Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion

Abstract

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca(2+) elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8-14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca(2+)-sensor, the Ca(2+) elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca(2+) elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca(2+). It is suggested that the photo-activation of ChRFR facilitated the release of Ca(2+) from intracellular Ca(2+) stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.

Figure 1. Genetic manipulation of presynaptic neurons innervating chick ciliary ganglion using in ovo electroporation method.

A, Schematic structure of pCAGGS-EGFP plasmid vector. B, Position of bipolar electrodes placed on the midbrain region (red ellipse) of E2 embryo. C and D, Bright-field and EGFP fluorescent images of isolated CG with oculomotor nerve of E14 embryo. E, A compiled image of a CG (E14) under confocal microscopy. Scale bars: 200 µm for D and 100 µm for E.

Figure 2. Mosaic expression of fluorescent proteins with Brainbow strategy. A

, Schematic structures of injected plasmid vectors, pCAGGS-mCherry-NCre (top) and pCAGGS-Brainbow1.1M (bottom). B, Compiled image of a sagittal section of the midbrain (E14) under confocal microscopy. The neurons, which are colored according to the combination of expressed m-XFPs, are clustered in the EW nucleus (center). C, An enlarged image of the EW nucleus. Note that each neuron expresses mCherry in the nucleus (arrows). D, Oculomotor axons (E14). E and F, a CG from E8 embryo. G and H, other CG from E10 embryo. I and J, other CG from E14 embryo. Arrows indicate debris of the axonal membrane. Scale bars: 100 µm for B, E, G and I; 50 µm for C and 20 µm for D, F, H and J.

Figure 3. Ca²⁺ imaging of the calyx-type presynaptic terminal. A

, Schematic structures of injected plasmid vectors, pCAGGS-ChRFR-EGFP (top) and pCAGGS-R-GECO1 (bottom). B, A confocal EGFP image of a calyx-type presynaptic terminal (E14) (optical slicing at 1.99 µm). C, A color-rated image of the ΔF/F of R-GECO1 immediately after electrical stimulation of the oculomotor nerve in the same optical slice. D, Overlay of B and C. Note that several hotspots are present in the synaptic face of the calyx. Scale bar, 10 µm. E, Time-dependent plots of bulky magnitudes of ΔF/F ([ΔF/F]_B). The concentration of the extracellular Ca²⁺ was 5 mM (red), 2.5 mM (blue) and 0 mM (green). The oculomotor nerve was electrically stimulated as indicated (arrow). F, Simultaneous recordings of the [ΔF/F]_B (blue) and the EPSC (red).

Figure 4. Direct optogenetic stimulation of calyx-type presynaptic terminals. A

, Calyx-type presynaptic terminals expressing ChRFR-EGFP. Scale bar, 20 µm. B, Direct photostimulation with laser pulses of 10 ms (blue) and 20 ms (red) in the presence of 4-AP (1 mM). The resting potential, −53 mV; the action potential, 43 mV; the input resistance, 74 MΩ.

Figure 5. The intracellular Ca²⁺ transient induced by direct optogenetic stimulation of the presynaptic terminal. A

, Sample [ΔF/F]_B of R-GECO1 responses evoked either by electrical stimulation of the oculomotor nerve (blue) or by direct photostimulation of the presynaptic terminal (red). The same calyx-type presynaptic terminal in the presence of 4-AP (1 mM). B–D, Quantitative comparison of Ca²⁺ transients between electrical stimulation and optogenetic stimulation: the peak [ΔF/F]_B (B), time constant of the rising phase (τ_R, C) and that of the decaying phase (τ_D, D). Each symbol indicates an individual presynaptic terminal.

Figure 6. Optogenetic Ca²⁺ mobilization.

A, Typical [ΔF/F]_B changes in the TTX-treated presynaptic terminal: the response to a single 20 ms laser pulse (blue), the response to a train of laser pulses (10 Hz) for 1 s (red) and the response to electrical stimulation (10 Hz, 1 s) to the oculomotor nerve (black). Each trace is an average of five consecutive records. B, Summary of peak [ΔF/F]_B changes (mean ± SEM) in the presence of TTX. Each column indicates (from left to right) the response to the train of electrical stimulations (10 Hz, 1 s), the single optical stimulation and the train of optical stimulations (10 Hz, 1 s). **, P<0.01 (n = 8). C, Sample [ΔF/F]_B responses of the same presynaptic terminal as shown in A, but with the extracellular Ca²⁺ being removed (EGTA, 1 mM). Each trace is an average of five consecutive records. D, The dependence of TTX-resistant [ΔF/F]_B changes (mean ± SEM) on the extracellular Ca²⁺ of 5 mM (left) and 0 mM (right): the response to single optical stimulation (left) and the response to a train of electrical stimulations (10 Hz, 1 s) (right). *P<0.05, two-tailed t-test (n = 5).

Figure 7. Involvement of Ca²⁺ store.

A, Typical [ΔF/F]_B response of a calyx to a single 20 ms laser pulse in the cation-free extracellular solution (black), the response with additional xestospongin C (blue), the response with additional dantrolene (red) and the response after repetitive photostimulation with additional thapsigargin (green). Each trace is an average of five consecutive records. B, Summary of peak [ΔF/F]_B changes (mean ± SEM) in the cation-free solution. Each column indicates the relative value to that without any pharmacological reagents. *, P<0.05 (n = 7). C, Sample [ΔF/F]_B responses of the same presynaptic terminal as shown in A, but in response to a train of electrical stimulations (10 Hz, 1 s); without any pharmacological reagents in cation-free solution (black), with additional xestospongin C (blue), with additional dantrolene (red) and after repetitive photostimulation with additional thapsigargin (green). Each trace is an average of five consecutive records. D, Summary of peak [ΔF/F]_B responses to a train of electrical stimulations (10 Hz, 1 s) (mean ± SEM) in the cation-free solution. Each column indicates the relative value to that without any pharmacological reagents. *, P<0.05 (n = 7). Note in A and C that the artifactual fluorescence was increased during photostimulation after treatment with dantrolene, which emits green-yellow fluorescence .