CSF-contacting neurons regulate locomotion by relaying mechanical stimuli to spinal circuits

Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate in vivo that CSF-cNs form an intraspinal mechanosensory organ that detects spinal bending. By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs.

Motion artifact removal for imaging calcium transients from CSF-cNs associated with active and passive tail bends. a, Sample traces of responsive dorsal ipsilateral CSF-cNs from the external pressure dataset. Shown are the ΔF/F traces of the red channel (tagRFP) and the green channel (GCaMP) as well as the ΔR/R of the combination of both channels to accounting for the motion artifact. The gap represents the time points immediately after the onset of stimulation where large movements preclude imaging of the cell. The ΔF/F is used only for illustration purposes and the ΔR/R is calculated from the raw, background corrected signal. b, Sample images of the three time points indicated in a. At t 1 , GCaMP fluorescence is very low while increased fluorescence can be seen at t 3 for the cell body that returns to the focal plane. The change in fluorescence due to shifts in focal plane is estimated from the tagRFP signal. The tagRFP signal is also used to track the position of the cell body in order to move the corresponding region of interest (ROI). During the stimulation (t 2 ) cells move out the focal plane and tracking fails. These data points are omitted in the analysis. The asterisk marks the ROI, which corresponds to the sample trace in a. The sample data shown is from passive external pressure experiments but the principle of analysis is the same for the tail free experiments. Scale bar: 20 µm.

Supplemental Figure 3
Additional behavioral parameters are not affected in pkd2l1 icm02/icm02 mutant larvae. a, Latency, duration, distance, speed, number of oscillations and C bend amplitude were not affected in the Escape duration and C bend amplitude were the only parameters not affected by this apparent habituation in our assay. c, Same as in b with wildtypes (240 escapes from 73 larvae) in red and pkd2l1 mutants (217 escapes from 66 larvae) in blue.

Supplemental Figure 4
Silencing of vesicular release in CSF-cNs by Botulinum toxin (BoTx) causes behavioral deficits reminiscent of pkd2l1 icm02/icm02 larvae, in particular, a reduction of TBF. a, TBF is significantly reduced in BoTxLC-GFP + larvae (p = 0.0042), as is C bend amplitude (sibling mean: 114.68 ± 1.17º, BoTxLC-GFP + mean: 106.13 ± 1.95º, p = 0.0001). b, Two minute inter trial intervals lead to a significant decrease in speed (p = 0.01) and TBF (p = 0.01). Other parameters exhibit trends suggesting habituation, but none are statistically significant. c, Same as in b, siblings represented in black (368 escapes from 128 larvae) and BoTxLC-GFP + larvae in green (177 escapes from 74 larvae). Accounting for the habituation effects observed in b, significance increases for the TBF effect (p = 0.0025).   Table 1 Transgenic lines and mutants generated for and used in this study.

Transgenic/mutant Affected gene Description Original reference
Tg(pkd2l1:GCaMP5G)icm07 A promoter fragment of the pkd2l1 gene drives expression of the calcium indicator GCaMP5G in CSF-cNs. Generated in this study

Tg(UAS:GCaMP6f;cryaa:mCherry)icm06
The expression of the calcium indicator GCaMP6f is dependent of the activation of a 5' upstream activator sequence (UAS). A crystalline promoter fragment (cryaa) induces expression of mCherry in the lens.

Generated in this study
Tg(pkd2l1:Gal4)icm10 A promoter fragment of the pkd2l1 gene drives expression of a GFF (optimized Gal4) transactivator in CSF-cNs The mutant pkd2l1 icm02 was generated with TALE nucleases engineered against the second exon of pkd2l1. +1 frameshift. Generated in this study The Tg(mnx1:gal4) line driving selective expression in spinal motor neurons based on the injection of the mnx1 construct 2 .

Generated in this study
Tg(UAS:tagRFP-caax;cmcl2:eGFP)icm22 The expression of tagRFP localized to the membrane with a CAAX motif is dependent of the activation of a 5' upstream activator sequence (UAS). A crystalline promoter fragment (cryaa) induces expression of eGFP in the lens.

Generated in this study
Tg(pkd2l1:tagRFP)icm17 A promoter fragment of the pkd2l1 gene drives expression of tagRFP in CSF-cNs. Generated in this study

Tg(UAS:BoTxBLC-GFP)icm21
Botulinum toxin light chain serotype B codon-optimized for zebrafish with C-terminal GFP fusion. Generated in this study