Cardiovascular and respiratory control systems undergo plastic changes in function that are critical to the maintenance of homeostasis. Central nervous system networks that control cardiorespiratory function exhibit significant plasticity in response to various stimuli and conditions, including hypertension (Guyenet et al., 2020; Johnson and Xue, 2018), heart failure (Zheng et al., 2022; Zucker et al., 1995), cardiovascular deconditioning (Moffitt et al., 1999), exercise (Chen et al., 2009; Martins-Pinge et al., 2000; Mischel et al., 2015; Mueller, 2010), and acute and chronic intermittent hypoxia (Devinney et al., 2013; Kline et al., 2007; Ma et al., 2008; Mitchell and Baker, 2022; Prabhakar et al., 2022; Shell et al., 2016a; Xing et al., 2013a). For example, exposure to repetitive bouts of hypoxia, even over a relatively short time period, elicits prolonged increases in both phrenic and sympathetic nerve activity, termed phrenic and sympathetic long-term facilitation (pLTF and sLTF), respectively (Dick et al., 2007; Fuller et al., 2000; Kim et al., 2016; Ostrowski et al., 2023; Xing et al., 2013b) as well as augmentation of peripheral chemoreflex responses (Dick et al., 2007; Fuller et al., 2000; Mateika and Sandhu, 2011; Ostrowski et al., 2023; Xing and Pilowsky, 2010). Plasticity associated with intermittent hypoxia involves alterations in carotid body chemoafferent function (Peng et al., 2003; Prabhakar et al., 2022) but changes in multiple cardiorespiratory brain regions also contribute (Mifflin et al., 2015; Mitchell and Baker, 2022; Shell et al., 2016).

The nucleus tractus solitarii (nTS) in the dorsomedial medulla receives input from visceral afferents, including chemoafferents, is critical to the integration of sensory afferent information, and sends reciprocal projections to forebrain and medullary regions to influence basal and reflex cardiorespiratory function (Andresen and Kunze, 1994; Kline et al., 2010; Mtui et al., 1993). The nTS contains numerous neurotransmitters, neuromodulators and receptor types. Furthermore, the nTS exhibits plasticity associated with these neuromodulators and altered afferent input, including chemoafferent input due to hypoxia (Accorsi-Mendonca et al., 2015; Bantikyan et al., 2009; Kline, 2008; Kline et al., 2010; Kline et al., 2007; Ostrowski, 2014; Shell et al., 2016; Yamamoto et al., 2015) and intermittent stimulation of nTS neurons alone, in the absence of chemoreceptor afferent activation, is sufficient to elicit LTF (Yamamoto et al., 2015). We recently showed that nTS activity is required for the production and maintenance of acute intermittent hypoxia (AIH)-induced LTF, although the specific mechanisms involved were not defined (Ostrowski et al., 2023). In the current study, we sought to determine the cellular mechanisms by which nTS neuronal activity contributes to AIH-induced LTF.

Reactive oxygen species (ROS) are important signaling molecules that modulate neuronal and synaptic function throughout the brain (Checa and Aran, 2020; Malard et al., 2021; Massaad and Klann, 2011; Peng et al., 2003) and their formation is increased by deoxygenation and reoxygenation as occurs in AIH (Griffioen et al., 2007). ROS play an important role in cardiorespiratory function, including dysregulation in cardiorespiratory diseases such as hypertension, heart failure and obstructive sleep apnea (Bozkurt et al., 2015; Camargo et al., 2024; Dey et al., 2018; Gillian Groeger and Cotter, 2009; Griendling et al., 2021; MacFarlane et al., 2009; Prabhakar, 2007). Elevated ROS produce sympathoexcitation and augment chemoreflex function in these conditions. While several ROS species exert CNS effects, hydrogen peroxide (H2O2) likely plays a critical role. Levels of H2O2 depend on the balance of H2O2 production by superoxide dismutase and its catabolism by glutathione peroxidase and catalase (Rice, 2011; Zimmerman and Davisson, 2002). Compared to other ROS, H2O2 is relatively stable, has a low molecular weight, and is highly diffusible across biological membranes and therefore has the potential to have substantial influence on multiple cells and subsequently physiological responses (Forman et al., 2010; Kamsler and Segal, 2004; Rice, 2011). H2O2 participates in neuronal and possibly glial signaling within the CNS (Rice, 2011) and modulates transmitter release in hippocampal slices (Pellmar et al., 1994; Pellmar et al., 1999). Nanoinjection of H2O2 into the nTS elicits dose-dependent hypotension and bradycardia, which is abolished by antioxidants and by blockade of excitatory glutamatergic transmission (Cardoso et al., 2009). In patch clamp recordings of nTS neurons, H2O2 initially reduces neuronal excitability but produces hyperexcitability after removal from the bath; these effects are abolished by exogenous catalase in the recording pipette (Ostrowski et al., 2014). Importantly, repeated application and washout of H2O2 produces sustained hyperexcitability of these neurons, in part via increased Ca2+ entry through voltage gated Ca2+ channels (Ostrowski et al., 2017; Ostrowski et al., 2014).

We hypothesized that, in response to AIH exposure, increased H2O2 within the nTS contributes to the induction and maintenance of phrenic and sympathetic LTF. To test this hypothesis, we nanoinjected the antioxidant catalase into the nTS to acutely reduce H2O2 either during AIH exposure or following the development of LTF and evaluated effects on phrenic and sympathetic nerve activity. In a separate set of experiments, we examined the effects of chronic nTS reduction of H2O2 by overexpressing catalase in the nTS using an adenovirus. Our results show that acute and chronic increases in nTS catalase attenuate the development of LTF. In contrast, catalase nanoinjection following the development of LTF had no effect on the magnitude of phrenic or sympathetic nerve activity. Together, the data indicate that nTS H2O2 is critical to the development of pLTF and sLTF but is not required for their maintenance.