Hippocampal sharp wave ripples underlie stress susceptibility in male mice.

The ventral hippocampus (vHC) is a core brain region for emotional memory. Here, we examined how the vHC regulates stress susceptibility from the level of gene expression to neuronal population dynamics in male mice. Transcriptome analysis of samples from stress-naïve mice revealed that intrinsic calbindin (Calb1) expression in the vHC is associated with susceptibility to social defeat stress. Mice with Calb1 gene knockdown in the vHC exhibited increased stress resilience and failed to show the increase in the poststress ventral hippocampal sharp wave ripple (SWR) rate. Poststress vHC SWRs triggered synchronous reactivation of stress memory-encoding neuronal ensembles and facilitated information transfer to the amygdala. Suppression of poststress vHC SWRs by real-time feedback stimulation or walking prevented social behavior deficits. Taken together, our results demonstrate that internal reactivation of memories of negative stressful episodes supported by ventral hippocampal SWRs serves as a crucial neurophysiological substrate for determining stress susceptibility.

Kaposi's sarcoma-associated herpesvirus is cell-intrinsically controlled in latency in microgravity.

In the next several decades, humans will explore deep space, including Mars. During long-term space flight, astronauts will be exposed to various physical stressors. Among these stressors, microgravity may compromise the immune system. Consistently, the reactivation of several latent herpesviruses has been reported in astronauts. Although herpesvirus infection status is determined by both cell-intrinsic and -extrinsic factors, it remains unclear which factors play major roles in the virus reactivation in microgravity. Here, using Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, we found that KSHV is cell-intrinsically controlled in latency in microgravity. Innate immunity appeared to be unaffected in microgravity, while the expression of some restriction factors against KSHV, such as CTCF and AMPK, was upregulated. Collectively, the infected cells in microgravity can control KSHV in latency, possibly by unimpaired innate immunity and upregulated KSHV restriction factors. This is the first pilot study of the conflicts between cell-intrinsic defense systems and viruses in microgravity and provides fundamental information regarding host-virus interactions in microgravity.

Sniffing behaviour-related changes in cardiac and cortical activity in rats.

KEY POINTS: High-frequency (HF) sniffing represents active odour sampling and an increase in the animal's motivation. We examined how HF sniffing affects the physiological activity of the brain-body system. During HF sniffing, heart rates and the ratio of theta to delta critical local field potential power were comparable to those observed during motion periods. Vagus nerve spike rates did not vary depending on HF sniffing. Our results suggest that physiological factors in the central nervous system and the periphery are not simply determined by locomotion but are crucially associated with HF sniffing. ABSTRACT: Sniffing is a fundamental behaviour for odour sampling, and high-frequency (HF) sniffing, generally at a sniff frequency of more than 6 Hz, is considered to represent an animal's increased motivation to explore external environments. Here, we examined how HF sniffing is associated with changes in physiological signals from the central and peripheral organs in rats. During HF sniffing while the rats were stationary, heart rates, the magnitude of dorsal neck muscle contraction, and the ratio of theta to delta local field potential power in the motor cortex were comparable to those observed during motion periods and were significantly higher than those observed during resting respiration periods. No pronounced changes in vagus nerve spike rates were detected in relation to HF sniffing. These results demonstrate that central and peripheral physiological factors are crucially associated with the emergence of HF sniffing, especially during quiescent periods. Behavioural data might be improved to more accurately evaluate an animal's internal psychological state if they are combined with a sniffing pattern as a physiological marker.

Cortical-wide functional correlations are associated with stress-induced cardiac dysfunctions in individual rats.

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.

Social defeat stress causes selective attenuation of neuronal activity in the ventromedial prefrontal cortex.

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.

[Analysis of mental states based on peripheral organ activity].

Behavioral tests using rodents are widely used for assessing mental states of animals and the effects of pharmacological drugs on psychiatric disorders. However, the results of behavioral tests are sometimes inconsistent due to individual differences. To evaluate animal's mental states based on internal organ activity, we recently developed a new electrophysiological method to simultaneously monitor cortical local field potentials, electrocardiograms, electromyograms, respiratory signals, and vagus nerve spikes in a freely moving rodent. Here, we introduce some results obtained from an elevated plus maze test. Both cortical activity patterns and vagus nerve spike patterns are crucially related to other peripheral organ activity rather than arm types of the maze in which animal were located, which demonstrates that combining behavioral tests with peripheral physiological makers enables a more accurate evaluation of rodent mental states. Moreover, we show that functional connection patterns across cortical regions could be predictive factors accounting for stress susceptibility defined based on the irregularity of heartbeat signals, demonstrating that cortical activity may be a mechanism that causes abnormal activity of peripheral organs in response to mental stress episodes. These observations from our recording technique are a new step for understanding of the neurophysiological correlates of mind-body associations in health and disease.

Collection of biochemical samples with brain-wide electrophysiological recordings from a freely moving rodent.

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.

Latent infection with Kaposi's sarcoma-associated herpesvirus enhances retrotransposition of long interspersed element-1.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a gamma-2 herpesvirus, is the causative agent of KS, primary effusion lymphoma (PEL), and a plasma cell variant of multicentric Castleman's disease. Although KSHV latency is detected in KS-related tumors, oncogenic pathways activated by KSHV latent infection are not fully understood. Here, we found that retrotransposition of long interspersed element-1 (L1), a retrotransposon in the human genome, was enhanced in PEL cells. Among the KSHV latent genes, viral FLICE-inhibitory protein (vFLIP) enhanced L1 retrotransposition in an NF-κB-dependent manner. Intracellular cell adhesion molecule-1 (ICAM-1), an NF-κB target, regulated the vFLIP-mediated enhancement of L1 retrotransposition. Furthermore, ICAM-1 downregulated the expression of Moloney leukemia virus 10 (MOV10), an L1 restriction factor. Knockdown of ICAM-1 or overexpression of MOV10 relieved the vFLIP-mediated enhancement of L1 retrotransposition. Collectively, during KSHV latency, vFLIP upregulates ICAM-1 in an NF-κB-dependent manner, which, in turn, downregulates MOV10 expression and thereby enhances L1 retrotransposition. Because active L1 retrotransposition can lead to genomic instability, which is commonly found in KS and PEL, activation of L1 retrotransposition during KSHV latency may accelerate oncogenic processes through enhancing genomic instability. Our results suggest that L1 retrotransposition may be a novel target for impeding tumor development in KSHV-infected patients.

Inhibition of LINE-1 Retrotransposition by Capsaicin.

Long interspersed nuclear element 1 (LINE-1 or L1) is a non-long terminal repeat (LTR) retrotransposon that constitutes approximately 17% of the human genome. Since approximately 100 copies are still competent for retrotransposition to other genomic loci, dysregulated retrotransposition of L1 is considered to be a major risk factor of endogenous mutagenesis in humans. Thus, it is important to find drugs to regulate this process. Although various chemicals are reportedly capable of affecting L1 retrotransposition, it is poorly understood whether phytochemicals modulate L1 retrotransposition. Here, we screened a library of compounds that were derived from phytochemicals for reverse transcriptase (RT) inhibition with an in vitro RT assay. We identified capsaicin as a novel RT inhibitor that also suppressed L1 retrotransposition. The inhibitory effect of capsaicin on L1 retrotransposition was mediated neither through its receptor, nor through its modulation of the L1 promoter and/or antisense promoter activity, excluding the possibility that capsaicin indirectly affected L1 retrotransposition. Collectively, capsaicin suppressed L1 retrotransposition most likely by inhibiting the RT activity of L1 ORF2p, which is the L1-encoded RT responsible for L1 retrotransposition. Given that L1-mediated mutagenesis can cause tumorigenesis, our findings suggest the potential of capsaicin for suppressing cancer development.

Characterization of Peripheral Activity States and Cortical Local Field Potentials of Mice in an Elevated Plus Maze Test.

Elevated plus maze (EPM) tests have been used to assess animal anxiety levels. Little information is known regarding how physiological activity patterns of the brain-body system are altered during EPM tests. Herein, we monitored cortical local field potentials (LFPs), electrocardiograms (ECGs), electromyograms (EMGs), and respiratory signals in individual mice that were repeatedly exposed to EPM tests. On average, mouse heart rates were higher in open arms. In closed arms, the mice occasionally showed decreased heart and respiratory rates lasting for several seconds or minutes, characterized as low-peripheral activity states of peripheral signals. The low-activity states were observed only when the animals were in closed arms, and the frequencies of the states increased as the testing days proceeded. During the low-activity states, the delta and theta powers of cortical LFPs were significantly increased and decreased, respectively. These results demonstrate that cortical oscillations crucially depend on whether an animal exhibits low-activity states in peripheral organs rather than the EPM arm in which the animal is located. These results suggest that combining behavioral tests with physiological makers enables a more accurate evaluation of rodent mental states.

Selective attenuation of electrophysiological activity of the dentate gyrus in a social defeat mouse model.

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.

Subcellular calcium dynamics during juvenile development in mouse hippocampal astrocytes.

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.

Subtle modulation of ongoing calcium dynamics in astrocytic microdomains by sensory inputs.

Astrocytes communicate with neurons through their processes. In vitro experiments have demonstrated that astrocytic processes exhibit calcium activity both spontaneously and in response to external stimuli; however, it has not been fully determined whether and how astrocytic subcellular domains respond to sensory input in vivo. We visualized the calcium signals in astrocytes in the primary visual cortex of awake, head-fixed mice. Bias-free analyses of two-photon imaging data revealed that calcium activity prevailed in astrocytic subcellular domains, was coordinated with variable spot-like patterns, and was dominantly spontaneous. Indeed, visual stimuli did not affect the frequency of calcium domain activity, but it increased the domain size, whereas tetrodotoxin reduced the sizes of spontaneous calcium domains and abolished their visual responses. The "evoked" domain activity exhibited no apparent orientation tuning and was distributed unevenly within the cell, constituting multiple active hotspots that were often also recruited in spontaneous activity. The hotspots existed dominantly in the somata and endfeet of astrocytes. Thus, the patterns of astrocytic calcium dynamics are intrinsically constrained and are subject to minor but significant modulation by sensory input.

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

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

Astrocyte calcium signalling orchestrates neuronal synchronization in organotypic hippocampal slices.

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

Intermolecular reductive radical addition to 2-(2,2,2-trifluoroethylidene)-1,3-dithiane 1-oxide: experimental and theoretical studies.

Tin hydride mediated radical addition of organic halide to 2-(2,2,2-trifluoroethylidene)-1,3-dithiane 1-oxide has been devised. The reaction is equivalent to an unrealizable radical addition to trifluoromethylketene, providing useful α-trifluoromethyl carbonyl equivalents. The trifluoromethyl and the sulfoxide groups of the substrate play key roles for the success of the radical addition, lowering the barrier of the radical addition step and controlling the stereoselectivity of the reaction, which DFT calculations have elucidated.