Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

<div><p>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 Ca²⁺ 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 <i>in ovo</i> 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 Ca²⁺-sensor, the Ca²⁺ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca²⁺ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca²⁺. It is suggested that the photo-activation of ChRFR facilitated the release of Ca²⁺ from intracellular Ca²⁺ 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.</p> </div

Both V3 interneurons and somatic motoneurons are generated from Nkx2.2-positive progenitors.

<p>A and B, <i>sim1</i> in situ hybridization (purple) followed by GFP immunohistochemistry (brown). Recombined cells were <i>sim1</i>-positive (arrow in A) or <i>sim1</i>-negatve (arrowhead in A). An arrow in B indicates recombined cell axon outside the spinal cord, suggesting it was a motoneuron axon. C, GFP-positive recombined cells in the HH35 spinal cord, showing a GFP-positive axon extending outside the spinal cord. D-F, Double staining with HB9 and GFP immunohistochemistry, demonstrating a HB9-positive recombind cell. Scale bars in A and B = 50 µm; in C = 200 µm; in F = 20 µm.</p

Expression of the murine retroviral receptor is specific to Nkx2.2-positive progenitors.

<p>A, A schematic diagram of the lineage tracing method of Nkx2.2-positive progenitors. It consists of the electroporation of the retroviral receptor followed by infection by the murine retrovirus. B–G, Double staining of spinal cord sections with anti-Olig2 and anti-Nkx2.2 antibodies at HH 14 (B–D) and HH 17 (E–G). H–J, Specific expression of mCAT1-<i>myc</i> in the p3 domain. pNkx2.2-mCAT1-<i>myc</i> was introduced by in ovo electroporation at HH 14, and 24 h after the electroporation, the spinal cord sections were immunostained using Myc (H, arrow) and Nkx2.2 antibodies (I). A merged image of H and I was shown in J. K and L, Expression of <i>mCAT1</i> mRNA was shown by in situ hybridization (K and L; purple, arrows) followed by immunohistochemistry using Nkx2.2 (K; brown) or Olig2 (L; brown). Scale bars indicate 50 µm.</p

Optogenetic Ca²⁺ mobilization.

<p><b>A</b>, Typical [<i>ΔF/F</i>]_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>B</b>, Summary of peak [<i>ΔF/F</i>]_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). <b>C</b>, Sample [<i>ΔF/F</i>]_B responses of the same presynaptic terminal as shown in <b>A</b>, but with the extracellular Ca²⁺ being removed (EGTA, 1 mM). Each trace is an average of five consecutive records. <b>D</b>, The dependence of TTX-resistant [<i>ΔF/F</i>]_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 <i>t</i>-test (n = 5).</p

Distribution of Nkx2.2 lineage motoneurons in the spinal cord.

<p>pNkx2.2-Cre and cAct-xStopx-nLacZ were electroporated at HH 14 in the chick neural tube. A and B, LacZ-positive cells of one chick spinal cord at HH 32 (E7) were superimposed and marked with dots on a schematic diagram. Three independent experiments were performed in the brachial spinal cord (A) or thoracic spinal cord (B). C and D, A LacZ-positive cell in the ventral horn in which most of the motoneurons were retrogradely labeled with fluorogold (FG). E–G, GFP-positive recombined cells with brainbow system in the ventral horn were retrogradely labeled with FG (G; arrowheads). GFP-positive axons were extending toward the ventral root (arrows). H and L, Sections of HH 42 spinal cord were subjected to LacZ-staining followed by immunohistochemistry using ChAT antibody. LacZ-positive cells in the ventral horn (H; somatomotor neuron) and close to the central canal (L; preganglionic CT neuron) are shown. Arrowheads indicate double positive cells and the asterisk shows the location of the central canal. I–K and M–O, Expression of ChAT in LacZ-positive cells was also analyzed by double fluorescent labeling (I–K, M–O). P, LacZ/ChAT-positive cells of one chick spinal cord were counted and divided by the total number of LacZ-positive cells and were shown as percentages (P). Five chick embryos were examined in this study. Scale bars in A and L = 200 µm; D, F and O = 50 µm.</p

Involvement of Ca²⁺ store.

<p><b>A</b>, Typical [<i>ΔF/F</i>]_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>B</b>, Summary of peak [<i>ΔF/F</i>]_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). <b>C</b>, Sample [<i>ΔF/F</i>]_B responses of the same presynaptic terminal as shown in <b>A</b>, 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. <b>D</b>, Summary of peak [<i>ΔF/F</i>]_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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059179#pone.0059179-Dehpour1" target="_blank">[72]</a>.</p

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

<p>, Schematic structures of injected plasmid vectors, pCAGGS-ChRFR-EGFP (top) and pCAGGS-R-GECO1 (bottom). <b>B</b>, A confocal EGFP image of a calyx-type presynaptic terminal (E14) (optical slicing at 1.99 µm). <b>C</b>, A color-rated image of the <i>ΔF</i>/<i>F</i> of R-GECO1 immediately after electrical stimulation of the oculomotor nerve in the same optical slice. <b>D</b>, Overlay of B and C. Note that several hotspots are present in the synaptic face of the calyx. Scale bar, 10 µm. <b>E</b>, Time-dependent plots of bulky magnitudes of <i>ΔF/F</i> ([<i>ΔF/F</i>]_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). <b>F</b>, Simultaneous recordings of the [<i>ΔF/F</i>]_B (blue) and the EPSC (red).</p

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

<p>, Sample [<i>ΔF/F</i>]_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>B</b>–<b>D</b>, Quantitative comparison of Ca²⁺ transients between electrical stimulation and optogenetic stimulation: the peak [<i>ΔF/F</i>]_B (<b>B</b>), time constant of the rising phase (τ_R, <b>C</b>) and that of the decaying phase (τ_D, <b>D</b>). Each symbol indicates an individual presynaptic terminal.</p

Mosaic expression of fluorescent proteins with Brainbow strategy. A

<p>, Schematic structures of injected plasmid vectors, pCAGGS-mCherry-NCre (top) and pCAGGS-Brainbow1.1M (bottom). <b>B</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). <b>C</b>, An enlarged image of the EW nucleus. Note that each neuron expresses mCherry in the nucleus (arrows). <b>D</b>, Oculomotor axons (E14). <b>E</b> and <b>F</b>, a CG from E8 embryo. <b>G</b> and <b>H</b>, other CG from E10 embryo. <b>I</b> and <b>J</b>, other CG from E14 embryo. Arrows indicate debris of the axonal membrane. Scale bars: 100 µm for <b>B</b>, <b>E</b>, <b>G</b> and <b>I</b>; 50 µm for <b>C</b> and 20 µm for <b>D</b>, <b>F</b>, <b>H</b> and <b>J</b>.</p

Direct optogenetic stimulation of calyx-type presynaptic terminals. A

<p>, Calyx-type presynaptic terminals expressing ChRFR-EGFP. Scale bar, 20 µm. <b>B</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.</p