Characterizing the effect of serotonergic input on medullary Phox2b neurons

Biological functions take place within tightly controlled parameters, including pH, which is managed in part through the ventilatory chemoreflex. This reflex is mediated by central respiratory chemoreceptors (CRCs) specialized to detect blood pH/CO2. Two neuronal populations are thought to mediate this response: the serotonergic (5-HT) neurons of the medullary raphé, and the Phox2b expressing neurons of the retrotrapezoid nucleus (RTN). These groups are both responsive to CO2 stimuli in vivo and in vitro. There are also apparent one-way connections from the raphé to the RTN, which is sensitive to 5-HT. Due to its complex innervation, study of RTN neurons while isolated from other cells, especially 5-HT neurons, has been limited. Here, we developed a culture model that simplifies this circuit, limiting cell types to those found in the rostral ventral medulla. This protocol yielded healthy RTN and 5-HT neurons in vitro, as well as other cell types from that area. Upon study with patch-clamp electrophysiology, cultured RTN neurons responded to CO2 and 5-HT in similar ways to what is reported for different RTN neuron preparations. Using this model, RTN neuron chemosensitivity was significantly decreased during application of 5-HT7 antagonists (SB258719, SB269970) and a 5-HT2A antagonist (MDL 11,939). The effect of 5-HT7 antagonists was recapitulated in slice recordings. Therefore, signaling at 5-HT7 and 5-HT2A receptors is necessary for RTN neuron chemosensitivity. Exogenous 5-HT application also increased RTN neuron firing rate without potentiating the response to CO2, most likely indicating that the necessary 5-HT stimulation must come from neurons that can alter their activity during acidosis. We conclude that RTN neuron chemosensitivity is largely driven by chemosensitive 5-HT neurons, and should be considered an integrative or relay center, rather than an independently chemosensitive one.