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Abstract Objective.Characterizing the relationship between neuron spiking and the signals that electrodes record is vital to defining the neural circuits driving brain function and informing clinical brain-machine interface design. However, high electrode biocompatibility and precisely localizing neurons around the electrodes are critical to defining this relationship.Approach.Here, we demonstrate consistent localization of the recording site tips of subcellular-scale (6.8µm diameter) carbon fiber electrodes and the positions of surrounding neurons. We implanted male rats with carbon fiber electrode arrays for 6 or 12+ weeks targeting layer V motor cortex. After explanting the arrays, we immunostained the implant site and localized putative recording site tips with subcellular-cellular resolution. We then 3D segmented neuron somata within a 50µm radius from implanted tips to measure neuron positions and health and compare to healthy cortex with symmetric stereotaxic coordinates.Main results.Immunostaining of astrocyte, microglia, and neuron markers confirmed that overall tissue health was indicative of high biocompatibility near the tips. While neurons near implanted carbon fibers were stretched, their number and distribution were similar to hypothetical fibers placed in healthy contralateral brain. Such similar neuron distributions suggest that these minimally invasive electrodes demonstrate the potential to sample naturalistic neural populations. This motivated the prediction of spikes produced by nearby neurons using a simple point source model fit using recorded electrophysiology and the mean positions of the nearest neurons observed in histology. Comparing spike amplitudes suggests that the radius at which single units can be distinguished from others is near the fourth closest neuron (30.7 ± 4.6µm, $\bar{\text{X}}$± S) in layer V motor cortex.Significance.Collectively, these data and simulations provide the first direct evidence that neuron placement in the immediate vicinity of the recording site influences how many spike clusters can be reliably identified by spike sorting. 

Current standards for safe delivery of electrical stimulation to the central nervous system are based on foundational studies which examined post-mortem tissue for histological signs of damage. This set of observations and the subsequently proposed limits to safe stimulation, termed the “Shannon limits,” allow for a simple calculation (using charge per phase and charge density) to determine the intensity of electrical stimulation that can be delivered safely to brain tissue. In the three decades since the Shannon limits were reported, advances in molecular biology have allowed for more nuanced and detailed approaches to be used to expand current understanding of the physiological effects of stimulation. Here, we demonstrate the use of spatial transcriptomics (ST) in an exploratory investigation to assess the biological response to electrical stimulation in the brain. Electrical stimulation was delivered to the rat visual cortex with either acute or chronic electrode implantation procedures. To explore the influence of device type and stimulation parameters, we used carbon fiber ultramicroelectrode arrays (7 μm diameter) and microwire electrode arrays (50 μm diameter) delivering charge and charge density levels selected above and below reported tissue damage thresholds (range: 2–20 nC, 0.1–1 mC/cm 2 ). Spatial transcriptomics was performed using Visium Spatial Gene Expression Slides (10x Genomics, Pleasanton, CA, United States), which enabled simultaneous immunohistochemistry and ST to directly compare traditional histological metrics to transcriptional profiles within each tissue sample. Our data give a first look at unique spatial patterns of gene expression that are related to cellular processes including inflammation, cell cycle progression, and neuronal plasticity. At the acute timepoint, an increase in inflammatory and plasticity related genes was observed surrounding a stimulating electrode compared to a craniotomy control. At the chronic timepoint, an increase in inflammatory and cell cycle progression related genes was observed both in the stimulating vs. non-stimulating microwire electrode comparison and in the stimulating microwire vs. carbon fiber comparison. Using the spatial aspect of this method as well as the within-sample link to traditional metrics of tissue damage, we demonstrate how these data may be analyzed and used to generate new hypotheses and inform safety standards for stimulation in cortex. 

Ultrasmall microelectrode arrays have the potential to improve the spatial resolution of microstimulation. Carbon fiber (CF) microelectrodes with cross-sections of less than 8 μm have been demonstrated to penetrate cortical tissue and evoke minimal scarring in chronic implant tests. In this study, we investigate the stability and performance of neural stimulation electrodes comprised of electrodeposited platinum-iridium (PtIr) on carbon fibers. We conducted pulse testing and characterized charge injection in vitro and recorded voltage transients in vitro and in vivo. Standard electrochemical measurements (impedance spectroscopy and cyclic voltammetry) and visual inspection (scanning electron microscopy) were used to assess changes due to pulsing. Similar to other studies, the application of pulses caused a decrease in impedance and a reduction in voltage transients, but analysis of the impedance data suggests that these changes are due to surface modification and not permanent changes to the electrode. Comparison of scanning electron microscope images before and after pulse testing confirmed electrode stability.