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HectoSTAR μLED Optoelectrodes for Large-Scale, High-Precision In Vivo Opto-Electrophysiology.

ABSTRACT: Dynamic interactions within and across brain areas underlie behavioral and cognitive functions. To understand the basis of these processes, the activities of distributed local circuits inside the brain of a behaving animal must be synchronously recorded while the inputs to these circuits are precisely manipulated. Even though recent technological advances have enabled such large-scale recording capabilities, the development of the high-spatiotemporal-resolution and large-scale modulation techniques to accompany those recordings has lagged. A novel neural probe is presented in this work that enables simultaneous electrical monitoring and optogenetic manipulation of deep neuronal circuits at large scales with a high spatiotemporal resolution. The "hectoSTAR" micro-light-emitting-diode (μLED) optoelectrode features 256 recording electrodes and 128 stimulation μLEDs monolithically integrated on the surface of its four 30-µm thick silicon micro-needle shanks, covering a large volume with 1.3-mm × 0.9-mm cross-sectional area located as deep as 6 mm inside the brain. The use of this device in behaving mice for dissecting long-distance network interactions across cortical layers and hippocampal regions is demonstrated. The recording-and-stimulation capabilities hectoSTAR μLED optoelectrodes enables will open up new possibilities for the cellular and circuit-based investigation of brain functions in behaving animals.

SUBMITTER: Voroslakos M

PROVIDER: S-EPMC9218760 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Objective.Non-human primates (NHPs) are critical for development of translational neural technologies because of their neurological and neuroanatomical similarities to humans. Large-scale neural interfaces in NHPs with multiple modalities for stimulation and data collection poise us to unveil network-scale dynamics of both healthy and unhealthy neural systems. We aim to develop a large-scale multi-modal interface for NHPs for the purpose of studying large-scale neural phenomena including neural disease, damage, and recovery.Approach.We present a multi-modal artificial dura (MMAD) composed of flexible conductive traces printed into transparent medical grade polymer. Our MMAD provides simultaneous neurophysiological recordings and optical access to large areas of the cortex (∼3 cm2) and is designed to mitigate photo-induced electrical artifacts. The MMAD is the centerpiece of the interfaces we have designed to support electrocorticographic recording and stimulation, cortical imaging, and optogenetic experiments, all at the large-scales afforded by the brains of NHPs. We performed electrical and optical experiments bench-side andin vivowith macaques to validate the utility of our MMAD.Main results.Using our MMAD we present large-scale electrocorticography from sensorimotor cortex of three macaques. Furthermore, we validated surface electrical stimulation in one of our animals. Our bench-side testing showed up to 90% reduction of photo-induced artifacts with our MMAD. The transparency of our MMAD was confirmed both via bench-side testing (87% transmittance) and viain vivoimaging of blood flow from the underlying microvasculature using optical coherence tomography angiography.Significance.Our results indicate that our MMAD supports large-scale electrocorticography, large-scale cortical imaging, and, by extension, large-scale optical stimulation. The MMAD prepares the way for both acute and long-term chronic experiments with complimentary data collection and stimulation modalities. When paired with the complex behaviors and cognitive abilities of NHPs, these assets prepare us to study large-scale neural phenomena including neural disease, damage, and recovery.

Project description:Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100?ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1?Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.

Project description:AimsCardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models.Methods and resultsFirst, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63-138) cm/s vs. 51 (41-64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217-254) ms vs. 192 (182-233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min-1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA.ConclusionsIn vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.

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HectoSTAR μLED Optoelectrodes for Large-Scale, High-Precision In Vivo Opto-Electrophysiology.

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