A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex.

Identification of structural connections between neurons is a prerequisite to understanding brain function. Here we developed a pipeline to systematically map brain-wide monosynaptic input connections to genetically defined neuronal populations using an optimized rabies tracing system. We used mouse visual cortex as the exemplar system and revealed quantitative target-specific, layer-specific and cell-class-specific differences in its presynaptic connectomes. The retrograde connectivity indicates the presence of ventral and dorsal visual streams and further reveals topographically organized and continuously varying subnetworks mediated by different higher visual areas. The visual cortex hierarchy can be derived from intracortical feedforward and feedback pathways mediated by upper-layer and lower-layer input neurons. We also identify a new role for layer 6 neurons in mediating reciprocal interhemispheric connections. This study expands our knowledge of the visual system connectomes and demonstrates that the pipeline can be scaled up to dissect connectivity of different cell populations across the mouse brain.

Automated high-throughput light-sheet fluorescence microscopy of larval zebrafish.

Light sheet fluorescence microscopy enables fast, minimally phototoxic, three-dimensional imaging of live specimens, but is currently limited by low throughput and tedious sample preparation. Here, we describe an automated high-throughput light sheet fluorescence microscope in which specimens are positioned by and imaged within a fluidic system integrated with the sheet excitation and detection optics. We demonstrate the ability of the instrument to rapidly examine live specimens with minimal manual intervention by imaging fluorescent neutrophils over a nearly 0.3 mm3 volume in dozens of larval zebrafish. In addition to revealing considerable inter-individual variability in neutrophil number, known previously from labor-intensive methods, three-dimensional imaging allows assessment of the correlation between the bulk measure of total cellular fluorescence and the spatially resolved measure of actual neutrophil number per animal. We suggest that our simple experimental design should considerably expand the scope and impact of light sheet imaging in the life sciences.

Passive and Active Microrheology of the Intestinal Fluid of the Larval Zebrafish.

The fluids of the intestine serve as a physical barrier to pathogens, a medium for the diffusion of nutrients and metabolites, and an environment for commensal microbes. The rheological properties of intestinal mucus have therefore been the subject of many investigations, thus far limited to in vitro studies due to the difficulty of measurement in the natural context of the gut. This limitation especially hinders our understanding of how the gut microbiota interact with the intestinal space, since examination of this calls not only for in vivo measurement techniques, but for techniques that can be applied to model organisms in which the microbial state of the gut can be controlled. We have addressed this challenge with two complementary approaches. We performed passive microrheological measurements using thermally driven nanoparticles and active microrheology using micron-scale ellipsoidal magnetic microparticles, in both cases using light-sheet fluorescence microscopy to optically access the intestinal bulb of the larval zebrafish, a model vertebrate. We present viscosity measurements in germ-free animals (devoid of gut microbes), animals colonized by a single bacterial species, and conventionally reared animals, and find that in all cases, the mucin-rich intestinal liquid is well described as a Newtonian fluid. Surprisingly, despite known differences in the number of secretory cells in germ-free zebrafish and their conventional counterparts, the fluid viscosity for these two groups is very similar, as measured with either technique. Our study provides, to our knowledge, the first in vivo microrheological measurements of the intestinal space in living animals, and we comment on its implications for timescales of host-microbe interactions in the gut.

A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

Spatial and temporal features of the growth of a bacterial species colonizing the zebrafish gut.

UNLABELLED: The vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of an Aeromonas species, representing an abundant genus in the zebrafish gut. Using light sheet fluorescence microscopy to obtain three-dimensional images spanning the gut, we quantified the entire bacterial load, as founding populations grew from tens to tens of thousands of cells over several hours. The data yield the first ever measurements of the growth kinetics of a microbial species inside a live vertebrate intestine and show dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were nonuniformly distributed throughout the gut, and bacterial aggregates showed considerably higher growth rates than did discrete individuals. The form of aggregate growth indicates intrinsically higher division rates for clustered bacteria, rather than surface-mediated agglomeration onto clusters. Thus, the spatial organization of gut bacteria both relative to the host and to each other impacts overall growth kinetics, suggesting that spatial characterizations will be an important input to predictive models of host-associated microbial community assembly. IMPORTANCE: Our intestines are home to vast numbers of microbes that influence many aspects of health and disease. Though we now know a great deal about the constituents of the gut microbiota, we understand very little about their spatial structure and temporal dynamics in humans or in any animal: how microbial populations establish themselves, grow, fluctuate, and persist. To address this, we made use of a model organism, the zebrafish, and a new optical imaging technique, light sheet fluorescence microscopy, to visualize for the first time the colonization of a live, vertebrate gut by specific bacteria with sufficient resolution to quantify the population over a range from a few individuals to tens of thousands of bacterial cells. Our results provide unprecedented measures of bacterial growth kinetics and also show the influence of spatial structure on bacterial populations, which can be revealed only by direct imaging.

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space.

Investigating bacterial-animal symbioses with light sheet microscopy.

Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high-resolution imaging of bacterial colonization of the intestine of Danio rerio, the zebrafish. The method allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional data sets generated by these imaging approaches require new strategies for image analysis. When integrated with other "omics" data sets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts.