Erratum: Modified Fear Conditioning for Inducing Flight Behaviors in Mice.

This corrects the article 10.3791/66266.

Spatiotemporal development of the neuronal accumulation of amyloid precursor protein and the amyloid plaque formation in the brain of 3xTg-AD mice.

The amyloid plaque is a hallmark of Alzheimer's disease. The accumulation of the amyloid precursor protein (APP) in the neuronal structure is assumed to lead to amyloid plaque formation through the excessive production of ß-amyloid protein. To study the relationship between the neuronal accumulation of APP and amyloid plaque formation, we histologically analyzed their development in the different brain regions in 3xTg-AD mice, which express Swedish mutated APP (APPSWE) in the neurons. Observation throughout the brain revealed APPSWE-positive somata in the broad regions. Quantitative model analysis showed that the somatic accumulation of APPSWE developed firstly in the hippocampus from a very early age (<1 month) and proceeded slower in the isocortex. In line with this, the hippocampus was the first region to form amyloid plaques at the age of 9-12 months, while amyloid plaques were rarely observed in the isocortex. Females had more APPSWE-positive somata and plaques than males. Furthermore, amyloid plaques were observed in the lateral septum and pontine grey, which did not contain APPSWE-positive somata but only the APPSWE-positive fibers. These results suggested that neuronal accumulation of APPSWE, both in somatodendritic and axonal domains, is closely related to the formation of amyloid plaques.

Disruption of sphingomyelin synthase 2 gene alleviates cognitive impairment in a mouse model of Alzheimer's disease.

The membrane raft accommodates the key enzymes synthesizing amyloid ß (Aß). One of the two characteristic components of the membrane raft, cholesterol, is well known to promote the key enzymes that produce amyloid-ß (Aß) and exacerbate Alzheimer's disease (AD) pathogenesis. Given that the raft is a physicochemical platform for the sound functioning of embedded bioactive proteins, the other major lipid component sphingomyelin may also be involved in AD. Here we knocked out the sphingomyelin synthase 2 gene (SMS2) in 3xTg AD model mice by hybridization, yielding SMS2KO mice (4S mice). The novel object recognition test in 9/10-month-old 4S mice showed that cognitive impairment in 3xTg mice was alleviated by SMS2KO, though performance in the Morris water maze (MWM) was not improved. The tail suspension test detected a depressive trait in 4S mice, which may have hindered the manifestation of performance in the wet, stressful environment of MWM. In the hippocampal CA1, hyperexcitability in 3xTg was also found alleviated by SMS2KO. In the hippocampal dentate gyrus of 4S mice, the number of neurons positive with intracellular Aß or its precursor proteins, the hallmark of young 3xTg mice, is reduced to one-third, suggesting an SMS2KO-led suppression of syntheses of those peptides in the dentate gyrus. Although we previously reported that large-conductance calcium-activated potassium (BK) channels are suppressed in 3xTg mice and their recovery relates to cognitive amelioration, no changes occurred by hybridization. Sphingomyelin in the membrane raft may serve as a novel target for AD drugs.

Action of GABAB receptor on local network oscillation in somatosensory cortex of oral part: focusing on NMDA receptor.

The balance of activity between glutamatergic and GABAergic networks is particularly important for oscillatory neural activities in the brain. Here, we investigated the roles of GABAB receptors in network oscillation in the oral somatosensory cortex (OSC), focusing on NMDA receptors. Neural oscillation at the frequency of 8-10 Hz was elicited in rat brain slices after caffeine application. Oscillations comprised a non-NMDA receptor-dependent initial phase and a later NMDA receptor-dependent oscillatory phase, with the oscillator located in the upper layer of the OSC. Baclofen was applied to investigate the actions of GABAB receptors. The later NMDA receptor-dependent oscillatory phase completely disappeared, but the initial phase did not. These results suggest that GABAB receptors mainly act on NMDA receptor, in which metabotropic actions of GABAB receptors may contribute to the attenuation of NMDA receptor activities. A regulatory system for network oscillation involving GABAB receptors may be present in the OSC.

Multiple factors contribute to flight behaviors during fear conditioning.

Shifting defensive mode from one to another by the imminence of threat is crucial for survival. The transition of defensive mode from freezing to flight is observed during the modified fear conditioning, however, the flight during fear conditioning is not well characterized. To characterize the flight behaviors during the fear conditioning, we conducted experiments in male mice focusing on the influence of the context, the intensity of the unconditioned stimulus and conditioned stimulus (CS), the schedule of conditioning, and the state of the subject. Flight behaviors triggered by salient CS showed characteristics of fear-potentiated defensive behaviors depending on the conditioned context, while repetitive conditioning enhanced the expression of the flight and developed an association between the CS and the flight. The salient auditory stimulus was the primary factor to trigger flight behaviors. Also, the spaced conditioning increased the expression of flight behaviors. Taken together, the flight behavior during fear conditioning is not a simple conditioned response nor simple fear-potentiated behavior, but a complicated mixture of multiple components of defensive behaviors. The transition of defensive mode could be induced by the integration of multiple innate and learned components of fear or anxiety.

IFNα primes cancer cells for Fusicoccin-induced cell death via 14-3-3 PPI stabilization.

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells. Among the identified 14-3-3 target proteins are THEMIS2, receptor interacting protein kinase 2 (RIPK2), EIF2AK2, and several members of the LDB1 complex. Biophysical and structural biology studies confirm these 14-3-3 PPIs as physical targets of FC stabilization, and transcriptome as well as pathway analyses suggest possible explanations for the observed synergistic effect of IFNα/FC treatment on cancer cells. This study elucidates the polypharmacological effects of FCs in cancer cells and identifies potential targets from the vast interactome of 14-3-3s for therapeutic intervention in oncology.

Pain-related neuronal ensembles in the primary somatosensory cortex contribute to hyperalgesia and anxiety.

The mechanism by which acute pain or itch information at the periphery is processed in the primary somatosensory cortex (S1) remains unclear. To elucidate this, we used a viral-mediated targeted-recombination-in-active population system to target S1 neuronal ensembles that are active during pain or itch sensations. We induced the expression of excitatory or inhibitory designer receptors exclusively activated by designer drugs in pain- or itch-related S1 neurons. We identified neuronal populations in mice that regulate the sensory components of pain and itch in the S1 hind paw region. Notably, the neuronal circuit between pain-related S1 neurons and the parafascicular nucleus contributed to hyperalgesia and anxiety-like behavior. We propose that S1 plays an essential role in sensory and affective responses to noxious stimuli, such as pain.

ATF6ß Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

Alternative strategy for driving voltage-oscillator in neocortex of rats.

Information integration in the brain requires functional connectivity between local neural networks. Here, we investigated the interregional coupling mechanism from the viewpoint of oscillations using optical recording methods. Low-frequency electrical stimulation of rat neocortical slices in a caffeine-containing medium induced oscillatory activity between the primary visual cortex (Oc1) and medial secondary visual cortex (Oc2M), in which the oscillation generator was located in the Oc2M and was triggered by a feedforward signal. During to-and-fro oscillatory activity, neural excitation was marked in layer II/III. When the upper layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the deep layer and switch on the oscillator in the Oc2M. When the lower layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the upper layer and switch on the oscillator in the Oc2M. In the backward direction, neither the upper layer cut nor the lower layer cut disrupted the propagation of the oscillations. In all cases, the horizontal and vertical pathways were used as needed. Fluctuations in the oscillatory waveforms of the local field potential at the upper and lower layers in the Oc2M were reversed, suggesting that the oscillation originated between the two layers. Thus, the neocortex may work as a safety device for interregional communications in an alternative way to drive voltage oscillators in the neocortex.

Modified Fear Conditioning for Inducing Flight Behaviors in Mice.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

[Characterization of the dorsal raphe-periaqueductal grey DAT neurons innervating onto the extended amygdala].

It has been known that a number of tyrosine hydroxylase (TH)-positive neurons, which are regarded as dopaminergic (DA) neurons, exist in the dorsal raphe (DR). These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are thought to belong to the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. The DA transporter (DAT) neurons, which are well overlapping with DA neurons, in the DR-PAG region are also expected to be heterogeneous. However, even though the heterogeneity of DA/DATDR-PAG neurons has been suggested, the characteristics of each DA/DATDR-PAG neuron subpopulation are not well investigated. In this paper, we summarize the previous reports investigating the heterogeneity of DA/DATDR-PAG neurons and the functional importance of DA/DATDR-PAG neurons on various affective behaviors and introduce our recent findings that DATDR-PAG neurons consist of two subpopulations: TH+/vasoactive intestinal peptide (VIP)- putative DA neurons and TH-/VIP+ putative glutamatergic neurons.

5-HT and α-m-5-HT attenuate excitatory synaptic transmissions onto the lateral amygdala principal neurons via presynaptic 5-HT1B receptors.

Accumulating evidence suggests that the serotonergic (5-HT) system in the amygdala has significant effects on affective states. Dysregulation of the 5-HT system in the basolateral amygdaloid complex causes affective disorders. To search for therapeutic targets, subtype specification of 5-HT receptors is crucial. The present study was undertaken to identify the 5-HT receptor subtype responsible for the 5-HT-mediated suppression of excitatory transmission to principal neurons (PNs) in the lateral amygdala (LA). Whole-cell recordings were performed to record excitatory post synaptic currents (EPSCs) in acute rat brain slices. We confirmed that 5-HT and α-m-5-HT, a broad 5-HT2 receptor agonist, attenuated EPSCs in LA PNs. The extent of suppressions by 5-HT and α-m-5-HT remained unchanged in the presence of ritanserin, a broad 5-HT2 receptor antagonist. In the presence of NAS-181, a selective 5-HT1B receptor antagonist, the extent of EPSC suppressions by 5-HT and α-m-5-HT was diminished. CP93129, a selective 5-HT1B receptor agonist, attenuated EPSCs in LA PNs, and this effect was abolished in the presence of NAS-181. Additionally, the paired-pulse ratio of EPSCs was increased by CP93129. Thus, our results indicate that 5-HT and α-m-5-HT attenuate excitatory transmissions to LA PNs via presynaptic 5-HT1B receptors.

Vocalization during agonistic encounter in Mongolian gerbils: Impact of sexual experience.

Behaviors and vocalizations associated with aggression are essential for animals to survive, reproduce, and organize social hierarchy. Mongolian gerbils (Meriones unguiculatus) are highly aggressive and frequently emit calls. We took advantage of these features to study the relationship between vocalizations and aggressive behaviors in virgin and sexually experienced male and female Mongolian gerbils through the same-sex resident-intruder test. Both sexes of resident gerbils exhibited aggressive responses toward intruders. Multiparous females exhibited the most aggressive responses among the four groups. We also confirmed two groups of vocalizations during the encounters: high-frequency (>24.6 kHz) and low-frequency (<24.6 kHz). At the timing of high-frequency vocalizations observed during the tests, the vast majority (96.2%) of the behavioral interactions were non-agonistic. While, at the timing of low-frequency vocalizations observed during the tests, around half (45%) of the behavioral interactions were agonistic. Low-frequency vocalizations were observed mainly during encounters in which multiparous females were involved. These results suggest that high- and low-frequency vocalizations relate to non-agonistic and agonistic interactions, respectively. In addition to affecting aggressive behavior, sexual experience also affects vocalization during encounters. These findings provide new insights into the modulatory effects of sex and sexual experience on vocalizations during agonistic encounters.

Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP- and TH-/VIP+ neurons. The DAT+/TH-/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH-/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP- and TH-/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

Impact of the COVID-19 Pandemic on Glycemic Control and Blood Pressure Control in Patients with Diabetes in Japan.

Objective In this study, we investigated whether and how the COVID-19 pandemic affected glycemic control and blood pressure (BP) control in patients with diabetes mellitus (DM). Methods DM patients whose HbA1c level was measured regularly before and after the declaration of a state of emergency were included in this study. Some patients were given questionnaires about changes in their lifestyle to determine the factors affecting glycemic control and BP control. Results The median HbA1c level of the 804 patients increased significantly from 6.8% before the state of emergency to 7.1% and 7.0% during and after the state of emergency, respectively. This was in contrast to the decrease one year earlier due to seasonal variations. In the 176 patients who responded to the questionnaire, the HbA1c level also increased significantly during and after the state of emergency. The worsening of glycemic control was more pronounced in the group that had achieved HbA1c of <7% before the state of emergency than in those with higher values. Unlike the rise in HbA1c, the BP did not rise during the state of emergency but did rise significantly afterwards. There was no marked decrease in HbA1c or BP after the state of emergency, even in patients who responded that they were much more careful with their diet, ate less, or exercised more. Conclusions The COVID-19 pandemic worsened glycemic control and BP control, even in patients who perceived no marked change in their diet or exercise, suggesting that more active lifestyle guidance is necessary for good treatment of DM patients.

Amyloid ß and Amyloid Precursor Protein Synergistically Suppress Large-Conductance Calcium-Activated Potassium Channel in Cortical Neurons.

Intracellular amyloid ß (Aß) injection suppresses the large-conductance calcium-dependent potassium (BK) channel in cortical pyramidal cells from wild-type (WT) mice. In 3xTg Alzheimer's disease (AD) model mice, intraneuronal Aß is genetically programed to accumulate, which suppresses the BK channel. However, the mode of BK channel suppression remained unclarified. The present report revealed that only one (11A1) of the three anti-Aß-oligomer antibodies that we examined, but not anti-monomer-Aß-antibodies, was effective in recovering BK channel activity in 3xTg neurons. Antibodies against amyloid precursor protein (APP) were also found to be effective, suggesting that APP plays an essential part in this Aß-oligomer-induced BK channel suppression in 3xTg neurons. In WT neurons, by contrast, APP suppressed BK channels by itself, which suggests that either APP or Aß is sufficient to block BK channels, thus pointing to a different co-operativity of Aß and APP in WT and 3xTg neurons. To clarify this difference, we relied on our previous finding that the scaffold protein Homer1a reverses the BK channel blockade in both WT and 3xTg neurons. In cortical neurons from 3xTg mice that bear Homer1a knockout (4xTg mice), neither anti-APP antibodies nor 11A1, but only the 6E10 antibody that binds both APP and Aß, rescued the BK channel suppression. Given that Homer1a expression is activity dependent and 3xTg neurons are hyperexcitable, Homer1a is likely to be expressed sufficiently in 3xTg neurons, thereby alleviating the suppressive influence of APP and Aß on BK channel. A unique way that APP modifies Aß toxicity is thus proposed.

The ATF6ß-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6ß remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6ß is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6ß deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6ß and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6ß-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

Characteristics of patients with emergency attendance for severe hypoglycemia and hyperglycemia in a general hospital in Japan.

ABSTRACT: Despite advances in treatments for diabetes mellitus (DM), severe acute glycemic crises still occur. In this study, the characteristics of patients who were transported to an emergency department due to acute glycemic crises were investigated.We enrolled patients who were transported to our hospital by ambulance due to hypoglycemia or hyperglycemia during the period from January 2015 to December 2019. Initial glucose levels below 70 mg/dL and above 250 mg/dL were defined as hypoglycemia and hyperglycemia, respectively.In the 5-year period, 16,910 patients were transported to our hospital by ambulance. Of those patients, 87 patients (0.51%) were diagnosed with hypoglycemia, 26 patients (0.15%) were diagnosed with hyperglycemia and 1 patient was diagnosed with lactic acidosis. Compared to patients with hypoglycemia, blood urea nitrogen, serum potassium and hemoglobin levels were higher in patients with hyperglycemia. Systolic blood pressure was lower and pulse rate was higher in patients with hyperglycemia, possibly reflecting dehydration in hyperglycemia. Patients with hyperglycemia were younger (63 vs 70 years old, median), more likely to be hospitalized (92.3% vs 23.0%) with poorer prognosis (23.1% vs 4.6%) than those with hypoglycemia. In 64 DM patients with hypoglycemia, 34 patients were treated with insulin and 24 patients were treated with sulfonylurea or glinide, and their medication was often inappropriate. Excessive alcohol intake and malnutrition were the main causes of hypoglycemia in 23 non-DM patients. The main reasons for hyperglycemia were interrupted treatment, forgetting insulin injection and infection.To avoid acute glycemic crises, optimization of anti-DM therapy and education of patients are needed.