Figure 4. Both TRIP8b-HCN binding sites are required for dendritic targeting of HCN channels.
A.) Confocal images demonstrating that unilateral injection of AAV-N13A causes a reduction in HCN1 staining in the SLM. Top panels show staining for HCN1 (red), middle panel shows staining for eGFP (green), and the bottom panel shows a composite image. Images in the left column are from the uninjected (control) hemisphere, while the right column shows the AAV-N13A injected hemisphere. B/C.) Unilateral injection of either AAV-Δ58 or AAV-Δ58/N13A fails to rescue HCN1 distal dendritic enrichment. Display of images is identical to that in A. Scale bar represents 100 µm. D.) Quantification of HCN1 distal enrichment. HCN1 staining intensity in regions of interest from the virally injected hemisphere were scaled by staining in the contralateral (uninjected) hemisphere. For comparison, the AAV-TRIP8b rescue experiment is reproduced from Figure 2. E.) Quantification of HCN1 staining in the SLM layer of CA1 (AAV-eGFP= 0.98±0.01, AAV-Δ58=0.98±0.04, AAV-N13A=0.78±0.04, AAV-Δ58/N13A=0.99±0.07). A one way ANOVA comparing staining intensity in the SLM of the different conditions (AAV-eGFP, AAV-Δ58, AAV-N13A, AAV-Δ58/N13A) was found to be significant (F(3,17)=22.375, p<0.05). Tukey’s post hoc tests found a difference between AAV-N13A and AAV-eGFP (p<0.05), AAV-N13A and AAV-Δ58 (p<0.05), and AAV-N13A and AAV-N13A/Δ58 (p<0.05). A one way ANOVA comparing HCN1 staining in the cell body layer of the different conditions (AAV-eGFP, AAV-N13A, AAV-Δ58, AAV-Δ58/N13A) was not significant (F(3,17)=0.59, p>0.5) hence post hoc tests were not performed. F.) TRIP8b KO mice bilaterally injected with AAV-N13A, but not AAV-Δ58, show less immobility time on TST and FST. A one way ANOVA examining the immobility time on FST found a significant difference (AAV-eGFP=148.42±18.8 sec, AAV-N13A=40.2±6.7 sec, AAV-Δ58=127.4±25.0 sec, F(2,18)=67.34, P<0.05), and follow up Tukey’s tests revealed a difference between AAV-eGFP and AAV-N13A as well as a difference between AAV-N13A and AAV-Δ58. G.) A one way ANOVA examining the immobility time on TST was significantly different (AAV-eGFP=130±12.2 sec, AAV-N13A=44.9±20.1 sec, AAV-Δ58=114±10.0 sec, F(2,17)=62.32, p<0.05) with Tukey’s test showing differences between AAV-eGFP and AAV-N13A and between AAV-Δ58 and AAV-N13A. *p<0.05 on Tukey’s HSD test following one way ANOVA. All error bars represent ± standard error of the mean and are described above as mean ± s.e.m.