Medullary serotonin neurons are CO₂ sensitive in situ

Abstract

Brainstem central chemoreceptors are critical to the hypercapnic ventilatory response, but their location and identity are poorly understood. When studied in vitro, serotonin-synthesizing (5-HT) neurons within the rat medullary raphé are intrinsically stimulated by CO2/acidosis. The contributions of these neurons to central chemosensitivity in vivo, however, are controversial. Lacking is documentation of CO2-sensitive 5-HT neurons in intact experimental preparations and understanding of their spatial and proportional distribution. Here we test the hypothesis that 5-HT neurons in the rat medullary raphé are sensitive to arterial hypercapnia. We use extracellular recording and hypercapnic challenge of spontaneously active medullary raphé neurons in the unanesthetized in situ perfused decerebrate brainstem preparation to assess chemosensitivity of individual cells. Juxtacellular labeling of a subset of recorded neurons and subsequent immunohistochemistry for the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) identify or exclude this neurotransmitter phenotype in electrophysiologically characterized chemosensitive and insensitive cells. We show that the medullary raphé houses a heterogeneous population, including chemosensitive and insensitive 5-HT neurons. Of 124 recorded cells, 16 cells were juxtacellularly filled, visualized, and immunohistochemically identified as 5-HT synthesizing, based on TPH-immunoreactivity. Forty-four percent of 5-HT cells were CO2 stimulated (increased firing rate with hypercapnia), while 56% were unstimulated. Our results demonstrate that medullary raphé neurons are heterogeneous and clearly include a subset of 5-HT neurons that are excited by arterial hypercapnia. Together with data identifying intrinsically CO2-sensitive 5-HT neurons in vitro, these results support a role for such cells as central chemoreceptors in the intact system.

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

“…Therefore, Ugt1a6 and Ugt1a7 in the brain may play important roles in detoxification and homeostasis to protect the brain. In the present study, APAP glucuronidation was increased by PB in the dmd.aspetjournals.org medulla oblongata and thalamus, where serotonin, a Ugt1a6 substrate, was found (Iceman et al, 2013;Chung et al, 2015). Thus, the concentrations of UGT substrates, including serotonin, may change locally, thereby altering efficacy.…”

Isoflurane abolishes spontaneous firing of serotonin neurons and masks their pH/CO2chemosensitivity

“…As previously reported, 5-HT neurons were chemosensitive to acidosis in culture (Wang et al 1998(Wang et al , 2001Wang and Richerson 1999), and this property was retained in a perfused brain preparation (Iceman et al 2013). In culture and in the perfused brain stem, 1% isoflurane abolished the firing of 5-HT neurons under control conditions and prevented the increase in firing frequency normally induced by hypercapnic acidosis.…”

“…Briefly, the mean interspike interval ( x, in ms) and the SD of the interspike interval (s, in ms) were derived as independent variables and used to solve the discriminant function y͑x , s͒ ϭ 146 ms Ϫ x ϩ 0.98s A value resulting from this function Ͻ0 indicates that the cell is likely to be serotonergic (putative 5-HT cell), while a value Ͼ0 indicates the cell is not likely to be serotonergic. We and others have characterized recordings as putative 5-HT neurons by firing pattern analysis and confirmed that it is accurate at least 90% of the time compared with juxtacellular labeling and immunohistochemical neurotransmitter phenotyping (Iceman et al 2013;Mulkey et al 2004).…”

“…Recorded neurons were filled with biotinamide by applying positive-current pulses (400-ms duration, 50% duty cycle) of gradually increasing intensity (0 -10 nA max in 0.2-nA steps) until entrainment of cell discharge to the current pulse was achieved (Iceman et al 2013). Cell entrainment was maintained for at least 30 s. Current pulses trigger the iontophoretic ejection of biotinamide, and entrainment facilitates uptake of this marker by the entrained cell.…”

Selective optogenetic stimulation of the retrotrapezoid nucleus in sleeping rats activates breathing without changing blood pressure or causing arousal or sighs

“…Lesions of serotonergic or the locus coeruleus noradrenergic neurons attenuate the CO 2 -induced arousal and the HCVR but have minimal effects on the hypoxia-induced arousal or the hypoxic ventilatory reflex (13,14,21,41,45). To date, the interpretation of this evidence is that subsets of these aminergic neurons are directly responsive to protons (i.e., central chemoreceptors) and unaffected by hypoxia (20).…”