 significantly impacted (Control – Nec3 + Nec1-shRNA: p = 0.0014, Apical Control – Nec3 + Nec1-shRNA: p = 0.0014, Basal Control – Nec3 + Nec1-shRNA: p = 0.07063, Additional file 3: Figure S3). These results indicate that developing apical dendrites may be more sensitive to reduced nectin-3 expression than basal dendrites. Differences in dendritic spine densities between double knockdown and control neurons at P21 were present despite seeing no difference in dendrite complexity, as analyzed by Sholl analysis (Additional file 3: Figure S3f, g). By P35, the dendritic spine densities of Nec3-shRNA neurons were no longer significantly different from control neurons (Fig. 2i). Collectively, these results are consistent with a model where the extended knockdown of nectin-3 leads to a transient overproduction of weak spines after eye opening, which are readily pruned over the critical period for ocular dominance plasticity (P21 – P35) due to reduced stability.
Early (E15.5) shRNA knockdown of nectin-3 yielded an overproduction of dendritic spines after eye opening, significantly increasing dendritic spine densities at P21 relative to control. On the other hand, dendritic spine densities between Nec3-shRNA and control neurons were not different at P35, indicating that the extra spines produced at P21 may have been removed over the critical period for ODP (P21 – P35). We next wanted to test whether the initial overproduction of spines at P21 after nectin-3 knockdown directly led to compensatory spine pruning in the following weeks. To accomplish this, we selectively knocked down nectin-3 near the start of the critical period for ODP and assessed dendritic spine densities at P35. For this experiment, we co-electroporated the same Cre-dependent Nec3-shRNA constructs (or control scramble shRNA) and a Cre-dependent tdTomato construct into transgenic CaMKII-Cre mice at E15.5 (Fig. 3a, c). In a previous study, CaMKII-Cre driven Cre/loxP recombination was first observed in cortex and hippocampus at P19 and expression was described as robust by P23 [42]. From this, we concluded that the knockdown of nectin-3 likely occurred in most cells around the start of the critical period for ocular dominance plasticity (P21). Both apical and basal dendritic spine densities were analyzed at P35, to allow time for full shRNA expression and knockdown of nectin-3 in L2/3 cortical neurons (Fig. 3b, c).
P19) significantly increased dendritic spine densities at P35 relative to control (p = 0.0107, Fig. 3d, e). While both apical and basal dendritic spine densities appeared to increase with nectin-3 knockdown, when considered separately, only apical dendrites showed a statistically significant increase relative to control (p = 0.0207, Fig. 3d, e, Additional file 5). The critical period for ODP is typically associated with experience dependent spine pruning in V1, though different dendrite types have been shown to undergo different degrees of pruning [19, 60]. While our results could indicate that nectin-3 knockdown disrupts pruning over the critical period for ODP, the increased spine densities observed could equally be the result of increased spinogenesis. It is also possible that, as with early (E15.5) knockdown, the increased spine densities observed with late knockdown of nectin-3 may be transient. We did not take measurements of spine densities after P35, but other studies have found that nectin-3 knockdown in adult hippocampus decreases dendritic spine densities [35, 36]. Further studies are necessary to determine whether the developmental knockdown of nectin-3 has long-term effects in L2/3 cortical neurons.
We next examined the effect of overexpressing nectin-3 in L2/3 cortical neurons over development. We generated a newly designed nectin-3 expression plasmid, which dramatically increased nectin-3 expression after transfection into HEK-293 cells (Fig. 4a, c). This nectin-3 overexpression construct was electroporated into developing L2/3 neurons at E15.5 (with a Cre-dependent tdTomato construct and a Cre-expression plasmid, Fig. 4a, b), and apical and basal dendritic spine densities were analyzed at P14, P21, and P35 (Fig. 4d). Dendritic spine densities on both control (scramble shRNA) and nectin-3 overexpression (Nec3-OE) neurons increased significantly between P14 and P21 (Control, P14 – P21: p = 2.47e-05 Nec3-OE, P14 – P21: p = 0.00017, Additional file 5). In addition, when all time points were considered together, Nec3-OE significantly decreased dendritic spine densities relative to control neurons (p = 0.0019, pooled apical and basal dendrites, Additional file 5, Fig. 4e). The greatest difference between Nec3-OE and control dendritic spine densities was observed at P35, and as with nectin-3 knockdown, spine densities were most significantly impacted on apical dendrites (pooled apical and basal, P35: p = 0.0492, apical dendrites alone, P35: p = 0.00136, Fig. 4i). Consistent with our shRNA experiments, these data further indicate that nectin-3 expression may restrict spinogenesis over development and/or facilitate pruning over the critical period for ODP (P21 – P35).
Our study indicates that the apical and basal dendrites we analyzed may experience different degrees of spinogenesis after eye opening. Individual t-tests were performed to determine whether apical and basal dendritic spine densities may differ within a single condition at a given age. Both control and Nec3-OE neurons demonstrated potential differences between apical and basal dendritic spine densities at P21 (Control, p = 0.0038 Nec3-OE, p = 0.029 Additional file 5, Metadata). When these datasets were combined in our statistical model (see methods), differences between apical and basal dendrites were highly significant (p = 4.14e-05, Additional file 5, Fig. 4g). This result suggests that the apical dendrites sampled in this study may not increase their spine densities after eye opening to the same degree as basal dendrites under the conditions of normal or above normal nectin-3 expression (Fig. 4e, h). It is important to note that this effect does not seem dependent on the overexpression of nectin-3, since no significant difference between Nec3-OE and control conditions was observed at P21 (Fig. 4g). Dendritic spine densities on apical dendrites continued to increase between P21 and P35 in the control condition alone. This indicates that, unlike other dendrite types [22, 61], proximal secondary apical dendrites (distinct from apical tufts) may be slower to increase dendritic spine densities after eye opening and may not undergo pruning over the critical period for ODP. Overexpression of nectin-3 most significantly decreased apical dendritic spine densities at P35, further indicating that the unique development of this dendrite type may be dependent on precise levels of nectin-3 expression. From this we conclude that dendrite type may also be an important consideration when evaluating developmental changes in dendritic spine densities following eye opening.
Nectin proteins consist of a three extracellular Ig domains, a transmembrane fragment, and multiple intracellular C-terminal domains with distinct and independent functions [11, 30, 62,63,64]. Most nectins, including nectin-3, have a conserved motif of four amino acids at their cytoplasmic tail that binds the PDZ domain of afadin [11, 27, 30, 40]. Nectin-3/afadin binding is required for the interaction of nectin-3 with the actin cytoskeleton and the organization of PAJs in cooperation with N-cadherin (Fig. 1a) [11, 26,27,28,29, 31, 65]. Nectins can also recruit and activate c-Src leading to the downstream activation of Cdc42 and Rac, which facilitate the formation of filopodia and lamellipodia, respectively [63, 66,67,68]. The nectin-1 C-terminus, but not the afadin binding site, was necessary for the activation of Cdc42 and Rac [66]. The extracellular domain of nectin-3 has also been shown to act independently of its C-terminus to bind nectin-1, leading to nectin-1 intracellular signaling and cadherin recruitment [63, 66, 69]. It is unclear which nectin-3 domain may regulate dendritic spine formation during critical periods of postnatal development.
We next tested whether the decreased spine densities observed with nectin-3 overexpression required an interaction between nectin-3 and afadin. For this experiment, we used in utero electroporation to overexpress a nectin-3 protein lacking the four C-terminal amino acids required to bind afadin (Nec3 Δafadin ) [11, 27, 30, 40]. Developing L2/3 neurons were electroporated with Nec3 Δafadin (plus Cre and Cre-dependent tdTomato plasmids, Fig. 5a) at E15.5, and dendritic spine densities were assayed at P14, P21, and P35 (Fig. 5b, c). Note that the Nec3 Δafadin protein should still bind nectin-1 and activate Cdc42 and Rac signaling [63, 66,67,68]. Similar to the Nec3-OE condition, Nec3 Δafadin produced an overall decrease in dendritic spine densities relative to