Mice

PirB^fl/fl, PirB^+/+, and PirB^−/− mice were generated by Syken et al. (2006). Briefly, PirB^fl/fl mice were generated by electroporating a construct with loxP sites flanking exons 10–13 of PirB into the 129 J1 ES line derived from agouti 129S4/SvJae mice. Exons 10–13 of PirB code for the transmembrane domain of PirB, as well as part of the intracellular domain encompassing the signaling immunoreceptor tyrosine-based inhibitory motifs. Thus, Cre-mediated excision of exons 10–13 from the PirB gene in PirB^fl/fl mice results in a truncated protein that is unable to signal, as shown previously by anti-phosphotyrosine immunoprecipitation experiments (Syken et al., 2006). To generate mice with germ line deletion of PirB (i.e., PirB^−/−), a deleter strain that targets Cre-recombinase expression to early mouse embryo via adenovirus EIIa promoter (B6.FVB-TGN(EIIa-cre)C5379Lmgd; The Jackson Laboratory) was crossed to PirB^fl/fl mice. Heterozygote sibling matings were then used to generate both control PirB^+/+ line and a homozygous PirB^−/− line (Syken et al., 2006). PirB^fl/fl, PirB^+/+, and PirB^−/− mice were maintained as three separate lines on the same mixed genetic background (C57BL/6 × SV/129J). Previous studies have shown that the excision of PirB from PirB^fl/fl by Cre recombinase under control of the UbC promoter occurs within 1 week (Bochner et al., 2014), and is accompanied by a complete loss of PirB protein after ∼3 weeks from the onset of Cre recombinase expression (Fig. 1; Bochner et al., 2014). Cre recombinase expression via the GFP.Cre construct under the phosphoglycerate kinase promoter used here for the electroporation experiments described below should be even more rapid and efficient (Qin et al., 2010).

Density of dendritic spines on L2/3 pyramidal neurons is greater in visual cortex of germline PirB^−/− mice than in PirB^+/+ mice at P30. A, A low-magnification fluorescence micrograph of P30 mouse visual cortex showing GFP expression (green) in cells in L2/3 after GFP.Cre electroporation at E15.5. B, C, Higher-magnification views of boxed region shown in A; DAPI nuclear counterstain (B) shows that most of the GFP⁺ neurons (C) are in layer 2 and upper layer 3. Soluble GFP fills the cells: cell bodies and dendrites, as well as descending axons clustering within layer 5 are all clearly visible. D, High-magnification maximum intensity projection of boxed region shown in C. Dendritic spines and axonal boutons are visible. E–H, High-magnification fluorescent micrographs showing apical (E, G) and basal (F, H) dendritic spines in PirB^+/+ and PirB^−/− mice. I, Apical dendritic spine density on L2/3 pyramidal neurons of PirB^−/− visual cortex is elevated compared with PirB^+/+ (PirB^+/+ GFP⁺;Cre⁺: 7.0 ± 0.6 dendritic spines/10 μm of dendrite length, n = 9 cells, 5 mice; PirB^−/−: GFP⁺;Cre⁺: 11.5 ± 1.0, n = 13 cells, 5 mice; p = 0.004^a, one-way ANOVA with post hoc Bonferroni’s multiple comparisons). J, Basal dendritic spine density is also increased in PirB^−/− mice compared with PirB^+/+ mice (PirB^+/+ GFP⁺;Cre⁺: 6.6 ± 0.6 dendritic spines/10 μm of dendrite length, n = 9 cells, 5 mice; PirB^−/− GFP⁺;Cre⁺: 11.4 ± 1.1, n = 10 cells, 5 mice; p = 0.002^b, one-way ANOVA with post hoc Bonferroni’s multiple comparisons). K, L, Sholl analysis reveals no significant changes in apical (K) or basal (L) dendritic branching between PirB^+/+ and PirB^−/− L2/3 neurons (PirB^+/+ GFP⁺;Cre⁺: n = 5 cells, 3 mice; PirB^−/− GFP⁺;Cre⁺: n = 8 cells, 5 mice; p = 0.2443^c (K), p = 0.0574^d (L), two-way ANOVA with repeated measures). **p < 0.01. Scale bars: A, 0.2 mm; B, C, 50 μm; D, 25 μm; E–H, 3 μm.

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Stanford University Institutional Animal Care and Use Committee. Experimental methods are also in accordance with the policies of the Society for Neuroscience on the Use of Animals and Humans in Neuroscience Research. All mice were maintained in a pathogen-free environment.