M-current regulates the speed of locomotion

To study the role of I_M in the locomotor behavior of juvenile rats (15–21 days old), potent and selective Kv7 channel modulators were injected intraperitoneally at a concentration commonly used for a systemic modulation of I_M [31–33]. The regular pattern of walking was captured with the CatWalk system before and after 30 min of drug administration (Fig 1). Control experiments with DMSO discarded potential effects of the vehicle (P > 0.05, in gray, Fig 1A–1C). Rats walked faster when the broad-spectrum activation of Kv7.2–Kv7.5 channels occurred by injecting retigabine (5 mg/kg; see [34]), which was related to a shorter stance phase (P < 0.05, in green, Fig 1A–1C). The broad-spectrum inhibition of Kv7 channels by linopirdine (3 mg/kg; [35]) had opposite effects, i.e., rats walked slower, attributable to a longer stance phase (P < 0.05, in yellow, Fig 1A–1C). A second set of experiments focused on the role of Kv7.2/3 subunits by means of more selective drugs. The potent Kv7.2/3 channel opener N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73) (5 mg/kg; [36]) (P < 0.05, in blue, Fig 1A–1C) and the Kv7.2/3 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) (5 mg/kg; [35]) (P < 0.05, in red, Fig 1A–1C) reproduced retigabine and linopirdine effects, respectively. Noteworthy, none of the Kv7 modulators affected the interlimb coordination or gait (P > 0.05; S1A–S1D Fig).

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Fig 1. I_M sets the speed of locomotion.

(A,D) Representative footfall diagrams during CatWalk locomotion of juvenile rats (A, 15–21 days old) before (black) and 30 min after acute i.p. administration of DMSO (gray, n = 6 rats), retigabine (5 mg/kg, green, n = 7 rats), linopirdine (3 mg/kg, yellow, n = 6 rats), ICA73 (5 mg/kg, blue, n = 7 rats), or XE991 (5 mg/kg, red, n = 6 rats) or in adult rats (D) before (black) and 5–10 min after acute i.t. administration at the L1–L2 level of saline (purple, n = 10 rats), DMSO (gray, n = 5 rats), ICA73 (0.05 mg/kg, blue, n = 8 rats), or XE991 (0.025 mg/kg, red, n = 6 rats). The stance phase is indicated by horizontal bars and the swing phase by open spaces. (B,E) Normalized changes of the body speed induced in juvenile (B) and adult (E) rats by the abovementioned drugs. Dotted lines indicate the 95% confidence intervals of control values. *P < 0.05, comparing animals before and after drug administration; Wilcoxon paired test. (C,F) Normalized changes of swing and stance phases induced in juvenile (C) and adult (F) rats by the abovementioned drugs and expressed as a percentage of the total step cycle. *P < 0.05, comparing data collected before and after drug administration; Wilcoxon paired test. Data in C and F are mean ± SEM. Underlying numerical values can be found in the S1 Data. ICA73, N-(2-chloro-5-pyrimidinyl)-3,4-diflurobenzamide; i.p., intraperitoneal; i.t., intrathecal; LF, left forelimb; LH, left hindlimb; ns, not significant; RF, right forelimb; RH, right hindlimb; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g001

Modulation of the step cycle through systemic administration of drugs may involve changes in the excitability of midbrain circuits setting the speed of locomotion [37, 38]. To investigate a putative modulation of I_M at the level of the spinal locomotor CPG, the opener ICA73 (0.05 mg/kg) and the blocker XE991 (0.025 mg/kg) were injected intrathecally at the L1–L2 level in adult rats. The intrathecal delivery of drugs replicated the effects observed with an intraperitoneal injection. Specifically, rats walked faster with ICA73 (P < 0.05, in blue, Fig 1D–1F) and slower with XE991 (P < 0.05, in red, Fig 1D–1F) without affecting interlimb coordination or gait (P > 0.05; S1E–S1H Fig). Note that control experiments with DMSO or saline discarded potential effects of vehicles (P > 0.05, in gray and in purple, Fig 1D–1F).

Altogether, these results support the concept that I_M, presumably mediated by Kv7.2 and/or Kv7.3 channels, is an important and physiologically relevant regulator of the spinal locomotor network without interfering with the dynamic postural control.

Ubiquitous expression of Kv7.2-containing channels in interneurons from the locomotor CPG region

We then studied the expression of Kv7.2/3 channels in ventromedial interneurons from upper lumbar segments (L1–L2), where components of the locomotor rhythm-generating network are located [1, 2]. Immunostaining substantiates the presence of Kv7.2 channels in the soma of all ventromedial interneurons and in the distal part of their axonal initial segments (AISs), identified by their expression of voltage-gated sodium (Na_v) channels (Fig 2A–2C, 2G, and 2H). A similar Kv7.2-immunostaining profile was observed in lumbar motoneurons (S2A–S2C, S2G, and S2H Fig). Conversely to Kv7.2, the Kv7.3 subunit was weakly expressed in the soma and virtually undetectable in AISs of most ventromedial interneurons (Fig 2D–2G). On the other hand, Kv7.3 immunostaining distinctively labeled the distal part of AISs in almost half of lumbar motoneurons (S2D–S2H Fig). In sum, L1–L2 ventromedial interneurons ubiquitously express Kv7.2-containing channels, suggestive of the existence of I_M in the locomotor CPG. Note that we validated the specificity the antibodies against Kv7.2 and Kv7.3 on Chinese hamster ovary (CHO) cells stably expressing Kv7.2 or Kv7.3 channels (S3 Fig).

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Fig 2. Lumbar interneurons within the locomotor CPG region ubiquitously express Kv7.2-containing channels.

(A–F) Immunostaining of lumbar (L1–L2) ventromedial interneurons from juvenile rats (n = 3 rats) against Kv7.2 (A, n = 216 cells) or Kv7.3 (D, n = 170 cells) along the AIS labeled by the pan-Na_v antibody (B,E). Kv7.2 and pan-Na_v are merged in (C), and Kv7.3 and pan-Na_v are merged in (F). Asterisks indicate the nucleus position and arrowheads the AIS. Scale bars = 10 μm. (G) Group means quantification of the proportion of pan-Na_v positive interneurons expressing Kv7.2 or Kv7.3 channels. (H) Group means quantification of the start and end positions of pan-Na_v and Kv7.2 immunolabeling along the axonal process from the soma (n = 10 cells). ***P < 0.001, comparing start or end positions between groups; Mann–Whitney test. Data are mean ± SEM. Underlying numerical values can be found in the S1 Data. AIS, axonal initial segment; CPG, central pattern generator; Na_v, voltage-gated sodium channel.

https://doi.org/10.1371/journal.pbio.3000738.g002

Characterization of I_M in the rhythm-generating kernel for locomotion

To assess whether interneurons from the CPG region exhibit a functional I_M, we isolated I_M by whole-cell patch-clamp recordings under tetrodotoxin (TTX) (1 μM). By using the standard relaxation protocol, we measured I_M as the amplitude of the tail inward current evoked by stepping down voltage from −10 mV (Fig 3A). All L1–L2 ventromedial neurons displayed an electrophysiological signature of I_M [39]. From the current–voltage (I–V) relationship fitted with a standard Boltzmann function (in black, Fig 3B), the threshold for activation (V_T) was positive to −67.3 ± 1.9 mV, and its amplitude increased steeply (slope factor k: 4.4 ± 0.6) for larger voltage steps with a midpoint of activation (V_1/2) at −43.6 ± 1.5 mV and then plateaued above −30 mV. The peak amplitude of I_M was in the mean of 79.2 ± 7.4 pA. We further characterized I_M pharmacologically (Fig 3C). The I_M enhancer ICA73 (10 μM) increased the holding current and the magnitude of I_M and hyperpolarized its V_T and V_1/2 activation (P < 0.05, in blue, Fig 3B–3D). The I_M enhancer retigabine (100 nM) replicated the effects of ICA73 on I_M (S4A–S4F Fig). The I_M blocker XE991 (10 μM) markedly reduced the holding current and virtually abolished I_M (P < 0.05, in red, Fig 3B–3D). Note that similar biophysical properties of I_M, sensitive to XE991, were recorded from transgenic mice Hb9:green fluorescent protein (GFP)-positive ventromedial interneurons that are considered as components of the locomotor rhythm-generating circuit (S5 Fig) [40, 41]. Given the strong expression of Kv7.2 in interneurons, we evaluated the contribution of Kv7.2 to I_M with knock-in mice bearing the T274M Kv7.2 mutation associated with Ohtahara syndrome [42]. Because this mutation is homozygous lethal, we studied I_M on Kv7.2^Thr274Met/+ animals. We found in heterozygous Kv7.2^Thr274Met/+ mice that I_M was about halved in amplitude relative to Kv7.2^+/+ littermates (Fig 3E). These results indicated that I_M was at least mediated by K_v7.2-containing channels. To further investigate which Kv7 subunits are involved, we exploited the differential sensitivity of Kv7 channels to tetraethylammonium (TEA) [43–46]. Thus, Kv7.2 homomers are almost fully blocked at 1 mM, whereas K_v7.3, K_v7.4, and K_v7.5 homomers are blocked with IC₅₀ values of >200, approximately 3.0, and approximately 70 mM, respectively. K_v7.2/7.3 and K_v7.2/7.5 heteromeric channels show intermediate sensitivities to TEA block with IC₅₀ values near approximately 3 mM and approximately 200 mM. We found that bath application of 1 mM TEA blocked approximately 80% of I_M in L1–L2 ventromedial interneurons from neonatal rats (P < 0.05, in purple, Fig 3F).

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Fig 3. Characterization of I_M in interneurons of the locomotor CPG region.

(A) Representative inward current relaxation recorded in L1–L2 ventromedial interneurons from neonatal rats and induced by the standard I_M deactivation voltage protocol. The current relaxation (I_M) was measured as the difference between the fast transient (I_VH) and the steady-state current (Iss). (B) Boltzmann-fitted I–V relationships of I_M normalized to control (black) values and established before and after bath-applying XE991 (10 μM, red) or ICA73 (10 μM, blue). Error bars represent ± SEM. (C) Representative deactivation of I_M before and after adding the abovementioned drugs. (D) Boxplots quantification of biophysical properties of I_M. (n = 6 cells). Dashed lines with gray shading indicate the 95% confidence intervals of control values. *P < 0.05, ***P < 0.001, comparing data collected before and after adding the abovementioned drugs; Wilcoxon paired test. (E) Representative deactivation (left), Boltzmann-fitted I–V relationships (middle), and amplitude (right) of I_M recorded in interneurons from wild-type (n = 11 cells, gray) and Kv7.2^Thr274Met/+ mutant mice (n = 8 cells, purple). *P < 0.05; Mann–Whitney test. (F) Representative deactivation (left), Boltzmann-fitted I–V relationships (middle), and amplitude (right) of I_M recorded in interneurons from neonatal rat before and after adding TEA (1 mM, n = 6 cells). *P < 0.05; Mann–Whitney test. Underlying numerical values can be found in the S1 Data. CPG, central pattern generator; ICA73, N-(2-chloro-5-pyrimidinyl)-3,4-diflurobenzamide; I–V, current–voltage; TEA, tetraethylammonium; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g003

Altogether, these results support the expression of a noninactivating K⁺ current corresponding to I_M in the locomotor CPG, presumably carried by Kv7.2-containing channels mostly in a homomeric form.

M-current sets the neuronal excitability and gates pacemaker bursting mode

We characterized the role of I_m in membrane properties of L1–L2 ventromedial interneurons recorded from neonatal rats. As a first observation, the I_m enhancer ICA73 hyperpolarized cells (in blue, S6A Fig; routinely compensated to −60 mV) along with a drop of the input resistance (P < 0.05 Table 1). Therefore, interneurons became less excitable (higher rheobase; P < 0.05, Table 1) and produced fewer spikes (P < 0.05, in blue, Fig 4A) without any changes in parameters of the action potential (P > 0.05, Table 1). The frequency–intensity (f–I) curve was thus shifted to the right (Fig 4B). The reversibility of these effects when the I_m blocker XE991 was applied emphasized the dependence of ICA73 on Kv7 channels (S6A–S6D Fig). Consistent with this, the Kv7 opener retigabine reproduced effects of ICA73 on electroresponsive properties (S4G and S4H Fig and Table 1). We further tested the functional implication of I_m by using the I_m blocker XE991 alone. The I_m blocker per se neither depolarized the resting membrane potential nor increased the neuronal excitability or the firing rate even 30 min after the bath application of the drug (P > 0.05, in red, Fig 4A and 4B and Table 1). Thus, the basal excitability was not affected by XE991. However, I_m blockers such as XE991 have the peculiarity to be voltage-dependent blockers, with higher affinity at positive potentials, and therefore are very poor inhibitors at perithreshold potentials [47]. Furthermore, the inhibition develops slowly upon depolarization within a timescale of minutes [48]. To overcome this experimental limitation, we used the Kv7.2^Thr274Met/+ mutant mice for which I_m was halved (see above). We showed that L1–L2 ventromedial interneurons from mutant mice displayed a higher spiking frequency (P < 0.001, Fig 4C and 4D) and a more depolarized resting membrane potential (P < 0.05, Fig 4E).

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Table 1. Effects of drugs targeting Kv7 channels on passive and active membrane properties of interneurons.

https://doi.org/10.1371/journal.pbio.3000738.t001

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Fig 4. I_M mediated by Kv7.2-containing channels regulates both the excitability and the spiking activity of L1–L2 ventromedial interneurons.

(A,B) Spiking activity of ventromedial interneurons (L1–L2) to a near-threshold depolarizing pulse (A) with its respective frequency–current relationships (B) before (black) and after bath-applying ICA73 (10 μM, n = 7 cells, blue) or XE991 (10 μM, n = 7 cells, red). Continuous lines represent the best-fitting linear regression. ***P < 0.001 comparison of the fits before and after adding the abovementioned drugs. (C–E) Representative spiking activity (C), frequency–current relationships (D), and resting membrane potential (E) of ventromedial interneurons (L1–L2) recorded from wild-type (n = 10 cells) and Kv7.2^Thr274Met/+ mutant mice (n = 8 cells). Continuous lines represent the best-fitting linear regression. ***P < 0.001 comparison of the fits. *P < 0.05, comparing resting membrane potentials; Mann–Whitney test. (F) Spiking activity from a single-neuron model at 3 different values of g_M. The injected current pulses in A, C, and F are indicated below voltage traces. (G) Mean plots of the firing rate, rheobase, and resting membrane potential as function of g_M (in the heterogeneous population model of 50 neurons). The continuous line is the best-fitting linear regression. ***P < 0.001, Spearman correlation test. R² indicates the correlation index. Data are mean ± SEM. Underlying numerical values can be found in the S1 Data. ICA73, N-(2-chloro-5-pyrimidinyl)-3,4-diflurobenzamide; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g004

We also studied the theoretical effect of I_m by modeling I_m in an Hb9 cell model that we previously used [10]. The model was supplemented by I_m derived from our voltage-clamp recordings. A population of 50 uncoupled interneurons was simulated with a randomized normal distribution of neuronal parameters (see Materials and methods). Simulated neurons reproduced key features of the biological responses to stepwise depolarizing currents with firing rate in the range of our experimental data (Fig 4F and 4G). The increase of the M-conductance (g_M) in the model qualitatively captured the modulation of neuronal excitability observed experimentally with I_m enhancers ICA73 or retigabine; firing rate decreased, the resting membrane potential hyperpolarized, and the rheobase increased (P < 0.001, Fig 4F and 4G). However, in contrast to our electrophysiological recordings with the I_m blocker XE991, decreasing g_M in the model predicted a depolarization of V_rest, whereas the firing rate and the rheobase would go up and down, respectively (P < 0.001, Fig 4F and 4G). On the other hand, these computational data were similar to those recorded in Kv7.2^Thr274Met/+ mutant mice, making them suitable to use as a tool to explore how I_m might shape the firing pattern of interneurons.

A remarkable effect of modeling a decrease of g_M was the gradual transfer to a bursting mode in a small proportion of neurons to reach 17% of bursters when g_M was switched off (Fig 5A and 5B). Bursts disappeared when I_NaP was zeroed (Fig 5A). To evaluate this computational prediction, we tested XE991 on cells intracellularly recorded and constantly depolarized with a suprathreshold current (Fig 5C). In this condition, XE991 caused a transition from tonic spiking to bursting in approximately 18% of the interneurons recorded (Fig 5C and 5D). Bursts were abolished by the I_NaP blocker riluzole (Fig 5C). The insensitivity of bursts to kynurenic acid (1.5 mM; blocker of the fast-glutamatergic transmission) and the lack of rhythmic currents in voltage-clamp recordings precluded a role of network inputs in the emergence of bursts (Fig 5C).

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Fig 5. I_M regulates the firing pattern of L1–L2 ventromedial interneurons.

(A) Typical switch of the firing pattern from spiking to bursting in a single-neuron model when g_M was switched “off”; simulated blockade of I_NaP (g_NaP = 0) abolished bursting activity. (B) Dependence of the percentage of bursting cells on g_M in the heterogeneous population model of 50 neurons. (C) On top, representative voltage traces of a ventromedial interneuron (L1–L2) intracellularly recorded in the presence of kynurenate (1.5 mM) before (left) and after (middle) bath-applying XE991 (10 μM). A subsequent application of riluzole (5 μM) abolished bursts (right). At bottom, current traces of the cell illustrated and voltage clamped at −80 mV. (D) Proportion of burster and nonburster interneurons before and after bath-applying XE991 (n = 22 cells). Data are mean ± SEM. Underlying numerical values can be found in the S1 Data. aCSF, artificial cerebrospinal fluid; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g005

Overall, these data indicate an important role of I_M in setting the excitability and firing properties of interneurons within the locomotor CPG region, notably by impeding the initiation of bursts mediated by I_NaP.

Interneurons balance I_M and I_NaP to trigger pacemaker bursting mode

The emergence of I_NaP-dependent bursting cells when Kv7 channels are blocked suggests that I_M might counteract I_NaP to regulate pacemaker properties. To test this possibility, voltage-clamp recordings were performed to examine the degree of interaction between the 2 currents. In response to very slow voltage ramps, ventromedial interneurons from neonatal rats displayed a large inward current attributable to I_NaP (Fig 6A; see [5]). In the presence of I_M blocker XE991, I_NaP was higher in amplitude, whereas V_T and V_1/2 did not change (P < 0.05, Fig 6A and 6B). Similar results were obtained from Kv7.2^Thr274Met/+ mutant mice (P < 0.05, Fig 6C and 6D). These data show that biophysical properties of I_M are well-suited to counteract the depolarizing drive furnished by I_NaP. Here, we speculate that the combination of I_M and I_NaP currents is a possible mechanism in controlling bursting dynamics. We evaluated this assumption by varying the maximal conductance levels of g_M and g_NaP in the heterogeneous population model composed of 50 uncoupled Hb9-type interneurons (Fig 6E). None of the neurons exhibited bursting at base levels of g_M (0.8 nS) and g_NaP (0.4 nS). We found that a population bursting activity could be triggered by either a reduction of g_M or an increase of g_NaP in turn (Fig 6E). However, the striking observation was the synergistic effect of reducing g_M and increasing g_NaP conductance on the generation of bursts (P < 0.001, Fig 6F). Compared with nonbursters, bursters were distinguished by a more negative V_1/2 of I_NaP but displayed a similar V_1/2 of I_M (P < 0.001, Fig 6G).

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Fig 6. A balance of I_M and I_NaP gates bursting mode of CPG interneurons.

(A,C) Raw traces of leak-subtracted persistent sodium current (I_NaP) evoked in L1–L2 ventromedial interneuron by a slow voltage ramp and recorded from neonatal rats (A) under control condition (n = 15 cells, black) or in the presence of XE991 (10 μM, n = 11 cells, red) or from neonatal mice (C) wild-type (n = 10 cells, gray) and Kv7.2^Thr274Met/+ mutant mice (n = 11 cells, pink). (B,D) Boxplot quantification of the biophysical properties of I_NaP. *P < 0.05; Mann–Whitney test. (E) Dependence of the percentage of bursting pacemaker cells on g_M and g_NaP in the heterogeneous population model of 50 neurons. Results are pooled from 10 simulations. (F) Percentage of bursting cells as function of the g_NaP/g_M ratio. Each dot represents average values of 10 simulations for a fixed g_NaP/g_M ratio. Continuous line is the best-fitting linear regression. ***P < 0.001, Spearman correlation test. R² indicates the correlation index. (G) Boxplot quantification of V_1/2 of I_NaP and I_M for burster and nonburster groups. Each dot represents 1 simulation in the heterogeneous population model of 50 neurons. Parameters values for g_NaP and g_M were set at 0.8 nS and 0.1 nS, respectively. ***P < 0.001; Mann–Whitney test. Underlying numerical values can be found in the S1 Data. CPG, central pattern generator; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g006

Taken together, these results suggest that most CPG interneurons are endowed with the intrinsic ability to switch from spiking to bursting behavior through a sliding balance between I_NaP and I_M.

M-current controls bursting dynamics of pacemaker cells

Our modeling study, combined with electrophysiological data, supports fine modulation of g_M as a key mechanism for the emergence of pacemaker cells. Here, we used the model as a tool to delineate the role of I_m in bursting dynamics. To study the dependence of bursting characteristics on I_M, we simulated a negative-voltage shift of I_NaP activation (V_1/2 = −54 mV) to convert a tonic cell into burster as a result of reducing [Ca²⁺]_o [10]. As previously described, the burst period and the burst duration decreased as the neuron was depolarized (Fig 7A). When g_M was omitted, the burst duration as well as the interburst interval increased (P < 0.001, Fig 7B and 7C). Opposite effects on burst timing were observed when g_M was increased (P < 0.001, Fig 7C). Notably, at high levels of g_m, a subset of bursting cells in the heterogeneous population model switched to tonic spiking (S7A and S7B Fig). To determine the dynamic contributions of I_M and I_NaP to the bursting activity, we examined changes of g_M and g_NaP at specific time points during the burst itself and during interburst intervals (Fig 7D). During the beginning of each burst, g_NaP preceded the activation of g_m. Over the course of the burst, a slow decrease of g_NaP was observed, whereas g_m slightly increased. At the end of the burst, g_m and g_NaP relaxed to a baseline level over the duration of an interburst interval. Altogether, these results suggest a scenario in which I_NaP initiates the burst and I_m contributes to the normal oscillatory activity of pacemakers by counteracting I_NaP during the burst.

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Fig 7. I_M controls burst dynamics.

(A,B) Firing behavior from a bursting pacemaker neuron model in response to incrementing depolarizing current injections (A) or for 2 different values of g_M (B). The injected current pulses are indicated below voltage traces V_1/2 I_NaP = −54 mV. (C) Duration and frequency of bursts as function of g_M in the heterogeneous population model of 50 neurons. Results are pooled from 10 simulations. R² indicates the correlation index. (D) Dynamics of the neuronal membrane potential (black), the I_NaP (green), and the I_M (red) during bursting activity of a single pacemaker neuron model. The broken-line box highlights dynamics of I_NaP and I_M at the onset of bursts. (E,F) [Ca²⁺]_o-free-saline–induced bursting activity recorded intracellularly from L1–L2 ventromedial interneurons before and after XE991 (10 μM, n = 7 cells) (E) or ICA73 (10 μM, n = 7 cells) (F). (G) Normalized changes of burst parameters. Dashed lines with gray shading indicate the 95% confidence intervals of control values. *P < 0.05, comparing data before and after the abovementioned drugs; Wilcoxon paired test. (H) [Ca²⁺]_o-free-saline–induced bursting activity recorded intracellularly in L1–L2 ventromedial interneurons from wild-type (n = 13 cells) and Kv7.2^Thr274Met/+ mutant mice (n = 18 cells). (I) Boxplot quantification of burst parameters. *P < 0.05, comparing wild-type versus mutant; Mann–Whitney test. Underlying numerical values can be found in the S1 Data. ICA73, N-(2-chloro-5-pyrimidinyl)-3,4-diflurobenzamide; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride.

https://doi.org/10.1371/journal.pbio.3000738.g007

The above predictions were explicitly tested by means of intracellular recordings of pacemaker cells driven by I_NaP and triggered by removing the Ca²⁺ from extracellular solution [5, 10, 49]. In this recording condition, the burst termination did not involve I_KCa but engaged a K⁺ current as the broad-spectrum K⁺ channel blocker TEA strongly increased the duration of bursts until ultimately, a plateau-like depolarization developed (P < 0.05, S7C and S7D Fig). This result led us to consider the involvement of a noninactivating TEA-sensitive K⁺ current such as I_M [44]. In line with computational predictions, the blockade of I_M by XE991 increased the duration and decreased the frequency of bursts (P < 0.05, Fig 7E and 7G). Similar results were obtained with linopirdine (S7E and S7F Fig). The ability of ICA73 to achieve the converse through the magnification of I_M was found (P < 0.05, Fig 7F and 7G). The effects of ICA73 on burst dynamics were reproduced with retigabine (S7G and S7H Fig). Interestingly, as predicted by the model, the enhancement of I_M by ICA73 converted rhythmic bursting into tonic spiking in a few cells (S7I and S7J Fig). Finally, to investigate the contribution of Kv7.2 channels, pacemaker cells were recorded from Kv7.2^Thr274Met/+ mice in [Ca²⁺]_o-free saline. Pacemaker properties displayed long-lasting and low-frequency bursts relative to those recorded in Kv7.2^+/+ littermates (P < 0.05, P < 0.01, Fig 7H and 7I).

Together, these data show that Im, at least mediated by Kv7.2-containing channels, takes a significant part in the timing/intensity control of bursts to regulate dynamics of oscillatory properties in pacemakers.

M-current mediated by Kv7.2-containing channels controls the locomotor cycle

To investigate the role of I_M in networkwide rhythmogenesis, we examined its role in the operation of the locomotor rhythm-generating network by using in vitro spinal cord preparations from neonatal rats. Rostral lumbar segments (L1–L2) have a more powerful rhythmogenic capacity than the caudal ones in neonatal rats [1, 2]. A Vaseline barrier was built at the L2–L3 level to selectively superfuse the 2 compartments with different drug cocktails (Fig 8A). During bath application of N-methyl-DL aspartate (NMA)/5-hydroxytryptamine (5-HT) on both sides of the barrier to induce locomotor-like activities, the addition of XE991 (10 μM) in the highly rhythmogenic L1–L2 compartment to decrease I_M led to an augmentation of the locomotor cycle period, burst duration, and burst amplitude (P < 0.05, Fig 8B). Also, when preincubated for 30 min before the application of NMA/5-HT, XE991 strongly decreased the latency for the emergence of fictive locomotion (P < 0.05, S8 Fig). By contrast, augmenting I_m with ICA73 sped up locomotor cycles and shortened burst duration without apparent effect on burst amplitude (P < 0.05, Fig 8C).

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Fig 8. I_M mediated by Kv7.2-containing channels modulates the locomotor cycle at the level of the CPG.

(A) Experimental setup. (B,C) Ventral-root recordings of NMA/5-HT–induced rhythmic activity generated before and after adding XE991 (10 μM, n = 5 spinal cords) (B) or ICA73 (10 μM, n = 6 spinal cords) (C) to rostral lumbar segments. Below traces, boxplot quantification of locomotor burst parameters. *P < 0.05, comparing data before and after the abovementioned drugs; Wilcoxon paired test. (D) Ventral-root recordings of NMA/5-HT–induced rhythmic activity generated in wild-type and Kv7.2^Thr274Met/+ mice. Below traces, boxplot quantification of locomotor burst parameters. *P < 0.05, **P < 0.01, comparing wild-type versus mutant; Mann–Whitney test. Underlying numerical values can be found in the S1 Data. CPG, central pattern generator; ICA73, N-(2-chloro-5-pyrimidinyl)-3,4-diflurobenzamide; NMA, N-methyl-DL aspartate; ns, not significant; XE991, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride; 5-HT, 5-hydroxytryptamine.

https://doi.org/10.1371/journal.pbio.3000738.g008

In some CNS neurons, M-channels have been reported to increase the glutamatergic release by acting at a presynaptic level [50, 51]. Because ionotropic glutamate receptors tune the locomotor network to perform at different speeds [52], we tested the possibility that XE991 and ICA93 affect central glutamatergic synaptic transmission at the CPG level. We analyzed miniature excitatory postsynaptic currents (mEPSCs) recorded from L1–L2 ventromedial interneurons in slice preparations (P > 0.05, S9 Fig). Neither the amplitudes nor the frequencies of mEPSPCs were affected by Kv7 modulators.

To study the contribution of Kv7.2 channels, fictive locomotion was also induced in neonatal Kv7.2^Thr274Met/+ mice. It appeared slower (P < 0.05), with longer burst duration (P < 0.01), compared with that induced in wild-type spinal cords (Fig 8D). However, when mice acquired a full-weight–bearing stepping, juvenile Kv7.2^Thr274Met/+ mutant displayed quite normal locomotor performances (S10 Fig), suggesting a compensatory mechanism during development.

Ethics statement

We made all efforts to minimize animal suffering and the number of animals used. All animal care and use conformed to the French regulations (Décret 2010–118) and were approved by the local ethics committee (Comité d’Ethique en Neurosciences INT-Marseille, CE Nb A1301404, authorization Nb 2018110819197361). Experiments were performed on Wistar rats, Hb9:eGFP mice, and heterozygous KCNQ2^T274M/+ mutant mice.

Surgery and microinjections

A chronic lumbar intrathecal catheter was implanted in young adult rats using a lumbar approach. Briefly, rats received an indwelling intrathecal catheter under anesthesia for spinal drug delivery (intraperitoneal injection of ketamine at 50 mg/kg (Imalgen, MERIAL, Lyon, France) and medetomidine at 0.25 mg/kg (Domitor, Orion Pharma, Espoo, Finland). After an incision of the skin and muscles facing the vertebrae from T11 to L6, a laminectomy of the L3 vertebra was performed. A small needle was used to perforate the dura mater, and an intrathecal polyurethane catheter (32 Ga, C08PU-RIT1301, Phymep, Paris, France) was inserted in the rostral direction until it reached the position allowing a perfusion at the height of L1–L2 spinal cord. The catheter was secured to the superficial muscle of the back, and the external end of the catheter was tunneled subcutaneously and exited at in the dorsal neck region and plugged with a piece of steel wire. The skin was stitched back with 3–0 silk sutures, and the rats were placed in individual cages for recovery. Only animals with no evidence of neurological deficits after catheter insertion were studied. Behavioral testing occurred 7 days after intrathecal catheter implantation.

The Kcnq2^Thr274Met/+ mouse model

The Kcnq2^Thr274Met/+ mouse model was generated by homologous recombination in embryonic stem (ES) cells using a targeting vector containing regions homologous to the genomic Kcnq2 sequences and the p.(Thr274Met) variant, a recurrent pathogenic variant identified in several patients suffering from developmental and epileptic encephalopathy. Correctly targeted 129Sv ES cell clones were injected into C57Bl/6N blastocysts implanted in pseudopregnant females. Chimerism rate was assessed in the progeny by coat color markers comparison, and the mice were bred with 129sv Cre-deleter mice to excise the neomycin selection cassette and to generate Kcnq2^Thr274Met/+ mice. Genotyping was performed using genomic DNA prepared from ear punch biopsies with the Direct DNA (Tail) (Viagen Biotech, Los Angeles, CA, USA). The Kcnq2^Thr274Met/+ animals were maintained and studied on the 129Sv genetic background. Wild-type and heterozygous knock-in animals express the same amount of Kcnq2 transcript; both alleles are equally expressed. The characterization of the Kcnq2^Thr274Met/+ mouse reveals that it faithfully reproduces what is expected based on the human phenotype: no gross morphological brain alterations and no neurosensory alterations before the onset of seizures occurring at P20, followed by a high rate of unexpected death in epilepsy and important cognitive difficulties [42].

Assessment of locomotor behaviors

Adults and juvenile animals (P15–P21) were tested when a mature pattern of locomotion occurred [96]. The CatWalkXT (v9.1, Noldus Information Technology, Wageningen, Netherlands) was employed to measure walking performance. Each animal walked freely through a corridor on a glass walkway illuminated with beams of light from below. A successful walking trial was defined as having the animal walk at a steady speed (no stopping, rearing, or grooming), and 3–5 successful trials were collected per animal. Experimental sessions typically lasted for 5–10 min. The footprints were recorded using a camera positioned below the walkway, and footprint classification was manually corrected to ensure accurate readings. The paw print parameters were then analyzed using the CatWalk software (see data analysis).

In vitro models

Details of the in vitro preparations have been previously described [97, 98] and are only summarized here. Experiments were performed on newborn rats or mice (1–5 days old). For the whole-spinal–cord preparation, the spinal cord was transected at T10, isolated, and transferred to the recording chamber perfused with oxygenated artificial cerebrospinal fluid (aCSF). For rats, the aCSF was composed of 120 mM NaCl, 4 mM KCl, 1.25 mM NaH₂PO₄, 1.3 mM MgSO₄, 1.2 mM CaCl₂, 25 mM NaHCO₃, 20 mM D-glucose (pH 7.4; 25°C–26°C). For mice, the aCSF was composed of 128 mM NaCl, 4 mM KCl, 0.5 mM NaH₂PO₄, 1 mM MgSO₄, 1.5 mM CaCl₂, 21 mM NaHCO₃, 30 mM D-Glucose (pH 7.4; 25°C–26°C). For the slice preparation, the lumbar spinal cord was isolated in ice-cold (<4°C) aCSF with the following composition: 232 mM sucrose, 3 mM KCl, 1.25 mM KH₂PO₄, 4 mM MgSO₄, 0.2 mM CaCl₂, 26 mM NaHCO₃, 25 mM D-glucose (pH 7.4). The lumbar spinal cord was then introduced into a 1% agar solution, quickly cooled, mounted in a vibrating microtome (Leica VT1000S; Leica Biosystems, Wetzlar, Germany), and sliced (350 μm) through lumbar segments. Slices were immediately transferred into the holding chamber filled with aCSF composed of 120 mM NaCl, 3 mM KCl, 1.25 mM NaH₂PO₄, 1.3 mM MgSO₄, 1.2 mM CaCl₂, 25 mM NaHCO₃, 20 mM D-glucose (pH 7.4; 32°C–34°C). Following a 1-h resting period, individual slices were transferred to a recording chamber that was continuously perfused with the same medium heated to approximately 27°C. Slices were visualized with epifluorescence and infrared differential interference contrast (IR-DIC) microscopy using a Nikon Eclipse E600FN upright microscope (Nikon, Tokyo, Japan) coupled with a 40× water immersion lens. The image was enhanced with an infrared-sensitive CCD camera and displayed on a video monitor. The temperature regulation was provided by the CL-100 bipolar temperature controller (Warner Instruments, Holliston, MA, USA).

In vitro recordings

For the whole-spinal–cord preparation, motor outputs were recorded from lumbar ventral roots (left/right L5) by means of glass suction electrodes connected to an AC-coupled amplifier. The ventral-root recordings were amplified (×2,000), high-pass filtered at 70 Hz, low-pass filtered at 3 kHz, and sampled at 10 kHz. Custom-built amplifiers enabled simultaneous online rectification and integration (100-ms time constant) of raw signals. Locomotor-like activity was induced by a bath application of NMA (10 μM) and 5-HT (5 μM). In some experiments, a Vaseline barrier was built at the L₂/L₃ level to superfuse the locomotor network located in the rostral lumbar cord independently from the more caudally located motoneurons. For the slice preparation, whole-cell patch-clamp recordings were performed from L1–L2 interneurons using a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA, USA). Interneurons located in the medial lamina VIII and adjacent to the central canal, a region proposed to contain a large part of the rhythm-generating locomotor network [2], were selected. In Hb9:eGFP transgenic mice, only GFP-positive interneurons compatible with the previously described electrophysiological profile of Hb9 interneurons—i.e., high input resistance, strong postinhibitory rebound, and absence of sag—were considered [99]. Motoneurons were visually identified as the largest cells located in layer IX. Patch electrodes (2–4 MΩ) were pulled from borosilicate glass capillaries (1.5 mm OD, 1.12 mm ID; World Precision Instruments, Sarasota, FL, USA) on a Sutter P-97 puller (Sutter Instruments Company, Novato, CA, USA) and filled with intracellular solution containing 140 mM K⁺-gluconate, 5 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 0.5 mM EGTA, 2 mM ATP, 0.4 mM GTP (pH 7.3; 280–290 mOsm). Pipette and neuronal capacitive currents were canceled, and after breakthrough, the series resistance was compensated and monitored. Recordings were digitized online and filtered at 10 kHz (Digidata 1322A, Molecular Devices). The main characterization of I_M was accomplished by holding the membrane potential at a relatively depolarized potential (V_H, −10 mV) to activate KCNQ channels and to inactivate many of the other K⁺ channels, notably Kv1.2 channels [20]. The membrane potential was then stepped down to more hyperpolarized potentials to deactivate the KCNQ channels giving rise to slow inward current relaxation. Stepping back to −10 mV led to the reactivation of the KCNQ channels to produce slow inward relaxations. All experiments were designed to gather data within a stable period (i.e., at least 5 min after establishing whole-cell access). Action-potential–independent mEPSCs were recorded in the presence of TTX (1 μM) at a holding potential of −70 mV. NMDA and non-NMDA receptor-mediated mEPSCs were recorded with a K⁺-gluconate–based intracellular solution (see above) and pharmacologically isolated with a combination of biccuculine (20 μM) and strychnine (1 μM) to fast GABAergic and glycinergic synapses, respectively.

Stable CHO cell lines expressing Kv7.2 or Kv7.3

CHO cells (CHO-K1 CCL-61; LGC Standards, Teddington, UK) were cultured at 37°C in a humidified atmosphere with 5% CO₂ with a Gibco F-12 Nutrient Mixture (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Fetal Bovine Serum) and 100 units/mL antibiotics/antimycotics (Life Technologies). 100,000 cells in suspension were transfected using the Neon Transfection System (Life Technologies) with 1 μg of pcDNA3.1/Hygro plasmid encoding Kv7.2 or pcDNA3/Neo plasmid encoding Kv7.3. Stable cell lines were subsequently established using antibiotic selection and serial dilutions in 96-well plates.

Immunostaining

For the slice preparation, spinal cords were immersion-fixed for 1 h in 0.25% PFA, washed in PBS, and cryoprotected overnight at 4°C in 20% sucrose in PBS. Lumbar spinal cords (L1–L2) were then frozen in OCT medium (Tissue Tec, Dormagen, Germany), cryosectioned (20 μm), and processed for immunohistochemistry. Slices were then (i) rehydrated in PBS at room temperature (15 min); (ii) permeated with 1% Bovin Serum Albumin (BSA), 2% Natural Goat Serum (NGS), and 0.2% Triton x-100 (1 h); (iii) incubated overnight at 4 °C in the following affinity-purified rabbit Kv7.2 (PA1-929, residues 1–70; 1:1,000; Thermo Fisher Scientific, Waltham, MA, USA), Kv7.3 (APC-051, residues 668–686; 1:400; Alomone, Jerusalem, Israel)-specific polyclonal antibody, and mouse pan-Na_v (residues 932–1,043, 1:1,000; Sigma-Aldrich, St Louis, MO, USA)-specific monoclonal antibody; (iv) washed in PBS (3 × 5 min); (v) incubated with fluorescent-conjugated secondary antibodies (Alexa 488- or 546-conjugated mouse- or rabbit-specific antibodies [1:800 and 1:400; Life Technologies]) used for visualization of the mouse monoclonal or rabbit polyclonal antibodies, respectively, in a solution containing 1% BSA and 2% NGS (1.5 h); (vi) washed in PBS 3 × 5 min; and (vii) coverslipped with a gelatinous aqueous medium. In control experiments, the primary antiserum was replaced with negative control mouse or rabbit immunoglobulin fraction during the staining protocol. Sections were scanned using a laser scanning confocal microscope (Zeiss LSM700; Carl Zeiss, Oberkochen, Germany) in stacks of 0.3- to 1-μm–thick optical sections at ×63 and/or ×20 magnification, respectively, and processed with the Zen 12.0 software (Zeiss). Each optical section resulted from 2 scanning averages. Each figure corresponds to a projection image from a stack of optical sections. For CHO cells stably expressing Kv7.2 or Kv7.3, cells were fixed with 4% paraformaldehyde and blocked with permeabilization buffer (0.2% Triton-x100, 1% BSA, 2% normal goat serum in PBS) for 30 min at room temperature. CHO cells were incubated with the appropriate primary antibody diluted in permeabilized buffer for 60 min. After extensive PBS washing, CHO cells were incubated in blocking buffer (1% BSA, 2% normal goat serum in PBS) with secondary antibodies for 45 min at room temperature, washed, and mounted (fluorSave Reagent; Calbiochem, San Diego, CA, USA). The following antibody dilutions were used: Thermo Fisher Scientific rabbit anti-Kv7.2 PA1-929 (1:1,000), Alomone rabbit anti-Kv7.3 APC-051 (1:100), and Thermo Fisher Scientific donkey anti-rabbit Alexa Fluor 488 (1:500). Imaging was performed with a Zeiss Apotome.2 microscope using 25× oil immersion objective.

Validation of Kv7.2 and Kv7.3 antibodies on CHO cells

We established the specificity of the Kv7.2 and Kv7.3 antibodies on CHO cells stably expressing Kv7.2 or Kv7.3. The CHO cells stably expressing Kv7.2 were strongly immunostained with the Kv7.2 antibody, and no labeling above background level was observed with the Kv7.3 antibody (S2 Fig). The CHO cells stably expressing Kv7.3 were immunostained with the Kv7.3 antibody, and no labeling above background level was observed with the Kv7.2 antibody (S2 Fig). In sum, these data provide solid evidence of the specificity of the antibodies against Kv7.2 and Kv7.3 channels with no cross-reactivity. Note that the specificity of the Kv7.3 antibody has been validated previously [61].

Cell model

The model of the pacemaker neuron is a typical somatic single-compartment model developed in the Hodgkin–Huxley style and was based on our previous study on Hb9 cells [10]. The neuronal membrane potential V was dynamically defined by a set of membrane ionic currents. The current balance equation is where C is neuronal membrane capacitance (pF) and t is time (ms). The modeled neuron included the following ionic currents: transient sodium current (I_Na with the maximal conductance ), the persistent sodium current (I_NaP with the maximal conductance ), the noninactivating M-current (I_M with the maximal conductance ), the delayed rectifier potassium current (I_K with the maximal conductance ), the sodium-dependent pump (I_Pump), leakage (I_L with the conductance g_L), and depolarizing injected (I_Inj) currents. These currents, except for I_Pump and I_Inj, are described as follows:

E_Na, E_K, and E_L are reversal potentials of the corresponding channels (in mV):

[Na⁺], [K⁺], and [Cl⁻] represent concentrations of sodium, potassium, and chloride ions, respectively. Indexes “o” and “i” define the concentrations of these ions outside and inside a cell, respectively: [Na₊]_o = 145 mM, [K₊]_o = 3 mM, [K₊]_i = 140 mM, [Cl⁻]_i = 8 mM, [Cl⁻]₀ = 130 mM, and [Na₊]_i is considered variable. Parameters p_Na/K and p_Cl/K represent the relative permeability of sodium and chloride ions (with respect to potassium ions): p_Na/K = 0.03, p_Cl/K = 0.1.

In our model, the intracellular sodium concentration [Na⁺]_i is accumulating because of I_Na and I_NaP currents and is pumped out by I_Pump. The contributions of these components are defined by the corresponding coefficients (α_Na, α_NaP, and α_Pump, respectively):

The pump current is described as I_Pump = R_Pump · (ϕ([Na⁺]_i) − ϕ([Na⁺]_ibase), where , [Na⁺]_ibase is the base intracellular sodium concentration, and R_Pump and K_Pump are the I_Pump parameters. The following parameters were used for sodium dynamics and pump current: α_Na = α_NaP = α_Pump = 10⁻⁵ mM/fC, R_Pump = 60 pA, [Na+]_ibase = 15 mM, and K_Pump = 18 mM.

The dynamics of the activation (m) or inactivation (h) variables for the above sodium and potassium channels are generally described by the differential equation dx/dt = (x_∞ − x)/τ_x, x = {m, h}, where x_∞ is the voltage-dependent steady-state value and τ_x is the voltage-dependent time constant of the variable x, which are described in the following form:

V_x1/2 and k_x are the half-activation voltage and the slope for variable x, τ_xmax is the maximum value of its time constant, and k_τ defines the slope of this time constant. The activation of sodium currents (I_Na and I_NaP) is considered instant, i.e., τ_mNa = τ_mNaP = 0; thus, m_Na and m_NaP are considered equal to their steady-state values.

To reproduce our experimental finding, the half-activation voltage for I_NaP, V_mNaP1/2, is made dependent on the outside calcium concentration: where V_mNaP1/2base corresponds to the half-activation voltage at [Ca²⁺]_o = [Ca²⁺]_obase = 1.2 mM and ΔV_Ca = 1 mV/mM defines a shift in V_mNaP1/2 occurring with changes in [Ca²⁺]_o.

In our simulations, we considered either a single neuron or a population of 50 uncoupled neurons. To provide a necessary heterogeneity in properties of neurons, we Gaussian-distributed the base values V_1/2 for I_M and I_NaP derived from our recordings. An additional heterogeneity was set by normal distribution of all conductances around appropriate base values (Table 2). Parameters for I_Na, I_K, and inactivation of I_NaP were taken from previous modeling studies with some modifications.

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Table 2.

https://doi.org/10.1371/journal.pbio.3000738.t002

Simulations were performed using MATLAB R2019b (The MathWorks, Natick, MA, USA). Differential equations were solved using a variable-order multistep differential equation solver ode15s available in MATLAB. In each simulation, a settling period of 30 s was allowed before data were collected. For population, each simulation was repeated 10 times, and demonstrated qualitatively similar behavior for particular values of parameters within the standard deviations.

Data analysis

Drug list and solutions

Normal aCSF was used in most cases for in vitro electrophysiological recordings. Ca²⁺-free solution was made by removing Ca²⁺ chloride from the recording solution and replacing it with an equimolar concentration of magnesium chloride. All solutions were oxygenated with 95% O₂/5% CO₂. For whole-cell voltage-clamp recordings of I_M, the aCSF was supplemented with 1 μM TTX. All salt compounds, TEA, kynurenic acid, strychnine, biccuculine, riluzole, NMA, and 5-HT were obtained from Sigma-Aldrich. Other drugs, including XE991 (IC₅₀ = 0.98 μM [43]), 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one dihydrochloride (linopirdine, IC₅₀ = 4.8 μM [43]), ICA-069673 or ICA73 (EC₅₀ = 0.69 μM [36]), ethyl [2-amino-4-[[(4-fluorophenyl)methyl]amino]phenyl] carbamate (retigabine, EC₅₀ = 0.6 μM [34]), 2- amino-6-trifluoromethoxybenzothia-zole hydrochloride (riluzole), and TTX were obtained from Tocris Bioscience (Nordenstadt, Germany). Riluzole, retigabine, and ICA73 were dissolved in DMSO and added to the aCSF (final concentration of DMSO: 0.05%). The other drugs were dissolved in water and added to the aCSF. Control experiments showed no effects of the vehicle.

Treatment design

Adult and juvenile rats (15–to 21 days old) were randomly treated with a single dose of linopirdine, retigabine, XE991, or ICA73 or its vehicle. The dose of drugs refers to previous reports [31–33]. All the drugs and their vehicle were administered intraperitoneally or intrathecally. The behavioral test was performed before drug treatment and 30 min after i.p. or 5–10 min after i.t. injection of drug or vehicle. The drugs i.t. administrated were delivery in strict respect of a final injected volume of 50 μl. The volume of ICA (0.05 mg/kg) and DMSO was 20 μl followed by 30 μl of saline, and that of XE991 was 10 μL (0.025 mg/kg) followed by 40 μl of saline.

Statistics

No statistical method was used to predetermine sample size. Group measurements were expressed as means ± SEM. We used Mann–Whitney test or Wilcoxon matched pairs test to compare 2 groups. For all statistical analyses, the data met the assumptions of the test, and the variance between the statistically compared groups was similar. The level of significance was set at P < 0.05. Statistical analyses were performed using Prism 5.0 software (GraphPad). Boxplots in figures show the distribution extremes (horizontal bars), 25th and 75th percentiles (box height), and median (center bar).