A role for primary cilia in glutamatergic synaptic integration of adult-born neurons

Abstract

The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule-based signaling center, is essential for vertebrate development, including the development of the CNS. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born mouse hippocampal neurons. Primary cilia were absent in young adult-born neurons, but assembled precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional deletion of cilia from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Deletion of primary cilia led to enhanced Wnt and β-catenin signaling, which may account for these developmental defects. Taken together, our findings identify the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.

Figure 1. Primary cilia assemble in developing adult-born neurons

(a) A schematic diagram of the retroviral vector (pCentrin-2), retroviral injection, a sample brain section and the parameters measured from reconstructed images. (b) Confocal images of cilia formation in developing adult-born neurons showing EGFP (Centrin-2), dTomato, DAPI and immunostaining for ACIII. Arrows point to primary cilia associated with centrosomes. Scale bar: 3 μm. (c) Quantification of percentage of labeled adult-born neurons with primary cilia (left) and the distribution of ciliary length (right) at 5, 14, 21 and 28 dpi. (d) Percentage of retrovirally labeled neurons with primary cilia and their relative position within the dentate gyrus granule layer at 5, 14, 21 and 28 dpi. Values represent mean±SEM (n=32–48 neurons; *: p<0.01, ANOVA or Kolmogorov-Smirnov test). The analyses in c and d are from the same group of cells.

Figure 2. Entorhinal cortical innervations of adult-born neurons accompany primary cilia assembly

(a) A schematic diagram showing the AAV-ChR2-EGFP injection sites in the contralateral hilus (Contra-Hi), lateral (LEnt) and medial (MEnt) entorhinal cortex. The sample image (EGFP) shows the MEnt injection site and axonal projections in the molecular layer of the dentate gyrus. Scale bar: 150 μm. (b) Laminar glutamatergic innervation pattern in the molecular layer of the dentate gyrus. The images, taken from the location marked by a rectangle in a, show axon terminals (EGFP) in the molecular layer originating from the contralateral hilus, medial and lateral entorhinal cortex, respectively. Scale bar: 50 μm. (c) Opto-evoked glutamatergic synaptic transmission in a randomly picked DGC using a 5ms pulse of 473 nm light stimulation. Shown to the left are MEnt axonal terminals (EGFP) and a recorded DGC (white). A recording trace is shown to the right, which could be completely blocked by 50 μm CNQX. Scale bar: 20 μm. (d) Functional glutamatergic synapse formation in the developing adult-born neurons. Shown are the percentages of recorded mature and adult-born DGCs at 7, 14, 21 or 28 dpi with detectable evoked eEPSCs after opto-stimulation of a single layer. (e) Correlations of functional entorhinal cortical innervations with primary cilia assembly. Shown is a summary of the contralateral, lateral and medial entorhinal cortical innervations an d cilia assembly of adult-bornDGCs at 7, 14, 21 and 28 dpi. Values represent mean±SEM (n=10–12 neurons). M, mature DGCs.

Figure 3. Primary cilia deletion caused by expressing dnKif3a severely disrupts functional glutamatergic synapse formation in adult-born neurons

(a) Primary cilia deletion in adult-born DGCs by dnKif3a ectopic expression. At top, a diagram of the inducible retroviral vector (dnKif3a), below, typical adult-born DGCs labeled with markers of cilia (White, ACIII staining), centrioles (Green, Centrin-2-EGFP) and adult-born DGCs (Red, dTomato). Arrows point to centrosome. Scale bar: 5 μm.At right, a comparison of cilia length in control and dnKif3a+ adult-born DGCs. (b) A schematic diagram of the electrophysiological testing and an image of a recorded newborn DGC filled with biocytin through the recording pipette. The stimulating electrode was placed in the outer molecular layer to excite principally the entorhinal cortical projections. The inset is an enlarged image of the recorded adult-born DGC with dTomato signal. GCL, granule cell layer. ML, molecular layer. Scale bar: 25 μm. (c–f) Glutamatergic synaptic transmission recorded from control and dnKif3a+ adult-born DGCs at 21 dpi. c) Sample traces of glutamatergic synaptic transmission in the presence of 5 μ bicuculline. Cells were held at Vm=-65 mV. Scale bars: 10 pA and 15 ms. d) The percent of recorded newborn DGCs with detectable synaptic transmission. e) eEPSCs amplitude. Shown are individual and averaged eEPSCs amplitudes from control and dnKif3a+ adult-born DGCs. f) Spontaneous glutamateric synaptic transmission, frequency and amplitude. In a, d–f, all values represent mean±SEM (n=7–12 neurons; *: p<0.01, ANOVA).

Figure 4. Primary cilia deletion in adult-born neurons results in defective dendritic refinement

(a) Defective dendritic refinement of dnKif3a+ DGCs. Shown on the left are the images of control and dnKif3a+ DGCs at 28 dpi. Solid lines mark the edges of the outer molecular layer. Shown to the right are drawings from the images. Scale bar: 20 μm. (b) A summary of total dendritic length for control and dnKif3a+ adult-born DGCs at 14, 21, and 28 dpi. (c) Dendritic branch number for control and dnKif3a+ adult-born DGCs at 14, 21, and 28 dpi. (d) Sholl analysis of the dendritic tree of control and dnKif3a adult-born neurons at 14 and 21dpi. (For b, c and d, n=25–38 neurons; for b and c, *: p<0.01, ANOVA; for d, statistical significance was determined by Student’s t-test). All values represent mean±SEM.

Figure 5. Altered Wnt/β-catenin signaling activity upon primary cilia deletion regulates the dendritic refinement of adult-born neurons

(a) Wnt/β-catenin signaling in developing adult-born DGCs. Shown are sample single plane images of β-gal signal in control and dnKif3a+ DGCS at 21 dpi using BAT-gal reporter mice. Arrows point to newborn DGCs. Scale bar: 10μm. (b, c) Enhanced β-gal signal upon primary cilia deletion at 21 dpi. Shown in (b) is a summary of the percent of the control or dnKif3a+ DGCs having β-gal signal. Shown in (c) is a summary of normalized intensity of the β-gal signal for control or dnKif3a+ adult-born DGCs at 21 dpi. All values represent mean±SEM (n=22–29; *: p<0.01, ANOVA). (d)Cre-mediated excision of β-catenin restores normal dendritic length in dnKif3a+ adult-born neurons at 21 dpi. β-catenin^fl/fl mice were injected with dnKif3a and inducible Cre retroviruses as described in the Methods and dendritic lengths measured at 21 dpi. S33Y β-catenin was induced at 14 dpi and dendritic lengths determined at 21 dpi. (e) Dendritic branch number for the same cohort of neurons shown in d. All values represent mean±SEM (n=35–40; *: p<0.01, ANOVA; ns represents no significance).