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. 2020 Oct 13;9:e59469. doi: 10.7554/eLife.59469

Figure 5. Local monoamine delivery restores calcium variations of ventral CSF-contacting neurons in scospondin mutants.

(A) Tg(pkd2l1:GCaMP5G) embryos we used to perform hindbrain ventricle injections at 30 hpf of artificial CSF (vehicle), epinephrine or norepinephrine (left). Intracellular calcium variations in ventral CSF-contacting neurons (v CSF-cNs, arrowhead) were recorded in the spinal cord 30 min after the injection to allow monoamines (pink dots) diffusing down the central can where bathes the Reissner fiber (RF) in control embryos. (B, C) Representative traces of calcium variations of individual ventral CSF-contacting neurons in control siblings (B) and scospondinicm15/icm15 mutants (C) after vehicle (left), epinephrine (middle) and norepinephrine injections (right). Sample traces from individual cells with integral ΔF/F values ranging around the median distribution of the imaged population are represented for each condition (n = 11). (D, E) Quantification of the normalized integrated intracellular calcium variation over time of ventral CSF-contacting neurons in control siblings (D) and scospondinicm15/icm15 mutants (E). Data were collected from three independent experiments and include 9, 11, and 12 control embryos recorded after vehicle, epinephrine and norepinephrine injections respectively (n = 131, 150, and 164 cells respectively) and 11, 10, and 10 scospondinicm15/icm15 mutants after vehicle, epinephrine and norepinephrine injections respectively (n = 168, 124, and 150 cells respectively). Each point represents a single cell. Bottom and top edges of the boxes indicate the 1st and 3rd quartiles. Dotted lines represent the distribution range around the 1st and 3rd quartiles of control embryos injected with a vehicle solution. Bold lines represent the median value for each distribution. ns p>0.05, ***p<0.001 (Kolmogorov-Smirnov test). See also Figure 5—figure supplement 1 and Figure 5-video 1.

Figure 5—source data 1. Data for Figure 5D–E.

Figure 5.

Figure 5—figure supplement 1. Exogenous norepinephrine injected in brain ventricles is transported to the spinal cord and saturates the central canal 60 min post-injection.

Figure 5—figure supplement 1.

(A) 30 hpf embryos were mounted laterally and injected in the hindbrain ventricle (HBV) with either aCSF or 3 mM norepinephrine (NE). Embryos from each group were fixed 30 and 60 min post-injection (mpi) and processed for an immunostaining against norepinephrine. Three regions in the spinal cord were imaged for each experimental condition (boxed regions): above the yolk (rostral, region 1), above the yolk extension (middle, region 2) and after the anal region (caudal, region 3). (B) Representative maximal z-projections of norepinephrine-positive signals detected after either the injection of aCSF (inj. aCSF) or exogenous norepinephrine (inj. NE), 30 and 60 min post-injection (mpi). The same imaging parameters and image processing parameters were applied to all experimental groups to avoid saturation for the most intense signals. Sagittal views of the three regions of the spinal cord shown in (A) are represented for a single representative embryo (n = 7 embryos 30 min post-aCSF injection; n = 7 embryos 30 min post-norepinephrine injection; n = 6 embryos 60 min post-aCSF injection; n = 8 embryos 60 min post-norepinephrine injection). Note that norepinephrine injected in the HBV saturates the central canal of the spinal cord (delineated by dotted lines) in the rostral most region 30 min post-injection, and the caudal most region 60 min post-injection (arrowheads). Embryos are oriented dorsal to the top and rostral to the left. Scale bars: 10 µm.
Figure 5—video 1. Epinephrine and norepinephrine restore intracellular calcium transients of ventral CSF-contacting neurons in scospondin mutants.
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Sagittal views of the spinal cord of Tg(pkd2l1:GCaMP5G) embryos at 30 hpf in a straight control sibling (top) and curled-down scospondinicm15/icm15 mutants after vehicle, epinephrine or norepinephrine injections in the hindbrain ventricle. Note that calcium transients are restored in a subset of ventral CSF-contacting neurons of scospondinicm15/icm15 embryos that received epinephrine and norepinephrine injections compared to mutants that received a vehicle injection. Data were collected at 4 Hz and displayed at 80 Hz. Scale bar: 30 µm.