ABSTRACT
Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.
Subject(s)
Cell Separation/methods , Fertility/physiology , Fertilization in Vitro/veterinary , Insemination, Artificial , Live Birth , Microfluidic Analytical Techniques/methods , Spermatozoa/physiology , Animals , Cattle , Female , Male , Pregnancy , Sperm Motility , Spermatozoa/cytologyABSTRACT
Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the ß subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.
Subject(s)
Blastocyst/cytology , Embryonic Development , Receptors, Glycine/physiology , Zygote/cytology , Amino Acid Sequence , Animals , Blastocyst/metabolism , Female , Glycine/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Pregnancy , Transcriptome , Zygote/metabolismABSTRACT
There is substantial interest in memory reconsolidation as a target for the treatment of anxiety disorders, such as post-traumatic stress disorder. However, its applicability is restricted by reconsolidation-resistant boundary conditions that constrain the initial memory destabilization. In this study, we investigated whether the induction of synaptic protein degradation through autophagy modulation, a major protein degradation pathway, can enhance memory destabilization upon retrieval and whether it can be used to overcome these conditions. Here, using male mice in an auditory fear reconsolidation model, we showed that autophagy contributes to memory destabilization and its induction can be used to enhance erasure of a reconsolidation-resistant auditory fear memory that depended on AMPAR endocytosis. Using male mice in a contextual fear reconsolidation model, autophagy induction in the amygdala or in the hippocampus enhanced fear or contextual memory destabilization, respectively. The latter correlated with AMPAR degradation in the spines of the contextual memory-ensemble cells. Using male rats in an in vivo LTP reconsolidation model, autophagy induction enhanced synaptic destabilization in an NMDAR-dependent manner. These data indicate that induction of synaptic protein degradation can enhance both synaptic and memory destabilization upon reactivation and that autophagy inducers have the potential to be used as a therapeutic tool in the treatment of anxiety disorders.SIGNIFICANCE STATEMENT It has been reported that inhibiting synaptic protein degradation prevents memory destabilization. However, whether the reverse relation is true and whether it can be used to enhance memory destabilization are still unknown. Here we addressed this question on the behavioral, molecular, and synaptic levels, and showed that induction of autophagy, a major protein degradation pathway, can enhance memory and synaptic destabilization upon reactivation. We also show that autophagy induction can be used to overcome a reconsolidation-resistant memory, suggesting autophagy inducers as a potential therapeutic tool in the treatment of anxiety disorders.
Subject(s)
Autophagy , Memory , Synaptic Transmission , Amygdala/metabolism , Amygdala/physiology , Animals , Endocytosis , Hippocampus/metabolism , Hippocampus/physiology , Male , Mice , Mice, Inbred C57BL , Proteolysis , Rats , Rats, Wistar , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
Glioma is an aggressive and incurable disease, and is frequently accompanied by augmented platelet-derived growth factor (PDGF) signaling. Overexpression of PDGF-B ligand characterizes a specific subclass of glioblastoma multiforme, but the significance of the ligand remains to be elucidated. For this end, we implanted a glioma-cell line transfected with PDGF-BB-overexpressing vector (GL261-PDGF-BB) or control vector (GL261-vector) into wild-type mouse brain, and examined the effect of glioma-derived PDGF on the tumor microenvironment. The volume of GL261-PDGF-BB rapidly increased compared with GL261-vector. Recruitment of many PDGF receptor (PDGFR)-α and Olig2-positive oligodendrocyte precursor cells and frequent hemorrhages were observed in GL261-PDGF-BB but not in GL261-vector. We then implanted GL261-PDGF-BB into the mouse brain with and without Pdgfra gene inactivation, corresponding to PDGFRα-knockout (KO) and Flox mice, respectively. The recruitment of oligodendrocyte precursor cells was largely suppressed in PDGFRα-KO than in Flox, whereas the volume of GL261-PDGF-BB was comparable between the two genotypes. Frequent hemorrhage and increased IgG-leakage were associated with aberrant vascular structures within the area where many recruited oligodendrocyte precursor cells accumulated in Flox. In contrast, these vascular phenotypes were largely normalized in PDGFRα-KO. Increased matrix metalloproteinase-9 in recruited oligodendrocyte precursor cells and decreased claudin-5 in vasculature may underlie the vascular abnormality. Glioma-derived PDGF-B signal induces cancer stroma characteristically seen in high-grade glioma, and should be therapeutically targeted to improve cancer microenvironment.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Oligodendroglia/physiology , Proto-Oncogene Proteins c-sis/physiology , Stem Cells/physiology , Animals , Antineoplastic Agents, Hormonal/pharmacology , Brain Neoplasms/drug therapy , Capillary Permeability/physiology , Cell Line, Tumor , Cerebral Hemorrhage/etiology , Collagen/physiology , Female , Gene Knockout Techniques , Genetic Vectors , Glioblastoma/drug therapy , Male , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Transplantation , Phenotype , Tamoxifen/pharmacology , Transfection , Tumor BurdenABSTRACT
BACKGROUND: The reflux of pancreatic-duodenal fluids is implicated in the pathophysiology of proton-pump inhibitor-resistant gastroesophageal reflux disease (GERD). Protease-activated receptor-2 (PAR-2) is activated by proteases, the pancreatic enzyme, trypsin, and the activated PAR-2 enhances transient receptor potential vanilloid 4 (TRPV4) function in neurons. TRPV4 stimulates ATP exocytosis in conjunction with the vesicular nucleotide transporter, which mediates mechano-transduction and vagal stimulation. The aim of the present study was to verify whether the activated PAR-2 up-regulates TRPV4 function in mouse esophageal keratinocytes, which may link to the pathophysiology in PPI-resistant GERD. METHODS: TRPV4 and PAR-2 expressions were detected by RT-PCR, immunostaining, and western blotting in mouse esophageal keratinocytes. The functional response of TRPV4 to esophageal keratinocytes was analyzed using a Ca(2+) imaging system. Cellular ATP release was examined by luciferase-luciferin reaction. TRPV4 phosphorylation was studied by immunoprecipitation and western blotting. RESULTS: PAR-2 and TRPV4 mRNAs and proteins were expressed in esophageal keratinocytes. Pre-treatment with trypsin significantly increased the responses to TRPV4 activator in esophageal keratinocytes, probably via the phosphorylation of serine residue of TRPV4 by protein kinase C and resulted in cellular ATP release from the cells. CONCLUSIONS: Activated PAR-2 with trypsin exposure up-regulated TRPV4 function and increased ATP release in mouse esophageal keratinocytes. This mechanism might be related to the pathophysiology of GERD, especially non-erosive GERD.
Subject(s)
Esophagus/cytology , Gastroesophageal Reflux/pathology , Keratinocytes/metabolism , Receptor, PAR-2/metabolism , TRPV Cation Channels/metabolism , Animals , Biopsy , Cells, Cultured , Esophagus/metabolism , Gastroesophageal Reflux/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, PAR-2/genetics , TRPV Cation Channels/genetics , Up-RegulationABSTRACT
Gastrointestinal ulcers and bleeding are serious complications of nonsteroidal anti-inflammatory drug (NSAID) use. Although administration of antibiotics and Toll-like receptor 4 knockdown mitigate NSAID-induced enteropathy, the molecular mechanism of these effects is poorly understood. Intestinal hyperpermeability is speculated to trigger the initial damage due to NSAID use. Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel expressed throughout the gastrointestinal tract epithelium that is activated by temperature, extension, and chemicals such as 5,6-epoxyeicosatrienoic acid (5,6-EET). The aim of this study was to investigate the possible role of TRPV4 in NSAID-induced intestinal damage. TRPV4 mRNA and protein expression was confirmed by RT-PCR and immunochemistry, respectively, in mouse and human tissues while TRPV4 channel activity of the intestinal cell line IEC-6 was assessed by Ca(2+)-imaging analysis. TRPV4 activators or the NSAID indomethacin significantly decreased transepithelial resistance (TER) in IEC-6 cells, and indomethacin-induced TER decreases were inhibited by specific TRPV4 inhibitors or small-interfering RNA TRPV4 knockdown, as well as by the epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, which decreased 5,6-EET levels. In TRPV4 knockout mice, indomethacin-induced intestinal damage was significantly reduced compared with WT mice. Taken together, these results show that TRPV4 activation in the intestinal epithelium caused epithelial hyperpermeability in response to NSAID-induced arachidonic acid metabolites and contributed to NSAID-induced intestinal damage. Thus, TRPV4 could be a promising new therapeutic target for the prevention of NSAID-induced intestinal damage.
Subject(s)
Indomethacin , Intestine, Small/metabolism , Peptic Ulcer/metabolism , TRPV Cation Channels/metabolism , Adult , Aged , Animals , Arachidonic Acid/metabolism , Calcium Signaling , Cell Line , Disease Models, Animal , Electric Impedance , Humans , Intestine, Small/drug effects , Intestine, Small/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Peptic Ulcer/chemically induced , Peptic Ulcer/genetics , Peptic Ulcer/pathology , Permeability , RNA Interference , Rats , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/deficiency , TRPV Cation Channels/drug effects , TRPV Cation Channels/genetics , Time Factors , TransfectionABSTRACT
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.
Subject(s)
Cognitive Dysfunction , Endophenotypes , Animals , Mice , Humans , Brain/metabolism , Cognitive Dysfunction/metabolism , Disease Models, Animal , Lactates/metabolism , Hydrogen-Ion ConcentrationABSTRACT
Glycine receptors (GlyRs) are ligand-gated chloride channels comprising alpha (α1-4) and ß subunits. The GlyR subunits play major roles in the mammalian central nervous system, ranging from regulating simple sensory information to modulating higher-order brain function. Unlike the other GlyR subunits, GlyR α4 receives relatively little attention because the human ortholog lacks a transmembrane domain and is thus considered a pseudogene. A recent genetic study reported that the GLRA4 pseudogene locus on the X chromosome is potentially involved in cognitive impairment, motor delay and craniofacial anomalies in humans. The physiologic roles of GlyR α4 in mammal behavior and its involvement in disease, however, are not known. Here we examined the temporal and spatial expression profile of GlyR α4 in the mouse brain and subjected Glra4 mutant mice to a comprehensive behavioral analysis to elucidate the role of GlyR α4 in behavior. The GlyR α4 subunit was mainly enriched in the hindbrain and midbrain, and had relatively lower expression in the thalamus, cerebellum, hypothalamus, and olfactory bulb. In addition, expression of the GlyR α4 subunit gradually increased during brain development. Glra4 mutant mice exhibited a decreased amplitude and delayed onset of the startle response compared with wild-type littermates, and increased social interaction in the home cage during the dark period. Glra4 mutants also had a low percentage of entries into open arms in the elevated plus-maze test. Although mice with GlyR α4 deficiency did not show motor and learning abnormalities reported to be associated in human genomics studies, they exhibited behavioral changes in startle response and social and anxiety-like behavior. Our data clarify the spatiotemporal expression pattern of the GlyR α4 subunit and suggest that glycinergic signaling modulates social, startle, and anxiety-like behaviors in mice.
Subject(s)
Central Nervous System , Receptors, Glycine , Mice , Humans , Animals , Receptors, Glycine/genetics , Receptors, Glycine/metabolism , Central Nervous System/metabolism , Mammals/metabolismABSTRACT
The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.
Subject(s)
CRISPR-Cas Systems , Electroporation/methods , Gene Editing/methods , Gene Targeting/methods , Mice, Knockout , Neurosciences/methods , Animals , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinase Type 1/genetics , Embryo Transfer , Exons/genetics , Gene Editing/economics , Gene Targeting/economics , Integrases , Mice , Mice, Inbred C57BL , Neurosciences/economics , TransgenesABSTRACT
BACKGROUND: The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples. OBJECTIVES: This study primarily aims to obtain baseline information on sorted spermatozoa according to its characteristics and in vitro life span. MATERIALS AND METHODS: Frozen-thawed bull semen was subjected to microfluidic sperm sorting using diffuser-type microfluidic sperm sorter (DMSS). After sorting, samples were collected as the sorted spermatozoa and unsorted residual spermatozoa and incubated at 37°C for subsequent evaluation. The samples were assessed at different time points (0 or 1, 6, and 24 h) in terms of motility, which was measured by computer-assisted sperm analysis (CASA), membrane integrity, mitochondrial function, and adenosine triphosphate (ATP) production after sorting (0 h). To determine the characteristics and efficiency of DMSS sorting, the sorted spermatozoa were compared with samples collected using the swim-up method, a conventional method in motile sperm selection. RESULTS: A comparison between the sorted and residual spermatozoa demonstrated significantly higher motility parameters, membrane integrity, and mitochondrial function of the sorted spermatozoa until 6 h after incubation. The time course decrement of membrane and mitochondrial status were subjected to curve fitting and theoretically supported. Sperm ATP production measured immediately after sorting showed higher ATP generation of the sorted spermatozoa compared with the unsorted, frozen-thawed spermatozoa. The motility parameters and mitochondrial activity of DMSS-sorted spermatozoa were higher than the swim-up-collected spermatozoa (p < 0.05). DISCUSSION AND CONCLUSION: These results indicate that DMSS sorting can strictly select highly motile spermatozoa with the ability to maintain its membrane integrity and mitochondrial function related to ATP production. We speculate that the device that is able to sort high-quality spermatozoa can have great potential in assisted reproduction.
Subject(s)
Lab-On-A-Chip Devices , Microfluidic Analytical Techniques , Sperm Motility , Spermatozoa/physiology , Adenosine Triphosphate/metabolism , Animals , Cattle , Male , Mitochondria/metabolismABSTRACT
Neurons and glia are highly polarized cells with extensive subcellular structures extending over large distances from their cell bodies. Previous research has revealed elaborate protein signaling complexes localized within intracellular compartments. Thus, exploring the function and the localization of endogenous proteins is vital to understanding the precise molecular mechanisms underlying the synapse, cellular, and circuit function. Recent advances in CRISPR/Cas9-based genome editing techniques have allowed researchers to rapidly develop transgenic animal models and perform single-cell level genome editing in the mammalian brain. Here, we introduce and comprehensively review the latest techniques for genome-editing in whole animals using fertilized eggs and methods for gene editing in specific neuronal populations in the adult or developing mammalian brain. Finally, we describe the advantages and disadvantages of each technique, as well as the challenges that lie ahead to advance the generation of methodologies for genome editing in the brain using the current CRISPR/Cas9 system.
ABSTRACT
The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.
Subject(s)
Embryo, Mammalian , Genetic Engineering/methods , Animals , CRISPR-Cas Systems , Cryopreservation , Electroporation , Fertilization in Vitro , Humans , Mice , Mice, TransgenicABSTRACT
Clustered protocadherins (Pcdhs), a large group of adhesion molecules, are important for axonal projections and dendritic spread, but little is known about how they influence neuronal activity. The Pcdhß cluster is strongly expressed in the hippocampus, and in vivo Ca2+ imaging in Pcdhß-deficient mice revealed altered activity of neuronal ensembles but not of individual cells in this region in freely moving animals. Specifically, Pcdhß deficiency increased the number of large-size neuronal ensembles and the proportion of cells shared between ensembles. Furthermore, Pcdhß-deficient mice exhibited reduced repetitive neuronal population activity during exploration of a novel context and were less able to discriminate contexts in a contextual fear conditioning paradigm. These results suggest that one function of Pcdhßs is to modulate neural ensemble activity in the hippocampus to promote context discrimination.
Subject(s)
CA1 Region, Hippocampal/physiology , Cadherins/physiology , Conditioning, Classical/physiology , Discrimination Learning/physiology , Fear/physiology , Animals , Cadherins/deficiency , Calcium/analysis , Electroshock , Exploratory Behavior , Genes, Reporter , Genetic Vectors , Male , Mice , Mice, Knockout , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Neurons/chemistry , Neurons/ultrastructure , Open Field Test , Protein Isoforms/deficiency , Protein Isoforms/physiologyABSTRACT
BACKGROUND: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. NEW METHOD: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing. RESULTS: Freeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks. COMPARISON WITH EXISTING METHOD (S): Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods. CONCLUSION: We developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.
Subject(s)
Animals, Genetically Modified/genetics , CRISPR-Cas Systems/genetics , Cryopreservation/methods , Electroporation/methods , Gene Targeting/methods , Animals , Embryo, Mammalian/physiology , Male , Mice, Inbred C57BL , MutationABSTRACT
Previous gain-of-function studies using an optogenetic technique showed that manipulation of the hippocampal dentate gyrus or CA1 cell ensembles is important for memory reactivation and to generate synthetic or false memory. However, gain-of-function study manipulating CA3 cell ensembles has not been reported. The CA3 area of the hippocampus comprises a recurrent excitatory circuit, which is thought to be important for the generation of associations among the stored information within one brain region. We investigated whether the coincident firing of cell ensembles in one brain region, hippocampal CA3, associates distinct events. CA3 cell ensembles responding to context exploration and during contextual fear conditioning were labeled with channelrhodopsin-2 (ChR2)-mCherry. The synchronous activation of these ensembles induced freezing behavior in mice in a neutral context, in which a foot shock had never been delivered. The recall of this artificial associative fear memory was context specific. In vivo electrophysiological recordings showed that 20-Hz optical stimulation of ChR2-mCherry-expressing CA3 neurons, which is the same stimulation protocol used in behavioral experiment, induced long-term potentiation at CA3-CA3 synapses. Altogether, these results demonstrate that the synchronous activation of ensembles in one brain region, CA3 of the hippocampus, is sufficient for the association of distinct events. The results of our electrophysiology potentially suggest that this artificial association of memory events might be induced by the strengthening of synaptic efficacy between CA3 ensembles via recurrent circuit.
Subject(s)
CA3 Region, Hippocampal/cytology , Memory/physiology , Optogenetics/methods , Animals , Long-Term Potentiation , Mice, Inbred C57BL , Neurons/physiologyABSTRACT
The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.
Subject(s)
Hippocampus/physiology , Memory/physiology , Neurons/physiology , Animals , Brain Mapping/methods , Female , Hippocampus/cytology , Intravital Microscopy/methods , Luminescent Proteins/chemistry , Male , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Microscopy, Fluorescence/methods , Models, Animal , Optical Imaging/methods , Optogenetics/methods , Sleep/physiologyABSTRACT
Memories are integrated into interconnected networks; nevertheless, each memory has its own identity. How the brain defines specific memory identity out of intermingled memories stored in a shared cell ensemble has remained elusive. We found that after complete retrograde amnesia of auditory fear conditioning in mice, optogenetic stimulation of the auditory inputs to the lateral amygdala failed to induce memory recall, implying that the memory engram no longer existed in that circuit. Complete amnesia of a given fear memory did not affect another linked fear memory encoded in the shared ensemble. Optogenetic potentiation or depotentiation of the plasticity at synapses specific to one memory affected the recall of only that memory. Thus, the sharing of engram cells underlies the linkage between memories, whereas synapse-specific plasticity guarantees the identity and storage of individual memories.
Subject(s)
Long-Term Potentiation/physiology , Memory/physiology , Synapses/physiology , Amnesia, Retrograde/physiopathology , Amnesia, Retrograde/psychology , Amygdala/physiology , Animals , Auditory Perception , Basolateral Nuclear Complex/physiology , Conditioning, Classical , Fear/psychology , Male , Mental Recall/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , OptogeneticsABSTRACT
Macrophages (MΦs) are involved in folliculogenesis and ovulation. However, it is unknown which type of MΦ, M1 or M2, plays a more essential role in the ovary. CD206 or CD11c diphtheria toxin receptor transgenic (DTR) mice, which enable depletion of CD206+ M2 MΦs and CD11c+ MΦ or CD11c+ Dendritic cells (DCs), respectively, were used. Oocytes were used for in vitro fertilization and embryo transfer. In vitro fertilized embryos derived from M2 MΦ depleted oocytes were transferred to pseudo pregnant wild type mice. CD11c DTR mice were also used to investigate the role of CD11c cells, M1 MΦ and DCs in folliculogenesis. In WT mice, the proportion of CD206+ M2-like MΦs was not increased in follicular induction, while that of CD11c+ M1-like MΦs was increased. In CD206 DTR mice, folliculogenesis was normal and the ovulation number, fertilization rate, and implantation rate were similar to those in WT mice. In CD11c DTR mice, folliculogenesis was impaired with ovarian hemorrhage and the staining of platelet derived growth factor-receptor ß (PDGF-Rß), a marker of pericytes, and CD34, a marker of endothelial cells, was reduced. CD11c+ cells, M1 MΦs or DCs, may be involved in folliculogenesis, while M2 MΦs are not involved in folliculogenesis.
Subject(s)
CD11c Antigen/metabolism , Macrophages/cytology , Macrophages/metabolism , Ovarian Follicle/physiology , Animals , Cell Count , Female , Fertilization , Lectins, C-Type/metabolism , Luteinization , Mannose Receptor , Mannose-Binding Lectins/metabolism , Mice , Oocytes/metabolism , Receptors, Cell Surface/metabolismABSTRACT
The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.
Subject(s)
Cardiovascular System/metabolism , Cell Lineage , Pluripotent Stem Cells/metabolism , Somites/cytology , Somites/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation , Cells, Cultured , Humans , Male , Mesoderm , Mice , Mice, Inbred ICR , Mice, Transgenic , T-Box Domain Proteins , Transcription Factors/geneticsABSTRACT
DBA/2J mice are among the oldest and most important inbred strains still used in many research fields. However, this strain has reproductive problems, which may consume considerable time and effort during experiments requiring a large population. Because the quality of DBA/2J embryos has not yet been described in detail, we compared DBA/2J mice with the reproductively efficient C57BL/6J strain. Compared with C57BL/6J embryos, DBA/2J embryos had a slower cleavage speed (mean ± 1 SD; first cleavage: C57BL/6J, 16.87 ± 1.32 ; DBA/2J, 19.64 ± 0.96 h; P < 0.01; second cleavage: C57BL/6J, 41.12 ± 2.02 h; DBA/2J, 46.20 ± 2.68 h, P < 0.01) and lower cell counts at the morula and blastocyst stages (morula stage: C57BL/6J, 15 ± 3 cells per embryo; DBA/2J, 9 ± 5 cells per embryo; P < 0.05; blastocyst stage: C57BL/6J, 52 ± 6 cells per embryo; DBA/2J, 35 ± 14 cells per embryo; P < 0.05). In addition, the results of reciprocal in vitro fertilization and male-female reciprocal crosses revealed that these phenotypes were not affected by the sperm genome and were recessively inherited. These findings likely will facilitate the production of DBA/2J mice and genetically modified mice with their background. Our results also suggest that, due to their slow cleavage speed, DBA/2J mice can serve as a new model for human infertility.