Genetic KCa3.1-Deficiency Produces Locomotor Hyperactivity and Alterations in Cerebral Monoamine Levels

Background The calmodulin/calcium-activated K+ channel KCa3.1 is expressed in red and white blood cells, epithelia and endothelia, and possibly central and peripheral neurons. However, our knowledge about its contribution to neurological functions and behavior is incomplete. Here, we investigated whether genetic deficiency or pharmacological activation of KCa3.1 change behavior and cerebral monoamine levels in mice. Methodology/Principal Findings In the open field test, KCa3.1-deficiency increased horizontal activity, as KCa3.1−/− mice travelled longer distances (≈145% of KCa3.1+/+) and at higher speed (≈1.5-fold of KCa3.1+/+). Working memory in the Y-maze was reduced by KCa3.1-deficiency. Motor coordination on the rotarod and neuromuscular functions were unchanged. In KCa3.1−/− mice, HPLC analysis revealed that turn-over rates of serotonin were reduced in frontal cortex, striatum and brain stem, while noradrenalin turn-over rates were increased in the frontal cortex. Dopamine turn-over rates were unaltered. Plasma catecholamine and corticosterone levels were unaltered. Intraperitoneal injections of 10 mg/kg of the KCa3.1/KCa2-activator SKA-31 reduced rearing and turning behavior in KCa3.1+/+ but not in KCa3.1−/− mice, while 30 mg/kg SKA-31 caused strong sedation in 50% of the animals of either genotypes. KCa3.1−/− mice were hyperactive (≈+60%) in their home cage and SKA-31-administration reduced nocturnal physical activity in KCa3.1+/+ but not in KCa3.1−/− mice. Conclusions/Significance KCa3.1-deficiency causes locomotor hyperactivity and altered monoamine levels in selected brain regions, suggesting a so far unknown functional link of KCa3.1 channels to behavior and monoaminergic neurotransmission in mice. The tranquilizing effects of low-dose SKA-31 raise the possibility to use KCa3.1/KCa2 channels as novel pharmacological targets for the treatment of neuropsychiatric hyperactivity disorders.


Introduction
The calcium/calmodulin-activated K + channel KCa3.1 [1,2] is voltage-independent and its primary cell biological role is to produce solid membrane hyperpolarization in response to increases in intracellular calcium concentrations. KCa3.1 is widely expressed in non-excitable tissues, e.g. erythrocytes (here known as the Gardos channel) [3,4], white blood cells [5], salivary glands [6], vascular endothelia [7,8] and intestinal and bronchial epithelia [9]. In these tissues KCa3.1 channels contribute to cell volume regulation, migration, and proliferation and thus play a role in modulating immune responses, fibrosis, restenosis disease, blood pressure, and fluid secretion [5,10]. Genetic deficiency of KCa3. 1 in mice has been shown to produce splenomegaly (likely caused by defective erythrocyte volume regulation) [3], endothelial dysfunction and mild systolic hypertension during locomotor activity [8,11], but no overt immunological deficits. In the brain, KCa3.1 channels are expressed in the blood brain barrier (i.e. cerebrovascular endothelium) [12] and in activated microglia [13,14] where they are involved in respiratory burst [14], nitric oxide production and inflammatory responses in the wake of ischemic stroke and traumatic brain injury [15,16]. Whether or not KCa3.1 is also expressed in central or peripheral neurons, is a matter of debate since the initial cloning papers reported that KCa3.1 is absent from neuronal tissue based on Northern blot analysis [1,17,18]. However, since then several studies reported expression of KCa3.1 in human dorsal root ganglia [19] and enteric neurons [20]. More recently, KCa3.1 expression has also been reported in cerebellar Purkinje cells of the rat in which the channel has been shown to contribute to after-hyperpolarizations and thereby regulation of excitatory postsynaptic potentials by suppressing low frequencies of parallel fiber input [21]. Despite these findings no behavioral phenotype has been reported in KCa3.1 2/2 mice.
In contrast to KCa3.1, the three related KCa2.X channels (KCa2.1, KCa2.2, and KCa2.3), which exhibit a smaller unitary conductance but a similar calmodulin-dependent activation and voltage-independence, are undoubtedly present in both the soma and dendrites of central neurons [22,23]. While KCa2.1 and KCa2.2 are most prominently expressed in the cortex and the hippocampus, KCa2.3 is enriched in subcortical areas like the striatum, thalamus and monoaminergic nuclei [24]. KCa2.X channels shape neurotransmission and firing frequency by underlying the apamin-sensitive medium after-hyperpolarization (AHP) current and have accordingly been implicated in the regulation of neuronal excitability, synaptic plasticity, learning, and memory [25,26]. For example, mice in which KCa2.3-expression can be suppressed by insertion of a tetracyclinesensitive genetic switch exhibited working/short-term memory deficits when treated with doxycycline (DOX) and an antidepressant-like phenotype in the forced swim test [27,28]. These changes were paralleled by enhanced dopamine and serotonin  signaling. Similarly, mice over-expressing KCa2.2 exhibit impaired hippocampal-dependent learning and memory in both the Morris water maze and a contextual fear-conditioning paradigm, while administration of the KCa2 channel blocker apamin improves the performance of rats or mice in the water maze or in the object recognition test (for recent complete review see [24]). However, higher doses of apamin induce seizures [24].
While KCa2.X channels thus clearly play an important role in the CNS, next to nothing is currently known about KCa3.1 in CNS functions and behavior. One exception is the recently reported observation that KCa3.1 2/2 mice are hyper-responsive to restrain-induced stress as concluded from an enhanced adrenocorticotropic hormone release from KCa3.1-deficient  Our behavioral studies demonstrate that genetic KCa3.1 deficiency enhances locomotor activity. These behavioral alterations were paralleled by changes in central, but not peripheral monoamine levels. In contrast, the KCa3.1/KCa2.X activator SKA-31 produced hypoactivity and sedation in a dose-dependent fashion.

Ethics Statement
Human tissue collection was performed by the Department of Pathology, Odense University Hospital and approved by the ethics committee of the University of Southern Denmark. Informed consent for such stored and anonymized material was not required and we obtained a waiver (CEIC No. 11/2012 and 13/2012) from our IRB to use this material. Animal protocols were approved by the Danish authorities (Dyreforsøgstilsynet, No. 2009/561-1740).

Behavioral tests
Open field test. OFT was performed with a non-transparent, squared plastic box (45645645 cm) over a period of 10 min. Movements were tracked using the SMART video tracking software (Panlab, Barcelona, Spain) connected to a video camera (SSC-DC378P, Biosite, Stockholm, Sweden). The distance travelled (meter), speed (cm/sec), resting time, turns, and the entries into the three zones (Wall, Inter peri and Center of the box) were recorded automatically. Rearing, grooming, and droppings were recorded manually and are given as number (n) of events [32].
Y-maze test. Spontaneous alteration behavior and hence working memory was evaluated in KCa3.1 +/+ and KCa3.1 2/2 , DOX-treated and untreated KCa2.3 T/T and KCa3.1 2/2 / KCa2.3 T/T mice using the Y-maze (arm length: 40 cm, arm bottom width: 7 cm, arm upper width: 8 cm, height of the wall: 16 cm). Each mouse was placed in the arm designated (a) of the Ymaze field. The number of entries, except for the first two, and alterations were recorded manually. Data were collected for 8 min.
Grip strength test. The grip strength meter (BIO-GT-3, BIOSEB, Sweden) was used to study the neuromuscular function by determining the maximum force required for KCa2.3 T/T , KCa2.3 T/T +DOX, KCa3.1 2/2 /KCa2.3 T/T and KCa3.1 2/2 / KCa2.3 T/T +DOX mice to release their grips. Mice were allowed to grasp the metal grid and then pulled backwards in the horizontal plane. The force applied to the grip was recorded as the peak tension. Total (both) front paw strengths were measured. Each mouse was tested in 5 sequential trials and the highest grip strength was recorded as the score [33].
The rotarod test. Was performed using a LE8200 system (Panlab Harvard Apparatus). The full test consisted of two parts; pre-training and 4 test trials (T1 -T4) [34,35]. Pre-training: mice were pre-trained to stay and walk on the rod for 30 seconds at 4 rounds per minute (rpm). Mice that were not able to do this were excluded from the subsequent test. All KCa3.1 2/2 /KCa2.3 T/T and KCa3.1 2/2 /KCa2.3 T/T +DOX mice fulfilled the criteria for the rotarod test, whereas 2 KCa2.3 T/T and 2 KCa2.3 T/T +DOX mice were excluded from the test due to failure of fulfilling the criteria in the pre-training. Trial: Prior to testing, the mice were allowed to acclimatize in the behavior room for at least 1 hour. Mice were placed on the rotarod and speed of the rotor was accelerated from 4 to 40 rpm over 5 minutes. The latency to fall from the rod was recorded automatically. Each mouse was tested in 4 trials with 20 min resting time between each trial.

Tissue processing for histology
Mice were given an overdose of pentobarbital and perfused through the left ventricle using 20 ml chilled 4% paraformalde-hyde (PFA) in 0.15 M phosphate-buffered saline, pH 7.4 (Lambertsen et al, 2009). Brains were quickly removed and post-fixated in 4% PFA for 1 hour and either immersed in 20% sucrose overnight, frozen and sectioned into a series of 8 parallel, 20 mm thick cryostat sections, which were stored at 280uC until further processing. Other brains were transferred to 1% PFA followed by 0.1% PFA, before serial cutting into 60 mm thick sections, which were stored in de Olmos cryoprotective solution at 212uC until further processing. One series of sections from each mouse was stained with Toluidine blue for visualization of the general histology [36].

Immunohistochemistry
Microglial CD11b and astroglial glial fibrillary acidic protein (GFAP) were visualized in vibratome sections using a three-step biotin-streptavidin-horseradish peroxidase (HRP) technique as described in detail elsewhere [36]. KCa3.1 and KCa2.3 immunohistochemistry (IHC) was performed on sections from paraffinembedded brain tissues. Sections were dewaxed with xylene, rehydrated through an alcohol gradient, treated with 1.5% H 2 O 2 , demasked with T-EG buffer, and incubated for 1 hour with primary antibody in antibody diluent S2022. The following primary antibodies (ABs) against KCa3.1 were used: Santa Cruz sc-32949 (1:500), Alomone ALM-051 (1:2000), and Sigma AV35098 (1:2000) followed by EnVision+polymer K4003(rabbit)/K4001(mouse). The ABs ALM-051 (1:2000) and Sigma AV35098 gave similar results (not shown). Sections were visualized with DAB and buffered Substrate Kit (Dako K3468), counterstained with hematoxylin and mounted with Aquatex. A second set of staining with a different antigen retrieval protocol, a different secondary antibody and the Vectastain kit was performed independently and gave similar results [15]. For IHC studies on KCa3.1 protein expression in human post mortem brain material, paraffin-embedded tissue was kindly provided by the Department of Pathology, Odense University Hospital and processed in the same way. As negative control we processed sections omitting the primary antibody (AV35098 against KCa3.1) or by substituting the primary antibody with rat IgG2a isotype control (CD11b) or rabbit serum (GFAP) as previously described [36].
High performance liquid chromatography analysis of monoamines in plasma and brain samples and corticosterone measurements Mice were scarified by decapitation and blood (about 300 ml) was collected in Eppendorf vials containing EDTA, centrifuged (1,000 g for 10 min, 4uC) and the plasma (about 150 ml per sample) was stored at 220uC until further processing. Prior to high Table 4. Radiographic assessment of hind limb composition of KCa2.3 T/T and KCa3.1 2/2 /KCa2.3 T/T mice.

KCa2.3 T/T (n = 11)
KCa3.   performance liquid chromatography (HPLC) analysis, plasma catecholamines were extracted using the ClinRepH complete kit (Recipe GmbH, Munich, Germany). After decapitation, the brain was quickly removed from the skull and different brain regions were rapidly dissected, placed on dry ice, weighed, and stored at 280uC. At the day of HPLC analysis, the brain tissue samples were briefly sonicated in Eppendorf vials containing 200-1000 ml (about 1:20 w/v) of 0.1 M perchloric acid (PCA) with antioxidants (0.2 g/l Na2S2O5, 0.05 g/l Na2-EDTA) and centrifuged at 20,627 g for 20 minutes at 4uC. The supernatant was used for HPLC analysis.
Levels of noradrenalin (NA), dopamine (DA), serotonin (5hydroxytryptamine, 5-HT) as well as the DA metabolites 3,4dihydroxyphenylacetic acid (DOPAC) and homovannilic acid (HVA), the NA metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) were assessed by reverse-phase HPLC with electrochemical detection, essentially as described previously [37], but using a mobile phase consisting of 10% methanol (v/v), 20 g/l citric acid monohydrate, 100 mg/l octane-1-sulfonic acid sodium salt, 40 mg/l EDTA dissolved in Milli-Q water and pH adjusted to 4.0 [38]. The Merck-Hitachi HPLC system consisted of a L-7100 pump a L-7200 autosampler, a D-7000 interface, and an electrochemical detector with in-built column oven (Decade, Antec, Leyden, Netherlands), connected to a computer equipped with D-7000 version 2.0 chromatography software. The mobile phase was pumped at a flow rate of 0.9 ml/min through a Waters Spherisorb S5 ODS2 guard column (4.6630 mm) and a Waters Spherisorb S3 ODS2 cartridge analytical column (4.66150 mm, Waters, MA, USA). A mixture of external standards was injected to identify and quantify the compounds of interest.
Plasma corticosterone levels were determined in mice sacrificed immediately after OFT and 40-45 minutes after treatment with either vehicle or 30 mg/kg SKA-31. Corticosterone was detected in 10 ml plasma samples (diluted 1:12) and quantified using the DetectXHCorticosterone CLIA Kit (Arbor Assays, Ann Arbor, Michigan) according to manufacturer's instructions.

Telemetry
Telemetric recording of voluntary diurnal home cage activity and analysis of data was performed after implanting a TA11-PAC10 transducer (Data Sciences International (DSI), St Paul, Minnesota, USA) as described previously [8].

Body composition measurements
Total tissue mass (g), fat mass (g), fat-%, lean tissue mass (g), lean-%, bone area (cm 2 ), bone mineral content (BMC, g), and bone mineral density (BMD, g/cm 2 ) were measured using dualenergy X-ray absorptiometry (DXA) using a PIXImus2 (Version 1.44; Lunar Corporation, Madison, WI, USA).  In DOX-treated and untreated KCa2.3 T/T and KCa3.1 2 / 2 /KCa2.3 T/T (right part of the graphs), 30 mg/kg, SKA-31 produced similar sedation. Representative SMART video tracking files show lasting immobilization in one mouse of either genotype. Numbers in columns (A) are the numbers of mice per group. Statistical comparisons were made between the different series of experiments (a-e) as indicated in (A) and by different column fillings. *P,0.05; **P,0.01; unpaired Student's T-test. (J) 24-hrs telemetric recording showed that KCa3.1 2/2 (n = 7) had higher diurnal home cage locomotor activity (counts/min) than KCa3.1 +/+ (n = 8). Note that the higher 24-hrs-average activity was due to a higher activity during the dark phase (activity phase of mice). Data are given as means 6 SEM. * P,0.05; unpaired Student's T-test. (K) SKA-31 (10 and 30 mg i.p at the end of the light phase) lowered night locomotor activity in KCa3.1 +/+ (10 mg, n = 5; 30 mg n = 3) but not KCa3.1 2/2 (10 mg, n = 5; 30 mg n = 3). * P,0.05, before vs. after SKA-31 injections in KCa3.1 +/+ ; paired Student's T-test. doi:10.1371/journal.pone.0047744.g004 reported previously [8]. Our radiographic assessment of total body mass, % lean mass, % fat mass, bone mineral density (BMD) and bone mineral content (BMC) (Tables 1 and 2) as well as of the same parameters in the hind limb (Tables 3 and 4) revealed no major differences between genotypes, with the exception of higher total body masses of male KCa3.1 2/2 mice (+3 g) compared to male KCa3-1 +/+ mice (Table 1) and of male KCa3.1 2/2 / KCa2.3 T/T mice (+5 g) compared to male KCa2.3 T/T mice ( Table 2) and a higher BMD and BMC in male KCa3.1 2/2 mice compared to male KCa3.1 +/+ mice (Table 1). General brain morphology and structures of major brain regions showed no abnormalities in any of the strains based on toluidine blue staining (data not shown). Immunohistochemical staining for GFAP + astrocytes and CD11b + microglia revealed no apparent differences in glial number or morphology in either genotype ( Figure 1A and B). Similar results were independently obtained when staining for IBA + microglia (data not shown). These results suggested that lifelong genetic deficiency of KCa3.1 or over-expression/suppression (using DOX) of KCa2.3 do not result in gross alterations of brain morphology. Specific immunoreactivity for KCa3.1 was not detectable in neurons, astrocytes, or microglia from any brain area of either genotype suggesting that KCa3.1 is not expressed at immunohistochemically detectable levels in the normal murine CNS ( Figure 1C). Similar observations were previously made in rat brain [15] and in our current study in human brain tissues, that were found to lack KCa3.1-immunoreactivity ( Figure 1D). In contrast, positive KCa3.1 immunoreactivity was observed in murine and human tissues such as endothelial cells (of human meningeal artery in the present study) and glioblastoma multiforme ( Figure 1D), which were previously shown to express KCa3.1 [7,8,39,40].

Analysis of motor coordination
KCa2.3 channels are expressed in dopaminergic neurons of the substantia nigra [22,26] and the cerebellum [24,26,41] while KCa3.1 channels have recently been reported to be present in rat cerebellar neurons [21]. We therefore tested whether untreated or DOX-treated KCa2.3 T/T and KCa3.1 2/2 /KCa2.3 T/T mice displayed alterations in motor coordination using the rotarod test ( Figure 3). The latencies to fall from the rotating rod in four consecutive trials did not differ between groups and all four groups spent similar total times on the rotarod, suggesting that expression changes in KCa3.1 and KCa2.3 do not have any overt effect on motor coordination ( Figure 3A, B). Note that HVA levels in the hypothalamus and brain stem were below the detection level in this analysis and DA turn-over rates are given as DOPAC/DA ratios for these tissues. (D) DA-turnover rates. * P,0.05, ** P,0.01, One-way ANOVA followed by Tukey's Multiple Comparison test. doi:10.1371/journal.pone.0047744.g007

Analysis of muscle force
We also studied whether the higher physical activity of KCa3.1 2/2 mice was paralleled by increased muscle force, using the grip strength test ( Figure 3C). However, a major difference in muscle force was not apparent, as grip strengths were comparable in all groups. Nonetheless, there was a trend towards slightly stronger grip strengths in untreated KCa2.3 T/T mice compared to untreated KCa3.1 2/2 /KCa2.3 T/T mice (P = 0.09). DOX treatment did not alter muscle force in DOX-treated KCa2.3 T/T mice when compared to untreated KCa2.3 T/T mice (P = 0.34). However, there was a trend for DOX treatment to reduce the grip strength in KCa3.1 2/2 /KCa2.3 T/T mice ( Figure 3C), even though the difference did not reach statistical significance (P = 0.09).
Behavioral impact of the KCa3.1-activator SKA-31 in the open field environment and the effect of KCa3.1deficiency and SKA-31 on home cage locomotor activity Since KCa3.1-deficiency produced hyperactivity, we hypothesized that pharmacological activation of KCa3.1/KCa2.X channels by SKA-31 produces hypoactivity (Figure 4). At a dose of 10 mg/kg administered intraperitoneally 30 min prior to the open field test, SKA-31 did not produce any overt differences in the total distance travelled ( Figure 4A) or the mean velocity ( Figure 4B) of KCa3.1 2/2 and KCa3.1 +/+ mice. Grooming behavior and the number of droppings were also unaffected ( Figure 4C and 4D). In contrast, SKA-31 produced low vertical explorative behavior (rearing) in the KCa3.1 +/+ mice but not in KCa3.1 2/2 mice (P,0.001; Figure 4E). Moreover, SKA-31-treated KCa3.1 +/+ mice made fewer turns than SKA-31-treated KCa3.1 2/2 mice (P,0.001; Figure 4F). Resting time, center/perimeter time ratios and entries into the different zones were unchanged by the treatment (Figure 4G-I). At the higher dose of 30 mg/kg, SKA-31 produced visible and lasting immobilization in 3 of 5 of the mice in both genotypes (see representative tracings in Figure 4 and for all tracings Figure S2), affecting all locomotor activity parameters ( Figure 4A-I), although if compared to vehicle-treated mice the differences did not reach statistical significance because two mice in each group did not respond to 30 mg/kg SKA-31 and produced high variance within groups. In DOX-treated and untreated KCa2.3 T/T and KCa3.1 2/2 /KCa2.3 T/T , 30 mg/kg, SKA-31 sedated 2 or 3 mice of each group (n = 3-5, see Figure S2 for all tracings) and e.g. rearing was nearly abolished (see right part of the graphs in Figure 4A-I). After 90 minutes the sedated mice recovered and freely travelled in the open field environment (see tracings in Figure S2, bottom panel).
These results suggested that SKA-31 produced low vertical and home cage activity levels over at least 12 hours in a KCa3.1dependent manner, while the strong immobilization caused by the high dose in some of the mice did not depend on KCa3.1 or expression levels of KCa2.3 and is most likely due to the activation of other KCa2 subtypes.

Discussion
Our study suggests a previously unrecognized but significant role of the KCa3.1 channel in the control of behavior and central monoaminergic transmission. We concluded this based on the following findings: 1. Genetic deficiency of KCa3.1 significantly increased locomotor activity without impairing neuromuscular functions or body physics. Locomotor hyperactivity due to KCa3.1-deficiency overcompensated for the locomotor hypoactivity normally observed in KCa2.3 T/T mice. 2. KCa3.1-deficiency produced slightly impaired working/short term memory. 3. Plasma catecholamine or corticosterone levels were not altered by KCa3.1 deficiency. 4. In contrast, central monoaminergic transmission was altered in several ways by KCa3.1 deficiency. KCa3.1 2/2 mice exhibited significantly reduced turn-over rates of 5-HT in frontal cortex, striatum, and brain stem. Moreover, KCa3.1 2/2 mice had reduced NA levels in brain stem and cerebellum and exhibited increased NA turn-over rates in the brain stem. 5. Administration of the selective KCa3.1/KCa2.Xactivator SKA-31 had opposite effects on behavior as it reduced rearing, turning behavior, and home cage activity in a KCa3.1dependent fashion and at a high dosages produced strong sedation in a KCa3.1/KCa2.3 independent manner, most likely related to KCa2.2 activation. These data suggested that KCa3.1-deficient mice show a type of attention-deficit hyperactivity disorder (ADHD) phenotype, which is related to lower 5-HT turn-over and lower NA levels in specifically the brain stem, but is not related to alterations in sympathetic drive and chronic distress. The efficacy of a selective KCa3.1/KCa2.X activator at reducing locomotor activity proposed KCa3.1/KCa2.X-channels as novel therapeutic targets for the treatment of human neurological syndromes characterized by physical hyperactivity such as ADHD.
Considering possible mechanisms underlying the behavioral alterations, our HPLC analysis of plasma and regional cerebral monoamine levels revealed normal plasma catecholamine levels, but several, brain-region specific changes in monoamine levels and turnover rates in KCa3.1 2/2 mice, including reduced NA levels and increased NA turn-over rate in the brain stem and frontal cortex as well as reduced 5-HT turnover rates in the striatum and brain stem. Particularly, the higher NA turn-over -pointing to increased NA transmission -may be linked to the observed hyperactivity in the KCa3.1 2/2 mice. However, whether these changes are causally related to the observed hyperactivity, remains unclear. A common finding in animal models of ADHD is hyperdopaminergic function (increased DA turnover)(for review see [42]), but this is apparently not the case in the KCa3.1deficient mice. However, because of cross-talk between the monoaminergic systems, changes in NA and 5-HT transmission can affect dopaminergic function and ADHD symptoms. Changes in noradrenergic transmission and action on inhibitory alpha-2 autoreceptors can either enhance or ameliorate ADHD symptoms. 5-HT may play a role in hyperactivity, either directly through 5-HT 2 receptors or indirectly by modulating dopaminergic transmission. Decreased activity of 5-HT has been reported in relation to impulsivity in borderline personality, aggression and suicide, while increased 5-HT activity and lower NA activity have been associated with ADHD (for references see [43]). However, changes in NA and/or 5-HT metabolism observed in clinical conditions or animal models may not be directly -causally -linked to hyperactivity, but rather reflect compensatory changes to counteract hyperactivity or dysfunction of other monoaminergic systems.
Recently, another KCa3.1 2/2 mouse has been reported to be hyper-responsive to stress due to increased release of adrenocorticotropic hormone (ACTH) and corticosterone, with no change in basal corticosterone levels [29]. We likewise did not observe higher plasma corticosterone or plasma levels of catecholamine suggesting that the hyperactivity of our KCa3.1-deficient mice is not related to higher chronic distress or overt sympathetic rush.
Unlike KCa3.1-deficient mice, KCa2.3 T/T mice exhibited significantly higher 5-HT levels in the frontal cortex and tended to also exhibit higher 5-HIAA levels in striatum with no change in 5-HIAA and 5-HIAA/5-HT turn-over rates. These increases, which are contrary to the decreases seen in KCa3.1 2/2 mice, could represent a compensatory mechanism to mitigate the overt hypoactivity in these animals. Higher 5-HT levels have been reported for this strain earlier [28] and the present study also showed that these alterations are presumably ''developmental'' as 2-week DOX-treatment and the resulting channel suppression had no impact on the 5-HT levels in these mice. It is, however, noteworthy that additional life-long KCa3.1-deficiency reduced levels to those measured in wild type mice.
While KCa3.1-deficiency produced hyperactivity, the brain penetrable KCa3.1/KCa2.X-activator SKA-31, with a 5-fold higher selectivity for KCa3.1 over KCa2.X channels, had opposite effects and produced hypoactivity and sedation in half of the animals, which at the higher dose was largely KCa3.1-and KCa2.3 independent as it occurred in KCa3.1 2/2 and KCa2.3 T/T with or without DOX treatment alike. This can be explained by activation of KCa2.1 and KCa2.2 channels with a more widespread expression in the murine brain [22] and is in line with findings that higher doses of SKA-31 are anticonvulsant presumably due to KCa2.2 activation [30] and acutely improve motor deficits in SCA3 mice, a model of spinocerebellar ataxia 3 [44]. Nonetheless, at a lower dose of SKA-31 the reduction of rearing and turning behavior depended on KCa3.1. Despite this poor KCa3.1-dependence of the acute SKA-31-effects, SKA-31 had a ''calming'' effect on spontaneous home cage activity in the wild-type, which was not observed in KCa3.1 2/2 mice, as expected. The acute treatment with the high dose of SKA-31 significantly increased 5-HT in the striatum and brain stem of wild type, but not in KCa3.1 2/2 mice, and significantly reduced 5-HIAA/5-HT turn-over rates in brain stem and hypothalamus in a KCa3.1-independent fashion, which suggested that these SKA-31effects are caused by activation of KCa2.X channels and involved a reduced 5-HT release as reflected by the lower 5-HT turn-over rate.
The mechanistic link between deficiency of KCa3.1 to the behavioral alterations and the changes of cerebral monoamine levels remains unclear. But we speculate that these alterations could be related to the endothelial dysfunction (severely impaired acetylcholine-induced vasodilation) and systolic hypertension during locomotor activity in the KCa3.1 2/2 mice [11,31]. In this regard KCa3.1 2/2 mice are similar to spontaneously hypertensive rats (SHR), which are also hyperactive, show memory deficits and alterations of monoaminergic neurotransmission, and thus neurological features similar to ADHD [42]. In contrast to our model, however, 5-HT levels are increased in several brain regions in the SHR model of ADHD. On the other hand, KCa3.1 2/2 (this study) and SHR [45] appear to have a concordant higher central NA activity.
In order to validate KCa3.1 deficient mice as an alternative model of ADHD, the behavioral effects of chronic methylphenidate treatment should be investigated in further studies.
In conclusion, the present study revealed a novel mechanistic link of the non-neuronal KCa3.1 channel to behavior, central monoamine levels, and hyperactivity in mice. From the clinical perspective, our pharmacological studies suggested that small molecule activators of KCa3.1 may have beneficial ''calming'' effects in hyperactivity disorders, including ADHD.