems unlikely to account for a substantial portion of the silent receptors.

A third and more interesting possibility is that nonfunctional receptors reflect regulatory mechanisms coming into play late in development to control the number of receptors available for activation. For example, a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism appears to increase the proportion of alpha 3*-AChRs that contribute to the whole cell response (Margiotta et al. 1987b), and developmentally the mechanism first appears at E10 (Margiotta and Gurantz 1989). Although the cAMP-dependent regulation does not itself explain the existence of functionally silent receptors, it may act by overcoming an inhibitory constraint expressed only late in development or it may exploit a subpopulation of alpha 3*-AChRs that appears late in development.

One alpha 3*-AChR subtype that might account for the late-term appearance of functionally silent receptors is a species containing alpha 5 subunits. Expression of the alpha 5 gene increases disproportionately late in development (Corriveau and Berg 1993; Levey and Jacob 1996; Levey et al. 1995), and the subunit appears in alpha 3*-AChRs (Conroy and Berg 1995; Vernallis et al. 1993) though it is not an obligatory component for receptor function (Couturier et al. 1990b; Duvoisin et al. 1989; Papke et al. 1989). The alpha 5 subunit has been reported to alter AChR dose-response curves and rates of desensitization (Ramirez-Latorre et al. 1996; Wang et al. 1996), and, together with other mechanisms, could alter the proportion of receptors functionally available on the neurons. The alpha 5 subunit, however, also contributes a mAb 35 binding site to the receptors (Conroy et al. 1992) and might increase the overall stoichiometry of antibody binding as a result. A change in the stoichiometry would inflate the estimated number of silent receptors. Definitive evidence for silent AChRs, apart from their occupying a reversibly desensitized state, will require different experimental approaches.

Whole cell recording and rapid application of agonist show that functional alpha 7-AChRs are present on the neurons at a time when the perisynaptic clusters are first forming. The receptors already display rapid desensitization at E8. The peak amplitude of the nicotine-induced current attributed to alpha 7-AChRs in freshly dissociated E14/15 neurons is about an order of magnitude greater than that of the alpha Bgt-resistant current attributed to alpha 3*-AChRs (Zhang et al. 1994). The number of alpha Bgt-binding sites in E15 CG homogenates is also about an order of magnitude greater than the number of mAb 35 binding sites (Corriveau and Berg 1994). Fluorescence imaging of alpha 7-AChRs in the present experiments shows that the receptors increase in number between E8 and E15 to the same extent as does the whole cell current generated by them. It is unclear whether any of the alpha 7-AChRs are functionally silent. The number of functional alpha 7-AChRs on CG neurons cannot be calculated both because their single-channel properties are unknown and because the receptors desensitize so rapidly. This latter feature introduces a large uncertainty into calculations of the maximal whole cell response produced by alpha 7-AChRs. It will be important in future work to examine the single channel properties of CG alpha 7-AChRs and to determine whether their function is regulated separately from changes in receptor number during development.

    ACKNOWLEDGEMENTS

  We thank Dr. Joseph F. Margiotta (Medical College of Ohio, Toledo) for comments on the manuscript, Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia) for providing monoclonal antibodies, and L. Ogden for excellent technical assistance.

  Grant support was provided by National Institute of Neurological Disorders and Stroke Grants NS-12601 and NS-35469, the Muscular Dystrophy Association, and the Tobacco-Related Diseases Research Program. E. M. Blumenthal is an MDA Postdoctoral Fellow.

    FOOTNOTES

   Present address of E. M. Blumenthal, Dept. of Biology, University of Virginia, Charlottesville, VA 22903.

  Address for reprint requests: D. K. Berg, Dept. of Biology, 035, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093.

  Received 22 May 1998; accepted in final form 25 September 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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