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  1. Abstract

    Mapping neuroanatomy is a foundational goal towards understanding brain function. Electron microscopy (EM) has been the gold standard for connectivity analysis because nanoscale resolution is necessary to unambiguously resolve synapses. However, molecular information that specifies cell types is often lost in EM reconstructions. To address this, we devise a light microscopy approach for connectivity analysis of defined cell types called spectral connectomics. We combine multicolor labeling (Brainbow) of neurons with multi-round immunostaining Expansion Microscopy (miriEx) to simultaneously interrogate morphology, molecular markers, and connectivity in the same brain section. We apply this strategy to directly link inhibitory neuron cell types with their morphologies. Furthermore, we show that correlative Brainbow and endogenous synaptic machinery immunostaining can define putative synaptic connections between neurons, as well as map putative inhibitory and excitatory inputs. We envision that spectral connectomics can be applied routinely in neurobiology labs to gain insights into normal and pathophysiological neuroanatomy.

  2. Abstract

    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–2irradiance. 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.

  3. 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 Spatialmore »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.« less
    Free, publicly-accessible full text available July 19, 2023
  4. Free, publicly-accessible full text available July 1, 2023
  5. Free, publicly-accessible full text available June 1, 2023
  6. Hippocampal assemblies and brain-state-dependent excitatory/inhibitory relationships are revealed using pulsed optogenetic stimulation.
    Free, publicly-accessible full text available February 4, 2023
  7. The incorporation of new information into the hippocampal network is likely to be constrained by its innate architecture and internally generated activity patterns. However, the origin, organization and consequences of such patterns remain poorly understood. In the present study we show that hippocampal network dynamics are affected by sequential neurogenesis. We birthdated CA1 pyramidal neurons with in utero electroporation over 4 embryonic days, encompassing the peak of hippocampal neurogenesis, and compared their functional features in freely moving adult mice. Neurons of the same birthdate displayed distinct connectivity, coactivity across brain states and assembly dynamics. Same-birthdate neurons exhibited overlapping spatial representations, which were maintained across different environments. Overall, the wiring and functional features of CA1 pyramidal neurons reflected a combination of birthdate and the rate of neurogenesis. These observations demonstrate that sequential neurogenesis during embryonic development shapes the preconfigured forms of adult network dynamics.
    Free, publicly-accessible full text available January 1, 2023
  8. The study of neuron morphology requires robust and comprehensive methods to quantify the differences between neurons of different subtypes and animal species. Several software packages have been developed for the analysis of neuron tracing results stored in the standard SWC format. The packages, however, provide relatively simple quantifications and their non-extendable architecture prohibit their use for advanced data analysis and visualization. We developed nGauge, a Python toolkit to support the parsing and analysis of neuron morphology data. As an application programming interface (API), nGauge can be referenced by other popular open-source software to create custom informatics analysis pipelines and advanced visualizations. nGauge defines an extendable data structure that handles volumetric constructions (e.g. soma), in addition to the SWC linear reconstructions, while remaining lightweight. This greatly extends nGauge’s data compatibility.
    Free, publicly-accessible full text available January 1, 2023
  9. 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.
    Free, publicly-accessible full text available December 3, 2022