Effects of genetic deletion of the Kv4.2 voltage-gated potassium channel on murine anxiety-, fear- and stress-related behaviors

The excitability and functional plasticity of specific brain circuits in cortical, limbic, and midbrain regions is thought to mediate behavioral responses to environmental threats, and to enable adaptation to stressors. At the cellular level, voltage- and calcium-activated potassium (K+) channels provide an important means of modulating neuronal activity and synaptic plasticity. Their function is further refined via the presence of multiple Kv primary and auxiliary subunits that differ in their voltage-dependence, post-translational regulation, sub-cellular localization and regional pattern of expression in the brain to accomplish distinct physiological functions.

A-type K+ channels composed of Kv4 subunits transmit a rapidly activating and inactivating current [1]. Kv4.2 mRNA is expressed in the periphery and in various brain regions involved in mediating stress-related behaviors, including the medial prefrontal cortex (mPFC), hippocampus and hypothalamus [2]. In CA1 hippocampal neurons, Kv4.2 protein is primarily localized on dendrites [3] where they exert their strongest functional effects [4]. Gene deletion of Kv4.2 (Kcnd2) in mice eliminates most of the A-type K+ current in hippocampal and cortical neurons [5–7], and increases back-propagation of the action potential from the axon to the somatodendritic region [6]. These and other effects of Kv4.2 knockout (KO) may be countered to some extent by compensatory increases in the expression of other Kv subunits [8], and increased inhibitory transmission in the hippocampus [9]. Notwithstanding, loss of Kv4.2 function produces a net increase in neuronal, and particularly dendritic, hyperexcitability in the mPFC and hippocampus, and possibly other brain regions in which this subunit is expressed.

Synaptic plasticity is also altered by Kv4.2 deletion, as evidenced by the enhanced CA1 hippocampal long-term potentiation (LTP) and increased threshold for long-term depression (LTD) seen in Kv4.2 (KO) mice [6, 10]. Further demonstrating an important contribution to hippocampal plasticity, Kv4.2 channels are internalized during LTP induction, and Kv4.2 overexpression or deletion alters synaptic expression of N-Methyl-D-aspartate (NMDA) receptor subunits (GluN2A, GluN2B) to respectively constrain or promote hippocampal LTP [11, 12]. Conversely, NMDAR throughput decreases hippocampal Kv4.2 through degradation [13, 14] whereas concurrently causing an increase in Kv4.2 translation [15]. The reciprocal relationship between Kv4.2 and NMDA receptor subunits is particularly intriguing in the context of prior studies showing that gene KO of GluN2A [16] or GluN2B [17] disrupts anxiety-related behaviors and adaptations to stress.

Behaviorally, Kv4.2 KO mice are viable and appear grossly normal [18]. However, Kv4.2 KO mice show augmented nociceptive responses to thermal and mechanical stimulation [8] and increased sensitivity to the pro-convulsive effects of kainate, with no apparent increase in spontaneous seizures [19]. Furthermore, in a recent study assessing Kv4.2 KO mice on a 129S6/SvEvTac (129S6) background on a range of behavioral assays, Lockridge et al. found that the KO mice had reduced grip strength on a wire-hang test and increased locomotor activity in a brightly illuminated open field, but not in a dimly illuminated one [20]. Although the KO mice displayed an increased tendency to enter into the aversive central area of the open field, consistent with an anxiolytic-like phenotype, the mutants were no different from the wild-type (WT) controls in the increased plus-maze test for anxiety-such as behavior. This study also found that Kv4.2 KO mice showed increased immobility in the forced-swim test (FST) (but not the tail-suspension test) for 'depression-related' behavior, and were insensitive to the anti-immobility effects of the antidepressant fluoxetine, but not imipramine or desipramine, in the FST. In addition, ex vivo slice physiological recordings indicated that a form of 5-hydroxytryptamine (5-HT)2A receptor-mediated excitatory synaptic transmission in mPFC pyramidal neurons was attenuated in Kv4.2 KO mice after a single forced swim exposure, but only after repeated exposures in WT controls. Based on these findings, Lockridge et al. concluded that Kv4.2 deletion produces abnormal behavioral responses to stress, and suggested that this might be related to excessive 5-HT release.

The aim of the present study was to confirm and extend the characterization of Kv4.2 KO mice in stress- and other 'emotion'-related phenotypes. KO mice and WT littermate controls were compared for a battery of sensory and neurological tests (including hot-plate nociception, acoustic startle and prepulse inhibition of startle, and home-cage locomotion), exploratory and anxiety-related behaviors (novel open field, light/dark exploration, elevated plus-maze tests), pavlovian fear conditioning and extinction, and 'depression-related' behavioral responses (single and repeated inescapable forced-swim exposure). Phenotyping was conducted after the mutants were backcrossed onto a C57BL/6J background strain. This is important because genetic background can strongly influence the phenotype of mutant mice [21, 22], and the aforementioned studies by Lockridge and colleagues tested the KO mice on a different genetic background (129S6).

Experimental procedures were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee.

The aim of the current study was to examine the effects of deleting the A-type voltage-gated K+ channel Kv4.2 on multiple stress- and other 'emotion'-related phenotypes. We found a complex set of significant phenotypic alterations in Kv4.2 mice. These included increased exploratory activity in novel environments, test-specific decreases in anxiety-like behavior, increased fear response to auditory stimuli, and an exaggerated corticosterone response to stress.

Consistent with previous studies [19], an initial observational battery showed no gross alterations in various measures of health or sensory function in the Kv4.2 knockout; however, this analysis did reveal increased rearing in the KO mice, suggestive of heightened levels of exploratory behavior. Similarly, exploratory locomotion in a novel open field was robustly increased in the KO mice, at least over a 30 minute session (longer sessions were not examined). These genotype effects did not seem to reflect generalized locomotor hyperactivity in the mutants, as evidenced by normal levels of activity in the familiar and low-stress environment of the home cage. Rather, these data suggest that Kv4.2 deletion produced an augmented behavioral reaction to novel and/or stressful environments.

This behavioral phenotype may be related to the neural abnormalities that have been reported in Kv4.2 KO mice. For example, these mice have been found to exhibit neuronal and dendritic hyperexcitability due to increased axonal back-propagation [6]. Interestingly in this context, we have previously identified a similar profile in the same behavioral assays after deletions of other genes involved in regulating neuronal excitability, such as components of the glutamate signaling system [16, 37, 38]. This overlap indicates that genetic loss of molecules involved in regulating neuronal excitability and synaptic plasticity produces some convergent phenotypic effects on novelty-driven behavioral reactivity. Although hyperlocomotion in a novel environment is often taken as a measure relevant to schizophrenia [39, 40], the Kv4.2 KO mice in the present study did not show changes in another schizophrenia-relevant measure, prepulse inhibition of startle, unlike our findings in previous studies of glutamate deletions. Therefore, without additional experiments to interrogate this issue further, it would be premature discuss the Kv4.2 KO phenotype in terms of potential relevance to that disease.

Novelty-induced hyperactivity can confound interpretation of behavior in tests for anxiety-like behavior due to non-specific increases in activity [41]. However, we found that Kv4.2 KO mice were no different from WT controls in the light/dark exploration test and showed increased open-arm exploration in the elevated plus maze, which was not associated with increased general locomotor activity in this test (as measured by the number of closed-arm entries). This suggests a test-specific anxiolytic-like phenotype in the KO mice, albeit with the caveat that the classic conflict-based assays for anxiety-like behavior cannot unequivocally parse an anxiety-like decrease in avoidance from a novelty-driven increase in approach [41]. Notwithstanding, the test-specific nature of this phenotype is notable, and echoes earlier studies showing that the elevated plus maze can be particularly sensitive to certain gene mutations, possibly because it is inherently more stressful than ostensibly similar tests such as the light/dark exploration [42]. This would generally fit with a profile of exaggerated behavioral responses of Kv4.2 KO mice, particularly under conditions of strong environmental 'provocation.'

Consistent with this interpretation, the KO mice showed increased unconditioned freezing to an auditory stimulus, and an augmented corticosterone response to a single exposure to the forced-swim test. By contrast, the behavioral response (immobility) to forced swim, a very stressful and 'provocative' situation, was normal in the KO mice. Increases in immobility produced by repeated, brief, forced-swim exposures were also similar between KO mice and WT controls. Thus, increased behavioral reactivity in the mutants does not generalize to altered immobility during various forms of forced-swim exposure. One explanation is enhanced tonic inhibitory transmission [9] served to mitigate the penetrance of the phenotype under forced-swim conditions.

The absence of changes in forced-swim tests in the current study differs from the previous finding by Lockridge et al. that these mice showed increased immobility in the forced-swim test [20]. Indeed, whereas the earlier report found, as we did, novel open-field hyperactivity, some other differences were seen in the earlier study, including no change in elevated plus maze anxiety-like behavior. Other than methodological variations, the principal salient factor potentially explaining these apparent discrepancies is genetic background. Kv4.2 KO mice were bred on a C57BL/6J background for the current study and a 129S6 background for the previous study. Genetic background can have a profound influence on the penetrance and expression of emotion-related phenotypes in mutant mice, because of epistatic interactions between a mutation and modifier genes [21, 22]. It seems likely that such interactions shaped the phenotypic profile of the Kv4.2 KO mice, and it would be interesting to explore this further by identifying the responsible modifiers.

Another important avenue for future studies will involve assessment of Kv4.2 KO mice for learning and memory. The mutants have enhanced hippocampal synaptic plasticity [6, 10], which is associated with altered synaptic expression of synaptic proteins that are strongly implicated in learning by mutant studies [23, 43–45], such as the NMDA receptor GluN2A and GluN2B subunits [11–13]. In the current study we found that the KO mice displayed normal amygdala-mediated fear learning and mPFC-mediated fear extinction. Given the localization of synaptic alterations to the hippocampus, it will be valuable to supplement these findings by assessing Kv4.2 KO mice for hippocampal-dependent forms of learning.

In summary, we found that constitutive deletion of the A-type voltage-gated K+ channel Kv4.2 produced a behavioral phenotype principally characterized by increased behavioral reactivity to exposure to some but not all novel environmental situations. This largely confirms earlier evaluation of these mice, although some discrepancies were found, possibly due to the influence of genetic background. These behavioral abnormalities extend the finding that deletion of Kv4.2 leads to exaggerated neuronal excitability in cortical and hippocampal regions involved in emotion regulation. These data shed further light on the role of Kv4.2 in regulating neural and behavioral functions.