Early Postnatal Mouse Research Articles

Reprogramming astroglia into neurons with hallmarks of fast-spiking parvalbumin-positive interneurons by phospho-site-deficient Ascl1.

Cellular reprogramming of mammalian glia to an induced neuronal fate holds the potential for restoring diseased brain circuits. While the proneural factor achaete-scute complex-like 1 (Ascl1) is widely used for neuronal reprogramming, in the early postnatal mouse cortex, Ascl1 fails to induce the glia-to-neuron conversion, instead promoting the proliferation of oligodendrocyte progenitor cells (OPC). Since Ascl1 activity is posttranslationally regulated, here, we investigated the consequences of mutating six serine phospho-acceptor sites to alanine (Ascl1SA6) on lineage reprogramming in vivo. Ascl1SA6 exhibited increased neurogenic activity in the glia of the early postnatal mouse cortex, an effect enhanced by coexpression of B cell lymphoma 2 (Bcl2). Genetic fate-mapping revealed that most induced neurons originated from astrocytes, while only a few derived from OPCs. Many Ascl1SA6/Bcl2-induced neurons expressed parvalbumin and were capable of high-frequency action potential firing. Our study demonstrates the authentic conversion of astroglia into neurons featuring subclass hallmarks of cortical interneurons, advancing our scope of engineering neuronal fates in the brain.

Electrophysiological properties of neurons in the visual sensory layers of the superior colliculus in early postnatal mice

Objective: To investigate the action potential firing patterns of neurons in the visual sensory layers of the superior colliculus in early postnatal mice and the electrophysiological characteristics of neurons with different firing patterns. Methods: This experimental study utilized whole-cell patch-clamp recordings performed on neurons in the visual sensory layers of the superior colliculus using brain slices from 57 healthy male C57BL/6J mice aged 14 to 20 days (weighing 5.0 to 8.9 g) using brain slices. In current-clamp mode, action potential characteristics were analyzed based on the first action potential generated by depolarizing current, and the firing patterns of neurons were recorded using step depolarizing currents. Neuronal firing patterns were analyzed using hierarchical clustering, and the active electrical properties of neurons with different firing patterns were compared. Results: A total of 135 neurons from the visual sensory layers of the superior colliculus were successfully recorded. Cluster analysis of the neuronal firing patterns identified three types of firing patterns: tonic firing (97, 72%), phasic firing (26, 19%), and single firing (12, 9%). The number of action potentials for each firing pattern was 13.30±7.38, 3.73±3.61, and 0.83±0.39, respectively, with significant differences (P<0.001). There was no significant difference in the membrane potential response to step currents among the three firing pattern types (P>0.05). The action potential amplitudes were (60.45±12.22), (53.67±13.20), and (44.04± 12.92) mV, and the afterhyperpolarization amplitudes were (13.45±13.79), (12.02±13.11), and (20.75±2.85) mV, respectively. The maximum rising slopes were (171.29±77.46), (130.14±61.83), and (78.89±37.08) V/s, and the maximum falling slopes were (-76.33±33.61), (-68.17±31.65), and (-47.97±13.92) V/s, respectively, with all differences being statistically significant (all P<0.05). There were no significant differences in the resting membrane potential, action potential threshold, half-width, and afterhyperpolarization duration among the three firing pattern types (all P>0.05). Conclusions: In the early postnatal mice, neurons in the visual sensory layers of the superior colliculus exhibit three distinct firing patterns: tonic, phasic, and single firing. These firing pattern types show significant differences in action potential amplitude, afterhyperpolarization amplitude, maximum rising slopes, and maximum falling slopes.

Exposure to Radiofrequency Induces Synaptic Dysfunction in Cortical Neurons Causing Learning and Memory Alteration in Early Postnatal Mice.

The widespread use of wireless communication devices has necessitated unavoidable exposure to radiofrequency electromagnetic fields (RF-EMF). In particular, increasing RF-EMF exposure among children is primarily driven by mobile phone use. Therefore, this study investigated the effects of 1850 MHz RF-EMF exposure at a specific absorption rate of 4.0 W/kg on cortical neurons in mice at postnatal day 28. The results indicated a significant reduction in the number of mushroom-shaped dendritic spines in the prefrontal cortex after daily exposure for 4 weeks. Additionally, prolonged RF-EMF exposure over 9 days led to a gradual decrease in postsynaptic density 95 puncta and inhibited neurite outgrowth in developing cortical neurons. Moreover, the expression levels of genes associated with synapse formation, such as synaptic cell adhesion molecules and cyclin-dependent kinase 5, were reduced in the cerebral cortexes of RF-EMF-exposed mice. Behavioral assessments using the Morris water maze revealed altered spatial learning and memory after the 4-week exposure period. These findings underscore the potential of RF-EMF exposure during childhood to disrupt synaptic function in the cerebral cortex, thereby affecting the developmental stages of the nervous system and potentially influencing later cognitive function.

The Notch-PDGFRβ axis suppresses brown adipocyte progenitor differentiation in early post-natal mice

De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively explored. Here, through invivo lineage tracing and mouse modeling, we observed that platelet-derived growth factor receptor beta (PDGFRβ)+ pericytes give rise to developmental brown adipocytes but not to those in adult homeostasis. By contrast, T-box 18 (TBX18)+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFRβ+ pericytes promotes brown adipogenesis by downregulating PDGFRβ. Furthermore, inhibition of Notch signaling in PDGFRβ+ pericytes mitigates high-fat, high-sucrose (HFHS)-induced glucose and metabolic impairment in mice during their development and juvenile phases. Collectively, these findings show that the Notch/PDGFRβ axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health.

The temporal and spatial pattern of leptin receptor-expressing cells in the developing mouse hypothalamus.

The arcuate nucleus is a crucial hypothalamic brain region involved in regulating body weight homeostasis. Neurons within the arcuate nucleus respond to peripheral metabolic signals, such as leptin, and relay these signals via neuronal projections to brain regions both within and outside the hypothalamus, ultimately causing changes in an animal's behaviour and physiology. There is a substantial amount of evidence to indicate that leptin is intimately involved with the postnatal development of arcuate nucleus melanocortin circuitry. Further, it is clear that leptin signalling directly in the arcuate nucleus is required for circuitry development. However, as leptin receptor long isoform (Leprb) mRNA is expressed in multiple nuclei within the developing hypothalamus, including the postsynaptic target regions of arcuate melanocortin projections, this raises the possibility that leptin also signals in these nuclei to promote circuitry development. Here, we used RT-qPCR and RNAscope® to reveal the spatio-temporal pattern of Leprb mRNA in the early postnatal mouse hypothalamus. We found that Leprb mRNA expression increased significantly in the arcuate nucleus, ventromedial nucleus and paraventricular nucleus of the hypothalamus from P8, in concert with the leptin surge. In the dorsomedial nucleus of the hypothalamus, increases in Leprb mRNA were slightly later, increasing significantly from P12. Using duplex RNAscope®, we found Leprb co-expressed with Sim1, Pou3f2, Mc4r and Bdnf in the paraventricular nucleus at P8. Together, these data suggest that leptin may signal in a subset of neurons postsynaptic to arcuate melanocortin neurons, as well as within the arcuate nucleus itself, to promote the formation of arcuate melanocortin circuitry during the early postnatal period.

Ontogenesis of the Mouse Ocular Surface Lymphatic Vascular Network.

Ocular lymphatic vessels play major physiological role in eye homeostasis and their dysfunction can contribute to the progression of several eye diseases. In this study, we characterized their spatiotemporal development and the cellular mechanisms occurring during their ontogenesis in the mouse eye. Whole mount immunofluorescent staining and imaging by standard or lightsheet fluorescence microscopy were performed on late embryonic and early postnatal eye mouse samples. We observed that the ocular surface lymphatic vascular network develops at the early postnatal stages (between P0 and P5) from two nascent trunks arising at the nasal side on both sides of the nictitating membrane. These nascent vessels further branch and encircle the whole eye surface by sprouting lymphangiogenesis. In addition, we got evidence for the existence of a transient lymphvasculogenesis process generating lymphatic vessel fragments that will mostly formed the corneolimbal lymphatic vasculature which further connect to the conjunctival lymphatic network. Our results also support that CD206-positive macrophages can transdifferentiate and then integrate into the lymphatic neovessels. Several complementary cellular processes participate in the development of the lymphatic ocular surface vasculature. This knowledge paves the way for the design of new therapeutic strategies to interfere with ocular lymphatic vessel formation when needed.

Sex-effects On Muscle Health Across A Range Of Physical Inactivity In Early Postnatal Mice

Sex-effects On Muscle Health Across A Range Of Physical Inactivity In Early Postnatal Mice

Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities.

In vivo direct neuronal reprogramming relies on the implementation of an exogenous transcriptional program allowing to achieve conversion of a particular neuronal or glial cell type towards a new identity. The transcription factor (TF) Fezf2 is known for its role in neuronal subtype specification of deep-layer (DL) subcortical projection neurons. High ectopic Fezf2 expression in mice can convert both upper-layer (UL) and striatal projection neurons into a corticofugal fate, even if at low efficiency. In this study, we show that Fezf2 synergizes with the nuclear co-adaptor Lmo4 to further enhance reprogramming of UL cortical pyramidal neurons into DL corticofugal neurons, at both embryonic and early postnatal stages. Reprogrammed neurons express DL molecular markers and project toward subcerebral targets, including thalamus, cerebral peduncle (CP), and spinal cord (SC). We also show that co-expression of Fezf2 with the reprogramming factors Neurog2 and Bcl2 in early postnatal mouse glia promotes glia-to-neuron conversion with partial hallmarks of DL neurons and with Lmo4 promoting further morphological complexity. These data support a novel role for Lmo4 in synergizing with Fezf2 during direct lineage conversion in vivo.

Upregulation of mesencephalic astrocyte-derived neurotrophic factor (MANF) expression offers protection against alcohol neurotoxicity.

Alcohol exposure has detrimental effects on both the developing and mature brain. Endoplasmic reticulum (ER) stress is one of the mechanisms that contributes to alcohol-induced neuronal damages. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-responsive protein and is neuroprotective in multiple neuronal injury and neurodegenerative disease models. MANF deficiency has been shown to exacerbate alcohol-induced ER stress and neurodegeneration. However, it is unknown whether MANF supplement is sufficient to protect against alcohol neurotoxicity. Alcohol alters MANF expression in the brain, but the mechanisms underlying alcohol modulation of MANF expression remain unclear. This study was designed to determine how alcohol alters MANF expression in neuronal cells and whether exogeneous MANF can alleviate alcohol neurotoxicity. We showed that alcohol increased MANF transcription and secretion without affecting MANF mRNA stability and protein degradation. ER stress was necessary for alcohol-induced MANF upregulation, as pharmacological inhibition of ER stress by 4-PBA diminished alcohol-induced MANF expression. In addition, the presence of ER stress response element II (ERSE-II) was required for alcohol-stimulated MANF transcription. Mutations or deletion of this sequence abolished alcohol-regulated transcriptional activity. We generated MANF knockout (KO) neuronal cells using CRISPR/Cas9. MANF KO cells exhibited increased unfolded protein response (UPR) and were more susceptible to alcohol-induced cell death. On the other hand, MANF upregulation by the addition of recombinant MANF protein or adenovirus gene transduction protected neuronal cells against alcohol-induced cell death. Further studies using early postnatal mouse pups demonstrated that enhanced MANF expression in the brain by intracerebroventricular (ICV) injection of MANF adeno-associated viruses ameliorated alcohol-induced cell death. Thus, alcohol increased MANF expression through inducing ER stress, which could be a protective response. Exogenous MANF was able to protect against alcohol-induced neurodegeneration.

Purification of retinal ganglion cells using low-pressure flow cytometry.

Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

Cell-autonomous and non-cell-autonomous roles of NKCC1 in regulating neural stem cell quiescence in the hippocampal dentate gyrus.

Quiescence is a hallmark of adult neural stem cells (NSCs) in the mammalian brain, and establishment and maintenance of quiescence is essential for life-long continuous neurogenesis. How NSCs in the dentate gyrus (DG) of the hippocampus acquire their quiescence during early postnatal stages and continuously maintain quiescence in adulthood is poorly understood. Here, we show that Hopx-CreERT2-mediated conditional deletion of Nkcc1, which encodes a chloride importer, in mouse DG NSCs impairs both their quiescence acquisition at early postnatal stages and quiescence maintenance in adulthood. Furthermore, PV-CreERT2-mediated deletion of Nkcc1 in PV interneurons in the adult mouse brain leads to activation of quiescent DG NSCs, resulting in an expanded NSC pool. Consistently, pharmacological inhibition of NKCC1 promotes NSC proliferation in both early postnatal and adult mouse DG. Together, our study reveals both cell-autonomous and non-cell-autonomous roles of NKCC1 in regulating the acquisition and maintenance of NSC quiescence in the mammalian hippocampus.

Bacterial meningitis in the early postnatal mouse studied at single-cell resolution.

Bacterial meningitis is a major cause of morbidity and mortality, especially among infants and the elderly. Here, we study mice to assess the response of each of the major meningeal cell types to early postnatal E. coli infection using single nucleus RNA sequencing (snRNAseq), immunostaining, and genetic and pharamacologic perturbations of immune cells and immune signaling. Flatmounts of the dissected leptomeninges and dura were used to facilitiate high-quality confocal imaging and quantification of cell abundances and morphologies. Upon infection, the major meningeal cell types - including endothelial cells (ECs), macrophages, and fibroblasts - exhibit distinctive changes in their transcriptomes. Additionally, ECs in the leptomeninges redistribute CLDN5 and PECAM1, and leptomeningeal capillaries exhibit foci with reduced blood-brain barrier integrity. The vascular response to infection appears to be largely driven by TLR4 signaling, as determined by the nearly identical responses induced by infection and LPS administration and by the blunted response to infection in Tlr4-/- mice. Interestingly, knocking out Ccr2, encoding a major chemoattractant for monocytes, or acute depletion of leptomeningeal macrophages, following intracebroventricular injection of liposomal clodronate, had little or no effect on the response of leptomeningeal ECs to E. coli infection. Taken together, these data imply that EC responses to infection are largely driven by the intrinsic EC response to LPS.

Identification of follicle-stimulating hormone-responsive genes in Sertoli cells during early postnatal mouse testis development.

In the mouse testis, Sertoli cells rapidly divide during a narrow window of time pre-pubertally and differentiate thereafter. The number of Sertoli cells determines the testis size and germ cell-carrying capacity. Follicle-stimulating hormone (FSH) binds its cognate FSH-receptors expressed on Sertoli cells and acts as a mitogen to regulate their proliferation. Fshb-/- mutant adult male mice have reduced Sertoli cell number and testis size and reduced sperm number and motility. However, FSH-responsive genes in early postnatal mouse Sertoli cells are unknown. To identify FSH-responsive genes in early postnatal mouse Sertoli cells. A fluorescence-activated cell sorting method was developed to rapidly purify Sertoli cells from control and Fshb-/- mice carrying a Sox9 GfpKI allele. These pure Sertoli cells were used for large-scale gene expression analyses. We show that mouse Sertoli cells rarely divide beyond postnatal day 7. Our in vivo BrdU labeling studies indicate loss of FSH results in a 30% reduction in Sertoli cell proliferation in mice at 5 days of age. Flowsorted GFP+ Sertoli cells with maximal Fshr expression were 97%-98% pure and mostly devoid of Leydig and germ cells as assessed by Taqman qPCR quantification of gene expression and immunolabeling of the corresponding cell-specific markers. Large-scale gene expression analysis identified several differentially regulated genes in flow-sorted GFP+ Sertoli cells obtained from testis of control and Fshb-/- mice at 5 days of age. The top 25 networks identified by pathway analysis include those related to the cell cycle, cell survival and most importantly, carbohydrate and lipid metabolism and molecular transport. Several of the FSH-responsive genes identified in this study could serve as useful markers for Sertoli cell proliferation in normal physiology, toxicant-induced Sertoli cell/testis injury, and other pathological conditions. Our studies reveal that FSH-regulates macromolecular metabolism and molecular transport networks of genes in early postnatal Sertoli cells most likely in preparation for establishment of functional associations with germ cells to successfully coordinate spermatogenesis.

Glutamatergic cerebellar neurons differentially contribute to the acquisition of motor and social behaviors

Insults to the developing cerebellum can cause motor, language, and social deficits. Here, we investigate whether developmental insults to different cerebellar neurons constrain the ability to acquire cerebellar-dependent behaviors. We perturb cerebellar cortical or nuclei neuron function by eliminating glutamatergic neurotransmission during development, and then we measure motor and social behaviors in early postnatal and adult mice. Altering cortical and nuclei neurons impacts postnatal motor control and social vocalizations. Normalizing neurotransmission in cortical neurons but not nuclei neurons restores social behaviors while the motor deficits remain impaired in adults. In contrast, manipulating only a subset of nuclei neurons leaves social behaviors intact but leads to early motor deficits that are restored by adulthood. Our data uncover that glutamatergic neurotransmission from cerebellar cortical and nuclei neurons differentially control the acquisition of motor and social behaviors, and that the brain can compensate for some but not all perturbations to the developing cerebellum.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1– microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

A276 CANNABINOID RECEPTOR 1 IS LOCALIZED IN ENTERIC NEURONS AND EPITHELIUM IN FETAL AND EARLY POSTNATAL MOUSE INTESTINE

Abstract Background The endocannabinoid system (ECS) is necessary for gastrointestinal (GI) homeostasis and serves as a target for exogenous medicinal and recreational cannabinoid compounds. While components of the ECS, including receptors, ligands, and biosynthetic/degradative enzymes, have been increasingly characterized in the adult GI tract of both humans and animal models, similar advances in understanding the role of the ECS in GI developmental programming have not yet been made. Purpose We tested the hypothesis that the ECS plays a role in the developmental programming of the GI tract. Method The developmental timeline of ECS component expression was investigated in the GI tract from fetal and early postnatal mice. Samples from timed-pregnant outbred mice were collected from embryonic days 14 (E14), E16, E18 and postnatal days 1 (P1), P7 and P28, representing a timeline of early colonization of the GI tract by enteric neurons (E14) to 1 week after weaning (P28). Localization of cannabinoid receptor 1 (CB1) expression was examined in fetal and postnatal small intestine by immunohistochemistry. Fetal intestine was processed for RNA extraction and relative expression of ECS components determined by RT-qPCR. Result(s) CB1 expression at E14 was observed in the outer wall of the gut and in the developing mucosa. By E18, expression of CB1 became more localized to the enteric plexuses and to mucosal epithelial cells. In postnatal ileum, at least &amp;gt;50% of myenteric ganglia were found to express CB1 from P1 to P28. CB1 expression continued to be present in the mucosal epithelium of ileum from P1 to P28, with a subset of CB1 expression in enterochromaffin cells, as identified by 5-HT immunolabeling. Significant decreases in expression of mRNA encoding CB1 (p&amp;lt;0.0001) and the ECS biosynthetic enzymes NAPE-PLD (p&amp;lt;0.05) and DAGLa (p&amp;lt;0.05) were found between E14 and E18. Conclusion(s) Components of the ECS are present in the fetal and early postnatal GI tract. Further research is needed to better characterize the roles and mechanisms of action of the ECS in GI development. The potential impact of exogenous cannabis exposure in early life, either by in utero exposure or via the use of medicinal cannabinoids in childhood, highlights the importance of identifying the contributions of the ECS to normal development. Disclosure of Interest None Declared

Muscle disuse as hindlimb unloading in early postnatal mice negatively impacts grip strength in adult mice: a pilot study.

Physical inactivity has many detrimental effects on health, yet the impact of physical inactivity in early life on muscle health in adulthood remains unknown. Early postnatal malnutrition has prolonged effects into adulthood and we propose that early postnatal (P) physical inactivity would have similar negative effects. To test this hypothesis, we exposed postnatal mice (∼P28, C57BL/6J) to 14 days of physical inactivity (shortly after weaning, from ∼P28 to P42 days of age) in the form of muscle disuse with hindlimb unloading (HU). After this early-life physical inactivity, they were allowed to normally ambulate until 5 mo of age (P140, adulthood) when they underwent 14 days of HU with and without 7-day recovery. They were then tested for physical function (grip strength) and muscles were extracted and weighed. Immunofluorescence was carried out on these muscle cross sections for analysis of myofiber cross-sectional area (fCSA), macrophage density (CD68+ cells), and extracellular matrix (ECM) area. Muscle weights and fCSA and myofiber diameter were used to quantify changes in muscle and fiber size. Compared with age-matched controls, no notable effects of early-life physical inactivity (HU) on skeletal muscle and myofiber size were observed. However, a significant reduction in adult grip strength was observed in those exposed to HU early in life. This was associated with reduced muscle macrophages and increased ECM area. Exposure to a short period of early life disuse has negative enduring effects into adulthood impacting grip strength, muscle macrophages, and muscle composition as low muscle quality.NEW & NOTEWORTHY We demonstrate that early life disuse resulted in less grip strength in adulthood. Analysis of muscle composition demonstrated no loss of whole muscle or myofiber size indicating lower muscle quality akin to premature aging. This poor muscle quality was characterized by altered muscle macrophages and extracellular matrix area. We demonstrate intriguing correlations between this loss of grip strength and muscle macrophages and also area of noncontractile tissue in the muscle.

Role of FGF10/FGFR2b Signaling in Homeostasis and Regeneration of Adult Lacrimal Gland and Corneal Epithelium Proliferation.

Fibroblast growth factor 10 (FGF10) is involved in eye, meibomian, and lacrimal gland (LG) development, but its function in adult eye structures remains unknown. This study aimed to characterize the role of FGF10 in homeostasis and regeneration of adult LG and corneal epithelium proliferation. Quantitative reverse transcription PCR was used for analysis of FGF10 expression in both early postnatal and adult mouse LG, and RNA sequencing was used to analyze gene expression during LG inflammation. FGF10 was injected into the LG of two mouse models of Sjögren's syndrome and healthy controls. Flow cytometry, BrdU cell proliferation assay, immunostaining, and hematoxylin and eosin staining were used to evaluate the effects of FGF10 injection on inflammation and cell proliferation in vivo. Mouse and human epithelial cell cultures were treated with FGF10 in vitro, and cell viability was assessed using WST-8 and adenosine triphosphate (ATP) quantification assays. The level of Fgf10 mRNA expression was lower in adult LG compared to early postnatal LG and was downregulated in chronic inflammation. FGF10 injection into diseased LGs significantly increased cell proliferation and decreased the number of B cells. Mouse and human corneal epithelial cell cultures treated with FGF10 showed significantly higher cell viability and greater cell proliferation. FGF10 appears to promote regeneration in damaged adult LGs. These findings have therapeutic potential for developing new treatments for dry eye disease targeting the ability of the cornea and LG to regenerate.

Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis.

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into threesubgroups based on location: (1) interneurons within 100μm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.