Fluoxetine treatment abolishes the in vitro respiratory response to acidosis in neonatal mice

Abstract

Background: To secure pH homeostasis, the central respiratory network must permanently adapt its rhythmic motor drive to environment and behaviour. In neonates, it is commonly admitted that the retrotrapezoid/parafacial respiratory group of neurons of the ventral medulla plays the primary role in the respiratory response to acidosis, although the serotonergic system may also contribute to this response.

Methodology/principal findings: Using en bloc medullary preparations from neonatal mice, we have shown for the first time that the respiratory response to acidosis is abolished after pre-treatment with the serotonin-transporter blocker fluoxetine (25-50 µM, 20 min), a commonly used antidepressant. Using mRNA in situ hybridization and immunohistology, we have also shown the expression of the serotonin transporter mRNA and serotonin-containing neurons in the vicinity of the RTN/pFRG of neonatal mice.

Conclusions: These results reveal that the serotonergic system plays a pivotal role in pH homeostasis. Although obtained in vitro in neonatal mice, they suggest that drugs targeting the serotonergic system should be used with caution in infants, pregnant women and breastfeeding mothers.

Figure 1. Acidosis increases the phrenic burst frequency of en bloc medullary preparations.

A – Schematic presentation of the en bloc preparation of neonatal mice (A1) and example of raw and integrated phrenic bursts (bottom and top traces, respectively) produced by the isolated respiratory rhythm generator (RRG) on the C4 ventral roots of en bloc preparations (A2). B – Columns of the histogram show the mean (and SEM) phrenic burst frequency (PBf; expressed as % of the control PBf) measured every min (one column  = 1 min) in 12 en bloc preparations when the control aCSF(7.4) (white columns) superfusing the preparations was replaced by aCSF(7.1) for 5 min (black columns). Note that acidosis significantly increased the PBf (asterisks indicate a p<0.05 statistical difference). C- As in B but aCSF(7.1) application for 10 min to 6 other preparations. Note that the PBf reached a plateau from the 5^th to the 10^th min of aCSF(7.1) application.

Figure 2. Fluoxetine and serotonin pre-treatments abolish the increase of phrenic burst frequency induced by acidosis.

A - Columns of the histogram show the mean (and SEM) PBf (expressed as % of the control PBf) measured every min (one column  = 1 min) in 10 preparations when the control aCSF(7.4) (white columns) superfusing the preparations was first replaced by aCSF(7.4) containing fluoxetine (10–25 µM) for 20 min to block the serotonin transporter SERT (grey columns) and thereafter by aCSF(7.1) containing the same amount of fluoxetine for 5 min (black columns). Note that pre-treatment with fluoxetine did not significantly increase the PBf but abolished the PBf increase under acidosis. B - PBf changes (expressed as % of control) every min (one column) but for 14 preparations superfused with control aCSF(7.4) (white columns), aCSF(7.4) containing serotonin, 5-HT (1–5 µM) for 20 min (grey columns) and thereafter aCSF(7.1) containing the same amount of 5-HT for 5 min (black columns). Note that pre-treatment with 5-HT significantly increased the PBf by about 50% and, similarly to fluoxetine treatment, abolished the PBf increase under acidosis.

Figure 3. SERT mRNA and 5-HT-containing neurons in the vicinity of the RTN/pFRG of neonatal mice.

A: Two serial coronal sections passing through the RTN/PFRG area of a neonatal mouse show SERT mRNA expression (A1) and Cresyl Violet staining (A2). Anatomical limits drawn from A2 Cresyl Violet section have been superimposed on A1 section (dotted line). Note SERT mRNA is expressed in three areas, the median Raphe Magnus (Rm), the para-pyramidal group (ppy) lateral to the pyramid tract (py) and a lateral spot (arrow) located in a medio-ventral position from the facial motor nucleus (n7), as defined from Cresyl Violet staining (in A2). B- 5-HT neurons in the RTN/pFRG area of a neonatal mouse. Note 5-HT neurons in the median raphe magnus (Rm), the parapyramidal group (ppy, doted circle) lateral to the pyramidal tract (py) and the lateral spot (arrow in B1) in the medio-ventral location of the facial nucleus (n7). In B2 and B3, note the superficial location of some 5-HT neurons (arrows). B3 is an enlargement of B1.