Cellular mechanisms of chemosensitivity in serotonergic raphe neurons

Abstract

Serotonergic neurons of the medullary raphe nuclei are strongly stimulated by acidosis [1,2], and are putative central respiratory chemoreceptors. We have examined the relationship between serotonergic neurons and blood vessels in the adult rat brain by using immunohistochemistry for tryptophan hydroxylase (TpOH) and injections of arteries with fluorescent albumin in gelatin. Serotonergic neurons within the raphe were closely associated with the basilar artery and its large penetrating branches. There were also homologous serotonergic neurons near large arteries of the rostral and caudal ventrolateral medullary surface (VLMS). We propose that serotonergic neurons on the VLMS contributed to the ventila-tory response to application of acidic solution in classical in vivo experiments that localized the chemoreceptors to that region. However, these neurons may not be specifically sensing CSF pH, but rather the PCO2 of blood in proximal arteries that happen to be located adjacent to the CSF space. In addition, a large number of homologous chemosensitive neurons are also located within the midline raphe nuclei. Perforated patch clamp recordings were made from raphe neurons in tissue culture, and the responses to respiratory acidosis, metabolic acidosis and isohydric hypercapnia were quantified. From a baselin firing rate of 0.2 Hz to 1.5 Hz, an increase in PCO2 from 5% to 9% and a decrease in pHo from 7.4 to 7.17 (at a constant [NaHCO3] of 26 mM) induced a mean increase in firing rate of 1.38 Hz (to 285% of control; n = 11). Metabolic acidosis (5% PCO2, 15 mM [NaHCO3], pHo 7.16) induced a mean increase in firing rate of 1.15 Hz (to 309% of control). Isohydric hypercapnia (9% PCO2, 40 mM [NaHCO3], at constant pHo) induced a mean increase in firing rate of 1.01 Hz (to 384% of control). These neurons also increased their firing rate in response to a decrease in pHo in the absence of CO2 and bicarbonate (in HEPES buffer).The only acid/base change that is shared by these four manipulations is a decrease in pHi, suggesting that this is the primary stimulus for these neurons – as is true for other chemosensitive neurons, including carotid body glomus cells. Using voltage clamp recordings, we have identified a novel ionic current in serotonergic neurons that is highly pH sensitive. This current was activated by calcium influx during depolarization. The major charge carrier was K+ under physiological conditions, but it also had a high permeability to Na+, with a permeability ratio for K+:Na+ of 4:1. It was not blocked by apamin (400 nM), charybdotoxin (240 nM), or TEA (15 mM). The current was extremely sensitive to changes in pH centered near 7.3. The current at pH7.61 was 95% of the maximum current, while the current at pH 6.99 was 2% of the maximum. To our knowledge, a current with these properties has not been reported previously. This current would be predicted to induce changes in membrane potential that have been observed during whole-cell current clamp recordings. Serotonergic medullary raphe neurons are strongly stimulated by a decrease in pHi due to the presence of a novel highly pH-sensitive calcium-activated cation channel. The degree of chemosensitivity of these neurons is large enough to make a major contribution to the changes in ventilation observed in whole animals in response to respiratory acidosis. The location of serotonergic neurons next to blood vessels and their widespread projections suggest that these neurons are chemoreceptors that modulate a variety of brain functions, including but not limited to respiratory control, and thus contribute to pH homeostasis.