{"paper_id":"183005d6-817f-419e-9fa7-fdb62baf2582","body_text":"0 \n \nTitle: Developmental mechanisms contributing to non-linear firing dynamics in spinal motoneurons of \nthe postnatal mouse \n \nAuthors: Simon A. Sharples1,2* and Gareth B. Miles1* \n \nAffiliation: 1School of Psychology and Neuroscience, University of St Andrews, Fife, United Kingdom, \nKY16 9JP; 2Division of Neurosurgery, Cincinnati Children’s Hospital, Cincinnati, Ohio, United States, \n45229 \n \nRunning Title: Mechanisms of firing hysteresis in spinal motoneurons \n \n*Corresponding Authors \n \nCorrespondence:  \nSimon A. Sharples \nDivision of Neurosurgery \nCincinnati Children’s Hospital \nCincinnati, Ohio, United States, 45229 \nSimon.sharples@cchmc.org \n \nProf. Gareth B. Miles \nSchool of Psychology and Neuroscience \nUniversity of St Andrews \nFife, United Kingdom, KY16 9JP \ngmb4@st-andrews.ac.uk \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n1 \n \nAbstract:  \nThe intrinsic properties of spinal motoneurons support flexible movement, including the maintenance of \npostural tone. Motoneurons can produce sustained action potential output that outlasts synaptic input, a \nphenomenon traditionally attributed to persistent inward currents (PICs) mediated by sodium and calcium \nchannels. Using whole -cell patch clamp electrophysiology, we examined how specific ion channels \ncontribute to PIC maturation and non-linear firing dynamics that allow motoneurons to sustain their output \nin fast and slow lumbar motoneurons across postnatal development in mice. PIC amplitude and non-linear \nfiring dynamics increased after weight bearing in fast but not slow motoneurons. Blocking Nav1.6 channels \nreduced PIC amplitude at both pre - and post -weight-bearing stages, whereas L -type calcium channel \nblockade only reduced PICs after weight bearing emerged. However, r educing PIC amplitude —either \nindividually or in combination —did not abolish sustained firing hysteresis. Unexpectedly, activation of \nmuscarinic receptors increased PIC amplitude while promoting adaptive firing dynamics, suggesting that \nPICs alone do not drive this behavior. Instead, pharmacological manipulation of potassium currents \nmediated by KCNQ and Kv1.2 channels, which oppose PICs, produced substantial changes in firing \ndynamics. Strikingly, blocking HCN channels promoted sustained firing dynamics and led to the emergence \nof self-sustained firing in fast motoneurons. These results indicate that while PICs and non-linear firing \ndynamics mature together, sustained firing relies on mechanisms beyond PICs, with potassium and HCN \nchannels playing key modulatory roles. \n \nKey Points \n● Persistent inward currents (PICs) and recruitment-derecruitment hysteresis increase in parallel in \nfast, but not slow, motoneurons following the onset of hindlimb weight bearing. \n \n● Increased expression or function of L-type calcium channels may contribute to enhanced PICs in \nfast motoneurons after weight bearing emerges. \n \n● Neither Nav1.6 nor L-type calcium channels are required for sustained firing hysteresis in fast \nmotoneurons. \n \n● KCNQ channels attenuate PICs and, together with Kv1.2 channels, shape recruitment–\nderecruitment asymmetry, thereby modulating firing hysteresis in fast motoneurons. \n \n● HCN channels generate a resting H-current that delays recruitment, modulates firing hysteresis, \nand prevents the emergence of self-sustained firing in fast motoneurons. \n \nKey Words: motoneurons, spinal cord, electrophysiology, intrinsic properties, persistent inward current, \nion channels \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n2 \n \nIntroduction \nThe development of complex motor control is a fundamental aspect of early life, enabling animals to \ninteract with their environment, maintain posture, and execute coordinated movements. In rodents, the \nemergence of hindlimb weight bearing represents a crit ical milestone in motor development, typically \noccurring during the second half of the second postnatal week (Altman & Sudarshan, 1975). This behavioral \ntransition reflects the maturation of spinal circuits and the intrinsic properties of motoneurons (Vinay et al., \n2000; Stifani, 2011; Quinlan et al. , 2011; Durand et al. , 2015; Jean -Xavier et al. , 2018; Smith & \nBrownstone, 2020; Sharples & Miles, 2021), which must generate sustained activation of muscle fibers to \nsupport postural stability and locomotion (Heckman et al., 2008b). \n \nPersistent inward currents (PICs) allow motoneurons to maintain firing once activated, which is believed \nto contribute to supporting postural tone. These currents, conducted by sodium (NaV1.6) and calcium \n(CaV1.3) channels, amplify excitatory inputs and help maintain the membrane potential above the threshold \nfor action potential generation, thereby supporting sustained firing beyond the initial synaptic input \n(Schwindt & Crill, 1977, 1980; Hounsgaard & Kiehn, 1989; Carlin et al., 2000; Powers & Binder, 2003; \nLi & Bennett, 2003; Li et al., 2004; Heckmann et al., 2005; Elbasiouny et al., 2006; Harvey et al., 2006a; \nManuel et al., 2009; Bouhadfane et al., 2013; Powers & Heckman, 2015; Mesquita et al., 2024). In other \nwords, a brief excitatory signal can produce ongoing neuronal activity because the inward current “holds” \nthe membrane potential near or above firing threshold. PICs also contribute to nonlinear membrane \nresponses, meaning that the relationship between input and output is not strictly proportional —small \nchanges in input can produce large changes in firing —and can create bistable firing patterns, in which a \nneuron can stably remain in either a low- or high-firing state depending on prior activity (Bos et al., 2018, \n2021; Drouillas et al., 2023; Harris-Warrick et al., 2024). \n \nA frequently studied feature associated with PICs is recruitment–derecruitment asymmetry (Bennett et al., \n2001; Li & Bennett, 2003; Harvey et al., 2006b; Button, 2008; Manuel et al., 2009; Meehan et al., 2010b, \n2010a; Quinlan et al., 2011; MacDonell et al., 2015; Huh et al., 2017). This is typically measured using a \ntriangular current injection, where the current is gradually increased and then decreased. Motoneurons often \nbegin firing at a higher current on the ascending limb of the ramp (recruitment) than the current at which \nfiring stops on the descending limb (derecruitment), producing a characteristic “hysteresis” in the firing –\ncurrent relationship (Bennett et al. , 2001) . In addition, PICs can support higher firing rates on the \ndescending limb compared with the ascending limb, believed to be reflecting the sustained depolarizing \ninfluence of inward currents. Similar patterns are observed in human motor units during triangular isometric \ncontractions (Gorassini et al., 2002; Udina et al., 2010), highlighting the translational relevance of these \nfiring behaviors. While PICs are believed to be central to these phenomena, other intrinsic mechanisms —\nincluding spike-frequency adaptation, spike-threshold accommodation, and potassium conductances—can \nalso influence recruitment, derecruitment, and firing rates independently of inward currents (Sawczuk et \nal., 1995; Button et al., 2007; Li & Bennett, 2007; Powers et al., 2008; Kalmar et al., 2009; Hamm et al., \n2010; Revill & Fuglevand, 2011; Vandenberk & Kalmar, 2014; Powers & Heckman, 2015; Leroy et al., \n2015; Bos et al., 2018; Sharples et al., 2023; Deutsch & Elbasiouny, 2024; Molkov et al., 2025). Together, \nthese mechanisms suggest that sustained firing and hysteresis arise from a dynamic balance of inward and \noutward currents. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n3 \n \nPrevious work has emphasized the role of dendritic CaV1.3 currents in mediating hysteresis under strong \nneuromodulatory drive (Heckman et al., 2003, 2008a), but other ion channels at the axon initial segment \n(AIS) are also likely to play a critical role. NaV1.6 channels generate a persistent sodium current, while \npotassium channels such as Kv1.2 and KCNQ are positioned to shape action potential initiation and \nrepetitive firing (Bos et al., 2018; Drouillas et al., 2023; Sharples et al., 2023; Harris-Warrick et al., 2024). \nThese AIS potassium channels may not simply oppose PICs (Verneuil et al., 2020), but likely actively \ndetermine the timing of recruitment and derecruitment, offering a complementary mechanism that can \ninfluence firing hysteresis. Understanding the relative contributions of inward versus outward conductances \nto the maturation of motoneuron firing properties is especially important during the postnatal period when \nhindlimb weight bearing emerges, as disruptions in these mechanisms may underlie motor disorders or \ndelayed motor development. \n \nIn this study, we investigated the maturation of PICs and recruitment–derecruitment asymmetry in fast and \nslow motoneurons across the postnatal period surrounding the onset of hindlimb weight bearing. Using \nwhole-cell patch-clamp recordings in mouse spinal cord slices combined with selective pharmacological \nmanipulation of NaV1.6, CaV1.3, KCNQ, Kv1.2, and HCN channels, we aimed to disentangle the relative \ncontributions of inward and outward currents to nonlinear firing behaviors. We hypothesized that while \nPIC amplitude and hysteresis would increase with development, other conductances would play a decisive \nrole in shaping recruitment–derecruitment asymmetry, providing a broader mechanistic basis for hysteresis \nthan inward currents alone. \n \nResults \nPICs and recruitment-derecruitment hysteresis increase in fast motoneurons around weight bearing stages. \nWhole cell patch -clamp electrophysiology was deployed to study persistent inward currents (PICs) and \nfiring hysteresis of lumbar motoneurons in transverse spinal cord slices (Figure 1A) obtained from mice \nduring the second and third postnatal weeks. In the se preparations, two motoneuron subtypes can be \nidentified based on delayed and immediate firing profiles (Figure 1B). These two motoneuron subtypes \ndisplay electrophysiological properties and molecular markers that are consistent with fast and slow \nmotoneurons respectively (Leroy et al., 2014; Bos et al., 2018), with functional differences between these \ntwo subtypes emerging sometime during the second postnatal week (Sharples & Miles, 2021; Sharples et \nal., 2025). This period of time coincides with drastic changes in locomotor behaviour, with the emergence \nof hindlimb weightbearing toward the end of the second postnatal week (Altman & Sudarshan, 1975).  \nPrevious work has suggested that increases in PICs in motoneurons may contribute to the emergence of \nhindlimb weight-bearing, toward the end of the second postnatal week, by supporting sustained activation \nof the fatigue resistant slow twitch motor units t hat maintain tone in postural muscles of the hindlimb \n(Brocard et al. , 1999; Vinay et al. , 2000; Clarac et al. , 2004; Jean -Xavier et al. , 2018) . To test this \nhypothesis, we reanalyzed a data set of 209 motoneurons from 81 mice from our previously published work \n(Sharples & Miles, 2021; Sharples et al., 2023), which measured PICs in voltage clamp during triangular \nvoltage ramps (Figure 1C, D). In our previous work, all motoneurons studied during week 2 were pooled. \nIn the current analysis, PICs were examined before and after the emergence of weight bearing dur ing the \nearly (P7-9) and late (P10-13) stages of the second and into the third (P14-20) postnatal weeks. In line with \nour hypothesis, PIC amplitude increased after weight bearing stages (P10 -13) but did not continue to \nincrease further into the third postnatal week (Figure 1E; Table 1a). However, in contrast to our hypothesis, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n4 \n \nand in line with previous work (Sharples & Miles, 2021; Harris-Warrick et al., 2023), PICs were larger in \nthe ‘fast-like’ delayed-firing motoneurons compared to ‘slow -like’ immediate firing motoneurons (Table \n2). This difference in PIC amplitude between delayed and immediate firing motoneurons emerged at the \nend of week 2 and was preserve d into week 3 (Figure 1E). Interestingly, the majority of the PIC was \nactivated below the spike threshold, however this proportion of PIC activated below spike threshold did not \ndiffer between motoneuron subtypes or change across weeks 2 and 3 (Table 2; Table 1b). PIC onset voltage \nwas more depolarized in delayed compared to immediate firing motoneurons but did not change across \nthese three developmental time points (Figure 1F; Table 2; Table1c).  \nIn addition to measuring PICs in voltage clamp, we also assessed recruitment-derecruitment and firing rate \nhysteresis in current clamp during slow triangular currents ramps (Figure 2A). This is an approach that is \noften used to estimate the influence of PICs on motoneuron firing, with negative values reflective of firing \npersisting at lower levels of current on the descending limb of the ramp compared to the ascending limb of \nthe ramp (Ioff<Ion) (Li & Bennett, 2003; Li et al., 2004; Harvey et al., 2006b; Quinlan et al., 2011; Durand \net al. , 2015; Steele et al. , 2020; Sharples & Miles, 2021; Harris -Warrick et al. , 2023) .  In line with \nmeasurements of PICs made in voltage clamp, recruitment-derecruitment hysteresis (delta I) became more \nnegative (increased) in delayed firing motoneurons but did not change in immediate firing motoneurons \nacross the second and third weeks of postnatal development (Figure 2B; Table 2; Table 1d). Delta I \nplateaued at the end of the second postnatal week, with no further change in to week 3 (Figure 2B; Table \n1d).  \nMotoneurons also present with 4 classes of firing rate hysteresis during triangular current ramps, with type \n1; linear, type 2; adapting/clockwise hysteresis, type 3; sustained, and type 4; counterclockwise hysteresis \n(Figure 2C). Of these classes, types 3  and 4 have been proposed to be mediated by a PIC (Bennett et al., \n2001; Durand et al., 2015; Sharples & Miles, 2021). Overall, these firing types did not significantly change \nwithin either subtype over the first three postnatal weeks, however significant differences were found \nbetween delayed and immediate firing motoneurons at all time points (Figure 2D; Table 1e). \nNav1.6 and L-type calcium channels contribute to PICs but do not underlie hysteresis in fast motoneurons \nHaving identified a time course that PIC, recruitment -derecruitment, and firing hysteresis mature during \npostnatal development, we next set out to determine which PIC -conducting ion channels contribute to the \nincrease in PICs and recruitment-derecruitment hysteresis in delayed firing motoneurons around weight \nbearing stages (P7-9 vs. P10-13).  \nRepetitive firing during slow depolarizing current injection is critically dependent on the availability of \npersistent sodium channels (INaP) and can be blocked with riluzole (Miles et al., 2005; Kuo et al., 2006). \nBecause persistent sodium current is a major contributor to PIC amplitude, reducing this current would be \nexpected to decrease PIC amplitude and consequently reduce delta I, reflecting a diminished contribution \nof PICs to firing hysteresis. Consistent wi th our previous findings (Sharples & Miles, 2021) and those of \nothers (Drouillas et al., 2023), blocking NaV1.6 channels produced a 34 ± 40% reduction in PIC amplitude \nat P7–9 and a 39 ± 35% reduction at P10–13, with no difference in the magnitude of this reduction between \ndevelopmental stages (Figure 3A,B; Table 3; Table 1f). Blocking NaV1.6 chann els also depolarized the \nonset voltage of the PIC at both early and late stages of week 2, again with no difference in the magnitude \nof this shift between stages (Figure 3C; Table 1g). Despite the reduction in PIC amplitude, however, delta \nI increased following NaV1.6 blockade (Figure 3E; Table 1h), contrary to the prediction that reducing PIC \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n5 \n \namplitude should decrease delta I. This increase in delta I was observed at both early and late stages of \nweek 2 and occurred without a change in the proportions of Type 1 -4 firing hysteresis (Figure 3F; Table \n1k). The increase in delta I was paralleled by  increases in both recruitment and derecruitment currents \n(Figure 3G, H; Table 1l,m). Together, these data indicate that NaV1.6 channels contribute to the amplitude \nand shape the onset voltage of the underlying PIC. However, changes in NaV1.6 function do n ot account \nfor the developmental increase in PIC amplitude during the second postnatal week and appear to contribute \nminimally to recruitment–derecruitment dynamics and firing hysteresis. \nWe next tested the contribution of L-type calcium channels to the PIC measured during the second postnatal \nweek to determine if changes in their contribution can account for the increase in PIC amplitude and \nrecruitment-derecruitment hysteresis during the second postnatal week. Blocking L-type calcium channels \nwith nifedipine did not alter PIC amplitude at P7 -9 (3±20%) but produced a 34±26% reduction in the \namplitude of the PIC at P10 -13 (Figure 4A, B; Table 3; Table 1q).  Consistent with previous reports \n(Sharples & Miles, 2021), nifedipine did not change the onset voltage of the PIC at either stage (Figure 4C; \nTable 1r). Despite the reduction in PIC amplitude, nifedipine did not significantly change delta I (Figure \n4D, E; Table 1s), proportions of firing hysteresis (Figure 4F; Table 1t), onset current (Figure 4G; Table 1u), \nor offset current (Figure 4H; Table 1v) at either time point. Together these data suggest that L-type calcium \nchannels contribute in part to PIC amplitude and an increase in their contribution toward the end o f the \nsecond postnatal week may account for the increase in PIC amplitude that we observed. However, in \ncontrast to what we expected, L -type calcium channels are not a key contributor to recruitment -\nderecruitment or firing hysteresis. \nWe next considered the possibility that blocking NaV1.6 or L -type calcium channels individually did not \nproduce a sufficient reduction in PIC amplitude to alter firing hysteresis. If delta I is strongly dependent on \nPIC magnitude, then a larger reduction in PIC amplitude should be expected to decrease delta I. To test this \npossibility, we simultaneously blocked NaV1.6 and L -type calcium channels using 4,9 -anhydro-\ntetrodotoxin and nifedipine. Although co-application of these antagonists (n = 8 MNs; P10–13) produced a \nsubstantial 58 ± 7.7% reduction in PIC amplitude (Table 3; Table 1w), this manipulation did not \nsignificantly alter delta I or firing hysteresis in the expected direction (Table 3; Table 1x). These findings \nfurther indicate that the magnitude of the PIC alone is not a primary determinant of recruitment –\nderecruitment hysteresis in developing motoneurons. \nParadoxical modulation of PIC amplitude and firing hysteresis in fast motoneurons \nPrevious work has suggested that the influence of PICs on motoneuron firing is critically dependent on the \npresence of endogenous sources of neuromodulation (Hounsgaard et al., 1984, 1988; Conway et al., 1988). \nIt has also been shown that multiple neuromodulators can increase PIC amplitude in hypoglossal \nmotoneurons (Revill et al., 2019). Here we tested the hypothesis that increasing PIC amplitude by activating \nmuscarinic acetylcholine receptors would produce a shift toward sustained firing hysteresis, as we found \nduring development. In line with previous reports (Revill and Funk, 2022), application of muscarine \nproduced a 174±82% increase in PIC amplitude (Figure 5A, B; Table 3; Table 1y) but did not alter the \nonset voltage of the PIC (Figure 5A, C; Table 1z). This increase in PIC amplitude produced by muscarine \nis equivalent to the mean increase that we observed between the early and late phase of the second postnatal \nweek. However, in contrast to what would be expected, muscarine decreased delta I (Figure 5D, E; Table \n1aa) and produced a general shift from types 3-4 to types 1-2 firing hysteresis (Figure 5F; Table 1bb)). The \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n6 \n \nreduction in delta I occurred without a change in onset current (Figure 5G; Table 1cc) and was instead \nattributable to a selective increase in the offset current in the presence of muscarine (Figure 5H; Table 1dd). \nFiring hysteresis in fast motoneurons is modulated by potassium channels \nAlthough muscarine increases PIC amplitude, it is also known to modulate multiple voltage -sensitive ion \nchannels. One current of particular interest is the M-type potassium current, conducted by slow-activating, \nnon-inactivating KCNQ channels that are largely active in the subthreshold voltage range (Alaburda et al., \n2002; Ghezzi et al., 2017, 2018; Verneuil et al., 2020; Sharples et al., 2023). Because these channels a \npersistent outward current, it can oppose the actions of PICs (Verneuil et al., 2020; Singh et al., 2025; \nGaudreau & Bui, 2026)  and was originally identified based on its inhibition by muscarinic receptor \nactivation (Brown & Adams, 1980) . In addition, our previous work demonstrated large M -currents in \ndelayed firing motoneurons and a selective role for this conductance in controlling recruitment (Sharples et \nal., 2023). \n \nBased on these observations, we hypothesized that M-currents shape firing hysteresis by counteracting PICs \nand by providing differential control of recruitment and derecruitment currents (Verneuil et al. , 2020; \nSharples et al. , 2023) . Under this framework, reducing M -current would be expected to enhance PIC \namplitude and potentially increase delta I by strengthening inward current amplification during firing. \nConsistent with the predicted effect on PICs, blocking M-currents with XE991 (10 μM) produced a 188 ± \n32% increase in PIC amplitude (Figure 6A, B; Table 3; Table 1ee) and hyperpolarized PIC onset voltage \nby 4.9 ± 2.8 mV (Figure 6C; Table 1ff). The magnitude of this increase in PIC amplitude was comparable \nto that observed developm entally and following muscarinic activation. However, despite the substantial \nincrease in PIC amplitude, blocking KCNQ channels reduced delta I (Figure 6D, E; Table 1gg) and shifted \nfiring behavior from Types 3 –4 toward Types 1 –3 patterns (Figure 6F; Table  1hh), contrary to the \nexpectation that larger PICs should increase firing hysteresis. Importantly, the mechanism underlying this \nreduction in delta I differed from that observed with muscarine. Following KCNQ channel blockade, the \nreduction in delta I res ulted from a decrease in the recruitment current (Figure 6G; Table 1ii), with no \nchange in the derecruitment current (Figure 6H; Table 1jj). Thus, whereas muscarine reduced delta I by \nincreasing the offset current, inhibition of KCNQ channels reduced delta I by lowering the onset current on \nthe ascending limb of the current ramp. \n \nConversely, activation of KCNQ channels with ICA73 increased delta I (Figure 6I, J; Table 3; Table 1kk) \nand produced a general shift toward Type 4 firing hysteresis (Figure 6K; Table 1ll). This increase in delta \nI resulted from an increase in recruitment current (Figure 6L; Table 1mm) with no change in derecruitment \ncurrent (Figure 6M; Table 1nn). Similar effects were observed with a second KCNQ channel activator, \nretigabine (Table 3; Table 1oo-rr). Moreover, these effects were reversed by subsequent application of the \nKCNQ blocker XE991, which reduced delta I to levels significantly below baseline and shifted firing \nbehavior toward Types 1 –2 patterns, consistent with blockade of KCNQ channels alone (Figure 6J –M; \nTable 1gg-jj). Together, these findings indic ate that KCNQ channels conduct an outward current that not \nonly opposes PICs but also plays a critical role in shaping recruitment –derecruitment hysteresis. Rather \nthan regulating derecruitment, KCNQ channels primarily influence firing hysteresis by contro lling the \nrecruitment current on the ascending limb of triangular current ramps. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n7 \n \nGiven that types 3-4 firing hysteresis and negative delta I values are a prominent feature of fast-type delayed \nfiring motoneurons (Figure 2), with delayed firing being produced by rapid activation and slow inactivation \nof Kv1.2 channels (Bos et al., 2018; Harris-Warrick et al., 2024), we next tested the hypothesis that slow \ninactivation of Kv1.2 channels might contribute to sustained firing hysteresis by allowing motoneurons to \nfire for longer on the descending aspect triangular current ramps. Blockade of Kv1.2 channels with \ntityustoxin (TsTx; 800 nM) blocked the slow depolarization of the membrane potential in response to a \nlong (5s) depolarizing current step applied near rheobase (Figure 7A). Interestingly, and in line with our \nhypothesis, blocking Kv1.2 channels decreased delta I (F igure 7B, C; Table 3; Table 1ss) and produced a \nshift from Types 3 and 4 to Types 1-3 firing hysteresis (Figure 7D; Table 1tt). However, in contrast to what \nwe hypothesized, the reduction of delta I was due to a significant decrease in the onset current (Figure 7E; \nTable 1uu) and no change in the offset current (Figure 7F; Table 1vv). These data suggest that the rapid \nactivation of Kv1.2 channels may influence firing hysteresis by shaping fast motoneuron recruitment, \nhowever slow inactivation of Kv1.2 channels may play less of a role in supporting sustained firing on the \ndescending limb of the ramp. \n \nHCN channels modulate firing hysteresis and prevent bistable firing in fast motoneurons  \nIn the preceding experiments, we found that manipulating PIC amplitude alone did not reliably predict \nchanges in recruitment –derecruitment hysteresis. Both NaV1.6 and KCNQ channel manipulations \nsubstantially altered PIC amplitude yet produced changes in de lta I that could not be explained solely by \nthe magnitude of the PIC. These findings suggested that additional conductances that shape the membrane \noperating range or input conductance may play an important role in determining whether PICs generate \nfiring hysteresis. \n \nIn a final set of experiments, we therefore assessed the contribution of hyperpolarization -activated cyclic \nnucleotide-gated (HCN) channels to recruitment–derecruitment and firing rate hysteresis in delayed-firing \nfast motoneurons. Our previous work demonstrated that by the third postnatal week (P14–20) the activation \nvoltage of HCN channels shifts in the depolarizing direction, resulting in an h-current that is active at resting \nmembrane potential. This resting h-current acts as a depolarizing shunt conductance that delays recruitment \nin response to depolarizing input (Sharples & Miles, 2021) . Because HCN channels deactivate slowly \nduring depolarization and reactivate during hyperpolarization, we hypothesized that this current could limit \nthe ability of PICs to generate bistability and thereby suppress recruitment–derecruitment hysteresis during \ntriangular current ramps. Consistent with this hypothesis, pharmacological blockade of HCN channels with \nZD7288 (Figure 8A, B; Table 3; Table 1ww) significantly increased delta I (Figure 8C, D; Table 1xx). \nNotably, following HCN channel blockade, 48% of motoneurons displayed self-sustained firing— a feature \nrarely observed in delayed - or immediate -firing motoneurons during the first three postnatal weeks \n(Sharples & Miles, 2021). This enhancement in sustained activity was reflected by a shift in firing hysteresis \nfrom type 4 at baseline to the emergence of a fifth firing pattern characterized by persistent self -sustained \nfiring (Figure 8E; Table 1yy). The increase in delta I was accompanied by significant reductions in both \nrecruitment (Figure 8F; Table 1zz) and derecruitment (Figure 8G; Table 1aaa) currents during triangular \ncurrent ramps. Moreover, after termination of the ramp (Figure 8H) or a brief depolarizing current step \n(Figure 8I), sustained discharge required hyperpolarizing current injection to terminate firing, consistent \nwith the emergence of bistable firing behavior following HCN channel blockade. To determine whether \nthis enhanced bistability resulted from changes in  the underlying PIC, we measured PIC amplitude in \nvoltage clamp in a subset of motoneurons (n = 4). Because PICs are believed to be a key contributor to self-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n8 \n \nsustained firing, we predicted removing the shunt current by blocking HCN channels might increase PIC \namplitude. However, in contrast to this prediction, HCN channel blockade produced no significant change \nin PIC amplitude (−10 ± 40%, Figure 8J, K; Table 1bbb). Instead, the onset voltage of the PIC became \nsignificantly hyperpolarized (−6.4 ± 5.1 mV, Figure 8L; Table 1ccc) in all cells examined. Together, these \nfindings suggest that HCN channels oppose the generation of self -sustained firing in fast motoneur ons at \nthe third postnatal week, likely by acting as a resting shunt conductance that stabilizes membrane potential \nand limits the voltage range over which PICs can generate bistability. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n9 \n \n \n \nFigure 1: PICs increase in delayed firing motoneurons after the emergence of hindlimb weight bearing. (A) Motoneurons \nvisualized under differential interference contrast were identified based on delayed (purple) and immediate (green) firing with long \n(5 second) depolarizing current steps applied near rheobase current (B). (C) Persistent inward currents (PI Cs) were measured in \nvoltage clamp from delayed (purple) and immediate firing (green) motoneurons at pre (Delayed n = 22; immediate n = 22;  P7 -9) \nand post- (Delayed n = 22; immediate n = 14; P10 -13) weight bearing stages and into the third postnatal week (Delayed n = 14; \nimmediate n = 13; P 14 -20) using a slow depolarizing voltage ramp (10mV/s; -90 to -10 mV). (F) PIC onset and amplitude were \nmeasured from  leak-subtracted, low pass filtered (5Hz Bessel) traces. A -D, adapted from (Sharples & Miles, 2021) . (G) PIC \namplitude increased in delayed firing motoneurons from pre- to post-weight bearing stages and did not change in immediate firing \nmotoneurons. (F) PIC onset voltage did not change in delayed or immediate firing motoneurons during the second and th ird \npostnatal week. Data are presented as mean ± SD with individual data points for each motoneuron displayed. Data were analyzed  \nwith a 2 factor ANOVA, with age and motoneuron type as factors. P values are reported when significant differences were detected. \nHedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n10 \n \n \nFigure 2:  Recruitment-derecruitment and firing hysteresis mature in parallel to PICs. (A) \nRepresentative traces of the membrane potential and repetitive firing from a delayed (purple) and immediate \nfiring (green) motoneuron during a triangular depolarizing current ramp up to an intensity of 2 x their \nrespective rheobase currents. (B) Recruitment-derecruitment hysteresis, measured as Delta I, represents the \ndifference between current at firing offset on the descending limb of the ramp and the current at firing onset \non the ascending limb of the ramp, increased in delayed firing motoneurons at pre- and post-weight bearing \nstages but did not change in immediate firing motoneurons. (C) Frequency current plots with firing rates \nderived from ascending (black) and descending  (red) limbs of triangular current ramps. Four patterns of \nfiring rate hysteresis can be identified as described by Li and Bennett, 2006; with types 3 and 4 suggestive \nof PIC actions. A&C, adapted from (Sharples & Miles, 2021). (D) Relative proportion of firing hysteresis \ntypes in delayed and immediate firing motoneurons across the second and third postnatal week. Data are \npresented as mean ± SD with individual data points for each motoneuron displayed. Data in B were analyzed \nwith a 2 factor ANOVA, with age and motoneuron type as factors. P values are reported when significant \ndifferences were detected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) \neffect sizes. (Delayed firing MNs: P7-9 n = 31, P10-13 n = 43, P14-20 n = 52; Immediate firing MNs: P7-\n9 n=29, P10-13 n = 26, P14-20 n = 27). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n11 \n \n \nFigure 3: Nav 1.6 channels contribute to PIC but not firing hysteresis.  (A) Representative traces of a \nleak-subtracted and filtered PIC measured in voltage clamp before (black) and after (red) application of the \nNaV1.6 blocker 4,9 Anhydro -tetrodotoxin (4,9 AH -TTX; 200 nM). (B) 4,9 AH -TTX decreased PIC \namplitude and depolarized PIC onset voltage (C) at both pre- (n=10; P7-9) and post- (n=12; P10-13) weight \nbearing stages. (D) Representative traces of the membrane potential and repetitive firing during a triangular \ndepolarizing current ramp before (black) and after (red) application of 4,9 AH -TTX. (E) 4,9 AH -TTX \nincreased recruitment-derecruitment hysteresis (Delta I) but did not alter the proportion of firing types (F) \nat pre- and post-weight bearing stages. 4,9 AH-TTX increased the current at firing onset on the ascending \nlimb of the ramp (G) and increased current at firing offset on descending limb of the ramp (H) at both pre-\nand post-weight bearing stages. Data are presented as individual data points and we re analyzed using a 2 \nfactor ANOVA with drug and stage as factors. P values are reported when significant differences were \ndetected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n12 \n \n \nFigure 4: L-type calcium channels contribute to PIC after the emergence of weight bearing but do \nnot contribute to firing hysteresis. (A) Representative traces of a leak -subtracted and filtered PIC \nmeasured in voltage clamp before (black) and after (red) application of the L-type calcium channel blocker \nNifedipine (20 µM). (B) Nifedipine decreased PIC amplitude at post weight-bearing stages but did not alter \nPIC onset voltage (C). (D) Representative traces of the membrane potential and repetitive firing dur ing a \ntriangular depolarizing current ramp before (black) and after (red) application of Nifedipine. (E) Nifedipine \ndid not affect recruitment -derecruitment hysteresis (Delta I) or alter the proportion of firing types (F) at \npre- (n= 12; P7-9) and post-weight bearing stages (n= 11; P10-13). Nifedipine did not affect current at firing \nonset on the ascending limb of the ramp (G) or current at firing offset on descending limb of the ramp (H) \nat both pre- or post-weight bearing stages. Data are presented as individual data points and were analyzed \nusing a 2 factor ANOVA with drug and stage as factors. P values are reported when significant differences \nwere detected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.  \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n13 \n \n \nFigure 5: Muscarine increases PIC but reduces firing hysteresis. (A) Representative traces of a leak -\nsubtracted and filtered PIC measured in voltage clamp before (black) and after (red) application of the \nMuscarine (10 µM; n = 10; P7 -13). (B) Muscarine increased PIC amplitude but did not alter PIC onset \nvoltage (C). (D) Representative traces of the membrane potential and repetitive firing during a triangular \ndepolarizing current ramp before (black) and after (red) application of muscarine. (E) Muscarine decreased \nrecruitment-derecruitment hysteresis (Delta I; n = 15) and produced a shift in firing types from types 3 and \n4 to types 1-4. These effects were reversed following a wash with regular aCSF. Muscarine did not affect \ncurrent at firing onset on the asce nding limb of the ramp (G) but increased the current of firing offset on \ndescending limb of the ramp (H). Data are presented as individual data points and were analyzed using a \npaired t -test (B & C) or repeated measures ANOVA (E - H). P values are reported  when significant \ndifferences were detected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) \neffect sizes.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n14 \n \n \nFigure 6: KCNQ channels attenuate PIC and promote firing hysteresis. (A) Representative traces of a leak -subtracted and \nfiltered PIC measured in voltage clamp before (black) and after (red) application of the KCNQ channel blocker XE991 (10 µM; n \n= 5; P10 -13). (B) XE991 increased PIC amplitude and hyperpolarized PIC onset vo ltage (C). (D) Representative traces of the \nmembrane potential and repetitive firing during a triangular depolarizing current ramp before (black) and after (red) application of \nXE991. (E) XE991 decreased recruitment-derecruitment hysteresis (Delta I; n = 10) and produced a shift in firing types from types \n3 and 4 to types 1 -3 (F). XE991 decreased the current at firing onset on the ascending limb of the ramp (G) but did not alter the \ncurrent of firing offset on the descending limb of the ramp (H). (I) Repres entative traces of the membrane potential and repetitive \nfiring during a triangular depolarizing current ramp before (black) and after (blue) application of the KCNQ channel activato r \nICA73 (n=11; 10 µM) and subsequent application of XE991 (red). (J) ICA73 increased recruitment-derecruitment hysteresis (Delta \nI) and produced a shift in firing types from types 3 and 4 to types 1 -3 (K). ICA73 increased the current at firing onset on the \nascending limb of the ramp (L) but did not alter the current of firing of fset on the descending limb of the ramp (M). These effects \nwere reversed with subsequent application of the KCNQ channel blocker, XE991 (n = 6). Data are presented as individual data \npoints and were analyzed using a paired t -test (B & C) or repeated measur es ANOVA (E - H; J - M). P values are reported when \nsignificant differences were detected. Hedge’s g values indicated small (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n15 \n \n \n \nFigure 7: Kv1.2 channels shape firing hysteresis. (A) Representative traces of the membrane potential \nduring a long (5 s) depolarizing current step applied just below rheobase before (black) and after (red) \napplication of the Kv1.2 channel blocker Tityustoxin (800 nM; TsTx; n = 13; P7 -13). (B) Representative \ntraces of the membrane potential and repetitive firing during a triangular depolarizing current ramp before \n(black) and after (red) application of TsTx. (C) TsTx decreased recruitment-derecruitment hysteresis (delta \nI) and produced a shift in firing types from types 1, 3 and 4 to types 2 -4. TsTx decreased the current at \nfiring onset on the ascending limb of the ramp (G) but did not affect the current of firing offset on \ndescending limb of the ramp (H). D ata are presented as individual data points and were analyzed using a \npaired t-test. P values are reported when significant differences were detected. Hedge’s g values indicated \nsmall (0.2-0.49), medium (0.5-0.79) and large (>0.8) effect sizes.  \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n16 \n \n \nFigure 8: HCN channels prevent self sustained firing at the third postnatal week.  (A) Representative voltage clamp traces \nillustrating the H-current measured in response to a 10 -mV hyperpolarizing voltage step applied from -60 mV before (black) and \nafter application of the HCN channel blocker ZD7288 (10 µM) in 10 delayed firing motoneu rons obtained from mice during the \nthird postnatal week (P14-20). (B) ZD7288 eliminates H current measured at -70 mV. (C) Representative traces of the membrane \npotential and repetitive firing during a triangular depolarizing current ramp before (black) and after (red) application of ZD7288. \nZD7288 increased recruitment-derecruitment hysteresis (D; Delta I) and produced a shift from Type 4 firing hysteresis in all cells \nto the emergence of a 5th firing type characterized by self sustained firing (E). ZD7288 decreased the current at firing onset on the \nascending limb of the ramp (F) decreased the current of firing offset on the descending limb of the ramp (G). Hyperpolarizing  \ncurrent was needed to terminate self-sustained firing elicited after triangular current ramps (H) or brief depolarizing current steps \n(I) in the presence of ZD7288. J) Representative traces of a leak -subtracted and filtered PIC measured in voltage clamp befor e \n(black) and after (red) application of the HCN channel blocker ZD7288 (10 uM; n = 4). ZD7288 did not change the amplitude (K) \nof the PIC but hyperpolarized the PIC onset voltage (L). Data are presented as individual data points and were analyzed using  a \npaired t-test. P values are reported when significant differences were detected. Hedge’s g values indicated small (0.2-0.49), medium \n(0.5-0.79) and large (>0.8) effect sizes.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n17 \n \nTable 1: Statistical summary table. Results from statistical tests performed in this study are annotated through the \nmanuscript and are indicated in the first column of the table.  \n  \n Source Data Condition Property Animal \nAge \nNo. of MNs \n \nStatistical Test d.f. Test \nstatistic \nP Value \na Figure 1E Development PIC Amp. P7-20 107 2W ANOV A 2,101 17.0 4.2e-7 \nb  text Development PIC at AP TH 7-20 104 2W ANOV A 2,98 0.9 0.9 \nc Figure 1F Development PIC On. V olt. P7-20 107 2W ANOV A 2,101 1.3 0.3 \nd Figure 2B Development Delta I P1-20 201 2W ANOV A 2,201 10.7 3.8e-5 \ne Figure 2D Development Firing Types P1-20 201 Kruskal-Wallis  115 1.0e-15 \nf Figure 3B 4,9 AH-TTX PIC Amp. P7-14 22 2W ANOV A 1,21 6.5 3.0e-4 \ng Figure 3C 4,9 AH-TTX PIC On. V olt. P7-14 22 2W ANOV A 1,21 25.1 6.6e-5 \nh Figure 3E 4,9 AH-TTX Delta I P7-14 21 2W ANOV A 1,20 15.4 9.1e-4 \nk Figure 3F 4,9 AH-TTX Firing Types P7-14 21 Kruskal-Wallis  5.9 0.1 \nl Figure 3G 4,9 AH-TTX Onset I P7-14 21 2W ANOV A 1,20 78.9 2.2e-8 \nm Figure 3H 4,9 AH-TTX Offset I P7-14 21 2W ANOV A 1,20 62.5 1.4e-7 \nq Figure 4B Nifedipine PIC Amp. P7-14 17 2W ANOV A 1,15 4.2 0.05 \nr Figure 4C Nifedipine PIC On. V olt. P7-14 17 2W ANOV A 1,15 1.5 0.23 \ns Figure 4E Nifedipine Delta I P7-14 23 2W ANOV A 1,22 0.9 0.35 \nt Figure 4F Nifedipine Firing Types P7-14 23 Kruskal-Wallis  9.3 0.02 \nu Figure 4G Nifedipine Onset I P7-14 23 2W ANOV A 1,22 1.8 0.2 \nv Figure 4H Nifedipine Offset I P7-14 23 2W ANOV A 1,22 0.6 0.45 \nw  text Nif. + TTX PIC Amp. 10-13 7 t-test 6 4.5 0.004 \nx  text Nif + TTX Delta I 10-13 8 t-test 7 1.1 0.3 \ny  Figure 5B Muscarine PIC Amp. P10-14 10 Paired t-test 9 5.2 5.1e-4 \nz  Figure 5C Muscarine PIC On. V olt. P10-14 10 Paired t-test 9 0.8 0.5 \naa  Figure 5E Muscarine Delta I P10-14 15 Mixed Effect ANOV A 1.9, 20.7 16.6 5.3e-5 \nbb  Figure 5F Muscarine Firing Types P10-14 15 Wilcoxon test  -78 4.9e-4 \ncc Figure 5G Muscarine Onset I P10-14 15 Mixed Effect ANOV A 0.007, 0.08 0.5 0.1 \ndd Figure 5H Muscarine Offset I P10-14 15 Mixed Effect ANOV A 1.1.12.4 5.6 0.03 \nee Figure 6B XE991 PIC Amp. P10-14 5 Paired t-test 4 7.5 0.002 \nff Figure 6C XE991 PIC On. V olt. P10-14 5 Paired t-test 4 3.8 0.002 \ngg Figure 6E XE991 Delta I P10-18 10 Paired t-test 9 2.3 0.04 \nhh Figure 6F XE991 Firing Types P10-18 10 Wilcoxon test  -45 0.004 \nii Figure 6G XE991 Onset I P10-18 10 Paired t-test 9 3.6 0.006 \njj Figure 6H XE991 Offset I P10-18 10 Paired t-test 9 0.4 0.7 \nkk Figure 6J ICA73 +XE991 Delta I P10-18 11 Mixed Effect ANOV A 1.1, 8.2 31.5 3.5e-5 \nll Figure 6K ICA73 +XE991 Firing Types P10-18 11 Kruskal-Wallis  18.6 9.3e-5 \nmm Figure 6L ICA73 +XE991 Onset I P10-18 11 Mixed Effect ANOV A 1.1,8.2 12.1 0.007 \nnn Figure 6M ICA73 +XE991 Offset I P10-18 11 Mixed Effect ANOV A 1.1, 8.2 2.5 0.15 \noo Text Retigabine Delta I P10-18 10 Paired t-test 9 4.2 0.002 \npp Text Retigabine Onset I P10-18 10 Paired t-test 9 3.2 0.01 \nqq Text Retigabine Offset I P10-18 10 Paired t-test 9 2.2 0.06 \nrr Text Retigabine Firing Types P10-18 10 Wilcoxon test  6 0.25 \nss Figure 7C Tityustoxin Delta I P10-14 13 Paired t-test 12 4.4 9.5e-4 \ntt Figure 7D Tityustoxin Firing Types P10-14 13 Wilcoxon test  -49 0.02 \nuu Figure 7E Tityustoxin Onset I P10-14 13 Paired t-test 12 2.5 0.03 \nvv Figure 7F Tityustoxin Offset I P10-14 13 Paired t-test 12 0.9 0.4 \nww Figure 8B ZD7288 Ih Amplitude P14-20 11 Paired t-test 10 3.6 0.005 \nxx Figure 8D ZD7288 Delta I P14-20 10 Paired t-test 9 2.9 0.02 \nyy Figure 8E ZD7288 Firing Types P14-20 10 Paired t-test  28 0.02 \nzz Figure 8F ZD7288 Onset I P14-20 10 Paired t-test 9 2.9 0.02 \naaa Figure 8G ZD7288 Offset I P14-20 10 Paired t-test 9 3.1 0.01 \nbbb Figure 8K ZD7288 PIC Amp. P14-20 10 Paired t-test 3 0.2 0.86 \nccc Figure 8L ZD7288 PIC On. V olt. P14-20 10 Paired t-test 3 2.5 0.04 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n18 \n \nTable 2: Developmental analysis of PIC and recruitment-derecruitment hysteresis. Data are presented as mean ± SD. \nSuperscript numbers indicate significant differences within motoneuron subtypes between P7-9 (1), P10-13 (2), or P14-20 \n(3). Asterisks denote significant differences between motoneuron subtypes within each developmental stage with *p<0.05, \n**p<0.01, ***p<0.001, ****p<0.00001.  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nParameter MN type P7-9 P10-13 P14-20 \nPIC Amplitude Delayed -119±1172,3 -395±2291 -476±2181 \n Immediate -119±74 -199±160** -293±170* \nPIC On V oltage Delayed -51±3.9 -51±4.3 -49±4.8 \n Immediate -58±4.3*** -58±5.9*** -58±4.9*** \nPIC Amp. At Spike TH Delayed -192±106 -300±218 -273±176 \n Immediate -102±95 -181±174 -238±123 \nProp. PIC at Spike TH Delayed 0.7±0.23 0.67±0.24 0.69±0.28 \n Immediate 0.68±0.28 0.62±0.35 0.72±0.34 \nDelta I (pA) Delayed -205±902,3 -294±1231 -318±1421 \n Immediate -38±99**** 14±31**** 27±126**** \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n19 \n \nTable 3: Pharmacological manipulation of PIC measured in voltage clamp and recruitment-derecruitment \nhysteresis measured in current clamp. Data are presented as mean ± SD. Asterisks denote significant differences \nbetween drug and baseline conditions with *p<0.05, **p<0.01, ***p<0.001, ****p<0.00001. \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nExperiment Parameter Age No. of MNs Baseline Drug \n4,9 AH-TTX PIC Amp P7-9 10 -341±225 -200±156** \n P10-13 12 -529±270 -348±313** \nPIC On. V olt. P7-9 10 -54±7.6 -46±5.3** \n P10-13 12 -50±3.3 -47±5.8* \nDelta I P7-9 10 -271±167 -328±190* \n P10-13 11 -300±152 -367±196* \nIon P7-9 10 657±370 934±442*** \n P10-13 11 884±474 1243±569*** \nIoff P7-9 10 385±266 607±332** \n P10-13 11 581±394 886±473*** \n      \nNifedipine PIC Amp P7-9 9 -198±146 -172±83.4 \n P10-13 8 -388±225 -243±183* \nPIC On. V olt. P7-9 9 -51±5.5 -51±8.1 \n P10-13 8 -51±5.9 -57±13 \nDelta I P7-9 12 -196±118 -182±79 \n P10-13 11 -283±112 -235±181 \nIon P7-9 12 603±331 558±231 \n P10-13 11 775±510 680±528 \nIoff P7-9 12 421±306 507±187 \n P10-13 11 453±463 448±401 \n      \nNifedipine + 4,9 \nAH-TTX \nPIC Amp P10-13 7 -392±108 -169±108* \nPIC On. V olt. P10-13 7 -50±5.8 -48±6.5** \nDelta I P10-13 8 -182±63 -212±89 \nIon P10-13 8 574±287 712±432* \nIoff P10-13 8 392±303 499±447 \n      \nMuscarine PIC Amp P10-13 10 -522±337 -796±404**** \nPIC On. V olt. P10-13 10 -42±9.9 -44±10.1 \nDelta I P10-13 15 -246±127 -56±150**** \nIon P10-13 15 568±355 577±316 \nIoff P10-13 15 323±284 521±290** \n      \nXE991 PIC Amp P10-13 5 -387±237 -670±266** \nPIC On. V olt. P10-13 5 -46±3.6 -51±4.4* \nDelta I P10-13 10 -179±180 -3.9±231* \nIon P10-13 10 702±470 477±370** \nIoff P10-13 10 523±326 473±425 \n      \nICA73 Delta I P10-13 11 -182±93 -271±135** \n Ion P10-13 11 750±397 838±432* \n Ioff P10-13 11 567±363 567±363 \n      \nRetigabine Delta I P10-13 10 -173±71 289±117** \nIon P10-13 10 825±463 1140±425* \nIoff P10-13 10 652±420 849±344 \n      \nTityustoxin Delta I P10-13 13 -305±193 -10±182**** \nIon P10-13 13 902±489 673±572* \nIoff P10-13 13 599±391 694±495 \n      \nZD7288 Ih Amp.  P14-20 11 -89±95 13.9±14.2** \nDelta I P14-20 10 -242±70 -300±94** \nIon P14-20 10 929±585 567±478* \nIoff P14-20 10 686±814 269±414* \nPIC Amp P14-20 4 -598±97 -558±109 \nPIC On. V olt. P14-20 4 -47±2.5 -54±3.3* \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n20 \n \nDiscussion \nThe present study examined the developmental maturation of persistent inward currents (PICs) and their \nrelationship to non-linear firing dynamics leading to recruitment–derecruitment and firing hysteresis in \nlumbar spinal motoneurons of the postnatal mouse. Although PICs are widely assumed to be the primary \ndeterminant of sustained firing hysteresis in motoneurons (Hounsgaard & Kiehn, 1989; Li & Bennett, 2003; \nHeckman et al., 2008b), alternative mechanisms have not been tested directly. This issue is particularly \nimportant because recruitment –derecruitment hysteresis measured in human motor units is commonly \nquantified using ΔF from paired motor unit recordings during triangular isom etric contractions and \ninterpreted as an index of PIC amplitude or neuromodulatory drive (Gorassini et al., 2002; Heckmann et \nal., 2005; Heckman et al., 2008a, 2008b; Goodlich et al., 2024; Mesquita et al., 2024). Here we addressed \nthree related hypotheses. First, that developmental increases in PIC amplitude underlie the emergence of \nrecruitment–derecruitment hysteresis during the onset of hindlimb weight bearing (Clarac et al., 2004; \nQuinlan et al., 2011; Sharples & Miles, 2021). Second, that altering PIC amplitude through pharmacological \nmanipulation of NaV1.6 and L -type calcium channels would produce predictable changes in firing \nhysteresis (Carlin et al. , 2000; Li & Bennett, 2003; Li et al. , 2004) . Third, that other conductances —\nparticularly those mediated by potassium and HCN channels —may shape hysteresis by modifying the \nbalance between recruitment and derecruitment currents (Manuel et al., 2007; Bos et al., 2018; Sharples & \nMiles, 2021; Sharples et al., 2023; Harris -Warrick et al., 2024). The conceptual advance of this study is \nthat it directly tests the long-standing assumption that PIC amplitude determines hysteretic firing behaviour. \n \nOur results partially supported the first hypothesis but challenged the latter two. PIC amplitude and \nrecruitment–derecruitment hysteresis increased in parallel in fast -type delayed firing motoneurons during \nthe onset of weight bearing, whereas slow-type immediate firing motoneurons showed little developmental \nchange. However, although NaV1.6 and L -type calcium channels contributed substantially to PIC \namplitude, manipulating these currents did not alter recruitment –derecruitment or firing hysteresis \n(Bouhadfane et al., 2013; Sharples & Miles, 2021; Drouillas et al., 2023). Moreover, pharmacological \nmanipulations that increased PIC amplitude did not produce the predicted increase in sustained firing \nhysteresis; notably, muscarine increased PIC amplitude while paradoxically reducing recruitment –\nderecruitment hysteresis and promoting adaptive firing hysteresis (Revill et al., 2019; Sharples et al., 2023). \nInstead, potassium channels—including KCNQ and Kv1.2 —strongly shaped hysteretic firing by altering \nthe balance of onset and offset currents during recruitment and derecruitment (Bos et al., 2018; Verneuil et \nal., 2020; Singh et al. , 2025) . Finally, HCN channels limited the emergence of bistable firing in fast \nmotoneurons by the third postnatal week (Manuel et al., 2007; Sharples & Miles, 2021) . Together these \nfindings indicate that although PICs and firing hysteresis mature together during development, sustained \nhysteretic firing cannot be attributed solely to PIC amplitude. Instead, hysteresis emerges from interactions \nbetween inward and outward currents that shape recruitment–derecruitment asymmetry. \n \nDevelopmental emergence of PICs and hysteresis \nHindlimb weight-bearing is a pivotal behavioral milestone in rodents, emerging toward the end of the \nsecond postnatal week (Altman & Sudarshan, 1975; Brocard et al., 1999). It requires sustained activation \nof postural motor units to support antigravity function, and it has long been proposed that maturation of \ndendritic PICs underlies this transition by enabling motoneurons to produce sustained action potential \ndischarge following transient excitatory inputs. Consistent with this view, we observed a developmental \nincrease in both PIC amplitude and recruitment –derecruitment hysteresis specifically in delayed firing \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n21 \n \nmotoneurons during the onset of weight bearing. Despite the increase in PIC amplitude and delta I, the \nproportion of Types 1 –4 firing hysteresis remained relatively stable across postnatal development. These \nfindings agree with prior work showing that PICs  become more prominent in motoneurons around the \nsecond postnatal week and contribute to enhanced excitability , however the changes in delta I before the \nend of the second week  are relatively subtle (Quinlan et al., 2011; Sharples & Miles, 2021) . However, a \nsurprising feature of our results, which is in line with recent findings from Harris -Warrick and colleagues \n(Harris-Warrick et al., 2024), is that the developmental increase in PIC amplitude was most pronounced in \ndelayed firing motoneurons, considered fast-type, rather than in immediate firing slow -type motoneurons \nthat innervate fatigue-resistant units thought to be essential for postural tone. Human studies similarly report \nlarger PIC estimates (ΔF ) in higher threshold motor units and in muscles enriched with fast motor units, \nsuch as tibialis anterior, compared with muscles more involved in postural control such as soleus (Orssatto \net al., 2021; Jenz et al., 2023; Škarabot et al., 2025). Although differences in firing rates between fast and \nslow muscle types could influence ΔF estimates in humans, our results suggest that the functional role of \nPICs extends beyond slow motor unit stabilization and may contribute to dynamic amplification or rapid \nrecruitment strategies in fast units. Thus, while PICs increase during the period when weight -bearing \nemerges, their distribution across motoneuron subtypes challenges classical assumptions and calls for a \nreassessment of their role in postural versus phasic motor control. \n \nPIC amplitude does not dictate firing hysteresis \nAlthough PIC amplitude and sustained firing hysteresis increased in parallel during development, our \npharmacological experiments demonstrate that PIC amplitude alone does not determine sustained hysteretic \nfiring. Blocking NaV1.6 or L-type calcium channels significantly reduced PIC amplitude but did not reduce \nimpact firing hysteresis (Sharples & Miles, 2021; Drouillas et al. , 2023) . Similarly, co -blockade that \nreduced PIC amplitude by nearly 60% failed to change firing hysteresis in a predictable way. Conversely, \nmuscarine nearly doubled PIC amplitude yet promoted adaptive firing hysteresis by selectively increasing \noffset current. Together these results indicate that hysteresis is not linearly —or even directly—related to \nPIC magnitude. This finding contrasts with the canonical model in which suggests that PIC activation drives \nsustained counterclockwise hysteresis in motoneuron firing (Hounsgaard et al., 1984; Hounsgaard & Kiehn, \n1989; Hultborn et al., 2003; Heckman et al., 2003). This model is supported by classic studies in cats and \nturtles showing bistable firing and plateau potentials dependent on dendritic L-type calcium channels under \nstrong neuromodulatory drive (Hounsgaard & Kiehn, 1989; Carlin et al., 2000; Hultborn et al., 2003; Bui \net al., 2006; Elbasiouny et al., 2006; Heckman et al., 2008a). Our data suggest that in neonatal mouse \nmotoneurons, particularly in slice preparations with reduced neuromodulatory tone, PIC amplitude alone is \ninsufficient to explain recruitment–derecruitment nonlinearities. Instead, other ionic mechanisms appear to \nprovide critical shaping of firing hysteresis (Manuel et al., 2007; Bos et al., 2018), which could account for \nexamples where motoneurons produce positive delta I values and adaptive firing hysteresis despite the \npresence of robust PICs (Revill et al., 2019). One interpretation of these findings is that PICs provide the \ninward drive necessary for sustained firing but do not themselves determine the asymmetry between \nrecruitment and derecruitment that defines hysteresis. Alternatively, computational studies in spired by \nexperimental work in the stomatogastric ganglion have elegantly demonstrated that degenerate circuit \nmechanisms can produce similar network outputs, such that distinct combinations of intrinsic and synaptic \nconductances generate comparable firing patterns (Prinz et al., 2004; Mellen, 2008). These findings raise \nthe possibility that similar degeneracy may exist in spinal motoneurons, where multiple ionic mechanisms \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n22 \n \ncould give rise to firing behaviors—such as hysteresis or sustained discharge—that are often interpreted as \nsignatures of persistent inward currents. \n \nPotassium channels shape recruitment–derecruitment asymmetry \nIf PICs alone do not determine firing hysteresis, then other conductances that influence membrane potential \nnear spike threshold may play a key role in shaping these firing behaviors. Potassium channels are strong \ncandidates in this regard because they regulate excitability in the subthreshold voltage range (Deardorff et \nal., 2021) and are well positioned to differentially influence onset and offset currents during recruitment \nand derecruitment. \n \nIn the present study, we identified KCNQ and Kv1.2 channels as modulators of hysteretic firing. KCNQ \nchannels, which conduct the M -type potassium current, are ideally positioned to counteract PICs due to \ntheir subthreshold activation and non-inactivating outward current (Brown & Adams, 1980; Verneuil et al., \n2020; Singh et al., 2025; Gaudreau & Bui, 2026). We found that blocking KCNQ channels increased PIC \namplitude but paradoxically reduced hysteresis, whereas activation of KCNQ channels enhanced hysteresis \nand shifted firing toward sustained counterclockwise patterns. These results highlight that outward currents, \nfar from simply opposing inward currents, can create the conditions for recruitment –derecruitment \nhysteresis by differentially influencing onset versus offset currents for firing. Kv1.2 channels provide an \nadditional complementary mechanism. The ir rapid activation and slow inactivation underlie the delayed \nfiring phenotype of fast motoneurons (Bos et al., 2018) and we hypothesized that their slow inactivation \nwould support sustained firing on the descending limb of the ramp. While blocking Kv1.2 channels reduced \ndelta I and shifted firing from sustained to more adaptive patterns as expected, it did so by decreasing onset \ncurrent without impacting the offset current. Nevertheless, these results point to potassium currents at the \naxon initial segment as critical determinants of hysteretic firing. These findings align with growing evidence \nthat motoneuron input–output nonlinearities are strongly influenced by potassium channel kinetics (Manuel \net al., 2014; Leroy et al., 2015; Deutsch & Elbasiouny, 2024; Molkov et al., 2025). Importantly, these \nresults shift the explanatory framework: rather than attributing firing hysteresis solely to dendritic PICs, \nour findings suggest that potassium channels —particularly those localized to the axon initial segment —\nshape recruitment–derecruitment asymmetry and therefore strongly influence the expression of sustained \nfiring behaviors. \n \nHCN channels limit bistable firing \nBy the third postnatal week, fast motoneurons develop a resting H -current that delays their recruitment \n(Sharples & Miles, 2021). Strikingly, blocking HCN channels in the present study produced bistable firing \nin nearly half of fast motoneurons, an observation that is consistent with previous reports of increased \nbistability in resonant motoneurons - a key property medicated by HCN channels (Manuel et al., 2007). In \na subset of cells, HCN channel blockade also hyperpolarized PIC onset voltage while producing minimal \nchange in PIC amplitude, suggesting that HCN channels may act as a shunt conductance near resting \nmembrane potential that delays PIC activation. It is therefore possible that reactivation of HCN channels \nduring the descending limb of triangular current ramps prevents bistable firing and maintains the temporal \nfidelity of synaptic inputs to fast motoneurons. Together these observations suggest that PICs, potassium \nchannels, and HCN channels act in concert to shape nonlinear motoneuron firing, with their relative \ninfluence depending on developmental stage, neuromodulatory state, and motoneuron subtype. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n23 \n \nNeuromodulation as a dynamic regulator of PIC and firing hysteresis \nNeuromodulators play a critical role in shaping motoneuron excitability, influencing both normal motor \nfunction and the emergence of aberrant firing in disease states such as spasticity, amyotrophic lateral \nsclerosis, and cerebral palsy. Traditionally, the ir effects have been attributed to modulation of PICs. \nHowever, the actions of neuromodulators extend beyond PICs, affecting multiple ionic conductances that \ntogether are likely to define recruitment–derecruitment dynamics and firing hysteresis. Our muscarinic \nexperiments illustrate this complexity. Muscarine robustly increased PIC amplitude yet paradoxically \npromoted adaptive firing hysteresis, an effect that can be explained, at least in part, by inhibition of KCNQ \nchannels (Revill et al., 2019; Sharples et al., 2023). Consistent with this idea, pharmacological blockade of \nKCNQ channels similarly increased PIC amplitude and decreased delta I. Importantly, the underlying \nmechanisms differed: KCNQ channel blockade reduced delta I by selectively decreasing onset current \nwithout affecting offset current, whereas muscarine reduced delta I by increasing offset current without \naltering onset. This divergence highlights that neuromodulators rarely act on a single channel type; instead, \nthey simultaneously modify multiple conduc tances, producing net effects that may diverge from simple \npredictions based on PIC amplitude alone (Marder, 2011; Perrier et al., 2013; Marder et al., 2014; Sharples \net al., 2014; Nascimento et al., 2020). Additional targets of neuromodulatory regulation likely include the \nsodium–potassium ATPase, which exerts activity-dependent inhibition of motoneuron excitability (Picton \net al., 2017; Hachoumi et al., 2022; Akkuratov et al., 2025; Sharples et al., 2025), which can be modulated \nby acetylcholine in the brain (Tiwari et al. , 2018; Mohan et al. , 2019, 2021) , and is a key target of \nneuromodulators in spinal networks (Picton et al., 2017; Hachoumi et al., 2022). It is therefore a reasonable \nhypothesis that increas ing sodium pump activity would promote adaptive firing hysteresis. In vivo, \nserotonergic, cholinergic, and noradrenergic systems converge to regulate both inward and outward \ncurrents, enabling flexible control of motoneuron gain and persistent firing in a behaviorally relevant \ncontext (Heckman et al., 2008a; Goodlich et al., 2023, 2024). Our results are particularly important in the \ncontext of injury and disease where PICs and their neuromodulatory control have been implicated in the \ngeneration of spasticity and motor dysfunction (Murray et al., 2010; ElBasiouny et al., 2010; Quinlan et \nal., 2011; D’Amico et al., 2013; Brocard et al., 2016; Steele et al., 2020; Marcantoni et al., 2020; Jiang et \nal., 2021; Reedich et al., 2023; Delestrée et al., 2023; Deutsch & Elbasiouny, 2024; Pagiazitis et al., 2025). \nThese observations therefore set the stage for our findings and future studies, which identify novel ionic \nmechanisms—including potassium and HCN channels —that contribute to neuromodulatory control and \nmay underlie dysfunction in injury and disease. \n \nMethodological considerations \nSeveral caveats should be considered when interpreting our results. Slice preparations limit dendritic \nintegrity and neuromodulatory input, potentially underestimating the contribution of dendritic PICs (Mousa \n& Elbasiouny, 2021) . Somatic current injection may also insufficiently activate distal channels that are \nrobustly engaged by synaptic inputs in vivo. Recordings were also conducted at sub physiological \ntemperature, which can not only alter channel kinetics (Bouhadfane et al., 2013), an important consideration \ngiven that temperature-sensitive TRPM5 channels are critical for generating bistability (Bos et al., 2021). \nStarting membrane potential also influences the likelihood of self -sustained discharge (Bouhadfane et al., \n2013; Mahrous et al., 2024; Molkov et al., 2025). Nevertheless, we still observed bistable firing at these \ntemperatures following HCN channel blockade from a starting membrane potential of -60 mV. Together, \nthese factors indicate that the relative contributions of PICs and outward conductances may vary under \nmore physiological conditions but do not negate our conclusions. Finally, developmental comparisons were \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n24 \n \nmade in transverse slices; complementary in vivo recordings could confirm whether the changes we \nobserved translate into functional differences during natural motor behavior (de Lourdes Martínez-Silva et \nal., 2025).  \n \nFunctional implications \nOur findings have important implications for understanding motor unit behavior in humans, where ΔF \nderived from paired motor unit recordings is widely interpreted as an estimate of PIC amplitude or \nneuromodulatory state (Powers et al., 2008; Udina et al., 2010; Revill & Fuglevand, 2011; Vandenberk & \nKalmar, 2014; Powers & Heckman, 2015) . Similarly, several other electrophysiological signatures \ncommonly interpreted as indicators of PIC activation may also arise from the combined influence of \nmultiple conductances. For example, analyses of motor unit firing -rate trajectories, including the  “brace-\nheight” metric used to quantify nonlinear acceleration of discharge during voluntary contractions, have \nbeen interpreted as evidence of PIC recruitment in human motoneurons (Beauchamp et al., 2023; Škarabot \net al., 2025). Further, quantification of secondary and tertiary firing ranges in motoneuron and motor unit \nfiring has also been used to infer the engagement of intrinsic depolarizing conductances that accelerate \ndischarge once firing is established (Lee & Heckman, 1998; Bennett et al., 1998; Meehan et al., 2010a; \nBinder et al., 2020; Afsharipour et al., 2020). Finally, intracellular recordings in animal models frequently \ndescribe a subthreshold acceleration of membrane potential preceding spike threshold, which has likewise \nbeen attributed to the activation of inward currents (Kuo et al., 2006; Delestrée et al., 2014; Jensen et al., \n2020; Sharples & Miles, 2021). However, the specific ionic mechanisms underlying these features have not \nbeen directly tested, and it remains unclear to what extent PICs versus other voltage -dependent \nconductances contribute to these dynamics. Together, these observations suggest that  many physiological \nproxies used to estimate PIC influence on motoneuron output —including ΔF, nonlinear firing -rate \nacceleration, and secondary-range discharge behavior—may reflect the emergent behavior of interacting \nchannel populations rather than the action of a single dominant inward current. Our results suggest that ΔF, \nand other non -linear firing properties, may reflect the integrated contribution of multiple intrinsic and \nneuromodulatory mechanisms —including potassium and HCN channel dynamics —that sh ape \nrecruitment–derecruitment asymmetry.  \n \nConclusion \nIn summary, this study demonstrates that while PIC amplitude and recruitment –derecruitment hysteresis \nincrease together during postnatal development, PIC magnitude alone does not determine sustained \nhysteretic firing. Instead, PICs may provide the inward d rive required for sustained firing, but potassium \nand HCN channels determine whether hysteretic firing actually emerges. These findings expand the \ntraditional PIC-centric view of motoneuron bistability and suggest that sustained firing behaviors arise from \nthe interaction of multiple inward and outward conductances. This broader framework also has important \nimplications for the interpretation of ΔF measurements in human motor unit studies, where hysteresis is \noften treated as a proxy for PIC amplitude or ne uromodulatory state. From our work, we suggest that ΔF \nmay reflect the integrated contribution of several intrinsic mechanisms that shape motoneuron input–output \nnonlinearities. \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n25 \n \nAdditional Information \nFunding: This work was supported by fellowships from The Royal Society (Newton International \nFellowship - NIF\\R1\\180091), Canadian Institute for Health Research (PDF - 202012MFE - 459188 - \n297534), and Wellcome Trust (ISSF - 204821/Z/16/Z) to SAS. \n \nContributions: Study Conception and Design: SAS, GBM; Data acquisition and analysis: SAS; \nPreparation of Figures: SAS; First draft and revision of manuscript: SAS, GBM; All authors approved the \nfinal version of the manuscript. \n \nAcknowledgements:  The authors reserve the right to apply a Creative Commons Attribution (CC BY) \nlicence to any Author Accepted manuscript version arising from this submission.  \n \nCompeting Interests: None of the authors have any conflicts of interests to declare. \n \nData Availability Statement: The research data supporting this publication will be made freely available \nin an open access data repository following acceptance to a peer-reviewed journal. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n26 \n \nMethods \nAnimals \nThis study included unpublished data in addition to reanalysis of experimental data summarised in (Sharples \n& Miles, 2021) , which included experiments performed on tissue obtained from 117 (male: n = 55; and \nfemale: n = 62) wild type C57Bl/6J mice at postnatal days (P) 7 -20. We also present unpublished data \nobtained from 21 C57Bl/6J mice (P7-13; n = 9 male, n = 12 female) that were included in (Sharples et al., \n2023). This study also included new experimental data obtained from 29 (male: n = 17; and female: n = 12) \nwild type C57Bl/6J mice at postnatal days P7 -13. All procedures were conducted in accordance with the \nUK Animals (Scientific Procedures) Act 1986. Experiments conducted at the University of St Andrews \nwere approved by the University of St Andrews Animal Welfare Ethics Committee and were covered under \na Project Licence (PP8253850) approved by the Home Office. All animals were provided with unrestricted \naccess to food and water and housed in climate-controlled conditions. \n \nTissue preparation \nAnimals were sourced from an in -house colony within the St Mary’s Animal Unit at the University of St \nAndrews. Animals were killed using Schedule 1 procedures defined by the Home Office by performing a \ncervical dislocation followed by rapid decapitation. Animals were then eviscerated and pinned ventral side \nup in a dissecting chamber lined with silicone elastomer (Sylguard), filled with ice -cold (1 -2 degrees \nCelsius) potassium gluconate based dissecting/slicing aCSF (containing in mM: 130 K-gluconate, 15 KCl, \n0.05 EGTA, 20 HEPES, 25 D-glucose, 3 kynurenic acid, 2 Na-pyruvate, 3 myo-inositol, 1 Na-L-ascorbate; \npH 7.4, adjusted with NaOH; osmolarity approximately 345 mOsm) that was continuously bubbled with \ncarbogen (95% oxygen, 5% carbon dioxide). Spinal cord s were exposed by performing a ventral \nvertebrectomy, cutting the dorsal roots and gently lifting the spinal cord from the spinal column. Spinal \ncords were removed within 3 - 5 minutes following cervical dislocation. Spinal cords were secured directly \nto an agar block (3 % agar) with VetBond surgical glue (3M) and glued to the base of the slicing chamber \nwith cyanoacrylate adhesive. The tissue was immersed in ice -cold dissecting/slicing aCSF and bubbled \nwith carbogen. Blocks of frozen slicing solution were also placed in the slicing chamber to keep the solution \naround 1-2 degrees Celsius. On average, the first slice was obtained within 10 minutes of decapitation \nwhich increased the likelihood of obtaining viable motoneurons in slices. 300 µm transverse slice s were \ncut at a speed of 10 um/s on the vibratome (Leica VT1200) to minimize tissue compression during slicing. \n3-4 slices were obtained from each animal. Slices were transferred to a recovery chamber filled with \ncarbogenated pre-warmed (35 degrees Celsius) recovery aCSF (containing in mM: 119 NaCl, 1.9 KCl, 1.2 \nNaH2PO4, 10 MgSO4, 1 CaCl, 26 NaHCO3, 20 glucose, 1.5 kynurenic acid, 3% dextran) for thirty minutes \nafter completion of the last slice which took 10 -15 minutes on average. Following recovery, slic es were \ntransferred to a chamber filled with warm (35 degrees Celsius) recording aCSF (containing in mM: 127 \nNaCl, 3 KCl, 1.25 NaH2PO4, 1 MgCl, 2 CaCl2, 26 NaHCO3, 10 glucose), bubbled with carbogen, and \nallowed to equilibrate at room temperature (maintain ed at 23 -25 degrees Celsius) for at least one hour \nbefore experiments were initiated. \n \nWhole cell patch clamp electrophysiology \nThis study includes data from whole -cell patch clamp recordings obtained from a total of 291 lumbar \nmotoneurons. 219 of these cells were reanalyzed using data published in (Sharples & Miles, 2021). We also \npresent novel data from motoneurons that were studied in (Sharples et al. , 2023)  and included new \nexperiments on 41 additional motoneurons. In these experiments, spinal cord slices were stabilized in a \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n27 \n \nrecording chamber with fine fibres secured to a platinum harp and visualized with a 40x objective with \ninfrared illumination and differential interference contrast (DIC) microscopy. A large proportion of the \nmotoneurons studied were identified based on location in the ventrolateral region with somata greater than \n20 µm. Recordings were obtained from a subset of motoneurons that had been retrogradely labelled with \nFluorogold (Fluorochrome, Denver, CO). Fluorogold was dissolved in sterile saline solution and 0.04 mg/g \ninjected intraperitoneally 24-48 hours prior to experiments(Miles et al., 2005). In addition to recording from \nlarger FG -positive cells, this approach allowed us to more confidently target smaller motoneurons. \nMotoneurons were visualized and whole cell recordings obtained under DIC illumination with pipettes (L: \n100 mm, OD: 1.5 mm, ID: 0.84 mm; World Precision Instruments) pulled on a Flaming Brown micropipette \npuller (Sutter instruments P97) to a resistance of 2.5 -3.5 MΩ. Pipettes were back -filled with intracellular \nsolution (containing in mM: 140 KMeSO4, 10 NaCl, 1 CaCl2, 10 HEPES, 1 EGTA, 3 Mg -ATP and 0.4 \nGTP-Na2; pH 7.2-7.3, adjusted with KOH). \n \nSignals were amplified and filtered (6 kHz low pass Bessel filter) with a Multiclamp 700 B amplifier, \nacquired at 20 kHz using a Digidata 1440A digitizer with pClamp Version 10.7 software (Molecular \nDevices) and stored on a computer for offline analysis. \n \nIdentification of fast and slow motoneuron types \nMotoneuron subtypes were identified using a protocol established by (Leroy et al. , 2014) , which \ndifferentiates motoneuron type based on the latency to the first spike when injecting a 5 second square \ndepolarizing current near the threshold for repetitive firing. Using this approach we were able to identify 2 \nmain firing profiles - a delayed repetitive firing profile with accelerating spike frequency, characteristic of \nfast-type motoneurons, and an immediate firing profile with little change in spike frequency, characteristic \nof slow-type motoneurons (Figure 1).  \n \nAll motoneuron intrinsic properties were studied by applying a bias current to maintain the membrane \npotential at -60 mV. Values reported are not liquid junction potential corrected to facilitate comparisons \nwith previously published data (Miles et al., 2007; Quinlan et al., 2011; Durand et al., 2015; Nascimento \net al., 2020, 2024; Smith & Brownstone, 2020; Özyurt et al., 2022; Pocratsky et al., 2023). Cells were \nexcluded from analysis if access resistance was greater than 20 MΩ or changed by more than 5 MΩ over \nthe duration of the recording, or if spike amplitude measured from threshold (described below) was less \nthan 60 mV. \n \nPharmacology \nNifedipine (20 µM; Tocris) was used to assess the contribution of L -type calcium channels, and 4,9 - \nAnhydro Tetrodotoxin (200 nM; Tocris) to assess the contribution of NaV1.6 channels to PIC and \nhysteresis. KCNQ channels that underlie the M current were a ctivated with ICA069673 (ICA73: 10 uM; \nTocris) or retigabine (10 uM; Tocris) or blocked with XE991 (10 uM; Tocris). Kv1.2 channels were blocked \nwith Tityustoxin (800 nM; Alomone). HCN Channels that underlie the H -current were blocked with \nZD7288 (10 uM; Tocris). \n \nData acquisition and analysis \nPassive properties including capacitance, membrane time constant (tau), and input resistance (Ri) were \nmeasured during a hyperpolarizing current pulse that brought the membrane potential from -60 to -70mV. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n28 \n \nInput resistance was measured from the initial voltage trough to minimize the impact of slower acting active \nconductances (eg. Ih, sag). The time constant was measured as the time it took to reach 2/3 of the peak \nvoltage change. Capacitance was calculated by dividing the time constant by the input resistance (C=T/R).  \n \nPICs were measured in voltage clamp during slow depolarizing voltage ramps (10 mV/s from -90 to -10 \nmV) over 8 seconds (Quinlan et al., 2011; Steele et al., 2020; Huh et al., 2021). PIC onset voltage and peak \ncurrent amplitude measured from post -hoc leak-subtracted traces as in previous studies (Quinlan et al., \n2011; Steele et al., 2020; Verneuil et al., 2020). \n \nRecruitment-derecruitment and firing hysteresis were measured in current clamp using triangular, \ndepolarizing current ramps with 5 second rise and fall times (Bennett et al., 2001; Li & Bennett, 2003; \nDurand et al., 2015; Steele et al., 2020). Triangular depolarizing current ramps were set to a peak current \nof 2 times repetitive firing threshold current (determined with a 100pA/s depolarizing current ramp initiated \nfrom -60 mV). Recruitment-derecruitment hysteresis was measured by calculating the difference (delta I) \nbetween the current at firing onset on the ascending component of the ramp and the current at derecruitment \non the descending component of the ramp. Firing hysteresis was also assessed by examining the frequency-\ncurrent trajectories on the ascending and descending components of the ramp. We subdivided cells into 1 \nof 4 types based on previously-defined criteria identifying the pattern of firing hysteresis on ascending and \ndescending portions of the ramp (Bennett et al. , 2001)  (Type 1: Linear, Type 2: Adapting clockwise \nhysteresis, Type 3: Linear Sustained, Type 4: Accelerating sustained Counter -clockwise hysteresis). In \naddition, we also identified a fifth firing type following blockade of HCN channels, characterized by \nsustained firing that continued beyond the end of the descending portion of the triangular current ramp. In \nthis fifth firing type, hyperpolarizing current was required to terminate sustained firing.  \n \nResearch design and statistical analysis \nTwo factor analysis of variance (ANOVA) was performed to study changes in PIC and recruitment -\nderecruitment hysteresis (delta I) in motoneuron subtypes across developmental time points or to determine \neffects of pharmacological agents at different developm ental stages. Paired or unpaired t -tests were \nperformed when comparing two conditions. Firing hysteresis (Types 1 -4) were analyzed with either a \nKruskal-Wallis test when comparing more than two conditions or Wilcoxon test when comparing two \nconditions. App ropriate and equivalent nonparametric tests (Mann -Whitney or Kruskal -Wallis) were \nconducted when data failed tests of normality or equal variance with Shapiro Wilk and Brown -Forsythe \ntests, respectively. Statistical tests were complemented with Hedge’s g to indicate effect sizes, categorized \nas small (0.2-0.49), medium (0.5-0.79) and large (>0.8). Individual data points for all cells are presented in \nfigures with mean ± SD. Statistical analyses were performed using Graph Pad Version 9.0 (Prism, San \nDiego, CA, USA). All statistical tests and results are summarized in Table 1 and are annotated in text where \nappropriate.  \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 15, 2026. ; https://doi.org/10.64898/2026.03.12.711366doi: bioRxiv preprint \n\n29 \n \nReferences \nAfsharipour B, Manzur N, Duchcherer J, Fenrich KF, Thompson CK, Negro F, Quinlan KA, Bennett DJ \n& Gorassini MA (2020). 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