Maturation of persistent and hyperpolarization-activated inward currents shapes the differential activation of motoneuron subtypes during postnatal development

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1. 

   Version published to 10.7554/elife.71385 on eLife

   Nov 16, 2021
2. 

   Version published to 10.1101/2021.06.16.448644v2 on bioRxiv

   Nov 11, 2021
3. 

   eLife

   Nov 3, 2021

   Author Response:

   Reviewer #1 (Public Review):

   1. The authors use a surrogate marker for "slow" vs "fast" MNs: immediate vs delayed firing in response to rectangular current injection. They switch their language to call these motoneurons slow and fast, but they should be more cautious about doing so, given that firing is a surrogate marker that has not been fully studied and characterised across this time period. We do not know if developmental changes in Kv1 might also contribute to the effects on spiking seen here. I agree, there is delayed firing from the outset, which is interesting in itself given the rather homogenous other properties. But that doesn't mean that Kv1 expression is stable and thus non-contributory to the changes in firing described.

   As suggested in the editorial summary, we have changed all reference to fast and …

   Author Response:

   Reviewer #1 (Public Review):

   1. The authors use a surrogate marker for "slow" vs "fast" MNs: immediate vs delayed firing in response to rectangular current injection. They switch their language to call these motoneurons slow and fast, but they should be more cautious about doing so, given that firing is a surrogate marker that has not been fully studied and characterised across this time period. We do not know if developmental changes in Kv1 might also contribute to the effects on spiking seen here. I agree, there is delayed firing from the outset, which is interesting in itself given the rather homogenous other properties. But that doesn't mean that Kv1 expression is stable and thus non-contributory to the changes in firing described.

   As suggested in the editorial summary, we have changed all reference to fast and slow motoneurons to delayed and immediate firing motoneurons up until the discussion. Furthermore, we have expanded our discussion highlighting potential roles for Kv1 in shaping motoneuron rheobase across development as an interesting direction for future study. Page 19, Lines 630-639.

   1. The focus of the manuscript is on MN recruitment, but recruitment is never defined, despite being used in the title, through the abstract and key points, as well as throughout the manuscript. What they are looking at is response to current injected at the soma vs recruitment during a behaviour when synaptic inputs are bombarding an extensive dendritic tree. Thus, this manuscript does not look at recruitment per se, but rather activation of action potentials in response to intra-somatic stimulation. Accordingly, the term "recruitment" would be best kept to the Discussion.

   We have changed the wording in the title from ‘recruitment’ to ‘active properties’ in the title, changed references to recruitment current to rheobase in the results section, and re-addressed recruitment in the discussion. Further, we have highlighted the importance of where synaptic inputs terminate on motoneurons and implications for recruitment with emphasis placed on the compartmental localization between synaptic terminals and compartmental clustering of voltage-sensitive ion channels on Page 21, Lines 688-692.

   I also note that the "size" principle relates more to electrical than physical size in the 21st century; I agree that the two are correlated, but the authors may not want to stick to arguments about physical size.

   We have tried to clarify when we are referring to electrical or physical size within the revised text.

   1. Figure 6 is problematic, possibly in the way it's presented. It seems to me, but not clear, that the authors suggest that Ih is active at rest, more so in "F" than "S," and that therefore "F" are more depolarised and have smaller mAHPs. So (a) how come RMP didn't seem to come out in the PC analysis earlier? (b) would that not suggest that "F" would be "recruited" earlier? (Or is it that there is reduced sodium channel availability because of the depolarisation - what are the differences in spike amplitude and rate of rise?) (c) shouldn't the mAHP be larger if the cell is more depolarised and further from the potassium equilibrium potential? On the last note, maybe it's that Ih makes the mAHP smaller, but with the kinetics of Ih, wouldn't the decay be faster, but in Fig 6 the decay seems faster when Ih is blocked? Finally, Fig 6E suggests changes in the fAHP and delayed depolarisation (and spike width?) - how to these come into the picture? If the fAHP is thought to result from a high conductance state, and if therefore one were to align the voltages based on this potential, then the mAHPs would be about the same amplitude? The authors likely have explanations, but I'm afraid that I can't follow it.

   We agree that some aspects of Figure 6 were not ideal, particularly those related to RMP and mAHP. For example, we had attempted to utilise measures of mAHP to help provide evidence that Ih was active at rest. However, upon reflection, the data provided in the original manuscript did not achieve this as clearly as the new data we have added, where we now measure Ih at resting membrane potential. Furthermore, the subset of data utilised in the previous Figure 6B misleadingly suggested a slightly more depolarised RMP in delayed MNs. However, as noted in supplementary table 1, delayed firing MNs in fact have a more hyperpolarised RMP compared to immediate firing MNs. We have therefore removed some of these problematic aspects of the figure that lacked clarity and have become less important towards the main points we were attempting to make, given the new data we have added. The simple explanation for RMP not coming out as a strong contributor in the PCA is likely because this parameter is not a prime contributor of variance across MN type or through development. We did not find any difference in spike amplitude or rise time between subtypes (these data are summarized in Supplementary File 1). As is common practice, we injected bias current to measure parameters included in the overall PCA from approximately -60mV. However, in hindsight, this could have reduced potential effects of RMP on some of the properties related to activation.

   1. A number of labs have looked at development of MN properties, and it would be useful to compare properties seen across different labs, for example Quinlan (e.g. PMID: 21486770) and Whelan (e.g. PMID: 20457856, which is only mentioned in the manuscript) (and for that matter, mine - e.g. PMID 10564356, PMID: 32851667 although I don't want to self-promote).

   We have highlighted the consistencies between our results and those reported by others, including Whelan, Heckman, Zytnicki, and Brownstone Labs. This statement can be found on page 3, Lines 104-106.

   We have also included a comparison table in the supplemental information (Supplementary File 3) and referenced this table in the discussion on Line 528.

   1. In the Discussion, the authors might want to discuss propensity of F vs S MNs to express PICs / sustained firing as described in the Heckman lab (albeit particularly in cat; see PMID 9705452, for e.g.). How do these data correspond?

   It is interesting to note that the properties of PICs that we measured (ie. onset and amplitude) in immediate and delayed firing motoneurons are similar to those of fully and partially bistable motoneurons described by Lee and Heckman. In particular, we find that higher input resistance, immediate firing motoneurons have smaller PIC amplitude than delayed firing motoneurons but their PICs are activated at more hyperpolarized membrane potentials. This is consistent with fully bistable motoneurons, which are higher input resistance, and have smaller PICs that are activated at more hyperpolarized voltages compared to the partially bistable motoneurons. While we did not see bistability in the samples of motoneurons that we studied, a key difference is that we studied intrinsic properties in the absence of neuromodulation, which is a key factor for promoting bistability in motoneurons. Modulation of PICs might contribute to this propensity for bistability; however, modulation of outward currents is also very likely. We have highlighted these similarities and differences in our discussion on PICs and can be found on Page 19, Line 591-596.

   Reviewer #4 (Public Review):

   1. During the 1st postnatal week, authors suggest that fast and slow MNs cannot be distinguished neither on their passive properties nor the rheobase and therefore their recruitment is mainly based on the size. The conclusion that the recruitment is not linked to MN functional differences is difficult to follow since the main distinction between MN subtypes is based upon the presence of a delayed firing, an active property that regulates the recruitment of MNs (Leroy et al., 2014). However, the square current pulse adopted to discriminate between delayed and immediate firing in Figure 1 was replaced with a ramped depolarization protocol on which the authors measured the rheobase (Figure 3A1). This suggests that the slow depolarization in immature motoneurons might minimize the activation of ionic conductance(s) responsible for the delayed firing and thus may bias the measure of the rheobase (the minimal current amplitude of infinite duration). In line with this, the recruitment of a motoneuron has been shown to depend on the rate of membrane potential depolarization preceding a spike (Krawitz et al., 2001). Rather than using a slow ramp depolarization, it therefore seems more appropriate to assess MN rheobase with the current pulse protocol used to distinguish between MN subtypes. With this kind of measure, differences in the excitability of MN subtypes related to active conductances may come out earlier during development.

   The reviewer raises an interesting point, which is consistent with one also raised by Reviewer 1. We have now included additional analysis in which we calculated rheobase values from the long (5 s) square current steps that were used to identify delayed and immediate firing MNs. These rheobase measurements made using current steps correlate strongly with rheobase measured from slow ramps (W1: r=0.87 p < 1.0 e-15; W2: r = 0.95 p < 1.0 e-15; W3: r= 0.92 p < 1.0 e-15). This is consistent with findings from Leroy et al. (2014) and Buisas et al., (2012), who both also demonstrated similar rheobase values in response to ramps and current steps. Importantly, we also find similar developmental changes in rheobase across motoneuron subtypes when assessed with a current step - showing no difference in rheobase values between delayed and immediate firing cells at week 1, with differences emerging in week 2 due to a progressive increase in rheobase in delayed firing motoneurons at 2nd and 3rd weeks. These findings are included as a new Supplementary Figure (Figure 3 – Figure Supplement 1) and summarized in the results on Page 6, lines 173-178.

   1. During the 2nd postnatal week, the study suggests that "PICs contribute to the emergence of orderly recruitment amongst MN subtypes". This interpretation appears too definitive because the study did not provide direct evidence for that.

   This is a good point. We did not directly test the contribution of PIC maturation to the staggering of rheobase currents between weeks 1 and 2. We have revised this statement to soften our claim, restricting differences in PIC activation to the second week. This revision can be found on Page 9, Line 319-329.

   The authors describe a more hyperpolarized activation of PICs in slow MNs suggesting that the early recruitment of PICs in slow MNs may help them to fire before fast MNs. By a pharmacological approach the authors show that the sodium PIC mediated by Nav1.6 channels sets the activation threshold of PICs and that their blockade increases the rheobase (recruitment current). However, since pharmacological investigations have been done only in fast MNs, it is not very informative on the putative role of the sodium PIC (and PICs in general) on the orderly recruitment of MN subtypes. Similar experiments should be extended to slow MNs to compare the effects with those observed on fast MNs. If sodium PIC plays a significant role in the differential recruitment of MN subtypes, its blockade should induce an overlap in the recruitment of slow and fast MNs.

   We initially focused specifically on the roles of PICs in shaping recruitment of delayed firing motoneurons during weeks 2 and 3, because we were trying to account for changes in rheobase that occur within delayed firing motoneurons during this period of postnatal development. However, in response to the useful comments here and above, we have now conducted an additional set of experiments to determine the relative contribution of NaV1.6 (n = 12 MNs, 7 animals) and L-type calcium channels (n = 10 MNs, 7 animals) to PIC and rheobase in immediate firing motoneurons. These results have been integrated into Figure 4, the results section, and discussion.

   Furthermore, voltage clamp recordings to characterize PICs in MN subtypes have been done without blocking potassium conductances. Therefore it is difficult to determine if differences in PICs between MN subtypes are related to inward currents or opposing outward currents.

   We agree that we cannot rule out contributions of other currents to our measures of PIC in voltage clamp given that we did not block potassium conductances. This is indeed an interesting point. However, our approaches are consistent with previously published approaches (Quinlan, et al., 2011, Verneuil, et al., 2020), which may be useful for the purposes of cross-study comparisons. Of note, Quinlan, et al., (2011), did measure PICs in the presence and absence of TEA (n = 18), with no differences in PIC amplitude or onset found. However, recent work from the Brocard Lab has found opposing contributions of M-currents to measures of PICs in voltage clamp in Hb9 interneurons. This would therefore be an interesting direction for future study amongst motoneuron subtypes. As highlighted below, and in our revised manuscript, it is quite possible that outward currents may oppose and diminish the actions of Ih. This is also likely true for PICs and would be an interesting direction for future study. We have included an additional statement in our discussion on Page 19, Lines 630-639 to acknowledge this potential interaction and contribution to maturation of motoneuron recruitment.

   1. During the 3rd postnatal week, the authors suggest that fast MNs display a prominent Ih current at rest that provides a depolarizing shunt delaying their recruitment compared to slow MNs. However, data appear not enough conclusive for such interpretation. First, the relationship between the resting membrane potential (RMP) and the amplitude of Ih (the larger the Ih, the more depolarized the RMP) depicted in Figure 6B from a small sample of MNs is not consistent with values reported in the supplementary table 1. Indeed, fast motoneurons supposed to have a prominent Ih current display a more hyperpolarized RMP compared to slow MNs. The opposite would be expected according to the authors' hypothesis. A similar concern can be raised regarding the strong relationship between the amplitude of Ih and that of the AHP illustrated in Figure 6F, which is not in line with the lack of difference in the amplitude of the AHP between slow and fast MNs in week 3 (see supplementary table 1).

   As discussed above in response to similar comments from another reviewer, we realise that the data presented in our original manuscript (Figure 6) regarding the relationships between RMP, Ih and mAHP lacked clarity and perhaps depicted a subset of recordings that was not representative of the complete dataset reported in our supplementary table (as pointed out by the current reviewer). Furthermore, these data did not achieve our main objective of supporting the existence of a resting Ih current as well as the new data we have included - where we directly measure Ih at resting membrane potentials. Given the addition of these new data, and the potential oversimplification of our attempts to relate RMP, Ih and mAHP (e.g. Ih is unlikely to be the main contributor to RMP), the latter has been removed from the revised manuscript.

   In addition, we have expanded our discussion to highlight potential roles for Ih in shaping recruitment of fast motoneurons during periods of inhibition, such as during rhythmic activity, where the membrane potential often dips below -70 mV. Fast fatigable (MMP9+) motoneurons have been shown to receive a greater density of inhibitory synaptic inputs, particularly those derived from V1 interneurons, compared to slow (ERRB+) motoneurons (Allodi et al., 2021), and this differential synaptic weighting may create greater opportunity for Ih to be engaged and contribute to staggering recruitment as our pharmacology data suggests. This addition can be found on Page 20 Lines 657-663.

   Second, the inward current recorded in fast MNs to hyperpolarization at -70 mV appears not significantly affected by the Ih blocker ZD7288 (Figure 5J, and 5L) suggesting that Ih is not recruited at rest in this class of MNs.

   We realize that displaying the full IV plots for Ih at weeks 2 and 3 in addition to before and after application of ZD7288 (at week 3) may not have effectively illustrated the magnitude of Ih measured at -70 mV given the wide range of current values, which vary 10-fold between measures made at -70 and -110mV. We have modified our graphs in Figure 5 to better illustrate the magnitude of Ih measured at -70 mV and -110 mV. We hope that these modified graphs better capture our observations. We have further simplified Figure 5 to reduce redundancy in results that are summarized in Supplementary File 2 and the Results text. We have also included additional traces and analysis in Figure 6 that highlight a significant ZD-sensitive sag potential detected in delayed but not immediate firing motoneurons when hyperpolarizing the membrane potential from -60 mV to resting potential. These data can be found on Page 13, Lines 434-439, and in figure 6A, B. These results have been further supported by an additional set of recordings of delayed (n= 13) and immediate (n = 11) firing motoneurons obtained from week 3 animals, where Ih was measured during a voltage step (in VC) from a holding potential of -50 mV down to the respective resting potential of each cell (Del: IhRMP = -96 ± 60 pA; Imm: IhRMP: -0.13 ± 11 pA; t(23) = 4.8, p = 8.7e-5). These results have been included on Page 13, Lines 431-434.

   On the other hand, ZD7288 hyperpolarizes the RMP in fast MNs (Figure 6A) and reduces the amplitude of their sags recorded at -70mV (Figure 5M). Similar discrepancies are more striking for slow MNs. Slow MNs did not display inward current sensitive to ZD7288 above -80 mV (Figure 5N). However, ZD7288 unexpectedly hyperpolarizes their RMP (Figure 6C). How the authors can explain such discrepancies? An interestinsting, but unexpected observation is the hyperpolarization of the RMP by ZD7288 in immediate firing motoneurons, even though, we were unable to detect measurable Ih or sag at potentials at resting membrane potential in immediate firing motoneurons. We have two explanations for these observations.

   1.) One possibility is that, as pointed out above regarding our measures of PICs, we did not block other conductances during our voltage clamp protocols for measuring Ih. It is therefore possible that immediate firing (and even delayed firing) motoneurons express other ion channels that oppose Ih and may mask its true magnitude and effects on membrane potential. Indeed, this has previously been demonstrated for a variety of currents (eg. Kjaerulff and Kiehn, 2001; MacLean et al., 2003; Picton et al., 2018; Buskila et al., 2019). If this is true, then it is possible that the relatively smaller Ih in immediate firing motoneurons may have been masked, whereas the relatively larger Ih in delayed firing motoneurons may have been more apparent. In support of this possibility, we note that many of the immediate firing motoneurons demonstrate a slow hyperpolarization of the membrane potential during current steps intended to measure sag. Interestingly we also find this phenomenon in delayed firing motoneurons (that demonstrated depolarizing sag potentials at baseline), in the presence of ZD7288. We have included an example in the modified figure 6 to highlight this phenomenon. We have included additional discussion to highlight these caveats and possibilities (Page 20 Lines 664-675).

   2.) Alternatively, voltage and space clamp errors, may have caused an underestimate of Ih. While we expect space clamp errors to be greater in the largest motoneurons, such as the delayed firing motoneurons, it is possible that such errors may have been sufficient to mask the small, albeit significant Ih in immediate firing motoneurons. It is possible that blockade of this small Ih by ZD7288 in immediate firing motoneurons may have hyperpolarized their RMP due to their high input resistance. This has been highlighted on Page 20 Lines675-679

   Finally, there is a mismatch in values reported in supplementary table 2 and figures 5G and 5I. In the table, both Ih amplitude and Ih density (at -70mV) appear significantly different between slow and fast MNs in week 3, but not in figures 5G and 5I. Altogether, these results appear inconsistent.

   We have modified our graphs to better illustrate the range of inward currents measured at -70 and -110 mV, which due to such high variance (in some cases 10 fold comparing those measured at -70 and -110 mV), were not as apparent when showing the full IV plot. We have modified our graphs in Figure 5 to better reflect the data summarized in Supp Table 2, and capture our observations. Both the data in the table and the plots have been analyzed using the same 2 way anova with cell type and age as factors.

   Regardless of inconsistencies, data should be replicated at least with a second Ih blocker such as Ivabradine hydrochloride or Zatebradine hydrochloride.

   We have performed an additional set of experiments in delayed (n = 8) and immediate (n = 3) firing motoneurons with ivabradine. These new results are included in text on Page 14, Lines 457-465. 10 µM Ivabradine produced a 45% reduction in Ih, and consistent with ZD7288, hyperpolarized the RMP of both delayed and immediate firing motoneurons and caused a significant decrease in rheobase of delayed firing motoneurons.

   Minor concerns:

   1. Does the pharmacological blockade of Nav1.6 channels 4,9-AH-TTX induce changes in the spiking threshold as already reported in cortical neurons (Hargus et al., 2013)? Such an effect may contribute to the higher rheobase observed in fast MNs under 4,9-AH-TTX (Figure 4M).

   We have included an analysis of spike threshold before and after application of 4,9-AH-TTX. In line with previous reports from cortical neurons (Hargus et al., 2013), 4,9-AH-TTX significantly depolarized the spike threshold of delayed and immediate firing motoneurons and could contribute to the higher rheobase observed in delayed firing motoneurons following blockade of Nav1.6. The results from this analysis have been included in text and can be found on page 9, Line 306-310.

   1. The study reported a more depolarized PIC in fast MNs during the 2nd postnatal week but the acceleration onset voltage in response to a current ramp depolarization (attributed to the activation of PICs), is similar between slow and fast MNs at the same age (Figure 4C). This is in discrepancy with figure 4G, where a significant effect on PIC onset voltage is shown within the same time points.

   Indeed, there are differences between delayed and immediate firing motoneurons in the onset voltage of the PIC measured in voltage clamp at week 2 and these findings are not mirrored by differences in the accelerating phase of the membrane potential depolarization as measured from depolarizing current ramps in current clamp mode. We believe that this difference likely reflects that measurements made in current clamp are indirect estimates of PICs, whereas voltage clamp protocols provide more direct and likely more sensitive measurements. This is highlighted on Page 8, Line 263-265.

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4. 

   eLife

   Oct 28, 2021

   Evaluation Summary:

   This manuscript will be of interest to those studying the neuroscience of movement, as it addresses a fundamental aspect of movement: motoneuron recruitment. The authors provide a comprehensive analysis of motoneuron intrinsic properties that mature in the early post-natal period in mice and may lead to differentiation into "slow" and "fast" phenotypes. The authors argue that these properties, studied in spinal cord slices, contribute to motoneuron recruitment. While the study provides insights on the maturation of electrophysiological properties in motoneuron subtypes, the claims related to ionic mechanisms involved in orderly recruitment require further justification.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …

   Evaluation Summary:

   This manuscript will be of interest to those studying the neuroscience of movement, as it addresses a fundamental aspect of movement: motoneuron recruitment. The authors provide a comprehensive analysis of motoneuron intrinsic properties that mature in the early post-natal period in mice and may lead to differentiation into "slow" and "fast" phenotypes. The authors argue that these properties, studied in spinal cord slices, contribute to motoneuron recruitment. While the study provides insights on the maturation of electrophysiological properties in motoneuron subtypes, the claims related to ionic mechanisms involved in orderly recruitment require further justification.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

   Read the original source
5. 

   eLife

   Oct 28, 2021

   Reviewer #1 (Public Review):

   In this paper, the authors study "recruitment" (see below) of spinal motoneurons over a 3 week developmental period. They show that properties change, and that properties of late-firing vs immediate firing MNs diverge over this period. With respect to firing, they show that the development of "persistent inward currents" and currents mediating sag could account for these changes. They suggest that the divergence of properties mediated by these currents leads to a wider range of recruitment and contributes to orderly recruitment of MNs.

   Overall, this is a very good manuscript with experiments well done. The large numbers of recordings are very helpful. The data are convincing that the range at which action potentials are recruited broadens over this time period, and that the currents studied contribute to …

   Reviewer #1 (Public Review):

   In this paper, the authors study "recruitment" (see below) of spinal motoneurons over a 3 week developmental period. They show that properties change, and that properties of late-firing vs immediate firing MNs diverge over this period. With respect to firing, they show that the development of "persistent inward currents" and currents mediating sag could account for these changes. They suggest that the divergence of properties mediated by these currents leads to a wider range of recruitment and contributes to orderly recruitment of MNs.

   Overall, this is a very good manuscript with experiments well done. The large numbers of recordings are very helpful. The data are convincing that the range at which action potentials are recruited broadens over this time period, and that the currents studied contribute to this broadening. This is of fundamental interest to neuroscientists studying movement.

   However, there are a few limitations:

   1. The authors use a surrogate marker for "slow" vs "fast" MNs: immediate vs delayed firing in response to rectangular current injection. They switch their language to call these motoneurons slow and fast, but they should be more cautious about doing so, given that firing is a surrogate marker that has not been fully studied and characterised across this time period. We do not know if developmental changes in Kv1 might also contribute to the effects on spiking seen here. I agree, there is delayed firing from the outset, which is interesting in itself given the rather homogenous other properties. But that doesn't meant that Kv1 expression is stable and thus non-contributory to the changes in firing described.

   2. The focus of the manuscript is on MN recruitment, but recruitment is never defined, despite being used in the title, through the abstract and key points, as well as throughout the manuscript. What they are looking at is response to current injected at the soma vs recruitment during a behaviour when synaptic inputs are bombarding an extensive dendritic tree. Thus, this manuscript does not look at recruitment per se, but rather activation of action potentials in response to intra-somatic stimulation. Accordingly, the term "recruitment" would be best kept to the Discussion. (I also note that the "size" principle relates more to electrical than physical size in the 21st century; I agree that the two are correlated, but the authors may not want to stick to arguments about physical size.)

   3. Figure 6 is problematic, possibly in the way it's presented. It seems to me, but not clear, that the authors suggest that Ih is active at rest, more so in "F" than "S," and that therefore "F" are more depolarised and have smaller mAHPs. So (a) how come RMP didn't seem to come out in the PC analysis earlier? (b) would that not suggest that "F" would be "recruited" earlier? (Or is it that there is reduced sodium channel availability because of the depolarisation - what are the differences in spike amplitude and rate of rise?) (c) shouldn't the mAHP be larger if the cell is more depolarised and further from the potassium equilibrium potential? On the last note, maybe it's that Ih makes the mAHP smaller, but with the kinetics of Ih, wouldn't the decay be faster, but in Fig 6 the decay seems faster when Ih is blocked? Finally, Fig 6E suggests changes in the fAHP and delayed depolarisation (and spike width?) - how to these come into the picture? If the fAHP is thought to result from a high conductance state, and if therefore one were to align the voltages based on this potential, then the mAHPs would be about the same amplitude? The authors likely have explanations, but I'm afraid that I can't follow it.

   4. A number of labs have looked at development of MN properties, and it would be useful to compare properties seen across different labs, for example Quinlan (e.g. PMID: 21486770) and Whelan (e.g. PMID: 20457856, which is only mentioned in the manuscript) (and for that matter, mine - e.g. PMID 10564356, PMID: 32851667 although I don't want to self-promote).

   5. In the Discussion, the authors might want to discuss propensity of F vs S MNs to express PICs / sustained firing as described in the Heckman lab (albeit particularly in cat; see PMID 9705452, for e.g.). How do these data correspond?

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6. 

   eLife

   Oct 28, 2021

   Reviewer #2 (Public Review):

   The manuscript deals with a fundamentally important tissue in motor control, the mechanism governing the orderly recruitment of spinal motoneurones. It is widely accepted that this is a consequence of excitability differences intrinsic to the different functional classes of motoneurones. For decades, the dogma has been that the recruitment order of spinal motoneurones is governed primarily by cell size which influences input resistant and this has been the standard textbook explanation. With an increasing number of reports not finding a convincing size difference between slow and fast motoneurones in rodents, it is now clear that this explanation does not suffice. This manuscript expertly addresses this issue with a set of elegant experiments to investigate how the intrinsic properties of slow and fast …

   Reviewer #2 (Public Review):

   The manuscript deals with a fundamentally important tissue in motor control, the mechanism governing the orderly recruitment of spinal motoneurones. It is widely accepted that this is a consequence of excitability differences intrinsic to the different functional classes of motoneurones. For decades, the dogma has been that the recruitment order of spinal motoneurones is governed primarily by cell size which influences input resistant and this has been the standard textbook explanation. With an increasing number of reports not finding a convincing size difference between slow and fast motoneurones in rodents, it is now clear that this explanation does not suffice. This manuscript expertly addresses this issue with a set of elegant experiments to investigate how the intrinsic properties of slow and fast motoneurones become tuned during development to ensure the later recruitment of fast motoneurones. They show that the size is only a primary determinant of recruitment currents at developmental stages. With further development by post natal week 3 the difference in recruitment currents of fast motoneurones have diverged from slow motoneurones as a consequence of increases in the activation threshold of sodium persistent inward currents combined with an active hyperpolarization activated inward current (Ih) at resting membrane potential in fast motoneurones.

   These experiments were expertly designed, executed, analysed, presented and discussed with the results clearly supporting the conclusions. The experiments carefully balance the need for the recordings to be made from functionally mature mice with the benefits of an in vitro preparation allowing pharmacological manipulations to test the hypotheses. This is made possible by the group's expertise in spinal cord slice preparations and the authors should be particularly commended for this as very few laboratories have succeeded in obtaining viable healthy slice preparations of adult spinal cords for motoneurone recording.

   The data convincingly demonstrate a casual role for the persistent sodium inward currents and Ih currents in determining the recruitment currents for fast versus slow motoneurones. The only minor caveat is that due to the potential loss of dendritic L-type calcium channels in a slice preparation combined with the use of intracellular current injection to the soma to recruit motoneurones, this makes it difficult to conclude that L-type calcium do not also play a role in influencing recruitment order under normal synaptic activation. However, the influence of the sodium and Ih conductance is so convincing that these results alone mean that we must finally re-write the text book explanation for the fundamentally basic principle of recruitment order of motor units.

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7. 

   eLife

   Oct 28, 2021

   Reviewer #3 (Public Review):

   There are different types of motoneurons based upon the muscles they innervate. The recruitment order of these motoneurons has long been believed to be due simply to differences in the size of these motoneurons, which leads to differences in input resistance and threshold currents. The authors used recent electrophysiological signatures of fast versus slow motoneurons to study whether active conductances in these neurons developed differently during the early postnatal stages of the mice when these animals start to weight bear and show more coordinated movements.

   The experiments consist of electrophysiological recordings of motoneurons in slice preparations of the spinal cord. Motoneurons are categorized based on several electrophysiological properties previously shown to differentiate motoneurons. The …

   Reviewer #3 (Public Review):

   There are different types of motoneurons based upon the muscles they innervate. The recruitment order of these motoneurons has long been believed to be due simply to differences in the size of these motoneurons, which leads to differences in input resistance and threshold currents. The authors used recent electrophysiological signatures of fast versus slow motoneurons to study whether active conductances in these neurons developed differently during the early postnatal stages of the mice when these animals start to weight bear and show more coordinated movements.

   The experiments consist of electrophysiological recordings of motoneurons in slice preparations of the spinal cord. Motoneurons are categorized based on several electrophysiological properties previously shown to differentiate motoneurons. The recordings are well done and the data analysis shows strong support for their conclusions that during the second week of development, fast motoneurons and slow motoneurons start to differentiate themselves due to the properties of a persistent inward current and a hyperpolarization-activated current. The main weakness of the paper is a confirmation of the type of motoneuron recorded with a technique that does not rely solely on the electrophysiological signature.

   As stated above, the results are convincing. There are clear differences in the properties of the currents that are being studied, and their analysis suggests that cell size is a weak predictor of possible recruitment order.

   The findings are likely to push those in the field of motor control to reconsider how the maturation of motor circuits can also happen at the level of intrinsic properties of spinal neurons

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8. 

   eLife

   Oct 28, 2021

   Reviewer #4 (Public Review):

   Motoneurons (MNs) are classified in 3 subtypes according to contractile properties of the innervated muscle fibers [Fast-contracting Fatigable (FF); Fast-contracting fatigue-Resistant (FR); Slow contracting fatigue resistant (S)]. To cover the full range of locomotor frequencies, MNs are recruited in the sequence S, FR and FF. According to Henneman, this orderly recruitment correlates to the size of MNs : S-MNs are the smallest, FRs-MN have an intermediate size and FFs-MN are the largest. The rationale is that the smallest MNs are activated prior to the largest as a consequence of their high input resistance. However, because the excitability of motoneurons is also regulated by nonlinear conductances of membrane ion channels, differences in ionic conductances between MN subtypes (related to differences in …

   Reviewer #4 (Public Review):

   Motoneurons (MNs) are classified in 3 subtypes according to contractile properties of the innervated muscle fibers [Fast-contracting Fatigable (FF); Fast-contracting fatigue-Resistant (FR); Slow contracting fatigue resistant (S)]. To cover the full range of locomotor frequencies, MNs are recruited in the sequence S, FR and FF. According to Henneman, this orderly recruitment correlates to the size of MNs : S-MNs are the smallest, FRs-MN have an intermediate size and FFs-MN are the largest. The rationale is that the smallest MNs are activated prior to the largest as a consequence of their high input resistance. However, because the excitability of motoneurons is also regulated by nonlinear conductances of membrane ion channels, differences in ionic conductances between MN subtypes (related to differences in the expression of channel subtypes) may contribute to the Henneman's principle. In this framework, the authors performed a longitudinal study of the electrical properties of MNs in mice to distinguish between passive and active properties that secure the orderly recruitment of MNs.

   Based on extensive analysis of electrophysiological intracellular recordings, the authors suggest that the recruitment of fast and slow MNs is similar and mainly based on size during the first postnatal week, then differs partly due to a differential maturation of active properties between motoneuron subtypes. Specifically, the authors show that fast motoneurons display a more depolarized threshold for persistent inward currents (PICs) during the 2nd postnatal week and a prominent hyperpolarization-activated inward current during the 3rd postnatal week, both delaying the recruitment of fast MNs relative to slow motoneurons. The present study provides significant insights on the maturation of electrophysiological properties in diverse MN subtypes, but key claims on ionic mechanisms contributing to the orderly recruitment of MN subtypes appear not enough supported by data presented. In particular:

   1. During the 1st postnatal week, authors suggest that fast and slow MNs cannot be distinguished neither on their passive properties nor the rheobase and therefore their recruitment is mainly based on the size. The conclusion that the recruitment is not linked to MN functional differences is difficult to follow since the main distinction between MN subtypes is based upon the presence of a delayed firing, an active property that regulates the recruitment of MNs (Leroy et al., 2014). However, the square current pulse adopted to discriminate between delayed and immediate firing in Figure 1 was replaced with a ramped depolarization protocol on which the authors measured the rheobase (Figure 3A1). This suggests that the slow depolarization in immature motoneurons might minimize the activation of ionic conductance(s) responsible for the delayed firing and thus may bias the measure of the rheobase (the minimal current amplitude of infinite duration). In line with this, the recruitment of a motoneuron has been shown to depend on the rate of membrane potential depolarization preceding a spike (Krawitz et al., 2001). Rather than using a slow ramp depolarization, it therefore seems more appropriate to assess MN rheobase with the current pulse protocol used to distinguish between MN subtypes. With this kind of measure, differences in the excitability of MN subtypes related to active conductances may come out earlier during development.

   2. During the 2nd postnatal week, the study suggests that "PICs contribute to the emergence of orderly recruitment amongst MN subtypes". This interpretation appears too definitive because the study did not provide direct evidence for that. The authors describe a more hyperpolarized activation of PICs in slow MNs suggesting that the early recruitment of PICs in slow MNs may help them to fire before fast MNs. By a pharmacological approach the authors show that the sodium PIC mediated by Nav1.6 channels sets the activation threshold of PICs and that their blockade increases the rheobase (recruitment current). However, since pharmacological investigations have been done only in fast MNs, it is not very informative on the putative role of the sodium PIC (and PICs in general) on the orderly recruitment of MN subtypes. Similar experiments should be extended to slow MNs to compare the effects with those observed on fast MNs. If sodium PIC plays a significant role in the differential recruitment of MN subtypes, its blockade should induce an overlap in the recruitment of slow and fast MNs. Furthermore, voltage clamp recordings to characterize PICs in MN subtypes have been done without blocking potassium conductances. Therefore it is difficult to determine if differences in PICs between MN subtypes are related to inward currents or opposing outward currents.

   3. During the 3rd postnatal week, the authors suggest that fast MNs display a prominent Ih current at rest that provides a depolarizing shunt delaying their recruitment compared to slow MNs. However, data appear not enough conclusive for such interpretation. First, the relationship between the resting membrane potential (RMP) and the amplitude of Ih (the larger the Ih, the more depolarized the RMP) depicted in Figure 6B from a small sample of MNs is not consistent with values reported in the supplementary table 1. Indeed, fast motoneurons supposed to have a prominent Ih current display a more hyperpolarized RMP compared to slow MNs. The opposite would be expected according to the authors' hypothesis. A similar concern can be raised regarding the strong relationship between the amplitude of Ih and that of the AHP illustrated in Figure 6F, which is not in line with the lack of difference in the amplitude of the AHP between slow and fast MNs in week 3 (see supplementary table 1). Second, the inward current recorded in fast MNs to hyperpolarization at -70 mV appears not significantly affected by the Ih blocker ZD7288 (Figure 5J,and 5L) suggesting that Ih is not recruited at rest in this class of MNs. On the other hand, ZD7288 hyperpolarizes the RMP in fast MNs (Figure 6A) and reduces the amplitude of their sags recorded at -70mV (Figure 5M). Similar discrepancies are more striking for slow MNs. Slow MNs did not display inward current sensitive to ZD7288 above -80 mV (Figure 5N). However, ZD7288 unexpectedly hyperpolarizes their RMP (Figure 6C). How the authors can explain such discrepancies? Finally, there is a mismatch in values reported in supplementary table 2 and figures 5G and 5I. In the table, both Ih amplitude and Ih density (at -70mV) appear significantly different between slow and fast MNs in week 3, but not in figures 5G and 5I. Altogether, these results appear inconsistent. Regardless of inconsistencies, data should be replicated at least with a second Ih blocker such as Ivabradine hydrochloride or Zatebradine hydrochloride.

   Minor concerns:

   1. Does the pharmacological blockade of Nav1.6 channels 4,9-AH-TTX induce changes in the spiking threshold as already reported in cortical neurons (Hargus et al., 2013)? Such an effect may contribute to the higher rheobase observed in fast MNs under 4,9-AH-TTX (Figure 4M).

   2. The study reported a more depolarized PIC in fast MNs during the 2nd postnatal week but the acceleration onset voltage in response to a current ramp depolarization (attributed to the activation of PICs), is similar between slow and fast MNs at the same age (Figure 4C). This is in discrepancy with figure 4G, where a significant effect on PIC onset voltage is shown within the same time points

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   Version published to 10.1101/2021.06.16.448644v1 on bioRxiv

   Jun 17, 2021