Molecular Changes in Neurons of Rat Nucleus Ambiguus after Axotomy, As Revealed by a Novel Method of In Vivo Fluorescence Neuronal Labeling Combined with Single-cell RT-PCR

Abstract

To elucidate the molecular changes of motoneurons which innervate laryngeal muscles after transection of rat recurrent laryngeal nerve, we have devised a new method of DiI labeling combined with single-cell RT-PCR, by which nucleus ambiguus (NA) neurons projecting to the intrinsic laryngeal muscles were labeled retrogradely by injecting the fluorescent dye DiI into bilateral thyroarytenoid muscles; then either one of the recurrent laryngeal nerves was transected, and the fluorescence-bright single neurons were microdissected, followed by single-cell reverse transcription (RT)-PCR. Immunohistochemically, many DiI-labeled motoneurons in NA were immunoreactive to phosphorylated Stat3 in the nucleus on 1, 7, 10 and 14 days after axotomy. Thirty μm-thick frozen sections of brain stem were mounted onto membrane slide, and DiI-labeled single neurons in NA were microdissected under a fluorescence microscope. By real-time quantitative RT-PCR for single motoneurons, the expression levels of GAP-43 and nNOS genes were upregulated at 7 days and 1 day after axotomy, respectively. The expression levels of Stat3, Reg-2, and Bcl-2 genes were upregulated at 7 days, whereas that of Bax was downregulated at 1 and 7 days after axotomy as compared with control levels. With this novel method in which single-cell RT-PCR was combined with neurotracer DiI labeling we could demonstrate a part of the molecular cascades involving Stat3 in neurons projecting to laryngeal muscles after axotomy.