It takes two to tango: M-current swings with the persistent sodium current to set the speed of locomotion

The central pattern generator (CPG) for locomotion is set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current, critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM); a subthreshold non-inactivating outward current blocked by XE991 and enhanced by ICA73. Immunostaining and mutant mice highlight an important role of axonal Kv7.2 channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities, and controls the speed of locomotion. Computational models captured these results and show howed an interplay between IM and INaP that endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

The central pattern generator (CPG) for locomotion is set of pacemaker neurons 4 endowed with inherent bursting driven by the persistent sodium current (I NaP ). How 5 they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal 6 rodents, we identified a persistent potassium current, critical in regulating pacemakers Locomotion requires a recurrent activation of muscles with variable rhythm to 4 adapt speed of movements as circumstances demand. In mammals, rhythmicity 5 appears to be ensured by a network mainly localized in the ventromedial grey matter 6 of upper lumbar segments [1,2]. The rhythm-generating network is set of pacemaker 7 cells endowed with intrinsic bursting activity in a frequency range similar to stepping 8 rhythms [3,4]. In exploring the ionic basis for rhythmogenesis, we identified the 9 persistent sodium current (I NaP ) as a critical current in burst-generating mechanism [5-10 7]. The immediate assumption was that the locomotor rhythm may emerge from 11 neurons incorporating I NaP as a "pacemaker" current. In line with this concept, inhibition 12 of I NaP abolishes locomotor-like activity in rodents [3,[8][9][10], salamanders [11] and 13 disrupts locomotion in zebrafish [7,12] or Xenopus laevis tadpoles [13]. Altogether, a 14 picture emerges that the locomotor rhythm arises from a dynamic interplay between 15 circuit-based activity and pacemaker burst-generating mechanisms with a critical role 16 of I NaP for the initiation of bursts [4,9]. 17 Among general principles of rhythmogenesis, outward conductances are 18 required to repolarize bursts [14]. In vertebrates, the Ca 2+ -activated K + current (I KCa ) 19 appears important in repolarizing bursts to regulate the locomotor rhythm [15][16][17][18], The present study characterizes the functional expression of I M in locomotor-6 related interneurons and identifies Kv7.2 channels as its molecular constituent. We 7 show that the modulation of I M adjusts I NaP amplitude, regulates the emergence of 8 pacemaker properties, their burst repolarization, the frequency regime of the locomotor 9 CPG and the speed of locomotion. In sum, we provide the first description of I M in a 10 vertebrate motor CPG and describe its dynamic interplay with I NaP as a fundamental 11 mechanism in shaping bursting activity of neurons and networks that control rhythmic 12 locomotor output. pattern of walking was captured with the CatWalk system before and after 30 min of 7 drug administration (Fig 1). Control experiments with DMSO discarded potential 8 effects of the vehicle (P > 0.05, in grey, Fig 1A-1C). Rats walked faster when the 9 broad-spectrum activation of Kv7.2-Kv7.5 channels occurred by injecting retigabine 10 [(5 mg/kg see [33]], which was related to a shorter stance phase (P < 0.05, in green, 11 Fig 1A-1C). The broad-spectrum inhibition of Kv7 channels by linopirdine (3 mg/kg) 12 [34] had opposite effects; i.e. rats walked slower, attributable to a longer stance phase 13 (P < 0.05, in yellow, Fig 1A-1C). A second set of experiments focused on the role of standard relaxation protocol, we measured I M as the amplitude of the tail inward current 23 evoked by stepping down voltage from -10 mV (Fig 3A). All L1-L2 ventromedial 24 neurons displayed an electrophysiological signature of I M . From the current-voltage (I-1 V) relationship fitted with a standard Boltzmann function (in black, Fig 3B), the 2 threshold for activation (V T ) was positive to -67.3 ± 1.9 mV, and its amplitude increased 3 steeply (slope factor k: 4.4 ± 0.6) for larger voltage steps with a mid-point of activation 4 (V 1/2 ) at -43.6 ± 1.5 mV and then plateaued above -30 mV. The peak amplitude of I M 5 was in mean of 79.2 ± 7.4 pA. We further characterized I M pharmacologically (Fig 3C). 6 The I M -enhancer ICA73 (10 µM) increased the holding current, the magnitude of I M and 7 hyperpolarized its V T and V 1/2 activation (P < 0.05, in blue, Fig 3B-3D). The  16 associated with Ohtahara syndrome [40]. Because this mutation is homozygous lethal 17 we studied I M on Kv7.2 Thr274Met/+ animals. We found in heterozygous Kv7.2 Thr274Met/+ 18 mice that I M was about halved in amplitude relative to Kv7.2 +/+ littermates (Fig 3F). 19 Altogether, these results support the expression of a non-inactivating K + current 20 corresponding to I M in the locomotor CPG, presumably carried by Kv7.2-containing 21 channels. with a drop of the input resistance (P < 0.05 Table 1). Therefore interneurons became 6 less excitable (higher rheobase; P < 0.05, Table 1) and produced fewer spikes (P < 7 0.05, in blue, Fig 4A) without any changes in parameters of the action potential (P > 8 0.05, Table 1). The f-I curve was thus shifted to the right (Fig 4B). The reversibility of 9 these effects when the I M -blocker XE991 was applied, emphasized the dependence of  Table 1). Thus, the basal excitability was not affected by XE991. 16 However, I M -blockers such as XE991 have the peculiarity to be voltage-dependent 17 blockers with higher affinity at positive potentials and thereby are very poor inhibitors 18 at perithreshold potentials [41]. Furthermore, the inhibition develops slowly upon 19 depolarization within a timescale of minutes [42].

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To overcome this experimental limitation, we studied the theoretical effect of I M 21 by modeling I M in a Hb9 cell model that we previously used [9]. The model was the biological responses to stepwise depolarizing currents with firing rate in the range 1 of our experimental data (Fig 4C and 4D). The increase of the M-conductance (g M ) in 2 the model qualitatively captured the modulation of neuronal excitability observed 3 experimentally with I M -enhancers ICA73 or retigabine; the resting membrane potential 4 hyperpolarized, the rheobase increased and firing rate decreased (P < 0.001, Fig 4C   5 and 4D). However, in contrast to our electrophysiological recordings with the I M -6 enhancers XE991, decreasing g M in the model predicted a depolarization of V rest , while 7 the firing rate and the rheobase would go up and down, respectively (P < 0.001, Fig   8   4C and 4D). Another remarkable effect of modeling a decrease of g M was the gradual 9 transfer to a bursting mode in a small proportion of neurons to reach 17 % of bursters 10 when g M was switched off (Fig 4E and 4F). Bursts disappeared when I NaP was zeroed 11 (Fig 4E). To evaluate this computational prediction, we tested XE991 on cells 12 intracellularly recorded and constantly depolarized with a suprathreshold current (Fig   13   4G). In this condition, XE991 caused a transition from tonic spiking to bursting in ~18% 14 of the interneurons recorded (Fig 4G and 4H). Bursts were abolished by the I NaP - 15 blocker riluzole (Fig 4G). The insensitivity of bursts to kynurenic acid (1.5 mM; blocker 16 of the fast glutamatergic transmission) or the lack of rhythmic currents in voltage clamp 17 recordings, precluded a role of network inputs in the emergence of bursts (Fig 4G). 18 Overall, these data indicate an important role of I M in setting the excitability and 19 firing properties of interneurons within the locomotor CPG region notably by impeding 20 the initiation of bursts mediated by I NaP .

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Interneurons balance I M and I NaP to trigger pacemaker bursting mode. 23 The emergence of I NaP -dependent bursting cells when Kv7 channels are 24 blocked suggests that I M might counteract I NaP to regulate pacemaker properties. To 1 test this possibility, voltage-clamp recordings were performed to examine the degree 2 of interaction between the two currents. In response to very slow voltage ramps, 3 ventromedial interneurons from neonatal rats displayed a large inward current 4 attributable to I NaP [Fig 5A; see [5]]. In the presence of I M -blocker XE991, I NaP was 5 higher in amplitude while V T and V 1/2 did not change (P < 0.05, Fig 5A and 5B). These 6 results show that biophysical properties of I M are well suited to counteract the 7 depolarizing drive furnished by I NaP . Here we speculate that the combination of I M and 8 I NaP currents is a possible mechanism in controlling bursting dynamics. We evaluated 9 this assumption, by varying the maximal conductance levels of g M and g NaP in the 10 heterogeneous population model composed of 50 uncoupled Hb9-type interneurons 11 (Fig 5C). None of the neurons exhibited bursting at base levels of g M (0.8 nS) and g NaP 12 (0.4 nS). We found that a population bursting activity could be triggered by either a 13 reduction of g M or an increase of g NaP in turn (Fig 5C). However, the striking 14 observation was the synergistic effect of reducing g M and increasing g NaP 15 conductances on the generation of bursts (Fig 5D). Compared to non-bursters, 16 bursters were distinguished by a more negative V 1/2 of I NaP but displayed a similar V 1/2 17 of I M (P < 0.001, Fig 5E). 18 Taken together, these results suggest that most of CPG interneurons are 19 endowed with the intrinsic ability to switch from spiking to bursting behavior through a 20 sliding balance between I NaP and I M .

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Our modeling study combined with electrophysiological data supports fine 24 modulation of g M as a key mechanism for the emergence of pacemaker cells. Here, 25 we used the model as a tool to delineate the role of I M in bursting dynamics. To study 1 dependence of bursting characteristics on I M , we simulated a negative-voltage shift of 2 I NaP activation (V 1/2 = -54 mV) to convert a tonic cell into burster as a result of reducing 3 [Ca 2+ ] o [9]. As previously described, the burst period and the burst duration decreased 4 as the neuron was depolarized (Fig 6A). When g M was omitted, the burst duration as 5 well as the interburst interval increased (P < 0.001, Fig 6B and 6C). Opposite effects 6 on burst timing was observed when g M was increased (P < 0.001, Fig 6C). Notably, 7 at high level of g M , a subset of bursting cells in the heterogeneous population model 8 switched to tonic firing (S5A and S5B Fig). To determine the dynamic contributions of 9 I M and I NaP to the bursting activity, we examined changes of g M and g NaP at specific time 10 points during the burst itself and during interburst intervals (Fig 6D). During the 11 beginning of each burst, g NaP preceded the activation of g M . Over the course of the 12 burst, a slow decrease of g NaP was observed whereas g M slightly increased. At the 13 end of the burst, g M and g NaP relaxed to a baseline level over the duration of an 14 interburst interval. Altogether, these results suggest a scenario in which I NaP initiates 15 the burst and I M contributes to the normal oscillatory activity of pacemakers by 16 counteracting I NaP during the burst. 17 The above predictions were explicitly tested by means of intracellular recordings 18 of pacemaker cells driven by I NaP and triggered by removing the Ca 2+ from extracellular 19 solution [5, 9,43]. In this recording condition the burst termination did not involve I KCa , 20 but engaged a K + current as the broad-spectrum K + channel blocker TEA strongly decreased the frequency of bursts (P < 0.05, Fig 6E and 6G). Similar results were 1 obtained with linopirdine (S5E and S5F Fig). The ability of ICA73 to achieve the 2 converse through the magnification of I M was found (P < 0.05, Fig 6F and 6G). The 3 effects of ICA73 on burst dynamics were reproduced with retigabine (S5G and S5H    barrier was built at the L2/L3 level to selectively superfuse the two compartments with 20 different drug cocktails (Fig 7A). During bath-application of NMA/5-HT in the two sides 21 of the barrier to induce locomotor-like activities, the addition of XE991 (10 µM) in the 22 highly-rhythmogenic L1/L2 compartment to decrease I M led to an augmentation of the 23 locomotor cycle period, burst duration and burst amplitude (P < 0.05, Fig 7B). Also, 24 when preincubated for 30 min before the application of NMA/5-HT, XE991 strongly 25 decreased the latency for the emergence of fictive locomotion (P < 0.05, S6 Fig). By 1 contrast, augmenting I M with ICA73 sped up locomotor cycles and shortened burst 2 duration without apparent effect on burst amplitude (P < 0.05, Fig 7C). To study the 3 contribution of Kv7.2 channels, fictive locomotion was induced in Kv7.2 Thr274Met/+ mice.

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It appeared slower (P < 0.05) with longer burst duration (P < 0.01) compared to that 5 induced in wild type spinal cords (Fig 7D). 6 In some CNS neurons, M-channels have been reported to increase the     Because I NaP functions as the primary mechanism for oscillatory burst 2 generation in CPG interneurons [3,5,8,9], it is conceivable that I M interacts with I NaP 3 to orchestrate bursting behavior. Several pieces of evidence support the existence of 4 a dynamic interplay between I M and I NaP . First, our voltage clamp recordings showed a 5 facilitation of I NaP once I M is reduced. Thus, even if the activation of Kv7 channels is too 6 slow to influence the transient sodium current associated with the spike generation 7 (see above), I M appears fast enough to interact with I NaP . Second, according to our 8 model that incorporates a heterogeneous distribution of I NaP and I M , the principal 9 distinguishing property between bursting pacemaker versus nonpacemaker behaviors 10 was the relative magnitude of I NaP to I M ; that is, bursting cells displayed a higher I NaP /I M 11 ratio compared to nonbursting cells. Third, a co-expression between sodium and Kv7.2 12 channels was found in interneurons from the CPG region at the AIS, supposed to be 13 the primary source for both I M and I NaP [61, 62, 67, 68]. Altogether, our data indicate 14 that I NaP and I M are ubiquitously expressed in CPG neurons, and that the core 15 biophysical mechanism for oscillatory activities relies on the spatial and temporal 16 dynamic interactions between the two conductances.

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Aside neuromodulation, we indicated that the propensity of a CPG neuron to 20 burst also depends on the ionic composition of the milieu in which it is embedded [3-5,   Declaration of Interest 13 The authors declare no competing financial interests.  software (see data analysis). 12 13 In vitro models. slices were transferred to a recording chamber that was continuously perfused with the 5 same medium heated to ~27°C. Slices were visualized with epifluorescence and 6 infrared differential interference contrast (IR-DIC) microscopy using a Nikon Eclipse 7 E600FN upright microscope coupled with a 40 X water immersion lens. The image was 8 enhanced with an infrared-sensitive CCD camera and displayed on a video monitor. 9 The temperature regulation was provided by the CL-100 bipolar temperature controller 10 (Warner Instruments). previous study on Hb9 cells [9]. The neuronal membrane potential V was dynamically 2 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 injected (I Inj ) currents. These currents, except for I Pump and I Inj are described as follows: 12 13 Na = Na ⋅ 3 Na ⋅ ℎ Na ⋅ ( -Na ); 15 16 E Na , E K , and E L are reversal potentials of the corresponding channels (in mV):  16 sodium dynamics and pump current: α Na = α NaP = α Pump = 10 -5 mM/fC ; R Pump = 60 pA, 17 [Na+] ibase = 15 mM, K Pump = 18 mM. To reproduce our experimental finding, the half-activation voltage for I NaP , 9 V mNaP1/2 , is made depended on the outside calcium concentration: recordings. An additional heterogeneity was set by normal distribution of all 20 conductances around appropriate base values (see Table below). Parameters for I Na , 21 I K , and inactivation of I NaP were taken from previous modeling studies with some 22 modifications.

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Dashed lines with grey shading indicate the 95% confidence intervals of control values.    after XE991 or ICA73 was bath-applied. n.s. P > 0.05, comparing data collected before