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.

Neocortical Rebound Depolarization Enhances Visual Perception.

Animals are constantly exposed to the time-varying visual world. Because visual perception is modulated by immediately prior visual experience, visual cortical neurons may register recent visual history into a specific form of offline activity and link it to later visual input. To examine how preceding visual inputs interact with upcoming information at the single neuron level, we designed a simple stimulation protocol in which a brief, orientated flashing stimulus was subsequently coupled to visual stimuli with identical or different features. Using in vivo whole-cell patch-clamp recording and functional two-photon calcium imaging from the primary visual cortex (V1) of awake mice, we discovered that a flash of sinusoidal grating per se induces an early, transient activation as well as a long-delayed reactivation in V1 neurons. This late response, which started hundreds of milliseconds after the flash and persisted for approximately 2 s, was also observed in human V1 electroencephalogram. When another drifting grating stimulus arrived during the late response, the V1 neurons exhibited a sublinear, but apparently increased response, especially to the same grating orientation. In behavioral tests of mice and humans, the flashing stimulation enhanced the detection power of the identically orientated visual stimulation only when the second stimulation was presented during the time window of the late response. Therefore, V1 late responses likely provide a neural basis for admixing temporally separated stimuli and extracting identical features in time-varying visual environments.

cAMP-Dependent Calcium Oscillations of Astrocytes: An Implication for Pathology.

Astrocytes in various brain regions exhibit spontaneous intracellular calcium elevations both in vitro and in vivo; however, neither the temporal pattern underlying this activity nor its function has been fully evaluated. Here, we utilized a long-term optical imaging technique to analyze the calcium activity of more than 4000 astrocytes in acute hippocampal slices as well as in the neocortex and hippocampus of head-restrained mice. Although astrocytic calcium activity was largely sparse and irregular, we observed a subset of cells in which the fluctuating calcium oscillations repeated at a regular interval of ∼30 s. These intermittent oscillations i) depended on type 2 inositol 1,4,5-trisphosphate receptors; ii) consisted of a complex reverberatory interaction between the soma and processes of individual astrocytes; iii) did not synchronize with those of other astrocytes; iv) did not require neuronal firing; v) were modulated through cAMP-protein kinase A signaling; vi) were facilitated under pathological conditions, such as energy deprivation and epileptiform hyperexcitation; and vii) were associated with enhanced hypertrophy in astrocytic processes, an early hallmark of reactive gliosis, which is observed in ischemia and epilepsy. Therefore, calcium oscillations appear to be associated with a pathological state in astrocytes.

Memory formation and retrieval of neuronal silencing in the auditory cortex.

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.

Long-delayed expression of the immediate early gene Arc/Arg3.1 refines neuronal circuits to perpetuate fear memory.

Fear memories typically persist for long time periods, and persistent fear memories contribute to post-traumatic stress disorder. However, little is known about the cellular and synaptic mechanisms that perpetuate long-term memories. Here, we find that mouse hippocampal CA1 neurons exhibit biphasic Arc (also known as Arg3.1) elevations after fear experience and that the late Arc expression regulates the perpetuation of fear memoires. An early Arc increase returned to the baseline after 6 h, followed by a second Arc increase after 12 h in the same neuronal subpopulation; these elevations occurred via distinct mechanisms. Antisense-induced blockade of late Arc expression disrupted memory persistence but not formation. Moreover, prolonged fear memories were associated with the delayed, specific elimination of dendritic spines and the reactivation of neuronal ensembles formed during fear experience, both of which required late Arc expression. We propose that late Arc expression refines functional circuits in a delayed fashion to prolong fear memory.

Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA.

Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

Operant conditioning of synaptic and spiking activity patterns in single hippocampal neurons.

Learning is a process of plastic adaptation through which a neural circuit generates a more preferable outcome; however, at a microscopic level, little is known about how synaptic activity is patterned into a desired configuration. Here, we report that animals can generate a specific form of synaptic activity in a given neuron in the hippocampus. In awake, head-restricted mice, we applied electrical stimulation to the lateral hypothalamus, a reward-associated brain region, when whole-cell patch-clamped CA1 neurons exhibited spontaneous synaptic activity that met preset criteria. Within 15 min, the mice learned to generate frequently the excitatory synaptic input pattern that satisfied the criteria. This reinforcement learning of synaptic activity was not observed for inhibitory input patterns. When a burst unit activity pattern was conditioned in paired and nonpaired paradigms, the frequency of burst-spiking events increased and decreased, respectively. The burst reinforcement occurred in the conditioned neuron but not in other adjacent neurons; however, ripple field oscillations were concomitantly reinforced. Neural conditioning depended on activation of NMDA receptors and dopamine D1 receptors. Acutely stressed mice and depression model mice that were subjected to forced swimming failed to exhibit the neural conditioning. This learning deficit was rescued by repetitive treatment with fluoxetine, an antidepressant. Therefore, internally motivated animals are capable of routing an ongoing action potential series into a specific neural pathway of the hippocampal network.

Synaptic plasticity associated with a memory engram in the basolateral amygdala.

Synaptic plasticity is a cellular mechanism putatively underlying learning and memory. However, it is unclear whether learning induces synaptic modification globally or only in a subset of neurons in associated brain regions. In this study, we genetically identified neurons activated during contextual fear learning and separately recorded synaptic efficacy from recruited and nonrecruited neurons in the mouse basolateral amygdala (BLA). We found that the fear learning induces presynaptic potentiation, which was reflected by an increase in the miniature EPSC frequency and by a decrease in the paired-pulse ratio. Changes occurred only in the cortical synapses targeting the BLA neurons that were recruited into the fear memory trace. Furthermore, we found that fear learning reorganizes the neuronal ensemble responsive to the conditioning context in conjunction with the synaptic plasticity. In particular, the neuronal activity during learning was associated with the neuronal recruitment into the context-responsive ensemble. These findings suggest that synaptic plasticity in a subset of BLA neurons contributes to fear memory expression through ensemble reorganization.

Blockade of stimulus convergence in amygdala neurons disrupts taste associative learning.

Humans and non-human animals learn associations of temporally contingent stimuli to better cope with the changing environment. In animal models of classical conditioning, a neutral conditioned stimulus (CS) predicts an aversive unconditioned stimulus (US). Several lines of indirect evidence indicate that this learning may rely on stimulus convergence in a subset of neurons, but this hypothesis has not been directly tested. In the current study, we tested this hypothesis using a pharmacogenetic approach, the cAMP response element-binding protein (CREB)/Allatostatin Receptor system, to target a subset of amygdala neurons receiving convergent stimuli in mice during conditioned taste aversion. Virally infected basolateral amygdala neurons with higher CREB levels were predominantly active during CS presentation. Blocking stimulus convergence in infected neurons by silencing them during US disrupted taste associative memory. Moreover, silencing infected neurons only during CS also disrupted associative memory formation. These results provide support for the notion that convergent inputs of CS and US in a subpopulation of neurons are critical for associative memory formation.

Layer III neurons control synchronized waves in the immature cerebral cortex.

Correlated spiking activity prevails in immature cortical networks and is believed to contribute to neuronal circuit maturation; however, its spatiotemporal organization is not fully understood. Using wide-field calcium imaging from acute whole-brain slices of rat pups on postnatal days 1-6, we found that correlated spikes were initiated in the anterior part of the lateral entorhinal cortex and propagated anteriorly to the frontal cortex and posteriorly to the medial entorhinal cortex, forming traveling waves that engaged almost the entire cortex. The waves were blocked by ionotropic glutamatergic receptor antagonists but not by GABAergic receptor antagonists. During wave events, glutamatergic and GABAergic synaptic inputs were balanced and induced UP state-like depolarization. Magnified monitoring with cellular resolution revealed that the layer III neurons were first activated when the waves were initiated. Consistent with this finding, layer III contained a larger number of neurons that were autonomously active, even under a blockade of synaptic transmission. During wave propagation, the layer III neurons constituted a leading front of the wave. The waves did not enter the parasubiculum; however, in some cases, they were reflected at the parasubicular border and propagated back in the opposite direction. During this reflection process, the layer III neurons in the medial entorhinal cortex maintained persistent activity. Thus, our data emphasize the role of layer III in early network behaviors and provide insight into the circuit mechanisms through which cerebral cortical networks maturate.

Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices.

Astrocytes are thought to detect neuronal activity in the form of intracellular calcium elevations; thereby, astrocytes can regulate neuronal excitability and synaptic transmission. Little is known, however, about how the astrocyte calcium signal regulates the activity of neuronal populations. In this study, we addressed this issue using functional multineuron calcium imaging in hippocampal slice cultures. Under normal conditions, CA3 neuronal networks exhibited temporally correlated activity patterns, occasionally generating large synchronization among a subset of cells. The synchronized neuronal activity was correlated with astrocyte calcium events. Calcium buffering by an intracellular injection of a calcium chelator into multiple astrocytes reduced the synaptic strength of unitary transmission between pairs of surrounding pyramidal cells and caused desynchronization of the neuronal networks. Uncaging the calcium in the astrocytes increased the frequency of neuronal synchronization. These data suggest an essential role of the astrocyte calcium signal in the maintenance of basal neuronal function at the circuit level.

Interneuron firing precedes sequential activation of neuronal ensembles in hippocampal slices.

Neuronal firing sequences that occur during behavioral tasks are precisely reactivated in the neocortex and the hippocampus during rest and sleep. These precise firing sequences are likely to reflect latent memory traces, and their reactivation is believed to be essential for memory consolidation and working memory maintenance. However, how the organized repeating patterns emerge through the coordinated interplay of distinct types of neurons remains unclear. In this study, we monitored ongoing spatiotemporal firing patterns using a multi-neuron calcium imaging technique and examined how the activity of individual neurons is associated with repeated ensembles in hippocampal slice cultures. To determine the cell types of the imaged neurons, we applied an optical synapse mapping method that identifies network connectivity among dozens of neurons. We observed that inhibitory interneurons exhibited an increase in their firing rates prior to the onset of repeating sequences, while the overall activity level of excitatory neurons remained unchanged. A specific repeating sequence emerged preferentially after the firing of a specific interneuron that was located close to the neuron first activated in the sequence. The times of repeating sequences could be more precisely predicted based on the activity patterns of inhibitory cells than excitatory cells. In line with these observations, stimulation of a single interneuron could trigger the emergence of repeating sequences. These findings provide a conceptual framework that interneurons serve as a key regulator of initiating sequential spike activity.

Interpyramid spike transmission stabilizes the sparseness of recurrent network activity.

Cortical synaptic strengths vary substantially from synapse to synapse and exhibit a skewed distribution with a small fraction of synapses generating extremely large depolarizations. Using multiple whole-cell recordings from rat hippocampal CA3 pyramidal cells, we found that the amplitude of unitary excitatory postsynaptic conductances approximates a lognormal distribution and that in the presence of synaptic background noise, the strongest fraction of synapses could trigger action potentials in postsynaptic neurons even with single presynaptic action potentials, a phenomenon termed interpyramid spike transmission (IpST). The IpST probability reached 80%, depending on the network state. To examine how IpST impacts network dynamics, we simulated a recurrent neural network embedded with a few potent synapses. This network, unlike many classical neural networks, exhibited distinctive behaviors resembling cortical network activity in vivo. These behaviors included the following: 1) infrequent ongoing activity, 2) firing rates of individual neurons approximating a lognormal distribution, 3) asynchronous spikes among neurons, 4) net balance between excitation and inhibition, 5) network activity patterns that was robust against external perturbation, 6) responsiveness even to a single spike of a single excitatory neuron, and 7) precise firing sequences. Thus, IpST captures a surprising number of recent experimental findings in vivo. We propose that an unequally biased distribution with a few select strong synapses helps stabilize sparse neuronal activity, thereby reducing the total spiking cost, enhancing the circuit responsiveness, and ensuring reliable information transfer.

Post-retrieval late process contributes to persistence of reactivated fear memory.

Several studies have demonstrated the mechanisms involved in memory persistence after learning. However, little is known about memory persistence after retrieval. In this study, a protein synthesis inhibitor, anisomycin, was infused into the basolateral amygdala of mice 9.5 h after retrieval of contextual conditioned fear. Anisomycin attenuated fear memory after 7 d, but not after 2 d. In contrast, infusion of anisomycin 5- or 24-h post-retrieval was ineffective. These findings indicate that anisomycin attenuates the persistence of reactivated fear memory in a time-dependent manner. We propose that late protein synthesis is required for memory persistence after retrieval.

Effects of axonal topology on the somatic modulation of synaptic outputs.

Depolarization of the neuronal soma augments synaptic output onto postsynaptic neurons via long-range, axonal cable properties. Here, we report that the range of this somatic influence is spatially restricted by not only axonal path length but also a branching-dependent decrease in axon diameter. Cell-attached recordings of action potentials (APs) from multiple axon branches of a rat hippocampal CA3 pyramidal cell revealed that an AP was broadened following a 20 mV depolarization of the soma and reverted to a normal width during propagation down the axon. The narrowing of the AP depended on the distance traveled by the AP and on the number of axon branch points through which the AP passed. These findings were confirmed by optical imaging of AP-induced calcium elevations in presynaptic boutons, suggesting that the somatic membrane potential modifies synaptic outputs near the soma but not long-projection outputs. Consistent with this prediction, whole-cell recordings from synaptically connected neurons revealed that depolarization of presynaptic CA3 pyramidal cells facilitated synaptic transmission to nearby CA3 pyramidal cells, but not to distant pyramidal cells in CA3 or CA1. Therefore, axonal geometry enables the differential modulation of synaptic output depending on target location.

Novel contribution of cell surface and intracellular M1-muscarinic acetylcholine receptors to synaptic plasticity in hippocampus.

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1-mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand-binding experiments with cell-permeable and -impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1-mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic-binding sites were abolished in M1-mAChR-gene-knockout mice. Activation of cell surface M1-mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long-term potentiation. On the other hand, activation of intracellular M1-mAChRs phosphorylated extracellular-regulated kinase 1/2 and gradually enhanced long-term potentiation. Our data thus demonstrate that M1-mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.

p38 MAPKs regulate the expression of genes in the dopamine synthesis pathway through phosphorylation of NR4A nuclear receptors.

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.

Circuit topology for synchronizing neurons in spontaneously active networks.

Spike synchronization underlies information processing and storage in the brain. But how can neurons synchronize in a noisy network? By exploiting a high-speed (500-2,000 fps) multineuron imaging technique and a large-scale synapse mapping method, we directly compared spontaneous activity patterns and anatomical connectivity in hippocampal CA3 networks ex vivo. As compared to unconnected pairs, synaptically coupled neurons shared more common presynaptic neurons, received more correlated excitatory synaptic inputs, and emitted synchronized spikes with approximately 10(7) times higher probability. Importantly, common presynaptic parents per se synchronized more than unshared upstream neurons. Consistent with this, dynamic-clamp stimulation revealed that common inputs alone could not account for the realistic degree of synchronization unless presynaptic spikes synchronized among common parents. On a macroscopic scale, network activity was coordinated by a power-law scaling of synchronization, which engaged varying sets of densely interwired (thus highly synchronized) neuron groups. Thus, locally coherent activity converges on specific cell assemblies, thereby yielding complex ensemble dynamics. These segmentally synchronized pulse packets may serve as information modules that flow in associatively parallel network channels.

Large-scale calcium waves traveling through astrocytic networks in vivo.

Macroscopic changes in cerebral blood flow, such as those captured by functional imaging of the brain, require highly organized, large-scale dynamics of astrocytes, glial cells that interact with both neuronal and cerebrovascular networks. However, astrocyte activity has been studied mainly at the level of individual cells, and information regarding their collective behavior is lacking. In this work, we monitored calcium activity simultaneously from hundreds of mouse hippocampal astrocytes in vivo and found that almost all astrocytes participated en masse in regenerative waves that propagated from cell to cell (referred to here as "glissandi"). Glissandi emerged depending on the neuronal activity and accompanied a reduction in infraslow fluctuations of local field potentials and a decrease in the flow of red blood cells. This novel phenomenon was heretofore overlooked, probably because of the high vulnerability of astrocytes to light damage; glissandi occurred only when observed at much lower laser intensities than previously used.

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.