Movement-dependent electrical stimulation for volitional strengthening of cortical connections in behaving monkeys.

Correlated activity of neurons can lead to long-term strengthening or weakening of the connections between them. In addition, the behavioral context, imparted by execution of physical movements or the presence of a reward, can modulate the plasticity induced by Hebbian mechanisms. In the present study, we have combined behavior and induced neuronal correlations to strengthen connections in the motor cortex of adult behaving monkeys. Correlated activity was induced using an electrical-conditioning protocol in which stimuli gated by voluntary movements were used to produce coactivation of neurons at motor-cortical sites involved in those movements. Delivery of movement-dependent stimulation resulted in small increases in the strength of associated cortical connections immediately after conditioning. Remarkably, when paired with further repetition of the movements that gated the conditioning stimuli, there were substantially larger gains in the strength of cortical connections, which occurred in a use-dependent manner, without delivery of additional conditioning stimulation. In the absence of such movements, little change was observed in the strength of motor-cortical connections. Performance of the motor behavior in the absence of conditioning also did not produce any changes in connectivity. Our results show that combining movement-gated stimulation with further natural use of the "conditioned" pathways after stimulation ends can produce use-dependent strengthening of connections in adult primates, highlighting an important role for behavior in cortical plasticity. Our data also provide strong support for combining movement-gated stimulation with use-dependent physical rehabilitation for strengthening connections weakened by a stroke or spinal cord injury.

Cortical Responses to Vagus Nerve Stimulation Are Modulated by Brain State in Nonhuman Primates.

Vagus nerve stimulation (VNS) has been tested as therapy for several brain disorders and as a means to modulate cortical excitability and brain plasticity. Cortical effects of VNS, manifesting as vagal-evoked potentials (VEPs), are thought to arise from activation of ascending cholinergic and noradrenergic systems. However, it is unknown whether those effects are modulated by brain state at the time of stimulation. In 2 freely behaving macaque monkeys, we delivered short trains of 5 pulses to the left cervical vagus nerve at different frequencies (5-300 Hz) while recording local field potentials (LFPs) from sites in contralateral prefrontal, sensorimotor and parietal cortical areas. Brain states were inferred from spectral components of LFPs and the presence of overt movement: active awake, resting awake, REM sleep and NREM sleep. VNS elicited VEPs in all sampled cortical areas. VEPs comprised early (<70 ms), intermediate (70-250 ms) and late (>250 ms) components. The magnitude of the intermediate and late components was largest during NREM sleep and smallest during wakefulness, whereas that of the early component was not modulated by brain state. VEPs during NREM were larger for stimuli delivered at the depolarized phase of ongoing delta oscillations. Higher pulsing frequencies generated larger VEPs. These short VNS trains did not affect brain state transitions during wakefulness or sleep. Our findings suggest that ongoing brain state modulates the evoked effects of VNS on cortical activity. This has implications for the role of ongoing cortical activity and brain state in shaping cortical responses to peripheral stimuli, for the modulation of vagal interoceptive signaling by cortical activity, and for the dose calibration of VNS therapies.

Paired Stimulation for Spike-Timing-Dependent Plasticity in Primate Sensorimotor Cortex.

Classic in vitro studies have described spike-timing-dependent plasticity (STDP) at a synapse: the connection from neuron A to neuron B is strengthened (or weakened) when A fires before (or after) B within an optimal time window. Accordingly, more recent in vivo works have demonstrated behavioral effects consistent with an STDP mechanism; however, many relied on single-unit recordings. The ability to modify cortical connections becomes useful in the context of injury, when connectivity and associated behavior are compromised. To avoid the need for long-term, stable isolation of single units, one could control timed activation of two cortical sites with paired electrical stimulation. We tested the hypothesis that STDP could be induced via prolonged paired stimulation as quantified by cortical evoked potentials (EPs) in the sensorimotor cortex of awake, behaving monkeys. Paired simulation between two interconnected sites produced robust effects in EPs consistent with STDP, but only at 2/15 tested pairs. The stimulation protocol often produced increases in global network excitability or depression of the conditioned pair. Together, these results suggest that paired stimulation in vivo is a viable method to induce STDP between cortical populations, but that factors beyond activation timing must be considered to produce conditioning effects.SIGNIFICANCE STATEMENT Plasticity of neural connections is important for development, learning, memory, and recovery from injury. Cellular mechanisms underlying spike-timing-dependent plasticity have been studied extensively in vitro Recent in vivo work has demonstrated results consistent with the previously defined cellular mechanisms; however, the output measure in these studies was typically an indirect assessment of plasticity at the neural level. Here, we show direct plasticity in recordings of neuronal populations in awake, behaving nonhuman primates induced by paired electrical stimulation. In contrast to in vitro studies, we found that plastic effects were only produced between specific cortical areas. These findings suggest that similar mechanisms drive plasticity in vitro and in vivo, but that cortical architecture may contribute significantly to site-dependent effects.

Targeted, activity-dependent spinal stimulation produces long-lasting motor recovery in chronic cervical spinal cord injury.

Use-dependent movement therapies can lead to partial recovery of motor function after neurological injury. We attempted to improve recovery by developing a neuroprosthetic intervention that enhances movement therapy by directing spike timing-dependent plasticity in spared motor pathways. Using a recurrent neural-computer interface in rats with a cervical contusion of the spinal cord, we synchronized intraspinal microstimulation below the injury with the arrival of functionally related volitional motor commands signaled by muscle activity in the impaired forelimb. Stimulation was delivered during physical retraining of a forelimb behavior and throughout the day for 3 mo. Rats receiving this targeted, activity-dependent spinal stimulation (TADSS) exhibited markedly enhanced recovery compared with animals receiving targeted but open-loop spinal stimulation and rats receiving physical retraining alone. On a forelimb reach and grasp task, TADSS animals recovered 63% of their preinjury ability, more than two times the performance level achieved by the other therapy groups. Therapeutic gains were maintained for 3 additional wk without stimulation. The results suggest that activity-dependent spinal stimulation can induce neural plasticity that improves behavioral recovery after spinal cord injury.

Direct control of paralysed muscles by cortical neurons.

A potential treatment for paralysis resulting from spinal cord injury is to route control signals from the brain around the injury by artificial connections. Such signals could then control electrical stimulation of muscles, thereby restoring volitional movement to paralysed limbs. In previously separate experiments, activity of motor cortex neurons related to actual or imagined movements has been used to control computer cursors and robotic arms, and paralysed muscles have been activated by functional electrical stimulation. Here we show that Macaca nemestrina monkeys can directly control stimulation of muscles using the activity of neurons in the motor cortex, thereby restoring goal-directed movements to a transiently paralysed arm. Moreover, neurons could control functional stimulation equally well regardless of any previous association to movement, a finding that considerably expands the source of control signals for brain-machine interfaces. Monkeys learned to use these artificial connections from cortical cells to muscles to generate bidirectional wrist torques, and controlled multiple neuron-muscle pairs simultaneously. Such direct transforms from cortical activity to muscle stimulation could be implemented by autonomous electronic circuitry, creating a relatively natural neuroprosthesis. These results are the first demonstration that direct artificial connections between cortical cells and muscles can compensate for interrupted physiological pathways and restore volitional control of movement to paralysed limbs.

Axonal thinning and extensive remyelination without chronic demyelination in spinal injured rats.

Remyelination following spinal cord injury (SCI) is thought to be incomplete; demyelination is reported to persist chronically and is proposed as a compelling therapeutic target. Yet most reports do not distinguish between the myelin status of intact axons and injury-severed axons whose proximal stumps persist but provide no meaningful function. We previously found full remyelination of spared, intact rubrospinal axons caudal to the lesion in chronic mouse SCI. However, the clinical concept of chronically demyelinated spared axons remains controversial. Since mouse models may have limitations in clinical translation, we asked whether the capacity for full remyelination is conserved in clinically relevant chronic rat SCI. We determined myelin status by examining paranodal protein distribution on anterogradely labeled, intact corticospinal and rubrospinal axons throughout the extent of the lesion. Demyelination was evident on proximal stumps of severed axons, but not on intact axons. For the first time, we demonstrate that a majority of intact axons exhibit remyelination (at least one abnormally short internode, <100 µm). Remarkably, shortened internodes were significantly concentrated at the lesion epicenter and individual axons were thinned by 23% compared with their rostral and caudal zones. Mathematical modeling predicted a 25% decrease in conduction velocity at the lesion epicenter due to short internodes and axonal thinning. In conclusion, we do not find a large chronically demyelinated population to target with remyelination therapies. Interventions may be better focused on correcting structural or molecular abnormalities of regenerated myelin.

Sensitivity of spinal neurons to GABA and glycine during voluntary movement in behaving monkeys.

GABAergic and glycinergic inhibition play key roles in the function of spinal motor pathways. However, there is little direct information on the extent to which inhibition controls the activity of spinal neurons during behavior or the relative effectiveness of GABA and glycine on cell activity under normal conditions. These issues were investigated in three macaque monkeys trained to perform voluntary ramp-and-hold wrist movements and grip. Pipettes with an extracellular recording electrode and iontophoresis barrels were used to eject GABA, glycine, and/or their respective antagonists, bicuculline and strychnine, as the activity of single neurons was recorded in the C6-T1 spinal segments during hand movements. The firing rate of the vast majority of neurons decreased when an inhibitory neurotransmitter was ejected from the electrode, suggesting that most movement-related spinal neurons are sensitive to both GABA and glycine. Most movement-related neurons exhibited increased activity during iontophoresis of an antagonist, suggesting that both GABAergic and glycinergic inhibition actively regulate the majority of spinal neurons during movement. These conclusions were supported by the responses of neurons tested with both agonists or both antagonists. Bicuculline and strychnine produced the largest increases in firing rate during dynamic movements (ramp phase), smaller increases during maintained torque/force (hold phase), and the smallest increase during the rest period. Since excitatory inputs also tend to increase progressively from rest to static to dynamic muscle contractions, this result is consistent with coupled excitatory and inhibitory inputs to spinal neurons during movement.

Responses of Cortical Neurons to Intracortical Microstimulation in Awake Primates.

Intracortical microstimulation (ICMS) is commonly used in many experimental and clinical paradigms; however, its effects on the activation of neurons are still not completely understood. To document the responses of cortical neurons in awake nonhuman primates to stimulation, we recorded single-unit activity while delivering single-pulse stimulation via Utah arrays implanted in primary motor cortex (M1) of three macaque monkeys. Stimuli between 5 and 50 µA delivered to single channels reliably evoked spikes in neurons recorded throughout the array with delays of up to 12 ms. ICMS pulses also induced a period of inhibition lasting up to 150 ms that typically followed the initial excitatory response. Higher current amplitudes led to a greater probability of evoking a spike and extended the duration of inhibition. The likelihood of evoking a spike in a neuron was dependent on the spontaneous firing rate as well as the delay between its most recent spike time and stimulus onset. Tonic repetitive stimulation between 2 and 20 Hz often modulated both the probability of evoking spikes and the duration of inhibition; high-frequency stimulation was more likely to change both responses. On a trial-by-trial basis, whether a stimulus evoked a spike did not affect the subsequent inhibitory response; however, their changes over time were often positively or negatively correlated. Our results document the complex dynamics of cortical neural responses to electrical stimulation that need to be considered when using ICMS for scientific and clinical applications.

Local field potentials and single unit dynamics in motor cortex of unconstrained macaques during different behavioral states.

Different sleep stages have been shown to be vital for a variety of brain functions, including learning, memory, and skill consolidation. However, our understanding of neural dynamics during sleep and the role of prominent LFP frequency bands remain incomplete. To elucidate such dynamics and differences between behavioral states we collected multichannel LFP and spike data in primary motor cortex of unconstrained macaques for up to 24 h using a head-fixed brain-computer interface (Neurochip3). Each 8-s bin of time was classified into awake-moving (Move), awake-resting (Rest), REM sleep (REM), or non-REM sleep (NREM) by using dimensionality reduction and clustering on the average spectral density and the acceleration of the head. LFP power showed high delta during NREM, high theta during REM, and high beta when the animal was awake. Cross-frequency phase-amplitude coupling typically showed higher coupling during NREM between all pairs of frequency bands. Two notable exceptions were high delta-high gamma and theta-high gamma coupling during Move, and high theta-beta coupling during REM. Single units showed decreased firing rate during NREM, though with increased short ISIs compared to other states. Spike-LFP synchrony showed high delta synchrony during Move, and higher coupling with all other frequency bands during NREM. These results altogether reveal potential roles and functions of different LFP bands that have previously been unexplored.

Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues.

Recent advances in tissue processing, labeling, and fluorescence microscopy are providing unprecedented views of the structure of cells and tissues at sub-diffraction resolutions and near single molecule sensitivity, driving discoveries in diverse fields of biology, including neuroscience. Biological tissue is organized over scales of nanometers to centimeters. Harnessing molecular imaging across three-dimensional samples on this scale requires new types of microscopes with larger fields of view and working distance, as well as higher imaging throughput. We present a new expansion-assisted selective plane illumination microscope (ExA-SPIM) with diffraction-limited and aberration-free performance over a large field of view (85 mm 2 ) and working distance (35 mm). Combined with new tissue clearing and expansion methods, the microscope allows nanoscale imaging of centimeter-scale samples, including entire mouse brains, with diffraction-limited resolutions and high contrast without sectioning. We illustrate ExA-SPIM by reconstructing individual neurons across the mouse brain, imaging cortico-spinal neurons in the macaque motor cortex, and tracing axons in human white matter.

A cerebellar origin of feedforward inhibition to the motor cortex in non-human primates.

Voluntary movements are driven by coordinated activity across a large population of motor cortical neurons. Formation of this activity is controlled by local interactions and long-range inputs. How remote areas of the brain communicate with motor cortical neurons to effectively drive movement remains unclear. We address this question by studying the cerebellar-thalamocortical system. We find that thalamic input to the motor cortex triggers feedforward inhibition by contacting inhibitory cells via highly effective GluR2-lacking AMPA receptors and that, during task performance, the activity of parvalbumin (PV) and pyramidal cells exhibits relations comparable with movement parameters. We also find that the movement-related activity of PV interneurons precedes firing of pyramidal cells. This counterintuitive sequence of events, where inhibitory cells are recruited more strongly and before excitatory cells, may amplify the cortical effect of cerebellar signals in a way that exceeds their sheer synaptic efficacy by suppressing other inputs.

Transcriptome of rat subcortical white matter and spinal cord after spinal injury and cortical stimulation.

Spinal cord injury disrupts ascending and descending neural signals causing sensory and motor dysfunction. Neuromodulation with electrical stimulation is used in both clinical and research settings to induce neural plasticity and improve functional recovery following spinal trauma. However, the mechanisms by which electrical stimulation affects recovery remain unclear. In this study we examined the effects of cortical electrical stimulation following injury on transcription at several levels of the central nervous system. We performed a unilateral, incomplete cervical spinal contusion injury in rats and delivered stimulation for one week to the contralesional motor cortex to activate the corticospinal tract and other pathways. RNA was purified from bilateral subcortical white matter and 3 levels of the spinal cord. Here we provide the complete data set in the hope that it will be useful for researchers studying electrical stimulation as a therapy to improve recovery from the deficits associated with spinal cord injury.

Cortical Stimulation Paired With Volitional Unimanual Movement Affects Interhemispheric Communication.

Cortical stimulation (CS) of the motor cortex can cause excitability changes in both hemispheres, showing potential to be a technique for clinical rehabilitation of motor function. However, previous studies that have investigated the effects of delivering CS during movement typically focus on a single hemisphere. On the other hand, studies exploring interhemispheric interactions typically deliver CS at rest. We sought to bridge these two approaches by documenting the consequences of delivering CS to a single motor cortex during different phases of contralateral and ipsilateral limb movement, and simultaneously assessing changes in interactions within and between the hemispheres via local field potential (LFP) recordings. Three macaques were trained in a unimanual reaction time (RT) task and implanted with epidural or intracortical electrodes over bilateral motor cortices. During a given session CS was delivered to one hemisphere with respect to movements of either the contralateral or ipsilateral limb. Stimulation delivered before contralateral limb movement onset shortened the contralateral limb RT. In contrast, stimulation delivered after the end of contralateral movement increased contralateral RT but decreased ipsilateral RT. Stimulation delivered before ipsilateral limb movement decreased ipsilateral RT. All other stimulus conditions as well as random stimulation and periodic stimulation did not have consistently significant effects on either limb. Simultaneous LFP recordings from one animal revealed correlations between changes in interhemispheric alpha band coherence and changes in RT, suggesting that alpha activity may be indicative of interhemispheric communication. These results show that changes caused by CS to the functional coupling within and between precentral cortices is contingent on the timing of CS relative to movement.

Comparative neuroanatomy of the lumbosacral spinal cord of the rat, cat, pig, monkey, and human.

The overall goal of this work was to create a high-resolution MRI atlas of the lumbosacral enlargement of the spinal cord of the rat (Sprague-Dawley), cat, domestic pig, rhesus monkey, and human. These species were chosen because they are commonly used in basic and translational research in spinal cord injuries and diseases. Six spinal cord specimens from each of the studied species (total of 30 specimens) were fixed, extracted, and imaged. Sizes of the spinal cord segments, cross-sectional dimensions, and locations of the spinal cord gray and white matter were quantified and compared across species. The lumbar enlargement spans spinal cord levels L3-S1 in rats, L4-S1 in cats, L3-S1 in pigs, L2/L3-L7/S1 in monkeys, and T12/L1-S1/S2 in humans. The enlargements in pigs and humans are largest and most similar in size (length and cross-sectional area); followed by monkeys and cats; and followed by rats. The obtained atlas establishes a neuroanatomical reference for the intact lumbosacral spinal cord in these species. It can also be used to guide the planning of surgical procedures of the spinal cord and technology design and development of spinal cord neuroprostheses, as well as precise delivery of cells/drugs into target regions within the spinal cord parenchyma.

Neurochip3: An Autonomous Multichannel Bidirectional Brain-Computer Interface for Closed-Loop Activity-Dependent Stimulation.

Toward addressing many neuroprosthetic applications, the Neurochip3 (NC3) is a multichannel bidirectional brain-computer interface that operates autonomously and can support closed-loop activity-dependent stimulation. It consists of four circuit boards populated with off-the-shelf components and is sufficiently compact to be carried on the head of a non-human primate (NHP). NC3 has six main components: (1) an analog front-end with an Intan biophysical signal amplifier (16 differential or 32 single-ended channels) and a 3-axis accelerometer, (2) a digital control system comprised of a Cyclone V FPGA and Atmel SAM4 MCU, (3) a micro SD Card for 128 GB or more storage, (4) a 6-channel differential stimulator with ±60 V compliance, (5) a rechargeable battery pack supporting autonomous operation for up to 24 h and, (6) infrared transceiver and serial ports for communication. The NC3 and earlier versions have been successfully deployed in many closed-loop operations to induce synaptic plasticity and bridge lost biological connections, as well as deliver activity-dependent intracranial reinforcement. These paradigms to strengthen or replace impaired connections have many applications in neuroprosthetics and neurorehabilitation.

Intraoperative Characterization of Subthalamic Nucleus-to-Cortex Evoked Potentials in Parkinson's Disease Deep Brain Stimulation.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a clinically effective tool for treating medically refractory Parkinson's disease (PD), but its neural mechanisms remain debated. Previous work has demonstrated that STN DBS results in evoked potentials (EPs) in the primary motor cortex (M1), suggesting that modulation of cortical physiology may be involved in its therapeutic effects. Due to technical challenges presented by high-amplitude DBS artifacts, these EPs are often measured in response to low-frequency stimulation, which is generally ineffective at PD symptom management. This study aims to characterize STN-to-cortex EPs seen during clinically relevant high-frequency STN DBS for PD. Intraoperatively, we applied STN DBS to 6 PD patients while recording electrocorticography (ECoG) from an electrode strip over the ipsilateral central sulcus. Using recently published techniques, we removed large stimulation artifacts to enable quantification of STN-to-cortex EPs. Two cortical EPs were observed - one synchronized with DBS onset and persisting during ongoing stimulation, and one immediately following DBS offset, here termed the "start" and the "end" EPs respectively. The start EP is, to our knowledge, the first long-latency cortical EP reported during ongoing high-frequency DBS. The start and end EPs differ in magnitude (p < 0.05) and latency (p < 0.001), and the end, but not the start, EP magnitude has a significant relationship (p < 0.001, adjusted for random effects of subject) to ongoing high gamma (80-150 Hz) power during the EP. These contrasts may suggest mechanistic or circuit differences in EP production during the two time periods. This represents a potential framework for relating DBS clinical efficacy to the effects of a variety of stimulation parameters on EPs.

A 0.0023 mm 2/ch. Delta-Encoded, Time-Division Multiplexed Mixed-Signal ECoG Recording Architecture With Stimulus Artifact Suppression.

This article demonstrates a scalable, time-division multiplexed biopotential recording front-end capable of real-time differential- and common-mode artifact suppression. A delta-encoded recording architecture exploits the power spectral density (PSD) characteristics of Electrocorticography (ECoG) recordings, combining an 8-bit ADC, and an 8-bit DAC to achieve 14 bits of dynamic range. The flexibility of the digital feedback architecture is leveraged to time-division multiplex 64 differential input channels onto a shared mixed-signal front-end, reducing channel area by 2x compared to the state-of-the-art. The feedback DAC used for delta-encoding also serves to cancel differential artifacts with an off-chip adaptive loop. Analysis of this architecture and measured silicon performance of a 65 nm CMOS test-chip implementation, both on the bench and in-vivo, are included with this paper.

No evidence for chronic demyelination in spared axons after spinal cord injury in a mouse.

The pattern of remyelination after traumatic spinal cord injury remains elusive, with animal and human studies reporting partial to complete demyelination followed by incomplete remyelination. In the present study, we found that spared rubrospinal tract (RST) axons of passage traced with actively transported dextrans and examined caudally to the lesion 12 weeks after mouse spinal cord contusion injury were fully remyelinated. Spared axons exhibited a marginally reduced myelin thickness and significantly shorter internodes. CASPR (contactin-associated protein) and K(v)1.2 channels were used to identify internodes and paranodal protein distribution properties were used as an index of myelin integrity. This is the first time the CNS myelin internode length was measured in a mouse. To better understand the significance of shortened internodes and thinner myelin in spared axons, we modeled conduction properties using McIntyre's et al. model of myelinated axons. Mathematical modeling predicted a 21% decrease in the conduction velocity of remyelinated RST axons attributable to shortened internodes. To determine whether demyelination could be present on axons exhibiting a pathological transport system, we used the retroviral reporter system. Virally delivered green fluorescent protein unveiled a small population of dystrophic RST axons that persist chronically with evident demyelination or abnormal remyelination. Collectively, these data show that lasting demyelination in spared axons is rare and that remyelination of axons of passage occurs in the chronically injured mouse spinal cord.

Functional organization of motor networks in the lumbosacral spinal cord of non-human primates.

Implantable spinal-cord-neuroprostheses aiming to restore standing and walking after paralysis have been extensively studied in animal models (mainly cats) and have shown promising outcomes. This study aimed to take a critical step along the clinical translation path of these neuroprostheses, and investigated the organization of the neural networks targeted by these implants in a non-human primate. This was accomplished by advancing a microelectrode into various locations of the lumbar enlargement of the spinal cord, targeting the ventral horn of the gray matter. Microstimulation in these locations produced a variety of functional movements in the hindlimb. The resulting functional map of the spinal cord in monkeys was found to have a similar overall organization along the length of the spinal cord to that in cats. This suggests that the human spinal cord may also be organized similarly. The obtained spinal cord maps in monkeys provide important knowledge that will guide the very first testing of these implants in humans.