Developmental Neurobiology

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.

The FoxP family of transcription factors has been linked to neurodevelopmental disorders. FoxP1 and FoxP2 were identified as genes associated with autism spectrum disorders (ASD). FoxP1, FoxP2, and FoxP4 are expressed in many brain regions, including the cerebellum that has been of particular interest in the context of ASD. However, their role in the development of the peripheral nervous system (PNS) has been neglected. In view of the fact that a majority of ASD patients show some signs of sensory perception deficits that could go beyond the central nervous system, we focused on neural circuit formation in the PNS. Our in vivo and in vitro studies revealed a role of all three FoxP transcription factors in neural circuit formation in the hindlimb of the chicken embryo. Silencing of FoxP genes altered motor axon guidance and also impacted neural crest cell migration. This in turn resulted in aberrant formation of dorsal root ganglia and deficits in neural circuits in the PNS. Taken together, our results are in line with the hypothesis that some of the sensory perception deficits in ASD patients, either hyper- or hyposensitivity, are due to aberrant neural circuits in the PNS.

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.

Following nervous system injury, surviving bystander neurons may rewire and form synapses with new postsynaptic targets to restore circuit function. This protocol quantifies compensatory motor neuron sprouting at the Drosophila larval neuromuscular junction. We describe larval dissection, immunostaining, Airyscan imaging, image masking, muscle-specific region-of-interest selection, and Fiji-based quantification of Dlg-positive area, bouton number, mean Dlg-positive area per bouton, NMJ arbor length, axonal versus NMJ branch points, ectopic muscle acquisition, and sprouting penetrance. For complete details on the use and execution of this protocol, please refer to Olsen et al.1

Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation.

Brain microRNA (miRNA) expression can shift in response to the social environment, translating external cues into regulatory action that can influence brain structure, neural activity, and behavior. miRNAs are key drivers of behavioral flexibility, regulating gene networks involved in neuroplasticity. We manipulated social rearing conditions of brilliant-thighed poison frog tadpoles (Allobates femoralis) to test whether isolation-linked behavioral changes are associated with altered miRNA expression in the midbrain and eyes, tissues linked by the retinotectal tract, a known connection for trafficked precursor miRNAs. We asked whether isolation alters visually guided behavior using a light/dark preference assay and found that isolated tadpoles spent less of the trial on the dark side than did group-housed tadpoles. In a social place preference assay, group-housed tadpoles preferred a zone near a conspecific over an object control when provided with multimodal sensory access, but not when access was restricted to vision alone. We next tested whether social rearing conditions influenced miRNA expression and found that isolated tadpoles exhibited upregulation of miR-15-P1c_3p*, the canonically non-dominant arm of an understudied miRNA, in the midbrain and eyes. We detected a shift in arm dosing for miR-124, a miRNA with conserved roles in neural differentiation and plasticity. Finally, social isolation during development was associated with downregulation of parathyroid hormone 2 (pth2), a peptide associated with social isolation and mechanosensation in other aquatic larvae. This work contributes to a growing body of literature implicating miRNAs in developmental neuroplasticity, presenting precedented and novel signatures of the transcriptional and regulatory response to isolation.

Amacrine cells (ACs) are the most diverse neuron in the retina, with more than sixty different subtypes, each contributing to the detection of specific visual features. Despite their importance in visual feature detection, the genetic basis for this diversity is still not well understood. Recent single-cell transcriptomic datasets have identified several potential genes that may regulate cellular diversity, but the underlying mechanisms have not been investigated. For example, the transcription factor Meis2 is selectively expressed in the GABAergic subclass of ACs. In this study, we focused on how Meis2 regulates the development of GABAergic ACs. We hypothesized that Meis2 is required for specification and survival of GABAergic ACs. To test this, we used the Meis2 conditional knockout mouse (Meis2-CKO) where Meis2 was deleted from either the entire retina or specifically from AC precursor cells. The deletion of Meis2 led to a ~60% reduction in total GABAergic ACs in the retina. This loss of GABAergic ACs occurred only after postnatal day 5 (P5), suggesting Meis2 is not required for early specification but may be required for survival. To further investigate cell death and survival, we quantified cleaved caspase-3 positive cells in the retina at several developmental timepoints. In Meis2-CKO mice, we found an increase in cleaved caspase-3 immunolabeling at P5 and P6 timepoints coinciding with the reduction of GABAergic ACs. Further analysis of GABAergic AC subtypes demonstrated a subtype-specific cell death in Meis2-CKO retinas. For example, the total number of dopaminergic (TH) ACs were significantly reduced, whereas starburst (ChAT) ACs remained unchanged. These findings suggest that Meis2 is important for the survival of a subset of GABAergic AC subtypes in the mammalian retina. In addition, Meis2 may control the expression of specific survival factors that prevent apoptosis during developmental cell death in the retina.

REV-ERB{beta} is a transcriptional repressor of nuclear receptors that regulates the circadian rhythm and plays an important role in regulation of the proliferation, differentiation, and maturation of neurons. Dysregulation of the circadian rhythm has been associated with neuropsychiatric disorders, and activation of REV-ERBs can induce anxiolytic behavior in mice. Furthermore, hippocampal neurogenesis is important in the effects of antidepressants. However, the role of REV-ERB{beta} in adult hippocampal neurogenesis in vivo at the single-cell level is not known. In this study, protein localization of REV-ERB{beta} in the subgranular zone of the hippocampal dentate gyrus (DG) was mainly shown in NeuN-positive neurons, and the effect of expressing a dominant negative form of REV-ERB{beta} lacking the C-terminal region on newborn neurons in the hippocampal DG of adult mice was examined to investigate the role of REV-ERB{beta} in neurogenesis. A retroviral vector containing the dominant negative REV-ERB{beta} or a control vector was injected into the mouse DG. At 4 weeks after injection, the morphology of dendrites and dendritic spines of newborn neurons labeled by the virus was examined. Expression of the dominant negative form of REV-ERB{beta} inhibited dendrite outgrowth and branching and decreased dendritic spine formation in newborn neurons in the adult mouse hippocampal DG. This study revealed a new role for REV-ERB&{beta} in adult hippocampal neurogenesis at the single cell level, and the results will provide insight into neurogenesis in the adult brain and its relationship with psychiatric disorders.

Abstract The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) controls behaviours that are essential for the parasitic lifestyle of plant parasitic nematodes (PPNs). Here, we used serotonin immunochemistry and in situ hybridization for the serotonin synthesis genes, Gpa-tph-1 and Gpa-bas-1, to map its signalling pathways in Globodera pallida J2 infective stages. We identified fourteen serotonin-immunoreactive cell bodies around the pharyngeal median bulb and revealed that eleven of them co-express Gpa-tph-1 and Gpa-bas-1, confirming these are serotonin-synthesising neurons. Serotonin immunoreactivity was also localised in structures consistent with labelling of amphids, and in sixteen non-neuronal cells located in the middle and posterior region of the nematode. Depletion of serotonin with reserpine reduced the serotonin immunoreactivity in all fourteen cell bodies confirming their neuronal identity, but not in non-neuronal tissue. We identified additional, but infrequent expression of Gpa-tph-1 in two cells and expression of Gpa-bas-1 in one pair of symmetrically bilateral cells anterior to the median bulb, that lacked serotonin immunoreactivity. The results reveal distinct pools of serotonin distribution in G. pallida and indicate serotonin signalling is more complex than in other parasitic nematodes and C. elegans, highlighting evolutionary divergence in neurotransmitter identity.

The adult size of the vertebrate telencephalon typically impedes our interpreting its functions within the context of brain-wide circuit dynamics. Danionella, a genus of miniature and transparent teleost fishes closely related to zebrafish (Danio rerio), holds promise for whole-brain imaging and connectomics in an adult vertebrate. These advantages result from progenesis, a form of heterochrony characterized by early developmental onset of sexual maturation and cessation of growth leading to the retention of larval or juvenile characters in adults. Using molecular markers, tract tracing, and quantitative comparisons of cellular distribution patterns, we report a detailed neuroanatomical atlas of the adult Danionella dracula (Dracula fish) telencephalon. The results demonstrate that adults of Dracula fish and other Danionella species show extreme reduction in cellular migration from periventricular zones and topographic reorganization of major pallial divisions, including a compartment-like caudal pallium reminiscent of insect glomeruli (a dense neuropil surrounded by a thin layer of somata). We further document these characters in larval zebrafish, providing essential evidence corroborating a larval-like telencephalon in Danionella adults. Despite this phenotype, most canonical regions of the adult teleost telencephalon are identified. We also generate a transgenic GCaMP Dracula fish line, an annotated 3-dimensional atlas, and align functional imaging data to demonstrate the usefulness of this atlas as a resource for deciphering how the novel progenetic cytoarchitecture of the adult Danionella telencephalon can support its rich behavioral repertoire.

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

Background: Late preterm (LP) children (34 0/7 - 36 6/7 weeks of gestation) are at risk for neurocognitive challenges, yet the neuroanatomic and functional disruptions underlying these developmental differences remain poorly understood. Purpose or Hypothesis: Investigate (1) regional differences in cortical surface area and thalamic nuclei volume and (2) structural variations that may explain heterogeneity in neurocognitive functioning in LP versus full term (FT) school-aged children, using two complementary approaches: a vertex-wise analysis of cortical surface area and a region-of-interest volumetric analysis of thalamic nuclei. Study Type: Secondary analysis of a prospective observational cohort study. Population or Subjects: 48 LP and 72 FT children aged 6-13 years. Field Strength/Sequence: 3T; Sagittal T1-weighted MP-RAGE and axial T2-weighted 3D SPACE. Assessment: FreeSurfer 7.4.1 was used to parcellate cortical regions (Destrieux atlas) and segment thalamic nuclei (25 nuclei). Neurocognitive functioning was assessed across four domains: hyperactivity/inattention (Pediatric Behavior Scale), processing speed (WISC-IV), working memory (WISC-III), and visual-spatial perception (Benton JLO). Statistical Tests: Independent samples t-tests for group comparisons; vertex-wise GLMs for cortical surface analyses with false discovery rate (FDR) correction; and multiple linear regression for thalamic nuclei analyses with FDR-corrected q < 0.05. Results: Vertex-wise analyses revealed regional differences in cortical surface area between LP and FT children in both hemispheres, along with associations between surface area in several cortical regions and hyperactivity/inattention, working memory, and visual-spatial perception. In contrast, using an ROI approach, thalamic nuclei volumes did not differ between groups and were not associated with any neurocognitive domains. Conclusion: LP birth may be associated with subtle, region-specific bidirectional left and right hemispheric cortical surface area differences rather than widespread structural alterations in middle childhood, with regional macrostructural measures associated with individual differences in neurocognitive functioning.

SNRNP70 is a core spliceosome RNA-binding protein best known for its essential role in nuclear pre-mRNA splicing. Although traditionally associated with nuclear RNA processing, previous studies have identified important extranuclear functions for SNRNP70 in neurons, including roles in mRNA stability, localisation, and axonal transport. Yet, much of our understanding of SNRNP70 localisation has relied on overexpression or transgenic approaches, leaving a critical gap in our knowledge of where endogenous SNRNP70 resides and how it behaves in living neurons under physiological expression conditions. Here, we address this limitation by establishing and validating a novel zebrafish SNRNP70-eGFP CRISPR knock-in line, enabling direct visualisation of the endogenous protein. eGFP was fused to the C-terminus of endogenous SNRNP70 while retaining the native 3' untranslated region, preserving key regulatory features of the endogenous locus. We demonstrate that the knock-in faithfully reports endogenous SNRNP70 expression and reveals widespread physiological localisation throughout the developing nervous system, including prominent enrichment within axonal and synaptic compartments. Crucially, live in vivo imaging reveals that endogenous SNRNP70 is dynamically localised within neuronal mRNP granules, providing direct evidence of its physiological behaviour in these structures without the confounding effects of protein overexpression. Proximity ligation analyses further demonstrates associations between endogenous SNRNP70 and PABPC1B, FUS, and UPF1, which are established neuronal mRNP granule components. Together, our work provides a validated genetic and imaging resource for investigating SNRNP70 at endogenous levels in the living nervous system. By overcoming key limitations of conventional transgenic and overexpression-based approaches, the SNRNP70-eGFP knock-in enables physiological analysis of SNRNP70 localisation and dynamics and reveals its prominent and dynamic organisation within neuronal mRNP granules. More broadly, this work highlights how endogenous fluorescent tagging can provide a versatile platform for resolving the spatial organisation of RNA-binding proteins in living neurons under physiological expression conditions and provide new insight into the regulation of neuronal mRNA fate.

For more than a century we have pictured the dendritic spine as the postsynaptic point where a single presynaptic bouton delivers its message. That picture is correct but incomplete, since it overlooks other neuronal elements surrounding each spine. Here we characterize that neighborhood for the first time by counting and measuring the proximity of the elements encircling each postsynaptic spine. We call this neighborhood the vicinome: the full set of neuronal and glial elements surrounding a dendritic spine. We used ground-truth three-dimensional meshes from the H01 petavoxel dataset, a reconstruction of the human cerebral cortex. We analyzed 4550 neighboring elements across 322 spines from 17 pyramidal neurons in cortical layers 2 through 5, with each spine classified as apical or basal. Neighbor number followed a depth gradient, falling from 17.2 {+/-} 4.2 elements per spine in layer 2 apical spines to 12.5 {+/-} 3.1 in layer 5 basal spines. A linear mixed model with a random intercept per neuron confirmed a strong effect of layer (p = 4.3 x 10-5) with large effect sizes (Cohen's d up to 1.45) and no effect of polarity. The minimum spine-to-neighbor distance stayed uniform, near 30 nm in every group, and although some depth-related contrasts were significant, all effect sizes were negligible (Cohen's d below 0.21). We conclude that cortical depth, and not pyramidal neuron polarity, was related to the number of neuronal elements neighboring the spine. We suggest that vicinome crowding could offer a measurable observable for comparison across regions, ages, species, and disease.

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.

Aim: ROR{gamma}t-transgenic mice provide a model of sustained T helper 17-cell bias, but the associated hippocampal transcriptional profile is incompletely characterized. We investigated gene-level and gene-set expression differences in this model. Methods: Bilateral hippocampal samples from 10-week-old male ROR{gamma}t-transgenic mice and wild-type littermates (n = 3 per genotype) underwent bulk RNA sequencing. Gene-level differential expression was evaluated using the Empirical Analysis of DGE tool in CLC Genomics Workbench. Preranked gene set enrichment analysis examined coordinated expression differences after expression filtering and exclusion of Rorc (13,813 genes). Results: Sixteen genes met a false discovery rate < 0.05 (nine higher and seven lower in transgenic mice), including Rorc, C4b, Xbp1, Nptx2 and Mt2. Of 212 candidates selected using unadjusted P < 0.05 and a twofold-change threshold, 155 (73.1%) had a mean expression below 0.5 reads per kilobase of transcript per million mapped reads. Hallmark interferon--response, interferon-{gamma}-response, and unfolded-protein-response gene sets showed positive enrichment. Thirteen Gene Ontology biological-process sets met a false discovery rate < 0.05, including negatively enriched sets related to excitatory postsynaptic potential, glutamatergic transmission, and calcineurin-mediated signaling. Il17a and Il17f had zero reported counts, and none of the displayed leukocyte or central nervous system cell-marker genes met false discovery rate < 0.05. Conclusion: This exploratory dataset identifies interferon-response, unfolded-protein-response and synaptic transcriptional signatures associated with the ROR{gamma}t-transgenic genotype. The cellular sources, causal mediators and functional consequences remain unresolved.

Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) exhibit largely complementary distributions and have been proposed as molecular markers of descending modulatory and ascending driver-like pathways, respectively. However, this correspondence has rarely been tested directly in anatomically identified projections. We combined anterograde Phaseolus vulgaris leucoagglutinin tracing with simultaneous VGluT1 and VGluT2 immunofluorescence to characterize glutamatergic boutons arising from the primary and secondary visual cortices, lateral geniculate nucleus, lateral posterior thalamic nucleus, and superior colliculus in adult mice. Among 2,178 singly labelled boutons, VGluT1 predominated in corticothalamic, corticopontine, corticotectal, corticostriatal, and corticocortical feedback projections. Conversely, thalamocortical, tectothalamic, thalamostriatal, and tectopontine projections were almost exclusively VGluT2-positive. Projection direction did not fully predict transporter phenotype: ascending corticocortical projections remained predominantly VGluT1-positive, whereas the descending tectopontine projection was exclusively VGluT2-positive. Only 12 of 2,190 VGluT-immunoreactive boutons exhibited detectable VGluT1/VGluT2 colocalization. Morphometric analyses further revealed an interaction between VGluT isoform and projection direction. Within ascending projections, VGluT2-positive boutons and puncta were larger than their VGluT1-positive counterparts, whereas no isoform-related difference in bouton area was detected within descending projections. These findings demonstrate an association between VGluT phenotype and projection class in the mouse visual system. VGluT isoforms therefore provide informative markers of pathway organization, but neither transporter identity nor terminal size alone constitutes an invariant molecular indicator of projection direction or driver-modulator function.

Drosophila s-LNv clock neurons promote nighttime sleep by releasing the neuropeptide sNPF to activate sNPF receptors (sNPF-Rs) on l-LNv clock neurons. Behavior is controlled by synaptic transmission, but s-LNv and l-LNv neurons are not connected directly by chemical synapses. To investigate the basis of sNPF/sNPF-R communication between LNv neurons, the spread of sNPF was imaged in the adult brain. We report the daily midmorning burst of sNPF released from s-LNv terminals does not reach l-LNv neurons or s-LNv somata. Instead, sNPF released by the s-LNv soma late at night in response to sleep-promoting IP3 signaling reaches l-LNv somata, but not their terminals. In addition to communication by neuropeptide diffusion, analysis of fly connectomes revealed that adult s-LNv and l-LNv neurons form non-synaptic direct contacts mediated by cytonemes. Remarkably, genetically perturbing LNv neuron cytonemes alters sleep latency, but not nighttime sleep, the target of somatic sNPF release, or circadian behavior, which depends on PDF neuropeptide released by LNv terminals. Therefore, three distinct aspects of adult rhythmic behavior are produced by terminals, the soma and cytonemes, with the latter possibly acting via contacts that are not currently annotated in the connectome.

The serotonergic system originates from a small population of brainstem neurons whose axons form one of the most extensive projection networks in the vertebrate nervous system. Although a number of molecular regulators of serotonergic development have been identified, the core principles that organize this widespread axonal architecture remain poorly understood. Classical neuroanatomical studies have proposed that serotonergic axons may navigate by growing along pre-existing fiber tracts, a process termed epiphytic guidance, but this hypothesis has remained largely untested. Here, we used organotypic transplantation assays to investigate how embryonic serotonergic axons navigate within developing and adult neural tissue. Rostral raphe explants from Tph2-GFP embryos were grafted onto embryonic hindbrain flat-mounts or adult brain slices, allowing donor-derived axons to be visualized in relation to genetically labelled endogenous serotonergic pathways and host tissue architecture. Across different grafting configurations, developmental stages, and both homotopic and heterotopic host territories, donor-derived axons consistently aligned with pre-existing serotonergic pathways adopting local trajectories rather than growing independently of them. This substrate-dependent behavior extended to adult tissue, where transplanted embryonic axons preferentially followed pre-existing serotonergic axon directions. These observations reveal substantial navigational plasticity and indicate that axonal and tissue architectures can provide permissive and potentially instructive substrates for the dispersal of serotonergic fibers. Together, our findings provide experimental support for epiphytic guidance and suggest that serotonergic pathway assembly relies, at least in part, on a pioneer-follower mechanism of progressive self-scaffolding. Such a strategy may help explain how a small population of raphe neurons generates an extensive and spatially coherent neuromodulatory system.

Tackling developmental changes in long-term potentiation (LTP) expression during adolescence is crucial to understand disease susceptibility in neurodevelopmental disorders, epileptogenesis or drug abuse. This paper investigated the alterations in the resting phosphorylation state of synaptic proteins essential for synaptic transmission and hippocampal LTP expression from weaning (3 weeks) to adulthood (12 weeks). Synaptic AMPA GluA1 and GluA2 subunit levels increased, yet to different extents, as the GluA1/GluA2 ratio also increased. Conversely, GluA1 phosphorylation in both Ser831 and Ser845 progressively decreased from weaning to adulthood, suggesting an enhanced availability of these sites for activity-dependent phosphorylation. In accordance, LTP induced by different theta-burst stimulation (TBS) intensities in the CA1 area of rat hippocampal slices enhanced gradually in this developmental period. In contrast, basal phosphorylation of GluN1, GluN2B and CaMKII increased during adolescence. Altogether, these findings suggest that alterations in AMPA receptor subunit composition and basal phosphorylation are crucial for the maturation of hippocampal LTP during adolescence, while a mild enhancement in synaptic CaMKII levels may further provide the necessary structural support to LTP expression and stability. Given the reported involvement of GluA1 phosphorylation changes in epileptogenesis and neurodevelopmental disorders, these findings provide important insights into hippocampal synaptic plasticity in normal and altered brain development and epileptogenesis.