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The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviors such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus in the modulation of laryngeal motoneuron (LMN) activity. The present study investigated the anatomical nature of cholinergic input to inspiratory- (ILM) and expiratory-modulated (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus. Using combined in vivo intracellular recording, dye filling, and immunohistochemistry, we demonstrate that LMNs identified in Sprague-Dawley rat receive several close appositions from vesicular acetylcholine transporter-immunoreactive (VAChT-ir) boutons. ELMs receive a significantly greater number of close appositions (mean ± standard deviation [SD]: 47 ± 11; n = 5) than ILMs (32 ± 9; n = 8; t-test P < 0.05). For both LMN types, more close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two-way analysis of variance [ANOVA], P < 0.0001). Using fluorescence confocal microscopy, almost 90% of VAChT-ir close appositions (n = 45 boutons on n = 4 ELMs) were colocalized with the synaptic marker synaptophysin. These results support a strong influence of cholinergic input on LMNs and may have implications in the differential modulation of laryngeal muscle activity.
Many respiration-related interneurons and motoneurons receive a catecholaminergic input, but the extent and distribution of this input to recurrent laryngeal motoneurons that innervate intrinsic muscles of the larynx are not clear. In the present study, we examined the catecholaminergic input to expiratory laryngeal motoneurons in the caudal nucleus ambiguus by combining intracellular labeling of single identified motoneurons, with immunohistochemistry to reveal tyrosine hydroxylase immunoreactive (catecholaminergic) terminal varicosities. Close appositions were found between the two structures, with 18 ± 5 close appositions per motoneuron (n = 7). Close appositions were more frequently observed on distal rather than proximal dendrites. Axosomatic appositions were not seen. In order to determine the source of this input, microinjections of cholera toxin B subunit (1%, 20 nl) were made into the caudal nucleus ambiguus. Retrogradely labeled neurons, located in the ipsilateral nucleus tractus solitarius and the area postrema, were tyrosine hydroxylase-positive. Our results not only demonstrate details of the extent and distribution of potential catecholamine inputs to the expiratory laryngeal motoneuron, but further indicate that the inputs, at least in part, originate from the dorsomedial medulla, providing a potential anatomical basis for previously reported catecholaminergic effects on the laryngeal adductor reflex.
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