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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.

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.

Diverse transmitter systems (e.g. acetylcholine, dopamine, endocannabinoids, endorphins, glutamate, histamine, 5-hydroxytryptamine, substance P) have been implicated in the pathways by which nausea and vomiting are induced and are targets for anti-emetic drugs (e.g. 5-hydroxytryptamine3 and tachykinin NK1 antagonists). The involvement of TRPV1 in emesis was discovered in the early 1990s and may have been overlooked previously as TRPV1 pharmacology was studied in rodents (mice, rats) lacking an emetic reflex. Acute subcutaneous administration of resiniferatoxin in the ferret, dog and Suncus murinus revealed that it had “broad–spectrum” anti-emetic effects against stimuli acting via both central (vestibular system, area postrema) and peripheral (abdominal vagal afferents) inputs. One of several hypotheses discussed here is that the anti-emetic effect is due to acute depletion of substance P (or another peptide) at a critical site (e.g. nucleus tractus solitarius) in the central emetic pathway. Studies in Suncus murinus revealed a potential for a long lasting (one month) effect against the chemotherapeutic agent cisplatin. Subsequent studies using telemetry in the conscious ferret compared the anti-emetic, hypothermic and hypertensive effects of resiniferatoxin (pungent) and olvanil (non-pungent) and showed that the anti-emetic effect was present (but reduced) with olvanil which although inducing hypothermia it did not have the marked hypertensive effects of resiniferatoxin. The review concludes by discussing general insights into emetic pathways and their pharmacology revealed by these relatively overlooked studies with TRPV1 activators (pungent an non-pungent; high and low lipophilicity) and antagonists and the potential clinical utility of agents targeted at the TRPV1 system.

Center for Information and Neural Networks, Suita City,Osaka 565-0871, JapanSummaryThe frontal association cortex (FrA) is implicated in higherbrain function [1]. Aberrant FrA activity is likely to beinvolved in dementia pathology [2–4]. However, the func-tional circuits both within the FrA and with other regionsare unclear. A recent study showed that inactivation of theFrAimpairsmemoryconsolidationofanauditoryfearcondi-tioninginyoungmice[5].Inaddition,dendriticspineremod-eling of FrA neurons is sensitive to paired sensory stimulithat produce associative memory [5]. These findings sug-gest that the FrA is engaged in neural processes criticalto associative learning. Here we characterize stimulus inte-gration in the mouse FrA during associative learning. Weexperimentally separated contextual fear conditioning intocontext exposure and shock, and found that memory forma-tion requiresprotein synthesisassociatedwith bothcontextexposure and shock in the FrA. Both context exposure andshock trigger Arc, an activity-dependent immediate-earlygene, expression in the FrA, and a subset of FrA neuronswas dually activated by both stimuli. In addition, we foundthat the FrA receives projections from the perirhinal (PRh)and insular (IC) cortices and basolateral amygdala (BLA),which are implicated in context and shock encoding [6–8].PRh and IC neurons projecting to the FrA were activatedby context exposure and shock, respectively. Arc expres-sion in the FrA associated with context exposure and shockdepended on PRh activity and both IC and BLA activities,respectively.ThesefindingsindicatethattheFrAisengagedinstimulusintegrationandcontributestomemoryformationin associative learning.ResultsThe Frontal Association Cortex Is Required for MemoryFormation in Contextual Fear ConditioningAsamodelforassociativelearning,weusedacontextualfear-conditioning task, which establishes an association betweencontext and shock. To test whether the frontal associationcortex (FrA) is involved in memory formation in contextualfear conditioning, we infused (2R)-amino-5-phosphonovalericacid(APV),anN-methyl-D-aspartate(NMDA)receptorantago-nist, anisomycin, a protein synthesis inhibitor, or vehicle intothe FrA. APV and anisomycin were infused 30 min prior to, orimmediately after, contextual fear conditioning, respectively(experiment 1; Figures 1A and 1B). Contextual fear memorywas assessed by measuring the percentage of freezing timein the conditioning context 1 day after conditioning. BothAPV and anisomycin infusions disrupted freezing behavior.When anisomycin infusions were administered into the dorso-medial prefrontal cortex, which is close to the FrA (experiment2; Figure S1A available online), freezing behavior was compa-rable to that of mice administered vehicle infusions (Fig-ureS1B).TheseresultsindicatethatNMDAreceptoractivationandproteinsynthesisintheFrAarerequiredforcontextualfearconditioning.ProteinSynthesisintheFrAIsRequiredforEncodingBothContext and ShockWe aimed to determine whether the FrA encodes context,shock, or both. To this end, we separated 10 min of contextexposureandimmediateshockbya1-dayintervalandinfusedanisomycinintotheFrAaftereithercontextexposureorshock(experiment 3; Figures 1C and 1D). Anisomycin infusions intothe FrA immediately after both context exposure and immedi-ate shock disrupted freezing behavior during the test. How-ever, when anisomycin infusions were administered into theFrA 6 hr after context exposure (Figure 1E), freezing behaviorwas comparable to that of mice administered vehicle infu-sions. These results indicate that protein synthesis in the FrAis required for encoding both context and shock.FrA Neurons Receive Convergent Information RegardingContext and Shock during Fear ConditioningBecause protein synthesis in the FrA is required for encodingboth context and shock, we hypothesized that paired stimuliconverge in a subset of FrA neurons to potentially contributeto the memory trace. To visualize stimulus convergence, weanalyzed the temporal dynamics of nuclear versus cyto-plasmic Arc localization by fluorescent in situ hybridization[9]. Arc is an activity-dependent immediate-early gene thatis essential for synaptic plasticity and long-term memory[10–13]. Transcribed Arc mRNA first appears in neuronalnuclei, and processed Arc mRNA then accumulates in thecytoplasm. Thus, an analysis of the subcellular localization ofArc enabled us to identify active neuronal ensembles duringtwo behavioral tasks [9, 14, 15]. We first examined the timecourse of the nuclear and cytoplasmic Arc signal after neuralactivity in the FrA. Mice were exposed to a context for 5 minand sacrificed either immediately or 30 min later (experiment4; Figures S2A and S2B). We observed more nuclear Arc

The mechanism of response of hippocampal neurons to a specific feature in sensory stimuli is not fully understood, although the hippocampus is well known to contribute to the formation of episodic memory in the multisensory world. Using in-vivo voltage-clamp recordings from awake mice, we found that sound pulses induced a transient increase in inhibitory, but not excitatory, conductance in hippocampal CA1 pyramidal cells. In local field potentials, sound pulses induced a phase resetting of the θ oscillations, one of the major oscillatory states of the hippocampus. Repetitive sound pulses at 7 Hz (θ rhythm) increased the θ oscillation power, an effect that was abolished by a surgical fimbria-fornix lesion. Thus, tone-induced inhibition is likely of subcortical origin. It may segment hippocampal neural processing and render temporal boundaries in continuously ongoing experiences.

The primary sensory neocortex generates an internal representation of the environment, and its circuit reorganization is thought to lead to a modification of sensory perception. This reorganization occurs primarily through activity-dependent plasticity and has been well documented in animals during early developmental stages. Here, we describe a new method for the noninvasive induction of long-term plasticity in the mature brain: simple transient visual stimuli (i.e., flashing lights) can be used to induce prolonged modifications in visual cortical processing and visually driven behaviors. Our previous studies have shown that, in the primary visual cortex (V1) of mice, a flashing light stimulus evokes a long-delayed response that persists for seconds. When the mice were repetitively presented with drifting grating stimuli (conditioned stimuli) during the flash stimulus–evoked delayed response period, the V1 neurons exhibited a long-lasting decrease in responsiveness to the conditioned stimuli. The flash stimulus–induced underrepresentation of the grating motion was specific to the direction of the conditioned stimuli and was associated with a decrease in the animal’s ability to detect the motion of the drifting gratings. The neurophysiological and behavioral plasticity both persisted for at least several hours and requiredN-methyl-d-aspartate receptor activation in the visual cortex. We propose that flashing light stimuli can be used as an experimental tool to investigate the visual function and plasticity of neuronal representations and perception after a critical period of neocortical plasticity.

Spontaneous neuronal activity is present in virtually all brain regions, but neither its function nor spatiotemporal patterns are fully understood. Ex vivo organotypic slice cultures may offer an opportunity to investigate some aspects of spontaneous activity, because they self-restore their networks that collapsed during slicing procedures. In hippocampal networks, we compared the levels and patterns of in vivo spontaneous activity to those in acute and cultured slices. We found that the firing rates and excitatory synaptic activity in the in vivo hippocampus are more similar to those in slice cultures compared to acute slices. The soft confidence-weighted algorithm, a machine learning technique without human bias, also revealed that hippocampal slice cultures resemble the in vivo hippocampus in terms of the overall tendency of the parameters of spontaneous activity.

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.

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.

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre- and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity-dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization-induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization-induced branch formation was blocked by the uncoupler p-trifluoromethoxyphenylhydrazone, which suggests that mitochondria-derived ATP mediates the observed phenomena. Real-time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP-activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity-dependent formation of axon branches.

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.

The development of sophisticated fluorescent probes has contributed to the elucidation of the molecular mechanisms of many complex biological phenomena. In particular, fluorescence imaging of the calcium ion (Ca) has become an essential technique for the investigation of signaling pathways involving Ca as a second messenger. For example, changes in the intracellular Ca concentration have been found to be related to physiological responses in obesity, as well as immune responses and pathological responses in Alzheimer s disease. Because Ca signaling is involved in so many biological phenomena, it is expected that the simultaneous visualization of Ca and other biomolecules, that is, multicolor imaging, would be particularly informative. For this purpose, we require a fluorescent probe for Ca that operates in a different color window from that of probes for other molecules. Fluorescent Ca sensors can be categorized into twomain classes: those based on genetically encoded fluorescent proteins and those based on fluorescent small organic molecules. Although both types of sensors have certain advantages and drawbacks, small-molecule-based probes have the particular advantage that their AM ester form (cell-permeable acetoxymethyl ester derivative) can be readily bulk loaded into live cells with no need for transfection. Most currently used small-molecular fluorescent probes for Ca are fluorescein-based, such as Fluo-3, Fluo-4, Calcium Green-1, and Oregon Green 488 BAPTA-1, and emit green fluorescence (ca. 527 nm). There are also some redemitting fluorescent probes for Ca, such as Rhod-2 (ca. 576 nm), which is based on the rhodamine scaffold. These red-emitting fluorescent probes for Ca, including Rhod-2, are also widely used for biological studies; however, the cationic nature of the rhodamine scaffold generally causes Rhod-2 AM to localize into mitochondria. Although this behavior is useful for monitoring the Ca dynamics of mitochondria, the visualization of cytoplasmic Ca is much more important for research on Ca signaling. The influx of Ca into the cytoplasm from the extracellular environment and/or from intracellular stores (including the endoplasmic reticulum) triggers numerous cellular responses mediated by the interaction of Ca with various Ca-binding proteins, such as calmodulin and troponin C. Fura Red is a representative near-infrared fluorescent probe for Ca that is often used in biological research. However, it has extremely low fluorescence quantum efficiency (Ffl 0.013). Accordingly, the fluorescence signal is very small unless a high concentration of Fura Red or a high-powered laser is used. However, the use of a high dye concentration has a buffering effect on Ca, whereas the use of a high laser power causes rapid photobleaching of the dye and phototoxicity to the cells. Thus, a novel fluorescent probe for cytoplasmic Ca with strong emission in the long-wavelength region would be extremely useful, especially for multicolor imaging. In the present study, we designed and synthesized a novel and practical red-fluorescence-emitting probe suitable for monitoring cytoplasmic Ca and confirmed its usefulness for the visualization of stimulus-induced Ca oscillation in HeLa cells. As a fluorophore that emits in the red region, we chose TokyoMagenta (TM). The absorption and fluorescence wavelengths of this fluorescein analogue are 90 nm longer than those of fluorescein. TM was also expected to retain the advantages of the fluorescein scaffold, including cytoplasmic localization. For the development of the red fluorescent probe, we chose a combination of 2-Me-substituted TM as the fluorescent moiety and 1,2-bis(o-aminophenoxy)ethaneN,N,N’,N’-tetraacetic acid (BAPTA) as a specific chelator for Ca, and synthesized CaTM-1 (Figure 1; see also Scheme S1 in the Supporting Information). The fluorescence-activation ratio of CaTM-1 in the presence/absence of Ca is 5.6:1 (Figure 2a,b, Table 1). To further improve this ratio, we decided on the strategy of decreasing the energy of the highest occupied molecular orbital (HOMO) of the fluorophore to obtain a high level of [*] T. Egawa, K. Hirabayashi, Dr. Y. Koide, C. Kobayashi, Dr. N. Takahashi, Dr. T. Terai, Dr. T. Ueno, Dr. T. Komatsu, Dr. Y. Ikegaya, Prof. N. Matsuki, Prof. T. Nagano, Dr. K. Hanaoka Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) E-mail: khanaoka@mol.f.u-tokyo.ac.jp

Antihistamines are widely used to treat allergy symptoms. First-generation antihistamines have adverse effects on the central nervous system (CNS), such as hypnotic and amnesic effects, whereas second-generation antihistamines have poor brain penetration, and therefore, have fewer CNS-related adverse effects. Memory consists of several phases, including acquisition, consolidation, expression, and extinction. It remains unclear whether these phases are affected by antihistamines. We investigated the effects of diphenhydramine, a first-generation antihistamine, and levocetirizine and olopatadine, second-generation antihistamines, on memory phases. Mice were subjected to fear conditioning on day 1 and tested on day 2. Antihistamines were administered before conditioning, immediately after conditioning, or before the test session. Diphenhydramine (30 mg/kg) decreased freezing time when administered immediately after conditioning or before the test session. These effects were not attributable to a change in locomotor activity. Levocetirizine (0.1, 1, 10 mg/kg) and olopatadine (1, 10, 20 mg/kg) had no effects on conditioned fear. We also examined the effect of diphenhydramine and levocetirizine on the expression of an activity-dependent gene associated with the test session. Diphenhydramine, but not levocetirizine, increased Arc transcription in the central nucleus of the amygdala. These data indicate that diphenhydramine, but not levocetirizine or olopatadine, impairs the consolidation and expression of conditioned fear.

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.

Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

Hippocampal granule cells (GCs) are generated throughout the lifetime and are properly incorporated into the innermost region of the granule cell layer (GCL). Hypotheses for the well-regulated lamination of newly generated GCs suggest that polysialic acid (PSA) is present on the GC surface to modulate GC-to-GC interactions, regulating the process of GC migration; however, direct evidence of this involvement is lacking. We show that PSA facilitates the migration of newly generated GCs and that the activity of N-acetyl-α-neuraminidase 1 (NEU1, sialidase 1) cleaves PSA from immature GCs, terminating their migration in the innermost GCL. Developing a migration assay of immature GCs in vitro, we found that the pharmacological depletion of PSA prevents the migration of GCs, whereas the inhibition of PSA degradation with a neuraminidase inhibitor accelerates this migration. We found that NEU1 is highly expressed in immature GCs. The knockdown of NEU1 in newly generated GCs in vivo increased PSA presence on these cells, and attenuated the proper termination of GC migration in the innermost GCL. In conclusion, this study identifies a novel mechanism that underlies the proper lamination of newly generated GCs through the modulation of PSA presence by neuronal NEU1.

The hippocampus plays a critical role in contextual fear conditioning. Population activity in the hippocampal CA1 encoding the surrounding environment is thought to be responsible for retrieval of contextual fear memory. However, the characteristics of CA1 neuronal ensemble activity during retrieval of contextual fear memory remain unclear. Here, we examined CA1 ensemble activity during contextual fear memory expression in male C57Bl/6J mice, using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization. The “Shock” group was conditioned with a footshock in two separate chambers, whereas the “No shock” group was not exposed to shocks in the chamber. Animals were then re-exposed to either the same chamber twice or two different conditioning chambers. In the No shock group, exposure to the same chamber twice activated a more significantly overlapping neuronal population than exposure to two different chambers. In the Shock group, exposure to the same conditioning chamber twice activated a similarly overlapping neuronal population as exposure to two different chambers, with overlap smaller than in nonshocked mice exposed to the same chamber twice. Thus, population activity in the hippocampal CA1 encoding the surrounding environment is detected during spatial exploration, but absent during contextual fear memory expression. Even the variable ensemble activity of CA1 may contribute to retrieval of contextual fear memory.

Neural activity and de novo protein synthesis during a rest period following memory retrieval in the amygdala is necessary for stabilization of reactivated fear memory. Arc/Arg3.1 (Arc) expression is regulated by neural activity and is a critical protein for memory reconsolidation. However, it remains unclear whether memory retrieval alters Arc transcription during subsequent rest. In this study, the populations of mouse lateral amygdala neurons that transcribe Arc during memory retrieval and at rest were detected using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (Arc catFISH). Results demonstrated that memory retrieval alters the composition of neuronal populations, which activate Arc transcription during subsequent rest. Approximately 50% of neurons that transcribe Arc at subsequent rest, transcribed Arc during memory retrieval, whereas only approximately 10% of neurons that transcribed Arc during a rest period prior to memory retrieval transcribe Arc during memory retrieval. In contrast, re-exposure to the chamber induced less preferential Arc transcription in latent inhibited mice that received shocks but recalled less conditioned fear. Taken together, these findings indicate that neuronal subpopulations activated during fear memory retrieval preferentially transcribe Arc during subsequent rest in the lateral amygdala. This preferential Arc transcription may contribute to memory reconsolidation.

otC), is the kernel for respiratory rhythm generation. Despite previous extensive studies focusing on neurons,themechanismofhowrespiratoryrhythmisgeneratedhasnotbeenfullyunderstood. • Here we show that non-neuronal glial cells (a subset of putative astrocytes) in the preB¨ are periodically activated preceding inspiratory neuronal activity, periodic activity of putative astrocytes persists during blockade of neuronal activity, and stimulation of astrocytes in the preB¨ otC induces inspiratory neuronal firings. • These findings together with the previous report that blockade of astrocytic metabolism abolishes inspiratory neural output suggest that astrocytes are functionally involved in respiratory rhythm generation. • These results will help us better understand how respiratory rhythm is generated and how respiratory output is disturbed in various pathological conditions.

The variation of individual synaptic transmission impacts the dynamics of complex neural circuits. We performed whole-cell recordings from monosynaptically connected hippocampal neurons in rat organotypic slice cultures using a synapse mapping method. The amplitude of unitary excitatory postsynaptic current (uEPSC) varied from trial to trial and was independent of the physical distance between cell pairs. To investigate the source of the transmission variability, we obtained patch-clamp recordings from intact axons. Axonal action potentials (APs) were reliably transmitted throughout the axonal arbour and showed modest changes in width. In contrast, calcium imaging from presynaptic boutons revealed that the amplitude of AP-evoked calcium transients exhibited large variations both among different boutons at a given trial and among trials in a given bouton. These results suggest that a factor contributing to the uEPSC fluctuations is the variability in calcium dynamics at presynaptic terminals. Finally, we acquired triple whole-cell recordings from divergent circuit motifs with one presynaptic neuron projecting to two postsynaptic neurons. Consistent with the independency of calcium dynamics among axonal boutons, a series of uEPSC fluctuations was not correlated between the two postsynaptic cells, indicating that different synapses even from the same neuron act independently.We conclude that the intra-bouton and inter-bouton variability in AP-induced calcium dynamics determine the heterogeneity and independency of uEPSCs.

Understanding the physiology of axons in the central nervous system requires experimental access to intact axons. This protocol describes how to perform cell-attached recordings from narrow axon fibers (ϕ

At early developmental stages, immature neuronal networks of the neocortex and hippocampus spontaneously exhibit synchronously oscillating activities, which are believed to play roles in normal circuit maturation. The tissue development of the dentate gyrus (DG) in the hippocampal formation is exceptionally late compared with other brain regions and persists until postnatal periods. Using patch-clamp recording and functional multineuron calcium imaging, we found that the DG networks of postnatal day (P)3-7 mice spontaneously generated traveling waves of action potentials, which were initiated at the upper blade of the granule cell layer and propagated to the lower blade. The propagation was dependent on glutamatergic and electrical synapses, but not on GABAergic receptor activity. Remarkably, the DG waves were almost completely abolished in offspring born to female rats exposed to restraint stress during pregnancy. In the prenatally stressed offspring, DG granule cell dendrites developed normally until P3 and showed atrophy by P9. Thus, the DG waves may be required for the maturation of DG granule cells.

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.

Coordinating Synapses Cortical microcircuits produce cell assemblies that emit spatiotemporally orchestrated spiking activity. These activity patterns are decoded by the dendrites of downstream neurons. Whether synaptic inputs are clustered or dispersed over target dendrites at a given time is critical for determining dendritic computational power. However, such subcellular dynamics are poorly understood. In rodent organotypic slice cultures, Takahashi et al. (p. 353 ) found that dendritic spine activities were frequently synchronized within a group of spines in the immediate vicinity of one another. This local synchronization seems to reflect convergent synaptic inputs from intrinsically synchronized presynaptic neuron populations.

Perceptual memory needs slow-wave sleep We know little about the mechanisms by which the brain consolidates nondeclarative (perceptual) memories. In a series of behavioral, optogenetic, and electrophysiological experiments, Miyamoto et al. show that coordinated neuronal information flow during sleep is required for perceptual memory formation. Activity spreading from the secondary motor area (brain area M2) to the primary sensory region S1 is necessary for this particular kind of memory consolidation. Disturbing this coordinated input during slow-wave sleep immediately after memory acquisition prevented mice from learning a simple texture discrimination task. Science , this issue p. 1315

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.

Vagus nerve stimulation (VNS) is an approved treatment for epilepsy and depression and has cognition-enhancing effects in patients with Alzheimer's disease. The hippocampus is widely recognized to be related to epilepsy, depression, and Alzheimer's disease. One possible mechanism of VNS involves its effect on the hippocampus; i.e. it increases the release of noradrenaline in the hippocampus. However, the effect of VNS on synaptic transmission in the hippocampus is unknown. To determine whether VNS modulates neurotransmission in the hippocampus, we examined the effects of VNS on perforant path (PP)-CA3 synaptic transmission electrophysiologically in anaesthetized rats. VNS induces a persistent enhancement of PP-CA3 field excitatory post-synaptic potentials (fEPSPs). Arc, an immediate early gene, was used to identify active brain regions after VNS. The locus coeruleus (LC), which contains the perikarya of noradrenergic projections, harboured more Arc-positive cells, as measured by in-situ hybridization, after 10-min VNS. In addition, electrical lesions of LC neurons or intraventricular administration of the β-adrenergic receptor antagonist timolol prevented the enhancement of PP-CA3 responses by VNS. In conclusion, the protracted increase in PP-CA3 synaptic transmission that is induced by VNS entails activation of the LC and β-adrenergic receptors. Our novel findings suggest that information from the periphery modulates synaptic transmission in the CA3 region of the hippocampus.

Homer1a, an activity-dependent induced member of the scaffold protein family Homer, plays an essential role in synaptic reorganization and is widely used as a neuronal activity marker. However, the cell type transcribing Homer1a remains unidentified. Here, we investigated the main cell types of the amygdala, hippocampus, primary somatosensory cortex, and dorsal striatum that express Homer1a. Homer1a expression relevant to the baseline neural activity as well as exposure to unfamiliar and conditioned contexts was preferentially detected in the excitatory neurons within the basolateral amygdala, hippocampus, and neocortex, and in inhibitory neurons within the central amygdala and dorsal striatum. These findings indicate that Homer1a is expressed in the principal neurons of each region regardless of whether they are excitatory or inhibitory neurons.

One of the problems in imaging from brain tissues is light-scattering. Thus, multiphoton laser scanning microscopy is widely used to optically access fluorescent signals located deeply in tissues. Here we report that Nipkow-type spinning-disk one-photon confocal microscopy, which embodies high temporal resolution and slow photobleaching, is also capable of imaging tissues to a depth of up to 150 μm. Using a Nipkow-disk microscope, we conducted functional multi-cell calcium imaging of CA3 neurons from in toto intact hippocampal preparations and astrocytes from in vivo neocortical layer 1. This novel application of Nipkow-disk microscopy expands the potential usefulness of this type of microscopy and will contribute to our understanding of natural neuronal microcircuitry.

Astrocytes exhibit spontaneous calcium fluctuations. These activities have not been captured by large-scale recordings, and little is known about their collective dynamics. In situ and in vivo calcium imaging from hundreds (up to 2195) of astrocytes in the mouse hippocampus and neocortex revealed that neighboring astrocytes spontaneously exhibited synchronous calcium elevations and formed locally correlated cell groups ("clusters" of 2 to 5 astrocytes within a diameter of 81 ± 45 μm). Cluster activity accounted for approximately 10% of the astrocytic calcium events, and 44% of the clusters appeared repetitively during our observation period of 60 min. Astrocytic clusters emerged through metabotropic glutamate receptor activation, independently of neuronal activity. Neurons were depolarized by 1.5 mV when clusters appeared near their dendrites. This depolarization was mediated by non-N-methyl-D-aspartate (NMDA) glutamate receptor channels and was replicated by calcium uncaging activation of multiple astrocytes. Importantly, the activation of single astrocytes alone could not depolarize neurons but readily elicited NMDA-dependent slow inward currents in depolarized neurons. Thus, these novel ensemble dynamics of astrocytes, which cannot be captured by conventional small-scale imaging techniques, play a different role in neuronal modulation than does the sporadic activity of single astrocytes.

The dentate gyrus (DG), a part of the hippocampal formation, is a candidate target of antidepressants and may play a role in the development of depressive syndrome; however, there is no direct neurobiological evidence supporting this theory. Here, we examined whether DG integrity is necessary for the behavioural effects of acute or chronic antidepressant treatment. Microinjection of colchicine into DG severely damaged the granule cells, as confirmed by morphological, electrophysiological, and behavioural analyses. Acute treatment with desipramine and fluoxetine decreased the immobility of saline-treated rats in the forced swimming test, whereas this decrease was inhibited in colchicine-treated rats. Chronic treatment with desipramine and fluoxetine also decreased the immobility of saline-treated rats; however, the extensive DG damage induced by colchicine had no effect on this decrease. In the novelty-suppressed feeding test, chronic treatment with desipramine and fluoxetine decreased the latency to feed in saline-treated rats while, once again, the extensive DG damage caused by colchicine had no effect on this decrease. Thus, we concluded that DG integrity was required for the behavioural effects of acute but not chronic antidepressant treatment; this disparity was not due to the time interval between surgery and behavioural tests. These findings indicate that treatment duration determines the influence of DG integrity on antidepressant effects.

The activity-regulated cytoskeleton-associated gene (Arc, also known as Arg3.1) is an effector immediate-early gene rapidly induced by strong neural activity. Although a number of studies have revealed significant functions of Arc and Arc has come into widespread use as a neural activity marker in behavioral studies, the mechanisms regulating Arc transcription remain unclear. Here, we examined the conditions of Arc transcription in acute slices of dentate gyrus. Surprisingly, kainic acid (1 μM to 10 mM) application to slices did not induce Arc transcription, although intraperitoneal injection of kainic acid (20 mg/kg) induced robust Arc transcription. No types of high-frequency stimulation examined induced Arc transcription in acute slices. These findings indicate that Arc transcription is dramatically suppressed in acute slices of the dentate gyrus, in which background neural activity is markedly reduced. Burst stimulation increased the number of Arc-expressing cells in the presence of picrotoxin, in which excitation was maintained even after the end of stimulation. Moreover, the involvement of background neural activity in Arc transcription was tested by application of carbachol, a muscarinic receptor agonist. Carbachol also increased the number of Arc-expressing cells, which was blocked by atropine, a muscarinic receptor antagonist. Taken together, these findings suggest that persistent background activity is critical for Arc transcription.