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A role for silent synapses in the development of the pathway from layer 2/3 to 5 pyramidal cells in the neocortex.
The integration of neurons within the developing cerebral cortex is a prolonged process dependent on a combination of molecular and physiological cues. To examine the latter we used laser scanning photostimulation (LSPS) of caged glutamate in conjunction with whole-cell patch-clamp electrophysiology to probe the integration of pyramidal cells in the sensorimotor regions of the mouse neocortex. In the days immediately after postnatal day 5 (P5) the origin of the LSPS-evoked AMPA receptor (AMPAR)-mediated synaptic inputs were diffuse and poorly defined with considerable variability between cells. Over the subsequent week this coalesced and shifted, primarily influenced by an increased contribution from layers 2/3 cells, which became a prominent motif of the afferent input onto layer 5 pyramidal cells regardless of cortical region. To further investigate this particular emergent translaminar connection, we alternated our mapping protocol between two holding potentials (-70 and +40 mV) allowing us to detect exclusively NMDA receptor (NMDAR)-mediated inputs. This revealed distal MK-801-sensitive synaptic inputs that predict the formation of the mature, canonical layer 2/3 to 5 pathway. However, these were a transient feature and had been almost entirely converted to AMPAR synapses at a later age (P16). To examine the role of activity in the recruitment of early NMDAR synapses, we evoked brief periods (20 min) of rhythmic bursting. Short intense periods of activity could cause a prolonged augmentation of the total input onto pyramidal cells up until P12; a time point when the canonical circuit has been instated and synaptic integration shifts to a more consolidatory phase.
Studies of cortical connectivity using optical circuit mapping methods.
An important consideration when probing the function of any neuron is to uncover the source of synaptic input onto the cell, its intrinsic physiology and efferent targets. Over the years, electrophysiological approaches have generated considerable insight into these properties in a variety of cortical neuronal subtypes and circuits. However, as researchers explore neuronal function in greater detail, they are increasingly turning to optical techniques to bridge the gap between local network interactions and behaviour. The application of optical methods has increased dramatically over the past decade, spurred on by the optogenetic revolution. In this review, we provide an account of recent innovations, providing researchers with a primer detailing circuit mapping strategies in the cerebral cortex. We will focus on technical aspects of performing neurotransmitter uncaging and channelrhodopsin-assisted circuit mapping, with the aim of identifying common pitfalls that can negatively influence the collection of reliable data.
Changing Metabolism in Differentiating Cardiac Progenitor Cells-Can Stem Cells Become Metabolically Flexible Cardiomyocytes?
The heart is a metabolic omnivore and the adult heart selects the substrate best suited for each circumstance, with fatty acid oxidation preferred in order to fulfill the high energy demand of the contracting myocardium. The fetal heart exists in an hypoxic environment and obtains the bulk of its energy via glycolysis. After birth, the "fetal switch" to oxidative metabolism of glucose and fatty acids has been linked to the loss of the regenerative phenotype. Various stem cell types have been used in differentiation studies, but most are cultured in high glucose media. This does not change in the majority of cardiac differentiation protocols. Despite the fact that metabolic state affects marker expression and cellular function and activity, the substrate composition is currently being overlooked. In this review we discuss changes in cardiac metabolism during development, the various protocols used to differentiate progenitor cells to cardiomyocytes, what is known about stem cell metabolism and how consideration of metabolism can contribute toward maturation of stem cell-derived cardiomyocytes.
Trilayer scaffold with cardiosphere-derived cells for heart valve tissue engineering.
Natural polymers collagen, glycosaminoglycans, and elastin are promising candidate materials for heart valve tissue engineering scaffolds. This work produced trilayer scaffolds that resembled the layered structures of the extracellular matrices of native heart valves. The scaffolds showed anisotropic bending moduli (in both dry and hydrated statuses) depending on the loading directions (lower in the With Curvature direction than in the Against Curvature direction), which mimicked the characteristic behavior of the native heart valves. The interactions between cardiosphere-derived cells and the scaffolds were characterized by multiphoton microscopy, and relatively similar cell distributions were observed on different layers (a cell density of 3,000-4,000 mm-3 and a migration depth of 0.3-0.4 mm). The trilayer scaffold has represented a forwarding step from the previous studies, in attempting to better replicate a native heart valve structurally, mechanically, and biologically.
Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering.
Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 μm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.
Influence of propofol on isolated neonatal rat carotid body glomus cell response to hypoxia and hypercapnia.
In humans the intravenous anaesthetic propofol depresses ventilatory responses to hypoxia and CO2. Animal studies suggest that this may in part be due to inhibition of synaptic transmission between chemoreceptor glomus cells of the carotid body and the afferent carotid sinus nerve. It is however unknown if propofol can also act directly on the glomus cell. Here we report that propofol can indeed inhibit intracellular Ca2+ responses to hypoxia and hypercapnia in isolated rat glomus cells. Neither this propofol effect, nor the glomus cell response to hypoxia in the absence of propofol, were influenced by GABA receptor activation (using GABA, muscimol and baclofen) or inhibition (using bicuculline and 5-aminovaleric acid). Suggesting that these effects of propofol are not mediated through GABA receptors. Propofol inhibited calcium responses to nicotine in glomus cells but the nicotinic antagonists vecuronium and methyllycaconitine did not inhibit calcium responses to hypoxia. TASK channel activity was not altered by propofol. The glomus cell Ca2+ response to depolarisation with 30 mM K+ was however modestly inhibited by propofol. In summary we conclude that propofol does have a direct effect upon hypoxia signalling in isolated type-1 cells and that this may be partially due to its ability to inhibit voltage gated Ca2+v channels. We also note that propofol has the capacity to supress glomus cell excitation via nicotinic receptors and may therefore also interfere with paracrine/autocrine cholinergic signalling in the intact organ. The effects of propofol on chemoreceptor function are however clearly complex and require further investigation.
Functional Properties of Mitochondria in the Type-1 Cell and Their Role in Oxygen Sensing.
The identity of the oxygen sensor in arterial chemoreceptors has been the subject of much speculation. One of the oldest hypotheses is that oxygen is sensed through oxidative phosphorylation. There is a wealth of data demonstrating that arterial chemoreceptors are excited by inhibitors of oxidative phosphorylation. These compounds mimic the effects of hypoxia inhibiting TASK1/3 potassium channels causing membrane depolarisation calcium influx and neurosecretion. The TASK channels of Type-I cells are also sensitive to cytosolic MgATP. The existence of a metabolic signalling pathway in Type-1 cells is thus established; the contentious issue is whether this pathway is also used for acute oxygen sensing. The main criticism is that because cytochrome oxidase has a high affinity for oxygen (P50 ≈ 0.2 mmHg) mitochondrial metabolism should be insensitive to physiological hypoxia. This argument is however predicated on the assumption that chemoreceptor mitochondria are analogous to those of other tissues. We have however obtained new evidence to support the hypothesis that type-1 cell mitochondria are not like those of other cells in that they have an unusually low affinity for oxygen (Mills E, Jobsis FF, J Neurophysiol 35(4):405-428, 1972; Duchen MR, Biscoe TJ, J Physiol 450:13-31, 1992a). Our data confirm that mitochondrial membrane potential, NADH, electron transport and cytochrome oxidase activity in the Type-1 cell are all highly sensitive to hypoxia. These observations not only provide exceptionally strong support for the metabolic hypothesis but also reveal an unknown side of mitochondrial behaviour.
TASK channels in arterial chemoreceptors and their role in oxygen and acid sensing.
Arterial chemoreceptors play a vital role in cardiorespiratory control by providing the brain with information regarding blood oxygen, carbon dioxide, and pH. The main chemoreceptor, the carotid body, is composed of sensory (type 1) cells which respond to hypoxia or acidosis with a depolarising receptor potential which in turn activates voltage-gated calcium entry, neurosecretion and excitation of adjacent afferent nerves. The receptor potential is generated by inhibition of Twik-related acid-sensitive K(+) channel 1 and 3 (TASK1/TASK3) heterodimeric channels which normally maintain the cells' resting membrane potential. These channels are thought to be directly inhibited by acidosis. Oxygen sensitivity, however, probably derives from a metabolic signalling pathway. The carotid body, isolated type 1 cells, and all forms of TASK channel found in the type 1 cell, are highly sensitive to inhibitors of mitochondrial metabolism. Moreover, type1 cell TASK channels are activated by millimolar levels of MgATP. In addition to their role in the transduction of chemostimuli, type 1 cell TASK channels have also been implicated in the modulation of chemoreceptor function by a number of neurocrine/paracrine signalling molecules including adenosine, GABA, and serotonin. They may also be instrumental in mediating the depression of the acute hypoxic ventilatory response that occurs with some general anaesthetics. Modulation of TASK channel activity is therefore a key mechanism by which the excitability of chemoreceptors can be controlled. This is not only of physiological importance but may also offer a therapeutic strategy for the treatment of cardiorespiratory disorders that are associated with chemoreceptor dysfunction.
TASK channels in arterial chemoreceptors and their role in oxygen and acid sensing
© 2015, The Author(s). Arterial chemoreceptors play a vital role in cardiorespiratory control by providing the brain with information regarding blood oxygen, carbon dioxide, and pH. The main chemoreceptor, the carotid body, is composed of sensory (type 1) cells which respond to hypoxia or acidosis with a depolarising receptor potential which in turn activates voltage-gated calcium entry, neurosecretion and excitation of adjacent afferent nerves. The receptor potential is generated by inhibition of Twik-related acid-sensitive K<sup>+</sup> channel 1 and 3 (TASK1/TASK3) heterodimeric channels which normally maintain the cells’ resting membrane potential. These channels are thought to be directly inhibited by acidosis. Oxygen sensitivity, however, probably derives from a metabolic signalling pathway. The carotid body, isolated type 1 cells, and all forms of TASK channel found in the type 1 cell, are highly sensitive to inhibitors of mitochondrial metabolism. Moreover, type1 cell TASK channels are activated by millimolar levels of MgATP. In addition to their role in the transduction of chemostimuli, type 1 cell TASK channels have also been implicated in the modulation of chemoreceptor function by a number of neurocrine/paracrine signalling molecules including adenosine, GABA, and serotonin. They may also be instrumental in mediating the depression of the acute hypoxic ventilatory response that occurs with some general anaesthetics. Modulation of TASK channel activity is therefore a key mechanism by which the excitability of chemoreceptors can be controlled. This is not only of physiological importance but may also offer a therapeutic strategy for the treatment of cardiorespiratory disorders that are associated with chemoreceptor dysfunction.
Moderate inhibition of mitochondrial function augments carotid body hypoxic sensitivity.
A functional role for the mitochondria in acute O2 sensing in the carotid body (CB) remains undetermined. Whilst total inhibition of mitochondrial activity causes intense CB stimulation, it is unclear whether this response can be moderated such that graded impairment of oxidative phosphorylation might be a mechanism that sets and modifies the O2 sensitivity of the whole organ. We assessed NADH autofluorescence and [Ca2+]i in freshly dissociated CB type I cells and sensory chemoafferent discharge frequency in an intact CB preparation, in the presence of varying concentrations of nitrite (NO2 −), a mitochondrial nitric oxide (NO) donor and a competitive inhibitor of mitochondrial complex IV. NO2 − increased CB type I cell NADH in a manner that was dose-dependent and rapidly reversible. Similar concentrations of NO2 − raised type I cell [Ca2+]i via L-type channels in a PO2-dependent manner and increased chemoafferent discharge frequency. Moderate inhibition of the CB mitochondria by NO2 − augmented chemoafferent discharge frequency during graded hypoxia, consistent with a heightened CB O2 sensitivity. Furthermore, NO2 − also exaggerated chemoafferent excitation during hypercapnia signifying an increase in CB CO2 sensitivity. These data show that NO2 − can moderate the hypoxia sensitivity of the CB and thus suggest that O2 sensitivity could be set and modified in this organ by interactions between NO and mitochondrial complex IV.
A method for continuous and stable perfusion of tissue and single cell preparations with accurate concentrations of volatile anaesthetics.
BACKGROUND: It is difficult to design a system to reliably deliver volatile anaesthetics such as halothane or isoflurane to in vitro preparations such as tissues or cells cultures: the very volatility of the drugs means that they can rapidly dissipate from even carefully-prepared solutions. Furthermore, many experiments require the control of other gases (such as oxygen or carbon dioxide) which requires constant perfusion. NEW METHOD: We describe a constant perfusion system that is air-tight (i.e., allows the accurate administration of hypoxic or hypercapnic gas mixtures), in which volatile anaesthetic is delivered via calibrated vaporisers by constant bubbling into the perfusing solution (and continuously monitored for stability by infrared spectroscopy in the headspace above the solution). RESULTS: We have confirmed the accuracy (i.e., linear relationship of dissolved concentrations with vapour dial settings) and stability (i.e., over time) of the anaesthetic concentrations in solutions in samples taken from the bottles into which anaesthetic is bubbled, and from samples taken from the tissue perfusion bath, using gas chromatrography-mass spectrometry (GC-MS). CONCLUSIONS: It is possible to deliver volatile anaesthetics in accurate concentrations to cell/tissue preparations whilst adjusting ambient air composition rapidly, stable over sustained time periods.
Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells.
The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O2) on mitochondrial function in neonatal rat type-1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P50 of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca(2+)-free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (m, measured using rhodamine 123) with a P50 of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca(2+)-free Tyrode and Tyrode containing the Ca(2+) channel antagonist Ni(2+), respectively. In the presence of oligomycin and low carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) m is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised m/inhibited electron transport with a P50 of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl-p-phenylenediamine. Under these conditions m is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised m) with a P50 of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P50 = 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type-1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained.