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.

Spinal motoneurons respond aberrantly to serotonin in a rabbit model of cerebral palsy.

Cerebral palsy (CP) is caused by a variety of factors that damage the developing central nervous system. Impaired motor control, including muscle stiffness and spasticity, is the hallmark of spastic CP. Rabbits that experience hypoxic-ischaemic (HI) injury in utero (at 70%-83% gestation) are born with muscle stiffness, hyperreflexia and, as recently discovered, increased 5-HT in the spinal cord. To determine whether serotonergic modulation of spinal motoneurons (MNs) contributes to motor deficits, we performed ex vivo whole cell patch clamp in neonatal rabbit spinal cord slices at postnatal day (P) 0-5. HI MNs responded to the application of α-methyl 5-HT (a 5-HT1 /5-HT2 receptor agonist) and citalopram (a selective 5-HT reuptake inhibitor) with increased amplitude and hyperpolarization of persistent inward currents and hyperpolarized threshold voltage for action potentials, whereas control MNs did not exhibit any of these responses. Although 5-HT similarly modulated MN properties of HI motor-unaffected and motor-affected kits, it affected sag/hyperpolarization-activated cation current (Ih ) and spike frequency adaptation only in HI motor-affected MNs. To further explore the differential sensitivity of MNs to 5-HT, we performed immunostaining for inhibitory 5-HT1A receptors in lumbar spinal MNs at P5. Fewer HI MNs expressed the 5-HT1A receptor compared to age-matched control MNs. This suggests that HI MNs may lack a normal mechanism of central fatigue, mediated by 5-HT1A receptors. Altered expression of other 5-HT receptors (including 5-HT2 ) likely also contributes to the robust increase in HI MN excitability. In summary, by directly exciting MNs, the increased concentration of spinal 5-HT in HI-affected rabbits can cause MN hyperexcitability, muscle stiffness and spasticity characteristic of CP. Therapeutic strategies that target serotonergic neuromodulation may be beneficial to individuals with CP. KEY POINTS: We used whole cell patch clamp electrophysiology to test the responsivity of spinal motoneurons (MNs) from neonatal control and hypoxia-ischaemia (HI) rabbits to 5-HT, which is elevated in the spinal cord after prenatal HI injury. HI rabbit MNs showed a more robust excitatory response to 5-HT than control rabbit MNs, including hyperpolarization of the persistent inward current and threshold voltage for action potentials. Although most MN properties of HI motor-unaffected and motor-affected kits responded similarly to 5-HT, 5-HT caused larger sag/hyperpolarization-activated cation current (Ih ) and altered repetitive firing patterns only in HI motor-affected MNs. Immunostaining revealed that fewer lumbar MNs expressed inhibitory 5-HT1A receptors in HI rabbits compared to controls, which could account for the more robust excitatory response of HI MNs to 5-HT. These results suggest that elevated 5-HT after prenatal HI injury could trigger a cascade of events that lead to muscle stiffness and altered motor unit development.

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

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into three subgroups based on location: (1) interneurons within 100 µm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1 mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.

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.

Conservation of locomotion-induced oculomotor activity through evolution in mammals.

Efference copies are neural replicas of motor outputs used to anticipate the sensory consequences of a self-generated motor action or to coordinate neural networks involved in distinct motor behaviors.1 An established example of this motor-to-motor coupling is the efference copy of the propulsive motor command, which supplements classical visuo-vestibular reflexes to ensure gaze stabilization during amphibian larval locomotion.2 Such feedforward replica of spinal pattern-generating circuits produces a spino-extraocular motor coupled activity that evokes eye movements, spatiotemporally coordinated to tail undulation independently of any sensory signal.3,4 Exploiting the developmental stages of the frog,1 studies in metamorphing Xenopus demonstrated the persistence of this spino-extraocular motor command in adults and its developmental adaptation to tetrapodal locomotion.5,6 Here, we demonstrate for the first time the existence of a comparable locomotor-to-ocular motor coupling in the mouse. In neonates, ex vivo nerve recordings of brainstem-spinal cord preparations reveal a spino-extraocular motor coupled activity similar to the one described in Xenopus. In adult mice, trans-synaptic rabies virus injections in lateral rectus eye muscle label cervical spinal cord neurons closely connected to abducens motor neurons. Finally, treadmill-elicited locomotion in decerebrated preparations7 evokes rhythmic eye movements in synchrony with the limb gait pattern. Overall, our data are evidence for the conservation of locomotor-induced eye movements in vertebrate lineages. Thus, in mammals as in amphibians, CPG-efference copy feedforward signals might interact with sensory feedback to ensure efficient gaze control during locomotion.

Time Course of Alterations in Adult Spinal Motoneuron Properties in the SOD1(G93A) Mouse Model of ALS.

Although amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, motoneuron electrical properties are already altered during embryonic development. Motoneurons must therefore exhibit a remarkable capacity for homeostatic regulation to maintain a normal motor output for most of the life of the patient. In the present article, we demonstrate how maintaining homeostasis could come at a very high cost. We studied the excitability of spinal motoneurons from young adult SOD1(G93A) mice to end-stage. Initially, homeostasis is highly successful in maintaining their overall excitability. This initial success, however, is achieved by pushing some cells far above the normal range of passive and active conductances. As the disease progresses, both passive and active conductances shrink below normal values in the surviving cells. This shrinkage may thus promote survival, implying the previously large values contribute to degeneration. These results support the hypothesis that motoneuronal homeostasis may be "hypervigilant" in ALS and a source of accumulating stress.

Suboptimal Discontinuous Current-Clamp Switching Rates Lead to Deceptive Mouse Neuronal Firing.

Intracellular recordings using sharp microelectrodes often rely on a technique called discontinuous current-clamp (DCC) to accurately record the membrane potential while injecting current through the same microelectrode. It is well known that a poor choice of DCC switching rate can lead to underestimation or overestimation of the cell potential; however, its effect on the cell firing is rarely discussed. Here, we show that suboptimal switching rates lead to an overestimation of cell excitability. We performed intracellular recordings of mouse spinal motoneurons and recorded their firing in response to pulses and ramps of current in Bridge and DCC mode at various switching rates. We demonstrate that using an incorrect (too low) DCC frequency leads not only to an underestimation of the input resistance, but also, paradoxically, to an artificial overestimation of the firing of these cells: neurons fire at lower current, and at higher frequencies than at higher DCC rates, or than the same neuron recorded in Bridge mode. These effects are dependent on the membrane time constant of the recorded cell, and special care needs to be taken in large cells with very short time constants. Our work highlights the importance of choosing an appropriate DCC switching rate to obtain not only accurate membrane potential readings but also an accurate representation of the firing of the cell.

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.

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis.

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

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.

Scaling of Motor Output, From Mouse to Humans.

Appropriate scaling of motor output from mouse to humans is essential. The motoneurons that generate all motor output are, however, very different in rodents compared with humans, being smaller and much more excitable. In contrast, feline motoneurons are more similar to those in humans. These scaling differences need to be taken into account for the use of rodents for translational studies of motor output.

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.

Liver X Receptor exerts a protective effect against the oxidative stress in the peripheral nerve.

Reactive oxygen species (ROS) modify proteins and lipids leading to deleterious outcomes. Thus, maintaining their homeostatic levels is vital. This study highlights the endogenous role of LXRs (LXRα and ß) in the regulation of oxidative stress in peripheral nerves. We report that the genetic ablation of both LXR isoforms in mice (LXRdKO) provokes significant locomotor defects correlated with enhanced anion superoxide production, lipid oxidization and protein carbonylation in the sciatic nerves despite the activation of Nrf2-dependant antioxidant response. Interestingly, the reactive oxygen species scavenger N-acetylcysteine counteracts behavioral, electrophysical, ultrastructural and biochemical alterations in LXRdKO mice. Furthermore, Schwann cells in culture pretreated with LXR agonist, TO901317, exhibit improved defenses against oxidative stress generated by tert-butyl hydroperoxide, implying that LXRs play an important role in maintaining the redox homeostasis in the peripheral nervous system. Thus, LXR activation could be a promising strategy to protect from alteration of peripheral myelin resulting from a disturbance of redox homeostasis in Schwann cell.

Decerebrate mouse model for studies of the spinal cord circuits.

The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons. The protocol also describes an example application for the protocol: the evocation of spontaneous and actively driven stepping, including optimization of these behaviors in decerebrate mice. The time taken to prepare the animal and perform a decerebration takes ∼2 h, and the mice are viable for up to 3-8 h, which is ample time to perform most short-term procedures. These protocols can be modified for those interested in cardiovascular or respiratory function in addition to motor function and can be performed by trainees with some previous experience in animal surgery.

PICs in motoneurons do not scale with the size of the animal: a possible mechanism for faster speed of muscle contraction in smaller species.

The majority of studies on the electrical properties of neurons are carried out in rodents, and in particular in mice. However, the minute size of this animal compared with humans potentially limits the relevance of the resulting insights. To be able to extrapolate results obtained in a small animal such as a rodent, one needs to have proper knowledge of the rules governing how electrical properties of neurons scale with the size of the animal. Generally speaking, electrical resistances of neurons increase as cell size decreases, and thus maintenance of equal depolarization across cells of different sizes requires the underlying currents to decrease in proportion to the size decrease. Thus it would generally be expected that voltage-sensitive currents are smaller in smaller animals. In this study, we used in vivo preparations to record electrical properties of spinal motoneurons in deeply anesthetized adult mice and cats. We found that PICs do not scale with size, but instead are constant in their amplitudes across these species. This constancy, coupled with the threefold differences in electrical resistances, means that PICs contribute a threefold larger depolarization in the mouse than in the cat. As a consequence, motoneuronal firing rate sharply increases as animal size decreases. These differences in firing rates are likely essential in allowing different species to control muscles with widely different contraction speeds (smaller animals have faster muscle fibers). Thus from our results we have identified a possible new mechanism for how electrical properties are tuned to match mechanical properties within the motor output system.NEW & NOTEWORTHY The small size of the mouse warrants concern over whether the properties of their neurons are a scaled version of those in larger animals or instead have unique features. Comparison of spinal motoneurons in mice to cats showed unique features. Firing rates in the mouse were much higher, in large part due to relatively larger persistent inward currents. These differences likely reflect adaptations for controlling much faster muscle fibers in mouse than cat.

Absence of UCHL 1 function leads to selective motor neuropathy.

OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.