The transcriptomic and spatial organization of telencephalic GABAergic neuronal types.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

Impaired drainage through capillary-venous networks contributes to age-related white matter loss.

The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Microscale visualization of cellular features in adult macaque visual cortex.

Expansion microscopy and light sheet imaging enable fine-scale resolution of intracellular features that comprise neural circuits. Most current techniques visualize sparsely distributed features across whole brains or densely distributed features within individual brain regions. Here, we visualize dense distributions of immunolabeled proteins across early visual cortical areas in adult macaque monkeys. This process may be combined with multiphoton or magnetic resonance imaging to produce multimodal atlases in large, gyrencephalic brains.

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

Shigella flexneri Adapts to Niche-Specific Stresses through Modifications in Cell Envelope Composition and Decoration.

Shigella flexneri is the primary causative agent of worldwide shigellosis. As the pathogen transverses the distinct niches of the gastrointestinal tract it necessitates dynamic adaptation strategies to mitigate host antimicrobials such as dietary fatty acids (FAs) and the bile salt, deoxycholate (DOC). This study investigates the dynamics of the S. flexneri cell envelope, by interrogating adaptations following FA or DOC exposure. We deciphered the effects of FAs and DOC on bacterial membrane fatty acid and lipopolysaccharide (LPS) compositions. We identified novel LPS-based strategies by the pathogen to support resistance to these host compounds. In particular, expression of S. flexneri very-long O antigen (VL-Oag) LPS was found to play a central role in stress mitigation, as VL-Oag protects against antimicrobial FAs, but its presence rendered S. flexneri susceptible to DOC stress. Collectively, this work underpins the importance for S. flexneri to maintain appropriate regulation of cell envelope constituents, in particular VL-Oag LPS, to adequately adapt to diverse stresses during infection.

Deep Imaging to Dissect Microvascular Contributions to White Matter Degeneration in Rodent Models of Dementia.

The increasing socio-economic burden of Alzheimer disease (AD) and AD-related dementias has created a pressing need to define targets for therapeutic intervention. Deficits in cerebral blood flow and neurovascular function have emerged as early contributors to disease progression. However, the cause, progression, and consequence of small vessel disease in AD/AD-related dementias remains poorly understood, making therapeutic targets difficult to pinpoint. Animal models that recapitulate features of AD/AD-related dementias may provide mechanistic insight because microvascular pathology can be studied as it develops in vivo. Recent advances in in vivo optical and ultrasound-based imaging of the rodent brain facilitate this goal by providing access to deeper brain structures, including white matter and hippocampus, which are more vulnerable to injury during cerebrovascular disease. Here, we highlight these novel imaging approaches and discuss their potential for improving our understanding of vascular contributions to AD/AD-related dementias.

Laminar distribution and arbor density of two functional classes of thalamic inputs to primary visual cortex.

Motion/direction-sensitive and location-sensitive neurons are the two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It is under debate whether motion/direction-sensitive inputs preferentially target the superficial layers in V1, as opposed to the location-sensitive inputs, which preferentially target the middle layers. Here, by using calcium imaging to measure the activity of motion/direction-sensitive and location-sensitive axons in V1, we find evidence against these cell-type-specific laminar biases at the population level. Furthermore, using an approach to reconstruct axon arbors with identified in vivo response types, we show that, at the single-axon level, the motion/direction-sensitive axons project more densely to the middle layers than the location-sensitive axons. Overall, our results demonstrate that motion/direction-sensitive thalamic neurons project extensively to the middle layers of V1 at both the population and single-cell levels, providing further insight into the organization of thalamocortical projection in the mouse visual system.

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

To Make or Take: Bacterial Lipid Homeostasis during Infection.

Bacterial fatty acids are critical components of the cellular membrane. A shift in environmental conditions or in the bacterium's lifestyle may result in the requirement for a distinct pool of fatty acids with unique biophysical properties. This can be achieved by the modification of existing fatty acids or via de novo synthesis. Furthermore, bacteria have evolved efficient means to acquire these energy-rich molecules from their environment. However, the balance between de novo fatty acid synthesis and exogenous acquisition during pathogenesis is poorly understood. Here, we studied the mouse fatty acid landscape prior to and after infection with Acinetobacter baumannii, a Gram-negative, opportunistic human pathogen. The lipid fluxes observed following infection revealed fatty acid- and niche-specific changes. Lipidomic profiling of A. baumannii isolated from the pleural cavity of mice identified novel A. baumannii membrane phospholipid species and an overall increased abundance of unsaturated fatty acid species. Importantly, we found that A. baumannii relies largely upon fatty acid acquisition in all but one of the studied niches, the blood, where the pathogen biosynthesizes its own fatty acids. This work is the first to reveal the significance of balancing the making and taking of fatty acids in a Gram-negative bacterium during infection, which provides new insights into the validity of targeting fatty acid synthesis as a treatment strategy. IMPORTANCE Acinetobacter baumannii is one of the world's most problematic superbugs and is associated with significant morbidity and mortality in the hospital environment. The critical need for new antimicrobial strategies is recognized, but our understanding of its behavior and adaptation to a changing environment during infection is limited. Here, we investigated the role of fatty acids at the host-pathogen interface using a mouse model of disease. We provide comprehensive insights into the bacterial membrane composition when the bacteria colonize the pleural cavity. Furthermore, we show that A. baumannii heavily relies upon making its own fatty acids when residing in the blood, whereas the bacterium favors fatty acid acquisition in most other host niches. Our new knowledge aids in understanding the importance of host fatty acids in infectious diseases. Furthermore, fatty acid synthesis is an attractive target for the development of new antimicrobial strategies, but our work emphasizes the critical need to understand microbial lipid homeostasis before this can be deemed suitable.

Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases.

Treatments for 'superbug' infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

Relationship between simultaneously recorded spiking activity and fluorescence signal in GCaMP6 transgenic mice.

Fluorescent calcium indicators are often used to investigate neural dynamics, but the relationship between fluorescence and action potentials (APs) remains unclear. Most APs can be detected when the soma almost fills the microscope's field of view, but calcium indicators are used to image populations of neurons, necessitating a large field of view, generating fewer photons per neuron, and compromising AP detection. Here, we characterized the AP-fluorescence transfer function in vivo for 48 layer 2/3 pyramidal neurons in primary visual cortex, with simultaneous calcium imaging and cell-attached recordings from transgenic mice expressing GCaMP6s or GCaMP6f. While most APs were detected under optimal conditions, under conditions typical of population imaging studies, only a minority of 1 AP and 2 AP events were detected (often <10% and ~20-30%, respectively), emphasizing the limits of AP detection under more realistic imaging conditions.

Neurons, the cells that make up the nervous system, transmit information using electrical signals known as action potentials or spikes. Studying the spiking patterns of neurons in the brain is essential to understand perception, memory, thought, and behaviour. One way to do that is by recording electrical activity with microelectrodes. Another way to study neuronal activity is by using molecules that change how they interact with light when calcium binds to them, since changes in calcium concentration can be indicative of neuronal spiking. That change can be observed with specialized microscopes know as two-photon fluorescence microscopes. Using calcium indicators, it is possible to simultaneously record hundreds or even thousands of neurons. However, calcium fluorescence and spikes do not translate one-to-one. In order to interpret fluorescence data, it is important to understand the relationship between the fluorescence signals and the spikes associated with individual neurons. The only way to directly measure this relationship is by using calcium imaging and electrical recording simultaneously to record activity from the same neuron. However, this is extremely challenging experimentally, so this type of data is rare. To shed some light on this, Huang, Ledochowitsch et al. used mice that had been genetically modified to produce a calcium indicator in neurons of the visual cortex and simultaneously obtained both fluorescence measurements and electrical recordings from these neurons. These experiments revealed that, while the majority of time periods containing multi-spike neural activity could be identified using calcium imaging microscopy, on average, less than 10% of isolated single spikes were detectable. This is an important caveat that researchers need to take into consideration when interpreting calcium imaging results. These findings are intended to serve as a guide for interpreting calcium imaging studies that look at neurons in the mammalian brain at the population level. In addition, the data provided will be useful as a reference for the development of activity sensors, and to benchmark and improve computational approaches for detecting and predicting spikes.

The Effect of Inclusion Criteria on the Functional Properties Reported in Mouse Visual Cortex.

Neurophysiology studies require the use of inclusion criteria to identify neurons responsive to the experimental stimuli. Five recent studies used calcium imaging to measure the preferred tuning properties of layer 2/3 pyramidal neurons in mouse visual areas. These five studies employed different inclusion criteria and reported different, sometimes conflicting results. Here, we examine how different inclusion criteria can impact reported tuning properties, modifying inclusion criteria to select different subpopulations from the same dataset of almost 17,000 layer 2/3 neurons from the Allen Brain Observatory. The choice of inclusion criteria greatly affected the mean tuning properties of the resulting subpopulations; indeed, the differences in mean tuning because of inclusion criteria were often of comparable magnitude to the differences between studies. In particular, the mean preferred temporal frequencies (TFs) of visual areas changed markedly with inclusion criteria, such that the rank ordering of visual areas based on their TF preferences changed with the percentage of neurons included. It has been suggested that differences in TF tuning support a hierarchy of mouse visual areas. These results demonstrate that our understanding of the functional organization of the mouse visual cortex obtained from previous experiments critically depends on the inclusion criteria used.

Sources of widefield fluorescence from the brain.

Widefield fluorescence microscopy is used to monitor the spiking of populations of neurons in the brain. Widefield fluorescence can originate from indicator molecules at all depths in cortex and the relative contributions from somata, dendrites, and axons are often unknown. Here, I simulate widefield illumination and fluorescence collection and determine the main sources of fluorescence for several GCaMP mouse lines. Scattering strongly affects illumination and collection. One consequence is that illumination intensity is greatest ~300-400 µm below the pia, not at the brain surface. Another is that fluorescence from a source deep in cortex may extend across a diameter of 3-4 mm at the brain surface, severely limiting lateral resolution. In many mouse lines, the volume of tissue contributing to fluorescence extends through the full depth of cortex and fluorescence at most surface locations is a weighted average across multiple cortical columns and often more than one cortical area.

Superficial Bound of the Depth Limit of Two-Photon Imaging in Mouse Brain.

Two-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. Two-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been postulated that the two-photon depth limit, beyond which results become inaccurate, is where in-focus and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ∼600 µm in mouse cortex. Neither the two-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth of two-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous two-photon and three-photon excitation. Two-photon and three-photon results from superficial locations were almost identical. two-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for two-photon excitation. However, the two-photon depth limit and balance depth were at 450 µm, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the two-photon depth limit in mouse visual cortex.

A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.

To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, large-scale physiological survey of activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes the cortical activity of nearly 60,000 neurons from six visual areas, four layers, and 12 transgenic mouse lines in a total of 243 adult mice, in response to a systematic set of visual stimuli. We classify neurons on the basis of joint reliabilities to multiple stimuli and validate this functional classification with models of visual responses. While most classes are characterized by responses to specific subsets of the stimuli, the largest class is not reliably responsive to any of the stimuli and becomes progressively larger in higher visual areas. These classes reveal a functional organization wherein putative dorsal areas show specialization for visual motion signals.

Dual-plane 3-photon microscopy with remote focusing.

3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples. To date, 3-photon excitation has been restricted to scanning a single focus, limiting the speed of volume acquisition. Here, for the first time to our knowledge, we implemented and characterized dual-plane 3-photon microscopy with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes beyond 600 µm deep in the cortex of a pan-excitatory GCaMP6s transgenic mouse with a per-plane framerate of 7 Hz and an effective 2 MHz laser repetition rate. This method is a straightforward and generalizable modification to single-focus 3PE systems, doubling the rate of volume (column) imaging with off-the-shelf components and minimal technical constraints.

Identification of genetic markers for cortical areas using a Random Forest classification routine and the Allen Mouse Brain Atlas.

The mammalian neocortex is subdivided into a series of cortical areas that are functionally and anatomically distinct and are often distinguished in brain sections using histochemical stains and other markers of protein expression. We searched the Allen Mouse Brain Atlas, a database of gene expression, for novel markers of cortical areas. To screen for genes that change expression at area borders, we employed a random forest algorithm and binary region classification. Novel genetic markers were identified for 19 of 39 areas and provide code that quickly and efficiently searches the Allen Mouse Brain Atlas. Our results demonstrate the utility of the random forest algorithm for cortical area classification and we provide code that may be used to facilitate the identification of genetic markers of cortical and subcortical structures and perhaps changes in gene expression in disease states.

Biological variation in the sizes, shapes and locations of visual cortical areas in the mouse.

Visual cortex is organized into discrete sub-regions or areas that are arranged into a hierarchy and serves different functions in the processing of visual information. In retinotopic maps of mouse cortex, there appear to be substantial mouse-to-mouse differences in visual area location, size and shape. Here we quantify the biological variation in the size, shape and locations of 11 visual areas in the mouse, after separating biological variation and measurement noise. We find that there is biological variation in the locations and sizes of visual areas.

Visual physiology of the layer 4 cortical circuit in silico.

Despite advances in experimental techniques and accumulation of large datasets concerning the composition and properties of the cortex, quantitative modeling of cortical circuits under in-vivo-like conditions remains challenging. Here we report and publicly release a biophysically detailed circuit model of layer 4 in the mouse primary visual cortex, receiving thalamo-cortical visual inputs. The 45,000-neuron model was subjected to a battery of visual stimuli, and results were compared to published work and new in vivo experiments. Simulations reproduced a variety of observations, including effects of optogenetic perturbations. Critical to the agreement between responses in silico and in vivo were the rules of functional synaptic connectivity between neurons. Interestingly, after extreme simplification the model still performed satisfactorily on many measurements, although quantitative agreement with experiments suffered. These results emphasize the importance of functional rules of cortical wiring and enable a next generation of data-driven models of in vivo neural activity and computations.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

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.