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.

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.

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.

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.