Freezing Behavior Research Articles

Static magnetic field alters properties of confined alkylammonium nitrate ionic liquids

Ethylammonium nitrate (EAN) and propylammonium nitrate (PAN) ionic liquids confined between polar glass plates and exposed to a strong magnetic field of 9.4 T demonstrate gradually slowing diffusivity, a process that can be reversed by removing the sample from the magnetic field. The process can be described well by the Avrami equation, which is typical for autocatalytic (particularly, nucleation controlled) processes. The transition can be stopped by freezing the sample. Cooling and heating investigations showed differences in the freezing and melting behavior of the sample depending on whether it had been exposed to the magnetic field. After exposure to the magnetic field, the sample demonstrated decrease in the 1H NMR signal of residual water. 1H NMR spectroscopy with presaturation demonstrates that the most probable mechanism of the decrease of the bulk water signal is adsorption of water on polar surfaces of glass plates. Generally, our findings confirm our previous suggestion that alteration of the dynamic properties of confined alkylammonium nitrate ionic liquids exposed to a magnetic field is related to the alteration of real physical-chemical phases.

Tuning magnetocaloric effect of Gd–Er–Al–Co metallic glass through crystallization

Magnetization and magnetocaloric effect (MCE) of as-cast Gd-based bulk metallic glass (BMG) Gd24Er32Co24Al20 and the crystallized samples were investigated. Reentrant-spin-glass-like behavior was observed in the as-cast sample. With increasing degree of crystallization, the magnetic frustration increased, which could be seen from the increased divergence of the zero-field-cooled and field-cooled magnetization behavior (i.e., the spin-glass-like freezing behavior). The Curie temperature and magnetization decreased after crystallization. The as-cast BMG showed a large maximum magnetic entropy change (− ΔSm) of 9.9 (J kg−1 K−1) under a field change of 5 T, which was comparable with that of Gd metal and other Gd-based metallic glasses. The maximum − ΔSm reduced to 6.7 (J kg−1 K−1) after crystallization, which was still superior to the Fe-/Co-based amorphous alloys. Their good MCE combining with high electrical resistivity, outstanding mechanical properties, tunable nature, and sufficiently soft magnetic property makes them to be attractive candidate for magnetic refrigerants in the low-temperature range.

Quantitative assessment of cypermethrin induced behavioural and biochemical anomalies in adult zebrafish

Cypermethrin is one of the top five pesticides used globally. Although the effect of cypermethrin on the embryonic stages of zebrafish is well characterized, its toxic effect on the behaviour of adult zebrafish is largely unknown. Here we used videogram and automated tracking approach to quantitatively assess behavioural toxicity induced by the short exposure of cypermethrin to adult zebrafish. We observed that cypermethrin at 25 ppb level induced behavioural toxicity in adult zebrafish. Motor activity of the treated group was significantly retarded which affected their overall exploratory behaviour including their visit to the central arena of the open-field test. Furthermore, the treated group showed erratic movements (covered less distance per unit time) without affecting their angle based behavioural endpoints. In contrast to the control group, the cypermethrin exposed group showed frequent freezing behaviour. However, their freezing episodes were characterized by constant drift-like movement caused by the loss of their voluntary control over the motor coordination. These behavioural changes are similar to typical anxiety-like behaviour. Though, cypermethrin exposure at ppb level for just half an hour was sufficient to induce behavioural toxicity, it failed to alter brain superoxide dismutase and acetylcholine esterase enzyme activity. Our data indicates that acute short-term exposure of cypermethrin induces behavioural anomalies in adult zebrafish through a mechanism distinct from alteration of brain superoxide dismutase and the acetylcholine esterase activity.

Frozen State of Sephadex® Gels of Different Crosslink Density Analyzed by X-ray Computed Tomography and X-ray Diffraction.

Water in Sephadex® (crosslinked dextran) gels is known to indicate different freezing behavior which is dependent on the density of the crosslinks, and water in a Sephadex® G25 gel remains partially unfrozen during cooling and crystallizes during rewarming. The mechanism of anomalous ice crystallization during rewarming is still unclear. The objective of this study is to observe the ice grains that form in Sephadex® beads and to comprehend their frozen state with a focus on the ice crystallization during rewarming. Sephadex® beads containing 50 wt % water were prepared and used for the measurements. The observation of the ice grains was carried out by using synchrotron radiation-sourced X-ray CT (computed tomography). XRD (X-ray diffraction) analysis was also conducted to investigate the frozen state. As a result, ice grains that were larger than ~1 μm were hardly observed after the slow cooling of Sephadex® beads, except in the G25 beads. However, at the occurrence of ice crystallization during rewarming, ice grains that were larger than 10 μm appeared in the G25 beads. Using XRD, it was found that small incomplete ice crystals were formed in G25 beads and the presence of glassy water was indicated in the gel. In conclusion, the size and distribution of ice grains that formed in Sephadex® beads were different depending on the density of the crosslinks.

Investigation of the Stability of Human Freezing-Like Responses to Social Threat From Mid to Late Adolescence.

Freezing behavior, a commonly observed defensive stress response, shows relatively high stability over time in animals. Given the relevance of freezing for stress-coping and human psychopathology, it is relevant to know whether freezing behavior is also stable in humans, particularly during adolescence, when most affective symptoms develop. In a prospective longitudinal study, we investigated freezing-like behavior in response to social threat in 75 adolescents at age 14, repeated 3 years later at age 17. We used a well-established method combining electrocardiography (ECG; heart rate) and posturography (body sway) in response to emotional picture-viewing of angry, happy, and neutral faces. We hypothesized that individual differences in freezing-like behavior in response to social threat—operationalized by contrasting angry vs. neutral faces—would be relatively stable over time. Our results indeed showed relative stability between ages 14 and 17 in individual differences in freezing-like behavior in heart rate (r = 0.82), as well as in combined heart rate and body sway measures (r = 0.65). These effects were not specific for the angry vs. neutral contrast; they were also visible in other emotion contrasts. Exploratory analysis in males and females separately showed stability in body sway specifically for angry vs. neutral faces only in females. Together, these results suggest moderate to strong stability in human freezing-like behavior in response to social threat from mid to late adolescence (with exception for the body sway measure in males). This relative stability was not specific for threat-induction and may reflect a general stability that is particularly strong for heart rate. The fact that this relative stability was found over a relatively long time range of 3 years is promising for studies aiming to use freezing-like behavior as a marker for internalizing symptoms in adolescent development.

Olfactory inputs modulate respiration-related rhythmic activity in the prefrontal cortex and freezing behavior

Respiration and airflow through the nasal cavity are known to be correlated with rhythmic neural activity in the central nervous system. Here we show in rodents that during conditioned fear-induced freezing behavior, mice breathe at a steady rate (~4 Hz), which is correlated with a predominant 4-Hz oscillation in the prelimbic prefrontal cortex (plPFC), a structure critical for expression of conditioned fear behaviors. We demonstrate anatomical and functional connections between the olfactory pathway and plPFC via circuit tracing and optogenetics. Disruption of olfactory inputs significantly reduces the 4-Hz oscillation in the plPFC, but leads to prolonged freezing periods. Our results indicate that olfactory inputs can modulate rhythmic activity in plPFC and freezing behavior.

Signals from the Gut Influence Mouse Behavior: Examination of Mechanisms

The intestinal microflora can have significant influences on brain activity. Gut microbiota release neurotransmitters that are hypothesized to send signals via the vagal nerve back to the brain. However, such afferent interactions have never been proven. The current experiments utilized optogenetic vectors (AAV‐CaMKIIa‐eNpHR3.0‐EYFP) placed within the jejunum to label vagal nerves. These vectors induce the insertion of light sensitive chloride channels into neuronal membranes that when stimulated with light deactivate the neuron. By placing these vectors in the jejunum, the experiment selectively deactivated only the select fibers that run from the jejunum to the brain. Control animals were injected with saline.In all animals, normal animal behavior was recorded for anxiety measures (elevated plus maze), open field behaviors, and forced swim tests. Each test was performed with both light deactivation of the vagus, and no‐light conditions. Results indicated that mouse behavior was not affected for the control animals nor for animals in the no‐light vector conditions. However, the animal behavior was dramatically altered for each animal in the optogenetic light deactivation condition. In each behavioral test, animals showed a freezing behavior for the extent of time that the vagus was deactivated. Within the forced swim experiments, frozen animals did not keep their head above water and tended to sink to the bottom of the tank (requiring multiple rescues during each session).The results support the hypothesis that the jejunum can influence brain activity via vagal nerve efferents. I conclude that optogenetic deactivation of the vagal afferents of the jejunum has a direct influence on animal behavior and thus brain circuits. Therefore, this pathway provides an optimal mechanism by which future nutritional therapies can be devised for a variety of brain pathologies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockdown of Corticotropin Releasing Hormone Receptor 1 (CRF1) in the Rostral Peri‐Cellular Region of the Locus Coeruleus (LC) Normalizes Anxiety‐Like Behavior in Female Rats Subjected to Coronary Ischemia and Reperfusion (IR)

After myocardial infarction (MI), patients often develop post‐traumatic stress disorder (PTSD), an anxiety disorder characterized by deficits in extinction of learned fear. Women develop more severe symptoms of PTSD, and severity of Post‐MI PTSD symptoms is predictive of further cardiovascular morbidity. Excessive noradrenergic drive in the medial prefrontal cortex (mPFC) has been implicated in PTSD. Neural projections from the amygdala release CRF in the rostral peri‐cellular region of the LC, and CRF stimulates LC neurons to release norepinephrine in mPFC to modulate fear extinction. Here, we examined the role of peri‐LC CRF in PTSD‐like behavior in a rat model of MI. Female and male rats were trained to fear a tone and then subjected to IR or sham surgery 1 week later. Two weeks later, rats were given bilateral LC injections of a viral vector encoding a shRNA sequence targeting the rat CRF1 receptor, or a scrambled sequence. Six weeks later, cumulative freezing behavior was measured during 10 extinction trials. One day later, rats were anesthetized with ketamine and perfused for in situ detection of CRF1 mRNA. IR impaired left ventricular (LV) function more in males than females (23.3±3.1 [n=17] vs 33.7±4.4% [n=18] fractional shortening). Females with more severe LV damage (fractional shortening ≤40%) took longer to extinguish fear than sham females (99.7±12.3 [n=4] vs. 41.6±7.3 seconds [n=6], P<0.01). Treatment with anti‐CRF1 shRNA reversed deficits in extinction among IR females with more severe LV damage (43.3±7.7 seconds [n=6], P<0.05). Males showed similar trends that were not significant (82.9±16.0 [n=9] vs. 57.1±9.7 [n=10] and 58.6±14.4 seconds [n=9]). Rostral Peri‐LC CRF1 mRNA was elevated by 66% (P<0.01) in sham females compared to sham males. ShRNA treatment reduced rostral peri‐LC CRF1 mRNA by 22.9% (P<0.05) and 42% (P<0.01) in female and male sham rats respectively, but did not affect caudal LC CRF1 mRNA levels. Female and male IR rats showed reduced rostral (but not caudal) peri‐LC CRF1 mRNA levels compared to their respective sham controls (−58%, P<0.01 and −38%, P<0.05 respectively). ShRNA treatment did not further reduce peri‐LC CRF1 mRNA levels in IR rats. Rostral peri‐LC CRF1 mRNA levels were inversely related to cumulative freezing in females treated with the scrambled sequence (P<0.01). Together, the data indicate that rostral peri‐LC CRF contributes to deficits in fear extinction in female rats after IR. The data are also consistent with the possibility that fear‐induced release of CRF in the rostral peri‐LC region acutely suppresses CRF1 gene expression.Support or Funding InformationAmerican Heart Association GRNT20380765, Cardiovascular Research Institute, Loyola University ChicagoThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nucleation of Aqueous Salt Solutions on Solid Surfaces

The freezing of water in the presence of salts is very common and widely investigated phenomena. However, the role of the substrate during crystallization, and in particular the molecular-level resolution of nucleation mechanism, is still not well-understood. In this work, we investigated the freezing behavior of supercooled water and aqueous lithium chloride solutions on smooth graphitic surfaces. We illustrate the role of solid surfaces in the freezing process of aqueous solutions as a function of mole fraction of a salt exhibiting lowering of freezing temperature, irrespective of a water-binding affinity to the surface. Our molecular dynamics simulations show that the hydrophobic surface is a better nucleating surface when the mole fraction of the salt is over 5%. Our findings reveal that nucleation of ice occurs heterogeneously at the liquid–solid interface. Consequently, propagation of the ice front yields phase-segregated brine near the liquid–vapor interface. Furthermore, we have investigated the effect of salt–surface interaction on the freezing process. We observe lowering of the freezing point with an increase in the water–surface interaction. The simulations demonstrate that nucleation of ice occurs heterogeneously at the liquid–solid interface for low values of interaction, whereas homogeneous nucleation of ice takes place away from the substrate at higher interaction strengths.

Bidirectional influence of amygdala β1-adrenoceptors blockade on cannabinoid signaling in contextual and auditory fear memory.

The basolateral amygdala (BLA) is a major target and modulator of stress and has a critical role in the neural circuitry presenting learned fear behaviors. On the other hand, both the endocannabinoid and noradrenergic systems may be involved in regulating the stress responses, fear, and anxiety. Considering the aforementioned, we have investigated the involvement of the BLA β1-adrenoceptors in conditioned fear responses induced by ACPA, a CB1 receptor (CB1R) agonist. In adult male NMRI mice, freezing responses to context and cue were measured using a Pavlovian fear conditioning apparatus. Pre-training intra-BLA microinjection of xamoterol (0.01 and 0.02 µg/mouse), a partial β1-adrenoceptor agonist, or atenolol (0.5 µg/mouse), a β1-adrenoceptor antagonist, decreased freezing behavior, which suggests an impairment of contextual and auditory fear retrieval. Similar results were found with pre-training intraperitoneal administration of ACPA (0.5 mg/kg). A sub-threshold dose of xamoterol, infused into the BLA, decreased ACPA (0.005 and 0.05 mg/kg) effect on both memories, while atenolol increased ACPA response to the context at the middle dose and decreased ACPA response to the tone at the lower dose. It can be concluded that the blockade of BLA β1-adrenoceptors differentially affects ACPA response on the contextual and auditory conditioned fear memories.

Impact of Buffer, Protein Concentration and Sucrose Addition on the Aggregation and Particle Formation during Freezing and Thawing

PurposeThis study addresses the effect of freezing and thawing on a therapeutic monoclonal antibody (mAb) solution and the corresponding buffer formulation. Particle formation, crystallization behaviour, morphology changes and cryo-concentration effects were studied after varying the freezing and thawing rates, buffer formulation and protein concentration. The impact of undergoing multiple freeze/thaw (FT)-cycles at controlled and uncontrolled temperature rates on mAb solutions was investigated in terms of particle formation.MethodsPhysicochemical characteristics were analysed by Differential Scanning Calorimetry whereas morphology changes are visualized by cryomicroscopy measurements. Micro Flow Imaging, Archimedes and Dynamic Light Scattering were used to investigate particle formation.ResultsData retrieved in the present study emphasizes the damage caused by multiple FT-cyles and the need for sucrose as a cryoprotectant preventing cold-crystallization specifically at high protein concentrations. Low protein concentrations cause an increase of micron particle formation. Low freezing rates lead to a decreased particle number with increased particle diameter.ConclusionThe overall goal of this research is to gain a better understanding of the freezing and thawing behaviour of mAb solutions with the ultimate aim to optimize this process step by reducing the unwanted particle formation, which also includes protein aggregates.

Interaction of basolateral amygdala, ventral hippocampus and medial prefrontal cortex regulates the consolidation and extinction of social fear

BackgroundFollowing a social defeat, the balanced establishment and extinction of aversive information is a beneficial strategy for individual survival. Abnormal establishment or extinction is implicated in the development of mental disorders. This study investigated the time course of the establishment and extinction of aversive information from acute social defeat and the temporal responsiveness of the basolateral amygdala (BLA), ventral hippocampus (vHIP) and medial prefrontal cortex (mPFC) in this process.MethodsMouse models of acute social defeat were established by using the resident–intruder paradigm. To evaluate the engram of social defeat, the intruder mice were placed into the novel context at designated time to test the social behavior. Furthermore, responses of BLA, vHIP and mPFC were investigated by analyzing the expression of immediate early genes, such as zif268, arc, and c-fos.ResultsThe results showed after an aggressive attack, aversive memory was maintained for approximately 7 days before gradually diminishing. The establishment and maintenance of aversive stimulation were consistently accompanied by BLA activity. By contrast, vHIP and mPFC response was inhibited from this process. Additionally, injecting muscimol (Mus), a GABA receptor agonist, into the BLA alleviated the freezing behavior and social fear and avoidance. Simultaneously, Mus treatment decreased the zif268 and arc expression in BLA, but it increased their expression in vHIP.ConclusionOur data support and extend earlier findings that implicate BLA, vHIP and mPFC in social defeat. The time courses of the establishment and extinction of social defeat are particularly consistent with the contrasting BLA and vHIP responses involved in this process.

Role of dopamine D3 receptor in alleviating behavioural deficits in animal models of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a complicated psychiatric disorder, which occurs after exposure to a traumatic event. The main clinical manifestation of PTSD includes fear and stress dysregulation. In both animals and humans, dysregulation of dopamine function appears to be related to conditioned fear responses. Previous studies show that the dopamine D3 receptor (D3R) is involved in schizophrenia, autism, and substance use disorders and is related to emotional disorders. However, few studies have investigated the role of the D3R in the pathogenesis and aetiology of PTSD. In the current study, we have reported that D3R knockout (D3R−/−) mice displayed decreased freezing time of contextual fearing and anxiolytic effects following training sessions consisting of exposure to inescapable electric foot-shocks. Similarly, highly selective blockade of D3Rs by YQA14, a novel D3R antagonist, significantly ameliorated freezing and anxiogenic-like behaviours in the single-prolonged stress (SPS) model of PTSD in rats. And more, YQA14 selectively alleviated the symptoms of PTSD in WT mice but not in D3R−/− mice. In summary, this study demonstrates the anti-PTSD effects of blockade or knockout of the D3R, suggesting that the D3R might play an important role in the pathogenesis and aetiology of PTSD, and might be a potential target for the clinical management of PTSD.

Influence of Pore Wall Hydrophobicity on Freezing and Melting of Confined Water

To examine the influence of pore wall hydrophobicity on freezing and melting behavior of a confined water, we measured the X-ray diffraction pattern of water in the nearly cylindrical pores of orde...

Numerical analysis of the freeze-thaw performance of cementitious composites that contain phase change material (PCM)

We employ a numerical methodology to predict the freeze-thaw performance of cementitious composites containing lightweight aggregates (LWAs) impregnated with phase change material (PCM) as thermal energy storage (TES) agent. The governing equation for heat transfer is combined with homogenization techniques to predict the temperature and associated heat flow for cementitious materials during the phase transition of the PCM. The material properties of the cementitious composite and heat dissipation behavior during thermal cycling were characterized using a small-scale longitudinal guarded comparative calorimeter (LGCC) test. Phase transitions associated with freezing/melting of PCM occurs gradually over a narrow temperature range. Pore size effect of LWA on freezing and melting behavior of PCM was found to be relatively small. This approach is extended to the prediction of the thermal energy storage capacity of PCM on improving the freeze-thaw performance of concrete specimens exposed to realistic thermal conditions at various locations in the United States.

The Activating Transcription Factor 3 (Atf3) Homozygous Knockout Mice Exhibit Enhanced Conditioned Fear and Down Regulation of Hippocampal GELSOLIN.

The genetic and molecular basis underlying fear memory formation is a key theme in anxiety disorder research. Because activating transcription factor 3 (ATF3) is induced under stress conditions and is highly expressed in the hippocampus, we hypothesize that ATF3 plays a role in fear memory formation. We used fear conditioning and various other paradigms to test Atf3 knockout mice and study the role of ATF3 in processing fear memory. The results demonstrated that the lack of ATF3 specifically enhanced the expression of fear memory, which was indicated by a higher incidence of the freeze response after fear conditioning, whereas the occurrence of spatial memory including Morris Water Maze and radial arm maze remained unchanged. The enhanced freezing behavior and normal spatial memory of the Atf3 knockout mice resembles the fear response and numbing symptoms often exhibited by patients affected with posttraumatic stress disorder. Additionally, we determined that after fear conditioning, dendritic spine density was increased, and expression of Gelsolin, the gene encoding a severing protein for actin polymerization, was down-regulated in the bilateral hippocampi of the Atf3 knockout mice. Taken together, our results suggest that ATF3 may suppress fear memory formation in mice directly or indirectly through mechanisms involving modulation of actin polymerization.

Proton NMR study of extra Virgin Olive Oil with temperature: Freezing and melting kinetics

The thermal properties of an extra Virgin Olive Oil (eVOO) depend on its composition and indeed characterize its quality. Many studies have shown that the freezing and melting behaviors of eVOOs can serve for geographical or chemical discrimination. We use Nuclear Magnetic Resonance spectroscopy to study the evolution of the fatty acids bands as a function of temperature during freezing and melting processes. In such a way we can follow separately the variations in the thermal properties of the different molecular groups during these thermodynamic phase transitions. The data indicate that the methyl group which is at the end of every fatty chain displays the major changes during both freezing and melting processes.

Interleukin-6 is both necessary and sufficient to produce perioperative neurocognitive disorder in mice

BackgroundPerioperative neurocognitive disorders (PND) result in long-term morbidity and mortality with no effective interventions available. Because interleukin-6 (IL-6), a pro-inflammatory cytokine, is consistently up-regulated by trauma, including after surgery, we determined whether IL-6 is a putative therapeutic target for PND in a mouse model. MethodsFollowing institutional approval, adult (12–14 weeks) male C57/Bl6 mice were pretreated with the IL-6 receptor (IL6R) blocking antibody tocilizumab prior to open tibia fracture with internal fixation under isoflurane anaesthesia. Inflammatory and behavioural responses in a trace fear conditioning (TFC) paradigm were assessed postoperatively. Separately, the effects of IL-6 administration or of depletion of bone marrow-derived monocytes (BM-DMs) with clodrolip on the inflammatory and behavioural responses were assessed. Blood brain barrier disruption, hippocampal microglial activation, and infiltration of BM-DMs were each assessed following IL-6 administration. ResultsThe surgery-induced decrement in freezing time in the TFC assay, indicative of cognitive decline, was attenuated by tocilizumab (P<0.01). The surgery-induced increase in pro-inflammatory mediators was significantly reduced by tocilizumab. Exogenously administered IL-6 significantly impaired freezing behaviour (P<0.05) and up-regulated pro-inflammatory cytokines; both responses were prevented by depletion of BM-DMs. IL-6 disrupted the blood brain barrier, and increased hippocampal activation of microglia and infiltration of BM-DMs. ConclusionsIL-6 is both necessary and sufficient to produce cognitive decline. Following further preclinical testing of its perioperative safety, the IL6R blocker tocilizumab is a candidate for prevention and/or treatment of PND.

Yokukansan, a traditional Japanese herbal medicine, enhances the anxiolytic effect of fluvoxamine and reduces cortical 5-HT2A receptor expression in mice

Ethnopharmacological relevanceYokukansan is a traditional Japanese herbal medicine that has been approved in Japan as a remedy for neurosis, insomnia, and irritability in children. It has also been reported to improve behavioral and psychological symptoms in patients with various forms of dementia. Aim of the studyTo evaluate the usefulness of co-treatment with an antidepressant and an herbal medicine in the psychiatric field, the current study examined the effect of yokukansan on the anxiolytic-like effect of fluvoxamine in mice. Materials and methodsThe anxiolytic-like effect in mice was estimated by the contextual fear conditioning paradigm. Contextual fear conditioning consisted of two sessions, i.e., day 1 for the conditioning session and day 2 for the test session. The expression levels of 5-HT1A and 5-HT2A receptor in the mouse brain regions were quantified by western blot analysis. ResultsA single administration of fluvoxamine (5–20 mg/kg, i.p.) before the test session dose-dependently and significantly suppressed freezing behavior in mice. In the combination study, a sub-effective dose of fluvoxamine (5 mg/kg, i.p.) significantly suppressed freezing behavior in mice that had been repeatedly pretreated with yokukansan (0.3 and 1 g/kg, p.o.) once a day for 6 days after the conditioning session. Western blot analysis revealed that the expression level of 5-HT2A receptor was specifically decreased in the prefrontal cortex of mice that had been administered yokukansan and fluvoxamine. Furthermore, microinjection of the 5-HT2A receptor antagonist ketanserin (5 nmol/mouse) into the prefrontal cortex significantly suppressed freezing behavior. ConclusionThe present findings indicate that repeated treatment with yokukansan synergistically enhances the anxiolytic-like effect of fluvoxamine in the contextual fear conditioning paradigm in mice in conjunction with a decrease in 5-HT2A receptor-mediated signaling in the prefrontal cortex. Therefore, combination therapy with fluvoxamine and yokukansan may be beneficial for the treatment of anxiety disorders.

Ice Nucleation Activity in Plants: The Distribution, Characterization, and Their Roles in Cold Hardiness Mechanisms.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.