iScience

H2O2 released to the extracellular space (ECS) regulates diverse physiological processes, yet its concentrations and spatial distribution in tissues remain poorly defined. This uncertainty hampers mechanistic understanding of redox signaling. Here, we used reaction-diffusion modeling to estimate extracellular H2O2 concentrations and transport ranges in various scenarios. Idealized analytical models were combined with numerical models incorporating localized NADPH oxidase (NOX) clusters, ECS microstructure, membrane permeability, and the thioredoxin- and GSH-dependent clearance systems. Using maximal neutrophil and NOX superoxide/H2O2 release rates, we obtained upper bounds for extracellular H2O2. Adjacent to isolated average-sized, fully active NOX2 clusters H2O2 peaked at ~540 nM at adhesion cell-cell separations, and decreased radially over ~50-100 nm. At the receptor cell surface, peak concentration decreased inversely with intercellular separation, to <5 nM at 1 m separation. Radial decrease here, for this wide separation, was over ~2.5 m. Even the former maximal extracellular concentrations induce just a minimal, highly localized oxidation of the intracellular Prdx, Trx and GSH pools. In turn, maximally activated neutrophils carry ~2000 such NOX2 clusters, inducing 10s of M peak ECS H2O2 concentrations. These cause extensive Prdx and Trx oxidation near the exposed membranes. However, the GSH-dependent system still sustains a strong transmembrane gradient if the permeation barrier remains intact, and ECS H2O2 concentrations decay to sub-M within a few m of the source cell. Extracellular H2O2 concentrations scaled linearly with source flux in all the examined conditions. These results establish stringent constraints on autocrine, juxtacrine and next-cell paracrine H2O2 signaling.

Electrical synapses are prevalent throughout nervous systems, including the mammalian brain. Several lines of evidence implicate these gap junction connections in several important roles in neural networks. Yet, progress has been hampered by a shortage of experimental tools to investigate their function with sufficient precision. In an effort to address this deficit, we here take advantage of a curious species-difference in electrical coupling in tuberoinfundibular dopamine (TIDA) neurons of the rodent hypothalamus. In rats, these cells exhibit strong electrical coupling, generating stereotyped, slow oscillations that are synchronized across the population. In contrast, mouse TIDA neurons lack gap junctions and display diverse, faster, and asynchronous oscillations. Motivated by this natural discrepancy, we designed a gain-of-function strategy to induce localized and robust expression of the main pore-forming protein of neuronal gap junctions, connexin-36 (Cx36), in mouse TIDA neurons by viral vectors. This perturbation yielded electrical coupling that was weaker than the rat TIDA system, but on par with many other brain populations connected by gap junctions. Cx36 overexpression resulted in significantly increased auto- and cross-correlation, as well as augmented functional connectivity, in agreement with the induction of electrical synapses in the population. Yet, these measures were modest in comparison with rat TIDA neurons, and Cx36 overexpression failed to alter serum levels of the hormone prolactin, which is controlled by this neuroendocrine system. These findings highlight the potential and limitations of experimentally inducing electrical synapses in a natively uncoupled system, and suggest directions for future improvements.

Understanding how the brain integrates internal bodily signals to shape behavior is essential for characterizing the neural basis of interoception. Here, we investigate whether the nucleus of the solitary tract (NTS), a brainstem hub for viscerosensory input, communicates with the locus coeruleus (LC), a noradrenergic nucleus involved in arousal and cognition, to influence anxiety-like behavior. We utilized viral-induced optogenetics combined with electrocardiography and diaphragmatic electromyography to monitor heart rate and respiratory frequency. Prior to behavioral testing we photoactivated transduced NTS neurons either at their somata, or at their axonal terminals in the LC. Anxiety-related behaviors were assessed using the elevated plus maze (EPM) and novelty-suppressed feeding test (NSFT). Stimulation of the NTS somata, or their terminals in the LC, increased aspects of anxiety-like behaviors in the EPM and NSFT, including freezing time during the EPM and latency to eat in the center of the NSFT. Importantly, only NTS somal activation suppressed post-NSFT feeding. This suggests that the anxiety-like behaviors observed from activating NTS projections to the LC are not secondary to activating feeding circuits. Additionally, activation of NTS terminals in the LC altered heart rate and its variability following food deprivation. These findings reveal that the NTS to LC circuit enhances anxiety-like behavior and modulates cardiorespiratory dynamics state dependently, thereby identifying a likely brainstem pathway through which viscerosensory signals influence interoception.

Simulated microgravity provides a controlled perturbation for testing how loss of mechanical load affects human tissues. After 24 hours in rotating-wall vessels, neural, cardiac and tonsil organoids developed tissue-specific transcriptional responses that overlapped published spaceflight datasets, accompanied by higher transcriptomic-age estimates in neural and cardiac models, altered cardiac-organoid contraction dynamics and modified tonsil-organoid antiviral gene-expression signatures. These findings establish a multi-organoid framework for studying acute responses to gravitational unloading.

Sex-specific differences in COVID-19 outcomes are well documented, with male sex being a key risk factor for severe disease. Multiple factors, including differences in the expression of Interferons (IFNs) and Interferon stimulated genes (ISGs) between the sexes have been identified as contributing to these differences. Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR enzymes, is an important component of antiviral innate immune signaling. While previous studies, including us, have identified altered expression and activity of ADARs in response to SARS-CoV-2 infection, it remains unknown whether sex-specific differences in expression and activity of ADARs exist during infection with SARS-CoV-2. We address this question by analyzing a publicly available whole blood RNA sequencing dataset from males and females, matched for age and comorbidity status across pre-, mid-, and post-infection stages of SARS-CoV-2 infection. Our results show distinct patterns of ADAR expression between the sexes across different stages of infection, with significant differences in the expression of ADAR and ADAR2 in pre- and mid- infection that were not observed post-viral clearance. Interestingly, expression of predominantly neural isoform ADAR3 was significantly different and higher in females, mid-infection. Furthermore, these differences in ADAR expression were likely amplified downstream with significant differences in the global editing levels. Correlation analysis revealed that the relationship between ADAR expression and global editing activity differed by sex and stage of infection, indicating that ADAR editing is dynamically regulated and is influenced by factors other than ADAR transcript abundance. Noteworthy, sex-specific differences in ADAR editing were observed within genes that escape X-chromosome inactivation (XCI), a gene set enriched in immune related functions and known to be involved in sex-specific outcomes to viral infections. ADAR editing within these genes differed at baseline and changed across stages of infection in a manner independent of gene expression. Altogether, our findings establish ADAR editing as an additional layer of immune regulation that may be contributing, in part, to the differential clinical manifestations of COVID-19 observed between males and females.

Obesity is a major health concern worldwide and is caused by an excessive fat accumulation in the body due to adipogenesis. Recent studies suggest that excessive stress or disruption of circadian rhythms can enhance adipogenic commitment and lead to obesity. Moreover, this adipogenic commitment occurs at a specific time of day, highlighting a correlation with the circadian rhythm. However, the dynamical nature of this kind of stress-induced adipogenic differentiation remains an unresolved puzzle. Herein, we proposed a mathematical model centred on the key transcription factor PPAR{gamma}, associated with adipogenic differentiation, and demonstrated that adipogenic commitment is a bistable, irreversible differentiation process in which slow positive feedback regulation of PPAR{gamma} primarily controls the differentiation commitment threshold. The model reproduced the experimental observations regarding the extent of adipogenesis induced by constant and pulsatile stress input signals and explained the correlation between adipogenic differentiation time and circadian phase. It further predicts how to control the adipogenic commitment even under stressful conditions. Overall, our study elucidates how stress signalling dynamically governs adipogenesis.

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.

Inter-organelle contact between mitochondria and lipid droplets (LDs) is crucial for hepatic lipid homeostasis, but the tethering complex underlying this interaction remains unclear. To address this, we aimed to characterize the molecular machinery governing mitochondria-LD coupling and evaluate the evolutionary conservation of its metabolic functions. Using hepatocyte models from multiple species and in vivo experiments in yellow catfish with differential lipid and creatine diets, we explored the mechanism and evolutionary conservation of mitochondria-LD coupling. The key findings were: (1) A novel complex formed by mitofusin 2 on mitochondria and hormone-sensitive lipase on lipid droplets via specific residues orchestrates efficient hepatic lipid catabolism by mediating mitochondria-LD coupling; (2) Creatine enhances this coupling by transcriptionally upregulating mfn2 via hnf4, thereby promoting peridroplet mitochondria formation and alleviating hepatic lipid accumulation; (3) The Mfn2-phosphorylated HSL axis is evolutionarily conserved across fish and mammals, representing a potential therapeutic target for hepatic steatosis.

Tardigrades, key ecdysozoan organisms, have poorly characterized daily behavioral rhythms linked to their molting (ecdysial) cycle. We continuously tracked the locomotor activity of the model tardigrade Hypsibius exemplaris using infrared videography and deep learning-based automated tracking under three photic regimes: a light/dark cycle, constant darkness following entrainment, and constant darkness without prior entrainment. We identified a robust endogenous circadian-like rhythm in locomotor activity that persisted in constant darkness, independent of photic input. Although the light/dark cycle enhanced the rhythm amplitude and the overall animal activity, it was not required for rhythmicity. Individual phases varied widely, indicating a lack of population-level synchronization. Furthermore, we developed a behavioral classification system based on activity level, which enables categorizing distinct behaviors. Behavioral patterns were modulated by distinct ecdysial stages, with quiescence gating premolt and time-of-day dependent patterns in feeding and wandering. These findings provide evidence consistent with an endogenous, circadian-like timing system in tardigrades that integrates daily activity with molting cycles and establish H. exemplaris as a promising model for studying ecdysozoan chronobiology.

Sensorimotor synchronization, the ability to align motor behaviour with the temporal structure of external stimuli, is thought to emerge gradually during development, but its origins remain unclear. Here, we investigated whether newborns can adapt their sucking behaviour and synchronise it to the temporal structure of auditory stimuli within the first days of life. Forty-one healthy full-term newborns (24-72 hours old) were exposed to two three-minute auditory streams differing in temporal structure: a highly structured stream with a stable temporal pattern and a highly variant stream with greater temporal variability, while their non-nutritive sucking (NNS) was continuously recorded. We assessed changes in NNS frequency and pressure and quantified NNS-sound coupling using magnitude-squared coherence at frequencies distinctive to each auditory stream. Structured stimulation promoted a more organised NNS, with more suck bursts than the variant stream, and increased sucking vigour, with a transiently higher suck peak rate during the first minute relative to the variant stream and greater suck pressure relative to silence. Coherence analyses revealed that coherence was selectively higher at the frequencies distinctive to each auditory stream, both for the structured and the highly variant stimulus. These findings show that, within hours of birth, NNS behaviour can selectively align with stimulus-specific temporal regularities, even when these are weakly predictable and lack a dominant periodicity. This early capacity to extract temporal structure and coordinate motor behaviour may provide a foundation for the subsequent development of sensorimotor synchronisation.

A central question in developmental biology is how spatiotemporal embryo morphological events and cell differentiation are precisely coordinated to eventually develop into a mature organism. Previous research has shown cell positions and asymmetric division stages are related to cell lineage specification since morula stage. However, the detailed spatiotemporal dynamics and histories of embryonic cells and their relations with cell lineage specification remain incompletely understood from the onset of embryo development onward. My study on in-vitro embryos showed that continuous live cell tracking mapped community-like patterns in cell origin and differentiation. Cell positions, cell temporal factors (such as cell division order and cell cycle) and their histories intricately interacted over pre- and peri-implantation stages. These interactions of spatiotemporal cellular activities directed embryo morphological events and asymmetric cell lineage origin and differentiation throughout 2-cell to around 100-cell stage. In conclusion, this study provides comprehensive insights into the cellular spatiotemporal dynamics of cell inheritance and differentiation during pre- and peri-implantation morphological events and cell lineage specification. By longitudinally integrating spatial and temporal parameters relating to early embryogenesis, the findings not only bridge divergent explanations stemmed from different studies in the field but also offers a refined foundation for evaluating embryo potential and improving outcomes in assisted reproductive technologies, stem cell research, and regenerative medicine.

Intrinsically light sensitive retinal ganglion cells through melanopsin photoreception (mRGCs) are important for fundamental regulatory brain processes such as circadian rhythms, mood, acute sleep, and pupillary constriction. While previous research in animal models indicates temporal differences in maturation of mRGC mediated projections, corresponding developmental patterns have not been studied in human infants. To elucidate the roles of mRGCs in early development of regulation of pupillary constriction and sleep/wake patterns, we here explore the maturation of mRGC mediated photoentrainment in human infants aged 1 to 12 months, using non-invasive eye tracking pupillometry and sleep diaries. Although the mRGCs relay rod/cone activation in a range of light intensities, it is possible to separate rod/cone/mRGC activation due to the photoreceptors' color sensitivity and temporal responsiveness. We therefore used sustained red and blue light stimulation after partial dark adaptation to characterize developmental changes in wavelength-dependent mRGC-relayed pupillary responses. Infants' pupil responses indicate a prolonged post-natal mRGC maturation to the shell of the olivary pretectal nucleus during the first year. This response was separate from infants' sleep/wake patterns, which parallels findings from cellular research on murines: the mRGCs have separate pathways for photoentrainment of pupillary constriction and sleep/wake patterns, which develop with different temporal dynamics. Together, these findings provide the first characterization of developmental changes in mRGC-relayed pupillary responses in human infants, and an initial step towards bridging human mRGC network development with murine and human adult mRGC research.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.

End-stage kidney disease (ESKD) is a major driver of cardiovascular disease (CVD) and requires dialysis treatment leading to a unique metabolic phenotype with cyclic transitions between accumulation and clearance of uremic toxins. Although ESKD patients display the highest cardiovascular mortality among CKD patients, the effect of cyclic uremia on cardiovascular disease remains subject to discussion. Existing mouse models rely on induction of kidney injury and do not resolve the interconnected pathophysiological effects of metabolic, hypertensive and endocrine renal failure. They are therefore unable to distinguish uremia-driven effects from well-established drivers of cardiovascular disease such as hypertension. Here, we established a simple, reproducible, and sex-inclusive mouse model of uremia in absence of kidney injury or hypertension leveraging a bistable vesico-peritoneal shunt (VPS). Strikingly, we identify cyclic uremia as an independent driver of ESKD-induced CVD, that induces heart failure with preserved ejection fraction (HFpEF), vascular inflammation and immunosenescence. As such, the VPS represents the first preclinical model that enables investigation of cyclic uremia as a driver of cardiovascular disease.

Neurodegenerative disorders manifest with markedly different phenotypes, with motor- and memory impairments dominating both clinical and preclinical phenotyping efforts. However, neurodegeneration in unlikely to be confined to these two functional domains alone, and its broader behavioral consequences, including social alterations remain underexplored. Here, we present a non-invasive, high-content home-cage phenotyping framework to investigate social and exploratory dysfunction in SPTAN1 R1098Q mice, a model of early infantile epileptic encephalopathy (EIEE5). Our pipeline integrates three open-source AI engines: SLEAP.ai for markerless multi-animal pose estimation, DeepOF for automated classification of autonomous and social behaviors, and SAM2 for segmentation and quantification of nest-building material usage. Long-term monitoring revealed that SPTAN1 R1098Q mutants exhibit impaired spatial habituation in mutant-mutant (MUT - MUT) pairs. Mutants also displayed persistent, unreciprocated trailing (following) of cage mates and a pronounced reduction of nest-building behavior alongside. Notably, co-housing with a wild-type littermate (MUT - WT) attenuated mutant hyperactivity and restored more typical rest pattern, suggesting that social context can partially buffer these behavioral abnormalities. By revealing social and exploratory alterations that expand conventional phenotyping, this computational ethology pipeline advances 3R principles while revealing that intrinsic executive state-switching failures in neurodegenerative models can be functionally buffered by social dynamics.

Microglia are essential immune cells of the brain parenchyma which are responsible for maintaining homeostasis and responding to brain injury. When activated, they induce immune signaling and become phagocytic. In the context of neurodegenerative diseases, they exhibit both pro- and anti-inflammatory properties. Human endogenous retroviruses (HERVs) are the remnants of ancient retroviral germline infections that integrated into the human genome, and their expression has been directly linked to microglial-mediated neurodegenerative diseases. Despite this, HERV expression is not consistently included in analyses characterizing microglia. Investigating the function of microglia across different diseases is critical for elucidating mechanisms of disease progression and for the development of effective therapeutic interventions. Approaches to studying microglia include isolation from post-mortem brain tissues, differentiation from peripheral blood monocytes or differentiation of induced pluripotent stem cells (iPSC). Ex vivo brain derived microglia (BdMG) are considered most physiologically relevant, but constraints including the unpredictable timing of availability, labor-intensive protocols, and low yields reduce their practicality. Existing alternatives are iPSC-derived microglia (iMG) and monocyte-derived microglia (MDMi) models that circumvent sample accessibility and scalability issues. Here, we explored the transcriptome and proteome of these three microglia sources and identified a common microglia surface marker expression profile, establishing that MDMi and iMG exhibit similar phenotypes relative to BdMG. We also identified a common HERV expression profile across BdMG, iMG and MDMi, indicating deeper similarities across different sources. Altogether, our findings indicate that given the phenotypic similarities of the 2 models, MDMi represent a practical and reliable model for investigating microglial function with iMG models serving for more molecular based approaches, such as CRISPRa/i.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

Hematopoiesis sustains lifelong blood production and immunity and is strongly influenced by biological sex. Males and females differ in their hematopoietic composition and susceptibility to infection and hematologic disease. Because current models cannot readily disentangle the independent contributions of sex chromosomes versus hormones, the mechanisms underlying sexual dimorphism remain poorly understood. Zebrafish have proven to be a powerful, translatable system for studying hematopoiesis, and offer a unique opportunity to isolate hormonal contributions due to their absence of sex chromosomes. However, whether sex is regulated in the zebrafish hematopoietic system remained unknown. Here, we discovered pronounced sexual dimorphism in the adult zebrafish marrow, with compositional differences peaking at reproductive age. Transcriptome analysis of marrows revealed conservation of sex-specific programs between zebrafish and human datasets. Upon immune challenge with lipopolysaccharide, all major blood lineages exhibited sex-biased changes in their composition and transcriptomes with the largest responses in erythroid and myeloid cell types. Among these sex differences was an unexpected immunoregulation of the erythroid lineage across maturation states. These findings establish biological sex as a major regulator of zebrafish hematopoiesis and immunity, paralleling humans, and lay the groundwork necessary for future mechanistic studies on sexual dimorphism in hematologic health and disease.

Aims: Seizure generation has been strongly linked to the accumulation of extracellular potassium. As astrocytes play a key role in potassium homeostasis, astrocyte targeted optogenetic modulation of extracellular potassium has been proposed as a novel strategy for seizure control. The effectiveness of such modulation is expected to depend strongly on the ion selectivity of the employed opsin. This study aims to mechanistically compare potassium-selective (WiChR) and cation-conducting (ChR2-like, ChR2*) opsins for astrocyte targeted modulation of extracellular potassium, and to determine how astrocytic properties and stimulation parameters influence modulation efficacy. Methods: A conductance-based computational framework was used to model astrocyte-extracellular space interactions, incorporating ion-specific photocurrents, Na+/K+-ATPase activity, and gap junction-mediated coupling within the astrocytic syncytium. Systematic parameter sweeps were performed to evaluate the influence of ion selectivity, astrocytic parameters, and stimulation paradigms on potassium dynamics. Results: WiChR-based modulation consistently produced more effective and robust reductions in [K+]e than ChR2*, via direct K+ inward currents and reduced dependence on pump activity. In contrast, ChR2* primarily limited potassium accumulation rather than reversing it and exhibited greater sensitivity to parameter variations, particularly Na+/K+-ATPase activity. The effectiveness of astrocyte-targeted modulation was strongly dependent on syncytial integrity, with reduced gap junction coupling impairing potassium clearance. Optimal stimulation performance for WiChR was observed for a pulse repetition period of 20 ms, with outcomes determined by the interaction between opsin kinetics and stimulation parameters. Conclusions: These results demonstrate that activation of WiChR opsins in astrocytes can reduce extracellular potassium levels and thereby modulate neuronal excitability. Ion selectivity emerges as a key determinant of modulation efficacy at the astrocytic level, with potassium-selective opsins providing a more direct and reliable approach than non-selective cation-conducting alternatives. Together, these findings establish a mechanistic basis for optogenetic activation of astrocytic potassium buffering as a potential strategy for seizure suppression and other neurological disorders associated with potassium dysregulation.

Hypothalamic supramammillary nucleus (SuM) neurons project to the dentate gyrus and co-release glutamate and GABA, thereby regulating hippocampal information processing. However, how synapses mediating glutamate/GABA co-transmission are established during development remains unclear. Here, we investigated the functional and structural maturation of SuM inputs onto adult-born granule cells (GCs). We found that most newborn GCs received GABAergic responses without detectable glutamatergic responses, whereas a smaller population exhibited glutamate/GABA co-transmission. Newborn GCs with glutamate/GABA co-transmission showed more mature anatomical and physiological properties than GABA-only neurons, with greater dendritic complexity, longer dendritic length, lower input resistance, and stronger medial perforant path inputs. Postsynaptic depolarization failed to induce synapse unsilencing in GABA-only neurons but induced robust long-term potentiation in newborn GCs with glutamatergic transmission, indicating that plasticity at SuM inputs emerges after functional glutamatergic transmission is established. Morphological analysis showed that SuM terminals apposed to newborn GCs contained both VGluT2 and VIAAT, whereas postsynaptic receptor organization remained immature. Moreover, environmental enrichment and chemogenetic activation of SuM neurons increased the proportion of newborn GCs receiving glutamate/GABA co-transmission, suggesting that SuM activity promotes the emergence of glutamatergic transmission. Together, these results suggest that SuM inputs onto newborn GCs undergo sequential maturation from initial GABAergic transmission to glutamate/GABA co-transmission, a process likely supported by SuM activity-dependent postsynaptic maturation.