Mouse Cerebral Cortex Research Articles

Fut9 Deficiency Causes Abnormal Neural Development in the Mouse Cerebral Cortex and Retina

α1,3-Fucosyltransferase 9 (Fut9) is responsible for the synthesis of Lewis X [LeX, Galβ1-4(Fucα1-3)GlcNAc] carbohydrate epitope, a marker for pluripotent or multipotent tissue-specific stem cells. Although Fut9-deficient mice show anxiety-related behaviors, structural and cellular abnormalities in the brain remain to be investigated. In this study, using in situ hybridization and immunohistochemical techniques in combination, we clarified the spatiotemporal expression of Fut9, together with LeX, in the brain and retina. We found that Fut9-expressing cells are positive for Ctip2, a marker of neurons residing in layer V/VI, and TLE4, a marker of corticothalamic projection neurons (CThPNs) in layer VI, of the cortex. A birthdating analysis using 5-ethynyl-2'-deoxyuridine at embryonic day (E)11.5, 5-bromo-2'-deoxyuridine at E12.5, and in utero electroporation of a GFP expression plasmid at E14.5 revealed a reduction in the percentage of neurons produced at E11.5 in layer VI/subplate of the cortex and in the ganglion cell layer of the retina in P0 Fut9-/- mice. Furthermore, this reduction in layer VI/subplate neurons persisted into adulthood, leading to a reduction in the number of Ctip2strong/Satb2- excitatory neurons in layer V/VI of the adult Fut9-/- cortex. These results suggest that Fut9 plays significant roles in the differentiation, migration, and maturation of neural precursor cells in the cortex and retina.

Brain injury environment critically influences the connectivity of transplanted neurons.

Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.

In Vivo Dynamic Movement of Polymerized Amyloid β in the Perivascular Space of the Cerebral Cortex in Mice.

Disposition of amyloid β (Aβ) into the perivascular space of the cerebral cortex has been recently suggested as a major source of its clearance, and its disturbance may be involved in the pathogenesis of cerebral amyloid angiopathy and Alzheimer’s disease. Here, we explored the in vivo dynamics of Aβ in the perivascular space of anesthetized mice. Live images were obtained with two-photon microscopy through a closed cranial window. Either fluorescent-dye-labeled Aβ oligomers prepared freshly or Aβ fibrils after 6 days of incubation at 37 °C were placed over the cerebral cortex. Accumulation of Aβ was observed in the localized perivascular space of the penetrating arteries and veins. Transportation of the accumulated Aβ along the vessels was slow and associated with changes in shape. Aβ oligomers were transported smoothly and separately, whereas Aβ fibrils formed a mass and moved slowly. Parenchymal accumulation of Aβ oligomers, as well as Aβ fibrils along capillaries, increased gradually. In conclusion, we confirmed Aβ transportation between the cortical surface and the deeper parenchyma through the perivascular space that may be affected by the peptide polymerization. Facilitation of Aβ excretion through the system can be a key target in treating Alzheimer’s disease.

In vivo Single Cell Optical Ablation of Brain Pericytes.

Pericytes have myriad functions in cerebrovascular regulation but remain understudied in the living brain. To dissect pericyte functions in vivo, prior studies have used genetic approaches to induce global pericyte loss in the rodent brain. However, this leads to complex outcomes, making it challenging to disentangle the physiological roles of pericytes from the pathophysiological effects of their depletion. Here, we describe a protocol to optically ablate individual pericytes of the mouse cerebral cortex in vivo for fine-scale studies of pericyte function. The strategy relies on two-photon microscopy and cranial window-implanted transgenic mice with mural cell-specific expression of fluorescent proteins. Single pericyte somata are precisely targeted with pulsed infrared laser light to induce selective pericyte death, but without overt blood-brain barrier leakage. Following pericyte ablation, the changes to the local capillary network and remaining pericytes can be examined longitudinally. The approach has been used to study pericyte roles in capillary flow regulation, and the structural remodeling of pericytes involved in restoration of endothelial coverage after pericyte loss.

Cover Image

Gross morphology of a neonatal brain dissected from Yap-cKO by crossing Yapflox/flox and Nestin-Cre. While it appears to be normal, intermediate progenitor cells and upper-layer projection neurons in the cerebral cortex are decreased. From: YAP maintains the production of intermediate progenitor cells and upper-layer projection neurons in the mouse cerebral cortex; Yi-Ching Su, Tzu-Heng Hung, Tzu-Fang Wang, Ying-Hsuan Lee, Tsu-Wei Wang, and Jenn-Yah Yu; DevDyn 251:5:846-863. https://doi.org/10.1002/dvdy.448

Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy.

.SignificanceThree-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to studying multicellular interactions and dynamics in the intact brain.AimWe characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain.ApproachWe used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225- to 1360-nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP.ResultsWe find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have a high signal-to-background ratio in the 1300- to 1360-nm range, consistent with enhanced 3P cross-sections.ConclusionsThese results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy.

Protective effect of Eucommia ulmoides Oliver male flowers on ethanol-induced DNA damage in mouse cerebellum and cerebral cortex.

Ethanol is a principal ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol‐associated oxidative DNA damage in the central nervous system is well documented. Natural product may offer new options to protect the brain against ethanol‐induced neurotoxicity. The male flower of Eucommia ulmoides (EUF) Oliver has been extensively utilized as the tea, the healthy hot drink on the market. In this study, 19 constituents in the effective fraction of EUF were identified by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). In the single‐cell gel electrophoresis assay, EUF was observed to ameliorate DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration, which was further confirmed by the morphological observation. The protective effects of EUF were associated with increasing total superoxide dismutase (T‐SOD) and glutathione peroxidase (GSH‐PX) activities, and a decrease in nitric oxide (NO), malondialdehyde (MDA), 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), and kelch‐like ECH‐associated protein‐1 (Keap1) levels. Molecular docking results demonstrated that compounds 4, 7, 9, and 16 from EUF have a strong affinity to the Keap1 Kelch domain to hinder the interaction of nuclear factor‐erythroid 2‐related factor 2 (Nrf2) with Keap1. These findings suggest that EUF is a potent inhibitor of ethanol‐induced brain injury possibly via the inhibition of oxidative stress.

Sevoflurane-Induced Apoptosis in the Mouse Cerebral Cortex Follows Similar Characteristics of Physiological Apoptosis.

General anesthetics are capable of inducing neuronal apoptosis during the rapid synaptogenesis of immature mammalian brains. In this vulnerable time window, physiological apoptosis also occurs to eliminate excess and inappropriately integrated neurons. We previously showed that physiological and ketamine-induced apoptosis in mouse primary somatosensory cortex (S1) followed similar developmental patterns. However, since sevoflurane is more widely used in pediatric anesthesia, and targets mainly on different receptors, as compared with ketamine, it is important to determine whether sevoflurane-induced apoptosis also follows similar developmental patterns as physiological apoptosis or not. Mice at postnatal days 5 (P5) and P9 were anesthetized with 1.5% sevoflurane for 4 h, and the apoptotic neurons in S1 were quantitated by immunohistochemistry. The results showed that sevoflurane raised the levels of apoptosis in S1 without interfering with the developmental patterns of physiological apoptosis. The cells more vulnerable to both physiological and sevoflurane-induced apoptosis shifted from layer V pyramidal neurons at P5 to layers II–IV GABAergic neurons by P9. The magnitude of both sevoflurane-induced and physiological apoptosis was more attenuated at P9 than P5. To determine whether the Akt-FoxO1-PUMA pathway contributes to the developmental decrease in magnitude of both physiological and sevoflurane-induced apoptosis, Western blot was used to measure the levels of related proteins in S1 of P5 and P9 mice. We observed higher levels of antiapoptotic phosphorylated Akt (p-Akt) and phosphorylated FoxO1 (p-FoxO1), and lower levels of the downstream proapoptotic factor PUMA in control and anesthetized mice at P9 than P5. In addition, the Akt-FoxO1-PUMA pathway may also be responsible for sevoflurane-induced apoptosis. Together, these results suggest that magnitude, lamination pattern and cell-type specificity to sevoflurane-induced apoptosis are age-dependent and follow physiological apoptosis pattern. Moreover, The Akt-FoxO1-PUMA pathway may mediate the developmental decreases in magnitude of both physiological and sevoflurane-induced apoptosis in neonatal mouse S1.

Intranasal metformin treatment ameliorates cognitive functions via insulin signaling pathway in ICV-STZ-induced mice model of Alzheimer's disease

AimsThe relationship between type 2 diabetes and Alzheimer's disease (AD) provides evidence that insulin and insulin sensitizers may be beneficial for the treatment of AD. The present study investigated the effect and mechanism of action of intranasal metformin treatment on impaired cognitive functions in an experimental mice model of AD. Main methodsIntracerebroventricularly (ICV) streptozotocin (STZ)-injected mice were treated with intranasal or oral metformin for 4 weeks. Learning and memory functions were evaluated using Morris water maze. Metformin and Aβ42 concentrations were determined by liquid chromatography tandem mass spectrometry and ELISA respectively. The expressions of insulin receptor, Akt and their phosphorylated forms were determined in the hippocampi and cerebral cortices of mice. Key findingsICV-STZ-induced AD mice displayed impaired learning and memory functions which were improved by metformin treatment. ICV-STZ injection or intranasal/oral metformin treatments had no effect on blood glucose concentrations. Intranasal treatment yielded higher concentration of metformin in the hippocampus and lower in the plasma compared to oral treatment. ICV-STZ injection and metformin treatments did not change amyloid β-42 concentration in the hippocampus of mice. In hippocampal and cortical tissues of ICV-STZ-induced AD mice, insulin receptor (IR) and Akt expressions were unchanged, while phosphorylated insulin receptor (pIR) and pAkt expressions decreased compared to control. Metformin treatments did not change IR and Akt expressions but increased pIR and pAkt expressions. SignificanceThe present study showed for the first time that intranasal metformin treatment improved the impaired cognitive functions through increasing insulin sensitivity in ICV-STZ-induced mice model of AD.

Contribution of Opioid and Nitrergic Systems to m-Trifluoromethyl diphenyl Diselenide Attenuates Morphine-Induced Tolerance in Mice

m-Trifluoromethyl diphenyl diselenide (TFDD) has antinociceptive and antidepressant-like properties and attenuates morphine withdrawal signs in mice. This study investigated if TFDD affects the development of morphine tolerance to its antinociceptive and antidepressant-like effects in mice. We also investigated whether TFDD modulates signaling pathways related to morphine tolerance, including the opioid receptors and some parameters of the nitrergic system. Male adult Swiss mice received morphine alone (5 mg/kg, subcutaneous) and in combination with TFDD (10 mg/kg, intragastric) for 7 days. Mice were subjected to hot plate and forced swim tests on days 1, 3, 5, and 7 of the experimental protocol. Repeated TFDD administrations avoided tolerance development mediated by morphine, including its antinociceptive and antidepressant-like effects. A single morphine dose increased MOR and NOx but decreased iNOS contents in the mouse cerebral cortex. In turn, single morphine and TFDD co-administration restored the MOR and iNOS protein levels. On the other hand, morphine repeated doses enhanced DOR and reduced MOR and NOx contents, whereas the morphine and TFDD association reestablished DOR and NOx levels in the mouse cerebral cortex. In conclusion, some opioid and nitrergic system parameters might contribute to TFDD attenuation of antinociceptive and antidepressant-like tolerance induced by morphine in mice.

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy.

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns.

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.

The lncRNA-AK046375 Upregulates Metallothionein-2 by Sequestering miR-491-5p to Relieve the Brain Oxidative Stress Burden after Traumatic Brain Injury.

We previously discovered that traumatic brain injury (TBI) induces significant perturbations in long noncoding RNA (lncRNA) levels in the mouse cerebral cortex, and lncRNA-AK046375 is one of the most significantly changed lncRNAs after TBI. lncRNA-AK046375 overexpression and knockdown models were successfully constructed both in vitro and in vivo. In cultured primary cortical neurons and astrocytes, lncRNA-AK046375 sequestered miR-491-5p, thereby enhancing the expression of metallothionein-2 (MT2), which ameliorated oxidative-induced cell injury. In addition, upregulated lncRNA-AK046375 promoted the recovery of motor, learning, and memory functions after TBI in C57BL/6 mice, and the underlying mechanism may be related to ameliorated apoptosis, inhibited oxidative stress, reduced brain edema, and relieved loss of tight junction proteins at the blood-brain barrier in the mouse brain. Therefore, we conclude that lncRNA-AK046375 enhances MT2 expression by sequestering miR-491-5p, ultimately strengthening antioxidant activity, which ameliorates neurological deficits post-TBI.

Tau nuclear translocation is a leading step in tau pathology process through P53 stabilization and nucleolar dispersion

Tau protein abnormalities are associated with various neurodegenerative disorders, including Alzheimer's disease (AD) and traumatic brain injury (TBI). In tau-overexpressing SHSY5Y cells and iPSC-derived neuron models of frontotemporal dementia (FTD), axonal tau translocates into the nuclear compartment, resulting in neuronal dysfunction. Despite extensive research, the mechanisms by which tau translocation results in neurodegeneration remain elusive thus far. We studied the nuclear displacement of different P-tau species [Cis phosphorylated Thr231-tau (cis P-tau), phosphorylated Ser202/Thr205-tau (AT8 P-tau), and phosphorylated Thr212/Ser214-tau (AT100 P-tau)] at various time points using starvation in primary cortical neurons and single severe TBI (ssTBI) in male mouse cerebral cortices as tauopathy models. While all P-tau species translocated into the somatodendritic compartment in response to stress, cis P-tau did so more rapidly than the other species. Notably, nuclear localization of P-tau was associated with p53 apoptotic stabilization and nucleolar stress, both of which resulted in neurodegeneration. In summary, our findings indicate that P-tau nuclear translocation results in p53-dependent apoptosis and nucleolar dispersion, which is consistent with neurodegeneration.

Balanced Expression of G Protein-coupled Receptor Subtypes in the Mouse, Macaque, and Human Cerebral Cortex

G-protein coupled receptors (GPCRs) modulate brain function by signaling through heterotrimeric Gq/11, Gs, and Gi/o protein subtypes. Researchers frequently study neuromodulation via these GPCR-subtypes on a 'cell-by-cell' basis. Although useful to explore a small number of interactions among neuromodulatory systems under controlled settings, this approach fails to account for a global organization of GPCRs in the brain. Furthermore, because multiple receptors and signal transduction pathways are present in single cells, neuromodulation is controlled by groups of GPCRs rather than by individual receptors. Using an integrative approach, the present study examined how large GPCR-subtype communities (ensembles) are expressed in different anatomical regions. Using the Allen Brain Atlas (http://www.brain-map.org/), we analyzed the mRNA expression energy of hundreds of GPCR-subtypes located in mouse, macaque, and human brains. We found that although there was a heterogeneous expression of GPCR-mRNA across all cortical regions, there were strong spatial correlations among congregated Gq/11-, Gs-, and Gi/o-linked systems. Correlation strength increased with age but dropped when randomly removing genes from their corresponding groups. These findings suggest that the expression patterns of GPCR subtypes and receptor families are intricately intertwined. Well-orchestrated interactions by neuromodulatory-GPCR ensembles could be crucial for the brain to function as a highly integrated complex system.

Features of the cytoprotective effect of selenium nanoparticles on primary cortical neurons and astrocytes during oxygen\u2013glucose deprivation and reoxygenation

The study is aimed at elucidating the effect of selenium nanoparticles (SeNPs) on the death of cells in the primary culture of mouse cerebral cortex during oxygen and glucose deprivation (OGD). A primary cell culture of the cerebral cortex containing neurons and astrocytes was subjected to OGD and reoxygenation to simulate cerebral ischemia-like conditions in vitro. To evaluate the neuroprotective effect of SeNPs, cortical astrocytes and neurons were incubated for 24 h with SeNPs, and then subjected to 2-h OGD, followed by 24-h reoxygenation. Vitality tests, fluorescence microscopy, and real-time PCR have shown that incubation of primary cultured neurons and astrocytes with SeNPs at concentrations of 2.5–10 µg/ml under physiological conditions has its own characteristics depending on the type of cells (astrocytes or neurons) and leads to a dose-dependent increase in apoptosis. At low concentration SeNPs (0.5 µg/ml), on the contrary, almost completely suppressed the processes of basic necrosis and apoptosis. Both high (5 µg/ml) and low (0.5 µg/ml) concentrations of SeNPs, added for 24 h to the cells of cerebral cortex, led to an increase in the expression level of genes Bcl-2, Bcl-xL, Socs3, while the expression of Bax was suppressed. Incubation of the cells with 0.5 µg/ml SeNPs led to a decrease in the expression of SelK and SelT. On the contrary, 5 µg/ml SeNPs caused an increase in the expression of SelK, SelN, SelT, SelP. In the ischemic model, after OGD/R, there was a significant death of brain cells by the type of necrosis and apoptosis. OGD/R also led to an increase in mRNA expression of the Bax, SelK, SelN, and SelT genes and suppression of the Bcl-2, Bcl-xL, Socs3, SelP genes. Pre-incubation of cell cultures with 0.5 and 2.5 µg/ml SeNPs led to almost complete inhibition of OGD/R-induced necrosis and greatly reduced apoptosis. Simultaneously with these processes we observed suppression of caspase-3 activation. We hypothesize that the mechanisms of the protective action of SeNPs involve the activation of signaling cascades recruiting nuclear factors Nrf2 and SOCS3/STAT3, as well as the activation of adaptive pathways of ESR signaling of stress arising during OGD and involving selenoproteins SelK and SelT, proteins of the Bcl-2 family ultimately leading to inactivation of caspase-3 and inhibition of apoptosis. Thus, our results demonstrate that SeNPs can act as neuroprotective agents in the treatment of ischemic brain injuries.

Establishment of a system for regulating the gene expression of embryonic mouse cerebral cortex neural stem cells by in utero electroporation

To establish a system for regulating the gene expression of embryonic mouse cerebral cortex neural stem cells (NSCs) using in utero electroporation (IUE). At embryonic day 14.5, the mouse cerebral cortex NSCs were electro-transfected with the pCIG plasmid injected into the ventricle of the mouse embryo. At embryonic day 16.5 or day 17.5, embryonic mouse brain tissues were collected to prepare frozen sections. Immunofluorescence staining was used to observe the proliferation, apoptosis, division, directional differentiation, migration, and maturation of NSCs. The differentiation of NSCs into intermediate progenitors, the proliferation and apoptosis of NSCs, and the morphological development of radial axis of radial glial cells were observed at embryonic day 16.5. The differentiation of NSCs into neurons in layers V-VI of the cerebral cortex, the migration of NSCs to the lateral cerebral cortex, the development of dendrites of migrating neurons, and the maturation of neurons were observed at embryonic day 17.5. The system for regulating the gene expression of embryonic mouse cerebral cortex NSCs can be established using IUE, which is useful for the study of neural development related to the proliferation, apoptosis, division, directional differentiation, migration and maturation of NSCs in the cerebral cortex.

Recursive splicing is a rare event in the mouse brain.

Recursive splicing (RS) is a splicing mechanism to remove long introns from messenger RNA precursors of long genes. Compared to the hundreds of RS events identified in humans and drosophila, only ten RS events have been reported in mice. To further investigate RS in mice, we analyzed RS in the mouse brain, a tissue that is enriched in the expression of long genes. We found that nuclear total RNA sequencing is an efficient approach to investigate RS events. We analyzed 1.15 billion uniquely mapped reads from the nuclear total RNA sequencing data in the mouse cerebral cortex. Unexpectedly, we only identified 20 RS sites, suggesting that RS is a rare event in the mouse brain. We also identified that RS is constitutive between excitatory and inhibitory neurons and between sexes in the mouse cerebral cortex. In addition, we found that the primary sequence context is associated with RS splicing intermediates and distinguishes RS AGGT site from non-RS AGGT sites, indicating the importance of the primary sequence context in RS sites. Moreover, we discovered that cryptic exons may use an RS-like mechanism for splicing. Overall, we provide novel findings about RS in long genes in the mouse brain.

The Two-Pore Domain Potassium Channel TREK-1 Promotes Blood-Brain Barrier Breakdown and Exacerbates Neuronal Death After Focal Cerebral Ischemia in Mice.

Earlier studies have shown the neuroprotective role of TWIK-related K+ channel 1 (TREK-1) in global cerebral and spinal cord ischemia, while its function in focal cerebral ischemia has long been debated. This study used TREK-1-deficient mice to directly investigate the role of TREK-1 after focal cerebral ischemia. First, immunofluorescence assays in the mouse cerebral cortex indicated that TREK-1 expression was mostly abundant in astrocytes, neurons, and oligodendrocyte precursor cells but was low in myelinating oligodendrocytes, microglia, or endothelial cells. TREK-1 deficiency did not affect brain weight and morphology or the number of neurons, astrocytes, or microglia but did increase glial fibrillary acidic protein (GFAP) expression in astrocytes of the cerebral cortex. The anatomy of the major cerebral vasculature, number and structure of brain micro blood vessels, and blood-brain barrier integrity were unaltered. Next, mice underwent 60min of focal cerebral ischemia and 72h of reperfusion induced by the intraluminal suture method. TREK-1-deficient mice showed less neuronal death, smaller infarction size, milder blood-brain barrier (BBB) breakdown, reduced immune cell invasion, and better neurological function. Finally, the specific pharmacological inhibition of TREK-1 also decreased infarction size and improved neurological function. These results demonstrated that TREK-1 might play a detrimental rather than beneficial role in focal cerebral ischemia, and inhibition of TREK-1 would be a strategy to treat ischemic stroke in the clinic.

Effects of ring finger and tryptophan-aspartic acid 2 on dendritic spines and synapse formation in cerebral cortex neurons of mice

To clarify the functional effects of differential expression of ring finger and tryptophan-aspartic acid 2 (RFWD2) on dendritic development and formation of dendritic spines in cerebral cortex neurons of mice. Immunofluorescent staining was used to identify the location and global expression profile of RFWD2 in mouse brain and determine the co-localization of RFWD2 with the synaptic proteins in the cortical neurons. We also examined the effects of RFWD2 over-expression (RFWD2-Myc) and RFWD2 knockdown (RFWD2-shRNA) on dendritic development, dendritic spine formation and synaptic function in cultured cortical neurons. RFWD2 is highly expressed in the cerebral cortex and hippocampus of mice, and its expression level was positively correlated with the development of cerebral cortex neurons and dendrites. RFWD2 expression was detected on the presynaptic membrane and postsynaptic membrane of the neurons, and its expression levels were positively correlated with the length, number of branches and complexity of the dendrites. In cultured cortical neurons, RFWD2 overexpression significantly lowered the expressions of the synaptic proteins synaptophysin (P < 0.01) and postsynapic density protein 95 (P < 0.01), while RFWD2 knockdown significantly increased their expressions (both P < 0.05). Compared with the control and RFWD2-overexpressing cells, the neurons with RFWD2 knockdown showed significantly reduced number of dendritic spines (both P < 0.05). RFWD2 can regulate the expression of the synaptic proteins, the development of the dendrites, the formation of the dendritic spines and synaptic function in mouse cerebral cortex neurons through ubiquitination of Pea3 family members and c-Jun, which may serve as potential treatment targets for neurological diseases.