High mucosal serotonin availability in neonatal guinea pig ileum is associated with low serotonin transporter expression

Abstract

Background & aims: 5-Hydroxytryptamine (5-HT) is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterochromaffin cells (EC), which release 5-HT, has not been studied in neonates. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs.

Methods: 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and high-performance liquid chromatography with electrochemical detection.

Results: An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface, and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 microM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs adult tissues. There was no difference in 5-HT content between neonates and adults but 5-hydroxyindole acetic acid/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number, but EC cells were found in the crypts in neonatal and along the villi in adult tissues.

Conclusions: SERT expression is low in neonates, and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may be important for development of neurohumoral control of intestinal motor reflexes.

Figure 1

Increased 5-HT availability in the mucosa of neonatal guinea pigs. (A) Baseline and mechanically-evoked 5-HT oxidation currents in adult and neonatal intestine in vitro. Bars labeled “Baseline” indicate when the electrode was positioned near the mucosa. The hatched area labeled “Stimulated” indicates when a glass probe was pressed against the mucosa near the recording electrode. (B) Analysis of experiments similar to those shown in “A”. The baseline oxidation current in tissues from neonatal guinea pigs (n=6) was larger (#, P <0.001) than that measured in tissues from adult guinea pigs (n=6). The stimulated current was significantly larger in neonatal (#, P < 0.001) vs. adult tissues and stimulation produced a significant increase in the oxidation current over baseline in adult and neonatal tissues (&, P < 0.01). All currents were significantly reduced in low extracellular Ca²⁺ solutions (0.25 mM)(*, P <0.05 compared to control). Tetrodotoxin (TTX) reduced all currents (*, P <0.05 compared to control) except the stimulated current in adult tissues. Data are mean ± S.E.M.

Figure 2

Fluoxetine potentiated 5-HT oxidation currents in adult but not neonatal tissues. (A) Baseline and stimulated 5-HT oxidation currents were increased in the presence of the SERT inhibitor fluoxetine. (B) Baseline and stimulated 5-HT oxidation currents were larger in the neonatal tissue compared to the adult tissue (see A) but fluoxetine did not change current amplitude in the neonatal tissue. (C) Data from experiments similar to those shown in “A” and “B”. Baseline currents were larger in neonatal (n=6) vs. adult tissues (n=6) but the current was increased significantly by fluoxetine only in adult tissues (*, P <0.05). Stimulation produced a significant increase in oxidation current over baseline levels in adult and neonatal tissues (&, P <0.01). The stimulated current was also increased significantly by fluoxetine in adult tissues but not in neonatal tissues (*, P < 0.05). Data are mean ± S.E.M.

Figure 3

Immunohistochemical localization of SERT in section neonatal and adult intestine. (A) Section from adult intestine showing SERT-ir in enterocytes lining several villi. (B) A similar section from the neonatal intestine showing absence of SERT-ir. Omitting the primary antibody removes SERT labeling from an adult (C) and neonatal (D) sections.

Figure 4

Western blot analysis of SERT expression in the neonatal and adult intestine. (A) Western blot showing multiple bands recognized by the SERT antibody in samples from 3 neonatal guinea pigs (lanes 1–3) and 3 adult guinea pigs (lanes 4–6). There is a faint band that corresponds to a prominent band near 75 kD in a protein extract from adult guinea pig brain. A prominent band near 60 kD was detected in adult extracts but this band was present at lower levels in neonatal extracts. (B) Intensity of the 60 and 75 kD bands was normalized to the total protein present in each lane. There was no difference in the intensity of the 75 kD bands between adult and neonatal tissues but the intensity of the 60 kD was significantly greater in adult compared to neonatal tissues (*, P < 0.05). Data are mean ± S.E.M.

Figure 5

5-HT and 5-HIAA levels in the mucosa of neonatal and adult intestine. (A) There was no difference in 5-HT concentration in the mucosa of neonatal (n=6) vs. adult (n=5) intestine. (B) 5-HIAA levels were lower in neonatal intestine (*, P < 0.05). (C) The 5-HT/5-HIAA ratio was also smaller in neonatal compared to adult intestine (*, P <0.05). Data are mean ± S.E.M.

Figure 6

Immunohistochemical localization of 5-HT-ir in sections of adult and newborn intestine. (A) A cross section of the adult intestine showing 5-HT-ir EC cells distributed in the crypt region and along the mucosal villi. (B) A cross section of neonatal intestine showing that 5-HT-ir EC cells are localized preferentially to the crypt region.

Figure 7

Analysis of the distribution of EC cells in the crypts and villi of neonatal and adult guinea pigs. (A) The total number of EC cells in the mucosa did not differ between adult (n=4) and neonatal (n=4) guinea pigs. However, the number of EC cells in the crypt of neonatal intestine was significantly greater (*, P < 0.05) than that in adult intestine. Alternatively, the number of EC cells in adult villi was significantly greater (*, P < 0.05) than that in the neonatal intestine. (B) The EC cell crypt-villus ratio was significantly larger in the neonatal intestine (*, P <0.05). Data are mean ± S.E.M.