Hypoxia-ischemia in the immature rodent brain impairs serotonergic neuronal function in certain dorsal raphé nuclei

Abstract

Neonatal hypoxia-ischemia (HI) results in losses of serotonergic neurons in specific dorsal raphé nuclei. However, not all serotonergic raphé neurons are lost and it is therefore important to assess the function of remaining neurons in order to understand their potential to contribute to neurological disorders in the HI-affected neonate. The main objective of this study was to determine how serotonergic neurons, remaining in the dorsal raphé nuclei after neonatal HI, respond to an external stimulus (restraint stress). On postnatal day 3 (P3), male rat pups were randomly allocated to one of the following groups: (i) control + no restraint (n = 5), (ii) control + restraint (n = 6), (iii) P3 HI + no restraint (n = 5) or (iv) P3 HI + restraint (n = 7). In the two HI groups, rat pups underwent surgery to ligate the common carotid artery and were then exposed to 6% O₂ for 30 minutes. Six weeks after P3 HI, on P45, rats were subjected to restraint stress for 30 minutes. Using dual immunolabeling for Fos protein, a marker for neuronal activity, and serotonin (5-hydroxytrypamine; 5-HT), numbers of Fos-positive 5-HT neurons were determined in five dorsal raphé nuclei. We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphé nuclei compared to control animals. However, following P3 HI, the number of stress-induced Fos-positive 5-HT neurons was decreased significantly in the dorsal raphé ventrolateral, interfascicular and ventral nuclei compared with control animals exposed to restraint stress. In contrast, numbers of stress-induced Fos-positive 5-HT neurons in the dorsal raphé dorsal and caudal nuclei were not affected by P3 HI. These data indicate that not only are dorsal raphé serotonergic neurons lost after neonatal HI, but also remaining dorsal raphé serotonergic neurons have reduced differential functional viability in response to an external stimulus. Procedures were approved by the University of Queensland Animal Ethics Committee (UQCCR958/08/NHMRC) on February 27, 2009.

Keywords: Fos; dorsal raphénuclei; hypoxia-ischemia; neonate; newborn brain injury; preterm; restraint stress; serotonin.

Figure 1

Effects of P3 HI and restraint stress on numbers of 5-HT-positive neurons in dorsal raphé nuclei. Schematics present the approximate location (mm relative to bregma) of counts in each of the five dorsal raphé nuclei (A–D). The total numbers of 5-HT-positive neurons in each dorsal raphé nucleus on the non-ligated (E) and ligated side (F) are presented. There were no effects of restraint stress or P3 HI on numbers of 5-HT neurons on the non-ligated side (E). On the ligated side in P3 HI animals, there was a significant reduction in the numbers of 5-HT-positive neurons in the DRd, DRvl, and DRv nuclei compared to control animals (F). Values are presented as mean ± SEM (n = 5 and 6 in the control + no restraint and control + restraint groups, respectively). *P < 0.05, **P < 0.01, vs. control + no restraint animals; †P < 0.05, ††P < 0.01, vs. control + restraint (one-way analysis of variance followed by Student’s t-test). 5-HT: 5-Hydroxytryptamine; Aq: aqueduct (Sylvius); DRc: dorsal raphé caudal nucleus; DRd: dorsal raphé dorsal nucleus; DRif: dorsal raphé interfascicular nucleus; DRv: dorsal raphé ventral nucleus; DRvl: dorsal raphé ventrolateral nucleus; P3 HI: postnatal day 3 hypoxia-ischemia; xcsp: decussation of the superior cerebellar peduncle.

Figure 2

Effects of P3 HI and restraint stress on numbers of Fos-positive 5-HT dorsal raphé neurons. Photomicrographs of brainstem sections through the dorsal raphé ventrolateral nucleus (A, mm relative to bregma) from a control + no restraint (B), control + restraint (C) and P3 HI + restraint animals (D). Brainstem sections have been immunolabeled for Fos protein (black nucleus) and 5-HT (amber cytoplasm). Scale bars represent 25 μm. The histograms present the total numbers of Fos-positive 5-HT neurons in each dorsal raphé nucleus on the non-ligated (E) and ligated side (F). Restraint stress in control animals significantly increased the number of Fos-positive 5-HT neurons in comparison to control animals with no restraint (E and F). In P3 HI animals subject to restraint stress, the numbers of Fos-positive 5-HT neurons on the ligated side were significantly reduced in the DRvl, Drif, and DRv nuclei (F). Values are presented as mean ± SEM (n = 5 and 6 in the control + no restraint and control + restraint groups, respectively). **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. control + no restraint animals; †P < 0.05, ††P < 0.01, vs. control + restraint (one-way analysis of variance followed by Student’s t-test). 5-HT: 5-Hydroxytryptamine; Aq: aqueduct (Sylvius); DRc: dorsal raphé caudal nucleus; DRd: dorsal raphé dorsal nucleus; DRif: dorsal raphé interfascicular nucleus; DRv: dorsal raphé ventral nucleus; DRvl: dorsal raphé ventrolateral nucleus; P3 HI: postnatal day 3 hypoxia-ischemia; xcsp: decussation of the superior cerebellar peduncle.