Figure 6. MEA recordings from InSyn1 KO neurons exhibit increased neuronal network activity.

(A) Experimental design to record spontaneous neuronal activity. Cultured cortical neurons were prepared from InSyn1 WT or KO neonatal pups at P0, and spontaneous neuronal activities were recorded at DIV8, 11, and 14. (B) Representative raster plots of four electrodes from each genotype recorded at DIV8, 11 and 14. Black ticks indicate the time of a spike occurred, and blue ticks indicate the spikes are part of single-electrode burst activity. (C) KO neurons showed increased spontaneous activity compare to WT at early time points. Bar graphs showing normalized average of burst frequency (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.0010. DIV11, WT n = 38, KO n = 45, p=0.0026. DIV14, WT n = 39, KO n = 43, p=0.1064), normalized average of burst duration (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.1689. DIV11, WT n = 38, KO n = 45, p=0.1195. DIV14, WT n = 39, KO n = 43, p=0.0021), and normalized average of number of spikes per burst (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.1460. DIV11, WT n = 38, KO n = 45, p=0.0093. DIV14, WT n = 39, KO n = 43, p=0.0038). **p<0.001. (D) Measurements of synchronous neuronal activities. Bar graphs showing the normalized average of network burst frequency (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.1253. DIV11, WT n = 38, KO n = 45, p=0.8531. DIV14, WT n = 39, KO n = 43 from three plates, p=0.0395.), normalized average of network burst duration (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.0742. DIV11, WT n = 38, KO n = 45, p=0.7676. DIV14, WT n = 39, KO n = 43, p=0.1380), and normalized average number of spikes per network burst (Two-tailed t-test. DIV8, WT n = 38, KO n = 46, p=0.0104. DIV11, WT n = 38, KO n = 45, p=0.0003. DIV14, WT n = 39, KO n = 43, p=0.0745). *p<0.05. ***p<0.001.

Figure 6—figure supplement 1. Developmental expression of InSyn1 in hippocampal neurons.

(A and C) Hippocampal neurons from H11 Cas9 neonatal pups were transduced at DIV0 either with AAV-InSyn1-HITI or AAV-mTubb3 to tag endogenous InSyn1 or β-III tubulin, respectively. Scale bars, 15 µm. (B and D) Graphs depicting the quantification of labeled neurons at each developmental time point.

Figure 6—figure supplement 2. InSyn1 KO neurons showed increased network activities but no change at the network synchrony recorded from MEA.

(A) InSyn1 KO neurons exhibited an increased firing rate at early developmental stages. Bar graphs showing normalized average of mean firing rate (Two-tailed t-test, DIV8, WT n = 38, KO n = 46 from three plates, p=0.0017. DIV11, WT n = 38, KO n = 45 from three plates, p=0.0324. DIV14, WT n = 39, KO n = 43 from three plates, n.s.) *p<0.05, **p<0.001. (B) Examples of cross-correlation histogram from each time points. X-axis; time delay (s), y-axis; the strength of the correlation. (C) Graph showing cross-correlation between WT and InSyn1 KO neurons (Two-tailed t-test, DIV8, WT n = 38, KO n = 46 from three plates, p=0.0829. DIV11, WT n = 38, KO n = 45 from three plates, p=0.3555. DIV14, WT n = 39, KO n = 43 from three plates, p=0.0729). (D) Experimental design of recording network activity from optogenetically activated neurons. Cultured cortical neurons were prepared from InSyn1 WT or KO neonatal pups at P0, and light-evoked neuronal activities were recorded at DIV8, 11, and 14. (E) Graph showing normalized evoked spike count in response to light stimulation from 0% to 75%. (n = 19–49 from three independent MEA plates. Two-way ANOVA followed by Bonferroni’s comparisons test. DIV8 genotype effect, F (1, 467)=6.059, p<0.0142, 75% p<0.0001. DIV11 genotype effect, F (1, 456)=16.43, p<0.0001, 10% p=0.0287, 25% p=0.0007. DIV14 genotype effect, F (1, 482)=0.0146) *p<0.05, **p<0.0001. (F) Graph showing the normalized duration of first spike response to light stimulation (n = 17–49 from three independent MEA plates. Light stimulation from 5% to 75% was analyzed. Two-way ANOVA followed by Bonferroni’s comparisons test. DIV8 genotype effect, F (1, 387)=0.8015, p=0.3712. DIV11 genotype effect, F (1, 380)=1.139, p=0.2865. DIV14 genotype effect, F (1, 400)=2.928, p=0.0878). Inset graphs represent the light intensity response between 0 and 10%.

Figure 6—figure supplement 3. Flurothyl-induced seizure test showed no difference between WT and InSyn1 KO mice.

Graph of the latency to myoclonic seizure onset (Two-tailed t-test, WT n = 12, KO n = 12, p=0.9362) and generalized seizure (Two-tailed t-test, WT n = 12, KO n = 12, p=0.7148) were measured.

Figure 6—figure supplement 4. InSyn1- /- mice showed normal memory in novel object recognition test.

(A) The preference score for the novel object training (Train), short-term (STM), and long-term (LTM) tests is shown (ANOVA with repeated measure, WT n = 21, KO n = 19, no significant main effects of genotypes, F(1,38)= 4.023, p=0.052, or time x genotype interaction, F(2,76) =0.282, p=0.755). (B) Total time spent with the objects were similar between the genotypes. (ANOVA with repeated measure, WT n = 21, KO n = 19, no significant main effects of genotypes, F(1,38)= 0.180, p=0.674, or time x genotype interaction, F(2,76) =0.485, p=0.618).

Figure 6—figure supplement 5. Spatial memory during the Morris Water Maze learning test of InSyn1- /- mice.

(A) A schematic of the water maze test showing the acquisition phase during days 1–8, reverse acquisition phase during days 9–16. NE, northeast; NW, northwest; SE, southeast; SW, southwest. (B) InSyn1 KO mice showed similar performance to WT mice in spatial learning as measured by total time spent to reach the platform during the acquisition phase or reverse phase. (C) Swim time in specific quadrants of the maze for WT and KO mice during acquisition probe tests (days 2, 4, 6, and 8) and reversal probe tests (days 10, 12, 14, and 16). At DIV10, KO mice showed a slight but a significant preference toward the new location (SW) compared to the previous location (NE) while WT mice did not.