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.

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.

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.

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.

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.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis.

Rap1, a Ras-like small GTPase, plays a crucial role in cell-matrix adhesive interactions, cell-cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell-cell junctions, disruption of paxillin and ß1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical-basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

Rap1 signaling prevents L-type calcium channel-dependent neurotransmitter release.

The small GTPase Rap1 contributes to fear learning and cortico-amygdala plasticity by inhibiting glutamate release from cortical neurons, but mechanisms of this inhibition remain unknown. Conversely, L-type calcium channels (LTCCs) become involved in glutamate release after fear learning and LTP induction. Here, we show that Rap1 deletion in mouse primary cortical neurons increases synaptic vesicle exocytosis without altering endocytosis or vesicle pool size in an LTCC-dependent manner. We identify Erk1/2 as the downstream effector of Rap1 and show that its inhibition increases plasma membrane expression of LTCCs near presynaptic terminals. We propose that the Rap1 signaling enables plasticity and fear learning by regulating LTCCs at cortico-amygdala synapses.

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.

Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine.

Dopamine (DA) in the basolateral amygdala (BLA) promotes fear learning by disinhibiting principal neurons (PNs) and enabling synaptic plasticity in their sensory inputs. While BLA interneurons (INs) are heterogeneous, it is unclear which interneuron subtypes decrease GABAergic input to PNs in the presence of DA. Here, using cell type-selective photostimulation by channelrhodopsin 2 in BLA slices from mouse brain, we examined the role of parvalbumin-positive INs (PV-INs), the major interneuronal subpopulation in BLA, in the disinhibitory effect of DA. We found that DA selectively suppressed GABAergic transmission from PV-INs to PNs by acting on presynaptic D(2) receptors, and this effect was mimicked by Rp-cAMP, an inhibitor of cAMP-dependent signaling. In contrast, DA did not alter GABA release from PV-INs to INs. Furthermore, neither suppressing cAMP-dependent signaling by Rp-cAMP nor enhancing it by forskolin altered GABA release from PV-INs to BLA INs. Overall, DA disinhibits BLA, at least in part, by suppressing GABA release from PV-INs in the target cell-specific manner that results from differential control of this release by cAMP-dependent signaling.

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.

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.

Erk1/2 inhibit synaptic vesicle exocytosis through L-type calcium channels.

L-type calcium channels play only a minor role in basal neurotransmitter release in brain neurons but contribute significantly after induction of plasticity. Very little is known about mechanisms that enable L-type calcium channel participation in neurotransmitter release. Here, using mouse primary cortical neurons, we found that inhibition of Erk1/2 (extracellular signal-regulated kinases 1 and 2) enhanced synaptic vesicle exocytosis by increasing calcium influx through L-type calcium channels. Furthermore, inhibition of Erk1/2 increased the surface fraction of these channels. These findings indicate a novel inhibitory effect of Erk1/2 on synaptic transmission through L-type calcium channels.

Selective suppression of plasticity in amygdala inputs from temporal association cortex by the external capsule.

GABAergic neurons in the external capsule (EC) provide feedforward inhibition in the lateral amygdala (LA), but how EC affects synaptic transmission and plasticity in inputs from specific cortical areas remains unknown; this is because axonal fibers from different cortical areas are intermingled in the amygdala and cannot be activated selectively using conventional electrical stimulation. Here, we achieved selective activation of fibers from the temporal association cortex (TeA) or the anterior cingulate cortex (ACC) by using channelrhodopsin-2. Long-term potentiation (LTP) in the TeA-LA pathway, which runs through EC, was enabled by cutting connections between EC and LA or by blocking GABA(A) receptor-mediated transmission. In contrast, LTP in the ACC-LA pathway, which bypasses EC, was GABA(A) receptor independent. The EC transection shifted balance between inhibitory and excitatory responses in the TeA-LA pathway toward excitation, but had no effect on the ACC-LA pathway. Thus, EC provides pathway-specific suppression of amygdala plasticity.

Conditional deletion of neuronal cyclin-dependent kinase 5 in developing forebrain results in microglial activation and neurodegeneration.

Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders.

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.

Enhanced generalization of auditory conditioned fear in juvenile mice.

Increased emotionality is a characteristic of human adolescence, but its animal models are limited. Here we report that generalization of auditory conditioned fear between a conditional stimulus (CS+) and a novel auditory stimulus is stronger in 4-5-wk-old mice (juveniles) than in their 9-10-wk-old counterparts (adults), whereas nonassociative sensitization induced by foot shock (US) and the ability to discriminate CS+ from an explicitly unpaired stimulus (CS-) are not dependent on age. These results suggest that aversive associations are less precise in juvenile mice and can more easily produce conditional responses to stimuli different from CS+. Yet, through the explicit unpairing of CS- from US during training, juveniles are able to overcome this greater fear generalization and learn that CS- is not associated with foot shock.

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.

Gene expression profiling of facilitated L-LTP in VP16-CREB mice reveals that BDNF is critical for the maintenance of LTP and its synaptic capture.

Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. We then explored the contribution of the most prominent candidate genes revealed by our screening, namely prodynorphin, BDNF, and MHC class I molecules, to the facilitated LTP of VP16-CREB mice. We found that the overexpression of brain-derived neurotrophic factor accounts for an important component of this phenotype.

Enhanced cortico-amygdala efficacy and suppressed fear in absence of Rap1.

Auditory fear conditioning, a model for fear learning, is thought to be mediated by synaptic changes in the cortical and thalamic inputs to the lateral amygdala (LA); however, the specific roles of both pathways are still debated. Here, we report that a CaMKII-alpha-Cre-mediated knock-out (KO) of the rap1a and rap1b genes impaired synaptic plasticity and increased basal synaptic transmission in the cortical but not thalamic input to the LA via presynaptic changes: increases in glutamate release probability and the number of glutamate quanta released by a single action potential. Moreover, KO mice with alterations in the cortico-LA pathway had impaired fear learning, which could be rescued by training with a more aversive unconditional stimulus. These results suggest that Rap1-mediated suppression of synaptic transmission enables plasticity in the cortico-amygdala pathway, which is required for fear learning with a moderately aversive unconditional stimulus.

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.