Dendritome Mapping Unveils Spatial Organization of Striatal D1/D2-Neuron Morphology.

Morphology is a cardinal feature of a neuron that mediates its functions, but profiling neuronal morphologies at scale remains a formidable challenge. Here we describe a generalizable pipeline for large-scale brainwide study of dendritic morphology of genetically-defined single neurons in the mouse brain. We generated a dataset of 3,762 3D-reconstructed and reference-atlas mapped striatal D1- and D2- medium spiny neurons (MSNs). Integrative morphometric analyses reveal distinct impacts of anatomical locations and D1/D2 genetic types on MSN morphologies. To analyze striatal regional features of MSN dendrites without prior anatomical constraints, we assigned MSNs to a lattice of cubic boxes in the reference brain atlas, and summarized morphometric representation ("eigen-morph") for each box and clustered boxes with shared morphometry. This analysis reveals 6 modules with characteristic dendritic features and spanning contiguous striatal territories, each receiving distinct corticostriatal inputs. Finally, we found aging confers robust dendritic length and branching defects in MSNs, while Huntington's disease (HD) mice exhibit selective length-related defects. Together, our study demonstrates a systems-biology approach to profile dendritic morphology of genetically-defined single-neurons; and defines novel striatal D1/D2-MSN morphological territories and aging- or HD-associated pathologies.

Gossamer: Scaling Image Processing and Reconstruction to Whole Brains.

Neuronal reconstruction-a process that transforms image volumes into 3D geometries and skeletons of cells- bottlenecks the study of brain function, connectomics and pathology. Domain scientists need exact and complete segmentations to study subtle topological differences. Existing methods are diskbound, dense-access, coupled, single-threaded, algorithmically unscalable and require manual cropping of small windows and proofreading of skeletons due to low topological accuracy. Designing a data-intensive parallel solution suited to a neurons' shape, topology and far-ranging connectivity is particularly challenging due to I/O and load-balance, yet by abstracting these vision tasks into strategically ordered specializations of search, we progressively lower memory by 4 orders of magnitude. This enables 1 mouse brain to be fully processed in-memory on a single server, at 67× the scale with 870× less memory while having 78% higher automated yield than APP2, the previous state of the art in performant reconstruction.

The mouse cortico-basal ganglia-thalamic network.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

Organization of the inputs and outputs of the mouse superior colliculus.

The superior colliculus (SC) receives diverse and robust cortical inputs to drive a range of cognitive and sensorimotor behaviors. However, it remains unclear how descending cortical input arising from higher-order associative areas coordinate with SC sensorimotor networks to influence its outputs. Here, we construct a comprehensive map of all cortico-tectal projections and identify four collicular zones with differential cortical inputs: medial (SC.m), centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). Further, we delineate the distinctive brain-wide input/output organization of each collicular zone, assemble multiple parallel cortico-tecto-thalamic subnetworks, and identify the somatotopic map in the SC that displays distinguishable spatial properties from the somatotopic maps in the neocortex and basal ganglia. Finally, we characterize interactions between those cortico-tecto-thalamic and cortico-basal ganglia-thalamic subnetworks. This study provides a structural basis for understanding how SC is involved in integrating different sensory modalities, translating sensory information to motor command, and coordinating different actions in goal-directed behaviors.

Connectivity characterization of the mouse basolateral amygdalar complex.

The basolateral amygdalar complex (BLA) is implicated in behaviors ranging from fear acquisition to addiction. Optogenetic methods have enabled the association of circuit-specific functions to uniquely connected BLA cell types. Thus, a systematic and detailed connectivity profile of BLA projection neurons to inform granular, cell type-specific interrogations is warranted. Here, we apply machine-learning based computational and informatics analysis techniques to the results of circuit-tracing experiments to create a foundational, comprehensive BLA connectivity map. The analyses identify three distinct domains within the anterior BLA (BLAa) that house target-specific projection neurons with distinguishable morphological features. We identify brain-wide targets of projection neurons in the three BLAa domains, as well as in the posterior BLA, ventral BLA, posterior basomedial, and lateral amygdalar nuclei. Inputs to each nucleus also are identified via retrograde tracing. The data suggests that connectionally unique, domain-specific BLAa neurons are associated with distinct behavior networks.

Brainwide Genetic Sparse Cell Labeling to Illuminate the Morphology of Neurons and Glia with Cre-Dependent MORF Mice.

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 cell clusters 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.

Precise segmentation of densely interweaving neuron clusters using G-Cut.

Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects.

Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks.

Understanding the organization of the hippocampus is fundamental to understanding brain function related to learning, memory, emotions, and diseases such as Alzheimer's disease. Physiological studies in humans and rodents have suggested that there is both structural and functional heterogeneity along the longitudinal axis of the hippocampus. However, the recent discovery of discrete gene expression domains in the mouse hippocampus has provided the opportunity to re-evaluate hippocampal connectivity. To integrate mouse hippocampal gene expression and connectivity, we mapped the distribution of distinct gene expression patterns in mouse hippocampus and subiculum to create the Hippocampus Gene Expression Atlas (HGEA). Notably, previously unknown subiculum gene expression patterns revealed a hidden laminar organization. Guided by the HGEA, we constructed the most detailed hippocampal connectome available using Mouse Connectome Project ( http://www.mouseconnectome.org ) tract tracing data. Our results define the hippocampus' multiscale network organization and elucidate each subnetwork's unique brain-wide connectivity patterns.

The mouse cortico-striatal projectome.

Different cortical areas are organized into distinct intracortical subnetworks. The manner in which descending pathways from the entire cortex interact subcortically as a network remains unclear. We developed an open-access comprehensive mesoscale mouse cortico-striatal projectome: a detailed connectivity projection map from the entire cerebral cortex to the dorsal striatum or caudoputamen (CP) in rodents. On the basis of these projections, we used new computational neuroanatomical tools to identify 29 distinct functional striatal domains. Furthermore, we characterized different cortico-striatal networks and how they reconfigure across the rostral-caudal extent of the CP. The workflow was also applied to select cortico-striatal connections in two different mouse models of disconnection syndromes to demonstrate its utility for characterizing circuitry-specific connectopathies. Together, our results provide the structural basis for studying the functional diversity of the dorsal striatum and disruptions of cortico-basal ganglia networks across a broad range of disorders.

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats: implications for affective pain.

OBJECTIVE: The rostral anterior cingulate cortex (rACC) is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors (NMDARs) are highly expressed in the rACC and mediate pain-related affect by activating a signaling pathway that involves cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and/or extracellular regulated kinase (ERK)/cAMP-response element-binding protein (CREB). The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. METHODS: Immunohistochemistry and Western blot analysis were used to separately assess the expression of phospho-ERK (pERK) and phospho-CREB (pCREB) in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. RESULTS: Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antagonist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine-binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up-regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. CONCLUSION: Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.