Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats
Introduction
In recent years, much progress was made in understanding mechanisms that give rise to respiratory neuroplasticity in adult animals (Fuller and Mitchell, 2017), with the idea that these mechanisms may be harnessed to improve breathing in clinical disorders characterized by insufficient respiratory neural output. Little is known whether similar mechanisms underlie respiratory neuroplasticity in neonates. One well-characterized form of respiratory neuroplasticity is phrenic long-term facilitation (LTF), a long-lasting increase in phrenic inspiratory motor output following intermittent hypoxia (Devinney et al., 2013; Xing et al., 2013; Dale et al., 2014). While LTF is robustly expressed in adult rodents (Baker-Herman and Mitchell, 2008), it is less reliably triggered in newborn and pre-weaned rodents less than 20 days old. For example, intermittent hypoxia induced a long-lasting increase in diaphragm EMG in only 7 of 15 anesthetized P14 rats (Reid and Solomon, 2014). Similarly, intermittent hypoxia triggered increases in tidal volume in neonatal rats > P10 (Julien et al., 2008) or P15 (Fuller et al., 2009), but not in younger animals. The lack of intermittent hypoxia-induced LTF in newborn rodents may also be due their high tolerance to hypoxia (Singer, 1999) or decreased metabolism due to hypoxia (Mortola, 2004). Despite the paucity of intermittent hypoxia-induced LTF in newborn rodents, long-lasting increases in inspiratory motor output are induced in isolated neonatal (P0-P3) rat brainstem-spinal cords by spinal serotonin application (Lovett-Barr et al., 2006). This suggests that the newborn rat respiratory control system has the capacity to express phrenic inspiratory motor facilitation, but there may be endogenous brakes that limit its ability to be induced by intermittent hypoxia.
In adult rats, intermittent hypoxia induces LTF by at least two different mechanisms, depending on hypoxia severity. Moderate hypoxia leads to the preferential activation of Gq protein‐coupled serotonin receptors (“Q-pathway”), resulting in synthesis of brain‐derived neurotrophic factor (BDNF) and subsequent tyrosine kinase B (TrkB) receptor activation (Baker-Herman et al., 2004; Dale et al., 2017). In contrast, severe hypoxia preferentially activates Gs protein‐coupled adenosine A2a receptors (“S-pathway”), increasing immature TrkB receptor synthesis and TrkB receptor transactivation (Hoffman and Mitchell, 2011; Nichols et al., 2012; Devinney et al., 2013). S-pathway activation attenuates Q-pathway LTF in adult rats (and vice versa), a phenomenon referred to as cross-talk inhibition (Hoffman et al., 2010; Dale-Nagle et al., 2010; Devinney et al., 2013; Hoffman and Mitchell, 2013; Xing et al., 2013). The precise location within the signaling pathways where the interactions take place are still under investigation (Perim et al., 2018, 2019; Turner et al., 2018; Perim et al., 2020). In adult animals, phrenic motor facilitation can be elicited pharmacologically by exogenous application of key molecules involved in the Q- or S-pathways (MacFarlane and Mitchell, 2009; Hoffman and Mitchell, 2011), but whether similar phrenic motor facilitation can be induced in neonates is poorly understood. It is also unknown whether the Q- and S- signaling pathways interact via cross-talk inhibition in neonates.
Since neonatal (P0-P2) rat brainstems express TrkB receptors (Liu and Wong-Riley, 2013) and adenosine A2a receptors (Zimmer and Goshgarian, 2007), we tested the hypothesis that exogenous activation of the Q- or S- pathways in P0-P3 neonates is sufficient to induce long-lasting increases in inspiratory motor output in spinal regions associated with the phrenic motor pool. In addition, we tested the hypothesis that activation of the S-pathway constrains Q-pathway-induced inspiratory motor facilitation. We report that similar to adults, pharmacological activation of the Q- or S-pathway induces inspiratory motor facilitation, and that these pathways interact via cross-talk inhibition. These data indicate that although neonatal rats do not express hypoxia-induced plasticity at this age, it can be evoked by exogenous manipulation of key signal transduction pathways underlying important forms of spinal respiratory plasticity in adult animals and raise the intriguing suggestion that ongoing adenosine A2a receptor activation acts as a brake on neuroplasticity.
Section snippets
Methods
All experimental procedures followed National Institutes of Health (NIH) Guidelines for Use of Laboratory Animals and were approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee.
Vehicle (DMSO) time controls
To quantify time-dependent changes in respiratory burst amplitude and frequency following vehicle application, respiratory-related motor activity was recorded from the C4 or C5 ventral root of the neonatal brainstem-spinal cord preparation (n = 15; 7 male, 8 female). Baseline firing activity was measured for 5 min, then DMSO was bath-applied for 15 min at concentrations (0.7–2.8 μM; 0.005−0.02% v/v) used to dissolve DHF and CGS21680. This amount of DMSO is only 10–40 % of the [DMSO] that alters
Discussion
The main findings were that TrkB receptor activation induced a long-lasting increase in respiratory-related burst amplitude in neonatal rat brainstem-spinal cord preparations, whereas respiratory plasticity was not consistently observed following adenosine A2a receptor activation. This finding contrasts with adult animals, which express plasticity following both TrkB and A2a receptor activation. It is notable that not all neonatal preparations expressed plasticity following TrkB receptor
Acknowledgements
This work was supported by the National Institutes of Health (NS085226, 2T35OD011078-06), and the Department of Comparative Biosciences in the School of Veterinary Medicine at the University of Wisconsin-Madison.
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