Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development
- School of Psychology and Neuroscience, University of St Andrews, United Kingdom
Figures
Figure 1
Delayed and immediate firing motoneurons exhibit intrinsic properties consistent with fast- and slow-type motoneurons, respectively.
(A) Whole-cell patch-clamp recordings were obtained from motoneurons identified in the ventrolateral horn of the lumbar spinal cord with a subset retrogradely labelled with Fluoro-Gold injected intraperitoneally. Two motoneuron subtypes were identified based on the onset latency for repetitive firing and subsequent changes in firing rates during a 5 s depolarizing current step applied at rheobase (B): a delayed onset of repetitive firing and an accelerating firing rate (B1; purple) and an immediate onset for repetitive firing with a stable or adapting firing rate (B2; green). Delayed and immediate firing motoneuron subtypes display intrinsic properties that are consistent with those of fast- and slow-type motoneurons illustrated by broader single-action potentials (C), longer medium afterhyperpolarizations (D), and lower rheobase currents (E) in immediate compared to delayed firing motoneurons.
Figure 2
Principal component analysis (PCA) reveals divergent maturation of the intrinsic properties of delayed and immediate firing motoneuron subtypes during postnatal development.
(A) Scree plot illustrating 10 principal components (PCs) identified in the PCA, with the first six accounting for greater than 75% of variance. (B) Variable loadings for PC1 and PC2 with arrow length representing loading score for each variable denoted in red text. (C) Variable loadings for PC1 and PC3 with arrow length representing loading score for each variable (red). (D–F) PC scores for PCs1–3 for delayed (purple) and immediate firing (green) motoneurons across the first three weeks of postnatal development. Individual PC scores are displayed for each cell, black bars represent mean ± SD. Statistical analysis was conducted using a two-way ANOVA and Holm–Sidak post hoc analysis. Asterisks denote significant differences from pairwise comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G - I) 3D scatterplots between PCs 1, 2, and 3 for delayed (purple) and immediate firing (green) motoneurons across weeks 1–3.
-
Figure 2—source data 1
Eigenvalues (Figure 2A), loading scores (Figure 2B-C), principal component scores (Figure 2D-F), and scatterplot data (Figure 2G-I) derived from principal component analysis on 25 intrinsic properties studied from motoneuron subtypes across postnatal development.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig2-data1-v2.xlsx
Figure 3 with 1 supplement
Passive properties contribute to maturation of rheobase currents between weeks 1 and 2 but additional factors contribute from weeks 2–3.
The rheobase current of delayed (purple) and immediate firing (green) motoneurons was assessed using slow (100 pA/s) depolarizing current ramps. (A1–C1) Representative traces of rheobase and repetitive firing of delayed and immediate firing motoneurons at weeks 1–3. (A2–C2) First action potential produced upon recruitment for delayed and immediate firing motoneurons at weeks 1–3. (D) Rheobase increases in delayed but not immediate firing motoneurons across weeks 1–3. (E) Cumulative proportion histograms of rheobase currents of motoneurons sampled during weeks 1 (black), 2 (red), and 3 (blue) indicate a progressive decrease in the recruitment gain. (F) Whole-cell capacitance increases, and input resistance decreases (G) in delayed but not immediate firing motoneurons between weeks 1 and 2 but not further into week 3. Individual data points are displayed, black bars represent mean ± SD. Statistical analysis was conducted using a two-way ANOVA and Holm–Sidak post hoc analysis. Asterisks denote significant differences from pairwise comparisons *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001. Scatterplots of rheobase current and passive properties (H1-H3: whole-cell capacitance; I1-I3: input resistance) (Gustafsson and Pinter, 1984) for delayed (purple) and immediate firing (green) motoneurons during postnatal weeks 1–3. Pearson correlations were performed for each type at each time point with outcomes displayed in purple text for delayed and green text for immediate firing motoneurons. A simple linear regression comparing the regression slopes for each motoneuron subtype at each time point was performed to determine the degree of similarities between subtypes, with outcomes displayed in black text.
-
Figure 3—source data 1
Rheobase (Figure 3D), recruitment gain (Figure 3E), capacitance (Figure 3F), and input resistance (Figure 3G) values, and correlations (Figure 3H-I) from motoneuron subtypes studied across postnatal development.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Rheobase is correlated and shows similar changes during postnatal development when measured during depolarizing current ramps or square depolarizing current steps.
(A–C) Scatterplots for rheobase measured during depolarizing current ramps (x-axis) and square depolarizing current steps (y-axis) in delayed (purple) and immediate (green) firing motoneurons during the first (A), second (B), and third (C) postnatal weeks. Data were analysed with a Pearson correlation. (D, E) Rheobase increases in delayed but not immediate firing motoneurons across postnatal development when measured using depolarizing current ramps (D) or depolarizing current steps (E). Statistical analyses were conducted with one- or two-way ANOVA and Holm–Sidak post hoc analysis when significant effects were detected. Asterisks denote significant differences from Holm–Sidak post hoc analysis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as individual points for each motoneuron studied with black bars depicting mean ± SD.
-
Figure 3—figure supplement 1—source data 1
Rheobase measured from motoneuron subtypes across development on depolarizing ramps (A) and current steps (B) are correlated during the first (C), second (D), and third (E) postnatal weeks.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Nav1.6-conducting persistent inward currents (PICs) shape motoneuron activation.
(A) Rheobase was assessed using slow depolarizing current ramps (100 pA/s from –60 mV). During these ramps, the trajectory of the membrane potential approaching the spike threshold is characterized by a passive, linear phase (DV: grey line) and an accelerating phase. The impact of the underlying current producing the membrane potential acceleration, termed ePIC (A1, B), is estimated as the difference between the extrapolated passive and actual rheobase currents. The acceleration onset voltage (A2, C) is the voltage at which the membrane potential deviates by greater than 1 mV from the linear function fitted to the initial (passive) depolarization of the membrane potential during the ramp. The amplitude of this acceleration phase is calculated from the onset voltage to the threshold of the first action potential (A3, D). (E) PICs were measured in voltage clamp from delayed (purple) and immediate (green) firing motoneurons of 2-week-old (n = 25 delayed; n = 14 immediate) and 3-week-old (n = 14 delayed; n = 13 immediate) mice using a slow depolarizing voltage ramp (10 mV/s; –90 to –10 mV). (F) PIC onset and amplitude were measured from leak-subtracted, low-pass filtered (5 Hz Bessel) traces. (G) PICs were larger in amplitude in delayed compared to immediate firing motoneurons at both 2 and 3 weeks. (H) PIC onset was significantly more depolarized in delayed compared to immediate firing motoneurons and did not change between weeks 2 and 3. (I) Representative traces of a PIC measured in voltage clamp from a delayed firing motoneuron. The relative contribution of Nav1.6 and L-type calcium channels to PICs in delayed and immediate firing motoneurons was tested with 4,9-anhydrotetrodotoxin (4,9 AH-TTX; 200 nM, delayed: n = 22; immediate: n = 12) and nifedipine (20 μM, delayed: n = 16; immediate: n = 10), respectively (black traces). 4,9 AH-TTX reduced PIC amplitude of delayed and immediate firing motoneurons (J), whereas nifedipine only reduced the PIC amplitude of delayed firing motoneurons (K). 4,9 AH-TTX depolarized PIC onset voltage of delayed firing motoneurons (L); nifedipine did not change PIC onset voltage in either subtype (M). (N) Representative traces from depolarizing current ramps of delayed firing motoneurons used to investigate changes in rheobase before (red) and after (black) blocking Nav1.6 or L-type calcium channels. (O) Blocking Nav1.6 increased the rheobase of delayed but not immediate firing motoneurons. (P) Cumulative proportion histograms for rheobase currents of delayed and immediate firing motoneurons sampled before (red) and after (black) blocking Nav1.6 channels. Blocking L-type calcium channels did not alter rheobase in either motoneuron subtype (Q) or change the recruitment gain (R). Individual data points are displayed, black bars represent mean ± SD. Statistical analysis was conducted using a two-way ANOVA and Holm–Sidak post hoc analysis. Asterisks denote significant differences from pairwise comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
-
Figure 4—source data 1
Estimated persistent inward current (PIC) (Figure 4B), acceleration onset voltage (Figure 4C), and spike threshold (Figure 4D) measured during depolarizing current ramps.
PIC onset voltage (Figure 4H) and amplitude (Figure 4J) measured in voltage clamp during depolarizing voltage ramps. PIC amplitude and onset voltage measured in voltage clamp (Figure 4), and rheobase and recruitment gain (Figure 4O-R) in motoneuron subtypes before and after blockade of Nav1.6 or L-type calcium channels.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Maturation of recruitment-derecruitment and firing hysteresis during postnatal development.
Representative traces of the membrane potential and repetitive firing from a delayed (A: purple) and immediate firing (B: green) motoneuron during a triangular depolarizing current ramp up to an intensity of 2× their respective recruitment currents. Recruitment-derecruitment hysteresis (ePIC), characterized by a lower current at derecruitment on the descending limb compared to recruitment on the ascending limb, producing a negative Delta I, is suggestive of the activation of a persistent inward current (PIC). Delta I is correlated with input resistance (C), increases after week 2 (D), but only in delayed firing motoneurons (E). Statistical analyses were conducted with one- or two-way ANOVA and Holm–Sidak post hoc analysis when significant effects were detected. Asterisks denote significant differences from Holm–Sidak post hoc analysis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (F) Four patterns of firing rate hysteresis can be identified as described by Li and Bennett, 2003, with types 3 and 4 suggestive of PIC action. (G) Relative proportion of firing hysteresis types in fast and slow motoneurons across 3 weeks of postnatal development.
-
Figure 4—figure supplement 1—source data 1
Recruitment-derecruitment (C–E) and firing rate hysteresis (G) measured from motoneuron subtypes across postnatal development.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig4-figsupp1-data1-v2.xlsx
Figure 5
Ih increases and becomes activated at more depolarized potentials in delayed firing motoneurons by week 3 .
(A) Sag potentials were measured from delayed (purple) and immediate firing (green) motoneurons of mice aged P1–4 (n = 54), P7–12 (n = 122), and P14–20 (n = 81). (B) The sag conductance (gSag) was estimated (eSag Conductance) during a series of hyperpolarizing current steps (1 s duration). Mean ± SD sag potentials plotted as a function of trough of membrane potential for delayed (C) and immediate firing (D) motoneurons at P1–4 (black), P7–12 (red), and P14–20 (blue). (E) Ih was measured in delayed and immediate firing motoneurons during weeks 2 (Delayed: n = 15; Immediate: n = 12) and 3 (Delayed: n = 19; Immediate: n = 19) in voltage clamp using a series of incremental hyperpolarizing voltage steps (−60 to –110 mV, 1 s duration, 10 mV increments). Ih measured at –70 mV (F) and –110 mV (G) increased in delayed firing motoneurons between weeks 2 and 3 and was larger in delayed compared to immediate firing motoneurons at week 3 (H). Representative trace of Ih recorded in voltage clamp from a delayed firing motoneuron before (red trace) and after (black trace) bath application of the HCN channel blocker ZD7288 (10 µM). ZD7288 blocked Ih in delayed firing motoneurons measured at –70 mV (I) and Ih measured at –110 mV in both delayed (n = 13) and immediate firing (n = 9) motoneurons (J). Data are presented as individual points for each cell studied. Statistical analyses were conducted using two-way ANOVA and Holm–Sidak post hoc analysis when significant effects were detected. Asterisks denote significant differences from Holm–Sidak post hoc analysis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
-
Figure 5—source data 1
Depolarizing sag potentials measured in current clamp from motoneuron subtypes (Figure 5B-D) and underlying Ih measured in voltage clamp (Figure 5F-G) from motoneuron subtypes at postnatal weeks 2 and 3.
Ih measured in voltage clamp before and after application of ZD7288 (Figure 5I-J).
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig5-data1-v2.xlsx
Figure 6
A resting Ih increases rheobase of delayed firing motoneurons at week 3.
(A) Representative trace of depolarizing sag potentials in a delayed firing motoneuron during negative current steps that brought the membrane potential from –60 mV to resting potential (–67 mV) for this cell, which could be blocked by the HCN channel blocker ZD7288 (black trace). The individual blue traces and arrows highlight a depolarizing sag from resting potential at baseline which turns into a slow hyperpolarizing potential following application of ZD7288. (B) ZD7288-sensitive depolarizing sag potentials were found at resting membrane potential (RMP) in delayed (n = 13; purple) but not immediate (n = 9; green) firing motoneurons. (C) Representative traces of the RMP of delayed and immediate firing motoneurons from lumbar slices of P14–20 mice during blockade of HCN channels with ZD7288. (D) ZD7288 hyperpolarized the RMP of both delayed and immediate firing motoneurons; however, the magnitude of the hyperpolarization was larger in delayed firing motoneurons (E: unpaired t-test). (F) Representative voltage trace of a delayed firing motoneuron during a slow depolarizing (100 pA/s) current ramp before (red) and after (black) ZD7288. (G) ZD7288 reduced the rheobase current of delayed but not immediate firing motoneurons. Data are presented as individual points for each cell studied. Statistical analyses were conducted using two-way ANOVA and Holm–Sidak post hoc analysis when significant effects were detected. Asterisks denote significant differences from Holm–Sidak post hoc analysis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (H) Recruitment gain represented by cumulative proportion histograms of rheobase currents of motoneurons sampled before (red) and after (black) blocking HCN channels.
-
Figure 6—source data 1
Depolarizing sag measured at resting potential (Figure 6B), resting membrane potential (Figure 6D-E), rheobase (Figure 6G), and recruitment gain (Figure 6H) before and after ZD7288.
- https://cdn.elifesciences.org/articles/71385/elife-71385-fig6-data1-v2.xlsx
Figure 7
Schematic summarizing changes in the physical properties, recruitment, and underlying currents in motoneuron subtypes that shape the orderly recruitment and recruitment gain of motoneurons during postnatal development as complex locomotor behaviours emerge.
(A) Illustrates the time course over which locomotor behaviours mature during the first three weeks of postnatal development (adapted from Jean-Xavier et al., 2018). (B) depicts the increase in the size of fast, but not slow motoneurons during the first two postnatal weeks, with no further increases into the third. (C) The range of inputs over which a motor pool is recruited expands across the first three weeks of postnatal development. (D) The expansion of the recruitment range coincides with the maturation of persistent (PIC) and hyperpolarization-activated (Ih) inward currents. Fast motoneurons and their underlying currents are depicted in purple and slow motoneurons, and their underlying currents depicted in green.
Additional files
-
Supplementary file 1
Passive, recruitment, repetitive firing, and single spike properties of delayed and immediate firing motoneuron subtypes across three postnatal weeks.
Number of motoneurons (n) included in each subtype (Del: delayed; Imm: immediate) across postnatal weeks 1–3. Passive properties include whole-cell capacitance, tau, input resistance, and resting membrane potential (RMP). Recruitment properties: rheobase, derecruitment current, current firing range, voltage threshold of the first spike, and relative distance between RMP and spike threshold. Repetitive firing properties measured following recruitment on slow depolarizing current ramps include minimum firing rate, firing rate at 2× rheobase, maximum firing rate, and the slope of the sub-primary range (SPR). Single spike (action potential) properties including spike amplitude, rise time, and half width were measured from threshold, and medium afterhyperpolarization (mAHP) amplitude and half width, were elicited with a 10 ms square current pulse applied at 1.25× rheobase. Statistics: data were analysed using two-factor ANOVA with MN type (Del and Imm) and developmental week (weeks 1–3) as factors. Statistical analyses addressed three core questions: (1) Was there a difference between subtypes? (2) Did it change during development? (3) Do the subtypes mature differentially during development? Statistical results listing F and p values from two-factor ANOVA addressing these questions are listed sequentially: (1) main effect of subtype; (2) main effect of development; and (3) subtype × development interaction. All data are presented as mean ± SD (min, max). p-Values are derived from Holm–Sidak post hoc comparisons within weeks, between MN types. Superscript numbers denote significant differences from Holm–Sidak within MN types, between weeks (1: week 1; 2: week 2; 3: week 3).
- https://cdn.elifesciences.org/articles/71385/elife-71385-supp1-v2.docx
-
Supplementary file 2
Active properties of delayed and immediate firing motoneuron subtypes across three postnatal weeks.
Characteristics of the subthreshold membrane potential trajectory during slow depolarizing recruitment current ramps. Presented are the acceleration onset voltage, acceleration amplitude, passive recruitment current estimated based on the initial linear trajectory of the membrane potential, actual measured recruitment current, and an estimate of the underlying persistent inward current (ePIC) that produces the membrane potential acceleration. Delta I reflects the difference in derecruitment and recruitment currents on triangular depolarizing current ramps and provides an additional estimate of PIC. PICs were measured in voltage clamp during weeks 2–3 with the onset voltage, peak voltage, current amplitude, and densities measured. Sag conductance was estimated during weeks 1–3 and underlying h current (Ih) measured with amplitude and density at –70 mV and –110 mV presented. Time constant (tau) of Ih. Statistics: data were analysed using two-factor ANOVA with MN type (delayed and immediate) and developmental week (weeks 1–3) as factors. Statistical analyses addressed three core questions: (1) Was there a difference between subtypes? (2) Did it change during development? (3) Do the subtypes mature differentially during development? Statistical results listing F and p values from two-factor ANOVA addressing these questions are listed sequentially: (1) main effect of subtype; (2) main effect of development; (3) subtype × development interaction. All data are presented as mean ± SD (min, max). Number of cells (n) are included in brackets for each group. p-Values are derived from Holm–Sidak post hoc comparisons within weeks, between MN types. Superscript numbers denote significant differences from Holm–Sidak within MN types, between weeks (1: week 1; 2: week 2; 3: week 3).
- https://cdn.elifesciences.org/articles/71385/elife-71385-supp2-v2.docx
-
Supplementary file 3
A comparison of studies on lumbar motoneuron intrinsic properties recorded with whole-cell patch-clamp electrophysiology across the first three weeks of postnatal development in mice.
Comparison of intrinsic properties measured in studies on motoneuron subtypes (current work; Leroy et al., 2014) and those that pooled all lumbar motoneurons studied (Quinlan et al., 2011; Nakanishi and Whelan, 2010; Smith and Brownstone, 2020). Data are presented as mean ± SD; Nakanishi and Whelan, 2010 are presented as mean ± SEM.
- https://cdn.elifesciences.org/articles/71385/elife-71385-supp3-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/71385/elife-71385-transrepform1-v2.docx
-
Source data 1
Values for intrinsic properties studied in motoneuron subtypes across the first, second, and third postnatal weeks and are summarized in Supplementary file 1.
- https://cdn.elifesciences.org/articles/71385/elife-71385-supp4-v2.xlsx
-
Source data 2
Values for active properties studied in motoneuron subtypes across the first, second, and third postnatal weeks and are summarized in Supplementary file 2.
- https://cdn.elifesciences.org/articles/71385/elife-71385-supp5-v2.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development
eLife 10:e71385.
https://doi.org/10.7554/eLife.71385
