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1
Effect of size on expression of bistability in mouse spinal 2
motoneurons 3
4
Ronald M Harris-Warrick1, Emilie Pecchi2, Benoît Drouillas2, Frédéric Brocard2,*, Rémi 5
Bos2,* 6
7
1: Department of Neurobiology and Behavior, Cornell University, Ithaca NY 14850, 8
United States; 2: Aix Marseille Univ, CNRS, Institut de Neurosciences de la Timone 9
(INT), UMR 7289, Marseille, France 10
11
12
13
14
15
16
17
18
19
*: These authors contributed equally 20
21
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Abstract 22
23
Bistability in spinal motoneurons supports tonic spike acvity in the absence of 24
excitatory drive. Earlier work in adult preparaons suggested that smaller motoneurons 25
innervang slow angravity muscle fibers are more likely to generate bistability for postural 26
maintenance. However, whether large motoneurons innervang fast-fagable muscle fibers 27
display bistability is sll controversial. To address this, we examined the relaonship between 28
soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of ChAT-GFP and Hb9-29
GFP mice during the first four weeks of life. We found that as neuron size increases, the 30
prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast α-31
motoneurons (MMP-9+/Hb9+) with a soma area ≥ 400µm2 exhibit significantly higher bistability. 32
Ionic currents associated with bistability, including the persistent Nav1.6 current, 33
thermosensive Trpm5 Ca2+-acvated Na+ current and the slowly inacvang Kv1.2 current, also 34
scale with cell size. Serotonin evokes full bistability in large motoneurons with paral bistable 35
properes, but not in small motoneurons. Our study provides important insights into the neural 36
mechanisms underlying bistability and how motoneuron size correlates with bistability in mice. 37
38
Keywords: Motoneuron, Spinal cord, Bistability, Current, Development, Posture, Serotonin, 39
Neuromodulaon 40
41
42
43
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New and Noteworthy 44
Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow 45
motoneurons but present in most large, fast motoneurons. This difference results from 46
differenal expression of ionic currents that enable bistability, which are highly expressed in 47
large motoneurons but small or absent in small motoneurons. These results support a possible 48
role for fast motoneurons in maintenance of tonic posture in addion to their known roles in 49
fast movements. 50
51
52
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INTRODUCTION 53
54
Spinal motoneurons not only transmit central commands for movement to muscles but 55
also shape motor output and muscle contracon through nonlinear firing properes (1). One 56
such property is bistability, where the motoneuron can switch between stable silent and acve 57
states, depending on transient synapc input or current injecon. Originally detected as plateau 58
potenals in invertebrate neurons (2-9), bistability was soon found in vertebrates (10), 59
specifically in spinal motoneurons (11-16). While oen induced by neuromodulators such as 60
serotonin (13, 15-17), bistability can also arise when motoneurons are depolarized to near 61
threshold and recorded under natural ionic and temperature condions (18, 19). 62
The acve state in bistable motoneurons is mainly mediated by slow ionic currents 63
including persistent Cav1.3 (13-16) and Nav1.6 currents (20, 21), and the Trpm5-mediated Ca2-64
acvated Na+ current (18, 19, 22). Drawing from a series of our previous invesgaons, we can 65
summarize the process as follows: The inial depolarizaon, caused by the slow inacvaon of 66
Kv1.2 potassium channels (23), acvates the persistent Nav1.6 current leading to spiking acvity 67
(24). This then prompts Ca2+ entry through the recruitment of Cav1.3 channels, iniang a Ca2+-68
induced Ca2+-release process (18). This process ulmately acvates thermosensive Trpm5 69
channels, which are the primary source of the plateau depolarizaon to sustain repeve firing 70
(19). Other currents such as the HCN-type hyperpolarizaon-acvated inward current, Ih (25, 71
26) and reducon of calcium-acvated outward currents (17, 25) are also involved in different 72
neurons. 73
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α-motoneurons, which drive extrafusal muscle contracons, can be subdivided 74
funconally into three classes based on size and the muscle type they innervate: large fast-75
fagable (FF), medium fast fague-resistant (FR), and small slow (S) motoneurons [reviewed by 76
(27)]. In decerebrate cats, small motoneurons characterized by slow conducon velocies and 77
low acvaon thresholds, appear to have more pronounced bistability than large ones (15, 16). 78
This observaon suggests that S or FR α-motoneurons show higher bistability than those linked 79
to FF muscle fibers. Notably, S motoneurons, primarily associated with postural adjustments 80
and slow movements, have been hypothesized to use their bistability for efficient postural 81
maintenance, ensuring minimal energy expenditure (15, 16, 28). In mice, the staonary posture 82
also involves the acvaon of fast motor units (57). Interesngly, our recent findings indicate 83
that large motoneurons exhibit bistable firing paerns, which are crucial for the control of 84
hindlimb movement (19). From as early as the 2nd postnatal week, large motoneurons in mice 85
present an electrophysiological profile that is disnct from that of smaller ones, marked by a 86
lower input resistance, a higher rheobase, more depolarized spike thresholds, narrower acon 87
potenals, and shorter aerhyperpolarizaons (27, 29, 30). They also exhibit delayed onset of 88
firing and firing acceleraon during a step depolarizaon, and receive a consistently higher level 89
of recurrent excitaon (23, 31-33). Despite this extensive characterizaon, the comparison of 90
bistability between large and small motoneurons in mice remains unexplored. While 91
comparisons with cat studies might lead us to hypothesize increased bistability in smaller 92
mouse motoneurons, cauon is warranted due to the disnct characteriscs of mouse spinal 93
motoneurons that are not seen in cats (34). 94
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In the present study, we examined the bistability as a funcon of size in genecally 95
labeled L4-L5 α-motoneurons in young mice (postnatal day (P) 1-25). Our data indicate that the 96
largest α-motoneurons showed stronger bistable firing properes, while the smallest 97
motoneurons were rarely bistable. There was a strong correlaon between bistability and the 98
amplitudes of the ionic currents known to support it. Motoneurons of intermediate size oen 99
showed paral bistability, which could be converted to full bistability by serotonin. These 100
unexpected results suggest new hypotheses regarding the contribuon of larger motoneurons 101
in bistability and postural control. 102
103
MATERIALS AND METHODS 104
105
Animals and ethical standards 106
Hb9-GFP mice were kindly provided by B. Pemann and Jackson Laboratories (strain 005029). 107
ChAT-Cre mice were obtained from Jackson Laboratories (strain 007902) and crossed to Rosa-108
26-floxed GFP mice. Mice of both sexes were used in all experiments. Animals were housed on 109
a 12 hr day/night cycle with ad libitum access to water and food. The room temperature was 110
maintained between 20 and 22°C. 111
Cornell: All animal protocols were approved by the Cornell Instuonal Animal Use and Care 112
Commiee and were in accordance with NIH guidelines. Marseille: All animal care and use 113
conformed to French regulaons (Décret 2010-118) and were approved by the local ethics 114
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commiee (Comité d’Ethique en Neurosciences INT-Marseille, CE71 Nb A1301404, 115
authorizaon Nb 2018110819197361). 116
117
Slice Preparaon 118
For electrophysiological experiments, mice were cryoanaesthezed (P2-P7) or anaesthezed 119
(P8-P25) with intraperitoneal injecon of a mixture of ketamine/xylazine (100mg/kg and 10 120
mg/kg, respecvely). They were then decapitated, eviscerated and the spinal cord removed by 121
laminectomy, and placed in a Sylgard-lined petri dish with ice cold (1-2°) aCSF containing (in 122
mM): 252 sucrose, 3 KCl, 1.25 KH2PO4, 4 MgSO4, 0.2 CaCl2, 26 NaHCO3, 25 D-glucose, pH 7.4, 123
bubbled with 95% O2 and 5% CO2. The meninges were removed and the posterior cord (L3-S5) 124
imbedded in agarose. The same soluon was used for slicing. 325-350µm secons were 125
prepared from the L4-L5 region and superfused at 3 ml/min with recording aCSF containing (in 126
mM): 120 NaCl, 3 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 1.2 CaCl2, 25 NaHCO3, 20 D-glucose, pH 7.4, 127
32-34°C (19, 23). In most experiments (P1-P14), blockers of fast synapc transmission (CNQX 128
and D,L-AP5 or kynurenic acid, strychnine, and bicuculline) were added in the aCSF to minimize 129
synapc contribuons to bistability. To isolate the persistent Na
+ currents (INaP) during 130
voltage-clamp experiments we used a modified aCSF containing (in mM): 100 NaCl, 3 KCl, 1.25 131
NaH2PO4, 1.3 MgSO4, 3.6 MgCl2, 1.2 CaCl2, 25 NaHCO3, 40 D-glucose, 10 TEA-Cl and 0.1 CdCl2 132
(24). 133
134
Mature mice (P21-25) were treated as the younger mice, except that the cord was sliced in an 135
ice cold slicing soluon (1-2°) containing (in mM): 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, 136
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25 D-glucose, 3 kynurenic acid, and pH 7.4 with NaOH (33). Aer a resng period of 30-60 min, 137
slices were transferred to the recording chamber and superfused with recording aCSF at 32°C 138
(35) without addion of fast synapc transmission blockers. 139
140
Electrophysiological recordings 141
142
Hb9-GFP and ChAT-GFP posive neurons were visualized in the ventrolateral region of lamina IX 143
in L4-L5 horizontal slices using an Olympus BX51 microscope with appropriate filters. Whole-cell 144
patch-clamp recordings from visualized fluorescent GFP+ neurons were made with electrodes 145
(2-6MΩ) pulled from borosilicate glass capillaries (1.5 mm OD, 1.12 mm ID; World Precision 146
Instruments). They were filled with a pipee soluon containing (in mM): 140 K+-gluconate, 5 147
NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP, 0.4 GTP, pH 7.3. Patch clamp recordings were made 148
using a Mulclamp 700B amplifier driven by PClamp 10 soware (Molecular Devices). 149
Recordings were digized on-line and filtered at 10 kHz (Digidata 1322A or 1440A, Molecular 150
Devices). Pipee and neuronal capacive currents were canceled, and, aer breakthrough, 151
series access resistance was compensated. The recording was allowed to stabilize for at least 2 152
min aer establishing whole-cell access before recording was started (19, 23). Unless otherwise 153
indicated, all neurons were held at -70 mV to ensure reproducibility in expression of voltage-154
sensive currents. 155
156
Drug list: All soluons were oxygenated with 95% O
2/5% CO2. All salt compounds, as well as 157
tetraethylammonium chloride (TEA; #T2265), Triphenylphosphine oxide (TPPO; #T84603), 158
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Serotonin creanine sulfate monohydrate (5-HT; #H7752), 6-Cyano-7-nitroquinoxaline-2,3-159
dione (CNQX, #5.04914), D-(-)-2-Amino-5-phosphonopentanoic Acid (DL-AP5 ; #165304), 160
strychnine (#S0532), bicuculline (#5.05875) , Kynurenic Acid (#K3375) and Amphotericin B 161
(#A4888) were obtained from Sigma-Aldrich. Tetrodotoxin (TTX; #1078) was obtained from 162
Tocris Bioscience. 163
164
Immunohistochemistry 165
To idenfy neurons expressing specific proteins, spinal cords from 10-12-day-old Hb9-GFP mice 166
were removed in ice cold (1-2°) aCSF containing (in mM): 252 sucrose, 3 KCl, 1.25 KH2PO4, 4 167
MgSO4, 0.2 CaCl2, 26 NaHCO3, 25 D-glucose, pH 7.4, bubbled with 95% O2 and 5% CO
2. The 168
spinal cords were fixed for 5-6 h in 4% paraformaldehyde (PFA) prepared in phosphate buffer 169
saline (PBS), then rinsed in PBS and cryoprotected overnight in 20% sucrose-PBS at 4°C. Spinal 170
cords were frozen in OCT medium (Tissue Tek), and 30 μm cryosecons were collected from the 171
L4-L5 segments (23). Aer washing in PBS 3×5 min, the slides were incubated for 1 h in a 172
blocking soluon (BSA 1%, Normal Donkey Serum 3% in PBS) with 0.2% Triton X-100 and for 12 173
h at 4 °C in a humidified chamber with the knockout-validated primary anbody: mouse-an-174
NeuN (Neuronal Nuclei, Sigma-Aldrich MAB377) (36) or goat-an-MMP-9 (Matrix 175
metallopepdase 9, Sigma-Aldrich M9570)(30, 37). Both anbodies were diluted in the blocking 176
soluon with 0.2% Triton X-100 (1:1000 and 1:500 for an-NeuN and an-MMP-9, respecvely). 177
Slides were washed 3×5 min in PBS and incubated for 2 h with an Alexa Fluor® Plus 555- 178
conjugated secondary anbody (Invitrogen A32816) diluted in the blocking soluon. Aer 3 179
washes of 5 min in PBS, they were mounted with a gelanous aqueous medium. 180
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181
Data analysis 182
Electrophysiological recording: 183
Electrophysiological data were analyzed with Clampfit 10 soware (Molecular Devices). Several 184
basic criteria were set to ensure opmum quality of intracellular recordings. Only cells with a 185
stable membrane potenal below -60 mV, stable access resistance (no more than 20% 186
variaon), and acon potenal amplitude larger than 60 mV were analyzed. Reported 187
membrane potenals were not corrected for liquid juncon potenals. 188
Confocal imaging:
189
Immunofluorescent staining was quanfied from confocal images acquired with the 40X 190
objecve (LSM700 microscope, Zeiss, Germany) on at least two slices per mouse and within the 191
ventro-lateral area (300 X 300 µm
2) from each slice. The cell body cross-seconal area was 192
manually segmented and quanfied from stacked confocal images (5 steps ; Z-step, 3 µm) with 193
Zen soware (Zeiss). An intensity threshold in each slice was applied to determine the presence 194
of NeuN or MMP9 staining from each segmented GFP+ motoneuron. 195
196
197
Stascs 198
No stascal method was used to predetermine sample size. Stascal analysis was carried out 199
using GraphPad Prism and Matlab (MathWorks) soware. When two groups were compared we 200
used, the Mann-Whitney test. Fisher’s exact test was used for comparing cell proporons 201
between two groups. In some cases, we also compared the slope of the simple linear 202
regressions between two groups. The level of significance was set at p <0.05. Each stascal test 203
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is indicated in the figure legends and provided in more detail in the Source Data File. In the 204
figures and in the main text, data are presented as mean ± SD for the histograms. Median and 205
quarles are represented in each violin plot. 206
Source Data File: 207
hps://github.com/remibos/Source-Data-File-HW.git 208
209
210
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RESULTS 211
212
Immunohistochemical idenficaon of fast α-motoneurons 213
214
Motoneurons in the ventrolateral spinal cord were inially idenfied in unfixed L4-L5 215
horizontal slices by their GFP fluorescence in choline acetyltransferase (ChAT)-tagged ChAT-GFP 216
mice. We found a number of labeled neurons of many sizes (from <200 µm2 to >800 µm2). 217
However, these include both α-motoneurons driving extrafusal muscle fibers and smaller γ-218
motoneurons innervang intrafusal muscles that regulate muscle spindle responsiveness to 219
stretch. These motoneuron subtypes can be disnguished by differenal expression of a 220
number of proteins. While the transcripon factor Hb9 is a marker for both α- and γ-221
motoneuron in Hb9-nls-LacZ animals (38), only α-motoneurons express GFP labeling in the Hb9-222
GFP mice (38). We thus used Hb9-GFP mice to idenfy α-motoneurons in the L4-L5 223
ventrolateral spinal cord, preferenally linked to hindlimb muscles (39, 40), from postnatal day 1 224
(P1) to P25. 225
The transcripon factor NeuN is commonly used to disnguish neurons from glial cells. 226
In the spinal cord, α-motoneurons express NeuN, whereas 2/3 of γ-motoneurons do not (41). 227
We examined co-expression of Hb9-GFP and NeuN immunolabeling in fixed ssues and, 228
regardless of age, we found that virtually all ventrolateral Hb9-GFP-labeled neurons exhibited 229
NeuN labeling (98 ± 2.4%, n = 144 cells), indicang they are likely α-motoneurons (Fig. 1A, B1). 230
We observed a preponderance of small labeled α-motoneurons during P4-6 (n = 571 neurons; 231
Fig. 1B2) and P8-10 (n = 425 neurons; Fig. 1B2), with 80.5% and 79.6% respecvely having a 232
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maximal cross seconal area of 400 µm2 or less. The percentage of large α-motoneurons 233
increased with age (38, 41), and by P12, only 50.4% of them remained under 400µm2 (n = 288 234
neurons; Fig. 1B2). 235
Only fast motoneurons express the marker matrix metalloproteinase-9 (MMP-9) (30, 236
37). Our observaons confirmed that larger motoneurons co-expressed Hb9-GFP and MMP-9, 237
while the smaller ones did not (Fig. 1 C1-C4). The mean cross-seconal area of MMP-9-negave 238
neurons was 280 ± 12 µm2, while that of MMP-9-posive neurons was 907 ± 26 µm2 (P<0.001, n 239
= 288; Fig. 1C2). Consistent with previous observaons (38), we noted a fairly clear demarcaon 240
in motoneuron characteriscs around the 400 µm² cross-seconal area. Specifically, most of the 241
motoneurons smaller than 400µm2 were HB-9+/MMP-9- (86.75%, n=144/166), which can be 242
presumed to be slow motoneurons. Conversely, those with a cross-seconal area exceeding 243
400µm2 were virtually all HB-9+/MMP-9+ (99.2%, n=121/122; Fig. 1C3, C4), suggesng their 244
classificaon as putave fast motoneurons. This paern led us to adopt the 400 µm² threshold 245
as a praccal and empirically supported cut-off, allowing us to grossly disnguish between fast 246
and slow motoneurons in our study. 247
248
Features of bistable motoneurons 249
We previously showed that under experimental condions mimicking natural in vivo 250
condions, most large motoneurons exhibit bistability in the absence of added 251
neuromodulators. This was achieved by maintaining in vivo calcium concentraons (1.2 mM) 252
(42) and keeping the preparaon temperature above 30°C (18, 19, 43, 44). Four disnct features 253
characterized bistable motoneurons, which we have previously characterized (18, 23) (Fig. 2): 1) 254
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self-sustained firing triggered by a brief (2 sec) excitaon when the motoneuron was pre-255
depolarized near the spike threshold (Fig. 2A1); 2) a slow aerdepolarizaon (sADP) following 256
the current step if the motoneuron was not pre-depolarized sufficiently to trigger the self-257
sustained firing (Fig. 2A1); 3) negave hysteresis during slow triangular current ramp injecons, 258
where spiking stopped at lower currents than where it began (Fig. 2A2); 4) a slowly depolarizing 259
potenal causing delayed spiking acceleraon in response to a near-threshold depolarizing step 260
(Fig. 2A3). We assigned each feature one point, with fully bistable motoneurons scoring 4 261
points. To be considered bistable, the motoneuron scored at least 3 points, including the 262
presence of a self-sustained firing and sADP, and either negave hysteresis or delayed firing. 263
Nearly a third of motoneurons (36%, n=71/199) did not express any of the bistability 264
criteria, scoring 0. They typically showed a decelerang firing rate during the 2 sec depolarizing 265
pulse, leading to a post-step aerhyperpolarizaon (Fig. 2B1). Their acvity during ramp current 266
injecons showed posive hysteresis, where spiking ceased at higher current values than where 267
it started (Fig. 2B2). They also began spiking immediately during a suprathreshold long step, 268
with connuous firing deceleraon (Fig. 2B3). Some motoneurons, despite having one scoring 269
feature, lacked self-sustained firing and were also considered as non-bistable. A significant 270
number of motoneurons also displayed intermediate characteriscs, scoring 2. The vast 271
majority of them (90 %, n=22/25) were not bistable, lacking self-sustained spiking, but meeng 272
two other criteria. This shows that bistability is not an all-or none property, but can manifest 273
with somewhat different properes. 274
275
Effects of size and age on bistability 276
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As previously described, bistability in motoneurons can emerge early in development 277
(18). Fig. 2C1 shows the average bistability scores of Hb9-GFP+ α-motoneurons during the inial 278
postnatal month, comparing small neurons (<400µm2) to large neurons (>400µm
2). Small 279
motoneurons consistently showed minimal bistability scores across all developmental ages (Fig. 280
2C1), rising up to a mean of 1.14 ± 0.31 by P9-14, then nearly vanishing by early adulthood 281
(P21-25). 54 out of 81 small cells (<400 µm2) displayed a bistability score of 0, and only 9 out of 282
81 small cells showed bistability (scores 3-4) at any age, with 55% appearing aer P9. In 283
contrast, large motoneurons (>400 µm2) exhibited increasing bistability, reaching near maximal 284
levels around P9 with a mean score of 3.24 ± 0.28. Remarkably, 83% (n = 24) of these large 285
motoneurons exhibited high bistability scores (3-4) at P9-P14, with all displaying bistability by 286
P21-25 (n = 7). Note that the bistability scores of large motoneurons were significantly higher 287
compared to small ones when we pooled all ages (P<0.001, n=199). 288
These data demonstrate a correlaon between motoneuron soma size and the 289
emergence of bistability in young mice. Fig. 2C2 shows the bistability score distribuon by size 290
across all ages. As described above, the large majority of small neurons (<400 µm2) were not 291
bistable, averaging a score of 0.67 ± 1.21 (n = 81); only 11 % achieved scores of 3-4. In contrast, 292
larger neurons (>400 µm2) consistently had high bistability scores (mean 2.54± 1.43, n = 118; 293
P<0.001); the largest (>800µm2) were the most bistable (mean 3.07 ± 1.11, n = 44) with 75% 294
scoring 3-4. Intermediate size neurons (400-800 µm2) showed largely a mixture of bistable and 295
intermediate bistability scores (mean 2.33 ± 1.51; n = 75). A clear gradient emerged: as neuron 296
size increased, the proporon of bistable neurons rose, while the proporon of non-bistable 297
cells decreased, especially above 400 µm2. Another way to see this correlaon was to measure 298
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bistability score as a funcon of input resistance, which was lower in large motoneurons and 299
higher in small motoneurons. A strong negave correlaon between input resistance and 300
bistability score was seen (Fig. 2C3; R2 = 0.99). As a further control for the possible effects of 301
cell dialysis during whole cell recordings, we used the amphotericin B perforated patch method 302
(35) to record bistability from HB9-GFP neurons, whose contents were less disturbed. This 303
method reaffirmed the trend of increasing bistability with α-motoneuron size (n = 26; p<0.05; 304
Fig. 2C4). 305
306
Similar results collected from ChAT-GFP mice reinforce the size-related nature of bistable 307
properes (Fig 2C5). Before P7, small motoneurons (<400 µm2) showed no bistability, with an 308
average score of 0.56 ± 1.13, n = 9. In contrast, larger neurons (>400µm2) showed higher 309
bistability (score 2.1 ± 1.69, n = 16; p = 0.02). 310
311
Currents associated with bistability 312
The fact that large mouse motoneurons showed greater levels of bistability than small 313
motoneurons is unlikely to arise from the size difference alone, as larger neurons inherently 314
show lower overall excitability owing to their lower input resistance (45). Instead, we proposed 315
that large neurons selecvely express higher levels of ionic currents that sustain the bistable 316
state than small neurons. Several ionic currents have been demonstrated to support the acve 317
state during bistability. Among others, these include a persistent inward current (PIC) that may 318
comprise both sodium and calcium components (16, 21, 46, 47), a thermosensive Trpm5 319
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calcium-acvated inward current (19), and a slow inacvaon of the Kv1.2 potassium current 320
(23). 321
We measured the PIC by delivering a slow ramp depolarizaon in voltage clamp (Fig. 322
3A). The PIC amplitude was measured at the inward peak from the extrapolated passive 323
component at the same voltage (See Fig. 3A). The PIC amplitude was very small or absent in 324
small Hb9-GFP+ motoneurons (<400µm2: 33 ± 58 pA , n = 17; Fig. 3B), but much larger in large 325
motoneurons (165 ± 172 pA, n = 24; p = 0.014), with its amplitude correlang posively with 326
cell size without any change in the acvaon threshold (Fig. 3C). Moreover, the amplitude of the 327
PIC was also found to be proporonal to the bistability score (Fig. 3D). Specifically, non-bistable 328
neurons (scores 0-1) had small to absent PICs (mean 11.7 ± 19.4 pA; n = 15, 9 with no PIC), 329
whereas bistable neurons (scores 3-4), especially those that are fully bistable (score 4), 330
exhibited a dramac rise in the PIC amplitude (mean 198 ± 160.6 pA, n = 22, 0 with no PIC; p < 331
0.001). . These findings highlighted the strong associaon between PICs and bistability in large 332
α-motoneurons and the absence or low amplitude of PICs in smaller non-bistable neurons. 333
To further understand the contribuon of the sodium component of PICs in bistability, 334
we added 10 mM TEA-Cl and 100 µM CdCl2 to the recording ACSF (24). Large α-motoneurons 335
displayed a more pronounced persistent sodium current (INaP; Fig. 3E). Although the 336
motoneuron size did not influence the INaP threshold acvaon (Fig. 3G), the peak amplitude of 337
INaP was significantly greater in large α-motoneurons (>400 µm2, 1126 ± 530 pA, n = 11) 338
compared to small motoneurons (<400 µm2, 221 ± 104 pA, n = 10; p<0.001, Fig. 3F). This 339
increase is not merely a reflecon of the greater surface area of the bigger neurons that could 340
express more channels, as the PIC density (corrected by the cellular capacitance) was also larger 341
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in large than in small α-motoneurons (4.84 ± 1.51 pA/pF, n = 11 vs. 2.38 ± 1.09 pA/pF, n = 342
10;Fig. 3H; P < 0.001). These findings highlighted the significant contribuon of INaP in 343
bistability from large α-motoneurons and its diminished presence in smaller, non-bistable 344
neurons. 345
A calcium-acvated inward current was shown to be pivotal in sustaining the plateau 346
depolarizaon underlying the tonically firing acve state in bistable crab (22) and turtle 347
motoneurons (48); this current has recently been shown to be mediated by Trpm5 channels in 348
mice (19). To measure the effect of this current, we induced a 2-sec depolarizaon in the 349
presence of tetrodotoxin (TTX, 1 µM) and tetraethylammonium-chloride (TEA, 10 mM) to 350
minimize sodium and potassium currents. This depolarizaon was large enough to elicit a series 351
of slow calcium-driven spikes to fully acvate Trpm5. Subsequently, we measured the resulng 352
aerdepolarizaon (sADP; Fig. 4A), which slowly declined as the Trpm5 current decayed (19). 353
Notably, the sADP was parally blocked by Triphenylphosphine oxide (TPPO, 50 µM Fig. 4A), a 354
known Trpm5 channel blocker (19, 49). The residual TPPO-insensive sADP appeared to 355
predominantly arise from channels that are not yet idenfied (see discussion in (19)). Thus, to 356
accurately esmate the Trpm5 component of the sADP, we subtracted the sADP measured in 357
TPPO from the total sADP. The resulng Trpm5 sADP was only detectable in one of 15 smaller 358
neurons (<400 µm2: 0.2 ± 0.92 mV, n = 15), but was evident in most of the larger neurons (>400 359
µm2: 4.9 ± 5.47 mV, n = 9; p = 0.0017; Fig. 4A-B). Neurons with lower bistability scores (scores 0-360
1) lacked Trpm5 sADP (n=10), but this current was present in most bistable neurons (scores 3-4: 361
4.6 ± 5.32 mV, n = 10; p = 0.0108; Fig. 4C). We isolated the Trpm5 calcium-acvated inward 362
current in response to a 2 sec depolarizing voltage step in presence of TTX (1µM) and TEA 363
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(10mM) (Fig.4D). We confirmed that the Trpm5 inward current amplitude and density 364
(corrected by cell capacitance) were significantly bigger in large α-motoneurons (>400 µm2, 324 365
± 146.3 pA & 2.7 ± 1.38 pA/pF, n = 8) compared to the small ones (<400 µm2, 26 ± 7.51 pA & 366
0.7 ± 0.67 pA/pF, n = 3) (Fig. 4E-F; P<0.05). Thus, the calcium-acvated inward current mediated 367
by Trpm5 channels (19) appeared associated with bistability in large α-motoneurons, but was 368
low or absent in smaller non-bistable neurons. 369
We further assessed the Kv1.2 potassium current, whose slow inacvaon delays the 370
iniaon and acceleraon of firing of bistable neurons during long current steps (23). 371
Interesngly, in our earlier work, only bistable neurons showed the delayed iniaon and 372
acceleraon of firing during long current steps, induced by closure of the Kv1.2 channels; non-373
bistable neurons showed immediate spike onset with spike deceleraon (23). We tested 374
whether the Kv1.2 inacvang current was larger in large, bistable neurons than small neurons. 375
This was measured in voltage clamp as the amplitude of the inward current over a 7 sec step, 376
measured aer inacvaon of the transient potassium current, in the presence of TTX (1µM) 377
and TEA (10mM) (Fig. 4G). Again, the amplitude of the Kv1.2 current scaled with cell size. 378
Smaller α-motoneurons (<400 µm2) had a lower current amplitude (68 ± 86.9 pA; n = 19) than 379
the larger neurons (159.2 ± 155.5 pA, n = 26; P < 0.02; Fig. 4H). Moreover, the smaller 380
motoneurons had a lower current density (2.19 ± 0.33 pA/pF; n = 3) compared to larger 381
motoneurons (3.61 ± 0.94 pA/pF, n = 7; P < 0.05; Fig. 4 I). 382
All together, our findings highlight that INaP, the Trpm5 mediated calcium-acvated 383
inward current, and the slow inacvang Kv1 current are pivotal indicators of bistability in large 384
α-motoneurons. 385
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386
Serotonin effects on bistability in parally bistable neurons 387
We found that bistability is not strictly binary, as 26% (52 out 199) of α-motoneurons 388
displayed some, but not all, of the defining features described above, especially the absence of 389
persistent firing aer the brief depolarizaon. It has been known for many years that 390
neuromodulators such as serotonin (5-HT) can evoke bistability in α-motoneurons which show 391
only paral bistable properes (14, 17, 25, 50). 392
We analyzed the effects of 10 µM 5-HT on α-motoneurons (n = 45) that had bistability scores 393
below 4 and lacked the self-sustained spiking. Fig. 5A shows an example of an α-motoneuron 394
that, under control condions (i.e. recording aCSF), did not show acceleraon of spiking during 395
a 2-sec depolarizaon, and only showed a modest sADP at the end of the depolarizaon. Seven 396
minutes aer applicaon of 5-HT, this motoneuron demonstrated accelerang spiking during 397
the current step, and self-sustained spiking acvity aer the end of the step. This acvity was 398
terminated only by a hyperpolarizing step, indicang that the neuron became fully bistable only 399
in the presence of 5-HT. Fig. 5B shows the ability of 5-HT to evoke bistability in α-motoneurons 400
of different sizes. Notably, small α-motoneurons (< 400µm2; n = 19) remained non-bistable even 401
with 5-HT. In contrast, larger neurons exhibited the potenal for a transion to bistability upon 402
5-HT exposure. Specifically, of non-bistable α-motoneurons of larger size (>400µm2), 38.5% 403
switched to full bistability with 5-HT (n = 26; p < 0.001). It is noteworthy that the majority of 404
neurons transioning to bistability with 5-HT already expressed an sADP before 5-HT addion, 405
as illustrated in Fig. 5A. Thus, 5-HT was able to evoke full bistability in a subset of neurons 406
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already leaning towards a bistable state, but could not evoke bistability in smaller or less 407
bistable neurons. 408
409
DISCUSSION 410
411
Our study reveals a size-based gradient in the bistable ability of GFP+ α-motoneurons 412
from Hb9-GFP and ChAT-GFP mice lines. Specifically, the likelihood of α-motoneurons being 413
bistable increases with the cell body cross-seconal area and over me. The currents, Trpm5 414
and INaP, which contribute to the acve state during bistability, and Kv1, whose inacvaon 415
helps to iniate sustained firing, are more pronounced in larger fast α-motoneurons which can 416
be reliably idenfied with the MMP-9 marker. Conversely, these currents are low or absent in 417
smaller MMP-9- α-motoneurons. We also showed that serotonin evokes full bistability only in 418
large motoneurons. These findings highlight an important correlaon between motoneuron size 419
and bistability. 420
Research on bistability has primarily focused on larger α-motoneurons, idenfied by 421
their ventrolateral locaon or by retrograde smulaon from ventral nerves (18, 27). This might 422
have led to a failure to record the properes of smaller α-motoneurons. Neurons recorded in 423
this study are motoneurons, as evidenced by their ventrolateral locaon and the expression of 424
ChAT and Hb9 markers (Fig. 1A-B). Furthermore, they are α−motoneurons since most γ-425
motoneurons do not express NeuN (41), and do not express GFP in Hb9-GFP mice (38, 41). 426
Finally, virtually all of the neurons larger than 400µm2 also expressed MMP-9, indicave of fast 427
α-motoneurons (30, 37). While there are no markers available to differenate between the fast 428
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fague-resistant (FR) and fast fagable (FF) α-motoneurons, we conclude that the largest 429
neurons are fast motoneurons. The smallest neurons are presumably slow (S) α-motoneurons. 430
However, size alone is not a reliable indicator to separate these classes (51-53). 431
Electrophysiologically, large fast α-motoneurons differ from small slow α-motoneurons 432
by exhibing a delayed and accelerated firing during prolonged smulaon (27, 29, 52). Our 433
experiments confirm this observaon, as near-threshold current steps resulted in a slow 434
depolarizaon in larger α-motoneurons leading to delayed firing acceleraon (Fig. 2). In 435
contrast, smaller α-motoneurons fired immediately upon reaching spike threshold followed by a 436
spike frequency deceleraon (27, 30). The depolarizaon and spike delay in large fast α-437
motoneurons were aributed to the slow inacvaon of a Kv1 current (23, 30). 438
We now add bistability as a predominant property of large fast L4-L5 α-motoneurons in 439
young mice. Over 75% of neurons over 800µm2 showed bistability, while only ~10% of neurons 440
less than 400 µm2 were bistable. Consistent with this, a large majority of fast α-motoneurons 441
showed negave hysteresis during triangular ramp steps, where the derecruitment current was 442
lower than the recruitment current, while far fewer of the slow α-motoneurons displayed 443
negave hysteresis (30) (Fig. 2). The frequency of bistability in large α-motoneurons increased 444
during the first weeks of postnatal development, as previously described (18); (Fig. 2C1). By P21, 445
all large α-motoneurons were fully bistable, while smaller neurons connued to lack bistability 446
features. 447
The disnct firing behaviors observed between large and small α-motoneurons can be 448
aributed to a specific set of ionic currents that are more highly expressed in large fast α-449
motoneurons compared to small slow α-motoneurons. One key contributor is the acvaon of 450
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slow persistent inward currents (PICs), which may be carried by calcium or sodium. This 451
acvaon has been linked to negave hysteresis and bistability (15, 16, 19). Expression of the 452
PIC increases with the size of the α-motoneurons (Fig. 3B), and with the bistability score (Fig. 453
3D). Furthermore, our observaons demonstrate the relaonship between the size of the α-454
motoneurons and the sodium component of PICs (INaP) (Fig. 3E-H). 455
456
Recently, the thermosensive Trpm5 current has been idenfied as a calcium-acvated 457
sodium current sustaining the plateau potenal in bistable mouse α-motoneurons (19). We 458
found that the Trpm5-evoked aerdepolarizaon was present in virtually all large bistable α-459
motoneurons, but not detectable in small non-bistable α-motoneurons (Fig. 4A-C). Finally, the 460
slowly inacvang Kv1.2-mediated current responsible for the delayed firing acceleraon in 461
large motoneurons (23) (Fig. 2), is less expressed in smaller motoneurons (Fig. 4G-I). 462
Interesngly, this current posively correlates with the degree of bistability (Fig. 4G-I; (23)). It is 463
likely, that the slow inacvaon of Kv1 will shi the balance of currents towards depolarizaon, 464
and will help to sustain connued firing in the bistable state. In sum, the lack of sufficient 465
expression of these three pivotal currents renders small α-motoneurons non-bistable. In 466
contrast, large α-motoneurons, which robustly express these currents, are bistable. Note that 467
there may be some duplicaon of effort in these currents. Indeed, among neurons that showed 468
connued bistable firing, 20% either did not show a marked negave hysteresis during ramps, 469
or did not show a delayed firing acceleraon. This suggest that bistability might not require the 470
full spectrum of these currents. 471
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Bistability in α-motoneurons was observed in earlier experiments only in the presence 472
of neuromodulators (14-16, 54). However, we have revealed inherent bistability in most large α-473
motoneurons, provided the recording temperature is sufficiently high (above 30°C) (18), to 474
unmask the thermosensive Trpm5 current responsible for the plateau potenal (19). In our 475
preparaons, some α-motoneurons expressed intermediate properes and were not fully 476
bistable under these condions. Addion of 5-HT evoked full bistability in ~30% and ~60% of 477
the intermediate and large neurons, respecvely (Fig. 5). Interesngly, α-motoneurons 478
completely lacking baseline bistability characteriscs (bistability score 0) never became bistable 479
with 5-HT. We propose that intermediate α-motoneurons express some of the essenal 480
currents for bistability, but at lower levels (Fig. 5). The known modulatory acons of 5-HT can 481
then enhance these currents to sustain prolonged firing in the bistable state. 482
483
Our demonstraon of a size principle for bistability contrasts with the groundbreaking 484
work by Lee and Heckman (15, 16), who studied α-motoneurons in adult cats. In their study, 485
they achieved a full bistability in about one-third of the α-motoneurons, which interesngly 486
exhibited characteriscs of smaller motoneurons. There are several potenal explanaons for 487
the differences between our findings. First, it is uncertain whether the smallest α-motoneurons 488
in the cat were recorded because of the use of sharp electrode recordings. Consequently, the 489
bistability in the smallest slow α-motoneurons in cats remains unknown. Second, mouse α-490
motoneurons are inherently more excitable than cat α-motoneurons, primarily due to their 491
smaller size (34, 55). Despite this, the PIC amplitude is relavely similar across both species, 492
suggesng that PIC has a more significant impact on the firing rate in mice. Third, our 493
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observaons were made on mouse α-motoneurons during the first 4 weeks of life, while Lee 494
and Heckman’s experiments were made on adult cats. However, bistability in the larger 495
neurons became more pronounced with age in the mouse neurons. In young adult mice (P21-496
P25) all large motoneurons exhibited bistability, whereas none of the smaller ones did. Finally, 497
our results were made ex vivo from slices in the presence of blockers of fast synapc 498
transmission (though not at P21/P25) and mostly without neuromodulators. On the other hand, 499
the cat neurons were recorded in vivo in the presence of methoxamine to maximize the 500
incidence of bistability. 501
The role of bistability in spinal α-motoneurons remains unclear. It has been recorded 502
during quiet standing in both rats and cats (56), suggesng a potenal significance for postural 503
control (15, 16, 28, 50). The Henneman Size Principle (57) posits that smaller α-motoneurons, 504
due to their lower input conductance, are the first to be recruited, potenally playing a role in 505
maintaining posture. This can be consistent with earlier work in cats, where smaller neurons 506
showed full bistability and were possibly acve during quiet standing (15, 16, 58). However, we 507
here demonstrate, in younger mice, that small α-motoneurons are not bistable. Instead, 508
bistability increases with the size of motoneurons (Fig. 2). Rier et al. (58) provided evidence 509
that large fast α-motoneurons may also be tonically recruited during quiet standing in mice. An 510
intriguing study by Bos et al. (19) showed that mice lacking Trpm5 channels in lumbar 511
motoneurons exhibited compromised postural control. Given the central role of Trpm5 in 512
bistable properes of larger α-motoneurons (19) (Fig 4), it is possible that fast α-motoneurons 513
may play a role in postural maintenance. Alternavely, the currents responsible for bistability 514
may play a more important role in recruing the less excitable large motoneurons. The PIC 515
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plays in important role in the iniaon of bistable firing, but is also crical for repeve firing in 516
α-motoneurons (59), and in synapc amplificaon due to their dendric locaon (1). Thus, the 517
currents which together lead to bistability may individually play mulple roles in motor control 518
in the mouse. These assumpons should be further invesgated in the future using more 519
integrated in vivo preparaons. 520
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FIGURE LEGENDS 521
522
Figure 1: Idenficaon and size distribuon of α-motoneurons in the ventrolateral spinal cord 523
at L4-L5. A: Ventrolateral cord at P12 showing Hb9-GFP (green), NeuN (red) and double labeling 524
(yellow). B1: The vast majority (98%, n = 141/144) of Hb9-GFP- labeled neurons co-express 525
NeuN. B2: Cross-seconal area distribuon of Hb9-GFP neurons at P4-P6 (light green, n=571 526
neurons), P8-P10 (medium green, n=475 neurons) and P12 (dark green, n=288 neurons). C1: 527
Ventrolateral cord at P12 showing Hb9-GFP (green), MMP-9 (red) and dual labeling (yellow). C2: 528
Size distribuon of Hb9-GFP labeled neurons expressing (n=122) or not expressing (n=166) 529
MMP-9. C3: Histogram of size distribuon of GFP+-MMP-9- (green) or HB9
+-MMP-9+ (yellow) 530
neurons. C4: Double-labeled Hb9-GFP+/MMP-9+ neurons are predominantly larger than 400 531
µm2 (yellow, top) while most Hb9-GFP+/MMP-9- neurons are smaller than 400 µm
2 (green, 532
boom). C2-C4: n=288 neurons. *** p<0.001 (two-tailed Mann-Whitney test for C2; Fisher’s 533
exact test for C4). Mean ± S.D. for B1. Median (red solid line) and quarles (red dashed lines) 534
are represented for C2. 535
536
Figure 2: Bistability score in spinal motoneurons varies by age and size. A: Fully bistable 537
neuron (bistability score 4). A1: Baseline depolarizaon towards spike threshold: a 2-sec 538
suprathreshold smulaon evokes a prolonged aerdepolarizaon (ADP: 1 point). Slightly 539
higher baseline depolarizaon followed by a 2 sec smulaon leads to prolonged firing which is 540
only terminated by hyperpolarizing current step (1 point); trace has been offset to be more 541
easily seen. A2: Negave hysteresis during ramp smulaon. The current threshold for onset of 542
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spiking is higher than the threshold for offset of spiking (-ΔI; 1 point). A3: A small subthreshold 543
current step leads to a slow depolarizaon (trace has been offset to be more easily seen). A 544
slightly higher current step leads to a larger depolarizaon leading to delayed spike onset and 545
acceleraon of spike frequency during the step (1 point). B: Completely non-bistable neuron 546
(bistability score 0). B1: Current steps to near threshold lead to decelerang spike frequency 547
during a 2 sec suprathreshold pulse, and an aerhyperpolarizaon at the end of the step (0 548
point). B2: Posive hysteresis during ramp smulaon. The current threshold for onset of 549
spiking is lower than the threshold for offset of spiking (+ΔI; 0 point). B3: A small subthreshold 550
current step does not evoke a slow depolarizaon. A slightly higher current step evokes 551
immediate onset firing with decelerang spike frequency during the step (0 point). Size 552
markers: 1 sec, 20 mV. C1: Bistability score as a funcon of postnatal age for smaller (<400µm2, 553
blue, n=81) and larger (>400µm2, red, n=118) Hb9-GFP+ neurons. Stascal comparison was 554
done for each age and when all ages were pooled. C2: Distribuon of bistability as a funcon of 555
neuronal cross-seconal area combining all ages. Smaller neurons predominantly have 556
bistability scores of 0-1 (blue, n=95) while larger neurons predominantly have bistability scores 557
of 3-4 (red, n=80). C1-C2: n= 199 neurons. C3: Bistability score as a funcon of neuronal input 558
resistance. A strong negave correlaon is seen (R
2= 0.99, n=176). C4: Effect of size on 559
bistability in neurons (P1/9) recorded by perforated patch-clamp. Large neurons show 560
significantly higher bistability scores than small neurons (p<0.05; n=27) C5: Post-natal (P2/6) 561
motoneurons idenfied by expression of ChAT-GFP show similar bistability scores that rise with 562
cross-seconal area (blue for <400µm2, n=16 and red for >400µm2, n=9; p=0.02). * p<0.05; *** 563
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p<0.001 (two-tailed Mann-Whitney test for C1, C4 and C5; Fisher test and slope comparison of 564
simple linear regressions for C2). Mean ± S.D. 565
566
Figure 3: Persistent inward current (PIC), and its sodium component, is larger in bistable 567
neurons. A: Voltage clamp measure of PIC acvaon during slow voltage ramp in a bistable but 568
not in a non-bistable neuron. B-C: PIC amplitude (B), and threshold (C) as a funcon of cell 569
cross-seconal area of Hb9-GFP+ neurons (blue circles, <400µm2,n=17 (B), n=9 (C) and red 570
circles, >400µm2, n=24 (B), n=17 (C); P=0.014 (B); P=0.72 (C)). D: PIC amplitude increases with 571
increasing bistability (Green: bistability scores 0-1, n=15; yellow: bistabiity scores 3-4, N=22; 572
P=0.001). E: Superimposed leak-subtracted sodium persistent current (INaP) recorded from 573
Hb9-GFP+ motoneurons in the presence of 10 mM TEA and 100µM CdCl2 (area <400µm2 or 574
>400µm2 for the blue and red trace, respecvely) in L4-L5 regions at P9 in response to a slow 575
ramp depolarizaon. F-H: INaP peak amplitude (F), threshold (G) and density (H) as a funcon 576
of cell cross-seconal area of Hb9-GFP+ neurons (blue circles , <400µm2, n= 10 and red circles, 577
>400µm2 n=11; P=0.001). ns, no significance; *p<0,05; ***p<0,001 (two-tailed Mann-Whitney 578
test for B-D and F-H). Median (solid line) and quarles (dashed lines) are represented in each 579
violin plot. 580
581
Figure 4: Trpm5-mediated current and slowly inacvang Kv1 current are signatures of 582
bistability. A: Neurons are depolarized for 2 sec in presence of 1µM TTX and 10 mM TEA. 583
Above a threshold, large calcium-dependent oscillaons are evoked. In bistable motoneurons 584
(red trace), these elicit a large, slow aerdepolarizaon which is parally blocked by 50µM 585
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TPPO (black trace). Non-bistable neurons (blue trace) do not express this slow 586
aerdepolarizaon. B: Amplitude of the Trpm5-induced, TPPO-resistant aerdepolarizaon is 587
larger in large motoneurons (>400 µm2, n=9) than small motoneurons (<400 µm2, n=15; 588
p=00017). C: Trpm5-induced, TPPO-resistant aerdepolarizaon is larger in bistable 589
motoneurons (score 3-4, n=10) than nonbistable motoneurons (score 0-1, n=10; p=0.0108). D: 590
TPPO-sensive Trpm5 current is isolated in response to a voltage step of 2 sec in presence of 591
1µM TTX and 10 mM TEA. Superimposed traces of Trpm5 isolated current from large (>400µm2, 592
red) vs small (<400µm2, blue) Hb9-GFP+ motoneurons. Note that the current trace during the 593
depolarizing step has been truncated for improved visualizaon of the post-step current. E-F: 594
Amplitude (E) and density (F) of the Trpm5 current increases with cross-seconal area of Hb9-595
GFP+ neurons (blue circles, <400µm2, n=3, and red circles, >400µm2, n=8; p=0,0121 and p=0242, 596
respecvely) G: Superimposed traces of the slowly inacvang Kv1 current in bistable (red) and 597
non-bistable (blue) neuron in response to a long depolarizing voltage step in presence of 1µM 598
TTX and 10 mM TEA. H-I: Amplitude (H) and density (I) of Kv1 current increases with cross-599
seconal area of Hb9-GFP+ neurons (blue circles, <400µm2, n=19 (H), n=3 (I) and red circles, 600
>400µm2, n=26 (H) and n=7 (I); p=0.0216 and p=0.0167). *p<0,05; **p<0,01 (two-tailed Mann-601
Whitney test for B-C, E-F and H-I). Median (solid line) and quarles (dashed lines) are 602
represented in each violin plot. 603
604
Figure 5: Inducon of full bistability by serotonin. A: Non-bistable neuron’s response (top, 605
black trace) to 2 sec depolarizing pulse (boom). Only a small aerdepolarizaon is recorded 606
aer the step. During 10µM serotonin (5-HT), this neuron became fully bistable, with 607
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connuous firing (top, red trace) following the 2 sec depolarizing step (boom). B: Not all non-608
bistable neurons respond to serotonin (n=45). Smaller neurons (<400 µm2,n=19 of 19) fail to 609
show full bistability (score 4) with 5-HT. 38% of larger (>400 µm2) non-bistable neurons became 610
bistable during 5-HT. *** p<0.001 (Fisher’s exact test for B). 611
612
DISCLOSURES 613
614
No conflicts of interest, financial or otherwise, are declared by the authors. 615
616
AUTHOR CONTRIBUTIONS: 617
618
R. H-W, R.B. and F.B. generated the hypotheses for the paper. R. H-W, B.D. and R.B. performed 619
electrophysiological experiments and analysis; E.P. performed immuno-histochemical 620
experiments and analysis. R. H-W draed the manuscript, and R. H-W, R.B. and F.B. revised the 621
manuscript and approved the final version of the manuscript. 622
623
ACKNOWLEDGEMENTS 624
625
This work was supported by NIH grant NS17323 (R. H-W), by Fonds d’invesssement INT 626
FI_INT_JCJC_2019 (R.B.), by Centre Naonal de la Recherche Scienfique (CNRS) (R.B. and F.B.) 627
and by Agence Naonale de la Recherche Scienfique ANR-16-CE16-0004 (F.B.). 628
629
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AUTHOR NOTES 630
631
Correspondence: R. M. Harris-Warrick, rmh4@cornell.edu; F. Brocard, frederic.brocard@univ-
632
amu.fr; R. Bos, remi.bos@univ-amu.fr
633
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Hb9-GFP MMP-9 Merge
0.0
0.5
1.0
1.5
2.0
Hb9-GFP+cell body
cross-sectional area (µm2 x 1000)
A
C4C3C2
C1
B1 B2
P8-P10
0
10
20
40
30
P4-P6
Cell body cross-sectional area (μm2)
% of Hb9-GFP+ cells
51/100
151-200
401-450
301-350
251-300
351-400
501-550
451-500
601-650
551-600
651-700
>701
P12
201-250
101/150
Hb9- GFP NeuN Merge
MMP9+
MMP9-
0
450 1350900 18000
10
20
30
40
50
GFP+ MMP9-
GFP+ MMP9+
Nb of Hb9 cells
Cell body cross-sectional area (μm2)
Figure 1
***
0
10
20
30
40
50
60
70
80
90
100
110
100μm
100μm
50μm
50μm
GFP+/MMP9+
(n=122)
GFP+/MMP9-
(n=166)
***
>400μm2
<400μm2
>400μm2
<400μm2
99%
87%
13%
1%
% of Hb9-GFP
+
cells
(n=141/144)
NeuN+
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
0
C3
C1
0-1 3-4
-ΔI
+ΔI
A3A2A1
B3
1
2
3
P0/2 P3/4 P5/6 P7/8 P9/14 P21/25
Age (days)
<400 µm2
>400 µm2
Bistability score:
Bistability score
Bistability score
% of Hb9-GFP+cells
P2/6
Hb9-GFP+cells (n=199) Hb9-GFP+cells (n=199)
<400 µm2
>400 µm2
ChAT-GFP+ cells (n=25)
Figure 2
*
I
V
R2 = 0.93, slope = -21.4
R2 = 0.93, slope = 17.6
***
***
0
1
2
3
4
C2
0
20
40
60
80
100
*** *** ** *** *** ***
<200 201/400 401/600 601/800 >801
Cell body cross-sectional area (μm2)
C5C4
B1 B2
1s
20mV
1s
20mV
1s
20mV
1s
20mV
1s
20mV
1s
20mV
0
1
2
3
4
Perforated-patch clamp
Hb9-GFP+ cells (n=27)
*
<400 µm2
>400 µm2
P1/9
0
1
2
3
4
5
Bistability score
Bistability score
Hb9-GFP+ cells (n=176)
Input Resistance (MΩ)
<50 51/100 101/200 201/300 >301
R2 = 0.99, slope = -0.63
400pA 200pA 200pA
100pA 100pA 100pA
I
V
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
E
-80
-60
-40
-20
INap Threshold (mV)
ns
0
500
1000
1500
2000
2500
INaP Peak Amplitude (pA)
***
0
2
4
6
8
INaP density (pA.pF
-1
)
***
1 s
200 pA
Large MN
Small MN
-70 mV
-10 mV
F G H
A
-200
0
200
400
600
800
PIC Amplitude (pA)
-80
-60
-40
-20
0
PIC Threshold (mV)
-200
0
200
400
600
800
PIC Amplitude (pA)
Large MNs
Small MNs
Large MN
Small MN
Large MNs
Small MNs
Bist. score 3-4
Bist. score 0-1
*
ns
***
Large MNs
Small MNs
Large MNs
Small MNs
Large MNs
Small MNs
B C D
Figure 3
2s
200 pA
-70 mV
-10 mV
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
1s
20mV
500pA
-70mV
-200
0
200
400
600
800
*
Large MNs
Small MNs
IKv1 Amplitude (pA)
0
2
4
6
IKv1 density (pA.pF
-1
)
Large MN
Small MN
+ TPPO
20mV
20mV
1s
sADP Amplitude (mV)
Large MNs
Small MNs
Large MNs
Small MNs
-10
0
10
20
30
sADP Amplitude (mV)
Bist. score 3-4
Bist. score 0-1
A B C
**
*
Figure 4
400 pA
1s
-200
0
200
400
600
800
TRPM5
Current Amplitude (pA)
-2
0
2
4
6
8
TRPM5
Current density (pA.pF
-1
)
0pA
-60mV
*
*
Large MNs
Small MNs
Large MNs
Small MNs
D E F
G H I
*
Large MN
Small MN
Large MN
Small MN
-10
0
10
20
30
60mV
0pA
300pA 900pA
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
I
V
A
+ 5-HT (10μM)
Control
20mV
1s
B
<400 >400
Cell body cross-sectional area (μm2)
100pA
Switch to full bistability in resp. to 5-HT
No Switch to full bistability in resp. to 5-HT
38.5%
(n=10)
61.5%
(n=16)
100%
(n=19)
***
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.