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.

Transcranial electric stimulation modulates firing rate at clinically relevant intensities.

BACKGROUND: Notwithstanding advances with low-intensity transcranial electrical stimulation (tES), there remain questions about the efficacy of clinically realistic electric fields on neuronal function. OBJECTIVE: To measure electric fields magnitude and their effects on neuronal firing rate of hippocampal neurons in freely moving rats, and to establish calibrated computational models of current flow. METHODS: Current flow models were calibrated on electric field measures in the motor cortex (n = 2 anesthetized rats) and hippocampus. A Neuropixels 2.0 probe with 384 channels was used in an in-vivo rat model of tES (n = 4 freely moving and 2 urethane anesthetized rats) to detect effects of weak fields on neuronal firing rate. High-density field mapping and computational models verified field intensity (1 V/m in hippocampus per 50 µA of applied skull currents). RESULTS: Electric fields of as low as 0.35 V/m (0.25-0.47) acutely modulated average firing rate in the hippocampus. At these intensities, firing rate effects increased monotonically with electric field intensity at a rate of 11.5 % per V/m (7.2-18.3). For the majority of excitatory neurons, firing increased for soma-depolarizing stimulation and diminished for soma-hyperpolarizing stimulation. While more diverse, the response of inhibitory neurons followed a similar pattern on average, likely as a result of excitatory drive. CONCLUSION: In awake animals, electric fields modulate spiking rate above levels previously observed in vitro. Firing rate effects are likely mediated by somatic polarization of pyramidal neurons. We recommend that all future rodent experiments directly measure electric fields to insure rigor and reproducibility.

Interaction of acetylcholine and oxytocin neuromodulation in the hippocampus.

A postulated role of subcortical neuromodulators is to control brain states. Mechanisms by which different neuromodulators compete or cooperate at various temporal scales remain an open question. We investigated the interaction of acetylcholine (ACh) and oxytocin (OXT) at slow and fast timescales during various brain states. Although these neuromodulators fluctuated in parallel during NREM packets, transitions from NREM to REM were characterized by a surge of ACh but a continued decrease of OXT. OXT signaling lagged behind ACh. High ACh was correlated with population synchrony and gamma oscillations during active waking, whereas minimum ACh predicts sharp-wave ripples (SPW-Rs). Optogenetic control of ACh and OXT neurons confirmed the active role of these neuromodulators in the observed correlations. Synchronous hippocampal activity consistently reduced OXT activity, whereas inactivation of the lateral septum-hypothalamus path attenuated this effect. Our findings demonstrate how cooperative actions of these neuromodulators allow target circuits to perform specific functions.

Brain-implanted conductors amplify radiofrequency fields in rodents: Advantages and risks.

Over the past few decades, daily exposure to radiofrequency (RF) fields has been increasing due to the rapid development of wireless and medical imaging technologies. Under extreme circumstances, exposure to very strong RF energy can lead to heating of body tissue, even resulting in tissue injury. The presence of implanted devices, moreover, can amplify RF effects on surrounding tissue. Therefore, it is important to understand the interactions of RF fields with tissue in the presence of implants, in order to establish appropriate wireless safety protocols, and also to extend the benefits of medical imaging to increasing numbers of people with implanted medical devices. This study explored the neurological effects of RF exposure in rodents implanted with neuronal recording electrodes. We exposed freely moving and anesthetized rats and mice to 950 MHz RF energy while monitoring their brain activity, temperature, and behavior. We found that RF exposure could induce fast onset firing of single neurons without heat injury. In addition, brain implants enhanced the effect of RF stimulation resulting in reversible behavioral changes. Using an optical temperature measurement system, we found greater than tenfold increase in brain temperature in the vicinity of the implant. On the one hand, our results underline the importance of careful safety assessment for brain-implanted devices, but on the other hand, we also show that metal implants may be used for neurostimulation if brain temperature can be kept within safe limits.

Autonomous animal heating and cooling system for temperature-regulated magnetic resonance experiments.

Temperature is a hallmark parameter influencing almost all magnetic resonance properties (e.g., T1 , T2 , proton density, and diffusion). In the preclinical setting, temperature has a large influence on animal physiology (e.g., respiration rate, heart rate, metabolism, and oxidative stress) and needs to be carefully regulated, especially when the animal is under anesthesia and thermoregulation is disrupted. We present an open-source heating and cooling system capable of regulating the temperature of the animal. The system was designed using Peltier modules capable of heating or cooling a circulating water bath with active temperature feedback. Feedback was obtained using a commercial thermistor, placed in the animal rectum, and a proportional-integral-derivative controller was used to modulate the temperature. Its operation was demonstrated in a phantom as well as in mouse and rat animal models, where the standard deviation of the temperature of the animal upon convergence was less than a 10th of a degree. An application where brain temperature of a mouse was modulated was demonstrated using an invasive optical probe and noninvasive magnetic resonance spectroscopic thermometry measurements.

Transcranial electric stimulation modulates firing rate at clinically relevant intensities.

Notwithstanding advances with low-intensity transcranial electrical stimulation (TES), there remain questions about the efficacy of clinically realistic electric fields on neuronal function. We used Neuropixels 2.0 probe with 384 channels in an in-vivo rat model of TES to detect effects of weak fields on neuronal firing rate. High-density field mapping and computational models verified field intensity (1 V/m in hippocampus per 50 µA of applied skull currents). We demonstrate that electric fields below 0.5 V/m acutely modulate firing rate in 5% of neurons recorded in the hippocampus. At these intensities, average firing rate effects increased monotonically with electric field intensity at a rate of 7 % per V/m. For the majority of excitatory neurons, firing increased for cathodal stimulation and diminished for anodal stimulation. While more diverse, the response of inhibitory neurons followed a similar pattern on average, likely as a result of excitatory drive. Our results indicate that responses to TES at clinically relevant intensities are driven by a fraction of high-responder excitatory neurons, with polarity-specific effects. We conclude that transcranial electric stimulation is an effective neuromodulator at clinically realistic intensities.

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.

ThermoMaze: A behavioral paradigm for readout of immobility-related brain events.

Brain states fluctuate between exploratory and consummatory phases of behavior. These state changes affect both internal computation and the organism's responses to sensory inputs. Understanding neuronal mechanisms supporting exploratory and consummatory states and their switching requires experimental control of behavioral shifts and collecting sufficient amounts of brain data. To achieve this goal, we developed the ThermoMaze, which exploits the animal's natural warmth-seeking homeostatic behavior. By decreasing the floor temperature and selectively heating unmarked areas, mice avoid the aversive state by exploring the maze and finding the warm spot. In its design, the ThermoMaze is analogous to the widely used water maze but without the inconvenience of a wet environment and, therefore, allows the collection of physiological data in many trials. We combined the ThermoMaze with electrophysiology recording, and report that spiking activity of hippocampal CA1 neurons during sharp-wave ripple events encode the position of the animal. Thus, place-specific firing is not confined to locomotion and associated theta oscillations but persist during waking immobility and sleep at the same location. The ThermoMaze will allow for detailed studies of brain correlates of immobility, preparatory-consummatory transitions and open new options for studying behavior-mediated temperature homeostasis.

Autonomous animal heating and cooling system for temperature-regulated MR experiments.

Temperature is a hallmark parameter influencing almost all magnetic resonance properties (e.g., T\textsubscript{1}, T\textsubscript{2}, proton density, diffusion and more). In the pre-clinical setting, temperature has a large influence on animal physiology (e.g., respiration rate, heart rate, metabolism, cellular stress, and more) and needs to be carefully regulated, especially when the animal is under anesthesia and thermoregulation is disrupted. We present an open-source heating and cooling system capable of stabilizing the temperature of the animal. The system was designed using Peltier modules capable of heating or cooling a circulating water bath with active temperature feedback. Feedback was obtained using a commercial thermistor, placed in the animal rectum, and a proportional{\text -}integral{\text -}derivative (PID) controller capable of locking the temperature. Operation was demonstrated in a phantom as well as mouse and rat animal models, where the standard deviation of the temperature of the animal upon convergence was less than a tenth of a degree. An application where brain temperature of a mouse was modulated was demonstrated using an invasive optical probe and non-invasive magnetic resonance spectroscopic thermometry measurements.

Brain rhythms have come of age.

Neuronal oscillations offer access to neuronal operations, bringing microscopic and macroscopic mechanisms, experimental methods, and explanations to a common platform. The field of brain rhythms has become the agora of discussions from temporal coordination of neuronal populations within and across brain regions to cognitive phenomena, including language and brain diseases.

Multilayer Arrays for Neurotechnology Applications (MANTA): Chronically Stable Thin-Film Intracortical Implants.

Flexible implantable neurointerfaces show great promise in addressing one of the major challenges of implantable neurotechnology, namely the loss of signal connected to unfavorable probe tissue interaction. The authors here show how multilayer polyimide probes allow high-density intracortical recordings to be combined with a reliable long-term stable tissue interface, thereby progressing toward chronic stability of implantable neurotechnology. The probes could record 10-60 single units over 5 months with a consistent peak-to-peak voltage at dimensions that ensure robust handling and insulation longevity. Probes that remain in intimate contact with the signaling tissue over months to years are a game changer for neuroscience and, importantly, open up for broader clinical translation of systems relying on neurotechnology to interface the human brain.

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.

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.

Brain temperature affects quantitative features of hippocampal sharp wave ripples.

Biochemical mechanisms are temperature dependent. Brain temperature shows wide variations across brain states, and such changes may explain quantitative changes in network oscillations. Here, we report on the relationship between various hippocampal sharp wave ripple features to brain temperature. Ripple frequency, occurrence rate, and duration correlated with temperature dynamics. By focal manipulation of the brain temperature in the hippocampal CA1 region, we show that ripple frequency can be increased and decreased by local heating and cooling, respectively. Changes of other parameters, such as the rate of sharp wave-ripple complex (SPW-R) and ripple duration were not consistently affected. Our findings suggest that brain temperature in the CA1 region plays a leading role in affecting ripple frequency, whereas other parameters of SPW-Rs may be determined by mechanisms upstream from the CA1 region. These findings illustrate that physiological variations of brain temperature exert important effects on hippocampal circuit operations.NEW & NOTEWORTHY During physiological conditions, brain temperature fluctuates approximately 3°C between sleep and active waking. Here, we show that features of hippocampal ripples, including the rate of occurrence, peak frequency, and duration are correlated with brain temperature variations. Focal bidirectional manipulation of temperature in the hippocampal CA1 region in awake rodents show that ripple frequency can be altered in the direction expected from the correlational observations, implying that temperature plays a significant role.

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.

Neuronal activity under transcranial radio-frequency stimulation in metal-free rodent brains in-vivo.

As the use of Radio Frequency (RF) technologies increases, the impact of RF radiation on neurological function continues to receive attention. Whether RF radiation can modulate ongoing neuronal activity by non-thermal mechanisms has been debated for decades. However, the interactions between radiated energy and metal-based neural probes during experimentation could impact neural activity, making interpretation of the results difficult. To address this problem, we modified a miniature 1-photon Ca2+ imaging device to record interference-free neural activity and compared the results to those acquired using metal-containing silicon probes. We monitored the neuronal activity of awake rodent-brains under RF energy exposure (at 950 MHz) and in sham control paradigms. Spiking activity was reliably affected by RF energy in metal containing systems. However, we did not observe neuronal responses using metal-free optical recordings at induced local electric field strengths up to 230 V/m. Our results suggest that RF exposure higher than levels that are allowed by regulatory limits in real-life scenarios do not affect neuronal activity.

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.

Metal microdrive and head cap system for silicon probe recovery in freely moving rodent.

High-yield electrophysiological extracellular recording in freely moving rodents provides a unique window into the temporal dynamics of neural circuits. Recording from unrestrained animals is critical to investigate brain activity during natural behaviors. The use and implantation of high-channel-count silicon probes represent the largest cost and experimental complexity associated with such recordings making a recoverable and reusable system desirable. To address this, we have designed and tested a novel 3D printed head-gear system for freely moving mice and rats. The system consists of a recoverable microdrive printed in stainless steel and a plastic head cap system, allowing researchers to reuse the silicon probes with ease, decreasing the effective cost, and the experimental effort and complexity. The cap designs are modular and provide structural protection and electrical shielding to the implanted hardware and electronics. We provide detailed procedural instructions allowing researchers to adapt and flexibly modify the head-gear system.

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.