Primary cilia TRP channel regulates hippocampal excitability

Significance Primary cilia are antenna-like organelles that represent a frontier of knowledge in neuroscience research. They were first reported by Duncan and Dahl more than 60 years ago, and although they are implicated in neurodevelopmental diseases, our understanding of their function in neurons is limited. In this manuscript, the authors demonstrate that the primary cilium is an excitable organelle richly populated with PKD2L1 ion channels. Using microelectrode electrophysiology and mouse genetics, the authors find that these channels in the cilia contribute to high-frequency action potential firing and their loss of function primarily impacts interneuron excitability. Loss of PKD2L1 expression impairs ciliary maturation in mice, which behaviorally exhibits autism-like features and seizure susceptibility that may have implications to human neuronal ciliopathy conditions.


Fig S1. PKD2L1 knockout mice exhibit ASD-like behaviors.
A) Results comparing WT and PKD2L1 -/mice using the open field behavioral test. Top, lines plotting locomotor trajectories during the 5 minute test period. Bottom, bar graphs comparing total distance and percent time spent at the center of the cage as readouts for locomotor activity and anxiety, respectively. B) Results comparing mouse genotypes using the marble burying assay designed to assess repetitive-like behaviors. The number of marbles buried in 30 min of testing is measured across the different genotypes. C) Results from the elevated zero maze assay to measure anxiety-like behavior. The percentage of time spent in the open arms of the maze were measured during 5 min of testing. D) Results from the threechamber social interaction test used to assay sociability in the mice. In a three-chambered box, the amount of time mice spent sniffing a novel mouse compared to a novel object was monitored and the total amount of time was quantified for each subject mouse during 10 min trials. E) There were no differences in spontaneous alternation between WT and PKD2L1 -/mice in the Y maze spontaneous alternation test, a commonly used assay to assess short term spatial working memory. Sample sizes are indicated in the parenthesis.

Fig S2. Confirming of loss of neuronal primary cilia from PKD2L1 -/hippocampus and cortex.
A) Confocal images of CA1 brain sections from PKD2L1 +/+ :ARL13B-EGFP and PKD2L1 -/-:ARL13B-EGFP mice treated with DAPI (nucleus) and immunolabeled for the centrosome component, -tubulin. Right, Pearson's coefficient analysis of colocalization of ARL13B and -tubulin. Number of images analyzed are indicated within the parenthesis. Average coefficient is indicated by red lines. B) Confocal images of CA1 brain sections from PKD2L1 +/+ :ARL13B-EGFP and PKD2L1 -/-:ARL13B-EGFP mice treated with DAPI (nucleus) and immunolabeled for acetylated-tubulin (primary cilia), confirming the loss of primary cilia from knockout animals. C) Loss of PKD2L1 expression results in immature primary cilia in the cortex. Left, example confocal images of cortex brain sections from PKD2L1 +/+ :ARL13B-EGFP and PKD2L1 -/-:ARL13B-EGFP mice treated with 4′,6-diamidino-2-phenylindole (DAPI). Right, scatter plots of primary cilia length. Number of images analyzed are indicated within the parenthesis. Average length is indicated by red lines.

Fig S3. Loss of PKD2L1 does not impair primary cilia maturation in non-neuronal tissues.
A-D) Example confocal images and respective cilia length analysis from tissues commonly impacted by ciliopathy disease. All example images are from tissues harvested from PKD2L1 +/+ :ARL13B-EGFP mice. P-values resulting from Student's t-tests are indicated above each graph. Average length is indicated by red lines. Number of cilia imaged in each data set is indicated in the parenthesis.

Fig. S4. Identification of excitatory and inhibitory neurons after current clamp experiments.
A, B) Example confocal images of fixed cultured neurons used to identify inhibitory/GABAergic interneurons for the analysis in Figure 3. Patch electrodes were loaded with biocytin to identify the current clamped neuron under the red emission. Green fluorescence identifies the transgenic ARL13B-EGFP protein in the primary cilia (arrows) and the immunofluorescence labeled glutamic acid decarboxylase 67 (GAD67), which is highly expressed in the soma (asterisks) of inhibitory neurons. Merged confocal images of neurons include nuclei labeled with DAPI or microtubule associated protein 2 (MAP2).

Fig S5. PKD2L1-mCherry localizes to neuronal primary cilia in intact hippocampal brain slices.
A) Global stitched confocal images of a fixed hippocampal brain slice harvested from a 3 month old PKD2L1-mCherry:ARL13B-EGFP mouse visualizing primary cilia (transgene ARL13B-EGFP, green), nuclei (DAPI staining, blue) and location of mCherry labeled channels (Red). B-D) Confocal images of the CA2, CA3 and dentate gyrus regions of the hippocampus. Inset, expanded views of mature and immature primary cilia taken from PKD2L1 +/+ or PKD2L1 -/mice. Right, corresponding analysis of the mCherry channel colocalization with the EGFP ciliary signal. E) Pearson's coefficient analysis of colocalization of PKD2L1-mCherry with the ciliary ARL13B-EGFP fluorescence. Horizontal red line indicated the average coefficient and sample size was 25 cilia imaged per brain location Inward single channel currents were integrated during the 300 ms phase after repolarization and normalized for each cilia recording. The resulting relationship is fit to a single exponential equation (red). B) Top, exemplar PKD2L1 whole cell currents activated by membrane repolarization (-80 mV) after variable 30 mV depolarization times (gray). Bottom, analogous to the analysis performed in B, normalized integrated inward tail currents were fit to a single exponential equation (red).