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In presymptomatic amyotrophic lateral sclerosis (ALS), spinal motoneurons (MNs) have reduced firing patterns and synaptic excitation levels. Preliminary data indicated that in the SOD1 G93A mouse model of ALS, monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in spinal MN by Ia proprioceptive afferent stimulation could be facilitated by trans-spinal direct current stimulation (tsDCS). However, which element of the Ia afferent-MN circuit is affected by tsDCS, and whether tsDCS-induced EPSP facilitation is a general phenomenon or specific to the superoxide dismutase type-1 (SOD1) Glycine to Alanine substitution at position 93 (G93A) mutation, remain to be determined. In this study, we have applied 15-minute tsDCS to the lumbar segments of presymptomatic SOD1 and wildtype (WT) mice and explored its impact on MN passive membrane properties, EPSP amplitude, and Ia afferent activity. While anodal tsDCS induced short-lasting EPSP facilitation in both SOD1 and WT mice, Ia afferent activity and passive membrane properties were altered only in SOD1 mice. Interestingly, EPSP amplitudes of SOD1 mice remained facilitated for at least 1 h after current application, but no long-lasting effect was observed in WT mice. Moreover, anodal tsDCS failed to induce any long-lasting changes in MN passive membrane properties in both SOD1 and WT mice. Conversely, cathodal tsDCS decreased Ia afferent induced EPSP amplitudes only during current application in SOD1 MNs, and no significant effects on Ia afferents excitability were observed. Our findings indicate the high susceptibility of SOD1 MNs to tsDCS and highlight the potential of this neuromodulation technique for the treatment of ALS.

Several studies have reported differences in the morphological characteristics of motoneurons and the contractile properties of motor units of male and female rats. However, differences in spinal motoneuron activity between the sexes are not well understood. This study investigates the electrophysiological properties of spinal α-motoneurons in male and female Wistar rats under pentobarbital anaesthesia. Fast and slow types of tibial motoneurons were recorded intracellularly in 15 male and 15 female rats, and the measured parameters were compared statistically using two-way ANOVA and Tukey post hoc tests. The membrane properties, action potential parameters and firing characteristics were not different between sexes, though significant differences were observed in the properties of fast and slow motoneuron types within both sex groups. We conclude that the sex-related differences observed in motor performance between male and female rats are largely due to differences in muscle mass, the proportion of muscle fibre types and the related motor unit contractile properties, while the mechanisms of motor control dependent on the electrophysiological activity of motoneurons are similar between the sexes. These findings are significant, as they indicate that results of experiments investigating electrophysiological properties can be reliably compared between sexes.

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.

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.

Od kilku lat obserwujemy znaczny wzrost zainteresowania nową techniką neuromodulacyjną, tj. polaryzacją układu nerwowego. Polaryzacja opiera się na indukowaniu pól elektrycznych w obrębie wybranych elementów układu nerwowego, przez zewnętrznie umiejscowione elektrody polaryzujące, co skutkuje zwiększeniem bądź zmniejszeniem aktywności sieci neuronalnych w zależności od zastosowanego bieguna elektrody czynnej. Obecnie istnieją dwie podstawowe formy aplikowania polaryzacji: polaryzacja przezczaszkowa (tDCS) i polaryzacja przezrdzeniowa (tsDCS). Będąc bardziej rozpowszechnioną, tDCS jest szeroko stosowana jako wspomaganie terapii w szeregu schorzeń neurologicznych, od terapii poudarowej, do terapii bólu i terapii schorzeń psychiatrycznych kończąc. tsDCS jest znacznie rzadziej stosowana, a efekty jej działania na poszczególne elementy układu nerwowego są wciąż badane w celu znalezienia optymalnych i bezpiecznych warunków aplikacji. Celem tego artykułu jest przybliżenie zjawiska polaryzacji układu nerwowego i stosowanych technik polaryzacji, ze szczególnym uwzględnieniem techniki tsDCS, jak również analiza możliwych mechanizmów fizjologicznych leżących u podstaw działania polaryzacji. Ponadto artykuł wskazuje możliwe zastosowanie tsDCS w zakresie wspomagania treningu sportowego, jak i terapii chorób neurodegeneracyjnych.

Recently, it has been shown that spinal cord polarization considerably modulates motoneuron activity, with certain observed changes in motoneuron membrane and firing properties outlasting the duration of polarization. The purpose of this study was to determine whether repeated sessions of transcutaneous transspinal direct current stimulation (tsDCS) induce adaptive changes in motoneuron properties. In this study, adult male Wistar rats under isoflurane anesthesia were subjected to anodal (n = 6) or cathodal (n = 6) tsDCS (100 μA, 15 min) 5 days per week for 5 wk. Sham control group rats (n = 6) served as a reference. Intracellular recordings from lumbar spinal motoneurons were performed under pentobarbital anesthesia 1 day after the final tsDCS session to analyze membrane and firing properties. Anodal polarization appeared to be effective in evoking significant adaptive changes toward the facilitation of motoneuron firing. When compared with the sham polarization group, these adaptations were expressed by the increased input resistance (P = 0.0077), decreased voltage threshold for spike generation (P = 0.0248) and doublet threshold (P = 0.0311), and increased maximum steady-state firing (SSF) frequency (P = 0.0073), SSF frequency range (P = 0.0075) and slope of the frequency-current relationship (P = 0.0111). However, the effects of cathodal polarization were modest and generally not significant in regard to the sham control. These novel findings support the existing knowledge on alterations in spinal neuronal network excitability in response to polarization and provide the direct evidence of adaptive neuroplasticity of spinal motoneurons in response to chronically applied tsDCS.NEW & NOTEWORTHY Transcutaneous spinal direct current stimulation applied systematically for 5 wk evoked polarity-dependent adaptations in the electrophysiological properties of rat spinal motoneurons. After anodal polarization sessions, motoneurons became more excitable and could evoke higher maximum discharge frequencies during repetitive firing than motoneurons in the sham polarization group. However, no significant adaptive changes of motoneuron properties were observed after repeated cathodal polarization in comparison with the sham control group.

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.

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.

Spinal polarization evoked by direct current stimulation [trans-spinal direct current stimulation (tsDCS)] is a novel method for altering spinal network excitability; however, it remains not well understood. The aim of this study was to determine whether tsDCS influences spinal motoneuron activity. Twenty Wistar rats under general pentobarbital anesthesia were subjected to 15 min anodal ( n = 10) or cathodal ( n = 10) tsDCS of 0.1 mA intensity, and the electrophysiological properties of their motoneurons were intracellularly measured before, during, and after direct current application. The major effects of anodal intervention included increased minimum firing frequency and the slope of the frequency-current ( f-I) relationship, as well as decreased rheobase and currents evoking steady-state firing (SSF). The effects of cathodal polarization included decreased maximum SSF frequency, decreased f-I slope, and decreased current evoking the maximum SSF. Notably, the majority of observed effects appeared immediately after the current onset, developed during polarization, and outlasted it for at least 15 min. Moreover, the effects of anodal polarization were generally more pronounced and uniform than those evoked by cathodal polarization. Our study is the first to present polarity-dependent, long-lasting changes in spinal motoneuron firing following tsDCS, which may aid in the development of more safe and accurate application protocols in medicine and sport. NEW & NOTEWORTHY Trans-spinal direct current stimulation induces significant polarity-dependent, long-lasting changes in the threshold and firing properties of spinal motoneurons. Anodal polarization potentiates motoneuron firing whereas cathodal polarization acts mainly toward firing inhibition. The alterations in rheobase and rhythmic firing properties are not restricted to the period of current application and can be observed long after the current offset.

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., Motoneuron activity is disrupted during Amyotrophic lateral sclerosis (ALS), depriving these cells of activity‐dependent neuroprotection. Anodal tsDCS can modify motoneuron excitability promising a new approach for ALS management.

This study demonstrates that, in a mouse model of ALS, excitatory synapses on spinal motoneurons are disrupted. Activation of the cAMP/PKA pathway reverts the functional and structural synaptic deficits and ameliorates disease markers by increasing motoneuron firing., 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., Graphical Abstract

Intracellular recording of spinal motoneurons in vivo provides a "gold standard" for determining the cells' electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

Direct current (DC) potently increases the excitability of myelinated afferent fibers in the dorsal columns, both during DC polarization of these fibers and during a considerable (>1 h) postpolarization period. The aim of the present study was to investigate whether similarly long-lasting changes in the excitability of myelinated nerve fibers in the dorsal columns may be evoked by field potentials following stimulation of peripheral afferents and by subthreshold epidurally applied current pulses. The experiments were performed in deeply anesthetized rats. The effects were monitored by changes in nerve volleys evoked in epidurally stimulated hindlimb afferents and in the synaptic actions of these afferents. Both were found to be facilitated during as well as following stimulation of a skin nerve and during as well as following epidurally applied current pulses of 5- to 10-ms duration. The facilitation occurring ≤2 min after skin nerve stimulation could be linked to both primary afferent depolarization and large dorsal horn field potentials, whereas the subsequent changes (up to 1 h) were attributable to effects of the field potentials. The findings lead to the conclusion that the modulation of spinal activity evoked by DC does not require long-lasting polarization and that relatively short current pulses and intrinsic field potentials may contribute to plasticity in spinal activity. These results suggest the possibility of enhancing the effects of epidural stimulation in human subjects by combining it with polarizing current pulses and peripheral afferent stimulation and not only with continuous DC. NEW & NOTEWORTHY The aim of this study was to define conditions under which a long-term increase is evoked in the excitability of myelinated nerve fibers. The results demonstrate that a potent and long-lasting increase in the excitability of afferent fibers traversing the dorsal columns may be induced by synaptically evoked intrinsic field as well as by epidurally applied intermittent current pulses. They thus provide a new means for the facilitation of the effects of epidural stimulation.

Trans-spinal direct current stimulation (tsDCS) is a novel neuromodulatory technique that has been used during neurological rehabilitation and sports to modulate muscle activation. However, the physiological mechanisms that underly the long-lasting functional effects of polarization are not yet fully understood, nor are their relationships with specific neuronal populations. The acute facilitatory and depressive effects of anodal and cathodal polarization on motoneurons have been recently demonstrated, and the aim of this study was to determine whether tsDCS-evoked modulations of motoneuron properties are able to persist over several hours. Intracellular recordings from multiple antidromically identified rat motoneurons were performed both before and after the application of tsDCS (0.1 mA for 15 min), at various time points up to 180 min after the offset of anodal or cathodal tsDCS. The examined effects of anodal polarization included decreased rheobase, voltage threshold, the minimum and maximum currents necessary for rhythmic firing, increased rhythmic firing frequencies and the slope of the f-I relationship. The majority of these facilitatory changes to threshold and firing properties were sustained for 30-60 min after polarization. In contrast, the significant effects of cathodal polarization were absent, except the short-lasting decreased ability for motoneurons to induce rhythmic activity. This study provides direct evidence that a single polarization session can alter the electrophysiological properties of motoneurons for at least one hour and provides a basis for the further use of tsDCS techniques under conditions where the sustained modification of motoneuron firing is desired.

Transcranial direct current stimulation (tDCS) affects neurons at both cortical and subcortical levels. The subcortical effects involve several descending motor systems but appeared toberelativelyweak,asonlysmallincreasesintheamplitudeofsubcorticallyinitiateddescending volleys and a minute shortening of latencies of these volleys were found. The aim of the present study was therefore to evaluate the consequences of facilitation of these volleys on the ensuing muscle activation. The experiments were carried out on deeply anaesthetized rats without neuro- muscular blockade. Effects of tDCS were tested on EMG potentials recorded from neck muscles evoked by weak (20-60 μA) single, double or triple stimuli applied in the medial longitudinal fascicle (MLF) or in the red nucleus (RN). Short latencies of these potentials were compatible with monosynaptic or disynaptic actions of reticulospinal and disynaptic or trisynaptic actions of rubrospinal neurons on neck motoneurons. Despite only weak effects on indirect descending volleys, the EMG responses from both the MLF and the RN were potently facilitated by cathodal tDCSanddepressedbyanodaltDCS.Boththefacilitationandthedepressiondevelopedrelatively rapidly (within the first minute) but both outlasted tDCS and were present for up to 1 h after tDCS.Thestudythusdemonstrateslong-lastingeffectsoftDCSonsubcorticalneuronsintherat, albeit evoked by an opposite polarity of tDCS to that found to be effective on subcortical neurons in the cat investigated in the preceding study, or for cortical neurons in the humans.

The main aim of the present study was to examine to what extent long-lasting subcortical actions of transcranial direct current stimulation (tDCS) may be related to its presynaptic actions. This was investigated in the red nucleus, where tDCS was recently demonstrated to facilitate transmission between interpositorubral and rubrospinal neurons. Changes in the excitability of preterminal axonal branches of interpositorubral neurons close to rubrospinal neurons were investigated during and after tDCS (0.2 mA) applied over the sensorimotor cortical area in deeply anaesthetized rats and cats. As a measure of the excitability, we used the probability of antidromic activation of individual interpositorubral neurons by electrical stimuli applied in the red nucleus. Our second aim was to compare effects of weak (≤1 μA) direct current applied within the red nucleus with effects of tDCS to allow the use of local depolarization in a further analysis of mechanisms of tDCS instead of widespread and more difficult to control depolarization evoked by distant electrodes. Local cathodal polarization was found to replicate all effects of cathodal tDCS hitherto demonstrated in the rat, including long-lasting facilitation of trans-synaptically evoked descending volleys and trisynaptically evoked EMG responses in neck muscles. It also replicated all effects of anodal tDCS in the cat. In both species, it increased the excitability of preterminal axonal branches of interpositorubral neurons up to 1 h post-tDCS. Local anodal polarization evoked opposite effects. We thus show that presynaptic actions of polarizing direct current may contribute to both immediate and prolonged effects of tDCS.

Ipsilateral actions of pyramidal tract (PT) neurons are weak but may, if strengthened, compensate for deficient crossed PT actions following brain damage. The purpose of the present study was to examine whether transcranial direct current stimulation (tDCS) can strengthen ipsilateral PT (iPT) actions; in particular, those relayed by reticulospinal neurons co-excited by axon collaterals of fibres descending in the iPT and contralateral PT (coPT) and of reticulospinal neurons descending in the medial longitudinal fascicle (MLF). The effects of tDCS were assessed in acute experiments on deeply anaesthetized cats by comparing postsynaptic potentials evoked in hindlimb motoneurons and discharges recorded from their axons in a ventral root, before, during and after tDCS. tDCS was consistently found to facilitate joint actions of the iPT and coPT, especially when they were stimulated together with the MLF. Both excitatory postsynaptic potentials and inhibitory postsynaptic potentials evoked in motoneurons and the ensuing ventral root discharges were facilitated, even though the facilitatory effects of tDCS were not sufficient for activation of motoneurons by iPT neurons alone. Facilitation outlasted single tDCS periods by at least a few minutes, and the effects evoked by repeated tDCS by up to 2 h. The results of this study thus indicate that tDCS may increase the contribution of iPT actions to the recovery of motor functions after injuries to coPT neurons, and thereby assist rehabilitation, provided that corticoreticular and reticulospinal connections are preserved.

The purpose of the study was to determine the effects of 5-week whole-body vibration (WBV) on contractile parameters and force–frequency relationship of functionally isolated motor units of the rat medial gastrocnemius muscle: fast fatigable (FF), fast fatigue-resistant (FR), and slow (S). Moreover, myosin heavy chain isoform content was quantified. Following WBV, the maximum tetanic force of FF units was increased by ∼25%. The twitch half-relaxation time in all types of motor units and the twitch contraction time in FR units were shortened. The twitch-to-tetanus force ratio was decreased and the force–frequency curves were shifted rightwards in S and FR units. Myosin heavy chain distribution was not changed. These findings suggest modifications of the excitation–contraction coupling towards shortening of a twitch contraction. The observed increase in force of FF units may contribute to gains in muscle dynamic strength reported following WBV treatment.

The study aimed at determining the influence of a whole body vibration (WBV) on electrophysiological properties of spinal motoneurons. The WBV training was performed on adult male Wistar rats, 5 days a week, for 5 wk, and each daily session consisted of four 30-s runs of vibration at 50 Hz. Motoneuron properties were investigated intracellularly during experiments on deeply anesthetized animals. The experimental group subjected to the WBV consisted of seven rats, and the control group of nine rats. The WBV treatment induced no significant changes in the passive membrane properties of motoneurons. However, the WBV-evoked adaptations in excitability and firing properties were observed, and they were limited to fast-type motoneurons. A significant decrease in rheobase current and a decrease in the minimum and the maximum currents required to evoke steady-state firing in motoneurons were revealed. These changes resulted in a leftward shift of the frequency-current relationship, combined with an increase in slope of this curve. The functional relevance of the described adaptive changes is the ability of fast motoneurons of rats subjected to the WBV to produce series of action potentials at higher frequencies in a response to the same intensity of activation. Previous studies proved that WBV induces changes in the contractile parameters predominantly of fast motor units (MUs). The data obtained in our experiment shed a new light to possible explanation of these results, suggesting that neuronal factors also play a substantial role in MU adaptation.