Developmental Neurobiology

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

Dendritic spines are the principal postsynaptic sites of excitatory transmission. For over a century, their shape has been sorted into discrete categories such as filopodia, thin, long thin, stubby, mushroom, and branched, largely by Golgi-Cox impregnation and, more recently, confocal microscopy. However, both approaches share a fundamental limitation. The histological sectioning and single-viewpoint imaging that these methods rely on cannot control the orientation of a spine relative to the observer. Because a spine is a three-dimensional object, the projection seen depends on how its parent dendrite lies within the section. Here, using the publicly available H01 petavoxel reconstruction of human temporal cortex imaged by serial-section electron microscopy (EM), we show that spine-shape classification depends strongly on viewing angle. A total of 445 spines on layer 4 basal dendrites of five pyramidal neurons were classified from an initial viewpoint (Angle 1), then reclassified after rotation in Neuroglancer (Angle 2). Only 20.9% kept their category, so chance-corrected agreement was negligible (Cohens kappa = 0.027). These observations provide direct evidence that the rigid Golgi-Cox and confocal taxonomies conflate true spine morphology with the arbitrary angle of view. Our results, therefore, support recasting spine shape as a three-dimensional continuum, measurable in petavoxel reconstructions such as H01 through free rotation in Neuroglancer. Significance statementThe classification of dendritic spines into discrete shape classes underpins a vast literature on synaptic plasticity, development, and disease. Yet it rests on two-dimensional images whose viewing angle is not controlled. By rotating the same human spines in a nanoscale EM reconstruction, this study shows that four out of five spines change category with viewpoint alone. The finding exposes a systematic bias in Golgi-Cox and confocal classifications. It argues that spine morphology should be treated as a measurable three-dimensional continuum rather than a set of fixed labels.

SNRNP70 is a core spliceosomal protein that localises to both the nucleus and cytoplasm. Previous studies have implicated SNRNP70 in regulating axonal stability and the transport of specific mRNAs during motor neuron development in zebrafish. Although the molecular functions and protein interactions of SNRNP70 in pre-mRNA splicing are well established, the mechanisms underlying its cytoplasmic functions remain poorly understood. Here, we show that SNRNP70 and TDP-43 exhibit similar localisation patterns in developing and mature neurons and co-associate in both nuclear and non-nuclear compartments, including axonal projections. We identify a functional interaction between SNRNP70 and TDP-43 that is essential for motor neuron development and demonstrate that the recruitment of SNRNP70 to cytoplasmic ribonucleoprotein (RNP) granules depends on TDP-43. These findings identify a previously unrecognised cytoplasmic function of TDP-43 in directing SNRNP70-containing RNP granule assembly, thereby linking TDP-43 to the splicing-independent functions of SNRNP70 during motor neuron development.

During development, many neurons prune their neurites. While many pruning events are activity-dependent, some neurons undergo stereotyped and developmentally regulated neurite pruning, and our understanding of the signaling pathways that mediate this form of pruning remains limited. In this study, using the PDB motor neuron in C. elegans, we show that the Wnt-calcium signaling pathway is required for stereotyped neurite pruning during development. We found that mutants of itr-1/IP3 receptor and two calcium-dependent kinases, unc-43/CaMKII and pkc-2/PKC, exhibit neurite pruning defects. Genetic analysis suggested that they function downstream of lin-44/Wnt in neurite pruning. Human CaMKIIA can induce neurite pruning in C. elegans, and mutations in CaMKII genes in patients with intellectual disabilities affect its pruning function. In vivo calcium imaging revealed that PDB neurites exhibit calcium transients during neurite pruning, which are regulated at least in part by lin-44 and itr-1. Furthermore, we demonstrate that pkc-2 regulates neurite pruning through clathrin-mediated endocytosis. Together, our work reveals the critical functions of Wnt-calcium signaling in neurite pruning.

Neuronal dendrites can be injured by a number of insults, but the cellular mechanism by which dendrites respond to tissue injury and undergo repair is poorly understood. Much of the fields progress has evaluated dendrite regeneration following laser injury. While precise, laser injury does not accurately model the real-world damage to surrounding tissue that would accompany neuronal injury. Here, we modify a pinch injury technique to injure both the dendrites and their surrounding tissues in Drosophila melanogaster larvae, more similar to what is observed in real-world injury. We refined this technique such that only half of a sensory neurons dendrites are injured, leaving the other half uninjured. Our data indicate that both dynamic and stable dendritic arbors regrow dendrites following pinch injury. Neurons primarily engage in compensatory regeneration whereby new branches are added on the uninjured half of the arbor. Comparing the regenerative response following pinch versus laser injury revealed that dendrites preferentially regrew into areas where the surrounding tissue was left intact, and not into areas where the surrounding tissue was damaged by pinch. These results prompted us to evaluate the damage sustained to surrounding tissue. In examining non-neuronal tissues after pinch injury, we found damage to epidermal cells and the ECM, but not glia. We also observed a robust immune response on the pinched half of the arbor. We conclude that the sustained damage to surrounding tissue and the initiation of an immune response create a non-permissive environment for dendrite regeneration following pinch injury. Significance StatementNeuronal dendrites are injured in clinical conditions, such as stroke, traumatic brain injury, and neonatal hypoxia. Dendrites also degenerate in the early stages of a number of neurodegenerative diseases. The role of surrounding tissues in dendrite regeneration is poorly characterized, especially considering that neuronal injury is typically accompanied by broad tissue damage. Our data evaluates dendrite regeneration following an injury that better mirrors real-world conditions and demonstrates that broad tissue damage diminishes a neurons capacity to regenerate its dendrites. Our findings show that neurons preferentially regrow into intact, undamaged tissue environments, addressing a large gap in the fields knowledge: how damage to the surrounding tissue limits neuron regeneration after injury. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/738747v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1b3f41forg.highwire.dtl.DTLVardef@160284dorg.highwire.dtl.DTLVardef@1f5f6b5org.highwire.dtl.DTLVardef@118208d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Axon collateral branching is a fundamental determinant of neuronal connectivity, enabling individual neurons to innervate multiple targets and establish functional neural circuits. Although branch initiation requires local actin remodelling within the axon shaft, the mechanisms that restrict actin assembly to prevent excessive branching remain poorly understood. Here, we identify Kaptin (Kptn) as a conserved negative regulator of axon collateral formation that limits the maturation of axonal actin patches. Using primary neuronal cultures, quantitative live-cell imaging, and zebrafish genetics, we show that loss of Kptn enhances the conversion of actin patches into filopodial protrusions, leading to excessive collateral branching. Kptn-deficient zebrafish exhibit increased motor axon arborisation, elevated neuromuscular junction density and impaired motor behaviour. Mechanistically, Kptn antagonises the actin elongation factor Formin-2 (Fmn2) to regulate actin filament barbed-end dynamics, thereby controlling the threshold for productive branch initiation. Importantly, these branching defects occur independently of Kptns established role in mTORC1 signalling, revealing a distinct physiological function during neuronal development. Our findings identify Kptn as a key molecular brake that constrains axon collateral branching and establish negative regulation of actin patch maturation as a fundamental mechanism controlling neuronal circuit assembly. This work provides a mechanistic framework for understanding how KPTN mutations associated with intellectual disability and epilepsy disrupt neuronal connectivity.

Valproic acid (VPA) is a widely used antiepileptic drug that also increases the risk of neurodevelopmental disorders in the offspring of exposed mothers. Prenatal exposure to VPA is a widely used rodent model of autism spectrum disorder (ASD). Anatomical, functional and molecular alterations in the retinas of various ASD model animals have been described in the literature, but the impact on the neural composition of the retina remains unclear. We examined whether and how the density and spatial regularity of selected retinal neurons are altered in the VPA induced model of ASD. Whole-mount retinas of 2-month-old VPA-treated and control animals were immunolabeled for S-cones, horizontal cells, AII amacrine cells, and parvalbumin-positive wide-field amacrines (PV-wfACs), and the positions of labelled cells mapped in various regions of interest (n = 39 for treated, n = 32 for control animals) across the retinas. Multivariate analysis of variance revealed a significant overall effect of VPA on cell densities (p = 6.1x10-7, 2 = 0.43), driven mainly by reduced AII amacrine density, while horizontal cells showed a modest reduction and S-cones were unaffected. After adjusting for retinal location, analysis of covariance indicated a 7% decrease in AII cells and a 15% increase in PV-wfACs. Regularity indices calculated from nearest neighbor distances or Voronoi-domain areas of cell mosaics were largely unchanged. These findings suggest that prenatal VPA exposure selectively alters inhibitory inner retinal circuitry in the rat ASD model at the time of cell differentiation, but self-organizing mechanisms responsible for spatial order are not affected. Lay SummaryValproic acid (VPA) is a medicine for epilepsy, but it can also raise the risk of autism in children when taken during pregnancy. In rats exposed to VPA before birth, we found changes in certain nerve cells of the retina: one type of cell important for night vision was reduced, while another type increased slightly, while most other cells stayed the same. This suggests that the changes in development that lead to autism may also be reflected in the structure and function of the eye.

Despite major advances in the study of cerebellar neurogenesis, cerebellar angiogenesis during embryogenesis remains poorly described. Recent advances in tissue clearing, light-sheet microscopy, and artificial intelligence have increasingly enabled detailed 3D modelling of cerebellar vasculature at early developmental stages. Here, vascular networks in mouse embryos from E11 to birth (P0) were labelled with podocalyxin, SMA, and PECAM-1 antibodies together with the nuclear marker TO-PRO-3 iodide, cleared, imaged by light-sheet microscopy, and finally modelled and quantitatively analyzed using Imaris and VesselVio. Our mapping reveals that the three main paired cerebellar arteries--the superior (SCA), anterior inferior (AICA), and posterior inferior (PICA) cerebellar arteries--emerge sequentially between E11 and E13 and display significant topographical variability comparable to that observed in humans. Morphometric analysis demonstrates distinct developmental dynamics, with SCA growth proportional to cerebellar expansion, whereas the AICA and PICA exhibit accelerated extension during later embryonic stages. Interestingly, the PICA does not reach the cerebellum before birth, highlighting the question of its contribution to embryonic cerebellar vascularization. The intrinsic vascular network evolves from a rudimentary bilayer at E11 into a highly branched architecture organized around radial penetrating vessels, giving rise to collaterals that progressively colonized the cerebellar parenchyma during foliation and lobulation. These vascular changes temporally coincided with the successive stages of cerebellar neurogenesis, supporting an interplay between vascular and neuronal development. Together, our findings provide the first spatio-temporal three-dimensional atlas of cerebellar vascularization during mouse embryogenesis, establishing a reference framework for investigating cerebellar angiogenesis in developmental and pathological conditions. HighlightsO_LIThis work is the first 3D modelling of the cerebellar vasculature in mouse embryo. C_LIO_LISCA, AICA, PICA develop through distinct spatial and temporal growth programs. C_LIO_LIPICA does not contribute to cerebellar vascularization before birth. C_LIO_LIThe intra-cerebellar vascularization evolves at E11 from a simple vessel bilayer. C_LIO_LIBetween E13 and P0, radial vessels form collaterals colonizing cerebellum. C_LIO_LIThe vascular changes temporally coincided with cerebellar neurogenesis. C_LI

The human brainstem is a complex division of the brain comprised of more than 200 nuclei and fiber tracts. The brainstem is essential for the functioning of the entire body. We introduce here the most detailed human brainstem Atlas across the human lifespan: fetus, child, adult. ANCHOR, the Atlas of Neurochemical Characterization of the Human Brainstem, is an online platform that includes more than 800 serial histological sections, stained for Nissl and seven immunochemical (IHC) markers, from the human brainstem of three ages: 25 fetal gestational weeks (GW), 9 years old, and 54 years old. This makes ANCHOR the most comprehensive human brainstem Atlas to date. In these three brainstems, we identified and manually annotated over 200 structures. We further characterized these structures with the seven IHC markers. We specifically describe the catecholaminergic groups in the human brainstem across all three age groups. In addition, we identified the protoplasmic commissural dendrites of the hypoglossal nucleus and we describe the pretectal nuclei in the Nissl-stained fetal 25 GW brainstem. ANCHOR includes an online viewer that integrates multimodal data, from magnetic resonance imaging and block face imaging to Nissl- and IHC-stained serial sections and 3D reconstruction of the entire brainstem. For the 9-year-old specimen, the online viewer allows simultaneous navigation of annotated sections with corresponding IHC, for viewing the specific region-wise cellular features accessible at https://anchor.humanbrain.in/.

A recent study (Manjarrez et al., 2026) showed that the classification of cortical dendritic spines into stubby, thin, and mushroom subtypes is unstable under rotation. That result criticizes the categorical scheme but leaves an open question. What is the actual structure of spine morphology once the viewing angle is controlled? Here we answer it. We analyzed 228 spines from layer II pyramidal neurons in the H01 nanometer-resolution reconstruction of human temporal cortex. We first quantified the source of instability. We found that rotating dendritic segments by 90 degrees about their axes shifted the apparent spine height and head width in opposite directions across the population, thereby confirming orientation-dependent measurement error. Furthermore, to obtain measurements free of this artifact, we developed the Spine Morphometry Hub (SMH), a 12-point anatomical landmark framework that characterizes each spine in all three orthogonal planes and extracts geometric, voxel-based, and mesh-based metrics. All morphometric distributions were unimodal and right-skewed. Density-based clustering assigned most spines to noise, and a Monte-Carlo test against a discrete two-type null model confirmed that this pattern is incompatible with categorical subtypes. We also confirmed that apical and basal spines were statistically indistinguishable. Unlike previous reports of a spine continuum, all based on orientation-dependent measurements, our framework removes the viewing-angle confound itself, so the continuum we observe cannot be attributed to a projection artifact. Hence, our framework will be useful to quantify dendritic-spine remodeling in neurological disorders, in which spine shape has long been observed but never measured against an orientation-invariant morphometric standard. HighlightsO_LISpine Morphometry Hub (SMH) measures spines free of viewing-angle error C_LIO_LISMH was validated as an orientation-invariant morphometry framework C_LIO_LIRotating dendrites by 90{degrees} shifts spine height and head width oppositely C_LIO_LIAll morphometric distributions are unimodal and right-skewed, not categorical C_LIO_LISMH could be used to quantify dendritic-spine remodeling in neurological disorders C_LI

Neural crest (NC) multipotent stem cells give rise to many tissues including most of the peripheral nervous system, pigment cells and the craniofacial mesenchyme and skeleton. During gastrulation and early neurulation, cranial NC cells are specified in the ectoderm territory located between the anterior neural plate ectoderm and the future pre-placodal and lateral non-neural ectoderm. At the end of neurulation, NC cells undergo an epithelial-to-mesenchymal transition and migrate to various locations in the developing embryo where they differentiate. While the fine-tuning of NC specification is increasingly being elucidated, many questions remain, including how microRNAs may govern expression of gene programs during these processes. MicroRNAs are short non-coding 20-22 nucleotides-long RNAs which regulate gene expression through post-transcriptional repression. We have identified miR-219 as a candidate regulator of Xenopus NC development. Here, miR-219-dependent molecular pathways were investigated by morpholino knock-down and reveal NC phenotypes. The development of the NC and adjacent ectoderm was evaluated using whole mount in situ hybridization of key markers (pax3, zic1, xhe2, sox10, snai2, sox2), alcian blue cartilage staining, phenotype analysis, RNA sequencing of microdissected dorsal ectoderm and microRNA rescue experiments. While neural induction is mainly unaffected, miR-219 depletion alters gene expression programs associated with neural border development, resulting in loss of NC specification. HighlightsO_LImiR-219 depletion expands the neural border territory and disrupts neural crest specification. C_LIO_LImiR-219 depletion phenotypes are rescued with miRNA mimics. C_LIO_LImiR-219 morphant neural border expansion is rescued by pax3 depletion. C_LIO_LIRNA-seq reveals specific gene program modulation in miR-219 morphant neural crest. C_LIO_LImiR-219 is predicted to directly downregulate the neural gene Hes5.3. C_LI

Structural remodeling of neuronal projections in response to developmental cues, injury, or disease is essential for adaptive circuit rewiring. This dynamic process, characterized by pruning and regrowth phases, requires the coordinated execution of neurite regrowth and branching to establish functional neuronal circuits. Yet, how these processes are regulated in space and time at the post-transcriptional level remains poorly understood. Here, we identify the conserved RNA-binding protein Imp (IGF2BP) as a central regulator of developmental axonal remodeling in Drosophila CCAP/Bursicon neurons. We show that Imp acts within a restricted time window during late metamorphosis to control both late regrowth and branching of adult CCAP/Bursicon axons. Combining functional approaches, high-resolution imaging and single-molecule mRNA detection, we further show that Imp controls these temporally distinct programs through genetically separable regulatory mechanisms. While axonal elongation is mediated by Imp-dependent stabilization of profilin mRNA, axonal branching is mediated by an independent mechanism that may involve local regulation in axons. Together, our findings demonstrate that axonal regrowth and branching, two morphogenetic events essential for neuronal circuit maturation in vivo, are controlled independently, yet coordinated through a common and conserved post-transcriptional framework. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/740905v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca291corg.highwire.dtl.DTLVardef@15108daorg.highwire.dtl.DTLVardef@11c5741org.highwire.dtl.DTLVardef@1a4773a_HPS_FORMAT_FIGEXP M_FIG C_FIG

In the zebrafish visual system, accurate retinotopic mapping occurs throughout life as new neurons are generated and integrated into existing circuitry in both the retina and optic tectum (OT). To explore how OT development changes relative to innervation from the retina, we examined cell death and proliferation in genetic and surgical models that disrupt retinal innervation of the OT. Specifically, we compared lakritz (lak) mutants, which have no optic nerves due to a lesion in the atoh7 gene, with either wild-type or one-eyed fish generated through surgical eye removal. We observed elevated cell death, fewer proliferating progenitors, and fewer sox2+ OT neuroepithelial stem cells in lak mutant and denervated OT lobes. To examine whether light-mediated vision contributes to proliferation and survival in the optic tectum, we reared fish in constant darkness and then compared survival and proliferation of OT cells in innervated and non-innervated tecta. We found that OT cells were still more likely to survive and proliferate in the presence of optic nerve innervation even when fish were reared in the dark. To identify molecular pathways that could regulate OT growth, we examined the expression of known mitogens in the zebrafish optic tectum and found evidence that Wnt/{beta}-catenin pathway activity could promote innervation-dependent proliferation in lak mutant tecta. Expression of both wnt3a and the Wnt/{beta}-catenin target gene axin2, as detected by in situ hybridization and RT-qPCR, is decreased in non-innervated tectal lobes. Further supporting an innervation-dependent role for Wnt/{beta}-catenin pathway activation in the zebrafish OT, we found that lak mutants treated with a Wnt-pathway agonist, BIO, exhibited levels of OT cell proliferation that were indistinguishable from wild-type. Together these findings suggest that progenitor cells in the optic tectum produce Wnt3a in response to innervation by the optic nerve, providing new insight into how a vertebrate visual system coordinates growth across its sensory and recipient tissues.

Transient plasma membrane domains called lipid rafts have emerged as important regulators of signal transduction. These territories are formed by lipid-lipid and lipid-protein interactions, and these local interactions can be scaffolded by resident lipid raft organizing protein family members. While roles for lipid rafts have been described for multiple signaling pathways in many contexts, their in vivo prevalence and role during embryonic development remains incompletely understood. Here we examined gene expression for the Raftlin family of lipid raft organizing proteins, Raftlin (RFTN1) and Raftlin-2 (RFTN2), over the course of early vertebrate development, with a focus on neural crest cell dynamics. By analyzing transcriptomic data across vertebrate species, we identified conserved patterns of RFTN1 and RFTN2 expression across species, where RFTN1 is broadly expressed at low levels, while RFTN2 is distinctly enriched in neural crest cells. We used fluorescent in situ hybridization to spatially define Raftlin gene expression patterns in the early avian embryo. Our results show that RFTN1 is broadly expressed with periods of enrichment in the developing paraxial mesoderm. In contrast, RFTN2 expression is strongly enriched in neural crest cells, beginning during specification and persisting through migration, with additional expression in both the cranial and intermediate mesoderm. Together, these patterns suggest that Raftlins may play important roles in regulating signaling during development with specific roles in somitogenesis and in neural crest and mesodermal cell migrations.

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

It has been previously shown that chronic social defeat stress caused by paired agonistic interactions between male mice is accompanied by the development of depression-like state and immune deficiency. The aim of this study was to investigate changes in the expression of C1qtnf superfamily genes (encoding the complement component related with tumor necrosis factor) in the hypothalamus, thymus and lungs against the background of the Lewis lung adenocarcinoma growth. In the experiments, on the 5th day of social stress, male mice were injected with tumor cells into the tail vein. Chronic social stress continued for the next two weeks. The transcriptomes of the hypothalamus, thymus and lungs of mice were sequenced at the Genoanalytica Collective Center (http://genoanalytica.ru/, Moscow). Changes in the expression of the C1qtnf genes in the tissues of stressed mice were studied compared with the control and mice that were additionally injected with tumor cells. Overall, significant correlations were found between expression of most genes in each tissue of the experimental groups. In the hypothalamus of stressed animals, when tumor cells were introduced, an increase in the expression of the genes C1qtnf1, C1qtnf2, C1qtnf3, C1qtnf6 and C1qtnf7 was observed compared to controls. In the thymus of these animals, tumor cell injection increased expression of the C1qtnf1, C1qtnf5, and C1qtnf6 genes. In the lung of tumor-injected stressed mice, expression of the C1qtnf1, C1qtnf2, C1qtnf7, and C1qtnf9 genes was decreased relative to controls and non-tumor-injected depressed mice, reaching near-zero levels in some mice. Analysis of C1qtnf superfamily gene expression in the all tissues revealed negative correlations between the expression of the C1qtnf1, C1qtnf2, and C1qtnf7 genes in the hypothalamus and lungs indicating synchronization of processes against the background of social stress and Levis lung adenocarcinoma.

Queuosine (Q) modification is a hypermodified nucleoside derived from guanine on tRNAs that enhances the decoding of codons and equilibrates translational speed. Q is biosynthesized in bacteria, and eukaryotes salvage Q and the nucleobase queuine from the diet and the gut microbiome. In animals, Q deficiency causes impaired proteostasis, mitochondrial dysfunction, and neurological phenotypes, possibly due to the longevity and high metabolic demand of neurons. Yet, how Q affects isolated neurons has not been explored yet. Here, primary rat cortical neurons were cultured in Q-free synthetic medium to directly modulate Q modification levels independently of genetic perturbation, enabling assessment of its effects on neuronal development, survival, morphology, synaptic organization, and activity. Importantly, we found that the presence of Q modification facilitated neuronal arborization, decreased inhibitory synaptic density, and increased the frequency of spontaneous calcium transients, showing that tRNA Q modification enhances neuronal structural maturation and synaptic activity. Thus, the fine-tuning of neuronal translation programs by Q-tRNAs is required for proper network development and may influence neuronal resilience and synaptic function.

Aquatic vertebrates are increasingly used in neuroscience research, yet underwater visual stimulation remains a challenge. Commonly used monochromatic stimuli have been shown to be inadequate to activate many visual neurons properly, and underwater refraction artifacts are prone to ruin stimulus designs. Here, we present MARINER - a visual stimulator, which remedies these issues and integrates concurrent behavioral and neurophysiological two-photon calcium imaging recordings. MARINERs full-field visual stimulation combined with receptive field mapping reveals that the visual field of larval zebrafish is larger than previously thought, extending almost down below the fish, and is spatially biased to better utilize motion content in naturalistic visual scenes. Using chromatic motion nulling, we further show that behavioral responses and task-associated sensory neurons are colorblind for "red" and "green" during the larvas optokinetic response. The MARINER stimulator facilitates naturalistic stimulation and faithful presentation of colored visual underwater stimuli for small aquatic species.

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival. ARTICLE SUMMARYUsing Drosophila as a model organism, we investigated the early molecular and cellular response to traumatic brain injury. Our findings substantiate the requirement of a functional glial associated AP-1 transcriptional activation response for survival. Using colocalization studies, we characterized the AP-1 glial response in six morphologically and functionally distinct glia subtypes. After TBI, we find high levels of AP-1 activation in glia of the hemolymph brain barrier, cortex glia, and ensheathing glia. We further show the importance of AP-1 transcription within the neuropil ensheathing glia subtype for optimal survival following TBI.

Basal ganglia-thalamo-cortical circuits are essential for learning complex motor sequences, yet their roles in controlling flexible motor behavior remain poorly understood. The homologous songbird Anterior Forebrain Pathway (AFP) drives song motor learning and was previously thought not to play a role in song performance, as early lesion studies reported no detectable effect. This perspective is now debated, as newer results demonstrate effects in subsets of songbirds. Here, we revisit this question in adult canaries by performing bilateral excitotoxic lesions targeting the lateral and medial subdivisions of the AFP. To quantify behavioral changes across thousands of recorded canary songs, we developed a high-throughput annotation pipeline that extends a self-supervised vision transformer (TweetyBERT) with a supervised classification head, eliminating the memory bottleneck of UMAP-based clustering. This model enables phrase-level analysis across thousands of songs per bird. We find that lesions involving the medial AFP produce a stuttering-like behavior, defined here as a prolonged and variable syllable repetition before transition, resulting in a significant increase in the variability of phrase duration. This effect was strongest in birds with medial+lateral AFP involvement, and was not observed in birds with lateral-only AFP lesions. Phrase duration variability remained elevated across much of the post-lesion recording period and was accompanied by detectable changes in syllable acoustic structure. Our results implicate the medial AFP in the ongoing control of phrase duration in adult canary song, challenging the view that the AFP is dispensable once song is learned. These findings position the medial AFP as a tractable model for understanding how basal ganglia and cortical dynamics jointly maintain complex learned motor sequences.