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Effect of size on expression of bistability in mouse spinal motoneurons

Authors:

Abstract

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar (L4-L5) ventrolateral a-motoneurons of ChAT-GFP and Hb9-GFP mice during the first four weeks of life. We found that as neuron size increases, the prevalence of bistability rises. Smaller a-motoneurons lack bistability, while larger fast a-motoneurons (MMP-9 ⁺ /Hb9 ⁺ ) with a soma area ≥ 400µm ² exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, thermosensitive Trpm5 Ca ²⁺ -activated Na ⁺ current and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties, but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.
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 acvity in the absence of 24
excitatory drive. Earlier work in adult preparaons suggested that smaller motoneurons 25
innervang slow angravity muscle fibers are more likely to generate bistability for postural 26
maintenance. However, whether large motoneurons innervang fast-fagable muscle fibers 27
display bistability is sll controversial. To address this, we examined the relaonship 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
thermosensive Trpm5 Ca2+-acvated Na+ current and the slowly inacvang Kv1.2 current, also 34
scale with cell size. Serotonin evokes full bistability in large motoneurons with paral bistable 35
properes, 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
Neuromodulaon 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
differenal 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 addion 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 contracon through nonlinearring properes (1). One 56
such property is bistability, where the motoneuron can switch between stable silent and acve 57
states, depending on transient synapc input or current injecon. Originally detected as plateau 58
potenals in invertebrate neurons (2-9), bistability was soon found in vertebrates (10), 59
specifically in spinal motoneurons (11-16). While oen 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 condions (18, 19). 62
The acve 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
acvated Na+ current (18, 19, 22). Drawing from a series of our previous invesgaons, we can 65
summarize the process as follows: The inial depolarizaon, caused by the slow inacvaon of 66
Kv1.2 potassium channels (23), acvates the persistent Nav1.6 current leading to spiking acvity 67
(24). This then prompts Ca2+ entry through the recruitment of Cav1.3 channels, iniang a Ca2+-68
induced Ca2+-release process (18). This process ulmately acvates thermosensive Trpm5 69
channels, which are the primary source of the plateau depolarizaon to sustain repeve firing 70
(19). Other currents such as the HCN-type hyperpolarizaon-acvated inward current, Ih (25, 71
26) and reducon of calcium-acvated outward currents (17, 25) are also involved in different 72
neurons. 73
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α-motoneurons, which drive extrafusal muscle contracons, can be subdivided 74
funconally into three classes based on size and the muscle type they innervate: large fast-75
fagable (FF), medium fast fague-resistant (FR), and small slow (S) motoneurons [reviewed by 76
(27)]. In decerebrate cats, small motoneurons characterized by slow conducon velocies and 77
low acvaon thresholds, appear to have more pronounced bistability than large ones (15, 16). 78
This observaon 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 staonary posture 82
also involves the acvaon of fast motor units (57). Interesngly, our recent findings indicate 83
that large motoneurons exhibit bistablering 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 disnct from that of smaller ones, marked by a 86
lower input resistance, a higher rheobase, more depolarized spike thresholds, narrower acon 87
potenals, and shorter aerhyperpolarizaons (27, 29, 30). They also exhibit delayed onset of 88
firing and firing acceleraon during a step depolarizaon, and receive a consistently higher level 89
of recurrent excitaon (23, 31-33). Despite this extensive characterizaon, 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, cauon is warranted due to the disnct characteriscs 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 funcon of size in genecally 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 properes, while the smallest 97
motoneurons were rarely bistable. There was a strong correlaon between bistability and the 98
amplitudes of the ionic currents known to support it. Motoneurons of intermediate size oen 99
showed paral bistability, which could be converted to full bistability by serotonin. These 100
unexpected results suggest new hypotheses regarding the contribuon 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. Pemann 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 Instuonal Animal Use and Care 112
Commiee and were in accordance with NIH guidelines. Marseille: All animal care and use 113
conformed to French regulaons (Décret 2010-118) and were approved by the local ethics 114
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commiee (Comité d’Ethique en Neurosciences INT-Marseille, CE71 Nb A1301404, 115
authorizaon Nb 2018110819197361). 116
117
Slice Preparaon 118
For electrophysiological experiments, mice were cryoanaesthezed (P2-P7) or anaesthezed 119
(P8-P25) with intraperitoneal injecon of a mixture of ketamine/xylazine (100mg/kg and 10 120
mg/kg, respecvely). 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 secons 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 synapc transmission (CNQX 128
and D,L-AP5 or kynurenic acid, strychnine, and bicuculline) were added in the aCSF to minimize 129
synapc contribuons 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 soluon (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). Aer a resng period of 30-60 min, 137
slices were transferred to the recording chamber and superfused with recording aCSF at 32°C 138
(35) without addion of fast synapc transmission blockers. 139
140
Electrophysiological recordings 141
142
Hb9-GFP and ChAT-GFP posive 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 pipee soluon 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 Mulclamp 700B amplifier driven by PClamp 10 soware (Molecular Devices). 149
Recordings were digized on-line and filtered at 10 kHz (Digidata 1322A or 1440A, Molecular 150
Devices). Pipee 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 aer 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
sensive 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 creanine 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 idenfy 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 werexed 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 cryosecons 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 soluon (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 anbody: mouse-an-174
NeuN (Neuronal Nuclei, Sigma-Aldrich MAB377) (36) or goat-an-MMP-9 (Matrix 175
metallopepdase 9, Sigma-Aldrich M9570)(30, 37). Both anbodies were diluted in the blocking 176
soluon with 0.2% Triton X-100 (1:1000 and 1:500 for an-NeuN and an-MMP-9, respecvely). 177
Slides were washed 3×5 min in PBS and incubated for 2 h with an Alexa Fluor® Plus 555- 178
conjugated secondary anbody (Invitrogen A32816) diluted in the blocking soluon. Aer 3 179
washes of 5 min in PBS, they were mounted with a gelanous aqueous medium. 180
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181
Data analysis 182
Electrophysiological recording: 183
Electrophysiological data were analyzed with Clampfit 10 soware (Molecular Devices). Several 184
basic criteria were set to ensure opmum quality of intracellular recordings. Only cells with a 185
stable membrane potenal below -60 mV, stable access resistance (no more than 20% 186
variaon), and acon potenal amplitude larger than 60 mV were analyzed. Reported 187
membrane potenals were not corrected for liquid juncon potenals. 188
Confocal imaging:
189
Immunofluorescent staining was quanfied from confocal images acquired with the 40X 190
objecve (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 quanfied from stacked confocal images (5 steps ; Z-step, 3 µm) with 193
Zen soware (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
Stascs 198
No stascal method was used to predetermine sample size. Stascal analysis was carried out 199
using GraphPad Prism and Matlab (MathWorks) soware. When two groups were compared we 200
used, the Mann-Whitney test. Fisher’s exact test was used for comparing cell proporons 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 stascal 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
quarles are represented in each violin plot. 206
Source Data File: 207
hps://github.com/remibos/Source-Data-File-HW.git 208
209
210
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RESULTS 211
212
Immunohistochemical idenficaon of fast α-motoneurons 213
214
Motoneurons in the ventrolateral spinal cord were inially idenfied 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 innervang intrafusal muscles that regulate muscle spindle responsiveness to 219
stretch. These motoneuron subtypes can be disnguished by differenal expression of a 220
number of proteins. While the transcripon 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, preferenally linked to hindlimb muscles (39, 40), from postnatal day 1 224
(P1) to P25. 225
The transcripon factor NeuN is commonly used to disnguish 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 inxedssues 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), indicang 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% respecvely having a 232
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maximal cross seconal 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 observaons 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-seconal area of MMP-9-negave 238
neurons was 280 ± 12 µm2, while that of MMP-9-posive neurons was 907 ± 26 µm2 (P<0.001, n 239
= 288; Fig. 1C2). Consistent with previous observaons (38), we noted a fairly clear demarcaon 240
in motoneuron characteriscs around the 400 µm² cross-seconal 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-seconal area exceeding 243
400µm2 were virtually all HB-9+/MMP-9+ (99.2%, n=121/122; Fig. 1C3, C4), suggesng their 244
classificaon as putave fast motoneurons. This paern led us to adopt the 400 µm² threshold 245
as a praccal and empirically supported cut-off, allowing us to grossly disnguish between fast 246
and slow motoneurons in our study. 247
248
Features of bistable motoneurons 249
We previously showed that under experimental condions 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 preparaon temperature above 30°C (18, 19, 43, 44). Four disnct features 253
characterized bistable motoneurons, which we have previously characterized (18, 23) (Fig. 2): 1) 254
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self-sustainedring triggered by a brief (2 sec) excitaon when the motoneuron was pre-255
depolarized near the spike threshold (Fig. 2A1); 2) a slow aerdepolarizaon (sADP) following 256
the current step if the motoneuron was not pre-depolarized sufficiently to trigger the self-257
sustained firing (Fig. 2A1); 3) negave hysteresis during slow triangular current ramp injecons, 258
where spiking stopped at lower currents than where it began (Fig. 2A2); 4) a slowly depolarizing 259
potenal causing delayed spiking acceleraon 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 negave 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 decelerang firing rate during the 2 sec depolarizing 265
pulse, leading to a post-step aerhyperpolarizaon (Fig. 2B1). Their acvity during ramp current 266
injecons showed posive 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 connuousring deceleraon (Fig. 2B3). Some motoneurons, despite having one scoring 269
feature, lacked self-sustainedring and were also considered as non-bistable. A significant 270
number of motoneurons also displayed intermediate characteriscs, scoring 2. The vast 271
majority of them (90 %, n=22/25) were not bistable, lacking self-sustained spiking, but meeng 272
two other criteria. This shows that bistability is not an all-or none property, but can manifest 273
with somewhat different properes. 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 inial 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 aer 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 correlaon between motoneuron soma size and the 289
emergence of bistability in young mice. Fig. 2C2 shows the bistability score distribuon 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 proporon of bistable neurons rose, while the proporon of non-bistable 297
cells decreased, especially above 400 µm2. Another way to see this correlaon was to measure 298
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bistability score as a funcon of input resistance, which was lower in large motoneurons and 299
higher in small motoneurons. A strong negave correlaon 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
properes (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 selecvely express higher levels of ionic currents that sustain the bistable 316
state than small neurons. Several ionic currents have been demonstrated to support the acve 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 thermosensive Trpm5 319
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calcium-acvated inward current (19), and a slow inacvaon of the Kv1.2 potassium current 320
(23). 321
We measured the PIC by delivering a slow ramp depolarizaon 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 correlang posively with 326
cell size without any change in the acvaon threshold (Fig. 3C). Moreover, the amplitude of the 327
PIC was also found to be proporonal 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 dramac 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 associaon 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 contribuon 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 acvaon (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 reflecon 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-acvated inward current was shown to be pivotal in sustaining the plateau 346
depolarizaon underlying the tonically firing acve 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 depolarizaon in the 349
presence of tetrodotoxin (TTX, 1 µM) and tetraethylammonium-chloride (TEA, 10 mM) to 350
minimize sodium and potassium currents. This depolarizaon was large enough to elicit a series 351
of slow calcium-driven spikes to fully acvate Trpm5. Subsequently, we measured the resulng 352
aerdepolarizaon (sADP; Fig. 4A), which slowly declined as the Trpm5 current decayed (19). 353
Notably, the sADP was parally blocked by Triphenylphosphine oxide (TPPO, 50 µM Fig. 4A), a 354
known Trpm5 channel blocker (19, 49). The residual TPPO-insensive sADP appeared to 355
predominantly arise from channels that are not yet idenfied (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 resulng 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-acvated 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 conrmed 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-acvated 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 inacvaon delays the 370
iniaon and acceleraon of firing of bistable neurons during long current steps (23). 371
Interesngly, in our earlier work, only bistable neurons showed the delayed iniaon and 372
acceleraon of firing during long current steps, induced by closure of the Kv1.2 channels; non-373
bistable neurons showed immediate spike onset with spike deceleraon (23). We tested 374
whether the Kv1.2 inacvang 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 aer inacvaon 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-acvated 383
inward current, and the slow inacvang Kv1 current are pivotal indicators of bistability in large 384
α-motoneurons. 385
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386
Serotonin effects on bistability in parally 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 aer the brief depolarizaon. 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 paral bistable properes (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 condions (i.e. recording aCSF), did not show acceleraon of spiking during 395
a 2-sec depolarizaon, and only showed a modest sADP at the end of the depolarizaon. Seven 396
minutes aer applicaon of 5-HT, this motoneuron demonstrated accelerang spiking during 397
the current step, and self-sustained spiking acvity aer the end of the step. This acvity was 398
terminated only by a hyperpolarizing step, indicang 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 potenal for a transion 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 transioning to bistability with 5-HT already expressed an sADP before 5-HT addion, 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-seconal area and overme. The currents, Trpm5 414
and INaP, which contribute to the acve state during bistability, and Kv1, whose inacvaon 415
helps to iniate sustained firing, are more pronounced in larger fast α-motoneurons which can 416
be reliably idened 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 correlaon between motoneuron size 419
and bistability. 420
Research on bistability has primarily focused on larger α-motoneurons, idenfied by 421
their ventrolateral locaon or by retrograde smulaon from ventral nerves (18, 27). This might 422
have led to a failure to record the properes of smaller α-motoneurons. Neurons recorded in 423
this study are motoneurons, as evidenced by their ventrolateral locaon 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, indicave of fast 427
α-motoneurons (30, 37). While there are no markers available to differenate between the fast 428
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fague-resistant (FR) and fast fagable (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 exhibing a delayed and accelerated firing during prolonged smulaon (27, 29, 52). Our 433
experiments confirm this observaon, as near-threshold current steps resulted in a slow 434
depolarizaon in larger α-motoneurons leading to delayed firing acceleraon (Fig. 2). In 435
contrast, smaller α-motoneurons fired immediately upon reaching spike threshold followed by a 436
spike frequency deceleraon (27, 30). The depolarizaon and spike delay in large fast α-437
motoneurons were aributed to the slow inacvaon 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 negave 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
negave 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 connued to lack bistability 446
features. 447
The disnct firing behaviors observed between large and small α-motoneurons can be 448
aributed to a specic 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 acvaon of 450
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slow persistent inward currents (PICs), which may be carried by calcium or sodium. This 451
acvaon has been linked to negave 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 observaons demonstrate the relaonship between the size of the α-454
motoneurons and the sodium component of PICs (INaP) (Fig. 3E-H). 455
456
Recently, the thermosensive Trpm5 current has been idenfied as a calcium-acvated 457
sodium current sustaining the plateau potenal in bistable mouse α-motoneurons (19). We 458
found that the Trpm5-evoked aerdepolarizaon 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 inacvang Kv1.2-mediated current responsible for the delayed firing acceleraon in 461
large motoneurons (23) (Fig. 2), is less expressed in smaller motoneurons (Fig. 4G-I). 462
Interesngly, this current posively correlates with the degree of bistability (Fig. 4G-I; (23)). It is 463
likely, that the slow inacvaon of Kv1 will shi the balance of currents towards depolarizaon, 464
and will help to sustain connued 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 duplicaon of effort in these currents. Indeed, among neurons that showed 468
connued bistable firing, 20% either did not show a marked negave hysteresis during ramps, 469
or did not show a delayed firing acceleraon. 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
preparaons, some α-motoneurons expressed intermediate properes and were not fully 476
bistable under these condions. Addion of 5-HT evoked full bistability in ~30% and ~60% of 477
the intermediate and large neurons, respecvely (Fig. 5). Interesngly, α-motoneurons 478
completely lacking baseline bistability characteriscs (bistability score 0) never became bistable 479
with 5-HT. We propose that intermediate α-motoneurons express some of the essenal 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 demonstraon 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 interesngly 486
exhibited characteriscs of smaller motoneurons. There are several potenal explanaons 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 relavely similar across both species, 492
suggesng that PIC has a more signicant impact on thering rate in mice. Third, our 493
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observaons 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), suggesng a potenal 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, potenally 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 acve 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). Rier 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 properes of larger α-motoneurons (19) (Fig 4), it is possible that fast α-motoneurons 513
may play a role in postural maintenance. Alternavely, the currents responsible for bistability 514
may play a more important role in recruing the less excitable large motoneurons. The PIC 515
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plays in important role in the iniaon of bistable firing, but is also crical for repeve firing in 516
α-motoneurons (59), and in synapc amplificaon due to their dendric locaon (1). Thus, the 517
currents which together lead to bistability may individually play mulple roles in motor control 518
in the mouse. These assumpons should be further invesgated in the future using more 519
integrated in vivo preparaons. 520
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FIGURE LEGENDS 521
522
Figure 1: Idenficaon and size distribuon 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-seconal area distribuon 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 distribuon of Hb9-GFP labeled neurons expressing (n=122) or not expressing (n=166) 529
MMP-9. C3: Histogram of size distribuon 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
boom). 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 quarles (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 depolarizaon towards spike threshold: a 2-sec 538
suprathreshold smulaon evokes a prolonged aerdepolarizaon (ADP: 1 point). Slightly 539
higher baseline depolarizaon followed by a 2 sec smulaon 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: Negave hysteresis during ramp smulaon. 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 depolarizaon (trace has been offset to be more easily seen). A 544
slightly higher current step leads to a larger depolarizaon leading to delayed spike onset and 545
acceleraon 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 decelerang spike frequency 547
during a 2 sec suprathreshold pulse, and an aerhyperpolarizaon at the end of the step (0 548
point). B2: Posive hysteresis during ramp smulaon. 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 decelerang spike frequency during the step (0 point). Size 552
markers: 1 sec, 20 mV. C1: Bistability score as a funcon of postnatal age for smaller (<400µm2, 553
blue, n=81) and larger (>400µm2, red, n=118) Hb9-GFP+ neurons. Stascal comparison was 554
done for each age and when all ages were pooled. C2: Distribuon of bistability as a funcon of 555
neuronal cross-seconal 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 funcon 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 idenfied by expression of ChAT-GFP show similar bistability scores that rise with 562
cross-seconal 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 acvaon 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 funcon of cell 569
cross-seconal 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, respecvely) in L4-L5 regions at P9 in response to a slow 575
ramp depolarizaon. F-H: INaP peak amplitude (F), threshold (G) and density (H) as a funcon 576
of cell cross-seconal 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 quarles (dashed lines) are represented in each 579
violin plot. 580
581
Figure 4: Trpm5-mediated current and slowly inacvang 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 oscillaons are evoked. In bistable motoneurons 584
(red trace), these elicit a large, slow aerdepolarizaon which is parally blocked by 50µM 585
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
TPPO (black trace). Non-bistable neurons (blue trace) do not express this slow 586
aerdepolarizaon. B: Amplitude of the Trpm5-induced, TPPO-resistant aerdepolarizaon 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 aerdepolarizaon 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-sensive 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 visualizaon of the post-step current. E-F: 594
Amplitude (E) and density (F) of the Trpm5 current increases with cross-seconal area of Hb9-595
GFP+ neurons (blue circles, <400µm2, n=3, and red circles, >400µm2, n=8; p=0,0121 and p=0242, 596
respecvely) G: Superimposed traces of the slowly inacvang 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
seconal 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 quarles (dashed lines) are 602
represented in each violin plot. 603
604
Figure 5: Inducon of full bistability by serotonin. A: Non-bistable neuron’s response (top, 605
black trace) to 2 sec depolarizing pulse (boom). Only a small aerdepolarizaon is recorded 606
aer the step. During 10µM serotonin (5-HT), this neuron became fully bistable, with 607
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
connuous firing (top, red trace) following the 2 sec depolarizing step (boom). 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 draed 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 dinvesssement INT 626
FI_INT_JCJC_2019 (R.B.), by Centre Naonal de la Recherche Scienfique (CNRS) (R.B. and F.B.) 627
and by Agence Naonale de la Recherche Scienfique ANR-16-CE16-0004 (F.B.). 628
629
Downloaded from journals.physiology.org/journal/jn at CNRS (193.054.110.055) on February 26, 2024.
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+
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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.
... Finally, some motoneurons can continue to fire action potentials after receiving a brief excitatory synaptic input (Fig. 2, panel C). This self-sustained firing seems to be more evident in smaller motoneurons innervating fatigue-resistant muscle fibres, as suggested in previous studies (36)(37)(38), though one recent study in young mice does not support this observation (39). This PIC-induced effect was initially referred to as bistability but it is now typically called self-sustained firing (23). ...
... These include the previously mentioned effects of monoamines on other intrinsic properties and spike-frequency adaptation, a time-dependent reduction in motoneuron firing rate during a period of constant input (41). Additional intrinsic properties are also emerging from studies in mouse motoneurons (39,42,43) 3), with permission): firing behaviour of a cat gastrocnemius medialis motoneuron (upper graph) with a medium-to-strong PIC during slowly increasing and decreasing current (lower graph). This was an intracellular recording obtained in a decerebrate cat preparation in which the noradrenergic agonist methoxamine was added. ...
Article
The manner in which motoneurons respond to excitatory and inhibitory inputs depends strongly on how their intrinsic properties are influenced by the neuromodulators serotonin and noradrenaline. These neuromodulators enhance the activation of voltage‑gated channels that generate persistent (long-lasting) inward sodium and calcium currents (PICs) into the motoneurons. PICs are crucial for initiating, accelerating, and maintaining motoneuron firing. A greater accessibility to state-of-the-art techniques that allows both the estimation and examination of PIC modulation in tens of motoneurons in vivo has rapidly evolved our knowledge of how motoneurons amplify and prolong the effects of synaptic input. We are now in a position to gain substantial mechanistic insight into the role of PICs in motor control at an unprecedented pace. The present review briefly describes the effects of PICs on motoneuron firing and the methods available for estimating them before presenting the emerging evidence of how PICs can be modulated in health and disease. Our rapidly developing knowledge of the potent effects of PICs on motoneuron firing has the potential to improve our understanding of how we move, and points to new approaches to improve motor control. Finally, gaps in our understanding are highlighted and methodological advancements suggested to encourage readers to explore outstanding questions to further elucidate PIC physiology.
... All experiments were designed to gather data within a stable period (i.e., at least 2 min after establishing whole-cell access). Because (i) the cell size influences neuronal excitability [39] and because (ii) we recorded non-identified neurons, we only compared the electrophysiological parameters of cells with the same size in both conditions to avoid any recording bias related to their morphological features. ...
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Background Non-invasive photobiomodulation therapy (PBMT), employing specific infrared light wavelengths to stimulate biological tissues, has recently gained attention for its application to treat neurological disorders. Here, we aimed to uncover the cellular targets of PBMT and assess its potential as a therapeutic intervention for multiple sclerosis (MS). Methods We applied daily dorsoventral PBMT in an experimental autoimmune encephalomyelitis (EAE) mouse model, which recapitulates key features of MS, and revealed a strong positive impact of PBMT on the sensorimotor deficits. To understand the cellular mechanisms underlying these striking effects, we used state-of-the-art tools and methods ranging from two-photon longitudinal imaging of triple fluorescent reporter mice to histological investigations and patch-clamp electrophysiological recordings. Results We found that PBMT induced anti-inflammatory and neuroprotective effects in the dorsal spinal cord. PBMT prevented peripheral immune cell infiltration, glial reactivity, as well as the EAE-induced hyperexcitability of spinal interneurons, both in dorsal and ventral areas, which likely underlies the behavioral effects of the treatment. Thus, aside from confirming the safety of PBMT in healthy mice, our preclinical investigation suggests that PBMT exerts a systemic and beneficial effect on the physiopathology of EAE, primarily resulting in the modulation of the inflammatory processes. Conclusion PBMT may therefore represent a new valuable therapeutic option to treat MS symptoms.
... In fast vs. slow mouse motoneurons, there are differences in the expression of channels that contribute to membrane voltage bistability, including Nav1.6, Trpm5, and K V 1.2. 58 Fast motoneurons have a higher density of C-boutons 59 ; it is possible that there are clustering/expression differences in K V 2 channels at these sites between slow and fast motoneurons, although none have been reported. In any case, taking the electrophysiological data together with the behavioral data together with the known high density of C-boutons on motoneurons, it is unlikely that a bias toward the larger motoneurons significantly impacted our conclusions. ...
Article
The increased muscular force output required for some behaviors is achieved via amplification of motoneuron output via cholinergic C-bouton synapses. Work in neonatal mouse motoneurons suggested that modulation of currents mediated by post-synaptically clustered KV2.1 channels is crucial to C-bouton amplification. By focusing on more mature motoneurons, we show that conditional knockout of KV2.1 channels minimally affects either excitability or response to exogenously applied muscarine. Similarly, unlike in neonatal motoneurons or cortical pyramidal neurons, pharmacological blockade of KV2 currents has minimal effect on mature motoneuron firing in vitro. Furthermore, in vivo amplification of electromyography activity and high-force task performance was unchanged following KV2.1 knockout. Finally, we show that KV2.2 is also expressed by spinal motoneurons, colocalizing with KV2.1 opposite C-boutons. We suggest that the primary function of KV2 proteins in motoneurons is non-conducting and that KV2.2 can function in this role in the absence of KV2.1.
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Spasticity, a prevalent motor issue characterized by network hyperexcitability, causes pain and discomfort, with existing treatments offering limited relief. While past research has focused on neuronal factors, the role of astrocytes in spasticity has been overlooked. This study explores the potential of restoring astrocytic potassium (K ⁺ ) uptake to reduce spasticity following SCI. Astrocytes buffer extracellular K ⁺ via Kir4.1 channels, preventing neuronal hyperexcitability. Following spinal cord injury (SCI), Kir4.1 levels decrease at the injury site, though the consequences and mechanisms of this reduction within the motor output area have not been investigated. Utilizing advanced techniques, we demonstrate that lumbar astrocytes in a juvenile thoracic SCI mouse model switch to reactive phenotype, displaying morpho-functional and pro-inflammatory changes. These astrocytes also experience NBCe1-mediated intracellular acidosis, leading to Kir4.1 dysfunction and impaired K ⁺ uptake. Enhancing Kir4.1 function reduces spasticity in SCI mice, revealing new therapeutic targets for neurological diseases associated with neuronal hyperexcitability. Highlights Lumbar astrocytes adopt a reactive phenotype following a thoracic SCI NBCe1-mediated acidosis in astrocytes disrupts Kir4.1 function post-SCI. Impaired K+ uptake leads to motoneuron hyperexcitability post-SCI. Enhanced astroglial Kir4.1 function reduces spastic-like symptoms in SCI mice.
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Motoneuron properties and their firing patterns undergo significant changes throughout development and in response to neuromodulators such as serotonin. Here, we examined the age‐related development of self‐sustained firing and general excitability of tibialis anterior motoneurons in a young development (7–17 years), young adult (18–28 years) and adult (32–53 years) group, as well as in a separate group of participants taking selective serotonin reuptake inhibitors (SSRIs, aged 11–28 years). Self‐sustained firing, as measured by Δ F , was larger in the young development (∼5.8 Hz, n = 20) compared to the young adult (∼4.9 Hz, n = 13) and adult (∼4.8 Hz, n = 8) groups, consistent with a developmental decrease in self‐sustained firing mediated by persistent inward currents (PIC). Δ F was also larger in participants taking SSRIs (∼6.5 Hz, n = 9) compared to their age‐matched controls (∼5.3 Hz, n = 26), consistent with increased levels of spinal serotonin facilitating the motoneuron PIC. Participants in the young development and SSRI groups also had higher firing rates and a steeper acceleration in initial firing rates (secondary ranges), consistent with the PIC producing a steeper acceleration in membrane depolarization at the onset of motoneuron firing. In summary, both the young development and SSRI groups exhibited increased intrinsic motoneuron excitability compared to the adults, which, in the young development group, was also associated with a larger unsteadiness in the dorsiflexion torque profiles. We propose several intrinsic and extrinsic factors that affect both motoneuron PICs and cell discharge which vary during development, with a time course similar to the changes in motoneuron firing behaviour observed in the present study. image Key points Neurons in the spinal cord that activate muscles in the limbs (motoneurons) undergo increases in excitability shortly after birth to help animals stand and walk. We examined whether the excitability of human ankle flexor motoneurons also continues to change from child to adulthood by recording the activity of the muscle fibres they innervate. Motoneurons in children and adolescents aged 7–17 years (young development group) had higher signatures of excitability that included faster firing rates and more self‐sustained activity compared to adults aged ≥18 years. Participants aged 11–28 years of age taking serotonin reuptake inhibitors had the highest measures of motoneuron excitability compared to their age‐matched controls. The young development group also had more unstable contractions, which might partly be related to the high excitability of the motoneurons.
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Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identify the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The inhibition of Nav1.6, but not of Nav1.1, in motoneurons impairs INaP, bistability, postural tone, and locomotor performance. In interneurons of the rhythmogenic CPG region, both Nav1.6 and Nav1.1 equally mediate INaP. Inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG.
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Mixed electrical-chemical synapses potentially complicate electrophysiological interpretations of neuronal excitability and connectivity. Here, we disentangle the impact of mixed synapses within the spinal locomotor circuitry of larval zebrafish. We demonstrate that soma size is not linked to input resistance for interneurons, contrary to the biophysical predictions of the ‘size principle’ for motor neurons. Next, we show that time constants are faster, excitatory currents stronger, and mixed potentials larger in lower resistance neurons, linking mixed synapse density to resting excitability. Using a computational model, we verify the impact of weighted electrical synapses on membrane properties, synaptic integration and the low-pass filtering and distribution of coupling potentials. We conclude differences in mixed synapse density can contribute to excitability underestimations and connectivity overestimations. The contribution of mixed synaptic inputs to resting excitability helps explain ‘violations’ of the size principle, where neuron size, resistance and recruitment order are unrelated.
Chapter
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Although they share the common function of controlling muscle fiber contraction, spinal motoneurons display a remarkable diversity. Alpha-motoneurons are the “final common pathway”, which relay all the information from spinal and supraspinal centers and allow the organism to interact with the outside world by controlling the contraction of muscle fibers in the muscles. On the other hand, gamma-motoneurons are specialized motoneurons that do not generate force and instead specifically innervate muscle fibers inside muscle spindles, which are proprioceptive organs embedded in the muscles. Beta-motoneurons are hybrid motoneurons that innervate both extrafusal and intrafusal muscle fibers. Even among alpha-motoneurons, there exists an exquisite diversity in terms of motoneuron electrical and molecular properties, physiological and structural properties of their neuromuscular junctions, and molecular and contractile properties of the innervated muscle fibers. This diversity, across species, across muscles, and across muscle fibers in a given muscle, underlie the vast repertoire of movements that one individual can perform.KeywordsMotoneuronMotor unitPhysiological typeElectrophysiologyNeuromuscular junctionsMuscle fibersContractile properties
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In vitro spinal cord preparations have been extensively used to study microcircuits involved in the control of movement. By allowing precise control of experimental conditions coupled with state-of-the-art genetics, imaging, and electrophysiological techniques, isolated spinal cords from mice have been an essential tool in detailing the identity, connectivity, and function of spinal networks. The majority of the research has arisen from in vitro spinal cords of neonatal mice, which are still undergoing important postnatal maturation. Studies from adults have been attempted in transverse slices, however, these have been quite challenging due to the poor motoneuron accessibility and viability, as well as the extensive damage to the motoneuron dendritic trees. In this work, we describe two types of coronal spinal cord preparations with either the ventral or the dorsal horn ablated, obtained from mice of different postnatal ages, spanning from preweaned to 1 mo old. These semi-intact preparations allow recordings of sensory-afferent and motor-efferent responses from lumbar motoneurons using whole cell patch-clamp electrophysiology. We provide details of the slicing procedure and discuss the feasibility of whole cell recordings. The in vitro dorsal and ventral horn-ablated spinal cord preparations described here are a useful tool to study spinal motor circuits in young mice that have reached the adult stages of locomotor development. NEW & NOTEWORTHY In the past 20 years, most of the research into the mammalian spinal circuitry has been limited to in vitro preparations from embryonic and neonatal mice. We describe two in vitro longitudinal lumbar spinal cord preparations from juvenile mice that allow the study of motoneuron properties and respective afferent or efferent spinal circuits through whole cell patch clamp. These preparations will be useful to those interested in the study of microcircuits at mature stages of motor development.
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Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na ⁺ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na ⁺ -permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca ²⁺ release and turned off by Ca ²⁺ reuptake through the sarco/endoplasmic reticulum Ca ²⁺ -ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.
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The size principle underlies the orderly recruitment of motor units; however, motoneuron size is a poor predictor of recruitment amongst functionally defined motoneuron subtypes. Whilst intrinsic properties are key regulators of motoneuron recruitment, the underlying currents involved are not well defined. Whole-cell patch-clamp electrophysiology was deployed to study intrinsic properties, and the underlying currents, that contribute to the differential activation of delayed and immediate firing motoneuron subtypes. Motoneurons were studied during the first three postnatal weeks in mice to identify key properties that contribute to rheobase and may be important to establish orderly recruitment. We find that delayed and immediate firing motoneurons are functionally homogeneous during the first postnatal week and are activated based on size, irrespective of subtype. The rheobase of motoneuron subtypes become staggered during the second postnatal week, which coincides with the differential maturation of passive and active properties, particularly persistent inward currents. Rheobase of delayed firing motoneurons increases further in the third postnatal week due to the development of a prominent resting hyperpolarization-activated inward current. Our results suggest that motoneuron recruitment is multifactorial, with recruitment order established during postnatal development through the differential maturation of passive properties and sequential integration of persistent and hyperpolarization-activated inward currents.
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All movements are generated by the activation of motoneurons, and hence their input-output properties define the final step in processing of all motor commands. A major challenge to understanding this transformation has been the striking nonlinear behavior of motoneurons conferred by the activation of persistent inward currents (PICs) mediated by their voltage-gated Na and Ca 2 channels. In this review, we focus on the contribution that these PICs make to motoneuronal discharge and how the nonlinearities they engender impede the construction of a comprehensive model of motor control. motoneuron; persistent inward currents; input-output function; nonlinear behavior
Chapter
Beginning about half a century ago, the rules that determine how motor units are recruited during movement have been deduced. These classical experiments led to the formulation of the ‘size principle’. It is now clear that motoneuronal size is not the only indicator of recruitment order. In fact, motoneuronal passive, active and synaptic conductances are carefully tuned to achieve sequential recruitment. More recent studies, over the last decade or so, show that the premotor circuitry is also functionally specialized and differentially recruited. Modular sub networks of interneurons and their post-synaptic motoneurons have been shown to drive movements with varying intensities. In addition, these modular networks are under the influence of neuromodulators, which are capable of acting upon multiple motor and premotor targets, thereby altering behavioral outcomes. We discuss the recruitment patterns of motoneurons in light of these new and exciting studies.KeywordsSize principleIntrinsic propertiesSynaptic propertiesNeuromodulationInterneuronsForceCircuitsDopamineSerotoninBistabilityExcitability
Chapter
Motoneurons are the ‘final common path’ between the central nervous system (that intends, selects, commands, and organises movement) and muscles (that produce the behaviour). Motoneurons are not passive relays, but rather integrate synaptic activity to appropriately tune output (spike trains) and therefore the production of muscle force. In this chapter, we focus on studies of mammalian motoneurons, describing their heterogeneity whilst providing a brief historical account of motoneuron recording techniques. Next, we describe adult motoneurons in terms of their passive, transition, and active (repetitive firing) properties. We then discuss modulation of these properties by somatic (C-boutons) and dendritic (persistent inward currents) mechanisms. Finally, we briefly describe select studies of human motor unit physiology and relate them to findings from animal preparations discussed earlier in the chapter. This interphyletic approach to the study of motoneuron physiology is crucial to progress understanding of how these diverse neurons translate intention into behaviour.KeywordsRepetitive firingSpike frequency adaptationC-boutonsPersistent inward currentsModulation
Article
Appropriate scaling of motor output from mouse to humans is essential. The motoneurons that generate all motor output are, however, very different in rodents compared with humans, being smaller and much more excitable. In contrast, feline motoneurons are more similar to those in humans. These scaling differences need to be taken into account for the use of rodents for translational studies of motor output.