Marcin Bączyk

Najwa Ouali Alami

Nicolas Delestrée

Clémence Martinot

Linyun Tang

Barbara Commisso

David Bayer

Nicolas Doisne

Wayne Frankel

Marin Manuel

Francesco Roselli

Daniel Zytnicki
email: daniel.zytnicki@parisdescartes.fr

Superoxide Sod1

Dismutase

Ts

Surae Triceps

Vesicular Vglut

Glutamate Transporter

Synaptic restoration by cAMP/PKA drives activity-dependent neuroprotection to motoneurons in ALS

Keywords: Amyotrophic Lateral Sclerosis, Chemogenetics, In vivo electrophysiological recordings, Ia proprioceptive afferents, Medial longitudinal fasciculus, GluR subunits ActPSAM, Activator PSAM ALS, Amyotrophic Lateral Sclerosis AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AP, Antero-Posterior AU, Arbitrary Units cAMPS-Sp, Specifically purified synperiplanar-isomer NB, NeuroBiotin™ OCT, Optimal Cutting Temperature compound PB, Posterior Biceps PKA, Protein Kinase A PSAM, Pharmacologically Selective Actuator Module PSD, PostSynaptic Density PSEM, Pharmacologically Selective Effector Molecule

Introduction

In Amyotrophic Lateral Sclerosis (ALS), the "excitotoxic model" hypothesized that pathologic increase in glutamatergic input at synaptic level and/or neuronal hyperexcitability would cause Ca 2+ overload, disruption of mitochondria and ultimately lead to motoneuron (MN) death [START_REF] Rothstein | Current hypotheses for the underlying biology of amyotrophic lateral sclerosis[END_REF]. In spinal muscular atrophy (SMA), a distinct MN disease, the reduced efficacy of the presynaptic terminals of Ia afferents (which originate from muscle spindles and provide direct, powerful excitation to MNs [START_REF] Binder | How different afferent inputs control motoneuron discharge and the output of the motoneuron pool[END_REF] disrupts MN firing [START_REF] Mentis | Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy[END_REF] and causes MN hyperexcitability [START_REF] Fletcher | Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy[END_REF]. Likewise, in a Drosophila model of SMA, loss of the SMN gene in interneurons projecting to MNs is sufficient to cause MN degeneration [START_REF] Imlach | SMN is required for sensory-motor circuit function in Drosophila[END_REF], confirming that disruption of synaptic inputs can drive MN vulnerability.

Interestingly, fast-fatigable (FF) MNs, the subpopulation most vulnerable in ALS, display the highest density of Ia afferents [START_REF] Basaldella | Multisensory Signaling Shapes Vestibulo-Motor Circuit Specificity[END_REF]. Moreover, genetic ablation of gamma MNs (which likely reduces Ia firing) results in delayed disease onset, suggesting a detrimental effect of Ia activity [START_REF] Lalancette-Hebert | Gamma motor neurons survive and exacerbate alpha motor neuron degeneration in ALS[END_REF]. However, degeneration of spindle sensory endings starts before symptoms onset [START_REF] Vaughan | Degeneration of proprioceptive sensory nerve endings in mice harboring amyotrophic lateral sclerosis-causing mutations[END_REF], rather suggesting that reduced Ia activity may be pathogenic. Furthermore, in contrast to SMA, vulnerable MNs in ALS become hypoexcitable just prior to the denervation onset of neuromuscular junctions [START_REF] Martínez-Silva M De | Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS[END_REF]. Thus, if Ia fibers are involved in ALS as well, pathogenic and compensatory pathways may be not necessarily the same in the two conditions.

To date, direct in vivo proof of increased or decreased functionality of Ia synapses (and, more broadly, of any excitatory synapse) on MNs in presymptomatic ALS mice is lacking and, if any, it is unclear if restoration of synaptic inputs may correlate with reduced disease burden. Here, we performed in vivo electrophysiological recordings to demonstrate that MN response to Ia synapses is actually disrupted in presymptomatic mutSOD1 mice, in correlation with the breakdown of the postsynaptic receptor clusters and scaffold proteins. We then used multiplexed chemogenetics to show that synaptic impairment could be acutely and chronically reversed by activation of the cAMP/PKA pathway in MNs, with activity-dependent beneficial effects on disease burden. Moreover, we show that functional and structural synaptic impairment is detected also in monosynaptic excitatory postsynaptic potentials (EPSPs) on MNs from the descending medio-longitudinal fasciculus (MLF). Thus, our work shows that dysfunction of excitatory synapses, largely of post-synaptic origin, reduces the excitatory drive to MNs, and may contribute to MN degeneration in ALS through decreased MN firing.

Results

The monosynaptic excitatory pathway between Ia afferents and motoneurons is impaired in presymptomatic SOD1(G93A) mice

We investigated the Ia synaptic responses in MNs, in vivo, in 45-55 days-old anesthetized SOD1(G93A) mice (hereafter, mutSOD1), before FF-MN denervation [START_REF] Pun | Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF[END_REF]. We activated the spindle primary endings in vivo using high frequency vibrations of the Achilles tendon while intracellularly recording the EPSP elicited in a triceps surae (TS) MN (Figure 1A). The vibration-induced compound EPSPs (resulting from the summation of one EPSP per vibration cycle, insert in B 2 ) were on average 30 % smaller in MNs from mutant mutSOD1 animals than in MNs from animals expressing the human wt SOD1 protein (wtSOD1) (Figure 1B-C).

Since the reduction of the EPSPs induced by tendon vibration could be caused by disruption of the spindles themselves, we bypassed the spindles by recording the monosynaptic EPSPs elicited by a direct electrical stimulation of Ia afferents in the TS nerve (Figure 1D) at an intensity that recruits all group I fibers (1.8-2.0× threshold of the most excitable afferents). At this intensity (and up to 5× threshold) no disynaptic EPSPs were visible, and disynaptic IPSPs, if present, appear only after the peak of the monosynaptic EPSP where measurements were made (Supplemental Figure 1A-C). The EPSPs elicited by a single electrical shock (no summation) were again, on average, 33 % smaller in MNs from mutSOD1 animals than in MNs from wtSOD1 animals (Figure 1E-F), indicating that the reduction was not due to spindle impairments. Input resistances (Figure 1G), membrane time constants (Figure 1H) and resting membrane potentials (Figure 1I) were all comparable between wtSOD1 and mutSOD1 animals ruling out additional confounders and establishing that the impairment originates in the synapses themselves.

Since activation of presynaptic axo-axonic GABAergic synapses may reduce the size of the EPSPs [START_REF] Rudomin | Presynaptic inhibition in the vertebrate spinal cord revisited[END_REF], we investigated whether presynaptic inhibition of Ia terminals is stronger in mutSOD1 than in wtSOD1 mice. As in cats [START_REF] Rudomin | Presynaptic inhibition in the vertebrate spinal cord revisited[END_REF], mouse group I fibers from the posterior biceps elicit some presynaptic inhibition of Ia afferents from the triceps surae (Supplemental Figure 1D-G). However, the level of presynaptic inhibition was not significantly different between mutSOD1 and wtSOD1 mice (Supplemental Figure 1H). Therefore the reduction in the size of EPSPs in mutSOD1 MNs cannot be explained by an increase of presynaptic inhibition of Ia terminals. We then studied the paired-pulse facilitation of Ia synapses on MNs. In wtSOD1 mice, the second Ia EPSP evoked by paired-pulse stimulation was systematically larger than the first one (paired-pulse facilitation; Figure 1J-L), a finding compatible with the contribution of presynaptic inhibition [START_REF] Stuart | Mechanisms of presynaptic inhibition studied using paired-pulse facilitation[END_REF][START_REF] Carlsen | Purines released from astrocytes inhibit excitatory synaptic transmission in the ventral horn of the spinal cord[END_REF]. However, in mutSOD1 mice, many MNs displayed a pairedpulse ratio of reduced magnitude, with half of the MNs actually displaying a net paired pulse depression (Figure 1K-L), despite of the fact that the level of presynaptic inhibition was the same as in wtSOD1 mice (Supplemental Figure 1H). This suggests that the SOD1 mutation elicits a depressing effect on the second EPSP that sometimes surpasses the facilitation generated by presynaptic inhibition. In summary, we show that, in mutSOD1 mice, Ia-MN synapses display abnormal physiological properties not only in terms of evoked EPSP but also of short term synaptic plasticity.

The postsynaptic side of Ia synapses on motoneurons is structurally disrupted in mutant mice

We investigated the origin of synaptic dysfunction using immunolabeling of pre-and postsynaptic proteins. Ia boutons onto MNs were identified by the expression of vesicular glutamate transporter 1 (VGluT1, [START_REF] Alvarez | Permanent central synaptic disconnection of proprioceptors after nerve injury and regeneration. I. Loss of VGLUT1/IA synapses on motoneurons[END_REF]. Because VGluT1+ synapse density is higher close to the soma and decreases with distance [START_REF] Rotterman | Normal distribution of VGLUT1 synapses on spinal motoneuron dendrites and their reorganization after nerve injury[END_REF], we first estimated the density of VGluT1+ terminals separately on cell bodies, proximal dendrites and distal dendrites of intracellularly labeled MNs (Supplemental We then focused on the postsynaptic side of the VGluT1+ synapses on MNs. We first analyzed the expression of the postsynaptic density proteins Shank1 and Homer1b (Figure 2A-D), critical organizers of the postsynaptic density [START_REF] Sheng | The postsynaptic architecture of excitatory synapses: a more quantitative view[END_REF]. In wt MNs, both Shank1 and Homer1b formed large, continuous clusters juxtaposed and parallel to the VGluT1+ puncta. However, in mutSOD1 mice, VGluT1+ synapses displayed smaller and fragmented clusters of Shank1 (Figure 2A-B) and Homer1b (Figure 2C-D). We then explored the distribution of AMPA receptor subunit GluR4, the most abundant subunit expressed in MNs [START_REF] Williams | volley recorded at the cervical level. Middle trace: volley recorded at the lumbar level. Bottom trace: membrane potential of the MN. Stimulation artefacts were truncated. B. Comparison of the amplitude of the first (monosynaptic) EPSP[END_REF][START_REF] Ragnarson | Glutamate and AMPA receptor immunoreactivity in Ia synapses with motoneurons and neurons of the central cervical nucleus[END_REF] at VGluT1+ synapses. In wt MNs, GluR4 formed multiple, discrete and round clusters juxtaposed to each VGluT1+ puncta (Figure 2E). MutSOD1 MNs, on the other hand, displayed a significant decrease in the total GluR4 cluster area (Figure 2H), and in their immunostaining intensity (Figure 2I). We further explored the disruption of the postsynaptic structures by assessing the level of T840-phosphorylated GluR1 (Figure 2F), whose levels reflect AMPAR with increased conductance [START_REF] Delgado | NMDA receptor activation dephosphorylates AMPA receptor glutamate receptor 1 subunits at threonine 840[END_REF][START_REF] Jenkins | Regulation of GluA1 α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor function by protein kinase C at serine-818 and threonine-840[END_REF][START_REF] Babiec | Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons[END_REF], and S880-phosphorylated GluR2 (Figure 2G), a marker of GluR2 subunits stably retained in synapses [START_REF] States | Stable synaptic retention of serine-880phosphorylated GluR2 in hippocampal neurons[END_REF]. The size and fluorescence intensity of both pGluR1 and pGluR2 clusters juxtaposed to VGluT1+ terminals were significantly decreased in mutSOD1 mice (Figure 2J-M). Taken together, these data show a significant disruption of the structure of the postsynaptic density (PSD) of VGluT1+ synapses and support the view of a substantial postsynaptic component in driving the physiological disturbance of MN responses to Ia afferents.

We further investigated if the disruption of Ia PSDs could be associated with other structural abnormalities. We exploited neurobiotin ability to fill the dendrites cytoplasm of the neurons, negatively highlighting intraneuronal dendritic structures. This approach revealed large vacuoles located in the varicosities along dendritic branches (Supplemental Figure 3). In addition, many VGluT1+ synapses were located in close apposition to these vacuoles (Supplemental Figure 2B). Vacuoles were already visible around P50 in mutSOD1 mice (Supplemental Figure 3C) (but not in wt and in wtSOD1 mice; Supplemental Figure 3A-B) and imparted a significant distortion in the geometry of the dendrite. The size of the vacuoles and their impact on dendritic architectures increased with disease progression (Supplemental Figure 3C). Thus, substantial structural disruption of dendritic architecture takes place in close proximity of VGluT1+ synapses already at early stages and increases with disease progression.

Postsynaptic activation of the PKA pathway elicits a partial functional rescue of the

Ia-MN synapses

Next, we reasoned that reduced EPSP size might be ameliorated by increasing the insertion of AMPA receptors in the postsynaptic membrane. Since phosphorylation of GluR4 by PKA on Ser842 is sufficient to relieve a retention signal in the C-terminus and allows its synaptic insertion [START_REF] Esteban | PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity[END_REF], we explored if activation of PKA in MNs might restore Ia-MN EPSP size. We applied the PKA activator cAMPS-Sp by intracellular iontophoretic injection through the recording microelectrode [START_REF] Lalley | cAMP-dependent protein kinase modulates expiratory neurons in vivo[END_REF] while recording electrically-evoked Ia EPSPs before and after the injection. In mutSOD1 MNs, PKA activation resulted in the progressive increase of EPSP size in all MNs tested (Figures 3A 2 and 3C 2 ; 14 % on average, i.e. about half of the average EPSP size reduction in the mutSOD1 animals). This restoration took place in the first 5 minutes and remained stable over the duration of the recording session (which may last 20 min). Furthermore, iontophoretic injection of cAMPS-Sp also partially restored the paired-pulse facilitation (Figures 3B 2 and 3D 2 ). On the other hand, the stimulation protocol alone (without cAMPS-Sp injection) did not modify EPSP size in 3 independent MNs. In contrast, in wtSOD1 mice, cAMPS-Sp induced only a small increase of the EPSP size (7% on average; Figures 3A 1 and 3C 1 ) but had no significant effect on the pairedpulse ratio (Figures 3B 1 and 3D 1 ). These results not only further confirm that the functional impairment of the Ia-MN synapses has a major post-synaptic origin, but they also show that it is acutely reversible. Furthermore, since the cAMPS-Sp was applied post-synaptically, these results confirm a major role of post-synaptic mechanisms in the origin of depressed paired-pulse response of mutSOD1 MNs.

Chemogenetic activation of PKA pathway in MNs enhances GluR4 content and

Homer1b in Ia synapses

Since iontophoretic delivery only allows the study of the effects of PKA activation for about 20 min (limited by the stability of the preparation), we sought to manipulate the cAMP/PKA pathway over hours/days through the use of Gs-coupled DREADD [START_REF] Farrell | A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons[END_REF][START_REF] Aldrin-Kirk | DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor[END_REF][START_REF] Roth | DREADDs for Neuroscientists[END_REF]. To this aim, 25 days-old mutSOD1/ChATcre double-transgenic mice were intraspinally injected with AAV9 encoding for doublefloxed DREADD(Gs)-mCherry (henceforth D(Gs)), (thus, D(Gs) expression was restricted to ChAT-cre+ cells). A first group of mice was administered with a single dose of CNO 17 days after the viral injection (acute CNO; Figure 3E 2 ) and sacrificed 3h later; a second group was administered CNO daily starting 10 days after virus injection and for 7 days (chronic CNO; Figure 3E 3 ) and sacrificed 8h after the last injections (thus, 17 days after viral injection) and a third group was treated only with saline for 7 days (control group; Figure 3E 1 ). For each mouse, we quantified GluR4 and Homer1b cluster area in VGluT1+ synapses on MNs expressing D(Gs) (mCherry+) as well as on contralateral non-infected (mCherry-) MNs. Extending the intracellular injection data, acute (3h) activation of D(Gs) resulted in the significant increase in GluR4 (Figure 3F 2 and 3H) as well as of Homer1b cluster area (Figure 3G 2 and 3I) in VGluT1+ terminals.

Long-term (7 days) activation of D(Gs) also resulted in a significant restoration of GluR4 (Figure 3F 3 and 3H) and Homer1b cluster area in Ia synapses (Figure 3G 3 and 3I) compared to contralateral MN (which were exposed to the same dose of CNO but did not express any chemogenetic receptor). No effect on GluR4 (Figure 3F 1 and 3H) or Homer1b (Figure 3G 1 and 3I) were detected in mice expressing D(Gs) but administered with vehicle alone compared to contralateral non-infected MNs, indicating that intraspinal injection and D(Gs) expression did not produce any substantial effect. Thus, cAMP/PKA activation is sufficient to restore the structure and the function of Ia synapses, increasing GluR4 and Homer1b content in the postsynaptic side.

PKA effects on the postsynaptic side of Ia terminals do not require MN firing but are mimicked by chemogenetic excitation of MNs

Hypoexcitability of vulnerable MNs is an early event in the pathogenic cascades leading to MN degeneration [START_REF] Martínez-Silva M De | Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS[END_REF] and restoration of MN firing produces beneficial effects on multiple disease markers [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF]. Therefore, we set out to verify if synaptic restoration could be also obtained by enhancing MN firing with chemogenetic cation-permeable (activator) actPSAM [START_REF] Magnus | Chemical and Genetic Engineering of Selective Ion Channel-Ligand Interactions[END_REF][START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF]. AAV9 encoding for actPSAM under double-inverted orientation was injected intraspinally in mutSOD1/ChAT-cre mice at P25. The PSAM agonist PSEM 308 was then administered 10 days later for 7 days (Supplemental Figure 4B). When compared to contralateral non-infected MNs, PSAM-activated MNs displayed a significant increase in both GluR4 and Homer1b cluster area in correspondence of VGluT1+ synapses (Supplemental Figure 4C-F).

We then wondered whether the effect of cAMP/PKA on synapses could be due to an effect on MN firing. To test this hypothesis, we designed a multiplexed chemogenetic experiment in which PKA signaling and MN firing could be independently controlled.

We co-injected an AAV9 encoding D(Gs)-mCherry together with a second AAV9 encoding the anion-permeable (inactivator) PSAM-GlyR (inhPSAM, [START_REF] Magnus | Chemical and Genetic Engineering of Selective Ion Channel-Ligand Interactions[END_REF] (both under double-floxed inverted orientation) in the spinal cord of mutSOD1/ChATcre mice (Figure 4A). We reasoned that if the effect of cAMP/PKA on synaptic integrity is secondary to changes in MN activity, it would be significantly decreased upon concomitant decrease in MN firing. Ten days after AAV injection, we started the administration of CNO and PSEM 308 , for 7 days (Figure 4A). We considered four populations of MNs: i) contralateral, non-infected, ii) expressing only the inhPSAM, iii) expressing only D(Gs) and iv) MNs expressing both D(Gs) and inhPSAM, and we quantified GluR4 (Figure 4B and4D) and Homer1b (Figure 4C and4E) levels in VGluT1+ synapses. Compared to non-infected MNs, inactivation of MNs by inhPSAM did not result in a further decrease in the already low levels of GluR4 (Figure 4D) or Homer1b (Figure 4E) which would have been anticipated since MN inactivation worsen disease progression [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF]) (a small increase in GluR4 content in Ia synapses was detected). As shown above, D(Gs) activation alone did result in a significant increase of synaptic GluR4 and Homer1b cluster area (Figure 4D-E).

However, D(Gs) effects on GluR4 and Homer1b were not modified by concomitant MN inactivation by inhPSAM (Figure 4D-E). These results indicate that restoration of synaptic integrity by D(Gs) is not a consequence of increased MN firing, but rather caused by direct synaptic effects, even If chemogenetic stimulation of MN firing can also induce by itself some synaptic restoration.

Chemogenetic activation of PKA ameliorates disease burden in MNs through firing

Next we assessed whether the activation of the cAMP/PKA pathway, which increased functional and structural integrity of Ia synapses, was associated with a significant effect on MN disease markers. We repeated the D(Gs) experiments with the same experimental groups (Figure 5A): acute CNO treatment (one dose, sacrifice after 3h), chronic CNO treatment (7 days treatment) and control group (saline 7 days). We considered the burden of misfolded SOD1 (henceforth misfSOD1; using the B8H10 conformation-specific monoclonal antibody), the build-up of LC3A and the accumulation of p62+ inclusions as disease markers [START_REF] Rudnick | Distinct roles for motor neuron autophagy early and late in the SOD1(G93A) mouse model of ALS[END_REF][START_REF] Martínez-Silva M De | Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS[END_REF][START_REF] Alami | NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS[END_REF]. Acute activation of D(Gs) did not reduce levels of misfSOD1 or p62 inclusion burden (in agreement with the slow turnover of these markers; Figure 5E-F), but was sufficient to decrease the build-up of LC3A+ autophagic structures (Figure 5G). Most importantly, chronic activation of D(Gs) resulted in a robust amelioration of all disease markers (misfSOD, p62 inclusions and LC3A levels; Figure 5E-G). On the other hand, saline-treated D(Gs)+ MNs were comparable to non-infected counterparts in all read-out measures.

Next we examined whether the effects of the cAMP/PKA pathway on disease markers required the firing of MNs, once again exploiting multiplexed chemogenetics to decrease MN firing (by inhPSAM) while activating PKA (by D(Gs); Figure 6A). We evaluated the burden of misfolded SOD1 and LC3A in distinct sets of MNs expressing, as before, either no chemogenetic receptor, either one of D(Gs) or inhPSAM, or both.

Whereas MNs expressing inhPSAM showed an increased burden of LC3A and misfSOD1 accumulation (although the latter did not quite reach significance in the present work) compared to non-infected MNs [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF], MNs expressing D(Gs) displayed a reduced load of both disease markers (Figure 6D-E). Most notably, and in contrast to what was observed for synaptic GluR4 and Homer1b, MNs expressing both inhPSAM and D(Gs), in which neuronal firing was reduced while PKA was stimulated, showed no improvements in disease burden compared with non-infected MNs (Figure 6D-E).

In order to demonstrate that the chemogenetic manipulations used above (D(Gs), inhPSAM, actPSAM and double-chemogenetics inhPSAM+D(Gs)) have a significant impact on the overall activity of the infected MNs, we measured the induction of the immediate-early genes c-Fos as a proxy of neuronal activity [START_REF] Hardingham | Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression[END_REF][START_REF] Ruediger | Learning-related feedforward inhibitory connectivity growth required for memory precision[END_REF][START_REF] Lacar | Nuclear RNA-seq of single neurons reveals molecular signatures of activation[END_REF] following an acute injection of the chemogenic agonist (single dose of CNO and PSEM administered 2h before sacrifice; Figure 7A).

Compared to uninfected contralateral MNs (Figure 7B), reduction of MN firing by inhPSAM did not decrease c-Fos expression (Figure 7C, 7G), probably because of the very low level in basal conditions. On the other hand, activation of actPSAM (Figure 7D) or D(Gs) (Figure 7E) resulted in a robust increase in c-Fos levels in the nucleus (Figure 7G). Remarkably, concomitant activation of inhPSAM and D(Gs) (Figure 7F) significantly decreased c-Fos levels compared to D(Gs) alone (Figure 7G), indicating a substantial decrease in neuronal firing. These data verify that the chemogenetic systems are delivering the expected change in MN activity.

Thus, whereas the effect of PKA on synaptic structures is independent of MN firing, reducing MN firing largely blocks any beneficial effect of the cAMP/PKA pathway on disease markers.

The excitatory transmission from descending fibers to motoneurons is also depressed

Having extensively characterized the postsynaptic dysfunction at the Ia synapse, we asked whether this phenomenon was restricted to this particular input or rather generalized. We considered as an alternative source of input, the medial longitudinal fasciculus (MLF), which is composed of descending reticulospinal and vestibulospinal fibers, some of which make direct synaptic contacts (VGluT2+, [START_REF] Basaldella | Multisensory Signaling Shapes Vestibulo-Motor Circuit Specificity[END_REF] devoid of presynaptic inhibition [START_REF] Rudomin | Presynaptic inhibition in the vertebrate spinal cord revisited[END_REF] onto lumbar MNs [START_REF] Liang | Projections from the lateral vestibular nucleus to the spinal cord in the mouse[END_REF][START_REF] Basaldella | Multisensory Signaling Shapes Vestibulo-Motor Circuit Specificity[END_REF]. In different mice than those used for recording Ia EPSPs, stereotaxic electrical stimulation of MLF in the brainstem evoked a mixture of monosynaptic and disynaptic EPSPs (Figure 8A). We focused first on the response to the first shock in a train at 200Hz, as it was practically uncontaminated by the disynaptic component (see methods and Supplemental Figure 5). Quantification of MLF-evoked EPSPs in MNs revealed a 33 % decrease in size in mutSOD1 mice compared to wtSOD1 mice (Figure 8B). Second, we quantified the complex response after the fifth shock (that included disynaptic components, unfilled arrowhead in Figure 8A). That response was similarly reduced (36 % on average; Figure 8C), suggesting that most of the decrease could be attributed to the last order synapses contacting MNs (larger reductions should be expected if other intervening synapses were also affected). As before, the reduction in EPSP size was not due to changes in MN intrinsic properties (Figure 8D-F). The similar disruption of Ia synapses and MLF synapses suggests a generalized postsynaptic dysfunction of excitatory synapses on MNs.

Furthermore, we verified that, in correspondence to VGluT2+ terminals (which account for the majority of descending inputs to MN, [START_REF] Basaldella | Multisensory Signaling Shapes Vestibulo-Motor Circuit Specificity[END_REF], the size of the immunolabeled clusters of GluR4, phosphorylated GluR1 and phosphorylated GluR2 were significantly decreased in mutSOD1 MNs (Figure 8G-I) compared to wt controls.

Similarly to VGluT1+ synapses, we also assessed the integrity of the VGluT2+ postsynaptic structure. Since Shank1 and Homer1b were poorly enriched in VGluT2+ synapses (compared to VGluT1+ synapses) we explored the clustering of the Shank2 scaffold protein. Shank2 displayed a punctate pattern with each cluster juxtaposed to a VGluT2+ terminal; and the size of Shank2 clusters was significantly reduced in mutSOD1 MN compared to wt counterparts (Figure 8J). In line with the model of a substantial post-synaptic site disruption, the size and immunostaining intensity of VGluT2+ terminals demonstrated a small compensatory increase (Figure 8K).

Discussion

Our data show that excitatory synaptic inputs to MNs in the mutSOD1 ALS mouse model are functionally reduced and display impaired plasticity already in presymptomatic stages. Structural disruption of the post-synaptic site was identified as a major contributor for these functional disorders. We further show that activation of the cAMP/PKA pathway enhances physiological responses and restores GluR4 content in post-synaptic structures. Importantly, we used a multiplexed-chemogenetic approach to demonstrate that cAMP/PKA effects on synaptic integrity are not a consequence of changes in MN firing, whereas the beneficial effects on disease markers are dependent on neuronal firing. Furthermore, we show that synaptic dysfunction is likely a generalized phenomenon: decrease in EPSPs size characterizes proprioceptive Ia synapses as well as monosynaptic inputs from the vestibulo and reticulospinal tracts (MLF).

Disruption of the post-synaptic side is a major cause of excitatory synaptic failure

The glutamatergic excitotoxicity model predicts a combination of increased intrinsic MN excitability and excessive synaptic or extrasynaptic excitatory stimulation [START_REF] Rothstein | Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis[END_REF][START_REF] Lewinski | Ca2+, mitochondria and selective motoneuron vulnerability: implications for ALS[END_REF][START_REF] Bading | Therapeutic targeting of the pathological triad of extrasynaptic NMDA receptor signaling in neurodegenerations[END_REF] driving abnormal cytoplasmic Ca 2+ loads and neuronal death. A substantial fraction of clinical trials based on preventing these conditions have failed to deliver beneficial effects [START_REF] Petrov | ALS Clinical Trials Review: 20 Years of Failure. Are We Any Closer to Registering a New Treatment? Front[END_REF]. Contradicting these assumptions, we have previously demonstrated that vulnerable MNs lose their ability to fire repetitively in presymptomatic adult mice (a form of intrinsic hypoexcitability, [START_REF] Delestrée | Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis[END_REF][START_REF] Martínez-Silva M De | Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS[END_REF], while resistant MNs are intrinsically hyperexcitable at an early stage (Leroy et al, 2014, 201). In these conditions, hyper-active glutamatergic synapses might still drive neurotoxicity by causing large cytoplasmic Ca 2+ loads even in a condition of normo-or hypo-excitability (Le [START_REF] Masson | A Computational Model of Motor Neuron Degeneration[END_REF]. Now we demonstrate that EPSPs are actually decreased in ALS MNs. The reduced clustering of GluR4, phosphorylated GluR1 and GluR2 subunits (in agreement with [START_REF] Ruegsegger | Impaired mTORC1-Dependent Expression of Homer-3 Influences SCA1 Pathophysiology[END_REF] and of postsynaptic scaffold proteins (Shank1 and Homer1b at Ia synapses, Shank2 at VGluT2+ synapses) point to a substantial postsynaptic contribution to the failure of excitatory synapses on ALS MNs. The involvement of a post-synaptic mechanism is further supported by the partial recovery of synaptic structure and function following PKA-pathway activation in the post-synaptic neuron. In contrast, wtSOD1 and mutSOD1 animals exhibited the same level of the classical axoaxonic presynaptic inhibition of Ia afferents, demonstrating that this pathway cannot account for the changes in EPSP size and paired-pulse ratio in mutSOD1 MNs. Moreover, presynaptic inhibition is restricted to Ia terminals and would not affect MLF terminals [START_REF] Rudomin | Presynaptic inhibition in the vertebrate spinal cord revisited[END_REF][START_REF] Fink | Presynaptic inhibition of spinal sensory feedback ensures smooth movement[END_REF]. Another presynaptic mechanism, the postactivation depression, which is restricted to homonymous Ia afferents [START_REF] Hultborn | On the mechanism of the post-activation depression of the H-reflex in human subjects[END_REF], was previously shown to be smaller in mutant SOD1 mice [START_REF] Hedegaard | Postactivation depression of the Ia EPSP in motoneurons is reduced in both the G127X SOD1 model of amyotrophic lateral sclerosis and in aged mice[END_REF] and thereby cannot be responsible for the reduced EPSP size. Finally, there are no gross abnormalities in number or constitutive elements of Ia boutons, as shown by immunohistochemical methods. Therefore, the contribution of presynaptic mechanisms, if any, on the synaptic dysfunction might be due to more subtle abnormalities and may be limited in comparison with substantial post-synaptic effects.

We then considered whether Ia synaptic dysfunction may be due to abnormal activity in corticospinal projections [START_REF] Eisen | Cortical influences drive amyotrophic lateral sclerosis[END_REF]. Cortical hyperexcitability has been reported in human patients [START_REF] Vucic | Novel threshold tracking techniques suggest that cortical hyperexcitability is an early feature of motor neuron disease[END_REF], in cultures [START_REF] Pieri | Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis[END_REF] and in slices [START_REF] Saba | Altered Functionality, Morphology, and Vesicular Glutamate Transporter Expression of Cortical Motor Neurons from a Presymptomatic Mouse Model of Amyotrophic Lateral Sclerosis[END_REF]. In mutSOD1 mice, the hyperexcitability of corticospinal neurons is restricted to the first postnatal week, or to the symptomatic phase (even though, in the latter, hyperexcitability did not result in altered pattern of calcium events in awake animals), whereas at the age of P40-P50, the excitability of the corticospinal neurons is normal [START_REF] Kim | Changes in the Excitability of Neocortical Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by Advancing Disease[END_REF]. Moreover, in mice, the projections from the motor cortex to spinal MNs are not direct and are relayed through the reticulospinal pathway included in the MLF [START_REF] Alstermark | In vivo recordings of bulbospinal excitation in adult mouse forelimb motoneurons[END_REF]. Notably, these MLF-MN synapses are actually depressed, as shown in the present work. Thus, cortical hyperexcitability, if any, may not translate into excessive excitation to MNs. Taken together, these findings consistently draw a new model in which vulnerable MNs are not only hypoexcitable but also receive reduced excitation likely because of post-synaptic dysfunction.

At this presymptomatic stage (around P50), the reduced strength of excitatory synapses is not large enough to impair electromyographic activity or motor performance in all but the most challenging motor tasks (such as walking on an incline, [START_REF] Akay | Degradation of mouse locomotor pattern in the absence of proprioceptive sensory feedback[END_REF][START_REF] Quinlan | Chronic electromyograms in treadmill running SOD1 mice reveal early changes in muscle activation[END_REF]. Nevertheless, the simultaneous decrease in synaptic strength and intrinsic excitability of MNs could be speculated to interfere with activity-dependent neuroprotective transcriptional programs, leading to vulnerability to disease [START_REF] Bading | Nuclear calcium signalling in the regulation of brain function[END_REF][START_REF] Bading | Therapeutic targeting of the pathological triad of extrasynaptic NMDA receptor signaling in neurodegenerations[END_REF][START_REF] Roselli | From intrinsic firing properties to selective neuronal vulnerability in neurodegenerative diseases[END_REF].

cAMP/PKA activation and neuronal firing contribute to synaptic restoration and have a beneficial impact on the ALS-related pathobiochemistry

We show that before muscle denervation, the disruption of Ia synapses (both EPSP size, and GluR4 and Homer1b content) is reversible by postsynaptic PKA activation. This is in agreement with the critical role of PKA in phosphorylating the Ser842 (corresponding to Ser862 in the mouse) on GluR4 C-terminus, enhancing its synaptic insertion [START_REF] Esteban | PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity[END_REF][START_REF] Gomes | PKC anchoring to GluR4 AMPA receptor subunit modulates PKC-driven receptor phosphorylation and surface expression[END_REF][START_REF] Diering | PKA-GluA1 coupling via AKAP5 controls AMPA receptor phosphorylation and cell-surface targeting during bidirectional homeostatic plasticity[END_REF]. In addition, the restoration of Homer1b clusters appears to be a direct effect of cAMP/PKA, in agreement with the enhanced Homer1b clustering induced by phosphodiesterase inhibition (an alternative approach to increase cAMP levels, [START_REF] Shiraishi | Coincidence in dendritic clustering and synaptic targeting of homer proteins and NMDA receptor complex proteins NR2B and PSD95 during development of cultured hippocampal neurons[END_REF] and may hint at broader consequences on synaptic structures regulated by PKA [START_REF] Lu | A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density[END_REF].

Furthermore, we show that the beneficial effects of cAMP/PKA activation extend to multiple markers of mSOD1/ALS pathobiochemistry. We have exploited a multiplexed chemogenetic strategy, based on the simultaneous expression and activation of the mutually orthogonal PSAM/PSEM system [START_REF] Magnus | Chemical and Genetic Engineering of Selective Ion Channel-Ligand Interactions[END_REF]) and the DREADD system [START_REF] Roth | DREADDs for Neuroscientists[END_REF] to show that decreasing MN excitability largely prevents the recovering actions of cAMP/PKA on disease markers but not on synaptic composition. It should, however, be noted that reducing MN firing by inhPSAM resulted in a small increase in synaptic GluR4 (but not Homer1b) levels. We hypothesize that such effect may bear similarity to the upregulation of AMPAR occurring as homeostatic scaling upon chronic blockade of neuronal firing [START_REF] O'brien | Activity-Dependent Modulation of Synaptic AMPA Receptor Accumulation[END_REF][START_REF] Turrigiano | Activitydependent scaling of quantal amplitude in neocortical neurons[END_REF][START_REF] Diering | PKA-GluA1 coupling via AKAP5 controls AMPA receptor phosphorylation and cell-surface targeting during bidirectional homeostatic plasticity[END_REF]. Notably, Homer1b levels are unaffected, suggesting that the hypothesized homeostatic response may be weak. Although it has been previously shown that cAMP/PKA promotes proteasomal degradation of misfolded SOD1 by phosphorylating the Rpn6 subunit of the 26S proteasome [START_REF] Lokireddy | cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins[END_REF][START_REF] Verplank | 26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation[END_REF], the activity-dependent nature of the effect of PKA on misfolded SOD1, LC3A and p62 suggests that it may be mediated by excitation-controlled transcription factors controlling the expression of autophagy and proteasome proteins, e.g. CREB [START_REF] Seok | Transcriptional regulation of autophagy by an FXR-CREB axis[END_REF][START_REF] Wang | A pivotal role of FOS-mediated BECN1/Beclin 1 upregulation in dopamine D2 and D3 receptor agonist-induced autophagy activation[END_REF]. Increasing MN firing by chemogenetically-controlled cation channels [START_REF] Magnus | Chemical and Genetic Engineering of Selective Ion Channel-Ligand Interactions[END_REF]) also results in a degree of restoration of GluR4 and Homer1b content in synapses and was previously reported to evoke similar beneficial effects on disease markers [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF]. Taken together, these findings support a model in which cAMP/PKA activation restores synaptic integrity which, in turn, improves MN recruitment and firing, reducing the burden of disease markers (Figure 9). These findings suggest the existence of a "vicious circle" at work: the pathogenic "primum movens" may cause an initial disruption of synapses and, in parallel, reduced MN excitability [START_REF] Delestrée | Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis[END_REF][START_REF] Martínez-Silva M De | Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS[END_REF]. We hypothesize that disturbed intracellular trafficking by misfSOD1 [START_REF] Hadano | Loss of ALS2/Alsin exacerbates motor dysfunction in a SOD1-expressing mouse ALS model by disturbing endolysosomal trafficking[END_REF][START_REF] Van Dis | Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons[END_REF][START_REF] Soo | Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUSassociated ALS[END_REF]) may be at the origin of the large dendritic vacuoles and these abnormal structures may weaken synapses by sequestering glutamate receptors. These two events would reduce MN firing, which would in turn further worsen cellular abnormalities (including trafficking impairment, autophagy overload, endoplasmic reticulum stress) and further accelerate the disruption of synapses and firing, precipitating the demise of the MN itself. The demonstration that PKA activation restores synaptic integrity and then leads to the activity-dependent beneficial effects on disease markers shows that this vicious circle can be interrupted acting at a singlepathway level. However, the focal nature of intraspinal injections and the comparatively small number of MN affected has limited the assessment of long-term effects of PKA activation on mouse survival. Nevertheless, we show the impact of PKA activation on different disease markers whose decrease has been repeatedly correlated with prolonged survival (e.g., [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF][START_REF] Alami | NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS[END_REF].

Together with the previously reported hypoexcitability of MNs, the early impairment of excitatory synaptic function depicts a new conceptual framework for excitation-dependent processes in ALS, in which loss of activity-dependent neuroprotection, rather than excitotoxicity, shapes MN vulnerability. Interventions at motoneuronal synaptic level, potentially aimed at the cAMP/PKA pathway, emerge as viable new strategies for early ALS management.

Materials and methods

Animals

Animals for electrophysiological experiments were bred and housed in the BioMedTech animal facility at Université de Paris. Electrophysiological procedures were approved by the Paris Descartes University ethics committee (CEEA34; authorization number CEEA34.MM.064.12) and followed the European Directives (86/609/CEE and 2010-63-UE) and the French legislation on the protection of animals used for scientific purposes. Two lines of transgenic mice were used: B6SJL-Tg(SOD1*G93A)1Gur/J (called mutSOD1 here), who carry a mutated human SOD1 gene and have a phenotype similar to late onset of ALS in humans, and B6SJL-Tg(SOD1)2Gur/J (called wtSOD1 here), who carry the non-mutated human SOD1 gene and display no pathological phenotype. In both lines the transgene copy number was measured by qPCR and found to be >20. Both male and female mice were used for electrophysiological recordings. MNs recorded from mutSOD1 mice were compared to the ones recorded from wtSOD1 mice.

Experiments were performed on mice at age P45-P55, i.e. presymptomatic young adult animals, unless otherwise specified.

The chemogenetic experiments and the quantitative immunohistological analysis were performed at Ulm University in compliance with institutional guidelines (Tierforschungszentrum, Ulm) and German animal protection laws, approved by Regierungspräsidium Tübingen (Tübingen, Germany), under the licence no. 1404. The breeding and the sacrifice of mutSOD1 mice in Ulm was approved by the local veterinary service and animal experimentation oversight committee under the licence no. 1312. For the chemogenetic experiments, high-copy B6SJL-Tg(SOD1*G93A)1Gur/J mice (obtained from Jackson Laboratories) were crossed with homozygous ChAT-cre females (kind gift of Prof. Pico Caroni) and the F1 offspring mutSOD1/ChAT-cre doubletg mice were used for experiments. Only male mice were used in chemogenetics and histological experiments in order to minimize the between-subject variability (Ouali [START_REF] Alami | NF-κB activation in astrocytes drives a stage-specific beneficial neuroimmunological response in ALS[END_REF]. Gria4 knockout mice (B6.129P2-Gria4t m1Dgen ; Gria4 -/-for brevity), were backcrossed to the C57BL/6J strain for ten generations and then maintained as a homozygous colony; these mice have been previously characterized [START_REF] Beyer | Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4[END_REF].

Wild-type mice C57BL/6J (Gria +/+ ) were used as controls in the experiment involving Gria4 -/-mice.

Surgery for electrophysiological experiments

The surgical procedures have been described previously [START_REF] Manuel | Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons[END_REF][START_REF] Delestrée | Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis[END_REF]. Briefly, atropine (0.20 mg/kg; Aguettant) and methylprednisolone (0.05 mg; Solu-Medrol; Pfizer) were given subcutaneously at the onset of experiment, to prevent salivation and oedema, respectively. Fifteen minutes later, anaesthesia was induced with an intraperitoneal injection of sodium pentobarbitone (70 mg/kg; Pentobarbital; Sanofi-Aventis). A tracheotomy was performed, and the mouse was artificially ventilated with pure oxygen (SAR-830/AP ventilator; CWE). The end tidal CO 2 level was maintained around 4 % (MicroCapstar; CWE). The heart rate was monitored (CT-1000; CWE), and the central temperature was kept at 37°C using an infrared heating lamp and an electric blanket. A catheter was introduced in the external jugular vein, allowing us to supplement the anaesthesia whenever necessary (usually every 20-30 min) by intravenous injections (sodium pentobarbitone, 6 mg/kg). The adequacy of anaesthesia was assessed on lack of noxious reflexes and on the stability of the heart rate (usually 400-500 bpm) and end-tidal PCO 2 .

A slow intravenous infusion (50 μL/h) of a 4 % glucose solution containing NaHCO 3 (1 %) and gelatine (14 %; Plasmagel; Roger Bellon) helped maintain the physiological parameters. The animal was paralyzed after the surgery with pancuronium bromide (Pavulon; Organon; initial bolus was 0.1 mg, followed by a continuous infusion 0.01 mg/h). Additional doses of anaesthetics were then provided at the same frequency as before the paralysis, and adequacy of anaesthesia was assessed on the stability of the heart rate and of PCO 2 . The vertebral column was immobilized with two pairs of horizontal bars (Cunningham Spinal Adaptor; Stoelting) applied on the Th12 and L2 vertebral bodies, and the L3-L4 spinal segments were exposed by a laminectomy at the Th13-L1 level. The tissues in the hindlimb and the spinal cord were covered with pools of mineral oil.

Stimulation and recording

The MNs were impaled with micropipettes (tip diameter, 1.0-1.5 μm) filled with a mixture of 2M K-Acetate and 0.1M QX-314 (sodium channel blocker, Sigma). Recordings were made using an Axoclamp 2B amplifier (Molecular Devices) connected to a Power1401 interface (sampling rate 20 kHz) and using the Spike2 software (CED). After impalement, identification of MNs rested on the observation of antidromic action potentials in response to the electrical stimulation of their axon in the triceps nerve. This method of identification was still possible despite the QX-314 as it takes approximately 30-90 s for the compound to diffuse from the tip of the electrode to the cell body to block the sodium channels. All MNs retained for analysis had a resting membrane potential more hyperpolarized than -50 mV and an overshooting action potential >65 mV. As fully described in [START_REF] Manuel | Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons[END_REF], the input resistance was measured using the peak response of a series of small-amplitude square current pulses (-3 to +3 nA, 500 ms) and the membrane time constant was measured on the relaxation of the membrane potential after injection of small hyperpolarizing current pulses (-5 nA, 1 ms). All recordings were performed in Bridge mode, except for the input resistance, which was recorded using the discontinuous current clamp mode (7-9 kHz).

All care was taken to compensate for the microelectrode resistance and capacitance. At the end of the experiments, animals were killed with a lethal intravenous injection of pentobarbitone (200 mg/kg).

Vibration experiments

Following initial surgery, the triceps nerve (composed of the nerves innervating medial gastrocnemius (MG), lateral gastrocnemius and soleus (LG) muscles) was separated from the surrounding tissues and mounted on a silver bipolar electrode for stimulation and electroneurogram (ENG) recording. The triceps muscle belly was separated from the surrounding tissues and the achilles tendon was cut free at the triceps insertion point as close to the heel as possible. The tendon was connected with a rigid suture to the center of a loudspeaker membrane driven by a function generator.

Prior to cutting the tendon the mouse paw was dorsiflexed to the maximum flexion angle and a small mark was made on the bone to indicate the muscle maximum physiological length. Following the transfer of the tendon to the vibrator, the muscle was stretched to mimic the maximum length during dorsiflection. The achilles tendon was vibrated at frequencies ranging from 300 to 500 Hz. For each MN, we searched for the vibration frequency that elicited the largest EPSP (370 Hz on average). The obtained signal was smoothed using root mean square amplitude (Spike2, time constant 4 ms) and then peak amplitude of the response was measured.

Electrically-evoked EPSPs and paired-pulse protocol experiments

In these experiments the surgery was similar to the previously described vibration experiments with the exception of achilles tendon dissection. Following the MN penetration and identification, the triceps nerve was stimulated with constant current pulses of 20 μs duration repeated at 3 Hz. The intensity of stimulation was gradually increased from the threshold (minimal intensity to recruit the most excitable afferents as seen by the apparition of a volley response recorded at the surface of the spinal cord) up to the point when maximum amplitude of EPSP was reached (Supplemental Figure 1A-B). 10-30 successive sweeps at the stimulation intensity that elicited the maximal EPSP (usually between 1.8×-2.0× threshold) were averaged offline and measurements were obtained from these averages. In the paired-pulse experiments, the nerve was stimulated with a pair of constant current pulses separated by 10 ms intervals at an intensity that elicited submaximal EPSPs amplitudes. The sequence was repeated at 3 Hz at least 10 times. For each MN, we computed the paired-pulse ratio for each individual pair and then we averaged the individual ratios. To investigate the presynaptic inhibition of Ia terminals, the Ia EPSP in Triceps Surae (TS) MNs elicited by stimulation of the TS nerve (test response) was conditionned by stimulating group I afferents in the nerve branches innervating the posterior part of the Biceps Femoris (3 shocks at 200 Hz; 2× threshold of the most excitable afferents; conditioning stimulation applied 23 ms before the test stimulation). Renshaw inhibition was observed in separate experiments in which the L4-L5 dorsal roots were cut in order to supress the sensory EPSPs. In these conditions, stimulation of the TS nerve produces only an IPSP elicited by the motor axon collaterals.

Stimulation of the descending tracts

These experiments were performed on a separate set of animals than the Ia EPSP experiments above. Following initial surgery (laminectomy and nerve dissection as described above), a craniotomy was performed over the cerebellum, approximately 1 mm caudal with respect to lambda landmark and an additional laminectomy over C7-Th1 spinal segments was performed for descending volley recording. A tungsten stimulating electrode (WPI, tip 1 µm, resistance 1 MΩ) was inserted into the mouse brainstem through the cerebellum, at the stereotaxic coordinates AP -7.2, ML +0.1 with reference to bregma landmark using Neurostar robot stereotaxic instrument (Neurostar, De). At this location the medial longitudinal fasciculus (MLF) contains reticulospinal, tectospinal and vestibulospinal fibers. The vestibulospinal and reticulospinal fibers make direct monosynaptic connections with triceps surae MNs in mice [START_REF] Liang | Projections from the lateral vestibular nucleus to the spinal cord in the mouse[END_REF]. The MLF was stimulated with single constant-current shocks of 150 µs duration delivered through the descending tungsten electrode against the reference electrode located in the temporal muscles. The smallest stimulation intensity (threshold intensity) to elicit a volley in the cervical cord and in the lumbar cord was never stronger than 25 µA (average 14.3 µA, SD 5.7 µA). Correct placement of the electrode was assessed while monitoring the descending volley at the C7 and L3 spinal levels. The electrode was placed at the depth at which the descending volley was maximal (Supplemental Figure 5A-B).

Upon successful penetration of a MN, the MLF was stimulated with a train of five pulses at 200 Hz, repeated at 3 Hz. We then recorded EPSPs in spinal MNs at 2.2× threshold intensity (the final intensity was always smaller than 60 µA; intensity at which the current spread from the tip of the electrode was less than 0.25 mm [START_REF] Gustafsson | Direct and indirect activation of nerve cells by electrical pulses applied extracellularly[END_REF] only activating MLF). We recorded 10-30 sweeps, which were averaged, and measurements were made on these averages. In a few cells ( 4wtSOD1 and 6 mutSOD1), the MLF stimulation only evoked a monosynaptic response, as exemplified in Supplemental Figure 5D. The latency between the descending volley recorded at the lumbar level and the EPSP onset was short (0.3 ms in the example of Supplemental Figure 5D). In such a short delay, there is no time for the activation of an interposed interneuron, indicating that the descending fibers synapse directly onto the MN. In extremely rare instances (1 wtSOD1 and 1 mutSOD1 cells not included in analysis of monosynaptic inputs), we observed solely a disynaptic EPSPs (Supplemental Figure 5E). Disynaptic EPSPs appeared only after the second shock (unfilled arrow, no EPSP visible in response to the first shock) and they grew in response to the following stimulations, indicating a progressive temporal facilitation of an increasing number of interposed excitatory interneurons. The central latency with respect to the fastest component in the volley was 1.4 ms, i.e. about 1 ms longer than the monosynaptic response (this supplemental delay is caused by the activation of the synapses to interneurons, the time for interneurons to reach their firing threshold, and the axonal conduction velocity of interneurons which might be slower than the axonal conduction velocity of descending fibers). However, in most of MNs (15 wtSOD1 and 14 mutSOD1), the responses were mixed (Figure 8A): after the peak of the monosynaptic component (filled arrowheads), a bump was visible reflecting the apparition of the disynaptic component (unfilled arrowhead). However, this disynaptic component was negligible in the response to the first stimulation of the train.

At the end of the experiment the location of the electrode was marked by an electrolytic lesion, the mouse was perfused intracardially with 4 % Paraformaldehyde (PFA), the brainstem was removed, cut at 100 µm on a vibrating microtome (Model 7000smz-2, Campden Instruments), stained with Cresyl violet and the lesion location was verified with light microscopy (Supplemental Figure 5C).

PKA activation experiments

Initial surgery, and MN stimulation and recording were performed as described above. In order to measure the impact of PKA activation on MN synaptic excitation levels, the recording microelectrode was filled with a mixture of 2M K-Acetate, 0.1 M QX-314, and 4 mM cAMP analog (S)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium (cAMPS-Sp). Next, EPSP amplitude and paired-pulse ratio were measured before, immediately after, and in successive 2 min intervals after iontophoretic injection of cAMPS-Sp through the microelectrode (500 ms pulses of -2 to -4 nA, repeated at 1 Hz, total amount injected -120--180 nA•s, [START_REF] Lalley | cAMP-dependent protein kinase modulates expiratory neurons in vivo[END_REF].

Intracellular labelling of motoneurons and immunostaining of Ia synaptic contacts

The surgery and intracellular recordings of TS MNs were performed as above. The microelectrode was filled with a mixture of 3 M KCl and 2 % Neurobiotin (Vector Laboratories). A series of current pulses (2-5 nA, 600 ms, 1 Hz), was used to transfer the neurobiotin into the MN. 45 minutes after injection, to allow the neurobiotin to diffuse in distal dendrites, the mouse was perfused intracardially, using 50 mL of 1× Phosphate-buffered saline (PBS) and 100 mL of 4 % PFA. The spinal cord was postfixed for 1 h and then preserved in 1x PBS . The spinal cord was embedded in 4 % agarose, then cut in 50 μm slices with a vibratome (VT1000S, Leica GmbH, Wetzlar, Germany).

Slices were incubated 1 h in 1× PBS, 3 % Triton, Bovine Serum Albumin (BSA; 5 mg/mL), and 10 % Donkey serum, then rinsed 3×10 min with PBS. The neurobiotin was revealed by incubation of the slices in 1× PBS, 1 % Triton, BSA (5 mg/mL) and Streptavidin-Cy3 (1:500, Sigma-Aldricht). After rinsing 3×10 min, slices were incubated overnight at 4°C in 1× PBS, 1 % Triton, BSA (5 mg/mL) with primary antibodies anti-VGluT1 (guinea pig, 1:4000, Merck Millipore). Slices were rinsed 3×10 min again, and secondary antibodies Alexa-647-conjugated donkey anti-guinea pig (1:500, Jackson Immunoresearch Laboratories) were applied in 1× PBS, 1 % Triton, BSA (5 mg/mL) for 3 h at room temperature. Finally, slices were washed 3×10 min with PBS and mounted on slides with Fluoroshield. Slides were then analysed using a confocal microscope for fluorescence (LSM710; Carl Zeiss, Oberkochen, Germany). For the somatic synaptic density, Cholera Toxin subunit B coupled to Alexa Fluor 488 (10 μL, 2 % CTb-488 in sterile water) was injected into the right lateral gastrocnemius muscles at P50 (N=3 wt and 3 mutSOD1 mice). Four to five days later, the animals were perfused with 4 % PFA and immunolabeling against VGluT1 was performed as above. Z-stacks of somas were acquired on a LSM710 confocal microscope with a 63× oil immersion objective. The images were processed in Neurolucida and a 3D model of the soma surface was constructed while counting the number of boutons in juxtaposition to the soma.

Chemogenetic experiments

The AAV9 encoding pAAV(9)-pCAG-A7-floxed-PSAM(L141F, Y115F)5HT3-WPRE and pAAV(9)-pCAG-A7-floxed-PSAM(L141F, Y115F)-GlyR-GFP-WPRE [START_REF] Magnus | Chemical and Genetic Engineering of Selective Ion Channel-Ligand Interactions[END_REF] were prepared by Vector Biolabs. The plasmid encoding pAAV-hSyn-DIO-rM3D(Gs)-mCherry was obtained from Addgene (Plasmid #50458) and the plasmids encoding the capsid protein for wt AAV9 and the rep gene were a kind gift of Oliver Müller (Kiel University); the AAV9 preparation was performed as previously reported [START_REF] Commisso | Stage-dependent remodeling of projections to motor cortex in ALS mouse model revealed by a new variant retrograde-AAV9[END_REF]. All AAV vectors were prepared in stock suspensions at the titer of 1⋅10 13 genomes/mL.

The intraspinal injection of single AAV9 vectors for the expression of the chemogenetic PSAM or DREADD receptors and the chemogenetic agonist treatment were performed as previously reported [START_REF] Saxena | Neuroprotection through Excitability and mTOR Required in ALS Motoneurons to Delay Disease and Extend Survival[END_REF]. This approach was chosen because it yields a large number of infected MNs at the injection site (Supplemental Figure 4A). However, the volume injected was purposefully kept small in order to i) minimize the surgical and injection injury to the juvenile spinal cord, and ii) minimize the discomfort to the experimental animal that may result from excessive or reduced excitability of a number of motoneurons. Consequently, only a spinal cord segment 200-300 µm-long contained infected MNs. Briefly, animals were placed in a stereotaxic frame under buprenorphine (0.1 mg/kg)/meloxicam (1.0 mg/kg) premedication and continuous 4 % sevoflurane anesthesia (in 96 % O 2 at 0.8 L/min). After incision of the skin and the subcutaneous fascia, the paraspinal muscles were blunt-dissected and the dorsal laminae of the T11-T12 vertebrae were incised with a corneal-limb scissor (Fine Science Instruments) and the bone flap was removed to expose the dorsal aspect of the spinal cord. Using the dorsal artery as reference, a pulled glass capillary was lowered using the stereotaxic frame at the coordinates y=+0.25 mm z=-0.4 mm at one location in the lumbar spinal cord. Approximately 0.5 µl in total of AAV suspension (titer 1⋅10 13 genomes/ml) was mixed with 0.5 µl of 1.5 % Fast-green and injected (at 0.1 µl/min) using a PicoSprizer III (Parker) microfluidic device. For the double-chemogenetic experiments, 0.5 µl of AAV9-PSAM suspension and 0.5 µl of AAV2-9-DREADD Gs suspension were mixed together with 1 µl of 1.5% Fast-green; a volume of 1 µl from the mix was then injected. The capillary was left in place for 10 min after the end of the injection. After retraction of the capillary, the paraspinal muscles were sutured on the midline using Prolene 6/0 surgical thread, the fascia was positioned on the midline and overlying skin was sutured with Prolene 6/0. Mice were administered buprenorphine (0.05 mg/kg) twice per day and meloxicam (1.0 mg/kg) daily for three consecutive days after surgery. The chemogenetic agonist PSEM 308 was obtained from Apex Scientific and administered subcutaneously at a dose of 5 mg/kg in saline. Clozapine-N-Oxide (CNO) was obtained from Tocris and administered at a dose of 5 mg/kg diluted in saline. For chronic experiments, the agonist was injected once per day starting 10 days after AAV injection for 7 days. For acute experiments, a single dose was administred on the 17th day after AAV injection. Mice were sacrificed 3 h after the last administration of the agonist.

Immunostaining of pre-and postsynaptic structures

All histological evaluations were performed on mutSOD1 or non-transgenic littermates (called wt here). Mice were terminally anesthetized with cloralium hydrate and transcardially perfused with 50 mL ice-cold PBS followed by 2.5-3 mL/g of 4 % PFA in PBS (pH 7.4). Spinal cord samples were quickly dissected and post-fixed in 4 % PFA in PBS at 4°C for 18 h, washed in PBS and cryoprotected in 30 % sucrose in PBS for 36 h. Samples were then snap-frozen in OCT and sectioned at -18°C in a cryostat (Leica CM1950); 40 µm-thick sections were washed in PBS and used for immunostaining.

Free-floating spinal cord sections were incubated in blocking buffer (3 % BSA + 0.3 % Triton), followed by the incubation with opportune primary antibody (diluted in blocking buffer): anti-VAChT 1:300, Millipore ABN100 (for goat anti-VAChT immunostaining we diluted the primary antibody directly in a solution BSA-free made with PBS + 0.3 % Triton X-100 (Thermo Fisher), avoiding the blocking step); anti-VAChT 1:300, SYSY 139105; anti-misfolded SOD1 B8H10 1:500, Biomol MM-0070-P; anti-LC3A 1:300, Cell Signaling D50G8 #4599, anti-RFP Atto 565 1:500, Nanotag Biotechnologies At565-S; anti-GFP 1:500, Abcam ab13970; α-Bungarotoxin CF®405S Conjugate 1:500, Biotium 00002; anti-pGluR1 1:100, Abcam ab12108; anti-pGluR2 1:200, Abcam ab52180; anti-VGluT1 1:500, SYSY 135304; anti-Bassoon 1:500, Enzo VAM-PS003; anti-Synaptophysin 1:200, SYSY 101002; anti-Homer1b/c 1:500, SYSY 160023; anti-Shank1 1:200 Novus Cat# NB300-167.500; anti-Shank2 1:500 (previously reported [START_REF] Peter | Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice[END_REF] kind gift from prof. Tobias Boeckers, Institute of Anatomy and Cell Biology, Ulm University); anti-GluR4 1:500, Cell Signaling Technologies D41A11 #8070; anti c-Fos [2H2] 1:500, Abcam ab208942 for 48 h at 4°C. The specificity of the antibody against GluR4 was tested on cerebellar sections of WT and Gria4 -/-mice. The molecular layer was brightly stained by the anti-GluR4 antibody in WT animals but the staining was reduced to background levels in Gria4 -/-mice (Supplemental Figure 4G-H excluded from the analysis. For a better display, the contrast of the representative pictures has been adjusted to highlight mainly the bright C-boutons rather than the relatively dimmer cytoplasmic VAChT staining (see Supplemental Figure 2E-F for examples where the VAChT signal was intentionally boosted to highlight the cytoplasmic labeling). MNs for analysis were selected randomly in the lateral pool of the ventral horn from spinal cord sections (at L3-L5). Confocal stacks were acquired starting at a depth of 4 µm below the surface of the section; 10-15 optical sections were acquired for each MN. Spinal cord sections displaying cutting or staining artifacts, uneven staining or background intensity were not considered for imaging. MNs smaller than 20 µm in diameter or displaying no VGluT1+ synapses were not included in the analysis. Cutting, immunostaining and imaging was performed in batches which included samples from both genotypes; freshly cut and immunostained samples from the same wt mice were also routinely included and analyzed to ensure batch-to-batch reproducibility.

Image quantification was performed in ImageJ. For the evaluation of the size of presynaptic terminals (VGluT1 and Synaptophysin), 3-5 consecutive optical sections were collapsed in maximum-intensity projection, subject to rolling-ball background subtraction and thresholded for the segmentation of the synaptic terminals. The orientation of the synaptic structures (i.e., the identification of the post-synaptic structures belonging to a selected MN) was performed using the cytoplasmic VAChT staining and the shape of the nucleus as references, as described above. The contour of the region of interest (ROI) including the area positive for VGluT1, Synaptophysin or Bassoon were traced manually in ImageJ and the surface and mean intensity values were logged. For c-Fos quantification, the contour of the nucleus was delineated manually on maximum-intensity projections of confocal stacks and the mean fluorescence intensity was logged.

Statistics

All values are provided as mean ± SD. Box-and-whiskers plots are defined such that the boxes extend from the lower to upper quartile values of the data, with a thick line at the median. The whiskers extend from the boxes up to the last point within the interval ±1.5× the interquartile range. Individual data points are overlaid on top of the boxes (note that, for the sake of clarity, at most 100 randomly-chosen points are shown). ✻ corresponds to p<0.05, ✻✻ to p<0.01, ✻✻✻ p<0.001 and ✻✻✻✻ p<0.0001. Statistical tests were performed in Prism v.6.0.7 (GraphPad Software, San Diego, California USA), and SciPy Python v.1.4.1 [START_REF] Virtanen | SciPy 1.0: fundamental algorithms for scientific computing in Python[END_REF]. Figures were generated using matplotlib v.3.1.3 [START_REF] Hunter | Matplotlib: A 2D Graphics Environment[END_REF], seaborn v.0.10.0 [START_REF] Waskom | mwaskom/seaborn: v0.10.0[END_REF], and ImageJ v.2.0.0 [START_REF] Schindelin | The ImageJ ecosystem: An open platform for biomedical image analysis[END_REF].

Electrophysiology

Results obtained from wtSOD1 MNs were compared to the ones from mutSOD1 animals using appropriate statistical tests. The tests were chosen based on data distribution (Shapiro-Wilk normality test) and variance (F-test). For data with normal distribution we used the Student t-test, and for data with non-normal distribution we used Wilcoxon-Mann-Whitney Rank Sum Test. In all cases the significance level was set at p<0.05.

Synaptic coverage

For the synapses on the soma, the synaptic density was calculated as the number of VGluT1 appositions divided by the surface of the reconstructed 3D model of the MN soma using Neurolucida (number of synapses per 100 μm² of soma). For dendrite analysis, we considered dendrites to be proximal if (i) they were located in the section containing the soma, (ii) the path of the dendrite could be traced back to the soma, and (iii) the dendritic path length was shorter than 100 μm from the soma. Dendrites segments from at least two 50 μm-thick sections (>100 µm radial distance) away from the soma were considered as distal dendrites (spread in 4 to 7 different spinal cord sections per animal). The density of synapses was computed for each dendritic segment (number of synapses per 100 μm). Differences were analyzed using Wilcoxon-Mann-Whitney Rank Sum Test. The significance level was set at p<0.05.

Synaptic proteins analysis

For the analysis of synaptic proteins, confocal stacks were manipulated in ImageJ; the same manipulations were applied to all images across replicates and genotypes. For fluorescence intensity quantification, small stacks (3-5 optical sections) were collapsed in maximum-intensity projection and on the resulting projected images region of interest (ROI) were manually traced either overlapping the presynaptic marker or in the volume adjacent to it (for the analysis of postsynaptic proteins). Each image was thresholded (different threshold for each synaptic protein, [START_REF] Roselli | Disassembly of shank and homer synaptic clusters is driven by soluble beta-amyloid(1-40) through divergent NMDAR-dependent signalling pathways[END_REF] in order to standardize the identification of pre-and postsynaptic clusters. For each presynaptic protein, the average fluorescence intensity value was obtained and individually plotted.

For postsynaptic proteins, whenever multiple postsynaptic clusters were detected, they were measured separately and the fluorescence intensity value was averaged. For the determination of the cluster area, the cumulative area of GluR4 or Homer1b clusters was determined by measuring the area of individual clusters (after thresholding) and summing the area of all clusters corresponding to a given presynaptic terminal.

Background fluorescence was measured locally and subtracted. Mann-Whitney nonparametric tests were used to compare wt and mutSOD1 MNs. For chemogenetic experiments, one-or two-way ANOVA, followed by Tukey HSD tests were used to test the effects of infection vs. genotype. The significance level was set at p<0.05.

Supplemental Material

Figure S1 shows how EPSP is growing with the stimulation intensity and that EPSP amplitude measurements are not distorted by disynaptic inhibition. It also shows that the level of presynaptic inhibition is similar in wtSOD1 and mutSOD1 mice. Figure S2 shows that the presynaptic elements are not affected by the mutation. Figure S3 shows that dendrites of mutSOD1 MNs become vacuolized and that the size of the vacuoles increase with disease progression. Figure S4 shows a representative image of a spinal cord after intraspinal injection of an AAV, illustrating the high rate of infection that can be achieved at the site of injection. In addition, the figure shows that increasing MN activity via actPSAM restores the quantity of GluR4 and Homer1b proteins at VGluT1+ synapses. It also includes data demonstrating the specificity of the GluR4 antibody. increased by 14 ± 5 % on average, N=6; paired t-test p=0.007. In mutant animals, the temporal facilitation. Note that it was not possible to isolate Ib inhibition or group II inhibition from Ia excitation, but these inhibitions are di-synaptic and thus cannot occur faster than Renshaw inhibition. Therefore, the peak measurement of the monosynaptic EPSP is not contaminated by any inhibition. D-H. Presynaptic inhibition in mouse MNs.

We have investigated whether Ia terminals from the triceps surae (TS) are subject to presynaptic inhibition following a conditioning stimulation of group I afferents from Posterior Biceps (PB), i.e. the most classical pathway for presynaptic inhibition [START_REF] Rudomin | Presynaptic inhibition in the vertebrate spinal cord revisited[END_REF]

Figure

  Figure 2A-B). Density of VGluT1+ terminals in these three compartments was

  ). Spinal cord sections were thereafter washed 3×45 min in PBS + 0.1 % Triton, incubated for 2 h at room temperature with the opportune secondary antibody combination (all diluted at 1:500: Donkey anti-Goat Alexa-405, Abcam ab175664; Donkey anti-Guinea pig CF-633, Biotium 20171; Donkey anti-Rabbit Alexa-488, Life Technologies A-21206; Donkey anti-Rabbit Alexa-647, Life Technologies A-31573; Donkey anti-Mouse Alexa-488, Alexa-568 and Alexa-647, Life Technologies A-21202, A-10037 and A-31571, Donkey anti-chicken Alexa-488, Jackson Immunoresearch 703-545-155) and mounted in ProLong Gold Antifade (ThermoFisher). Fluorescent images were acquired with a Zeiss 710 confocal microscope, with a 63× oil-immersion objective (NA 1.40) and 2.5 added optical zoom; optical section thickness was set at 200 nm; images were acquired at 12 bit depth. Acquisition parameters were adjusted so as to maintain fluorescence intensity from target structures below saturation and above zero values; imaging parameters were kept constant across replicates and genotypes. α-MNs were identified by their VAChT-positive cytoplasm surrounded by bright VAChT-positive C-boutons and displaying a VAChT-negative area at the centre of the cell corresponding to the nucleus. This strategy allows the exclusion of γ-MNs, which are devoid of C-boutons. The combined use of cytoplasmic VAChT immunostaining, and C-boutons lining, together with the identification of the nuclear halo, allows the definition of the cellular contour and the establishment of the spatial orientation of synaptic structures. The post-synaptic structures located between the presynaptic terminal and the cytoplasm of the cell are considered belonging to the identified MN, whereas those located in proximity of the presynaptic terminal but away from the cytoplasm are considered belonging to other structures or cells and are

Figure

  Figure S5 explains the method used for MLF stimulation and shows examples of atypical

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Alterations in the postsynaptic structure of VGluT1+ synapses onto motoneurons.

Figure 3 .

 3 Figure 3. Postsynaptic activation of the PKA pathway enhances Ia EPSPs and restores synaptic structures.

  . D. The left panel shows the unconditioned Ia monosynaptic EPSP on a wtSOD1 TS motoneuron while the right panel shows the same Ia EPSP conditioned by group I afferents from the PB. The conditioning stimulation (red dots) is a train of 3 electrical stimulation at 200 Hz that precedes the stimulation of the TS by 23 ms. The conditioning stimulation reduces the test EPSP. We repeated this experiment in 14 wtSOD1 MNs (F) where the conditioning stimulation elicited an average reduction of the test Ia EPSP of 6 ± 8 %; paired t-test p=0.004. Asterisk points to the example in D. E. Presynaptic inhibition in a mutSOD1 MNs. Same arrangement as in D. We repeated this experiment in 13 mutSOD1 MNs (G) on which the conditioning stimulation elicited an average reduction of the test Ia EPSP of 5 ± 6 %; paired t-test p=0.006. Asterisk points to the example in E. H. The effect of the conditioning stimulation on the size of Ia EPSPs was the same in wtSOD1 and mutSOD1 MNs (t-test p=0.50) indicating that the strength of presynaptic inhibition is similar in the two phenotypes. Experiments were carried out in 4 wtSOD1 mice and 6 mutSOD1 mice.the last stimulation. E. Example of recording where only disynaptic excitation (unfilled arrowhead) was visible. In this cell, the stimulation of descending fibers did not evoke monosynaptic EPSP indicating that they did not project directly to this motoneuron. The disynaptic EPSP started to appear in response to the second stimulation (unfilled arrowhead) and grew upon stimulation repetition indicating temporal summation of the excitatory interneurons activated by the descending fibers. The latency of disynaptic response is about 1 ms longer than for the monosynaptic response. Note that since this motoneuron did not display any monosynaptic component, it was not included in our analysis.

Acknowledgements

We would like to thank Professor Philippe Ascher and professor Tobias Böckers for helpful discussions on the data and Professor Albert Ludolph and Professor George Mentis for critically reviewing an early version of the manuscript. This work has benefited from the support and expertise of the animal facility and microscopy platform of BioMedTech Facilities at Université de Paris (INSERM US36/CNRS UMS2009). We thank Rebecca D. Imhoff-Manuel for expert mouse breeding and genotyping in Paris, Sabrina Petri for processing of Gria4 -/-samples at Columbia, Professor Ignatius for the use of the histology lab equipment, and Professor Frank Kirchhoff for the use of the Zeiss 710 confocal microscope. This work was financed by NIH-NINDS R01NS077863, TARGET-ALS, the Thierry Latran Foundation project "SPIN-ALS" and project "TRiALS", Association pour la Recherche sur la SLA et autres maladies du motoneurone (ARSLA), the Association Française contre les Myopathies (AFM) project "HYPERTOXIC", Radala Foundation for ALS Research, and Programme Hubert Curien "Polonium" for scientific exchanges. C.M. and N.Del. were supported by doctoral grants from Paris Descartes University. F.R. is supported by the Ulm University-Medical School Baustein program, by the Synapsis Foundation, by the Deutsche Forschungsgemeinschaft (DFG) as part of the SFB1149 and with the individual grant no. 431995586 (RO-5004/8-1), by the Cellular and Molecular Mechanisms in Aging (CEMMA) Research Training Group and by BMBF (FKZ 01EW1705A, as member of the ERANET-NEURON consortium "MICRONET"). D.B. is supported by the Cellular and Molecular Mechanisms in Aging (CEMMA) Research Training Group. B.C. and N.O.A. are supported by the International Graduate School in Molecular Medicine at Ulm University. L.T. is supported by the Chinese Science Council.

The authors declare no conflict of interest.

experiments were conducted in four MNs: Ia EPSPs coming from either of the two TS branches were tested in two MNs, whereas only one source of Ia excitation was tested in the remaining two MNs (see different symbols and line styles in C 2 ). The difference persists if we consider only EPSPs elicited by stimulation of the LG nerve in each MN.

The EPSP size increased by 14 ± 6 % on average, N=4; paired t-test p=0.02. D.

Quantification of the change in the paired-pulse ratio after cAMPS-Sp injection. Same organization as in C. (D 1 ) In wtSOD1 animals (4 mice), cAMPS-Sp did not significantly change the paired-pulse ratio (average difference 1 ± 2 %; N=6, paired t-test p=0.23), while it caused an increased by 5 ± 5 %, N=6 in mutSOD1 animals (D 2 , 2 mice, pairedpulse ratio before: 1.00 ± 0.06, N=6 vs. after 1.05 ± 0.08, N=6; paired t-test p=0.038). As before, the difference persists if we consider only EPSPs elicited by the LG nerve in each MN. cAMPS-Sp increased the paired-pulse ratio from 1.00 ± 0.07 to 1.05 ± 0.10, N=4; paired t-test p=0.048. E 1-3 . Experimental design for DREADD experiments. F 1-3 . MNs expressing D(Gs) and immunostained for VGluT1 and GluR4 under either vehicle (F 1 ), acute CNO (F 2 ) or chronic CNO treatment (F 3 ). The dotted line represents the approximate outline of the MNs. MNs are identified by VAChT staining. Scale bars: 20 µm (inset: 1 µm). G 1-3 . Same organization as F, but immunostained for VGluT1 and Homer1b. H. Significant increase in GluR4 cluster area in VGlut1 synapses of D(Gs)+ MNs (magenta) compared to contralateral D(Gs)-MNs (grey) upon acute (4.3 ± 2.6 μm², N=43 vs. 2.3 ± 1.7 μm², N=27; from 3 mice; two-way ANOVA followed by Tukey HSD p=0.001) and chronic (3.9 ± 2.5 μm², N=144 vs. 2.3 ± 1.5 μm², N=128; from 4 mice; Tukey HSD p=0.001) CNO treatment, but not in vehicle-treated mice (2.1 ± 1.6 μm², N=119 vs. 2.3 ± 1.6 μm², N=111; from 3 mice; Tukey HSD p=0.9). I. Significant increase in Homer1b cluster area in D(Gs)+ MNs compared to contralateral D(Gs)-MNs upon acute (2.4 ± 1.6 μm², N=109 vs. 1.8 ± 1.2 μm² N=83; two-way ANOVA followed by Tukey HSD p=0.0147) and chronic (2.4 ± 1.5, N=88 vs. 1.6 ± 1.4 μm², N=69; from 4 mice; Tukey HSD p=0.0019) CNO treatment, but not in vehicle-treated mice (1.4 ± 1.2 μm² in D(Gs)+ MNs, N=51 vs. 1.3 ± 1.6 μm² in D(Gs)-MNs, N=117; from 3 mice; Tukey HSD p=0.9). (2.10 ± 1.41 μm²; N=106; ANOVA followed by Tukey HSD p=0.003). Notably, inhPSAM by itself did not have any effect on Homer1b cluster area (2.23 ± 1.27 μm²; N=52; Tukey HSD p=0.9) and did not diminish the effect of D(Gs) activation (3.46 ± 2.12 μm²; N=91; Tukey HSD p=0.68). This experiment was conducted on 5 mutSOD1/ChAT-cre mice. The burden of p62 inclusion in D(Gs)+ MNs was not modified by vehicle (90.6 ± 91.7 % of contralateral uninfected) or acute CNO treatment (116.2 ± 84.8 % of uninfected), but was reduced compared to contralateral non-infected MNs upon chronic CNO treatment (42.9 ± 50.6 % of contralateral; Tukey HSD p<0.01). G. LC3A immunostaining intensity in MNs was unaltered in D(Gs)+ neurons compared to contralateral non-infected MNs upon vehicle (104.7 ± 22.2 % of uninfected) whereas acute D(Gs) activation (84.8 ± 18.6 % of uninfected; Tukey HSD p=0.001), or chronic D(Gs) activation (84.8 ± 22.9 % of uninfected; Tukey HSD p=0.001) resulted in the significant decrease in LC3A intensity. Scale bars: 20 µm. D. Reduction of MN firing by inhPSAM did not significantly affect the misfSOD1 burden (112.9 ± 24.5 % of control non-infected; one-way ANOVA followed by Tukey HSD p=0.087). On the other hand, D(Gs) activation significantly reduced misfSOD1 burden (74.2 ± 29.0 % of control uninfected; Tukey HSD p=0.001). However, reduction of MN firing combined with D(Gs) activation abolished the beneficial effect of the D(Gs) alone (95.9 ± 31.6 % of uninfected; Tukey HSD p=0.001). E. The accumulation of LC3A in MNs was significantly increased upon reduced firing by inhPSAM (116.7 ± 18.3 % of control; one-way ANOVA followed by Tukey HSD p=0.001) and significantly decreased by D(Gs) activation (90.1 ± 16.8 % of control non-infected; Tukey HSD p=0.001); the reduction of MN firing significantly decreased the effect of concomitant D(Gs) activation (103.8 ± 18.0 % of control; Tukey HSD p=0.001). Data from 5 mutSOD1/ChAT-cre animals per group.