Pannexin-1 Deficiency Decreases Epileptic Activity in Mice

Abstract

Objective: Pannexin-1 (Panx1) is suspected of having a critical role in modulating neuronal excitability and acute neurological insults. Herein, we assess the changes in behavioral and electrophysiological markers of excitability associated with Panx1 via three distinct models of epilepsy. Methods Control and Panx1 knockout C57Bl/6 mice of both sexes were monitored for their behavioral and electrographic responses to seizure-generating stimuli in three epilepsy models-(1) systemic injection of pentylenetetrazol, (2) acute electrical kindling of the hippocampus and (3) neocortical slice exposure to 4-aminopyridine. Phase-amplitude cross-frequency coupling was used to assess changes in an epileptogenic state resulting from Panx1 deletion.

Results: Seizure activity was suppressed in Panx1 knockouts and by application of Panx1 channel blockers, Brilliant Blue-FCF and probenecid, across all epilepsy models. In response to pentylenetetrazol, WT mice spent a greater proportion of time experiencing severe (stage 6) seizures as compared to Panx1-deficient mice. Following electrical stimulation of the hippocampal CA3 region, Panx1 knockouts had significantly shorter evoked afterdischarges and were resistant to kindling. In response to 4-aminopyridine, neocortical field recordings in slices of Panx1 knockout mice showed reduced instances of electrographic seizure-like events. Cross-frequency coupling analysis of these field potentials highlighted a reduced coupling of excitatory delta-gamma and delta-HF rhythms in the Panx1 knockout.

Significance: These results suggest that Panx1 plays a pivotal role in maintaining neuronal hyperexcitability in epilepsy models and that genetic or pharmacological targeting of Panx1 has anti-convulsant effects.

Keywords: 4-AP; PTZ; epilepsy; kindling; panx1; seizure.

Figure 1

Response to 80 mg/kg pentylenetetrazol (PTZ) injection in each of the four animal groups, WT (normal pannexin-1 expression), KO (genetic knockout of pannexin-1) and WT-treated BB (pretreated with Brilliant Blue-FCF) or PB (pretreated with probenecid) mice. (a,b) male and female (respectively) mice demonstrated altered distributions of time spent manifesting behaviors of different seizure stages. (c) Survival curve of both sexes of animals following PTZ injection. (d,e) Average time to first stage 5 seizure. No significant differences exist between the two sexes of the same experimental group. KO Males took significantly longer to reach stage 5 seizures than any other group except KO females. (f) The average concentration of BB-FCF, as measured through spectrophotometry of homogenized brain tissue, was approximately 0.88 ± 0.26 µM. In all cases, * p < 0.05, ** p < 0.01, *** p < 0.001, Two-way ANOVA with Bonferroni post-hoc test.

Figure 2

Results from the kindling of 12 Panx1 +/+ (WT) and 10 Panx1 −/− (KO) mice. (a,b) Sample recordings of the CA3 following electrical stimulation to induce kindling in WT (a) and KO (b) mice, demonstrating the difference in the afterdischarge (AD). These examples are reflective of the stimulus experienced by each mouse. (c) The maximal behavioral outcome experienced, as measured by the Racine score, averaged by group. A significant decrease in cumulative AD was observed in KO animals as compared to their WT counterparts. (d) The average duration of ADs following each stimulus showed no significant trial-dependent trends but strongly indicated a reduced AD duration in KO mice. Dashed lines show the mean AD duration and dotted lines represent the standard error of the mean (SEM). (e) Kindling power by frequency band. Significant differences are reported by band, between the WT and KO groups (f) Percent total power by frequency band. * p < 0.05, *** p < 0.001, Student’s t-test.

Figure 3

Seizure-like events in vitro in WT, KO, BB (Brilliant Blue-FCF-treated) and PB (probenecid-treated) groups. (a–d) Sample recordings of local field potentials from each of the four experimental groups, along with pie charts summarizing seizure incidence with 4-AP. Recordings from WT mice often had one or more seizure-like events (78%), whereas KO, BB and PB mice often had only small synchronous bursting events (20%, 10% and 8% respectively). All traces share the calibrations defined in (d). (e,f) High-energy activity in the WT and KO traces is zoomed in to reveal a SLE in the WT slice and bursting in the KO slice, indicative of the most common outcomes to 4-AP for each group. Both traces share the calibrations defined in (f). (g,h) Comparison of seizure duration and inter-seizure interval of SLEs in both WT and KO slices. Despite a reduced incidence, once initiated, KO SLEs remained similar to WT SLEs.

Figure 4

Frequency-based analysis of the extracellular recordings of Panx1 KO mice and their WT counterparts. (a) The relative increase by frequency band in neocortical field potentials after bath application of 4-AP. As 4-AP induces broadband increased spectral power, the relative increase in power of each band is normalized to the overall increase in spectral power. While changes in Delta through Beta bands were not significant, the increase in spectral power in Gamma (p = 0.0009), HF (p = 0.0011) and vHF (p = 0.0008) bands were significantly lower in Panx1 deficient animals following 4-AP. (b,c) Traces of WT and KO extracellular recordings under 4-AP. (d,e) Phase-amplitude coupling comodulograms of 10-s windows during baseline (blue) and 4-AP (orange) conditions, as highlighted in subfigures b and c respectively, highlighting periods of enhanced PAC in the WT 4-AP response which are absent in the KO. ** p < 0.01, *** p < 0.001.

Figure 5

Median phase-amplitude cross frequency coupling comodulograms for the in vitro 4-AP slice model (a,b,e,f) and in vivo kindling model (c,d) following z-score normalization to baseline conditions. Cortical slices from mice in the KO group demonstrated reduced phase-amplitude coupling following incubation with 4-AP (b) or electrical kindling (c) as compared to WT counterparts (a and d respectively). Coupling of delta phase to HF amplitude was observed in both models of hyper-excitability in control animals but was absent in Panx1 deficient mice, including those pretreated with Panx1 blockers BB-FCF (e) and probenecid (f). This finding is indicative of reduced epileptiform activity in the absence of functional Panx1.