Molecular and Cellular Neuroscience

Electrical transmission is mediated by intercellular channels that cluster into structures known as gap junctions (GJ). In vertebrates, GJ channels are encoded by the gene family of connexin (Cx) proteins that assemble as hexamers, termed hemichannels, in the pre- and postsynaptic membranes, and that subsequently dock to form GJ channels. Auditory contacts on the fish Mauthner cells serve as model to study the properties and organization of vertebrate electrical synapses. Electrical transmission at these synapses is mediated by multiple co-existing GJs at which the presence of intercellular channels is regulated by a molecular scaffold. Zebrafish contain four homologs of the neuronal Cx36: Cx35.5 and Cx35.1 (gjd2a and b, respectively), and Cx34.1 and Cx34.7 (gjd1a and b). Cx mutations suggested that GJs are formed by heterotypic channels made of presynaptic Cx35.5 and postsynaptic Cx34.1. Using transgenic fish in which Cxs were tagged, we found that a second Cx, Cx34.7, is present together with Cx34.1 on the postsynaptic side at some but not all GJs at these terminals. When exogenously expressed, both Cx34.1 and Cx34.7 formed heterotypic functional channels with Cx35.5, each with substantially different voltage-dependent properties, indicating they can serve differential functions. However, we previously demonstrated that electrical transmission is lost in Cx34.1 but not Cx34.7 null mutants, suggesting that Cx34.7 cannot compensate for the loss of Cx34, despite the intrinsic ability of Cx34.1 and Cx34.7 to create functional channels. The findings reveal an unanticipated functional organization in the electrical synapse, where Cx34.1 is obligatory and Cx34.7 accessory, roles that appear to be defined by the postsynaptic molecular scaffold, with two postsynaptic Cxs possibly assembling under specific functional contexts. Thus, our results indicate that electrical synapses share an organizational motif with chemical synapses, akin to how they combine postsynaptic receptor types to modify synaptic function.

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

Vestibular neurons are core elements of the pathways involved in vestibulo-motor functions, such as vestibulo-spinal and vestibulo-ocular reflexes. To meet behavioral needs, electrophysiological neuronal properties are adequately adapted to the sensory-motor computation sustaining these distinct vestibular reflexes. During frog metamorphosis, there is a complete reorganization of the posturo-locomotor system while the oculomotor system remains minimally changed, probably associated to so far unknown changes in vestibular neuronal properties. We used this unique model to investigate the central developmental mechanisms underlying such a reconfiguration of vestibular-associated behaviors. Central vestibular neurons exhibit two types of electrophysiological phenotypes: tonic neurons with a continuous discharge and phasic neurons with a transitory discharge mainly due to the activation of Kv1.1 channel. Electrophysiological recordings and Kv1.1 immunolabeling of vestibulospinal (VS) and vestibulo-ocular (VO) neurons at both larval and juvenile stages revealed that the majority of VS neurons exhibited a tonic discharge in larvae but a phasic discharge in juvenile, while VO neurons remained mainly tonic throughout development. Changes in phasic and tonic neurons proportions in VS population are partly explained by neurogenesis. But we provide evidences that an electrophysiological phenotype switch is a concomitant developmental mechanism participating in the maturation of these central vestibular neurons. All together our results showed that the maturation process in central vestibular neuronal groups is highly related to the metamorphosis-induced remodeling of vestibulo-motor functions they are involved in, with the ultimate purpose of ensuring an adequate adaptation of neuronal elements properties to the developmental changes of behavioral constrains.

Lack of social interaction results in various behavioral abnormalities in rodents, including increased anxiety levels, altered sociability, and impaired cognitive ability. Epigenetic factors regulate gene expression, however, how they contribute to juvenile social isolation (jSI)-induced behavioral alterations remains largely unknown. Here, we focused on the nucleus accumbens (NAc), a critical brain region of the reward system that regulates motivation-related behaviors. We first performed RNA-seq on neuronal nuclei and found alterations in genes related to neuronal function, as well as in transcriptional and epigenetic regulation. Protein-protein interaction (PPI) analysis of differentially expressed genes (DEGs) showed that top key nodes among down-regulated genes include membrane receptors (Ntrk2, Grin3a, and Grik1) and an apoptosis regulator (Bcl2). To further investigate whether jSI-induced gene expression alterations are mediated by histone modifications, we next performed CUT&Tag for four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3), and the results implied that epigenetic alterations may also play a role in neuronal function as well as transcriptional regulation. Reanalysis of previously published RNA-seq data on the manipulation of histone modification-associated factors (including Kdm6b, Brd4, and Setd1a) suggested that these enzymes were probably involved in jSI-induced gene expression alterations. Taken together, our comprehensive analysis implies the involvement of histone modification regulation in jSI-related alterations of gene expression in NAc.

Plasma Membrane Calcium ATPase (PMCA) is essential for maintaining intracellular calcium homeostasis. Previously, we used constitutive PMCA downregulation in Drosophila melanogaster dopaminergic neurons as a model to increase intracellular calcium and mimic early neuronal alterations associated with Parkinsons disease. Here, we examined the mechanisms underlying the effects mediated by the conditional, adult-specific downregulation of PMCA in dopaminergic neurons in Drosophila melanogaster, both in vivo and in primary neuronal cultures. Adult-specific conditional silencing of PMCA in dopaminergic neurons reduced lifespan but to a lesser extent than the constitutive model and impaired locomotor performance. At the cellular level, PMCA-downregulated dopaminergic neurons exhibited elevated basal calcium, indicating disrupted calcium regulation. This was associated with a progressive increase in presynaptic vesicles and extracellular dopamine levels, suggesting enhanced neurotransmitter release. Notably, the synaptic active zone structure was preserved, indicating primarily functional rather than structural alterations. In primary neuronal cultures, PMCA downregulation reduced dopaminergic neuron survival and induced transient increases in neurite branching. Together, these findings show that PMCA downregulation leads to calcium dysregulation and presynaptic dysfunction without overt neurodegeneration in vivo, while promoting premature neuronal death in culture, indicating increased vulnerability and supporting a pre-degenerative state in which synaptic alterations precede neuronal loss.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

BackgroundKATNIP (Katanin-interacting protein), also known as KIAA0556, is one of the human genes with pathogenic variants linked to Joubert syndrome, an archetypal neurodevelopmental ciliopathy. KATNIP is a scaffolding protein with a critical role in ciliogenesis. In this study, we characterized the ciliopathy phenotypes due to KATNIP gene deletion. ResultsWe produced a Katnip null mouse model using CRISPR-Cas12a (Cpf1). The null heterozygotes appeared normal while the homozygotes died around postnatal day 9, showing severe hydrocephalus and deficiency in neuroprogenitor cell proliferation. Katnip-deficient cells in the brain have a higher rate of cilia formation and longer cilia than wild type cells. ConclusionKATNIP loss of function gives rise to hydrocephalus found in Joubert syndrome. The results indicate that KATNIP restricts ciliogenesis and cilia extension and supports proliferation of neuroprogenitor cells in the brain.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

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

Parkinsons disease (PD) is associated with motor impairment and cortical synaptic dysfunction, which involve altered glutamate receptor trafficking, yet the underlying mechanisms remain incompletely understood. VPS26B, a component of the retromer complex, regulates GluA1 recycling in the trans-entorhinal cortex region. However, its role in the primary motor cortex (M1) under Parkinsonian conditions has not been explored. Here, we show that VPS26B levels are reduced in the M1 of an MPTP-induced PD mouse model, accompanied by decreased surface GluA1 and synaptic protein levels. VPS26B overexpression partially attenuated these alterations. In the accelerating rotarod test, VPS26B-deficient mice exhibited unstable motor performance following MPTP administration, whereas VPS26B overexpression was associated with improved performance in both wild-type and knockout mice. These findings suggest that cortical VPS26B may contribute to maintaining glutamate receptor surface expression and synaptic protein levels, especially under Parkinsonian conditions, with potential implications for motor learning.

The effect of sortilin inhibition on acute inner retinal neurodegeneration induced by optic nerve crush was investigated. Pharmacological sortilin inhibition using intravitreal delivery of a polyclonal antibody or a small-molecule inhibitor was evaluated in C57BL/6JRj male mice subjected to unilateral crush. Inner retinal thickness was evaluated by optical coherence tomography, and retinal ganglion cell density was determined in retinal flat mounts. Furthermore, the effect of constitutive sortilin deficiency was examined using Sort1-/- mice. Changes in protein and mRNA levels of sortilin, p75NTR, and associated injury markers were analyzed. Neither pharmacological inhibition or constitutive loss of sortilin protected against inner retinal thinning or retinal ganglion cell loss following optic nerve crush. A transient 1.4-fold increase in p75NTR mRNA was observed early after injury, accompanied by a two-fold increase in protein levels. While sortilin expression remained largely unchanged, sortilin deficiency was associated with an altered baseline retinal state, including increased GFAP, p75NTR, and proBDNF levels. Following optic nerve crush, the induction of p75NTR was significantly attenuated in sortilin-deficient retinas compared with wild type, without affecting the extent of RGC degeneration. In summary, sortilin inhibition does not preserve inner retinal structure following optic nerve crush, but modulates glial activation, inflammatory signaling, and proneurotrophin dynamics. These findings indicate that sortilin-dependent pathways are not key drivers of optic nerve crush-induced neurodegeneration but may be more relevant in disease contexts characterized by chronic stress and neuroinflammation.

Sympathetic neuronal (SN) activity critically regulates the development and function of peripheral organs and tissues. Activity-dependent plasticity has been shown to modulate SN output, suggesting that compensatory forms of plasticity could contribute to maintaining stability of sympathetic circuits. Early SN hyperactivity drives the development of hypertension in humans and in the spontaneously hypertensive rat (SHR). In this study we used chemogenetic and pharmacological approaches, and took advantage of the enhanced activity of SHR SNs, to examine how long-term changes in activity impact synaptic properties in neonatal SN cultures. We showed that bidirectional changes in SN activity result in compensatory shifts in synaptic density that counteract long-term activity manipulations. These changes were mediated by satellite glial cells (SGCs), a non-neuronal cell in the sympathetic ganglia that has been shown to influence cholinergic synaptic sites during development. In the absence of SGCs there was no induction of homeostatic plasticity. Further, direct chemogenetic activation of SGCs was sufficient to drive compensatory plasticity, while glial inhibition blocked SN plasticity. We found that SGCs respond to cholinergic signaling by downregulating the expression of the synaptic regulators NGF and TNF, suggesting that neurons and glia interact to stabilize sympathetic output during long-term changes in circuit activity. Finally, we investigated whether these plasticity mechanisms are present in neonatal SHR SNs. We demonstrated that SHR SNs have an attenuated response to glia, both during synapse formation and activity-dependent plasticity. Taken together, this work outlines a novel homeostatic activity-dependent plasticity mechanism in the peripheral nervous system.

Motoneurons are under strong pressure to maintain stable motor output throughout an individual life, through homeostatic regulation of their electrical properties. Dysregulated spinal motoneuron excitability has long been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent work in SOD1G93A mice suggests that the homeostatic response of motoneurons becomes dysregulated as cellular processes are disrupted by the disease, causing fluctuations in motoneuron electrical properties. Yet, few studies directly test whether ALS motoneurons respond differently than wild type motoneurons to a common chronic perturbation. Here, we used in vivo electrophysiology to test whether motoneurons from pre-symptomatic SOD1G93A mice modulate excitability differently than wild type motoneurons in response to the same homeostatic perturbation: chronic inhibition exerted by the benzodiazepine diazepam. Using linear mixed-effects statistical models, we assessed whether diazepam treatment differentially modulated passive properties, firing behavior, spike properties, and/or synaptic inputs in SOD1G93A versus wild type motoneurons. We identified a significant genotype x treatment interaction effect selectively for properties related to passive membrane integration and spike initiation, including membrane time constant, peak input resistance, and recruitment current. In contrast, firing gain, spike waveform characteristics, and synaptic inputs were largely unaffected. These findings indicate that sustained inhibitory perturbation selectively triggered overactive intrinsic compensatory mechanisms in SOD1G93A motoneurons rather than inducing widespread changes in firing or synaptic transmission. Together, our results provide direct evidence for over-active homeostatic control of motoneuron excitability and support a view of motoneuron dysfunction in ALS as a problem of altered feedback regulation rather than simply hyper- or hypo-excitability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/725609v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@25f125org.highwire.dtl.DTLVardef@faf2c9org.highwire.dtl.DTLVardef@15993a8org.highwire.dtl.DTLVardef@1ed006a_HPS_FORMAT_FIGEXP M_FIG C_FIG

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon (PAH), is a widespread environmental toxicant and potent ligand of the aryl hydrocarbon receptor (AHR). Yet, how early developmental exposure to BaP influences human neurodevelopment remains poorly understood. We first examined AHR expression dynamics during human embryonic stem cell (ESC)-derived cerebral organoid development and found that AHR expression was highest at the ESC stage and declined during subsequent differentiation, suggesting a potential window of heightened susceptibility to AHR-mediated environmental perturbations. Based on this observation, ESCs were exposed to BaP (0.1, 1 M) for 7 days prior to organoid generation. BaP exposure did not alter proliferation, cell death, or global transcription of ESCs but increased expression of a subset of AHR target genes. Remarkably, however, organoids derived from BaP-exposed ESCs exhibited profound morphological defects resulting from premature neurogenesis, characterized by disrupted neural rosette organization, reduced EOMES intermediate progenitors, and increased BCL11B neurons. Pharmacological inhibition of AHR with CH-223191 attenuated AHR activation and rescued the progenitor-neuron imbalance. These findings identify AHR signaling as a critical upstream mediator of BaP-induced developmental neurotoxicity and highlight the vulnerability of early pluripotent stages to environmental insults.

Traumatic brain injury (TBI) is a condition of high incidence worldwide, but remains mostly undertreated. Previous observations in preclinical studies pointed to a beneficial effect of insulin-like growth factor 1 (IGF-1) in TBI. As brain injury is associated to loss of IGF-1 sensitivity, we tested the therapeutic potential of AIK3a305 (AIK3), a novel IGF-1 sensitizer. Twenty-four hours after mild TBI induced by controlled impact, mice received daily intraperitoneal injections of AIK3 during 4 weeks. We found that TBI-associated sensorimotor disturbances measured with the adhesive-removal test were reverted by AIK3 treatment. In addition, neurological and cognitive disturbances measured by the neurological severity score and Y maze respectively, were also ameliorated by treatment with the IGF-1 sensitizer, whereas increased anxiety after mild TBI was also normalized by AIK3. Circulating levels of IGF-1 were increased after AIK3 treatment in TBI mice, while serum IL-6 levels, a biomarker of inflammation associated to TBI were similar to control mice treated with AIK3. Transcriptomic analysis determined that treatment with AIK3 widely affected gene expression in TBI brains, showing a general reduction in both up- and down-regulated genes. Collectively, these data support the use of IGF-1 sensitizers such as AIK3 for treatment of TBI.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Clinically, burn severity is reported as the size (and depth) of burn wounds relative to total body surface area (TBSA). This nomenclature is also often used in rodent models of burns. Accordingly, accurate determination and reporting of rodent TBSA is required to ensure the rigor and reproducibility of preclinical burn research. Rodent TBSA is typically estimated indirectly as a function of body mass. Further, empirical quantification of rodent TBSA through pelt dissection does not consider differences in rodent and human anatomy, making comparison of relative burn size in rodents and humans a challenge. Here, we compared commonly used approaches to directly determine or indirectly estimate rodent TBSA to demonstrate the impact different approaches can have on the calculation of relative burn size. A total of n=48 C57BL/6J background mice (55% male) ranging from 4 to 45 weeks of age and 17 to 40 grams were used. Mice were weighed prior to euthanasia. After euthanasia, mouse length was measured from the nose to anus. Mice were then placed into clear polypropylene sheet protectors (21.6 x 27.9 cm) to trace the areas of both the dorsal and ventral surfaces as well as all four limbs (dorsal-ventral (DV) tracing). Next, the pelt was carefully excised from the body through cutting a lateral line from the mouth to the genitalia, then again proximally to distally on all four limbs. The pelt was gently placed on a sheet protector and traced when both relaxed and stretched. The ears and tail were removed and traced separately. Photographs were taken of all tracings next to a ruler for scale and analyzed in ImageJ. Stretched pelt measurements of TBSA were 34% (79.4{+/-}7.6 vs. 57.5{+/-}7.5 cm2, P<0.001) and 30% (70.6{+/-}10.9 vs. 52.7{+/-}8.1 cm2, P<0.001) greater than relaxed pelt TBSA measurements in male and female mice respectively. TBSA estimated by DV tracing was 9% greater in males (62.5{+/-}10.9 vs. 57.5{+/-}7.5 cm2) and 15% in females (60.6{+/-}12.3 vs. 52.7{+/-}8.1 cm2) compared to TBSA measurements made on relaxed pelts. Accordingly, empirically derived Meeh constants (k) from DV tracing were greater than those derived from relaxed pelt measurements for both males (7.14{+/-}0.59 vs. 6.58{+/-}0.72) and females (7.72{+/-}0.58 vs. 6.78{+/-}0.80). In contrast k values derived from stretched pelt measures of TBSA were significantly greater than those determined in relaxed pelts for males (8.91{+/-}0.87 vs. 6.58{+/-}0.72, P<0.001) and females (8.85{+/-}1.25 vs. 6.78{+/-}0.80, P>0.001). The combined ears and tail represent approximately 7% and 8% of the TBSA measured by the relaxed pelt approach, respectively. Exclusion of the tail and ears from the calculated TBSA results in derived k values that are [~]16-17% lower. The approach used to determine TBSA in mice significantly influences measured areas and thus derived k values. We suggest that stretching the pelt prior to tracing inflates TBSA values, where measurements made from relaxed pelts or by DV tracing likely provide more accurate estimates of actual TBSA. Further, exclusion of the tail and ears (the latter of which is not typically considered in estimates of TBSA in humans) may be a useful approach relating relative burn sizes of mice to those of humans.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.