Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H+ and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2, a pH-sensitive K+ channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel and essential central mediators of respiratory chemosensitivity.
Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO 2 /Hϩ via an unidentified pH-sensitive background K ϩ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K ϩ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or -galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n ϭ 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2 Ϫ/Ϫ mice (n ϭ 49 of 88) could be classified as pH sensitive (Ͼ30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2 Ϫ/Ϫ mice selected based on -galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K ϩ currents were reduced in amplitude in RTN neurons from TASK-2 Ϫ/Ϫ mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart-brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2 Ϫ/Ϫ mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold.
Central respiratory chemoreceptors sense changes in CO2/H+ and initiate the adjustments to ventilation required to preserve brain and tissue pH. The cellular nature of the sensors (neurons and/or glia) and their CNS location are not conclusively established but the glutamatergic, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) are strong candidates. However, a direct demonstration that RTN neurons are intrinsically sensitive to CO2/H+, required for designation as a chemosensor, has been lacking. To address this, we tested the pH sensitivity of RTN neurons that were acutely dissociated from two lines of Phox2b-GFP BAC transgenic mice. All GFP-labeled cells assayed by RT-PCR (n=40) were Phox2b+, VGlut2+, TH− and ChAT−, the neurochemical phenotype previously defined for chemosensitive RTN neurons in vivo. We found that most dissociated RTN neurons from both lines of mice were CO2/H+-sensitive (~79%), with discharge increasing during acidification and decreasing during alkalization. The pH-sensitive cells could be grouped into two populations characterized by similar pH sensitivity but different basal firing rates, as previously observed in recordings from GFP-labeled RTN neurons in slice preparations. In conclusion, these data indicate that RTN neurons are inherently pH-sensitive, as expected for a respiratory chemoreceptor.
Angiotensin II (AngII) is a major culprit in essential hypertension. Based on a genetic rodent model of hypertension, we review here evidence that AngII may signal across the blood brain barrier to affect neuronal circuits within the nucleus tractus solitarii (NTS) of the brainstem, a pivotal region regulating both the baroreceptor reflex and set point control of arterial pressure. We have termed this form of signalling as vascular-neuronal signalling. We describe that the depressant action of AngII in NTS on the baroreceptor reflex is mediated via activation of endothelial nitric oxide synthase (eNOS) releasing NO that promotes release of the inhibitory transmitter-GABA. This could shunt the incoming excitatory baroreceptor afferent traffic impinging on NTS neurones. Chronic studies recording arterial pressure in conscious unrestrained rats using radio-telemetry have revealed that eNOS in NTS plays an endogenous physiological role in the homeostatic regulation of the gain of the cardiac baroreceptor reflex. However, in the spontaneously hypertensive rat, eNOS mRNA was higher (compared to normotensive rats), and its chronic blockade in NTS restored the abnormally depressed cardiac baroreceptor reflex to levels akin to normotensive rats, improved heart rate variability and lowered arterial pressure. Hence, it seems that excessive eNOS activity in NTS of the SHR contributes to the pathological state of this animal model's cardiovascular autonomic nervous system. We speculate on why eNOS activity may be up regulated in the NTS of the SHR and propose that it is a consequence of high cerebral vascular resistance and inadequate blood perfusion of the brainstem.
The cellular mechanisms mediating nitric oxide (NO) modulation of the inhibitory transmission in the nucleus tractus solitarii (NTS) remain unclear, even though this could be extremely important for various physiological and pathological processes. Specifically, in the NTS NO-evoked glutamate and gamma-aminobutyric acid (GABA) release might contribute to pathological hypertension. In cultured rat brainstem slices, NTS GABAergic neurons were targeted using an adenoviral vector to express enhanced green fluorescent protein and studied with a combination of patch clamp and confocal microscopy. Low nanomolar concentrations of NO increased intracellular Ca2+ concentration ([Ca2+]i) in somata, dendrites, and putative axons of GABAergic neurons, with axons being the most sensitive compartment. This effect was cGMP mediated and not related to depolarization or indirect presynaptic effects on glutamatergic transmission. Blockade of the cyclic adenosine diphosphate ribose (cADPR)/ryanodine-sensitive stores but not the inositol triphosphate-sensitive stores, inhibited NO effect. Since cADPR/ryanodine-sensitive stores are implicated in the Ca2+-induced Ca2+ release, NO can be expected to potentiate GABA release. In support of this notion, a cADPR antagonist abolished the NO-induced potentiation of GABAergic inhibitory postsynaptic potentials in the NTS. Thus, the NO-cGMP-cADPR-Ca2+ pathway, previously described in sea urchin eggs, also operates in mammalian GABAergic neurons. Potentiation of GABA release by NO may have implications for numerous brain functions.
During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.
We employ viral vectors to address questions related to the function of specific types of neurones in the central control of blood pressure. Adenoviral vectors (AVVs) or lentiviral vectors (LVVs) can be used to visualize specifically living GABAergic or noradrenergic (NAergic) neurones or to interfere with intracellular signalling within these cell types. Here, we review recent in vitro, in situ and in vivo applications of these vectors in the rat brainstem as performed in our laboratories. In organotypic slice cultures prepared from defined cardiovascular brainstem areas, viral vectors were used to study the electrophysiological properties, intracellular signalling and gene expression in selected neuronal phenotypes. In vivo, vectors were microinjected into brainstem nuclei to inhibit specific aspects of cell signalling by expression of dominant negative proteins, for example. Outcomes for cardiovascular control were measured either acutely in situ or chronically in vivo with radio telemetry in freely moving rats. We showed that AVVs and LVVs have distinct properties that need to be considered prior to their application. For example, LVVs can be manufactured very quickly, have no immunogenicity and can be pseudotyped to display higher tropism for neurones than glia. However, comparatively lower production yields of LVVs may limit their use for some types of applications. In contrast, AVVs require a lengthy construction period, are easy to amplify to high yields at moderate cost but may trigger an immune response when used at high titres in vivo. These features make AVVs particularly suitable for in vitro applications. As the two vector types complement each other in several ways we generated a shuttle system that simplifies transfer of transgene cassettes between the backbones of AVVs and LVVs. Thus, AVVs and LVVs are powerful experimental tools that can be used in a variety of experimental designs in vivo, in situ and in vitro.
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