Morphological Evidence Of Pre-b(?)tC D?H-ir Nerve Fibers Regulate Respiration In VLM Of Rats

ObjectivePre-B?tzinger complex(pre-B?tC) is the key part of the respiratory rhythm generation. Its position largely overlap with catecholaminergic C1 neurons' that closely related with cardiovascular function in the ventrolateral medulla(VLM). Previous study shown that the activation of catecholaminergic neurons can promote the occurrence of respiratory rhythm and enhance the excitability of respiratory and cardiovascular activity. This implies that C1 and pre-B?tC neurons may transmit neurotransmitter during respiratory activity and participate the regulation of respiratory together. However, there is no morphological evidence at present. Pre-B?tC neurons can express high level neurokinin 1 receptor(NK1R). More than 80% of the bilateral NK1 R immunoreactivity(ir) positive neurons were damaged at the same time, which could seriously interfere the normal respiratory rhythm and lead to blood gas abnormalities. So in this experiment, we apply NK1 R as the marker of pre-B?tC to lable the respiratory neurons. C1 cell group is double phenotypes neurons with catecholamine and glutamate(Glu), which distribute in the VLM and involve in cardiovascular sympathetic nervous regulation, as well as the respiratory-sympathetic stress sensitivity regulation in the pathological condition such as hypoxia. It has been shown that norepinephrine(NE) can affect the respiratory activity via the ?1 adrenoreceptor(AR) in the postsynaptic membrane. Dopamine beta hydroxylase(D?H) is a key enzyme during the synthesis of NE, also be used to reflect the changing of NE. D?H-ir positive can determine not only the nature and distribution of noradrenergic neurons but also D?H level as well as quantity and changing of NE neurons according to the amount and dye of immunoreactive products. Therefore, we apply D?H as a specific marker of C1 neurons, and investigate the synaptic phenotypes between D?H-ir positive cardiovascular-related C1 neurons and NK1R-ir positive pre-B?tzinger respiratory-related neurons. From the point of morphology, this article describes C1 neurons regulatory effect on respiratory.MethodsOwing to the routine pre-embedding immunoelectron microscopy, we found that the methods only label few numbers of D?H-ir positive neurons and the processes are sparse. Considering the difference of immune markers about same antibody to apply with light and electron microscopy, the results may be related with different thickness of slice and the ability of the antibody penetration for different tissues. Therefore, we used different times with liquid nitrogen freezing and different concentration of Triton X-100 to treat slices, compared with different effect of methods on the catecholamine products in A1/C1 of rat. Basis on the pre-experiment, immunofluorescence histochemistry was used to reveal immunoreactivities of D?H and NK1 R in the VLM of co-localization. The distribution and synaptic phenotypes of neurons were examined with double-labeling immunoelectronic microscopy.ResultsUnder the light microscope of the pre-experiment, D?H-ir product distribute in somas and processes of neurons, clearly outlining C1 neurons. D?H-ir neurons are distinctly different in the number and the shape in freeze-thaw groups as compared to Triton X-100 groups, especially in 0.05 % Triton X-100 groups that neurons show dense and long processes, whereas short and thin in freeze-thaw groups. Electron microscopy results also showed gold particles of D?H immunoreactivity were localized to somas, axonal terminals in C1 neurons. D?H-ir terminals were found in contacts with somas and dendrites. Asymmetric synaptic contacts were visualized between them. Immunofluorescence of the experiment showed that D?H-ir neurons were distributed in ventrolateral area of NK1R-ir neurons groups and some of them overlapped and were intermingled in close associations. The close contact located between the processes of D?H-ir positive and the processes and neurons of NK1R-ir positive. The immunoreactivity of D?H and NK1 R was not co-localized. The golden particles indicative of D?H immunoreactivity were distributed in somas, dendrites and axon terminals in the pre-B?tC. The density of immunogold particles was various among different profiles, as that the most dense particles were found in terminals. D?H-ir terminals contained small lucent spherical synaptic vesicles, which intermingled with the dense immunogold particles. Immunoperoxidase reaction product indicative of NK1 R immunoreactivity was localized mainly along the inner surface of the plasma membrane of somas, dendrites, and in the cytoplasm of the pre-B?tC neurons. D?H-ir terminals formed asymmetric synapses, and occasionally, symmetric synapses in the pre-B?tC, featuring the local circuitry. D?H-ir terminals were visualized in contacts with NK1R-ir dendrites. Asymmetric synapses were identified between them, which represent excitatory transmission of glutaminergic neurotransmitter. Symmetric synapses usually receive inhibitory neurotransmitter ?-aminobutyric acid(GABA) regulation. It is suggests that the C1 neurons were mainly transmitted excitatory Glu and the inhibition of synapse played a very important role in the regulation of respiratory rhythm. Under the electron microscope observed some asymmetric synapses between D?H-ir axon terminals and NK1R-ir neurons, especially with small fusiform NK1R-ir positive neurons, generating respiratory rhythm pacemaker neurons, formed synaptic connections. It showes that the generation of respiratory rhythm also receive the regulation of catecholaminergic nerve fibers. This is the first evidence that record the characteristic of synapses connecting the cardiovascular-related catecholaminergic C1 neurons and respiratory-related pre-B?tC neurons. This study provides structural evidence supporting the functional connection of C1 neurons in respiratory regulation in the VLM. Close appositions between NK1R-ir processes and D?H-ir somas or dendrites identified by immunofluorescent analysis were complemented with the result of electron microscope, showing that NK1R-ir dendrites could not be in direct contact with D?H-ir somas and dendrites, and it was separated by membranes between them. Paracrine may be contributed to the transmission of cardiorespiratory activities.ConclusionGold particles of the pre-experiment were much denser in D?H-ir neurons in Triton X-100 groups than those in freeze-thaw groups, being in consistence with the light microscope observation. Asymmetic synapses are predominant in the integration of information between C1 and pre-B?tC neurons in the VLM, elaborating excitatory transmission driving the coupling of cardiorespiratory activities in the experiment.