Frontiers in Molecular Neuroscience

Spiral ganglion neurons (SGNs) are the primary auditory afferents in the inner ear. These neurons degenerate in response to a number of conditions, including auditory neuropathies, concussions, and aging. Research to assess the extent of degeneration and to test the efficacy of protective or rehabilitative strategies requires quantification of SGNs from tissue sections. However, manual counting of SGNs can be arduous and time-consuming due to dense crowding and the lack of reliable nuclear-specific labels. SGNs receive afferent input via GluA2-containing AMPA receptors. As the Gria2 transcripts that code for GluA2 must undergo RNA editing to ensure calcium impermeability, we hypothesized that SGNs would express high levels of the adenosine deaminase acting on RNA (ADAR) enzyme ADARB1. Here we confirm enriched expression of Adarb1 in SGNs via in situ hybridization and show that anti-ADARB1 antibodies robustly label the nuclei of both type I and type II SGNs in cochlear sections from young and aged mice. Neuronal specificity was confirmed using antibodies against neurofilament heavy chain (NFH), human antigen D (HuD), GATA binding protein 3 (GATA3), and SRY-box 2 (SOX2). A blinded investigator manually counted SGNs via NFH staining, and these were compared to automated counts of ADARB1-positive nuclei using the analyze particles function in ImageJ. A concordance correlation coefficient and Bland-Altman analysis demonstrated strong agreement between the manual and automated counts. Additionally, immunolabeling of ADARB1 in macaque and human temporal bone sections confirm robust labeling of SGN nuclei, suggesting broad utility of ADARB1 immunolabeling for automated counts of SGNs across species.

The nasal epithelium is a complex tissue composed of both respiratory and olfactory tissue, and is constantly exposed to environmental insults, including toxins and pathogens. The main olfactory epithelium (MOE) serves as the critical site for olfaction, or sense of smell. Dysfunction at this critical barrier tissue can result in partial or total loss of olfactory function, resulting in significant impact to quality of life. The MOE is heterogeneous, comprised of many cell types including olfactory sensory neurons, support cells, and immune cells. It is not well understood how these diverse cell types in the MOE interact to regulate this tissue during homeostasis, and during times of injury and inflammation. We investigated how environmental olfactory exposures impact cell type specific transcriptional responses in the mouse MOE. We performed single-cell RNA sequencing (scRNA-seq) of the MOE following controlled environmental exposure to both well-known odorants and allergens. We identified major cell types and subtypes within the MOE, and identified transcriptional changes in response to the olfactory exposures. We identified transcriptional changes in OSNs, sustentacular cells, and resident immune cells to each condition. This indicated that environmental olfactory exposures drive changes to multiple cell types in the MOE. To our knowledge, this is the first study to identify effects of environmental olfactory exposures on cell-type specific transcription at homeostasis. These findings highlight the potential importance of multi-cellular interactions and communication in regulation of the olfactory epithelium.

The spinocerebellar ataxia type 5 (SCA5) L253P mutation in {beta}-III-spectrin causes high-affinity actin binding. Here we developed a CRISPR knock-in mouse to determine the in vivo impact of L253P on Purkinje neurons and motor activity, and to establish a model for future testing of SCA5 therapeutics. Significantly, the knock-in mouse shows impaired motor activity on elevated beam assays at 20 weeks. In the cerebellum, L253P causes a subcellular redistribution of {beta}-III-spectrin in Purkinje neurons. This is marked by loss of {beta}-III-spectrin in distal dendrites, accumulation of {beta}-III-spectrin at the plasma membrane of the soma and proximal dendrites, and formation of inclusions in the soma. The inclusions additionally contain F-actin and -II-spectrin, accumulate around the nucleus, form at an early age, and are larger in homozygous {beta}-III-spectrinL253P/L253P compared to heterozygous {beta}-III-spectrinL253P/+ mice. In contrast, neurons of the hippocampus and cerebral cortex, where {beta}-III-spectrin is also known to be expressed, abnormally accumulate {beta}-III-spectrin at the plasma membrane but do not form inclusions. To gain greater insight into disease mechanisms, unbiased proteomics identified over 150 cerebellar proteins that physically associate with {beta}-III-spectrin. Of these, cluster analysis revealed a group of 41 proteins, including glutamate receptors, SERCA2, and CaMKII, linked to synaptic transmission. Thus, the effect of the L253P to alter {beta}-III-spectrin localization, including decreased levels in distal dendrites, is likely associated with a disruption of {beta}-III-spectrin function in postsynaptic signaling. Consistent with this, and in agreement with prior findings in knockout mice, the L253P {beta}-III-spectrin knock-in mouse here shows that CaMKII, a calcium sensor and key mediator of glutamate signaling, is ~2-fold activated. Further, the abundance of EAAT4, a glutamate transporter, is significantly reduced. The L253P knock-in mouse primes future preclinical testing of SCA5 therapeutics, such as small molecule modulators of spectrin-actin binding, and glutamate and calcium signaling pathways.

WAC is an autism-associated gene involved in neurodevelopment. However, the effects of reduced WAC function on behavior and synaptic regulation in vivo remain unclear. Taking cues from the previous studies on the wac gene and the C. elegans model of ASD, we elucidated the effects of wac gene deletion on food-leaving behavior, a known parameter linked to ASD associated genes along with the cholinergic pathway. wac-deficient worms exhibited curtailed food-leaving behavior. Notably, observed phenotype was similar to that exhibited by nematodes with mutation in ASD related gene, neuroligin. In addition, wac-deficient worms showed impaired growth, reduced pharyngeal pumping, and lifespan. To examine potential synaptic mechanisms, we analyzed expression of genes related to cholinergic signaling across all developmental stages (L1-L4) through young adult (YA). Stage-specific transcriptional changes were observed, with increased expression of ace-1 and acr-3 at L1, acr-3 at L3, and acr-3, cha-1, lev-1, and lev-10 at L4. The transcriptomic alteration was most prominent at YA stage, exhibiting upregulation of ace-1, cha-1, cho-1, lev-1, lev-10, unc-17, unc-29, unc-38, and unc-50. To identify specific suppressor of upmodulated Ach signaling, RNAi of the upregulated genes was performed. cho-1 was identified as a specific suppressor of elevated Ach signaling. cho-1 encodes a high-affinity choline transporter responsible for choline uptake in the pre-synapse. These studies identify the molecular mechanisms pertaining to up-modulation of cholinergic signaling in wac mutant worms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/719318v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13b9510org.highwire.dtl.DTLVardef@b74e11org.highwire.dtl.DTLVardef@6676e0org.highwire.dtl.DTLVardef@1068f35_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lysosomal trafficking and homeostasis are biological functions that are pivotal for DRG neurons, given their metabolic demands and extremely long axons. Previous studies indicate that lysosomal signaling is altered in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and that blocking mitogen activated protein kinase-associated kinase (MNK1/2) signaling can alleviate pain behaviors in CIPN. Here, we investigated lysosome dynamics and lysosome-associated signaling in a mouse model of CIPN induced by paclitaxel (PTX), a chemotherapeutic agent used for various types of cancer. Using spinning disk super-resolution microscope (SPINSR), we demonstrate that PTX treatment in vivo causes reduced lysosome motility observed in vitro. PTX likewise drives the accumulation of Sequestosome 1 (SQSTM1), also known as P62, in cultured mouse DRG neurons, indicating lysosomal dysfunction in DRG neurons. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, was also upregulated in the nucleus of cultured mouse DRG neurons treated with PTX. In line with this, increased lysosomal-associated membrane protein 1 (LAMP1) expression was observed in PTX-treated mice. Given that our previous work demonstrated PTX treatment increases MNK1/2-eIF4E signaling in DRG neurons, we examined whether MNK1/2 inhibition could rescue lysosomal dysfunction. Treatment with Tomivosertib (eFT508), a potent MNK1/2 inhibitor, restored P62 levels in DRG neurons of PTX-treated mice and reduced TFEB in DRG treated in vitro. To establish translation relevance, we further show that PTX elevates phosphorylated eiF4E (p-eIF4E) in human DRG neurons, and concurrent eFT508 administration attenuates this effect. Collectively, these findings indicated that PTX disrupts lysosome trafficking and biogenesis, and that MNK inhibition with eFT508 restores lysosomal signaling and can serve as a neuroprotective strategy for CIPN.

Primary neuronal cultures derived from mouse tissue serve as an essential model for investigating neuronal development and function. Despite this, comprehensive developmental and sex-specific transcriptomic profiles in primary neurons have not been defined. Here, we performed multiplexed RNA-sequencing of neurons derived from male and female cortices across time. We validated this approach by assessing established neuronal maturation genes and we identified highly stable genes to serve as controls across development. Next, using linear modeling and temporal regression, we defined developmentally regulated transcripts, longitudinal expression dynamics, and gene signatures associated with transition states throughout development. Unexpectedly, this also revealed sex-specific effects on autosomal genes that emerge only after neuronal maturation even in the absence of in vivo cues. Most notably, neuropeptide genes Cortistatin and Neurokinin A are more highly expressed in female neurons. Furthermore, exposure to these neuropeptides elicited distinct transcriptional responses in male-versus female-derived cultures. These findings provide a valuable resource and reveal sex-specific autosomal transcriptional signatures that emerge in neurons maintained ex vivo. HighlightsO_LIMultiplexed RNA-sequencing of primary cultured neurons across neuronal maturation provides a new resource for the field. C_LIO_LIGene signatures associated with transition states are identified through linear modeling. C_LIO_LISex-specific regulation of autosomal genes encoding neuropeptides emerge even in the absence of in vivo cues. C_LIO_LIExposure to neuropeptides elicit distinct transcriptional responses in male and female primary neurons. C_LI

Age-related hearing loss (ARHL) is a rapidly growing public health concern, affecting two-thirds of adults over 65 years old, with no effective therapeutics available. As the aging population grows at an unprecedented rate, the burden of ARHL will only increase. The causes of ARHL are multifactorial, but an understudied major contributor is glial dysfunction. The auditory nerve (AN) conducts sound from the cochlea to the brainstem and holds a diverse population of immune cells and myelinating glia. As the AN fibers bundle together within the cochlea to project to the brainstem, they are first myelinated by Schwann cells in the peripheral AN, then myelinated by oligodendrocytes in the central AN. The region where myelination shifts from Schwann cells to oligodendrocytes is the glial transition zone (GTZ), located in the cochlear modiolus, creating a unique biological niche. While central-peripheral interfaces are recognized in other cranial nerves, the AN GTZ is understudied. This region integrates the peripheral and central microenvironments within the confined bony cochlea, positioning it as a niche for glial dysfunction in pathological conditions, such as aging. We hypothesize that the GTZ is a site of enhanced glial dysfunction contributing to age-related AN demyelination, an important contributor to ARHL. We evaluated this in an ARHL mouse model combining RNA-sequencing, quantitative immunohistochemistry, and 3D high-resolution imaging. We examined the AN GTZ from human temporal bone donors. RNA-sequencing of the AN revealed age-associated increases in abnormal myelination/glial function and inflammation. There was a significant age-dependent increase in Iba1+ macrophages/microglia, with accumulation at the AN GTZ, and an increase in cellular volume and surface area, suggesting greater age-related activation. Macrophages/microglia contained significantly more internalized myelin debris in the AN (peripheral, central, and GTZ) with aging. More importantly, we found structurally intact myelin within macrophages/microglia only at the GTZ, suggesting a unique microenvironment at the GTZ altering phagocytic activity in aging. Together, our data suggest that the GTZ, a previously unrecognized central-peripheral interface, is a critical site of immune-glial interactions and especially vulnerable to age-related demyelination and neuroinflammation. This study highlights the GTZ as a potential target for preserving AN myelination and mitigating ARHL.

Although commonly known as rapid and easy to use methodology, Golgi staining requires a range of staining solutions, impregnation periods, concentrations and slicing variables. The use of this methodology can help researchers identify and label individual neuronal components within the extended circuitry. The original Golgi stain technique, developed by Camillo Golgi in 1873, is a silver staining method that enabled scientists to visualize individual neurons in their entirety within nervous tissue for the first time. publications featuring the Golgi staining technique utilise cryostat or microtome slicing, with the combination of a readily purchased kit which comes with a cost and limited morphological detail. Here, we describe an optimised Golgi staining methodology that specifically targets the major drawbacks of traditional protocols; prolonged and inconsistent impregnation, slice fragility during sectioning, and variable visualization of fine dendritic structures. Through modest adjustments to impregnation duration and temperature, fixation, and vibratome sectioning conditions, this low-cost and simple protocol improves staining reliability, facilitates robust slicing without specialized embedding, and supports detailed analysis of neuronal morphology throughout the central nervous system. We validate our optimised protocol using tissue from on-going animal studies of pain and treatment. Representative images illustrate typical staining patterns, characterised by sparse background and high signal-to-noise ratio, facilitating unbiased neuronal tracing and analysis.

Phagocytic and immune-like cells have been observed in the satellite envelope of neuronal somata in peripheral sensory ganglia of many species for several decades. These cells likely play an important role in normal function of sensory neurons and they may also play an important role in neuronal dysfunction and neurodegeneration seen with neuropathy. Recent findings have described a satellite macrophage population transcriptomically similar to microglia in peripheral ganglia of some mammalian species. The function of these cells, and the mechanisms by which they may influence neurons in neuropathy are unclear. We sought to understand the phenotype and localization of these cells in the human dorsal root ganglion (hDRG) using large-scale single nucleus and spatial transcriptomic datasets from individuals with and without a history of peripheral diabetic neuropathy. We observed a large population of macrophages that express classical microglia makers such as TMEM119 and P2RY12 in the hDRG, as previously described. Our findings confirm that these microglia-like cells (MLCs) localize to the satellite envelope around neuronal somata, yet are transcriptomically distinct from all glial cell types characterized in the hDRG. These MLCs exhibit changes in abundance and localization with diabetic painful neuropathy (DPN) in both the hDRG and sural nerves suggesting that they are not exclusively localized to the DRG. We conclude that microglia-like cells are likely the resident tissue macrophage (RTM) of the hDRG, and perhaps the peripheral nervous system (PNS) given their localization to the sural nerve and other ganglia, where they are predicted to regulate homeostatic neuronal functions and response to injury. HighlightsO_LIMLCs are likely the RTM of hDRGs C_LIO_LIMLCs localize to the satellite envelope and recede with Nageotte nodule formation C_LIO_LIMLC activation state and signaling shift with diabetic neuropathy C_LIO_LIMLCs are also present in other ganglia and sural nerve C_LI

Hypoxic-ischemic injury is a major cause of olfactory dysfunction, yet the cellular and morphological mechanisms underlying this sensory loss remain poorly understood. Here, we investigated the structural, cellular, and functional effects of acute hypoxic exposure on the olfactory system of adult zebrafish (Danio rerio) of both sexes, a model organism with remarkable neuroregenerative capacity. Fish were subjected to 15 minutes of acute severe hypoxia (0.8 mg/L dissolved oxygen) and assessed at 1 and 5 days post-hypoxia (dph). We evaluated olfactory function by means of cadaverine-evoked aversive behavioral assays. Structural and morphological integrity and inflammation of the olfactory epithelium (OE) and olfactory bulb (OB) were characterized using immunohistochemistry, histological stainings, and a 2,3,5-triphenyltetrazolium chloride (TTC) colorimetric assay. Acute hypoxic exposure impaired olfactory-mediated behaviors without affecting locomotion or exploratory behavior. In the peripheral OE, hypoxia caused neurodegeneration, disruption of the nasal mucus layer, and robust leukocytic infiltration. We observed reduced mitochondrial dehydrogenase activity in the olfactory bulb (OB) along with reactive astrogliosis. Olfactory function recovered by 5 days, coinciding with full restoration of OE morphology, and supported by a strong proliferative response. These findings reveal a coordinated degenerative and regenerative response to hypoxia across the olfactory axis, with implications for understanding hypoxia-induced sensory loss and neural repair. SIGNIFICANCEThis work addresses an important gap in knowledge regarding the mechanisms linking hypoxic insult and olfactory dysfunction. By using adult zebrafish, an extraordinarily regenerative vertebrate, it also provides insight into neuronal repair and regenerative processes supporting olfactory recovery. The novelty of our study resides in that, to our knowledge, there are no studies that provide a comprehensive characterization of the effects of hypoxia in the olfactory system across molecular, histological, and functional levels. These findings advance our understanding of hypoxia-induced sensory neurodegeneration and regeneration, and highlight the zebrafish olfactory system as a powerful model for investigating neural repair mechanisms relevant to hypoxic-ischemic brain injury.

The hippocampal formation (HPF) provides neural substrates integrating disparate sensory cues into episodic memories and coherent action. Whereas HPF structures are formed by birth, the functional circuits evolve over postnatal development. Our previous studies showed that transient perinatal expression of the serotonin (5-HT) transporter SERT/Slc6a4 in CA3 pyramidal neurons, which do not synthesize 5-HT but take up extracellular 5-HT thus termed "5-HT-absorbing neurons", exerts sex-biased effects on long-term activity-dependent HPF synaptic plasticity and behavior in mice. This study investigates SERT impact on circuit development, through single-nucleus transcriptomics of postnatal HPF from CA3-pyramidal neuron SERT knockout (SERTPyramid{Delta}) mice. We demonstrate that SERTPyramid{Delta} mice preserve cell identities across the HPF but alter gene expression in specific neuronal types in a sex-biased manner. We observed SERTPyramid{Delta} male-biased upregulation of genes preferentially in glutamatergic neurons, particularly affecting the CA2 and parasubiculum (PaS) when they develop social novelty and spatial representations, respectively. In both the CA2 and PaS, altered genes center on two categories -- modulators of gene expression patterning including chromatin plasticity, RNA processing and ubiquitin-dependent protein degradation, and aspects of synaptic transmission. >20% of the dysregulated genes in the CA2 and PaS are associated with Autism and engaged in cell-type distinct functional networks, showing CA3 SERT regulation of ASD-vulnerable genes in intersecting biological processes in specific neurons during social and spatial circuits development. The data, available at https://scviewer.shinyapps.io/hippocampus_sertKO, provide an entry map for further deducing anatomical neuronal origin and the molecular and cellular pathways impaired by 5-HT dysfunction during HPF circuits development leading to lifetime cognitive deficits.

Autism spectrum disorder (ASD) is associated with deficits in synaptic plasticity across brain regions. While striatal dysfunction is observed in various mouse models of ASD, the effect of ASD-associated genes on striatal plasticity has not been well characterised. We previously showed that overexpression of the SFARI ASD risk gene eIF4E in transgenic (eIF4E-TG) mice produces ASD-like behaviours and impairs dorsal striatal dopamine release. Here, we examined whether eIF4E overexpression alters striatal synaptic transmission and plasticity. Using microscopy, whole-cell electrophysiology, optogenetics and fast-scan cyclic voltammetry, we assessed dendritic morphology and excitatory synaptic properties of spiny projection neurons (SPNs). The eIF4E-TG mice exhibited higher dendritic spine density, elevated AMPA and NMDA receptor-mediated mEPSC frequency, and reduced AMPA mEPSC amplitude. We also observed an increased induction rate and magnitude of long-term potentiation (LTP) in SPNs, which is NMDA receptor-dependent but is not prevented by pharmacological D1 or D2 receptor antagonism under the conditions tested. Finally, we found that somatic and dendritic Ca2+ signals evoked by brief depolarisation are altered in SPNs from eIF4E-TG mice. Together, these findings are consistent with eIF4E overexpression promoting an NMDA receptor-dependent form of striatal LTP that is not prevented by D1/D2 receptor antagonism.

The human olfactory epithelium (OE) represents a lifelong source of neural progenitor cells and has been proposed as an accessible model to investigate molecular alterations associated with neurodevelopmental disorders in postnatal individuals. Globose basal cells are considered the immediate neuronal progenitors within the OE, and several studies have attempted to culture these cells from nasal exfoliates. However, the actual contribution of neurogenic lineages in these cultures remains largely unquantified. Here, we cultured human nasal explants using an established protocol and characterized the resulting cell populations by immunohistochemistry and single-cell RNA sequencing. Integration with primary in vivo OE datasets revealed that these cultures are predominantly composed of mesenchymal-like cells, with limited representation of globose basal cells and neurons, and low expression of canonical neuronal markers. Using curated gene sets associated with neurodevelopmental disorders and malformations of cortical development, we assessed the extent to which disease-relevant transcriptional programs are captured in OE-derived cultures. While disease-associated genes are enriched in neurogenic lineages in vivo, their representation in mesenchymal cells is reduced. Together, our results challenge the assumption that standard OE culture systems faithfully model neurogenic compartments and suggest that current approaches may need refinement to recover neurogenic lineages.

Motor performance and coordination deficits are hallmarks of spinocerebellar ataxias, yet effective disease-modifying therapies remain limited. Here, we investigate the expression of the interleukin 17 receptor subunit A (IL-17RA) in cerebellum and assess the therapeutic potential of its ligand in a mouse model for Spinocerebellar Ataxia Type 2 (SCA2). We found that IL-17RA is highly enriched in cerebellar molecular layer interneurons (MLIs), which provide inhibitory input to Purkinje neurons. In-vitro electrophysiological recordings revealed that symptomatic SCA2 mice exhibited increased spontaneous inhibitory synaptic input onto Purkinje neurons, which was normalized by IL-17A application to control levels. Concomitantly, IL-17A application restored Purkinje neuron firing, a parameter characteristically reduced in SCA2 mice. Behaviorally, intranasal administration of IL-17A restored motor performance of symptomatic SCA2 mice to control levels in both rotarod and beam-crossing assays. Collectively, our results indicate that IL-17A rescues Purkinje neuron dysfunction and motor deficits in SCA2 mice, highlighting IL-17A signaling as a promising therapeutic target for spinocerebellar ataxia.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are mechanosensory cells in the spinal cord that detect compression and regulate locomotion, posture, and morphogenesis. Although CSF-cNs respond to changes in pH, neurotransmitters and metabolites, their chemosensory repertoire is not fully understood. Using hybridization chain reaction, we investigated the distribution of expression of chemoreceptors in CSF-cNs and neighboring cells in the spinal cord. We found that CSF-cNs express receptors for glutamate (grm2), somatostatin (sstr2) and low-density lipoprotein (LDL) (ldlrad2), indicating roles in detecting glutamate, somatostatin and LDL in the CSF. High LDL receptor expression in CSF-contacting cells suggests CSF lipid capture. Most receptors were enriched but not exclusive to CSF-cNs and also appeared in ependymal radial glial cells. Our findings indicate multiple chemosensory pathways can sustain long-distance communication between neurons and glia through the cerebrospinal fluid.

TECPR2-related disorder is a rare, autosomal recessive neurodevelopmental and neurodegenerative disease characterized by early-onset motor dysfunction, sensory- and autonomic neuropathy, and progressive neurological decline with early mortality. Currently, there are no effective treatments for individuals affected by this debilitating condition. To advance our understanding of disease mechanisms and explore therapeutic strategies, we developed and then characterized a knock-in (KI) mouse model carrying the human TECPR2 c.1319delC frameshift mutation. TECPR2-KI mice exhibit a subset of disease-relevant phenotypes, most prominently abnormal gait, along with reduced body weight and altered tactile sensitivity. We additionally observe a reduction in acoustic startle responses, consistent with dysfunction of brainstem-associated sensorimotor pathways. Histopathological analyses reveal progressive accumulation of axonal spheroids in the dorsal column nuclei, together with abnormalities in autophagy-related markers, features previously reported in individuals with TECPR2-related disorder. To assess the therapeutic potential of gene replacement, we delivered TECPR2 via intracisternal infusion of AAV9/TECPR2 in neonatal KI mice. Gene therapy restored mechanosensory function, normalized gait and startle responses, maintain autophagic homeostasis, and partially reduced axonal pathology. These findings demonstrate that TECPR2-associated deficits are not only replicable in this new mouse model but are also amenable to postnatal intervention. Our study introduces a genetically accurate murine model of TECPR2 deficiency, identifies brainstem-associated phenotypes, and provides preliminary evidence supporting the feasibility of AAV9-mediated TECPR2 gene delivery, establishing a foundation for future translational research in a currently untreatable disease. One-Sentence Key MessageTECPR2 deficiency disrupts brainstem sensory-motor circuits, impairing autophagy and tactile, gait, and startle function and is prevented by neonatal AAV9.

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

AO_SCPLOWBSTRACTC_SCPLOWSpiral ganglion neurons (SGNs) constitute the sole afferent connection between cochlear hair cells and central auditory nuclei. SGNs die during postnatal developmental pruning, and also following hair cell death, which can be triggered by ototoxic agents such as aminoglycoside antibiotics, including kanamycin. After hair cell loss, animal models show extensive SGN degeneration occurring gradually over a period of weeks to months. Here, we compared spatial and temporal patterns of SGN loss and immune cell involvement in these two cases of cell death in rats. Developmental SGN pruning occurred from postnatal day 5 (P5) to P8 in the basal half of the cochlea, and from P5 to P12 in the apical half. This was accompanied by a transient increase in spiral ganglion macrophages temporally and spatially correlated with SGN death, consistent with a role clearing degenerating neurons. After deafening neonatal rats with kanamycin injections, SGN death became evident at approximately 5.5 weeks of age and persisted throughout the ganglion, with greatest loss in the middle regions; less in the base and apex. Macrophage numbers also increased but neither temporally nor spatially correlated with SGN death. Rather, increased macrophage number and activation began approximately three weeks before SGN death and was highest in the apex. Additionally, T-cells and NK cells appeared in the ganglion concurrently with SGN degeneration. These observations suggest fundamentally different roles for macrophages post-deafening than during developmental pruning and, with prior observations that anti-inflammatory drugs reduce SGN death, support a causal role for immune responses in SGN death post-deafening.

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity; many of which are implicated in neuropsychiatric disorders. Members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and post-synaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a powerful tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by a broad spectrum of behavioral impairments. While multiple genetic and environmental factors are attributed to its cause, biological underpinnings are still poorly understood. We investigated an ASD-associated gene, WAC, for its neurobehavioral aspects using C. elegans and mice. Studies of C. elegans with wac gene deletions (wac-1.1 and wac-1.2) showed enhanced acetylcholine-associated behavior, as indicated by the aldicarb assay. No alteration in acetylcholine levels or acetylcholinesterase activity was observed. Upon further investigation, we found that the elevated cholinergic transmission resulted from increased activity of nicotinic acetylcholine receptors (nAChRs). Additionally, we observed reduced motility and dopamine-associated behaviors, along with a reduced ability to switch from crawling to swimming, a serotonin-dependent behavior. Upregulation in mRNA expression of the lev-1 gene was observed. Conversely, a feedback-counterbalancing response in the form of downregulated genes, acr-2, unc-17, unc-63, and unc-50, was also observed. Surprisingly, lev-1 RNAi did not reverse the enhanced cholinergic transmission in PHX2587 worms, indicating the involvement of other players. To validate our findings, we also assessed CHRNA7 levels in Wac+/- mice. While some genetic compensation was observed in heterozygous mice, we found a direct, inverse correlation between Wac mRNA expression and CHRNA7 levels in the mouse brain cortex, corroborating our findings from C. elegans. Overall, these studies indicate that wac gene deletion in C. elegans exhibits a neurotransmitter alteration that is relatable to ASD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/709202v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1771dc4org.highwire.dtl.DTLVardef@1434b4eorg.highwire.dtl.DTLVardef@10525ecorg.highwire.dtl.DTLVardef@fcd8a9_HPS_FORMAT_FIGEXP M_FIG C_FIG