Figure 1.
Characterization of Panx1−/− mice.
a, RT-PCR and western blot analysis of 4 adult Panx1+/+ control and Panx1−/− animals. Note the lack of the 287 bp PCR amplicon representing the deleted exon4 in the Panx1 mRNA and the loss of the Panx1 protein in Panx1−/− mice. b, Immunohistochemistry demonstrating loss of Panx1 protein expression in CA1 region of the hippocampus and cerebellum. Abbreviations: M, muscle, H, heart, B, brain, R, retina, OR, stratum oriens, PY, stratum pyramidale, RAD, stratum radiatum, GCL, granular cell layer, PCL, Purkinje cell layer, ML, molecular layer. Scale bars: 100 µm.
Figure 2.
Loss of Panx1 alters postsynaptic responses in the hippocampal CA1 area.
a, IO-relations demonstrate increased excitability of acute Panx1−/− hippocampal slices (−/−, solid line; +/+, dashed line). b, fEPSP sample traces at 10, 50, 100% of the maximum input stimulus intensity. At high input stimuli, a characteristic oscillatory component appears in Panx1−/− but not Panx1+/+ mice. c, Panx1−/− animals show increased LTP responses during the early (0–5 min) and persistent phase (25–30 min). Insets in (C) depict sample traces before (black) and 30 min post (grey) high frequency stimulation (HFS). Note that inhibition of Panx1 by 50 nM mefloquine (MEQ) does not fully emulate the knock out effect. c, Comparing fEPSP amplitudes reveals significant increased early and persistent LTP in Panx1−/− slices. MEQ does not reconstitute the Panx1+/+ response to Panx1−/− levels. Statistics: (a) two tailed Mann-Whitney test (non-parametric), (c) ANOVA (early: F3,64 = 573.7; P<0.0001; late: F3,64 = 259.1; P<0.0001) and Holm-Sidack post-hoc test.
Figure 3.
Pre- and postsynaptic components of altered Panx1−/− LTP.
a, Evoked LTP in presence of 3 µM adenosine: Panx1−/− derived fEPSPs during early and persistent LTP phases are restored towards LTP levels of the untreated controls. Figure insets in (a) indicate −/− and +/+ original responses under blocking conditions before and 30 min post HFS (scale horizontal: 10 ms, vertical: 0.5 mV). b, Comparison for early phase (0–5 min) and persistent phase LTP (25–30 min). All conditions differ significantly from the Panx1−/− (see Figure 1). c, IO- correlation of the adenosine-treated Panx1−/− in comparison to Panx1−/− and Panx1+/+ under ACSF conditions. Adenosine treatment normalized the input sensitivity of the Panx1−/− towards values of the untreated controls (Panx1+/+). d, fEPSP sample traces of the Panx1−/−, revealing a decrease in oscillatory activity (arrow) and amplitudes. e, NMDA dependence of −/− and +/+ derived LTP after application of 50 µM D-AP5. NMDA inhibition leads to decrease of the signal during early and persistent phase of LTP, as demonstrated in (f). f, All conditions differ significantly from the Panx1−/−. Statistics: (b) ANOVA (early: F3,64 = 157.9; P<0.0001; late: F3,64 = 458.7; P<0.0001), (f) ANOVA (early: F3,64 = 184.9; P<0.0001; late: F3,64 = 547.4; P<0.0001).
Figure 4.
Upregulation of metabotropic glutamate receptor 4 in Panx1−/− mice.
a, Volcano plot representing the expression of 84 genes relevant for synaptic plasticity (Panx1+/+/Panx1−/−, n = 4). b, Inhibition of grm4 by bath application of 100 µM UBP1112. Fifteen minutes post-HFS, UBP1112 induced impaired persistent LTP in Panx1−/−s. Panx1+/+-LTP steadily increased during the entire post-HFS period without reaching the level of untreated Panx1−/−s. Insets in (b) indicate Panx1−/− and Panx1+/+ original responses under blocking conditions before and 30 min post-HFS (scale horizontal: 10 ms, vertical: 0.5 mV). c, No differences were found for Panx1−/−s during the early LTP phase (0–5 min) compared to the ACSF treated Panx1−/−s. Persistent phase analysis reveals alignment of the fEPSP levels of both the UBP1112-treated +/+ and −/−. Statistics: (c) ANOVA (early: F3,64 = 637.1; P<0.0001; late: F3,64 = 105.9; P<0.0001).
Figure 5.
Behavioral dysfunctions of Panx1−/− mice.
a, Pre-pulse inhibition of the acoustic startle response (PPI) showing a tendency towards lower levels in Panx1−/− (n = 8) compared to Panx1+/+ (n = 6) mice at the intensities of 62 and 64 dB. At the intensity of 72 dB, a statistically significant reduction of the PPI is detected. b, Object recognition was equivalent in both Panx1+/+ and Panx1−/− mice when the animals were allowed to explored two novel objects (A and B) for 5 minutes. c, One hour later control Panx1+/+ mice explored the now familiar object A significantly less than the novel object C. By contrast, Panx1−/− animals explored object C with significantly less intensity than object A. d, Cookie finding test: Training trials were performed on seven subsequent days, where in trial 1 and 2 a large cookie was used (500 mg), in trial 4 and 5 a smaller cookie (50 mg), and in trial 6 and 7 replaced by a very weak-odorous mouse chow. The time till the mice held the treat in their front paws is depicted in the bar diagram. Panx1−/− performed equal to Panx1+/+ mice in this experiment (P>0.05, Student’s t-test) e, A further trial was performed after training trails where no treat was hidden (Movie S1). Bar graphs depict the mean distance of the mice to the location where the cookie was hidden during training trials for the first 60 s. Error bars represent SEM. n = 9 for each mouse group. Panx1 knock out mice have an impaired memory, as they spent less time searching at the correct position (P = 0.02). However, they still remembered the former location to some extent, as they spent significantly more time searching at the correct position as untrained animals (P<0.001, Student’s t-test).