Nicotinic acetylcholine receptors containing alpha7 subunits are required for reliable synaptic transmission in situ

Abstract

Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system. The receptors are cation-selective, relatively permeable to calcium, and avid binders of alpha-bungarotoxin. Although the receptors can act both pre- and postsynaptically, their physiological significance is unclear. Using whole-cell patch-clamp analysis of chick ciliary ganglion neurons in situ, we show that the receptors are required for reliable synaptic transmission early in development. Stimulation of the presynaptic nerve root elicited a biphasic synaptic current, including a large rapidly decaying component generated by alpha7-containing receptors. Selective blockade of alpha7-containing receptors by perfusing the ganglion with alpha-bungarotoxin induced failures in synaptic transmission. One-half of the ciliary neurons that were tested failed when stimulated synaptically at 1 Hz, and two-thirds failed at 25 Hz. Failing cells missed, on average, 80% of the trials during a test train of stimuli. The ability to fire synaptically evoked action potentials after toxin treatment was correlated positively with the amplitude of the remaining synaptic current, suggesting that alpha7-containing receptors were needed to augment synaptic responses. Consistent with patch-clamp analysis, toxin blockade reduced the amplitude of the synaptically evoked compound action potential in the postganglionic nerve; it also desynchronized the firing of the remaining units. Methyllycaconitine, another antagonist of alpha7-containing receptors, mimicked alpha-bungarotoxin blockade. Toxin blockade had less impact on transmission in ganglia at the end of embryogenesis. The ability of the receptors to synchronize and sustain population firing, together with their ability to deliver calcium, may influence early developmental events such as target innervation and neuronal survival.

Fig. 1.

Responses of a neuron before and after α7-AChR blockade. Shown are conventional whole-cell patch-clamp recordings from a ciliary neuron synaptically stimulated before (left column) and after (right column) a 5 min exposure to 50 nm αBgt. A, Recordings in voltage-clamp mode (−70 mV) showing that the toxin treatment blocked the large rapidly decaying synaptic current caused by α7-nAChRs.B, Evoked APs recorded under current clamp during 10 pulses of presynaptic stimulation at 1 Hz. C, Evoked APs under current clamp during 25 Hz presynaptic stimulation. The toxin treatment abolished synaptically evoked APs at both stimulation frequencies. The small residual synaptic current, produced by α3*-AChRs, was inadequate to generate any APs in this neuron.D, Repetitive APs elicited by constant current injection, showing that the toxin treatment did not alter the excitability of the neuron.

Fig. 2.

αBgt effects on ciliary responses monitored with perforated patch-clamp recording. Shown are synaptically evoked responses in a ciliary neuron before (left column) and after (right column) exposure to 50 nmαBgt (50 nm) for 5 min, recorded with the amphotericin B perforated patch-clamp method. A, Synaptic currents, voltage-clamp mode. B, Synaptically evoked APs at 25 Hz, current-clamp mode. Insets, Individual AP with preceding postsynaptic potential on an expanded time scale. The toxin exposure substantially reduced the reliability of synaptic transmission, as also seen with conventional patch-clamp recording, and increased the time to threshold for firing an AP.

Fig. 3.

Positive correlation between the reliability of synaptically evoked APs and the synaptic current density remaining in the presence of αBgt. The proportion of times an individual cell successfully fired a synaptically driven AP (from a train of 10 stimuli) was graphed as a function of peak synaptic current density (pA/pF). Results were pooled from conventional and perforated patch-clamp recordings performed on cells after toxin treatment. Presynaptic stimulation was delivered either at 1 Hz (A) or 25 Hz (B). The results indicate a strong correlation between the likelihood of reliable firing and the size of the postsynaptic current after αBgt treatment.

Fig. 4.

Effects of α7-nAChR blockade on synaptically evoked compound APs in the postganglionic nerve of E13/E14 ganglia.A, Extracellular recording of compound APs in the ciliary nerve root before and after perfusion of the ganglion with vehicle alone (left), 200 nm αBgt for 40 min (middle), or 40 nm MLA for 50 min (right). αBgt and MLA each decreased the peak amplitude of the compound AP and increased its duration. In theright panel a small fast electrical component is observed before the onset of the chemical component of the synaptic response; the electrical component was not decreased either by 100 μmd-tubocurarine or by 40 nmMLA. B, Time dependence of αBgt and MLA effects on the compound AP. The peak amplitude of the synaptically evoked compound AP was measured at 10 min intervals for ganglia perfused with vehicle alone (filled diamonds), with 200 nmαBgt (open circles), or with 40 nm MLA (filled squares). αBgt and MLA perfusion was initiated after 30 min and continued as indicated; results were normalized to those obtained 30 min after the initiation of perfusion, immediately before the addition of toxin or MLA. Values represent the mean ± SEM of four ganglia for each condition at each time point. Baseline for the compound AP was taken as the point immediately before the rising phase of the chemical response in each case.