Large-scale intramuscular electrode system for chronic electromyography and functional electrical stimulation.

To understand how the central nervous system (CNS) enacts movements, it seems important to monitor the activities of the many muscles involved. Likewise, to restore complex movements to paralyzed limbs with electrical stimulation requires access to most limb muscles. Intramuscular electrodes are needed to obtain isolated recordings or stimulation of individual muscles. As such, we developed and tested the stability of large arrays of implanted intramuscular electrodes. We implanted 58 electrodes in 29 upper limb muscles in each of three macaques. Electrode connectors were protected within a skull-mounted chamber. During surgery, wires were tunneled subcutaneously to target muscles, where gold anchors were crimped onto the leads. The anchors were then deployed with an insertion device. In two monkeys, the chamber was fixed to the skull with a titanium baseplate rather than acrylic cement. In multiple sessions up to 15 wk after surgery, electromyographic (EMG) signals were recorded while monkeys made the same reaching movement. EMG signals were stable, with an average (SD) coefficient of variation across sessions of 0.24 ± 0.15. In addition, at 4, 8, and 16 wk after surgery, forces to incrementing stimulus pulses were measured for each electrode. The threshold current needed to evoke a response at 16 wk was not different from that at 4 wk. Likewise, peak force evoked by 16 mA of current at 16 wk was not different from 4 wk. The stability of this system implies it could be effectively used to monitor and stimulate large numbers of muscles needed to understand the control of natural and evoked movements.NEW AND NOTEWORTHY A new method was developed to enable long-lasting recording and stimulation of large numbers of muscles with intramuscular electrodes. Electromyographic signals and evoked force responses in 29 upper limb muscles remained stable over several months when tested in nonhuman primates. This system could be used effectively to monitor and stimulate numerous muscles needed to more fully understand the control of natural and evoked movements.

A motor unit-based model of muscle fatigue.

Muscle fatigue is a temporary decline in the force and power capacity of skeletal muscle resulting from muscle activity. Because control of muscle is realized at the level of the motor unit (MU), it seems important to consider the physiological properties of motor units when attempting to understand and predict muscle fatigue. Therefore, we developed a phenomenological model of motor unit fatigue as a tractable means to predict muscle fatigue for a variety of tasks and to illustrate the individual contractile responses of MUs whose collective action determines the trajectory of changes in muscle force capacity during prolonged activity. An existing MU population model was used to simulate MU firing rates and isometric muscle forces and, to that model, we added fatigue-related changes in MU force, contraction time, and firing rate associated with sustained voluntary contractions. The model accurately estimated endurance times for sustained isometric contractions across a wide range of target levels. In addition, simulations were run for situations that have little experimental precedent to demonstrate the potential utility of the model to predict motor unit fatigue for more complicated, real-world applications. Moreover, the model provided insight into the complex orchestration of MU force contributions during fatigue, that would be unattainable with current experimental approaches.

Inhibition linearizes firing rate responses in human motor units: implications for the role of persistent inward currents.

KEY POINTS: Motor neurons are the output neurons of the central nervous system and are responsible for controlling muscle contraction. When initially activated during voluntary contraction, firing rates of motor neurons increase steeply but then level out at modest rates. Activation of an intrinsic source of excitatory current at recruitment onset may underlie the initial steep increase in firing rate in motor neurons. We attempted to disable this intrinsic excitatory current by artificially activating an inhibitory reflex. When motor neuron activity was recorded while the inhibitory reflex was engaged, firing rates no longer increased steeply, suggesting that the intrinsic excitatory current was probably responsible for the initial sharp rise in motor neuron firing rate. ABSTRACT: During graded isometric contractions, motor unit (MU) firing rates increase steeply upon recruitment but then level off at modest rates even though muscle force continues to increase. The mechanisms underlying such firing behaviour are not known although activation of persistent inward currents (PICs) might be involved. PICs are intrinsic, voltage-dependent currents that activate strongly when motor neurons (MNs) are first recruited. Such activation might cause a sharp escalation in depolarizing current and underlie the steep initial rise in MU firing rate. Because PICs can be disabled with synaptic inhibition, we hypothesized that artificial activation of an inhibitory pathway might curb this initial steep rise in firing rate. To test this, human subjects performed slow triangular ramp contractions of the ankle dorsiflexors in the absence and presence of tonic synaptic inhibition delivered to tibialis anterior (TA) MNs by sural nerve stimulation. Firing rate profiles (expressed as a function of contraction force) of TA MUs recorded during these tasks were compared for control and stimulation conditions. Under control conditions, during the ascending phase of the triangular contractions, 93% of the firing rate profiles were best fitted by rising exponential functions. With stimulation, however, firing rate profiles were best fitted with linear functions or with less steeply rising exponentials. Firing rate profiles for the descending phases of the contractions were best fitted with linear functions for both control and stimulation conditions. These results seem consistent with the idea that PICs contribute to non-linear firing rate profiles during ascending but not descending phases of contractions.

Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 µm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

Distinguishing intrinsic from extrinsic factors underlying firing rate saturation in human motor units.

During voluntary contraction, firing rates of individual motor units (MUs) increase modestly over a narrow force range beyond which little additional increase in firing rate is seen. Such saturation of MU discharge may be a consequence of extrinsic factors that limit net synaptic excitation acting on motor neurons (MNs) or may be due to intrinsic properties of the MNs. Two sets of experiments involving recording of human biceps brachii MUs were carried out to evaluate saturation. In the first set, the extent of saturation was quantified for 136 low-threshold MUs during isometric ramp contractions. Firing rate-force data were best fit by a saturating function for 90% of MUs recorded with a maximum rate of 14.8 ± 2.0 impulses/s. In the second set of experiments, to distinguish extrinsic from intrinsic factors underlying saturation, we artificially augmented descending excitatory drive to biceps MNs by activation of muscle spindle afferents through tendon vibration. We examined the change in firing rate caused by tendon vibration in 96 MUs that were voluntarily activated at rates below and at saturation. Vibration had little effect on the discharge of MUs that were firing at saturation frequencies but strongly increased firing rates of the same units when active at lower frequencies. These results indicate that saturation is likely caused by intrinsic mechanisms that prevent further increases in firing rate in the presence of increasing synaptic excitation. Possible intrinsic cellular mechanisms that limit firing rates of motor units during voluntary effort are discussed.

Prediction of muscle activity during loaded movements of the upper limb.

BACKGROUND: Accurate prediction of electromyographic (EMG) signals associated with a variety of motor behaviors could, in theory, serve as activity templates needed to evoke movements in paralyzed individuals using functional electrical stimulation. Such predictions should encompass complex multi-joint movements and include interactions with objects in the environment. METHODS: Here we tested the ability of different artificial neural networks (ANNs) to predict EMG activities of 12 arm muscles while human subjects made free movements of the arm or grasped and moved objects of different weights and dimensions. Inputs to the trained ANNs included hand position, hand orientation, and thumb grip force. RESULTS: The ability of ANNs to predict EMG was equally as good for tasks involving interactions with external loads as for unloaded movements. The ANN that yielded the best predictions was a feed-forward network consisting of a single hidden layer of 30 neural elements. For this network, the average coefficient of determination (R2 value) between predicted and actual EMG signals across all nine subjects and 12 muscles during movements that involved episodes of moving objects was 0.43. CONCLUSION: This reasonable accuracy suggests that ANNs could be used to provide an initial estimate of the complex patterns of muscle stimulation needed to produce a wide array of movements, including those involving object interaction, in paralyzed individuals.

TMS-derived short afferent inhibition discriminates cognitive status in older adults without dementia.

Aging is a complex and diverse biological process characterized by progressive molecular, cellular, and tissue damage, resulting in a loss of physiological integrity and heightened vulnerability to pathology. This biological diversity corresponds with highly variable cognitive trajectories, which are further confounded by genetic and environmental factors that influence the resilience of the aging brain. Given this complexity, there is a need for neurophysiological indicators that not only discern physiologic and pathologic aging but also closely align with cognitive trajectories. Transcranial Magnetic Stimulation (TMS) may have utility in this regard as a non-invasive brain stimulation tool that can characterize features of cortical excitability. Particularly, as a proxy for central cholinergic function, short-afferent inhibition (SAI) dysfunction is robustly associated with cognitive deficits in the latter stages of Alzheimer's Disease and Related Dementia (ADRD). In this study, we evaluated SAI in healthy young adults and older adults who, though absent clinical diagnoses, were algorithmically classified as cognitively normal (CN) or cognitively impaired (CI) according to the Jak/Bondi actuarial criteria. We report that SAI is preserved in the Old-CN cohort relative to the young adults, and SAI is significantly diminished in the Old-CI cohort relative to both young and CN older adults. Additionally, diminished SAI was significantly associated with impaired sustained attention and working memory. As a proxy measure for central cholinergic deficits, we discuss the potential value of SAI for discerning physiological and pathological aging.

Interoceptive Signals Bias Decision Making in Rhesus Macaques.

Several influential theories have proposed that interoceptive signals, sent from the body to the brain, contribute to neural processes that coordinate complex behaviors. Using pharmacological agents that do not cross the blood-brain barrier, we altered interoceptive states and evaluated their effect on decision-making in rhesus monkeys. We used glycopyrrolate, a non-specific muscarinic (parasympathetic) antagonist, and isoproterenol, a beta-1/2 (sympathetic) agonist, to create a sympathetic-dominated physiological state indexed by increased heart rate. Rhesus monkeys were trained on two variants of an approach-avoidance conflict task, where they chose between enduring mildly aversive stimuli in exchange for a steady flow of rewards, or cancelling the aversive stimuli, forgoing the rewards. The delay to interrupt the aversive stimuli and the reward were used as a measure of the cost-benefit estimation that drove the monkeys' decisions. Both drugs altered approach-avoidance decisions, substantially reducing the delay to interrupt the aversive stimuli. To determine whether this autonomic state lowered tolerance to aversive stimuli or reduced the subjective value of the reward, we tested the effects of glycopyrrolate on a food preference task. Food preference was unaltered, suggesting that the sympathetic dominated state selectively reduces tolerance for aversive stimuli without altering reward-seeking behaviors. As these drugs have no direct effect on brain physiology, interoceptive afferents are the most likely mechanism by which decision making was biased toward avoidance.

A context-dependent switch from sensing to feeling in the primate amygdala.

The skin transmits affective signals that integrate into our social vocabulary. As the socio-affective aspects of touch are likely processed in the amygdala, we compare neural responses to social grooming and gentle airflow recorded from the amygdala and the primary somatosensory cortex of non-human primates. Neurons in the somatosensory cortex respond to both types of tactile stimuli. In the amygdala, however, neurons do not respond to individual grooming sweeps even though grooming elicits autonomic states indicative of positive affect. Instead, many show changes in baseline firing rates that persist throughout the grooming bout. Such baseline fluctuations are attributed to social context because the presence of the groomer alone can account for the observed changes in baseline activity. It appears, therefore, that during grooming, the amygdala stops responding to external inputs on a short timescale but remains responsive to social context (or the associated affective states) on longer time scales.

Short-term synchrony in diverse motor nuclei presumed to receive different extents of direct cortical input.

Motor units within human muscles usually exhibit a significant degree of short-term synchronization. Such coincident spiking typically has been attributed to last-order projections that provide common synaptic input across motor neurons. The extent of branched input arising directly from cortical neurons has often been suggested as a critical factor determining the magnitude of short-term synchrony. The purpose of this study, therefore, was to quantify motor unit synchrony in a variety of human muscles differing in the presumed extent of cortical input to their respective motor nuclei. Cross-correlation histograms were generated from the firing times of 551 pairs of motor units in 16 human muscles. Motor unit synchrony tended to be weakest for proximal muscles and strongest for more distal muscles. Previous work in monkeys and humans has shown that the strength of cortical inputs to motor neurons also exhibits a similar proximal-to-distal gradient. However, in the present study, proximal-distal location was not an exclusive predictor of synchrony magnitude. The muscle that exhibited the least synchrony was an elbow flexor, whereas the greatest synchrony was most often found in intrinsic foot muscles. Furthermore, the strength of corticospinal inputs to the abductor hallucis muscle, an intrinsic foot muscle, as assessed through transcranial magnetic stimulation, was weaker than that projecting to the tibialis anterior muscle, even though the abductor hallucis muscle had higher synchrony values compared with the tibialis anterior muscle. We argue, therefore, that factors other than the potency of cortical inputs to motor neurons, such as the number of motor neurons innervating a muscle, significantly affects motor unit synchrony.

Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure.

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4ß2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4ß2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

The role of the amygdala in processing social and affective touch.

The amygdala plays a central role in emotion and social behavior, yet its role in processing social and affective touch is not well established. Longitudinal studies reveal that touch-deprived infants show later in life exaggerated emotional reactivity related to structural and functional changes in the amygdala and other brain structures. The internal organization and connectivity of the amygdala is well-suited to process the sensory features of tactile stimuli and also the socio-cognitive dimensions of the received touch. The convergent processing of bottom-up and top-down pathways that carry information about touch results in the elaboration of context appropriate autonomic responses. Indeed, the positive value of affective touch in humans and social grooming in non-human primates is correlated with vagal tone and the release of oxytocin and endogenous opioids. Grooming, the non-human primate equivalent of affective touch in humans, reduces vigilance, that depends on the amygdala. During touch-induced vagal tone and low vigilance, neural activity in the amygdala is substantially different from activity corresponding to the attentive processing of tactile stimuli. Under these circumstances neurons no longer respond phasically to each touch stimulus, rather they signal a sustained functional state in which the amygdala appears decoupled from monitoring the external environment.

Restoration of complex movement in the paralyzed upper limb.

Objective.Functional electrical stimulation (FES) involves artificial activation of skeletal muscles to reinstate motor function in paralyzed individuals. While FES applied to the upper limb has improved the ability of tetraplegics to perform activities of daily living, there are key shortcomings impeding its widespread use. One major limitation is that the range of motor behaviors that can be generated is restricted to a small set of simple, preprogrammed movements. This limitation stems from the substantial difficulty in determining the patterns of stimulation across many muscles required to produce more complex movements. Therefore, the objective of this study was to use machine learning to flexibly identify patterns of muscle stimulation needed to evoke a wide array of multi-joint arm movements.Approach. Arm kinematics and electromyographic (EMG) activity from 29 muscles were recorded while a 'trainer' monkey made an extensive range of arm movements. Those data were used to train an artificial neural network that predicted patterns of muscle activity associated with a new set of movements. Those patterns were converted into trains of stimulus pulses that were delivered to upper limb muscles in two other temporarily paralyzed monkeys.Main results. Machine-learning based prediction of EMG was good for within-subject predictions but appreciably poorer for across-subject predictions. Evoked responses matched the desired movements with good fidelity only in some cases. Means to mitigate errors associated with FES-evoked movements are discussed.Significance. Because the range of movements that can be produced with our approach is virtually unlimited, this system could greatly expand the repertoire of movements available to individuals with high level paralysis.

Mechanical properties and neural control of human hand motor units.

Motor units serve both as the mechanical apparatus and the final stage of neural processing through which motor behaviours are enacted. Therefore, knowledge about the contractile properties and organization of the neural inputs to motor units supplying finger muscles is essential for understanding the control strategies underlying the diverse motor functions of the human hand. In this brief review, basic contractile properties of motor units residing in human hand muscles are described. Hand motor units are not readily categorized into the classical physiological types as established in the cat gastrocnemius muscle. In addition, the distribution of descending synaptic inputs to motor nuclei supplying different hand muscles is outlined. Motor neurons innervating intrinsic muscles appear to have relatively independent lines of input from supraspinal centres whereas substantial divergence of descending input is seen across motor nuclei supplying extrinsic hand muscles. The functional significance of such differential organizations of descending inputs for the control of hand movements is discussed.

Effects of persistent inward currents, accommodation, and adaptation on motor unit behavior: a simulation study.

Motor neurons are often assumed to generate spikes in proportion to the excitatory synaptic input received. There are, however, many intrinsic properties of motor neurons that might affect this relationship, such as persistent inward currents (PICs), spike-threshold accommodation, or spike-frequency adaptation. These nonlinear properties have been investigated in reduced animal preparation but have not been well studied during natural motor behaviors because of the difficulty in characterizing synaptic input in intact animals. Therefore, we studied the influence of each of these intrinsic properties on spiking responses and muscle force using a population model of motor units that simulates voluntary contractions in human subjects. In particular, we focused on the difference in firing rate of low-threshold motor units when higher threshold motor units were recruited and subsequently derecruited, referred to as ΔF. Others have used ΔF to evaluate the extent of PIC activation during voluntary behavior. Our results showed that positive ΔF values could arise when any one of these nonlinear properties was included in the simulations. Therefore, a positive ΔF should not be considered as exclusive evidence for PIC activation. Furthermore, by systematically varying contraction duration and speed in our simulations, we identified a means that might be used experimentally to distinguish among PICs, accommodation, and adaptation as contributors to ΔF.

Synchronization of presynaptic input to motor units of tongue, inspiratory intercostal, and diaphragm muscles.

The respiratory central pattern generator distributes rhythmic excitatory input to phrenic, intercostal, and hypoglossal premotor neurons. The degree to which this input shapes motor neuron activity can vary across respiratory muscles and motor neuron pools. We evaluated the extent to which respiratory drive synchronizes the activation of motor unit pairs in tongue (genioglossus, hyoglossus) and chest-wall (diaphragm, external intercostals) muscles using coherence analysis. This is a frequency domain technique, which characterizes the frequency and relative strength of neural inputs that are common to each of the recorded motor units. We also examined coherence across the two tongue muscles, as our previous work shows that, despite being antagonists, they are strongly coactivated during the inspiratory phase, suggesting that excitatory input from the premotor neurons is distributed broadly throughout the hypoglossal motoneuron pool. All motor unit pairs showed highly correlated activity in the low-frequency range (1-8 Hz), reflecting the fundamental respiratory frequency and its harmonics. Coherence of motor unit pairs recorded either within or across the tongue muscles was similar, consistent with broadly distributed premotor input to the hypoglossal motoneuron pool. Interestingly, motor units from diaphragm and external intercostal muscles showed significantly higher coherence across the 10-20-Hz bandwidth than tongue-muscle units. We propose that the lower coherence in tongue-muscle motor units over this range reflects a larger constellation of presynaptic inputs, which collectively lead to a reduction in the coherence between hypoglossal motoneurons in this frequency band. This, in turn, may reflect the relative simplicity of the respiratory drive to the diaphragm and intercostal muscles, compared with the greater diversity of functions fulfilled by muscles of the tongue.

Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons.

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.

Number of motor units in human abductor hallucis.

Motor unit number was estimated for the human abductor hallucis (AH) muscle in 11 subjects by counting the number of increments in surface electromyographic responses to progressive increases in current-pulse amplitude applied to the muscle-nerve. The average motor unit count for AH (43) was substantially smaller than that estimated for other human muscles. Consequently, motor unit activity should be readily recordable up to high forces in AH, making it well suited for studies of recruitment and rate coding.

Transcranial magnetic stimulation reveals diminished homoeostatic metaplasticity in cognitively impaired adults.

Homoeostatic metaplasticity is a neuroprotective physiological feature that counterbalances Hebbian forms of plasticity to prevent network destabilization and hyperexcitability. Recent animal models highlight dysfunctional homoeostatic metaplasticity in the pathogenesis of Alzheimer's disease. However, the association between homoeostatic metaplasticity and cognitive status has not been systematically characterized in either demented or non-demented human populations, and the potential value of homoeostatic metaplasticity as an early biomarker of cognitive impairment has not been explored in humans. Here, we report that, through pre-conditioning the synaptic activity prior to non-invasive brain stimulation, the association between homoeostatic metaplasticity and cognitive status could be established in a population of non-demented human subjects (older adults across cognitive spectrums; all within the non-demented range). All participants (n = 40; age range, 65-74, 47.5% female) underwent a standardized neuropsychological battery, magnetic resonance imaging and a transcranial magnetic stimulation protocol. Specifically, we sampled motor-evoked potentials with an input/output curve immediately before and after repetitive transcranial magnetic stimulation to assess neural plasticity with two experimental paradigms: one with voluntary muscle contraction (i.e. modulated synaptic activity history) to deliberately introduce homoeostatic interference, and one without to serve as a control condition. From comparing neuroplastic responses across these experimental paradigms and across cohorts grouped by cognitive status, we found that (i) homoeostatic metaplasticity is diminished in our cohort of cognitively impaired older adults and (ii) this neuroprotective feature remains intact in cognitively normal participants. This novel finding suggests that (i) future studies should expand their scope beyond just Hebbian forms of plasticity that are traditionally assessed when using non-invasive brain stimulation to investigate cognitive ageing and (ii) the potential value of homoeostatic metaplasticity in serving as a biomarker for cognitive impairment should be further explored.