Mild Transient Hypercapnia as a Novel Fear Conditioning Stimulus Allowing Re-Exposure during Sleep

Introduction:Studies suggest that sleep plays a role in traumatic memories and that treatment of sleep disorders may help alleviate symptoms of posttraumatic stress disorder. Fear-conditioning paradigms in rodents are used to investigate causal mechanisms of fear acquisition and the relationship between sleep and posttraumatic behaviors. We developed a novel conditioning stimulus (CS) that evoked fear and was subsequently used to study re-exposure to the CS during sleep.Methods:Experiment 1 assessed physiological responses to a conditioned stimulus (mild transient hypercapnia, mtHC; 3.0% CO2; n = 17)+footshock for the purpose of establishing a novel CS in male FVB/J mice. Responses to the novel CS were compared to tone+footshock (n = 18) and control groups of tone alone (n = 17) and mild transient hypercapnia alone (n = 10). A second proof of principle experiment re-exposed animals during sleep to mild transient hypercapnia or air (control) to study sleep processes related to the CS.Results:Footshock elicited a response of acute tachycardia (30-40 bpm) and increased plasma epinephrine. When tone predicted footshock it elicited mild hypertension (1-2 mmHg) and a three-fold increase in plasma epinephrine. When mtHC predicted footshock it also induced mild hypertension, but additionally elicited a conditioned bradycardia and a smaller increase in plasma epinephrine. The overall mean 24 hour sleep-wake profile was unaffected immediately after fear conditioning.Discussion:Our study demonstrates the efficacy of mtHC as a conditioning stimulus that is perceptible but innocuous (relative to tone) and applicable during sleep. This novel model will allow future studies to explore sleep-dependent mechanisms underlying maladaptive fear responses, as well as elucidate the moderators of the relationship between fear responses and sleep. © 2013 McDowell et al

Functional Modifications of Acid-Sensing Ion Channels by Ligand-Gated Chloride Channels

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABAA receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABAA receptor-mediated currents. Moreover, activation of the GABAA receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABAA receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABAA receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABAA receptors, also modified ASICs in spinal neurons. We conclude that GABAA receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

The Interaction between the First Transmembrane Domain and the Thumb of ASIC1a Is Critical for Its N-Glycosylation and Trafficking

Acid-sensing ion channel-1a (ASIC1a), the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1) and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function

Experimental and computational study of trace element distribution between orthopyroxene and anhydrous silicate melts: substitution mechanisms and the effect of iron

Although orthopyroxene (Opx) is present during a wide range of magmatic differentiation processes in the terrestrial and lunar mantle, its effect on melt trace element contents is not well quantified. We present results of a combined experimental and computational study of trace element partitioning between Opx and anhydrous silicate melts. Experiments were performed in air at atmospheric pressure and temperatures ranging from 1,326 to 1,420°C in the system CaO-MgO-A

High-Density Expression of Ca2+-Permeable ASIC1a Channels in NG2 Glia of Rat Hippocampus

NG2 cells, a fourth type of glial cell in the mammalian CNS, undergo reactive changes in response to a wide variety of brain insults. Recent studies have demonstrated that neuronally expressed acid-sensing ion channels (ASICs) are implicated in various neurological disorders including brain ischemia and seizures. Acidosis is a common feature of acute neurological conditions. It is postulated that a drop in pH may be the link between the pathological process and activation of NG2 cells. Such postulate immediately prompts the following questions: Do NG2 cells express ASICs? If so, what are their functional properties and subunit composition? Here, using a combination of electrophysiology, Ca2+ imaging and immunocytochemistry, we present evidence to demonstrate that NG2 cells of the rat hippocampus express high density of Ca2+-permeable ASIC1a channels compared with several types of hippocampal neurons. First, nucleated patch recordings from NG2 cells revealed high density of proton-activated currents. The magnitude of proton-activated current was pH dependent, with a pH for half-maximal activation of 6.3. Second, the current-voltage relationship showed a reversal close to the equilibrium potential for Na+. Third, psalmotoxin 1, a blocker specific for the ASIC1a channel, largely inhibited proton-activated currents. Fourth, Ca2+ imaging showed that activation of proton-activated channels led to an increase of [Ca2+]i. Finally, immunocytochemistry showed co-localization of ASIC1a and NG2 proteins in the hippocampus. Thus the acid chemosensor, the ASIC1a channel, may serve for inducing membrane depolarization and Ca2+ influx, thereby playing a crucial role in the NG2 cell response to injury following ischemia

Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice — Refinement of Animal Welfare?

Carbon dioxide (CO2) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the “second gas” effect. We therefore evaluated the addition of Nitrous Oxide (N2O) to a rising CO2 concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N2O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O2 (95%); Isoflurane (5%)+N2O (75%)+O2 (25%) and N2O (75%)+O2 (25%) with a total flow rate of 3l/min (into a 7l induction chamber). The addition of N2O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO2 (20% of the chamber volume.min−1); CO2+N2O (20 and 60% of the chamber volume.min−1 respectively); or CO2+Nitrogen (N2) (20 and 60% of the chamber volume.min−1). Arterial partial pressure (Pa) of O2 and CO2 were measured as well as blood pH and lactate. When compared to the gradually rising CO2 euthanasia, addition of a high concentration of N2O to CO2 lowered the time to loss of righting reflex by 10.3% (P<0.001), lead to a lower PaO2 (12.55±3.67 mmHg, P<0.001), a higher lactataemia (4.64±1.04 mmol.l−1, P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO2 during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia

Unstable Maternal Environment, Separation Anxiety, and Heightened CO2 Sensitivity Induced by Gene-by-Environment Interplay

Background: In man, many different events implying childhood separation from caregivers/unstable parental environment are associated with heightened risk for panic disorder in adulthood. Twin data show that the occurrence of such events in childhood contributes to explaining the covariation between separation anxiety disorder, panic, and the related psychobiological trait of CO2 hypersensitivity. We hypothesized that early interference with infant-mother interaction could moderate the interspecific trait of response to CO2 through genetic control of sensitivity to the environment. Methodology: Having spent the first 24 hours after birth with their biological mother, outbred NMRI mice were crossfostered to adoptive mothers for the following 4 post-natal days. They were successively compared to normally-reared individuals for: number of ultrasonic vocalizations during isolation, respiratory physiology responses to normal air (20%O2), CO2-enriched air (6% CO2), hypoxic air (10%O2), and avoidance of CO2-enriched environments. Results: Cross-fostered pups showed significantly more ultrasonic vocalizations, more pronounced hyperventilatory responses (larger tidal volume and minute volume increments) to CO2-enriched air and heightened aversion towards CO2- enriched environments, than normally-reared individuals. Enhanced tidal volume increment response to 6%CO2 was present at 16–20, and 75–90 postnatal days, implying the trait’s stability. Quantitative genetic analyses of unrelated individuals, sibs and half-sibs, showed that the genetic variance for tidal volume increment during 6%CO2 breathing was significantly higher (Bartlett x = 8.3, p = 0.004) among the cross-fostered than the normally-reared individuals, yielding heritability of 0.37 and 0.21 respectively. These results support a stress-diathesis model whereby the genetic influences underlying the response to 6%CO2 increase their contribution in the presence of an environmental adversity. Maternal grooming/licking behaviour, and corticosterone basal levels were similar among cross-fostered and normally-reared individuals. Conclusions: A mechanism of gene-by-environment interplay connects this form of early perturbation of infant-mother interaction, heightened CO2 sensitivity and anxiety. Some no

Transcranial Magnetic Stimulation Intensities in Cognitive Paradigms

BACKGROUND: Transcranial magnetic stimulation (TMS) has become an important experimental tool for exploring the brain's functional anatomy. As TMS interferes with neural activity, the hypothetical function of the stimulated area can thus be tested. One unresolved methodological issue in TMS experiments is the question of how to adequately calibrate stimulation intensities. The motor threshold (MT) is often taken as a reference for individually adapted stimulation intensities in TMS experiments, even if they do not involve the motor system. The aim of the present study was to evaluate whether it is reasonable to adjust stimulation intensities in each subject to the individual MT if prefrontal regions are stimulated prior to the performance of a cognitive paradigm. METHODS AND FINDINGS: Repetitive TMS (rTMS) was applied prior to a working memory task, either at the 'fixed' intensity of 40% maximum stimulator output (MSO), or individually adapted at 90% of the subject's MT. Stimulation was applied to a target region in the left posterior middle frontal gyrus (pMFG), as indicated by a functional magnetic resonance imaging (fMRI) localizer acquired beforehand, or to a control site (vertex). Results show that MT predicted the effect size after stimulating subjects with the fixed intensity (i.e., subjects with a low MT showed a greater behavioral effect). Nevertheless, the individual adaptation of intensities did not lead to stable effects. CONCLUSION: Therefore, we suggest assessing MT and account for it as a measure for general cortical TMS susceptibility, even if TMS is applied outside the motor domain