Genetic studies have established that potassium channel dysfunction is responsible for multiple pediatric epilepsy disorders (Brenner and Wilcox, 2012; Geisheker et al., 2017; Niday and Tzingounis, 2018; Oyrer et al., 2018). KCNQ2 and KCNQ3 channels, in particular, have emerged as fundamental regulators of normal brain activity (Brenner and Wilcox, 2012; Geisheker et al., 2017). Pathogenic variants of these channels are strongly associated with early-onset neonatal epileptic encephalopathy and developmental disorders (Millichap et al., 2017; Mulkey et al., 2017; Oyrer et al., 2018). However, the network mechanisms by which KCNQ2/3 variants lead to hyperexcitability are not fully understood.

KCNQ2/3 channels are expressed in both pyramidal neurons and interneurons (Cooper et al., 2001). In pyramidal neurons, KCNQ2/3 channels primarily control spike frequency adaptation, a quiescence period neurons enter following a brief train of activity (Peters et al., 2005; Soh et al., 2014). However, our knowledge of KCNQ2/3 function in interneurons is limited. This gap in knowledge is partly because KCNQ2/3 function is most easily observed in neurons that undergo pronounced spike frequency adaptation, a characteristic not traditionally found in interneurons (Pelkey et al., 2017).

It is currently unknown whether loss of KCNQ2/3 function in interneurons would have effects on overall network activity, and if it does, whether it would lead to a dampening or an increase in excitability. Previous work suggests that loss of KCNQ2/3 function in interneurons would likely elevate their excitability (Lawrence et al., 2006; Pelkey et al., 2017), which in turn would increase GABAergic inhibition and decrease network excitability. However, at early times in development GABA is depolarizing due to shifted chloride equilibrium potential (Le Magueresse and Monyer, 2013). Previous work found that neonatal administration of bumetanide, which changes GABA receptor activity from depolarizating to hyperpolarizing, prevents seizures in mice with a global loss of KCNQ2 channels (Marguet et al., 2015). This may be evidence that loss of KCNQ2/3 function at this time increases network excitability through changes in interneuron GABA signaling, but the authors did not directly examine synaptic activity. Further, it is entirely unknown what effects KCNQ2/3 dysfunction in interneurons has on network excitability in juvenile and mature mice.

In this work, we developed mice lacking KCNQ2 and KCNQ3 specifically in interneurons to address their possible role at the cellular and network activity. We found that KCNQ2/3 channels regulate interneuron properties in a cell type-specific manner. Deletion of KCNQ2/3 channels primarily impacts the firing properties of PV⁺ interneurons, but not SST⁺ interneurons. We also find that interneuronal loss of KCNQ2/3 or KCNQ2 channels increases excitatory transmission between pyramidal neurons, perhaps as homeostatic compensation for the increased GABAergic signaling we observe.

To investigate the role of KCNQ2/3 channels in interneurons, we developed mouse lines that lack KCNQ2/3 channels specifically in parvalbumin-positive (PV⁺) and somatostatin-positive (SST⁺) interneurons, cell types known to express KCNQ2/3 channels (Cooper et al., 2001; Lawrence et al., 2006). We crossed Kcnq2 or Kcnq3 floxed mice (Kcnq2^f/f and Kcnq3^f/f) to Nkx2-1^cre mice (Xu et al., 2008). The Nkx2-1^cre driver is expressed starting early in development (~embryonic day 10.5) in SST⁺ and PV⁺ interneurons, allowing us to study the impact of KCNQ2/3 channel ablation in young and juvenile neurons. In order to identify the Cre-expressing PV⁺ and SST⁺ interneurons, we crossed these mice to a reporter line (Ai9) that expresses the fluorescent protein tdTomato in cells in which Cre recombinase has been active. We designated these mice as IN:Kcnq2/3 null (Nkx2-1^cre;Kcnq2^f/f;Kcnq3^f/f;Ai9).

We first examined whether ablation of KCNQ2/3 channels from interneurons led to changes in the excitatory and inhibitory drive of CA1 pyramidal neurons. Based on previous work (Pelkey et al., 2017), we predicted that loss of KCNQ2/3 channels from PV⁺/SST⁺ interneurons would lead to increased inhibitory but not excitatory transmission. We recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and excitatory postsynaptic currents (sEPSCs) from CA1 pyramidal neurons of IN:Kcnq2/3 null mice. These measurements are commonly used to detect synaptic input. We first focused our analysis on the second week of life, as KCNQ2/3 dysfunction is primarily associated with neonatal epilepsy (Oyrer et al., 2018). Consistent with KCNQ2/3 channel expression in PV⁺/SST⁺ interneurons (Pelkey et al., 2017), the loss of these channels increased sIPSC frequency (Figure 1a; mean frequency: control 6.4 ± 0.5 Hz, n = 15; IN:Kcnq2/3 null 8.8 ± 1.1 Hz, n = 10; df = 23 t = −2.22 p=0.036 two-tailed Student’s t-test). Importantly, ablation of Kcnq2/3 from PV⁺/SST⁺ also increased sEPSC frequency in CA1 pyramidal neurons (Figure 1a; mean frequency: control 2.01 ± 0.16 Hz, n = 14; IN:Kcnq2/3 null 3.01 ± 0.35 Hz, n = 8; df = 20 t = −2.92 p=0.0085 Student’s t-test). This frequency increase was accompanied by a three-fold change in the miniature EPSC (mEPSC) frequency (Figure 1b; mean frequency: control 0.71 ± 0.11 Hz, n = 9; IN:Kcnq2/3 null 2.2 ± 0.4 Hz, n = 6; p=0.002 df = 13 t = −3.86 Student’s t-test), suggesting that elevating interneuron excitability led to secondary changes in excitatory synaptic drive. We did not find any changes in the miniature IPSC (mIPSC) frequency (Figure 1b; mean frequency: control 5.25 ± 0.48 Hz, n = 12; IN:Kcnq2/3 null 5.8 ± 0.6 Hz, n = 9; df = 19 t = −0.81 p=0.43 Student’s t-test), suggesting that the effect on sIPSCs was due to elevated interneuron excitability. The effects on the mEPSC frequency were likely a result of synaptic homeostasis in an effort to maintain a constant excitation and inhibition ratio. This is because an increase in mEPSCs, and consequently sEPSCs, would counteract the increases in the sIPSC frequency. Indeed, the sEPSC/sIPSC ratio (mean ratio: control 0.34 ± 0.03, n = 14; IN:Kcnq2/3 null 0.36 ± 0.04, n = 8), unlike the mEPSC/mIPSC ratio (mean ratio control 0.17 ± 0.04, n = 9; IN:Kcnq2/3 null 0.38 ± 0.05, n = 5; df = 12 t = −3.51 p=0.004 unpaired Student’s t-test), remained the same in neurons with or without PV⁺/SST⁺ KCNQ2/3 channels. Importantly, the effects on the mEPSCs were specific to changes in interneuron excitability, as we did not find any significant changes in the mEPSC frequency in CA1 pyramidal neurons from Kcnq2 pyramidal neuron knockout mice (Emx1^Cre;Kcnq2^f/f, designated as PYR:Kcnq2 null) (Figure 1). Rather, in PYR:Kcnq2 null neurons only the spontaneous frequency was elevated (Figure 1; mean frequency: control 2.01 ± 0.16 Hz, n = 14; PYR:Kcnq2 null 3.3 ± 0.46 Hz, n = 10; df = 22 t = −2.99 p=0.0067 Student’s t-test), consistent with the role of KCNQ2/3 channels in controlling pyramidal neurons’ axonal excitability (Battefeld et al., 2014).

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Are the synaptic changes due to loss of KCNQ2/3 from PV⁺, SST⁺, or both types of interneurons? To address this question, we tested whether ablation of KCNQ2/3 channels alters the excitability of PV⁺ and SST⁺ cells in mouse CA1. We distinguished these neurons based on their intrinsic excitability and firing properties (Figure 2). We found that ablation of Kcnq2/3 from PV⁺-like interneurons led to an increase in the number of action potentials following suprathreshold depolarizing current pulses (Figure 2a). In contrast, ablation of Kcnq2/3 from SST⁺-like interneurons did not change their firing properties (Figure 2a). We obtained similar results for L2/3 interneurons, suggesting that KCNQ2/3 function is critical for PV⁺ interneuron properties across multiple forebrain regions (Figure 2a). To confirm our finding that KCNQ2/3 alters PV⁺ interneuron excitability, we tested the effect of ablating Kcnq2 and Kcnq3 from PV⁺ interneurons by developing Pvalb^cre;Kcnq2^f/f/Kcnq3^f/f;Ai9 (designated as PV:Kcnq2/3 null) mice. Indeed, ablation of Kcnq2 and Kcnq3 increased the firing rate of PV⁺ neurons in the CA1 region (Figure 2b). The increased excitability in PV⁺ interneurons might have been due to their increased input resistance of Kcnq2/3 null neurons (Figure 2b; mean R_IN: control 166 ± 16 MΩ, n = 15; PV:Kcnq2/3 null 257 ± 33 MΩ, n = 14; df = 27 t = −2.54 p=0.017 unpaired Student’s t-test). However, as application of the pan-KCNQ blocker XE-991 did not change the input resistance in control PV⁺ interneurons (Figure 2—figure supplement 3; R_IN: control 169 ± 20 MΩ,+XE-991 143 ± 20 MΩ, n = 6; t = 1.355 df = 5 p=0.233), it’s unclear whether the change in input resistance in Kcnq2/3 null neurons is due to direct loss of KCNQ2/3 activity or an indirect effect. Next, we repeated our experiments in SST^cre;Kcnq2^f/f/Kcnq3^f/f;Ai9 neurons (Figure 2b; refer to as SST:Kcnq2/3 null). Consistent with our earlier finding, ablation of Kcnq2/3 from SST⁺ interneurons did not change their firing properties or input resistance (mean R_IN: control 360.28 ± 34 MΩ, n = 6; SST:Kcnq2/3 null 327 ± 64 MΩ, n = 8; df = 12 t=−0.42 p=0.68 unpaired Student’s t-test).

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Our data suggest that ablation of KCNQ2/3 channel activity from PV⁺ interneurons results in elevated interneuron excitability, which in turn could lead to remodeling of fast excitatory synaptic activity. As KCNQ2 pathogenic variants are much more frequent than KCNQ3 variants (Brenner and Wilcox, 2012), we also examined whether ablation of Kcnq2 alone could lead to similar effects. Indeed, we found that loss of KCNQ2 channels from PV⁺ interneurons increased their intrinsic excitability (Figure 3a) and the frequency of sEPSCs in pyramidal neurons (Figure 3c; Pvalb-Cre;Kcnq2^+/+;Ai9 1.165 ± 0.12 Hz, n = 13; Pvalb-Cre;Kcnq2^f/f;Ai9: 2.108 ± 0.22 Hz, n = 19; p=0.0098 Mann-Whitney test). The observed changes in the sEPSC frequency were not due to changes in the intrinsic excitability of pyramidal neurons (Figure 3b). To determine whether ablation of Kcnq2 from PV⁺-expressing interneurons affected the excitatory drive in vivo, we administered picrotoxin, a GABA_A receptor antagonist. We implanted mice with bilateral frontal and parietal electrodes and validated generalized epileptiform activity coinciding with Racine scale Stage five seizures induced by intraperitoneal injection of picrotoxin (10 mg/kg) (Figure 4a). Thereafter, we video-recorded the latency to seizure onset without implantation. All of the mice lacking Kcnq2, including Pvalb;Kcnq2 heterozygous mice, reached Stage five seizure activity within 30 min, with a significantly reduced latency to seizure onset when compared to Pvalb;Kcnq2^+/+ mice (Figure 4b). These data confirm that loss of KCNQ2 channel activity from interneurons can lead to excitatory network hyperexcitability in vivo.

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In summary, our ex vivo and in vivo work reveals that changes in interneuron excitability induced by loss of KCNQ2/3 channel activity in PV⁺ interneurons could change the course of excitatory network development. Multiple studies have shown that early in development GABA-mediated transmission partly drives excitatory synapse maturation and formation in pyramidal neurons. This is because early in development pyramidal neurons have a depolarized GABA equilibrium potential(Le Magueresse and Monyer, 2013; Wang and Kriegstein, 2011), allowing GABA to provide a much-needed depolarizing signal to relieve magnesium block from NMDA receptors and in turn promote AMPA receptor unsilencing and synaptic input maturation. Indeed, recent work showed that pharmacologically blocking potassium channels with 4-AP to drive GABA release on newborn neurons led to robust formation of excitatory synaptic inputs, allowing for the integration of newborn cells to excitatory synaptic networks (Chancey et al., 2013). We thus speculate a similar mechanism might in play in our mice. Future work is needed to directly test this hypothesis.

Importantly, our work suggests a unique mechanism compared to earlier work showing that loss of potassium channel activity critical for interneuron firing behavior leads only to depolarization block (Lau et al., 2000) and hyperexcitability through disinhibition. Therefore, our work raises the possibility that potassium channel dysfunction that elevates GABAergic transmission could also lead to remodeling of excitatory transmission in the cortex. Such changes might contribute to the severe neurodevelopmental effects seen in patients with potassium channel epileptic encephalopathy.

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Author details

1. Heun Soh

   Department of Physiology and Neurobiology, University of Connecticut, Connecticut, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing—original draft

   Contributed equally with

   Suhyeorn Park

   Competing interests

   No competing interests declared

2. Suhyeorn Park

   Department of Neurology, Baylor College of Medicine, Texas, United States

   Contribution

   Formal analysis, Investigation

   Contributed equally with

   Heun Soh

   Competing interests

   No competing interests declared

3. Kali Ryan

   Department of Physiology and Neurobiology, University of Connecticut, Connecticut, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Kristen Springer

   Department of Physiology and Neurobiology, University of Connecticut, Connecticut, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

5. Atul Maheshwari

   Department of Neurology, Baylor College of Medicine, Texas, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   atul.maheshwari@bcm.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3045-7901

6. Anastasios V Tzingounis

   Department of Physiology and Neurobiology, University of Connecticut, Connecticut, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   anastasios.tzingounis@uconn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4605-3437

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