EDTP enhances and protects the fluorescent signal of GFP in cleared and expanded tissues.

Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue.

Morphological analysis of descending tracts in mouse spinal cord using tissue clearing, tissue expansion and tiling light sheet microscopy techniques.

Descending tracts carry motor signals from the brain to spinal cord. However, few previous studies show the full view of the long tracts from a 3D perspective. In this study, we have followed five less well-known tracts that project from midbrain, hindbrain, and cerebellum to the mouse spinal cord, using the tissue clearing method in combination with tiling light sheet microscopy. By tracing axons in spinal cord, we found several notable features: among the five tracts the collateral "sister" branches occurred only in the axons originating from the cerebellospinal tracts; the axons from the spinal trigeminal nucleus crossed the midline of spinal cord to the contralateral side; those arising in the medullary reticular formation ventral part gave many branches in both cervical and lumbar segments; the axons from superior colliculus terminated only at upper cervical but with abundant branches in the hindbrain. Furthermore, we investigated the monosynaptic connections between the tracts and motor neurons in the spinal cord through hydrogel-based tissue expansion, and found several monosynaptic connections between the medullary reticular formation ventral part axons and spinal motor neurons. We believe that this is the first study to show the full 3D scope of the projection patterns and axonal morphologies of these five descending tracts to the mouse spinal cord. In addition, we have developed a new method for future study of descending tracts by three-dimensional imaging.

Structure of the human sodium leak channel NALCN in complex with FAM155A.

NALCN, a sodium leak channel expressed mainly in the central nervous system, is responsible for the resting Na+ permeability that controls neuronal excitability. Dysfunctions of the NALCN channelosome, NALCN with several auxiliary subunits, are associated with a variety of human diseases. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 angstroms. FAM155A forms extensive interactions with the extracellular loops of NALCN that may help stabilize NALCN in the membrane. A Na+ ion-binding site, reminiscent of a Ca2+ binding site in Cav channels, is identified in the unique EEKE selectivity filter. Despite its 'leaky' nature, the channel is closed and the intracellular gate is sealed by S6I, II-III linker and III-IV linker. Our study establishes the molecular basis of Na+ permeation and voltage sensitivity, and provides important clues to the mechanistic understanding of NALCN regulation and NALCN channelosome-related diseases.

High level of CTP synthase induces formation of cytoophidia in cortical neurons and impairs corticogenesis.

De novo synthesis of the nucleotide CTP is catalyzed by the essential pyrimidine biosynthesis enzyme CTP synthase (CTPs), which forms large-scale filamentous structures consisting of CTPs termed cytoophidia in prokaryotes and in eukaryotes. Recent studies have shown that cytoophidia are abundant in neuroepithelial stem cells in Drosophila optic lobes and that overexpression of CTPs impairs optic lobe development. Whether CTPs and cytoophidia also play a role in the development of the mammalian cortex remains elusive. Here, we show that overexpression of CTPs by in utero electroporation in the embryonic mouse brain induces formation of cytoophidia in developing cortical neurons and impairs neuronal migration. In addition, the increase of cytoophidia accelerates neuronal differentiation and inhibits neural progenitor cell proliferation by reducing their mitotic activity. Furthermore, we discovered that the cytoophidia diffused during the early G1-phase of the cell cycle. Together, our findings show, for the first time, that CTPs play a significant role in the development of the mammalian cortex.

The toxic influence of paraquat on hippocampal neurogenesis in adult mice.

Paraquat, a fast-acting non-selective contact herbicide, is considered an etiological factor related to Parkinson's disease. This study investigated its effects on hippocampal neurogenesis and cognition in adult mice as well as possible mechanisms for the effects. We administered paraquat (1.25 mg/kg, intraperitoneal injection, i.p.) and an equal volume of normal saline for 3 weeks to adult male C57BL/6J mice. The results showed that hippocampus-dependent spatial learning and memory was significantly impaired in paraquat-treated mice. Moreover, paraquat administration inhibited the proliferation of neural progenitor cells, and impaired the survival and altered the fate decision of newly generated cells in the hippocampus. The expression levels of caspase-3 and glial fibrillary acidic protein were significantly higher in paraquat-treated mice than in control mice. Interestingly, paraquat reduced the phosphorylation of Akt, but did not affect the total amount of Akt. In conclusion, our findings suggest that paraquat negatively affected adult hippocampal neurogenesis and cognition function.

Aberrant expression of LIMK1 impairs neuronal migration during neocortex development.

Neuronal migration is essential for the formation of cortical layers, and proper neuronal migration requires the coordination of cytoskeletal regulation. LIMK1 is a serine/threonine protein kinase that mediates actin dynamics by regulating actin depolymerization factor/cofilin. However, the role of LIMK1 in neuronal migration and its potential mechanism remains elusive. Here, we found that using the in utero electroporation to overexpress LIMK1 and its mutants, constitutively active LIMK1 (LIMK1-CA) and dominant-negative LIMK1 (LIMK1-DN), impaired neuronal migration in the embryonic mouse brain. In addition, the aberrant expression of LIMK1-WT and LIMK1-CA induced abnormal branching and increased the length of the leading process, while LIMK1-DN-transfected neurons gave rise to two leading processes. Furthermore, the co-transfection of LIMK1-CA and cofilin-S3A partially rescued the migration deficiency and fully rescued the morphological changes in migrating neurons induced by LIMK1-CA. Our results indicated that LIMK1 negatively regulated neuronal migration by affecting the neuronal cytoskeleton and that its effects were partly mediated by cofilin phosphorylation.

Neuronal maturation and laminar formation in the chicken optic tectum are accompanied by the transition of phosphorylated cofilin from cytoplasm to nucleus.

Laminar formation in the chicken optic tectum requires processes that coordinate proliferation, migration and differentiation of neurons, in which the dynamics of actin filaments are crucial. Cofilin plays pivotal roles in regulating actin arrangement via its phosphorylation on Ser3. Given poor studies on the profile of phosphorylated cofilin (p-cofilin) in the developing tectum, we investigated its expression pattern. As determined by immunofluorescence histochemistry and western blotting, p-cofilin could be detected in most tectal layers except for the neural epithelium. In addition, we found p-cofilin was expressed both in the cytoplasm and the nucleus. During development, the expression of the cytoplasmic p-cofilin was decreasing and the nuclear p-cofilin was gradually increasing, but the total level of p-cofilin was down regulated. Double-labeling experiments revealed that the nuclear p-cofilin could be labeled in mature neurons but undetected in immature neurons. Furthermore, the number of cells co-stained with nuclear p-cofilin and NeuN was up-regulated during lamination and 60% cells were detected to be mature neurons that can express nuclear p-cofilin just at the first appearance of completed laminae. Our results demonstrate that the maturation of neurons is accompanied by this cytoplasm-to-nucleus transition of p-cofilin, and the nuclear p-cofilin can work effectively as a marker in the laminar formation of the chicken optic tectum.

Expression of macrophage migration inhibitory factor in the mouse neocortex and posterior piriform cortices during postnatal development.

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in a vast array of cellular and biological processes. Abnormal expression of MIF has been implicated in many neurological diseases, including Parkinson's disease, epilepsy, Alzheimer's Disease, stroke, and neuropathic pain. However, the expression patterns of mif transcript and MIF protein from the early postnatal period through adulthood in the mouse brain are still poorly understood. We therefore investigated the temporal and spatial expression of MIF in the mouse neocortex during postnatal development in detail and partially in posterior piriform cortices (pPC). As determined by quantitative real-time PCR (qPCR), mif transcript gradually increased during development, with the highest level noted at postnatal day 30 (P30) followed by a sharp decline at P75. In contrast, Western blotting results showed that MIF increased constantly from P7 to P75. The highest level of MIF was at P75, while the lowest level of MIF was at P7. Immunofluorescence histochemistry revealed that MIF-immunoreactive (ir) cells were within the entire depth of the developed neocortex, and MIF was heterogeneously distributed among cortical cells, especially at P7, P14, P30, and P75; MIF was abundant in the pyramidal layer within pPC. Double immunostaining showed that all the mature neurons were MIF-ir and all the intensely stained MIF-ir cells were parvalbumin positive (Pv +) at adult. Moreover, it was demonstrated that MIF protein localized in the perikaryon, processes, presynaptic structures, and the nucleus in neurons. Taken together, the developmentally regulated expression and the subcellular localization of MIF should form a platform for an analysis of MIF neurodevelopmental biology and MIF-related nerve diseases.