Isocitrate supplementation promotes breathing generation, gasping, and autoresuscitation in neonatal mice

Abstract

Breathing is a vital function generated and controlled by a brainstem neural network, which is able to adjust its function to fit different metabolic demands. For instance, the pre-Bötzinger complex (preBötC) can respond to low oxygen availability (hypoxia) by an initial increase in rhythm frequency followed by a decrease in respiratory efforts that leads to gasping generation. Gasping is essential for autoresuscitation, which has motivated studies of the cellular mechanisms involved in these processes. Hypoxia has different effects on enzymes that participate in the Krebs cycle. In particular, aconitase is downregulated, whereas isocitrate dehydrogenase is unaffected or upregulated under hypoxic conditions. We hypothesized that the application of isocitrate, the product of aconitase and the substrate of isocitrate dehydrogenase as well as an alternative metabolic substrate, might enhance breathing and render it more resistant to hypoxic insult. We tested the effects of isocitrate applied on brainstem slices containing the preBötC as well as its central effects in vivo using plethysmography. Our results show that isocitrate increases the frequency of fictive eupnea and fictive gasping produced by the preBötC in vitro. Moreover, isocitrate increases the amplitude of ventilation in vivo in normoxia, increases ventilation during gasping, and favors autoresuscitation when animals were subjected to asphyxiation. In conclusion, we have found that isocitrate improves ventilation under both normoxic and hypoxic conditions through a mechanism that involves the preBötC and possibly other respiratory neural networks. Thus, isocitrate would be useful to avoid the failure of gasping generation and autoresuscitation in pathological conditions.

Change in network connectivity during fictive-gasping generation in hypoxia: prevention by a metabolic intermediate

“…In contrast, when ACSF was bubbled with 95% N2 and 5% CO2, the PO2 in the recording chamber was 38 ± 28 Torr, while 300 μm deep in the slices PO2 was 5 ± 6 Torr, which can be considered hypoxic (Hill et al, 2011). Isocitrate (Sigma-Aldrich, St. Louis, MO) was bath applied at a final concentration of 3 mM for 1 h, as previously reported using the same preparation (Hülsmann et al, 2000; Rivera-Angulo and Peña-Ortega, 2014). Fresh, 1000× isocitrate stock solution was prepared by dissolving it in distilled water.…”

“…The preBötC is able to adjust its function to fit different metabolic demands by acquiring different configurations (Richter et al, 1991; Ballanyi et al, 1994; England et al, 1995; Ramirez et al, 1998; Peña, 2009; Rivera-Angulo and Peña-Ortega, 2014). The network focused studies of the respiratory network have started to reveal that the preBötC forms dense clusters of respiratory cells with occasional connections between them (Hartelt et al, 2008; Mironov, 2009; Gaiteri and Rubin, 2011) that can be reconfigured in a cycle-by-cycle manner (Carroll et al, 2013; Carroll and Ramirez, 2013; Koshiya et al, 2014).…”

“…These results suggest that the study of circuit configurations and reconfigurations is key to understand the flexibility of breathing generation during different metabolic states (Mironov, 2009; Galan et al, 2010). One of the extreme examples of respiratory network flexibility is the reconfiguration process that this network undergoes under extreme hypoxic conditions (Lieske et al, 2000; Peña and Ramirez, 2005; Rivera-Angulo and Peña-Ortega, 2014): Lieske et al (2000) documented that this process allows the preBötC to change its burst pattern from fictive-eupnea in normoxia to fictive-gasping in hypoxia. Since the changes in the burst pattern as well as in firing patterns of respiratory neurons observed in the brainstem slice preparation closely resemble those observed in vivo during the transition from actual eupnea to actual gasping, we adopted the term “fictive” to refer to those patterns generated by the preBötC in vitro (Lieske et al, 2000; Lieske and Ramirez, 2003).…”

“…With the help of multi-electrode array (MEA) recordings (Lindsey et al, 2000; Segers et al, 2008; Galan et al, 2010; Ott et al, 2011; Carroll and Ramirez, 2013; Carroll et al, 2013), we wanted to establish whether the respiratory network reconfiguration in hypoxia involves changes in the number of active respiratory elements, in the functional connections among the elements, or in the strength of these functional connections. The other aim of this study was to characterize the changes in respiratory network configuration upon the application of a citric acid cycle intermediate (isocitrate), particularly during fictive-gasping generation (Rivera-Angulo and Peña-Ortega, 2014). Since a failure to generate gasping has been linked to Sudden Infant Death Syndrome (SIDS; Lijowska et al, 1985; Poets et al, 1999; Peña and García, 2006; Peña, 2009), the study of gasping generation, as well as the identification of pharmacological approaches to promote it, may have clinical relevance as a preventive intervention in babies at risk for SIDS (Peña and García, 2006; Peña, 2009).…”

“…We have recently shown that supplementation of the respiratory network with the metabolic intermediate isocitrate increases preBötC activity in normoxia and favors gasping generation in hypoxia both in vitro and in vivo (Rivera-Angulo and Peña-Ortega, 2014). Several metabolic intermediates can modulate both the intrinsic and the synaptic properties (Dehaven and Carpenter, 1964; Böhmer et al, 1976; Chaplain et al, 1976; Dinse et al, 1976; Shoji, 1992) of neurons in different networks, including those in the respiratory network (Dehaven and Carpenter, 1964; Böhmer et al, 1976; Chaplain et al, 1976; Dinse et al, 1976), therefore we hypothesized that isocitrate could influence respiratory network configuration and also modulate its reconfiguration in hypoxia.…”