Search

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic 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. 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, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

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.

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

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.

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.

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.

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.

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.

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.

Quantitative analysis of neuronal morphology is critical in cell type classification and for deciphering how structure gives rise to function in the brain. Most current approaches to imaging and tracing neuronal 3D morphology are data intensive. We introduce SmartScope2, the first open source, automated neuron reconstruction machine integrating online image analysis with automated multiphoton imaging. SmartScope2 takes advantage of a neuron’s sparse morphology to improve imaging speed and reduce image data stored, transferred and analyzed. We show that SmartScope2 is able to produce the complex 3D morphology of human and mouse cortical neurons with six-fold reduction in image data requirements and three times the imaging speed compared to conventional methods.

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.

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.

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.

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).