Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter ...transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
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•23 new driver lines and 26 new reporter lines for a wide range of applications•TIGRE2.0 reporters have viral-like transgene expression level in diverse cell types•New calcium- or voltage-sensing reporters with the former functionally characterized•Comparative analysis of new optogenetic effectors with complementary properties
An expanded toolkit of transgenic mouse lines for exploring the organization, function, and development of mammalian neural circuits.
Vascular calcification is common in chronic kidney disease, where cardiovascular mortality remains the leading cause of death. Patients with kidney disease are often prescribed vitamin D receptor ...agonists (VDRAs) that confer a survival benefit, but the underlying mechanisms remain unclear. Here we tested two VDRAs in a mouse chronic kidney disease model where dietary phosphate loading induced aortic medial calcification. Mice were given intraperitoneal calcitriol or paricalcitol three times per week for 3 weeks. These treatments were associated with half of the aortic calcification compared to no therapy, and there was no difference between the two agents. In the setting of a high-phosphate diet, serum parathyroid hormone and calcium levels were not significantly altered by treatment. VDRA therapy was associated with increased serum and urine klotho levels, increased phosphaturia, correction of hyperphosphatemia, and lowering of serum fibroblast growth factor-23. There was no effect on elastin remodeling or inflammation; however, the expression of the anticalcification factor, osteopontin, in aortic medial cells was increased. Paricalcitol upregulated osteopontin secretion from mouse vascular smooth muscle cells in culture. Thus, klotho and osteopontin were upregulated by VDRA therapy in chronic kidney disease, independent of changes in serum parathyroid hormone and calcium.
Learning from experience depends at least in part on changes in neuronal connections. We present the largest map of connectivity to date between cortical neurons of a defined type (layer 2/3 L2/3 ...pyramidal cells in mouse primary visual cortex), which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects (250 × 140 × 90 μm
volume). We used the map to identify constraints on the learning algorithms employed by the cortex. Previous cortical studies modeled a continuum of synapse sizes by a log-normal distribution. A continuum is consistent with most neural network models of learning, in which synaptic strength is a continuously graded analog variable. Here, we show that synapse size, when restricted to synapses between L2/3 pyramidal cells, is well modeled by the sum of a binary variable and an analog variable drawn from a log-normal distribution. Two synapses sharing the same presynaptic and postsynaptic cells are known to be correlated in size. We show that the binary variables of the two synapses are highly correlated, while the analog variables are not. Binary variation could be the outcome of a Hebbian or other synaptic plasticity rule depending on activity signals that are relatively uniform across neuronal arbors, while analog variation may be dominated by other influences such as spontaneous dynamical fluctuations. We discuss the implications for the longstanding hypothesis that activity-dependent plasticity switches synapses between bistable states.
Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the average connectivity of several ...interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells, and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type-specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together, these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.
Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and ...degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.
Serial-section electron microscopy (ssEM) is the method of choice for studying macroscopic biological samples at extremely high resolution in three dimensions. In the nervous system, nanometer-scale ...images are necessary to reconstruct dense neural wiring diagrams in the brain, so -called
connectomes
. The data that can comprise of up to 10
8
individual EM images must be assembled into a volume, requiring seamless 2D registration from physical section followed by 3D alignment of the stitched sections. The high throughput of ssEM necessitates 2D stitching to be done at the pace of imaging, which currently produces tens of terabytes per day. To achieve this, we present a modular volume assembly software pipeline
ASAP
(Assembly Stitching and Alignment Pipeline) that is scalable to datasets containing petabytes of data and parallelized to work in a distributed computational environment. The pipeline is built on top of the
Render
Trautman and Saalfeld (2019) services used in the volume assembly of the brain of adult
Drosophila melanogaster
(Zheng et al. 2018). It achieves high throughput by operating only on image meta-data and transformations. ASAP is modular, allowing for easy incorporation of new algorithms without significant changes in the workflow. The entire software pipeline includes a complete set of tools for stitching, automated quality control, 3D section alignment, and final rendering of the assembled volume to disk. ASAP has been deployed for continuous stitching of several large-scale datasets of the mouse visual cortex and human brain samples including one cubic millimeter of mouse visual cortex (Yin et al. 2020); Microns Consortium et al. (2021) at speeds that exceed imaging. The pipeline also has multi-channel processing capabilities and can be applied to fluorescence and multi-modal datasets like array tomography.
The high metabolic demand of brain tissue is supported by a constant supply of blood flow through dense microvascular networks. Capillaries are the smallest class of vessels in the brain and their ...lumens vary in diameter between ~2 and 5 μm. This diameter range plays a significant role in optimizing blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if the architecture of the endothelial wall also contributes to capillary diameter. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical mm3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of two to six interlocked endothelial cells, while the capillaries intervening these zones are composed of either one or two endothelial cells, with roughly equal proportions. The luminal area and diameter are on average slightly larger with capillary segments composed of two interlocked endothelial cells vs one endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, including pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.
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