Self-Assembled Origami Neural Probes for Scalable, Multifunctional, Three-Dimensional Neural Interface.

Flexible intracortical neural probes have drawn attention for their enhanced longevity in high-resolution neural recordings due to reduced tissue reaction. However, the conventional monolithic fabrication approach has met significant challenges in: (i) scaling the number of recording sites for electrophysiology; (ii) integrating of other physiological sensing and modulation; and (iii) configuring into three-dimensional (3D) shapes for multi-sided electrode arrays. We report an innovative self-assembly technology that allows for implementing flexible origami neural probes as an effective alternative to overcome these challenges. By using magnetic-field-assisted hybrid self-assembly, multiple probes with various modalities can be stacked on top of each other with precise alignment. Using this approach, we demonstrated a multifunctional device with scalable high-density recording sites, dopamine sensors and a temperature sensor integrated on a single flexible probe. Simultaneous large-scale, high-spatial-resolution electrophysiology was demonstrated along with local temperature sensing and dopamine concentration monitoring. A high-density 3D origami probe was assembled by wrapping planar probes around a thin fiber in a diameter of 80∼105 µm using optimal foldable design and capillary force. Directional optogenetic modulation could be achieved with illumination from the neuron-sized micro-LEDs (µLEDs) integrated on the surface of 3D origami probes. We could identify angular heterogeneous single-unit signals and neural connectivity 360° surrounding the probe. The probe longevity was validated by chronic recordings of 64-channel stacked probes in behaving mice for up to 140 days. With the modular, customizable assembly technologies presented, we demonstrated a novel and highly flexible solution to accommodate multifunctional integration, channel scaling, and 3D array configuration.

Simultaneous electrophysiology and optogenetic perturbation of the same neurons in chronically implanted animals using µLED silicon probes.

Micro-light-emitting-diode (µLED) silicon probes feature independently controllable miniature light-emitting-diodes (LEDs) embedded at several positions in each shank of a multi-shank probe, enabling temporally and spatially precise optogenetic neural circuit interrogation. Here, we present a protocol for performing causal and reproducible neural circuit manipulations in chronically implanted, freely moving animals. We describe steps for introducing optogenetic constructs, preparing and implanting a µLED probe, performing simultaneous in vivo electrophysiology with focal optogenetic perturbation, and recovering a probe following termination of an experiment. For complete details on the use and execution of this protocol, please refer to Watkins de Jong et al. (2023).1.

A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis.

Patients with triple-negative breast cancer remain at risk for metastatic disease despite treatment. The acquisition of chemoresistance is a major cause of tumor relapse and death, but the mechanisms are far from understood. We have demonstrated that breast cancer cells (BCCs) can engulf mesenchymal stem/stromal cells (MSCs), leading to enhanced dissemination. Here, we show that clinical samples of primary invasive carcinoma and chemoresistant breast cancer metastasis contain a unique hybrid cancer cell population coexpressing pancytokeratin and the MSC marker fibroblast activation protein-α. We show that hybrid cells form in primary tumors and that they promote breast cancer metastasis and chemoresistance. Using single-cell microfluidics and in vivo models, we found that there are polyploid senescent cells within the hybrid cell population that contribute to metastatic dissemination. Our data reveal that Wnt Family Member 5A (WNT5A) plays a crucial role in supporting the chemoresistance properties of hybrid cells. Furthermore, we identified that WNT5A mediates hybrid cell formation through a phagocytosis-like mechanism that requires BCC-derived IL-6 and MSC-derived C-C Motif Chemokine Ligand 2. These findings reveal hybrid cell formation as a mechanism of chemoresistance and suggest that interrupting this mechanism may be a strategy in overcoming breast cancer drug resistance.

Power-Efficient LFP-Adaptive Dynamic Zoom-and-Track Incremental ΔΣ Front-End for Dual-Band Subcortical Recordings.

We report a power-efficient analog front-end integrated circuit (IC) for multi-channel, dual-band subcortical recordings. In order to achieve high-resolution multi-channel recordings with low power consumption, we implemented an incremental ΔΣ ADC (IADC) with a dynamic zoom-and-track scheme. This scheme continuously tracks local field potential (LFP) and adaptively adjusts the input dynamic range (DR) into a zoomed sub-LFP range to resolve tiny action potentials. Thanks to the reduced DR, the oversampling rate of the IADC can be reduced by 64.3% compared to the conventional approach, leading to significant power reduction. In addition, dual-band recording can be easily attained because the scheme continuously tracks LFPs without additional on-chip hardware. A prototype four-channel front-end IC has been fabricated in 180 nm standard CMOS processes. The IADC achieved 11.3-bit ENOB at 6.8 µW, resulting in the best Walden and SNDR FoMs, 107.9 fJ/c-s and 162.1 dB, respectively, among two different comparison groups: the IADCs reported up to date in the state-of-the-art neural recording front-ends; and the recent brain recording ADCs using similar zooming or tracking techniques to this work. The intrinsic dual-band recording feature reduces the post-processing FPGA resources for subcortical signal band separation by >45.8%. The front-end IC with the zoom-and-track IADC showed an NEF of 5.9 with input-referred noise of 8.2 µVrms, sufficient for subcortical recording. The performance of the whole front-end IC was successfully validated through in vivo animal experiments.

Optogenetics reveals paradoxical network stabilizations in hippocampal CA1 and CA3.

Recurrent connectivity between excitatory neurons and the strength of feedback from inhibitory neurons are critical determinants of the dynamics and computational properties of neuronal circuits. Toward a better understanding of these circuit properties in regions CA1 and CA3 of the hippocampus, we performed optogenetic manipulations combined with large-scale unit recordings in rats under anesthesia and in quiet waking, using photoinhibition and photoexcitation with different light-sensitive opsins. In both regions, we saw striking paradoxical responses: subsets of cells increased firing during photoinhibition, while other cells decreased firing during photoexcitation. These paradoxical responses were more prominent in CA3 than in CA1, but, notably, CA1 interneurons showed increased firing in response to photoinhibition of CA3. These observations were recapitulated in simulations where we modeled both CA1 and CA3 as inhibition-stabilized networks in which strong recurrent excitation is balanced by feedback inhibition. To directly test the inhibition-stabilized model, we performed large-scale photoinhibition directed at (GAD-Cre) inhibitory cells and found that interneurons in both regions increased firing when photoinhibited, as predicted. Our results highlight the often-paradoxical circuit dynamics that are evidenced during optogenetic manipulations and indicate that, contrary to long-standing dogma, both CA1 and CA3 hippocampal regions display strongly recurrent excitation, which is stabilized through inhibition.

Simultaneous Electrophysiology and Optogenetic Perturbation of the Same Neurons in Chronically Implanted Animals using µLED Silicon Probes.

Optogenetics are a powerful tool for testing how a neural circuit influences neural activity, cognition, and behavior. Accordingly, the number of studies employing optogenetic perturbation has grown exponentially over the last decade. However, recent studies have highlighted that the impact of optogenetic stimulation/silencing can vary depending on the construct used, the local microcircuit connectivity, extent/power of illumination, and neuron types perturbed. Despite these caveats, the majority of studies employ optogenetics without simultaneously recording neural activity in the circuit that is being perturbed. This dearth of simultaneously recorded neural data is due in part to technical difficulties in combining optogenetics and extracellular electrophysiology. The recent introduction of µLED silicon probes, which feature independently controllable miniature LEDs embedded at several levels of each of multiple shanks of silicon probes, provides a tractable method for temporally and spatially precise interrogation of neural circuits. Here, we provide a protocol addressing how to perform chronic recordings using µLED probes. This protocol provides a schematic for performing causal and reproducible interrogations of neural circuits and addresses all phases of the recording process: introduction of optogenetic construct, implantation of the µLED probe, performing simultaneous optogenetics and electrophysiology in vivo , and post-processing of recorded data. SUMMARY: This method allows a researcher to simultaneously perturb neural activity and record electrophysiological signal from the same neurons with high spatial specificity using silicon probes with integrated µLEDs. We outline a procedure detailing all stages of the process for performing reliable µLED experiments in chronically implanted rodents.

EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression.

Triple-negative breast cancers (TNBCs) are frequently poorly differentiated with high propensity for metastasis. Enhancer of zeste homolog 2 (EZH2) is the lysine methyltransferase of polycomb repressive complex 2 that mediates transcriptional repression in normal cells and in cancer through H3K27me3. However, H3K27me3-independent non-canonical functions of EZH2 are incompletely understood. We reported that EZH2 phosphorylation at T367 by p38α induces TNBC metastasis in an H3K27me3-independent manner. Here, we show that cytosolic EZH2 methylates p38α at lysine 139 and 165 leading to enhanced p38α stability and that p38 methylation and activation require T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2-T367, H3K27me3, and p-p38 pathways in vivo and reduces TNBC growth and metastasis. These data uncover a cooperation between EZH2 canonical and non-canonical mechanisms and suggest that inhibition of these pathways may be a potential therapeutic strategy.

Ultraflexible and Stretchable Intrafascicular Peripheral Nerve Recording Device with Axon-Dimension, Cuff-Less Microneedle Electrode Array.

Peripheral nerve mapping tools with higher spatial resolution are needed to advance systems neuroscience, and potentially provide a closed-loop biomarker in neuromodulation applications. Two critical challenges of microscale neural interfaces are 1) how to apply them to small peripheral nerves, and 2) how to minimize chronic reactivity. A flexible microneedle nerve array (MINA) is developed, which is the first high-density penetrating electrode array made with axon-sized silicon microneedles embedded in low-modulus thin silicone. The design, fabrication, acute recording, and chronic reactivity to an implanted MINA, are presented. Distinctive units are identified in the rat peroneal nerve. The authors also demonstrate a long-term, cuff-free, and suture-free fixation manner using rose bengal as a light-activated adhesive for two time-points. The tissue response is investigated at 1-week and 6-week time-points, including two sham groups and two MINA-implanted groups. These conditions are quantified in the left vagus nerve of rats using histomorphometry. Micro computed tomography (micro-CT) is added to visualize and quantify tissue encapsulation around the implant. MINA demonstrates a reduction in encapsulation thickness over previously quantified interfascicular methods. Future challenges include techniques for precise insertion of the microneedle electrodes and demonstrating long-term recording.

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

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.

Probing subthreshold dynamics of hippocampal neurons by pulsed optogenetics.

Understanding how excitatory (E) and inhibitory (I) inputs are integrated by neurons requires monitoring their subthreshold behavior. We probed the subthreshold dynamics using optogenetic depolarizing pulses in hippocampal neuronal assemblies in freely moving mice. Excitability decreased during sharp-wave ripples coupled with increased I. In contrast to this "negative gain," optogenetic probing showed increased within-field excitability in place cells by weakening I and unmasked stable place fields in initially non-place cells. Neuronal assemblies active during sharp-wave ripples in the home cage predicted spatial overlap and sequences of place fields of both place cells and unmasked preexisting place fields of non-place cells during track running. Thus, indirect probing of subthreshold dynamics in neuronal populations permits the disclosing of preexisting assemblies and modes of neuronal operations.

A 3.1-5.2GHz, Energy-Efficient Single Antenna, Cancellation-Free, Bitwise Time-Division Duplex Transceiver for High Channel Count Optogenetic Neural Interface.

We report an energy-efficient, cancellation-free, bit-wise time-division duplex (B-TDD) transceiver (TRX) for real-time closed-loop control of high channel count neural interfaces. The proposed B-TDD architecture consists of a duty-cycled ultra-wide band (UWB) transmitter (3.1-5 GHz) and a switching U-NII band (5.2 GHz) receiver. An energy-efficient duplex is realized in a single antenna without power-hungry self-interference cancellation circuits which are prevalently used in the conventional full-duplex, single antenna transceivers. To suppress the interference between up- and down-links and enhance the isolation between the two, we devised a fast-switching scheme in a low noise amplifier and used 5× oversampling with a built-in winner-take-all voting in the receiver. The B-TDD transceiver was fabricated in 65 nm CMOS RF process, achieving low energy consumption of 0.32 nJ/b at 10 Mbps in the receiver and 9.7 pJ/b at 200 Mbps in the transmitter, respectively. For validation, the B-TDD TRX has been integrated with a µLED optoelectrode and a custom analog frontend integrated circuit in a prototype wireless bidirectional neural interface system. Successful in-vivo operation for simultaneously recording broadband neural signals and optical stimulation was demonstrated in a transgenic rodent.

A Miniaturized 256-Channel Neural Recording Interface With Area-Efficient Hybrid Integration of Flexible Probes and CMOS Integrated Circuits.

We report a miniaturized, minimally invasive high-density neural recording interface that occupies only a 1.53 mm2 footprint for hybrid integration of a flexible probe and a 256-channel integrated circuit chip. To achieve such a compact form factor, we developed a custom flip-chip bonding technique using anisotropic conductive film and analog circuit-under-pad in a tiny pitch of 75 µm. To enhance signal-to-noise ratios, we applied a reference-replica topology that can provide the matched input impedance for signal and reference paths in low-noise aimpliers (LNAs). The analog front-end (AFE) consists of LNAs, buffers, programmable gain amplifiers, 10b ADCs, a reference generator, a digital controller, and serial-peripheral interfaces (SPIs). The AFE consumes 51.92 µW from 1.2 V and 1.8 V supplies in an area of 0.0161 mm2 per channel, implemented in a 180 nm CMOS process. The AFE shows > 60 dB mid-band CMRR, 6.32 µVrms input-referred noise from 0.5 Hz to 10 kHz, and 48 MΩ input impedance at 1 kHz. The fabricated AFE chip was directly flip-chip bonded with a 256-channel flexible polyimide neural probe and assembled in a tiny head-stage PCB. Full functionalities of the fabricated 256-channel interface were validated in both in vitro and in vivo experiments, demonstrating the presented hybrid neural recording interface is suitable for various neuroscience studies in the quest of large scale, miniaturized recording systems.

Monitoring Spontaneous Quiescence and Asynchronous Proliferation-Quiescence Decisions in Prostate Cancer Cells.

The proliferation-quiescence decision is a dynamic process that remains incompletely understood. Live-cell imaging with fluorescent cell cycle sensors now allows us to visualize the dynamics of cell cycle transitions and has revealed that proliferation-quiescence decisions can be highly heterogeneous, even among clonal cell lines in culture. Under normal culture conditions, cells often spontaneously enter non-cycling G0 states of varying duration and depth. This also occurs in cancer cells and G0 entry in tumors may underlie tumor dormancy and issues with cancer recurrence. Here we show that a cell cycle indicator previously shown to indicate G0 upon serum starvation, mVenus-p27K-, can also be used to monitor spontaneous quiescence in untransformed and cancer cell lines. We find that the duration of spontaneous quiescence in untransformed and cancer cells is heterogeneous and that a portion of this heterogeneity results from asynchronous proliferation-quiescence decisions in pairs of daughters after mitosis, where one daughter cell enters or remains in temporary quiescence while the other does not. We find that cancer dormancy signals influence both entry into quiescence and asynchronous proliferation-quiescence decisions after mitosis. Finally, we show that spontaneously quiescent prostate cancer cells exhibit altered expression of components of the Hippo pathway and are enriched for the stem cell markers CD133 and CD44. This suggests a hypothesis that dormancy signals could promote cancer recurrence by increasing the proportion of quiescent tumor cells poised for cell cycle re-entry with stem cell characteristics in cancer.

3D-printed Recoverable Microdrive and Base Plate System for Rodent Electrophysiology.

Extracellular recordings in freely moving animals allow the monitoring of brain activity from populations of neurons at single-spike temporal resolution. While state-of-the-art electrophysiological recording devices have been developed in recent years (e.g., µLED and Neuropixels silicon probes), implantation methods for silicon probes in rats and mice have not advanced substantially for a decade. The surgery is complex, takes time to master, and involves handling expensive devices and valuable animal subjects. In addition, chronic silicon neural probes are practically single implant devices due to the current low success rate of probe recovery. To successfully recover silicon probes, improve upon the quality of electrophysiological recording, and make silicon probe recordings more accessible, we have designed a miniature, low cost, and recoverable microdrive system. The addition of a novel 3D-printed skull baseplate makes the surgery less invasive, faster, and simpler for both rats and mice. We provide detailed procedural instructions and print designs, allowing researchers to adapt and flexibly customize our designs to their experimental usage.

Preexisting hippocampal network dynamics constrain optogenetically induced place fields.

Memory models often emphasize the need to encode novel patterns of neural activity imposed by sensory drive. Prior learning and innate architecture likely restrict neural plasticity, however. Here, we test how the incorporation of synthetic hippocampal signals is constrained by preexisting circuit dynamics. We optogenetically stimulated small groups of CA1 neurons as mice traversed a chosen segment of a linear track, mimicking the emergence of place fields. Stimulation induced persistent place field remapping in stimulated and non-stimulated neurons. The emergence of place fields could be predicted from sporadic firing in the new place field location and the temporal relationship to peer neurons before the optogenetic perturbation. Circuit modification was reflected by altered spike transmission between connected pyramidal cells and inhibitory interneurons, which persisted during post-experience sleep. We hypothesize that optogenetic perturbation unmasked sub-threshold place fields. Plasticity in recurrent/lateral inhibition may drive learning through the rapid association of existing states.

Needle-compatible miniaturized optoelectronic sensor for pancreatic cancer detection.

Pancreatic cancer is one of the deadliest cancers, with a 5-year survival rate of <10%. The current approach to confirming a tissue diagnosis, endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), requires a time-consuming, qualitative cytology analysis and may be limited because of sampling error. We designed and engineered a miniaturized optoelectronic sensor to assist in situ, real-time, and objective evaluation of human pancreatic tissues during EUS-FNA. A proof-of-concept prototype sensor, compatible with a 19-gauge hollow-needle commercially available for EUS-FNA, was constructed using microsized optoelectronic chips and microfabrication techniques to perform multisite tissue optical sensing. In our bench-top verification and pilot validation during surgery on freshly excised human pancreatic tissues (four patients), the fabricated sensors showed a comparable performance to our previous fiber-based system. The flexibility in source-detector configuration using microsized chips potentially allows for various light-based sensing techniques inside a confined channel such as a hollow needle or endoscopy.

Single-Cell Transcriptome Analysis of Colon Cancer Cell Response to 5-Fluorouracil-Induced DNA Damage.

DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.

Novel diamond shuttle to deliver flexible neural probe with reduced tissue compression.

The ability to deliver flexible biosensors through the toughest membranes of the central and peripheral nervous system is an important challenge in neuroscience and neural engineering. Bioelectronic devices implanted through dura mater and thick epineurium would ideally create minimal compression and acute damage as they reach the neurons of interest. We demonstrate that a three-dimensional diamond shuttle can be easily made with a vertical support to deliver ultra-compliant polymer microelectrodes (4.5-µm thick) through dura mater and thick epineurium. The diamond shuttle has 54% less cross-sectional area than an equivalently stiff silicon shuttle, which we simulated will result in a 37% reduction in blood vessel damage. We also discovered that higher frequency oscillation of the shuttle (200 Hz) significantly reduced tissue compression regardless of the insertion speed, while slow speeds also independently reduced tissue compression. Insertion and recording performance are demonstrated in rat and feline models, but the large design space of these tools are suitable for research in a variety of animal models and nervous system targets.

Early Prediction of Single-Cell Derived Sphere Formation Rate Using Convolutional Neural Network Image Analysis.

Functional identification of cancer stem-like cells (CSCs) is an established method to identify and study this cancer subpopulation critical for cancer progression and metastasis. The method is based on the unique capability of single CSCs to survive and grow to tumorspheres in harsh suspension culture environment. Recent advances in microfluidic technology have enabled isolating and culturing thousands of single cells on a chip. However, tumorsphere assay takes a relatively long period of time, limiting the throughput of this assay. In this work, we incorporated machine learning with single-cell analysis to expedite tumorsphere assay. We collected 1,710 single-cell events as the database and trained a convolutional neural network model that predicts whether a single cell could grow to a tumorsphere on Day 14 based on its Day 4 image. With this future-telling model, we precisely estimated the sphere formation rate of SUM159 breast cancer cells to be 17.8% based on Day 4 images. The estimation was close to the ground truth of 17.6% on Day 14. The preliminary work demonstrates not only the feasibility to significantly accelerate tumorsphere assay but also a synergistic combination between single-cell analysis with machine learning, which can be applied to many other biomedical applications.

Artifact-free and high-temporal-resolution in vivo opto-electrophysiology with microLED optoelectrodes.

The combination of in vivo extracellular recording and genetic-engineering-assisted optical stimulation is a powerful tool for the study of neuronal circuits. Precise analysis of complex neural circuits requires high-density integration of multiple cellular-size light sources and recording electrodes. However, high-density integration inevitably introduces stimulation artifact. We present minimal-stimulation-artifact (miniSTAR) µLED optoelectrodes that enable effective elimination of stimulation artifact. A multi-metal-layer structure with a shielding layer effectively suppresses capacitive coupling of stimulation signals. A heavily boron-doped silicon substrate silences the photovoltaic effect induced from LED illumination. With transient stimulation pulse shaping, we reduced stimulation artifact on miniSTAR µLED optoelectrodes to below 50 µVpp, much smaller than a typical spike detection threshold, at optical stimulation of >50 mW mm-2 irradiance. We demonstrated high-temporal resolution (<1 ms) opto-electrophysiology without any artifact-induced signal quality degradation during in vivo experiments. MiniSTAR µLED optoelectrodes will facilitate functional mapping of local circuits and discoveries in the brain.