Articles, Cellular/Molecular

α7-Containing and Non-α7-Containing Nicotinic Receptors Respond Differently to Spillover of Acetylcholine

Doychin Stanchev and Peter B. Sargent
Journal of Neuroscience 19 October 2011, 31 (42) 14920-14930; https://doi.org/10.1523/JNEUROSCI.3400-11.2011

Abstract

We explored whether nicotinic acetylcholine receptors (nAChRs) might participate in paracrine transmission by asking if they respond to spillover of ACh at a model synapse in the chick ciliary ganglion, where ACh activates diffusely distributed α7- and α3-containing nAChRs (α7-nAChRs and α3*-nAChRs). Elevating quantal content lengthened EPSC decay time and prolonged both the fast (α7-nAChR-mediated) and slow (α3*-nAChR-mediated) components of decay, even in the presence of acetylcholinesterase. Increasing quantal content also prolonged decay times of pharmacologically isolated α7-nAChR- and α3*-nAChR-EPSCs. The effect upon EPSC decay time of changing quantal content was 5–10 times more pronounced for α3*-nAChR- than α7-nAChR-mediated currents and operated over a considerably longer time window: ∼20 vs ∼2 ms. Control experiments rule out a presynaptic source for the effect. We suggest that α3*-nAChR currents are prolonged at higher quantal content because of ACh spillover and postsynaptic potentiation (Hartzell et al., 1975), while α7-nAChR currents are prolonged probably for other reasons, e.g., increased occupancy of long channel open states. α3*-nAChRs report more spillover when α7-nAChRs are competitively blocked than under native conditions; this could be explained if α7-nAChRs buffer ACh and regulate its availability to activate α3*-nAChRs. Our results suggest that non-α7-nAChRs such as α3*-nAChRs may be suitable for paracrine nicotinic signaling but that α7-nAChRs may not be suitable. Our results further suggest that α7-nAChRs may buffer ACh and regulate its bioavailability.

Introduction

Nicotinic acetylcholine receptors (nAChRs) mediate rapid synaptic transmission in the peripheral nervous system. In the CNS nAChRs are thought to serve a modulatory role (Gotti and Clementi, 2004; Dani and Bertrand, 2007; Albuquerque et al., 2009). This thinking is based on several findings: cholinergic boutons are not usually located at synaptic sites (Umbriaco et al., 1994), nAChRs are often nonsynaptic (Fabian-Fine et al., 2001; Jones et al., 2001; Jones and Wonnacott, 2004), and activation of nAChRs by ACh modulates synaptic transmission or excitability (Mansvelder et al., 2002; Genzen and McGehee, 2003; Kawai et al., 2007; Wanaverbecq et al., 2007; Zhang and Berg, 2007).

The failure to find widespread evidence of colocalization of cholinergic boutons and nAChRs in the CNS has lead to the suggestion that nAChRs are involved in paracrine transmission, in much the same way that muscarinic AChRs function (Descarries et al., 1997; Vizi and Lendvai, 1999). However, at least two issues challenge acceptance of this notion: (1) the presence of acetylcholinesterase (AChE) at many sites of cholinergic projection (Mizukawa et al., 1986; but see Kawaja et al., 1990), and (2) the fact that nAChRs may not have a suitably low EC50 for ACh. While volume transmission seems unlikely generally (Sarter et al., 2009), paracrine transmission may be feasible given the possible presence in brain of nAChRs with micromolar affinity for ACh (Zwart and Vijverberg, 1998; Nelson et al., 2003; Moroni et al., 2006).

Neuronal nAChRs comprise a diverse set of pentameric Cys-loop receptors, which include heteromeric “α-β” nAChRs of several types as well as homomeric α7-nAChRs (Albuquerque et al., 2009). Recent evidence points to the participation of non-α7-nAChRs in paracrine transmission in the brain (Ren et al., 2011). α7-nAChRs might not be as well suited, since they desensitize rapidly (Bouzat et al., 2008); however, α7-nAChRs can be activated when singly bound (Rayes et al., 2009; Williams et al., 2011) and this, coupled with the presence of five presumptive binding sites for ACh, may allow them to respond to low concentrations of ACh and participate in paracrine signaling (Williams et al., 2011).

To explore the potential for α7-nAChRs and non-α7-nAChRs to participate in paracrine transmission we asked whether they respond to spillover of nerve-released ACh at a model calyciform synapse in the chick ciliary ganglion, where ACh acts on both α7-nAChRs and non-α7-nAChRs (α3-containing nAChRs, or α3*-nAChRs). We find that α3*-nAChRs readily “report” spillover of ACh, as evidenced by a broadening of synaptic currents at elevated quantal content (Hartzell et al., 1975), while α7-nAChR-mediated currents broaden only slightly. Curiously, when both nAChRs are active, α3*-nAChRs report less spillover than when α7-nAChRs are competitively blocked. Our results suggest that non-α7-nAChRs may be suitable for paracrine transmission, even in the presence of acetylcholinesterase, but that α7-nAChRs may not be suitable; our results further suggest that α7-nAChRs may buffer ACh and regulate its availability to non-α7-nAChRs.

Materials and Methods

Animals and materials.

Animal procedures were done in accordance with the University of California, San Francisco Institutional Animal Care and Use Committee. Chick embryos of either sex were obtained from the Avian Facility at University of California, Davis and were housed in a forced draft incubator at 37−38°C and 50–55% relative humidity until 14–15 d of incubation (Hamilton-Hamburger stages 39–41). Embryos were killed by decapitation, and ciliary ganglia were dissected in HEPES-buffered saline (containing, in mm: 147.5 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4, milliosmolarity: 310–315). Most experiments were performed in bicarbonate-buffered chick saline (containing, in mm: 130 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 1 Na2HPO4, 25 NaHCO3, pH buffered to 7.4 by bubbling with 95% O2-5% CO2, milliosmolarity: 310–315). Peptide neurotoxins were applied in HEPES-buffered saline without oxygenation and in the presence of 0.1 mg/ml protease-free bovine serum albumin. Internal solution contained the following (in mm): 120 Cs methanesulfonate, 15 TEA-Cl, 10 EGTA, 10 HEPES, 2 ATP, 0.2 GTP, and 5 QX-314, pH 7.2 (adjusted with CsOH), milliosmolarity: 295–305. Quantal content was varied by recording EPSCs at high [Ca2+]e (5 mm) and then by adding 25–50 μm CdCl2 or ω-agatoxin GIVA to the bath.

α-Conotoxin MII (α-CTx-MII) was kindly provided by Dr. Michael McIntosh (University of Utah, Salt Lake City, UT). Echothiophate (ECHO) was kindly provided by Wyeth-Ayerst (now Pfizer). Other reagents were purchased from Sigma-Aldrich except methyllycaconitine (MLA) and QX-314 (Tocris Bioscience) and ω-conotoxin GIVA (Invitrogen).

Electrophysiology.

Neurons were prepared for whole-cell recording as described previously (Sargent, 2009) in a process that entails perfusion via a coarse patch pipette with a combination of collagenase and thermolysin (Liberase III, Roche). Because collagenase can release AChE from the extracellular matrix and prolong nicotinic synaptic currents (Hall and Kelly, 1971; Betz and Sakmann, 1973), we explored the extent to which currents at this synapse were prolonged by inhibitors of AChE and compared it with results from other nicotinic synapses that were not enzyme-treated. The decay time (defined as τw, see below) of native EPSCs, produced by the activation of both α7-nAChRs and α3*-nAChRs, was prolonged 2.9-fold by 10 μm ECHO (n = 8 cells, p = 0.02). The decay time of pharmacologically isolated α3*-nAChR EPSCs (produced by blocking α7-nAChRs with 50 nm MLA) was increased 3.6-fold (n = 12, p < 0.0001). The broadening of EPSCs seen here is similar to the 2.5- to 3.5-fold broadening of synaptic currents reported in rat submandibular cells after inhibition of AChE by Rang (1981), who used sharp electrodes and did not expose cells to enzyme. This suggests that enzyme treatment of ciliary neurons does not remove enough AChE from the synaptic cleft to have obvious effects on EPSC time course.

Whole-cell recordings in voltage-clamp mode were made from ciliary neurons according to standard procedures using an Axopatch 200B amplifier (Molecular Devices) as described previously (Sargent, 2009). Neurons were usually held at −60 mV (−74 mV, when corrected for the calculated liquid junction potential of −14 mV). Series resistance was monitored during the recording, and experiments were discontinued if total series resistance exceeded 10 MΩ or if the current required to hold the cell at −60 mV exceeded −100 pA. Series resistance compensation (80%) was used using the Axopatch 200B, and in a few instances the remaining series resistance error was eliminated subsequently using the method of Traynelis (1998) as implemented in ChannelLab (Synaptosoft) and using a correction for a nonlinear IV curve (Sargent, 2009). Synaptic currents were analyzed using Neuromatic (www.neuromatic.thinkrandom.com, Jason Rothman) implemented in Igor Pro (Wavemetrics). Experiments were done at room temperature (21−23°C).

EPSC “rise time” in the text is 10–90% rise time. EPSC decay was calculated as the time constant (τ) of a mono- or biexponentially decaying function. For the biexponential function, I = Af · (e(−(t − t0)/τf)) + As · (e(−(t − t0)/τs)), where Af and As are the amplitudes of the rapidly and slowly decaying components of current, τf and τs are the time constants of decay, and t0 is the time from which the fit begins. The weighted τ for biexponential fits, τw, is equal to τf (Af/A) + τs (As/A), where A = Af + As.

The “sensitivity” of EPSC decay to changes in quantal content was assessed as Δτw/ΔA, with τw and A defined as above, before and after EPSCs were partially blocked by 25–50 μm cadmium, with each measurement derived from an average of 3–5 consecutive EPSCs. When only one component of decay was present, sensitivity was measured as Δτf/ΔAf or Δτs/ΔAs. By using current amplitude as a measure of quantal content we are assuming that quantal content is linearly related to peak response. Spillover may result in a nonlinear relationship between quantal content and current amplitude, and thus our assumption about linearity may not strictly be true; however, this does not change the interpretation of our results.

AChE was inhibited by exposure to either 10 μm ECHO or 0.1 μm neostigmine for >10 min. Native EPSCs treated with ECHO or with neostigmine had similar amplitudes (p = 0.6), rise times (p = 0.1), and decay times (p = 0.9; all comparisons by unpaired t test); in the Results section, we treat ECHO and neostigmine as equivalent. ECHO blocks ∼90% of AChE in the ganglion under the conditions used (Rogers and Sargent, 2003).

Statistical tests were made using PAST.exe (http://folk.uio.no/ohammer/past/index.html). Sample errors are given as SD, and samples were compared using the paired Student's t test, unless otherwise noted.

Control experiments.

One of the principal findings in Results is that reducing the quantal content of EPSCs with cadmium shortens decay time, and we will argue that this is the result of a reduction in quantal content mediated by blocking calcium channels in the nerve terminal. The possibility that the quantal response itself is altered in time course by cadmium is unlikely, since uniquantal evoked responses recorded in cadmium are similar in shape to mEPSCs recorded in the absence of cadmium (Sargent, 2009). This same finding argues against the possibility that the effect is due to a direct action of cadmium on nAChR channels; moreover, we get the same effect on EPSC decay time when we block calcium channels with ω-agatoxin GIVA instead of cadmium (data not shown). The possibility that the latency function (release probability vs time) is altered in cadmium is unlikely, since changes in decay time are not accompanied by a change in native EPSC rise time. To explore whether EPSC decay is affected by changing quantal size, unaccompanied by changes in quantal content, we reduced the holding potential from −60 mV to −20 mV (from −74 mV to −34 mV, when a correction is made for the liquid junction potential); this reduced EPSCs by 56 ± 2% but had no effect on EPSC decay (n = 4, p = 0.2). In addition, partially blocking α3*-nAChR-EPSCs (EPSCs recorded in 50 nm MLA) with the competitive nAChR antagonist dihydro-β-erythroidine did not change EPSC decay (data not shown). These results suggest that EPSC decay is influenced by changes in quantal content, but not by changes in quantal size.

To demonstrate that the broadening of kinetically fast α7-nAChR-EPSCs (recorded in α-CTx-MII) at high quantal content is not due to the residual 20% series resistance error, we corrected off-line for the remainder of the error (0.5–1 MΩ) using ChannelLab (Traynelis, 1998). Full correction of α7-nAChR-EPSCs increased their amplitude and shortened their decay time constants (data not shown). Fully corrected α7-nAChR-EPSCs still had a faster τ values for decay at low quantal content than at high quantal content (data not shown). Thus, residual series resistance errors do not contribute to the effect of changing quantal content upon τf. Finally, to demonstrate that the broadening of α7-nAChR-EPSCs at high quantal content is not due to a contribution from residual α3*-nAChR-mediated current that survives 0.3 μm α-CTx-MII (see Fig. 2C1), we calculated the residual α3-nAChR current likely to be present in 0.3 μm α-CTx-MII and subtracted it from total current to generate “pharmacologically cleaned” α7-nAChR-mediated currents. Pharmacologically cleaned α7-nAChR-EPSCs still showed shorter decay time constants in cadmium compared with high quantal conditions (data not shown).

Results

We explored whether α7-nAChRs and α3*-nAChRs respond to spillover of ACh at the calyciform synapse by noting whether changing quantal content produced systematic changes in EPSC decay. We define “spillover” as the ability of ACh to reach nAChRs not directly opposite sites of release (non-focal sites). Our approach mimics that of Hartzell et al. (1975), who found that synaptic currents decay more slowly at elevated quantal content when ACh released in separate quanta reaches common areas of the postsynaptic membrane. This effect, which Hartzell et al. (1975) called “postsynaptic potentiation,” results from the fact that the interaction of ACh at low concentration with its receptor operates in the supralinear part of the dose–response curve.

Figure 1 shows a schematic of the chick calyciform synapse at embryonic day 15. Our current understanding of this synapse, perhaps the best characterized interneuronal nicotinic synapse, comes from a combination of anatomical, physiological, and modeling studies (Jacob and Berg, 1983; Loring and Zigmond, 1987; Horch and Sargent, 1995; Zhang et al., 1996; Ullian et al., 1997; Shoop et al., 1999; Conroy et al., 2003; Coggan et al., 2005; Sargent, 2009). The synapse contains ∼500 contacts, of which ∼10% are located on spine mats, which are widely distributed over the cell surface. α3*-nAChRs are clustered at PSDs, but these nAChRs do not contribute significantly to the EPSC. Most of the response to nerve-released ACh is generated by the coactivation of α7-nAChRs and α3*-nAChRs; since α7-nAChRs are found preferentially on spines, most of the synaptic current is presumably generated by ACh release onto spines, where it acts on diffusely distributed α7-nAChRs and α3*-nAChRs (Fig. 1).

Figure 1.
Figure 1.

Schematic of the calyciform synapse in the chick ciliary ganglion. Low-magnification view (bottom) is of a ciliary neuron with its calyciform (calyx-like) input. High-magnification view (top) is of a spine mat, showing a clump of interdigitating spines cut in cross-section (Shoop et al., 2002). One synaptic contact is shown on the soma and a second on a spine. α3*-nAChRs (light gray triangles) are clustered at PSDs, and both α3*-nAChRs and α7-nAChRs (dark gray ovals) are diffusely distributed in the extrasynaptic membrane on spines. Transmitter release may be active zone based (Nguyen and Sargent, 2002) and/or ectopic (Coggan et al., 2005); in either case the functional receptors recognized by ACh are largely extrasynaptic and located on spines (Sargent, 2009).

α7-nAChRs and α3*-nAChRs differ in their response to changes in quantal content

To explore whether α7-nAChRs and α3*-nAChRs respond differently to changes in quantal content, we took advantage of differences in their channel kinetics (Zhang et al., 1996; Ullian et al., 1997; McNerney et al., 2000; Nai et al., 2003) and fit EPSC decays to the sum of two exponentially decaying functions, fast (α7-nAChR) and slow (α3*-nAChR), under conditions of high and low quantal content. Figure 2A1 shows a biexponential fit (dotted gray line) to an EPSC recorded in 5 mm [Ca2+]e; in this example, the time constants for the fast (τf, red) and slow (τs, green) phases of decay were 1.3 ms and 9.5 ms. Over a sample of 8 cells, τf and τs were 1.42 ± 0.25 ms and 10.5 ± 1.5 ms and similar to those reported earlier (Zhang et al., 1996; Ullian et al., 1997). After addition of 50 μm Cd2+, EPSC amplitude was reduced >7-fold (Fig. 2A2a, inset), and both the fast and slow components of decay were shortened: τf was reduced from 1.3 to 0.8 ms, and τs was reduced from 9.5 to 8.0 ms (Fig. 2A2b). Over 8 experiments, both τf and τs were reduced significantly when quantal content was lowered: τf was reduced by 13 ± 15% (p = 0.04, Fig. 2A3, left) and τs was reduced by 20 ± 5% (p = 0.0001; Fig. 2A3, right). Fitted τ values are summarized in Table 1 along with the current amplitudes (A). Rise time was not affected (0.73 ± 0.06 ms at high quantal content, p = 0.8). This suggests that currents mediated by both α7-nAChRs and α3*-nAChRs decay more rapidly when quantal content is reduced. In the presence of cadmium, EPSCs were generally 0.5–1.0 nA in amplitude and multiquantal, since mEPSCs are ∼50 pA in amplitude (Sargent, 2009). Spontaneously occurring mEPSCs and uniquantal evoked responses are yet faster that the multiquantal EPSCs recorded in cadmium and shown here (Sargent, 2009).

Figure 2.
Figure 2.

Changing quantal content alters EPSC decay mediated by both α3*-nAChRs and α7-nAChRs. A–C show results for native EPSCs (A), α3*-nAChR-EPSCs (B), and α7-nAChR-EPSCs (C). A1 shows a native EPSC (each trace is the average of 3–5 consecutive responses) with a decay that is well fit by the sum of two exponential functions: a fast τf of 1.3 ms (red, α7-nAChRs) and a slow τs of 9.5 ms (green, α3*-nAChRs). A2a and A2b show normalized traces at high and low quantal content, illustrating that both decay components are prolonged at high quantal content (inset in A2a shows traces before normalization). A2b shows fits on a semilog graph with the currents reversed in sign. The legend for A2 and τ values for the fits in A2b are shown in the box beneath the figure. A3 shows data summary for τf (left) and τs (right), both of which are reduced significantly at low quantal content (low m). B shows the same set of graphs as in A, except that recordings made in 50 nm MLA to block α7-nAChRs; EPSCs are thus α3*-nAChR-EPSCs. EPSC decay is usually well fit by a single exponentially decaying function (τs), and thus there is no graph in B3 for fast decay. C shows the same set of graphs as in A, except that recordings were made in 0.3 μm α-CTx-MII, which blocks ∼90% of α3*-nAChRs. EPSCs recorded in α-CTx-MII show a small amount of residual α3*-nAChR-mediated current (green trace in C1). Both τf and τs are again reduced at low quantal content, as for native EPSCs (A). C3, left, contains 5 data pairs but appears to contain only 3, due to data point superposition.

View this table:
Table 1.

Fitted decay time constants (τf and τs) and weights (Af and As) for fast (f) and slow (s) phases of EPSC decay under conditions of high and low quantal content (m)

To improve the resolving power of our approach, we repeated the experiments after blocking α7-nAChRs or α3*-nAChRs to produce pharmacologically enriched “α3*-nAChR-EPSCs” or “α7-nAChR-EPSCs,” respectively. When α7-nAChRs are fully blocked by 50 nm MLA, α3*-nAChR-EPSC decay in 5 mm [Ca2+]e was well fit usually with a single exponential function having a time constant of 8.9 ± 1.0 ms (in 10 of 12 cells; in two cells a second, longer τ was present); this τ was comparable to the slow phase of decay, τs, in native EPSCs. At low quantal content this τ was reduced by 16 ± 6% (p < 0.0001; Fig. 2B3). Figure 2, B1 and B2, shows an experiment where this τ was shorted from 10.1 ms to 7.8 ms by reducing quantal content. Addition of cadmium also shortened the rise time of α3*-nAChR-EPSCs by 24 ± 13% (from 2.38 ± 0.41 ms, p = 0.003).

When α3*-nAChRs were 90% blocked by 0.3 μm α-CTx-MII, resultant α7-nAChR EPSCs decayed biexponentially, with a small component of slowly decaying current arising from residual, unblocked α3*-nAChRs (Fig. 2C1, green; compare with Fig. 2A1). At high quantal content these α7-nAChR-EPSCs had a τf of 1.40 ± 0.17 ms and a τs of 10.6 ± 2.8 ms (not different from τ values for native EPSCs, p > 0.8 by unpaired t test). Reducing quantal content significantly reduced both τf and τs (Fig. 2C3); τf was reduced by 41 ± 14% (p = 0.001), and τs was reduced by 47 ± 19% (p = 0.003). Figure 2, C1 and C2, shows an example from a cell where cadmium reduced τf from 1.4 to 1.0 ms and τs from 10.5 ms to 8.1 ms. Addition of cadmium did not affect the rise time (p = 0.11), which is consistent with results on native EPSCs, where rise time is dominated by the α7-nAChR current. In summary, changing quantal content alters the decay of synaptic currents generated by activation of both α7-nAChRs and α3*-nAChRs (Table 1). Rise time is altered for α3*-nAChR-EPSCs but not for α7-nAChR-EPSCs.

One possible basis for the systematic change in the decay of currents mediated by α7-nAChRs and α3*-nAChRs is spillover; by analogy with the work of Hartzell et al. (1975), elevating quantal content allows ACh from separate quanta to reach overlapping areas of postsynaptic membrane; this, coupled with the nonlinear dose–response curve for nAChRs, produces postsynaptic potentiation (Hartzell et al., 1975). In control experiments (see Materials and Methods) we rule out a presynaptic source for the changes in EPSC time course produced by changes in quantal content.

The sensitivity of synaptic currents to changes in quantal content, expressed as change in decay τ (ms) per unit change in amplitude (nA), is markedly greater for α3*-nAChRs than for α7-nAChRs, as can be seen qualitatively by comparing the normalized traces at high and low quantal content (Fig. 2B2a vs Fig. 2C2a). The sensitivity of τs (α3*-nAChRs) to changes in quantal content is 0.76 ± 0.28 ms/nA when measured on native EPSCs and 1.20 ± 0.84 ms/nA when measured on α3*-nAChR EPSCs recorded in 50 nm MLA (p = 0.2 by unpaired t test). The sensitivity of τf (α7-nAChRs) is 5–10 times smaller: 0.09 ± 0.11 ms/nA when measured on native EPSCs (p = 0.0005) and 0.18 ± 0.10 ms/nA when measured on α7-nAChR EPSCs recorded in 0.3 μm α-CTx-MII (p = 0.02, unpaired t test).

Prolongation of EPSCs at high quantal content is not due to delayed release

We performed three experiments to address the possibility that delayed/asynchronous release, which is calcium dependent (Atluri and Regehr, 1998; Oleskevich and Walmsley, 2002), underlies the prolongation of EPSC decay time at high quantal content. In the first, we looked for evidence of delayed release late in the falling phase of individual EPSCs and at the end of a stimulus train. Delayed release is difficult to detect during the steeply decaying part of the EPSC. Between 20 and 40 ms after the EPSC peak, however, when the current approaches baseline and when individual quantal events are readily detectable, we found that delayed release events were uncommon and occurred at an overall frequency of 1.0 Hz (Fig. 3A1,A2; n = 8 cells, 15–100 trials per cell).

Figure 3.
Figure 3.

Delayed/asynchronous release is not prominent at the calyciform synapse in the chick ciliary ganglion. Traces A1 and A2 show typical EPSCs at both low and high gain (scale is in A2). Arrow in A2 identifies a delayed release event, assumed to be monoquantal. MEPSC frequency normally is <0.1 Hz (Sargent, 2009). B1 and B2 show responses to trains of 40 stimuli delivered at 50 Hz (examples from two different cells), with insets showing higher gain at the end of the train (a) and after the end of the train (b). Transmitter release remains largely synchronous at the end of the train (inset a), and asynchronous events after the end of the train are not common (inset b). C1 shows peak EPSC amplitude (evoked at 0.033 Hz) as EGTA-AM is added to the bath; peak response declines, but time course is not altered substantially (C2). EPSCs in C2 were generated from averages of five consecutive responses, indicated by boxes in C1. EPSC decay time, measured as τw, is not altered over a sample of five cells (C3). A 10% reduction in decay time would be expected on the basis of the effect of EGTA-AM on quantal content (see Results for details).

Delayed release is enhanced by increasing the number of stimuli (Atluri and Regehr, 1998; Hagler and Goda, 2001); we therefore noted the effect of trains of 40 stimuli at 50 Hz on the appearance of delayed release, which should be especially evident at and after the end of the train. Figure 3B shows typical responses, collected in 2 mm [Ca2+]e and 1 mm [Mg2+]e, to 50 Hz stimulation. Virtually all release during the train is synchronous, although a few asynchronous events are evident (Fig. 3B, insets). Similar results were obtained in six other cells. These findings suggest that delayed release is not a prominent feature of transmission at this synapse.

If delayed release contributes significantly to the falling phase of EPSCs, then reducing bulk calcium in the nerve terminal should shorten EPSC decay. We thus explored the effect of EGTA-AM, which should block delayed release more readily than synchronous release after permeating the terminal and being hydrolyzed to EGTA (Atluri and Regehr, 1998; Hagler and Goda, 2001; Otsu et al., 2004). Addition of 50 μm EGTA-AM to cells bathed in 5 mm [Ca2+]e reduced EPSC size (Fig. 3C1) but did not shorten decay time significantly (Fig. 3C2,C3, n = 5 cells, p = 0.3). Since EGTA-AM reduced synchronous release, we expected to see a reduction in EPSC decay solely because of its reduction of quantal content; however, the expected reduction in decay is only ∼10%, which may explain why it was not detected. (The expected shortening was calculated from the change in current amplitude and from the average sensitivity of τw to changes in quantal content: 0.28 ms/nA.) We conclude that delayed release does not contribute detectably to EPSC decay at the calyciform synapse.

Blocking AChE enhances the sensitivity of synaptic currents to changes in quantal content

If spillover underlies the prolongation of EPSC decay with elevated quantal content, then inhibiting AChE within the synaptic cleft at this synapse (Olivieri-Sangiacomo et al., 1983) should increase the sensitivity of EPSC decay to changes in quantal content, since it should afford greater opportunity for ACh released in separate quanta to reach overlapping areas of the postsynaptic membrane (Hartzell et al., 1975). We thus repeated the cadmium protocol after inhibiting AChE with either 0.1 μm neostigmine or 10 μm ECHO (similar results were obtained with the two inhibitors; see Materials and Methods). Inhibiting AChE prolonged synaptic currents (Zhang et al., 1996); decay time, measured as τw, was lengthened from 4.4 ± 1.2 ms to 14.4 ± 10.6 ms, while rise time was not changed (Fig. 4A1,A2, n = 8 cells, p = 0.02). To explore the effects of inhibiting AChE separately on α7-nAChR- and α3*-nAChR-mediated currents, we studied the effect of ECHO on τf and τs of biexponentially fitted EPSC decays. Blocking AChE with ECHO increased τs from 9.7 ± 1.8 ms to 13.6 ± 5.2 ms (n = 9, p = 0.02) but had no effect on τf (p = 1.0), as reported originally by Zhang et al. (1996). To increase the resolving power of these experiments, we treated pharmacologically enriched α7-nAChR-EPSCs and α3*-nAChR-EPSCs with AChE inhibitors, and here we saw an effect on both components of decay; τs measured in α3*-nAChR-EPSCs, was prolonged from 8.0 ± 2.0 ms to 13.0 ± 5.6 ms after blocking AChE (n = 12, p = 0.01; not illustrated), and τf, measured as the fast component of α7-nAChR-EPSC decay, was prolonged from 1.5 ± 0.3 ms to 2.0 ± 0.4 ms (n = 5, p = 0.0001; not illustrated). These results extend the original findings of Zhang et al. (1996) and suggest that currents produced by both α7-nAChRs and α3*-nAChRs are prolonged by inhibiting AChE at the calyciform synapse.

Figure 4.
Figure 4.

Inhibition of AChE prolongs EPSC decay time and increases sensitivity of decay time to changes in quantal content. A shows effect on EPSC time course of inhibiting AChE with ECHO. A1 shows an example from one cell, where decay time is prolonged but rise time is not evidently changed, and A2 shows the data summary (n = 8 cells). B shows that reducing quantal content shortens decay time of EPSCs recorded in neostigmine (B1), as is true for native EPSCs (Fig. 2), and that the sensitivity of decay time to changes in peak amplitude is greater after treatment with neostigmine (neo.) than in its absence (control) (B2). C is similar to B, except that recordings were made 50 nm MLA to produce α3*-nAChR-EPSCs. Again, decay time showed more sensitivity to changes in quantal content after inhibition of AChE (neo.) than in control conditions (C2).

The decay time of EPSCs showed more sensitivity to changes in quantal content after inhibition of AChE than in control conditions. In the example shown in Figure 4B1 decay time of native EPSCs was reduced by 0.76 ms per nA of peak current change by cadmium. Overall, the sensitivity of EPSC decay was enhanced more than fivefold compared with conditions with intact AChE (Fig. 4B2, “control” vs “neo.,” p = 0.001). We repeated these experiments on α3*-nAChR EPSCs recorded in the presence of MLA (Fig. 4C); again, sensitivity after blocking AChE was greater than sensitivity in controls (Fig. 4C2, p < 0.0001). Thus, the sensitivity of EPSC decay time to changes in quantal content is increased after inhibition of AChE, both for native EPSCs and for α3*-nAChR-EPSCs. This is consistent with the hypothesis that altering quantal content affects EPSC decay by changing the consequences of spillover.

Channel reopenings cannot explain increased EPSC decay times at high quantal content

If transmitter release is multivesicular, then elevating quantal content may increase peak ACh concentration and prolong currents due to the repeated opening of nAChR channels near sites of release (Giniatullin et al., 1993). Thus, EPSC prolongation might be caused by reopenings of channels that lie physically close to release sites rather than by openings of naive channels that lie some distance away, i.e., true spillover. Repeated activation of channels should be antagonized by chlorisondamine (el-Bizri and Clarke, 1994; Amador and Dani, 1995), which preferentially blocks open channels and should prevent them from recycling rapidly.

To explore whether channel reopenings contribute to the prolongation of EPSC decay at high quantal content, we asked whether chlorisondamine is more effective at reducing EPSC decay at high quantal content than at low quantal content. We performed these experiments on pharmacologically isolated α3*-nAChR EPSCs, since these currents show more sensitivity to changes in quantal content than do α7-nAChR-mediated currents. To account for the possibility that chlorisondamine might wash out only slowly after receptor block, the low quantal content conditions were performed first. Figure 5A shows the results of a typical experiment; EPSCs were first recorded at low quantal content, both before and after addition of chlorisondamine; then, after washing out the cadmium and chlorisondamine, they were measured at high quantal content, both before and after addition of chlorisondamine. In this trial chlorisondamine shortened EPSC decay 57% at low quantal content, and it subsequently shortened EPSC decay at high quantal content by 47% (Fig. 5B1,B2). Over 6 experiments chlorisondamine shortened EPSC decay by a similar extent at low and at high quantal content (47 ± 15% vs 43 ± 15% block, respectively, Fig. 5B3, p = 0.7). These results indicate that channel reopening does not contribute detectably to the prolongation of EPSCs at high quantal content; EPSCs decay more slowly at higher quantal content because of the delayed opening of naive channels, as would be expected from spillover of ACh and its action on distant nAChRs. These findings are equally compatible with the possibility that there are no channel reopenings whatsoever and with the possibility that there is a comparable degree of reopening at low and high quantal content.

Figure 5.
Figure 5.

The open channel blocker chlorisondamine reduces α3*-nAChR EPSC decay times equally well at low and at high quantal content. A monitors EPSC amplitude (top) and decay time (τ, bottom) as chlorisondamine is washed in at low quantal content and then, after washout of it and cadmium, after chlorisondamine is reapplied at high quantal content. The reduction of EPSC decay at low (B1) and high (B2) quantal content (low m, high m) was not different across a sample of 6 cells (B3).

Contributions to synaptic currents from α7-nAChRs and from α3*-nAChRs do not add linearly

The decay of α3*-nAChR-EPSCs shows 5- to 10-fold more sensitivity to changes in quantal content, measured as ms/nA, than that of α7-nAChR-EPSCs, as reported above. Surprisingly, when both α7-nAChRs and α3*-nAChRs are activated by transmitter, as occurs in native conditions, the sensitivity of EPSC decay (0.28 ± 0.17 ms/nA, measured as Δτw/ΔA) is modest and only marginally greater than that of the α7-nAChR-mediated τf (p = 0.03 when compared with τf sensitivity for native EPSCs and p = 0.25 when compared with τf sensitivity for α7-nAChR-enriched EPSCs recorded in α-CTx-MII). By contrast, the sensitivity of native EPSC decay was significantly less than that of the α3*-nAChR τs, measured either on native EPSCs (p = 0.003) or on α3*-nAChR-EPSCs recorded in MLA (p = 0.007). If α7-nAChRs and α3*-nAChRs function independently, then the sensitivity of native EPSC decay time to changes in quantal content should be intermediate between that of α7-nAChR-EPSCs (∼0.2 ms/nA) and that of α3*-nAChR-EPSCs (∼1.0 ms/nA), but it was evidently not. To confirm that native EPSC decay should show an intermediate degree of sensitivity, assuming linear addition of currents from the two nAChRs, we generated fits to representative α7-nAChR- and α3*-nAChR-EPSCs using a modification of the fitting function described by Bekkers and Clements (1999), collected at both low and high quantal content, and then summed the fits (Fig. 6). The decay of summed waveforms (Fig. 6C) indeed showed an intermediate degree of sensitivity to changes in quantal content, 0.57 ± 0.17 ms/nA (n = 3), which was greater than the sensitivity of native EPSC decay (p = 0.04 by unpaired t test). The sensitivity of summed EPSCs (Fig. 6) to changes in quantal content are not different from the sensitivity of the α3*-nAChR-mediated τs of native EPSCs (p = 0.3) or of the α3*-nAChR-EPSC τs recorded in MLA (p = 0.2). Thus, the sensitivity of α3*-nAChR current to changes in quantal content should be pronounced in native EPSCs, but it's barely detectable. This can be visualized graphically by comparing Figure 6C, right (summed EPSC), and 6D, right (actual EPSC); the native condition (Fig. 6D) does not replicate the “simulated” one (Fig. 6C). It is as if α3*-nAChRs, which contribute significantly to EPSC broadening when acting on their own (Fig. 6C, right), contribute less when α7-nAChRs are also available to bind ACh (Fig. 6D, right). This may occur because α7-nAChRs buffer ACh and prevent it from reaching α3*-nAChRs: a prediction made by MCell modeling of this synapse (Coggan et al., 2005).

Figure 6.
Figure 6.

The decay time of simulated EPSCs shows more sensitivity to changes in quantal content than that of native EPSCs. A–D, Simulated EPSCs were generated by taking pharmacologically isolated α7-nAChR (A) and α3*-nAChR EPSCs (B), with τ values that fell within 20% of the sample median, scaling them to the sample mean amplitude at low and high quantal content, and by then adding the currents to produce “summed” native EPSCs (C). The decay time of summed native EPSCs shows more sensitivity to changes in quantal content than native EPSCs (compare C, right, to D, right). This comparison suggests that synaptic currents generated by α7-nAChRs and α3*-nAChRs do not add linearly when both nAChRs are active. See text for additional discussion.

Discussion

EPSCs at the chick calyciform synapse decay more slowly at higher quantal content. Control experiments rule out a presynaptic basis for this effect, and for α3*-nAChRs we suggest that the broadening of the EPSC is caused by spillover; by analogy with findings first reported at the neuromuscular junction, ACh released in separate exocytotic events reaches overlapping areas of the postsynaptic membrane, activates nAChRs there, and prolongs and potentiates the response (Hartzell et al., 1975; Magleby and Terrar, 1975). This effect differs from the original reports at the neuromuscular junction in that it occurs in the presence of acetylcholinesterase. An alternative possibility, that the prolongation results from a lengthening of channel open times, seems unlikely given that the time window over which currents are potentiated, ∼20 ms (Fig. 2B), is considerably longer than the longest population of channel opening times observed for α3*-nAChRs on ciliary neurons, which is 3–4 ms (McNerney et al., 2000; Nai et al., 2003). These findings extend our previous work (Sargent, 2009), which showed that extrasynaptic nAChRs dominate the response to nerve-released ACh. It is not clear whether extrasynaptic receptors at this synapse are activated focally by ectopic release (Coggan et al., 2005) and/or by spillover following active zone-based release (Nguyen and Sargent, 2002). An active zone-based model of release requires spillover of ACh to explain transmission at this synapse, and here we show that spillover indeed occurs.

The effect of changing quantal content upon α7-nAChR-mediated current decay is barely one-tenth of the magnitude of the effect upon α3*-nAChR-mediated current, and it occurs without a detectable change in rise time. The potentiation of α7-nAChR-mediated currents is confined to a 1–2 ms time window (Fig. 2C2a): approximately a tenth of the time window over which α3*-nAChR-mediated currents are potentiated. We suggest that spillover does not underlie the broadening of current over this restricted time frame. A more likely possibility is that the prolongation of α7-nAChR-mediated current results from increased occupancy of longer channel opening states; α7-nAChR channels on chick ciliary neurons display multiple populations of open times, including intermediate and long states with lifetimes of ∼0.7 and ∼2 ms (Nai et al., 2003); possibly, average open time is prolonged when the receptor is activated by more agonist molecules (Beato et al., 2002; Rayes et al., 2009; Williams et al., 2011), as might occur when quantal content is elevated. Additional study will be required to learn whether elevated [ACh] is likely to result from elevated quantal content at this synapse; this would be expected if release is active zone based and multivesicular at high probability of release (Wadiche and Jahr, 2001).

The ability of α3*-nAChRs, but less so α7-nAChRs, to report spillover of ACh at higher quantal content is not likely to have arisen because of differences in the distribution of nAChRs, but we cannot rule out this possibility with certainty. Both α3*-nAChRs and α7-nAChRs are intermixed and appear to be distributed diffusely on somatic spines (Fig. 1) (Horch and Sargent, 1995; Conroy et al., 2003); these receptors, and not those located at PSDs, are responsible for the bulk of the synaptic current (Sargent, 2009).

The anatomy of the chick calyciform synapse (Fig. 1) is well suited for spillover of ACh to feature prominently in transmission (Barbour and Häusser, 1997): (1) the area of apposition between terminal and target cell is substantial, which will force ACh to travel a considerable distance before escaping the cleft (Kinney et al., 1997); (2) nAChRs are widely distributed on spines (Horch and Sargent, 1995; Shoop et al., 1999; Conroy et al., 2003); and (3) release sites may be widely distributed over the spine membrane (Coggan et al., 2005). Given this, it is worth exploring why α7-nAChRs show less tendency to respond to spillover. A complete picture of the channel properties of native α7-nAChRs has yet to emerge; however, chimeric α7-nAChR-5-HT3A receptors, and perhaps native α7-nAChRs, are efficiently activated only when three of their binding sites are occupied (Rayes et al., 2009), which means that their ability to respond to ACh release should decline steeply with distance from a release site.

Our findings replicate those reported for multisite glutamatergic synapses in the CNS (Takahashi et al., 1995; Otis et al., 1996; Silver et al., 1996) and those where independent inputs to a target cell are close enough to allow for cooperative activation of receptors (Carter and Regehr, 2000; Arnth-Jensen et al., 2002; Balakrishnan et al., 2009). The time course of nicotinic synaptic currents recorded from rat submandibular ganglion cells is not altered upon the addition of sufficient cadmium to reduce the EPSC by ∼90% (Callister and Walmsley, 1996), suggesting that at this synapse cooperation between independent release events does not occur. This difference could arise because the relationship between release sites and nAChR clusters is more conventional in the rat system, with active zones aligned with high density nAChR clusters (McCann et al., 2008).

Giniatullin et al. (1993) found that EPSC decay at the frog neuromuscular junction lengthened with increasing quantal content, but only after quantal content exceeded ∼100; they argued that the prolongation in EPSC decay is driven by the onset of multivesicular release and is caused by repeated opening of focal nAChR channels. However, at the calyciform synapse our results with the open channel blocker chlorisondamine suggest that reopening of nAChR channels cannot explain EPSC broadening at high quantal content; rather, the broadening presumably represents naive receptor channels that open late, presumably because they are distant from release sites.

α7-nAChRs may not report spillover in our system; to the contrary, they appear to reduce the tendency of α3*-nAChRs to report spillover. This property was predicted by an MCell modeling study of transmission at this synapse (Coggan et al., 2005); α7-nAChRs may be optimized in this system to buffer ACh and regulate its availability to α3*-nAChRs because of their high copy number (Chiappinelli and Giacobini, 1978; Conroy et al., 1992; Pugh et al., 1995), because each α7-nAChR oligomer may bind five ACh molecules (Palma et al., 1996; Rayes et al., 2009), and because α7-nAChRs and α3*-nAChRs are intermixed on the surface of spines (Fig. 1) (Horch and Sargent, 1995; Conroy et al., 2003). The possibility that α7-nAChR activation negatively regulates α3*-nAChR function via a calcium-mediated intracellular signaling pathway, as it does GABAA receptors in other systems (Wanaverbecq et al., 2007; Zhang and Berg, 2007), is unlikely, since our recordings are made with elevated EGTA in the internal solution, which blocks the pathway regulating GABAA receptor function. We speculate that α7-nAChRs, which are found in perisynaptic locations elsewhere in the nervous system (Fabian-Fine et al., 2001; Jones and Wonnacott, 2004) and which are numerous in the brain, may buffer ACh and regulate its availability to non-α7-nAChRs. This proposed role is analogous to that demonstrated for the acetylcholine binding protein of the mollusc Lymnea stagnalis (Smit et al., 2001), a secreted protein with high homology to the extracellular domain of α7-nAChRs.

What is the relevance of this work to the ongoing dialogue about paracrine nicotinic transmission in the CNS? One of the enduring questions regarding central nAChRs is how they are activated by endogenous transmitter. An accompanying set of questions includes whether ACh is the endogenous ligand for nAChRs (Papke et al., 1996; Alkondon et al., 1997, 2004) and whether modulation of neuronal signaling in adult brain is the principal function of nAChRs (McLaughlin et al., 2003; Chernyavsky et al., 2004; Liu et al., 2006; Morishita et al., 2010). The number of instances of functionally characterized fast nicotinic synapses in the CNS is modest, and paracrine mechanisms may be at play (Descarries et al., 1997; Dani and Bertrand, 2007; Ren et al., 2011). Might ACh diffuse short distances (∼1–10 μm) from its release site to act on nearby nAChRs? In conjunction with a sufficient number of nAChRs, even micromolar ACh may be capable of opening a sufficient number of nAChR channels to produce a meaningful signal. This scenario has been illustrated in the medial habenula by Guo and Lester (2007); there, non-α7 nAChRs with a subunit composition similar to nAChRs in the ciliary ganglion (with α3, α5, β2, and β4 possibly present in individual nAChR pentamers) respond to low concentrations of ACh, and possibly to choline, in a sustained fashion.

Many cholinergic boutons in brain have no associated postsynaptic elements (Descarries et al., 1997), and many immunocytochemically demonstrable nAChRs have no apposing presynaptic elements (Jones and Wonnacott, 2004); the details of what lies between looms as a “missing link” in our understanding of endogenous nicotinic signaling in brain. Further work should be done on the anatomical arrangement of cholinergic boutons and “nonsynaptic” nAChRs: 3D reconstructions are needed, combined with high resolution visualization of nAChRs (Jones and Wonnacott, 2004). This, coupled with additional information about the levels of AChE present, would then permit a model to be constructed. Finally, to gain acceptability, one will need to stimulate cholinergic boutons and demonstrate the consequences of ACh release on nearby cells bearing nAChRs (Wanaverbecq et al., 2007; Ren et al., 2011).

Footnotes

  • This work was supported by NIH Grant R01 MH068690. We thank Philippe Ascher for many stimulating discussions, Steven Traynelis for assistance with ChannelLab, Michael McIntosh for the gift of α-conotoxin MII, Jackie Pisenti and the Avian Facility at University of California, Davis for supplying chick embryos, Tom Babcock for making Figure 1, and Philippe Ascher, Bill Betz, Tom Bartol, Jay Coggan, and Joseph Margiotta for comments on this manuscript.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Dr. Peter B. Sargent, School of Dentistry Dean's Office, Box 0430, University of California, San Francisco, CA 94143-0430. peter.sargent{at}ucsf.edu

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α7-Containing and Non-α7-Containing Nicotinic Receptors Respond Differently to Spillover of Acetylcholine
Doychin Stanchev, Peter B. Sargent
Journal of Neuroscience 19 October 2011, 31 (42) 14920-14930; DOI: 10.1523/JNEUROSCI.3400-11.2011
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