Neonatal hypoxia-ischemia(HI)results in losses of serotonergic neurons in specific dorsal raphe nuclei.However,not all serotonergic raphe 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 raphe 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 raphe nuclei.We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphe 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 raphe 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 raphe dorsal and caudal nuclei were not affected by P3 HI.These data indicate that not only are dorsal raphe serotonergic neurons lost after neonatal HI,but also remaining dorsal raphe 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.
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