Figure 4.
Figure 4. Knockdown of Farp1 reduces spine numbers and abrogates SynCAM-mediated increases in spine density. (A) Farp1 is postsynaptic in developing and mature neurons. Confocal images show dissociated hippocampal neurons at 14 (top) and 21 div (bottom) coexpressing GFP-Farp1 (green) and Cherry. Postsynaptic PSD-95 was detected by immunostaining. Circles, dendritic protrusions where Farp1 and PSD-95 colocalize. Grayscale, individual channels. Bars, 2 µm. (B and C) Knockdown of Farp1 lowers spine density and precludes the synaptogenic effect of SynCAM 1. (B) Confocal images of dissociated hippocampal neurons at 21 div expressing shScramble control or shFarp1 vectors. GFP (green)-filled transfected neurons. SynCAM 1 was overexpressed where indicated. Bars, 2 µm. (C) Mushroom spine densities imaged as in B (shScramble, 550 spines; shScramble + SynCAM 1, 437; shFarp1, 275; shFarp1 + SynCAM 1, 274; three independent experiments). (D) No effect on mushroom spine length in dissociated neurons expressing shFarp1 (shScramble, 220 spines; shFarp1, 132; three independent experiments). n.s., not significant. (E–G) Farp1 is required for normal spine density in organotypic slice culture. (E) Confocal overview of a CA1 pyramidal neuron expressing the shScramble vector in slice culture at 14 div. Neurons were visualized by GFP expressed from the knockdown vector. Bar, 100 µm. (F) 3D renderings of dendrites of CA1 neurons expressing shScramble (top) or shFarp1 (bottom). m, mushroom; s, stubby; t, thin spines. Bars, 2 µm. (G) Spine densities imaged as in F (mushroom spines, shScramble 2.1 ± 0.25 per 10 µm dendrite, shFarp1 1.0 ± 0.13; stubby, shScramble 0.47 ± 0.08, shFarp1 0.21 ± 0.04; thin, shScramble 0.60 ± 0.10, shFarp1 0.32 ± 0.09; n = 22 neurons for shScramble, 20 for shFarp1; two independent experiments). Error bars indicate mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Knockdown of Farp1 reduces spine numbers and abrogates SynCAM-mediated increases in spine density. (A) Farp1 is postsynaptic in developing and mature neurons. Confocal images show dissociated hippocampal neurons at 14 (top) and 21 div (bottom) coexpressing GFP-Farp1 (green) and Cherry. Postsynaptic PSD-95 was detected by immunostaining. Circles, dendritic protrusions where Farp1 and PSD-95 colocalize. Grayscale, individual channels. Bars, 2 µm. (B and C) Knockdown of Farp1 lowers spine density and precludes the synaptogenic effect of SynCAM 1. (B) Confocal images of dissociated hippocampal neurons at 21 div expressing shScramble control or shFarp1 vectors. GFP (green)-filled transfected neurons. SynCAM 1 was overexpressed where indicated. Bars, 2 µm. (C) Mushroom spine densities imaged as in B (shScramble, 550 spines; shScramble + SynCAM 1, 437; shFarp1, 275; shFarp1 + SynCAM 1, 274; three independent experiments). (D) No effect on mushroom spine length in dissociated neurons expressing shFarp1 (shScramble, 220 spines; shFarp1, 132; three independent experiments). n.s., not significant. (E–G) Farp1 is required for normal spine density in organotypic slice culture. (E) Confocal overview of a CA1 pyramidal neuron expressing the shScramble vector in slice culture at 14 div. Neurons were visualized by GFP expressed from the knockdown vector. Bar, 100 µm. (F) 3D renderings of dendrites of CA1 neurons expressing shScramble (top) or shFarp1 (bottom). m, mushroom; s, stubby; t, thin spines. Bars, 2 µm. (G) Spine densities imaged as in F (mushroom spines, shScramble 2.1 ± 0.25 per 10 µm dendrite, shFarp1 1.0 ± 0.13; stubby, shScramble 0.47 ± 0.08, shFarp1 0.21 ± 0.04; thin, shScramble 0.60 ± 0.10, shFarp1 0.32 ± 0.09; n = 22 neurons for shScramble, 20 for shFarp1; two independent experiments). Error bars indicate mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal