Modeling the cell-type-specific mesoscale murine connectome with anterograde tracing experiments.

The Allen Mouse Brain Connectivity Atlas consists of anterograde tracing experiments targeting diverse structures and classes of projecting neurons. Beyond regional anterograde tracing done in C57BL/6 wild-type mice, a large fraction of experiments are performed using transgenic Cre-lines. This allows access to cell-class-specific whole-brain connectivity information, with class defined by the transgenic lines. However, even though the number of experiments is large, it does not come close to covering all existing cell classes in every area where they exist. Here, we study how much we can fill in these gaps and estimate the cell-class-specific connectivity function given the simplifying assumptions that nearby voxels have smoothly varying projections, but that these projection tensors can change sharply depending on the region and class of the projecting cells. This paper describes the conversion of Cre-line tracer experiments into class-specific connectivity matrices representing the connection strengths between source and target structures. We introduce and validate a novel statistical model for creation of connectivity matrices. We extend the Nadaraya-Watson kernel learning method that we previously used to fill in spatial gaps to also fill in gaps in cell-class connectivity information. To do this, we construct a "cell-class space" based on class-specific averaged regionalized projections and combine smoothing in 3D space as well as in this abstract space to share information between similar neuron classes. Using this method, we construct a set of connectivity matrices using multiple levels of resolution at which discontinuities in connectivity are assumed. We show that the connectivities obtained from this model display expected cell-type- and structure-specific connectivities. We also show that the wild-type connectivity matrix can be factored using a sparse set of factors, and analyze the informativeness of this latent variable model.

Endogenous pathology in tauopathy mice progresses via brain networks.

Neurodegenerative tauopathies are hypothesized to propagate via brain networks. This is uncertain because we have lacked precise network resolution of pathology. We therefore developed whole-brain staining methods with anti-p-tau nanobodies and imaged in 3D PS19 tauopathy mice, which have pan-neuronal expression of full-length human tau containing the P301S mutation. We analyzed patterns of p-tau deposition across established brain networks at multiple ages, testing the relationship between structural connectivity and patterns of progressive pathology. We identified core regions with early tau deposition, and used network propagation modeling to determine the link between tau pathology and connectivity strength. We discovered a bias towards retrograde network-based propagation of tau. This novel approach establishes a fundamental role for brain networks in tau propagation, with implications for human disease.

Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

Regional, Layer, and Cell-Type-Specific Connectivity of the Mouse Default Mode Network.

The evolutionarily conserved default mode network (DMN) is a distributed set of brain regions coactivated during resting states that is vulnerable to brain disorders. How disease affects the DMN is unknown, but detailed anatomical descriptions could provide clues. Mice offer an opportunity to investigate structural connectivity of the DMN across spatial scales with cell-type resolution. We co-registered maps from functional magnetic resonance imaging and axonal tracing experiments into the 3D Allen mouse brain reference atlas. We find that the mouse DMN consists of preferentially interconnected cortical regions. As a population, DMN layer 2/3 (L2/3) neurons project almost exclusively to other DMN regions, whereas L5 neurons project in and out of the DMN. In the retrosplenial cortex, a core DMN region, we identify two L5 projection types differentiated by in- or out-DMN targets, laminar position, and gene expression. These results provide a multi-scale description of the anatomical correlates of the mouse DMN.

Network structure of the mouse brain connectome with voxel resolution.

Fine-grained descriptions of brain connectivity are required to understand how neural information is processed and relayed across spatial scales. Previous investigations of the mouse brain connectome have used discrete anatomical parcellations, limiting spatial resolution and potentially concealing network attributes critical to connectome organization. Here, we provide a voxel-level description of the network and hierarchical structure of the directed mouse connectome, unconstrained by regional partitioning. We report a number of previously unappreciated organizational principles in the mammalian brain, including a directional segregation of hub regions into neural sink and sources, and a strategic wiring of neuromodulatory nuclei as connector hubs and critical orchestrators of network communication. We also find that the mouse cortical connectome is hierarchically organized along two superimposed cortical gradients reflecting unimodal-transmodal functional processing and a modality-specific sensorimotor axis, recapitulating a phylogenetically conserved feature of higher mammals. These findings advance our understanding of the foundational wiring principles of the mammalian connectome.

Hierarchical organization of cortical and thalamic connectivity.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

High-resolution data-driven model of the mouse connectome.

Knowledge of mesoscopic brain connectivity is important for understanding inter- and intraregion information processing. Models of structural connectivity are typically constructed and analyzed with the assumption that regions are homogeneous. We instead use the Allen Mouse Brain Connectivity Atlas to construct a model of whole-brain connectivity at the scale of 100 µm voxels. The data consist of 428 anterograde tracing experiments in wild type C57BL/6J mice, mapping fluorescently labeled neuronal projections brain-wide. Inferring spatial connectivity with this dataset is underdetermined, since the approximately 2 × 105 source voxels outnumber the number of experiments. To address this issue, we assume that connection patterns and strengths vary smoothly across major brain divisions. We model the connectivity at each voxel as a radial basis kernel-weighted average of the projection patterns of nearby injections. The voxel model outperforms a previous regional model in predicting held-out experiments and compared with a human-curated dataset. This voxel-scale model of the mouse connectome permits researchers to extend their previous analyses of structural connectivity to much higher levels of resolution, and it allows for comparison with functional imaging and other datasets.

Whole brain imaging reveals distinct spatial patterns of amyloid beta deposition in three mouse models of Alzheimer's disease.

A variety of Alzheimer's disease (AD) mouse models overexpress mutant forms of human amyloid precursor protein (APP), producing high levels of amyloid ß (Aß) and forming plaques. However, the degree to which these models mimic spatiotemporal patterns of Aß deposition in brains of AD patients is unknown. Here, we mapped the spatial distribution of Aß plaques across age in three APP-overexpression mouse lines (APP/PS1, Tg2576, and hAPP-J20) using in vivo labeling with methoxy-X04, high throughput whole brain imaging, and an automated informatics pipeline. Images were acquired with high resolution serial two-photon tomography and labeled plaques were detected using custom-built segmentation algorithms. Image series were registered to the Allen Mouse Brain Common Coordinate Framework, a 3D reference atlas, enabling automated brain-wide quantification of plaque density, number, and location. In both APP/PS1 and Tg2576 mice, plaques were identified first in isocortex, followed by olfactory, hippocampal, and cortical subplate areas. In hAPP-J20 mice, plaque density was highest in hippocampal areas, followed by isocortex, with little to no involvement of olfactory or cortical subplate areas. Within the major brain divisions, distinct regions were identified with high (or low) plaque accumulation; for example, the lateral visual area within the isocortex of APP/PS1 mice had relatively higher plaque density compared with other cortical areas, while in hAPP-J20 mice, plaques were densest in the ventral retrosplenial cortex. In summary, we show how whole brain imaging of amyloid pathology in mice reveals the extent to which a given model recapitulates the regional Aß deposition patterns described in AD.

Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons.

Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.

Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK.

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

Metabotropic glutamate receptors promote disinhibition of olfactory bulb glomeruli that scales with input strength.

Increasing evidence indicates that the neural circuitry within glomeruli of the olfactory bulb plays a major role in affecting information flow between olfactory sensory neurons (OSNs) and output mitral cells (MCs). Glutamatergic external tufted (ET) cells, located at glomeruli, can act as intermediary cells in excitation between OSNs and MCs, whereas activation of MCs by OSNs is, in turn, suppressed by inhibitory synapses onto ET cells. In this study, we used patch-clamp recordings in rat olfactory bulb slices to examine the function of metabotropic glutamate receptors (mGluRs) in altering these glomerular signaling mechanisms. We found that activation of group II mGluRs profoundly reduced inhibition onto ET cells evoked by OSN stimulation. The mGluRs that mediated disinhibition were located on presynaptic GABAergic periglomerular cells and appeared to be activated by glutamate transients derived from dendrites in glomeruli. In terms of glomerular output, the mGluR-mediated reduction in GABA release led to a robust increase in the number of action potentials evoked by OSN stimulation in both ET cells and MCs. Importantly, however, the enhanced excitation was specific to when a glomerulus was strongly activated by OSN inputs. By being selective for strong vs. weak glomerular activation, mGluR-mediated disinhibition provides a mechanism to enhance the contrast in odor signals that activate OSN inputs into a single glomerulus at varying intensities.

Role of cortactin in dynamic actin remodeling events in gonadotrope cells.

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHß mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

Information for decision-making and stimulus identification is multiplexed in sensory cortex.

In recordings from anterior piriform cortex in awake behaving mice, we found that neuronal firing early in the olfactory pathway simultaneously conveyed fundamentally different information: odor value (is the odor rewarded?) and identity (what is the smell?). Thus, this sensory system performs early multiplexing of information reflecting stimulus-specific characteristics with that used for decision-making.

Interglomerular lateral inhibition targeted on external tufted cells in the olfactory bulb.

Lateral inhibition between neurons occurs in many different sensory systems, where it can perform such functions as contrast enhancement. In the olfactory bulb, lateral inhibition may occur between odorant receptor-specific glomeruli that are linked anatomically by GABAergic granule cells (GCs) and cells within the glomerular layer, although evidence supporting lateral inhibition at a functional level is modest. Here, we used patch-clamp, imaging, and glutamate uncaging methods in rat olfactory bulb slices to test for the presence of interglomerular lateral inhibition, as well as its underlying mechanisms. We found that a conditioning stimulus applied at one or a small group of glomeruli could suppress stimulus-evoked excitation of output mitral cells (MCs) at another glomerulus for interstimulus intervals of 20-50 ms and glomerular separations of up to 600 µm. The observed lateral inhibition was entirely dependent on circuitry within the glomerular layer, rather than GCs, and it involved GABAergic synaptic inputs that were targeted mainly onto tufted cells, which act as intermediaries in the excitation between olfactory sensory neurons and MCs. The key cell type responsible for mediating lateral interactions between glomeruli were GABAergic short-axon cells. These results suggest a functional segregation of GABAergic cells within the bulb, with one set located in the glomerular layer mediating suppression of MC spiking across glomeruli, and a second set, the GCs, synchronizing different glomeruli.

Mitral cells in the olfactory bulb are mainly excited through a multistep signaling path.

Within the olfactory system, information flow from the periphery onto output mitral cells (MCs) of the olfactory bulb (OB) has been thought to be mediated by direct synaptic inputs from olfactory sensory neurons (OSNs). Here, we performed patch-clamp measurements in rat and mouse OB slices to investigate mechanisms of OSN signaling onto MCs, including the assumption of a direct path, using electrical and optogenetic stimulation methods that selectively activated OSNs. We found that MCs are in fact not typically activated by direct OSN inputs and instead require a multistep, diffuse mechanism involving another glutamatergic cell type, the tufted cells. The preference for a multistep mechanism reflects the fact that signals arising from direct OSN inputs are drastically shunted by connexin 36-mediated gap junctions on MCs, but not tufted cells. An OB circuit with tufted cells intermediate between OSNs and MCs suggests that considerable processing of olfactory information occurs before its reaching MCs.

Does a nonclassical signaling mechanism underlie an increase of estradiol-mediated gonadotropin-releasing hormone receptor binding in ovine pituitary cells?

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

From the top down: flexible reading of a fragmented odor map.

Animals that depend on smell for communication and survival extract multiple pieces of information from a single complex odor. Mice can collect information on sex, genotype, health and dietary status from urine scent marks, a stimulus made up of hundreds of molecules. This ability is all the more remarkable considering that natural odors are encountered against varying olfactory backgrounds; the olfactory system must therefore provide some mechanism for extracting the most relevant information. Here we discuss recent data indicating that the readout of olfactory input by mitral cells in the olfactory bulb can be modified by behavioral context. We speculate that the olfactory cortex plays a key role in tuning the readout of olfactory information from the olfactory bulb.

Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes.

The delayed-rectifier voltage-gated K(+) channel (Kv) 2.1 underlies the cardiac slow K(+) current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile (tau = 252 +/- 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (tau = 479 +/- 178 s), although its mobility is slower than Kv1.4 (tau(1) = 18.9 +/- 2.3 s; tau(2) = 305 +/- 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.

The stargazin C terminus encodes an intrinsic and transferable membrane sorting signal.

Activity-dependent plasticity of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors is regulated by their auxiliary subunit, stargazin. Association with stargazin enhances alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor surface expression and modifies the receptor's biophysical properties. Fusing the cytoplasmic C terminus of stargazin to the C-terminal domains of either GluR1 or the gonadotropin-releasing hormone receptor permits efficient trafficking from the endoplasmic reticulum and sorting to the basolateral membrane without altering other properties of either receptor.

Neuroendocrine plasticity in the anterior pituitary: gonadotropin-releasing hormone-mediated movement in vitro and in vivo.

The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived alphaT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.