Neurons In Motor Cortex Research Articles

Pathological Comparison of TDP-43 Between Motor Neurons and Interneurons Expressed by a Tetracycline Repressor System on the Mouse Artificial Chromosome.

Cytoplasmic mislocalization of TAR-DNA binding protein of 43 kDa (TDP-43) is a major hallmark of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation is detected in the cortical and spinal motor neurons in most ALS cases; however, pathological mechanism of this mislocalized TDP-43 remains unknown. We generated a tetracycline-inducible TDP-43 A315T system on a mouse artificial chromosome (MAC) vector to avoid transgene-insertional mutagenesis, established a mouse embryonic stem (ES) cell line holding this MAC vector system, and investigated whether overexpressed exogenous TDP-43 A315T was mislocalized in the cytoplasm of the ES cell-derived neurons and triggered the neurotoxic effects on these cells. Inducible TDP-43 A315T system was successfully loaded onto the MAC and introduced into the mouse ES cells. These ES cells could differentiate into motor neurons and interneurons. Overexpression of TDP-43 A315T by addition of doxycycline in both neurons resulted in mislocalization to cytoplasm. Mislocalized TDP-43 caused cell death of motor neurons, but not interneurons. Vulnerability to cytoplasmic mislocalized TDP-43 is selective on neuronal types, whereas mislocalization of overexpressed TDP-43 occurs in even insusceptible neurons. This inducible gene expression system using MAC remains useful for providing critical insights into appearance of TDP-43 pathology.

Motor training promotes both synaptic and intrinsic plasticity of layer V pyramidal neurons in the primary motor cortex.

Here we examined motor training-induced synaptic and intrinsic plasticity of layer V pyramidal neurons in primary motor cortex. The training reduced presynaptic GABA release in early phase, but strengthened AMPA receptor-mediated excitatory synapses in later phase: acquired motor performance after training correlated with the strength of excitatory synapses rather than inhibitory synapses. As to the intrinsic property, the training transiently decreased firing rate in early phase, but returned to pre-training levels in later phase. Western-blot analysis detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in acute phase, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in later phase. Live-imaging analysis of Thy1-YFP transgenic mice showed rapid and long-term spine plasticity in M1 layer V neurons, suggesting training-induced increases in self-entropy per spine. Layer V neurons in primary motor cortex (M1) are important for motor skill learning. Since pretreatment of either CNQX or APV in rats M1 layer V impaired rotor rod learning, we analyzed training-induced synaptic plasticity by whole-cell patch-clamp technique in acute brain slices. One-day trained rats showed a decrease in small inhibitory postsynaptic current (mIPSC) frequency and an increase in the paired-pulse ratio of evoked IPSCs, suggesting a transient decrease in presynaptic GABA release in early phase. Two-days trained rats showed an increase in miniature excitatory postsynaptic current (mEPSC) amplitudes/frequency and elevated AMPA/NMDA ratios, suggesting a long-term strengthening of AMPA receptor-mediated excitatory synapses. Importantly, rotor rod performance in trained rats was correlated with the mean mEPSC amplitude and the frequency obtained from that animal. In current-clamp analysis, 1-day-trained rats transiently decreased current-induced firing rate, while 2-day-trained rats returned to pre-training levels, suggesting a dynamic changes in intrinsic properties. Furthermore, western-blot analysis of layer V detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in 1-day trained rats, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in 2-days trained rats. Finally, live-imaging analysis of Thy1-YFP transgenic mice showed that the training rapidly recruited a substantial number of spines for long-term plasticity in M1 layer V neurons. Taken together, these results indicate that motor training induces complex and diverse plasticity in M1 layer V pyramidal neurons. Abstract figure legend Schematic of motor training and rapid spine dynamics in M1 layer V neurons. Exercise training promotes physiological and morphological spine diversity. Red: glutamate and AMPA receptors. Blue: GABA and GABAA receptors. This article is protected by copyright. All rights reserved.

Learning in a closed-loop brain-machine interface with distributed optogenetic cortical feedback

Objective. Distributed microstimulations at the cortical surface can efficiently deliver feedback to a subject during the manipulation of a prosthesis through a brain-machine interface (BMI). Such feedback can convey vast amounts of information to the prosthesis user and may be key to obtain an accurate control and embodiment of the prosthesis. However, so far little is known of the physiological constraints on the decoding of such patterns. Here, we aimed to test a rotary optogenetic feedback that was designed to encode efficiently the 360° movements of the robotic actuators used in prosthetics. We sought to assess its use by mice that controlled a prosthesis joint through a closed-loop BMI. Approach. We tested the ability of mice to optimize the trajectory of a virtual prosthesis joint in order to solve a rewarded reaching task. They could control the speed of the joint by modulating the activity of individual neurons in the primary motor cortex. During the task, the patterned optogenetic stimulation projected on the primary somatosensory cortex continuously delivered information to the mouse about the position of the joint. Main results. We showed that mice are able to exploit the continuous, rotating cortical feedback in the active behaving context of the task. Mice achieved better control than in the absence of feedback by detecting reward opportunities more often, and also by moving the joint faster towards the reward angular zone, and by maintaining it longer in the reward zone. Mice controlling acceleration rather than speed of the joint failed to improve motor control. Significance. These findings suggest that in the context of a closed-loop BMI, distributed cortical feedback with optimized shapes and topology can be exploited to control movement. Our study has direct applications on the closed-loop control of rotary joints that are frequently encountered in robotic prostheses.

Ischemic stroke rehabilitation through optogenetic modulation of parvalbumin neurons in the contralateral motor cortex

BackgroundBased on the theory of interhemispheric inhibition and the bimodal balance–recovery model in stroke, we explored the effects of excitation/inhibition (E/I) of parvalbumin (PV) neurons in the contralateral primary motor cortex (cM1) connecting the ipsilateral M1 (iM1) via the corpus callosum (cM1-CC-iM1) of ischemic stroke rats by optogenetic stimulation. MethodsWe tested this by injecting anterograde and retrograde virus in rats with middle cerebral artery occlusion (MCAO), and evaluated the neurological scores, motor behavior, volume of cerebral infarction and the E/I balance of the bilateral M1 two weeks after employing optogenetic treatment. ResultsWe found that concentrations of Glu and GABA decreased and increased, respectively, in the iM1 of MCAO rats, and that the former increased in the cM1, suggesting E/I imbalance in bilateral M1 after ischemic stroke. Interestingly, optogenetic stimulation improved M1 E/I imbalance, as illustrated by the increase of Glu in the iM1 and the decrease of GABA in both iM1 and cM1, which were accompanied by an improvement in neurological deficit and motor dysfunction. In addition, we observed a reduced infarct volume, an increase in the expression of the NMDAR and AMPAR, and a decrease in GAD67 in the iM1 after intervention. ConclusionsOptogenetic modulation of PV neurons of the iM1-CC-cM1 improve E/I balance, leading to reduced neurological deficit and improved motor dysfunction following ischemic stroke in rats.

Moderate-Intensity Treadmill Exercise Promotes mTOR-Dependent Motor Cortical Neurotrophic Factor Expression and Functional Recovery in a Murine Model of Crush Spinal Cord Injury (SCI).

Treadmill exercise is widely considered an effective strategy for restoration of skilled motor function after spinal cord injury (SCI). However, the specific exercise intensity that optimizes recovery and the underlying mechanistic basis of this recovery remain unclear. To that end, we sought to investigate the effect of different treadmill exercise intensities on cortical mTOR activity, a key regulator of functional recovery following CNS trauma, in an animal model of C5 crush spinal cord injury (SCI). Following injury, animals were subjected to treadmill exercise for 4 consecutive weeks at three different intensities (low intensity [LEI]; moderate intensity [MEI]; and high intensity [HEI]). Motor function recovery was assessed by horizontal ladder test, cylinder rearing test, and electrophysiology, while neurotrophic factors and cortical mechanistic target of rapamycin (mTOR) pathway-related proteins were assessed by Western blotting. The activation of the cortical mTOR pathway and axonal sprouting was evaluated by immunofluorescence and the changes of plasticity in motor cortex neurons were assessed by Golgi staining. In keeping with previous studies, we found that 4weeks of treadmill training resulted in improved skilled motor function, enhanced nerve conduction capability, increased neuroplasticity, and axonal sprouting. Importantly, we also demonstrated that when compared with the LEI group, MEI and HEI groups demonstrated elevated expression of brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), phosphorylated ribosomal S6 protein (p-S6), and protein kinase B (p-Akt), consistent with an intensity-dependent activation of the mTOR pathway and neurotrophic factor expression in the motor cortex. We also observed impaired exercise endurance and higher mortality during training in the HEI group than in the LEI and MEI groups. Collectively, our findings suggest that treadmill exercise following SCI is an effective means of promoting recovery and highlight the importance of the cortical mTOR pathway and neurotrophic factors as mediators of this effect. Importantly, our findings also demonstrate that excessive exercise can be detrimental, suggesting that moderation may be the optimal strategy. These findings provide an important foundation for further investigation of treadmill training as a modality for recovery following spinal cord injury and of the underlying mechanisms.

Calcium activity is a degraded estimate of spikes

Recording action potentials extracellularly during behavior has led to fundamental discoveries regarding neural function-hippocampal neurons respond to locations in space,1 motor cortex neurons encode movement direction,2 and dopamine neurons signal reward prediction errors3-observations undergirding current theories of cognition,4 movement,5 and learning.6 Recently it has become possible to measure calcium flux, an internal cellular signal related to spiking. The ability to image calcium flux in anatomically7,8 or genetically9 identified neurons can extend our knowledge of neural circuit function by allowing activity to be monitored in specific cell types or projections, or in the same neurons across many days. However, while initial studies were grounded in prior unit recording work, it has become fashionable to assume that calcium is identical to spiking, even though the spike-to-fluorescence transformation is nonlinear, noisy, and unpredictable under real-world conditions.10 It remains an open question whether calcium provides a high-fidelity representation of single-unit activity in awake, behaving subjects. Here, we have addressed this question by recording both signals in the lateral orbitofrontal cortex (OFC) of rats during olfactory discrimination learning. Activity in the OFC during olfactory learning has been well-studied in humans,11,12,13,14 nonhuman primates,15,16 and rats,17,18,19,20,21 where it has been shown to signal information about both the sensory properties of odor cues and the rewards they predict. Our single-unit results replicated prior findings, whereas the calcium signal provided only a degraded estimate of the information available in the single-unit spiking, reflecting primarily reward value.

Large-volume and deep brain imaging in rabbits and monkeys using COMPACT two-photon microscopy

In vivo imaging has been widely used for investigating the structure and function of neurons typically located within ~ 800 μm below the cortical surface. Due to light scattering and absorption, it has been difficult to perform in-vivo imaging of neurons in deep cortical and subcortical regions of large animals with two-photon microscopy. Here, we combined a thin-wall quartz capillary with a GRIN lens attached to a prism for large-volume structural and calcium imaging of neurons located 2 mm below the surface of rabbit and monkey brains. The field of view was greatly expanded by rotating and changing the depth of the imaging probe inside a quartz capillary. Calcium imaging of layer 5/6 neurons in the rabbit motor cortex revealed differential activity of these neurons between quiet wakefulness and slow wave sleep. The method described here provides an important tool for studying the structure and function of neurons located deep in the brains of large animals.

A novel assessment of fine-motor function reveals early hindlimb and detectable forelimb deficits in an experimental model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the loss of cortical and spinal motor neurons (MNs) and muscle degeneration (Kiernan et al. in Lancet 377:942–955, 2011). In the preclinical setting, functional tests that can detect early changes in motor function in rodent models of ALS are critical to understanding the etiology of the disease and treatment development. Here, we established a string-pulling paradigm that can detect forelimb and hindlimb motor deficits in the SOD1 mouse model of ALS earlier than traditional motor performance tasks. Additionally, our findings indicate that early loss of forelimb and hindlimb function is correlated with cortical and spinal MN loss, respectively. This task is not only ecological, low-cost, efficient, and non-onerous, it also requires little animal handling and reduces the stress placed on the animal. It has long been a concern in the field that the SOD1 mouse does not display forelimb motor deficits and does not give researchers a complete picture of the disease. Here, we provide evidence that the SOD1 model does in fact develop early forelimb motor deficits due to the task’s ability to assess fine-motor function, reconciling this model with the various clinical presentation of ALS. Taken together, the string-pulling paradigm may provide novel insights into the pathogenesis of ALS, offer nuanced evaluation of prospective treatments, and has high translational potential to the clinic.

Synaptic dysfunction in ALS and FTD: anatomical and molecular changes provide insights into mechanisms of disease.

Synaptic loss is a pathological feature of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is a disease of the cortical and spinal motor neurons resulting in fatal paralysis due to denervation of muscles. FTD is a form of dementia that primarily affects brain regions controlling cognition, language and behavior. Once classified as two distinct diseases, ALS and FTD are now considered as part of a common disease spectrum based on overlapping clinical, pathological and genetic evidence. At the cellular level, aggregation of common proteins and overlapping gene susceptibilities are shared in both ALS and FTD. Despite the convergence of these two fields of research, the underlying disease mechanisms remain elusive. However, recent discovers from ALS and FTD patient studies and models of ALS/FTD strongly suggests that synaptic dysfunction is an early event in the disease process and a unifying hallmark of these diseases. This review provides a summary of the reported anatomical and cellular changes that occur in cortical and spinal motor neurons in ALS and FTD tissues and models of disease. We also highlight studies that identify changes in the proteome and transcriptome of ALS and FTD models and provide a conceptual overview of the processes that contribute to synaptic dysfunction in these diseases. Due to space limitations and the vast number of publications in the ALS and FTD fields, many articles have not been discussed in this review. As such, this review focuses on the three most common shared mutations in ALS and FTD, the hexanucleuotide repeat expansion within intron 1 of chromosome 9 open reading frame 72 (C9ORF72), transactive response DNA binding protein 43 (TARDBP or TDP-43) and fused in sarcoma (FUS), with the intention of highlighting common pathways that promote synaptic dysfunction in the ALS-FTD disease spectrum.

Microglial homeostasis disruption modulates non-rapid eye movement sleep duration and neuronal activity in adult female mice

Sleep is a natural physiological state, tightly regulated through several neuroanatomical and neurochemical systems, which is essential to maintain physical and mental health. Recent studies revealed that the functions of microglia, the resident immune cells of the brain, differ along the sleep-wake cycle. Inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mainly produced by microglia in the brain, are also well-known to promote sleep. However, the contributing role of microglia on sleep regulation remains largely elusive, even more so in females. Given the higher prevalence of various sleep disorders in women, we aimed to determine the role of microglia in regulating the sleep-wake cycle specifically in female mice. Microglia were depleted in adult female mice with inhibitors of the colony-stimulating factor 1 receptor (CSF1R) (PLX3397 or PLX5622), which is required for microglial population maintenance. This led to a 65–73% reduction of the microglial population, as confirmed by immunofluorescence staining against IBA1 (marker of microglia/macrophages) and TMEM119 (microglia-specific marker) in the reticular nucleus of the thalamus and primary motor cortex. The spontaneous sleep-wake cycle was evaluated at steady-state, during microglial homeostasis disruption and after complete microglial repopulation, upon cessation of treatment with the inhibitors of CSF1R, using electroencephalography (EEG) and electromyography (EMG). We found that microglia-depleted female mice spent more time in non-rapid eye movement (NREM) sleep and had an increased number of NREM sleep episodes, which was partially restored after microglial total repopulation. To determine whether microglia could regulate sleep locally by modulating synaptic transmission, we used patch clamp to record spontaneous activity of pyramidal neurons in the primary motor cortex, which showed an increase of excitatory synaptic transmission during the dark phase. These changes in neuronal activity were modulated by microglial depletion in a phase-dependent manner. Altogether, our results indicate that microglia are involved in the sleep regulation of female mice, further strengthening their potential implication in the development and/or progression of sleep disorders. Furthermore, our findings indicate that microglial repopulation can contribute to normalizing sleep alterations caused by their partial depletion.

Increased glutamatergic synaptic transmission during development in layer II/III mouse motor cortex pyramidal neurons.

Postnatal maturation of the motor cortex is vital to developing a variety of functions, including the capacity for motor learning. The first postnatal weeks involve many neuronal and synaptic changes, which differ by region and layer, likely due to different functions and needs during development. Motor cortex layer II/III is critical to receiving and integrating inputs from somatosensory cortex and generating attentional signals that are important in motor learning and planning. Here, we examined the neuronal and synaptic changes occurring in layer II/III pyramidal neurons of the mouse motor cortex from the neonatal (postnatal day 10) to young adult (postnatal day 30) period, using a combination of electrophysiology and biochemical measures of glutamatergic receptor subunits. There are several changes between p10 and p30 in these neurons, including increased dendritic branching, neuronal excitability, glutamatergic synapse number and synaptic transmission. These changes are critical to ongoing plasticity and capacity for motor learning during development. Understanding these changes will help inform future studies examining the impact of early-life injury and experiences on motor learning and development capacity.

Deep learning for neural decoding in motor cortex

Objective. Neural decoding is an important tool in neural engineering and neural data analysis. Of various machine learning algorithms adopted for neural decoding, the recently introduced deep learning is promising to excel. Therefore, we sought to apply deep learning to decode movement trajectories from the activity of motor cortical neurons. Approach. In this paper, we assessed the performance of deep learning methods in three different decoding schemes, concurrent, time-delay, and spatiotemporal. In the concurrent decoding scheme where the input to the network is the neural activity coincidental to the movement, deep learning networks including artificial neural network (ANN) and long-short term memory (LSTM) were applied to decode movement and compared with traditional machine learning algorithms. Both ANN and LSTM were further evaluated in the time-delay decoding scheme in which temporal delays are allowed between neural signals and movements. Lastly, in the spatiotemporal decoding scheme, we trained convolutional neural network (CNN) to extract movement information from images representing the spatial arrangement of neurons, their activity, and connectomes (i.e. the relative strengths of connectivity between neurons) and combined CNN and ANN to develop a hybrid spatiotemporal network. To reveal the input features of the CNN in the hybrid network that deep learning discovered for movement decoding, we performed a sensitivity analysis and identified specific regions in the spatial domain. Main results. Deep learning networks (ANN and LSTM) outperformed traditional machine learning algorithms in the concurrent decoding scheme. The results of ANN and LSTM in the time-delay decoding scheme showed that including neural data from time points preceding movement enabled decoders to perform more robustly when the temporal relationship between the neural activity and movement dynamically changes over time. In the spatiotemporal decoding scheme, the hybrid spatiotemporal network containing the concurrent ANN decoder outperformed single-network concurrent decoders. Significance. Taken together, our study demonstrates that deep learning could become a robust and effective method for the neural decoding of behavior.

CBP/p300 activation promotes axon growth, sprouting, and synaptic plasticity in chronic experimental spinal cord injury with severe disability.

The interruption of spinal circuitry following spinal cord injury (SCI) disrupts neural activity and is followed by a failure to mount an effective regenerative response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here, we have investigated whether the epigenetic stimulation of the regenerative gene expression program can overcome the current inability to promote neurological recovery in chronic SCI with severe disability. We delivered the CBP/p300 activator CSP-TTK21 or vehicle CSP weekly between week 12 and 22 following a transection model of SCI in mice housed in an enriched environment. Data analysis showed that CSP-TTK21 enhanced classical regenerative signalling in dorsal root ganglia sensory but not cortical motor neurons, stimulated motor and sensory axon growth, sprouting, and synaptic plasticity, but failed to promote neurological sensorimotor recovery. This work provides direct evidence that clinically suitable pharmacological CBP/p300 activation can promote the expression of regeneration-associated genes and axonal growth in a chronic SCI with severe neurological disability.

Organization of Cortical and Thalamic Input to Inhibitory Neurons in Mouse Motor Cortex.

Intracortical inhibition in motor cortex (M1) regulates movement and motor learning. If cortical and thalamic inputs target different inhibitory cell types in different layers, then these afferents may play different roles in regulating M1 output. Using mice of both sexes, we quantified input to two main classes of M1 interneurons, parvalbumin+ (PV+) cells and somatostatin+ (SOM+) cells, using monosynaptic rabies tracing. We then compared anatomic and functional connectivity based on synaptic strength from sensory cortex and thalamus. Functionally, each input innervated M1 interneurons with a unique laminar profile. Different interneuron types were excited in a distinct, complementary manner, suggesting feedforward inhibition proceeds selectively via distinct circuits. Specifically, somatosensory cortex (S1) inputs primarily targeted PV+ neurons in upper layers (L2/3) but SOM+ neurons in middle layers (L5). Somatosensory thalamus [posterior nucleus (PO)] inputs targeted PV+ neurons in middle layers (L5). In contrast to sensory cortical areas, thalamic input to SOM+ neurons was equivalent to that of PV+ neurons. Thus, long-range excitatory inputs target inhibitory neurons in an area and a cell type-specific manner, which contrasts with input to neighboring pyramidal cells. In contrast to feedforward inhibition providing generic inhibitory tone in cortex, circuits are selectively organized to recruit inhibition matched to incoming excitatory circuits.SIGNIFICANCE STATEMENT M1 integrates sensory information and frontal cortical inputs to plan and control movements. Although inputs to excitatory cells are described, the synaptic circuits by which these inputs drive specific types of M1 interneurons are unknown. Anatomical results with rabies tracing and physiological quantification of synaptic strength shows that two main classes of inhibitory cells (PV+ and SOM+ interneurons) both receive substantial cortical and thalamic input, in contrast to interneurons in sensory areas (where thalamic input strongly prefers PV+ interneurons). Further, each input studied targets PV+ and SOM+ interneurons in a different fashion, suggesting that separate, specific circuits exist for recruitment of feedforward inhibition.

Functionally distinct NPAS4-expressing somatostatin interneuron ensembles critical for motor skill learning

During motor learning, dendritic spines on pyramidal neurons (PNs) in the primary motor cortex (M1) undergo reorganization. Intriguingly, the inhibition from local somatostatin-expressing inhibitory neurons (SST-INs) plays an important role in regulating the PN plasticity and thus new motor skill acquisition. However, the molecular mechanisms underlying this process remain unclear. Here, we identified that the early-response transcription factor, NPAS4, is selectively expressed in SST-INs during motor learning. By utilizing invivo two-photon imaging in mice, we found that cell-type-specific deletion of Npas4 in M1 disrupted learning-induced spine reorganization among PNs and impaired motor learning. In addition, NPAS4-expressing SST-INs exhibited lower neuronal activity during task-related movements, and chemogenetically increasing the activity of NPAS4-expressing ensembles was sufficient to mimic the effects of Npas4 deletion. Together, our results reveal an instructive role of NPAS4-expressing SST-INs in modulating the inhibition to downstream task-related PNs to allow proper spine reorganization that is critical for motor learning.

Functional implication of ubiquitinating and deubiquitinating mechanisms in TDP-43 proteinopathies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which motor neurons in spinal cord and motor cortex are progressively lost. About 15% cases of ALS also develop the frontotemporal dementia (FTD), in which the frontotemporal lobar degeneration (FTLD) occurs in the frontal and temporal lobes of the brain. Among the pathologic commonalities in ALS and FTD is ubiquitin-positive cytoplasmic aggregation of TDP-43 that may reflect both its loss-of-function and gain-of-toxicity from proteostasis impairment. Deep understanding of how protein quality control mechanisms regulate TDP-43 proteinopathies still remains elusive. Recently, a growing body of evidence indicates that ubiquitinating and deubiquitinating pathways are critically engaged in the fate decision of aberrant or pathological TDP-43 proteins. E3 ubiquitin ligases coupled with deubiquitinating enzymes may influence the TDP-43-associated proteotoxicity through diverse events, such as protein stability, translocation, and stress granule or inclusion formation. In this article, we recapitulate our current understanding of how ubiquitinating and deubiquitinating mechanisms can modulate TDP-43 protein quality and its pathogenic nature, thus shedding light on developing targeted therapies for ALS and FTD by harnessing protein degradation machinery.

MeCP2 deficiency impairs motor cortical circuit flexibility associated with motor learning

Loss of function mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2) cause Rett syndrome (RTT), a postnatal neurological disorder. The loss of motor function is an important clinical feature of RTT that manifests early during the course of the disease. RTT mouse models with mutations in the murine orthologous Mecp2 gene replicate many human phenotypes, including progressive motor impairments. However, relatively little is known about the changes in circuit function during the progression of motor deficit in this model. As the motor cortex is the key node in the motor system for the control of voluntary movement, we measured firing activity in populations of motor cortical neurons during locomotion on a motorized wheel-treadmill. Different populations of neurons intermingled in the motor cortex signal different aspects of the locomotor state of the animal. The proportion of running selective neurons whose activity positively correlates with locomotion speed gradually decreases with weekly training in wild-type mice, but not in Mecp2-null mice. The fraction of rest-selective neurons whose activity negatively correlates with locomotion speed does not change with training in wild-type mice, but is higher and increases with the progression of locomotion deficit in mutant mice. The synchronization of population activity that occurs in WT mice with training did not occur in Mecp2-null mice, a phenotype most clear during locomotion and observable across all functional cell types. Our results could represent circuit-level biomarkers for motor regression in Rett syndrome.

The Existence of the StartReact Effect Implies Reticulospinal, Not Corticospinal, Inputs Dominate Drive to Motoneurons during Voluntary Movement.

Reaction time is accelerated if a loud (startling) sound accompanies the cue—the “StartReact” effect. Animal studies revealed a reticulospinal substrate for the startle reflex; StartReact may similarly involve the reticulospinal tract, but this is currently uncertain. Here we trained two female macaque monkeys to perform elbow flexion/extension movements following a visual cue. The cue was sometimes accompanied by a loud sound, generating a StartReact effect in electromyogram response latency, as seen in humans. Extracellular recordings were made from antidromically identified corticospinal neurons in primary motor cortex (M1), from the reticular formation (RF), and from the spinal cord (SC; C5–C8 segments). After loud sound, task-related activity was suppressed in M1 (latency, 70–200 ms after cue), but was initially enhanced (70–80 ms) and then suppressed (140–210 ms) in RF. SC activity was unchanged. In a computational model, we simulated a motoneuron pool receiving input from different proportions of the average M1 and RF activity recorded experimentally. Motoneuron firing generated simulated electromyogram, allowing reaction time measurements. Only if ≥60% of motoneuron drive came from RF (≤40% from M1) did loud sound shorten reaction time. The extent of shortening increased as more drive came from RF. If RF provided <60% of drive, loud sound lengthened the reaction time—the opposite of experimental findings. The majority of the drive for voluntary movements is thus likely to originate from the brainstem, not the cortex; changes in the magnitude of the StartReact effect can measure a shift in the relative importance of descending systems.SIGNIFICANCE STATEMENT Our results reveal that a loud sound has opposite effects on neural spiking in corticospinal cells from primary motor cortex, and in the reticular formation. We show that this fortuitously allows changes in reaction time produced by a loud sound to be used to assess the relative importance of reticulospinal versus corticospinal control of movement, validating previous noninvasive measurements in humans. Our findings suggest that the majority of the descending drive to motoneurons producing voluntary movement in primates comes from the reticulospinal tract, not the corticospinal tract.

Changes in electrophysiological properties of pyramidal neuron in motor cortex during the postnatal early development of mice

To investigate the electrophysiological properties of pyramidal neurons in mouse motor cortex during the early postnatal development. Thirty-six mice were randomly divided into postnatal 1-, 2-, 3-Week and 1-, 2-,3-Month groups (n=6). Membrane properties, action potentials (AP) and spontaneous excitatory postsynaptic currents (sEPSCs) of motor cortex pyramidal neurons were recorded to evaluate the changes in the intrinsic electrophysilogical characteristics by using whole cell patch clamp. Pyramidal neurons and interneurons were distinguished according to the AP firing patterns. Comparing with interneurons, pyramidal neurons exhibited regular spiking (RS) with smaller frequency. During the period of postnatal 1 Week-3 Months, some of the intrinsic membrane properties of motor cortex pyramidal neurons changed. Compared to the 1-Week mice, the resting membrane potential (RMP) of 2-Week decreased significantly (P＜0.01), and the membrane input resistance (Rin) of 1-Month got a hyperpolarization (P＜0.01), and they showed no significant change in the next period, while the membrane capacitance (Cm) showed no significant changes during the whole postnatal development. The AP dynamic properties changed significantly during this period. Compared to the 1-Week mice, the absolute value of the AP threshold and the AP amplitude of the 3-Week increased significantly (P＜0.01), while the spike half width of the 2-Week decreased substantially (P＜0.05), and they showed no significant change in the next period. The sEPSCs frequency and amplitude of 1- Month increased significantly compared to the 1-Week mice(P＜0.01), while during the period of next 1 Month-3 Months, the amplitude and frequency showed no significant change. These results suggest that the motor cortex pyramidal neurons have time-specific eletrophysilogical properties during the postnatal development. The electrophysiological properties can be used as a functional index to detect the degree of neurons maturity, and as a marker to distinguish the pyramidal neurons and interneurons.

High-Throughput Neurite Outgrowth Assay Using GFP-Labeled iPSC-Derived Neurons.

The potential neurotoxicity from an increasing number of drugs and untested environmental chemicals creates a need to develop reliable and efficient in vitro methods for identifying chemicals that may adversely affect the nervous system. An important process in neurodevelopment is neurite outgrowth, which can be affected by developmental neurotoxicity. Currently, neurite outgrowth assays rely mainly on staining, which requires multiple sample processing steps, particularly washing steps, that may introduce variation and limit throughput. Here, we describe a neurite outgrowth assay that uses induced pluripotent stem cell (iPSC)-derived human cortical glutamatergic neurons and/or spinal motor neurons labeled with green fluorescent protein (GFP) to test compounds in a high-content and high-throughput format. This method enables live and time-lapse imaging of GFP-labeled neurons using an assay plate that is continuously imaged at multiple times after chemical treatment. In this article, we describe how to thaw frozen GFP-labeled neurons, culture them, treat them with a compound of interest, and analyze neurite outgrowth using a high-content imaging platform. In this assay, GFP-labeled iPSC-derived human neurons represent a promising tool for identifying and prioritizing compounds with potential developmental neurotoxicity for further hazard characterization. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Thawing and seeding of iPSC-derived neurons Basic Protocol 2: Compound plate preparation and treatment of neurons Basic Protocol 3: High-content imaging and analysis.