Gating small conductance calcium‐activated potassium channels in the thalamic reticular nucleus

Small conductance calcium‐activated potassium (SK) channels are well‐known regulators of neuronal excitability. In the thalamic hub, SK2 channels act as pacemakers of thalamic reticular neurons, which play a key role in the thalamocortical circuit. Several disease‐linked genes are highly enriched in these neurons, including genes known to be associated with schizophrenia and attentional disorders, which could affect neuronal firing. The present study assessed the effect of pharmacological modulation of SK channels in the firing pattern and intrinsic properties of thalamic reticular neurons by performing whole cell patch clamp recordings in brain slices. Two SK positive allosteric modulators and one negative allosteric modulator were used: CyPPA, NS309, and NS8593, respectively. By acting on the burst afterhyperpolarization (AHP), negative modulation of SK channels resulted in increased action potential (AP) firing, increased burst duration, and decreased intervals between bursts. Conversely, both CyPPA and NS309 increased the afterburst AHP, prolonging the interburst interval, which additionally resulted in reduced AP firing in the case of NS309. Alterations in SK channel activity would be expected to alter functioning of thalamocortical circuits. Targeting SK channels could be promising in treating disorders involving thalamic reticular dysfunction such as psychiatric and neurodevelopmental disorders.

F I G U R E 1 SK modulators.Positive allosteric modulators of SK channels are shown in blue (NS309, CyPPA), and negative modulators are shown in red (NS8593).The color code reflects the potency of the target; the darker the color the more potent on the target.Source: Modified from Hougaard et al. (2007), Strøbaek et al. (2006), and Strøbaek et al. (2004).
Small conductance Ca 2+ -activated K + channels (SK) play a crucial role in regulating neuronal excitability by mediating the medium afterhyperpolarization (AHP) following a single or multiple action potentials (APs).Three SK channel subunits (SK1, SK2, and SK3) have been cloned so far (Köhler et al., 1996), and their expression is widely distributed in the nervous system (for detailed reviews, see Rimini et al., 2000;Stocker & Pedarzani, 2000).In the brain, although SK channels are predominantly expressed in neurons, intermediate conductance K + channels (IK) are mainly located in microglia and endothelial cells (Kshatri et al., 2018;Stocker & Pedarzani, 2000).TRN neurons express mainly SK2 channels, and SK1 channels to a lesser extent.Importantly, neuronal activity in the TRN is paced by the coordinated action of low threshold Ca 2+ channels (CaV 3 ) and SK channels.
CaV 3.3 channels mediate burst firing, and Ca 2+ ions entering through these channels stimulate SK2 channels, which are responsible for the AHP (Astori et al., 2011;Fernandez & Lüthi, 2020).The AHP allows TRN cells to be ready to fire again and therefore ensures that the burst can occur repeatedly over time.This is crucial for the generation of thalamocortical oscillations during sleep and wakefulness.Reduced SK2 channel function in the TRN has been described in models of attentional disorder and the epileptic encephalopathy called Dravet syndrome (Ritter-Makinson et al., 2019).On the contrary, SK2 channel-overexpressing mice showed increased bursting activity in TRN neurons, prolonged thalamic oscillations, and less fragmented sleep compared with their WT littermates (Wimmer et al., 2012).However, less is known regarding how pharmacological modulation of SK channels affects TRN activity.In this study, we sought to address the impact of pharmacological modulators of SK channels on AP firing and intrinsic properties of TRN neurons.

Animals
All experimental procedures were performed in strict accordance with the Danish legislation regulating animal experiments; Law and Order on Animal experiments; Act No. 474 of 15/05/2014 and Order No. 12 of 07/01/2016, and with the specific license for this experiment issued by the National Authority.Two-three-week male Sprague-Dawley rats were housed under standard temperature (22 ± 1.5 • C) and humidity (55%-65%) in controlled laboratory conditions.Food and water were available ad libitum in a 12 h light/dark cycle (lights on at 7:00 h).

Chemicals and reagents
Compounds were chosen based on their selectivity profile and potency on SK channels (Figure 1).Two positive allosteric modulators (PAMs) and one negative allosteric modulator were used: CyPPA, NS309, and NS8593, respectively.Concentrations of compounds were picked based on previous literature.NS309 is a nonselective activator of SK1-3 (EC 50 s = .6,.8 and .9µM, respectively, according to Kasumu et al., 2012) and IK (EC 50 = .01µM according to Strøbaek et al., 2004).NS8593 inhibits all SK1-3 subtypes in a Ca 2+ -dependent manner (Kds of .42,.60,and .73µM at .5 µM Ca 2+ , respectively).CyPPA acts preferentially on SK3 (EC 50 = 6 µM), and SK2 (EC 50 = 14 µM) channels, and it is inactive for SK1 and IK (Hougaard et al., 2007).Several concentrations of NS8593 were tested in the slice (1, 3, and 10 µM), data not shown.We selected the lowest dose (1 µM) as it was enough to see a clear effect on TRN firing.We did not test higher doses for CyPPA (>10 µM) as it is shown that this compound inhibits Na V channel currents at slightly higher concentrations (Herrik et al., 2012;Hougaard et al., 2007).All compounds were prepared as a 10 mM stock solution in dimethylsulfoxide (DMSO) and stored in aliquots at −20 • C. The final concentration of DMSO in the experimental solutions was ≤.1%.

Electrophysiological recordings
Whole cell patch clamp recordings in current clamp configuration were performed in acute horizontal brain slices (300 µm) containing TRN of male Sprague-Dawley rats (P15-21).Individual slices were transferred to a recording chamber and continually perfused with fresh oxygenated (95% O 2 , 5% CO 2 ) artificial cerebrospinal fluid (ACSF) containing 3 mM Ca 2+ /.5 mM Mg 2+ (Astori et al., 2011) at a flow rate of 1.5 mL/min.TRN was visually identified by differential contrast optics with an Olympus BX51WI microscope and an infrared video camera (Olympus XM10

RESULTS
TRN neurons typically exhibit two distinct firing patterns (tonic or burst firing) depending on their resting membrane potential (RMP).Burst firing is characterized by low-threshold T-type Ca 2+ spikes elicited at hyperpolarized membrane potentials, followed by high-frequency Na + spike trains.
When depolarized, TRN neurons fire regular Na + spikes in a tonic firing pattern.TRN can be visually identified as a dense layer of cells located between the internal capsule and the thalamus, and cells typically display an elongated soma (Figure 2a).In response to hyperpolarizing current step injections, TRN neurons exhibited a characteristic rebound burst (Figure 2b) followed by an afterburst AHP.Under the current experimental conditions, TRN cells showed an average RMP of −73.3 ± 5 mV, input resistance (Rin) of 202.9 ± 10 MΩ and AP threshold of −43 ± 1 mV (n = 31; N = 11).Although recordings were obtained in P15-21 animals, firing profiles agree with what is shown in the literature for rodent TRN neurons (Clemente-Perez et al., 2017;Lee et al., 2007).
A number of APs were studied as a function of increasing current injection (from −200 to 200 pA; step size 25 pA) before (baseline) and 10 min after each compound was administered to the bath.Only depolarizing current steps (from 0 to 200 pA) are shown (Figure 3).Results obtained showed that opposite effects were exerted on TRN firing by NS309 and NS8593 (Figure 3b), whereas NS8593 (1 µM) significantly increased the number of APs compared to baseline (F [1, 18] = 12.29; p = .003),NS309 (3 µM) reduced it (F [1, 22] = 20.57;p = .0002).No significant effect was observed when applying CyPPA (10 µM), which acts less potently on SK2 channels (F [1, 10] = .26;p = .62).Similar results were obtained when observing the effect of SK modulators across time (Figure 3c).In this situation, APs were activated by the injection of a squared current pulse (150 pA, 1 s) repeated every 5 min.None of the compounds showed a washout effect when switching back to regular ACSF in the bath for more than 5 min (Figure 3c).NS8593-treated cells displayed a significant increased number of APs compared to DMSO time-matched control cells at 10, 15, and 20 min after bath application of the compound (F [1, 84] = 45.20 p < .0001),and the opposite effect was observed when applying NS309, which reduced significantly the number of APs across time (F [1, 14] = 13.96;p = .002).No significant effects were observed when CyPPA was bath applied to the slice (F [1, 8] = .31;p = .59)compared to DMSO.
Afterburst AHP regulates neuronal excitability and ensures that the burst firing in TRN neurons can occur repeatedly.To gain insight into how SK modulators affect intrinsic excitability and burst properties in TRN neurons, we assessed the effect of SK modulators on the amplitude of AHP, burst duration, intraburst firing frequency, and time between bursts (interburst interval) elicited by a depolarizing current injection (1 s, 150 pA), before (baseline) and 10 min after compound administration (Figure 4).Afterburst AHP was defined as the hyperpolarizing phase after the first burst, and it was measured as the difference in voltage compared to the AP threshold.In line with the results shown previously, when applying NS8593 to the slice TRN cells exhibited a clear reduction in the AHP (t(7) = 8.8, p < .0001)and a shorter interburst interval (t(6) = 4.3, p = .005).Conversely, both SK PAMs increased the afterburst AHP (CyPPA t(5) = 4.99, p = .004;NS309 t(11) = 2.8, p = .02)and the time between bursts (CyPPA t(5) = 3.35, p = .02;NS309 t(9) = 9.6, p < .0001).Additionally, NS8953-treated cells showed a longer burst duration (t(7) = 6.4,p = .0004)and lower intraburst burst firing frequency (t(7) = 2.98, p = .02).Even though the overall intraburst frequency was reduced in the presence of NS8593, the traces show an increase in the frequency during the first spikes and a reduced spike frequency by the end of the burst (Figure 4a).Applying DMSO to the bath did not show a significant effect on any of the parameters studied.

DISCUSSION
When taken together, results show that SK channel modulators affect firing pattern and frequency in TRN neurons.By reducing the AHP as a consequence of SK negative modulation, NS8593 increases excitability on TRN cells, prolongs the burst duration, and shortens the refractory period between bursts, leading to a higher firing frequency.On the other hand, positive modulation of SK channels increases the AHP in TRN neurons, which reduces intrinsic excitability and increases the time between bursts.This effect is particularly remarkable for NS309 that showed a significant reduction in firing frequency.Thus, targeting SK channels could be promising in treating disorders involving TRN dysfunction.Based on previous literature, this might be particularly important in the context of neurodevelopmental and neuropsychiatric disorders.For example, reduced TRN function which includes marked deficits in TRN-generated sleep spindles has been noted in schizophrenia that supports genetic findings that a spontaneous mutation leading to suppression of SK channel currents is present in human schizophrenia patients (Miller et al., 2001).Accordingly, pharmacological agents that enhance SK function offer hope of normalization of TRN firing and restoration of more normal attentional processes and sleep spindle behavior, which has been suggested to be a novel treatment target for improving cognitive symptoms of schizophrenia (Manoach et al., 2016).Future studies using SK modulators in animal models will be key in finding the potential of these compounds as therapeutical tools.

AUTHOR CONTRIBUTIONS
Ágata Silván and Charlotte Hougaard designed the study.Ágata Silván carried out the experiments and performed analyses of the data.Charlotte

F
I G U R E 2 Thalamic reticular nucleus (TRN) neurons exhibited rebound bursts in response to a hyperpolarizing current.(a) Differential contrast microscopy images depicting TRN.Left: TRN was visually identified as a dense layer of cells located in between the internal capsule (ic) and the thalamus (Thal).Right: under higher magnification, TRN cells display an elongated/oval shape.(b) Typical rebound burst firing of a TRN neuron (1) followed by the afterburst afterhyperpolarization (AHP) (2).
Data are presented, given as means ± SEM.Normality tests (Kolmogorov-Smirnov/Shapiro-Wilk) were run in advance.Parametric tests (paired ttest, two-way repeated measures ANOVA) or nonparametric tests were performed when appropriate.Multiple comparisons were carried out using post hoc Holm-Šídák tests.Data were considered statistically significant when p < .05.N = number of animals; n = number of cells = number of slices recorded.As no washout effect was shown in the 5 min after recordings, only one cell per slice was recorded.DMSO time-matched control recordings were performed for comparison.