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## Results: 5

1.

CASPR2 KD decreases synaptic transmission without altering neuronal excitability. (

*A*) Sample traces (*Upper*) and summary graphs of the amplitudes (*Lower*) of evoked NMDA-R– and AMPA-R–mediated EPSCs and of IPSCs as indicated, measured in neurons infected with control lentivirus (Control) or lentiviruses expressing the CASPR2 shRNA without (CASPR2 KD) or with a CASPR2 rescue cDNA (KD + Rescue). (*B*) Sample traces (*Left*) and frequency summary graphs (*Right*) of spontaneous miniature mEPSCs (*Upper*) or mIPSCs (*Lower*) measured in neurons infected with lentiviruses described in*A*, in the presence of 0.5 μM tetrodotoxin. (*C*) Sample traces (*Left*) and spiking frequency summary graphs (*Right*) of action potentials induced by current injections in neurons obtained as described for*A*(see also Fig. S2). Data shown are means ± SEMs; number of cells/independent cultures analyzed are depicted in the bars. Statistical significance was evaluated by Student’s*t*test: **P*< 0.05; ***P*< 0.01.2.

CASPR2 KD acts cell-autonomously in impairing synaptic network activity. (

*A*) Representative images of mCherry-expressing transfected neurons. (*B*) Sample traces (*Left*) and amplitude summary graphs (*Right*) of NMDA-R–mediated EPSCs evoked in neurons transfected with control mCherry-expressing vector (Control) or mCherry expression vector coexpressing the CASPR2 shRNA without (CASPR2 KD) or with a CASPR2 rescue cDNA (KD + Rescue). (*C*and*D*) Membrane capacitance (*C*) and input resistance (*D*) recorded from neurons described in*B*. (*E*–*G*) Sample traces of mEPSCs (*E*), cumulative plot of mEPSC interevent intervals (*Inset*, summary graph of event frequency) (*F*), and summary graph of averaged mEPSC amplitudes (*G*) recorded from neurons described in*B*. (*H*–*J*) Same as*E*–*G*, but for mIPSCs. Data shown are means ± SEMs; number of cells/independent cultures analyzed are depicted in the bars. Statistical significance was evaluated by Student’s*t*test (**P*< 0.05; ***P*< 0.01) except for the cumulative probabilities that were analyzed by the Kolmogorov–Smirnov test (****P*< 0.001).3.

CASPR2 and CNTN2 KD bidirectional effects on excitatory network activity is restored upon coexpression of rescue control. (

*A*and*B*) Example (*A*) and summary (*B*) graphs of somatic Ca^{2+}transients monitored in cultured neurons infected with control lentivirus (Control) or lentiviruses expressing the CASPR2 shRNA without (CASPR2 KD) or with the CASPR2 rescue cDNA (KD + Rescue). (*B*) Frequency (*Left*), amplitude (*Center*), and decay time constants (*Right*) of Ca^{2+}signals as assessed with the Ca^{2+}indicator dye Fluo4-AM were quantified. (*C*and*D*) Sample (*C*) and summary (*D*) graphs of the frequency (*Left*) and magnitude (quantified as synaptic charge transfer) (*Right*) of spontaneous EPSC bursts measured during picrotoxin-induced neuronal network activity in neurons obtained as described for*A*and*B*. (*E*and*F*) Same as*A*and*B*, except that the neurons were infected with control lentiviruses (Control) or lentiviruses expressing the contactin-2 KD shRNA without (CNTN2 KD) or with a contactin-2 rescue cDNA (KD + Rescue). Data shown are means ± SEMs; number of cells (or fields of view for Ca^{2+}Spikes/Min)/independent cultures analyzed are depicted in the bars. Statistical significance was evaluated by Student’s*t*test: **P*< 0.05, ***P*< 0.01, ****P*< 0.001.4.

CASPR2 KD impairs dendritic arbor and spine growth. (

*A*–*D*) Representative images (*A*) and summary graphs of neuronal morphological properties [total dendritic length (*B*); dendritic branch points (*C*); somatic area (*D*)] of neurons transfected with control plasmid (Control) or plasmids expressing the CASPR2 shRNA without (CASPR2 KD) or with a CASPR2 rescue cDNA (KD + Rescue). (*E*–*H*) Representative images of dendrites of mCherry-expressing transfected neurons stained for synapsin (*E*) and summary graphs of the density (*F*), synapsin staining intensity (*G*), and size (*H*) of synapsin-positive puncta along a single dendrite. (*I*–*N*) Representative images of mCherry fluorescence in dendritic branches (*I*, magnified in*J*to visualize individual spines) and summary graphs of the density (*K*), average height (*L*) and head width (*M*) of spines along a single dendrite, and cumulative probability plots of the spine head width (*N*) in transfected neurons as described for*A*–*D*. Data shown are means ± SEMs; number of cells/independent cultures analyzed are depicted in the bars. Statistical significance was evaluated by Student’s*t*test (**P*< 0.05; ***P*< 0.01; ****P*< 0.001) except for*N*, which was analyzed by the Kolmogorov–Smirnov test (****P*< 0.001).5.

Autism gene survey identifies CASPR2 function in maintaining normal neural network activity.

*(A*) Representative image of cultured cortical neurons loaded with the Ca^{2+}indicator Fluo-4 AM, under basal resting conditions (*Left*) and upon addition of 50 μM picrotoxin (PTX) to elicit large spontaneous transient increases in fluorescence intensity (*Right*). (*B*and*C*) Sample traces (*B*) and summary graphs (*C*) of spontaneous somatic Ca^{2+}transients monitored in cultured cortical neurons before and after treatment with 50 μM picrotoxin, followed by coapplication of 0.5 μM TTX, 50 μM AP5, or 20 μM CNQX to silence picrotoxin induced network activity. (*D*and*E*) Validation of Ca^{2+}-imaging analysis of neural network activity. Sample traces (*Left*) and summary graphs (*Right*) of somatic Ca^{2+}transients were monitored in cultured cortical neurons from conditional RIM1/2 double KO neurons that were infected with lentiviruses expressing inactive (Control) or active cre-recombinase (RIM1&2 DKO) (*D*) or wild-type neurons infected with a control lentivirus (control) or lentiviruses expressing an shRNA to synaptotagmin-1 alone (Syt1 KD) or together with a synaptotagmin-1 rescue cDNA (Syt1 KD + rescue) (*E*). (*F*) Design of the lentiviral vector (L309) expressing shRNAs under the control of H1 promoter and mCherry under the control of ubiquitin (Ub) promoter (*Top*), representative images of cultured cortical neurons after lentiviral expression of mCherry (*Middle*), and measurements of the mRNA KD efficiency obtained for the indicated targets (*Bottom*). (*G*) Cultured cortical neurons were infected with control lentiviruses and lentiviruses knocking down the indicated genes, and pictrotoxin induced network activity in the infected neurons was analyzed by Fluo-4 Ca^{2+}imaging. Both the frequency of Ca^{2+}transients (*Upper*) and maximal amplitudes (*Lower*) were evaluated. Data shown are means ± SEMs (for*C*,*D*, and*E*, number of cells/independent cultures analyzed are depicted in the bars; for F,*n*= 2–4 cultures; for G, for the Ca^{2+}spikes/min graphs,*n*= 8–14 fields of view/3–5 independent cultures, and for the data shown in the ΔF/F_{sat}maximal amplitude graphs,*n*= 50–186 neurons/3–5 independent cultures). Statistical significance was evaluated by Student’s*t*test comparing the various test conditions to their respective control (**P*< 0.05; ***P*< 0.01; ****P*< 0.001).