Summary The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells and their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

While much is known about the effects of cocaine use on the cellular structure and function of neurons and synapses within the brain’s reward circuitry, relatively little is known about the effects of cocaine on astrocytes. Given the significant role that astrocytes play in modulating neuronal and synaptic function, this lack of knowledge regarding the role of astroglial adaptations in the neuropathology of drug abuse represents an important investigative need. We recently showed that astrocytes within the nucleus accumbens (NAc) core exhibit decreased volume, surface area, and synaptic colocalization following cocaine self-administration and extinction, compared to NAc astrocytes from saline-administering animals (Scofield et al., 2016b). However, it is unknown whether these cocaine-dependent changes in astrocytes are ubiquitous throughout the brain’s reward circuitry, or represent specific adaptations within the NAc. It is also not known whether the extinction period is necessary for the retracted phenotype, or whether self-administration alone is sufficient to drive these changes. In the current study, we have extended our assessment of the effects of cocaine self-administration on morphometric properties and synaptic colocalization of astrocyte peripheral processes in the prelimbic region of the medial prefrontal cortex (PL) and basolateral nucleus of the amygdala (BLA), both known to also contribute significantly to motivated behaviors. In addition, in order to pinpoint the temporal dimension of previously observed effects, we also examined astrocytes within the NAc following the last self-administration session. While a reduction of astrocyte size and synaptic colocalization was observed in the NAc core of cocaine-extinguished rats as previously shown, no differences in PL or BLA astrocytes were observed between saline- and cocaine-extinguished rats. Moreover, decreased synaptic colocalization of peripheral processes in the NAc was observed with a post-synaptic marker, instead of a presynaptic marker as used previously. In contrast, no significant changes were found in NAc astrocytes after self-administration alone. These results provide insights into the influence of cocaine use on astrocytes within the brain reward circuitry, and inform both regional heterogeneity as well as temporal dynamics of astrocyte responsiveness to cocaine self-administration.

Background & AimTiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Methods & ResultsFluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface.ConclusionThese results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired – as for efficient photodynamic cancer therapy.

Astrocytes play numerous vital roles in the central nervous system. Accordingly, it is of merit to identify structural and functional properties of astrocytes in both health and disease. The majority of studies examining the morphology of astrocytes have employed immunoassays for markers such as glial fibrillary acidic protein, which are insufficient to encapsulate the considerable structural complexity of these cells. Herein, we describe a method utilizing a commercially available and validated, genetically encoded membrane‐associated fluorescent marker of astrocytes, AAV5‐GfaABC1D‐Lck‐GFP. This tool and approach allow for visualization of a single isolated astrocyte in its entirety, including fine peripheral processes. Astrocytes are imaged using confocal microscopy and reconstructed in three dimensions to obtain detailed morphometric data. We further provide an immunohistochemistry procedure to assess colocalization of isolated astrocytes with synaptic markers throughout the z‐plane. This technique, which can be utilized via a standard laboratory confocal microscope and Imaris software, allows for detailed analysis of the morphology and synaptic colocalization of astrocytes in fixed tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Microinjection of AAV5‐GfaABC1D‐Lck‐GFP into the nucleus accumbens of rats Basic Protocol 2: Tissue processing and immunohistochemistry for post‐synaptic density‐95 Basic Protocol 3: Single‐cell image acquisition Basic Protocol 4: Three‐dimensional reconstruction of single cells Basic Protocol 5: Three‐dimensional colocalization analysis

It is well established that astrocytes play pivotal roles in neuronal synapse formation and maturation as well as in the modulation of synaptic transmission. Despite their general importance for brain function, relatively little is known about the maturation of astrocytes during normal postnatal development, especially during adolescence, and how that maturation may influence astroglial-synaptic contact. The medial prefrontal cortex (mPFC) and dorsal hippocampus (dHipp) are critical for executive function, memory, and their effective integration. Further, both regions undergo significant functional changes during adolescence and early adulthood that are believed to mediate these functions. However, it is unclear the extent to which astrocytes change during these late developmental periods, nor is it clear whether their association with functional synapses shifts as adolescent and young adult maturation proceeds. Here we utilize an astrocyte-specific viral labeling approach paired with high resolution single cell astrocyte imaging and threedimensional reconstruction to determine whether mPFC and dHipp astrocytes have temporally distinct maturation trajectories. mPFC astrocytes, in particular, continue to mature well into emerging adulthood (postnatal day 70). Moreover, this ongoing maturation is accompanied by a substantial increase in colocalization of astrocytes with the postsynaptic neuronal marker, PSD-95. Taken