High-throughput volumetric mapping of synaptic transmission.

Volumetric imaging of synaptic transmission in vivo requires high spatial and high temporal resolution. Shaping the wavefront of two-photon fluorescence excitation light, we developed Bessel-droplet foci for high-contrast and high-resolution volumetric imaging of synapses. Applying our method to imaging glutamate release, we demonstrated high-throughput mapping of excitatory inputs at >1,000 synapses per volume and >500 dendritic spines per neuron in vivo and unveiled previously unseen features of functional synaptic organization in the mouse primary visual cortex.

Simultaneous cortex-wide fluorescence Ca2+ imaging and whole-brain fMRI.

Achieving a comprehensive understanding of brain function requires multiple imaging modalities with complementary strengths. We present an approach for concurrent widefield optical and functional magnetic resonance imaging. By merging these modalities, we can simultaneously acquire whole-brain blood-oxygen-level-dependent (BOLD) and whole-cortex calcium-sensitive fluorescent measures of brain activity. In a transgenic murine model, we show that calcium predicts the BOLD signal, using a model that optimizes a gamma-variant transfer function. We find consistent predictions across the cortex, which are best at low frequency (0.009-0.08 Hz). Furthermore, we show that the relationship between modality connectivity strengths varies by region. Our approach links cell-type-specific optical measurements of activity to the most widely used method for assessing human brain function.

Tumor-associated macrophages promote the metastasis and growth of non-small-cell lung cancer cells through NF-κB/PP2Ac-positive feedback loop.

Non-small-cell lung cancer (NSCLC), with its aggressive biological behavior, is one of the most diagnosed cancers. Tumor-associated inflammatory cells play important roles in the interaction between chronic inflammation and lung cancer, however the mechanisms involved are far from defined. In the present study, by developing an orthotopic NSCLC mouse model based on chronic inflammation, we proved that an inflammatory microenvironment accelerated the growth of orthotopic xenografts in vivo. Tumor-associated macrophages, the most abundant population of inflammatory cells, were identified. Treatment with macrophage-conditioned medium (MCM) promoted the growth and migration of NSCLC cells. Using bioinformatics analysis, we identified downregulated PP2Ac expression in NSCLC cells upon treatment with MCM. We further confirmed that this downregulation was executed in an NF-κB pathway-dependent manner. As IκB kinase (IKK) has been proved to be a substrate of PP2Ac, inhibition on PP2Ac could result in amplification of NF-κB pathway signaling. Overexpression of PP2Ac, or the dominant-negative forms of IKK or IκB, attenuated the acceleration of growth and metastasis by MCM. Using bioinformatics analysis, we further identified that CXCL1 and COL6A1 could be downstream of NF-κB/PP2Ac pathway. Luciferase assay and ChIP assay further confirmed the location of response elements on the promoter regions of CXCL1 and COL6A1. Elevated CXCL1 facilitated angiogenesis, whereas upregulated COL6A1 promoted proliferation and migration.

Identification of seven tumor-educated platelets RNAs for cancer diagnosis.

BACKGROUND: Tumor-educated platelets (TEPs) may enable blood-based cancer diagnosis. This study aimed to identify diagnostic TEPs genes involved in carcinogenesis. MATERIALS AND METHODS: The TEPs differentially expressed genes (DEGs) between healthy samples and early/advanced cancer samples were obtained using bioinformatics. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were used to identify the pathways and functional annotation of TEPs DEGs. Protein-protein interaction of these TEPs DEGs was analyzed based on the STRING database and visualized by Cytoscape software. The correlation analysis and diagnostic analysis were performed to evaluate the diagnostic value of TEPs mRNAs expression for early/advanced cancers. Quantitative real-time PCR (qRT-PCR) was applied to validate the role of DEGs in cancers. RESULTS: TEPs mRNAs were mostly involved in protein binding, extracellular matrix, and cellular protein metabolic process. RSL24D1 was negatively correlated to early-stage cancers compared to healthy controls and may be potentially used for early cancer diagnosis. In addition, HPSE, IFI27, LGALS3BP, CRYM, HBD, COL6A3, LAMB2, and IFITM3 showed an upward trend in the expression from early to advanced cancer stages. Moreover, ARL2, FCGR2A, and KLHDC8B were positively associated with advanced, metastatic cancers compared to healthy controls. Among the 12 selected DEGs, the expression of 7 DEGs, including RSL24D1, IFI27, CRYM, HBD, IFITM3, FCGR2A, and KLHDC8B, were verified by the qRT-PCR method. CONCLUSION: This study suggests that the 7-gene TEPs liquid-biopsy biomarkers may be used for cancer diagnosis and monitoring.

The values of applying classification and counts of white blood cells to the prognostic evaluation of resectable gastric cancers.

BACKGROUND: The classifications and counts of white blood cells (WBCs) have been proved to be able to be used as prognostic markers in cancer cases. The present study investigated the potential values of the classifications and counts of WBC, including lymphocyte (LY), monocyte (MO), neutrophil (NE), eosinophil (EO), and basophil (BA) in the prognosis of resectable gastric cancers (GCs). METHODS: This retrospective study recruited 104 resectable GC cases which were pathologically confirmed. The patients were divided into two groups according to the median pre-treatment values. To evaluate the changes in WBC counts and classification after treatment, we introduced the concept of post/pre-treatment ratios (≤ 1 indicated count was not increased after therapy, while > 1 suggested increased count). RESULTS: Pre-treatment NE and total WBC counts were negatively correlated with overall survival (OS). Surgery significantly decreased the level of NE count, but increased the level of EO, whereas had no effect on the levels of LY, MO, BAor total WBC. Adjuvant chemotherapy significantly decreased the level of BA. Whole course of treatment (surgery combined with adjuvant chemotherapy) had no significant effect on the counts of LY, MO, NE, EO, BA or total WBC. Post/pre-treatment ratios of LY, MO NE, EO, BA and total WBC levels had no effects on OS. Univariate analysis indicated that AJCC stage (III) and higher level of pre-treatment total WBC count were prognostic factors affecting OS. Multivariate Cox regression analysis revealed that AJCC stage (III) and higher level of pre-treatment total WBC count were independent prognostic factors. CONCLUSIONS: Pre-treatment NE count and pre-treatment total WBC count may be potential prognostic factors for the prognostic evaluation of GCs.

Retinal Wave Patterns Are Governed by Mutual Excitation among Starburst Amacrine Cells and Drive the Refinement and Maintenance of Visual Circuits.

Retinal waves are correlated bursts of spontaneous activity whose spatiotemporal patterns are critical for early activity-dependent circuit elaboration and refinement in the mammalian visual system. Three separate developmental wave epochs or stages have been described, but the mechanism(s) of pattern generation of each and their distinct roles in visual circuit development remain incompletely understood. We used neuroanatomical,in vitroandin vivoelectrophysiological, and optical imaging techniques in genetically manipulated mice to examine the mechanisms of wave initiation and propagation and the role of wave patterns in visual circuit development. Through deletion of ß2 subunits of nicotinic acetylcholine receptors (ß2-nAChRs) selectively from starburst amacrine cells (SACs), we show that mutual excitation among SACs is critical for Stage II (cholinergic) retinal wave propagation, supporting models of wave initiation and pattern generation from within a single retinal cell type. We also demonstrate that ß2-nAChRs in SACs, and normal wave patterns, are necessary for eye-specific segregation. Finally, we show that Stage III (glutamatergic) retinal waves are not themselves necessary for normal eye-specific segregation, but elimination of both Stage II and Stage III retinal waves dramatically disrupts eye-specific segregation. This suggests that persistent Stage II retinal waves can adequately compensate for Stage III retinal wave loss during the development and refinement of eye-specific segregation. These experiments confirm key features of the "recurrent network" model for retinal wave propagation and clarify the roles of Stage II and Stage III retinal wave patterns in visual circuit development. SIGNIFICANCE STATEMENT: Spontaneous activity drives early mammalian circuit development, but the initiation and patterning of activity vary across development and among modalities. Cholinergic "retinal waves" are initiated in starburst amacrine cells and propagate to retinal ganglion cells and higher-order visual areas, but the mechanism responsible for creating their unique and critical activity pattern is incompletely understood. We demonstrate that cholinergic wave patterns are dictated by recurrent connectivity within starburst amacrine cells, and retinal ganglion cells act as "readouts" of patterned activity. We also show that eye-specific segregation occurs normally without glutamatergic waves, but elimination of both cholinergic and glutamatergic waves completely disrupts visual circuit development. These results suggest that each retinal wave pattern during development is optimized for concurrently refining multiple visual circuits.

A modified hyaluronic acid hydrogel with strong bacterial capture and killing capabilities for drug-resistant bacteria-infected diabetic wound healing.

Management of diabetic wounds becomes increasingly challenging as bacterial infections intensify the inflammation. Employing polysaccharide hydrogels with inherent antibacterial qualities can significantly reduce the need for antibiotics to manage infections in diabetic wounds. The typical approach to achieving antibacterial outcomes with hydrogels relies on the penetration of bacteria into their porous architecture. Such penetration not only takes time but can also prolong inflammation, thus impeding the healing of wounds. Hence, the quick capture and eradication of bacteria are essential for optimizing the hydrogel's antibacterial performance. Herein, we introduce a multifunctional polysaccharide hydrogel dressing-designated as HAQ-created for managing bacterial infections in diabetic wounds. This dressing is based on hyaluronic acid, which is modified with methacrylic anhydride, and special functional groups are added to the modified hyaluronic acid matrix: phenylboronic acid for capturing bacteria and quaternary ammonium chitosan for bacterial destruction. As expected, the HAQ system exhibits robust antibacterial effectiveness against both methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa in vitro and in vivo. Consequently, HAQ stands as a promising hydrogel dressing with intrinsic antibacterial capabilities and offers significant potential for managing diabetic wounds infected by drug-resistant bacteria.

Essential role of glycoprotein Ibα in platelet activation.

ABSTRACT: Glycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα-elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα-14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbß3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa-/-) decreased platelet aggregation, integrin αIIbß3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIbα axis.

A cuttlefish ink nanoparticle-reinforced biopolymer hydrogel with robust adhesive and immunomodulatory features for treating oral ulcers in diabetes.

Oral ulcers can be managed using a variety of biomaterials that deliver drugs or cytokines. However, many patients experience minimal benefits from certain medical treatments because of poor compliance, short retention times in the oral cavity, and inadequate drug efficacy. Herein, we present a novel hydrogel patch (SCE2) composed of a biopolymer matrix (featuring ultraviolet-triggered adhesion properties) loaded with cuttlefish ink nanoparticles (possessing pro-healing functions). Applying a straightforward local method initiates the formation of a hydrogel barrier that adheres to mucosal injuries under the influence of ultraviolet light. SCE2 then demonstrates exceptional capabilities for near-infrared photothermal sterilization and neutralization of reactive oxygen species. These properties contribute to the elimination of bacteria and the management of the oxidation process, thus accelerating the healing phase's progression from inflammation to proliferation. In studies involving diabetic rats with oral ulcers, the SCE2 adhesive patch significantly quickens recovery by altering the inflamed state of the injured area, facilitating rapid re-epithelialization, and fostering angiogenesis. In conclusion, this light-sensitive hydrogel patch offers a promising path to expedited wound healing, potentially transforming treatment strategies for clinical oral ulcers.

Multimodal measures of spontaneous brain activity reveal both common and divergent patterns of cortical functional organization.

Large-scale functional networks have been characterized in both rodent and human brains, typically by analyzing fMRI-BOLD signals. However, the relationship between fMRI-BOLD and underlying neural activity is complex and incompletely understood, which poses challenges to interpreting network organization obtained using this technique. Additionally, most work has assumed a disjoint functional network organization (i.e., brain regions belong to one and only one network). Here, we employ wide-field Ca2+ imaging simultaneously with fMRI-BOLD in mice expressing GCaMP6f in excitatory neurons. We determine cortical networks discovered by each modality using a mixed-membership algorithm to test the hypothesis that functional networks exhibit overlapping organization. We find that there is considerable network overlap (both modalities) in addition to disjoint organization. Our results show that multiple BOLD networks are detected via Ca2+ signals, and networks determined by low-frequency Ca2+ signals are only modestly more similar to BOLD networks. In addition, the principal gradient of functional connectivity is nearly identical for BOLD and Ca2+ signals. Despite similarities, important differences are also detected across modalities, such as in measures of functional connectivity strength and diversity. In conclusion, Ca2+ imaging uncovers overlapping functional cortical organization in the mouse that reflects several, but not all, properties observed with fMRI-BOLD signals.

Ferric Iron/Shikonin Nanoparticle-Embedded Hydrogels with Robust Adhesion and Healing Functions for Treating Oral Ulcers in Diabetes.

Oral ulcers can be addressed using various biomaterials designed to deliver medications or cytokines. Nevertheless, the effectiveness of these substances is frequently limited in many patients due to poor adherence, short retention time in the mouth, and less-than-optimal drug efficacy. In this study, a new hydrogel patch (FSH3) made of a silk fibroin/hyaluronic acid matrix with light-sensitive adhesive qualities infused with ferric iron/shikonin nanoparticles to enhance healing effects is presented. Initially, this hydrogel forms an adhesive barrier over mucosal lesions through a straightforward local injection, solidifying when exposed to UV light. Subsequently, FSH3 demonstrates superior reactive oxygen species elimination and near-infrared photothermal bactericidal activity. These characteristics support bacterial elimination and regulate oxidative levels, promoting a wound's progression from inflammation to tissue regeneration. In a diabetic rat model mimicking oral ulcers, FSH3 significantly speeds up healing by adjusting the inflammatory environment of the injured tissue, maintaining balance in oral microbiota, and promoting faster re-epithelialization. Overall, the light-sensitive FSH3 hydrogel shows potential for rapid wound recovery and may transform therapeutic methods for managing oral ulcers in diabetes.

Molecular and cellular dynamics of the developing human neocortex at single-cell resolution.

The development of the human neocortex is a highly dynamic process and involves complex cellular trajectories controlled by cell-type-specific gene regulation1. Here, we collected paired single-nucleus chromatin accessibility and transcriptome data from 38 human neocortical samples encompassing both the prefrontal cortex and primary visual cortex. These samples span five main developmental stages, ranging from the first trimester to adolescence. In parallel, we performed spatial transcriptomic analysis on a subset of the samples to illustrate spatial organization and intercellular communication. This atlas enables us to catalog cell type-, age-, and area-specific gene regulatory networks underlying neural differentiation. Moreover, combining single-cell profiling, progenitor purification, and lineage-tracing experiments, we have untangled the complex lineage relationships among progenitor subtypes during the transition from neurogenesis to gliogenesis in the human neocortex. We identified a tripotential intermediate progenitor subtype, termed Tri-IPC, responsible for the local production of GABAergic neurons, oligodendrocyte precursor cells, and astrocytes. Remarkably, most glioblastoma cells resemble Tri-IPCs at the transcriptomic level, suggesting that cancer cells hijack developmental processes to enhance growth and heterogeneity. Furthermore, by integrating our atlas data with large-scale GWAS data, we created a disease-risk map highlighting enriched ASD risk in second-trimester intratelencephalic projection neurons. Our study sheds light on the gene regulatory landscape and cellular dynamics of the developing human neocortex.

[The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells].

OBJECTIVE: To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. METHODS: SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. RESULTS: We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. CONCLUSION: Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

An Immunomodulatory Hydrogel by Hyperthermia-Assisted Self-Cascade Glucose Depletion and ROS Scavenging for Diabetic Foot Ulcer Wound Therapeutics.

Current therapeutic protocols for diabetic foot ulcers (DFUs), a severe and rapidly growing chronic complication in diabetic patients, remain nonspecific. Hyperglycemia-caused inflammation and excessive reactive oxygen species (ROS) are common obstacles encountered in DFU wound healing, often leading to impaired recovery. These two effects reinforce each other, forming an endless loop. However, adequate and inclusive methods are still lacking to target these two aspects and break the vicious cycle. This study proposes a novel approach for treating DFU wounds, utilizing an immunomodulatory hydrogel to achieve self-cascade glucose depletion and ROS scavenging to regulate the diabetic microenvironment. Specifically, AuPt@melanin-incorporated (GHM3) hydrogel dressing is developed to facilitate efficient hyperthermia-enhanced local glucose depletion and ROS scavenging. Mechanistically, in vitro/vivo experiments and RNA sequencing analysis demonstrate that GHM3 disrupts the ROS-inflammation cascade cycle and downregulates the ratio of M1/M2 macrophages, consequently improving the therapeutic outcomes for dorsal skin and DFU wounds in diabetic rats. In conclusion, this proposed approach offers a facile, safe, and highly efficient treatment modality for DFUs.

Multimodal measures of spontaneous brain activity reveal both common and divergent patterns of cortical functional organization.

Large-scale functional networks have been characterized in both rodent and human brains, typically by analyzing fMRI-BOLD signals. However, the relationship between fMRI-BOLD and underlying neural activity is complex and incompletely understood, which poses challenges to interpreting network organization obtained using this technique. Additionally, most work has assumed a disjoint functional network organization (i.e., brain regions belong to one and only one network). Here, we employed wide-field Ca2+ imaging simultaneously with fMRI-BOLD in mice expressing GCaMP6f in excitatory neurons. We determined cortical networks discovered by each modality using a mixed-membership algorithm to test the hypothesis that functional networks are overlapping rather than disjoint. Our results show that multiple BOLD networks are detected via Ca2+ signals; there is considerable network overlap (both modalities); networks determined by low-frequency Ca2+ signals are only modestly more similar to BOLD networks; and, despite similarities, important differences are detected across modalities (e.g., brain region "network diversity"). In conclusion, Ca2+ imaging uncovered overlapping functional cortical organization in the mouse that reflected several, but not all, properties observed with fMRI-BOLD signals.

[Main Factors Influencing the Platelet Spreading].

OBJECTIVE: To explore the main factors of platelet spreading and provide the foundation for related research. METHODS: Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 µg/ml, 30 µg/ml, 100 µg/ml) and kinds of agonists ï¼»thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 µmol/L）, U46619（0.125,0.25,0.5 µmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. RESULTS: Compared to the group which coated with 10 µg/ml and 100 µg/ml fibrinogen, the platelet density is optimal when coated with 30 µg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. CONCLUSION: The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 µmol/L ADP and 0.5 µmol/L U46619 on the slide coated with 30 µg/ml fibrinogen.

Retinal waves prime visual motion detection by simulating future optic flow.

The ability to perceive and respond to environmental stimuli emerges in the absence of sensory experience. Spontaneous retinal activity prior to eye opening guides the refinement of retinotopy and eye-specific segregation in mammals, but its role in the development of higher-order visual response properties remains unclear. Here, we describe a transient window in neonatal mouse development during which the spatial propagation of spontaneous retinal waves resembles the optic flow pattern generated by forward self-motion. We show that wave directionality requires the same circuit components that form the adult direction-selective retinal circuit and that chronic disruption of wave directionality alters the development of direction-selective responses of superior colliculus neurons. These data demonstrate how the developing visual system patterns spontaneous activity to simulate ethologically relevant features of the external world and thereby instruct self-organization.

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression.

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Visual Cortex Gains Independence from Peripheral Drive before Eye Opening.

Visual spatial perception in the mammalian brain occurs through two parallel pathways: one reaches the primary visual cortex (V1) through the thalamus and another the superior colliculus (SC) via direct projections from the retina. The origin, development, and relative function of these two evolutionarily distinct pathways remain obscure. We examined the early functional development of both pathways by simultaneously imaging pre- and post-synaptic spontaneous neuronal activity. We observed that the quality of retinal activity transfer to the thalamus and superior colliculus does not change across the first two postnatal weeks. However, beginning in the second postnatal week, retinal activity does not drive V1 as strongly as earlier wave activity, suggesting that intrinsic cortical activity competes with signals from the sensory periphery as the cortex matures. Together, these findings bring new insight into the function of the SC and V1 and the role of peripheral activity in driving both circuits across development.