Postnatal changes in tryptophan hydroxylase and serotonin transporter immunoreactivity in multiple brainstem nuclei of the rat: implications for a sensitive period

Abstract

Previously, we found that the brainstem neuronal network in normal rats undergoes abrupt neurochemical, metabolic, and physiological changes around postnatal days (P) 12-13, a critical period when the animal's response to hypoxia is also the weakest. This has special implications for sudden infant death syndrome (SIDS), insofar as seemingly normal infants succumb to SIDS when exposed to respiratory stressors (e.g., hypoxia) during a narrow postnatal window. Because an abnormal serotonergic system has recently been implicated in SIDS, we conducted a large-scale investigation of the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) and serotonin transporter (SERT) with semiquantitative immunohistochemistry in multiple brainstem nuclei of normal rats aged P2-21. We found that 1) TPH and SERT immunoreactivity in neurons of raphé magnus, obscurus, and pallidus and SERT in the neuropil of the pre-Bötzinger complex, nucleus ambiguus, and retrotrapezoid nucleus were high at P2-11 but decreased markedly at P12 and plateaued thereafter until P21; 2) SERT labeling in neurons of the lateral paragigantocellular nucleus (LPGi) and parapyramidal region (pPy) was high at P2-9 but fell significantly at P10, followed by a gradual decline until P21; 3) TPH labeling in neurons of the ventrolateral medullary surface was stable except for a significant fall at P12; and 4) TPH and SERT immunoreactivity in a number of other nuclei was relatively stable from P2 to P21. Thus, multiple brainstem nuclei exhibited a significant decline in TPH and SERT immunoreactivity during the critical period, suggesting that such normal development can contribute to a narrow window of vulnerability in postnatal animals.

Fig. 1

Tryptophan hydroxylase (TPH) immunoreactive (-ir) neurons in the nucleus raphé magnus (RM) (A), nucleus raphé obscurus (ROb) (B), nucleus raphé pallidus (RP) (C), and the ventrolateral medullary surface (VLMS) (D) at P2 (A1–D1), P7 (A2–D2), P12 (A3–D3), and P21 (A4–D4). The insets in A1–D1 indicate diagrammatically the locations of these four nuclear groups. TPH expression in the RM, ROb, and RP showed relatively high level at P2 and P7, but relatively low at P12 and P21, whereas that in the VLMS exhibited significantly low level at P12, compared with those in P2, P7, and P21 samples. Scale bar: 20 μm for all.

Fig. 2

TPH-ir neurons in the lateral paragigantocellular nucleus (LPGi) (A), parapyramidal region (pPy) (B), and the commissural subnucleus of the solitary tract nucleus (NTS_COM) (C) at P2 (A1–C1), P7 (A2–C2), P12 (A3–C3), and P21 (A4–C4). The insets in A1–C1 indicate diagrammatically the locations of these three nuclear groups. TPH expression in these three nuclei groups exhibited relatively constant level from P2 to P21. Scale bar: 20 μm for all.

Fig. 3

Optical densitometric measurements of TPH-ir product in the cytoplasm of individual neurons in the RM (A), ROb (B), RP (C), VLMS (D), LPGi (E), pPy (F) and NTS_COM (G) from P2 to P21. Data points were presented as mean ± SEM. Labeling in the first three nuclear groups (A–C) followed a similar trend of development, with initial high levels of expression from P2 to P11, followed by a marked reduction at P12 and remained low thereafter until P21. The developmental pattern in VLMS (D) was relatively stable during the first 3 postnatal weeks, with a distinct and significant fall in TPH expression at P12. On the other hand, the expression in LPGi (E), pPy (F), and NTS_COM (G) remained relatively constant from P2 to P21. ANOVA revealed significant differences in TPH expression among ages in the RM, ROb, and VLMS (P < 0.01), and Tukey's Studentized test showed a significant reduction in these three nuclei at P12, as compared with their adjacent younger age groups (P11). *, P < 0.05; **, P < 0.01 (Tukey's Studentized test).

Fig. 4

Serotonin transporter (SERT) expression in neurons of the RM (A), ROb (B), RP (C), LPGi (D), pPy (E), and NTS_COM (F) at P2 (A1–F1), P7 (A2–F2), P12 (A3–F3), and P21 (A4–F4). The insets in A1–F1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla or pons. SERT expression in the RM, ROb, RP, LPGi, and pPy were relatively high at P2 and P7, but were much lower at P12 and P21; On the other hand, the level in NTS_COM was quite stable from P2 to P21. Scale bar: 20 μm for all.

Fig. 5

SERT expression in the neuropil of the pre-Bötzinger complex (PBC) (A), nucleus ambiguus (Amb) (B), retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG) (C), hypoglossal nucleus (XII) (D), ventrolateral subnucleus of the solitary tract nucleus (NTS_VL) (E), and cuneate nucleus (CN) (F) at P2 (A1–F1), P7 (A2–F2), P12 (A3–F3), and P21 (A4–F4). The insets in A1–F1 indicate diagrammatically the locations of these six nuclear groups. All arrowheads point to SERT-ir terminal fibers. In these six nuclear groups, the majority of neurons exhibited SERT-negative expression. Scale bar in A1: 20 μm for all except 8 μm for all high magnification insets. See text for details.

Fig. 6

Optical densitometric measurements of immunoreactive (-ir) product of SERT in the cytoplasm of individual neurons in the RM (A), ROb (B), RP (C), LPGi (D), pPy (E), and NTS_COM (F), as well as in the neuropil of the PBC (G), Amb (H), RTN/pFRG (I), XII (J), NTS_VL (K), and CN (L) from P2 to P21. Data points were presented as mean ± SEM. The expressions of SERT in neurons of the RM (A), ROb (B), and RP (C), as well as in the neuropil of the PBC (G), Amb (H), and RTN/pFRG (I) were relatively high during the first 1 to 1 ½ postnatal weeks, then fell significantly or noticeably at P12, followed by a plateau of relatively low levels until P21. In the LPGi (D) and pPy (E) neurons, SERT expression was relatively high at P2–P9, then markedly reduced at P10, followed by a plateau and another decline at P17–P21, except for a small, statistically insignificant peak at P15–P16 in LPGi. On the other hand, labeling in neurons in the NTS_COM (F) and in the neuropil of the XII (J), NTS_VL (K), and CN (L) was quite stable with only minor fluctuations from P2 to P21. ANOVA yielded significant differences in SERT expressions among the ages in the RM, ROb, RP, LPGi, pPy, PBC, Amb, and RTN/pFRG (P < 0.01). Tukey's Studentized test revealed a significant reduction in SERT expression in the ROb, PBC, and Amb at P12, as compared with their adjacent younger age groups (P11), and a significant reduction at P10 in the LPGi and pPy (compared to their adjacent younger age groups P9). *, P < 0.05; **, P < 0.01 (Tukey's Studentized test).