Dysregulation of persistent inward and outward currents in spinal motoneurons of symptomatic SOD1-G93A mice.

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.

PICking out progressive PIC alterations in amyotrophic lateral sclerosis.

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.

Intrinsic motoneuron properties in typical human development.

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.

Muscle Spasms after Spinal Cord Injury Stem from Changes in Motoneuron Excitability and Synaptic Inhibition, Not Synaptic Excitation.

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.

Relationship between disturbances of CO2 homeostasis and force output characteristics during isometric knee extension.

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.

Intrinsic motor neuron excitability is increased after resistance training in older adults.

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.

A geometric approach to quantifying the neuromodulatory effects of persistent inward currents on individual motor unit discharge patterns.

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.

The effects of α-lactalbumin supplementation and handgrip contraction on soleus motoneuron excitability.

INTRODUCTION: 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.

The Cellular Basis for the Generation of Firing Patterns in Human Motor Units.

Motor units, which comprise a motoneuron and the set of muscle fibers it innervates, are the fundamental neuromuscular transducers for all motor commands. The one to one relationship between a motoneuron and its innervated muscle fibers allow motoneuron firing patterns to be readily measured in humans. In this chapter, we summarize the current understanding of the cellular basis for the generation of firing patterns in human motor units. We provide a brief review of landmark insights from classic studies and then proceed to consider the features of motor unit firing patterns that are most likely to be sensitive estimators of motoneuron inputs and properties. In addition, we discuss recent advances in technology for recording human motor unit firing patterns and highly realistic computer simulations of motoneurons. The final section presents our recent efforts to use the power of supercomputers for implementation of the motoneuron models, with a goal of achieving a true "reverse engineering" approach that maximizes the insights from motor unit firing patterns into the synaptic structure of motor commands.

Electrical Properties of Adult Mammalian Motoneurons.

Motoneurons are the 'final common path' between the central nervous system (that intends, selects, commands, and organises movement) and muscles (that produce the behaviour). Motoneurons are not passive relays, but rather integrate synaptic activity to appropriately tune output (spike trains) and therefore the production of muscle force. In this chapter, we focus on studies of mammalian motoneurons, describing their heterogeneity whilst providing a brief historical account of motoneuron recording techniques. Next, we describe adult motoneurons in terms of their passive, transition, and active (repetitive firing) properties. We then discuss modulation of these properties by somatic (C-boutons) and dendritic (persistent inward currents) mechanisms. Finally, we briefly describe select studies of human motor unit physiology and relate them to findings from animal preparations discussed earlier in the chapter. This interphyletic approach to the study of motoneuron physiology is crucial to progress understanding of how these diverse neurons translate intention into behaviour.

Cholinergic modulation of persistent inward currents is mediated by activating muscarinic receptors of serotonergic neurons in the brainstem of ePet-EYFP mice.

Persistent inward currents (PICs) play important roles in regulating neural excitability. Results from our previous studies showed that serotonergic (5-HT) neurons of the brainstem expressed PICs. However, little is known about cholinergic (ACh) modulation of PICs in the 5-HT neurons. The whole-cell patch-clamp recordings were performed in the brainstem slices of ePet-EYFP mice to investigate the electrophysiological properties of PICs with cholinergic modulation. PICs in 5-HT neurons were activated at - 51.4 ± 3.7 mV with the amplitude of - 171.6 ± 48.9 pA (n = 71). Bath application of 20-25 µM ACh increased the amplitude by 79.1 ± 42.5 pA (n = 23, p < 0.001) and hyperpolarized the onset voltage by 2.2 ± 2.7 mV (n = 23, p < 0.01) and half-maximal activation by 3.6 ± 2.7 mV (n = 6, p < 0.01). Muscarine mimicked the effects of ACh on PICs, while bath application of nicotine (15-20 µM) did not induce substantial change in the PICs (n = 9). Muscarine enhanced the amplitude of PICs by 100.0 ± 27.4 pA (n = 28, p < 0.001) and lowered the onset voltage by 2.8 ± 1.2 mV (n = 28, p < 0.001) and the half-maximal activation by 2.9 ± 1.4 mV. ACh-induced increase of amplitude and hyperpolarization of onset voltage were blocked by 3-5 µM atropine. Furthermore, the muscarine-induced enhancement of the PICs was antagonized by 5 µM 4-DAMP, the antagonist of M3 receptor, while the antagonists of M1 (Telenzepine, 5 µM) and M5 (VU6008667, 5 µM) receptors did not significantly affect the PIC enhancement. This study suggested that ACh potentiated PICs in 5-HT neurons of the brainstem by activating muscarinic M3 receptor.

Firing rate trajectories of human motor units during activity-dependent muscle potentiation.

During activity-dependent potentiation (ADP), motor unit firing rates (MUFRs) are lower; however, the mechanism for this response is not known. During increasing torque isometric contractions at low contraction intensities, MUFR trajectories initially accelerate and saturate demonstrating a nonlinear response due to the activation of persistent inward currents (PICs) at the motoneuron. The purpose was to assess whether PICs are a factor in the reduction of MUFRs during ADP. To assess this, MUFR trajectories were fit with competing functions of linear regression and a rising exponential (i.e., acceleration and saturation). With fine-wire electrodes, discrete MU potential trains were recorded in the tibialis anterior during slowly increasing dorsiflexion contractions to 10% of maximal voluntary contraction following both voluntary [postactivation potentiation (PAP)] and evoked [posttetanic potentiation (PTP)] contractions. In eight participants, 25 MUs were recorded across both ADP conditions and compared with the control with no ADP effect. During PAP and PTP, the average MUFRs were 16.4% and 9.2% lower (both P ≤ 0.001), respectively. More MUFR trajectories were better fit to the rising exponential during control (16/25) compared with PAP (4/25, P < 0.001) and PTP (8/25, P = 0.03). The MU samples that had a rising exponential MUFR trajectory during PAP and PTP displayed an ∼11% lower initial acceleration compared with control (P < 0.05). Thus, presumed synaptic amplification and MUFR saturation due to PIC properties are attenuated during ADP regardless of the type of conditioning contraction. This response may contribute to lower MUFRs and likely occurred because synaptic input is reduced when contractile function is enhanced.NEW & NOTEWORTHY During activity-dependent muscle potentiation (ADP), initial motor unit firing rate (MUFR) acceleration and the occurrence of MUFR trajectory saturation as a function of increasing contraction intensity were assessed. With no ADP (control), trajectories were more likely to accelerate and saturate (16/25 units) compared with voluntary- and stimulated-induced ADP conditions (4/25 and 8/25 units, respectively) that were fit better linearly. Therefore, during ADP, an attenuated intrinsic response to voluntary synaptic inputs occurs.

Firing rate trajectories of human motor units during isometric ramp contractions to 10, 25 and 50% of maximal voluntary contraction.

During low torque graded isometric contractions, motor units (MU) exhibit initial firing rate acceleration followed by saturation demonstrating a non-linear response attributed to persistent inward currents (PICs) which contribute to the net excitatory input. Firing rate saturation studies have been done exclusively at recruitment thresholds of low firing threshold MUs below 10% of isometric maximal voluntary contraction(MVC). It remains unclear whether later recruited (i.e. higher-threshold) MUs follow a similar firing rate trajectory as low-threshold units. Thus, MU firing rate trajectories were explored in relation to MU recruitment threshold (RT) at contraction levels between 10 and 50% of MVC. During graded isometric contractions to 10, 25 and 50% of MVC, single MU potentials were recorded from the tibialis anterior from 5 participants using tungsten microelectrodes. To characterize the firing rate trajectory, each MU train was fit by competing functions of torque as an exponential (i.e. saturated) and simple linear regression, using previous analysis methods (Fuglevand et al. 2015). Throughout a RT range of 0.02-41% of MVC, 261 MUs were compared. In 87% of MUs the better fit was by a linear function, whereas the remaining MUs (13%) were fit better with an exponential (saturated) firing rate trajectory. There was no statistical difference in the number of MUs better fit by the exponential function between low (<10% MVC) and relatively higher threshold MUs (>10% MVC; both p < 0.05). Increasing RT and rate of torque development (RTD) of the ramps were correlated with increased firing rate variability (larger error) in both fits (r = 0.3 and r = 0.4, both p < 0.01). Additionally, there was a 4-fold increase in peak antagonist surface electromyography (EMG) from 10 to 50% MVC contraction ramps. When all MUs were plotted with a normalized firing onset (i.e. 0% MVC) the data visually displayed an initial firing rate acceleration followed by a linear response (biphasic trajectory). Increased synaptic drive and greater antagonist surface EMG during moderate torque outputs may dampen PIC activity as compared with MUs during lower torque (<10% MVC) recruitment levels.

Serotonergic Modulation of Persistent Inward Currents in Serotonergic Neurons of Medulla in ePet-EYFP Mice.

Serotonergic (5-HT) neurons in the medulla play multiple functional roles associated with many symptoms and motor activities. The descending serotonergic pathway from medulla is essential for initiating locomotion. However, the ionic properties of 5-HT neurons in the medulla remain unclear. Using whole-cell patch-clamp technique, we studied the biophysical and modulatory properties of persistent inward currents (PICs) in 5-HT neurons of medulla in ePet-EYFP transgenic mice (P3-P6). PICs were recorded by a family of voltage bi-ramps (10-s duration, 40-mV peak step), and the ascending and descending PICs were mirrored to analyze the PIC hysteresis. PICs were found in 77% of 5-HT neurons (198/258) with no significant difference between parapyramidal region (n = 107) and midline raphe nuclei (MRN) (n = 91) in either PIC onset (-47.4 ± 10 mV and -48.7 ± 7 mV; P = 0.44) or PIC amplitude (226.9 ± 138 pA and 259.2 ± 141 pA; P = 0.29). Ninety-six percentage (191/198) of the 5-HT neurons displayed counterclockwise hysteresis and four percentage (7/198) exhibited the clockwise hysteresis. The composite PICs could be differentiated as calcium component (Ca_PIC) by bath application of nimodipine (25 µM), sodium component (Na_PIC) by tetrodotoxin (TTX, 2 µM), and TTX- and dihydropyridine-resistance component (TDR_PIC) by TTX and nimodipine. Ca_PIC, Na_PIC and TDR_PIC all contributed to upregulation of excitability of 5-HT neurons. 5-HT (15 µM) enhanced the PICs, including a 26% increase in amplitude of the compound currents of Ca_PIC and TDR_PIC (P < 0.001, n = 9), 3.6 ± 5 mV hyperpolarization of Na_PIC and TDR_PIC onset (P < 0.05, n = 12), 30% increase in amplitude of TDR_PIC (P < 0.01), and 2.0 ± 3 mV hyperpolarization of TDR_PIC onset (P < 0.05, n = 18). 5-HT also facilitated repetitive firing of 5-HT neurons through modulation of composite PIC, Na_PIC and TDR_PIC, and Ca_PIC and TDR_PIC, respectively. In particular, the high voltage-activated TDR_PIC facilitated the repetitive firing in higher membrane potential, and this facilitation could be amplified by 5-HT. Morphological data analysis indicated that the dendrites of 5-HT neurons possessed dense spherical varicosities intensively crossing 5-HT neurons in medulla. We characterized the PICs in 5-HT neurons and unveiled the mechanism underlying upregulation of excitability of 5-HT neurons through serotonergic modulation of PICs. This study provided insight into channel mechanisms responsible for the serotonergic modulation of serotonergic neurons in brainstem.

Three-Week Treadmill Exercise Enhances Persistent Inward Currents, Facilitates Dendritic Plasticity, and Upregulates the Excitability of Dorsal Raphe Serotonin Neurons in ePet-EYFP Mice.

Exercise plays a key role in preventing or treating mental or motor disorders caused by dysfunction of the serotonergic system. However, the electrophysiological and ionic channel mechanisms underlying these effects remain unclear. In this study, we investigated the effects of 3-week treadmill exercise on the electrophysiological and channel properties of dorsal raphe nucleus (DRN). Serotonin (5-HT) neurons in ePet-EYFP mice, using whole-cell patch clamp recording. Treadmill exercise was induced in ePet-EYFP mice of P21-24 for 3 weeks, and whole-cell patch clamp recording was performed on EYFP-positive 5-HT neurons from DRN slices of P42-45 mice. Experiment data showed that 5-HT neurons in the DRN were a heterogeneous population with multiple firing patterns (single firing, phasic firing, and tonic firing). Persistent inward currents (PICs) with multiple patterns were expressed in 5-HT neurons and composed of Cav1.3 (Ca-PIC) and sodium (Na-PIC) components. Exercise hyperpolarized the voltage threshold for action potential (AP) by 3.1 ± 1.0 mV (control: n = 14, exercise: n = 18, p = 0.005) and increased the AP amplitude by 6.7 ± 3.0 mV (p = 0.031) and firing frequency by more than 22% especially within a range of current stimulation stronger than 70 pA. A 3-week treadmill exercise was sufficient to hyperpolarize PIC onset by 2.6 ± 1.3 mV (control: -53.4 ± 4.7 mV, n = 28; exercise: -56.0 ± 4.7 mV, n = 25, p = 0.050) and increase the PIC amplitude by 28% (control: 193.6 ± 81.8 pA; exercise: 248.5 ± 105.4 pA, p = 0.038). Furthermore, exercise hyperpolarized Na-PIC onset by 3.8 ± 1.8 mV (control: n = 8, exercise: n = 9, p = 0.049) and increased the Ca-PIC amplitude by 23% (p = 0.013). The exercise-induced enhancement of the PIC amplitude was mainly mediated by Ca-PIC and hyperpolarization of PIC onset by Na-PIC. Moreover, exercise facilitated dendritic plasticity, which was shown as the increased number of branch points by 1.5 ± 0.5 (p = 0.009) and dendritic branches by 2.1 ± 0.6 (n = 20, p = 0.001) and length by 732.0 ± 100.1 µm (p < 0.001) especially within the range of 50-200 µm from the soma. Functional analysis suggested that treadmill exercise enhanced Na-PIC for facilitation of spike initiation and Ca-PIC for regulation of repetitive firing. We concluded that PICs broadly existed in DRN 5-HT neurons and could influence serotonergic neurotransmission in juvenile mice and that 3-week treadmill exercise induced synaptic adaptations, enhanced PICs, and thus upregulated the excitability of the 5-HT neurons.

Estimation of self-sustained activity produced by persistent inward currents using firing rate profiles of multiple motor units in humans.

Persistent inward calcium and sodium currents (IP) activated during motoneuron recruitment help synaptic inputs maintain self-sustained firing until derecruitment. Here, we estimate the contribution of the IP to self-sustained firing in human motoneurons of varying recruitment threshold by measuring the difference in synaptic input needed to maintain minimal firing once the IP is fully activated compared with the larger synaptic input required to initiate firing before full IP activation. Synaptic input to ≈20 dorsiflexor motoneurons simultaneously recorded during ramp contractions was estimated from firing profiles of motor units decomposed from high-density surface electromyography (EMG). To avoid errors introduced when using high-threshold units firing in their nonlinear range, we developed methods where the lowest threshold units firing linearly with force were used to construct a composite (control) unit firing rate profile to estimate synaptic input to higher threshold (test) units. The difference in the composite firing rate (synaptic input) at the time of test unit recruitment and derecruitment (ΔF = Frecruit - Fderecruit) was used to measure IP amplitude that sustained firing. Test units with recruitment thresholds 1-30% of maximum had similar ΔF values, which likely included both slow and fast motor units activated by small and large motoneurons, respectively. This suggests that the portion of the IP that sustains firing is similar across a wide range of motoneuron sizes.NEW & NOTEWORTHY A new method of estimating synaptic drive to multiple, simultaneously recorded motor units provides evidence that the portion of the depolarizing drive from persistent inward currents that contributes to self-sustained firing is similar across motoneurons of different sizes.