Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis

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1. 

   eLife

   Dec 22, 2020

   Reviewer #3:

   General Assessment:

   The manuscript is well written and the methods are sound. The strengths of this manuscript are that this study is the first to systematically perform detailed electrophysiological measurements on inhibitory interneurons (INTs), in particular RC and non-RC INTs using the SOD1 mouse model for ALS. It is very interesting that they showed a dichotomy between reduced excitability in RC neurons (which could lead to an indirect increase in overall excitability of MNs) and non-RC INTs, which actually showed an increase in excitability which would have the opposite effect on MNs.

   Main comments:

   1. Most electrophysiological studies have focused on motor neurons and showed that they become hyperexcitable at very young ages, although there is controversy as to whether the hyperexcitability persists and is causative or …

   Reviewer #3:

   General Assessment:

   The manuscript is well written and the methods are sound. The strengths of this manuscript are that this study is the first to systematically perform detailed electrophysiological measurements on inhibitory interneurons (INTs), in particular RC and non-RC INTs using the SOD1 mouse model for ALS. It is very interesting that they showed a dichotomy between reduced excitability in RC neurons (which could lead to an indirect increase in overall excitability of MNs) and non-RC INTs, which actually showed an increase in excitability which would have the opposite effect on MNs.

   Main comments:

   1. Most electrophysiological studies have focused on motor neurons and showed that they become hyperexcitable at very young ages, although there is controversy as to whether the hyperexcitability persists and is causative or compensatory to disease progression.

   2. The dichotomy observed between RC and non-RC Inhibitory neurons is interesting. Given that many of the glycinergic non-RC interneurons are Ia-inhibitory interneurons responsible for reciprocal inhibition, their effects on the target motor neurons have opposite effects on MN excitability. At this point it is mere speculation as to how these changes actually exacerbate the progression of the disease and effects circuit function.

   3. This paper is mainly descriptive with no specific hypothesis other that what has been discuss often in the literature: Motor neuron hyperexcitability occurs from intrinsic alterations in MN ion channels, increased excitatory synaptic activity, or a decrease in inhibitory activity or all of the above. Although the authors are most likely the first to demonstrate changes in inhibitory interneuron excitability with direct electrophysiological recordings, it is unlikely that these findings will significantly move the field forward presently. The authors suggest that biomarkers could be developed, this is just a broad statement without concrete proposal for implementation. It would be useful to show a specific target that could be modified pharmacologically in animals over time to see if this changes the progression/survivability of the ALS animals.

   4. Furthermore, the functional significance of early hyperexcitability as either a cause or compensation of ALS is controversial at present. Numerous studies have addressed hyperexcitability but yet we are still far from understanding the bases for this disease and one cannot help question whether this avenue of investigation is fruitful.

   5. Does this change in interneuron excitability and the dichotomy between RC and non-RC demonstrated persist over the course of the disease? How relevant are these changes to disease progression?

   6. It will be necessary to use other animal models available for comparison since SOD1, although historically a well-studied mouse model, is an ectopic over expresser, and is not the predominate mechanism for ALS in humans. There are others probably more pertinent models, ie. C9ORF72. Whether such changes in inhibitory interneurons occur in those other models and in humans remains to be determined.

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2. 

   eLife

   Dec 22, 2020

   Reviewer #2:

   Amyotrophic lateral sclerosis (ALS) used to be considered primarily a disease of motoneurons. Recent work using mouse models of ALS has revealed that pathological changes can also be detected in spinal interneurons, particularly inhibitory interneurons, and that some of these changes can be detected before birth. The present paper is the first to directly examine the electrical properties of spinal inhibitory interneurons in a mouse model of ALS and show that some of these are altered in the neonatal period well before the mice start to exhibit symptoms. The authors show that SOD1 Lamina IX neurons are smaller than the Lamina IX WT neurons whereas no differences were found between WT and SOD1 neurons outside Lamina IX. They also use whole cell recordings to reveal that putative 'Renshaw cells' are less excitable in SOD1 …

   Reviewer #2:

   Amyotrophic lateral sclerosis (ALS) used to be considered primarily a disease of motoneurons. Recent work using mouse models of ALS has revealed that pathological changes can also be detected in spinal interneurons, particularly inhibitory interneurons, and that some of these changes can be detected before birth. The present paper is the first to directly examine the electrical properties of spinal inhibitory interneurons in a mouse model of ALS and show that some of these are altered in the neonatal period well before the mice start to exhibit symptoms. The authors show that SOD1 Lamina IX neurons are smaller than the Lamina IX WT neurons whereas no differences were found between WT and SOD1 neurons outside Lamina IX. They also use whole cell recordings to reveal that putative 'Renshaw cells' are less excitable in SOD1 mice than wild type animals whereas non-Renshaw inhibitory SOD1 neurons are more excitable.

   Major Comments

   1. The authors claim that Renshaw cells are in lamina IX, when they have been shown to be located mostly in the ventral part of lamina VII, ventromedial to the motor nucleus (Alvarez and Fyffe, 2007). In addition, not all calbindin+ neurons in lamina VII are Renshaw cells. From the location of the whole cell recordings shown in fig.2, it seems likely that most of the recorded neurons are not Renshaw cells because they are outside the classical 'Renshaw' area. It is not clear why the authors are focusing on glycinergic neurons in lamina IX, as there is no evidence that they belong to a unique class or that they are presynaptic to motoneurons.

   2. The concern about the identity of the Renshaw cells obviously undermines the statistical modeling to segregate Renshaw versus non-Renshaw cells. Furthermore, it was not clear from the text whether the model used both WT and SOD1 calbindin-positive neurons to define 'Renshaw cells'. Assuming it did, and given that there were changes in the electrical and morphological properties of the calbindin+ SOD1 neurons, is it not surprising that they could be grouped with the WT 'Renshaw cells'?

   In addition, the characteristics the 'Renshaw cell' population used for the model are not clear. On line 186 that it states that 15/23 of the whole cell recorded interneurons were positive for calbindin. Does this refer to 15 WT and 23 SOD1 neurons? Thus 38 neurons were calbindin positive. Of the remaining 21 neurons how many were calbindin-negative and how many were not tested? How many of the 38 calbindin-positive neurons had their dendrites reconstructed sufficiently from the intracellular fill to be used in the model? The model predicted that 80% of the 59 patched interneurons were Renshaw cells. How many of these were in the calbindin-negative group and how many were in the not-tested group? The spatial distribution of these groups should also be plotted. However, it seems very unlikely that 80% of the recorded cells are Renshaw based on their location as shown in fig.2B.

   Second it would have been useful apply the model to known non-Renshaw cells, to establish that it was not generating too many false positives. Another way the authors could test the model is to establish if it could distinguish WT and SOD1 neurons based on their morphology.

   1. The authors suggest that the reduced excitability of 'Renshaw cells' might contribute to the excitability changes seen in motoneurons. However, based on their own data, this is not a straightforward conclusion. They find that 'non-Renshaw cells' are hyperexcitable and since this population would include 1a inhibitory interneurons and other premotor inhibitory interneurons, it is not clear what the overall effect on motoneuron excitability would be. Additionally, because the authors suggest that 'Renshaw cells' are less excitable this would presumably lead to reduced inhibition of 1a inhibitory interneurons counteracting a potential loss of inhibition onto motoneurons from Renshaw cells.

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3. 

   eLife

   Dec 22, 2020

   Reviewer #1:

   In this study, the authors investigated whether the morphological and electrophysiological properties of glycinergic interneurons in the spinal ventral horn of GlyT2eGFP SOD1 G93A mice are altered compared with GlyT2eGFP WT mice at P6-P10 (the SOD1 G93A mice is the classic mouse model of amyotrophic lateral sclerosis). Such an investigation has never previously been done. The main body of results relies on a sample of 34 WT and 25 SOD1 patched interneurons located throughout the ventral horn. The authors found that soma sizes of patched interneurons are not significantly different in SOD1 animals than in WT animals but their dendrites are larger. The onset and the peak of persistent inward currents (PICs) are more depolarized in SOD1 interneurons suggesting that they are less excitable than in WT. Immunohistochemistry for …

   Reviewer #1:

   In this study, the authors investigated whether the morphological and electrophysiological properties of glycinergic interneurons in the spinal ventral horn of GlyT2eGFP SOD1 G93A mice are altered compared with GlyT2eGFP WT mice at P6-P10 (the SOD1 G93A mice is the classic mouse model of amyotrophic lateral sclerosis). Such an investigation has never previously been done. The main body of results relies on a sample of 34 WT and 25 SOD1 patched interneurons located throughout the ventral horn. The authors found that soma sizes of patched interneurons are not significantly different in SOD1 animals than in WT animals but their dendrites are larger. The onset and the peak of persistent inward currents (PICs) are more depolarized in SOD1 interneurons suggesting that they are less excitable than in WT. Immunohistochemistry for Calbindin was performed in a subset of the patched interneurons to identify Renshaw cells (7 cells in WT animals and 6 cells in SOD1 animals). Calbindin positive cells display more depolarized PICs onset and peak in SOD1 than in WT animals. A predictive statistical analysis was then performed in order to include in the Renshaw cells sample cells that were not tested for calbindin. This analysis suggested that the predicted Renshaw cells are less excitable in SOD1 mice than in WT mice whereas the predicted non-Renshaw cells are more excitable. The implications of these findings for the ALS pathophysiology are discussed.

   However, a number of major concerns substantially weaken the findings:

   1. Morphological properties Texas red allowed the authors to localize the patched cells in the ventral horn, to measure the soma and the dendrites and to investigate whether the patched cells were immunopositive to Calbindin. It appears that the soma volumes of the patched neurons are on average 2-3 times larger than the soma of the general population of GlyT2-GFP neurons in the ventral horn or in lamina IX (Table 1). No explanation is provided for this discrepancy. Does it mean that there is a systematic recording bias towards the largest interneurons ? Alternatively, is there a systematic swelling of the patched cells or a shrinking in the fixed spinal sections? Also, it is not clear what the dendritic parameters are? It is necessary in Figure 2 to show a reconstruction of dendrites in order to figure out which dendritic length, surface and volume are reported in Table 1.

   2. Electrophysiological properties The shift in the onset of the persistent inward currents onset is taken as an important indicator of a reduced excitability in SOD1 interneurons. However the measurement of the PIC onset is problematic. It is claimed in the Material and Methods section that "PIC onset was defined as the voltage at which the current began to deviate from the horizontal, leak substracted trace" (lines 374-375), which seems reasonable. However, in Figure 3A, the arrow for the PIC in the SOD1 motor neuron (red trace) does not point to the initial deviation from the horizontal which actually occurred at about -60mV, i.e. close to the PIC onset for the WT motoneuron (blue trace), in contradiction with the authors claim. The arrow points to a second component whose onset appears at a more depolarized voltage. Then the net current is likely to be complex and a pharmacological dissection of the currents at work is required both in WT and SOD1 neurons. Indeed, the net inward current might result from the summation of inward and outward currents. Are they outward currents at work? Are the inward currents Na+ or Ca++ currents? In the absence of such a pharmacological "dissection" it is difficult to fully interpret the data.

   3. Identification of the Renshaw cells The authors identified a subset of GlyT2 neurons as Renshaw cells because they expressed Calbindin-D-28K. This sole criteria does not allow a proper identification of Renshaw cells, particularly in P6-P10 mice. Indeed, many non-Renshaw cells in the ventral horn are calbindin-immunopositive during this post-natal maturation period in addition to the Renshaw cells (Siembab et al, J Comp Neurol, 2010). One distinguishing feature of Renshaw cells is that they are excited by recurrent motor axon collaterals. Then, the presence of VACht boutons on the GlyT2 cells would have been an interesting additional identification criteria. However, there is another source of VACht boutons than motor axon terminals in the spinal cord (Zagoraiou et al, Neuron 2009). Since this is an electrophysiological work, the authors had the possibility to unambiguously identify Renshaw cells: the presence of synaptic excitations in response to the stimulation of motor axons in a ventral rootlet (using oblique spinal cord slices, see for instance: Lamotte d'Incamps and Ascher, J Neurosci 2008; Bhumbra et al, J Neurosci 2014). The authors are advised to perform such an electrophysiological identification of Renshaw cells.

   4. Statistics and predictive model The number of patched cells identified as "Renshaw cells" on the basis of their Calbindin immunopositivity is low (7 WT and 6 SOD1). Indeed, I do not see any reason why the authors did not repeat the experiments in order to gather a more reasonable number of cells. Statistical analysis was performed on this low cell samples, in order to investigate whether each property under investigation differs or not in WT and SOD1 animals as reported in Table 3 (normality of the distribution was tested for each property and either ANOVA analysis or Kruskall Wallis analysis was performed). The validity of statistics on such low cell samples is questionable. The analysis was then extended to all patched cells using sophisticated random forest and principal components analysis in order to check whether some cells among those not tested for calbindin display enough similarities with the calbindin-positive cells to be considered as putative Renshaw cells. The model predicted that 80% of the 59 patched cells were "Renshaw cells", a percentage astonishingly larger than the percentage of calbindin-positive cells in the ventral horn (65%). This prediction is doubtful since the number of calbindin-positive cells is already higher at P6-P10 than the number of Renshaw cells (see bullet point 3). Nevertheless, the authors made statistics (not shown on the paper) on the basis of this prediction, and they found that the predicted Renshaw cells are less excitable in SOD1 mice than in WT mice whereas the predicted non-Renshaw cells are more excitable.

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4. 

   eLife

   Dec 22, 2020

   Summary: In the present study, the authors searched for early signs (during the neonatal period) of amyotrophic lateral sclerosis (ALS) disease focusing on a specific class of spinal interneurons; i.e.: glycinergic interneurons. In SOD1 mice, they aimed at testing whether these inhibitory neurons exhibit measurable changes at young age that could then contribute to the MN pathology known to develop later. The originality of this study is that, for the first time, it examines specifically inhibitory neurons. The authors investigated the morphological and electrophysiological properties of lumbar glycinergic interneurons in the spinal ventral horn in one model of SOD1 mice compared to WT P6-P10 mice. In addition, the authors more specifically considered Renshaw cells in this process and found that these cells were less excitable in SOD1 …

   Summary: In the present study, the authors searched for early signs (during the neonatal period) of amyotrophic lateral sclerosis (ALS) disease focusing on a specific class of spinal interneurons; i.e.: glycinergic interneurons. In SOD1 mice, they aimed at testing whether these inhibitory neurons exhibit measurable changes at young age that could then contribute to the MN pathology known to develop later. The originality of this study is that, for the first time, it examines specifically inhibitory neurons. The authors investigated the morphological and electrophysiological properties of lumbar glycinergic interneurons in the spinal ventral horn in one model of SOD1 mice compared to WT P6-P10 mice. In addition, the authors more specifically considered Renshaw cells in this process and found that these cells were less excitable in SOD1 mice.. Based on these experimental data they created a statistical model to make predictions on RC cells (and non-Renshaw cells found to be more excitable in SOD1 mice) to further demonstrate that early changes in their excitability could account for the disease.

   Despite the fact that this paper addresses the potential role of an unprecedentedly investigated class of neurons (inhibitory ones) in ALS disease, reviewers pointed to several concerns. First, there is a major problem with the identification of the Renshaw cells. Indeed arguments using the localization within the ventral horn of the spinal cord, the calbindin expression, the size and the number are questionable as it is done here. In addition, because the characteristics of this type of cell has been later used for the predictive statistical model, it importantly attenuates the validity of the model and credibility of the conclusions reached. Finally, because of the problems addressed above and because this paper is mainly descriptive without bringing real new hypotheses this paper might not participate in moving the field of ALS significantly forward. Thus, the three reviewers and I agree that the paper would be better suited for a specialised audience assuming detailed comments about the methodology are addressed.

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5. 

   Version published to 10.1101/2020.10.21.348359 on bioRxiv

   Oct 21, 2020