Background: While several mouse strains have recently been developed for tracing neural crest or oligodendrocyte lineages, each strain has inherent limitations. The connection between humanSOX10mutations and neural crest cell pathogenesis led us to focus on theSox10gene, which is critical for neural crest development. We generatedSox10-Venus BAC transgenic mice to monitor Sox10 expression in both normal development and in pathological processes.Results: Tissue fluorescence distinguished neural crest progeny cells and oligodendrocytes in theSox10-Venus mouse embryo. Immunohistochemical analysis confirmed that Venus expression was restricted to cells expressing endogenous Sox10. Time-lapse imaging of various tissues inSox10-Venus mice demonstrated that Venus expression could be visualized at the single-cell levelin vivodue to the intense, focused Venus fluorescence. In the adultSox10-Venus mouse, several types of mature and immature oligodendrocytes along with Schwann cells were clearly labeled with Venus, both before and after spinal cord injury.Conclusions: In the newly-developedSox10-Venus transgenic mouse, Venus fluorescence faithfully mirrors endogenous Sox10 expression and allows forin vivoimaging of live cells at the single-cell level. ThisSox10-Venus mouse will thus be a useful tool for studying neural crest cells or oligodendrocytes, both in development and in pathological processes.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca²⁺ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca²⁺, inhibits halothane- and isoflurane-induced Ca²⁺ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer’s disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

For normal neurogenesis and circuit formation, delamination of differentiating neurons from the proliferative zone must be precisely controlled; however, the regulatory mechanisms underlying cell attachment are poorly understood. Here, we show that Down syndrome cell adhesion molecule (DSCAM) controls neuronal delamination by local suppression of the RapGEF2–Rap1–N-cadherin cascade at the apical endfeet in the dorsal midbrain. Dscam transcripts were expressed in differentiating neurons, and DSCAM protein accumulated at the distal part of the apical endfeet. Cre-loxP–based neuronal labeling revealed that Dscam knockdown impaired endfeet detachment from ventricles. DSCAM associated with RapGEF2 to inactivate Rap1, whose activity is required for membrane localization of N-cadherin. Correspondingly, Dscam knockdown increased N-cadherin localization and ventricular attachment area at the endfeet. Furthermore, excessive endfeet attachment by Dscam knockdown was restored by co-knockdown of RapGEF2 or N-cadherin. Our findings shed light on the molecular mechanism that regulates a critical step in early neuronal development.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca ²⁺ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca ²⁺ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca ²⁺ imaging using the endoplasmic reticulum–targeted fluorescent probes demonstrates that the Ca ²⁺ burst originates from heat-induced Ca ²⁺ release (HICR) through RyR1-mutant channels because of the channels’ heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5′-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.