Sex and stress interactions in fear synchrony of mouse dyads.

Socially coordinated threat responses support the survival of animal groups. Given their distinct social roles, males and females must differ in such coordination. Here, we report such differences during the synchronization of auditory-conditioned freezing in mouse dyads. To study the interaction of emotional states with social cues underlying synchronization, we modulated emotional states with prior stress or modified the social cues by pairing unfamiliar or opposite-sex mice. In same-sex dyads, males exhibited more robust synchrony than females. Stress disrupted male synchrony in a prefrontal cortex-dependent manner but enhanced it in females. Unfamiliarity moderately reduced synchrony in males but not in females. In dyads with opposite-sex partners, fear synchrony was resilient to both stress and unfamiliarity. Decomposing the synchronization process in the same-sex dyads revealed sex-specific behavioral strategies correlated with synchrony magnitude: following partners' state transitions in males and retroacting synchrony-breaking actions in females. Those were altered by stress and unfamiliarity. The opposite-sex dyads exhibited no synchrony-correlated strategy. These findings reveal sex-specific adaptations of socio-emotional integration defining coordinated behavior and suggest that sex-recognition circuits confer resilience to stress and unfamiliarity in opposite-sex dyads.

Investigation of Tribological Characteristics of PEO Coatings Formed on Ti6Al4V Titanium Alloy in Electrolytes with Graphene Oxide Additives.

Coatings with a thickness from ~40 to ~50 µm on Ti6Al4V titanium alloys were formed by plasma electrolytic oxidation (PEO) in a silicate-hypophosphite electrolyte with the addition of graphene oxide. The PEO treatment was carried out in the anode-cathode mode (50 Hz) at a ratio of anode and cathode currents of 1:1; their sum density was 20 A/dm2, and the treatment's duration was 30 min. The effect of the graphene oxide's concentration in the electrolyte on the thickness, roughness, hardness, surface morphology, structure, composition, and tribological characteristics of the PEO coatings was studied. Wear experiments, under dry conditions, were carried out in a ball-on-disk tribotester with an applied load of 5 N, a sliding speed of 0.1 m·s-1, and a sliding distance of 1000 m. According to the obtained results, the addition of graphene oxide (GO) into the base silicate-hypophosphite electrolyte leads to a slight decrease in the coefficient of friction (from 0.73 to 0.69) and a reduction in the wear rate by more than 1.5 times (from 8.04 to 5.2 mm3/N·m), with an increase in the GO's concentration from 0 to 0.5 kg/m3, respectively. This occurs due to the formation of a GO-containing lubricating tribolayer upon contact with the coating of the counter-body in the friction pair. Delamination of the coatings during wear occurs due to contact fatigue; with an increase in the concentration of GO in the electrolyte from 0 to 0.5 kg/m3, this process slows down by more than four times.

Investigation of Phase Shifts Using AUC Diagrams: Application to Differential Diagnosis of Parkinson's Disease and Essential Tremor.

This study was motivated by the well-known problem of the differential diagnosis of Parkinson's disease and essential tremor using the phase shift between the tremor signals in the antagonist muscles of patients. Different phase shifts are typical for different diseases; however, it remains unclear how this parameter can be used for clinical diagnosis. Neurophysiological papers have reported different estimations of the accuracy of this parameter, which varies from insufficient to 100%. To address this issue, we developed special types of area under the ROC curve (AUC) diagrams and used them to analyze the phase shift. Different phase estimations, including the Hilbert instantaneous phase and the cross-wavelet spectrum mean phase, were applied. The results of the investigation of the clinical data revealed several regularities with opposite directions in the phase shift of the electromyographic signals in patients with Parkinson's disease and essential tremor. The detected regularities provide insights into the contradictory results reported in the literature. Moreover, the developed AUC diagrams show the potential for the investigation of neurodegenerative diseases related to the hyperkinetic movements of the extremities and the creation of high-accuracy methods of clinical diagnosis.

Social Synchronization of Conditioned Fear in Mice Requires Ventral Hippocampus Input to the Amygdala.

BACKGROUND: Social organisms synchronize behaviors as an evolutionary-conserved means of thriving. Synchronization under threat, in particular, benefits survival and occurs across species, including humans, but the underlying mechanisms remain unknown because of the scarcity of relevant animal models. Here, we developed a rodent paradigm in which mice synchronized a classically conditioned fear response and identified an underlying neuronal circuit. METHODS: Male and female mice were trained individually using auditory fear conditioning and then tested 24 hours later as dyads while allowing unrestricted social interaction during exposure to the conditioned stimulus under visible or infrared illumination to eliminate visual cues. The synchronization of the immobility or freezing bouts was quantified by calculating the effect size Cohen's d for the difference between the actual freezing time overlap and the overlap by chance. The inactivation of the dorsomedial prefrontal cortex, dorsal hippocampus, or ventral hippocampus was achieved by local infusions of muscimol. The chemogenetic disconnection of the hippocampus-amygdala pathway was performed by expressing hM4D(Gi) in the ventral hippocampal neurons and infusing clozapine N-oxide in the amygdala. RESULTS: Mice synchronized cued but not contextual fear. It was higher in males than in females and attenuated in the absence of visible light. Inactivation of the ventral but not dorsal hippocampus or dorsomedial prefrontal cortex abolished fear synchronization. Finally, the disconnection of the hippocampus-amygdala pathway diminished fear synchronization. CONCLUSIONS: Mice synchronize expression of conditioned fear relying on the ventral hippocampus-amygdala pathway, suggesting that the hippocampus transmits social information to the amygdala to synchronize threat response.

A Statistical Method for Exploratory Data Analysis Based on 2D and 3D Area under Curve Diagrams: Parkinson's Disease Investigation.

A statistical method for exploratory data analysis based on 2D and 3D area under curve (AUC) diagrams was developed. The method was designed to analyze electroencephalogram (EEG), electromyogram (EMG), and tremorogram data collected from patients with Parkinson's disease. The idea of the method of wave train electrical activity analysis is that we consider the biomedical signal as a combination of the wave trains. The wave train is the increase in the power spectral density of the signal localized in time, frequency, and space. We detect the wave trains as the local maxima in the wavelet spectrograms. We do not consider wave trains as a special kind of signal. The wave train analysis method is different from standard signal analysis methods such as Fourier analysis and wavelet analysis in the following way. Existing methods for analyzing EEG, EMG, and tremor signals, such as wavelet analysis, focus on local time-frequency changes in the signal and therefore do not reveal the generalized properties of the signal. Other methods such as standard Fourier analysis ignore the local time-frequency changes in the characteristics of the signal and, consequently, lose a large amount of information that existed in the signal. The method of wave train electrical activity analysis resolves the contradiction between these two approaches because it addresses the generalized characteristics of the biomedical signal based on local time-frequency changes in the signal. We investigate the following wave train parameters: wave train central frequency, wave train maximal power spectral density, wave train duration in periods, and wave train bandwidth. We have developed special graphical diagrams, named AUC diagrams, to determine what wave trains are characteristic of neurodegenerative diseases. In this paper, we consider the following types of AUC diagrams: 2D and 3D diagrams. The technique of working with AUC diagrams is illustrated by examples of analysis of EMG in patients with Parkinson's disease and healthy volunteers. It is demonstrated that new regularities useful for the high-accuracy diagnosis of Parkinson's disease can be revealed using the method of analyzing the wave train electrical activity and AUC diagrams.

Rap1 in the VMH regulates glucose homeostasis.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

Pre-Senescence Induction in Hepatoma Cells Favors Hepatitis C Virus Replication and Can Be Used in Exploring Antiviral Potential of Histone Deacetylase Inhibitors.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions-from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.

Disinhibition-assisted long-term potentiation in the prefrontal-amygdala pathway via suppression of somatostatin-expressing interneurons.

Significance: Natural brain adaptations often involve changes in synaptic strength. The artificial manipulations can help investigate the role of synaptic strength in a specific brain circuit not only in various physiological phenomena like correlated neuronal firing and oscillations but also in behaviors. High- and low-frequency stimulation at presynaptic sites has been used widely to induce long-term potentiation (LTP) and depression. This approach is effective in many brain areas but not in the basolateral amygdala (BLA) because the robust local GABAergic tone inside BLA restricts synaptic plasticity. Aim: We aimed at identifying the subclass of GABAergic neurons that gate LTP in the BLA afferents from the dorsomedial prefrontal cortex (dmPFC). Approach: Chemogenetic or optogenetic suppression of specific GABAergic neurons in BLA was combined with high-frequency stimulation of the BLA afferents as a method for LTP induction. Results: Chemogenetic suppression of somatostatin-positive interneurons (Sst-INs) enabled the ex vivo LTP by high-frequency stimulation of the afferent but the suppression of parvalbumin-positive interneurons (PV-INs) did not. Moreover, optogenetic suppression of Sst-INs with Arch also enabled LTP of the dmPFC-BLA synapses, both ex vivo and in vivo. Conclusions: These findings reveal that Sst-INs but not PV-INs gate LTP in the dmPFC-BLA pathway and provide a method for artificial synaptic facilitation in BLA.

Conserved features of anterior cingulate networks support observational learning across species.

The ability to observe, interpret, and learn behaviors and emotions from conspecifics is crucial for survival, as it bypasses direct experience to avoid potential dangers and maximize rewards and benefits. The anterior cingulate cortex (ACC) and its extended neural connections are emerging as important networks for the detection, encoding, and interpretation of social signals during observational learning. Evidence from rodents and primates (including humans) suggests that the social interactions that occur while individuals are exposed to important information in their environment lead to transfer of information across individuals that promotes adaptive behaviors in the form of either social affiliation, alertness, or avoidance. In this review, we first showcase anatomical and functional connections of the ACC in primates and rodents that contribute to the perception of social signals. We then discuss species-specific cognitive and social functions of the ACC and differentiate between neural activity related to 'self' and 'other', extending into the difference between social signals received and processed by the self, versus observing social interactions among others. We next describe behavioral and neural events that contribute to social learning via observation. Finally, we discuss some of the neural mechanisms underlying observational learning within the ACC and its extended network.

EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity.

Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.

Prefrontal-amygdala plasticity enabled by observational fear.

Observing fear in others (OF) is a form of social stress. In mice, it enhances inhibitory avoidance learning and causes the formation of silent synapses in the prefrontal-amygdala pathway. Here, we report that OF made that pathway prone to facilitation both ex vivo and in vivo. Ex vivo, OF enabled induction of long-term potentiation (LTP), expressed mostly postsynaptically and occluded by inhibitory avoidance training. In vivo, OF enabled facilitation of the dmPFC-BLA pathway by inhibitory avoidance training. The facilitation persisted during the first 4 h after the training when the prefrontal cortex and amygdala are involved in memory consolidation. Thus, the OF-generated silent synapses likely enable plasticity that may enhance the consolidation of inhibitory avoidance memories.

Social modulation of fear: Facilitation vs buffering.

Social behaviors largely constitute mutual exchanges of social cues and the responses to them. The adaptive response also requires proper interpretation of the current context. In fear behaviors, social signals have bidirectional effects-some cues elicit or enhance fear whereas other suppress or buffer it. Studies on the social facilitation and social buffering of fear provide evidence of competition between social cues of opposing meanings. Co-expression of opposing cues by the same animal may explain the contradicting outcomes from the interaction between naive and frightened conspecifics, which reflect the fine balance between fear facilitation and buffering. The neuronal mechanisms that determine that balance provide an exciting target for future studies to probe the brain circuits underlying social modulation of emotional behaviors.

Impaired social contacts with familiar anesthetized conspecific in CA3-restricted brain-derived neurotrophic factor knockout mice.

Familiarity is conveyed by social cues and determines behaviors toward conspecifics. Here, we characterize a novel assay for social behaviors in mice-contacts with anesthetized conspecific-which eliminates reciprocal interactions, including intermale aggression and shows behaviors that are independent of the demonstrator's activity. During the initial 10 minutes (phase-1), the wild-type (WT) subjects contacted the anesthetized conspecifics vigorously regardless of familiarity. During the subsequent 80 minutes (phase-2), however, they contacted more with familiar than unfamiliar conspecifics. We then applied this test to highly aggressive mice with a hippocampal CA3-restricted knockout (KO) of brain-derived neurotrophic factor (BDNF), in which aggression may mask other social behaviors. The KO mice showed less preference for contacting familiar conspecifics than did WT mice during phase-2 but no differences during phase-1. Among nonsocial behaviors, WT mice also spent less time eating in the presence of familiar than with unfamiliar conspecifics, which was not seen in KO mice. In addition, KO mice exhibited reduced pain sensitization. Altogether, these findings suggest that CA3-specific deletion of BDNF results in deficits in circuits that process social cues from familiar conspecifics as well as pain and may underlie empathy-like behaviors.

Behavioral Modulation by Social Experiences in Rodent Models.

The lasting behavioral changes elicited by social signals provide important adaptations for survival of organisms that thrive as a group. Unlike the rapid innate responses to social cues, such adaptations have been understudied. Here, the rodent models of the lasting socially induced behavioral changes are presented as either modulations or reinforcements of the distinct forms of learning and memory or non-associative changes of affective state. The purpose of this categorization is to draw attention to the potential mechanistic links between the neuronal pathways that process social cues and the neuronal systems that mediate the well-studied forms of learning and memory. © 2018 by John Wiley & Sons, Inc.

Overexpression of channelrhodopsin-2 interferes with the GABAb receptor-mediated depression of GABA release from the somatostatin-containing interneurons of the prefrontal cortex.

Region and cell-type restricted expression of light-activated ion channels is the indispensable tool to study properties of synapses in specific circuits and to monitor synaptic alterations by various stimuli including neuromodulators and behaviors, both ex vivo and in vivo. These analyses require the light-activated proteins or viral vectors for their delivery that do not interfere with the phenomenon under study. Here, we report a case of such interference in which the high-level expression of channelrhodopsin-2 introduced in the somatostatin-positive GABAergic neurons of the dorsomedial prefrontal cortex by an adeno-associated virus vector weakens the presynaptic GABAb receptor-mediated suppression of GABA release.

GABAb Receptor Mediates Opposing Adaptations of GABA Release From Two Types of Prefrontal Interneurons After Observational Fear.

The observational fear (OF) paradigm in rodents, in which the subject is exposed to a distressed conspecific, elicits contextual fear learning and enhances future passive avoidance learning, which may model certain behavioral traits resulting from traumatic experiences in humans. As these behaviors affected by the OF require dorso-medial prefrontal cortex (dmPFC), we searched for synaptic adaptations in dmPFC resulting from OF in mice by recording synaptic responses in dmPFC layer V pyramidal neurons elicited by repeated 5 Hz electrical stimulation of dmPFC layer I or by optogenetic stimulation of specific interneurons ex vivo 1 day after OF. OF increased depression of inhibitory postsynaptic currents (IPSCs) along IPSC trains evoked by the 5 Hz electrical stimulation, but, surprisingly, decreased depression of dendritic IPSCs isolated after blocking GABAa receptor on the soma. Subsequent optogenetic analyses revealed increased depression of IPSCs originating from perisomatically projecting parvalbumin interneurons (PV-IPSCs), but decreased depression of IPSCs from dendritically projecting somatostatin cells (SOM-IPSCs). These changes were no longer detectable in the presence of a GABAb receptor antagonist CGP52432. Meanwhile, OF decreased the sensitivity of SOM-IPSCs, but not PV-IPSCs to a GABAb receptor agonist baclofen. Thus, OF causes opposing changes in GABAb receptor mediated suppression of GABA release from PV-positive and SOM-positive interneurons. Such adaptations may alter dmPFC connectivity with brain areas that target its deep vs superficial layers and thereby contribute to the behavioral consequences of the aversive experiences.

Neuronal Rap1 Regulates Energy Balance, Glucose Homeostasis, and Leptin Actions.

The CNS contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in high-fat diet (HFD)-induced obesity. Genetic ablation of CNS Rap1 protects mice from dietary obesity, glucose imbalance, and insulin resistance in the periphery and from HFD-induced neuropathological changes in the hypothalamus, including diminished cellular leptin sensitivity and increased endoplasmic reticulum (ER) stress and inflammation. Furthermore, pharmacological inhibition of CNS Rap1 signaling normalizes hypothalamic ER stress and inflammation, improves cellular leptin sensitivity, and reduces body weight in mice with dietary obesity. We also demonstrate that Rap1 mediates leptin resistance via interplay with ER stress. Thus, neuronal Rap1 critically regulates leptin sensitivity and mediates HFD-induced obesity and hypothalamic pathology and may represent a potential therapeutic target for obesity treatment.

5-HT3a Receptors Modulate Hippocampal Gamma Oscillations by Regulating Synchrony of Parvalbumin-Positive Interneurons.

Gamma-frequency oscillatory activity plays an important role in information integration across brain areas. Disruption in gamma oscillations is implicated in cognitive impairments in psychiatric disorders, and 5-HT3 receptors (5-HT3Rs) are suggested as therapeutic targets for cognitive dysfunction in psychiatric disorders. Using a 5-HT3aR-EGFP transgenic mouse line and inducing gamma oscillations by carbachol in hippocampal slices, we show that activation of 5-HT3aRs, which are exclusively expressed in cholecystokinin (CCK)-containing interneurons, selectively suppressed and desynchronized firings in these interneurons by enhancing spike-frequency accommodation in a small conductance potassium (SK)-channel-dependent manner. Parvalbumin-positive interneurons therefore received diminished inhibitory input leading to increased but desynchronized firings of PV cells. As a consequence, the firing of pyramidal neurons was desynchronized and gamma oscillations were impaired. These effects were independent of 5-HT3aR-mediated CCK release. Our results therefore revealed an important role of 5-HT3aRs in gamma oscillations and identified a novel crosstalk among different types of interneurons for regulation of network oscillations. The functional link between 5-HT3aR and gamma oscillations may have implications for understanding the cognitive impairments in psychiatric disorders.

Exogenous Hsp70 delays senescence and improves cognitive function in aging mice.

Molecular chaperone Heat Shock Protein 70 (Hsp70) plays an important protective role in various neurodegenerative disorders often associated with aging, but its activity and availability in neuronal tissue decrease with age. Here we explored the effects of intranasal administration of exogenous recombinant human Hsp70 (eHsp70) on lifespan and neurological parameters in middle-aged and old mice. Long-term administration of eHsp70 significantly enhanced the lifespan of animals of different age groups. Behavioral assessment after 5 and 9 mo of chronic eHsp70 administration demonstrated improved learning and memory in old mice. Likewise, the investigation of locomotor and exploratory activities after eHsp70 treatment demonstrated a significant therapeutic effect of this chaperone. Measurements of synaptophysin show that eHsp70 treatment in old mice resulted in larger synaptophysin-immunopositive areas and higher neuron density compared with control animals. Furthermore, eHsp70 treatment decreased accumulation of lipofuscin, an aging-related marker, in the brain and enhanced proteasome activity. The potential of eHsp70 intranasal treatment to protect synaptic machinery in old animals offers a unique pharmacological approach for various neurodegenerative disorders associated with human aging.

The Fate of Exogenous Human HSP70 Introduced into Animal Cells by Different Means.

Over the last decade, it has become evident that in mammals, including humans, heat shock protein 70 (HSP70), apart from its intracellular localization, is found in extracellular space, where it may execute various protective functions. Furthermore, the upregulation of HSP70 family members can be beneficial in the prevention and treatment of various human neurodegenerative diseases and cancer. Here, we demonstrate that recombinant human HSP70 after intranasal administration can penetrate various brain regions of mice in its native form and subsequently undergo rapid degradation. It was also shown that labeled HSP70 added to culture medium of different human and mouse cell lines enters the cells with strikingly different kinetics, which positively correlates with the basic levels of membrane bound Toll-like receptors (TLR) that are characteristic of these cell lines. HSP70 administration does not significantly modulate the level of TLR expression at the protein or RNA level. The degradation of the introduced recombinant HSP70 after entering the cells is likely proteasome-dependent and varies significantly depending on the cells type and origin. These results should be considered when developing HSP70-based therapies.