Mesotrode chronic simultaneous mesoscale cortical imaging and subcortical or peripheral nerve spiking activity recording in mice.

Brain function originates from hierarchical spatial-temporal neural dynamics distributed across cortical and subcortical networks. However, techniques available to assess large-scale brain network activity with single-neuron resolution in behaving animals remain limited. Here, we present Mesotrode that integrates chronic wide-field mesoscale cortical imaging and compact multi-site cortical/subcortical cellular electrophysiology in head-fixed mice that undergo self-initiated running or orofacial movements. Specifically, we harnessed the flexibility of chronic multi-site tetrode recordings to monitor single-neuron activity in multiple subcortical structures while simultaneously imaging the mesoscale activity of the entire dorsal cortex. A mesoscale spike-triggered averaging procedure allowed the identification of cortical activity motifs preferentially associated with single-neuron spiking. Using this approach, we were able to characterize chronic single-neuron-related functional connectivity maps for up to 60 days post-implantation. Neurons recorded from distinct subcortical structures display diverse but segregated cortical maps, suggesting that neurons of different origins participate in distinct cortico-subcortical pathways. We extended the capability of Mesotrode by implanting the micro-electrode at the facial motor nerve and found that facial nerve spiking is functionally associated with the PTA, RSP, and M2 network, and optogenetic inhibition of the PTA area significantly reduced the facial movement of the mice. These findings demonstrate that Mesotrode can be used to sample different combinations of cortico-subcortical networks over prolonged periods, generating multimodal and multi-scale network activity from a single implant, offering new insights into the neural mechanisms underlying specific behaviors.

Water-Reaching Platform for Longitudinal Assessment of Cortical Activity and Fine Motor Coordination Defects in a Huntington Disease Mouse Model.

Huntington disease (HD), caused by dominantly inherited expansions of a CAG repeat results in characteristic motor dysfunction. Although gross motor defects have been extensively characterized in multiple HD mouse models using tasks such as rotarod and beam walking, less is known about forelimb deficits. We develop a high-throughput alternating reward/nonreward water-reaching task and training protocol conducted daily over approximately two months to simultaneously monitor forelimb impairment and mesoscale cortical changes in GCaMP activity, comparing female zQ175 (HD) and wild-type (WT) littermate mice, starting at ∼5.5 months. Behavioral analysis of the water-reaching task reveals that HD mice, despite learning the water-reaching task as proficiently as wild-type mice, take longer to learn the alternating event sequence as evident by impulsive (noncued) reaches and initially display reduced cortical activity associated with successful reaches. At this age gross motor defects determined by tapered beam assessment were not apparent. Although wild-type mice displayed no significant changes in cortical activity and reaching trajectory throughout the testing period, HD mice exhibited an increase in cortical activity, especially in the secondary motor and retrosplenial cortices, over time, as well as longer and more variable reaching trajectories by approximately seven months. HD mice also experienced a progressive reduction in successful performance. Tapered beam and rotarod tests as well as reduced DARPP-32 expression (striatal medium spiny neuron marker) after water-reaching assessment confirmed HD pathology. The water-reaching task can be used to inform on a daily basis, HD and other movement disorder onset and manifestation, therapeutic intervention windows, and test drug efficacy.

Towards a Visualizable, De-identified Synthetic Biomarker of Human Movement Disorders.

Human motion analysis has been a common thread across modern and early medicine. While medicine evolves, analysis of movement disorders is mostly based on clinical presentation and trained observers making subjective assessments using clinical rating scales. Currently, the field of computer vision has seen exponential growth and successful medical applications. While this has been the case, neurology, for the most part, has not embraced digital movement analysis. There are many reasons for this including: the limited size of labeled datasets, accuracy and nontransparent nature of neural networks, and potential legal and ethical concerns. We hypothesize that a number of opportunities are made available by advancements in computer vision that will enable digitization of human form, movements, and will represent them synthetically in 3D. Representing human movements within synthetic body models will potentially pave the way towards objective standardized digital movement disorder diagnosis and building sharable open-source datasets from such processed videos. We provide a perspective of this emerging field and describe how clinicians and computer scientists can navigate this new space. Such digital movement capturing methods will be important for both machine learning-based diagnosis and computer vision-aided clinical assessment. It would also supplement face-to-face clinical visits and be used for longitudinal monitoring and remote diagnosis.

Altered cortical processing of sensory input in Huntington disease mouse models.

Huntington disease (HD), a hereditary neurodegenerative disorder, manifests as progressively impaired movement and cognition. Although early abnormalities of neuronal activity in striatum are well established in HD models, there are fewer in vivo studies of the cortex. Here, we record local field potentials (LFPs) in YAC128 HD model mice versus wild-type mice. In multiple cortical areas, limb sensory stimulation evokes a greater change in LFP power in YAC128 mice. Mesoscopic imaging using voltage-sensitive dyes reveals more extensive spread of evoked sensory signals across the cortical surface in YAC128 mice. YAC128 layer 2/3 sensory cortical neurons ex vivo show increased excitatory events, which could contribute to enhanced sensory responses in vivo. Cortical LFP responses to limb stimulation, visual and auditory input are also significantly increased in zQ175 HD mice. Results presented here extend knowledge of HD beyond ex vivo studies of individual neurons to the intact cortical network.

MesoNet allows automated scaling and segmentation of mouse mesoscale cortical maps using machine learning.

Understanding the basis of brain function requires knowledge of cortical operations over wide spatial scales and the quantitative analysis of brain activity in well-defined brain regions. Matching an anatomical atlas to brain functional data requires substantial labor and expertise. Here, we developed an automated machine learning-based registration and segmentation approach for quantitative analysis of mouse mesoscale cortical images. A deep learning model identifies nine cortical landmarks using only a single raw fluorescent image. Another fully convolutional network was adapted to delimit brain boundaries. This anatomical alignment approach was extended by adding three functional alignment approaches that use sensory maps or spatial-temporal activity motifs. We present this methodology as MesoNet, a robust and user-friendly analysis pipeline using pre-trained models to segment brain regions as defined in the Allen Mouse Brain Atlas. This Python-based toolbox can also be combined with existing methods to facilitate high-throughput data analysis.

A three-dimensional virtual mouse generates synthetic training data for behavioral analysis.

We developed a three-dimensional (3D) synthetic animated mouse based on computed tomography scans that is actuated using animation and semirandom, joint-constrained movements to generate synthetic behavioral data with ground-truth label locations. Image-domain translation produced realistic synthetic videos used to train two-dimensional (2D) and 3D pose estimation models with accuracy similar to typical manual training datasets. The outputs from the 3D model-based pose estimation yielded better definition of behavioral clusters than 2D videos and may facilitate automated ethological classification.

Gamma frequency activation of inhibitory neurons in the acute phase after stroke attenuates vascular and behavioral dysfunction.

Alterations in gamma oscillations occur in several neurological disorders, and the entrainment of gamma oscillations has been recently proposed as a treatment for neurodegenerative disease. Optogenetic stimulation enhances recovery in models of stroke when applied weeks after injury; however, the benefits of acute brain stimulation have not been investigated. Here, we report beneficial effects of gamma-frequency modulation in the acute phase, within 1 h, after stroke. Transgenic VGAT-ChR2 mice are subject to awake photothrombotic stroke in an area encompassing the forelimb sensory and motor cortex. Optogenetic stimulation at 40 Hz in the peri-infarct zone recovers neuronal activity 24 h after stroke in motor and parietal association areas, as well as blood flow over the first week after stroke. Stimulation significantly reduces lesion volume and improves motor function. Our results suggest that acute-phase modulation of cortical oscillatory dynamics may serve as a target for neuroprotection against stroke.

Real-Time Selective Markerless Tracking of Forepaws of Head Fixed Mice Using Deep Neural Networks.

Here, we describe a system capable of tracking specific mouse paw movements at high frame rates (70.17 Hz) with a high level of accuracy (mean = 0.95, SD < 0.01). Short-latency markerless tracking of specific body parts opens up the possibility of manipulating motor feedback. We present a software and hardware scheme built on DeepLabCut-a robust movement-tracking deep neural network framework-which enables real-time estimation of paw and digit movements of mice. Using this approach, we demonstrate movement-generated feedback by triggering a USB-GPIO (general-purpose input/output)-controlled LED when the movement of one paw, but not the other, selectively exceeds a preset threshold. The mean time delay between paw movement initiation and LED flash was 44.41 ms (SD = 36.39 ms), a latency sufficient for applying behaviorally triggered feedback. We adapt DeepLabCut for real-time tracking as an open-source package we term DeepCut2RealTime. The ability of the package to rapidly assess animal behavior was demonstrated by reinforcing specific movements within water-restricted, head-fixed mice. This system could inform future work on a behaviorally triggered "closed loop" brain-machine interface that could reinforce behaviors or deliver feedback to brain regions based on prespecified body movements.

Mapping cortical mesoscopic networks of single spiking cortical or sub-cortical neurons.

Understanding the basis of brain function requires knowledge of cortical operations over wide-spatial scales, but also within the context of single neurons. In vivo, wide-field GCaMP imaging and sub-cortical/cortical cellular electrophysiology were used in mice to investigate relationships between spontaneous single neuron spiking and mesoscopic cortical activity. We make use of a rich set of cortical activity motifs that are present in spontaneous activity in anesthetized and awake animals. A mesoscale spike-triggered averaging procedure allowed the identification of motifs that are preferentially linked to individual spiking neurons by employing genetically targeted indicators of neuronal activity. Thalamic neurons predicted and reported specific cycles of wide-scale cortical inhibition/excitation. In contrast, spike-triggered maps derived from single cortical neurons yielded spatio-temporal maps expected for regional cortical consensus function. This approach can define network relationships between any point source of neuronal spiking and mesoscale cortical maps.

The Effect of Motor Cortex Stimulation on Central Poststroke Pain in a Series of 16 Patients With a Mean Follow-Up of 28 Months.

OBJECTIVE: To investigate the effect of motor cortex stimulation (MCS) on central poststroke pain (CPSP) and the outcome predictors associated with medium- to long-term results. MATERIALS AND METHODS: This is a retrospective review of 16 CPSP patients treated with MCS with a mean follow-up of 28.2 months. The pain intensity was assessed based on the visual analogue scale (VAS) before surgery and at various follow-up visits. An effective outcome was determined to be at least 40% pain relief. The type (hemorrhage or ischemia) and location (thalamus or non-thalamus) of the stroke, the location of the electrode (epidural or subdural), and preoperative repetitive transcranial magnetic stimulation (rTMS) results were analyzed to evaluate whether they are predictive of effective outcomes. RESULTS: The mean VAS before surgery was 8.0 ± 0.7 compared with 3.8 ± 2.1 one month after surgery and 5.3 ± 2.4 at the last follow-up. There were no differences in the analgesic effects between the types of stroke (hemorrhage or ischemia), stroke location (thalamus or non-thalamus), or the location of the electrode (epidural or subdural). The association between preoperative rTMS and effective outcomes was significant with a positive predictive value of 0.8571 and a negative predictive value of 0.7778. CONCLUSIONS: Our results suggest that MCS significantly reduces the pain intensity of CPSP. The types of stroke (hemorrhage or ischemia), stroke location (thalamus or nonthalamus), and the location of the electrode (epidural or subdural) were not significant predictors of the analgesic effects of MCS. Preoperative rTMS might be helpful for screening candidates for MCS.

Intact skull chronic windows for mesoscopic wide-field imaging in awake mice.

BACKGROUND: Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. NEW METHOD: We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. RESULTS: The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30min. A single set-screw provides a stable means of attachment (in relation to the measured lateral and axial resolution) for mesoscale assessment without obscuring the cortical field of view. COMPARISON WITH EXISTING METHODS: We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice in longitudinal studies of up to 2 months in duration. CONCLUSIONS: We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors or in transgenic mice expressing genetically encoded sensors of activity.

Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR.

Wide-field-of-view mesoscopic cortical imaging with genetically encoded sensors enables decoding of regional activity and connectivity in anesthetized and behaving mice; however, the kinetics of most genetically encoded sensors can be suboptimal for in vivo characterization of frequency bands higher than 1-3 Hz. Furthermore, existing sensors, in particular those that measure calcium (genetically encoded calcium indicators; GECIs), largely monitor suprathreshold activity. Using a genetically encoded sensor of extracellular glutamate and in vivo mesoscopic imaging, we demonstrate rapid kinetics of virally transduced or transgenically expressed glutamate-sensing fluorescent reporter iGluSnFR. In both awake and anesthetized mice, we imaged an 8 × 8 mm field of view through an intact transparent skull preparation. iGluSnFR revealed cortical representation of sensory stimuli with rapid kinetics that were also reflected in correlation maps of spontaneous cortical activities at frequencies up to the alpha band (8-12 Hz). iGluSnFR resolved temporal features of sensory processing such as an intracortical reverberation during the processing of visual stimuli. The kinetics of iGluSnFR for reporting regional cortical signals were more rapid than those for Emx-GCaMP3 and GCaMP6s and comparable to the temporal responses seen with RH1692 voltage sensitive dye (VSD), with similar signal amplitude. Regional cortical connectivity detected by iGluSnFR in spontaneous brain activity identified functional circuits consistent with maps generated from GCaMP3 mice, GCaMP6s mice, or VSD sensors. Viral and transgenic iGluSnFR tools have potential utility in normal physiology, as well as neurologic and psychiatric pathologies in which abnormalities in glutamatergic signaling are implicated. SIGNIFICANCE STATEMENT: We have characterized the usage of virally transduced or transgenically expressed extracellular glutamate sensor iGluSnFR to perform wide-field-of-view mesoscopic imaging of cortex in both anesthetized and awake mice. Probes for neurotransmitter concentration enable monitoring of brain activity and provide a more direct measure of regional functional activity that is less dependent on nonlinearities associated with voltage-gated ion channels. We demonstrate functional maps of extracellular glutamate concentration and that this sensor has rapid kinetics that enable reporting high-frequency signaling. This imaging strategy has utility in normal physiology and pathologies in which altered glutamatergic signaling is observed. Moreover, we provide comparisons between iGluSnFR and genetically encoded calcium indicators and voltage-sensitive dyes.

Formononetin protects neurons against hypoxia-induced cytotoxicity through upregulation of ADAM10 and sAßPPα.

Formononetin, an active constituent of the Chinese herb Astragali Radix, has been reported to have beneficial effects for Alzheimer's disease (AD). Yet the mechanism of this effect remains to be elucidated. The present study shows that formononetin increases soluble-AßPPα (sAßPPα) secretion and thus protects human-AßPP Swedish mutation cell (N2a-AßPP cell) from hypoxia-induced apoptosis. Using hypoxic N2a-AßPP cell as an in vitro model of AD-like pathology, we confirmed that regular treatment with formononetin could have neuroprotective effects, followed respectively by reduced caspase 3 activity and increased cell viability. Strikingly, our data revealed that the caspase 3-blocking effect of formononetin was largely mediated by stimulation of α-secretase cleavage of AßPP, and increasing the secretion of its soluble form, sAßPPα. Moreover, the protective effect of formononetin was totally inhibited by TAPI-2, an α-secretase complex inhibitor, suggesting the role of the sAßPPα pathway in the neuroprotective response to formononetin. We also found that the stimulative effect of formononetin on α-secretase activity was mainly conducted by upregulating ADAM10 expression at the transcriptional level. Altogether, our study provides novel insights into how formononetin mediates stimulation of the ADAM10-sAßPPα pathway and exerts a neuronal protective effect.

[Differential neuronal firing in globus pallidus internus of patients with Parkinson's disease and dystonia].

OBJECTIVE: To explore the neuronal activity in globus pallidus internus in patients with Parkinson's disease (PD) and dystonia. METHODS: Thirteen patients with Parkinson's disease and eight with dystonia undergoing stereotactic surgery of globus pallidus internus were studied. Microrecording in globus pallidus internus and electromyography on contralateral limbs to surgery were simultaneously performed. Single unit analysis, interspike intervals (ISI) and coefficient of variation of ISI were performed. ANOVA and Student t-test were employed to compare neuronal firing in globus pallidus internus between two patient groups. RESULTS: One hundred and eight neurons were identified from PD patients, including 19.4% tremor-related neuronal activity, 55.6% neurons with tonic firing and 25.0% neurons with irregular discharge. Further analysis revealed that neurons with tonic firing had a mean firing rate of (103.7 +/- 25.5) Hz; neurons with irregular discharge had a mean firing rate of (63.4 +/- 16.1) Hz. Forty-five neurons were identified from patients with dystonia, including 37.8% neurons with tonic firing and 62.2% neurons with irregular discharge. Further analysis showed that neurons with tonic firing had a mean firing rate of (50.2 +/- 19.1) Hz; neurons with irregular discharge had a mean firing rate of (28.5 +/- 10.5) Hz. ANOVA showed that a significance of firing rate of tonic firing and irregular discharge in globus pallidus internus was reached between two patient groups (P < 0.001). The mean firing rate of two patterns of neuronal activity in PD were significantly higher than those in dystonia (Bonferroni test, both P < 0.05). CONCLUSION: The data support the current view that an increased neuronal firing rate in globus pallidus internus is associated with PD whereas a decreased neuronal firing rate is associated with dystonia.