Non-canonical adrenergic neuromodulation of motoneuron intrinsic excitability through ß-receptors in wild-type and ALS mice.

Altered neuronal excitability and synaptic inputs to motoneurons are part of the pathophysiology of Amyotrophic Lateral Sclerosis. The cAMP/PKA pathway regulates both of them but therapeutic interventions at this level are limited by the lack of knowledge about suitable pharmacological entry points. Here we used transcriptomics on microdissected and in situ motoneurons to reveal the modulation of PKA-coupled receptorome in SOD1(G93A) ALS mice, vs WT, demonstrating the dysregulation of multiple PKA-coupled GPCRs, in particular on vulnerable MNs, and the relative sparing of ß-adrenergic receptors. In vivo MN electrophysiology showed that ß2/ß3 agonists acutely increase excitability, in particular the input/output relationship, demonstrating a non-canonical adrenergic neuromodulation mediated by ß2/ß3 receptors both in WT and SOD1 mice. The excitability increase corresponds to the upregulation of immediate-early gene expression and dysregulation of ion channels transcriptome. However the ß2/ß3 neuromodulation is submitted to a strong homeostasis, since a ten days delivery of ß2/ß3 agonists results in an abolition of the excitability increase. The homeostatic response is largely caused by a substantial downregulation of PKA-coupled GPCRs in MNs from WT and SOD1 mice. Thus, ß-adrenergic receptors are physiologically involved in the regulation of MN excitability and transcriptomics, but, intriguingly, a strong homeostatic response is triggered upon chronic pharmacologic intervention.

Spinal microcircuits go through multiphasic homeostatic compensations in a mouse model of motoneuron degeneration.

In neurological conditions affecting the brain, early-stage neural circuit adaption is key for long-term preservation of normal behaviour. We tested if motoneurons and respective microcircuits also adapt in the initial stages of disease progression in a mouse model of progressive motoneuron degeneration. Using a combination of in vitro and in vivo electrophysiology and super-resolution microscopy, we found that, preceding muscle denervation and motoneuron death, recurrent inhibition mediated by Renshaw cells is reduced in half due to impaired quantal size associated with decreased glycine receptor density. Additionally, higher probability of release from proprioceptive Ia terminals leads to increased monosynaptic excitation to motoneurons. Surprisingly, the initial impairment in recurrent inhibition is not a widespread feature of inhibitory spinal circuits, such as group I inhibitory afferents, and is compensated at later stages of disease progression. We reveal that in disease conditions, spinal microcircuits undergo specific multiphasic homeostatic compensations to preserve force output.

Diversity of Mammalian Motoneurons and Motor Units.

Although they share the common function of controlling muscle fiber contraction, spinal motoneurons display a remarkable diversity. Alpha-motoneurons are the "final common pathway", which relay all the information from spinal and supraspinal centers and allow the organism to interact with the outside world by controlling the contraction of muscle fibers in the muscles. On the other hand, gamma-motoneurons are specialized motoneurons that do not generate force and instead specifically innervate muscle fibers inside muscle spindles, which are proprioceptive organs embedded in the muscles. Beta-motoneurons are hybrid motoneurons that innervate both extrafusal and intrafusal muscle fibers. Even among alpha-motoneurons, there exists an exquisite diversity in terms of motoneuron electrical and molecular properties, physiological and structural properties of their neuromuscular junctions, and molecular and contractile properties of the innervated muscle fibers. This diversity, across species, across muscles, and across muscle fibers in a given muscle, underlie the vast repertoire of movements that one individual can perform.

From Physiological Properties to Selective Vulnerability of Motor Units in Amyotrophic Lateral Sclerosis.

Spinal alpha-motoneurons are classified in several types depending on the contractile properties of the innervated muscle fibers. This diversity is further displayed in different levels of vulnerability of distinct motor units to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). We summarize recent data suggesting that, contrary to the excitotoxicity hypothesis, the most vulnerable motor units are hypoexcitable and experience a reduction in their firing prior to symptoms onset in ALS. We suggest that a dysregulation of activity-dependent transcriptional programs in these motoneurons alter crucial cellular functions such as mitochondrial biogenesis, autophagy, axonal sprouting capability and re-innervation of neuromuscular junctions.

Synaptic disruption and CREB-regulated transcription are restored by K+ channel blockers in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity-related pathological features of iPSC-derived C9orf72-mutant motoneurons (MN). We found that human ALSC9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic "malactivation" of the transcription factor CREB. A similar phenotype was also found in TBK1-mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS-related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activity-dependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression.

Is there hope that transpinal direct current stimulation corrects motoneuron excitability and provides neuroprotection in amyotrophic lateral sclerosis?

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of largely unknown pathophysiology, characterized by the progressive loss of motoneurons (MNs). We review data showing that in presymptomatic ALS mice, MNs display reduced intrinsic excitability and impaired level of excitatory inputs. The loss of repetitive firing specifically affects the large MNs innervating fast contracting muscle fibers, which are the most vulnerable MNs in ALS. Interventions that aimed at restoring either the intrinsic excitability or the synaptic excitation result in a decrease of disease markers in MNs and delayed neuromuscular junction denervation. We then focus on trans-spinal direct current stimulation (tsDCS), a noninvasive tool, since it modulates the activity of spinal neurons and networks. Effects of tsDCS depend on the polarity of applied current. Recent work shows that anodal tsDCS induces long-lasting enhancement of MN excitability and synaptic excitation of spinal MNs. Moreover, we show preliminary results indicating that anodal tsDCS enhances the excitatory synaptic inputs to MNs in ALS mice. In conclusion, we suggest that chronic application of anodal tsDCS might be useful as a complementary method in the management of ALS patients.

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

Excessive excitation is hypothesized to cause motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS), but actual proof of hyperexcitation in vivo is missing, and trials based on this concept have failed. We demonstrate, by in vivo single-MN electrophysiology, that, contrary to expectations, excitatory responses evoked by sensory and brainstem inputs are reduced in MNs of presymptomatic mutSOD1 mice. This impairment correlates with disrupted postsynaptic clustering of Homer1b, Shank, and AMPAR subunits. Synaptic restoration can be achieved by activation of the cAMP/PKA pathway, by either intracellular injection of cAMP or DREADD-Gs stimulation. Furthermore, we reveal, through independent control of signaling and excitability allowed by multiplexed DREADD/PSAM chemogenetics, that PKA-induced restoration of synapses triggers an excitation-dependent decrease in misfolded SOD1 burden and autophagy overload. In turn, increased MN excitability contributes to restoring synaptic structures. Thus, the decrease of excitation to MN is an early but reversible event in ALS. Failure of the postsynaptic site, rather than hyperexcitation, drives disease pathobiochemistry.

Molecular and electrophysiological properties of mouse motoneuron and motor unit subtypes.

The field of motoneuron and motor unit physiology in mammals has deeply evolved the last decade thanks to the parallel development of mouse genetics and transcriptomic analysis and of in vivo mouse preparations that allow intracellular electrophysiological recordings of motoneurons. We review the efforts made to investigate the electrophysiological properties of the different functional subtypes of mouse motoneurons, to decipher the mosaic of molecular markers specifically expressed in each subtype, and to elucidate which of those factors drive the identity of motoneurons.

Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomotion.

Spinal motoneurons are endowed with nonlinear spiking behaviors manifested by a spike acceleration whose functional significance remains uncertain. Here, we show in rodent lumbar motoneurons that these nonlinear spiking properties do not rely only on activation of dendritic nifedipine-sensitive L-type Ca2+ channels, as assumed for decades, but also on the slow inactivation of a nifedipine-sensitive K+ current mediated by Kv1.2 channels that are highly expressed in axon initial segments. Specifically, the pharmacological and computational inhibition of Kv1.2 channels occluded the spike acceleration of rhythmically active motoneurons and the correlated slow buildup of rhythmic motor output recorded at the onset of locomotor-like activity. This study demonstrates that slow inactivation of Kv1.2 channels provides a potent gain control mechanism in mammalian spinal motoneurons and has a behavioral role in enhancing locomotor drive during the transition from immobility to steady-state locomotion.

Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS.

Hyperexcitability has been suggested to contribute to motoneuron degeneration in amyotrophic lateral sclerosis (ALS). If this is so, and given that the physiological type of a motor unit determines the relative susceptibility of its motoneuron in ALS, then one would expect the most vulnerable motoneurons to display the strongest hyperexcitability prior to their degeneration, whereas the less vulnerable should display a moderate hyperexcitability, if any. We tested this hypothesis in vivo in two unrelated ALS mouse models by correlating the electrical properties of motoneurons with their physiological types, identified based on their motor unit contractile properties. We found that, far from being hyperexcitable, the most vulnerable motoneurons become unable to fire repetitively despite the fact that their neuromuscular junctions were still functional. Disease markers confirm that this loss of function is an early sign of degeneration. Our results indicate that intrinsic hyperexcitability is unlikely to be the cause of motoneuron degeneration.

Force encoding in muscle spindles during stretch of passive muscle.

Muscle spindle proprioceptive receptors play a primary role in encoding the effects of external mechanical perturbations to the body. During externally-imposed stretches of passive, i.e. electrically-quiescent, muscles, the instantaneous firing rates (IFRs) of muscle spindles are associated with characteristics of stretch such as length and velocity. However, even in passive muscle, there are history-dependent transients of muscle spindle firing that are not uniquely related to muscle length and velocity, nor reproduced by current muscle spindle models. These include acceleration-dependent initial bursts, increased dynamic response to stretch velocity if a muscle has been isometric, and rate relaxation, i.e., a decrease in tonic IFR when a muscle is held at a constant length after being stretched. We collected muscle spindle spike trains across a variety of muscle stretch kinematic conditions, including systematic changes in peak length, velocity, and acceleration. We demonstrate that muscle spindle primary afferents in passive muscle fire in direct relationship to muscle force-related variables, rather than length-related variables. Linear combinations of whole muscle-tendon force and the first time derivative of force (dF/dt) predict the entire time course of transient IFRs in muscle spindle Ia afferents during stretch (i.e., lengthening) of passive muscle, including the initial burst, the dynamic response to lengthening, and rate relaxation following lengthening. Similar to acceleration scaling found previously in postural responses to perturbations, initial burst amplitude scaled equally well to initial stretch acceleration or dF/dt, though later transients were only described by dF/dt. The transient increase in dF/dt at the onset of lengthening reflects muscle short-range stiffness due to cross-bridge dynamics. Our work demonstrates a critical role of muscle cross-bridge dynamics in history-dependent muscle spindle IFRs in passive muscle lengthening conditions relevant to the detection and sensorimotor response to mechanical perturbations to the body, and to previously-described history-dependence in perception of limb position.

Potassium currents dynamically set the recruitment and firing properties of F-type motoneurons in neonatal mice.

In neonatal mice, fast- and slow-type motoneurons display different patterns of discharge. In response to a long liminal current pulse, the discharge is delayed up to several seconds in fast-type motoneurons and their firing frequency accelerates. In contrast, slow-type motoneurons discharge immediately, and their firing frequency decreases at the beginning of the pulse. Here, we identify the ionic currents that underlie the delayed firing of fast-type motoneurons. We find that the firing delay is caused by a combination of an A-like potassium current that transiently suppresses firing on a short time scale and a slowly-inactivating potassium current that inhibits the discharge over a much longer time scale. We then show how these intrinsic currents dynamically shape the discharge threshold and the frequency-input function of fast-type motoneurons. These currents contribute to the orderly recruitment of motoneurons in neonates and might play a role in the postnatal maturation of motor units.

MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance.

The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain (cysteine-rich domain [CRD]). Dysfunction of MuSK CRD in patients has been recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSKΔCRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSKΔCRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.

Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS) the large motoneurons that innervate the fast-contracting muscle fibers (F-type motoneurons) are vulnerable and degenerate in adulthood. In contrast, the small motoneurons that innervate the slow-contracting fibers (S-type motoneurons) are resistant and do not degenerate. Intrinsic hyperexcitability of F-type motoneurons during early postnatal development has long been hypothesized to contribute to neural degeneration in the adult. Here, we performed a critical test of this hypothesis by recording from identified F- and S-type motoneurons in the superoxide dismutase-1 mutant G93A (mSOD1), a mouse model of ALS at a neonatal age when early pathophysiological changes are observed. Contrary to the standard hypothesis, excitability of F-type motoneurons was unchanged in the mutant mice. Surprisingly, the S-type motoneurons of mSDO1 mice did display intrinsic hyperexcitability (lower rheobase, hyperpolarized spiking threshold). As S-type motoneurons are resistant in ALS, we conclude that early intrinsic hyperexcitability does not contribute to motoneuron degeneration.

The dendritic location of the L-type current and its deactivation by the somatic AHP current both contribute to firing bistability in motoneurons.

Spinal motoneurons may display a variety of firing patterns including bistability between repetitive firing and quiescence and, more rarely, bistability between two firing states of different frequencies. It was suggested in the past that firing bistability required that the persistent L-type calcium current be segregated in distal dendrites, far away from the spike generating currents. However, this is not supported by more recent data. Using a two compartment model of motoneuron, we show that the different firing patterns may also result from the competition between the more proximal dendritic component of the dendritic L-type conductance and the calcium sensitive potassium conductance responsible for afterhypolarization (AHP). Further emphasizing this point, firing bistability may be also achieved when the L-type current is put in the somatic compartment. However, this requires that the calcium-sensitive potassium conductance be triggered solely by the high threshold calcium currents activated during spikes and not by calcium influx through the L-type current. This prediction was validated by dynamic clamp experiments in vivo in lumbar motoneurons of deeply anesthetized cats in which an artificial L-type current was added at the soma. Altogether, our results suggest that the dynamical interaction between the L-type and afterhyperpolarization currents is as fundamental as the segregation of the calcium L-type current in dendrites for controlling the discharge of motoneurons.

Adult spinal motoneurones are not hyperexcitable in a mouse model of inherited amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS), an adult onset disease in which there is progressive degeneration of motoneurones, it has been suggested that an intrinsic hyperexcitability of motoneurones (i.e. an increase in their firing rates), contributes to excitotoxicity and to disease onset. Here we show that there is no such intrinsic hyperexcitability in spinal motoneurones. Our studies were carried out in an adult mouse model of ALS with a mutated form of superoxide dismutase 1 around the time of the first muscle fibre denervations. We showed that the recruitment current, the voltage threshold for spiking and the frequency-intensity gain in the primary range are all unchanged in most spinal motoneurones, despite an increased input conductance. On its own, increased input conductance would decrease excitability, but the homeostasis for excitability is maintained due to an upregulation of a depolarizing current that is activated just below the spiking threshold. However, this homeostasis failed in a substantial fraction of motoneurones, which became hypoexcitable and unable to produce sustained firing in response to ramps of current. We found similar results both in lumbar motoneurones recorded in anaesthetized mice, and in sacrocaudal motoneurones recorded in vitro, indicating that the lack of hyperexcitability is not caused by anaesthetics. Our results suggest that, if excitotoxicity is indeed a mechanism leading to degeneration in ALS, it is not caused by the intrinsic electrical properties of motoneurones but by extrinsic factors such as excessive synaptic excitation.

Alpha, beta and gamma motoneurons: functional diversity in the motor system's final pathway.

Since their discovery in the late 19th century our conception of motoneurons has steadily evolved. Motoneurons share the same general function: they drive the contraction of muscle fibers and are the final common pathway, i.e., the seat of convergence of all the central and peripheral pathways involved in motricity. However, motoneurons innervate different types of muscular targets. Ordinary muscle fibers are subdivided into three main subtypes according to their structural and mechanical properties. Intrafusal muscle fibers located within spindles can elicit either a dynamic, or a static, action on the spindle sensory endings. No less than seven categories of motoneurons have thereby been identified on the basis of their innervation pattern. This functional diversity has hinted at a similar diversity in the inputs each motoneuron receives, as well as in the electrical, or cellular, properties of the motoneurons that match the properties of their muscle targets. The notion of the diverse properties of motoneurons has been well established by the work of many prominent neuroscientists. But in today's scientific literature, it tends to fade and motoneurons are often thought of as a homogenous group, which develop from a given population of precursor cells, and which express a common set of molecules. We first present here the historical milestones that led to the recognition of the functional diversity of motoneurons. We then review how the intrinsic electrical properties of motoneurons are precisely tuned in each category of motoneurons in order to produce an output that is adapted to the contractile properties of their specific targets.

Mixed mode oscillations in mouse spinal motoneurons arise from a low excitability state.

We explain the mechanism that elicits the mixed mode oscillations (MMOs) and the subprimary firing range that we recently discovered in mouse spinal motoneurons. In this firing regime, high-frequency subthreshold oscillations appear a few millivolts below the spike voltage threshold and precede the firing of a full blown spike. By combining intracellular recordings in vivo (including dynamic clamp experiments) in mouse spinal motoneurons and modeling, we show that the subthreshold oscillations are due to the spike currents and that MMOs appear each time the membrane is in a low excitability state. Slow kinetic processes largely contribute to this low excitability. The clockwise hysteresis in the I-F relationship, frequently observed in mouse motoneurons, is mainly due to a substantial slow inactivation of the sodium current. As a consequence, less sodium current is available for spiking. This explains why a large subprimary range with numerous oscillations is present in motoneurons displaying a clockwise hysteresis. In motoneurons whose I-F curve exhibits a counterclockwise hysteresis, it is likely that the slow inactivation operates on a shorter time scale and is substantially reduced by the de-inactivating effect of the afterhyperpolarization (AHP) current, thus resulting in a more excitable state. This accounts for the short subprimary firing range with only a few MMOs seen in these motoneurons. Our study reveals a new role for the AHP current that sets the membrane excitability level by counteracting the slow inactivation of the sodium current and allows or precludes the appearance of MMOs.