Figure 4. Constitutive Cbln2 deletion occludes regulation of postsynaptic AMPAR- and NMDAR-EPSCs by presynaptic Nrxn1^SS4+ and Nrxn3^SS4+, respectively.

(A) Experimental strategy for testing whether the Cbln2 deletion blocks the effects of Nrxn1^SS4+ and Nrxn3^SS4+ signaling. Constitutive Cbln2 KO mice (Cbln2^KO) were crossed with Nrxn1^SS4+ and Nrxn3^SS4+ knockin mice that constitutively express Nrxn1^SS4+ and Nrxn3^SS4+ splice variants, but that are converted into constitutively expressing Nrxn1^SS4- and Nrxn3^SS4- splice variants by Cre-recombinase (Dai et al., 2019). Three groups of mice were compared: 1. Cbln2^WT mice expressing Nrxn1^SS4+ or Nrxn3^SS4+, 2. Cbln2^KO mice expressing Nrxn1^SS4+ and Nrxn3^SS4+ in which presynaptic CA1 neurons were infected stereotactically at P21 with AAVs expressing inactive ΔCre (retains presynaptic Nrxn1^SS4+ and Nrxn3^SS4+ genotype); and 3. Nrxn1^SS4+ and Nrxn3^SS4+ in which presynaptic CA1 neurons were infected stereotactically at P21 with AAVs expressing active Cre (generates presynaptic Nrxn1^SS4- and Nrxn3^SS4- genotype). CA1→subiculum synapses were then analyzed in acute slices from these mice at P35-42. (B) On the background of the Cbln2 KO, knockin of Nrxn3^SS4+ no longer suppresses AMPAR-ESPCs, nor does it reverse the increase in AMPAR-EPSCs induced by the Cbln2 KO at CA1→subiculum synapses (left, representative traces; middle, summary plot of the input/output relation; right, summary graph of the slope of the input/output relations). (C) Similarly, Nrxn1^SS4+ no longer enhances NMDAR-ESPCs on the background of the Cbln2 KO, nor does it reverse the decrease in NMDAR-EPSCs induced by the Cbln2 KO (left, representative traces; middle, summary plot of the input/output relation; right, summary graph of the slope of the input/output relations). (D & E) Constitutive expression of Nrxn1^SS4+ and Nrxn3^SS4+ alone or in combination with the Cbln2 KO have no effect on the paired-pulse ratio of evoked AMPAR-EPSCs (D) or NMDAR-EPSCs (E) at CA1→subiculum synapses (left, sample traces; right, summary plots of PPRs). (F) Alternative experimental strategy for testing whether the Cbln2 deletion blocks the effects of Nrxn1^SS4+ and Nrxn3^SS4+ signaling. Analysing the epistatic relation of neurexin alternative splicing at SS4 with the Cbln2 KO at CA1→subiculum synapses using viral overexpression of Nrxn1β^SS4+ or Nrxn3β^SS4+ in Cbln2 KO mice. The CA1 region of constitutive Cbln2 KO mice was bilaterally infected at P21 by stereotactic injections with AAVs expressing Nrxn1β^SS4+, Nrxn1β^SS4-, Nrxn3β^SS4+, or Nrxn3β^SS4-, and subiculum neurons were analyzed 2–3 weeks later. The representative image on the right depicts the signal for eGFP (which is co-expressed with the neurexins) in CA1 neurons after 2 weeks infection. (G) On the background of the Cbln2 KO, overexpression of Nrxn3β^SS4+ again no longer suppresses AMPAR-ESPCs, nor does it reverse the increase in AMPAR-EPSCs induced by the Cbln2 KO at CA1→subiculum synapses (left, representative traces; middle, summary plot of the input/output relation; right, summary graph of the slope of the input/output relations). (H) Similarly, overexpressed Nrxn1β^SS4+ no longer enhances NMDAR-ESPCs on the background of the Cbln2 KO, nor does it reverse the decrease in NMDAR-EPSCs induced by the Cbln2 KO (left, representative traces; middle, summary plot of the input/output relation; right, summary graph of the slope of the input/output relations). (I & J ) Overexpression of any neurexin has no effect on the paired-pulse ratio of evoked AMPAR-EPSCs (I) or NMDAR-EPSCs (J) (left, sample traces; right, summary plots of PPRs). Data are means ± SEM. Number of neurons/mice are indicated in bars. Statistical significance was assessed by unpaired two-tailed t-test comparing to control and two-way ANOVA (*p≤0.05, **p≤0.01, and ***p≤0.001).

Figure 4—figure supplement 1. Analysis of neurexin SS4 alternative splicing reveals that whereas all neurexins are almost exclusively expressed as SS4 +variants in the cerebellum, a mixture of SS4 +and SS4- variants is observed in other brain regions.

(A) Sample gels of amplified DNA obtained by RT-PCR of mRNAs from the indicated brain regions. RT-PCR was carried out with primers flanking SS4; as a result, in most brain regions two bands are observed that correspond to mRNAs containing (upper bands) and lacking SS4 (lower bands). Please also see original full-sized gels in Figure 4—figure supplement 1—source data 2. (B) Quantification of the prevalence of SS4 +variants of the three neurexins in the indicated brain regions. Data are means ± SEM (n=3).