Fig. 5.

Deletion of Cbln4 or Cbln2 in the MHb of juvenile mice differentially affects anxiety or passive avoidance learning behaviors. (A) Experimental strategy. The schematic shows the time course of stereotactic virus injections into the MHb of Cbln2 and Cbln4 cKO mice and of the electrophysiological, behavioral, and morphological analyses of these mice. (B) Representative coronal brain section showing expression of GFP (green) after stereotactic injection of AAV-Syn-Cre-GFP into the MHb. The bregma value denotes the anteroposterior position of the section from bregma in millimeters, as previously described (66). (Scale bar: 1 mm.) (C) High-magnification image demonstrating the efficacy of the Cre-mediated recombination following stereotactic injection of AAV-Syn-Cre-GFP (green) into the MHb of Cbln2-mVenus-tdTomato cKO mouse. Neurons in which Cbln2 has been knocked out express tdTomato (false-colored magenta). (Scale bars: 50 μm.) (D) Coronal sections showing the fasciculus retroflexus (FR) following stereotactic injection of AAV- ΔCre into the MHb of one hemisphere (Left) and AAV-Cre into the MHb of the other hemisphere (Right) in a single Cbln2-mVenus-tdTomato cKO mouse. Following Cre-mediated recombination, Cbln2 is deleted from the MHb, resulting in a loss of mVenus expression (green). (Magnification: D, 20×.) (E and F) No change in anxiety-like behavior or general activity in Cbln2-cKO mice (n = 10) compared with Cbln2-Ctl mice (n = 11) in the open field test (E) or elevated plus maze (F). (G) In an open field measuring 28 × 28 cm, there was no difference in total distance traveled or time spent in the center of the field between the Cbln4-Ctl (n = 12) and Cbln4-cKO mice (n = 10). (H) In an open field measuring 40 × 40 cm, Cbln4-cKO mice (n = 10) spent significantly less time in the center of the field (*P = 0.0135, Student’s t test) compared with Cbln4-Ctl mice (n = 12), although there was no significant difference between groups in the latency to enter the center of the field. (I) In the elevated plus maze, Cbln4-cKO mice (n = 10) spent significantly less time in the open arms (*P = 0.0198, Student’s t test), and had an increased latency to enter the open arms (*P = 0.0471, Student’s t test) compared with Cbln4-Ctl mice (n = 11). (J) In the light/dark exploration test, there was no difference in the total time spent in the light chamber or in the latency to enter the light chamber between Cbln4-Ctl (n = 12) and Cbln4-cKO mice (n = 10). (K) In the passive avoidance test, there was no difference in baseline exit latency (i.e., time to enter the dark chamber) between the Cbln2-Ctl (n = 11) and Cbln2-cKO (n = 11) mice. During the recall sessions, Cbln2-Ctl mice took significantly more time to enter the dark chamber (i.e., increased exit latency) than at baseline (vs. recall trial 1: ⁺adjusted P = 0.0304, Sidak’s post hoc test), and compared with Cbln2-cKO mice (time, *P = 0.0344; genotype, ***P = 0.0004, two-way ANOVA), whereas the Cbln2-cKO mice did not significantly increase their escape latency compared with baseline. (L) Same as K but for Cbln4. There was no difference between Cbln4-Ctl and Cbln4-cKO mice at any time point. and both groups demonstrated learning (time, ****P < 0.0001, two-way ANOVA). Data are shown as mean ± SEM.