Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy.

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.

Restoring the Sense of Touch Using a Sensorimotor Demultiplexing Neural Interface.

Paralyzed muscles can be reanimated following spinal cord injury (SCI) using a brain-computer interface (BCI) to enhance motor function alone. Importantly, the sense of touch is a key component of motor function. Here, we demonstrate that a human participant with a clinically complete SCI can use a BCI to simultaneously reanimate both motor function and the sense of touch, leveraging residual touch signaling from his own hand. In the primary motor cortex (M1), residual subperceptual hand touch signals are simultaneously demultiplexed from ongoing efferent motor intention, enabling intracortically controlled closed-loop sensory feedback. Using the closed-loop demultiplexing BCI almost fully restored the ability to detect object touch and significantly improved several sensorimotor functions. Afferent grip-intensity levels are also decoded from M1, enabling grip reanimation regulated by touch signaling. These results demonstrate that subperceptual neural signals can be decoded from the cortex and transformed into conscious perception, significantly augmenting function.

The science and engineering behind sensitized brain-controlled bionic hands.

Advances in our understanding of brain function, along with the development of neural interfaces that allow for the monitoring and activation of neurons, have paved the way for brain-machine interfaces (BMIs), which harness neural signals to reanimate the limbs via electrical activation of the muscles or to control extracorporeal devices, thereby bypassing the muscles and senses altogether. BMIs consist of reading out motor intent from the neuronal responses monitored in motor regions of the brain and executing intended movements with bionic limbs, reanimated limbs, or exoskeletons. BMIs also allow for the restoration of the sense of touch by electrically activating neurons in somatosensory regions of the brain, thereby evoking vivid tactile sensations and conveying feedback about object interactions. In this review, we discuss the neural mechanisms of motor control and somatosensation in able-bodied individuals and describe approaches to use neuronal responses as control signals for movement restoration and to activate residual sensory pathways to restore touch. Although the focus of the review is on intracortical approaches, we also describe alternative signal sources for control and noninvasive strategies for sensory restoration.

Proprioceptive feedback amplification restores effective locomotion in a neuromechanical model of lampreys with spinal injuries.

Spinal injuries in many vertebrates can result in partial or complete loss of locomotor ability. While mammals often experience permanent loss, some nonmammals, such as lampreys, can regain swimming function, though the exact mechanism is not well understood. One hypothesis is that amplified proprioceptive (body-sensing) feedback can allow an injured lamprey to regain functional swimming even if the descending signal is lost. This study employs a multiscale, integrative, computational model of an anguilliform swimmer fully coupled to a viscous, incompressible fluid and examines the effects of amplified feedback on swimming behavior. This represents a model that analyzes spinal injury recovery by combining a closed-loop neuromechanical model with sensory feedback coupled to a full Navier-Stokes model. Our results show that in some cases, feedback amplification below a spinal lesion is sufficient to partially or entirely restore effective swimming behavior.

A neural circuit for vocal production responds to viscerosensory input in the songbird.

Motor performance is monitored continuously by specialized brain circuits and used adaptively to modify behavior on a moment-to-moment basis and over longer time periods. During vocal behaviors, such as singing in songbirds, internal evaluation of motor performance relies on sensory input from the auditory and vocal-respiratory systems. Sensory input from the auditory system to the motor system, often referred to as auditory feedback, has been well studied in singing zebra finches (Taeniopygia guttata), but little is known about how and where nonauditory sensory feedback is evaluated. Here we show that brief perturbations in air sac pressure cause short-latency neural responses in the higher-order song control nucleus HVC (used as proper name), an area necessary for song learning and song production. Air sacs were briefly pressurized through a cannula in anesthetized or sedated adult male zebra finches, and neural responses were recorded in both nucleus parambigualis (PAm), a brainstem inspiratory center, and HVC, a cortical premotor nucleus. These findings show that song control nuclei in the avian song system are sensitive to perturbations directly targeted to vocal-respiratory, or viscerosensory, afferents and support a role for multimodal sensory feedback integration in modifying and controlling vocal control circuits.NEW & NOTEWORTHY This study presents the first evidence of sensory input from the vocal-respiratory periphery directly activating neurons in a motor circuit for vocal production in songbirds. It was previously thought that this circuit relies exclusively on sensory input from the auditory system, but we provide groundbreaking evidence for nonauditory sensory input reaching the higher-order premotor nucleus HVC, expanding our understanding of what sensory feedback may be available for vocal control.

Attenuation of muscle spindle firing with artificially increased series compliance during stretch of relaxed muscle.

Muscle spindles relay vital mechanosensory information for movement and posture, but muscle spindle feedback is coupled to skeletal motion by a compliant tendon. Little is known about the effects of tendon compliance on muscle spindle feedback during movement, and the complex firing of muscle spindles makes these effects difficult to predict. Our goal was to investigate changes in muscle spindle firing using added series elastic elements (SEEs) to mimic a more compliant tendon, and to characterize the accompanying changes in firing with respect to muscle-tendon unit (MTU) and muscle fascicle displacements (recorded via sonomicrometry). Sinusoidal, ramp-and-hold and triangular stretches were analysed to examine potential changes in muscle spindle instantaneous firing rates (IFRs) in locomotor- and perturbation-like stretches as well as serial history dependence. Added SEEs effectively reduced overall MTU stiffness and generally reduced muscle spindle firing rates, but the effect differed across stretch types. During sinusoidal stretches, peak and mean firing rates were not reduced and IFR was best-correlated with fascicle velocity. During ramp stretches, SEEs reduced the initial burst, dynamic and static responses of the spindle. Notably, IFR was negatively related to fascicle displacement during the hold phase. During triangular stretches, SEEs reduced the mean IFR during the first and second stretches, affecting the serial history dependence of mean IFR. Overall, these results demonstrate that tendon compliance may attenuate muscle spindle feedback during movement, but these changes cannot be fully explained by reduced muscle fascicle length or velocity, or MTU force.

Capitalizing on skin in orthotics design: the effects of texture on plantar intrinsic foot muscles during locomotion.

Foot orthoses (FO) are a commonly prescribed intervention to alter foot function during walking although their effects have been primarily studied in the extrinsic muscles of the foot. Furthermore, enhancing sensory feedback under the foot sole has been recently shown to alter extrinsic muscle activity during gait; however, the effects of FOs with enhanced sensory feedback on plantar intrinsic foot muscles (PIFMs) remain unknown. Thus, the purpose of this study was to investigate the effect of FOs with and without sensory facilitation on PIFM activity during locomotion. Forty healthy adults completed a series of gait trials in non-textured and textured FOs when walking over hard and soft flooring. Outcome measures included bilateral joint kinematics and electromyography (EMG) of four PIFMs. Results of this study highlight the distinct onset and cessations of each PIFM throughout the stance phase of gait. PIFMs remained active during mid-stance when wearing FOs and textured FOs facilitated muscle activity across the stance phase of gait. Increasing cutaneous input from foot sole skin, via the addition of texture under the foot sole, appears to alter motor-neuron pool excitation of PIFMs. Future academics are encouraged to increase our understanding on which pathologies, diseases, and/or medical conditions would best benefit from textured FOs.

COVID-19 and silent hypoxemia in a minimal closed-loop model of the respiratory rhythm generator.

Silent hypoxemia, or "happy hypoxia," is a puzzling phenomenon in which patients who have contracted COVID-19 exhibit very low oxygen saturation ( SaO 2 < 80%) but do not experience discomfort in breathing. The mechanism by which this blunted response to hypoxia occurs is unknown. We have previously shown that a computational model of the respiratory neural network (Diekman et al. in J Neurophysiol 118(4):2194-2215, 2017) can be used to test hypotheses focused on changes in chemosensory inputs to the central pattern generator (CPG). We hypothesize that altered chemosensory function at the level of the carotid bodies and/or the nucleus tractus solitarii are responsible for the blunted response to hypoxia. Here, we use our model to explore this hypothesis by altering the properties of the gain function representing oxygen sensing inputs to the CPG. We then vary other parameters in the model and show that oxygen carrying capacity is the most salient factor for producing silent hypoxemia. We call for clinicians to measure hematocrit as a clinical index of altered physiology in response to COVID-19 infection.

A computational neural model that incorporates both intrinsic dynamics and sensory feedback in the Aplysia feeding network.

Studying the nervous system underlying animal motor control can shed light on how animals can adapt flexibly to a changing environment. We focus on the neural basis of feeding control in Aplysia californica. Using the Synthetic Nervous System framework, we developed a model of Aplysia feeding neural circuitry that balances neurophysiological plausibility and computational complexity. The circuitry includes neurons, synapses, and feedback pathways identified in existing literature. We organized the neurons into three layers and five subnetworks according to their functional roles. Simulation results demonstrate that the circuitry model can capture the intrinsic dynamics at neuronal and network levels. When combined with a simplified peripheral biomechanical model, it is sufficient to mediate three animal-like feeding behaviors (biting, swallowing, and rejection). The kinematic, dynamic, and neural responses of the model also share similar features with animal data. These results emphasize the functional roles of sensory feedback during feeding.

Our research path toward the restoration of natural sensations in hand prostheses.

The human hand, with its intricate sensory capabilities, plays a pivotal role in our daily interactions with the world. This remarkable organ possesses a wide range of natural sensors that enrich our experiences, enabling us to perceive touch, position, and temperature. These natural sensors work in concert to provide us with a rich sensory experience, enabling us to distinguish between various textures, gauge the force of our grip, determine the position of our fingers without needing to see them, perceive the temperature of objects we come into contact with or detect if a cloth is wet or dry. This complex sensory system is fundamental to our ability to manipulate objects, explore our surroundings, and interact with the world and people around us. In this article, we summarize the research performed in our laboratories over the years and our findings to restore both touch, position, and temperature modalities. The combination of intraneural stimulation, sensory substitution, and wearable technology opens new possibilities for enhancing sensory feedback in prosthetic hands, promising improved functionality and a closer approximation to natural sensory experiences for individuals with limb differences.

The effect of types of sensory feedback on the acquisition and retention of squat performance: A randomized, double-blind, controlled trial.

Various sensory feedback methods are considered important for motor learning, but the effect of each sensory feedback method on effective squat learning still needs to be clarified. This study aimed to investigate the effect of sensory feedback types on the acquisition and retention of a squat. A double-blinded, randomized controlled trial was carried out. Thirty-healthy people were recruited and randomly assigned to the visual feedback group (VFG = 10), tactile feedback group (TFG = 10), and control group (CG = 10). VFG received visual feedback through video data of the participant performing squats, and TFG received tactile feedback through manual contact with a physical therapist. Both groups received feedback on the movements that needed correction after each set was completed. CG maintained rest without receiving any feedback. The retro-reflexive marker, force plate, and electromyography were used to measure body angle, foot center of pressure (COP), and muscle activity. All assessments were measured to confirm a squat acquisition. VFG and TFG showed significant differences in neutral knee position (NKP), trunk forward lean (TFL), anterior knee displacement (AKD), and anteroposterior (AP) foot COP (p < 0.050). In addition, the acquisition was retained until 3 days later for NKP and a week later for TFL, AKD, and AP foot COP in VFG (p < 0.050), while the acquisition was not retained in TFG (p > 0.050). There was no statistically significant change in CG (p > 0.050). This study demonstrated that visual feedback positively affects the acquisition and retention of squats. Therefore, we recommend the use of visual feedback for squat acquisition and retention in exercise novices.

Neural interfacing architecture enables enhanced motor control and residual limb functionality postamputation.

Despite advancements in prosthetic technologies, patients with amputation today suffer great diminution in mobility and quality of life. We have developed a modified below-knee amputation (BKA) procedure that incorporates agonist-antagonist myoneural interfaces (AMIs), which surgically preserve and couple agonist-antagonist muscle pairs for the subtalar and ankle joints. AMIs are designed to restore physiological neuromuscular dynamics, enable bidirectional neural signaling, and offer greater neuroprosthetic controllability compared to traditional amputation techniques. In this prospective, nonrandomized, unmasked study design, 15 subjects with AMI below-knee amputation (AB) were matched with 7 subjects who underwent a traditional below-knee amputation (TB). AB subjects demonstrated significantly greater control of their residual limb musculature, production of more differentiable efferent control signals, and greater precision of movement compared to TB subjects (P < 0.008). This may be due to the presence of greater proprioceptive inputs facilitated by the significantly higher fascicle strains resulting from coordinated muscle excursion in AB subjects (P < 0.05). AB subjects reported significantly greater phantom range of motion postamputation (AB: 12.47 ± 2.41, TB: 10.14 ± 1.45 degrees) when compared to TB subjects (P < 0.05). Furthermore, AB subjects also reported less pain (12.25 ± 5.37) than TB subjects (17.29 ± 10.22) and a significant reduction when compared to their preoperative baseline (P < 0.05). Compared with traditional amputation, the construction of AMIs during amputation confers the benefits of enhanced physiological neuromuscular dynamics, proprioception, and phantom limb perception. Subjects' activation of the AMIs produces more differentiable electromyography (EMG) for myoelectric prosthesis control and demonstrates more positive clinical outcomes.

Activity of Spinal Interneurons during Forward and Backward Locomotion.

Higher vertebrates are capable not only of forward but also backward and sideways locomotion. Also, single steps in different directions are generated for postural corrections. While the networks responsible for the control of forward walking (FW) have been studied in considerable detail, the networks controlling steps in other directions are mostly unknown. Here, to characterize the operation of the spinal locomotor network during FW and backward walking (BW), we recorded the activity of individual spinal interneurons from L4 to L6 during both FW and BW evoked by epidural stimulation (ES) of the spinal cord at L5-L6 in decerebrate cats of either sex. Three groups of neurons were revealed. Group 1 (45%) had a similar phase of modulation during both FW and BW. Group 2 (27%) changed the phase of modulation in the locomotor cycle depending on the direction of locomotion. Group 3 neurons were modulated during FW only (Group 3a, 21%) or during BW only (Group 3b, 7%). We suggest that Group 1 neurons belong to the network generating the vertical component of steps (the limb elevation and lowering) because it should operate similarly during locomotion in any direction, while Groups 2 and 3 neurons belong to the networks controlling the direction of stepping. Results of this study provide new insights into the organization of the spinal locomotor circuits, advance our understanding of ES therapeutic effects, and can potentially be used for the development of novel strategies for recuperation of impaired balance control, which requires the generation of corrective steps in different directions.SIGNIFICANCE STATEMENT Animals and humans can perform locomotion in different directions in relation to the body axis (forward, backward, sideways). While the networks that control forward walking have been studied in considerable detail, the networks controlling steps in other directions are unknown. Here, by recording the activity of the same spinal neurons during forward and backward walking, we revealed three groups of neurons forming, respectively, the network operating similarly during stepping in different directions, the network changing its operation with a change in the direction of stepping, and the network operating only during locomotion in a specific direction. These networks presumably control different aspects of the step. The obtained results provide new insights into the organization of the spinal locomotor networks.

Ventral wing hairs provide tactile feedback for aerial prey capture in the big brown bat, Eptesicus fuscus.

Bats rely on their hand-wings to execute agile flight maneuvers, to grasp objects, and cradle young. Embedded in the dorsal and ventral membranes of bat wings are microscopic hairs. Past research findings implicate dorsal wing hairs in airflow sensing for flight control, but the function of ventral wing hairs has not been previously investigated. Here, we test the hypothesis that ventral wing hairs carry mechanosensory signals for flight control, prey capture, and handling. To test this hypothesis, we used synchronized high-speed stereo video and audio recordings to quantify flight and echolocation behaviors of big brown bats (Eptesicus fuscus) engaged in an aerial insect capture task. We analyzed prey-capture strategy and performance, along with flight kinematics, before and after depilation of microscopic hairs from the bat's ventral wing and tail membranes. We found that ventral wing hair depilation significantly impaired the bat's prey-capture performance. Interestingly, ventral wing hair depilation also produced increases in the bat's flight speed, an effect previously attributed exclusively to airflow sensing along the dorsal wing surface. These findings demonstrate that microscopic hairs embedded in the ventral wing and tail membranes of insectivorous bats provide mechanosensory feedback for prey handling and flight control.

Hindlimb muscle spindles inform preparatory forelimb coordination prior to landing in toads.

Animals move across a wide range of surface conditions in real-world environments to acquire resources and avoid predation. To effectively navigate a variety of surfaces, animals rely on several mechanisms including intrinsic mechanical responses, spinal-level central pattern generators, and neural commands that require sensory feedback. Muscle spindle Ia afferents play a critical role in providing sensory feedback and informing motor control strategies across legged vertebrate locomotion, which is apparent in cases where this sensory input is compromised. Here, we tested the hypothesis that spindle Ia afferents from hindlimb muscles are important for coordinating forelimb landing behavior in the cane toad. We performed bilateral sciatic nerve reinnervations to ablate the stretch reflex from distal hindlimb muscles while allowing for motor neuron recovery. We found that toads significantly delayed the onset and reduced the activation duration of their elbow extensor muscle following spindle Ia afferent ablation in the hindlimbs. However, reinnervated toads achieved similar elbow extension at touchdown to that of their pre-surgery state. Our results suggest that while toads likely tuned the activation timing of forelimb muscles in response to losing Ia afferent sensation from the hindlimbs they were likely able to employ compensatory strategies that allowed them to continue landing effectively with reduced sensory information during take-off. These findings indicate muscle spindle Ia afferents may contribute to tuning complex movements involving multiple limbs.

Real-time vibrotactile pattern generation and identification using discrete event-driven feedback.

This study assesses human identification of vibrotactile patterns by using real-time discrete event-driven feedback. Previously acquired force and bend sensor data from a robotic hand were used to predict movement-type (stationary, flexion, contact, extension, release) and object-type (no object, hard object, soft object) states by using decision tree (DT) algorithms implemented in a field-programmable gate array (FPGA). Six able-bodied humans performed a 2- and 3-step sequential pattern recognition task in which state transitions were signaled as vibrotactile feedback. The stimuli were generated according to predicted classes represented by two frequencies (F1: 80 Hz, F2: 180 Hz) and two magnitudes (M1: low, M2: high) calibrated psychophysically for each participant; and they were applied by two actuators (Haptuators) placed on upper arms. A soft/hard object was mapped to F1/F2; and manipulating it with low/high force was assigned to M1/M2 in the left actuator. On the other hand, flexion/extension movement was mapped to F1/F2 in the right actuator, with movement in air as M1 and during object manipulation as M2. DT algorithm performed better for the object-type (97%) than the movement-type (88%) classification in real time. Participants could recognize feedback associated with 14 discrete-event sequences with low-to-medium accuracy. The performance was higher (76 ± 9% recall, 76 ± 17% precision, 78 ± 4% accuracy) for recognizing any one event in the sequences. The results show that FPGA implementation of classification for discrete event-driven vibrotactile feedback can be feasible in haptic devices with additional cues in the physical context.

The frequency of cortical microstimulation shapes artificial touch.

Intracortical microstimulation (ICMS) of the somatosensory cortex evokes vivid tactile sensations and can be used to convey sensory feedback from brain-controlled bionic hands. Changes in ICMS frequency lead to changes in the resulting sensation, but the discriminability of frequency has only been investigated over a narrow range of low frequencies. Furthermore, the sensory correlates of changes in ICMS frequency remain poorly understood. Specifically, it remains to be elucidated whether changes in frequency only modulate sensation magnitude-as do changes in amplitude-or whether they also modulate the quality of the sensation. To fill these gaps, we trained monkeys to discriminate the frequency of ICMS pulse trains over a wide range of frequencies (from 10 to 400 Hz). ICMS amplitude also varied across stimuli to dissociate sensation magnitude from ICMS frequency and ensure that animals could not make frequency judgments based on magnitude. We found that animals could consistently discriminate ICMS frequency up to ∼200 Hz but that the sensory correlates of frequency were highly electrode dependent: On some electrodes, changes in frequency were perceptually distinguishable from changes in amplitude-seemingly giving rise to a change in sensory quality; on others, they were not. We discuss the implications of our findings for neural coding and for brain-controlled bionic hands.

Wrist speed feedback improves elbow compensation and reaching accuracy for myoelectric transradial prosthesis users in hybrid virtual reaching task.

BACKGROUND: Myoelectric prostheses are a popular choice for restoring motor capability following the loss of a limb, but they do not provide direct feedback to the user about the movements of the device-in other words, kinesthesia. The outcomes of studies providing artificial sensory feedback are often influenced by the availability of incidental feedback. When subjects are blindfolded and disconnected from the prosthesis, artificial sensory feedback consistently improves control; however, when subjects wear a prosthesis and can see the task, benefits often deteriorate or become inconsistent. We theorize that providing artificial sensory feedback about prosthesis speed, which cannot be precisely estimated via vision, will improve the learning and control of a myoelectric prosthesis. METHODS: In this study, we test a joint-speed feedback system with six transradial amputee subjects to evaluate how it affects myoelectric control and adaptation behavior during a virtual reaching task. RESULTS: Our results showed that joint-speed feedback lowered reaching errors and compensatory movements during steady-state reaches. However, the same feedback provided no improvement when control was perturbed. CONCLUSIONS: These outcomes suggest that the benefit of joint speed feedback may be dependent on the complexity of the myoelectric control and the context of the task.

Automated calibration of somatosensory stimulation using reinforcement learning.

BACKGROUND: The identification of the electrical stimulation parameters for neuromodulation is a subject-specific and time-consuming procedure that presently mostly relies on the expertise of the user (e.g., clinician, experimenter, bioengineer). Since the parameters of stimulation change over time (due to displacement of electrodes, skin status, etc.), patients undergo recurrent, long calibration sessions, along with visits to the clinics, which are inefficient and expensive. To address this issue, we developed an automatized calibration system based on reinforcement learning (RL) allowing for accurate and efficient identification of the peripheral nerve stimulation parameters for somatosensory neuroprostheses. METHODS: We developed an RL algorithm to automatically select neurostimulation parameters for restoring sensory feedback with transcutaneous electrical nerve stimulation (TENS). First, the algorithm was trained offline on a dataset comprising 49 subjects. Then, the neurostimulation was then integrated with a graphical user interface (GUI) to create an intuitive AI-based mapping platform enabling the user to autonomously perform the sensation characterization procedure. We assessed the algorithm against the performance of both experienced and naïve and of a brute force algorithm (BFA), on 15 nerves from five subjects. Then, we validated the AI-based platform on six neuropathic nerves affected by distal sensory loss. RESULTS: Our automatized approach demonstrated the ability to find the optimal values of neurostimulation achieving reliable and comfortable elicited sensations. When compared to alternatives, RL outperformed the naïve and BFA, significantly decreasing the time for mapping and the number of delivered stimulation trains, while improving the overall quality. Furthermore, the RL algorithm showed performance comparable to trained experimenters. Finally, we exploited it successfully for eliciting sensory feedback in neuropathic patients. CONCLUSIONS: Our findings demonstrated that the AI-based platform based on a RL algorithm can automatically and efficiently calibrate parameters for somatosensory nerve stimulation. This holds promise to avoid experts' employment in similar scenarios, thanks to the merging between AI and neurotech. Our RL algorithm has the potential to be used in other neuromodulation fields requiring a mapping process of the stimulation parameters. TRIAL REGISTRATION: ClinicalTrial.gov (Identifier: NCT04217005).

The impact of walking on the perception of multichannel electrotactile stimulation in individuals with lower-limb amputation and able-bodied participants.

BACKGROUND: One of the drawbacks of lower-limb prostheses is that they do not provide explicit somatosensory feedback to their users. Electrotactile stimulation is an attractive technology to restore such feedback because it enables compact solutions with multiple stimulation points. This allows stimulating a larger skin area to provide more information concurrently and modulate parameters spatially as well as in amplitude. However, for effective use, electrotactile stimulation needs to be calibrated and it would be convenient to perform this procedure while the subject is seated. However, amplitude and spatial perception can be affected by motion and/or physical coupling between the residual limb and the socket. In the present study, we therefore evaluated and compared the psychometric properties of multichannel electrotactile stimulation applied to the thigh/residual limb during sitting versus walking. METHODS: The comprehensive assessment included the measurement of the sensation and discomfort thresholds (ST & DT), just noticeable difference (JND), number of distinct intervals (NDI), two-point discrimination threshold (2PD), and spatial discrimination performance (SD). The experiment involved 11 able-bodied participants (4 females and 7 males; 29.2 ± 3.8 years), 3 participants with transtibial amputation, and 3 participants with transfemoral amputation. RESULTS: In able-bodied participants, the results were consistent for all the measured parameters, and they indicated that both amplitude and spatial perception became worse during walking. More specifically, ST and DT increased significantly during walking vs. sitting (2.90 ± 0.82 mA vs. 2.00 ± 0.52 mA; p < 0.001 for ST and 7.74 ± 0.84 mA vs. 7.21 ± 1.30 mA; p < 0.05 for DT) and likewise for the JND (22.47 ± 12.21% vs. 11.82 ± 5.07%; p < 0.01), while the NDI became lower (6.46 ± 3.47 vs. 11.27 ± 5.18 intervals; p < 0.01). Regarding spatial perception, 2PD was higher during walking (69.78 ± 17.66 mm vs. 57.85 ± 14.87 mm; p < 0.001), while the performance of SD was significantly lower (56.70 ± 10.02% vs. 64.55 ± 9.44%; p < 0.01). For participants with lower-limb amputation, the ST, DT, and performance in the SD assessment followed the trends observed in the able-bodied population. The results for 2PD and JND were however different and subject-specific. CONCLUSION: The conducted evaluation demonstrates that electrotactile feedback should be calibrated in the conditions in which it will be used (e.g., during walking). The calibration during sitting, while more convenient, might lead to an overly optimistic (or in some cases pessimistic) estimate of sensitivity. In addition, the results underline that calibration is particularly important in people affected by lower-limb loss to capture the substantial variability in the conditions of the residual limb and prosthesis setup. These insights are important for the implementation of artificial sensory feedback in lower-limb prosthetics applications.