Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

Postnatal maturation of microglia is associated with alternative activation and activated TGFß signaling.

Microglia are involved in a widespread set of physiological and pathological processes and further play important roles during neurodevelopmental events. Postnatal maturation of microglia has been associated with the establishment of microglia-specific gene expression patterns. The mechanisms governing microglia maturation are only partially understood but Tgfß1 has been suggested to be one important mediator. In the present study, we demonstrate that early postnatal microglia maturation is associated with alternative microglia activation, increased engulfment of apoptotic cells as well as activated microglial Tgfß signaling. Interestingly, microglial Tgfß signaling preceded the induction of the microglia-specific gene expression indicating the importance of Tgfß1 for postnatal microglia maturation. Moreover, we provide evidence that Tgfß1 is expressed by neurons in postnatal and adult brains defining neuron-microglia communication via Tgfß1 as an important event. Finally, we introduce the recently identified microglia marker Tmem119 as a direct Tgfß1-Smad2 target gene. Taken together, the data presented here further increase the understanding of Tgfß1-mediated effects in microglia and place emphasis on the importance of Tgfß1 for microglia maturation and maintenance.

MicroRNAs modulate core-clock gene expression in pancreatic islets during early postnatal life in rats.

AIMS/HYPOTHESIS: Evidence continues to emerge detailing a fine-tuning of the regulation of metabolic processes and energy homeostasis by cell-autonomous circadian clocks. Pancreatic beta cell functional maturation occurs after birth and implies transcriptional changes triggered by a shift in the nutritional supply that occurs at weaning, enabling the adaptation of insulin secretion. So far, the developmental timing and exact mechanisms involved in the initiation of the circadian clock in the growing pancreatic islets have never been addressed. METHODS: Circadian gene expression was measured by quantitative RT-PCR in islets of rats at different postnatal ages up to 3 months, and by in vitro bioluminescence recording in newborn (10-day-old) and adult (3-month-old) islets. The effect of the microRNAs miR-17-5p and miR-29b-3p on the expression of target circadian genes was assessed in newborn rat islets transfected with microRNA antisense or mimic oligonucleotides, and luciferase reporter assays were performed on the rat insulin-secreting cell line INS832/13 to determine a direct effect. The global regulatory network between microRNAs and circadian genes was computationally predicted. RESULTS: We found up to a sixfold-change in the 24 h transcriptional oscillations and overall expression of Clock, Npas2, Bmal1, Bmal2, Rev-erbα, Per1, Per2, Per3 and Cry2 between newborn and adult rat islets. Synchronisation of the clock machinery in cultured islet cells revealed a delayed cell-autonomous rhythmicity of about 1.5 h in newborn compared with adult rats. Computational predictions unveiled the existence of a complex regulatory network linking over 40 microRNAs displaying modifications in their expression profiles during postnatal beta cell maturation and key core-clock genes. In agreement with these computational predictions, we demonstrated that miR-17-5p and miR-29b-3p directly regulated circadian gene expression in the maturing islet cells of 10-day-old rats. CONCLUSIONS/INTERPRETATION: These data show that the circadian clock is not fully operational in newborn islets and that microRNAs potently contribute to its regulation during postnatal beta cell maturation. Defects in this process may have long-term consequences on circadian physiology and pancreatic islet function, favouring the manifestation of metabolic diseases such as diabetes.

Mucin-Microbiota Interaction During Postnatal Maturation of the Intestinal Ecosystem: Clinical Implications.

The mucus layer and gut microbiota interplay contributes to host homeostasis. The mucus layer serves as a scaffold and a carbon source for gut microorganisms; conversely, gut microorganisms, including mucin degraders, influence mucin gene expression, glycosylation, and secretion. Conjointly they shield the epithelium from luminal pathogens, antigens, and toxins. Importantly, the mucus layer and gut microbiota are established in parallel during early postnatal life. During this period, the development of gut microbiota and mucus layer is coupled with that of the immune system. Developmental changes of different mucin types can impact the age-dependent patterns of intestinal infection in terms of incidence and severity. Altered mucus layer, dysbiotic microbiota, and abnormal mucus-gut microbiota interaction have the potential for inducing systemic effects, and accompany several intestinal diseases such as inflammatory bowel disease, colorectal cancer, and radiation-induced mucositis. Early life provides a pivotal window of opportunity to favorably modulate the mucus-microbiota interaction. The support of a health-compatible mucin-microbiota maturation in early life is paramount for long-term health and serves as an important opportunity for clinical intervention.

Deep and precise quantification of the mouse synaptosomal proteome reveals substantial remodeling during postnatal maturation.

During postnatal murine maturation, behavioral patterns emerge and become shaped by experience-dependent adaptations. During the same period, the morphology of dendritic spines, the morphological correlates of excitatory synapses, is known to change, and there is evidence of concurrent alterations of the synaptosomal protein machinery. To obtain comprehensive and quantitative insights in the developmental regulation of the proteome of synapses, we prepared cortical synaptosomal fractions from a total of 16 individual juvenile and adult mouse brains (age 3 or 8 weeks, respectively). We then applied peptide-based iTRAQ labeling (four pools of 4 animals) and high-resolution two-dimensional peptide fractionation (99 SCX fractions and 3 h reversed-phase gradients) using a hybrid CID-HCD acquisition method on a Velos Orbitrap mass spectrometer to identify a comprehensive set of synaptic proteins and to quantify changes in protein expression. We obtained a data set tracking expression levels of 3500 proteins mapping to 3427 NCBI GeneIDs during development with complete quantification data available for 3422 GeneIDs, which, to the best of our knowledge, constitutes the deepest coverage of the synaptosome proteome to date. The inclusion of biological replicates in a single mass spectrometry analysis demonstrated both high reproducibility of our synaptosome preparation method as well as high precision of our quantitative data (correlation coefficient R = 0.87 for the biological replicates). To evaluate the validity of our data, the developmental regulation of eight proteins identified in our analysis was confirmed independently using western blotting. A gene ontology analysis confirmed the synaptosomal nature of a large fraction of identified proteins. Of note, the set of the most strongly regulated proteins revealed candidates involved in neurological processes in health and disease states. This highlights the fact that developmentally regulated proteins can play additional roles in neurological disease processes. All data have been deposited to the ProteomeXchange with identifier PXD000552.

Dissecting gene expression networks in the developing hippocampus through the lens of NEIL3 depletion.

Gene regulation in the hippocampus is fundamental for its development, synaptic plasticity, memory formation, and adaptability. Comparisons of gene expression among different developmental stages, distinct cell types, and specific experimental conditions have identified differentially expressed genes contributing to the organization and functionality of hippocampal circuits. The NEIL3 DNA glycosylase, one of the DNA repair enzymes, plays an important role in hippocampal maturation and neuron functionality by shaping transcription. While differential gene expression (DGE) analysis has identified key genes involved, broader gene expression patterns crucial for high-order hippocampal functions remain uncharted. By utilizing the weighted gene co-expression network analysis (WGCNA), we mapped gene expression networks in immature (p8-neonatal) and mature (3 m-adult) hippocampal circuits in wild-type and NEIL3-deficient mice. Our study unveiled intricate gene network structures underlying hippocampal maturation, delineated modules of co-expressed genes, and pinpointed highly interconnected hub genes specific to the maturity of hippocampal subregions. We investigated variations within distinct gene network modules following NEIL3 depletion, uncovering NEIL3-targeted hub genes that influence module connectivity and specificity. By integrating WGCNA with DGE, we delve deeper into the NEIL3-dependent molecular intricacies of hippocampal maturation. This study provides a comprehensive systems-level analysis for assessing the potential correlation between gene connectivity and functional connectivity within the hippocampal network, thus shaping hippocampal function throughout development.

Hypoxic depression of PKG-mediated inhibition of serotonergic contraction in ovine carotid arteries.

Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O2-5% CO2 in the presence of nitro-l-arginine methyl ester (l-NAME) and N(G)-nitro-l-arginine (l-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of α-adrenergic receptors. The PKG activator 8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Twist2-driven chromatin remodeling governs the postnatal maturation of dermal fibroblasts.

Dermal fibroblasts lose stem cell potency after birth, which prevents regenerative healing. However, the underlying intracellular mechanisms are largely unknown. We uncover the postnatal maturation of papillary fibroblasts (PFs) driven by the extensive Twist2-mediated remodeling of chromatin accessibility. A loss of the regenerative ability of postnatal PFs occurs with decreased H3K27ac levels. Single-cell transcriptomics, assay for transposase-accessible chromatin sequencing (ATAC-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the postnatal maturation trajectory associated with the loss of the regenerative trajectory in PFs, which is characterized by a marked decrease in chromatin accessibility and H3K27ac modifications. Histone deacetylase inhibition delays spontaneous chromatin remodeling, thus maintaining the regenerative ability of postnatal PFs. Genomic analysis identifies Twist2 as a major regulator within chromatin regions with decreased accessibility during the postnatal period. When Twist2 is genetically deleted in dermal fibroblasts, the intracellular cascade of postnatal maturation is significantly delayed. Our findings reveal the comprehensive intracellular mechanisms underlying intrinsic postnatal changes in dermal fibroblasts.

Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation.

During embryonic development and adulthood, Reelin exerts several important functions in the brain including the regulation of neuronal migration, dendritic growth and branching, dendritic spine formation, synaptogenesis and synaptic plasticity. As a consequence, the Reelin signaling pathway has been associated with several human brain disorders such as lissencephaly, autism, schizophrenia, bipolar disorder, depression, mental retardation, Alzheimer's disease and epilepsy. Several elements of the signaling pathway are known. Core components, such as the Reelin receptors very low-density lipoprotein receptor (VLDLR) and Apolipoprotein E receptor 2 (ApoER2), Src family kinases Src and Fyn, and the intracellular adaptor Disabled-1 (Dab1), are common to most but not all Reelin functions. Other downstream effectors are, on the other hand, more specific to defined tasks. Reelin is a large extracellular protein, and some aspects of the signal are regulated by its processing into smaller fragments. Rather than being inhibitory, the processing at two major sites seems to be fulfilling important physiological functions. In this review, I describe the various cellular events regulated by Reelin and attempt to explain the current knowledge on the mechanisms of action. After discussing the shared and distinct elements of the Reelin signaling pathway involved in neuronal migration, dendritic growth, spine development and synaptic plasticity, I briefly outline the data revealing the importance of Reelin in human brain disorders.

Postnatal Increases in Axonal Conduction Velocity of an Identified Drosophila Interneuron Require Fast Sodium, L-Type Calcium and Shaker Potassium Channels.

During early postnatal life, speed up of signal propagation through many central and peripheral neurons has been associated with an increase in axon diameter or/and myelination. Especially in unmyelinated axons postnatal adjustments of axonal membrane conductances is potentially a third mechanism but solid evidence is lacking. Here, we show that axonal action potential (AP) conduction velocity in the Drosophila giant fiber (GF) interneuron, which is required for fast long-distance signal conduction through the escape circuit, is increased by 80% during the first day of adult life. Genetic manipulations indicate that this postnatal increase in AP conduction velocity in the unmyelinated GF axon is likely owed to adjustments of ion channel expression or properties rather than axon diameter increases. Specifically, targeted RNAi knock-down of either Para fast voltage-gated sodium, Shaker potassium (Kv1 homologue), or surprisingly, L-type like calcium channels counteracts postnatal increases in GF axonal conduction velocity. By contrast, the calcium-dependent potassium channel Slowpoke (BK) is not essential for postnatal speeding, although it also significantly increases conduction velocity. Therefore, we identified multiple ion channels that function to support fast axonal AP conduction velocity, but only a subset of these are regulated during early postnatal life to maximize conduction velocity. Despite its large diameter (∼7 µm) and postnatal regulation of multiple ionic conductances, mature GF axonal conduction velocity is still 20-60 times slower than that of vertebrate Aß sensory axons and α motoneurons, thus unraveling the limits of long-range information transfer speed through invertebrate circuits.

Role of Farnesoid X Receptor in the Determination of Liver Transcriptome during Postnatal Maturation in Mice.

The liver is a vital organ with critical functions in metabolism of various biologically useful materials, synthesis of several vital proteins, detoxification of toxic substances, and immune defense. Most liver functions are not mature at birth and many changes happen during postnatal liver development, which lead to differential vulnerabilities of the liver at different developmental stages. However, the details of what changes occur in liver after birth, at what developmental stages they occur, and molecular mechanisms in the regulation of the developmental process are not clearly known. The nuclear receptor Farnesoid X receptor (FXR) is an important transcriptional regulator in liver. Here, we used RNA-Sequencing to analyze the transcriptome of mouse liver from perinatal to adult ages in both C57BL/6 and Fxr-/- mice. We have defined a clear timeline of functional transition from prenatal through neonatal and adolescent to adult in C57BL/6 mice. Without FXR, activation of neonatal-specific pathways was prolonged and maturation of multiple metabolic pathways was delayed. The loss of FXR also led to increased expression of 27 other transcription regulators. Our data support a conclusion that developmental transcriptome revealed significant functional transition during postnatal liver development and FXR plays an important role in control of postnatal liver maturation.

Pre- and Postnatal Lung Development: An Updated Species Comparison.

The purpose of this review is to give an outline of respiratory tract morphological and functional development with an emphasis on perinatal and postnatal maturational processes. In view of the rising need for qualitative and quantitative data for the development of pediatric pharmaceuticals, a comparison of the human situation to experimental animal models is made, and functional data as well as suitable models for human airway diseases and functional testing are presented. Birth Defects Research 109:1519-1539, 2017. © 2017 Wiley Periodicals, Inc.

Reelin-Haploinsufficiency Disrupts the Developmental Trajectory of the E/I Balance in the Prefrontal Cortex.

The reelin gene is a strong candidate in the etiology of several psychiatric disorders such as schizophrenia, major depression, bipolar disorders, and autism spectrum disorders. Most of these diseases are accompanied by cognitive and executive-function deficits associated with prefrontal dysfunctions. Mammalian prefrontal cortex (PFC) development is characterized by a protracted postnatal maturation constituting a period of enhanced vulnerability to psychiatric insults. The identification of the molecular components underlying this prolonged postnatal development is necessary to understand the synaptic properties of defective circuits participating in these psychiatric disorders. We have recently shown that reelin plays a key role in the maturation of glutamatergic functions in the postnatal PFC, but no data are available regarding the GABAergic circuits. Here, we undertook a cross-sectional analysis of GABAergic function in deep layer pyramidal neurons of the medial PFC of wild-type and haploinsufficient heterozygous reeler mice. Using electrophysiological approaches, we showed that decreased reelin levels impair the maturation of GABAergic synaptic transmission without affecting the inhibitory nature of GABA. This phenotype consequently impacted the developmental sequence of the synaptic excitation/inhibition (E/I) balance. These data indicate that reelin is necessary for the correct maturation and refinement of GABAergic synaptic circuits in the postnatal PFC and therefore provide a mechanism for altered E/I balance of prefrontal circuits associated with psychiatric disorders.

Oral Feeding Assessment Predicts Length of Hospital Stay in Late Preterm Infants.

BACKGROUND: When compared with term infants, late preterm (LP) infants have greater morbidity and mortality, longer hospital stay, and greater rate of hospital readmission. Oral feeding difficulty is one of the prominent reasons for delayed discharge in LP infants. OBJECTIVE: To identify the maturity levels of LP infants' oral feeding skills (OFS) at the time of their first oral feeding and to determine the relationship between OFS maturity levels and length of hospital stay. METHODS: OFS was assessed in 48 LP infants born between 340/7 and 356/7 weeks gestational age at the time of their first oral feeding within 24 h of birth. The intake at 5 minutes, at completion of the feeding, and the duration of feeding a 15 mL prescribed volume of milk were tabulated. Proficiency expressed as percent mL consumed in the first 5 min/15 mL prescribed and rate of milk transfer over the entire feeding (mL/min) were recorded. OFS were assessed using a novel 4-level scale defined by the combined proficiency and rate of milk transfer. RESULTS: When compared with their 35-week counterparts, infants born at 34 weeks gestation had poorer OFS profiles (p = 0.035) and longer hospital stay (p < 0.001). Additionally, further analyses demonstrated that, independently, LOS was associated negatively with both GA and OFS. CONCLUSION: Assessment of OFS levels in LP infants at their first oral feeding can help identify infants at risk of oral feeding issues that may delay hospital discharge. For those infants, we speculate that provision of evidence-based efficacious interventions that improve OFS may shorten hospital stay and decrease hospital re-admissions.

Characteristics of Light-evoked Retinal Ganglion Cell Activity with Postnatal Maturation in SD Rat / 의학물리

As part of Korean retinal prosthesis project, we have provided preliminary experimental results regarding voltage parameters for the stimulation of chemically degenerated rabbit retina. Since our APB-treated chemically degenerated retina is only ON-pathway blocked, now we switch our experiments to more appropriate retinal degeneration model, genetically degenerated retina model (RD mouse: rd/rd (C3H/HeJ)). Before studying with RD mouse, we started control experiments with normal SD rat to understand characteristics of retinal ganglion cell activity with postnatal maturation in rodents. Ganglion cell activities were recorded with 8x8 multi-electrode array. Moving spontaneous bursts appeared until postnatal day of 15. During pre-eye opening period, no light evoked response appeared. After postnatal day of 2 weeks (post-eye opening period), ON-, OFF- and ON/OFF response appeared. The fractional distributions of ON, OFF, and ON/OFF ganglion cell is about 40%, 50%, and 5%. The percentage (%) of light evoked response in each dorso-temporal, ventral, and dorso-nasal area of eye is about 50%, 37.5% and 12.5%, respectively. We concluded that the optimal period for experiment in rodent is about postnatal day of 2~3 weeks.

Area-Specific Postnatal Maturation of the Calcium-Binding Proteins Parvalbumin- and Calbindin-Immunoreactive Neurons in the Rat Hippocampus

The present study was attempted to obtain the regional differences of development of the parvalbumin- and calbindin-ir neurons in the maturing rat hippocampus. Sprague-Dawley rats at 3, 7 days, 2, 3, 4, 5, 6, and 9 weeks of age were transcardially perfused with 4% paraformaldehyde fixative. The hippocampus was cut into 30 or 40(m sections with a cryostat. Immunohistochemical staining was performed for the parvalbumin and the calbindin using ABC method. The parvalbumin-ir neurons and their axon plexuses achieved the adult patterns as following sequences;CA2 and CA3 at ghe second postnatal week, CA1 at the third week, and dentate granule cell layer by fifth postnatal week. In addition, the calbindin-ir neurons showed the adult patterns by second postnatal week in CA2, CA3, mossy fibers, and the molecular layer of the dentate gyrus, the CA1 at the third week, and finally the dentate granular cell layer by fifth week. These results revealed that time course of the maturation of the parvalbumin- and calbindin-ir neurons in the rat hippocampus showed an area-specificity. The delayed maturation of CA1 and the latest maturation of the dentage granular cell layer suggested that the neurons in these locations seemed to exhibit differential responses to the conditions of increased intracellular calcium, when compared with other areas or with the adult hippocampus.