The Central Control Of Laryngeal Motoneurons On Respiration And Non Respiratory Activity Of Rat

Objective:The laryngeal chemoreflex, which is elicited by aspiration of fluid into the larynx, exerts a potent inhibition on breathing. Electrical stimulation of the superior laryngeal (SLN) nerve induces the apnoeic response, which stops central inspiration, and activate the expiratory laryngeal motoneurons (ELM). But the mechanism of the SLN-induced apnoeic response is unclear. We aim to study the neuronal pathway underlying the SLN-induced apnoea associated with inhibition of inspiration and activation of ELM.Methods:The apnoea was evoked by the stimulation of SLN (20Hz,0.2ms). Then, the GABAA receptor agonist, isoguvacine (10mM,20-40nl) subsequently was microinjected to ipsilateral Botzinger complex (BotC) and contralateral nucleus tractus solitaries (NTS). And isoguvacine was injected into contralateral NTS without a preceding BotC microinjection after 60 min recovery. The RLN was recorded to monitor the ELM burst activity. Effects of isoguvacine microinjections were recorded from the RLN and phrenic nerve two minutes after each microinjection.Results:20 Hz stimulation of the SLN caused an apnoeic response with total PND reduced to 12±9% of baseline. A microinjection of isoguvacine into the ipsilateral BotC significantly weakened the apnoeic response, and a further microinjection of isoguvacine into the contralateral NTS, no apnoeic response was seen during the SLN stimulation. However, neither the BotC nor NTS inactivation had an obvious effect on ELM burst activity. Little effect on the apnoeic response was observed when isoguvacine was injected into contralateral NTS without a preceding BotC microinjection.Conclusions:It almost entirely abolished the apnoeic response without affecting the ELM burst activity after microinjection of isoguvacine into the ipsilateral BotC and contralateral NTS. Our results suggest a bifurcating projection from NTS to both the caudal nucleus ambiguus and BotC, which mediates the ELM burst and apnoeic response respectively. Objective:Many motoneurons and respiratory neurons are demonstrated to receive tyrosine hydroxylase (TH) immunoreactive (ir) inputs, but the TH-ir inputs to laryngeal motoneurons in the caudal nucleus ambiguous (NA) still remain unknown. In the present study, we aim to:1) examine whether the close appositions exists or not between TH-ir boutons and ELMs by using intracellular labelling combined with immunohistochemistry, and, if yes,2) identify the potential origins of the catecholamine inputs by using tract-tracing study.Methods:In the present study, we examined TH-ir inputs to expiratory laryngeal motoneurons (ELMs) in anesthetized Sprague Dawley rats by using a combination of intracellular labelling and immunohistochemistry. ELMs were identified with the post-inspiratory firing pattern and the antidromic response to the stimulation of the recurrent laryngeal nerve. Further tract-tracing study was carried by the injection of cholera toxinβ(CTB) into the caudal NA. The retrogradely labeled neurons and the TH-ir structures were revealed by dual fluorescence immunohistochemistry.Results:TH-ir close appositions were found on all ELMs, with an average number of 18±5 per neuron (n=7, mean±SD). Close appositions were commonly observed on the distal dendrites rather than on proximal ones, but none on somata or axons. Further tract-tracing study by the injection of cholera toxinβ(CTB) into the caudal NA, revealed that a large number of CTB labelled neurons in the ipsilateral nucleus tractus solitarii (NTS) and area postrema (AP) were catecholaminergic, and the highest density appeared at 0.2-0.4 mm caudal to the obex.Conclusions:We demonstrated for the first time that the ELMs receive TH-ir close appositions, suggesting that the catecholamine inputs may play a role in the activity of ELMs. The afferents of the superior laryngeal nerve (SLN) project to the NTS neurons at the same longitudinal level, our further tract-tracing study suggest that catecholaminergic neurons located in the NTS at the obex level may play an important role in control of the SLN-mediated airway protective reflexes. Objective:Laryngeal motoneurons (LMN), located in the caudal nucleus ambiguus (NA), control the intrinsic laryngeal muscles to perform different laryngeal functions including respiration, phonation and airway protective reflexes, such as coughing, sneezing and swallowing. It is assumed that different chemical inputs to LMN are contributed to different functions of the larynx. Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto LMN were studied at the light microscope level, but the distribution of their terminals in the caudal NA, remains unclear. In addition, the identification of the relationship between the neurochemical immunoreactive (ir) terminals and LMN at the ultrastructural level is lacked, which is essential to confirm the existence of authentic synapses. Here we aimed to (1) estimate the distribution synaptic terminals that are SP, TH or serotonin-ir in the caudal NA; (2) identify the relationship of SP-ir terminals with LMN by using intracellular labelling, immunohistochemistry and electron microscopy.Methods:We applied multiple immunofluorescence and confocal microscopy, to simultaneously assess the distribution of SP, TH and serotonin-ir synaptic terminals in caudal NA. Motoneurons in the caudal NA were identified on the basis of their immunoreactivity to Choline acetyltransferase. The total neuronal terminal area was identified with presynaptic protein, synaptophysin (SYN). Areas where SYN was co-localized with SP, serotonin or TH were taken as a representative of their corresponding terminal area. The proportions of the SP/TH/serotonin-ir terminal area in the total terminal area were used to assess the distribution of different neurochemical-ir terminals in the caudal NA. On the basis of the assessment of SP, TH and serotonin-ir synaptic terminals distribution in caudal NA, we examined the relationship between SP-ir terminals and laryngeal motoneuron using electron microscopy. In one case, an inspiratory laryngeal motoneuron (ILM) was identified by intracellular recording, stimulation of the cervical vagus nerve, and its location in the caudal nucleus ambiguus. The neuron was filled with biotinamide and then stained to reveal, simultaneously, SP and the neuron by pre-embedding immunohistochemistry. Ultrathin sections containing laryngeal motoneurons and SP-ir structures were cut, stained, and observed under the transmission electron microscope.Results:We find that the proportions of SP, TH and serotonin-ir terminal densities relative to the total nerve terminal densities in caudal NA is no greater than 10% for each neurochemical, and less than 15% in total. SP-ir terminals appeared relatively higher density compared with TH-ir and serotonin-ir terminals in caudal NA. At the ultrastructural level,53.3% (114/206) of varicosities formed asymmetric synapses and 22.3%(46/206) formed symmetric synapses on the dendrites of ILM; the remaining varicosities formed contacts but without clear synaptic specializations. Thirty-three of the 206 varicosities observed were SP-ir (16%). Of the 33 SP-ir terminals that formed synapses on proximal dendrites of ILM,29 were asymmetric, and 4 symmetric. A few SP-ir terminals were observed to form synapses with ILM’s somata but none observed on axon. Non-immunoreactive synapses also occurred on dendritic spines, some with sub-synaptic specializations. Several large (～30×40μm), ambigual non-immunoreactive neurons were observed in the section where ILM was identified, which contain oval, triangular or polygonal somata with well-developed organelles, a large round homogeneous nucleus, and a prominent nucleolus.Conclusions:Our results demonstrate first, using fluorescence microscopy, which SP, TH and serotonin-ir terminals are few in number compared with the total number of SYN-ir terminals in caudal NA, suggesting that SP, TH, and serotonin-ir inputs play only a modest role in the control of LMN function. Secondly, we confirmed that SP-ir terminals make ultrastructurally identifiable synapses onto ILM. A single ILM, that was activated by stimulation of the cervical vagus nerve, and which was located in the caudal nucleus ambiguus, was examined ultrastructurally. This labelled ILM was found to receive many symmetric and asymmetric synapses. Thirty-three of the SP-ir terminals were found to form synapses with ILM dendrites. Among them, most (29) were asymmetric, and a few (4) were symmetric synapses, suggesting that SP positive neurons may directly enhance the excitability of ILM, through synaptic inputs onto proximal dendrites. Thirdly, we examined the general synaptology of large neurons in the area of the caudal NA. We find that large numbers of symmetric and asymmetric synapses are present on these neurons. Objective:The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviours such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus (NA) in the modulation of laryngeal motoneurons (LMN) activity. The present study aimed to investigate the anatomical nature of cholinergic input to expiratory (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus (AmbL).Methods:We applied in vivo intracellular recording, dye filling combined with immunohistochemistry to identify the relationship between the LMN and cholinergic immunoreactive terminals. Using fluorescence confocal microscopy and the colocalization analysis with the immunoreactivity of synaptic marker, synaptophysin (SYN), we further identified the proportion of close appositions of cholinergic-ir terminals on labelled LMN that also expressed SYN, that is, cholinergic-ir synaptic terminals.Results:We demonstrate that LMN received close appositions from vesicular acetylcholine transporter-immunoreactive (VAChT-ir) boutons. There are 9 ELMs that were identified by iintracellular recording and revealed by Neurobiotin injection. Among them, ELMs receive a significantly great number of close appositions (mean±SD:32±9; n=5), and most of the close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two-way ANOVA, P<0.0001). Fluorescence confocal microscopic study demonstrated that almost 90% of VAChT-ir close appositions (n=45 boutons on n=4 ELMs) were colocalized with the synaptic marker synaptophysin.Conclusions:The novel findings of the present study are that ELMs, functionally identified in vivo in the rat, are closely apposed by VAChT-ir (cholinergic) boutons over their entire somatodendritic tree. ELMs received many VAChT-ir close appositions which mainly were found on somata and proximal dendrites when compared to more distal regions. In this study, we investigated whether close appositions represent synapses by combining intracellular recording and dye labelling with fluorescence confocal microscopy using an antibody to a synaptic protein. We demonstrate that many, but not all, close appositions between VAChT-ir boutons and labelled LMN cell somata are also SYN-ir. In conclusion, our founding provides anatomical evidence for a potentially strong influence of cholinergic input on LMN behaviour. The nature of cholinergic receptor expression in and the origin of cholinergic input to, functionally characterized LMN remain to be resolved.