# Effect of size on expression of bistability in mouse spinal motoneurons

Ronald Harris-Warrick, Emilie Pecchi, Benoît Drouillas, Frédéric Brocard, Rémi Bos

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Ronald Harris-Warrick, Emilie Pecchi, Benoît Drouillas, Frédéric Brocard, Rémi Bos. Effect of size on expression of bistability in mouse spinal motoneurons. Journal of Neurophysiology, In press, 10.1152/jn.00320.2023. hal-04477199

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Effect of size on expression of bistability in mouse spinal motoneurons

Ronald M Harris-Warrick<sup>1</sup>, Emilie Pecchi<sup>2</sup>, Benoît Drouillas<sup>2</sup>, Frédéric Brocard<sup>2,*</sup>, Rémi Bos<sup>2,*</sup>

<sup>1</sup>: Department of Neurobiology and Behavior, Cornell University, Ithaca NY 14850,

United States; <sup>2</sup>: Aix Marseille Univ, CNRS, Institut de Neurosciences de la Timone
(INT), UMR 7289, Marseille, France

*: These authors contributed equally
22

**Abstract**

23

24 Bistability in spinal motoneurons supports tonic spike activity in the absence of
25 excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons
26 innervating slow antigravity muscle fibers are more likely to generate bistability for postural
27 maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers
28 display bistability is still controversial. To address this, we examined the relationship between
29 soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of ChAT-GFP and Hb9-
30 GFP mice during the first four weeks of life. We found that as neuron size increases, the
31 prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast α-
32 motoneurons (MMP-9+/Hb9+) with a soma area ≥ 400μm² exhibit significantly higher bistability.
33 Ionic currents associated with bistability, including the persistent Nav1.6 current,
34 thermosensitive Trpm5 Ca²⁺-activated Na⁺ current and the slowly inactivating Kv1.2 current, also
35 scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable
36 properties, but not in small motoneurons. Our study provides important insights into the neural
37 mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.

38

39 **Keywords:** Motoneuron, Spinal cord, Bistability, Current, Development, Posture, Serotonin,
40 Neuromodulation

41

42

43
45 Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow
46 motoneurons but present in most large, fast motoneurons. This difference results from
47 differential expression of ionic currents that enable bistability, which are highly expressed in
48 large motoneurons but small or absent in small motoneurons. These results support a possible
49 role for fast motoneurons in maintenance of tonic posture in addition to their known roles in
50 fast movements.

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53

# INTRODUCTION

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Spinal motoneurons not only transmit central commands for movement to muscles but also shape motor output and muscle contraction through nonlinear firing properties (1). One such property is bistability, where the motoneuron can switch between stable silent and active states, depending on transient synaptic input or current injection. Originally detected as plateau potentials in invertebrate neurons (2-9), bistability was soon found in vertebrates (10), specifically in spinal motoneurons (11-16). While often induced by neuromodulators such as serotonin (13, 15-17), bistability can also arise when motoneurons are depolarized to near threshold and recorded under natural ionic and temperature conditions (18, 19).

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The active state in bistable motoneurons is mainly mediated by slow ionic currents including persistent Cav1.3 (13-16) and Nav1.6 currents (20, 21), and the Trpm5-mediated Ca²⁺-activated Na⁺ current (18, 19, 22). Drawing from a series of our previous investigations, we can summarize the process as follows: The initial depolarization, caused by the slow inactivation of Kv1.2 potassium channels (23), activates the persistent Nav1.6 current leading to spiking activity (24). This then prompts Ca²⁺ entry through the recruitment of Cav1.3 channels, initiating a Ca²⁺-induced Ca²⁺-release process (18). This process ultimately activates thermosensitive Trpm5 channels, which are the primary source of the plateau depolarization to sustain repetitive firing (19). Other currents such as the HCN-type hyperpolarization-activated inward current, Iₕ (25, 26) and reduction of calcium-activated outward currents (17, 25) are also involved in different neurons.
α-motoneurons, which drive extrafusal muscle contractions, can be subdivided functionally into three classes based on size and the muscle type they innervate: large fast-fatigable (FF), medium fast fatigue-resistant (FR), and small slow (S) motoneurons [reviewed by (27)]. In decerebrate cats, small motoneurons characterized by slow conduction velocities and low activation thresholds, appear to have more pronounced bistability than large ones (15, 16). This observation 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 (15, 16, 28). In mice, the stationary posture also involves the activation of fast motor units (57). Interestingly, our recent findings indicate that large motoneurons exhibit bistable firing patterns, which are crucial for the control of hindlimb movement (19). From as early as the 2nd postnatal week, large motoneurons in mice present an electrophysiological profile that is distinct from that of smaller ones, marked by a lower input resistance, a higher rheobase, more depolarized spike thresholds, narrower action potentials, and shorter afterhyperpolarizations (27, 29, 30). They also exhibit delayed onset of firing and firing acceleration during a step depolarization, and receive a consistently higher level of recurrent excitation (23, 31-33). Despite this extensive characterization, 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, caution is warranted due to the distinct characteristics of mouse spinal motoneurons that are not seen in cats (34).
In the present study, we examined the bistability as a function of size in genetically labeled L4-L5 α-motoneurons in young mice (postnatal day (P) 1-25). Our data indicate that the largest α-motoneurons showed stronger bistable firing properties, while the smallest motoneurons were rarely bistable. There was a strong correlation between bistability and the amplitudes of the ionic currents known to support it. Motoneurons of intermediate size often showed partial bistability, which could be converted to full bistability by serotonin. These unexpected results suggest new hypotheses regarding the contribution of larger motoneurons in bistability and postural control.

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104 MATERIALS AND METHODS

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106 Animals and ethical standards

107 Hb9-GFP mice were kindly provided by B. Pettmann and Jackson Laboratories (strain 005029).
108 ChAT-Cre mice were obtained from Jackson Laboratories (strain 007902) and crossed to Rosa-
109 26-floxed GFP mice. Mice of both sexes were used in all experiments. Animals were housed on
110 a 12 hr day/night cycle with ad libitum access to water and food. The room temperature was
111 maintained between 20 and 22°C.

112 Cornell: All animal protocols were approved by the Cornell Institutional Animal Use and Care
113 Committee and were in accordance with NIH guidelines. Marseille: All animal care and use
114 conformed to French regulations (Décret 2010-118) and were approved by the local ethics
115 committee (Comité d'Ethique en Neurosciences INT-Marseille, CE71 Nb A1301404,
116 authorization Nb 2018110819197361).

117

118 *Slice Preparation*

119 For electrophysiological experiments, mice were cryoanaesthetized (P2-P7) or anaesthetized
120 (P8-P25) with intraperitoneal injection of a mixture of ketamine/xylazine (100mg/kg and 10
121 mg/kg, respectively). They were then decapitated, eviscerated and the spinal cord removed by
122 laminectomy, and placed in a Sylgard-lined petri dish with ice cold (1-2°) aCSF containing (in
123 mM): 252 sucrose, 3 KCl, 1.25 KH₂PO₄, 4 MgSO₄, 0.2 CaCl₂, 26 NaHCO₃, 25 D-glucose, pH 7.4,
124 bubbled with 95% O₂ and 5% CO₂. The meninges were removed and the posterior cord (L3-S5)
125 imbedded in agarose. The same solution was used for slicing. 325-350µm sections were
126 prepared from the L4-L5 region and superfused at 3 ml/min with recording aCSF containing (in
127 mM): 120 NaCl, 3 KCl, 1.25 NaH₂PO₄, 1.3 MgSO₄, 1.2 CaCl₂, 25 NaHCO₃, 20 D-glucose, pH 7.4,
128 32-34°C (19, 23). In most experiments (P1-P14), blockers of fast synaptic transmission (CNQX
129 and D,L-AP5 or kynurenic acid, strychnine, and bicuculline) were added in the aCSF to minimize
130 synaptic contributions to bistability. To isolate the persistent Na⁺ currents (iNaP) during
131 voltage-clamp experiments we used a modified aCSF containing (in mM): 100 NaCl, 3 KCl, 1.25
132 NaH₂PO₄, 1.3 MgSO₄, 3.6 MgCl₂, 1.2 CaCl₂, 25 NaHCO₃, 40 D-glucose, 10 TEA-Cl and 0.1 CdCl₂
133 (24).

134

135 Mature mice (P21-25) were treated as the younger mice, except that the cord was sliced in an
136 ice cold slicing solution (1-2°) containing (in mM): 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES,
137 25 D-glucose, 3 kynurenic acid, and pH 7.4 with NaOH (33). After a resting period of 30-60 min,
138 slices were transferred to the recording chamber and superfused with recording aCSF at 32°C
139 (35) without addition of fast synaptic transmission blockers.

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141 Electrophysiological recordings

142
143 Hb9-GFP and ChAT-GFP positive neurons were visualized in the ventrolateral region of lamina IX
144 in L4-L5 horizontal slices using an Olympus BX51 microscope with appropriate filters. Whole-cell
145 patch-clamp recordings from visualized fluorescent GFP+ neurons were made with electrodes
146 (2-6MΩ) pulled from borosilicate glass capillaries (1.5 mm OD, 1.12 mm ID; World Precision
147 Instruments). They were filled with a pipette solution containing (in mM): 140 K⁺-gluconate, 5
148 NaCl, 2 MgCl₂, 10 HEPES, 0.5 EGTA, 2 ATP, 0.4 GTP, pH 7.3. Patch clamp recordings were made
149 using a Multiclamp 700B amplifier driven by PClamp 10 software (Molecular Devices).
150 Recordings were digitized on-line and filtered at 10 kHz (Digidata 1322A or 1440A, Molecular
151 Devices). Pipette and neuronal capacitive currents were canceled, and, after breakthrough,
152 series access resistance was compensated. The recording was allowed to stabilize for at least 2
153 min after establishing whole-cell access before recording was started (19, 23). Unless otherwise
154 indicated, all neurons were held at -70 mV to ensure reproducibility in expression of voltage-
155 sensitive currents.

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157 Drug list: All solutions were oxygenated with 95% O₂/5% CO₂. All salt compounds, as well as
158 tetraethylammonium chloride (TEA; #T2265), Triphenylphosphine oxide (TPPO; #T84603),
159 Serotonin creatinine sulfate monohydrate (5-HT; #H7752), 6-Cyano-7-nitroquinoxaline-2,3-
dione (CNQX, #5.04914), D-(-)-2-Amino-5-phosphonopentanoic Acid (DL-AP5; #165304),
160
161 strychnine (#S0532), bicuculline (#5.05875), Kynurenic Acid (#K3375) and Amphotericin B
162 (#A4888) were obtained from Sigma-Aldrich. Tetrodotoxin (TTX; #1078) was obtained from
163 Tocris Bioscience.

164
165 Immunohistochemistry

166 To identify neurons expressing specific proteins, spinal cords from 10-12-day-old Hb9-GFP mice
167 were removed in ice cold (1-2°) aCSF containing (in mM): 252 sucrose, 3 KCl, 1.25 KH2PO4, 4
168 MgSO4, 0.2 CaCl2, 26 NaHCO3, 25 D-glucose, pH 7.4, bubbled with 95% O2 and 5% CO2. The
169 spinal cords were fixed for 5-6 h in 4% paraformaldehyde (PFA) prepared in phosphate buffer
170 saline (PBS), then rinsed in PBS and cryoprotected overnight in 20% sucrose-PBS at 4°C. Spinal
171 cords were frozen in OCT medium (Tissue Tek), and 30 µm cryosections were collected from the
172 L4-L5 segments (23). After washing in PBS 3x5 min, the slides were incubated for 1 h in a
173 blocking solution (BSA 1%, Normal Donkey Serum 3% in PBS) with 0.2% Triton X-100 and for 12
174 h at 4 °C in a humidified chamber with the knockout-validated primary antibody: mouse-anti-
175 NeuN (Neuronal Nuclei, Sigma-Aldrich MAB377) (36) or goat-anti-MMP-9 (Matrix
176 metallopeptidase 9, Sigma-Aldrich M9570)(30, 37). Both antibodies were diluted in the blocking
177 solution with 0.2% Triton X-100 (1:1000 and 1:500 for anti-NeuN and anti-MMP-9, respectively).
178 Slides were washed 3x5 min in PBS and incubated for 2 h with an Alexa Fluor® Plus 555-
179 conjugated secondary antibody (Invitrogen A32816) diluted in the blocking solution. After 3
180 washes of 5 min in PBS, they were mounted with a gelatinous aqueous medium.
181

182 **Data analysis**

183 Electrophysiological recording:

184 Electrophysiological data were analyzed with Clampfit 10 software (Molecular Devices). Several
185 basic criteria were set to ensure optimum quality of intracellular recordings. Only cells with a
186 stable membrane potential below -60 mV, stable access resistance (no more than 20%
187 variation), and action potential amplitude larger than 60 mV were analyzed. Reported
188 membrane potentials were not corrected for liquid junction potentials.

189 **Confocal imaging:**

190 Immunofluorescent staining was quantified from confocal images acquired with the 40X
191 objective (LSM700 microscope, Zeiss, Germany) on at least two slices per mouse and within the
192 ventro-lateral area (300 X 300 µm²) from each slice. The cell body cross-sectional area was
193 manually segmented and quantified from stacked confocal images (5 steps ; Z-step, 3 µm) with
194 Zen software (Zeiss). An intensity threshold in each slice was applied to determine the presence
195 of NeuN or MMP9 staining from each segmented GFP+ motoneuron.

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198 **Statistics**

199 No statistical method was used to predetermine sample size. Statistical analysis was carried out
200 using GraphPad Prism and Matlab (MathWorks) software. When two groups were compared we
201 used, the Mann-Whitney test. Fisher's exact test was used for comparing cell proportions
202 between two groups. In some cases, we also compared the slope of the simple linear
203 regressions between two groups. The level of significance was set at p <0.05. Each statistical test
204 is indicated in the figure legends and provided in more detail in the Source Data File. In the
205 figures and in the main text, data are presented as mean ± SD for the histograms. Median and
206 quartiles are represented in each violin plot.

207 Source Data File:

208 https://github.com/remibos/Source-Data-File-HW.git

209

210
211 **RESULTS**

212

213 **Immunohistochemical identification of fast α-motoneurons**

214

215 Motoneurons in the ventrolateral spinal cord were initially identified in unfixed L4-L5
216 horizontal slices by their GFP fluorescence in choline acetyltransferase (ChAT)-tagged ChAT-GFP
217 mice. We found a number of labeled neurons of many sizes (from <200 µm² to >800 µm²).

218 However, these include both α-motoneurons driving extrafusal muscle fibers and smaller γ-
219 motoneurons innervating intrafusal muscles that regulate muscle spindle responsiveness to
220 stretch. These motoneuron subtypes can be distinguished by differential expression of a
221 number of proteins. While the transcription factor Hb9 is a marker for both α- and γ-
222 motoneuron in Hb9-nls-LacZ animals (38), only α-motoneurons express GFP labeling in the Hb9-
223 GFP mice (38). We thus used Hb9-GFP mice to identify α-motoneurons in the L4-L5
224 ventrolateral spinal cord, preferentially linked to hindlimb muscles (39, 40), from postnatal day 1
225 (P1) to P25.

226 The transcription factor NeuN is commonly used to distinguish neurons from glial cells.
227 In the spinal cord, α-motoneurons express NeuN, whereas 2/3 of γ-motoneurons do not (41).
228 We examined co-expression of Hb9-GFP and NeuN immunolabeling in fixed tissues and,
229 regardless of age, we found that virtually all ventrolateral Hb9-GFP-labeled neurons exhibited
230 NeuN labeling (98 ± 2.4%, n = 144 cells), indicating they are likely α-motoneurons (Fig. 1A, B1).
231 We observed a preponderance of small labeled α-motoneurons during P4-6 (n = 571 neurons;
232 Fig. 1B2) and P8-10 (n = 425 neurons; Fig. 1B2), with 80.5% and 79.6% respectively having a
233 maximal cross sectional area of 400 µm² or less. The percentage of large α-motoneurons
234 increased with age (38, 41), and by P12, only 50.4% of them remained under 400µm² (n = 288
235 neurons; Fig. 1B2).

236 Only fast motoneurons express the marker matrix metalloproteinase-9 (MMP-9) (30,
237 37). Our observations confirmed that larger motoneurons co-expressed Hb9-GFP and MMP-9,
238 while the smaller ones did not (Fig. 1 C1-C4). The mean cross-sectional area of MMP-9-negative
239 neurons was 280 ± 12 µm², while that of MMP-9-positive neurons was 907 ± 26 µm² (P<0.001, n
240 = 288; Fig. 1C2). Consistent with previous observations (38), we noted a fairly clear demarcation
241 in motoneuron characteristics around the 400 µm² cross-sectional area. Specifically, most of the
242 motoneurons smaller than 400µm² were HB-9⁺/MMP-9⁻ (86.75%, n=144/166), which can be
243 presumed to be slow motoneurons. Conversely, those with a cross-sectional area exceeding
244 400µm² were virtually all HB-9⁺/MMP-9⁺ (99.2%, n=121/122; Fig. 1C3, C4), suggesting their
245 classification as putative fast motoneurons. This pattern led us to adopt the 400 µm² threshold
246 as a practical and empirically supported cut-off, allowing us to grossly distinguish between fast
247 and slow motoneurons in our study.

248

249 Features of bistable motoneurons

250 We previously showed that under experimental conditions mimicking natural *in vivo*
251 conditions, most large motoneurons exhibit bistability in the absence of added
252 neuromodulators. This was achieved by maintaining *in vivo* calcium concentrations (1.2 mM)
253 (42) and keeping the preparation temperature above 30°C (18, 19, 43, 44). Four distinct features
254 characterized bistable motoneurons, which we have previously characterized (18, 23) (Fig. 2): 1
self-sustained firing triggered by a brief (2 sec) excitation when the motoneuron was pre-
depolarized near the spike threshold (Fig. 2A1); 2) a slow afterdepolarization (sADP) following
the current step if the motoneuron was not pre-depolarized sufficiently to trigger the self-
sustained firing (Fig. 2A1); 3) negative hysteresis during slow triangular current ramp injections,
where spiking stopped at lower currents than where it began (Fig. 2A2); 4) a slowly depolarizing
potential causing delayed spiking acceleration 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 negative 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 decelerating firing rate during the 2 sec depolarizing
pulse, leading to a post-step afterhyperpolarization (Fig. 2B1). Their activity during ramp current
injections showed positive 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 continuous firing deceleration (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 characteristics, scoring 2. The vast
majority of them (90 %, n=22/25) were not bistable, lacking self-sustained spiking, but meeting
two other criteria. This shows that bistability is not an all-or none property, but can manifest
with somewhat different properties.

275

276 Effects of size and age on bistability
As previously described, bistability in motoneurons can emerge early in development (18). Fig. 2C1 shows the average bistability scores of Hb9-GFP+ α-motoneurons during the initial postnatal month, comparing small neurons (<400µm²) to large neurons (>400µm²). 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 µm²) 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 after P9. In contrast, large motoneurons (>400 µm²) 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).

These data demonstrate a correlation between motoneuron soma size and the emergence of bistability in young mice. Fig. 2C2 shows the bistability score distribution by size across all ages. As described above, the large majority of small neurons (<400 µm²) 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 µm²) consistently had high bistability scores (mean 2.54± 1.43, n = 118; P<0.001); the largest (>800µm²) were the most bistable (mean 3.07 ± 1.11, n = 44) with 75% scoring 3-4. Intermediate size neurons (400-800 µm²) 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 proportion of bistable neurons rose, while the proportion of non-bistable cells decreased, especially above 400 µm². Another way to see this correlation was to measure
299 bistability score as a function of input resistance, which was lower in large motoneurons and
300 higher in small motoneurons. A strong negative correlation between input resistance and
301 bistability score was seen (Fig. 2C3; R² = 0.99). As a further control for the possible effects of
302 cell dialysis during whole cell recordings, we used the amphotericin B perforated patch method
303 (35) to record bistability from HB9-GFP neurons, whose contents were less disturbed. This
304 method reaffirmed the trend of increasing bistability with α-motoneuron size (n = 26; p<0.05;
305 Fig. 2C4).

306
307 Similar results collected from ChAT-GFP mice reinforce the size-related nature of bistable
308 properties (Fig 2C5). Before P7, small motoneurons (<400 µm²) showed no bistability, with an
309 average score of 0.56 ± 1.13, n = 9. In contrast, larger neurons (>400µm²) showed higher
310 bistability (score 2.1 ± 1.69, n = 16; p = 0.02).

311
312
Currents associated with bistability

313
The fact that large mouse motoneurons showed greater levels of bistability than small
314 motoneurons is unlikely to arise from the size difference alone, as larger neurons inherently
315 show lower overall excitability owing to their lower input resistance (45). Instead, we proposed
316 that large neurons selectively express higher levels of ionic currents that sustain the bistable
317 state than small neurons. Several ionic currents have been demonstrated to support the active
318 state during bistability. Among others, these include a persistent inward current (PIC) that may
319 comprise both sodium and calcium components (16, 21, 46, 47), a thermosensitive Trpm5
calcium-activated inward current (19), and a slow inactivation of the Kv1.2 potassium current (23).

We measured the PIC by delivering a slow ramp depolarization 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 (<400µm²: 33 ± 58 pA, n = 17; Fig. 3B), but much larger in large motoneurons (165 ± 172 pA, n = 24; p = 0.014), with its amplitude correlating positively with cell size without any change in the activation threshold (Fig. 3C). Moreover, the amplitude of the PIC was also found to be proportional 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 dramatic rise in the PIC amplitude (mean 198 ± 160.6 pA, n = 22, 0 with no PIC; p < 0.001). These findings highlighted the strong association 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 contribution of the sodium component of PICs in bistability, we added 10 mM TEA-Cl and 100 µM CdCl₂ to the recording ACSF (24). Large α-motoneurons displayed a more pronounced persistent sodium current (INaP; Fig. 3E). Although the motoneuron size did not influence the INaP threshold activation (Fig. 3G), the peak amplitude of INaP was significantly greater in large α-motoneurons (>400 µm², 1126 ± 530 pA, n = 11) compared to small motoneurons (<400 µm², 221 ± 104 pA, n = 10; p<0.001, Fig. 3F). This increase is not merely a reflection 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 contribution of INaP in bistability from large α-motoneurons and its diminished presence in smaller, non-bistable neurons.

A calcium-activated inward current was shown to be pivotal in sustaining the plateau depolarization underlying the tonically firing active state in bistable crab (22) and turtle motoneurons (48); this current has recently been shown to be mediated by Trpm5 channels in mice (19). To measure the effect of this current, we induced a 2-sec depolarization in the presence of tetrodotoxin (TTX, 1 µM) and tetraethylammonium-chloride (TEA, 10 mM) to minimize sodium and potassium currents. This depolarization was large enough to elicit a series of slow calcium-driven spikes to fully activate Trpm5. Subsequently, we measured the resulting afterdepolarization (sADP; Fig. 4A), which slowly declined as the Trpm5 current decayed (19). Notably, the sADP was partially blocked by Triphenylphosphine oxide (TPPO, 50 µM Fig. 4A), a known Trpm5 channel blocker (19, 49). The residual TPPO-insensitive sADP appeared to predominantly arise from channels that are not yet identified (see discussion in (19)). Thus, to accurately estimate the Trpm5 component of the sADP, we subtracted the sADP measured in TPPO from the total sADP. The resulting Trpm5 sADP was only detectable in one of 15 smaller neurons (<400 µm²: 0.2 ± 0.92 mV, n = 15), but was evident in most of the larger neurons (>400 µm²: 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-activated 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 µm², 324 ± 146.3 pA & 2.7 ± 1.38 pA/pF, n = 8) compared to the small ones (<400 µm², 26 ± 7.51 pA & 0.7 ± 0.67 pA/pF, n = 3) (Fig. 4E-F; P<0.05). Thus, the calcium-activated inward current mediated by Trpm5 channels (19) 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 inactivation delays the initiation and acceleration of firing of bistable neurons during long current steps (23). Interestingly, in our earlier work, only bistable neurons showed the delayed initiation and acceleration of firing during long current steps, induced by closure of the Kv1.2 channels; non-bistable neurons showed immediate spike onset with spike deceleration (23). We tested whether the Kv1.2 inactivating 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 after inactivation 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 µm²) 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. 4I).

All together, our findings highlight that INaP, the Trpm5 mediated calcium-activated inward current, and the slow inactivating Kv1 current are pivotal indicators of bistability in large α-motoneurons.
387 Serotonin effects on bistability in partially bistable neurons

388 We found that bistability is not strictly binary, as 26% (52 out 199) of α-motoneurons
389 displayed some, but not all, of the defining features described above, especially the absence of
390 persistent firing after the brief depolarization. It has been known for many years that
391 neuromodulators such as serotonin (5-HT) can evoke bistability in α-motoneurons which show
392 only partial bistable properties (14, 17, 25, 50).

393 We analyzed the effects of 10 µM 5-HT on α-motoneurons (n = 45) that had bistability scores
394 below 4 and lacked the self-sustained spiking. Fig. 5A shows an example of an α-motoneuron
395 that, under control conditions (i.e. recording aCSF), did not show acceleration of spiking during
396 a 2-sec depolarization, and only showed a modest sADP at the end of the depolarization. Seven
397 minutes after application of 5-HT, this motoneuron demonstrated accelerating spiking during
398 the current step, and self-sustained spiking activity after the end of the step. This activity was
399 terminated only by a hyperpolarizing step, indicating that the neuron became fully bistable only
400 in the presence of 5-HT. Fig. 5B shows the ability of 5-HT to evoke bistability in α-motoneurons
401 of different sizes. Notably, small α-motoneurons (< 400μm²; n = 19) remained non-bistable even
402 with 5-HT. In contrast, larger neurons exhibited the potential for a transition to bistability upon
403 5-HT exposure. Specifically, of non-bistable α-motoneurons of larger size (>400μm²), 38.5%
404 switched to full bistability with 5-HT (n = 26; p < 0.001). It is noteworthy that the majority of
405 neurons transitioning to bistability with 5-HT already expressed an sADP before 5-HT addition,
406 as illustrated in Fig. 5A. Thus, 5-HT was able to evoke full bistability in a subset of neurons
already leaning towards a bistable state, but could not evoke bistability in smaller or less bistable neurons.

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-sectional area and over time. The currents, Trpm5 and INaP, which contribute to the active state during bistability, and Kv1, whose inactivation helps to initiate sustained firing, are more pronounced in larger fast α-motoneurons which can be reliably identified 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 correlation between motoneuron size and bistability.

Research on bistability has primarily focused on larger α-motoneurons, identified by their ventrolateral location or by retrograde stimulation from ventral nerves (18, 27). This might have led to a failure to record the properties of smaller α-motoneurons. Neurons recorded in this study are motoneurons, as evidenced by their ventrolateral location and the expression of ChAT and Hb9 markers (Fig. 1A-B). Furthermore, they are α-motoneurons since most γ-motoneurons do not express NeuN (41), and do not express GFP in Hb9-GFP mice (38, 41). Finally, virtually all of the neurons larger than 400µm² also expressed MMP-9, indicative of fast α-motoneurons (30, 37). While there are no markers available to differentiate between the fast

DISCUSSION
429 fatigue-resistant (FR) and fast fatigable (FF) α-motoneurons, we conclude that the largest
430 neurons are fast motoneurons. The smallest neurons are presumably slow (S) α-motoneurons.
431 However, size alone is not a reliable indicator to separate these classes (51-53).

432 Electrophysiologically, large fast α-motoneurons differ from small slow α-motoneurons
433 by exhibiting a delayed and accelerated firing during prolonged stimulation (27, 29, 52). Our
434 experiments confirm this observation, as near-threshold current steps resulted in a slow
435 depolarization in larger α-motoneurons leading to delayed firing acceleration (Fig. 2). In
436 contrast, smaller α-motoneurons fired immediately upon reaching spike threshold followed by a
437 spike frequency deceleration (27, 30). The depolarization and spike delay in large fast α-
438 motoneurons were attributed to the slow inactivation of a Kv1 current (23, 30).

439 We now add bistability as a predominant property of large fast L4-L5 α-motoneurons in
440 young mice. Over 75% of neurons over 800μm² showed bistability, while only ~10% of neurons
441 less than 400 μm² were bistable. Consistent with this, a large majority of fast α-motoneurons
442 showed negative hysteresis during triangular ramp steps, where the derecruitment current was
443 lower than the recruitment current, while far fewer of the slow α-motoneurons displayed
444 negative hysteresis (30) (Fig. 2). The frequency of bistability in large α-motoneurons increased
445 during the first weeks of postnatal development, as previously described (18); (Fig. 2C1). By P21,
446 all large α-motoneurons were fully bistable, while smaller neurons continued to lack bistability
447 features.

448 The distinct firing behaviors observed between large and small α-motoneurons can be
449 attributed to a specific set of ionic currents that are more highly expressed in large fast α-
450 motoneurons compared to small slow α-motoneurons. One key contributor is the activation of
slow persistent inward currents (PICs), which may be carried by calcium or sodium. This
activation has been linked to negative hysteresis and bistability (15, 16, 19). Expression of the
PIC increases with the size of the α-motoneurons (Fig. 3B), and with the bistability score (Fig.
3D). Furthermore, our observations demonstrate the relationship between the size of the α-
motoneurons and the sodium component of PICs (INaP) (Fig. 3E-H).

Recently, the thermosensitive Trpm5 current has been identified as a calcium-activated
sodium current sustaining the plateau potential in bistable mouse α-motoneurons (19). We
found that the Trpm5-evoked afterdepolarization was present in virtually all large bistable α-
motoneurons, but not detectable in small non-bistable α-motoneurons (Fig. 4A-C). Finally, the
slowly inactivating Kv1.2-mediated current responsible for the delayed firing acceleration in
large motoneurons (23) (Fig. 2), is less expressed in smaller motoneurons (Fig. 4G-I).
Interestingly, this current positively correlates with the degree of bistability (Fig. 4G-I; (23)). It is
likely, that the slow inactivation of Kv1 will shift the balance of currents towards depolarization,
and will help to sustain continued 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 duplication of effort in these currents. Indeed, among neurons that showed
continued bistable firing, 20% either did not show a marked negative hysteresis during ramps,
or did not show a delayed firing acceleration. 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 (14-16, 54). However, we have revealed inherent bistability in most large α-motoneurons, provided the recording temperature is sufficiently high (above 30°C) (18), to unmask the thermosensitive Trpm5 current responsible for the plateau potential (19). In our preparations, some α-motoneurons expressed intermediate properties and were not fully bistable under these conditions. Addition of 5-HT evoked full bistability in ~30% and ~60% of the intermediate and large neurons, respectively (Fig. 5). Interestingly, α-motoneurons completely lacking baseline bistability characteristics (bistability score 0) never became bistable with 5-HT. We propose that intermediate α-motoneurons express some of the essential currents for bistability, but at lower levels (Fig. 5). The known modulatory actions of 5-HT can then enhance these currents to sustain prolonged firing in the bistable state.

Our demonstration of a size principle for bistability contrasts with the groundbreaking work by Lee and Heckman (15, 16), who studied α-motoneurons in adult cats. In their study, they achieved a full bistability in about one-third of the α-motoneurons, which interestingly exhibited characteristics of smaller motoneurons. There are several potential explanations 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 α-motoneurons are inherently more excitable than cat α-motoneurons, primarily due to their smaller size (34, 55). Despite this, the PIC amplitude is relatively similar across both species, suggesting that PIC has a more significant impact on the firing rate in mice. Third, our
494 observations were made on mouse α-motoneurons during the first 4 weeks of life, while Lee 495 and Heckman's experiments were made on adult cats. However, bistability in the larger 496 neurons became more pronounced with age in the mouse neurons. In young adult mice (P21- 497 P25) all large motoneurons exhibited bistability, whereas none of the smaller ones did. Finally, 498 our results were made ex vivo from slices in the presence of blockers of fast synaptic 499 transmission (though not at P21/P25) and mostly without neuromodulators. On the other hand, 500 the cat neurons were recorded in vivo in the presence of methoxamine to maximize the 501 incidence of bistability.

502 The role of bistability in spinal α-motoneurons remains unclear. It has been recorded 503 during quiet standing in both rats and cats (56), suggesting a potential significance for postural 504 control (15, 16, 28, 50). The Henneman Size Principle (57) posits that smaller α-motoneurons, 505 due to their lower input conductance, are the first to be recruited, potentially playing a role in 506 maintaining posture. This can be consistent with earlier work in cats, where smaller neurons 507 showed full bistability and were possibly active during quiet standing (15, 16, 58). However, we 508 here demonstrate, in younger mice, that small α-motoneurons are not bistable. Instead, 509 bistability increases with the size of motoneurons (Fig. 2). Ritter et al. (58) provided evidence 510 that large fast α-motoneurons may also be tonically recruited during quiet standing in mice. An 511 intriguing study by Bos et al. (19) showed that mice lacking Trpm5 channels in lumbar 512 motoneurons exhibited compromised postural control. Given the central role of Trpm5 in 513 bistable properties of larger α-motoneurons (19) (Fig 4), it is possible that fast α-motoneurons 514 may play a role in postural maintenance. Alternatively, the currents responsible for bistability 515 may play a more important role in recruiting the less excitable large motoneurons. The PIC
516 plays in important role in the initiation of bistable firing, but is also critical for repetitive firing in
517 α-motoneurons (59), and in synaptic amplification due to their dendritic location (1). Thus, the
518 currents which together lead to bistability may individually play multiple roles in motor control
519 in the mouse. These assumptions should be further investigated in the future using more
520 integrated *in vivo* preparations.
521 FIGURE LEGENDS

522

523 **Figure 1: Identification and size distribution of α-motoneurons in the ventrolateral spinal cord at L4-L5.** A: Ventrolateral cord at P12 showing Hb9-GFP (green), NeuN (red) and double labeling (yellow). B1: The vast majority (98%, n = 141/144) of Hb9-GFP-labeled neurons co-express NeuN. B2: Cross-sectional area distribution of Hb9-GFP neurons at P4-P6 (light green, n=571 neurons), P8-P10 (medium green, n=475 neurons) and P12 (dark green, n=288 neurons). C1: Ventrolateral cord at P12 showing Hb9-GFP (green), MMP-9 (red) and dual labeling (yellow). C2: Size distribution of Hb9-GFP labeled neurons expressing (n=122) or not expressing (n=166) MMP-9. C3: Histogram of size distribution of GFP⁺-MMP-9⁻ (green) or HB9⁺-MMP-9⁺ (yellow) neurons. C4: Double-labeled Hb9-GFP⁺/MMP-9⁺ neurons are predominantly larger than 400 µm² (yellow, top) while most Hb9-GFP⁺/MMP-9⁻ neurons are smaller than 400 µm² (green, bottom). C2-C4: n=288 neurons. *** p<0.001 (two-tailed Mann-Whitney test for C2; Fisher's exact test for C4). Mean ± S.D. for B1. Median (red solid line) and quartiles (red dashed lines) are represented for C2.

536

537 **Figure 2: Bistability score in spinal motoneurons varies by age and size.** A: Fully bistable neuron (bistability score 4). A1: Baseline depolarization towards spike threshold: a 2-sec suprathreshold stimulation evokes a prolonged afterdepolarization (ADP: 1 point). Slightly higher baseline depolarization followed by a 2 sec stimulation leads to prolonged firing which is only terminated by hyperpolarizing current step (1 point); trace has been offset to be more easily seen. A2: Negative hysteresis during ramp stimulation. The current threshold for onset of
spiking is higher than the threshold for offset of spiking (-ΔI; 1 point). **A3:** A small subthreshold
current step leads to a slow depolarization (trace has been offset to be more easily seen). A
slightly higher current step leads to a larger depolarization leading to delayed spike onset and
acceleration of spike frequency during the step (1 point). **B:** Completely non-bistable neuron
(bistability score 0). **B1:** Current steps to near threshold lead to decelerating spike frequency
during a 2 sec suprathreshold pulse, and an afterhyperpolarization at the end of the step (0
point). **B2:** Positive hysteresis during ramp stimulation. 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 depolarization. A slightly higher current step evokes
immediate onset firing with decelerating spike frequency during the step (0 point). Size
markers: 1 sec, 20 mV. **C1:** Bistability score as a function of postnatal age for smaller (<400μm²,
blue, n=81) and larger (>400μm², red, n=118) Hb9-GFP⁺ neurons. Statistical comparison was
done for each age and when all ages were pooled. **C2:** Distribution of bistability as a function of
neuronal cross-sectional 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 function of neuronal input
resistance. A strong negative correlation is seen (R²= 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 identified by expression of ChAT-GFP show similar bistability scores that rise with
cross-sectional area (blue for <400μm², n=16 and red for >400μm², n=9; p=0.02). * p<0.05; ***
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:** Persistent inward current (PIC), and its sodium component, is larger in bistable neurons. **A:** Voltage clamp measure of PIC activation during slow voltage ramp in a bistable but not in a non-bistable neuron. **B-C:** PIC amplitude (B), and threshold (C) as a function of cell cross-sectional area of Hb9-GFP⁺ neurons (blue circles, <400µm², n=17 (B), n=9 (C) and red circles, >400µm², n=24 (B), n=17 (C); P=0.014 (B); P=0.72 (C)). **D:** PIC amplitude increases with increasing bistability (Green: bistability scores 0-1, n=15; yellow: bistability scores 3-4, N=22; P=0.001). **E:** Superimposed leak-subtracted sodium persistent current (INaP) recorded from Hb9-GFP⁺ motoneurons in the presence of 10 mM TEA and 100µM CdCl₂ (area <400µm² or >400µm² for the blue and red trace, respectively) in L4-L5 regions at P9 in response to a slow ramp depolarization. **F-H:** INaP peak amplitude (F), threshold (G) and density (H) as a function of cell cross-sectional area of Hb9-GFP⁺ neurons (blue circles, <400µm², n=10 and red circles, >400µm², n=11; P=0.001). ns, no significance; \*p<0.05; \*\*\*p<0.001 (two-tailed Mann-Whitney test for B-D and F-H). Median (solid line) and quartiles (dashed lines) are represented in each violin plot.

**Figure 4:** Trpm5-mediated current and slowly inactivating Kv1 current are signatures of bistability. **A:** Neurons are depolarized for 2 sec in presence of 1µM TTX and 10 mM TEA. Above a threshold, large calcium-dependent oscillations are evoked. In bistable motoneurons (red trace), these elicit a large, slow afterdepolarization which is partially blocked by 50µM
586 TPPO (black trace). Non-bistable neurons (blue trace) do not express this slow
587 afterdepolarization. B: Amplitude of the Trpm5-induced, TPPO-resistant afterdepolarization is
588 larger in large motoneurons (>400 µm², n=9) than small motoneurons (<400 µm², n=15;
589 p=00017). C: Trpm5-induced, TPPO-resistant afterdepolarization is larger in bistable
590 motoneurons (score 3-4, n=10) than nonbistable motoneurons (score 0-1, n=10; p=0.0108). D:
591 TPPO-sensitive Trpm5 current is isolated in response to a voltage step of 2 sec in presence of
592 1µM TTX and 10 mM TEA. Superimposed traces of Trpm5 isolated current from large (>400µm²,
593 red) vs small (<400µm², blue) Hb9-GFP+ motoneurons. Note that the current trace during the
594 depolarizing step has been truncated for improved visualization of the post-step current. E-F:
595 Amplitude (E) and density (F) of the Trpm5 current increases with cross-sectional area of Hb9-
596 GFP+ neurons (blue circles, <400µm², n=3, and red circles, >400µm², n=8; p=0,0121 and p=0242,
597 respectively) G: Superimposed traces of the slowly inactivating Kv1 current in bistable (red) and
598 non-bistable (blue) neuron in response to a long depolarizing voltage step in presence of 1µM
599 TTX and 10 mM TEA. H-I: Amplitude (H) and density (I) of Kv1 current increases with cross-
600 sectional area of Hb9-GFP+ neurons (blue circles, <400µm², n=19 (H), n=3 (I) and red circles,
601 >400µm², n=26 (H) and n=7 (I); p=0.0216 and p=0.0167). \*p\<0,05; \*\*p\<0,01 (two-tailed Mann-
602 Whitney test for B-C, E-F and H-I). Median (solid line) and quartiles (dashed lines) are
603 represented in each violin plot.

604

605 **Figure 5: Induction of full bistability by serotonin.** A: Non-bistable neuron's response (top, black trace) to 2 sec depolarizing pulse (bottom). Only a small afterdepolarization is recorded after the step. During 10µM serotonin (5-HT), this neuron became fully bistable, with
606
607
608 continuous firing (top, red trace) following the 2 sec depolarizing step (bottom). B: Not all non-
609 bistable neurons respond to serotonin (n=45). Smaller neurons (<400 µm², n=19) fail to
610 show full bistability (score 4) with 5-HT. 38% of larger (>400 µm²) non-bistable neurons became
611 bistable during 5-HT. *** p<0.001 (Fisher's exact test for B).

612

613 DISCLOSURES

614

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

616

617 AUTHOR CONTRIBUTIONS:

618

619 R. H-W, R.B. and F.B. generated the hypotheses for the paper. R. H-W, B.D. and R.B. performed
620 electrophysiological experiments and analysis; E.P. performed immuno-histochemical
621 experiments and analysis. R. H-W drafted the manuscript, and R. H-W, R.B. and F.B. revised the
622 manuscript and approved the final version of the manuscript.

623

624 ACKNOWLEDGEMENTS

625

626 This work was supported by NIH grant NS17323 (R. H-W), by Fonds d'investissement INT
627 FI_INT_JCJC_2019 (R.B.), by Centre National de la Recherche Scientifique (CNRS) (R.B. and F.B.)
628 and by Agence Nationale de la Recherche Scientifique ANR-16-CE16-0004 (F.B.).

629
630 AUTHOR NOTES

631
632 Correspondence: R. M. Harris-Warrick, rmh4@cornell.edu; F. Brocard, frederic.brocard@univ-amu.fr; R. Bos, remi.bos@univ-amu.fr

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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>
Effect of size on expression of bistability in mouse spinal motoneurons

<figure><img src="image_8.png" /></figure>

Large mouse spinal motoneurons exhibit sustained firing after stimulation due to expression of ionic currents supporting bistability. Small spinal motoneurons