Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors

Abstract

We analyzed the kinetics and pharmacology of EPSCs in two kinds of neurons in the embryonic avian ciliary ganglion. Whole-cell voltage-clamp recordings revealed that the singly innervated ciliary neurons had large-amplitude (1.5-8.0 nA) EPSCs that could be classified according to the kinetics of their falling phases. Most of the neurons responded with an EPSC the falling phase of which followed a double exponential time course with time constants of approximately 1 and 10 msec. The EPSCs of the remaining ciliary neurons followed a single time constant ( approximately 8 msec). Multiple innervated choroid neurons had smaller-amplitude responses (0.2-1.5 nA when all inputs were activated) that appeared to contain only a slowly decaying component (tau = 12 msec). The fast and slow components of EPSC decay seen in most ciliary neurons could be pharmacologically isolated with two toxins against nicotinic acetylcholine receptors (AChRs). The fast component was blocked by 50 nM alpha-bungarotoxin (alpha-BuTx), which binds alpha7-subunit-containing AChRs. The slow component was selectively blocked by 50 nM alpha-conotoxin MII (alpha-CTx-MII), which blocks mammalian AChRs containing an alpha3/beta2 subunit interface. A combination of both alpha-BuTx and alpha-CTx-MII abolished nearly all evoked current. Similar pharmacological results were found for ciliary neurons with monoexponentially decaying EPSCs and for choroid neurons. These results suggest that nerve-evoked transmitter acts on at least two different populations of AChRs on autonomic motor neurons in the ciliary ganglion.

Fig. 1.

Ciliary and choroid neurons can be distinguished by the extent of synaptic convergence. The two panels show multiple, superimposed records of evoked synaptic currents recorded from a ciliary and a choroid neuron. Inward current is represented by a downward deflection. Preganglionic axons within the oculomotor nerve were gradually recruited by increasing the stimulus intensity delivered to the nerve via a suction electrode. In the left panel, a stimulus intensity of 1–3 V caused no synaptic current, whereas a stimulus of >3 V caused a large synaptic current that fluctuated little from trial to trial. When the same paradigm was applied to a choroid neuron, the EPSC increased in several increments, each with a well-defined threshold. The choroid neuron record shows two responses at each of four stimuli, one of which elicited no response. This neuron had a minimum of three independently elicitable inputs.

Fig. 2.

Ciliary neuron EPSC decay is either biexponential or monoexponential. A, A biexponential ciliary EPSC. The EPSC falling phase was best fit with two exponentials as assessed by the maximum-likelihood estimation method (pClamp 6.0).B, The EPSC replotted in semilogarithmic coordinates. The two exponential components can be seen qualitatively as linear phases of the current decay. C, A monoexponential ciliary EPSC. The falling phase of the EPSC was best fit with a single exponential. D, The EPSC replotted in semilogarithmic coordinates. The single exponential component can be seen qualitatively as a linear phase.

Fig. 3.

The cumulative distribution of the relative amount of rapidly decaying current in ciliary EPSCs suggests two distinct ciliary neuron populations. All ciliary neuron EPSCs were fitted assuming two phases of decay, and the ratio of the amplitude of the fast component to the total current (at 90% of peak) is displayed in a cumulative distribution plot. The plot shows a break in the distribution of amplitude ratios, with one group showing 20% or more of the fast component and another group showing 5% or less of the fast component.

Fig. 4.

The time constant of decay for the slow phase of biexponential ciliary neurons differs from that of monoexponential ciliary neurons. Shown is a cumulative probability plot of the slow component of decay of biexponential ciliary EPSCs and the single component of decay of monoexponential ciliary EPSCs. The two populations of τ are significantly different (p = 0.016 by Student’s ttest). This indicates that the biexponential and monoexponential neurons constitute two distinct classes of neurons, on the basis of the time constant of the slow phase of decay.

Fig. 5.

Effect of 50 nm α-BuTx (A) and 50 nm α-CTx-MII (B) on ciliary neuron biexponential EPSCs. Theleft trace in both A and Bshows the EPSC from a ciliary neuron recorded before toxin addition. The middle trace shows the EPSC of the same cell after stabilization of the toxin effect. The right trace shows the semilog plots of the before- and after-toxin EPSCs. The rapidly decaying phase of current is eliminated by α-BuTx, whereas the slowly decaying phase is eliminated by α-CTx-MII.

Fig. 6.

α-CTx-MII and α-BuTx together block the ciliary EPSC. The left panel shows a biexponential ciliary EPSC, the middle panel shows the EPSC of the same cell after addition of 50 nm α-CTx-MII, and theright panel shows the EPSC of the same cell after further addition of 50 nm α-BuTx.

Fig. 7.

Effect of 50 nm α-BuTx (A) and 50 nm α-CTx-MII (B) on ciliary neuron monoexponential EPSCs. See legend to Figure 5 for complete description of traces. α-BuTx reduces the peak of the EPSC but does not change its time course of decay (A). α-CTx-MII reduces the EPSC size substantially, eliminates the slowly decaying phase of current, and reveals a small, rapidly decaying phase of current (B).

Fig. 8.

Choroid neuron EPSCs are best fit with a single exponential. The left trace shows a choroid EPSC. The right trace shows the same EPSC replotted in semilog coordinates. All choroid EPSCs examined (n = 15) were best fit with a single exponential.

Fig. 9.

Effect of 50 nm α-BuTx (A) and 50 nm α-CTx-MII (B) on choroid neuron EPSCs. See legend to Figure5 for complete description of traces. α-BuTx reduces the peak of the EPSC but does not change the rate of decay (A). α-CTx-MII substantially reduces the EPSC size, eliminates the slowly decaying phase of current, and reveals a small, rapidly decaying phase of current (B).