Fig 1: PCDH19 expression in the hippocampal CA1 and DG of epileptic mice. A Detection of the endogenous PCDH19 protein (red) in CA1 by immunofluorescent labeling. Neurons were labeled by the neuronal marker, NeuN (green). Scale bar, 100 µm. B Analysis of fluorescence intensity was performed using ImageJ. Differences in the relative fluorescence intensity (PCDH19 vs. NeuN) were analyzed with the Students t-test (Control: 0.987 ± 0.006, 60 days: 0.947 ± 0.012, n = 3, p = 0.015). C Detection of PCDH19 protein (red) and NeuN (green) in DG by immunofluorescent labeling. Scale bar, 100 µm. D Analysis of fluorescence intensity was performed using ImageJ. Differences in the relative fluorescence intensity (PCDH19 vs. NeuN) were analyzed with the Students t-test (Control: 0.967 ± 0.010, 60 days: 0.693 ± 0.024, n = 3, p < 0.0001). The data are expressed as mean ± SEM and analyzed with unpaired Student’s t-test. **p < 0.01, *p < 0.05, ****p < 0.0001. E The levels of PCDH19 protein at different time points following pilocarpine-induced SE. F Comparison of PCDH19 blots density between control mice and epileptic mice at each time point after SE (n = 3 per group). Bmal1 expression was significantly decreased at 14 days (0.480 ± 0.046) and 60 days (0.522 ± 0.119), compared with Ctrl (0.960 ± 0.028). The data are expressed as mean ± SEM and analyzed with one-way ANOVA, **p < 0.01
Fig 2: Brain structural abnormalities in pcdh19+/- larvae. (A) Representative confocal maximum intensity projections of a Z-stack taken from a 5dpf WT and a pcdh19+/- mutant crossed with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP::vglut2:DsRed) line show no gross morphological abnormalities in either the dsRed or the GFP expressing neuron population. Scale bar 200 µm. (B) Quantification of the number of inhibitory neurons within the tectum shows a reduction only in pcdh19+/- mutants compared to WT controls. n = 22 WT, n = 19 pcdh19+/-, n = 11 pcdh19-/-, n = 19 mosaic, n = 11 scrambled larvae, one-way ANOVA with Dunnett’s multiple comparisons test, p < 0.0001. (C) Representative confocal maximum intensity projections of a Z-stack of 2dpf WT, pcdh19+/-, pcdh19-/- and mosaic mutants showing an altered, immature neuronal pattern in the pcdh19+/- mutant compared to the WT larvae. tel., telencephalon; teO, tectum opticum; CCe, cerebellar corpus; tec, tectum; pTh, pre-thalamus, hy, hypothalamus.
Fig 3: Hyperexcitability in LFP recordings of pcdh19 mutant larval tectum. (A) Exemplary traces of LFP recordings for control and pcdh19 mutants showing large amplitude events (LAE) consisting of bursts of spikes present in pcdh19 mutants, which are rare in controls. (B) Quantification shows an increase in LAE rate in all pcdh19 mutant lines, compared to their respective controls; mosaics were compared to scrambled injected and a ‘control injected’ group representing larvae that were injected with pcdh19 sgRNA but had <10% indel frequencies. (C) The average LAE spike amplitude is increased in pcdh19+/- KO mutant lines compared to WT controls. (D) The average amount of bursts is increased in pcdh19+/- KO and mosaic mutants compared to their respective controls. n = 16 WT, n = 13 pcdh19+/-, n = 8 pcdh19-/-, n = 16 mosaic, n = 15 injection control, n = 11 scrambled injected. One-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: Bmal1 and PCDH19 expression in the hippocampal DG of patients with TLE. A, B Detection of the endogenous Bmal1 and PCDH19 protein (red) in DG of HS type I, HS type III, and no HS by immunofluorescent labeling. Neurons were labeled by the neuronal marker, NeuN (green). Scale bar, 100 µm. C Analysis of fluorescence intensity was performed using ImageJ. Differences in the relative fluorescence intensity (Bmal1 vs. NeuN) among the three groups were analyzed with one-way ANOVA, compared with no HS (HS I: 0.741 ± 0.060, HS III: 0.860 ± 0.651, no HS:1.059 ± 0.081, n = 5). D Differences in the relative fluorescence intensity (PCDH19 vs. NeuN) among the three groups were analyzed with one-way ANOVA, compared with no HS (HS I: 0.739 ± 0.079, HS III: 1.044 ± 0.093, no HS:1.160 ± 0.157, n = 5). E The levels of Bmal1 and PCDH19 protein in the hippocampus of HS type I, HS type III, and no HS by immunoblotting. F Differences in the levels of Bmal1 among the three groups were analyzed with one-way ANOVA, compared with no HS (For Bmal1, HS I: 0.350 ± 0.062, HS III: 0.494 ± 0.106, no HS:1.000 ± 0.135, n = 6; For PCDH19, HS I: 0.379 ± 0.037, HS III: 0.501 ± 0.102, no HS:1.000 ± 0.105, n = 6;). *p < 0.05, **p < 0.01
Fig 5: High-throughput detection of spontaneous and provoked seizure activity by behavioral and calcium fluorescence-based assays. (A) Quantification of the percentage of larvae with seizure events (left axis, green bar = 0.1 mM PTZ exposure blue bar = exposure to 37 °C warm water) and the number of seizures per larvae per minute (right axis, open circles) untreated (grey bars) or after exposure to 0.1 mM PTZ (green bars) or temperature increase up to 37 °C (blue bars). pcdh19+/- larvae treated with 0.1 mM PTZ showed an increased number of seizures compared WT. Untreated and 0.1 mM PTZ: n = 429 WT, n = 500 pcdh19+/-, n = 539 pcdh19-/-, n = 384 mosaic, n = 192 scrambled, Chi-squared test, ***p < 0.001. Untreated and temperature elevation: n = 258 WT, n = 96 pcdh19+/-, n = 163 pcdh19-/-, n = 150 mosaic, n = 192 scrambled, Chi-squared test, *p < 0.05, ***p < 0.001. (B) Quantification of seizure events in larvae treated with 2.5 mM PTZ (left axis, pink bars = 2.5 mM PTZ) induces seizures in almost all pcdh19+/- and pcdh19-/- mutant larvae, significantly more than in WT larvae. n = 447 WT, n = 225 pcdh19+/-, n = 620 pcdh19-/-, n = 192 mosaic, n = 192 scrambled, Chi-squared test, *p < 0.05, ***p < 0.001. The frequency of seizures in each animal is similar across genotypes (right axis, open circles) (C) Quantification of the average event rate of calcium transients in GCaMP6s larvae shows no difference between WT and mutants in the untreated condition; there is a significant increase in events in all groups after exposure to 10 mM PTZ, similar to WT. Comparing mosaic and scrambled injected larvae shows a significant increase in calcium events when treated with 1 mM PTZ. n = 155 WT, n = 264 pcdh19+/-, n = 145 pcdh19-/-, n = 128 mosaic, n = 61 scrambled, one-way ANOVA with Tukey’s multiple comparisons test for Pcdh19 KO mutants and two-way ANOVA with Sidak’s multiple comparisons test mosaic vs scrambled and PTZ vs no treatment, *p < 0.05, ***p < 0.001. (D) Average movement (distance moved in top panel and maximum velocity in lower panel) of larvae exposed to dark, 100% light (indicated by horizontal yellow lines), or 3 pulses, 3 s each, of strobe light (1 Hz, 2 Hz, and 5 Hz, indicated by vertical dashed lines) over 30 min did not change in response to the different light paradigms in pcdh19 mutants compared to WT. n = 223 WT, n = 227 pcdh19+/-, n = 134 pcdh19-/-, n = 261 mosaic.
Supplier Page from Abcam for Anti-PCDH19 antibody - C-terminal