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.

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.

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.

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.

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.

The ventral lateral geniculate nucleus (GLv) is a conserved retinorecipient structure positioned between visual and premotor systems, yet its role in visuomotor transformations remains poorly understood. Here, we examined whether the pigeon GLv contains a spatially organized motor representation linking visual-field locations to orienting movements. Anatomical tracing identified descending projections from neurons in the GLv inner lamina (GLv-li) through pretectal and tegmental regions toward pontine targets. GLv-li projection neurons are GABAergic, yet electrical microstimulation of GLv-li in awake pigeons evoked rapid head movements followed by smaller eye movements. Each stimulation locus produced a reproducible characteristic motor vector whose direction and duration were largely insensitive to stimulation parameters, whereas movement amplitude and velocity approached site-specific plateaus. Movements initiated from different head positions were better explained by repetition of a fixed displacement than by convergence toward a common spatial goal. Critically, motor-vector direction was systematically related to the direction of the visual receptive field represented at the same GLv locus, while motor-vector amplitude covaried with receptive-field eccentricity. These defining features persisted after optic tectum ablation. Together, these findings reveal a retinotopically-organized map of fixed-vector head movements in the avian GLv and identify a tectum-independent route through which visual spatial representations can access descending orienting circuitry.

The olfactory ventricle (OV), an anterior extension of the lateral ventricular system into the olfactory bulbs, is generally considered a transient developmental structure that closes during early postnatal life. However, this view largely derives from a limited number of laboratory models, and the evolutionary distribution and cellular organization of the adult OV remain poorly understood. Here, we combined a comparative survey of mammalian neuroanatomy with phylogenetic analyses and cellular characterization of the OV across species. We compiled anatomical data for 154 mammalian species spanning 26 orders and 80 families, revealing widespread persistence of a patent OV into adulthood. Phylogenetic analyses supported OV presence as the ancestral mammalian state and indicated that its absence is phylogenetically structured and consistent with repeated secondary losses. Chiroptera displayed multiple lineage-specific losses, whereas variation within Muridae extended to the intraspecific level, with contrasting OV persistence among Mus musculus and Rattus norvegicus populations. Comparative histological analyses in sheep, Peromyscus maniculatus, and CD1 mice revealed that, despite marked interspecific differences in OV morphology, the persistent ventricular region retains a conserved cellular organization characterized by progenitor- and glial-associated markers, proliferative cells, immature neuronal populations, and a differentiated apical/ciliary interface. Although these observations do not establish local neuronal production or demonstrate that the OV constitutes an autonomous neurogenic niche, their convergence across species indicates that the persistent adult OV is not simply an anatomically residual lumen but a specialized component of mammalian olfactory bulb organization. Together, our findings identify the OV as an ancestral and widely conserved feature of the mammalian brain and provide an evolutionary and cellular framework for investigating the developmental mechanisms controlling its persistence and its potential contribution to adult olfactory neurogenesis and plasticity.

The mouse superior colliculus (SC) regulates critical behaviors such as prey capture and visual threat avoidance, each of which depend on binocular visual processing. In contrast to our previous understanding, recent work has demonstrated widespread and diverse binocular interactions in the mouse SC. The SC receives direct input from contralaterally and ipsilaterally projecting retinal ganglion cells (contra- and ipsi-RGCs), but the ways in which eye-specific information is filtered in the SC remain unclear. Here, we utilized a trans-synaptic, intersectional viral tracing strategy to label SC neurons receiving input monocularly from contra-RGCs (SC-C), monocularly from ipsi-RGCs (SC-I), or binocularly from both (SC-B). We show that a surprising number of SC neurons receive ipsi-RGC input and that the proportions of SC-C, SC-I, and SC-B neurons are consistent throughout the SC. Morphometric analysis revealed few differences between neurons receiving different eye-specific patterns of input. Consistent with this, we show that SC-C, SC-I, and SC-B neurons are comprised of similar proportions of five morphologically distinct subtypes. And, the expression of candidate molecular markers appears similar across innervation patterns. Together, these data suggest that eye-specific information is widespread throughout the SC targeting overlapping populations of neurons.

Background: Infants with extremely low birth weight (ELBW) are at high risk for neurodevelopmental impairment, but the relationship between cerebellar structure and later developmental outcome remains incompletely defined. Methods: In this retrospective single-center study, we examined total cerebellar volume on clinically acquired brain magnetic resonance imaging (MRI) in ELBW infants while accounting for postmenstrual age (PMA) at imaging. Results: Of 65 infants screened, 42 were included. Mean gestational age at birth was 25.54 {+/-} 1.68 weeks, mean birth weight was 733.2 {+/-} 118.7 g, and mean PMA at MRI was 43.02 {+/-} 6.53 weeks (range, 31.57--68.00). Mean cerebellar volume was 22.17 {+/-} 9.63 cm. Independently adjudicated developmental outcomes comprised normal development in 16 infants, isolated or specific impairment in 12, and moderate/severe impairment in 14. Unadjusted cerebellar volumes did not differ across developmental groups (P = 0.490) or qualitative MRI categories (P = 0.875). However, the PMA-by-developmental-category interaction was significant (P = 0.002), with estimated cross-sectional slopes of 1.72, 1.56, and 0.39 cm per week, respectively. The interaction remained significant within 36--46 weeks PMA (n = 29; P = 0.011) and after adjustment for small-for-gestational-age (SGA) status. Sensitivity analyses indicated uncertainty in the stability of the interaction. Conclusions: These findings suggest that age-informed cerebellar volumetric patterns may provide developmental information not evident from average volumes alone and support prospective validation with standardized imaging and developmental assessments. As a result, cerebellar volumes obtained early in life may hold prognostic information for ELBW infants.

Neuroimaging studies identify the superior temporal sulcus (STS) as an anatomical and functional target in autism spectrum disorder (ASD). Yet, the STS is a heterogeneous structure containing unique subregions, such as the superior temporal asymmetrical pit (STAP), which is a recently identified human-specific landmark. To date, no study has compared the STAP between ASD individuals and neurotypical controls (NTs) in a large sample. Here, we analyzed the mean intra-hemispheric sulcal depth and inter-hemispheric laterality of the STAP, as well as the anterior and posterior portions of the STS (aSTS, pSTS), in 100 NTs and 100 ASD male individuals (ages 5-18) using a discovery-replication design. Across samples, the STAP and pSTS showed rightward asymmetry, while the aSTS showed leftward asymmetry within groups. All features of the STAP, aSTS, or pSTS did not differ significantly between groups. However, pSTS laterality related to individual differences in social cognition, which was further supported by a meta-analysis across over 200 studies showing that neurocognitive functional differences associated with social cognition and ASD are restricted to portions of the STS outside of the STAP. Together, these findings provide fine-grained insight into prior studies showing coarse morphological differences of the STS between individuals with ASD and NTs, as well as open up new questions and new neuroanatomical targets of the heterogeneous STS in future studies of brain structure, brain function, and cognition in ASD, as well as other neurodevelopmental disorders targeting the superior temporal cortex.

The sexual identity of specific tissues can be altered through cell-autonomous manipulation of the sex determination pathway in the nematode Caenorhabditis elegans. Previously described sexually mosaic strains carried multicopy transgenic arrays that are potentially subject to transgene silencing or misexpression artefacts. We used here the CRISPR/Cas9 system to engineer strains in which coding sequences for either a masculinizing agent (FEM-3) or a feminizing agent (intracellular domain of TRA-2) are inserted with a trans-splicing system at the 3 end of endogenous genetic loci that are known to be expressed in either a neuron-specific (rab-3) or gut-specific (elt-2) manner. We validate the tissue-specific sex-transformations induced by these genomic manipulations through expression analysis of known sex-specific genes and through assaying behavioral readouts. These strains may serve the worm community as standard reference tools to study the consequences of tissue-specific sex transformations.

Neurons extend axons toward spatially defined but often distant targets to establish brain circuits. Callosal projection neurons exemplify this organization, linking corresponding cortical territories between the two hemispheres. Growing axons rely on growth cones to regulate direction and speed through densely packed tissues. Although growth cone morphology and dynamics have been extensively characterized in cultured neurons and invertebrate models, how growth cones navigate the three-dimensional environment of the developing mammalian brain remains poorly explored. Here, we combined a genetically encoded electron microscopy (EM)-labeling strategy and zero-shot deep-learning-based tracing with high-resolution volume EM to reconstruct growth cones of identified callosal projection neurons in the developing mouse brain. Mediolaterally extending growth cones exhibited a planar, fan-shaped morphology, preferentially expanded along the dorsoventral axis. Filopodia extended from the edges of this plane and aligned predominantly with the same axis but showed no directional bias toward the growing direction. Longitudinal in vivo imaging revealed that increased growth cone complexity associated with filopodial extension coincided with lower instantaneous velocity but preceded accelerated extension. Together, these findings reveal a dorsoventrally oriented planar growth cone architecture in which filopodial extension precedes accelerated axon growth, suggesting a structural constraint that may restrict axon navigation to the callosal plane.

During mammalian nervous system development, multipotent progenitor cells differentiate into hundreds of distinct neuronal subtypes. Neuronal subtype terminal differentiation is regulated by transcription factors that directly control the acquisition and maintenance of terminal identity. Using the mouse retina as a model system, we show that the transcription factor ISL1 is required for the terminal differentiation and maintenance of starburst amacrine cells (SACs), the sole cholinergic neuron subtype in the retina. We conditionally deleted Isl1 selectively from amacrine and horizontal cell precursors, allowing us to assess its cell-autonomous role in SAC development without disrupting its critical function in bipolar cell and retinal ganglion cell development. We find that ISL1 is not required for initial SAC specification but is required for their terminal differentiation. Isl1-deficient SACs retain a GABAergic amacrine cell identity but fail to adopt many SAC-specific features including expression of genes required for cholinergic transmission. In the absence of ISL1, SACs fail to migrate properly, with both OFF- and ON- SACs localizing to the inner nuclear layer. Deletion of Isl1 from post-migratory SACs at later stages of development revealed that it is also required for maintaining SAC identity and proper dendrite stratification. Loss of Isl1 from SACs ultimately caused abnormal development of the direction selective circuit. Together, these findings establish that ISL1 is required for the induction and maintenance of SAC-specific identity from a generic amacrine state.

Heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) is a member of the hnRNP family of RNA-binding proteins that regulate neuronal function through multiple aspects of RNA metabolism. Mutations in the nuclear localisation signal (NLS) and quasi-RNA recognition motifs (qRRMs) of hnRNP H2 have recently been linked to a severe developmental syndrome, termed HNRNPH2-related neurodevelopmental disorder. Despite its emerging importance, the physiological roles of hnRNP H2 in neuronal development and synaptic maturation, and how disease-associated variants alter these processes to drive pathology, remain poorly understood. Here, we show that hnRNP H2 is expressed in hippocampal neurons and localises to the synaptic compartment. Knockdown of hnRNP H2 revealed essential roles of the protein in the regulation of neuronal morphology, dendritic spine maturation, synaptic transmission, and cortical neuronal migration in vivo. Disease-associated hnRNP H2 variants altered protein localisation and activity-dependent synaptic recruitment, resulting in defects in neuronal morphology, in vivo cortical neuronal migration and laminar orientation, dendritic spine maturation, and synaptic function. Together, these findings establish hnRNP H2 as a critical regulator of neurodevelopment and identify a molecular basis for HNRNPH2-related neurodevelopmental disorder.

Venezuelan equine encephalitis virus (VEEV) is a re-emerging neurotropic arbovirus, which affects the developing human brain, potentially leading to miscarriage, stillbirth, or long-term neurological sequelae. Currently, the mechanisms underlying these sequelae remain largely elusive. Using human induced pluripotent stem cell (iPSC)-derived neurospheres as a model for brain development, we show that both human neurons and glial cells are susceptible to VEEV infection. Microscopy-based quantification reveals that VEEV impairs neurite outgrowth in the human neurospheres. To mechanistically assess these observations, we performed multi-omics analyses of VEEV-infected neurospheres and demonstrate translational shutoff including downregulation of Rho GTPase pathways. The transcriptional and proteome profile is indicative of reduced actin cytoskeleton dynamics and consequently migratory defects of cells in the neurospheres. Shotgun secretomics moreover revealed downmodulation of neurite outgrowth associated proteins including midkine (MDK), amyloid-beta precursor protein (APP) and microtubule-associated protein 1B (MAP1B). Our findings reveal mechanistic insights into how VEEV infection impairs neuronal development and demonstrate that neurospheres are a powerful platform for assessing teratogenic effects of viruses on brain development.

Cell type designations label uniquely-identifiable groups of related cells that typically function in similar ways. Cell types divide the Drosophila melanogaster central nervous system into functional units, but remain challenging to target and identify. Previously we generated a driver line and image resource for targeting many neuron types (Meissner et al., 2025). Here we improve on identifying the targeted neurons by automated searching against electron microscopy datasets and manual scoring of search results to add 6,368 annotations across 1,772 driver lines in the collection. These annotations provide a potential training resource for future search algorithms. We then estimate the number of cell types and thus the fraction of the central nervous system labeled by the collection as a whole.

Oxytocin (OT) and vasopressin (AVP) are neuropeptide hormones involved in regulating animal social behavior and a broad spectrum of physiological processes. Although their distributions are well documented in neuroendocrine regions of the forebrain and midbrain, their expression in the hindbrain remains poorly understood. Here, we used immunohistochemistry to quantify OT and AVP immunoreactive puncta within three auditory brainstem nuclei, the lateral superior olive (LSO), the medial superior olive (MSO), and the medial nucleus of the trapezoid body (MNTB) in six wild-caught rodent species differing in sociality. We also quantified the volume of these nuclei and examined variation in total brain volume across species and sociality. OT and AVP puncta count differed among species and social groups. Group-living species exhibited higher OT and AVP puncta counts than monogamous and solitary species in the LSO and MNTB. In the MSO, OT puncta counts did not differ among social groups, whereas AVP puncta counts were higher in group-living than in monogamous and solitary species. Total brain volume and the volumes of the MNTB and MSO differed among species, but not across social groups, whereas LSO volume did not differ among species or sociality. These findings revealed sociality-related variation in OT and AVP immunoreactive puncta within auditory brainstem circuits and suggest that neuropeptide signaling within early auditory brainstem pathways may contribute to the neural integration of social and auditory information.

Background: Insufficient oxygen (hypoxia) or complete lack of oxygen (anoxia) causes cellular dysfunction and death in terrestrial animals. As a result, animals have developed homeostatic strategies, including changes in movement strategies to escape and survive stressful environments. Caenorhabditis elegans respond to changes in oxygen by increasing the frequency of reversals (backward movement) and increasing locomotor speed; these behaviors enable them to escape unfavorable environments and move to more suitable ones. The molecular and neural mechanisms underlying behavioral responses to low-oxygen environments are incomplete. Neuropeptides regulate C. elegans behaviors by modifying neural circuit function in response to hypoxia. Loss of function in C. elegans neuropeptide nlp-40 and its receptor aex-2 increased survival to anoxia [1]. However, the cells in which NLP-40 and AEX-2 function for anoxia, as well as the mechanisms underlying responses to anoxia, remain elusive. We hypothesized that NLP-40 and AEX-2 regulate anoxia by altering escape behaviors. Results: We found that neuropeptide NLP-40 functions in cholinergic neurons and AEX-2 functions in cholinergic or GLR-1 interneurons or motor neurons to regulate susceptibility to anoxia. We found that loss of either nlp-40 or aex-2 decreased locomotor activity and reversal frequency early during 48 hours of anoxia, suggesting that nlp-40 and aex-2 are required for the proper execution of these behaviors. We found that neuronal knockdown of nlp-40 recapitulated the behaviors observed in nlp-40 mutant animals and increased survival; however, restoring aex-2 function in glr-1 interneurons or motor neurons was sufficient to rescue behaviors to wild-type and restore vulnerability to anoxia. Conclusions: Overall, our results suggest that NLP-40 and AEX-2 likely function in cholinergic or glr-1 interneurons or motor neurons to regulate escape behaviors during anoxia. Escape behaviors are part of the homeostatic response and are typically protective in the wild; however, in our paradigm, we show that loss of either nlp-40 or aex-2 reduces escape responses and confers protection against 48 hours of anoxia. Collectively, these results highlight a new role for NLP-40 and AEX-2 in anoxia and shed light on mechanisms underlying the adaptive responses to anoxia.