The connections between the claustrum and the cortex in mouse are systematically investigated with adeno-associated virus (AAV), an anterograde viral tracer. We first define the boundary and the three-dimensional structure of the claustrum based on a variety of molecular and anatomical data. From AAV injections into 42 neocortical and allocortical areas, we conclude that most cortical areas send bilateral projections to the claustrum, the majority being denser on the ipsilateral side. This includes prelimbic, infralimbic, medial, ventrolateral and lateral orbital, ventral retrosplenial, dorsal and posterior agranular insular, visceral, temporal association, dorsal and ventral auditory, ectorhinal, perirhinal, lateral entorhinal, and anteromedial, posteromedial, lateroposterior, laterointermediate, and postrhinal visual areas. In contrast, the cingulate and the secondary motor areas send denser projections to the contralateral claustrum than to the ipsilateral one. The gustatory, primary auditory, primary visual, rostrolateral visual, and medial entorhinal cortices send projections only to the ipsilateral claustrum. Primary motor, primary somatosensory and subicular areas barely send projections to either ipsi- or contralateral claustrum. Corticoclaustral projections are organized in a rough topographic manner, with variable projection strengths. We find that the claustrum, in turn, sends widespread projections preferentially to ipsilateral cortical areas with different projection strengths and laminar distribution patterns and to certain contralateral cortical areas. Our quantitative results show that the claustrum has strong reciprocal and bilateral connections with prefrontal and cingulate areas as well as strong reciprocal connections with the ipsilateral temporal and retrohippocampal areas, suggesting that it may play a crucial role in a variety of cognitive processes. J. Comp. Neurol. 525:1317-1346, 2017. © 2016 Wiley Periodicals, Inc.

Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage-restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage. This Matters Arising paper is in response to Guo et al. (2013), published in Neuron. See also the Matters Arising Response paper by Eckler et al. (2015), published concurrently with this Matters Arising in Neuron.

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.

Type 1 estrogen receptor-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvlEsr1) play a causal role in the control of social behaviors, including aggression. Here we use six different viral-genetic tracing methods to systematically map the connectional architecture of VMHvlEsr1 neurons. These data reveal a high level of input convergence and output divergence ("fan-in/fan-out") from and to over 30 distinct brain regions, with a high degree (∼90%) of bidirectionality, including both direct as well as indirect feedback. Unbiased collateralization mapping experiments indicate that VMHvlEsr1 neurons project to multiple targets. However, we identify two anatomically distinct subpopulations with anterior vs. posterior biases in their collateralization targets. Nevertheless, these two subpopulations receive indistinguishable inputs. These studies suggest an overall system architecture in which an anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. This architecture differs from the "brain-inspired," hierarchical feed-forward circuits used in certain types of artificial intelligence networks.

Generating a comprehensive description of cortical networks requires a large-scale, systematic approach. To that end, we have begun a pipeline project using multipatch electrophysiology, supplemented with two-photon optogenetics, to characterize connectivity and synaptic signaling between classes of neurons in adult mouse primary visual cortex (V1) and human cortex. We focus on producing results detailed enough for the generation of computational models and enabling comparison with future studies. Here, we report our examination of intralaminar connectivity within each of several classes of excitatory neurons. We find that connections are sparse but present among all excitatory cell classes and layers we sampled, and that most mouse synapses exhibited short-term depression with similar dynamics. Synaptic signaling between a subset of layer 2/3 neurons, however, exhibited facilitation. These results contribute to a body of evidence describing recurrent excitatory connectivity as a conserved feature of cortical microcircuits.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.

The structural organization of neural circuits is strongly influenced by experience, but the underlying mechanisms are incompletely understood. We found that, in the developing dentate gyrus (DG), excitatory drive promotes the somatic innervation of principal granule cells (GCs) by parvalbumin (PV)-positive basket cells. In contrast, presynaptic differentiation of GCs and interneuron subtypes that inhibit GC dendrites is largely resistant to loss of glutamatergic neurotransmission. The networks of PV basket cells in the DG are regulated by vesicular release from projection entorhinal cortical neurons and, at least in part, by NMDA receptors in interneurons. Finally, we present evidence that glutamatergic inputs and NMDA receptors regulate these networks through a presynaptic mechanism that appears to control the branching of interneuron axons. Our results provide insights into how cortical activity tunes the inhibition in a subcortical circuit and reveal new principles of interneuron plasticity.

It is important to digitally reconstruct the 3D morphology of neurons and brain vasculatures. A number of previous methods have been proposed to automate the reconstruction process. However, in many cases, noise and low signal contrast with respect to the image background still hamper our ability to use automation methods directly. Here, we propose an adaptive image enhancement method specifically designed to improve the signal-to-noise ratio of several types of individual neurons and brain vasculature images. Our method is based on detecting the salient features of fibrous structures, e.g. the axon and dendrites combined with adaptive estimation of the optimal context windows where such saliency would be detected. We tested this method for a range of brain image datasets and imaging modalities, including bright-field, confocal and multiphoton fluorescent images of neurons, and magnetic resonance angiograms. Applying our adaptive enhancement to these datasets led to improved accuracy and speed in automated tracing of complicated morphology of neurons and vasculatures.

A comprehensive characterization of neuronal cell types, their distributions, and patterns of connectivity is critical for understanding the properties of neural circuits and how they generate behaviors. Here we review the experiences of the BRAIN Initiative Cell Census Consortium, ten pilot projects funded by the U.S. BRAIN Initiative, in developing, validating, and scaling up emerging genomic and anatomical mapping technologies for creating a complete inventory of neuronal cell types and their connections in multiple species and during development. These projects lay the foundation for a larger and longer-term effort to generate whole-brain cell atlases in species including mice and humans.

The dentate gyrus (DG), in addition to its role in learning and memory, is increasingly implicated in the pathophysiology of anxiety disorders. Here, we show that, dependent on their position along the dorsoventral axis of the hippocampus, DG granule cells (GCs) control specific features of anxiety and contextual learning. Using optogenetic techniques to either elevate or decrease GC activity, we demonstrate that GCs in the dorsal DG control exploratory drive and encoding, not retrieval, of contextual fear memories. In contrast, elevating the activity of GCs in the ventral DG has no effect on contextual learning but powerfully suppresses innate anxiety. These results suggest that strategies aimed at modulating the excitability of the ventral DG may be beneficial for the treatment of anxiety disorders.

The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universally responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in several Cre-driver lines, including new Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.

Brain cells communicate with one another via local and long-range synaptic connections. Structural connectivity is the foundation for neural function. Brain-wide connectivity can be described at macroscopic, mesoscopic and microscopic levels. The mesoscale connectome represents connections between neuronal types across different brain regions. Building a mesoscale connectome requires a detailed understanding of the cell type composition of different brain regions and the patterns of inputs and outputs that each of these cell types receives and forms, respectively. In this review, I discuss historical and contemporary tracing techniques in both anterograde and retrograde directions to map the input and output connections at population and individual cell levels, as well as imaging and network analysis approaches to build mesoscale connectomes for mammalian brains.

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.

Understanding how behavior emerges from brain electrical activity is one of the ultimate goals of neuroscience. To achieve this goal we require methods for large-scale recording of the electrical activity of specific neuronal circuits. A very promising approach is to use optical reporting of membrane voltage transients, particularly if the voltage reporter is genetically targeted to specific neuronal populations. Targeting in this way allows population signals to be recorded and interpreted without blindness to neuronal diversity. Here, we evaluated the voltage-sensitive fluorescent protein, VSFP Butterfly 2.1, a genetically encoded voltage indicator (GEVI), for monitoring electrical activity of layer 2/3 cortical pyramidal neurons in mouse brain slices. Standard widefield fluorescence and two-photon imaging revealed robust, high signal-to-noise ratio read-outs of membrane voltage transients that are predominantly synaptic in nature and can be resolved as discrete areas of synaptically connected layer 2/3 neurons. We find that targeted expression of this GEVI in the cortex provides a flexible and promising tool for the analysis of L2/3 cortical network function.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

To understand the function of cortical circuits, it is necessary to catalog their cellular diversity. Past attempts to do so using anatomical, physiological or molecular features of cortical cells have not resulted in a unified taxonomy of neuronal or glial cell types, partly due to limited data. Single-cell transcriptomics is enabling, for the first time, systematic high-throughput measurements of cortical cells and generation of datasets that hold the promise of being complete, accurate and permanent. Statistical analyses of these data reveal clusters that often correspond to cell types previously defined by morphological or physiological criteria and that appear conserved across cortical areas and species. To capitalize on these new methods, we propose the adoption of a transcriptome-based taxonomy of cell types for mammalian neocortex. This classification should be hierarchical and use a standardized nomenclature. It should be based on a probabilistic definition of a cell type and incorporate data from different approaches, developmental stages and species. A community-based classification and data aggregation model, such as a knowledge graph, could provide a common foundation for the study of cortical circuits. This community-based classification, nomenclature and data aggregation could serve as an example for cell type atlases in other parts of the body.

To achieve goals, organisms are often faced with complex tasks that require enhanced control of cognitive faculties for optimal performance. However, the neural circuit mechanisms underlying this ability are unclear. The claustrum is proposed to mediate a variety of functions ranging from sensory binding to cognitive control of action, but direct functional assessments of this telencephalic nucleus are lacking.

No abstract available

The advancement of single-cell RNA-sequencing technologies has led to an explosion of cell type definitions across multiple organs and organisms. While standards for data and metadata intake are arising, organization of cell types has largely been left to individual investigators, resulting in widely varying nomenclature and limited alignment between taxonomies. To facilitate cross-dataset comparison, the Allen Institute created the common cell type nomenclature (CCN) for matching and tracking cell types across studies that is qualitatively similar to gene transcript management across different genome builds. The CCN can be readily applied to new or established taxonomies and was applied herein to diverse cell type datasets derived from multiple quantifiable modalities. The CCN facilitates assigning accurate yet flexible cell type names in the mammalian cortex as a step toward community-wide efforts to organize multi-source, data-driven information related to cell type taxonomies from any organism.