Frontiers in Cellular Neuroscience

Peripheral neurons exhibit robust regeneration after nerve injury in adulthood; a process linked to the activation of regeneration-associated genes (RAGs). However, the mechanisms underlying nerve regeneration during early postnatal development remain poorly understood. In a previous work, we observed that motoneurons axotomized at early postnatal stages regenerate without overexpressing RAGs, contrasting with the expected RAG activation observed in postnatal day 30 (P30) mice. To determine whether this phenomenon extends to sensory neurones, we investigated two paradigmatic dorsal root ganglia populations: proprioceptive (PV) and nociceptive (TrpV1) neurones, which exhibit distinct regenerative capacities in adult mice. Following sciatic nerve crush injury at P4, P10, and P30, both neuronal subtypes demonstrated robust axonal regeneration across all postnatal stages, with nociceptors exhibiting significantly superior regeneration compared to proprioceptors. Remarkably, no substantial neuronal death was observed at any developmental timepoint. However, RNA sequencing revealed that P4 and P10 sensory neurones failed to upregulate RAGs following injury, whereas P30 neurones exhibited the expected pro-regenerative transcriptional programme. Pathway enrichment analysis confirmed that classical regeneration-associated signalling pathways were activated exclusively in young adults, whilst developmental and inflammatory pathways predominated in early postnatal stages. Using the conditioning lesion paradigm in primary dorsal root ganglia cultures, we demonstrated that only P30 neurones exhibited enhanced neurite outgrowth following conditioning lesion, whilst P4 and P10 neurones showed no conditioning effect or reduced outgrowth, respectively. These findings indicate that early postnatal sensory neurones possess intrinsic growth capacity independent of RAG activation, suggesting fundamentally distinct regenerative mechanisms across developmental stages.

Neuronal intracellular chloride concentration shapes the polarity and strength of GABAA receptor-mediated inhibition. ClC-2 is a hyperpolarization-activated chloride channel proposed to support chloride extrusion, but its acute contribution to EGABA and neuronal chloride homeostasis remains incompletely understood. Here, we used the selective ClC-2 inhibitor AK-42 to test how acute ClC-2 inhibition affects GABAergic chloride signaling in organotypic hippocampal slice cultures. We combined targeted single-interneuron optogenetic activation of stCoChR-expressing GABAergic interneurons with gramicidin-perforated patch recordings to measure optogenetically evoked inhibitory postsynaptic currents and calculate EGABA and we used SuperClomeleon imaging to measure neuronal intracellular chloride. AK-42 depolarized EGABA, with a larger effect when stimulation was delivered 2 s rather than 100 ms after voltage-step onset. This delay-dependent effect is consistent with greater engagement of voltage- and time-dependent ClC-2 activity during prolonged voltage steps. AK-42 also abolished the more hyperpolarized EGABA normally measured at the 2-s stimulation delay and shifted baseline holding currents at hyperpolarized command potentials without measurably changing estimated resting membrane potential. SuperClomeleon imaging showed that AK-42 increased neuronal intracellular chloride in the presence of TTX, supporting a role for ClC-2 in basal chloride regulation. Together, these findings show that acute ClC-2 inhibition depolarizes EGABA, increases neuronal intracellular chloride, and alters the temporal dynamics of GABAergic chloride signaling, identifying ClC-2 as an acute regulator of synaptic GABAergic chloride signaling.

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

Oligodendrocyte (OLG) injury and loss are increasingly recognized as major determinants of demyelination and remyelination failure in multiple sclerosis (MS), alongside conventional immune-mediated processes. However, the mechanism underlying OLG injury and remyelination failure remains incompletely understood. Astrocyte-oligodendrocyte interactions mediated by Connexin (Cx)-43-Cx47 gap junction (GJ) communication and leukemia inhibitory factor (LIF)-LIFR/gp130 signaling are critical for oligodendroglial survival, homeostasis, and myelination. Despite their established role, how these pathways are altered during neuroinflammatory diseases remains unclear. To address this, we integrated a murine {beta}-coronavirus (RSA59)-induced in vivo model with enriched primary oligodendrocyte precursor cell (OPC) and mature OLG cultures to investigate the effect of RSA59 infection on OLGs, Cx47-mediated GJ communication, and LIF-LIFR/gp130 Signaling. We observed that RSA59 directly targets oligodendroglial lineage cells, including OPCs and mature OLGs, and induces apoptosis in mature OLGs. Despite increased OPC abundance, RSA59 infection reduces the expression of mature and myelinating markers CNPase and MBP, raising the possibility of impaired oligodendrocyte maturation and myelination. RSA59 infection also differentially regulates Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling, crucial for OLG survival and homeostasis. Furthermore, our studies demonstrate that extracellular signal-regulated kinase (ERK), a downstream effector of LIF signaling, phosphorylates the Cx47 C-terminal domain (Cx47CT) at Serine-372 residue, identifying a previously unreported mechanism. Collectively, these findings reveal that RSA59 infection disrupts interconnected oligodendroglial communication and survival pathways, providing mechanistic insights into virus-induced chronic demyelination pathology. The study further identifies Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling as crucial therapeutic targets, and highlights ERK-dependent Cx47CT phosphorylation as a potential regulatory mechanism.

Axonal action potential propagation critically depends on tightly regulated intra and extracellular electrolyte concentrations and acid base homeostasis. Experimental studies have demonstrated that changes in extracellular sodium, potassium, and calcium concentrations alter axonal excitability and conduction properties. However, it remains unknown whether small variations in electrolyte concentrations within the physiological range are associated with measurable changes in nerve conduction velocity in humans. In this study, data from neurologically healthy children who underwent both nerve conduction studies and blood gas analysis were analyzed to investigate the relationship between electrolyte concentrations and median motor nerve conduction velocity. Nerve conduction velocity was significantly negatively correlated with ionized calcium concentration and positively correlated with sodium concentration, whereas no significant correlation was observed for potassium. In an a priori defined multiple linear regression model, ionized calcium, sodium, and bicarbonate remained significantly associated with nerve conduction velocity after correction for multiple testing. These findings indicate that even small variations in electrolyte concentrations within the physiological range are associated with measurable differences in peripheral nerve conduction velocity and suggest an additional contribution of acid base status to nerve conduction.

Charcot Marie Tooth type 1A (CMT1A) is the most common inherited peripheral neuropathy, caused by duplication of the peripheral myelin protein 22 (PMP22) gene and characterized by dysmyelination and defects in myelin maturation. Hypercellularity is a prominent feature of the disease, although its origin, persistence, and underlying mechanisms remain controversial. Here, we performed a longitudinal analysis of sciatic nerves from wild-type (WT) and CMT1A rats between 2 and 12 months of age. We demonstrated that CMT1A nerves exhibit persistent cellular proliferation extending into adulthood. This phenotype is confirmed by bulk RNA-seq data showing sustained enrichment of proliferation-associated gene signatures. Cell type signature analysis identifies overrepresentation of immature, neural crest related, and epithelial mesenchymal transitional (EMT) transcriptional programs in CMT1A nerves. Among the most upregulated genes, RUNX2 showed strong and prolonged expression at both transcript and protein levels. Consistently with the key role of RUNX2 in the activation of the injury response, CMT1A nerves also showed a marked enrichment in SOX10+JUN+ cells, that only partially overlap with SOX10+RUNX2+ cells, indicating a maladaptive activation of the injury-like Schwann cell (SC) program. Interestingly, we detected robust upregulation of SMOC2, an extracellular matrix protein selectively expressed by endoneurial fibroblasts, pointing to activation of non-canonical cell types and altered extracellular matrix remodeling. Together, these data demonstrate that CMT1A peripheral nerves are characterized by chronic hypercellularity driven by persistent proliferation, EMT, and dysregulated injury transcriptional programs, collectively pointing at progressive and multicellular nerve remodeling as a relevant pathomechanism in the disease.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Myelin formation requires extensive membrane lipid synthesis, yet how myelinating glia acquire the choline needed for this process remains incompletely understood. Choline transporter-like protein 1 (CTL1; SLC44A1) is highly expressed in Schwann cells, and CTL1 deficiency in oligodendrocytes impairs CNS myelination. Here, we examined the role of CTL1 in peripheral nerve myelination using Schwann cell-specific Ctl1 knockout mice. Unexpectedly, loss of Ctl1 did not alter Schwann cell differentiation, the number of myelinated axons, or myelin thickness, but increased myelin abnormalities, including in-foldings and out-foldings. Lipidomic analysis revealed selective alterations in myelin lipid composition, particularly among long-chain lipid species, together with triglyceride accumulation in whole nerves. CTL1 deficiency also increased mTORC1-associated S6 and mTORC2-associated AKT S473 phosphorylation. Transcriptomic analysis revealed downregulation of gene programs associated with fatty acid {beta}-oxidation, triglyceride catabolism, and oxidative phosphorylation. Following peripheral nerve injury, Ctl1-deficient Schwann cells generated a normal repair response and efficiently remyelinated regenerated axons despite altered mTOR signaling. Together, these findings demonstrate that CTL1 is not required for overall peripheral myelin production but contributes to maintaining normal myelin lipid composition and architecture, revealing an unexpected ability of Schwann cells to sustain myelination despite disruption of CTL1-dependent choline metabolism

The small GTPase Rem2 is a key signaling molecule involved in synaptic formation, dendritic complexity, spine formation, and regulation of CaMKII-dependent long-term potentiation (LTP). However, it remains unclear how Rem2 explicitly impacts learning and memory. To address this issue, we deleted Rem2 specifically in dorsal CA1 neurons of male and female mice and assessed spatial learning using a spatial object recognition (SOR) task. We found that males outperform females in this behavioral assay and that deleting Rem2 did not impact spatial learning in males. In contrast, Rem2 deletion significantly improved spatial learning in females enabling them to perform as well as males in the SOR task. Using an automated Western blot system to assess all known AMPA- and NMDA-mediated glutamate receptor (AMPAR and NMDAR) subunits in each mouse, we found that the sex-dependent change in SOR we observed was likely due to Rem2 increasing GluN2D expression in the interneurons of female mice only. Furthermore, using S-statistics to compare the overall AMPAR and NMDAR composition among groups, we found that males and females normally have divergent covariance structures but this divergence is eliminated when Rem2 is deleted from CA1 neurons. These results reveal an unexpected role of Rem2 in maintaining sex differences in glutamate receptor composition and spatial learning abilities. To our knowledge, this is the first demonstration of a signaling molecule that confers sexual dimorphism to excitatory synapses. As such, Rem2 may play a critical role in understanding how sex-dependent symptoms of neurodevelopmental and neurodegenerative disorders arise.

Microglial activation is a central component of neuroinflammatory responses in many brain pathologies. Increasing evidence indicates that microglial phenotype is tightly linked to cellular metabolism, with pro-inflammatory activation associated with enhanced glycolytic flux. Lactate, traditionally considered a metabolic substrate, has recently emerged as a signaling molecule capable of modulating immune responses. However, its direct impact on microglial inflammatory activation remains incompletely understood. In the present study, we investigated the effects of lactate on microglial phenotype under inflammatory conditions using primary rat microglial cultures stimulated with lipopolysaccharide (LPS). Microglial activation was assessed through the expression of phenotypic markers, cytokine production, and secreted chemokine profiles. LPS stimulation induced a strong pro-inflammatory response characterized by increased CD86 expression, elevated TNF-alpha secretion, and enhanced release of several pro-inflammatory chemokines. Post-treatment with sodium L-lactate significantly attenuated these inflammatory responses, reducing pro-inflammatory marker expression and cytokine secretion, while restoring the anti-inflammatory marker CD206. To explore the relevance of these findings in a pathological context, the effects of lactate were further examined in a neonatal rat model of hypoxia-ischemia. Sodium L-lactate administration after injury reduced microglial activation and promoted a shift toward an anti-inflammatory phenotype in cortical regions, whereas hippocampal microglia showed a more limited response. Together, these results demonstrate that lactate directly modulates microglial inflammatory activation and cytokine production in vitro and suggest that lactate-mediated metabolic signaling may contribute in vivo to the regulation of neuroinflammatory responses.

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

The mammillary bodies (MB) are a hypothalamic component of the limbic Papez circuit that plays a critical role in spatial and episodic memory in mammals. Degeneration of the MB occurs in disorders associated with cognitive impairment, including Korsakoff's syndrome and Alzheimer's disease, yet the molecular organization and intrinsic properties of MB neurons remain poorly understood. Recent single-cell RNA sequencing identified multiple transcriptionally distinct neuronal populations within the MB and suggested that they differentially express voltage-gated ion channels that regulate neuronal excitability. Here, we used fluorescence in situ hybridization (FISH) to define the anatomical organization of cluster-enriched molecular markers and determine the spatial distribution of transcripts encoding voltage-gated sodium (NaV), potassium (KV), and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels among defined subregions of the mouse MB. We found that marker transcripts occupy characteristic but partially overlapping spatial domains that broadly correspond to classical anatomical subdivisions. In addition, several ion channel transcripts, including Scn1a, Scn2a, Kcnq2, Kcnq3, and Hcn1, exhibited distinct patterns of enrichment across molecularly defined neuronal populations and MB subregions. Multiplex FISH further revealed unexpected co-expression of Scn1a and Scn2a within Pvalb-enriched neuronal populations, while whole-cell recordings demonstrated distinct intrinsic firing properties of neurons in the lateral and medial mammillary nuclei. Together, these findings establish a molecular framework linking neuronal population identity with voltage-gated ion channel expression in the MB and provide a foundation for future studies investigating how cell type-specific differences in intrinsic excitability contribute to memory function and neurological disease.

Spinal cord injury (SCI) is an incurable neurological condition in which post-traumatic edema contributes to secondary ischemic damage because the spinal cord is confined within the rigid vertebral canal. However, glymphatic transport in the healthy and injured spinal cord remains poorly characterized. We hypothesized that spinal cerebrospinal (CSF) and glymphatic transport is shaped by anatomy, administration route, and molecular size of the tracer; is disrupted after SCI; and is further impaired by loss of aquaporin-4 (AQP4). CSF tracers were administered by cisterna magna or lumbar intrathecal injection to anesthetized adult C57BL/6 mice. Dynamic contrast-enhanced MRI, ex vivo fluorescence imaging, and histology were used to characterize tracer distribution, influx, and clearance. Constrictions of the spinal subarachnoid space at C3-C7, T6-T12 and L5-S1 spinal segments accelerated contrast-agent dispersion, demonstrating that local anatomy regulates spinal CSF transport. Tracer influx into the cord itself occurred primarily along periarterial pathways.Tracer distribution depended on the administration route: cisterna magna injection preferentially labeled white matter in the upper spinal cord, whereas lumbar injection preferentially labeled grey matter in the lower spinal cord. Gadobutrol and ovalbumin penetrated and dispersed throughout the parenchyma, whereas fibrinogen remained confined to the meninges. Under physiological conditions, intraparenchymal tracers cleared within 24 h. SCI was modeled using a minimally invasive NMDA-induced lesion at T11 that produced focal necrosis while preserving the dura and vertebral structures. Glymphatic transport, lesion volume, and motor recovery were compared between wild-type and AQP4-knockout mice using imaging, the Basso Mouse Scale, and voluntary wheel running. After SCI, intrathecal contrast propagated more rapidly, especially at T7-T8 segments and at the S1 segment, but tissue clearance was profoundly impaired, with tracer retention persisting for up to seven days. AQP4-knockout mice showed greater clearance impairment, larger lesions, and poorer motor recovery than wild-type controls compared at 24 h. These findings define major determinants of spinal glymphatic transport and show that accelerated tracer propagation after SCI does not indicate effective clearance. Instead, SCI produces glymphatic dysfunction that is exacerbated by loss of AQP4 and associated with greater tissue damage and functional impairment. The results extend the glymphatic framework to the spinal cord and identify fluid-clearance pathways as potential therapeutic targets for limiting edema and secondary injury. Future studies should determine whether restoring glymphatic function improves neurological recovery after SCI.

Axons of substantia nigra dopaminergic neurons "die back" in Parkinson's disease (PD), exhibiting early degeneration relative to cell bodies, a key feature also observed in PD models. A critical step towards understanding the process of axonal neurodegeneration is to determine the physiological properties of dopaminergic axons that are altered during the early stages of cellular dysfunction. Here, we compared the electrophysiology and calcium dynamics of dopaminergic axons in the MitoPark model of PD with age-matched controls. Our experiments show a pronounced reduction in action potential (AP)-evoked calcium signals in MitoPark axons that emerges well before the onset of dopaminergic fiber loss and motor deficits. In addition, calcium imaging and direct axon recording revealed deficits in AP propagation accompanied by alterations in the axonal AP waveform. Therefore, our experimental findings identify the progression of axon-specific physiological deficits that occur long before neurodegeneration, more precisely defining the period that precedes axonal loss.

Drosophila glutathione S-transferase S1 (GstS1) is a unique member of the glutathione S-transferase family and is considered the ortholog of vertebrate hematopoietic prostaglandin D synthase based on amino acid sequence similarity. A previous forward genetic screen revealed that a null mutation in GstS1 dominantly suppresses genetically induced behavioral hyperexcitability in several seizure-prone Drosophila mutants, including paraShu, a gain-of-function mutant of the voltage-gated sodium channel gene paralytic (para), and eas2, a loss-of-function mutant of the ethanolamine kinase gene easily shocked (eas). Here, using the GAL4/UAS system combined with GstS1 RNAi, we show that hemocyte-specific GstS1 knockdown suppresses hyperexcitable phenotypes in both paraShu and eas2. Notably, suppression of adult paraShu phenotypes required GstS1 knockdown before adult eclosion, suggesting that GstS1 modulates adult neuronal excitability by influencing neural development through neuroimmune interactions. Consistent with this model, hemocyte-specific GstS1 knockdown rescued abnormal dendritic morphology in developing class IV dendritic arborization (C4da) neurons of paraShu larvae. Moreover, paraShu and eas2 larvae exhibited increased numbers of sessile hemocytes surrounding C4da neuron somata, a phenotype reversed by either GstS1 knockdown or dietary supplementation with the {omega}-3 polyunsaturated fatty acid -linolenic acid. Together, these findings reveal a previously unrecognized developmental role for GstS1 in promoting nervous system hyperexcitability through hemocyte-mediated neuroimmune interactions, potentially involving bioactive lipid signaling. Our results highlight neuroimmune crosstalk as an important mechanism linking genetic perturbations to altered neural development and seizure-associated hyperexcitability.

The ventral subiculum (vSub), a major output node of the hippocampal formation, has been implicated in diverse neurological disorders. Disruption of excitatory-inhibitory balance is a common feature of neurological disorders, with altered GABAergic signaling representing a critical component. More recently, the vSub has gained increasing attention as a component of neural circuits underlying emotional and motivational regulation, through its extensive connections with regions implicated in mental health disorders such as addiction and anxiety. Our previous work in models of alcohol use disorder and comorbid anxiety identified an important role for GABAergic inhibition in regulating vSub excitability, prompting us to investigate the functional consequences of recruiting the broader local GABAergic population. Although previous studies have examined individual interneuron subtypes, the network consequences of activating the global population of local GABAergic neurons remain poorly understood. To address this question, we expressed channelrhodopsin using an mDlx-dependent strategy to target GABAergic neurons in the ventral hippocampus and performed in vivo electrophysiological recordings from vSub neurons in anesthetized rats. Repeated activation of this GABAergic population at 20 Hz, a stimulation frequency commonly used in behavioral optogenetic studies, produced robust suppression of pyramidal neuron firing during stimulation. This direct inhibition was accompanied by network-dependent increases in post-stimulus activity that varied as a function of baseline activity, sex, and trial number. Complementary ex vivo electrophysiological experiments further supported network-dependent effects and revealed short-term synaptic plasticity that evolved across repeated stimulation trials, reflecting contributions from both pre- and postsynaptic mechanisms. Together, these findings demonstrate that recruitment of the broader GABAergic population produces effects extending beyond direct inhibition and can engage dynamic network responses that depend on stimulation history and circuit state. These findings highlight the importance of stimulation frequency, timing, and network context when interpreting the functional consequences of GABAergic circuit manipulation.

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.

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.

Pattern separation and pattern completion are core hippocampal computations linked to memory interference and overgeneralization in many neuropsychiatric disorders and to mnemonic discrimination deficits in neurodegenerative conditions. Astrocytes are increasingly recognized as important integrators and modulators of neuronal activity as many studies have shown changes in animal behavior and neural circuits induced by manipulation of astrocytic activity. The role of astrocytes in the dentate gyrus for learning and memory is, however, largely unknown. To investigate the functional significance of astrocytic activity in the dentate gyrus, we expressed the calcium extruding pump hPMCA2w/b (CalEx), resulting in inhibition of astrocytic calcium activity, the Gq-coupled receptor hM3Dq, or the Gi-coupled receptor hM4Di, allowing chronic activation of Gq- and Gi-dependent pathways by a designer drug, in dentate gyrus astrocytes. Our main finding is that inhibition of astrocytic calcium activity by CalEx resulted in improved pattern separation and impaired pattern completion. In -vivo calcium imaging revealed that CalEx expression in astrocytes altered the neuronal activity pattern in dentate gyrus neurons with an overall inhibitory effect. This suggests that in the dentate gyrus astrocytes promote the activity of these neurons. Furthermore, chronic activation of hM4Di resulted in impaired between-session fear extinction while chronic activation of hM3Dq resulted in impaired within-session fear extinction and improved pattern separation learning over multiple days, suggesting that activation of astrocytic activity enhances mechanistically separable forms of fear extinction in a Gq- or Gi-pathway-specific manner. Our findings reveal that dentate gyrus astrocytic calcium shifts the balance between pattern separation and pattern completion towards pattern completion and thus modulates an essential neuronal functional output of the dentate gyrus. Chronic activation of astrocytes inhibits fear extinction, and thus also affects management of memory interference. Our findings imply that astrocytic modulation may be beneficial to treat cognitive deficits in disorders in which overgeneralization is a problem.

Hearing loss and deafness as a result of hair cell loss cannot be restored due to the incapacity of spontaneous regeneration of these cells. Compounds that manipulate key signaling pathways can potentially regenerate cochlear hair cells. In order to test different compounds that promote hair cell regeneration in vitro models can be used. Three-dimensional cultures have allowed the expansion and experimentation of human and mouse inner ear organoids. This is mainly performed in the embryonic or the early postnatal developmental stage. However, since the majority of patients with hearing loss are adult, it is crucial to understand the adult inner ear regenerative capacity. Here, we evaluated progenitor cell markers in the adult human and mouse inner ear and the generation, expansion and differentiation of cochlear organoids derived from the adult human and mouse inner ear. Cochlear and vestibular sensory epithelium from adult humans; and from adult and aged mice express the progenitor markers SOX2 and LGR5; and can generate organoids in vitro. By optimizing the culture conditions, organoids derived from the cochlea and vestibular organ from adult human and adult and aged mouse differentiated to Myosin 7A positive cells. This indicates that the adult inner ear has regenerative capacity. These findings are encouraging for future regenerative therapies to cure hair cell-related hearing loss and deafness.