The Mechanism Of Endomorphin-2’s Respiratory Depression Function On The Pre-b(?)tzinger Complex

In mammals, breath is controlled by neural circuits existed in the central nerve system(CNS). The main nucleus related to respiration serially arrayed in brainstem. They interactwith each other and extend from the pons to the lower medulla. The key circuitcomponents generating respiratory rhythm and shaping inspiratory and expiratory motorpatterns are distributed among three structures in the ventrolateral medulla: the B(?)tzingercomplex (B(?)tC), Pre-B(?)tzinger complex (Pre-B(?)tC) and rostral ventral respiratory group(rVRG).The Pre-B(?)tC has intrinsic rhythm properties neurons, so it is recognized as the centerof respiratory rhythm generation. It lies ventral to medulla oblongata, ventral to thenucleus ambiguous and in reticular nucleus. The generation of respiratory rhythm andregulation are depended by multiple drives from more upper brainstem components,including the rostral ventral respiratory group, the retrotrapezoid nucleus and pons. Theycommonly control dynamic characteristics of respiratory network. The emerging view isthat the brainstem respiratory network has rhythmogenic capabilities in multiple levels. Itcan allow fexible, state-dependent expression of different rhythmogenic mechanismsunder different conditions.Respiratory rhythm is modulated by many neurotransmitters, including inhibitory and excitatory neurotransmitter. The principal excitatory neurotransmitters include glutamate(Glu). Glu primarily generate respiratory rhythm, it also involves in the transmission ofrespiratory rhythm to the phrenic motor neurons. The inhibitory transmitters include γ-aminobutyric acid (GABA) and glycine (Gly). GABA, as well as Gly, transmits phasicwaves of inhibition. In addition to the above classic neural active substances mentioned,5-hydroxtryptamine (5-HT) has also been implicated in regulating respiratory rhythm.Substance P (SP) largely augments respiration, by increasing respiratory rhythm. Opioidsare known to decrease respiration.Endomorphin (EM) are the endogenous opioid peptides which are found in1997.They are divided into two subtypes: Endomorphin1(EM1) and Endomorphin1(EM2).EM1is widely and intensively distributed in the brain and the upper brain stem. Itinvolves in pain information processing, cognitive, stress and other functions. EM2ismainly distributed in the spinal cord and brain stem, involving in pain transmission,regulation of respiratory depression and other side effects. They have amazing affinity andselectivity for the μ-opoid receptor (MOR). They have potent analgesic activity likeopioids. However, respiratory depression is one of the bad effects of EM. It limits theirclinical application. In order to exert EM’s potent analgesic activit, it is important toexpore its side-effect of respiratory depression. By using modern neuroscience methods,we study on the EM’s respiratory depression function in the following aspects:1. Are there some EM1or EM2-like immunoreactive structures in the Pre-B(?)tC? Aretheir distribution located in the presynaptic terminal or postsynaptic dendrites and cellin the body?2. If the EM positive structures exist in the presynaptic terminal, where do they comefrom? What do their property? If they have synaptic connections with each type ofneurons in the Pre-B(?)tC?3. What are the function of EM on the respiratory circuits? Through what mechanism?And what are the relationship between EM and other neurotransmitters?Results:1. We used the method of immunofluorescence and3,3’-diaminobenzidine (DAB) staining to observe there were a large number of EM2-like immunoreactive fibers andaxon terminals in the Pre-B(?)tC, but there were no EM1-like immunoreactive fibersand axon terminals, and no EM1、2-like immunoreactive neuronal body.2. Immunofluorescence histochemistry was combined with fluorescent retrograde tract-tracing methods, we found that EM2-like immunoreactive fibers and axon terminals inthe Pre-B(?)tC originated from the ipsilateral nucleus of the solitary tract (NTS).Statistical results showed that the percentage of EM2/FG double-labeled neurons toFG-labeled neurons was8%. The percentage of EM2/FG double-labeled neuron toEM2-labeled neurons was16.7%. Immunofluorescence double staining showed thatsome of these EM2-like immunoreactive fibers were colocalized with SP, and rarelywith glutamic acid decarboxylase (GAD) or vesicular glutamate transporter2(VGLUT2).3. Neurokinin-1receptor (NK1R)-positive neurons are a marker of the Pre-B(?)tC. Underthe laser confocal microscopy, immunofluorescence multiple staining results showedthat some of the EM2-like immunoreactive fibers and axon terminals in the Pre-B(?)tCwere closely apposed to the membrane of NK1R-or MOR-like immunoreactivesomata and dendrites. Some NK1R-positive neurons were also MOR-positive. EM2and SP-, GAD-or VGLUT2-like immunoreactive axon terminals were closelyapposed to the membrane of NK1R-like immunoreactive somata and dendrites. Someof these EM2-like immunoreactive fibers were colocalized with SP, and rarely withGAD or VGLUT2.4. Pre-embedding immune electron microscopy revealed that EM2positive axon terminalin DAB product evenly dispersed in the cytoplasm, mitochondria outer membrane andsynaptic vesicles on the outer membrane. Nanogold-labeled NK1R or MOR-likeimmumnoreactivities were detected by the presence of black round or oval particleswith high electron densities underneath the plasma membrane of the cell bodies andlarge dendritic processes of neurons in the Pre-B(?)tC. Symmetric synapses were mainlyfound between EM2-like immunoreactive terminals and NK1R-like immunoreactivePre-B(?)tC neurons. The percentage of symmetric synapses to total synapses was64%. And symmetric synapses were also found between EM2-like immunoreactiveterminals and MOR-like immunoreactive preB(?)tC neurons. The percentage ofsymmetric synapses to total synapses was79%.5. In view of the above morphological study, we further used the method of function toexplore possible respiratory depression function and its mechanism. Diaphragm recordresults in vivo showed that EM2induced respiratory depression byintracerebroventricular injection, however, there was no effect on breathing activity byEM2+-funaltrexamine (β-FNA) or D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2(CTOP) microinjection. It showed that EM2might be through MOR to exertrespiratory depression.0.5μl100μM Tyr-DAla-Gly-MePhe-Gly-ol （DAMGO） wasunilateral microinjected into Pre-B(?)tC, and breathing frequency (fB) decreased from79.7±1.5breath/min to65.5±1.5breath/min (17.8±2.1%decrease). Peak activitydecreased from24.9±1.8arbitrary units to19.5±1.5arbitrary units (21.6±1.4%decrease). No changes of fBand peak activity were observed after β-FNAmicroinjection. Microinjections of a10mM concentration of EM2(0.5μl, unilateral)into the Pre-B(?)tC produced a reduction in both fBand peak activity. fBdecreased from84.7±1.7breath/min to69±1.9breath/min (18.5±2.3%decrease). Peak activitydecreased from25.9±1.8arbitrary units to18.5±1.5arbitrary units (28.5±1.7%decrease). EM2was administrated followed β-FNA injection at the same site. EM2-induced reduction of breathing activity was no longer observed. It further proved thatEM2’s respiratory inhibition was likely to act on MOR-like immunoreactive Pre-B(?)tCneurons. At the same time, EM2could strengthen or weaken the otherneurotransmitters on fBand peak activity, influencing respiratory regulation.Microinjections of a2mM concentration of SP (0.5μl, unilateral) into the Pre-B(?)tCproduced an increase in both fBand peak activity. fBincreased from80.5±1.2breath/min to93.7±2.1breath/min (16.4±2.1%increase). Peak activity increasedfrom24.7±1.6arbitrary units to33.5±1.7arbitrary units (35.6±1.5%increase),then microinjections of EM2could weaken SP-induced increase of fBand peak activity.Microinjections of a100μM concentration of GABA (0.5μl, unilateral) into the Pre- B(?)tC produced a reduction in both fBand peak activity. fBdecreased from82.5±1.4breath/min to57±2.1breath/min (30.9±1.6%decrease). Peak activity decreasedfrom26.2±1.8arbitrary units to17.3±1.7arbitrary units (33.9±1.3%decrease),then microinjections of EM2could strengthen GABA-induced decrease of fBand peakactivity. Microinjections of a5mM concentration of L-glutamate (L-Glu,0.5μl,unilateral) into the Pre-B(?)tC produced an increase in both fBand peak activity. fBincreased from80.6±1.4breath/min to91.4±1.6breath/min (13.4±1.9%increase).Peak activity increased from24.4±1.8arbitrary units to30.5±1.2arbitrary units (25±1.2%increase), then microinjections of EM2could weaken L-Glu-induced increaseof fBand peak activity.Through the above results, we got the following conclusion:（1） There were a large number of EM2-like immunoreactive fibers and axonterminals in the Pre-B(?)tC, some of the EM2-like immunoreactive fibers and axonterminals in the Pre-B(?)tC were closely apposed to the membrane of MOR-likeimmunoreactive somata and dendrites.（2） EM2-like immunoreactive fibers and axon terminals in the Pre-B(?)tC mightoriginate from the ipsilateral NTS, and its nature might be peptidergic fiber.（3） The mechanism of EM2’ s respiratory depression was likely to act on MOR-likeimmunoreactive Pre-B(?)tC neurons.In a word, the present results suggested that EM2-like immunoreactive neuronalbody in the NTS sent axon to the Pre-B(?)tC. In the Pre-B(?)tC, EM2-likeimmunoreactive axon terminals exerted its function through MOR, and might bethrough postsynaptic inhibition. At the same time, EM2could strengthen or weakenthe other neurotransmitters on breathing frequency and peak activity, influencingrespiratory regulation.