Highly local activation of inhibition at the seizure wavefront in vivo.

The propagation of a seizure wavefront in the cortex divides an intensely firing seizure core from a low-firing seizure penumbra. Seizure propagation is currently thought to generate strong activation of inhibition in the seizure penumbra that leads to its decreased neuronal firing. However, the direct measurement of neuronal excitability during seizures has been difficult to perform in vivo. We used simultaneous optogenetics and calcium imaging (all-optical interrogation) to characterize real-time neuronal excitability in an acute mouse model of seizure propagation. We find that single-neuron excitability is decreased in close proximity to the seizure wavefront but becomes increased distal to the seizure wavefront. This suggests that inhibitory neurons of the seizure wavefront create a proximal circumference of hypoexcitability but do not influence neuronal excitability in the penumbra.

An in vitro whole-cell electrophysiology dataset of human cortical neurons.

BACKGROUND: Whole-cell patch-clamp electrophysiology is an essential technique for understanding how single neurons translate their diverse inputs into a functional output. The relative inaccessibility of live human cortical neurons for experimental manipulation has made it difficult to determine the unique features of how human cortical neurons differ from their counterparts in other species. FINDINGS: We present a curated repository of whole-cell patch-clamp recordings from surgically resected human cortical tissue, encompassing 118 neurons from 35 individuals (age range, 21-59 years; 17 male, 18 female). Recorded human cortical neurons derive from layers 2 and 3 (L2&3), deep layer 3 (L3c), or layer 5 (L5) and are annotated with a rich set of subject and experimental metadata. For comparison, we also provide a limited set of comparable recordings from 21-day-old mice (11 cells from 5 mice). All electrophysiological recordings are provided in the Neurodata Without Borders (NWB) format and are available for further analysis via the Distributed Archives for Neurophysiology Data Integration online repository. The associated data conversion code is made publicly available and can help others in converting electrophysiology datasets to the open NWB standard for general reuse. CONCLUSION: These data can be used for novel analyses of biophysical characteristics of human cortical neurons, including in cross-species or cross-lab comparisons or in building computational models of individual human neurons.

Implantable photonic neural probes for light-sheet fluorescence brain imaging.

Significance: Light-sheet fluorescence microscopy (LSFM) is a powerful technique for high-speed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. We demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes. Aim: Mass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components. Approach: We develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200-mm-diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed, in vitro, and in vivo mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose. Results: The probes had 5 to 10 addressable sheets and average sheet thicknesses < 16 µ m for propagation distances up to 300 µ m in free space. Imaging areas were as large as ≈ 240 µ m × 490 µ m in brain tissue. Image contrast was enhanced relative to epifluorescence microscopy. Conclusions: The neural probes can lead to new variants of LSFM for deep brain imaging and experiments in freely moving animals.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

A tale of two cousins: Ependymal cells, quiescent neural stem cells and potential mechanisms driving their functional divergence.

Recent work has suggested that stem cells exhibit far greater heterogeneity than initially thought. Indeed, their dynamic nature and shared traits with surrounding niche cells have made prospective identification of adult neural stem cells (NSCs) challenging. Refined fate mapping strategies and single-cell omics techniques have begun to clarify functionally distinct states within the adult NSC pool, the molecular signatures that govern these states, and the functional contributions/interactions with neighboring cells within the subventricular niche. Ependymal cells are the epithelial cells which line the ventricular system and reside in the same niche as NSCs. Our own work has revealed that, despite sharing similar embryonic origins with NSCs and close geographic proximity, ependymal cells are transcriptionally distinct and fail to exhibit stem cell function in vivo, even following injury. Intriguingly, comparison of ependymal cells with qNSCs revealed transcriptional signatures that are largely overlapping, suggesting that post-transcriptional regulation might underlie their divergent phenotypes. Additional analysis of ependymal versus qNSC gene regulatory network activation supports this notion. This Viewpoint summarizes the historical confusion regarding the identity of NSCs within the lateral ventricle niche and describes recent work that provides greater appreciation for the diverse functional states within the NSC niche.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

Purification and Characterization of Schwann Cells from Adult Human Skin and Nerve.

Despite its modest capacity for regeneration, peripheral nervous system injury often results in significant long-term disability. Supplementing peripheral nervous system injury with autologous Schwann cells (SCs) may serve to rejuvenate the postinjury environment to enhance regeneration and ultimately improve functional outcomes. However, human nerve-derived SC (hN-SC) collection procedures require invasive surgical resection. Here, we describe the characterization of SCs from adult human skin (hSk-SCs) of four male donors ranging between 27 and 46 years old. Within five weeks of isolating and culturing adherent mixed skin cells, we were able to obtain 3-5 million purified SCs. We found that hSk-SCs appeared transcriptionally indistinguishable from hN-SCs with both populations exhibiting expression of SC genes including: SOX10, SOX9, AP2A1, CDH19, EGR1, ETV5, PAX3, SOX2, CX32, DHH, NECL4, NFATC4, POU3F1, S100B, and YY1. Phenotypic analysis of hSk-SCs and hN-SCs cultures revealed highly enriched populations of SCs indicated by the high percentage of NES+ve, SOX10+ve, s100+ve and p75+ve cells, as well as the expression of a battery of other SC-associated proteins (PAX3, CDH19, ETV5, SOX2, POU3F1, S100B, EGR2, and YY1). We further show that both hSk-SCs and hN-SCs are capable of promoting axonal growth to similar degrees and that a subset of both associate with regenerating axons and form myelin following transplantation into the injured mouse sciatic nerve. Interestingly, although the majority of both hSk-SCs and hN-SCs maintained SOX10 immunoreactivity following transplant, only a subset of each activated the promyelinating factor, POU3F1, and were able to myelinate. Taken together, we demonstrate that adult hSk-SCs are genetically and phenotypically indistinguishable to hN-SCs.

Serum-free bioprocessing of adult human and rodent skin-derived Schwann cells: implications for cell therapy in nervous system injury.

Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

Macrophage polarization in nerve injury: do Schwann cells play a role?

In response to peripheral nerve injury, the inflammatory response is almost entirely comprised of infiltrating macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris and regulating the microenvironment to allow for efficient regeneration. There are several cells within the microenvironment that likely interact with macrophages to support their function - most notably the Schwann cell, the glial cell of the peripheral nervous system. Schwann cells express several ligands that are known to interact with receptors expressed by macrophages, yet the effects of Schwann cells in regulating macrophage phenotype remains largely unexplored. This review discusses macrophages in peripheral nerve injury and how Schwann cells may regulate their behavior.

The immunomodulatory properties of adult skin-derived precursor Schwann cells: implications for peripheral nerve injury therapy.

Skin-derived precursor Schwann cell (SKPSC) therapy has been identified as a potentially beneficial treatment for peripheral nerve injuries. One hypothesised mechanism by which SKPSCs enhance recovery is via the modulation of macrophages. In the present study, we investigated the immunomodulatory properties of adult rat SKPSCs, and demonstrated that these cells expressed a battery of cytokines, including interferon-γ (IFN-γ), interleukin (IL)-1ß, and, most abundantly, IL-6. Whereas macrophages exposed to depleted or fibroblast-conditioned medium secreted minimal amounts of tumor necrosis factor-α (TNF-α), in the presence of SKPSC-conditioned medium, macrophages secreted > 500 pg/mL TNF-α. Following the transplantation of SKPSCs into injured rat sciatic nerves, we observed an SKPSC density-dependent increase in the number of macrophages (Pearson's r = 0.66) and an SKPSC density-dependent decrease in myelin debris (Pearson's r = -0.68). To determine the effect of IL-6 in a proinflammatory context, macrophage cultures were primed with either lipopolysaccharide (LPS)/IFN-γ/IL-1ß or LPS/IFN-γ/IL-1ß + IL-6, and this showed a 212% and 301% increase in the number of inducible nitric oxide synthase (iNOS)-positive proinflammatory macrophages respectively. In contrast to neurons exposed to conditioned medium from unprimed macrophages, neurons treated with conditioned medium from proinflammatory-primed macrophages showed a 13-26% reduction in neurite outgrowth. Anti-IL-6 antibody combined with SKPSC transplant therapy following nerve injury did not alter macrophage numbers or debris clearance, but instead reduced iNOS expression as compared with SKPSC + IgG-treated rats. SKPSC + anti-IL-6 treatment also resulted in a two-fold increase in gastrocnemius compound muscle action potential amplitudes as compared with SKPSC + IgG treatment. Understanding the mechanisms underlying immunomodulatory aspects of SKPSC therapy and developing approaches to manipulate these responses are important for advancing Schwann cell-based therapies.