CO2-dependent opening of an inwardly rectifying K+ channel

CO2 chemosensing is a vital function for the maintenance of life that helps to control acid–base balance. Most studies have reported that CO2 is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO2 under conditions of constant extracellular pH. As PCO2 increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of –70 mV, which lay between the K+ and Cl– equilibrium potentials. This reversal potential was shifted by +61 mV following a tenfold increase in extracellular [K+] but was insensitive to variations of extracellular [Cl–]. The single-channel conductance increased with extracellular [K+]. We propose that this channel is a member of the Kir family. In addition to this K+ channel, we found that many of the excised patches also contained a conductance carried via a Cl–-selective channel. This CO2-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO2-dependent inhibition during hypercapnia

Your input is a breath of fresh air! A chemosensory microcircuit of medullary raphe and RTN neurons

Breathing is our first act upon birth and the last action we complete before death. The first to last breath taken, is in fact, how we define someone’s life. Since it was first reported that the blood concentration of CO2istightly controlled, and provides the dominant drive to breathe, the search for the cells that regulate it began. It took almost 60 years for the identification of the first central chemosensitive areas, regions within the brain that respond to specific chemical stimuli (such as CO2or its proxy H+), found at the ventrolateral surface of the medulla (VLM). Since then the debate over which cells in these areas are responsible for detectingCO2and signalling its fluctuations to the respiratory oscillators, has been extensive and heated. Chemosensitive cells are thought to have cell bodies located in, or close to, the VLM with dendrites in close apposition to blood vessels to better detect changes in blood gases. Several candidates fulfil this criteria, including the retrotrapezoid nucleus (RTN) and medullary raphe

ATP and mechanisms of central CO2 chemosensitivity

ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release

ATP and mechanisms of central CO2 chemosensitivity

ATP release from the surface of the ventro-lateral medulla (VLM) is integral to the hypercapnic response in vivo and can be seen in vitro. By employing horizontal slices of the ventral medulla containing the ventral chemosensitive nuclei, I have developed a model that consistently evokes hypercapnia-induced ATP release in vitro. Using this preparation I have studied CO2-triggered ATP release by means of microelectrode biosensors. I conclude that it is the change in PCO2 itself, and not associated pH changes that accompany it, that is directly responsible for eliciting ATP release from the surface of the VLM. In addition ATP release from this region may have a role in the response to hypocapnia as well as hypercapnia. Using pharmacological agents I have demonstrated that gating of connexin hemichannels mediates ATP release. The dorso-ventral distribution of Cx26 ascertained via quantitative PCR and immunofluorescence makes this hemichannel the most likely candidate. Dye loading the cells responsible for ATP release with carboxyfluorescein, which co-localised with Cx26, revealed these cells reside in the pia mater and subpial astrocytes. Application of gap-junction antagonists, with selectivity towards connexin 26, greatly reduced ATP release in response to elevated CO2 in vitro and in vivo and reduced the tone of ATP at the VLM surface. Moreover, by loading Cx26 expressing HeLa cells with ATP, I was able to recapitulate the entire in vivo response. Therefore I propose that ATP is released from sub-pial astrocytes and leptomeningeal cells through connexin 26 hemichannels in response to alterations in PCO2. Here Cx26 performs a dual role, as both the chemosensory transducer and the conduit for ATP release.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The role of parafacial neurons in the control of breathing during exercise

Neuronal cell groups residing within the retrotrapezoid nucleus (RTN) and C1 area of the rostral ventrolateral medulla oblongata contribute to the maintenance of resting respiratory activity and arterial blood pressure, and play an important role in the development of cardiorespiratory responses to metabolic challenges (such as hypercapnia and hypoxia). In rats, acute silencing of neurons within the parafacial region which includes the RTN and the rostral aspect of the C1 circuit (pFRTN/C1), transduced to express HM4D (Gi-coupled) receptors, was found to dramatically reduce exercise capacity (by 60%), determined by an intensity controlled treadmill running test. In a model of simulated exercise (electrical stimulation of the sciatic or femoral nerve in urethane anaesthetised spontaneously breathing rats) silencing of the pFRTN/C1 neurons had no effect on cardiovascular changes, but significantly reduced the respiratory response during steady state exercise. These results identify a neuronal cell group in the lower brainstem which is critically important for the development of the respiratory response to exercise and, determines exercise capacity

Interactions between respiratory oscillators in adult rats

Breathing in mammals is hypothesized to result from the interaction of two distinct oscillators: the preBötzinger Complex (preBötC) driving inspiration and the lateral parafacial region (pFL) driving active expiration. To understand the interactions between these oscillators, we independently altered their excitability in spontaneously breathing vagotomized urethane-anesthetized adult rats. Hyperpolarizing preBötC neurons decreased inspiratory activity and initiated active expiration, ultimately progressing to apnea, i.e., cessation of both inspiration and active expiration. Depolarizing pFL neurons produced active expiration at rest, but not when inspiratory activity was suppressed by hyperpolarizing preBötC neurons. We conclude that in anesthetized adult rats active expiration is driven by the pFL but requires an additional form of network excitation, i.e., ongoing rhythmic preBötC activity sufficient to drive inspiratory motor output or increased chemosensory drive. The organization of this coupled oscillator system, which is essential for life, may have implications for other neural networks that contain multiple rhythm/pattern generators

Imaging single-cell Ca2+ dynamics of brainstem neurons and glia in freely behaving mice

In vivo brain imaging, using a combination of genetically encoded Ca2+ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond

Identifying the recurrence of sleep apnea using a harmonic hidden Markov model

We propose to model time-varying periodic and oscillatory processes by means of a hidden Markov model where the states are defined through the spectral properties of a periodic regime. The number of states is unknown along with the relevant periodicities, the role and number of which may vary across states. We address this inference problem by a Bayesian nonparametric hidden Markov model assuming a sticky hierarchical Dirichlet process for the switching dynamics between different states while the periodicities characterizing each state are explored by means of a trans-dimensional Markov chain Monte Carlo sampling step. We develop the full Bayesian inference algorithm and illustrate the use of our proposed methodology for different simulation studies as well as an application related to respiratory research which focuses on the detection of apnea instances in human breathing traces

Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process

4D polycarbonates via stereolithography as scaffolds for soft tissue repair

3D printing has emerged as one of the most promising tools to overcome the processing and morphological limitations of traditional tissue engineering scaffold design. However, there is a need for improved minimally invasive, void-filling materials to provide mechanical support, biocompatibility, and surface erosion characteristics to ensure consistent tissue support during the healing process. Herein, soft, elastomeric aliphatic polycarbonate-based materials were designed to undergo photopolymerization into supportive soft tissue engineering scaffolds. The 4D nature of the printed scaffolds is manifested in their shape memory properties, which allows them to fill model soft tissue voids without deforming the surrounding material. In vivo, adipocyte lobules were found to infiltrate the surface-eroding scaffold within 2 months, and neovascularization was observed over the same time. Notably, reduced collagen capsule thickness indicates that these scaffolds are highly promising for adipose tissue engineering and repair