The Mind of a Mouse.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval.

Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Single-cell transcriptomic classification of rabies-infected cortical neurons.

Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear, and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets.

Context-dependent and dynamic functional influence of corticothalamic pathways to first- and higher-order visual thalamus.

Layer 6 (L6) is the sole purveyor of corticothalamic (CT) feedback to first-order thalamus and also sends projections to higher-order thalamus, yet how it engages the full corticothalamic circuit to contribute to sensory processing in an awake animal remains unknown. We sought to elucidate the functional impact of L6CT projections from the primary visual cortex to the dorsolateral geniculate nucleus (first-order) and pulvinar (higher-order) using optogenetics and extracellular electrophysiology in awake mice. While sustained L6CT photostimulation suppresses activity in both visual thalamic nuclei in vivo, moderate-frequency (10 Hz) stimulation powerfully facilitates thalamic spiking. We show that each stimulation paradigm differentially influences the balance between monosynaptic excitatory and disynaptic inhibitory corticothalamic pathways to the dorsolateral geniculate nucleus and pulvinar, as well as the prevalence of burst versus tonic firing. Altogether, our results support a model in which L6CTs modulate first- and higher-order thalamus through parallel excitatory and inhibitory pathways that are highly dynamic and context-dependent.

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors.

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or "leak" expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant "cross-over insensitive" recognition sites combined with an "ATG-out" design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex.

How specific features in the environment are represented within the brain is an important unanswered question in neuroscience. A subset of retinal neurons, called direction-selective ganglion cells (DSGCs), are specialized for detecting motion along specific axes of the visual field. Despite extensive study of the retinal circuitry that endows DSGCs with their unique tuning properties, their downstream circuitry in the brain and thus their contribution to visual processing has remained unclear. In mice, several different types of DSGCs connect to the dorsal lateral geniculate nucleus (dLGN), the visual thalamic structure that harbours cortical relay neurons. Whether direction-selective information computed at the level of the retina is routed to cortical circuits and integrated with other visual channels, however, is unknown. Here we show that there is a di-synaptic circuit linking DSGCs with the superficial layers of the primary visual cortex (V1) by using viral trans-synaptic circuit mapping and functional imaging of visually driven calcium signals in thalamocortical axons. This circuit pools information from several types of DSGCs, converges in a specialized subdivision of the dLGN, and delivers direction-tuned and orientation-tuned signals to superficial V1. Notably, this circuit is anatomically segregated from the retino-geniculo-cortical pathway carrying non-direction-tuned visual information to deeper layers of V1, such as layer 4. Thus, the mouse harbours several functionally specialized, parallel retino-geniculo-cortical pathways, one of which originates with retinal DSGCs and delivers direction- and orientation-tuned information specifically to the superficial layers of the primary visual cortex. These data provide evidence that direction and orientation selectivity of some V1 neurons may be influenced by the activation of DSGCs.

Diverse Representations of Olfactory Information in Centrifugal Feedback Projections.

UNLABELLED: Although feedback or centrifugal projections from higher processing centers of the brain to peripheral regions have long been known to play essential functional roles, the anatomical organization of these connections remains largely unknown. Using a virus-based retrograde labeling strategy and 3D whole-brain reconstruction methods, we mapped the spatial organization of centrifugal projections from two olfactory cortical areas, the anterior olfactory nucleus (AON) and the piriform cortex, to the granule cell layer of the main olfactory bulb in the mouse. Both regions are major recipients of information from the bulb and are the largest sources of feedback to the bulb, collectively constituting circuits essential for olfactory coding and olfactory behavior. We found that, although ipsilateral inputs from the AON were uniformly distributed, feedback from the contralateral AON had a strong ventral bias. In addition, we observed that centrifugally projecting neurons were spatially clustered in the piriform cortex, in contrast to the distributed feedforward axonal inputs that these cells receive from the principal neurons of the bulb. Therefore, information carried from the bulb to higher processing structures by anatomically stereotypic projections is likely relayed back to the bulb by organizationally distinct feedback projections that may reflect different coding strategies and therefore different functional roles. SIGNIFICANCE STATEMENT: Principles of anatomical organization, sometimes instantiated as "maps" in the mammalian brain, have provided key insights into the structure and function of circuits in sensory systems. Generally, these characterizations focus on projections from early sensory processing areas to higher processing structures despite considerable evidence that feedback or centrifugal projections often constitute major conduits of information flow. Our results identify structure in the organization of centrifugal feedback projections to the olfactory bulb that is fundamentally different from the organization of feedforward circuits. Our study suggests that understanding computations performed in the olfactory bulb, and more generally in the olfactory system, requires understanding interactions between feedforward and feedback "maps" both structurally and functionally.

Brain-Wide Maps of Synaptic Input to Cortical Interneurons.

Cortical inhibition is mediated by diverse inhibitory neuron types that can each play distinct roles in information processing by virtue of differences in their input sources, intrinsic properties, and innervation targets. Previous studies in brain slices have demonstrated considerable cell-type specificity in laminar sources of local inputs. In contrast, little is known about possible differences in distant inputs to different cortical interneuron types. We used the monosynaptic rabies virus system, in conjunction with mice expressing Cre recombinase in either parvalbumin-positive, somatostatin-positive (SST+), or vasoactive intestinal peptide-positive (VIP+) neurons, to map the brain-wide input to the three major nonoverlapping classes of interneurons in mouse somatosensory cortex. We discovered that all three classes of interneurons received considerable input from known cortical and thalamic input sources, as well as from probable cholinergic cells in the basal nucleus of Meynert. Despite their common input sources, these classes differed in the proportion of long-distance cortical inputs originating from deep versus superficial layers. Similar to their laminar differences in local input, VIP+ neurons received inputs predominantly from deep layers while SST+ neurons received mostly superficial inputs. These classes also differed in the amount of input they received. Cortical and thalamic inputs were greatest onto VIP+ interneurons and smallest onto SST+ neurons. SIGNIFICANCE STATEMENT: These results indicate that all three major interneuron classes in the barrel cortex integrate both feedforward and feedback information from throughout the brain to modulate the activity of the local cortical circuit. However, differences in laminar sources and magnitude of distant cortical input suggest differential contributions from cortical areas. More input to vasoactive intestinal peptide-positive (VIP+) neurons than to somatostatin-positive (SST+) neurons suggests that disinhibition of the cortex via VIP+ cells, which inhibit SST+ cells, might be a general feature of long-distance corticocortical and thalamocortical circuits.

Cortical representations of olfactory input by trans-synaptic tracing.

In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor has convergent axonal projections to two specific glomeruli in the olfactory bulb, thereby creating an odour map. However, it is unclear how this map is represented in the olfactory cortex. Here we combine rabies-virus-dependent retrograde mono-trans-synaptic labelling with genetics to control the location, number and type of 'starter' cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the olfactory bulb input differently. For example, the cortical amygdala preferentially receives dorsal olfactory bulb input, whereas the piriform cortex samples the whole olfactory bulb without obvious bias. These differences probably reflect different functions of these cortical areas in mediating innate odour preference or associative memory. The trans-synaptic labelling method described here should be widely applicable to mapping connections throughout the mouse nervous system.

Functional Local Input to Layer 5 Pyramidal Neurons in the Rat Visual Cortex.

Pyramidal neurons in layer 5 of the neocortex can be differentiated into 3 cell subtypes: 1) short regular spiking (SH), 2) tall regular spiking (TR), and 3) tall burst spiking (TB), based on their morphological and electrophysiological properties. We characterized the functional excitatory local input to these 3 cell subtypes in rat primary visual cortex using laser-scanning photostimulation. Although all cell types received significant input from all cortical layers, SH neurons received stronger input from layer 4 and weaker input from layer 5 than did tall pyramidal cells. However, the laminar input to the 2 populations of tall pyramidal cells was indistinguishable. Simultaneous paired recording were then used to calculate a correlation probability (CP) to infer the proportion of shared input based on the occurrence of simultaneous synaptic potentials. Tall pairs of matched type had significantly higher CPs compared with unmatched pairs, suggesting that subpopulations of layer 4, 5, and 6 neurons preferentially connect to each tall cell type. Hence, this study shows that unconnected but matching pairs of tall pyramidal neurons, but not short pyramidal neurons, receive functional input from different interconnected networks within layers 4, 5, and 6.

Anatomical identification of extracellularly recorded cells in large-scale multielectrode recordings.

This study combines for the first time two major approaches to understanding the function and structure of neural circuits: large-scale multielectrode recordings, and confocal imaging of labeled neurons. To achieve this end, we develop a novel approach to the central problem of anatomically identifying recorded cells, based on the electrical image: the spatiotemporal pattern of voltage deflections induced by spikes on a large-scale, high-density multielectrode array. Recordings were performed from identified ganglion cell types in the macaque retina. Anatomical images of cells in the same preparation were obtained using virally transfected fluorescent labeling or by immunolabeling after fixation. The electrical image was then used to locate recorded cell somas, axon initial segments, and axon trajectories, and these signatures were used to identify recorded cells. Comparison of anatomical and physiological measurements permitted visualization and physiological characterization of numerically dominant ganglion cell types with high efficiency in a single preparation.

Genetic dissection of an amygdala microcircuit that gates conditioned fear.

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ(+) neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ(-) neurons in CEl. Electrical silencing of PKC-δ(+) neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEl(off) units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.

Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus.

New neurons, which have been implicated in pattern separation, are continually generated in the dentate gyrus in the adult hippocampus. Using a genetically modified rabies virus, we demonstrated that molecular layer perforant pathway (MOPP) cells innervated newborn granule neurons in adult mouse brain. Stimulating the perforant pathway resulted in the activation of MOPP cells before the activation of dentate granule neurons. Moreover, activation of MOPP cells by focal uncaging of glutamate induced strong inhibition of granule cells. Together, these results indicate that MOPP cells located in the molecular layer of the dentate gyrus contribute to feed-forward inhibition of granule cells via perforant pathway activation.