Persistent inward currents (PICs) and persistent outward currents (POCs) regulate the excitability and firing behaviours of spinal motoneurons (MNs). Given their potential role in MN excitability dysfunction in amyotrophic lateral sclerosis (ALS), PICs have been previously studied in superoxide dismutase 1 (SOD1)-G93A mice (the standard animal model of ALS); however, conflicting results have been reported on how the net PIC changes during disease progression. Also, individual PICs and POCs have never been examined before in symptomatic ALS. To fill this gap, we measured the net and individual PIC and POC components of wild-type (WT) and SOD MNs in current clamp and voltage clamp during disease progression (assessed by neuroscores). We show that SOD MNs of symptomatic mice experience a much larger net PIC, relative to WT cells from age-matched littermates. Specifically, the Na+ and Ca2+ PICs are larger, whereas the lasting SK-mediated (SKL) POC is smaller than WT (Na+ PIC is the largest and SKL POC is the smallest components in SOD MNs). We also show that PIC dysregulation is present at symptom onset, is sustained throughout advanced disease stages and is proportional to SOD MN cell size (largest dysregulation is in the largest SOD cells, the most vulnerable in ALS). Additionally, we show that studying disease progression using neuroscores is more accurate than using SOD mouse age, which could lead to misleading statistics and age-based trends. Collectively, this study contributes novel PIC and POC data, reveals ionic mechanisms contributing to the vulnerability differential among MN types/sizes, and provides insights on the roles PIC and POC mechanisms play in MN excitability dysfunction in ALS. KEY POINTS: Individual persistent inward currents (PICs) and persistent outward currents (POCs) have never been examined before in spinal motoneurons (MNs) of symptomatic amyotrophic lateral sclerosis (ALS) mice. Thus, we contribute novel PIC and POC data to the ALS literature. Male SOD MNs of symptomatic mice have elevated net PIC, with larger Na+ and Ca2+ PICs but reduced SKL POC vs. wild-type littermates. Na+ PIC is the largest and SKL POC is the smallest current in SOD cells. The PIC/POC dysregulation is present at symptom onset. PIC dysregulation is sustained throughout advanced disease, and is proportional to SOD MN size (largest dysregulation is in the largest cells, the most vulnerable in ALS). Thus, we reveal ionic mechanisms contributing to the vulnerability differential among MN types/sizes in ALS. Studying disease progression using SOD mice neuroscores is more accurate than using age, which could distort the statistical differences between SOD and WT PIC/POC data and the trends during disease progression.

Motoneuron properties and their firing patterns undergo significant changes throughout development and in response to neuromodulators such as serotonin. Here, we examined the age-related development of self-sustained firing and general excitability of tibialis anterior motoneurons in a young development (7-17 years), young adult (18-28 years) and adult (32-53 years) group, as well as in a separate group of participants taking selective serotonin reuptake inhibitors (SSRIs, aged 11-28 years). Self-sustained firing, as measured by ΔF, was larger in the young development (∼5.8 Hz, n = 20) compared to the young adult (∼4.9 Hz, n = 13) and adult (∼4.8 Hz, n = 8) groups, consistent with a developmental decrease in self-sustained firing mediated by persistent inward currents (PIC). ΔF was also larger in participants taking SSRIs (∼6.5 Hz, n = 9) compared to their age-matched controls (∼5.3 Hz, n = 26), consistent with increased levels of spinal serotonin facilitating the motoneuron PIC. Participants in the young development and SSRI groups also had higher firing rates and a steeper acceleration in initial firing rates (secondary ranges), consistent with the PIC producing a steeper acceleration in membrane depolarization at the onset of motoneuron firing. In summary, both the young development and SSRI groups exhibited increased intrinsic motoneuron excitability compared to the adults, which, in the young development group, was also associated with a larger unsteadiness in the dorsiflexion torque profiles. We propose several intrinsic and extrinsic factors that affect both motoneuron PICs and cell discharge which vary during development, with a time course similar to the changes in motoneuron firing behaviour observed in the present study. KEY POINTS: Neurons in the spinal cord that activate muscles in the limbs (motoneurons) undergo increases in excitability shortly after birth to help animals stand and walk. We examined whether the excitability of human ankle flexor motoneurons also continues to change from child to adulthood by recording the activity of the muscle fibres they innervate. Motoneurons in children and adolescents aged 7-17 years (young development group) had higher signatures of excitability that included faster firing rates and more self-sustained activity compared to adults aged ≥18 years. Participants aged 11-28 years of age taking serotonin reuptake inhibitors had the highest measures of motoneuron excitability compared to their age-matched controls. The young development group also had more unstable contractions, which might partly be related to the high excitability of the motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motoneuron death. Alterations to motoneuron excitability in ALS are suspected to contribute to motoneuron degeneration. Therefore, mechanisms underlying changes in motoneuron excitability are being thoroughly investigated. A recent publication from Trajano et al. (Trajano GS, Orssatto LB, McCombe PA, Rivlin W, Tang L, Henderson RD. J Physiol 601: 4723-4735, 2023) examined temporal changes to persistent inward currents (PICs) in ALS patients. They show that delta frequency (ΔF, an estimate of PICs) has opposite temporal trends in stronger and weaker muscles of ALS patients. This study is very important to aid in the understanding of disease mechanisms. This Neuro Forum article explores some important considerations for interpreting the results of this study, including treatment effects, potential sex differences, and a lack of comparison to healthy individuals.

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Muscle spasms are common in chronic spinal cord injury (SCI), posing challenges to rehabilitation and daily activities. Pharmacological management of spasms mostly targets suppression of excitatory inputs, an approach known to hinder motor recovery. To identify better targets, we investigated changes in inhibitory and excitatory synaptic inputs to motoneurons as well as motoneuron excitability in chronic SCI. We induced either a complete or incomplete SCI in adult mice of either sex and divided those with incomplete injury into low or high functional recovery groups. Their sacrocaudal spinal cords were then extracted and used to study plasticity below injury, with tissue from naive animals as a control. Electrical stimulation of the dorsal roots elicited spasm-like activity in preparations of chronic severe SCI but not in the control. To evaluate overall synaptic inhibition activated by sensory stimulation, we measured the rate-dependent depression of spinal root reflexes. We found inhibitory inputs to be impaired in chronic injury models. When synaptic inhibition was blocked pharmacologically, all preparations became clearly spastic, even the control. However, preparations with chronic injuries generated longer spasms than control. We then measured excitatory postsynaptic currents (EPSCs) in motoneurons during sensory-evoked spasms. The data showed no difference in the amplitude of EPSCs or their conductance among animal groups. Nonetheless, we found that motoneuron persistent inward currents activated by the EPSCs were increased in chronic SCI. These findings suggest that changes in motoneuron excitability and synaptic inhibition, rather than excitation, contribute to spasms and are better suited for more effective therapeutic interventions.Significance Statement Neural plasticity following spinal cord injury is crucial for recovery of motor function. Unfortunately, this process is blemished by maladaptive changes that can cause muscle spasms. Pharmacological alleviation of spasms without compromising the recovery of motor function has proven to be challenging. Here, we investigated changes in fundamental spinal mechanisms that can cause spasms post-injury. Our data suggest that the current management strategy for spasms is misdirected toward suppressing excitatory inputs, a mechanism that we found unaltered after injury, which can lead to further motor weakness. Instead, this study shows that more promising approaches might involve restoring synaptic inhibition or modulating motoneuron excitability.

To determine whether disturbances of CO2 homeostasis alter force output characteristics of lower limb muscles, participants performed four isometric knee extension trials (MVC30 %, 10 s each with 20-s rest intervals) in three CO2 conditions (normocapnia [NORM], hypercapnia [HYPER], and hypocapnia [HYPO]). Respiratory frequency and tidal volume were matched between CO2 conditions. In each MVC30 %, the participants exerted a constant force (30 % of maximum voluntary contraction [MVC]). The force coefficient of variation (Fcv) during each MVC30 % and MVC before and after the four MVC30 % trials were measured. For the means of the four trials, Fcv was significantly lower in HYPER than in HYPO. However, within HYPER, a significant positive correlation was found between the increase in end-tidal CO2 partial pressure and the increase in Fcv. MVCs in NORM and HYPO decreased significantly over the four trials, while no such reduction was observed in HYPER. These results suggest that perturbed CO2 homeostasis influences the force output characteristics independently of breathing pattern variables.

This study investigated the effects of high-intensity resistance training on estimates of the motor neuron persistent inward current (PIC) in older adults. Seventeen participants (68.5 ± 2.8 yr) completed a 2-wk nonexercise control period followed by 6 wk of resistance training. Surface electromyographic signals were collected with two 32-channel electrodes placed over soleus to investigate motor unit discharge rates. Paired motor unit analysis was used to calculate delta frequency (ΔF) as an estimate of PIC amplitudes during 1) triangular-shaped contractions to 20% of maximum torque capacity and 2) trapezoidal- and triangular-shaped contractions to 20% and 40% of maximum torque capacity, respectively, to understand their ability to modulate PICs as contraction intensity increases. Maximal strength and functional capacity tests were also assessed. For the 20% triangular-shaped contractions, ΔF [0.58-0.87 peaks per second (pps); P ≤ 0.015] and peak discharge rates (0.78-0.99 pps; P ≤ 0.005) increased after training, indicating increased PIC amplitude. PIC modulation also improved after training. During the control period, mean ΔF differences between 20% trapezoidal-shaped and 40% triangular-shaped contractions were 0.09-0.18 pps (P = 0.448 and 0.109, respectively), which increased to 0.44 pps (P < 0.001) after training. Also, changes in ΔF showed moderate to very large correlations (r = 0.39-0.82) with changes in peak discharge rates and broad measures of motor function. Our findings indicate that increased motor neuron excitability is a potential mechanism underpinning training-induced improvements in motor neuron discharge rate, strength, and motor function in older adults. This increased excitability is likely mediated by enhanced PIC amplitudes, which are larger at higher contraction intensities.NEW & NOTEWORTHY Resistance training elicited important alterations in soleus intrinsic motor neuronal excitability, likely mediated by enhanced persistent inward current (PIC) amplitude, in older adults. Estimates of PICs increased after the training period, accompanied by an enhanced ability to increase PIC amplitudes at higher contraction intensities. Our data also suggest that changes in PIC contribution to self-sustained discharging may contribute to increases in motor neuron discharge rates, maximal strength, and functional capacity in older adults after resistance training.

Objective.All motor commands flow through motoneurons, which entrain control of their innervated muscle fibers, forming a motor unit (MU). Owing to the high fidelity of action potentials within MUs, their discharge profiles detail the organization of ionotropic excitatory/inhibitory as well as metabotropic neuromodulatory commands to motoneurons. Neuromodulatory inputs (e.g. norepinephrine, serotonin) enhance motoneuron excitability and facilitate persistent inward currents (PICs). PICs introduce quantifiable properties in MU discharge profiles by augmenting depolarizing currents upon activation (i.e. PIC amplification) and facilitating discharge at lower levels of excitatory input than required for recruitment (i.e. PIC prolongation).Approach. Here, we introduce a novel geometric approach to estimate neuromodulatory and inhibitory contributions to MU discharge by exploiting discharge non-linearities introduced by PIC amplification during time-varying linear tasks. In specific, we quantify the deviation from linear discharge (\'brace height\') and the rate of change in discharge (i.e. acceleration slope, attenuation slope, angle). We further characterize these metrics on a simulated motoneuron pool with known excitatory, inhibitory, and neuromodulatory inputs and on human MUs (number of MUs; Tibialis Anterior: 1448, Medial Gastrocnemius: 2100, Soleus: 1062, First Dorsal Interosseus: 2296).Main results. In the simulated motor pool, we found brace height and attenuation slope to consistently indicate changes in neuromodulation and the pattern of inhibition (excitation-inhibition coupling), respectively, whereas the paired MU analysis (ΔF) was dependent on both neuromodulation and inhibition pattern. Furthermore, we provide estimates of these metrics in human MUs and show comparable variability in ΔFand brace height measures for MUs matched across multiple trials.Significance. Spanning both datasets, we found brace height quantification to provide an intuitive method for achieving graded estimates of neuromodulatory and inhibitory drive to individual MUs. This complements common techniques and provides an avenue for decoupling changes in the level of neuromodulatory and pattern of inhibitory motor commands.

BACKGROUND: We tested two strategies that hypothetically increase serotonin availability (α-lactalbumin consumption and a remote submaximal handgrip contraction) on estimates of persistent inward currents (PICs) amplitude of soleus muscle in healthy participants.
METHODS: With a randomised, double-blind, and cross-over design, 13 healthy participants performed triangular-shaped ramp contractions with their plantar flexors (20% of maximal torque), followed by a 30-s handgrip sustained contraction (40% of maximal force) and consecutive repeated triangular-shaped contractions. This was performed before and after the consumption of either 40 g of α-lactalbumin, an isonitrogenous beverage (Zein) or an isocaloric beverage (Corn-starch). Soleus motor units discharge rates were analysed from high-density surface electromyography signals. PICs were estimated by calculating the delta frequency (ΔF) of motor unit train spikes using the paired motor unit technique.
RESULTS: ΔF (0.19 pps; p = 0.001; d = 0.30) and peak discharge rate (0.20 pps; p < 0.001; d = 0.37) increased after the handgrip contraction, irrespective of the consumed supplement. No effects of α-lactalbumin were observed.
CONCLUSIONS: Our results indicate that 40 g of α-lactalbumin was unable to modify intrinsic motoneuron excitability. However, performing a submaximal handgrip contraction before the plantar flexion triangular contraction was capable of increasing ΔF and discharge rates on soleus motor units. These findings highlight the diffused effects of serotonergic input, its effects on motoneuron discharge behaviour, and suggest a cross-effector effect within human motoneurons.