Microglia induce auditory dysfunction after status epilepticus in mice.

Auditory dysfunction and increased neuronal activity in the auditory pathways have been reported in patients with temporal lobe epilepsy, but the cellular mechanisms involved are unknown. Here, we report that microglia play a role in the disinhibition of auditory pathways after status epilepticus in mice. We found that neuronal activity in the auditory pathways, including the primary auditory cortex and the medial geniculate body (MGB), was increased and auditory discrimination was impaired after status epilepticus. We further demonstrated that microglia reduced inhibitory synapses on MGB relay neurons over an 8-week period after status epilepticus, resulting in auditory pathway hyperactivity. In addition, we found that local removal of microglia from the MGB attenuated the increase in c-Fos+ relay neurons and improved auditory discrimination. These findings reveal that thalamic microglia are involved in auditory dysfunction in epilepsy.

Astrocytic cAMP modulates memory via synaptic plasticity.

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.

Neuronal brain-derived neurotrophic factor manipulates microglial dynamics.

Brain-derived neurotrophic factor (BDNF), a main member of the neurotrophin family that is active in the brain, supports neuronal survival and growth. Microglial BDNF affects both the structural and functional properties of neurons. In contrast, whether and how neuronal BDNF affects microglial dynamics remain largely undetermined. Here, we examined the effects of BDNF on the properties of microglia in the CA3 region of the hippocampus. We chose this site because the axonal boutons of hippocampal mossy fibers, which are mostly formed in the CA3 region, contain the highest levels of BDNF in the rodent brain. We transfected mouse dentate granule cells with an adeno-associated virus that encodes both a BDNF short hairpin RNA (shRNA) and red fluorescent protein to examine the effects of mossy fiber-derived BDNF on microglia. Based on immunohistochemistry, BDNF knockdown with an shRNA resulted in an increase in microglial density in the mossy fiber pathway and increased engulfment of mossy fiber axons by microglia. In addition, we performed time-lapse imaging of microglial processes in hippocampal slice cultures to examine the effects of BDNF on microglial motility. Time-lapse imaging revealed increases in the motility of microglial processes and the engulfment of mossy fiber synapses by microglia when BDNF signaling was pharmacologically blocked. Thus, neuronal BDNF prevents microglia from engulfing mossy fiber synapses in the hippocampus.

Highly active neurons emerging in vitro.

Mean firing rates vary across neurons in a neuronal network. Although most neurons infrequently emit spikes, a small fraction of neurons exhibit extremely high frequencies of spikes; this fraction of neurons plays a pivotal role in information processing, however, little is known about how these outliers emerge and whether they are maintained over time. In primary cultures of mouse hippocampal neurons, we traced highly active neurons every 24 h for 7 wk by optically observing the fluorescent protein dVenus; the expression of dVenus was controlled by the promoter of Arc, an immediate early gene that is induced by neuronal activity. Under default-mode conditions, 0.3%-0.4% of neurons were spontaneously Arc-dVenus positive, exhibiting high firing rates. These neurons were spatially clustered, exhibited intermittently repeated dVenus expression, and often continued to express Arc-dVenus for approximately 2 wk. Thus, highly active neurons constitute a few select functional subpopulations in the neuronal network.NEW & NOTEWORTHY The overdispersion of neuronal activity levels can often be attributed to very few neurons exhibiting extremely high firing rates, but due to technical difficulty, no studies have examined how these outliers are selected during development and whether they are maintained over time. We optically monitored highly active neurons for as long as 7 wk in vitro and found that they constituted a unique population that was different from other "mediocre" neurons with normal firing rates.

The effects of microglia- and astrocyte-derived factors on neurogenesis in health and disease.

Hippocampal neurogenesis continues throughout life and has been suggested to play an essential role in maintaining spatial cognitive function under physiological conditions. An increasing amount of evidence has indicated that adult neurogenesis is tightly controlled by environmental conditions in the neurogenic niche, which consists of multiple types of cells including microglia and astrocytes. Microglia maintain the environment of neurogenic niche through their phagocytic capacity and interaction with neurons via fractalkine-CX3CR1 signaling. In addition, microglia release growth factors such as brain-derived neurotrophic factor (BDNF) and cytokines such as tumor necrosis factor (TNF)-α to support the development of adult born neurons. Astrocytes also manipulate neurogenesis by releasing various soluble factors including adenosine triphosphate and lactate. Whereas, under pathological conditions such as Alzheimer's disease, depression, and epilepsy, microglia and astrocytes play a leading role in inflammation and are involved in attenuating the normal process of neurogenesis. The modulation of glial functions on neurogenesis in these brain diseases are attracting attention as a new therapeutic target. This review describes how these glial cells play a role in adult hippocampal neurogenesis in both health and disease, especially focusing glia-derived factors.

Extracellular Vesicles Taken up by Astrocytes.

Extracellular vesicles (EVs) are composed of lipid bilayer membranes and contain various molecules, such as mRNA and microRNA (miRNA), that regulate the functions of the recipient cell. Recent studies have reported the importance of EV-mediated intercellular communication in the brain. The brain contains several types of cells, including neurons and glial cells. Among them, astrocytes are the most abundant glial cells in the mammalian brain and play a wide range of roles, from structural maintenance of the brain to regulation of neurotransmission. Furthermore, since astrocytes can take up EVs, it is possible that EVs originating from inside and outside the brain affect astrocyte function, which in turn affects brain function. However, it has not been fully clarified whether the specific targeting mechanism of EVs to astrocytes as recipient cells exists. In recent years, EVs have attracted attention as a cell-targeted therapeutic approach in various organs, and elucidation of the targeting mechanism of EVs to astrocytes may pave the way for new therapies for brain diseases. In this review, we focus on EVs in the brain that affect astrocyte function and discuss the targeting mechanism of EVs to astrocytes.

Microglia modulate the structure and function of the hippocampus after early-life seizures.

The hippocampus is a brain region well-known to exhibit structural and functional changes in temporal lobe epilepsy. Studies analyzing the brains of patients with epilepsy and those from animal models of epilepsy have revealed that microglia are excessively activated, especially in the hippocampus. These findings suggest that microglia may contribute to the onset and aggravation of epilepsy; however, direct evidence for microglial involvement or the underlying mechanisms by which this occurs remain to be fully discovered. To date, neuron-microglia interactions have been vigorously studied in adult epilepsy models; such studies have clarified microglial responses to excessive synchronous firing of neurons. In contrast, the role of microglia in the postnatal brain of patients with epileptic seizures remain largely unclear. Some early-life seizures, such as complex febrile seizures, have been shown to cause structural and functional changes in the brain, which is a risk factor for future development of epilepsy. Because brain structure and function are actively modulated by microglia in both health and disease, it is essential to clarify the role of microglia in early-life seizures and its impact on epileptogenesis.

Ischemic Brain Injury Leads to Brain Edema via Hyperthermia-Induced TRPV4 Activation.

Brain edema is characterized by an increase in net brain water content, which results in an increase in brain volume. Although brain edema is associated with a high fatality rate, the cellular and molecular processes of edema remain largely unclear. Here, we developed an in vitro model of ischemic stroke-induced edema in which male mouse brain slices were treated with oxygen-glucose deprivation (OGD) to mimic ischemia. We continuously measured the cross-sectional area of the brain slice for 150 min under macroscopic microscopy, finding that OGD induces swelling of brain slices. OGD-induced swelling was prevented by pharmacologically blocking or genetically knocking out the transient receptor potential vanilloid 4 (TRPV4), a member of the thermosensitive TRP channel family. Because TRPV4 is activated at around body temperature and its activation is enhanced by heating, we next elevated the temperature of the perfusate in the recording chamber, finding that hyperthermia induces swelling via TRPV4 activation. Furthermore, using the temperature-dependent fluorescence lifetime of a fluorescent-thermosensitive probe, we confirmed that OGD treatment increases the temperature of brain slices through the activation of glutamate receptors. Finally, we found that brain edema following traumatic brain injury was suppressed in TRPV4-deficient male mice in vivo Thus, our study proposes a novel mechanism: hyperthermia activates TRPV4 and induces brain edema after ischemia.SIGNIFICANCE STATEMENT Brain edema is characterized by an increase in net brain water content, which results in an increase in brain volume. Although brain edema is associated with a high fatality rate, the cellular and molecular processes of edema remain unclear. Here, we developed an in vitro model of ischemic stroke-induced edema in which mouse brain slices were treated with oxygen-glucose deprivation. Using this system, we showed that the increase in brain temperature and the following activation of the thermosensitive cation channel TRPV4 (transient receptor potential vanilloid 4) are involved in the pathology of edema. Finally, we confirmed that TRPV4 is involved in brain edema in vivo using TRPV4-deficient mice, concluding that hyperthermia activates TRPV4 and induces brain edema after ischemia.

Deep learning-based quality control of cultured human-induced pluripotent stem cell-derived cardiomyocytes.

Using bright-field images of cultured human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we trained a convolutional neural network (CNN), a machine learning technique, to decide whether the qualities of cell cultures are suitable for experiments. VGG16, an open-source CNN framework, resulted in a mean F1 score of 0.89 and judged the cell qualities at a speed of approximately 2000 images per second when run on a commercially available laptop computer equipped with Core i7. Thus, CNNs provide a useful platform for the high-throughput quality control of hiPSC-CMs.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

The Astrocytic cAMP Pathway in Health and Disease.

Astrocytes are major glial cells that play critical roles in brain homeostasis. Abnormalities in astrocytic functions can lead to brain disorders. Astrocytes also respond to injury and disease through gliosis and immune activation, which can be both protective and detrimental. Thus, it is essential to elucidate the function of astrocytes in order to understand the physiology of the brain to develop therapeutic strategies against brain diseases. Cyclic adenosine monophosphate (cAMP) is a major second messenger that triggers various downstream cellular machinery in a wide variety of cells. The functions of astrocytes have also been suggested as being regulated by cAMP. Here, we summarize the possible roles of cAMP signaling in regulating the functions of astrocytes. Specifically, we introduce the ways in which cAMP pathways are involved in astrocyte functions, including (1) energy supply, (2) maintenance of the extracellular environment, (3) immune response, and (4) a potential role as a provider of trophic factors, and we discuss how these cAMP-regulated processes can affect brain functions in health and disease.

Microglia engulf viable newborn cells in the epileptic dentate gyrus.

Microglia, which are the brain's resident immune cells, engulf dead neural progenitor cells during adult neurogenesis in the subgranular zone (SGZ) of the dentate gyrus (DG). The number of newborn cells in the SGZ increases significantly after status epilepticus (SE), but whether and how microglia regulate the number of newborn cells after SE remain unclear. Here, we show that microglia rapidly eliminate newborn cells after SE by primary phagocytosis, a process by which viable cells are engulfed, thereby regulating the number of newborn cells that are incorporated into the DG. The number of newborn cells in the DG was increased at 5 days after SE in the adult mouse brain but rapidly decreased to the control levels within a week. During this period, microglia in the DG were highly active and engulfed newborn cells. We found that the majority of engulfed newborn cells were caspase-negative viable cells. Finally, inactivation of microglia with minocycline maintained the increase in the number of newborn cells after SE. Furthermore, minocycline treatment after SE induced the emergence of hilar ectopic granule cells. Thus, our findings suggest that microglia may contribute to homeostasis of the dentate neurogenic niche by eliminating excess newborn cells after SE via primary phagocytosis. GLIA 2016;64:1508-1517.

Dentate Circuitry as a Model to Study Epileptogenesis.

Epileptogenesis, which can be initiated by brain insults or gene mutations in the normal brain, is defined as the gradual (months to years) process of epilepsy development that begins before the first epileptic seizure. Epileptogenic changes include induction of immediate early genes, post-translational modification of ion-channel functions, neuronal death, gliosis, and reorganization of neural circuits. Each of these changes alone or in combination can contribute to an epileptogenic focus, which is defined by the minimal cortical region that is necessary and sufficient to induce synchronized epileptic bursting activity in neurons. Therefore to discover and develop anti-epileptogenic drugs it is essential to unveil the cellular and molecular mechanisms underlying the development of epileptogenic foci. Among the epileptogenic changes, abnormally appended excitatory recurrent circuits can directly cause synchronized bursting of neuron activity. Here, I will introduce and discuss the mechanisms underlying the development of two representative abnormal neural circuits, namely, hippocampal mossy fiber sprouting and ectopic granule cells, which are found in the dentate gyrus of patients with mesial temporal lobe epilepsy and its animal models.

The use of organotypic slice cultures for the study of epileptogenesis.

Epilepsy is a nervous system disorder characterized by recurrent seizures. Among several types of epilepsy, which accounts for a significant portion of the disease worldwide, temporal lobe epilepsy (TLE) is one of the most common types of intractable epilepsy in adulthood. It has been suggested that complex febrile seizures in early life are associated with the development of TLE later in life; however, cellular and molecular links between febrile seizures and TLE remain unclear because of the lack of an appropriate in vitro system. Using rat hippocampal slice cultures, in which many features of native organotypic organization are retained, we found that the dentate granule cells exhibit aberrant migration in the dentate hilus via enhanced excitatory GABAA receptor (GABAA -R) signaling, which results in granule cell ectopia that persists into adulthood. We further found that the granule cell ectopia is associated with spontaneous limbic seizures in adulthood. Importantly, both of these phenomena were prevented by inhibiting Na(+) K(+) 2Cl(-) co-transporter (NKCC1) which mediates the excitatory action of GABA.

Microglia and GABA: Diverse functions of microglia beyond GABA-receiving cells.

Neurotransmitters modulate intracellular signaling not only in neurons but also in glial cells such as astrocytes, which form tripartite synapses, and oligodendrocytes, which produce the myelin sheath on axons. Another major glial cell type, microglia, which are often referred to as brain-resident immune cells, also express receptors for neurotransmitters. Recent studies have mainly focused on excitatory neurotransmitters such as glutamate, and few have examined microglial responses to the inhibitory neurotransmitter GABA. Microglia can also structurally and functionally modulate inhibitory neuronal circuits, but the underlying molecular mechanisms remain largely unknown. Since the well-regulated balance of excitatory/inhibitory (E/I) neurotransmission is believed to be the basis of proper brain function, understanding how microglia regulate and respond to inhibitory neurotransmission will help us deepen our knowledge of neuron-glia interactions. In this review, we discuss the mechanisms by which GABA alters microglial behavior and the possibility that microglia are more than just GABA-receiving cells.

[Synaptic phagocytosis by multiple glial cell types].

Neurons in the brain build circuits by synapsing with each other, and glial cells are involved in the formation and elimination of synapses. Glial cells include microglia, astrocytes, and oligodendrocytes, each with distinctive functions supported by different gene expression patterns and morphologies, but all have been shown to regulate the number of synapses in the neuronal circuits through a common function, synaptic phagocytosis. It has also been reported that specific glial cell types phagocytose specific synapses in different brain regions and at different times, and some of the molecular mechanisms involved in each phagocytotic process have been elucidated. For example, microglia, the most frequently reported glial cell type in relation to synaptic phagocytes, are known to recognize various "eat me signals" including complement and phagocytose synapses, contributing to the refinement of neuronal circuits during development. More recently, astrocytes and oligodendrocyte precursor cells have also been shown to be involved in synaptic phagocytosis. Interestingly, there are also reports of different types of glial cells phagocytosing the same types of synapses. And in some cases, it has been suggested that different glial cell types regulate each other's synaptic phagocytosis. In this review, we will discuss the significance of synaptic phagocytosis by multiple types of glial cells by presenting recent studies on synaptic phagocytosis by glial cells.

Microarray dataset of gene transcription in mouse microglia and peripheral monocytes in contextual fear conditioning.

The transcription profile of microglia related to fear conditioning remains unclear. Here, we used Illumina MouseWG-6v2 microarrays to investigate the gene transcription changes in microglia and peripheral monocytes after contextual fear conditioning of C57BL/6 J mice. Mice were trained with or without a single minimized footshock stimulation (0-s or 2-s, 0.4 mA) and re-exposed to the training context without footshock for three different durations 24 h later: 0 min (FS0), 3 min (FS3), or 30 min (FS30). Whole brain microglia and peripheral monocytes were prepared 24 h after re-exposure using a neural tissue dissociation kit, including non-footshock controls for two re-exposure durations (Con3 and Con30). The data can be valuable for researchers interested in glial cells and neurotransmission studies and are related to the research article "Contextual fear conditioning regulates synapse-related gene transcription in mouse microglia".

Prenatal stress inhibits neuronal maturation through downregulation of mineralocorticoid receptors.

Prenatal stress (PS) increases the risk of depressive disorders in adult offspring. The pathophysiology of depressive disorders has been linked to hippocampal dysfunction; however, whether and how PS attenuates the development and function of hippocampal networks remains unknown. Using a rat model of PS, in which pregnant mothers receive daily restraint stress during late gestation and their offspring exhibit depressive-like behavior later in life, we show that PS impairs the morphological and functional maturation of hippocampal granule cells in adult offspring via the downregulated expression of mineralocorticoid receptors. PS reduced the dendritic complexity and spine density of neonatal-generated granule cells, which persists into adulthood. These granule cells exhibited depressed synaptic responses to stimulation of the medial perforant path. We further revealed that the expression of mineralocorticoid receptors, which we found is necessary for proper dendritic maturation in this study, was significantly downregulated in granule cells after PS. These results suggest that PS-induced downregulation of mineralocorticoid receptors attenuates neuronal maturation, which results in dysfunction of neuronal network in adulthood.

[Microglial Response to Brain Temperature and Neuronal Circuit Reorganization].

Brain tissue is vulnerable to temperature changes, which are known to affect the structure and function of neural circuits. Owing to their dynamic ramified processes, microglia, which serve as immune cells in the brain, are associated with surveillance of the brain environment and mediate synaptic pruning to reorganize neural circuits. In this section, we discuss the possible role of microglia as temperature sensors in the brain via thermosensitive transient receptor potential channels and their role in reorganization of neural circuits.

Replacement of Mouse Microglia With Human Induced Pluripotent Stem Cell (hiPSC)-Derived Microglia in Mouse Organotypic Slice Cultures.

Microglia, the major immune cells in the brain, are reported to differ in gene expression patterns among species. Therefore, it would be preferable in some cases to use human microglia rather than mouse microglia in microglia-targeted disease research. In the past half a decade, researchers have developed in vivo transplantation methods in which human induced pluripotent stem cell-derived microglia (hiPSC-MG) are transplanted into a living mouse brain. However, in vivo transplantation methods are not necessarily accessible to all researchers due to the difficulty of obtaining the materials needed and the transplantation technique itself. In addition, for in vivo systems for microglia-targeted drug screening, it is difficult to control the pharmacokinetics, especially blood-brain barrier permeability. Therefore, in addition to existing in vivo transplantation systems, the development of an ex vivo transplantation system would help to further evaluate the properties of hiPSC-MG. In this study, we aimed to establish a method to efficiently transplant hiPSC-MG into cultured mouse hippocampal slices. We found that approximately 80% of the total microglia in a cultured slice were replaced by hiPSC-derived microglia when innate microglia were pharmacologically removed prior to transplantation. Furthermore, when neuronal death was induced by applying Kainic acid (KA) to slice cultures, transplanted hiPSC-MG changed their morphology and phagocytosed cell debris. Thus, this study provides a method to transplant hiPSC-MG into the mouse hippocampal slice cultures with a high replacement rate. Because the transplanted microglia survived and exerted phagocytic functions, this method will be useful for evaluating the properties of hiPSC-MG ex vivo.