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. 2022 Oct 7;11:e78649. doi: 10.7554/eLife.78649

Figure 5. Cbln1 and Cbln2 double KO in the subiculum phenocopies the Cbln2 single KO at CA1→subiculum synapses.

(A) Experimental strategy. AAVs encoding Cre or ΔCre (as a control) were stereotactically injected into the subiculum of conditional KO mice at P21, and mice were analyzed by slice physiology 2–3 weeks later. (B-E) Input/output measurements of evoked EPSCs recorded from combined burst- and regular-spiking neurons in acute subiculum slices reveal that the conditional Cbln2 KO enhances AMPAR-EPSCs (B) without changing the paired-pulse ratio of AMPAR-EPSCs (C) but suppresses NMDAR-EPSCs (D), again without changing the paired-pulse ratio of NMDAR-EPSCs (E). Sample traces are shown above the respective summary plots and graphs. (F-I) Analyses of mEPSCs recorded at –70 mV and +60 mV holding potentials from burst- and regular-firing neurons in the subiculum after deletion of both Cbln1 and Cbln2 reveal an increase in mEPSC frequency measured at both holding potentials, but a decrease in charge transfer only of mEPSCs monitored at a+60 mV holding potential consistent with the decreased NMDAR-EPSC amplitude detected during input/output measurements (F, sample traces; G, bar graphs of the mEPSC frequency and amplitude, respectively; H & I, same as F & G but for recordings at +60 mV). (J) Experimental strategy. The subiculum region of Cbln1/2cKO was bilaterally infected at P21 by stereotactic injections of AAVs expressing ΔCre-eGFP (Cbln1/2f/f) or Cre-eGFP (Cbln1/2cKO), and then two weeks later cohorts of mice injected with Cre were further injected into the CA1 region with AAVs expressing Nrxn1βSS4+ or Nrxn1βSS4-. Mice were then analyzed at P49-P56 by acute slice electrophysiology. (K) Overexpressed Nrxn1βSS4+ no longer enhances NMDAR-ESPCs on the background of the double Cbln1/2 cKO, nor does it reverse the decrease in NMDAR-EPSCs induced by the double Cbln1/2 cKO (left, representative traces; middle, summary plot of the input/output relation; right, summary graph of the slope of the input/output relations). (L) Conditional deletion of both Cbln1 and Cbln2 without or with presynaptic overexpression of Nrxn1βSS4+ or Nrxn1βSS4- does not alter paired-pulse ratios of NMDAR EPSCs. Left panels show sample traces; right panels summary plots of the paired-pulse ratio as a function of the interstimulus interval. Data are means ± SEMs; the number of cells/mice are depicted in the bars. Statistical analyses were performed by two-way ANOVA or unpaired two-tailed t-test comparing KOs to WT (*p≤0.05; **p≤0.01, and ***p≤0.001).

Figure 5—source data 1. Cbln1 and Cbln2 double KO in the subiculum phenocopies the Cbln2 single KO at CA1→subiculum synapses.

Figure 5.

Figure 5—figure supplement 1. Analyses of the region-specific expression patterns of Cbln1 and Cbln2 using single-molecule RNA in situ hybridization (A & B).

Figure 5—figure supplement 1.

(A & B) Single-molecule in-situ hybridization analysis of Cbln1 (A) and Cbln2 mRNAs (B) reveals highly restricted expression patterns in brain (left, overview of horizontal mouse brain sections hybridized for Cbln1 or Cbln2 mRNAs; right, representative images for Cbln1 and Cbln2 in the cerebellum (i), hippocampal formation (ii), and mPFC (iii)). Note that Cbln1 is highly expressed only in the cerebellum, whereas Cbln2 is most abundant in the subiculum and PFC (abbreviations used: RSA, retrosplenial agranular cortex; IC, inferior colliculus; PPT, posterior pretectal nucleus; sm, stria medullaris; Au1, primary autitory area; S1 & S2, primary and secondary somatosensory cortex; OB, olfactory bulb; gr, granular layer; mo, molecular layer; pcl, purkinje cell layer; S, subiculum; PrS, presubiculum; Pas, parasubiculum; CA1, 2, 3, cornu ammonis 1, 2, 3; DG, dentate gyrus; CPu, caudate putamen (striatum); cg, cingulum; IL, infralimbic cortex; VO, ventro orbital cortex; MO, medial orbital cortex; PrL, prelimbic cortex). The extremely high expression levels of Cbln1 in the cerebellum causes the Cbln1 signal to become dim in other brain regions if all images are analyzed with the same settings. To compensate for this, the Cbln1 signal strength was enhanced in the A (ii) and A (iii) images, and the strength of the Cbln1 signal in the images cannot be directly compared to that of A (i).
Figure 5—figure supplement 2. The constitutive Cbln2 deletion does not alter the overall expression levels of Nrxn1, Nrxn2, Nrxn3, Cbln1, Cbln2, GluD1 and GluD2 mRNAs in the PFC or subiculum.

Figure 5—figure supplement 2.

(A & B) Left, sample microdissection of the PFC and subiculum indicated by black dashed boxes from Cbln2 WT and KO brain sections; right, quantification of the relative mRNA levels of the three neurexins, two cerebellins, and two GluDs in the indicated brain regions using qRT-PCR (normalized to β-actin). Data are means ± SEM (n=3).
Figure 5—figure supplement 2—source data 1. The constitutive Cbln2 deletion does not alter the overall expression levels of Nrxn1, Nrxn2, Nrxn3, Cbln1, Cbln2, GluD1 and GluD2 mRNAs in the PFC or subiculum.