Acute exercise-induced activation of Phox2b-expressing neurons of the retrotrapezoid nucleus in rats may involve the hypothalamus
Introduction
The retrotrapezoid nucleus (RTN) within the ventrolateral medulla (VLM) is an important center of chemoreception and is characterized by neurons that express high levels of the transcriptor factor paired-like homeobox 2b but lack tyrosine hydroxylase (Phox2b+TH−). These CO2-activated excitatory neurons innervate the entire ventral respiratory column (Stornetta et al., 2006). Recently, we demonstrated that ventrolateral medullary neurons are activated after acute exercise in rodents (Barna et al., 2012). Although we presumed that these neurons were RTN chemosensitive neurons, we did not identify their exact phenotype.
In the present study, we refer to the chemosensitive neurons as the chemically-coded RTN neurons (ccRTN neurons) to distinguish them from other types of neurons in this area (Lazarenko et al., 2009, Marina et al., 2010). ccRTN neurons are activated by hypercapnia in anesthetized rats and are uniformly activated by acidification in slices; and the selective activation of ccRTN neurons in vivo stimulates breathing (Abbott et al., 2009, Lazarenko et al., 2009, Marina et al., 2010). Their acute inhibition or chronic destruction eliminates or markedly attenuates breathing and the ability of CO2 to elicit breathing in anesthetized or unrestrained awake rats (Takakura et al., 2006, Takakura et al., 2008, Takakura et al., 2013, Marina et al., 2010). A very elegant genetic study revealed the importance of ccRTN neurons in respiratory chemoreception (Ramanantsoa et al., 2011). The authors created a mouse model that expresses the Phox2b Congenital Central Hypoventilation Syndrome mutation in neurons of rhombomere 3 and 5 lineage (Phox2b27alacki; Egr2cre/+). This offspring lacked all Phox2b neurons but unexpectedly survived to adulthood, despite a complete loss of respiratory responses to CO2. Their survival was attributed to a respiratory compensation via peripheral chemoreceptors (Ramanantsoa et al., 2011). Collectively, this evidence suggests that the ccRTN neurons provide a substantial fraction of the excitatory drive to the central pattern generator (CPG) at rest and are required for the homeostatic regulation of breathing by CO2.
The ccRTN neurons presumably contribute to the increased respiratory activity associated with various behaviors and during hypothalamic stimulation, including central command, emotions, sleep and thermoregulation (Hilton and Redfern, 1986, Waldrop et al., 1988, Dimicco et al., 2002, Zhang et al., 2006, Tanaka and McAllen, 2008). Given that hypothalamic stimulation activates the ccRTN neurons vigorously (Fortuna et al., 2009) and that selective stimulation of the same neurons activates breathing (Abbott et al., 2009), the RTN is likely a relay for the central command of respiration during exercise. This view is also consistent with the notion that RTN drives active expiration (Janczewski and Feldman, 2006, Abdala et al., 2009, Takakura et al., 2013).
The present experiments were designed to test the hypothesis that acute exercise activates ccRTN neurons and this activation is mediated, at least in part, by exercise-induced activation of hypothalamic inputs to the RTN.
Section snippets
Activation of ccRTN Phox2b-expressing neurons after acute exercise
The first set of experiments was designed to test if ccRTN neurons are activated by acute treadmill exercise in rats. We used the proto-oncogene product c-Fos as a measure of cell activation. ccRTN neurons can be identified histologically as Phox2b+/TH− cells. All Phox2b+ cells in this region contain vesicular glutamate transporter 2 (VGLUT2) mRNA (Stornetta et al., 2006), and the Phox2b+/TH− cells are readily distinguished from ChAT+ neurons in the facial motor nucleus and serotonergic neurons
Discussion
The data presented support the view that chemosensory neurons in the RTN, which are involved in the pH-induced changes in breathing, are also involved in the control of breathing during acute exercise and that the PeF/LH may contribute to this mechanism.
Conclusion
Our experiments showed that acute exercise activates chemosensitive Phox2b+TH− RTN neurons and may lead to activation of the CPG controlling inspiratory and expiratory activity (Abbott et al., 2011, Barna et al., 2012, Pagliardini et al., 2012).
Moreover, the present data showed for the first time that the activity of RTN neurons is not simply from their classic intrinsic chemosensitive properties, but also by synaptic interactions, probably through hypothalamic neurons that are part of the
Animals
Surgical procedures and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of São Paulo, in compliance with the Ethical Principles in Animal Research of the Brazilian College of Animal Experimentation. All experiments were conducted using adult male Wistar rats weighing 250–350 g.
Exercise protocol
Rats were preselected for their ability to walk/run on a treadmill (KT-300; Inbramed, Porto Alegre, Brazil; 1.7 m/min, 0% gradient, 5–10 min for a period
Authors contribution
B.F.B., A.C.T. and T.S.M. designed research; B.F.B. and T.S.M. performed research; B.F.B. analyzed data and B.F.B., A.C.T. and T.S.M. wrote the paper.
Acknowledgments
This research was supported by public funding from São Paulo Research Foundation (FAPESP) (Grants: 10/12590-9 to B.F.B., 10/09776-3 to A.C.T. and 10/19336-0 to T.S.M.). We thank Dr. J.-F. Brunet for the donation of the Phox2b antibody. We would like to thanks Dr. Susan M. Barman for her help in the revision of the manuscript and Marilu Mazzaro for the technical support.
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