Ronald M Harris-Warrick

Emilie Pecchi

Benoît Drouillas

Frédéric Brocard

Rémi Bos

Effect of size on expression of bistability in mouse spinal motoneurons

Keywords: Motoneuron, Spinal cord, Bistability, Current, Development, Posture, Serotonin, Neuromodula on

Bistability in spinal motoneurons supports tonic spike ac vity in the absence of excitatory drive. Earlier work in adult prepara ons suggested that smaller motoneurons innerva ng slow an gravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innerva ng fast-fa gable muscle fibers display bistability is s ll controversial. To address this, we examined the rela onship between soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of ChAT-GFP and Hb9-GFP mice during the first four weeks of life. We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast αmotoneurons (MMP-9 + /Hb9 + ) with a soma area ≥ 400µm 2 exhibit significantly higher bistability.

Ionic currents associated with bistability, including the persistent Nav1.6 current, thermosensi ve Trpm5 Ca 2+ -ac vated Na + current and the slowly inac va ng Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with par al bistable proper es, but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.

INTRODUCTION

Spinal motoneurons not only transmit central commands for movement to muscles but also shape motor output and muscle contrac on through nonlinear firing proper es [START_REF] Binder | Nonlinear Input-Output Func ons of Motoneurons[END_REF]. One such property is bistability, where the motoneuron can switch between stable silent and ac ve states, depending on transient synap c input or current injec on. Originally detected as plateau poten als in invertebrate neurons [START_REF] Russell | Burs ng neural networks: a reexamina on[END_REF][START_REF] Russell | Slow ac ve poten als and burs ng motor pa erns in pyloric network of the lobster, Panulirus interruptus[END_REF][START_REF] Hartline | Cellular and synap c proper es in the crustacean stomatogastric nervous system[END_REF][START_REF] Arbas | Ionic conductances underlying the ac vity of interneurons that control heartbeat in medicinal leech[END_REF][START_REF] Katz | Motor pa ern modula on by serotonergic sensory cells in the stomatogastric nervous system[END_REF][START_REF] Chrachri | Fic ve locomo on in the fourth thoracic ganglion of the crayfish Procambarus clarkii[END_REF][START_REF] Straub | Endogenous and network proper es of Lymnaea feeding central pa ern generator interneurons[END_REF][START_REF] Mercer | Plateau poten als in developing antennal-lobe neurons of the moth, Manduca sexta[END_REF], bistability was soon found in vertebrates [START_REF] Llinas | Electrophysiological proper es of in vitro Purkinje cell somata in mammalian cerebellar slices[END_REF], specifically in spinal motoneurons [START_REF] Hultborn | Prolonged ac va on of soleus motoneurones following a condi oning train in soleus Ia afferents -A case for a reverbera ng loop?[END_REF][START_REF] Schwindt | Proper es of a persistent inward current in normal and TEAinjected motoneurons[END_REF][START_REF] Hounsgaard | Ca ++ dependent bistability induced by serotonin in spinal motoneurons[END_REF][START_REF] Hounsgaard | Serotonin-induced bistability of turtle motoneurons caused by a nifedipine-sensi ve calcium plateau poten al[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF]. While o en induced by neuromodulators such as serotonin [START_REF] Hounsgaard | Ca ++ dependent bistability induced by serotonin in spinal motoneurons[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Kiehn | 5-HT modula on of hyperpolariza on-ac vated inward current and calcium-dependent outward current in a crustacean motor neuron[END_REF], bistability can also arise when motoneurons are depolarized to near threshold and recorded under natural ionic and temperature condi ons [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF][START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF].

The ac ve state in bistable motoneurons is mainly mediated by slow ionic currents including persistent Cav1.3 [START_REF] Hounsgaard | Ca ++ dependent bistability induced by serotonin in spinal motoneurons[END_REF][START_REF] Hounsgaard | Serotonin-induced bistability of turtle motoneurons caused by a nifedipine-sensi ve calcium plateau poten al[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF] and Nav1.6 currents [START_REF] Lee | Essen al role of a fast persistent inward current in ac on poten al ini a on and control of rhythmic firing[END_REF][START_REF] Harvey | 5-HT2 receptor ac va on facilitates a persistent sodium current and repe ve firing in spinal motoneurons of rats with and without chronic spinal cord injury[END_REF], and the Trpm5-mediated Ca 2ac vated Na + current [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF][START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF][START_REF] Zhang | Calcium-dependent plateau poten als in a crab stomatogastric ganglion motoneuron. II. Calcium-ac vated slow inward current[END_REF]. Drawing from a series of our previous inves ga ons, we can summarize the process as follows: The ini al depolariza on, caused by the slow inac va on of Kv1.2 potassium channels [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF], ac vates the persistent Nav1.6 current leading to spiking ac vity [START_REF] Drouillas | Persistent Nav1.6 current drives spinal locomotor func ons through nonlinear dynamics[END_REF]. This then prompts Ca 2+ entry through the recruitment of Cav1.3 channels, ini a ng a Ca 2+induced Ca 2+ -release process [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF]. This process ul mately ac vates thermosensi ve Trpm5 channels, which are the primary source of the plateau depolariza on to sustain repe ve firing [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. Other currents such as the HCN-type hyperpolariza on-ac vated inward current, I h [START_REF] Kiehn | Serotonergic stretch receptors induce plateau proper es in a crustacean motor neuron by a dual-conductance mechanism[END_REF][START_REF] Thuault | Prefrontal cortex HCN1 channels enable intrinsic persistent neural firing and execu ve memory func on[END_REF] and reduc on of calcium-ac vated outward currents [START_REF] Kiehn | 5-HT modula on of hyperpolariza on-ac vated inward current and calcium-dependent outward current in a crustacean motor neuron[END_REF][START_REF] Kiehn | Serotonergic stretch receptors induce plateau proper es in a crustacean motor neuron by a dual-conductance mechanism[END_REF] α-motoneurons, which drive extrafusal muscle contrac ons, can be subdivided func onally into three classes based on size and the muscle type they innervate: large fastfa gable (FF), medium fast fa gue-resistant (FR), and small slow (S) motoneurons [reviewed by [START_REF] Manuel | Molecular and electrophysiological proper es of mouse motoneuron and motor unit subtypes[END_REF]]. In decerebrate cats, small motoneurons characterized by slow conduc on veloci es and low ac va on thresholds, appear to have more pronounced bistability than large ones [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF]. This observa on suggests that S or FR α-motoneurons show higher bistability than those linked to FF muscle fibers. Notably, S motoneurons, primarily associated with postural adjustments and slow movements, have been hypothesized to use their bistability for efficient postural maintenance, ensuring minimal energy expenditure [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Kiehn | Plateau poten als and ac ve integra on in the 'final common pathway' for motor behaviour[END_REF]. In mice, the sta onary posture also involves the ac va on of fast motor units [START_REF] Henneman | Func onal organiza on of motoneuron pool and its inputs[END_REF]. Interes ngly, our recent findings indicate that large motoneurons exhibit bistable firing pa erns, which are crucial for the control of hindlimb movement [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. From as early as the 2 nd postnatal week, large motoneurons in mice present an electrophysiological profile that is dis nct from that of smaller ones, marked by a lower input resistance, a higher rheobase, more depolarized spike thresholds, narrower ac on poten als, and shorter a erhyperpolariza ons [START_REF] Manuel | Molecular and electrophysiological proper es of mouse motoneuron and motor unit subtypes[END_REF][START_REF] Sharples | Matura on of persistent and hyperpolariza on-ac vated inward currents shapes the differen al ac va on of motoneuron subtypes during postnatal development[END_REF][START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF]. They also exhibit delayed onset of firing and firing accelera on during a step depolariza on, and receive a consistently higher level of recurrent excita on [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF][START_REF] Leroy | Is hyperexcitability really guilty in amyotrophic lateral sclerosis?[END_REF][START_REF] Durand | Developing electrical proper es of postnatal mouse lumbar motoneurons[END_REF][START_REF] Bhumbra | Recurrent excita on between motoneurones propagates across segments and is purely glutamatergic[END_REF]. Despite this extensive characteriza on, the comparison of bistability between large and small motoneurons in mice remains unexplored. While comparisons with cat studies might lead us to hypothesize increased bistability in smaller mouse motoneurons, cau on is warranted due to the dis nct characteris cs of mouse spinal motoneurons that are not seen in cats [START_REF] Huh | PICs in motoneurons do not scale with the size of the animal: a possible mechanism for faster speed of muscle contrac on in smaller species[END_REF]. In the present study, we examined the bistability as a func on of size in gene cally labeled L4-L5 α-motoneurons in young mice (postnatal day (P) . Our data indicate that the largest α-motoneurons showed stronger bistable firing proper es, while the smallest motoneurons were rarely bistable. There was a strong correla on between bistability and the amplitudes of the ionic currents known to support it. Motoneurons of intermediate size o en showed par al bistability, which could be converted to full bistability by serotonin. These unexpected results suggest new hypotheses regarding the contribu on of larger motoneurons in bistability and postural control.

MATERIALS AND METHODS

Animals and ethical standards

Hb9-GFP mice were kindly provided by B. Pe mann and Jackson Laboratories (strain 005029).

ChAT-Cre mice were obtained from Jackson Laboratories (strain 007902) and crossed to Rosa-26-floxed GFP mice. Mice of both sexes were used in all experiments. Animals were housed on a 12 hr day/night cycle with ad libitum access to water and food. The room temperature was maintained between 20 and 22°C. 25 D-glucose, 3 kynurenic acid, and pH 7.4 with NaOH [START_REF] Bhumbra | Recurrent excita on between motoneurones propagates across segments and is purely glutamatergic[END_REF]. A er a res ng period of 30-60 min, slices were transferred to the recording chamber and superfused with recording aCSF at 32°C [START_REF] Husch | Long dura on perforated patch recordings from spinal interneurons of adult mice[END_REF] without addi on of fast synap c transmission blockers.

Electrophysiological recordings

Hb9-GFP and ChAT-GFP posi ve neurons were visualized in the ventrolateral region of lamina IX in L4-L5 horizontal slices using an Olympus BX51 microscope with appropriate filters. Whole-cell patch-clamp recordings from visualized fluorescent GFP+ neurons were made with electrodes (2-6MΩ) pulled from borosilicate glass capillaries (1.5 mm OD, 1.12 mm ID; World Precision Instruments). They were filled with a pipe e solu on containing (in mM): 140 K + -gluconate, 5 NaCl, 2 MgCl 2 , 10 HEPES, 0.5 EGTA, 2 ATP, 0.4 GTP, pH 7.3. Patch clamp recordings were made using a Mul clamp 700B amplifier driven by PClamp 10 so ware (Molecular Devices).

Recordings were digi zed on-line and filtered at 10 kHz (Digidata 1322A or 1440A, Molecular Devices). Pipe e and neuronal capaci ve currents were canceled, and, a er breakthrough, series access resistance was compensated. The recording was allowed to stabilize for at least 2 min a er establishing whole-cell access before recording was started [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF][START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF]. Unless otherwise indicated, all neurons were held at -70 mV to ensure reproducibility in expression of voltagesensi ve currents.

Drug list: All solu ons were oxygenated with 95% O 2 /5% CO 2 . All salt compounds, as well as tetraethylammonium chloride (TEA; #T2265), Triphenylphosphine oxide (TPPO; #T84603), Serotonin crea nine sulfate monohydrate (5-HT; #H7752), 6-Cyano-7-nitroquinoxaline-2,3dione (CNQX, #5.04914), D-(-)-2-Amino-5-phosphonopentanoic Acid (DL-AP5 ; #165304), strychnine (#S0532), bicuculline (#5.05875) , Kynurenic Acid (#K3375) and Amphotericin B (#A4888) were obtained from Sigma-Aldrich. Tetrodotoxin (TTX; #1078) was obtained from Tocris Bioscience.

Immunohistochemistry

To iden fy neurons expressing specific proteins, spinal cords from 10-12-day-old Hb9-GFP mice were removed in ice cold (1-2°) aCSF containing (in mM): 252 sucrose, 3 KCl, 1.25 KH 2 PO 4 , 4 MgSO 4 , 0.2 CaCl 2 , 26 NaHCO 3 , 25 D-glucose, pH 7.4, bubbled with 95% O 2 and 5% CO 2 . The spinal cords were fixed for 5-6 h in 4% paraformaldehyde (PFA) prepared in phosphate buffer saline (PBS), then rinsed in PBS and cryoprotected overnight in 20% sucrose-PBS at 4°C. Spinal cords were frozen in OCT medium (Tissue Tek), and 30 μm cryosec ons were collected from the L4-L5 segments [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF]. A er washing in PBS 3×5 min, the slides were incubated for 1 h in a blocking solu on (BSA 1%, Normal Donkey Serum 3% in PBS) with 0.2% Triton X-100 and for 12 h at 4 °C in a humidified chamber with the knockout-validated primary an body: mouse-an -NeuN (Neuronal Nuclei, Sigma-Aldrich MAB377) [START_REF] Jacko | Rbfox Splicing Factors Promote Neuronal Matura on and Axon Ini al Segment Assembly[END_REF] or goat-an -MMP-9 (Matrix metallopep dase 9, Sigma-Aldrich M9570) [START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF][START_REF] Kaplan | Neuronal matrix metalloproteinase-9 is a determinant of selec ve neurodegenera on[END_REF]. Both an bodies were diluted in the blocking solu on with 0.2% Triton X-100 (1:1000 and 1:500 for an -NeuN and an -MMP-9, respec vely).

Slides were washed 3×5 min in PBS and incubated for 2 h with an Alexa Fluor® Plus 555conjugated secondary an body (Invitrogen A32816) diluted in the blocking solu on. A er 3 washes of 5 min in PBS, they were mounted with a gela nous aqueous medium.

Data analysis

Electrophysiological recording: Electrophysiological data were analyzed with Clampfit 10 so ware (Molecular Devices). Several basic criteria were set to ensure op mum quality of intracellular recordings. Only cells with a stable membrane poten al below -60 mV, stable access resistance (no more than 20% varia on), and ac on poten al amplitude larger than 60 mV were analyzed. Reported membrane poten als were not corrected for liquid junc on poten als.

Confocal imaging:

Immunofluorescent staining was quan fied from confocal images acquired with the 40X objec ve (LSM700 microscope, Zeiss, Germany) on at least two slices per mouse and within the ventro-lateral area (300 X 300 µm 2 ) from each slice. The cell body cross-sec onal area was manually segmented and quan fied from stacked confocal images (5 steps ; Z-step, 3 µm) with Zen so ware (Zeiss). An intensity threshold in each slice was applied to determine the presence of NeuN or MMP9 staining from each segmented GFP+ motoneuron.

Sta s cs

No sta s cal method was used to predetermine sample size. Sta s cal analysis was carried out using GraphPad Prism and Matlab (MathWorks) so ware. When two groups were compared we used, the Mann-Whitney test. Fisher's exact test was used for comparing cell propor ons between two groups. In some cases, we also compared the slope of the simple linear regressions between two groups. The level of significance was set at p <0.05. Each sta s cal test

RESULTS

Immunohistochemical iden fica on of fast α-motoneurons

Motoneurons in the ventrolateral spinal cord were ini ally iden fied in unfixed L4-L5 horizontal slices by their GFP fluorescence in choline acetyltransferase (ChAT)-tagged ChAT-GFP mice. We found a number of labeled neurons of many sizes (from <200 µm 2 to >800 µm 2 ).

However, these include both α-motoneurons driving extrafusal muscle fibers and smaller γmotoneurons innerva ng intrafusal muscles that regulate muscle spindle responsiveness to stretch. These motoneuron subtypes can be dis nguished by differen al expression of a number of proteins. While the transcrip on factor Hb9 is a marker for both α-and γmotoneuron in Hb9-nls-LacZ animals [START_REF] Friese | Gamma and alpha motor neurons dis nguished by expression of transcrip on factor Err3[END_REF], only α-motoneurons express GFP labeling in the Hb9-GFP mice [START_REF] Friese | Gamma and alpha motor neurons dis nguished by expression of transcrip on factor Err3[END_REF]. We thus used Hb9-GFP mice to iden fy α-motoneurons in the L4-L5 ventrolateral spinal cord, preferen ally linked to hindlimb muscles [START_REF] Ozyurt | In vitro longitudinal lumbar spinal cord prepara ons to study sensory and recurrent motor microcircuits of juvenile mice[END_REF][START_REF] Hadzipasic | Selec ve degenera on of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS[END_REF], from postnatal day 1 (P1) to P25.

The transcrip on factor NeuN is commonly used to dis nguish neurons from glial cells.

In the spinal cord, α-motoneurons express NeuN, whereas 2/3 of γ-motoneurons do not [START_REF] Shneider | Gamma motor neurons express dis nct gene c markers at birth and require muscle spindle-derived GDNF for postnatal survival[END_REF].

We examined co-expression of Hb9-GFP and NeuN immunolabeling in fixed ssues and, regardless of age, we found that virtually all ventrolateral Hb9-GFP-labeled neurons exhibited NeuN labeling (98 ± 2.4%, n = 144 cells), indica ng they are likely α-motoneurons (Fig. 1A,B1).

We observed a preponderance of small labeled α-motoneurons during P4-6 (n = 571 neurons; maximal cross sec onal area of 400 µm 2 or less. The percentage of large α-motoneurons increased with age [START_REF] Friese | Gamma and alpha motor neurons dis nguished by expression of transcrip on factor Err3[END_REF][START_REF] Shneider | Gamma motor neurons express dis nct gene c markers at birth and require muscle spindle-derived GDNF for postnatal survival[END_REF], and by P12, only 50.4% of them remained under 400µm 2 (n = 288 neurons; Fig. 1B2).

Only fast motoneurons express the marker matrix metalloproteinase-9 (MMP-9) [START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF][START_REF] Kaplan | Neuronal matrix metalloproteinase-9 is a determinant of selec ve neurodegenera on[END_REF]. Our observa ons confirmed that larger motoneurons co-expressed Hb9-GFP and MMP-9, while the smaller ones did not (Fig. 1 C1-C4). The mean cross-sec onal area of MMP-9-nega ve neurons was 280 ± 12 µm 2 , while that of MMP-9-posi ve neurons was 907 ± 26 µm 2 (P<0.001, n = 288; Fig. 1C2). Consistent with previous observa ons [START_REF] Friese | Gamma and alpha motor neurons dis nguished by expression of transcrip on factor Err3[END_REF], we noted a fairly clear demarca on in motoneuron characteris cs around the 400 µm² cross-sec onal area. Specifically, most of the motoneurons smaller than 400µm 2 were HB-9 + /MMP-9 -(86.75%, n=144/166), which can be presumed to be slow motoneurons. Conversely, those with a cross-sec onal area exceeding 400µm 2 were virtually all HB-9 + /MMP-9 + (99.2%, n=121/122; Fig. 1C3,C4), sugges ng their classifica on as puta ve fast motoneurons. This pa ern led us to adopt the 400 µm² threshold as a prac cal and empirically supported cut-off, allowing us to grossly dis nguish between fast and slow motoneurons in our study.

Features of bistable motoneurons

We previously showed that under experimental condi ons mimicking natural in vivo condi ons, most large motoneurons exhibit bistability in the absence of added neuromodulators. This was achieved by maintaining in vivo calcium concentra ons (1.2 mM) [START_REF] Jones | Brain fluid calcium concentra on and response to acute hypercalcaemia during development in the rat[END_REF] and keeping the prepara on temperature above 30°C [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF][START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF][START_REF] Brocard | Ac vity-dependent changes in extracellular Ca2+ and K+ reveal pacemakers in the spinal locomotor-related network[END_REF][START_REF] Fowler | Ontogeny of thermoregulatory mechanisms in the rat[END_REF]. Four dis nct features characterized bistable motoneurons, which we have previously characterized [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF][START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF] self-sustained firing triggered by a brief (2 sec) excita on when the motoneuron was predepolarized near the spike threshold (Fig. 2A1); 2) a slow a erdepolariza on (sADP) following the current step if the motoneuron was not pre-depolarized sufficiently to trigger the selfsustained firing (Fig. 2A1); 3) nega ve hysteresis during slow triangular current ramp injec ons, where spiking stopped at lower currents than where it began (Fig. 2A2); 4) a slowly depolarizing poten al causing delayed spiking accelera on in response to a near-threshold depolarizing step (Fig. 2A3). We assigned each feature one point, with fully bistable motoneurons scoring 4 points. To be considered bistable, the motoneuron scored at least 3 points, including the presence of a self-sustained firing and sADP, and either nega ve hysteresis or delayed firing.

Nearly a third of motoneurons (36%, n=71/199) did not express any of the bistability criteria, scoring 0. They typically showed a decelera ng firing rate during the 2 sec depolarizing pulse, leading to a post-step a erhyperpolariza on (Fig. 2B1). Their ac vity during ramp current injec ons showed posi ve hysteresis, where spiking ceased at higher current values than where it started (Fig. 2B2). They also began spiking immediately during a suprathreshold long step, with con nuous firing decelera on (Fig. 2B3). Some motoneurons, despite having one scoring feature, lacked self-sustained firing and were also considered as non-bistable. A significant number of motoneurons also displayed intermediate characteris cs, scoring 2. The vast majority of them (90 %, n=22/25) were not bistable, lacking self-sustained spiking, but mee ng two other criteria. This shows that bistability is not an all-or none property, but can manifest with somewhat different proper es. As previously described, bistability in motoneurons can emerge early in development [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF]. Fig. 2C1 shows the average bistability scores of Hb9-GFP + α-motoneurons during the ini al postnatal month, comparing small neurons (<400µm 2 ) to large neurons (>400µm 2 ). Small motoneurons consistently showed minimal bistability scores across all developmental ages (Fig. 2C1), rising up to a mean of 1.14 ± 0.31 by P9-14, then nearly vanishing by early adulthood (P21-25). 54 out of 81 small cells (<400 µm 2 ) displayed a bistability score of 0, and only 9 out of 81 small cells showed bistability (scores 3-4) at any age, with 55% appearing a er P9. In contrast, large motoneurons (>400 µm 2 ) exhibited increasing bistability, reaching near maximal levels around P9 with a mean score of 3.24 ± 0.28. Remarkably, 83% (n = 24) of these large motoneurons exhibited high bistability scores (3-4) at P9-P14, with all displaying bistability by P21-25 (n = 7). Note that the bistability scores of large motoneurons were significantly higher compared to small ones when we pooled all ages (P<0.001, n=199).

Effects of size and age on bistability

These data demonstrate a correla on between motoneuron soma size and the emergence of bistability in young mice. Fig. 2C2 shows the bistability score distribu on by size across all ages. As described above, the large majority of small neurons (<400 µm 2 ) were not bistable, averaging a score of 0.67 ± 1.21 (n = 81); only 11 % achieved scores of 3-4. In contrast, larger neurons (>400 µm 2 ) consistently had high bistability scores (mean 2.54± 1.43, n = 118; P<0.001); the largest (>800µm 2 ) were the most bistable (mean 3.07 ± 1.11, n = 44) with 75% scoring 3-4. Intermediate size neurons (400-800 µm 2 ) showed largely a mixture of bistable and intermediate bistability scores (mean 2.33 ± 1.51; n = 75). A clear gradient emerged: as neuron size increased, the propor on of bistable neurons rose, while the propor on of non-bistable cells decreased, especially above 400 µm 2 . Another way to see this correla on was to measure bistability score as a func on of input resistance, which was lower in large motoneurons and higher in small motoneurons. A strong nega ve correla on between input resistance and bistability score was seen (Fig. 2C3; R 2 = 0.99). As a further control for the possible effects of cell dialysis during whole cell recordings, we used the amphotericin B perforated patch method [START_REF] Husch | Long dura on perforated patch recordings from spinal interneurons of adult mice[END_REF] to record bistability from HB9-GFP neurons, whose contents were less disturbed. This method reaffirmed the trend of increasing bistability with α-motoneuron size (n = 26; p<0.05; Fig. 2C4).

Similar results collected from ChAT-GFP mice reinforce the size-related nature of bistable proper es (Fig 2C5). Before P7, small motoneurons (<400 µm 2 ) showed no bistability, with an average score of 0.56 ± 1.13, n = 9. In contrast, larger neurons (>400µm 2 ) showed higher bistability (score 2.1 ± 1.69, n = 16; p = 0.02).

Currents associated with bistability

The fact that large mouse motoneurons showed greater levels of bistability than small motoneurons is unlikely to arise from the size difference alone, as larger neurons inherently show lower overall excitability owing to their lower input resistance [START_REF] Menelaou | Mixed synapses reconcile viola ons of the size principle in zebrafish spinal cord[END_REF]. Instead, we proposed that large neurons selec vely express higher levels of ionic currents that sustain the bistable state than small neurons. Several ionic currents have been demonstrated to support the ac ve state during bistability. Among others, these include a persistent inward current (PIC) that may comprise both sodium and calcium components [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Harvey | 5-HT2 receptor ac va on facilitates a persistent sodium current and repe ve firing in spinal motoneurons of rats with and without chronic spinal cord injury[END_REF][START_REF] Harvey | Persistent sodium currents and repe ve firing in motoneurons of the sacrocaudal spinal cord of adult rats[END_REF][START_REF] Li | Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury[END_REF], a thermosensi ve Trpm5 calcium-ac vated inward current [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF], and a slow inac va on of the Kv1.2 potassium current [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF].

We measured the PIC by delivering a slow ramp depolariza on in voltage clamp (Fig. 3A). The PIC amplitude was measured at the inward peak from the extrapolated passive component at the same voltage (See Fig. 3A). The PIC amplitude was very small or absent in small Hb9-GFP + motoneurons (<400µm 2 : 33 ± 58 pA , n = 17; Fig. 3B), but much larger in large motoneurons (165 ± 172 pA, n = 24; p = 0.014), with its amplitude correla ng posi vely with cell size without any change in the ac va on threshold (Fig. 3C). Moreover, the amplitude of the PIC was also found to be propor onal to the bistability score (Fig. 3D). Specifically, non-bistable neurons (scores 0-1) had small to absent PICs (mean 11.7 ± 19.4 pA; n = 15, 9 with no PIC), whereas bistable neurons (scores 3-4), especially those that are fully bistable (score 4), exhibited a drama c rise in the PIC amplitude (mean 198 ± 160.6 pA, n = 22, 0 with no PIC; p < 0.001). . These findings highlighted the strong associa on between PICs and bistability in large α-motoneurons and the absence or low amplitude of PICs in smaller non-bistable neurons.

To further understand the contribu on of the sodium component of PICs in bistability, we added 10 mM TEA-Cl and 100 µM CdCl 2 to the recording ACSF [START_REF] Drouillas | Persistent Nav1.6 current drives spinal locomotor func ons through nonlinear dynamics[END_REF]. Large α-motoneurons displayed a more pronounced persistent sodium current (INaP; Fig. 3E). Although the motoneuron size did not influence the INaP threshold ac va on (Fig. 3G), the peak amplitude of INaP was significantly greater in large α-motoneurons (>400 µm 2 , 1126 ± 530 pA, n = 11) compared to small motoneurons (<400 µm 2 , 221 ± 104 pA, n = 10; p<0.001, Fig. 3F). This increase is not merely a reflec on of the greater surface area of the bigger neurons that could express more channels, as the PIC density (corrected by the cellular capacitance) was also larger in large than in small α-motoneurons (4.84 ± 1.51 pA/pF, n = 11 vs. 2.38 ± 1.09 pA/pF, n = 10;Fig. 3H; P < 0.001). These findings highlighted the significant contribu on of INaP in bistability from large α-motoneurons and its diminished presence in smaller, non-bistable neurons.

A calcium-ac vated inward current was shown to be pivotal in sustaining the plateau depolariza on underlying the tonically firing ac ve state in bistable crab [START_REF] Zhang | Calcium-dependent plateau poten als in a crab stomatogastric ganglion motoneuron. II. Calcium-ac vated slow inward current[END_REF] and turtle motoneurons [START_REF] Perrier | Ca 2+ -ac vated nonselec ve ca onic current (I CAN ) in turtle motorneurons[END_REF]; this current has recently been shown to be mediated by Trpm5 channels in mice [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. To measure the effect of this current, we induced a 2-sec depolariza on in the presence of tetrodotoxin (TTX, 1 µM) and tetraethylammonium-chloride (TEA, 10 mM) to minimize sodium and potassium currents. This depolariza on was large enough to elicit a series of slow calcium-driven spikes to fully ac vate Trpm5. Subsequently, we measured the resul ng a erdepolariza on (sADP; Fig. 4A), which slowly declined as the Trpm5 current decayed [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF].

Notably, the sADP was par ally blocked by Triphenylphosphine oxide (TPPO, 50 µM Fig. 4A), a known Trpm5 channel blocker [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF][START_REF] Palmer | Triphenylphosphine Oxide Is a Potent and Selec ve Inhibitor of the Transient Receptor Poten al Melasta n-5 Ion Channel[END_REF]. The residual TPPO-insensi ve sADP appeared to predominantly arise from channels that are not yet iden fied (see discussion in [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]). Thus, to accurately es mate the Trpm5 component of the sADP, we subtracted the sADP measured in TPPO from the total sADP. The resul ng Trpm5 sADP was only detectable in one of 15 smaller neurons (<400 µm 2 : 0.2 ± 0.92 mV, n = 15), but was evident in most of the larger neurons (>400 µm 2 : 4.9 ± 5.47 mV, n = 9; p = 0.0017; Fig. 4A-B). Neurons with lower bistability scores (scores 0-1) lacked Trpm5 sADP (n=10), but this current was present in most bistable neurons (scores 3-4: 4.6 ± 5.32 mV, n = 10; p = 0.0108; Fig. 4C). We isolated the Trpm5 calcium-ac vated inward current in response to a 2 sec depolarizing voltage step in presence of TTX (1µM) and TEA (10mM) (Fig. 4D). We confirmed that the Trpm5 inward current amplitude and density (corrected by cell capacitance) were significantly bigger in large α-motoneurons (>400 µm 2 , 324 ± 146.3 pA & 2.7 ± 1.38 pA/pF, n = 8) compared to the small ones (<400 µm 2 , 26 ± 7.51 pA & 0.7 ± 0.67 pA/pF, n = 3) (Fig. 4E-F; P<0.05). Thus, the calcium-ac vated inward current mediated by Trpm5 channels [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF] appeared associated with bistability in large α-motoneurons, but was low or absent in smaller non-bistable neurons.

We further assessed the Kv1.2 potassium current, whose slow inac va on delays the ini a on and accelera on of firing of bistable neurons during long current steps [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF].

Interes ngly, in our earlier work, only bistable neurons showed the delayed ini a on and accelera on of firing during long current steps, induced by closure of the Kv1.2 channels; nonbistable neurons showed immediate spike onset with spike decelera on [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF]. We tested whether the Kv1.2 inac va ng current was larger in large, bistable neurons than small neurons. This was measured in voltage clamp as the amplitude of the inward current over a 7 sec step, measured a er inac va on of the transient potassium current, in the presence of TTX (1µM) and TEA (10mM) (Fig. 4G). Again, the amplitude of the Kv1.2 current scaled with cell size. Smaller α-motoneurons (<400 µm 2 ) had a lower current amplitude (68 ± 86.9 pA; n = 19) than the larger neurons (159.2 ± 155.5 pA, n = 26; P < 0.02; Fig. 4H). Moreover, the smaller motoneurons had a lower current density (2.19 ± 0.33 pA/pF; n = 3) compared to larger motoneurons (3.61 ± 0.94 pA/pF, n = 7; P < 0.05; Fig. 4 I

Serotonin effects on bistability in par ally bistable neurons

We found that bistability is not strictly binary, as 26% (52 out 199) of α-motoneurons displayed some, but not all, of the defining features described above, especially the absence of persistent firing a er the brief depolariza on. It has been known for many years that neuromodulators such as serotonin (5-HT) can evoke bistability in α-motoneurons which show only par al bistable proper es [START_REF] Hounsgaard | Serotonin-induced bistability of turtle motoneurons caused by a nifedipine-sensi ve calcium plateau poten al[END_REF][START_REF] Kiehn | 5-HT modula on of hyperpolariza on-ac vated inward current and calcium-dependent outward current in a crustacean motor neuron[END_REF][START_REF] Kiehn | Serotonergic stretch receptors induce plateau proper es in a crustacean motor neuron by a dual-conductance mechanism[END_REF][START_REF] Hounsgaard | Bistability of -motoneurons in the decrebrate cat and in the acute spinal cat a er intravenous 5-hydroxytryptophan[END_REF].

We analyzed the effects of 10 µM 5-HT on α-motoneurons (n = 45) that had bistability scores below 4 and lacked the self-sustained spiking. Fig. 5A shows an example of an α-motoneuron that, under control condi ons (i.e. recording aCSF), did not show accelera on of spiking during a 2-sec depolariza on, and only showed a modest sADP at the end of the depolariza on. Seven minutes a er applica on of 5-HT, this motoneuron demonstrated accelera ng spiking during the current step, and self-sustained spiking ac vity a er the end of the step. This ac vity was terminated only by a hyperpolarizing step, indica ng that the neuron became fully bistable only in the presence of 5-HT. Fig. 5B shows the ability of 5-HT to evoke bistability in α-motoneurons of different sizes. Notably, small α-motoneurons (< 400µm 2 ; n = 19) remained non-bistable even with 5-HT. In contrast, larger neurons exhibited the poten al for a transi on to bistability upon 5-HT exposure. Specifically, of non-bistable α-motoneurons of larger size (>400µm 2 ), 38.5% switched to full bistability with 5-HT (n = 26; p < 0.001). It is noteworthy that the majority of neurons transi oning to bistability with 5-HT already expressed an sADP before 5-HT addi on, as illustrated in Fig. 5A. Thus, 5-HT was able to evoke full bistability in a subset of neurons

DISCUSSION

Our study reveals a size-based gradient in the bistable ability of GFP+ α-motoneurons from Hb9-GFP and ChAT-GFP mice lines. Specifically, the likelihood of α-motoneurons being bistable increases with the cell body cross-sec onal area and over me. The currents, Trpm5 and INaP, which contribute to the ac ve state during bistability, and Kv1, whose inac va on helps to ini ate sustained firing, are more pronounced in larger fast α-motoneurons which can be reliably iden fied with the MMP-9 marker. Conversely, these currents are low or absent in smaller MMP-9 -α-motoneurons. We also showed that serotonin evokes full bistability only in large motoneurons. These findings highlight an important correla on between motoneuron size and bistability.

Research on bistability has primarily focused on larger α-motoneurons, iden fied by their ventrolateral loca on or by retrograde s mula on from ventral nerves [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF][START_REF] Manuel | Molecular and electrophysiological proper es of mouse motoneuron and motor unit subtypes[END_REF]. This might have led to a failure to record the proper es of smaller α-motoneurons. Neurons recorded in this study are motoneurons, as evidenced by their ventrolateral loca on and the expression of ChAT and Hb9 markers (Fig. 1A-B). Furthermore, they are α-motoneurons since most γmotoneurons do not express NeuN [START_REF] Shneider | Gamma motor neurons express dis nct gene c markers at birth and require muscle spindle-derived GDNF for postnatal survival[END_REF], and do not express GFP in Hb9-GFP mice [START_REF] Friese | Gamma and alpha motor neurons dis nguished by expression of transcrip on factor Err3[END_REF][START_REF] Shneider | Gamma motor neurons express dis nct gene c markers at birth and require muscle spindle-derived GDNF for postnatal survival[END_REF].

Finally, virtually all of the neurons larger than 400µm 2 also expressed MMP-9, indica ve of fast α-motoneurons [START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF][START_REF] Kaplan | Neuronal matrix metalloproteinase-9 is a determinant of selec ve neurodegenera on[END_REF]. While there are no markers available to differen ate between the fast fa gue-resistant (FR) and fast fa gable (FF) α-motoneurons, we conclude that the largest neurons are fast motoneurons. The smallest neurons are presumably slow (S) α-motoneurons.

However, size alone is not a reliable indicator to separate these classes [START_REF] Baczyk | Diversity of Mammalian Motoneurons and Motor Units[END_REF][START_REF] Smith | Electrical Proper es of Adult Mammalian Motoneurons[END_REF][START_REF] Thirumalai | Recruitment of Motoneurons[END_REF].

Electrophysiologically, large fast α-motoneurons differ from small slow α-motoneurons by exhibi ng a delayed and accelerated firing during prolonged s mula on [START_REF] Manuel | Molecular and electrophysiological proper es of mouse motoneuron and motor unit subtypes[END_REF][START_REF] Sharples | Matura on of persistent and hyperpolariza on-ac vated inward currents shapes the differen al ac va on of motoneuron subtypes during postnatal development[END_REF][START_REF] Smith | Electrical Proper es of Adult Mammalian Motoneurons[END_REF]. Our experiments confirm this observa on, as near-threshold current steps resulted in a slow depolariza on in larger α-motoneurons leading to delayed firing accelera on (Fig. 2). In contrast, smaller α-motoneurons fired immediately upon reaching spike threshold followed by a spike frequency decelera on [START_REF] Manuel | Molecular and electrophysiological proper es of mouse motoneuron and motor unit subtypes[END_REF][START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF]. The depolariza on and spike delay in large fast αmotoneurons were a ributed to the slow inac va on of a Kv1 current [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF][START_REF] Leroy | Early intrinsic hyperexcitability does not contribute to motoneuron degenera on in amyotrophic lateral sclerosis[END_REF].

We now add bistability as a predominant property of large fast L4-L5 α-motoneurons in young mice. Over 75% of neurons over 800µm 2 showed bistability, while only ~10% of neurons less than 400 µm 2 were bistable. Consistent with this, a large majority of fast α-motoneurons showed nega ve hysteresis during triangular ramp steps, where the derecruitment current was lower than the recruitment current, while far fewer of the slow α-motoneurons displayed nega ve hysteresis (30) (Fig. 2). The frequency of bistability in large α-motoneurons increased during the first weeks of postnatal development, as previously described ( 18); (Fig. 2C1). By P21, all large α-motoneurons were fully bistable, while smaller neurons con nued to lack bistability features.

The dis nct firing behaviors observed between large and small α-motoneurons can be a ributed to a specific set of ionic currents that are more highly expressed in large fast αmotoneurons compared to small slow α-motoneurons. One key contributor is the ac va on of slow persistent inward currents (PICs), which may be carried by calcium or sodium. This ac va on has been linked to nega ve hysteresis and bistability [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. Expression of the PIC increases with the size of the α-motoneurons (Fig. 3B), and with the bistability score (Fig. 3D). Furthermore, our observa ons demonstrate the rela onship between the size of the αmotoneurons and the sodium component of PICs (INaP) (Fig. 3E-H).

Recently, the thermosensi ve Trpm5 current has been iden fied as a calcium-ac vated sodium current sustaining the plateau poten al in bistable mouse α-motoneurons [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. We found that the Trpm5-evoked a erdepolariza on was present in virtually all large bistable αmotoneurons, but not detectable in small non-bistable α-motoneurons (Fig. 4A-C). Finally, the slowly inac va ng Kv1.2-mediated current responsible for the delayed firing accelera on in large motoneurons (23) (Fig. 2), is less expressed in smaller motoneurons (Fig. 4G-I).

Interes ngly, this current posi vely correlates with the degree of bistability (Fig. 4G-I; [START_REF] Bos | Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomo on[END_REF]). It is likely, that the slow inac va on of Kv1 will shi the balance of currents towards depolariza on, and will help to sustain con nued firing in the bistable state. In sum, the lack of sufficient expression of these three pivotal currents renders small α-motoneurons non-bistable. In contrast, large α-motoneurons, which robustly express these currents, are bistable. Note that there may be some duplica on of effort in these currents. Indeed, among neurons that showed con nued bistable firing, 20% either did not show a marked nega ve hysteresis during ramps, or did not show a delayed firing accelera on. This suggest that bistability might not require the full spectrum of these currents. Bistability in α-motoneurons was observed in earlier experiments only in the presence of neuromodulators [START_REF] Hounsgaard | Serotonin-induced bistability of turtle motoneurons caused by a nifedipine-sensi ve calcium plateau poten al[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Hounsgaard | Ca++ dependent bistability induced by serotonin in spinal motoneurons[END_REF]. However, we have revealed inherent bistability in most large αmotoneurons, provided the recording temperature is sufficiently high (above 30°C) [START_REF] Bouhadfane | Sodium-mediated plateau poten als in lumbar motoneurons of neonatal rats[END_REF], to unmask the thermosensi ve Trpm5 current responsible for the plateau poten al [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF]. In our prepara ons, some α-motoneurons expressed intermediate proper es and were not fully bistable under these condi ons. Addi on of 5-HT evoked full bistability in ~30% and ~60% of the intermediate and large neurons, respec vely (Fig. 5). Interes ngly, α-motoneurons completely lacking baseline bistability characteris cs (bistability score 0) never became bistable with 5-HT. We propose that intermediate α-motoneurons express some of the essen al currents for bistability, but at lower levels (Fig. 5). The known modulatory ac ons of 5-HT can then enhance these currents to sustain prolonged firing in the bistable state.

Our demonstra on of a size principle for bistability contrasts with the groundbreaking work by Lee and Heckman [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF], who studied α-motoneurons in adult cats. In their study, they achieved a full bistability in about one-third of the α-motoneurons, which interes ngly exhibited characteris cs of smaller motoneurons. There are several poten al explana ons for the differences between our findings. First, it is uncertain whether the smallest α-motoneurons in the cat were recorded because of the use of sharp electrode recordings. Consequently, the bistability in the smallest slow α-motoneurons in cats remains unknown. Second, mouse αmotoneurons are inherently more excitable than cat α-motoneurons, primarily due to their smaller size [START_REF] Huh | PICs in motoneurons do not scale with the size of the animal: a possible mechanism for faster speed of muscle contrac on in smaller species[END_REF][START_REF] Manuel | Scaling of Motor Output, From Mouse to Humans[END_REF]. Despite this, the PIC amplitude is rela vely similar across both species, sugges ng that PIC has a more significant impact on the firing rate in mice. Third, our Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024. observa ons were made on mouse α-motoneurons during the first 4 weeks of life, while Lee and Heckman's experiments were made on adult cats. However, bistability in the larger neurons became more pronounced with age in the mouse neurons. In young adult mice (P21-P25) all large motoneurons exhibited bistability, whereas none of the smaller ones did. Finally, our results were made ex vivo from slices in the presence of blockers of fast synap c transmission (though not at P21/P25) and mostly without neuromodulators. On the other hand, the cat neurons were recorded in vivo in the presence of methoxamine to maximize the incidence of bistability.

The role of bistability in spinal α-motoneurons remains unclear. It has been recorded during quiet standing in both rats and cats [START_REF] Eken | Possible func ons of transmi er-controlled plateau poten als in alpha motoneurones[END_REF], sugges ng a poten al significance for postural control [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Kiehn | Plateau poten als and ac ve integra on in the 'final common pathway' for motor behaviour[END_REF][START_REF] Hounsgaard | Bistability of -motoneurons in the decrebrate cat and in the acute spinal cat a er intravenous 5-hydroxytryptophan[END_REF]. The Henneman Size Principle (57) posits that smaller α-motoneurons, due to their lower input conductance, are the first to be recruited, poten ally playing a role in maintaining posture. This can be consistent with earlier work in cats, where smaller neurons showed full bistability and were possibly ac ve during quiet standing [START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in rhythmic firing pa erns[END_REF][START_REF] Lee | Bistability in spinal motoneurons in vivo: systema c varia ons in persistent inward currents[END_REF][START_REF] Ri Er | Characteriza on of motor units in behaving adult mice shows a wide primary range[END_REF]. However, we here demonstrate, in younger mice, that small α-motoneurons are not bistable. Instead, bistability increases with the size of motoneurons (Fig. 2). Ri er et al. [START_REF] Ri Er | Characteriza on of motor units in behaving adult mice shows a wide primary range[END_REF] provided evidence that large fast α-motoneurons may also be tonically recruited during quiet standing in mice. An intriguing study by Bos et al. [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF] showed that mice lacking Trpm5 channels in lumbar motoneurons exhibited compromised postural control. Given the central role of Trpm5 in bistable proper es of larger α-motoneurons [START_REF] Bos | Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice[END_REF] plays in important role in the ini a on of bistable firing, but is also cri cal for repe ve firing in α-motoneurons [START_REF] Kuo | Essen al role of the persistent sodium current in spike ini a on during slowly rising inputs in mouse spinal neurones[END_REF], and in synap c amplifica on due to their dendri c loca on (1). Thus, the currents which together lead to bistability may individually play mul ple roles in motor control in the mouse. These assump ons should be further inves gated in the future using more integrated in vivo prepara ons. spiking is higher than the threshold for offset of spiking (-ΔI; 1 point). A3: A small subthreshold current step leads to a slow depolariza on (trace has been offset to be more easily seen). A slightly higher current step leads to a larger depolariza on leading to delayed spike onset and accelera on of spike frequency during the step (1 point). B: Completely non-bistable neuron (bistability score 0). B1: Current steps to near threshold lead to decelera ng spike frequency during a 2 sec suprathreshold pulse, and an a erhyperpolariza on at the end of the step (0 point). B2: Posi ve hysteresis during ramp s mula on. The current threshold for onset of spiking is lower than the threshold for offset of spiking (+ΔI; 0 point). B3: A small subthreshold current step does not evoke a slow depolariza on. A slightly higher current step evokes immediate onset firing with decelera ng spike frequency during the step (0 point). Size markers: 1 sec, 20 mV. C1: Bistability score as a func on of postnatal age for smaller (<400µm 2 , blue, n=81) and larger (>400µm 2 , red, n=118) Hb9-GFP + neurons. Sta s cal comparison was done for each age and when all ages were pooled. C2: Distribu on of bistability as a func on of neuronal cross-sec onal area combining all ages. Smaller neurons predominantly have bistability scores of 0-1 (blue, n=95) while larger neurons predominantly have bistability scores of 3-4 (red, n=80). C1-C2: n= 199 neurons. C3: Bistability score as a func on of neuronal input resistance. A strong nega ve correla on is seen (R 2 = 0.99, n=176). C4: Effect of size on bistability in neurons (P1/9) recorded by perforated patch-clamp. Large neurons show significantly higher bistability scores than small neurons (p<0.05; n=27) C5: Post-natal (P2/6) motoneurons iden fied by expression of ChAT-GFP show similar bistability scores that rise with cross-sec onal area (blue for <400µm 2 , n=16 and red for >400µm Above a threshold, large calcium-dependent oscilla ons are evoked. In bistable motoneurons (red trace), these elicit a large, slow a erdepolariza on which is par ally blocked by 50µM

TPPO (black trace).

Non-bistable neurons (blue trace) do not express this slow a erdepolariza on. B: Amplitude of the Trpm5-induced, TPPO-resistant a erdepolariza on is larger in large motoneurons (>400 µm 2 , n=9) than small motoneurons (<400 µm 2 , n=15; p=00017). C: Trpm5-induced, TPPO-resistant a erdepolariza on is larger in bistable motoneurons (score 3-4, n=10) than nonbistable motoneurons (score 0-1, n=10; p=0.0108). D: TPPO-sensi ve Trpm5 current is isolated in response to a voltage step of 2 sec in presence of 1µM TTX and 10 mM TEA. Superimposed traces of Trpm5 isolated current from large (>400µm 2 , red) vs small (<400µm 2 , blue) Hb9-GFP + motoneurons. Note that the current trace during the depolarizing step has been truncated for improved visualiza on of the post-step current. E-F: Amplitude (E) and density (F) of the Trpm5 current increases with cross-sec onal area of Hb9-GFP + neurons (blue circles, <400µm 2 , n=3, and red circles, >400µm 2 , n=8; p=0,0121 and p=0242, respec vely) G: Superimposed traces of the slowly inac va ng Kv1 current in bistable (red) and non-bistable (blue) neuron in response to a long depolarizing voltage step in presence of 1µM TTX and 10 mM TEA. H-I: Amplitude (H) and density (I) of Kv1 current increases with crosssec onal area of Hb9-GFP + neurons (blue circles, <400µm 2 , n=19 (H), n=3 (I) and red circles,

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

  Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

  Cornell: All animal protocols were approved by the Cornell Ins tu onal Animal Use and Care Commi ee and were in accordance with NIH guidelines. Marseille: All animal care and use conformed to French regula ons (Décret 2010-118) and were approved by the local ethics Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.commi ee (Comité d'Ethique en Neurosciences INT-Marseille, CE71 Nb A1301404, authoriza on Nb 2018110819197361). Slice Prepara on For electrophysiological experiments, mice were cryoanaesthe zed (P2-P7) or anaesthe zed (P8-P25) with intraperitoneal injec on of a mixture of ketamine/xylazine (100mg/kg and 10 mg/kg, respec vely). They were then decapitated, eviscerated and the spinal cord removed by laminectomy, and placed in a Sylgard-lined petri dish with ice cold (1-2°) aCSF containing (in mM): 252 sucrose, 3 KCl, 1.25 KH 2 PO 4 , 4 MgSO 4 , 0.2 CaCl 2 , 26 NaHCO 3 , 25 D-glucose, pH 7.4, bubbled with 95% O 2 and 5% CO 2 . The meninges were removed and the posterior cord (L3-S5) imbedded in agarose. The same solu on was used for slicing. 325-350µm sec ons were prepared from the L4-L5 region and superfused at 3 ml/min with recording aCSF containing (in mM): 120 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 1.3 MgSO 4 , 1.2 CaCl 2 , 25 NaHCO 3 , 20 D-glucose, pH 7.4, 32-34°C (19, 23). In most experiments (P1-P14), blockers of fast synap c transmission (CNQX and D,L-AP5 or kynurenic acid, strychnine, and bicuculline) were added in the aCSF to minimize synap c contribu ons to bistability. To isolate the persistent Na + currents (INaP) during voltage-clamp experiments we used a modified aCSF containing (in mM): 100 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 1.3 MgSO 4 , 3.6 MgCl 2 , 1.2 CaCl 2 , 25 NaHCO 3 , 40 D-glucose, 10 TEA-Cl and 0.1 CdCl 2 (24). Mature mice (P21-25) were treated as the younger mice, except that the cord was sliced in an ice cold slicing solu on (1-2°) containing (in mM): 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

Fig

  Fig.1B2) and P8-10 (n = 425 neurons; Fig.1B2), with 80.5% and 79.6% respec vely having a

  (Fig.2): 1) Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

  ). All together, our findings highlight that INaP, the Trpm5 mediated calcium-ac vated inward current, and the slow inac va ng Kv1 current are pivotal indicators of bistability in large α-motoneurons. Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

  (Fig 4), it is possible that fast α-motoneurons may play a role in postural maintenance. Alterna vely, the currents responsible for bistability may play a more important role in recrui ng the less excitable large motoneurons. The PIC Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

Figure 1 :

 1 Figure 1: Iden fica on and size distribu on of α-motoneurons in the ventrolateral spinal cord

Figure 2 :

 2 Figure 2: Bistability score in spinal motoneurons varies by age and size. A: Fully bistable

  p<0.001 (two-tailed Mann-Whitney test for C1, C4 and C5; Fisher test and slope comparison of simple linear regressions for C2). Mean ± S.D.

Figure 3 :

 3 Figure 3: Persistent inward current (PIC), and its sodium component, is larger in bistable

Figure 4 :

 4 Figure 4: Trpm5-mediated current and slowly inac va ng Kv1 current are signatures of

>400µm 2 ,

 2 n=26 (H) and n=7 (I); p=0.0216 and p=0.0167). *p<0,05; **p<0,01 (two-tailed Mann-Whitney test forB-C, E-F and H-I). Median (solid line) and quar les (dashed lines) are represented in each violin plot.

Figure 5 :

 5 Figure 5: Induc on of full bistability by serotonin. A: Non-bistable neuron's response (top,

  Cell body cross-sectional area (μm 2 )

NeuN+

  Figure 1

V

  Figure 2

  are also involved in different neurons.

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  Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

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  Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.is indicated in the figure legends and provided in more detail in the Source Data File. In the figures and in the main text, data are presented as mean ± SD for the histograms. Median and quar les are represented in each violin plot. Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

	Source Data File:
	h ps://github.com/remibos/Source-Data-File-HW.git

  Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.already leaning towards a bistable state, but could not evoke bistability in smaller or less bistable neurons.

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  2 , n=9; p=0.02). * p<0.05; *** Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

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ACKNOWLEDGEMENTS

This work was supported by NIH grant NS17323 (R. H-W), by Fonds d'inves ssement INT FI_INT_JCJC_2019 (R.B.), by Centre Na onal de la Recherche Scien fique (CNRS) (R.B. and F.B.) and by Agence Na onale de la Recherche Scien fique ANR-16-CE16-0004 (F.B.). Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.

AUTHOR CONTRIBUTIONS:

AUTHOR NOTES

Correspondence: R. M. Harris-Warrick, rmh4@cornell.edu; F. Brocard, frederic.brocard@univamu.fr; R. Bos, remi.bos@univ-amu.fr