Electrical microstimulation provides high-resolution control of neural circuits for causal studies and restoration of impaired functions, yet how responses to artificial activation evolve with learning remains unclear. Here, we deploy a detection task and pair ultraflexible electrodes for stable intracortical microstimulation (ICMS) with longitudinal imaging and recordings to track single-cell and population responses across weeks of learning. Detection thresholds decreased with learning, indicating plasticity. Chronic imaging showed that stimulus-evoked recruitment expanded at a fixed current, while a consistent number of neurons continued to underlie behavioral responses. A subset of learning-sensitive cells enhanced modulation and reduced latency. Electrophysiological recordings further distinguished two forms of adaptation: Directly activated, pulse-locked neurons strengthened their excitability, whereas polysynaptically recruited neurons expanded in number and were predictive of behavioral outcomes. These results show that learning in an ICMS task reshapes cortical circuits through activation-mechanism-dependent plasticity, underscoring the need for stimulation paradigms that adapt to both cell-intrinsic and network dynamics.

Advancing neural interfaces requires large-scale, high-density recording technologies capable of capturing full-spectrum neural activity across cortical and subcortical regions. Here, we present a scalable approach to integrate neural electrodes with advanced application-specific integrated circuits (ASICs). Specifically, we custom-designed an ASIC with 5376 simultaneous channels, each sampling at 20 kS/s and enabling >1.3 Gb/s total data streaming throughput. The ASIC incorporates in-pixel amplification, time-division multiplexed ADCs, and on-chip stimulation capabilities, ensuring precise signal acquisition with minimal power consumption while maintaining a low noise level of 5.5 µVrms. We further developed an interconnect strategy using gold bump bonding, which allows for high-density integration of the flexible probe and rigid chip. We demonstrate the capacity of this platform through the integration with a flexible μECoG array. The resulting device allows for the high-resolution mapping of in vivo field potentials on the cortical surfaces of rat brains, supported by the precise localization of evoked sensory activities. These results prove an effective approach towards highly integrated neural interfaces with applications in brain-computer interfaces, neuroprosthetics, and large-scale functional brain mapping.

We report a hybrid miniaturized achromatic objective for nonlinear endomicroscopy that combines two-photon polymerization (2PP)-fabricated polymer optics with a commercial glass lens. The objective achieves 0.6 numerical aperture with dual-wavelength correction at 775 and 860 nm and a 200 µm field of view, all within a 3 mm housing compatible with a hypodermic tube. Fabrication leverages sequential multi-step two-photon polymerization (2PP) to produce a monolithic multi-material triplet with submicron precision. The triplet incorporates two photoresists with different dispersion properties printed in successive steps with post-processing and marker-based realignment between steps, yielding a single monolithic optic that eliminates adhesives while correcting chromatic aberrations. Imaging tests with USAF resolution targets, biological samples, and two-photon fluorescence microscopy of pollen grains demonstrate near-diffraction-limited performance across the design wavelength range. The approach offers a practical method for compact optical systems in biomedical applications.

Advancing neural interfaces requires large-scale, high-density recording technologies capable of capturing full-spectrum neural activity across cortical and subcortical regions. Here, we present a scalable approach to integrate neural electrodes with advanced application-specific integrated circuits (ASICs). Specifically, we custom-designed an ASIC with 5,376 simultaneous channels, each sampling at 20 kS/s and enabling >1.3 Gb/s total data streaming throughput. The ASIC incorporates in-pixel amplification, time-division multiplexed ADCs, and on-chip stimulation capabilities, ensuring precise signal acquisition with minimal power consumption while maintaining a low noise level of 5.5 μVrms. We further developed an interconnect strategy using gold bump bonding, which allows for high-density integration of the flexible probe and rigid chip. We demonstrate the capacity of this platform through the integration with a flexible μECoG array. The resulting device allows for the high-resolution mapping of in vivo field potentials on the cortical surfaces of rat brains, supported by the precise localization of evoked sensory activities. These results prove an effective approach towards highly integrated neural interfaces with applications in brain-computer interfaces, neuroprosthetics, and large-scale functional brain mapping.

Transcranial focused ultrasound (tFUS) is a promising technique that has been shown to have high spatial precision, deep brain penetration, and cell-type specificity. Intracranial electrophysiological recordings can measure neural responses to tFUS with high spatial and temporal resolution, but conventional silicon-based multi-electrode arrays cause vibration artifacts induced by increased tFUS pressure. In this study, using an ultraflexible nanoelectric thread electrode, we demonstrate the cell-type selective effects of tFUS under high acoustic pressure with a broad range of ultrasound parameters. We observe that their flexibility mitigates vibrations, eliminating artifacts even at high pressure levels. We observed a positive nonlinear relationship between pressure levels and both time-locked and delayed spiking responses in multiple cell types. We show that higher pressure levels produce distinct response curves across independently varied ultrasound pulse repetition frequency and duty cycle, suggesting the need for further investigation of pressure effects.

Intracerebral Hemorrhagic (ICH) stroke is the second most common type of stroke and its aftermath is often more severe than ischemia. Recent population data has shown increasing trends of ICH in middle aged people, contributing to the economic burden of society. In order to improve post ICH outcome, immediate therapeutic interventions should be administered. Hence, our research is looking into the effects of electrical stimulation of the perihematomal cortex in the hyperacute/acute phase post ICH. The end goal is to test improvement in outcomes of subjects post the administration of such a stimulation paradigm by using the observations in experimental data to validate the theoretical model and hence design a closed loop stimulation paradigm. The two approaches in the work: Experimental: Hemorrhagic stroke is induced in minipigs by injecting 2cc of blood into the ventricular space beneath sensorimotor cortex to simulate an ICH. We insert ultra flexible microelectrodes in this region to record cortical neuronal activity and administer stimulation. Neural activity (Electrophysiological) data is collected pre/post stroke and during stimulation. The data collected is analyzed to understand changes in neural activity at different points in time. Through preliminary data analysis, we could clearly see the changes in the statistics of neural activity (pre/post ICH InterSpike Interval (ISI) histogram and firing frequency histogram). Local connectivity changes were observed in the data. Further removal of stimulation artifacts and analysis of neural data before, during and after stimulation shall be performed to understand the changes that occur during stimulation. Theoretical: We have mathematically modeled a neuron-astrocyte-vascular system in the cortical perihematoma by extending Hodgkin-Huxley biophysics to simulate pre/post acute ICH and electrical stimulation paradigms in such conditions. The results obtained help us understand the changes in the system dynamics post ICH with bifurcation analysis giving unique outlook of the complex system. The simulated data shows increase in excitability of perihematomal tissue during acute phase. Further, the model also showcases calcium ion dyshomeostasis and problems in ATP production and consumption. Normal stimulation paradigms are shown to not work in such settings. Further exploration of the intricacies of the system is at focus to better understand electrical stimulation in such cerebrovascular conditions.

Stably representing recurring visual scenes is crucial for behavior. However, previous studies report varying degrees of gradual neural activity changes over time in slow dynamic (1-5 seconds) firing rate code. Here we show that temporal codes, which capture structures in visually evoked fast (tens of milliseconds) spiking patterns, support the stability of visual representations. We tracked the spiking responses of the same visual cortical populations in male mice for 15 consecutive days using custom-developed, large-scale, ultraflexible electrode arrays. Across various stimuli, neurons exhibited different day-to-day stability in their firing rate-based tuning. The across day stability correlated with tuning reliability. Notably, temporal codes increased single neuron tuning stability, especially for less reliable neurons. Temporal coding further improved population representation discriminability and decoding accuracy. The stability of temporal codes was more correlated with network functional connectivity than rate coding. Thus, temporal coding may be essential in ensuring consistent sensory experiences over time.

We present a fully 3D-printed, broadband endomicroscopic objective optimized for near-infrared two-photon fluorescence imaging, featuring a compact, alignment-free design. The objective provides a numerical aperture of 0.6 in water immersion over a 200 µm field of view and maintains near diffraction-limited performance across the 720–950 nm excitation band. Monolithically fabricated with multi-material via two-photon polymerization (2PP), the three-element refractive objective achieves a 2 mm lens mechanical diameter, a mechanical housing diameter of 3 mm, and a total length of 2.1 mm with an approximately 10-hour build time. A self-aligning architecture integrates the printed half-housing with the optics to minimize assembly error, yielding sub-10 µm element decenter and <0.1° element tilt. Optical performance was validated using a resolution target and biological specimens, with broadband excitation behavior assessed by two-photon imaging of pollen grains across multiple excitation wavelengths, and with autofluorescence imaging of fresh mouse liver confirming feasibility for in situ tissue imaging. This work demonstrates the potential of fully printed broadband objectives for compact, high-performance endomicroscopy and integrated biomedical optics.

Neural representations arise from high-dimensional population activity, but current neuromodulation methods lack the precision to write information into the central nervous system at this complexity. In this perspective, we propose high-dimensional stimulation as an approach to better approximate natural neural codes for brain-machine interfaces. Key advancements in resolution, coverage, and safety are essential, with flexible microelectrode arrays offering a promising path toward precise synthetic neural codes.

Microinfarcts, the "invisible lesions", are prevalent in aged and injured brains and associated with cognitive impairments, yet their neurophysiological impact remains largely unknown. Using a multimodal chronic neural platform that combines functional microvasculature imaging with spatially resolved neural recording, the neurovascular effect of a single microinfarct is investigated. Unlike larger strokes, microinfarcts induced only temporary suppression of neural activity with minimal cell death, with recovery paralleling vasculature remodeling at the infarct core. Neural activity is more severely suppressed at the shallower cortical layer despite milder vascular damage compared to deeper layers, and the excitability of fast-spiking interneurons attenuation is accompanied by heightened bursting of regular spiking neurons. Spike phase locking at the low-gamma band is disrupted, indicating a lasting impairment of long-range assembly communication. These results highlight the subtle yet significant neurovascular disruptions of a single microinfarct.

Implantable electrode arrays are powerful tools for directly interrogating neural circuitry in the brain, but implementing this technology in the spinal cord in behaving animals has been challenging due to the spinal cord’s significant motion with respect to the vertebral column during behavior. Consequently, the individual and ensemble activity of spinal neurons processing motor commands remains poorly understood. Here, we demonstrate that custom ultraflexible 1-μm-thick polyimide nanoelectronic threads can conduct laminar recordings of many neuronal units within the lumbar spinal cord of unrestrained, freely moving mice. The extracellular action potentials have high signal-to-noise ratio, exhibit well-isolated feature clusters, and reveal diverse patterns of activity during locomotion. Furthermore, chronic recordings demonstrate the stable tracking of single units and their functional tuning over multiple days. This technology provides a path for elucidating how spinal circuits compute motor actions.

Peripheral nerve interfaces (PNIs) are electrical systems designed to integrate with peripheral nerves in patients, such as following central nervous system (CNS) injuries to augment or replace CNS control and restore function. We review the literature for clinical trials and studies containing clinical outcome measures to explore the utility of human applications of PNIs. We discuss the various types of electrodes currently used for PNI systems and their functionalities and limitations. We discuss important design characteristics of PNI systems, including biocompatibility, resolution and specificity, efficacy, and longevity, to highlight their importance in the current and future development of PNIs. The clinical outcomes of PNI systems are also discussed. Finally, we review relevant PNI clinical trials that were conducted, up to the present date, to restore the sensory and motor function of upper or lower limbs in amputees, spinal cord injury patients, or intact individuals and describe their significant findings. This review highlights the current progress in the field of PNIs and serves as a foundation for future development and application of PNI systems.

Neural stimulation has a variety of applications in neuroscience research and clinical therapies. Conventionally, stimulation is delivered by implanted electrodes, such as in intracortical microstimulation (ICMS) and deep brain stimulation (DBS). However, long-term reliability associated with the electrode-tissue interface remains a fundamental challenge [1], due to foreign body responses that can lead to glial scar formation and encapsulation of electrodes (Fig. 17.5.1). Magnetic stimulation has emerged as a promising alternative to overcome this challenge, as no direct contact is needed between the stimulation coil and tissues. Its efficacy has been clinically verified through FDA-approved transcranial magnetic stimulation (TMS) for treating neurological disorders. However, TMS devices are power-hungry and bulky (Fig. 17.5.1), which not only restricts patient accessibility but also limits the spatial resolution of the stimulation.

Penetrating neural electrodes provide a powerful approach to decipher brain circuitry by allowing for time-resolved electrical detections of individual action potentials. This unique capability has contributed tremendously to basic and translational neuroscience, enabling both fundamental understandings of brain functions and applications of human prosthetic devices that restore crucial sensations and movements. However, conventional approaches are limited by the scarce number of available sensing channels and compromised efficacy over long-term implantations. Recording longevity and scalability have become the most sought-after improvements in emerging technologies. In this review, we discuss the technological advances in the past 5–10 years that have enabled larger-scale, more detailed, and longer-lasting recordings of neural circuits at work than ever before. We present snapshots of the latest advances in penetration electrode technology, showcase their applications in animal models and humans, and outline the underlying design principles and considerations to fuel future technological development.

Intracortical microstimulation (ICMS) enables applications ranging from neuroprosthetics to causal circuit manipulations. However, the resolution, efficacy, and chronic stability of neuromodulation is often compromised by the adverse tissue responses to the indwelling electrodes. Here we engineer ultraflexible stim-Nanoelectronic Threads (StimNETs) and demonstrate low activation threshold, high resolution, and chronically stable ICMS in awake, behaving mouse models. In vivo two-photon imaging reveals that StimNETs remain seamlessly integrated with the nervous tissue throughout chronic stimulation periods and elicit stable, focal neuronal activation at low currents of 2 μA. Importantly, StimNETs evoke longitudinally stable behavioral responses for over eight months at markedly low charge injection of 0.25 nC/phase. Quantified histological analysis show that chronic ICMS by StimNETs induce no neuronal degeneration or glial scarring. These results suggest that tissue-integrated electrodes provide a path for robust, long-lasting, spatially-selective neuromodulation at low currents which lessen risks of tissue damage or exacerbation of off-target side-effects.

Conventional pseudo-resistors used in AC-coupled biopotential amplifiers suffer from intrinsic and photo-induced leakage currents, which may saturate the amplifier and induce an input-amplitude-dependent DC drift at the amplifier output. To mitigate these issues, this paper presents a new complementary pseudo-resistor with a leakage current self-compensation mechanism. As proof of concept, a capacitive feedback biopotential amplifier using the proposed pseudo-resistor is designed in the DB HiTek 180nm CMOS process. In the measurement, it achieves enhanced robustness against both input-amplitude-dependent DC drift and light exposure, compared to the amplifier using the conventional pseudo-resistor. It also demonstrates high signal fidelity during in vitro tests using pre-recorded neural signals.

Significance: Electrophysiological recording and optical imaging are two prevalent neurotechnologies with complementary strengths, the combined application of which can significantly improve our capacity in deciphering neural circuits. Flexible electrode arrays can support longitudinal optical imaging in the same brain region, but their mechanical flexibility makes surgical preparation challenging. Here, we provide a step-by-step protocol by which an ultraflexible nanoelectronic thread is co-implanted with a cranial window in a single surgery to enable chronic, dual-modal measurements. Aim: The method uses 1 - μ m -thick polymer neural electrodes which conform to the site of implantation. The mechanical flexibility of the probe allows bending without breaking and enables long-lasting electrophysiological recordings of single-unit activities and concurrent, high-resolution optical imaging through the cranial window. Approach: The protocol describes methods and procedures to co-implant an ultraflexible electrode array and a glass cranial window in the mouse neocortex. The implantation strategy includes temporary attachment of flexible electrodes to a retractable tungsten-microwire insertion shuttle, craniotomy, stereotaxic insertion of the electrode array, skull fixation of the cranial window and electrode, and installation of a head plate. Results: The resultant implant allows simultaneous interrogation of brain activity both electrophysiologically and optically for several months. Importantly, a variety of optical imaging modalities, including wide-field fluorescent imaging, two-photon microscopy, and functional optical imaging, can be readily applied to the specific brain region where ultraflexible electrodes record from. Conclusions: The protocol describes a method for co-implantation of ultraflexible neural electrodes and a cranial window for chronic, multimodal measurements of brain activity in mice. Device preparation and surgical implantation are described in detail to guide the adaptation of these methods for other flexible neural implants and cranial windows.

Background Anesthetized animal models are used extensively during neurophysiological and behavioral studies despite systemic effects from anesthesia that undermine both accurate interpretation and translation to awake human physiology. The majority of work examining the impact of anesthesia on cerebral blood flow (CBF) has been restricted to before and after measurements with limited spatial resolution. New Method We used multi-exposure speckle imaging (MESI), an advanced form of laser speckle contrast imaging (LSCI), to characterize the dynamics of isoflurane anesthesia induction on cerebral vasculature and blood flow in the mouse brain. Results The large anatomical changes caused by isoflurane are depicted with wide-field imagery and video highlighting the induction of general anesthesia. Within minutes of exposure, both vessel diameter and blood flow increased drastically compared to the awake state and remained elevated for the duration of imaging. An examination of the dynamics of anesthesia induction reveals that blood flow increased faster in arteries than in veins or parenchyma regions. Comparison with Existing Methods MESI offers robust hemodynamic measurements across large fields-of-view and high temporal resolutions sufficient for continuous visualization of cerebrovascular events featuring major changes in blood flow. Conclusion The large alterations caused by isoflurane anesthesia to the cortical vasculature and CBF are readily characterized using MESI. These changes are unrepresentative of normal physiology and provide further evidence that neuroscience experiments would benefit from transitioning to un-anesthetized awake animal models.

Flexible neural electrodes improve the recording longevity and quality of individual neurons by promoting tissue-electrode integration. However, the intracortical implantation of flexible electrodes inevitably induces tissue damage. Understanding the longitudinal neural and vascular recovery following the intracortical implantation is critical for the ever-growing applications of flexible electrodes in both healthy and disordered brains. Aged animals are of particular interest because they play a key role in modeling neurological disorders, but their tissue-electrode interface remains mostly unstudied. Here we integrate in-vivo two-photon imaging and electrophysiological recording to determine the time-dependent neural and vascular dynamics after the implantation of ultraflexible neural electrodes in aged mice. We find heightened angiogenesis and vascular remodeling in the first two weeks after implantation, which coincides with the rapid increase in local field potentials and unit activities detected by electrophysiological recordings. Vascular remodeling in shallow cortical layers preceded that in deeper layers, which often lasted longer than the recovery of neural signals. By six weeks post-implantation vascular abnormalities had subsided, resulting in normal vasculature and microcirculation. Putative cell classification based on firing pattern and waveform shows similar recovery time courses in fast-spiking interneurons and pyramidal neurons. These results elucidate how structural damages and remodeling near implants affecting recording efficacy, and support the application of ultraflexible electrodes in aged animals at minimal perturbations to endogenous neurophysiology.

Penetrating flexible electrode arrays can simultaneously record thousands of individual neurons in the brains of live animals. However, it has been challenging to spatially map and longitudinally monitor the dynamics of large three-dimensional neural networks. Here we show that optimized ultraflexible electrode arrays distributed across multiple cortical regions in head-fixed mice and in freely moving rats allow for months-long stable electrophysiological recording of several thousand neurons at densities of about 1,000 neural units per cubic millimetre. The chronic recordings enhanced decoding accuracy during optogenetic stimulation and enabled the detection of strongly coupled neuron pairs at the million-pair and millisecond scales, and thus the inference of patterns of directional information flow. Longitudinal and volumetric measurements of neural couplings may facilitate the study of large-scale neural circuits. Optimized ultraflexible electrode arrays enable months-long electrophysiological recordings of several thousand neurons at densities of up to 1,000 neural units per cubic millimetre.