Intelligent Beam Optimization for Light-Sheet Fluorescence Microscopy through Deep Learning

Light-sheet fluorescence microscopy (LSFM) provides the benefit of optical sectioning coupled with rapid acquisition times, enabling high-resolution 3-dimensional imaging of large tissue-cleared samples. Inherent to LSFM, the quality of the imaging heavily relies on the characteristics of the illumination beam, which only illuminates a thin section of the sample. Therefore, substantial efforts are dedicated to identifying slender, nondiffracting beam profiles that yield uniform and high-contrast images. An ongoing debate concerns the identification of optimal illumination beams for different samples: Gaussian, Bessel, Airy patterns, and/or others. However, comparisons among different beam profiles are challenging as their optimization objectives are often different. Given that our large imaging datasets (approximately 0.5 TB of images per sample) are already analyzed using deep learning models, we envisioned a different approach to the problem by designing an illumination beam tailored to boost the performance of the deep learning model. We hypothesized that integrating the physical LSFM illumination model (after passing it through a variable phase mask) into the training of a cell detection network would achieve this goal. Here, we report that joint optimization continuously updates the phase mask and results in improved image quality for better cell detection. The efficacy of our method is demonstrated through both simulations and experiments that reveal substantial enhancements in imaging quality compared to the traditional Gaussian light sheet. We discuss how designing microscopy systems through a computational approach provides novel insights for advancing optical design that relies on deep learning models for the analysis of imaging datasets

Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

ABSTRACT Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA + ependymal lining to 2,8-linked PSA + migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. IMPORTANCE Viruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain

A Knock-in Foxj1 CreERT2::GFP mouse for recombination in epithelial cells with motile cilia: Generation ofFoxj1CreERT2::GFPMICE

The transcription factor Foxj1 is expressed by cells destined to differentiate into epithelial cells projecting motile cilia into fluid- or air- filled cavities. Here we report the generation of an inducible knock-in Foxj1CreERT2::GFP mouse which we show reliably induces Cre-mediated recombination for genetic studies in epithelial cells with motile cilia throughout embryonic and postnatal development. Induction during embryonic stages revealed efficient recombination in the epithelial component of the choroid plexus in the developing brain as early as E12.5. Induction during late embryonic stages showed confined recombination not only in the choroid plexus, but also in the ventricular walls of the brain. Recombination induced during postnatal periods expanded to include epithelia of the lungs, testis, oviduct, and brain. Using these mice, we confirmed our recent discovery of a perinatally derived neuronal population in the mouse olfactory bulbs which is derived from the Foxj1 lineage. Our Foxj1CreERT2::GFP knock-in mouse will be a powerful tool for studying molecular mechanisms associated with the continuum of cells that form the Foxj1 lineage, and for assessing their physiological significance during development and aging

Clonal analysis of gliogenesis in the cerebral cortex reveals stochastic expansion of glia and cell autonomous responses to Egfr dosage

Development of the nervous system undergoes important transitions, including one from neurogenesis to gliogenesis which occurs late during embryonic gestation. Here we report on clonal analysis of gliogenesis in mice using Mosaic Analysis with Double Markers (MADM) with quantitative and computational methods. Results reveal that developmental gliogenesis in the cerebral cortex occurs in a fraction of earlier neurogenic clones, accelerating around E16.5, and giving rise to both astrocytes and oligodendrocytes. Moreover, MADM-based genetic deletion of the epidermal growth factor receptor (Egfr) in gliogenic clones revealed that Egfr is cell autonomously required for gliogenesis in the mouse dorsolateral cortices. A broad range in the proliferation capacity, symmetry of clones, and competitive advantage of MADM cells was evident in clones that contained one cellular lineage with double dosage of Egfr relative to their environment, while their sibling Egfr-null cells failed to generate glia. Remarkably, the total numbers of glia in MADM clones balance out regardless of significant alterations in clonal symmetries. The variability in glial clones shows stochastic patterns that we define mathematically, which are different from the deterministic patterns in neuronal clones. This study sets a foundation for studying the biological significance of stochastic and deterministic clonal principles underlying tissue development, and identifying mechanisms that differentiate between neurogenesis and gliogenesis.</jats:p

Deficient NRG1-ERBB signaling alters social approach: relevance to genetic mouse models of schizophrenia

Growth factor Neuregulin 1 (NRG1) plays an essential role in development and organization of the cerebral cortex. NRG1 and its receptors, ERBB3 and ERBB4, have been implicated in genetic susceptibility for schizophrenia. Disease symptoms include asociality and altered social interaction. To investigate the role of NRG1-ERBB signaling in social behavior, mice heterozygous for an Nrg1 null allele (Nrg1+/−), and mice with conditional ablation of Erbb3 or Erbb4 in the central nervous system, were evaluated for sociability and social novelty preference in a three-chambered choice task. Results showed that deficiencies in NRG1 or ERBB3 significantly enhanced sociability. All of the mutant groups demonstrated a lack of social novelty preference, in contrast to their respective wild-type controls. Effects of NRG1, ERBB3, or ERBB4 deficiency on social behavior could not be attributed to general changes in anxiety-like behavior, activity, or loss of olfactory ability. Nrg1+/− pups did not exhibit changes in isolation-induced ultrasonic vocalizations, a measure of emotional reactivity. Overall, these findings provide evidence that social behavior is mediated by NRG1-ERBB signaling

A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors

Summary Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs). Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1cre line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs

Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA(+) ependymal lining to 2,8-linked PSA(+) migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. IMPORTANCE Viruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain

To scratch an itch: Establishing a mouse model to determine active brain areas involved in acute histaminergic itch

Background: Strategies to efficiently control itch require a deep understanding of the underlying mechanisms. Several areas in the brain involved in itch and scratching responses have been postulated, but the central mechanisms that drive pruritic responses are still unknown. Histamine is recognized as a major mediator of itch in humans, and has been the most frequently used stimulus as an experimental pruritogen for brain imaging of itch. Objective: Histaminergic itch via histamine and the selective histamine H4 receptor (H4R) agonist, ST-1006, recruit brain nuclei through c-fos activation and activate specific areas in the brain. Methods: An acute itch model was established in c-fos-EGFP transgenic mice using ST-1006 and histamine. Coronal brain sections were stained for c-fos immunoreactivity and the forebrain was mapped for density of c-fos + nuclei. Results: Histamine and ST-1006 significantly increased scratching response in c-fos-EGFP mice compared to vehicle controls. Mapping c-fos immunostained brain sections revealed neuronal activity in the cortex, striatum, hypothalamus, thalamus, amygdala, and the midbrain. Conclusions: Histaminergic itch and selective H4R activation significantly increased the density of c-fos + nuclei in the medial habenula (MHb). Thus, the MHb may be a new target to investigate and subsequently develop novel mechanism-based strategies to treat itch and possibly provide a locus for pharmacological control of pruritus. Keywords: c-fos, Itch, Brain, Medial habenula, Histamine H4 recepto