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Brain circuits comprise vast numbers of intricately interconnected neurons with diverse molecular, anatomical and physiological properties. To allow “user-defined” targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases (SSRs) based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally-precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Identification of the structural connections between neurons is a prerequisite to understanding brain function. We developed a pipeline to systematically map brain-wide monosynaptic inputs to specific neuronal populations using Cre-driver mouse lines and the recombinant rabies tracing system. We first improved the rabies virus tracing strategy to accurately identify starter cells and to efficiently quantify presynaptic inputs. We then mapped brain-wide presynaptic inputs to different excitatory and inhibitory neuron subclasses in the primary visual cortex and seven higher visual areas. Our results reveal quantitative target-, layer- and cell-class-specific differences in the retrograde connectomes, despite similar global input patterns to different neuronal populations in the same anatomical area. The retrograde connectivity we define is consistent with the presence of the ventral and dorsal visual information processing streams and reveals further subnetworks within the dorsal stream. The hierarchical organization of the entire visual cortex can be derived from intracortical feedforward and feedback pathways mediated by upper- and lower-layer input neurons, respectively. This study expands our knowledge of the brain-wide inputs regulating visual areas and demonstrates that our improved rabies virus tracing strategy can be used to scale up the effort in dissecting connectivity of genetically defined cell populations in the whole mouse brain.

The motion/direction-sensitive and location-sensitive neurons are two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It has been proposed that the motion/direction-sensitive neurons mainly target the superficial layers in V1, in contrast to the location-sensitive neurons which mainly target the middle layers. Here, by imaging calcium activities of motion/direction-sensitive and location-sensitive axons in V1, we find no evidence for these cell-type specific laminar biases at population level. Furthermore, using a novel approach to reconstruct single-axon structures with identified in vivo response types, we show that, at single-axon level, the motion/direction-sensitive axons have middle layer preferences and 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, challenging the current view of the thalamocortical organizations in the mouse visual system.

SUMMARY 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., In brief Zhuang et al., investigate the functionally specific thalamocortical projection patterns in mouse primary visual cortex at both the population and single-axon levels. They find that the motion/direction-sensitive axons project extensively to the middle layers of primary visual cortex, challenging an existing hypothesis proposing a superficial targeting bias of these axons., Graphical Abstract

To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To systematically profile morpho-electric properties of mammalian neurons, we established a single-cell characterization pipeline using standardized patch-clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly accessible online database, the Allen Cell Types Database, to display these datasets. Intrinsic physiological properties were measured from 1,938 neurons from the adult laboratory mouse visual cortex, morphological properties were measured from 461 reconstructed neurons, and 452 neurons had both measurements available. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We established a taxonomy of morphologically and electrophysiologically defined cell types for this region of the cortex, with 17 electrophysiological types, 38 morphological types and 46 morpho-electric types. There was good correspondence with previously defined transcriptomic cell types and subclasses using the same transgenic mouse lines.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The highly distinctive morpho-electric properties of these neurons have mainly been described in rodents, where ET neurons can be labeled by retrograde tracers or transgenic lines. Similar labeling strategies are not possible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically-defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology and morphology are assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were also apparent but were often smaller than differences between cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy, but also highlight marked phenotypic divergence possibly related to functional specialization of human neocortex.

Ever since the seminal findings of Ramon y Cajal, dendritic and axonal morphology has been recognized as a defining feature of neuronal types. Yet our knowledge concerning the diversity of neuronal morphologies, in particular distal axonal projection patterns, is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale, we established a platform with five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling of a wide range of neuronal types by combining transgenic or viral Cre delivery with novel transgenic reporter lines. We acquired high-resolution whole-brain fluorescent images from a large set of sparsely labeled brains using fluorescence micro-optical sectioning tomography (fMOST). We developed a set of software tools for efficient large-volume image data processing, registration to the Allen Mouse Brain Common Coordinate Framework (CCF), and computer-assisted morphological reconstruction. We reconstructed and analyzed the complete morphologies of 1,708 neurons from the striatum, thalamus, cortex and claustrum. Finally, we classified these cells into multiple morphological and projection types and identified a set of region-specific organizational rules of long-range axonal projections at the single cell level. Specifically, different neuron types from different regions follow highly distinct rules in convergent or divergent projection, feedforward or feedback axon termination patterns, and between-cell homogeneity or heterogeneity. Major molecularly defined classes or types of neurons have correspondingly distinct morphological and projection patterns, however, we also identify further remarkably extensive morphological and projection diversity at more fine-grained levels within the major types that cannot presently be accounted for by preexisting transcriptomic subtypes. These insights reinforce the importance of full morphological characterization of brain cell types and suggest a plethora of ways different cell types and individual neurons may contribute to the function of their respective circuits.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits., Sparse labelling and whole-brain imaging are used to reconstruct and classify brain-wide complete morphologies of 1,741 individual neurons in the mouse brain, revealing a dependence on both brain region and transcriptomic profile.

Summary Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Neurons are frequently classified into distinct groups or cell types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 3,700 GABAergic mouse visual cortical neurons and reconstructed the local morphologies of 350 of those neurons. We found that most transcriptomic types (t-types) occupy specific laminar positions within mouse visual cortex, and many of those t-types exhibit consistent electrophysiological and morphological features. We observed that these properties could vary continuously between t-types, which limited the ability to predict specific t-types from other data modalities. Despite that, the data support the presence of at least 20 interneuron met-types that have congruent morphological, electrophysiological, and transcriptomic properties.HighlightsPatch-seq data obtained from >3,700 GABAergic cortical interneuronsComprehensive characterization of morpho-electric features of transcriptomic types20 interneuron met-types that have congruent properties across data modalitiesDifferent Sst met-types preferentially innervate different cortical layers

Neurons are frequently classified into distinct groups or cell types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 3,700 GABAergic mouse visual cortical neurons and reconstructed the local morphologies of 350 of those neurons. We found that most transcriptomic types (t-types) occupy specific laminar positions within mouse visual cortex, and many of those t-types exhibit consistent electrophysiological and morphological features. We observed that these properties could vary continuously between t- types, which limited the ability to predict specific t-types from other data modalities. Despite that, the data support the presence of at least 20 interneuron met-types that have congruent morphological, electrophysiological, and transcriptomic properties.

Location-sensitive and motion-sensitive units are the two major functional types of feedforward projections from lateral genicular nucleus (LGN) to primary visual cortex (V1) in mouse. The distribution of these inputs in cortical depth remains under debate. By measuring the calcium activities of LGN axons in V1 of awake mice, we systematically mapped their functional and structural properties. Although both types distributed evenly across cortical depth, we found that they differ significantly across multiple modalities. Compared to the location-sensitive axons, which possessed confined spatial receptive fields, the motion-sensitive axons lacked spatial receptive fields, preferred lower temporal, higher spatial frequencies and had wider horizontal bouton spread. Furthermore, the motion-sensitive axons showed a strong depth-dependent motion direction bias while the location-sensitive axons showed a depth-independent OFF dominance. Overall, our results suggest a new model of receptive biases and laminar structure of thalamic inputs to V1.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.

During starvation, intra-mitochondrial sirtuins, NAD+ sensitive deacylating enzymes that modulate metabolic homeostasis and survival, directly adjust mitochondrial function to nutrient availability; concomitantly, mitochondria elongate to escape autophagic degradation. However, whether sirtuins also impinge on mitochondrial dynamics is still uncharacterized. Here we show that the mitochondrial Sirtuin 5 (Sirt5) is essential for starvation induced mitochondrial elongation. Deletion of Sirt5 in mouse embryonic fibroblasts increased levels of mitochondrial dynamics of 51kDa protein and mitochondrial fission protein 1, leading to mitochondrial accumulation of the pro-fission dynamin related protein 1 and to mitochondrial fragmentation. During starvation, Sirt5 deletion blunted mitochondrial elongation, resulting in increased mitophagy. Our results indicate that starvation induced mitochondrial elongation and evasion from autophagic degradation requires the energy sensor Sirt5.

Summary Visual spatial perception in the mammalian brain occurs through two parallel pathways: one reaches the primary visual cortex (V1) through the thalamus and another the superior colliculus (SC) via direct projections from the retina. The origin, development, and relative function of these two evolutionarily distinct pathways remain obscure. We examined the early functional development of both pathways by simultaneously imaging pre- and post-synaptic spontaneous neuronal activity. We observed that the quality of retinal activity transfer to the thalamus and superior colliculus does not change across the first two postnatal weeks. However, beginning in the second postnatal week, retinal activity does not drive V1 as strongly as earlier wave activity, suggesting that intrinsic cortical activity competes with signals from the sensory periphery as the cortex matures. Together, these findings bring new insight into the function of the SC and V1 and the role of peripheral activity in driving both circuits across development.

Brain circuits are composed of vast numbers of intricately interconnected neurons with diverse molecular, anatomical and physiological properties. To allow highly specific “user-defined” targeting of individual neurons for structural and functional studies, we modified three site-specific DNA recombinases, Cre, Dre and Flp, by combining them with a fungal light-inducible protein, Vivid, to create light-inducible recombinases (named RecV). We generated viral vectors to express these light-inducible recombinases and demonstrated that they can induce genomic modifications in dense or sparse populations of neurons in superficial as well as deep brain areas of live mouse brains by one-photon or two-photon light induction. These light-inducible recombinases can produce highly targeted, sparse and strong labeling of individual neurons in multiple loci and species. They can be used in combination with other genetic strategies to achieve specific intersectional targeting of mouse cortical layer 5 or inhibitory somatostatin neurons. In mouse cortex sparse light-induced recombination allows whole-brain morphological reconstructions to identify axonal projection specificity. Furthermore these enzymes allow single cell targeted genetic modifications via soma restricted two-photon light stimulation in individual cortical neurons and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally-precise, targeted optogenomic modifications that could greatly facilitate detailed analysis of neural circuits at the single cell level by linking genetic identity, morphology, connectivity and function.

Dendritic and axonal morphology is a defining feature of neuronal types and their connectivity. Yet our knowledge concerning the diversity of neuronal morphology is extremely limited. To systematically obtain single neuron full morphology on a brain-wide scale in mice, we established a pipeline that encompasses five major components: sparse labeling, whole-brain imaging, reconstruction, registration, and classification. We achieved sparse, robust and consistent fluorescent labeling by combining transgenic or viral Cre delivery with novel transgenic reporter lines, and generated whole-brain fluorescent imaging datasets containing tens of thousands of reconstructable neurons. We developed software tools for large-volume image data processing and computer-assisted morphological reconstruction. For a proof-of-principle, we reconstructed the full morphologies of 96 neurons from the claustrum and cortex that belong to a single transcriptomically-defined subclass, and classified them into multiple morphological types, suggesting that they work in a targeted and coordinated manner to process cortical information over a large region.

Neuromodulators such as acetylcholine, noradrenaline (norepinephrine), and serotonin are released into the cortex by axons ascending from subcortical nuclei. These neuromodulators have been hypothesized to influence cortical function during behavioral periods such as arousal, locomotion, exploration, and attention. To determine when these neuromodulatory projections were active, we expressed the genetically-encoded calcium sensor GCaMP6 in neuromodulatory axons which project to the mouse primary visual cortex and performed two-photon microscopy to monitor their activity in vivo. We observed that the fluorescence of both cholinergic and noradrenergic axons increased during periods of pupil dilation, with the fluorescence of the axons rising less than one second before eye pupil dilation. We also observed increases in cholinergic and noradrenergic axon fluorescence periods of locomotion, which was accompanied by pupil dilation and nasal (forward) movement of both pupils. Locomotion was preceded by a rise in axonal fluorescence with a timing and amplitude that matched the subsequent pupil dilation, but axon fluorescence was more sustained than expected from the pupil dilation, suggesting that there is an additional physiological factor that affects cholinergic and noradrenergic axon activity in primary visual cortex during locomotion.

Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.

Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cellclusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To profile morpho-electric properties of mammalian neurons systematically, we established a single cell characterization pipeline using standardized patch clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly-accessible online database, the Allen Cell Types Database, to display these data sets. Intrinsic physiological and morphological properties were measured from over 1,800 neurons from the adult laboratory mouse visual cortex. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We establish a taxonomy of morphologically- and electrophysiologically-defined cell types for this region of cortex with 17 e-types and 35 m-types, as well as an initial correspondence with previously-defined transcriptomic cell types using the same transgenic mouse lines.

Neocortex contains a multitude of cell types segregated into layers and functionally distinct regions. To investigate the diversity of cell types across the mouse neocortex, we analyzed 12,714 cells from the primary visual cortex (VISp), and 9,035 cells from the anterior lateral motor cortex (ALM) by deep single-cell RNA-sequencing (scRNA-seq), identifying 116 transcriptomic cell types. These two regions represent distant poles of the neocortex and perform distinct functions. We define 50 inhibitory transcriptomic cell types, all of which are shared across both cortical regions. In contrast, 49 of 52 excitatory transcriptomic types were found in either VISp or ALM, with only three present in both. By combining single cell RNA-seq and retrograde labeling, we demonstrate correspondence between excitatory transcriptomic types and their region-specific long-range target specificity. This study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct regions of the mouse cortex.

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.

Single-cell characterization and perturbation of neurons provides knowledge critical to addressing fundamental neuroscience questions including the structure–function relationship and neuronal cell-type classification. Here we report a robot for efficiently performing in vivo single-cell experiments in deep brain tissues optically difficult to access. This robot automates blind (non-visually guided) single-cell electroporation (SCE) and extracellular electrophysiology, and can be used to characterize neuronal morphological and physiological properties of, and/or manipulate genetic/chemical contents via delivering extraneous materials (for example, genes) into single neurons in vivo. Tested in the mouse brain, our robot successfully reveals the full morphology of single-infragranular neurons recorded in multiple neocortical regions, as well as deep brain structures such as hippocampal CA3, with high efficiency. Our robot thus can greatly facilitate the study of in vivo full morphology and electrophysiology of single neurons in the brain., Single-cell characterization and perturbation of neurons is critical for revealing the structure-function relationship of brain cells. Here the authors develop a robot that performs single-cell electroporation and extracellular electrophysiology and can be used for performing in vivo single-cell experiments in deep brain tissues optically difficult to access.

Calcium imaging using two-photon scanning microscopy has become an essential tool in neuroscience. However, in its typical implementation, the tradeoffs between fields of view, acquisition speeds, and depth restrictions in scattering brain tissue pose severe limitations. Here, using an integrated systems-wide optimization approach combined with multiple technical innovations, we introduce a new design paradigm for optical microscopy based on maximizing biological information while maintaining the fidelity of obtained neuron signals. Our modular design utilizes hybrid multi-photon acquisition and allows volumetric recording of neuroactivity at single-cell resolution within up to 1 × 1 × 1.22 mm volumes at up to 17 Hz in awake behaving mice. We establish the capabilities and potential of the different configurations of our imaging system at depth and across brain regions by applying it to in vivo recording of up to 12,000 neurons in mouse auditory cortex, posterior parietal cortex, and hippocampus.

Although G-protein-coupled receptor kinase 2 (GRK2) is the most widely studied member of a family of kinases that has been shown to exert powerful influences on a variety of G-protein-coupled receptors, its role in the brain remains largely unknown. Here we report the localization of GRK2 in the mouse brain and generate novel conditional knock-out (KO) mice to assess the physiological importance of this kinase in cholinergic neurons. Mice with the selective deletion of GRK2 in this cell population (ChATIRES-creGrk2f/fKO mice) exhibit reduced behavioral responsiveness to challenge with oxotremorine-M (Oxo-M), a nonselective muscarinic acetylcholine receptor agonist. Specifically, Oxo-M-induced hypothermia, hypolocomotion, and salivation were markedly reduced in these animals, while analgesic responses were unaltered. In contrast, we found that GRK2 deficiency in cholinergic neurons does not alter cocaine-induced psychomotor activation, behavioral sensitization, or conditioned place preference. These results demonstrate that the elimination of GRK2 in cholinergic neurons reduces sensitivity to select muscarinic-mediated behaviors, while dopaminergic effects remain intact and further suggests that GRK2 may selectively impair muscarinic acetylcholine receptor-mediated functionin vivo.

It is well established that the cannabinoid and dopamine systems interact at various levels to regulate basal ganglia function. Although it is well known that acute administration of cannabinoids to mice can modify dopamine-dependent behaviors, the intraneuronal signaling pathways employed by these agents in the striatum are not well understood. Here we used knockout mouse models to examine the regulation of striatal extracellular-signal-regulated kinases 1 and 2 (ERK1/2) signaling by behaviorally relevant doses of cannabinoids. This cellular pathway has been implicated as a central mediator of drug reward and synaptic plasticity. In C57BL/6J mice, acute administration of the cannabinoid agonists, (-)-11-hydroxydimethylheptyl-Δ8-tetrahydrocannabinol (HU-210) and delta-9-tetrahydrocannabinol (Δ(9) -THC), promoted a dose- and time-dependent decrease in the phosphorylation of ERK1/2 in dorsal striatum. Co-administration of the CB1 cannabinoid receptor antagonist N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(AM251) with HU-210 prevented ERK1/2 inactivation, indicating a requirement for activation of this receptor. In dopamine D1 receptor knockout animals treated with HU-210, the magnitude of the HU-210-dependent decrease in striatal ERK1/2 signaling was greater than in wild-type controls. In contrast, HU-210 administration to N-methyl-D-aspartate receptor knockdown mice was ineffective at promoting striatal ERK1/2 inactivation. Genetic deletion of other potential ERK1/2 mediators, the dopamine D2 receptors or β-arrestin-1 or -2, did not affect the HU-210-induced modulation of ERK1/2 signaling in the striatum. These results support the hypothesis that dopamine D1 receptors and N-methyl-D-aspartate receptors act in an opposite manner to regulate striatal CB1 cannabinoid receptor signal transduction.

Morphine is a widely used analgesic in humans that is associated with multiple untoward effects, such as addiction and physical dependence. In rodent models, morphine also induces locomotor activity. These effects likely involve functionally selective mechanisms. Indeed, G protein-coupled receptor desensitization and adaptor protein β-arrestin 2 (βarr2) through its interaction with the μ-opioid receptor regulates the analgesic but not the rewarding properties of morphine. However, βarr2 is also required for morphine-induced locomotor activity in mice, but the exact cellular and molecular mechanisms that mediate this arrestin-dependent behavior are not understood. In this study, we show that βarr2 is required for morphine-induced locomotor activity in a dopamine D1 receptor (D1R)-dependent manner and that a βarr2/phospho-ERK (βarr2/pERK) signaling complex may mediate this behavior. Systemic administration of SL327, an MEK inhibitor, inhibits morphine-induced locomotion in wild-type mice in a dose-dependent manner. Acute morphine administration to mice promotes the formation of a βarr2/pERK signaling complex. Morphine-induced locomotor activity and formation of the βarr2/pERK signaling complex is blunted in D1R knockout (D1-KO) mice and is presumably independent of D2 dopamine receptors. However, D1Rs are not required for morphine-induced reward as D1-KO mice show the same conditioned place preference for morphine as do control mice. Taken together, these results suggest a potential role for a D1R-dependent βarr2/pERK signaling complex in selectively mediating the locomotor-stimulating but not the rewarding properties of morphine.

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.

Agonists stimulate CB1 receptor internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization – likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB1) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB1 receptor internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB1 receptors stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB1 receptors expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of βarrestin-2 recruitment to mutant CB1 receptors suggests that putative carboxy-terminal phosphorylation sites mediate βarrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB1 receptor internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB1 receptors.

The development of the living acute brain slice preparation for analyzing synaptic function roughly a half century ago was a pivotal achievement that greatly influenced the landscape of modern neuroscience. Indeed, many neuroscientists regard brain slices as the gold-standard model system for detailed cellular, molecular, and circuitry level analysis and perturbation of neuronal function. A critical limitation of this model system is the difficulty in preparing slices from adult and aging animals, and over the past several decades few substantial methodological improvements have emerged to facilitate patch clamp analysis in the mature adult stage. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled populations in slices derived from transgenic mice. This adult brain slice method is suitable for diverse experimental applications, including both monitoring and manipulating neuronal activity with genetically encoded calcium indicators and optogenetic actuators, respectively. We describe the application of this adult brain slice platform and associated methods for screening kinetic properties of Channelrhodopsin (ChR) variants expressed in genetically-defined neuronal subtypes.

GRK2 is a G protein-coupled receptor kinase (GRK) that is broadly expressed and is known to regulate diverse types of receptors. GRK2 null animals exhibit embryonic lethality due to a severe developmental heart defect, which has precluded the study of this kinase in the adult brain. To elucidate the specific role of GRK2 in the brain dopamine (DA) system, we used a conditional gene knockout approach to selectively delete GRK2 in DA D1 receptor (D1R)-, DA D2 receptor (D2R)-, adenosine 2A receptor (A2AR)-, or DA transporter (DAT)-expressing neurons. Here we show that select GRK2-deficient mice display hyperactivity, hyposensitivity, or hypersensitivity to the psychomotor effects of cocaine, altered striatal signaling, and DA release and uptake. Mice with GRK2 deficiency in D2R-expressing neurons also exhibited increased D2 autoreceptor activity. These findings reveal a cell-type-specific role for GRK2 in the regulation of normal motor behavior, sensitivity to psychostimulants, dopamine neurotransmission, and D2 autoreceptor function.

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or by directly interacting with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).

CENP-A mutations in Drosophila cause a BubR1-dependent early mitotic delay without kinetochore localization of Spindle Assembly Checkpoint components Michael D. Blower 1 , Tanya Daigle 2 , Thom Kaufman 3 , and Gary H. Karpen 1,4 Department of Molecular and Cell Biology, University of California, Berkeley, CA Department of Anesthesiology, University of Washington, Seatttle, WA 98195 Department of Biology, Indiana University, Bloomington, IN 47405, USA. Department of Genome Biology, Lawrence Berkeley National Lab, Berkeley, CA 94720 Correspondence should be addressed to GK (karpen@fruitfly.org)

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization., Synopsis Normal inheritance of genetic traits from one cell or organismal generation to the next depends on accurate chromosome replication and segregation. Defective chromosome segregation is associated with birth defects and cancer. The centromere is a single site on the chromosome that is responsible for assembling the kinetochore, which mediates chromosome attachment to the microtubule spindle and all chromosome movements. In addition, the spindle assembly checkpoint (SAC) ensures normal inheritance by delaying entry into anaphase when chromosome–spindle attachments are defective. Previous studies suggested that SAC function required kinetochore localization of key components. This study shows that elimination of a centromere-specific histone (CID) results in an early mitotic delay. Although this delay occurs earlier than the established time of SAC function (at the metaphase–anaphase transition), it depends on the presence of an essential SAC protein (BUBR1). Furthermore, the CID-mediated early mitotic delay occurs in the absence of kinetochore formation or localization of key SAC proteins. These results suggest that the fidelity of kinetochore–microtubule attachment is also monitored early in mitosis, and in the absence of kinetochore formation and localization of SAC components.

Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.