Increase In Transients Research Articles

Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

Mitochondrial Fission Protein Drp1 Regulates Platelet Function

Background: Mitochondria in platelets contribute to platelet function and are altered by diseases that involve dysregulated hemostasis or thrombosis. Mitochondrial integrity, governed by its size, number and distribution is modulated by fusion and fission proteins. Pharmacological inhibition of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) has been shown to inhibit granule release in human platelets and impair thrombus formation in a murine model of thrombosis. However, the mitochondrial roles for Drp1 in platelet function are still largely unknown. Objective: To determine the mitochondrial roles of Drp1 in regulating platelet function. Methods: PF4-Cre mediated MK/platelet specific Drp1 null mice (Drp1 -/-) were used. Platelet morphology in Drp1 -/- and wild type (WT) platelets was analyzed by confocal microscopy. Procoagulant platelet formation (PS +ve platelets) after thrombin and the GPVI agonist convulxin (THR+CVX) stimulation was measured using Annexin V staining. Mitochondrial potential (ΔΨm) and mass at baseline and after stimulation was measured using TMRM and mitotracker green. Washed platelet activation in WT and Drp1 -/- platelets in response to convulxin was measured by flow cytometry. Platelet cytoplasmic calcium transients were measured using FURA-2AM after stimulation with THR+CVX. Phosphorylation of Drp1 (pDrp1 S616) in washed WT platelets in response to dual agonist THR+CVX was measured by western blotting. Results: Drp1 -/- platelets were bigger in size than WT platelets as measured by microscopy and had increased mitochondrial mass as determined by flow cytometry. Absence of Drp1 reduced activation of the αIIb/β3 fibrinogen binding receptor (P=0.035; n=5-6 per group) and reduced alpha granule release (P=0.025; n=5-6 per group) in response to CVX.Dual agonist stimulation resulted in reduced PS +ve platelets in Drp1 -/- compared to WT platelets (P=0.0035; n=7 per group). Since depolarization precedes PS exposure, we measured ΔΨm using TMRM in resting and dual agonist stimulated WT and Drp1 -/- platelets. No difference in ΔΨm was observed in resting Drp1 -/- vs WT platelets whereas, stimulated Drp1 -/- platelets exhibited significantly higher ΔΨm (P=0.038; n=4 per group) than stimulated WT platelets. Interestingly, in response to dual agonists, Drp1 -/- platelets also showed a significant increase (P<0.001) in cytoplasmic calcium transients than WT platelets. Western blot analyses of washed WT platelets stimulated with dual agonist showed phosphorylation of Drp1 at S616 (S616), indicating Drp1 activation. Conclusion: These findings highlight a role for Drp1 in murine platelet mitochondrial function, GPVI activation, and procoagulant platelet formation.

Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5Gy, two doses per week, for 4weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700μm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

About ghost transients in spatial continuous media

The impact of space on ecosystem dynamics has been a matter of debate since the dawn of theoretical ecology. Several studies have revealed that space usually involves an increase in transients’ times, promoting the so-called supertransients. However, the effect of space and diffusion in transients close to bifurcations has not been thoroughly investigated. In non-spatial deterministic models such as those given by ordinary differential equations transients become extremely long in the vicinity of bifurcations. Specifically, for the saddle–node (s–n) bifurcation the time delay, τ, follows τ∼|μ−μc|−1/2; μ and μc being the bifurcation parameter and the bifurcation value, respectively. Such long transients are labeled delayed transitions and are governed by the so-called ghosts. Here, we explore a simple model with intra-specific cooperation (autocatalysis) and habitat loss undergoing a s–n bifurcation using a partial differential equations (PDE) approach. We focus on the effects of diffusion in the ghost extinction transients right after the tipping point found at a critical habitat loss threshold. Our results show that the bifurcation value does not depend on diffusion. Despite transients’ length typically increase close to the bifurcation, we have observed that at extreme values of diffusion, both small and large, extinction times remain long and close to the well-mixed results. However, ghosts lose influence at intermediate diffusion rates, leading to a dramatic reduction of transients’ length. These results, which strongly depend on the initial size of the population, are shown to remain robust for different initial spatial distributions of cooperators. A simple two-patch metapopulation model gathering the main results obtained from the PDEs approach is also introduced and discussed. Finally, we provide analytical results of the passage times and the scaling for the model under study transformed into a normal form. Our findings are discussed within the framework of ecological transients.

Calcium signaling in individual APP/PS1 mouse dentate gyrus astrocytes increases ex vivo with Aβ pathology and age without affecting astrocyte network activity.

Astrocytes are critical for healthy brain function. In Alzheimer’s disease, astrocytes become reactive, which affects their signaling properties. Here, we measured spontaneous calcium transients ex vivo in hippocampal astrocytes in brain slices containing the dentate gyrus of 6‐ (6M) and 9‐month‐old (9M) APPswe/PSEN1dE9 (APP/PS1) mice. We investigated the frequency and duration of calcium transients in relation to aging, amyloid‐β (Aβ) pathology, and the proximity of the astrocyte to Aβ plaques. The 6M APP/PS1 astrocytes showed no change in spontaneous calcium‐transient properties compared to wild‐type (WT) astrocytes. 9M APP/PS1 astrocytes, however, showed more hyperactivity compared to WT, characterized by increased spontaneous calcium transients that were longer in duration. Our data also revealed an effect of aging, as 9M astrocytes overall showed an increase in calcium activity compared to 6M astrocytes. Subsequent calcium‐wave analysis showed an increase in sequential calcium transients (i.e., calcium waves) in 9M astrocytes, suggesting increased network activity ex vivo. Further analysis using null models revealed that this network effect is caused by chance, due to the increased number of spontaneous transients. Our findings show that alterations in calcium signaling in individual hippocampal astrocytes of APP/PS1 mice are subject to both aging and Aβ pathology but these do not lead to a change in astrocyte network activity. These alterations in calcium dynamics of astrocytes may help to understand changes in neuronal physiology leading to cognitive decline and ultimately dementia.

Nitrosylation of cardiac CaMKII at Cys290 mediates mechanical-afterload induced increase in CA transients

Mechanical stress can affect Calcium (Ca) dynamics in cardiomyocytes and lead to cardiac remodeling, hypertrophy, arrhythmias, and heart failure. Both nitric oxide (NO) produced by NOS1 and Ca-calmodulin kinase II (CaMKII) signaling are required mediators of mechano-chemo-transduction (MCT) where mechanical afterload enhances Ca transients and contractility. Our group showed that S-nitrosylation of Cys290 in CaMKIIδ can promote CaMKII activity (Erickson et al. 2015). Here, we developed a novel CaMKIIδ knock-in (KI) mouse (C290A substitution) to test whether this site is essential for MCT-induced enhancement sarcoplasmic reticulum (SR) Ca release and reuptake.

Cyclooxygenase-2 inhibition prevents stress induced amygdala activation and anxiety-like behavior

Stress is a major risk factor for the development and exacerbation of mood and anxiety disorders, and recent studies have suggested inflammatory contributions to the pathogenesis of depression. Interestingly, pharmacological inhibition of cyclooxygenase-2 (COX-2) has shown promise in the treatment of affective disorders in small scale clinical studies; however, the mechanisms by which COX-2 inhibition affects behavioral domains relevant to affective disorders are not well understood. Here, we examined the effects of pharmacological inhibition of COX-2 with the highly selective inhibitor Lumiracoxib (LMX) on anxiety-like behavior and in vivo basolateral amygdala (BLA) neural activity in response to acute restraint stress exposure. In male mice, pretreatment with LMX prevented the increase in BLA calcium transients induced by restraint stress and prevented anxiogenic behavior seen after restraint stress exposure. Specifically, acute injection of LMX 5 mg kg−1 reduced anxiety-like behavior in the light–dark box (LD) and elevated-zero maze (EZM). In addition, in vivo fiber photometry studies showed that acute stress increased calcium transients and the predicted action potential frequency of BLA neurons, which was also normalized by acute LMX pretreatment. These findings indicate pharmacological inhibition of COX-2 can prevent acute stress-induced increase in BLA cellular activity and anxiety-like behavior and provides insights into the neural mechanisms by which COX-2 inhibition could affect anxiety domain symptoms in patients with affective disorders.

Flexible PMOS Inverter and NOR Gate Using Inkjet-Printed Dual-Gate Organic Thin Film Transistors

We report the fabrication of low voltage dual-gate organic thin film transistors, and their possible applications for PMOS inverters and NOR logic gates, on $125~\mu \text{m}$ -thick flexible polyethylene naphthalate (PEN) substrates using solution-processed techniques. Source, drain, and gate electrodes are made by inkjet-printing, while active semiconductor is printed by a dispenser system over parylene dielectric. Bottom and top channel interfaces have been characterized by taking long drain current transients. Formation of stable top channel interface, with low trap density, is a reason for higher top channel current than bottom channel. Increase in drain current transients, and positive threshold voltage shift is attributed to the accumulation of extra positive charge carriers induced by slow polarization of the dipoles in the parylene dielectric.

Beta-Adrenergic Signaling in Isolated Cardiomyocytes Propagates Spatially Over Time

Increased β-adrenergic tone is implicated in cardiac arrhythmias We have tested the hypothesis that the response to isoproterenol (ISO) spreads spatially in a time-dependent manner. Isolated rabbit cardiomyocytes were exposed to ISO locally via pipette placed at its downstream end. Calcium transients were measured along a confocal scan line extending from underneath the ISO pipette to the other end. The peak Ca transients underneath the pipette increased 88.5±11.3% and transient decline was accelerated. Peak transients got gradually smaller with a slower decline with distance from the local ISO exposure. Transients rose only 42.4±5.7% at 36.4 μM distal to the pipette. Time from ISO application to 50% peak Ca transients increased from 22.4±1.7 under the pipette to 29.2±2.3 s. 20 μM away (t-test, p<0.05, n = 7). To verify that delayed activation was a property of the adrenergic response we used 4 mM Ca instead of ISO in the pipette to increase the Ca transient magnitude without activating second messengers. Ca transients increased by 60.7±14.7% of pre-ISO levels underneath the pipette. As was the case with local perfusion of ISO, the Ca transient magnitude rose only 30.1±8.1% at 18.4±2.6 μM distal to the pipette. However, the time to 50% increase in peak calcium transients remained the same with increasing distance away from the site of local Ca perfusion (12.5±1.0 Vs. 13.3±0.7 s. at 20 μM distal from the pipette, t-test, n=6). We conclude that the magnitude of the response to local increases in Ca signaling propagates decrementally away from a focal site of stimulation. Further, the data suggest that there is an additional delay in the response to adrenergic stimulation due to the spatial spread of second messengers that must carry the response distally in the cell.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

In This Issue

In This Issue

Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐derived cardiomyocytes in serum‐free medium

Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemically defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibroblasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tissue displayed the typical fiber morphology with massive striation. An increase in contraction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemically defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro.

Engineering an in vitro organotypic model for studying cardiac hypertrophy.

Neonatal cardiomyocytes cultured on flat surfaces are commonly used as a model to study cardiac failure of diverse origin. A major drawback of such a system is that the cardiomyocytes do not exhibit alignment, organization and calcium transients, similar to the native heart. Therefore, there is a need to develop in vitro platforms that recapitulate the cellular microenvironment of the murine heart as organotypic models to study cardiovascular diseases. In this study, we report an engineered platform that mimics cardiac cell organization and function of the heart. For this purpose, microscale ridges were fabricated on silicon using ultraviolet lithography and reactive ion etching techniques. Physical characterization of the microstructures was done using scanning electron microscopy and atomic force microscopy. Cardiomyocytes grown on these micro-ridges showed global parallel alignment and elliptical nuclear morphology as observed in the heart. Interestingly, calcium currents traversed the engineered cardiomyocytes in a coordinated and directional manner. Moreover, the cardiomyocytes on the engineered substrates were found to be responsive to hypertrophic stimuli, as observed by the expression of a fetal gene, atrial natriuretic peptide and increase in calcium transients upon agonist treatment. Taken together, our work demonstrates that micro-ridges can be used to obtain cardiomyocyte response in vitro, which closely resembles mammalian heart.

How Important Is SERCA In Neuronal Calcium Homeostasis?

Maintaining intracellular calcium homeostasis is critical for excitation‐contraction coupling in the heart, and is essentially dependent on primary and secondary active transport systems. However, upregulating sarcoendoplasmic reticulum calcium ATPase (SERCA) in cardiomyocytes from patients with heart failure has recently proved inconclusive (CUPID2 trial). Neuronal calcium impairment is also seen in cardiovascular diseases resulting in heightened sympathetic responsiveness that further exacerbate the disease phenotype. We tested whether overexpression of SERCA in cardiac sympathetic neurons could modulate intracellular calcium transients. Isolated neurons were incubated with either Ad‐MCherry‐SERCA2A or Ad‐MCherry‐Empty (control) for 24 hours immediately post isolation. Calcium imaging using fura‐2AM (2.5μM) was carried out three days post transfection. SERCA2A transgene neurons showed greater depolarisation induced intracellular calcium transients, compared to control (Δ 380/340 ratio (au): SERCA 0.75±0.03, n=68; Empty 0.64±0.03, n=57. P&lt;0.01). SERCA2A expressing neurons also showed greater SR calcium storage compared to control, as demonstrated by significantly higher calcium responses to caffeine (10mM: Δ 380/340 ratio (au): SERCA 0.15±0.01, n=45; Empty 0.03±0.01, n=35. P&lt;0.0001) and thapsigargin stimulation (1μM: Δ 380/340 ratio (au): SERCA 0.12±0.02, n=42; Empty 0.03±0.01, n=33. P&lt;0.0001). Additionally mitochondrial calcium uncoupling with FCCP (1μM) resulted in a greater intracellular calcium change in the SERCA2A transgenic neurons (Δ 380/340 ratio (au): SERCA 0.13±0.01, n=22; Empty 0.05±0.01, n=22. P&lt;0.0001), indicating that altering neuronal calcium handling with upregulation of SERCA has also effected mitochondrial calcium handling and potentially metabolic function in these neurons. Within this study we demonstrated that a SERCA2A gene construct can be transfected in isolated sympathetic neurons from the stellate ganglia. Moreover, this transduction caused an increase in intracellular calcium transients that would be expected to facilitate neurotransmission and be potentially deleterious for cardiac function.Support or Funding InformationBritish Heart Foundation

Myocardial Infarction Causes Transient Cholinergic Transdifferentiation of Cardiac Sympathetic Nerves via gp130.

Sympathetic neurons normally make norepinephrine (NE), which increases heart rate and the contractility of cardiac myocytes. We found that, after myocardial infarction, the sympathetic neurons innervating the heart begin to make acetylcholine (ACh), which slows heart rate and decreases contractility. Several lines of evidence confirmed that the source of ACh was sympathetic nerves rather than parasympathetic nerves that are the normal source of ACh in the heart. Global application of NE with or without ACh to ex vivo hearts showed that ACh partially reversed the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. That suggests that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility.

Dopamine Modulates the Activity of Sensory Hair Cells.

The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of these mechanosensitive cells. The mechanism of dopamine activation requires voltage-gated calcium channels that are also present at hair-cell synapses.

The soluble guanylate cyclase stimulator riociguat and the soluble guanylate cyclase activator cinaciguat exert no direct effects on contractility and relaxation of cardiac myocytes from normal rats

In cardiovascular diseases, reduced responsiveness of soluble guanylate cyclase (sGC) to nitric oxide (NO) upon long-term application has led to the development of NO-independent sGC stimulators (heme-dependent) and sGC activators (heme-independent). Any direct inotropic or lusitropic effects of these compounds on isolated cardiac myocytes, however, remain to be elucidated.Here, we analyzed the dose-dependent effects of clinical relevant concentrations (10−10–10−5M) of the sGC activator cinaciguat and the sGC stimulator riociguat on the contraction, relaxation, and calcium transients of isolated field-stimulated cardiac myocytes from healthy rats. For comparison, we used isoproterenol, which induced a dose-dependent significant increase in cell contractility, relaxation, and calcium transients, verapamil that significantly decreased these parameters (both at 10−9–10−5M) and 8-(4-Chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT–cGMP) that induced a negative inotropic effect at 10−5M accompanied by a slight increase in relaxation. In contrast, neither cinaciguat nor riociguat significantly influenced any measured parameters. Furthermore, isoproterenol significantly increased intracellular cAMP levels that were not influenced by cinaciguat or riociguat (all at 10−6M). Otherwise, riociguat and cinaciguat (both at 10−6M) significantly enhanced intracellular cGMP generation. This accumulation was significantly augmented by cinaciguat in the presence of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 25µM), whereas ODQ blocked cGMP generation by riociguat. However, blocking of sGC did not influence cell contractility.Our results demonstrate that, in isolated cardiac myocytes from healthy rats, the increase in cGMP levels induced by cinaciguat and riociguat at clinical relevant concentrations is not associated with acute direct effects on cell contraction and relaxation.

Minimal Change in the Cytoplasmic Calcium Dynamics in Striatal GABAergic Neurons of a DYT1 Dystonia Knock-In Mouse Model

DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.

Spontaneous cluster activity in the inferior olivary nucleus in brainstem slices from postnatal mice

A distinctive property of the cerebellar system is olivocerebellar modules, where synchronized electrical activity in neurons in the inferior olivary nucleus (IO) evokes organized activity in the cerebellar cortex. However, the exact function of these modules, and how they are developed, is still largely unknown. Here we show that the IO in in vitro slices from postnatal mice spontaneously generates clusters of neurons with synchronous Ca(2+) transients. Neurons in the principal olive (PO), and the vestibular-related dorsomedial cell column (dmcc), showed an age-dependent increase in spontaneous calcium transients. The spatiotemporal activity pattern was occasionally organized in clusters of co-active neighbouring neurons,with regular (16 min-1) and irregular (2-3 min(-1)) repeating cluster activity in the dmcc and PO, respectively. IO clusters had a diameter of 100-170 μm, lasted~1 s, and increased in occurrence from postnatal day P5.5 to P12.5, followed by a sharp drop to near zero at P15.5. IO clusters were overlapping, and comprised nearly identical neurons at some time points, and a varied subset of neurons at others. Some neurons had hub-like properties, being co-active with many other neighbours, and some were co-active with separate clusters at different times. The coherence between calcium transients in IO neurons decreased with Euclidean distance between the cells reaching low values at 100-200 μm distances. Intracellular recordings from IO neurons during cluster formation revealed the presence of spikelet-like potentials, suggesting that electrical coupling between neighbouring IO neurons may serve as a synchronizing mechanism. In conclusion, the IO shows spontaneous cluster activity under in vitro conditions, coinciding with a critical postnatal period in olivocerebellar development. We propose that these clusters may be forerunners of the ensembles of IO neurons shown to be co-active in adult animals spontaneously and during motor acts.

Effect of Chemical Modification of Al-Si Alloys on Thermal Diffusivity and Contact Heat Transfer at the Casting–Chill Interface

Abstract The heat flow during the unidirectional downward solidification of Al-7Si and Al-12Si alloys was analyzed using thermal analysis technique and inverse modeling. Chills instrumented with thermocouples were brought into contact with a small pool of liquid metal so as to minimize the effect of convection caused by pouring and temperature gradients. Modification melt treatment resulted in an increase in the cooling rate of the solidifying casting near the casting–chill interfacial region. The corresponding interfacial heat flux transients were also found to be higher. The thermal diffusivities of alloys were measured using a laser pulse technique and were found to be higher for modified alloys. However, the increase in the heat flux transients was attributed mainly to the improvement in the casting–chill interfacial thermal contact condition brought about by the decrease in the surface tension of the liquid metal upon the addition of sodium.