Topographical and cell type-specific connectivity of rostral and caudal forelimb corticospinal neuron populations.

Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse assortment of cells with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted. Here, we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal neuron types, with notable exceptions that suggest that these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive highly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior.

Logic-based mechanistic machine learning on high-content images reveals how drugs differentially regulate cardiac fibroblasts.

Fibroblasts are essential regulators of extracellular matrix deposition following cardiac injury. These cells exhibit highly plastic responses in phenotype during fibrosis in response to environmental stimuli. Here, we test whether and how candidate anti-fibrotic drugs differentially regulate measures of cardiac fibroblast phenotype, which may help identify treatments for cardiac fibrosis. We conducted a high-content microscopy screen of human cardiac fibroblasts treated with 13 clinically relevant drugs in the context of TGFß and/or IL-1ß, measuring phenotype across 137 single-cell features. We used the phenotypic data from our high-content imaging to train a logic-based mechanistic machine learning model (LogiMML) for fibroblast signaling. The model predicted how pirfenidone and Src inhibitor WH-4-023 reduce actin filament assembly and actin-myosin stress fiber formation, respectively. Validating the LogiMML model prediction that PI3K partially mediates the effects of Src inhibition, we found that PI3K inhibition reduces actin-myosin stress fiber formation and procollagen I production in human cardiac fibroblasts. In this study, we establish a modeling approach combining the strengths of logic-based network models and regularized regression models. We apply this approach to predict mechanisms that mediate the differential effects of drugs on fibroblasts, revealing Src inhibition acting via PI3K as a potential therapy for cardiac fibrosis.

Topographical and cell type-specific connectivity of rostral and caudal forelimb corticospinal neuron populations.

Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse group of neurons with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, no studies have mapped the diversity of spinal cell types targeted by CSNs, let alone compare CFA and RFA populations. Here we use anatomical and RNA-sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal cell types, with notable exceptions that suggest these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive surprisingly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior.

Network model integrated with multi-omic data predicts MBNL1 signals that drive myofibroblast activation.

RNA-binding protein muscleblind-like1 (MBNL1) was recently identified as a central regulator of cardiac wound healing and myofibroblast activation. To identify putative MBNL1 targets, we integrated multiple genome-wide screens with a fibroblast network model. We expanded the model to include putative MBNL1-target interactions and recapitulated published experimental results to validate new signaling modules. We prioritized 14 MBNL1 targets and developed novel fibroblast signaling modules for p38 MAPK, Hippo, Runx1, and Sox9 pathways. We experimentally validated MBNL1 regulation of p38 expression in mouse cardiac fibroblasts. Using the expanded fibroblast model, we predicted a hierarchy of MBNL1 regulated pathways with strong influence on αSMA expression. This study lays a foundation to explore the network mechanisms of MBNL1 signaling central to fibrosis.

Logic-based mechanistic machine learning on high-content images reveals how drugs differentially regulate cardiac fibroblasts.

Fibroblasts are essential regulators of extracellular matrix deposition following cardiac injury. These cells exhibit highly plastic responses in phenotype during fibrosis in response to environmental stimuli. Here, we test whether and how candidate anti-fibrotic drugs differentially regulate measures of cardiac fibroblast phenotype, which may help identify treatments for cardiac fibrosis. We conducted a high content microscopy screen of human cardiac fibroblasts treated with 13 clinically relevant drugs in the context of TGFß and/or IL-1ß, measuring phenotype across 137 single-cell features. We used the phenotypic data from our high content imaging to train a logic-based mechanistic machine learning model (LogiMML) for fibroblast signaling. The model predicted how pirfenidone and Src inhibitor WH-4-023 reduce actin filament assembly and actin-myosin stress fiber formation, respectively. Validating the LogiMML model prediction that PI3K partially mediates the effects of Src inhibition, we found that PI3K inhibition reduces actin-myosin stress fiber formation and procollagen I production in human cardiac fibroblasts. In this study, we establish a modeling approach combining the strengths of logic-based network models and regularized regression models, apply this approach to predict mechanisms that mediate the differential effects of drugs on fibroblasts, revealing Src inhibition acting via PI3K as a potential therapy for cardiac fibrosis.

Corticospinal populations broadcast complex motor signals to coordinated spinal and striatal circuits.

Many models of motor control emphasize the role of sensorimotor cortex in movement, principally through the projections that corticospinal neurons (CSNs) make to the spinal cord. Additionally, CSNs possess expansive supraspinal axon collaterals, the functional organization of which is largely unknown. Using anatomical and electrophysiological circuit-mapping techniques in the mouse, we reveal dorsolateral striatum as the preeminent target of CSN collateral innervation. We found that this innervation is biased so that CSNs targeting different striatal pathways show biased targeting of spinal cord circuits. Contrary to more conventional perspectives, CSNs encode not only individual movements, but also information related to the onset and offset of motor sequences. Furthermore, similar activity patterns are broadcast by CSN populations targeting different striatal circuits. Our results reveal a logic of coordinated connectivity between forebrain and spinal circuits, where separate CSN modules broadcast similarly complex information to downstream circuits, suggesting that differences in postsynaptic connectivity dictate motor specificity.

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation.

Background A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early proinflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase-like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after transverse aortic constriction surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2 knockout mice displayed a proinflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, transverse aortic constriction surgery led to the death of all mice by day 6 that was associated with myocardial hyperinflammation and vascular leakage. Conclusions Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

Network model-based screen for FDA-approved drugs affecting cardiac fibrosis.

Cardiac fibrosis is a significant component of pathological heart remodeling, yet it is not directly targeted by existing drugs. Systems pharmacology approaches have the potential to provide mechanistic frameworks with which to predict and understand how drugs modulate biological systems. Here, we combine network modeling of the fibroblast signaling network with 36 unique drug-target interactions from DrugBank to predict drugs that modulate fibroblast phenotype and fibrosis. Galunisertib was predicted to decrease collagen and α-SMA expression, which we validated in human cardiac fibroblasts. In vivo fibrosis data from the literature validated predictions for 10 drugs. Further, the model was used to identify network mechanisms by which these drugs work. Arsenic trioxide was predicted to induce fibrosis by AP1-driven TGFß expression and MMP2-driven TGFß activation. Entresto (valsartan/sacubitril) was predicted to suppress fibrosis by valsartan suppression of ERK signaling and sacubitril enhancement of PKG activity, both of which decreased Smad3 activity. Overall, this study provides a framework for integrating drug-target mechanisms with logic-based network models, which can drive further studies both in cardiac fibrosis and other conditions.

Network Analysis Reveals a Distinct Axis of Macrophage Activation in Response to Conflicting Inflammatory Cues.

Macrophages are subject to a wide range of cytokine and pathogen signals in vivo, which contribute to differential activation and modulation of inflammation. Understanding the response to multiple, often-conflicting cues that macrophages experience requires a network perspective. In this study, we integrate data from literature curation and mRNA expression profiles obtained from wild type C57/BL6J mice macrophages to develop a large-scale computational model of the macrophage signaling network. In response to stimulation across all pairs of nine cytokine inputs, the model predicted activation along the classic M1-M2 polarization axis but also a second axis of macrophage activation that distinguishes unstimulated macrophages from a mixed phenotype induced by conflicting cues. Along this second axis, combinations of conflicting stimuli, IL-4 with LPS, IFN-γ, IFN-ß, or TNF-α, produced mutual inhibition of several signaling pathways, e.g., NF-κB and STAT6, but also mutual activation of the PI3K signaling module. In response to combined IFN-γ and IL-4, the model predicted genes whose expression was mutually inhibited, e.g., iNOS or Nos2 and Arg1, or mutually enhanced, e.g., Il4rα and Socs1, validated by independent experimental data. Knockdown simulations further predicted network mechanisms underlying functional cross-talk, such as mutual STAT3/STAT6-mediated enhancement of Il4rα expression. In summary, the computational model predicts that network cross-talk mediates a broadened spectrum of macrophage activation in response to mixed pro- and anti-inflammatory cytokine cues, making it useful for modeling in vivo scenarios.

Computational model predicts paracrine and intracellular drivers of fibroblast phenotype after myocardial infarction.

The fibroblast is a key mediator of wound healing in the heart and other organs, yet how it integrates multiple time-dependent paracrine signals to control extracellular matrix synthesis has been difficult to study in vivo. Here, we extended a computational model to simulate the dynamics of fibroblast signaling and fibrosis after myocardial infarction (MI) in response to time-dependent data for nine paracrine stimuli. This computational model was validated against dynamic collagen expression and collagen area fraction data from post-infarction rat hearts. The model predicted that while many features of the fibroblast phenotype at inflammatory or maturation phases of healing could be recapitulated by single static paracrine stimuli (interleukin-1 and angiotensin-II, respectively), mimicking the reparative phase required paired stimuli (e.g. TGFß and endothelin-1). Virtual overexpression screens simulated with either static cytokine pairs or post-MI paracrine dynamic predicted phase-specific regulators of collagen expression. Several regulators increased (Smad3) or decreased (Smad7, protein kinase G) collagen expression specifically in the reparative phase. NADPH oxidase (NOX) overexpression sustained collagen expression from reparative to maturation phases, driven by TGFß and endothelin positive feedback loops. Interleukin-1 overexpression had mixed effects, both enhancing collagen via the TGFß positive feedback loop and suppressing collagen via NFκB and BAMBI (BMP and activin membrane-bound inhibitor) incoherent feed-forward loops. These model-based predictions reveal network mechanisms by which the dynamics of paracrine stimuli and interacting signaling pathways drive the progression of fibroblast phenotypes and fibrosis after myocardial infarction.

Multiscale Coupling of an Agent-Based Model of Tissue Fibrosis and a Logic-Based Model of Intracellular Signaling.

Wound healing and fibrosis following myocardial infarction (MI) is a dynamic process involving many cell types, extracellular matrix (ECM), and inflammatory cues. As both incidence and survival rates for MI increase, management of post-MI recovery and associated complications are an increasingly important focus. Complexity of the wound healing process and the need for improved therapeutics necessitate a better understanding of the biochemical cues that drive fibrosis. To study the progression of cardiac fibrosis across spatial and temporal scales, we developed a novel hybrid multiscale model that couples a logic-based differential equation (LDE) model of the fibroblast intracellular signaling network with an agent-based model (ABM) of multi-cellular tissue remodeling. The ABM computes information about cytokine and growth factor levels in the environment including TGFß, TNFα, IL-1ß, and IL-6, which are passed as inputs to the LDE model. The LDE model then computes the network signaling state of individual cardiac fibroblasts within the ABM. Based on the current network state, fibroblasts make decisions regarding cytokine secretion and deposition and degradation of collagen. Simulated fibroblasts respond dynamically to rapidly changing extracellular environments and contribute to spatial heterogeneity in model predicted fibrosis, which is governed by many parameters including cell density, cell migration speeds, and cytokine levels. Verification tests confirmed that predictions of the coupled model and network model alone were consistent in response to constant cytokine inputs and furthermore, a subset of coupled model predictions were validated with in vitro experiments with human cardiac fibroblasts. This multiscale framework for cardiac fibrosis will allow for systematic screening of the effects of molecular perturbations in fibroblast signaling on tissue-scale extracellular matrix composition and organization.

Contactless Transport and Mixing of Liquids on Self-Sustained Sublimating Coatings.

Controlled handling of liquids and colloidal suspensions as they interact with surfaces, targeting a broad palette of related functionalities, is of great importance in science, technology, and nature. When small liquid volumes (drops on the order of microliters or nanoliters) need to be processed in microfluidic devices, contamination on the solid/liquid interface and loss of liquid due to adhesion on the transport channels are two very common problems that can significantly alter the process outcome, for example, the chemical reaction efficiency or the purity and the final concentration of a suspension. It is, therefore, no surprise that both levitation and minimal contact transport methods-including nonwetting surfaces-have been developed to minimize the interactions between liquids and surfaces. Here, we demonstrate contactless surface levitation and transport of liquid drops, realized by harnessing and sustaining the natural sublimation of a solid-carbon-dioxide-coated substrate to generate a continuous supporting vapor layer. The capability and limitations of this technique in handling liquids of extreme surface tension and kinematic viscosity values are investigated both experimentally and theoretically. The sublimating coating is capable of repelling many viscous and low-surface-tension liquids, colloidal suspensions, and non-Newtonian fluids as well, displaying outstanding omniphobic properties. Finally, we demonstrate how sublimation can be used for liquid transport, mixing, and drop coalescence, with a sublimating layer coated on an underlying substrate with prefabricated channels, conferring omniphobicity using a simple physical approach (i.e., phase change) rather than a chemical one. The independence of the surface levitation principle from material properties, such as electromagnetic, thermal or optical, surface energy, adhesion, or mechanical properties, renders this method attractive for a wide range of potential applications.

The Basal Forebrain and Motor Cortex Provide Convergent yet Distinct Movement-Related Inputs to the Auditory Cortex.

Cholinergic inputs to the auditory cortex from the basal forebrain (BF) are important to auditory processing and plasticity, but little is known about the organization of these synapses onto different auditory cortical neuron types, how they influence auditory responsiveness, and their activity patterns during various behaviors. Using intersectional tracing, optogenetic circuit mapping, and in vivo calcium imaging, we found that cholinergic axons arising from the caudal BF target major excitatory and inhibitory auditory cortical cell types, rapidly modulate auditory cortical tuning, and display fast movement-related activity. Furthermore, the BF and the motor cortex-another source of movement-related activity-provide convergent input onto some of the same auditory cortical neurons. Cholinergic and motor cortical afferents to the auditory cortex display distinct activity patterns and presynaptic partners, indicating that the auditory cortex integrates bottom-up cholinergic signals related to ongoing movements and arousal with top-down information concerning impending movements and motor planning.

A synaptic and circuit basis for corollary discharge in the auditory cortex.

Sensory regions of the brain integrate environmental cues with copies of motor-related signals important for imminent and ongoing movements. In mammals, signals propagating from the motor cortex to the auditory cortex are thought to have a critical role in normal hearing and behaviour, yet the synaptic and circuit mechanisms by which these motor-related signals influence auditory cortical activity remain poorly understood. Using in vivo intracellular recordings in behaving mice, we find that excitatory neurons in the auditory cortex are suppressed before and during movement, owing in part to increased activity of local parvalbumin-positive interneurons. Electrophysiology and optogenetic gain- and loss-of-function experiments reveal that motor-related changes in auditory cortical dynamics are driven by a subset of neurons in the secondary motor cortex that innervate the auditory cortex and are active during movement. These findings provide a synaptic and circuit basis for the motor-related corollary discharge hypothesized to facilitate hearing and auditory-guided behaviours.

A circuit for motor cortical modulation of auditory cortical activity.

Normal hearing depends on the ability to distinguish self-generated sounds from other sounds, and this ability is thought to involve neural circuits that convey copies of motor command signals to various levels of the auditory system. Although such interactions at the cortical level are believed to facilitate auditory comprehension during movements and drive auditory hallucinations in pathological states, the synaptic organization and function of circuitry linking the motor and auditory cortices remain unclear. Here we describe experiments in the mouse that characterize circuitry well suited to transmit motor-related signals to the auditory cortex. Using retrograde viral tracing, we established that neurons in superficial and deep layers of the medial agranular motor cortex (M2) project directly to the auditory cortex and that the axons of some of these deep-layer cells also target brainstem motor regions. Using in vitro whole-cell physiology, optogenetics, and pharmacology, we determined that M2 axons make excitatory synapses in the auditory cortex but exert a primarily suppressive effect on auditory cortical neuron activity mediated in part by feedforward inhibition involving parvalbumin-positive interneurons. Using in vivo intracellular physiology, optogenetics, and sound playback, we also found that directly activating M2 axon terminals in the auditory cortex suppresses spontaneous and stimulus-evoked synaptic activity in auditory cortical neurons and that this effect depends on the relative timing of motor cortical activity and auditory stimulation. These experiments delineate the structural and functional properties of a corticocortical circuit that could enable movement-related suppression of auditory cortical activity.

Characterizing biobank organizations in the U.S.: results from a national survey.

BACKGROUND: Effective translational biomedical research hinges on the operation of 'biobanks,' repositories that assemble, store, and manage collections of human specimens and related data. Some are established intentionally to address particular research needs; many, however, have arisen opportunistically, in a variety of settings and with a variety of expectations regarding their functions and longevity. Despite their rising prominence, little is known about how biobanks are organized and function beyond simple classification systems (government, academia, industry). METHODS: In 2012, we conducted the first national survey of biobanks in the U.S., collecting information on their origins, specimen collections, organizational structures, and market contexts and sustainability. From a list of 636 biobanks assembled through a multi-faceted search strategy, representatives from 456 U.S. biobanks were successfully recruited for a 30-minute online survey (72% response rate). Both closed and open-ended responses were analyzed using descriptive statistics. RESULTS: While nearly two-thirds of biobanks were established within the last decade, 17% have been in existence for over 20 years. Fifty-three percent listed research on a particular disease as the most important reason for establishment; 29% listed research generally. Other reasons included response to a grant or gift, and intent to centralize, integrate, or harmonize existing research structures. Biobank collections are extraordinarily diverse in number and types of specimens and in sources (often multiple) from which they are obtained, including from individuals, clinics or hospitals, public health programs, and research studies. Forty-four percent of biobanks store pediatric specimens, and 36% include postmortem specimens. Most biobanks are affiliated in one or multiple ways with other entities: 88% are part of at least one or more larger organizations (67% of these are academic, 23% hospitals, 13% research institutes). The majority of biobanks seem to fill a particular 'niche' within a larger organization or research area; a minority are concerned about competition for services, although many are worried about underutilization of specimens and long-term funding. CONCLUSIONS: Effective utilization of biobank collections and effective policies to govern their use will require understanding of the immense diversity found in organizational features, including the very different history and primary goals that many biobanks have.

New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking.

Rodents begin to use bilaterally coordinated, rhythmic sweeping of their vibrissae ("whisking") for environmental exploration around 2 weeks after birth. Whether (and how) the vibrissal control circuitry changes after birth is unknown, and the relevant premotor circuitry remains poorly characterized. Using a modified rabies virus transsynaptic tracing strategy, we labeled neurons synapsing directly onto vibrissa facial motor neurons (vFMNs). Sources of potential excitatory, inhibitory, and modulatory vFMN premotor neurons, and differences between the premotor circuitry for vFMNs innervating intrinsic versus extrinsic vibrissal muscles were systematically characterized. The emergence of whisking is accompanied by the addition of new sets of bilateral excitatory inputs to vFMNs from neurons in the lateral paragigantocellularis (LPGi). Furthermore, descending axons from the motor cortex directly innervate LPGi premotor neurons. Thus, neural modules that are well suited to facilitate the bilateral coordination and cortical control of whisking are added to the premotor circuitry in parallel with the emergence of this exploratory behavior.