Figure 5. Continuous SynCAM 1 Presence is Required to Maintain Increased Synapse Number.

(A) Experimental design. SynCAM 1 overexpression was either continuously repressed by Dox treatment (OE^never), or occurred continuously until P28 (OE^always). Cohorts were treated with Dox from P14 to repress overexpression (OE^early), or were removed from Dox at P14 (OE^late) to turn overexpression on.

(B) Hippocampal homogenates from animals treated as in (A) were probed by immunoblotting. At P14, SynCAM 1^flag was only detected in OE^always mice. At P28, SynCAM 1^flag is repressed to undetectable levels in both OE^early and OE^never conditions. SynCAM 1^flag amounts reach maximum by P28 even when overexpression was only initiated from P14 on (OE^late). Actin served as a loading control.

(C) Representative mEPSC traces from P28 hippocampal CA1 neurons after treatments as in (A).

(D) The SynCAM 1-induced increase in synapses requires its presence to be maintained. OE^always mice exhibit increased mEPSC frequency at P28 compared to OE^never controls. Overexpression of SynCAM 1 increases mEPSC frequencies at P14 (see Figure 3B), but subsequent repression returns frequencies to control levels by P28 (OE^early). When overexpression is turned on at P14 (OE^late), mEPSC frequencies are indistinguishable from OE^always mice. mEPSC frequencies in OE^never mice are identical to littermate controls carrying only the tTA transgene (see Figures S3A and S3B). For statistical comparisons, see Figures S3C and S3F.

(E) Maintaining the SynCAM 1-induced spine increase requires its presence. Overexpression of SynCAM 1 until P14 increases spines densities over tTA littermate controls. At P28, OE^always mice similarly show increased spine densities over tTA controls. Turning SynCAM 1 overexpression on at P14 (OE^late) results at P28 in spine densities that are identical to OE^always mice. Repression of SynCAM 1 overexpression from P14 (OE^early) reduces spine densities to control levels by P28 (dashed line). Spine densities in OE^never mice are identical to tTA littermate controls (see Figure S4A). For statistical comparisons, see Figures S4B.