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The reproductive performance of cattle can be suppressed by heat stress. Reproductive organ temperature, especially ovarian temperature, may affect follicle development and ovulation. The establishment of a technique for long-term measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperature-dependent changes in follicular development and subsequent ovulation in cows. Here we report a novel method facilitating long-term and continuous recording of ovarian parenchymal temperature in cows. The method revealed that the ovarian temperature in the luteal phase was constantly maintained lower than the vaginal temperature, and that the diurnal temperature variation in the ovary was significantly greater than that in the vagina, suggesting that the ovaries may require a lower temperature than other organs to maintain their functions. This novel method could be used for the further understanding of ovarian functions during estrous cycles in cows.

Senescence affects various aspects of sleep, and it remains unclear how sleep-related neuronal network activity is altered by senescence. Here, we recorded local field potential signals from multiple brain regions covering the forebrain in young (10-week-old) and aged (2-year-old) mice. Interregional LFP correlations across these brain regions showed smaller differences between awake and sleep states in aged mice. Multivariate analyses with machine learning algorithms with uniform manifold approximation and projection (UMAP) and robust continuous clustering (RCC) demonstrated that these LFP correlational patterns in aged mice less represented awake/sleep states than those in young mice. By housing aged mice in an enriched environment, the LFP patterns were restored to those observed in young mice. Our results demonstrate senescence-induced changes in neuronal activity at the network level and provide insight into the prevention of pathological symptoms associated with sleep disturbance in senescence.

The Argonaut octopus, commonly called the paper nautilus, has a spiral-coiled shell-like eggcase. As the main characteristics, the eggcase has no internal septum, is composed entirely of calcite with chitosan being the main polycarbonate and is reportedly formed by organic materials secreted from the membranes of the arms. Meanwhile, the biomineralized external “true” shells of the Mollusks, which includes the Cephalopods, are secreted from the mantle tissue. Therefore, the histological origin of the two shells is completely different. The question of how the Argonauts, which phylogenetically diverged from the completely shell-less octopuses, could form a converging shell-like external structure has thus intrigued biologists for a long time. To answer this question, we performed a multi-omics analysis of the transcriptome and proteome of the two congeneric Argonaut species,Argonauta argoandA. hians. Our result indicates that the shell-like eggcase is not a homolog of the shell, even at the protein level, because the Argonauts apparently recruited a different set of protein repertoires to as eggcase matrix proteins (EcMPs). However, we also found the homologs of three shell matrix proteins (SMPs) of the Conchiferan Mollusks, Pif-like, SOD, and TRX, in the eggcase matrix. The proteins were also found in the only surviving shelled Cephalopods, the nautiloidNautilus pompilius. Phylogenetic analysis revealed that homologous genes of the Conchiferan SMPs and EcMPs were found in the draft genome of shell-less octopuses. Our result reported here thus suggests that the SMP-coding genes are conserved in both shelled and shell-less Cephalopods. Meanwhile, the Argonauts adopted some of the SMP-coding genes and other non-SMP-coding genes, to form a convergent, non-homologous biomineralized external structure, the eggcase, which is autapomorphic to the group.

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

The central nervous system controls the activity states of the peripheral organs in response to various environmental changes. However, the physiological interactions across multiple organs remain largely unknown. Recently, we have developed an electrophysiological recording system that simultaneously captures neuronal population activity patterns in the brain, heartbeat signals, muscle contraction signals, respiratory signals, and vagus nerve action potentials in freely moving rodents. This paper summarizes several recent insights obtained from this recording system, including the observations that some but not all brain activity patterns are associated with peripheral organ activity in a behavioral test, and that functions across cortical networks can predict stress-induced changes in cardiac function in rats. The evidence suggests that adding information on peripheral physiological signals to behavioral data assists in a more accurate estimation of animals' mental states. The concept of such a research approach opens a new field of large-scale analysis of systemic physiological signals, termed "physiolomics," which is expected to unveil further physiological issues involving mind-body associations in health and disease.

The ventromedial prefrontal cortex (vmPFC) plays key roles in higher cognitive abilities, including mental representations and the regulation of emotion. Previous studies have reported that vmPFC activity is altered in depressed human patients, highlighting this subregion as a major site of dysfunction in neuropsychiatric diseases. To examine how neuronal activity at spike levels in the vmPFC is altered by social defeat stress, we performed electrophysiological multiunit recordings along the dorsoventral axis of the mPFC of freely moving mice. Chronic social defeat stress-susceptible mice showing an impairment in social interaction exhibited significant reductions in the overall spike frequencies of neurons in the vmPFC, but not in the dorsal mPFC. Analysis of local field potentials revealed that the vmPFC generated spatially constrained 20–40 Hz events lasting hundreds of milliseconds, with an average event frequency of 0.05 Hz; during these events, a subset of neurons were transiently inhibited. The frequency of 20–40 Hz events in the vmPFC was reduced in defeated stress-susceptible animals, and this decrease was reversed by systemic ketamine administration. The novel neurophysiological correlates of stress-induced changes in the vmPFC advance the understanding of the neural basis of stress-induced dysregulation of social behavior.

Human induced-pluripotent stem cell (hiPSC)-derived neurons develop organized neuronal networks under in vitro cultivation conditions. Here, using a multielectrode array system, we examined whether the spike patterns of hiPSC-derived neuronal populations differed in a manner that depended on the proportions of glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons in the cultures. Synchronous burst firing events spanning multiple electrodes became more frequent as the number of days in culture increased. However, at all developmental stages, the event rates of synchronous burst firing, the repertoires of synchronous burst firing, and the frequencies of sporadic spikes did not differ in cultures with different glutamatergic-to-GABAergic ratios. Pharmacological blockade of GABAergic synaptic transmission increased the frequencies of spike patterns specifically in cultures with lower glutamatergic-to-GABAergic ratios. These results demonstrate that a robust homeostatic property of developing hiPSC-derived neuronal networks in culture counteracts chronically imbalanced glutamatergic and GABAergic signaling.

Bridging accumulating insights from microscopic and macroscopic studies in neuroscience research requires monitoring of neuronal population dynamics and quantifying specific molecules or genes from the brain of identical animals. To this end, by minimizing the size and weight of an electrode array, we developed a method that records local field potential signals of multiple brain regions from one side of the hemisphere in a freely moving rodent. At the same time, extracellular cerebrospinal fluid for biochemical assays or a small part of brain tissue samples for gene expression assays are collected from the other side of the hemisphere. This method allows ongoing stable recordings and sample collections for at least two months. The methodological concept is applicable to a wide range of biological reactions at various spatiotemporal scales, allowing us to integrate an idea of physiolomics into existing omics analyses, leading to a new combination of multi-omics approaches. Keywords: Brain, Local field potential, Neuromodulator, Microdialysis, Genes

Aims/introduction Double C2 domain protein b (DOC2b), one of the synaptotagmins, has been shown to translocate to the plasma membrane, and to initiate membrane‐fusion processes of vesicles containing glucose transporter 4 proteins on insulin stimulation. However, the mechanism by which DOC2b is regulated remains unclear. Herein, we identified the upstream regulatory factors of DOC2b in insulin signal transduction. We also examined the role of DOC2b on systemic homeostasis using DOC2b knockout (KO) mice. Materials and Methods We first identified DOC2b binding proteins by immunoprecipitation and mutagenesis experiments. Then, DOC2b KO mice were generated by disrupting the first exon of the DOC2b gene. In addition to the histological examination, glucose metabolism was assessed by measuring parameters on glucose/insulin tolerance tests. Insulin‐stimulated glucose uptake was also measured using isolated soleus muscle and epididymal adipose tissue. Results We identified an isoform of atypical protein kinase C (protein kinase C iota) that can bind to DOC2b and phosphorylates one of the serine residues of DOC2b (S34). This phosphorylation is essential for DOC2b translocation. DOC2b KO mice showed insulin resistance and impaired oral glucose tolerance on insulin and glucose tolerance tests, respectively. Insulin‐stimulated glucose uptake was impaired in isolated soleus muscle and epididymal adipose tissues from DOC2b KO mice. Conclusions We propose a novel insulin signaling mechanism by which protein kinase C iota phosphorylates DOC2b, leading to glucose transporter 4 vesicle translocation, fusion and facilitation of glucose uptake in response to insulin. The present results also showed DOC2b to play important roles in systemic glucose homeostasis.

The human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmission, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified amygdala-specific changes in the expression of genes involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Acute alcohol exposure impairs hippocampus-dependent spatial memory. However, there is little evidence for the effects of ethanol on the spike patterns of hippocampal cell populations. Here, we examined how the spatial firing patterns of place cells, neurons that encode specific locations, were altered in rats that were intraperitoneally injected with 1.5 g/kg ethanol. Ethanol administration partly reduced or abolished place-selective spiking of a subset of place cells during running periods in a spatial task, whereas a subset of place fields newly emerged, suggesting a partial reorganization of hippocampal spatial maps by ethanol. On the other hand, ethanol administration did not significantly alter the frequency of hippocampal sharp-wave ripple (SWRs) and synchronous spike patterns during resting periods, suggesting that offline memory consolidation and retrieval mechanisms underpinned by hippocampal neuronal synchronization are not strongly affected by ethanol. These results indicate that acute ethanol intake mainly affects the encoding of external information but has little impact on internal memory processing.

Summary: Cooperative reactivation of hippocampal and prefrontal neurons is considered crucial for mnemonic processes. To directly record synaptic substances supporting the interregional interactions, we develop concurrent spike recordings of hippocampal neuronal ensembles and whole-cell patch-clamp recordings of medial prefrontal neurons in awake rats. We find that medial prefrontal neurons depolarize when hippocampal neurons synchronize. The depolarization in medial prefrontal neurons is larger when hippocampal place cells that encoded overlapping place fields and place cells that encoded a novel environment are synchronously reactivated. Our results suggest a functional circuit-synapse association that enables prefrontal neurons to read out specific memory traces from the hippocampus.

Cardiac 123I-metaiodobenzylguanidine (MIBG) scintigraphy is a promising biomarker for dementia with Lewy bodies (DLB). However, we experienced a patient with cognitive decline, parkinsonism, and a decreased MIBG uptake who turned out to have HIV dementia. Normal dopamine transporter single-photon emission computed tomography reduced the possibility of comorbid Lewy body pathology causing the patient' s parkinsonism. The decreased MIBG uptake was most likely due to postganglionic sympathetic nerve denervation, which can also be caused by HIV. This case further emphasizes the importance of excluding other causes of autonomic neuropathy, including HIV infection, before interpreting MIBG scans.

The olfactory piriform cortex (PC) is thought to participate in olfactory associative memory. Like the hippocampus, which is essential for episodic memory, it belongs to an evolutionally conserved paleocortex and comprises a three-layered cortical structure. During slow-wave sleep, the olfactory PC becomes less responsive to external odor stimuli and instead displays sharp wave (SPW) activity similar to that observed in the hippocampus. Neural activity patterns during hippocampal SPW have been intensively studied in terms of memory consolidation; however, little is known about the activity patterns of olfactory cortical neurons during olfactory cortex sharp waves (OC-SPWs). In this study, we recorded multi-unit neural activities in the anterior PC in urethane-anesthetized mice. We found that the activity patterns of olfactory cortical neurons during OC-SPWs were non-randomly organized. Individual olfactory cortical neurons varied in the timings of their peak firing rates during OC-SPW events. Moreover, specific pairs of olfactory cortical neurons were more frequently activated together than expected by chance. On the basis of these observations, we speculate that coordinated activation of specific subsets of olfactory cortical neurons repeats during OC-SPWs, thereby facilitating synaptic plasticity underlying the consolidation of olfactory associative memories.

A hippocampal mossy fiber synapse has a complex structure in which presynaptic boutons attach to the dendritic trunk by puncta adherentia junctions (PAJs) and wrap multiply-branched spines, forming synaptic junctions. It was previously shown that afadin regulates the formation of the PAJs cooperatively with nectin-1, nectin-3, and N-cadherin. Afadin is a nectin-binding protein with two splice variants, l-afadin and s-afadin: l-afadin has an actin filament-binding domain, whereas s-afadin lacks it. It remains unknown which variant is involved in the formation of the PAJs or how afadin regulates it. We showed here that re-expression of l-afadin, but not s-afadin, in the afadin-deficient cultured hippocampal neurons in which the PAJ-like structure was disrupted, restored this structure as estimated by the accumulation of N-cadherin and αΝ-catenin. The l-afadin mutant, in which the actin filament-binding domain was deleted, or the l-afadin mutant, in which the αΝ-catenin-binding domain was deleted, did not restore the PAJ-like structure. These results indicate that l-afadin, but not s-afadin, regulates the formation of the hippocampal synapse PAJ-like structure through the binding to actin filaments and αN-catenin. We further found here that l-afadin bound αN-catenin, but not γ-catenin, whereas s-afadin bound γ-catenin, but hardly αN-catenin. These results suggest that the inability of s-afadin to form the hippocampal synapse PAJ-like structure is due to its inability to efficiently bind αN-catenin.

Rebalancing mechanisms during sleep Synapses are often strengthened during wake periods and thus need to be homeostatically readjusted during sleep. During slow-wave sleep, synaptic depression is dominant. Sharp wave and ripple events are transient high-frequency field oscillations that occur spontaneously during slow-wave sleep in the brain. Norimoto et al. found that these events induced long-term depression of hippocampal synapses and may thus help to refine recently acquired memories (see the Perspective by Draguhn). Science , this issue p. 1524 ; see also p. 1461

Mental stress-induced biological responses considerably differ across animals, which may be explained by intrinsic brain activity patterns. To address this hypothesis, we recorded local field potential signals from six cortical areas, electrocardiograms, and electromyograms from freely moving rats. Based on their stress-induced changes in cardiac signals, individual defeated rats were classified into stress susceptible and resilient groups. Rats with lower correlations in theta power across wide ranges of cortical regions before the stress challenge had higher probability to be stress-susceptible rats as defined based on the irregularity of heartbeat signals. A combination of principal component analysis and the support vector machine algorithm revealed that functional connections across cortical regions could be predictive factors accounting for individual differences in future stress susceptibility. These results suggest that individual differences in cortical activity may be a mechanism that causes abnormal activity of peripheral organs in response to mental stress episodes. This evidence will advance the understanding of the neurophysiological correlates of mind-body associations during mental stress exposure.

The transplantation of human-induced pluripotent stem cell (hiPSC)-derived cells has emerged as a potential clinical approach for the treatment of brain diseases. Recent studies with animal disease models have shown that hiPSC-derived neurons transplanted into the brain, especially the nigrostriatal area, could restore degenerated brain functions. Further works are required to test whether hiPSC-derived neurons can also gain functional properties for other cortical areas. In this study, hiPSC-derived neurospheres were transplanted into the adult mouse hippocampus and sensory cortex. Most transplanted hiPSC-derived neurons expressed both Nestin and NeuN at 7 weeks after transplantation. Whole-cell patch-clamp recordings from brain slices indicated that transplanted cells showed no action potentials upon current injection and few small inward currents, indicating that hiPSC-derived neurons did not become functionally mature within these time periods.

Animals process temporal information in an ever-changing environment, but the neuronal mechanisms of this process, especially on timescales longer than seconds, remain unresolved. Here, we designed a hippocampus-dependent task in which rats prospectively increased their reward-seeking behavior over a duration of minutes. During this timing behavior, hippocampal and striatal neurons represented successive time points on the order of minutes by gradually changing their firing rates and transiently increasing their firing rates at specific time points. These minute-encoding patterns progressively developed as the rats learned a time-reward relationship, and the patterns underwent flexible scaling in parallel with timing behavior. These observations suggest a neuronal basis in the hippocampal-striatal circuits that enables temporal processing and formation of episodic memory on a timescale of minutes.

Tropical monoculture leguminous tree plantations are important nitrous oxide (N2O) sources, especially in the Asian tropics. We evaluated the effect of conversion from leguminous Acacia mangium to non-leguminous Eucalyptus pellita on N2O emissions. A 4-year monitoring experiment was conducted in three areas in South Sumatra, Indonesia. In each area, we established acacia and eucalyptus plantation sites adjacent to one another after the harvest of acacia trees. In contrast to our expectation, the averaged N2O flux at the eucalyptus stands (1.07 mg m−2 day−1 of N) was not significantly different from that at the acacia stands (0.96 mg m−2 day−1 of N). This result was attributable to the higher soil NO3 content in the eucalyptus stands than that in the acacia stands, which was due to the lower N requirement of eucalyptus than that of acacia. In conclusion, converting leguminous acacia to non-leguminous eucalyptus was not an effective option to mitigate N2O emissions in the short term.

A multidisciplinary approach reveals key insights into the principles of collective cell migration, which is involved in fundamental biological processes. The conformational plasticity of a single molecule, JRAB/MICAL-L2, provides “law and order” in collective cell migration., In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the “law and order” allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides “law and order” in collective cell migration.

Current research on stress pathology has revealed a set of molecular and cellular mechanisms through which psychosocial stress impairs brain function. However, there are few studies that have examined how chronic stress exposure alters neuronal activity patterns at a network level. Here, we recorded ensemble neuronal activity patterns of the cortico-hippocampal network from urethane-anesthetized mice that were subjected to repeated social defeat stress. In socially defeated mice, the magnitudes of local field potential signals, including theta, slow gamma, and fast gamma oscillations, were significantly reduced in the dentate gyrus, whereas they remained unchanged in the hippocampus and somatosensory cortex. In accordance with the vast majority of histological and biochemical studies, our evidence from electrophysiological investigations highlights the dentate gyrus as a key brain area that is primarily susceptible to stress-induced dysfunction.

Neuronal activity is highly sensitive to changes in oxygen tension. In this study, we examined the impact of hypoxic/ischemic conditions on neuronal ensemble activity patterns in the mouse brain using in vivo extracellular electrophysiological recordings from up to 8 sites in the thalamus, dorsal hippocampus, and neocortex, while cerebral hypoperfusion was induced by unilateral carotid artery occlusion. After a few minutes, the occlusion triggered a rapid change in the power of the local field oscillations. In the hippocampus, but not in the neocortex, the absolute power changes at all frequency ranges (relative to the baseline) became less pronounced with time, and no significant changes were observed 30min after the occlusion-induced hypoperfusion. We also tested whether continuous hypoperfusion induced by the occlusion for up to 1 week alters neuronal activity. In the hippocampus and the thalamus, the chronic occlusion did not lead to a reduction in the power of the local field oscillations. These results indicate that certain neuronal populations have the ability to maintain internal neurophysiological homeostasis against continuous hypoperfusion.

Astrocytes generate calcium signals throughout their fine processes, which are assumed to locally regulate neighbouring neurotransmission and blood flow. The intercellular morphological relationships mature during juvenile periods when astrocytes elongate highly ramified processes. In this study, we examined developmental changes in calcium activity patterns of single hippocampal astrocytes using a transgenic mouse line in which astrocytes selectively express a genetically encoded calcium indicator, Yellow Cameleon-Nano50. Compared with postnatal day 7, astrocytes at postnatal day 30 showed larger subcellular calcium events and a greater proportion of somatic events. At both ages, the calcium activity was abolished by removal of extracellular calcium ions. Calcium events in late juvenile astrocytes were not affected by spontaneously occurring sharp waves that trigger synchronized neuronal spikes, implying the independence of astrocyte calcium signals from neuronal synchronization. These results demonstrate that astrocytes undergo dynamic changes in their activity patterns during juvenile development.

Epithelial cell integrity and remodeling require proper actomyosin organization at adherens junctions through αE-catenin complexed with β-catenin. Sakakibara et al. show that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin–binding activity in a novel mechanism., Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with β-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with β-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with β-catenin. Although a “catch-bond model” partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin–binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with β-catenin and E-cadherin at adherens junctions.

The olfactory piriform cortex is thought to participate in olfactory associative memory. Like the hippocampus, which is essential for episodic memory, it belongs to an evolutionally conserved paleocortex and comprises a three-layered cortical structure. During slow-wave sleep, the olfactory piriform cortex becomes less responsive to external odor stimuli and instead displays sharp wave (SPW) activity similar to that observed in the hippocampus. Neural activity patterns during hippocampal SPW have been intensively studied in terms of memory consolidation; however, little is known about the activity patterns of olfactory cortical neurons during olfactory cortex sharp waves (OC-SPWs). In this study, we recorded multi-unit neural activities in the anterior piriform cortex in urethane-anesthetized mice. We found that the activity patterns of olfactory cortical neurons during OC-SPWs were non-randomly organized. Individual olfactory cortical neurons varied in the timings of their peak firing rates during OC-SPW events. Moreover, specific pairs of olfactory cortical neurons were more frequently activated together than expected by chance. On the basis of these observations, we speculate that coordinated activation of specific subsets of olfactory cortical neurons repeats during OC-SPWs, thereby facilitating synaptic plasticity underlying the consolidation of olfactory associative memories.

The hippocampal network generates synchronized spikes of a large population of pyramidal neurons associated with sharp-wave ripples in local field potential signals. Ample evidence demonstrates that the synchronized spikes are created by sequential activation of hippocampal place cells that correspond to the animal's past or future trajectories and are hypothesized to play instrumental roles in mnemonic functions. However, not all place-cell spike sequences are precisely organized, and some sequences are composed of spikes from non-spatial cells, implying that not all hippocampal synchronized events directly replicate learned behavioral episodes. While less attention has been given to such non-ordered spike sequences, variable and dynamic selection of active neuronal assemblies may be optimal mechanisms for rapidly reorganizing functional circuits and self-developing novel representations to enable flexible decision-making processes. We recently showed that specific neurons, including both spatial and non-spatial cells, are preferentially recruited in synchronous events for particular time periods, suggesting that there are temporally fluctuating background states of the hippocampal network that determine active neuronal ensembles. Based on recent reports, this review discusses potential roles of the low-fidelity, heterogeneous repertoire of synchronized spike sequences of hippocampal neurons.

Spike rates of a hippocampal place cell are not constant and vary even when an animal visits an identical place field with nearly identical behavior. As one potential neurophysiological source underlying place cell spiking variability, we focused on the temporally fluctuating activity states of neuronal ensembles. Spike patterns of hippocampal neurons were recorded from rats performing a linear track task. Within a single consummatory period, similar sets of neurons were more frequently recruited in synchronous firing events, whereas different synchronized firing patterns of neuronal populations tended to be identified in different consummatory periods. A linear regression analysis indicated that the time-varying activation patterns of neuronal populations during consummatory periods are correlated with the spike rates of a place cell within its place field during running. These findings suggest that place cell in-field spiking is not only triggered by static inputs that represent external environments but also strongly depends on the time-varying internal states of neuronal populations.

Complex spatial working memory tasks have been shown to require both hippocampal sharp-wave ripple (SWR) activity and dentate gyrus (DG) neuronal activity. We therefore asked whether DG inputs to CA3 contribute to spatial working memory by promoting SWR generation. Recordings from DG and CA3 while rats performed a dentate-dependent working memory task on an eight-arm radial maze revealed that the activity of dentate neurons and the incidence rate of SWRs both increased during reward consumption. We then found reduced reward-related CA3 SWR generation without direct input from dentate granule neurons. Furthermore, CA3 cells with place fields in not-yet-visited arms preferentially fired during SWRs at reward locations, and these prospective CA3 firing patterns were more pronounced for correct trials and were dentate-dependent. These results indicate that coordination of CA3 neuronal activity patterns by DG is necessary for the generation of neuronal firing patterns that support goal-directed behavior and memory. Fil: Sasaki, Takuya. University of California at San Diego; Estados Unidos. The University of Tokyo; Japón. Japan Science and Technology Agency; Japón Fil: Piatti, Veronica del Carmen. University of California at San Diego; Estados Unidos Fil: Hwaun, Ernie. University of California at San Diego; Estados Unidos Fil: Ahmadi, Siavash. University of California at San Diego; Estados Unidos Fil: Lisman, John E.. Brandeis University; Estados Unidos Fil: Leutgeb, Stefan. University of California at San Diego; Estados Unidos Fil: Leutgeb, Jill K.. University of California at San Diego; Estados Unidos

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant disease that is resistant to various chemotherapeutic agents and commonly relapses. Efficient elimination of metastasized PDA is critical for a positive post-surgical treatment outcome. The present study analyzed the effect of the B-cell lymphoma-2 (Bcl-2)/B-cell lymphoma extra-large (Bcl-xL) inhibitor, ABT-737, on paclitaxel-induced PDA cell death. A total of 8 PDA cell lines were subjected to immunoblotting to compare the expression of Bcl-2/Bcl-xL and other factors associated with taxane resistance, including myeloid cell leukemia 1 and βIII-tubulin (TUBB3). The viability of PDA cells was analyzed following treatment with paclitaxel alone or a combination treatment with ABT-737 and paclitaxel. Treatment with the ABT-737/paclitaxel combination induced PDA cell death at a lower concentration of paclitaxel compared with paclitaxel alone. In addition, the viable cell population at the saturation point of paclitaxel was also decreased by co-treatment with ABT-737. ABT-737 lowered the half maximal inhibitory concentration (IC50) by >2-fold in PDA cells with high Bcl-2/Bcl-xL expression, but not in PDA cells with low Bcl-2/Bcl-xL expression and high TUBB3 expression. Knockdown of Bcl-xL lowered the IC50 of paclitaxel, but knockdown of TUBB3 did not. ABT-737 sensitized PDA to paclitaxel-induced cell death, and Bcl-xL expression was a key determinant of its sensitivity. ABT-737 is potential candidate for combination chemotherapy of PDA with high Bcl-xL expression levels.

Astrocytes generate local calcium (Ca(2+)) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca(2+) indicator yellow Cameleon-Nano 50 (YC-Nano50) in astrocytes. In these mice, we detected a unique pattern of Ca(2+) signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca(2+) signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology.

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.

SummaryThe brain demands high-energy supply and obstruction of blood flow causes rapid deterioration of the healthiness of brain cells. Two major events occur upon ischemia: acidosis and liberation of excess glutamate, which leads to excitotoxicity. However, cellular source of glutamate and its release mechanism upon ischemia remained unknown. Here we show a causal relationship between glial acidosis and neuronal excitotoxicity. As the major cation that flows through channelrhodopsin-2 (ChR2) is proton, this could be regarded as an optogenetic tool for instant intracellular acidification. Optical activation of ChR2 expressed in glial cells led to glial acidification and to release of glutamate. On the other hand, glial alkalization via optogenetic activation of a proton pump, archaerhodopsin (ArchT), led to cessation of glutamate release and to the relief of ischemic brain damage in vivo. Our results suggest that controlling glial pH may be an effective therapeutic strategy for intervention of ischemic brain damage.

We describe a female patient with sporadic amyotrophic lateral sclerosis (ALS) with fused in sarcoma (FUS)-immunoreactive neuronal inclusions. She presented with weakness mainly in the upper extremities that had started at the age of 53 years. She died at the age of 61 years. Neuronal loss and gliosis accompanying FUS-positive neuronal cytoplasmic inclusions involved the upper and lower motor neurons, brainstem motor neurons, basal ganglia, substantia nigra, cerebellar dentate nuclei, thalamus, hypothalamic nuclei, and red nuclei. Most of these inclusions were positive for transportin 1, in a pattern similar to frontotemporal lobar degeneration with FUS and different from familial ALS-FUS. Mutational analysis showed no mutations in the FUS gene. Our findings show that transportin 1 accumulates in the FUS-positive inclusions in sporadic ALS-FUS.

In the mammalian cortex, axons are highly ramified and link an enormous number of neurons over large distances. The conventional view assumes that action potentials (APs) are initiated at the axon initial segment in an all-or-none fashion and are then self-propagated orthodromically along axon collaterals without distortion of the AP waveform. By contrast, recent experimental results suggest that the axonal AP waveform can be modified depending on the activation states of the ion channels and receptors on axonal cell membranes. This AP modulation can regulate neurotransmission to postsynaptic neurons. In addition, the latest studies have provided evidence that cortical axons can integrate somatic burst firings and promote activity-dependent ectopic AP generation, which may underlie the oscillogenesis of fast rhythmic network activity. These seminal observations indicate that axons can perform diverse functional operations that extend beyond the prevailing model of axon physiology.

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.

Dynamic activity of glia has repeatedly been demonstrated, but if such activity is independent from neuronal activity, glia would not have any role in the information processing in the brain or in the generation of animal behavior. Evidence for neurons communicating with glia is solid, but the signaling pathway leading back from glial-to-neuronal activity was often difficult to study. Here, we introduced a transgenic mouse line in which channelrhodopsin-2, a light-gated cation channel, was expressed in astrocytes. Selective photostimulation of these astrocytes in vivo triggered neuronal activation. Using slice preparations, we show that glial photostimulation leads to release of glutamate, which was sufficient to activate AMPA receptors on Purkinje cells and to induce long-term depression of parallel fiber-to-Purkinje cell synapses through activation of metabotropic glutamate receptors. In contrast to neuronal synaptic vesicular release, glial activation likely causes preferential activation of extrasynaptic receptors that appose glial membrane. Finally, we show that neuronal activation by glial stimulation can lead to perturbation of cerebellar modulated motor behavior. These findings demonstrate that glia can modulate the tone of neuronal activity and behavior. This animal model is expected to be a potentially powerful approach to study the role of glia in brain function.

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.

Rubicon, a subunit of the Beclin 1-PI3-kinase complex and its homologue, PLEKHM1, negatively regulate endocytic pathway through the interaction with Rab7. Synchronous association with the Beclin 1–PI3-kinase complex and Rab7 is necessary for the function of Rubicon, but not PLEKHM1., The endocytic and autophagic pathways are involved in the membrane trafficking of exogenous and endogenous materials to lysosomes. However, the mechanisms that regulate these pathways are largely unknown. We previously reported that Rubicon, a Beclin 1–binding protein, negatively regulates both the autophagic and endocytic pathways by unidentified mechanisms. In this study, we performed database searches to identify potential Rubicon homologues that share the common C-terminal domain, termed the RH domain. One of them, PLEKHM1, the causative gene of osteopetrosis, also suppresses endocytic transport but not autophagosome maturation. Rubicon and PLEKHM1 specifically and directly interact with Rab7 via their RH domain, and this interaction is critical for their function. Furthermore, we show that Rubicon but not PLEKHM1 uniquely regulates membrane trafficking via simultaneously binding both Rab7 and PI3-kinase.

Recent research has suggested that epigenetic mechanisms, which exert lasting control over gene expression without altering the genetic code, could mediate stable changes in brain function. A growing body of evidence supports the idea that epigenetic changes play a role in the etiology of aging and its associated brain dysfunction. The present study was undertaken to evaluate the age-related changes in the expression of doublecortin, which is a marker for neuronal precursors, along with epigenetic modification in the hippocampus of aged mice. In the present study, the doublecortin-positive cells were almost completely absent from the dentate gyrus of the hippocampus of 28-month-old mice. Furthermore, the expression level of doublecortin mRNA was significantly decreased in the hippocampus of aged mice. Under these conditions, a significant decrease in H3K4 trimethylation and a significant increase in H3K27 trimethylation at doublecortin promoters were observed with aging without any changes in the expression of their associated histone methylases and demethylases in the hippocampus. These findings suggest that aging produces a dramatic decrease in the expression of doublecortin along with epigenetic modifications in the hippocampus.

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.