Mitochondria modulate programmed neuritic retraction.

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon "neuritosis." Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

In vivo microstimulation with cathodic and anodic asymmetric waveforms modulates spatiotemporal calcium dynamics in cortical neuropil and pyramidal neurons of male mice.

Electrical stimulation has been critical in the development of an understanding of brain function and disease. Despite its widespread use and obvious clinical potential, the mechanisms governing stimulation in the cortex remain largely unexplored in the context of pulse parameters. Modeling studies have suggested that modulation of stimulation pulse waveform may be able to control the probability of neuronal activation to selectively stimulate either cell bodies or passing fibers depending on the leading polarity. Thus, asymmetric waveforms with equal charge per phase (i.e., increasing the leading phase duration and proportionately decreasing the amplitude) may be able to activate a more spatially localized or distributed population of neurons if the leading phase is cathodic or anodic, respectively. Here, we use two-photon and mesoscale calcium imaging of GCaMP6s expressed in excitatory pyramidal neurons of male mice to investigate the role of pulse polarity and waveform asymmetry on the spatiotemporal properties of direct neuronal activation with 10-Hz electrical stimulation. We demonstrate that increasing cathodic asymmetry effectively reduces neuronal activation and results in a more spatially localized subpopulation of activated neurons without sacrificing the density of activated neurons around the electrode. Conversely, increasing anodic asymmetry increases the spatial spread of activation and highly resembles spatiotemporal calcium activity induced by conventional symmetric cathodic stimulation. These results suggest that stimulation polarity and asymmetry can be used to modulate the spatiotemporal dynamics of neuronal activity thus increasing the effective parameter space of electrical stimulation to restore sensation and study circuit dynamics.

Calcium activation of cortical neurons by continuous electrical stimulation: Frequency dependence, temporal fidelity, and activation density.

Electrical stimulation of the brain has become a mainstay of fundamental neuroscience research and an increasingly prevalent clinical therapy. Despite decades of use in basic neuroscience research and the growing prevalence of neuromodulation therapies, gaps in knowledge regarding activation or inactivation of neural elements over time have limited its ability to adequately interpret evoked downstream responses or fine-tune stimulation parameters to focus on desired responses. In this work, in vivo two-photon microscopy was used to image neuronal calcium activity in layer 2/3 neurons of somatosensory cortex (S1) in male C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J mice during 30 s of continuous electrical stimulation at varying frequencies. We show frequency-dependent differences in spatial and temporal somatic responses during continuous stimulation. Our results elucidate conflicting results from prior studies reporting either dense spherical activation of somas biased toward those near the electrode, or sparse activation of somas at a distance via axons near the electrode. These findings indicate that the neural element specific temporal response local to the stimulating electrode changes as a function of applied charge density and frequency. These temporal responses need to be considered to properly interpret downstream circuit responses or determining mechanisms of action in basic science experiments or clinical therapeutic applications.

Isoflurane and ketamine differentially influence spontaneous and evoked laminar electrophysiology in mouse V1.

General anesthesia is ubiquitous in research and medicine, yet although the molecular mechanisms of anesthetics are well characterized, their ultimate influence on cortical electrophysiology remains unclear. Moreover, the influence that different anesthetics have on sensory cortexes at neuronal and ensemble scales is mostly unknown and represents an important gap in knowledge that has widespread relevance for neural sciences. To address this knowledge gap, this work explored the effects of isoflurane and ketamine/xylazine, two widely used anesthetic paradigms, on electrophysiological behavior in mouse primary visual cortex. First, multiunit activity and local field potentials were examined to understand how each anesthetic influences spontaneous activity. Then, the interlaminar relationships between populations of neurons at different cortical depths were studied to assess whether anesthetics influenced resting-state functional connectivity. Lastly, the spatiotemporal dynamics of visually evoked multiunit and local field potentials were examined to determine how each anesthetic alters communication of visual information. We found that isoflurane enhanced the rhythmicity of spontaneous ensemble activity at 10-40 Hz, which coincided with large increases in coherence between layer IV with superficial and deep layers. Ketamine preferentially increased local field potential power from 2 to 4 Hz, and the largest increases in coherence were observed between superficial and deep layers. Visually evoked responses across layers were diminished under isoflurane, and enhanced under ketamine anesthesia. These findings demonstrate that isoflurane and ketamine anesthesia differentially impact sensory processing in V1. NEW & NOTEWORTHY We directly compared electrophysiological responses in awake and anesthetized (isoflurane or ketamine) mice. We also proposed a method for quantifying and visualizing highly variable, evoked multiunit activity. Lastly, we observed distinct oscillatory responses to stimulus onset and offset in awake and isoflurane-anesthetized mice.

A Materials Roadmap to Functional Neural Interface Design.

Advancement in neurotechnologies for electrophysiology, neurochemical sensing, neuromodulation, and optogenetics are revolutionizing scientific understanding of the brain while enabling treatments, cures, and preventative measures for a variety of neurological disorders. The grand challenge in neural interface engineering is to seamlessly integrate the interface between neurobiology and engineered technology, to record from and modulate neurons over chronic timescales. However, the biological inflammatory response to implants, neural degeneration, and long-term material stability diminish the quality of interface overtime. Recent advances in functional materials have been aimed at engineering solutions for chronic neural interfaces. Yet, the development and deployment of neural interfaces designed from novel materials have introduced new challenges that have largely avoided being addressed. Many engineering efforts that solely focus on optimizing individual probe design parameters, such as softness or flexibility, downplay critical multi-dimensional interactions between different physical properties of the device that contribute to overall performance and biocompatibility. Moreover, the use of these new materials present substantial new difficulties that must be addressed before regulatory approval for use in human patients will be achievable. In this review, the interdependence of different electrode components are highlighted to demonstrate the current materials-based challenges facing the field of neural interface engineering.

Ultra-miniature ultra-compliant neural probes with dissolvable delivery needles: design, fabrication and characterization.

Stable chronic functionality of intracortical probes is of utmost importance toward realizing clinical application of brain-machine interfaces. Sustained immune response from the brain tissue to the neural probes is one of the major challenges that hinder stable chronic functionality. There is a growing body of evidence in the literature that highly compliant neural probes with sub-cellular dimensions may significantly reduce the foreign-body response, thereby enhancing long term stability of intracortical recordings. Since the prevailing commercial probes are considerably larger than neurons and of high stiffness, new approaches are needed for developing miniature probes with high compliance. In this paper, we present design, fabrication, and in vitro evaluation of ultra-miniature (2.7 µm x 10 µm cross section), ultra-compliant (1.4 × 10-2 µN/µm in the axial direction, and 2.6 × 10-5 µN/µm and 1.8 × 10-6 µN/µm in the lateral directions) neural probes and associated probe-encasing biodissolvable delivery needles toward addressing the aforementioned challenges. The high compliance of the probes is obtained by micron-scale cross-section and meandered shape of the parylene-C insulated platinum wiring. Finite-element analysis is performed to compare the strains within the tissue during micromotion when using the ultra-compliant meandered probes with that when using stiff silicon probes. The standard batch microfabrication techniques are used for creating the probes. A dissolvable delivery needle that encases the probe facilitates failure-free insertion and precise placement of the ultra-compliant probes. Upon completion of implantation, the needle gradually dissolves, leaving behind the ultra-compliant neural probe. A spin-casting based micromolding approach is used for the fabrication of the needle. To demonstrate the versatility of the process, needles from different biodissolvable materials, as well as two-dimensional needle arrays with different geometries and dimensions, are fabricated. Further, needles incorporating anti-inflammatory drugs are created to show the co-delivery potential of the needles. An automated insertion device is developed for repeatable and precise implantation of needle-encased probes into brain tissue. Insertion of the needles without mechanical failure, and their subsequent dissolution are demonstrated. It is concluded that ultra-miniature, ultra-compliant probes and associated biodissolvable delivery needles can be successfully fabricated, and the use of the ultra-compliant meandered probes results in drastic reduction in strains imposed in the tissue as compared to stiff probes, thereby showing promise toward chronic applications.

Elastomeric and soft conducting microwires for implantable neural interfaces.

Current designs for microelectrodes used for interfacing with the nervous system elicit a characteristic inflammatory response that leads to scar tissue encapsulation, electrical insulation of the electrode from the tissue and ultimately failure. Traditionally, relatively stiff materials like tungsten and silicon are employed which have mechanical properties several orders of magnitude different from neural tissue. This mechanical mismatch is thought to be a major cause of chronic inflammation and degeneration around the device. In an effort to minimize the disparity between neural interface devices and the brain, novel soft electrodes consisting of elastomers and intrinsically conducting polymers were fabricated. The physical, mechanical and electrochemical properties of these materials were extensively characterized to identify the formulations with the optimal combination of parameters including Young's modulus, elongation at break, ultimate tensile strength, conductivity, impedance and surface charge injection. Our final electrode has a Young's modulus of 974 kPa which is five orders of magnitude lower than tungsten and significantly lower than other polymer-based neural electrode materials. In vitro cell culture experiments demonstrated the favorable interaction between these soft materials and neurons, astrocytes and microglia, with higher neuronal attachment and a two-fold reduction in inflammatory microglia attachment on soft devices compared to stiff controls. Surface immobilization of neuronal adhesion proteins on these microwires further improved the cellular response. Finally, in vivo electrophysiology demonstrated the functionality of the elastomeric electrodes in recording single unit activity in the rodent visual cortex. The results presented provide initial evidence in support of the use of soft materials in neural interface applications.

Activation and depression of neural and hemodynamic responses induced by the intracortical microstimulation and visual stimulation in the mouse visual cortex.

Objective. Intracortical microstimulation (ICMS) can be an effective method for restoring sensory perception in contemporary brain-machine interfaces. However, the mechanisms underlying better control of neuronal responses remain poorly understood, as well as the relationship between neuronal activity and other concomitant phenomena occurring around the stimulation site.Approach. Different microstimulation frequencies were investigatedin vivoon Thy1-GCaMP6s mice using widefield and two-photon imaging to evaluate the evoked excitatory neural responses across multiple spatial scales as well as the induced hemodynamic responses. Specifically, we quantified stimulation-induced neuronal activation and depression in the mouse visual cortex and measured hemodynamic oxyhemoglobin and deoxyhemoglobin signals using mesoscopic-scale widefield imaging.Main results. Our calcium imaging findings revealed a preference for lower-frequency stimulation in driving stronger neuronal activation. A depressive response following the neural activation preferred a slightly higher frequency stimulation compared to the activation. Hemodynamic signals exhibited a comparable spatial spread to neural calcium signals. Oxyhemoglobin concentration around the stimulation site remained elevated during the post-activation (depression) period. Somatic and neuropil calcium responses measured by two-photon microscopy showed similar dependence on stimulation parameters, although the magnitudes measured in soma was greater than in neuropil. Furthermore, higher-frequency stimulation induced a more pronounced activation in soma compared to neuropil, while depression was predominantly induced in soma irrespective of stimulation frequencies.Significance. These results suggest that the mechanism underlying depression differs from activation, requiring ample oxygen supply, and affecting neurons. Our findings provide a novel understanding of evoked excitatory neuronal activity induced by ICMS and offer insights into neuro-devices that utilize both activation and depression phenomena to achieve desired neural responses.

Neuronal functional connectivity is impaired in a layer dependent manner near chronically implanted intracortical microelectrodes in C57BL6 wildtype mice.

Objective.This study aims to reveal longitudinal changes in functional network connectivity within and across different brain structures near chronically implanted microelectrodes. While it is well established that the foreign-body response (FBR) contributes to the gradual decline of the signals recorded from brain implants over time, how the FBR affects the functional stability of neural circuits near implanted brain-computer interfaces (BCIs) remains unknown. This research aims to illuminate how the chronic FBR can alter local neural circuit function and the implications for BCI decoders.Approach.This study utilized single-shank, 16-channel,100µm site-spacing Michigan-style microelectrodes (3 mm length, 703µm2 site area) that span all cortical layers and the hippocampal CA1 region. Sex balanced C57BL6 wildtype mice (11-13 weeks old) received perpendicularly implanted microelectrode in left primary visual cortex. Electrophysiological recordings were performed during both spontaneous activity and visual sensory stimulation. Alterations in neuronal activity near the microelectrode were tested assessing cross-frequency synchronization of local field potential (LFP) and spike entrainment to LFP oscillatory activity throughout 16 weeks after microelectrode implantation.Main results. The study found that cortical layer 4, the input-receiving layer, maintained activity over the implantation time. However, layers 2/3 rapidly experienced severe impairment, leading to a loss of proper intralaminar connectivity in the downstream output layers 5/6. Furthermore, the impairment of interlaminar connectivity near the microelectrode was unidirectional, showing decreased connectivity from Layers 2/3 to Layers 5/6 but not the reverse direction. In the hippocampus, CA1 neurons gradually became unable to properly entrain to the surrounding LFP oscillations.Significance. This study provides a detailed characterization of network connectivity dysfunction over long-term microelectrode implantation periods. This new knowledge could contribute to the development of targeted therapeutic strategies aimed at improving the health of the tissue surrounding brain implants and potentially inform engineering of adaptive decoders as the FBR progresses. Our study's understanding of the dynamic changes in the functional network over time opens the door to developing interventions for improving the long-term stability and performance of intracortical microelectrodes.

Electrode sharpness and insertion speed reduce tissue damage near high-density penetrating arrays.

Objective. Over the past decade, neural electrodes have played a crucial role in bridging biological tissues with electronic and robotic devices. This study focuses on evaluating the optimal tip profile and insertion speed for effectively implanting Paradromics' high-density fine microwire arrays (FµA) prototypes into the primary visual cortex (V1) of mice and rats, addressing the challenges associated with the 'bed-of-nails' effect and tissue dimpling.Approach. Tissue response was assessed by investigating the impact of electrodes on the blood-brain barrier (BBB) and cellular damage, with a specific emphasis on tailored insertion strategies to minimize tissue disruption during electrode implantation.Main results.Electro-sharpened arrays demonstrated a marked reduction in cellular damage within 50µm of the electrode tip compared to blunt and angled arrays. Histological analysis revealed that slow insertion speeds led to greater BBB compromise than fast and pneumatic methods. Successful single-unit recordings validated the efficacy of the optimized electro-sharpened arrays in capturing neural activity.Significance.These findings underscore the critical role of tailored insertion strategies in minimizing tissue damage during electrode implantation, highlighting the suitability of electro-sharpened arrays for long-term implant applications. This research contributes to a deeper understanding of the complexities associated with high-channel-count microelectrode array implantation, emphasizing the importance of meticulous assessment and optimization of key parameters for effective integration and minimal tissue disruption. By elucidating the interplay between insertion parameters and tissue response, our study lays a strong foundation for the development of advanced implantable devices with a reduction in reactive gliosis and improved performance in neural recording applications.