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1.
Science ; 383(6685): 890-897, 2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38386755

RESUMO

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.


Assuntos
Cálcio , Fenômenos Fisiológicos Celulares , Células , Coloração e Rotulagem , Animais , Corantes , Perfilação da Expressão Gênica , Peixe-Zebra , Células/química , Domínios e Motivos de Interação entre Proteínas
2.
Brain ; 147(3): 1011-1024, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-37787057

RESUMO

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal ∼1.5 mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.


Assuntos
Epilepsias Parciais , Ácido Glutâmico , Humanos , Convulsões , Cognição , Ácido gama-Aminobutírico
3.
Neuron ; 111(10): 1547-1563.e9, 2023 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-37015225

RESUMO

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.


Assuntos
Enzima de Conversão de Angiotensina 2 , Rodopsina , Camundongos , Animais , Potenciais de Ação/fisiologia , Rodopsina/genética , Neurônios/fisiologia , Mutação/genética
4.
Nature ; 615(7954): 884-891, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36922596

RESUMO

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.


Assuntos
Sinalização do Cálcio , Cálcio , Calmodulina , Neurônios , Óxido Nítrico Sintase Tipo III , Fragmentos de Peptídeos , Cálcio/análise , Cálcio/metabolismo , Calmodulina/metabolismo , Neurônios/metabolismo , Cinética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fatores de Tempo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo
5.
Curr Biol ; 33(7): 1249-1264.e7, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-36921605

RESUMO

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA "sniffer" and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks.


Assuntos
Hipocampo , Transmissão Sináptica , Transmissão Sináptica/fisiologia , Neurônios , Interneurônios/fisiologia , Ácido gama-Aminobutírico
6.
Elife ; 112022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36355598

RESUMO

A wide range of techniques in neuroscience involve placing individual probes at precise locations in the brain. However, large-scale measurement and manipulation of the brain using such methods have been severely limited by the inability to miniaturize systems for probe positioning. Here, we present a fundamentally new, remote-controlled micropositioning approach composed of novel phase-change material-filled resistive heater micro-grippers arranged in an inchworm motor configuration. The microscopic dimensions, stability, gentle gripping action, individual electronic control, and high packing density of the grippers allow micrometer-precision independent positioning of many arbitrarily shaped probes using a single piezo actuator. This multi-probe single-actuator design significantly reduces the size and weight and allows for potential automation of microdrives. We demonstrate accurate placement of multiple electrodes into the rat hippocampus in vivo in acute and chronic preparations. Our robotic microdrive technology should therefore enable the scaling up of many types of multi-probe applications in neuroscience and other fields.


Assuntos
Neurônios , Procedimentos Cirúrgicos Robóticos , Animais , Ratos , Eletrofisiologia/métodos , Eletrodos Implantados , Encéfalo
7.
Bio Protoc ; 11(14): e4085, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34395724

RESUMO

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly (< 30 s), resulting in a high yield of whole-cell recording success rate (> 90%) for over 100 reuses of a single pipette. For most patch-clamp experiments (< 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract: Rapid enzymatic cleaning for reuse of patch-clamp pipettes.

8.
ACS Sens ; 5(7): 1959-1968, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32571014

RESUMO

Genetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.


Assuntos
Cálcio , Neurônios , Peixe-Zebra , Animais , Linhagem Celular , Células Cultivadas , Peixe-Zebra/genética
9.
J Neurosci Methods ; 328: 108442, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562888

RESUMO

BACKGROUND: Recent advancements with induced pluripotent stem cell-derived (iPSC) retinal pigment epithelium (RPE) have made disease modeling and cell therapy for macular degeneration feasible. However, current techniques for intracellular electrophysiology - used to validate epithelial function - are painstaking and require manual skill; limiting experimental throughput. NEW METHOD: A five-stage algorithm, leveraging advances in automated patch clamping, systematically derived and optimized, improves yield and reduces skill when compared to conventional, manual techniques. RESULTS: The automated algorithm improves yield per attempt from 17% (manually, n = 23) to 22% (automated, n = 120) (chi-squared, p = 0.004). Specifically for RPE, depressing the local cell membrane by 6 µm and electroporating (buzzing) just prior to this depth (5 µm) maximized yield. COMPARISON WITH EXISTING METHOD: Conventionally, intracellular epithelial electrophysiology is performed by manually lowering a pipette with a micromanipulator, blindly, towards a monolayer of cells and spontaneously stopping when the magnitude of the instantaneous measured membrane potential decreased below a predetermined threshold. The new method automatically measures the pipette tip resistance during the descent, detects the cell surface, indents the cell membrane, and briefly buzzes to electroporate the membrane while descending, overall achieving a higher yield than conventional methods. CONCLUSIONS: This paper presents an algorithm for high-yield, automated intracellular electrophysiology in epithelia; optimized for human RPE. Automation reduces required user skill and training while, simultaneously, improving yield. This algorithm could enable large-scale exploration of drug toxicity and physiological function verification for numerous kinds of epithelia.


Assuntos
Algoritmos , Eletrofisiologia/métodos , Epitélio Pigmentado da Retina/fisiologia , Humanos , Técnicas de Patch-Clamp
10.
Nat Methods ; 16(8): 763-770, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308547

RESUMO

Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.


Assuntos
Técnicas Biossensoriais/métodos , Encéfalo/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Anestesia , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismo , Convulsões/patologia , Peixe-Zebra
11.
J Neurophysiol ; 121(6): 2341-2357, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969898

RESUMO

Patch clamping is the gold standard measurement technique for cell-type characterization in vivo, but it has low throughput, is difficult to scale, and requires highly skilled operation. We developed an autonomous robot that can acquire multiple consecutive patch-clamp recordings in vivo. In practice, 40 pipettes loaded into a carousel are sequentially filled and inserted into the brain, localized to a cell, used for patch clamping, and disposed. Automated visual stimulation and electrophysiology software enables functional cell-type classification of whole cell-patched cells, as we show for 37 cells in the anesthetized mouse in visual cortex (V1) layer 5. We achieved 9% yield, with 5.3 min per attempt over hundreds of trials. The highly variable and low-yield nature of in vivo patch-clamp recordings will benefit from such a standardized, automated, quantitative approach, allowing development of optimal algorithms and enabling scaling required for large-scale studies and integration with complementary techniques. NEW & NOTEWORTHY In vivo patch-clamp is the gold standard for intracellular recordings, but it is a very manual and highly skilled technique. The robot in this work demonstrates the most automated in vivo patch-clamp experiment to date, by enabling production of multiple, serial intracellular recordings without human intervention. The robot automates pipette filling, wire threading, pipette positioning, neuron hunting, break-in, delivering sensory stimulus, and recording quality control, enabling in vivo cell-type characterization.


Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Robótica , Córtex Visual/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa
12.
J Neural Eng ; 16(4): 046003, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30970335

RESUMO

OBJECTIVE: Intracellular patch-clamp electrophysiology, one of the most ubiquitous, high-fidelity techniques in biophysics, remains laborious and low-throughput. While previous efforts have succeeded at automating some steps of the technique, here we demonstrate a robotic 'PatcherBot' system that can perform many patch-clamp recordings sequentially, fully unattended. APPROACH: Comprehensive automation is accomplished by outfitting the robot with machine vision, and cleaning pipettes instead of manually exchanging them. MAIN RESULTS: the PatcherBot can obtain data at a rate of 16 cells per hour and work with no human intervention for up to 3 h. We demonstrate the broad applicability and scalability of this system by performing hundreds of recordings in tissue culture cells and mouse brain slices with no human supervision. Using the PatcherBot, we also discovered that pipette cleaning can be improved by a factor of three. SIGNIFICANCE: The system is potentially transformative for applications that depend on many high-quality measurements of single cells, such as drug screening, protein functional characterization, and multimodal cell type investigations.


Assuntos
Encéfalo/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Técnicas de Patch-Clamp/métodos , Robótica/métodos , Animais , Encéfalo/citologia , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/instrumentação , Robótica/instrumentação
13.
IEEE Trans Image Process ; 27(4): 1847-1861, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29346099

RESUMO

Differential interference contrast (DIC) microscopy is widely used for observing unstained biological samples that are otherwise optically transparent. Combining this optical technique with machine vision could enable the automation of many life science experiments; however, identifying relevant features under DIC is challenging. In particular, precise tracking of cell boundaries in a thick ( ) slice of tissue has not previously been accomplished. We present a novel deconvolution algorithm that achieves the state-of-the-art performance at identifying and tracking these membrane locations. Our proposed algorithm is formulated as a regularized least squares optimization that incorporates a filtering mechanism to handle organic tissue interference and a robust edge-sparsity regularizer that integrates dynamic edge tracking capabilities. As a secondary contribution, this paper also describes new community infrastructure in the form of a MATLAB toolbox for accurately simulating DIC microscopy images of in vitro brain slices. Building on existing DIC optics modeling, our simulation framework additionally contributes an accurate representation of interference from organic tissue, neuronal cell-shapes, and tissue motion due to the action of the pipette. This simulator allows us to better understand the image statistics (to improve algorithms), as well as quantitatively test cell segmentation and tracking algorithms in scenarios, where ground truth data is fully known.

14.
J Neurosci ; 38(7): 1821-1834, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29279309

RESUMO

Repeated sequences of neural activity are a pervasive feature of neural networks in vivo and in vitro In the hippocampus, sequential firing of many neurons over periods of 100-300 ms reoccurs during behavior and during periods of quiescence. However, it is not known whether the hippocampus produces longer sequences of activity or whether such sequences are restricted to specific network states. Furthermore, whether long repeated patterns of activity are transmitted to single cells downstream is unclear. To answer these questions, we recorded intracellularly from hippocampal CA1 of awake, behaving male mice to examine both subthreshold activity and spiking output in single neurons. In eight of nine recordings, we discovered long (900 ms) reoccurring subthreshold fluctuations or "repeats." Repeats generally were high-amplitude, nonoscillatory events reoccurring with 10 ms precision. Using statistical controls, we determined that repeats occurred more often than would be expected from unstructured network activity (e.g., by chance). Most spikes occurred during a repeat, and when a repeat contained a spike, the spike reoccurred with precision on the order of ≤20 ms, showing that long repeated patterns of subthreshold activity are strongly connected to spike output. Unexpectedly, we found that repeats occurred independently of classic hippocampal network states like theta oscillations or sharp-wave ripples. Together, these results reveal surprisingly long patterns of repeated activity in the hippocampal network that occur nonstochastically, are transmitted to single downstream neurons, and strongly shape their output. This suggests that the timescale of information transmission in the hippocampal network is much longer than previously thought.SIGNIFICANCE STATEMENT We found long (≥900 ms), repeated, subthreshold patterns of activity in CA1 of awake, behaving mice. These repeated patterns ("repeats") occurred more often than expected by chance and with 10 ms precision. Most spikes occurred within repeats and reoccurred with a precision on the order of 20 ms. Surprisingly, there was no correlation between repeat occurrence and classical network states such as theta oscillations and sharp-wave ripples. These results provide strong evidence that long patterns of activity are repeated and transmitted to downstream neurons, suggesting that the hippocampus can generate longer sequences of repeated activity than previously thought.


Assuntos
Região CA1 Hipocampal/fisiologia , Sinapses/fisiologia , Vigília/fisiologia , Algoritmos , Animais , Comportamento Animal/fisiologia , Cinética , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Transmissão Sináptica/fisiologia
15.
J Neurophysiol ; 116(4): 1564-1578, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27385800

RESUMO

Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight "gigaseal" connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments.


Assuntos
Algoritmos , Automação Laboratorial , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Técnicas de Patch-Clamp/métodos , Animais , Automação Laboratorial/instrumentação , Calibragem , Gráficos por Computador , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp/instrumentação , Fatores de Tempo , Técnicas de Cultura de Tecidos , Interface Usuário-Computador , Córtex Visual/citologia , Córtex Visual/fisiologia
16.
J Neurophysiol ; 113(4): 1275-82, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25429119

RESUMO

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 µV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas de Patch-Clamp/instrumentação , Animais , Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Sensibilidade e Especificidade
17.
Dysphagia ; 29(3): 346-54, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24562508

RESUMO

Paralysis of the structures in the head and neck due to stroke or other neurological disorder often causes dysphagia (difficulty in swallowing). Patients with dysphagia have a significantly higher incidence of aspiration pneumonia and death. The recurrent laryngeal nerve (RLN), which innervates the intrinsic laryngeal muscles that control the vocal folds, travels superiorly in parallel to the trachea in the tracheoesophageal groove. This study tests the hypothesis that functional electrical stimulation (FES) applied via transtracheal electrodes can produce controlled vocal fold adduction. Bipolar electrodes were placed at 15° intervals around the interior mucosal surface of the canine trachea, and current was applied to the tissue while electromyography (EMG) from the intrinsic laryngeal muscles and vocal fold movement visualization via laryngoscopy were recorded. The lowest EMG thresholds were found at an average location of 100° to the left of the ventral midsagittal line and 128° to the right. A rotatable pair of bipolar electrodes spaced 230° apart were able to stimulate bilaterally both RLNs in every subject. Laryngoscopy showed complete glottal closure with transtracheal stimulation in six of the eight subjects, and this closure was maintained under simultaneous FES-induced laryngeal elevation. Transtracheal stimulation is an effective tool for minimally invasive application of FES to induce vocal fold adduction, providing an alternative mechanism to study airway protection.


Assuntos
Estimulação Elétrica , Movimento/fisiologia , Nervo Laríngeo Recorrente/fisiologia , Prega Vocal/fisiologia , Animais , Cães , Eletrodos , Eletromiografia , Traqueia
18.
J Neural Eng ; 10(4): 046013, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23838089

RESUMO

OBJECTIVE: Laryngeal elevation protects the airway and assists opening of the esophagus during swallowing. The GH, thyrohyoid, and MH muscles provide a majority of this elevatory motion. This study applied functional electrical stimulation to the XII/C1 nerve complex using a nerve cuff electrode to determine the capabilities of neural stimulation to induce laryngeal elevation. APPROACH: Multi-contact FINE electrodes were implanted onto the XII/C1 nerve complex at locations proximal and distal to the thyrohyoid branching point in five anesthetized canines. Motion of the thyroid cartilage and the hyoid bone was recorded during stimulation of nerve cuffs and intramuscular electrodes. MAIN RESULTS: Nerve stimulation induced 260% more laryngeal elevation than intramuscular stimulation (18.8 mm versus 5.2 mm, p ≪ 0.01), and 228% higher velocity (143.8 versus 43.9 mm s(-1), p ≪ 0.01). While stimulation at all cuff and electrode locations elevated the larynx, only the proximal XII/C1 nerve cuff significantly elicited both thyroid-hyoid approximation and hyoid elevation. In all proximal XII/C1 nerve cuffs (n = 7), stimulation was able to obtain selectivity of greater than 75% of at least one elevatory muscle. SIGNIFICANCE: These results support the hypothesis that an implanted neural interface system can produce increased laryngeal elevation, a significant protective mechanism of deglutition.


Assuntos
Deglutição/fisiologia , Estimulação Elétrica/métodos , Nervo Hipoglosso/fisiologia , Laringe/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Animais , Cães , Músculo Esquelético/inervação
19.
Artigo em Inglês | MEDLINE | ID: mdl-23366435

RESUMO

Protection of the airway during swallow is often compromised following stroke and other neurological diseases. If a patient fails to recover airway protection with standard therapy, they are often left with few if any options to avoid repeated pneumonia resulting from aspiration. For them, the only option is no food by mouth and a PEG-tube for nutrition. Functional electrical stimulation offers possible solutions for restoring airway protection. Here we report the capabilities of transtracheal stimulation for vocal fold closure and selective stimulation of the XII cranial nerve to produce elevation of the hyolaryngotracheal complex. These stimulation locations add to our toolbox for managing swallowing difficulties and allow patients to maintain oral feeding.


Assuntos
Deglutição/fisiologia , Estimulação Elétrica/instrumentação , Sucção/instrumentação , Animais , Transtornos de Deglutição/cirurgia , Cães , Frequência Cardíaca , Pneumonia Aspirativa/prevenção & controle , Acidente Vascular Cerebral/complicações , Temperatura
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