Role Of Glial Cells In The Effects Of Doxapram On The Respiratory Rhythmical Discharge Activity Of Brainstem Slices And Expressing Of GlyT1, GDNF In The Medial Area Of Nucleus Retrofacialis

In 1986, we proved that the medial area of nucleus retrofacialis (mNRF) may be the core site which can generate the basic rhythmical respiration in brainstem in mammals. In 1991,Smith demonstrated that the pre-Botzinger complex (pre-BotC, PBC) is the crucial site in generating the rhythmical respiration. Though the two sites are all located in the ventrolateral of medulla, they overlaping each other. Now it is generally believed that the respiratory center is located in the rostral ventrolateral medulla (RVLM).There are two hypothesis for the mechanism of respiratory rhythmogenesis, pacemaker neuron hypothesis and neurons network hypothesis. Pacemaker neuron hypothesis is supported by majority of the present findings. We had found Expiratory-Inspiratory phase spanning (E-I PS) neuron characteristic of pacemaker on mNRF in vivo and in vitro in neonate rats. The type of E-I PS neurons begin to discharge before inspiration, burst to initiation inspiration by constant frequency, continue and terminate simultaneously with discharge of inspiration. The type of E-I PS neurons may be the pacemaker neurons.In the subsequent studies, we found that many respiratory neurons, neurotransmitters, receptors in mNRF are all involved in the generating and modulating of the the respiratory rhythmical discharge activity (RRDA) in brainstem slice.As a safe, effective respiratory stimulant, doxapram has been used to treat some central respiratory failure diseases. It is generally believed that the small doseage (0.05-0.25 mg/kg) of doxapram mainly action upon the glomus cells (Type I cells) in carotid bodies and larger dosage can excit both respiratory and non-respiratory medullary neurons. It stimulates the release of calcium (Ca2+)-dependent neurotransmitters from Type I cells by inhibiting a baseline membrane K+ conductance. The precise mechanism of the central effects, whether the glial cells are involved in the exciting procedure are still unclear.It is generally believed that the role of glial cell is a supporter in central nervous system (CNS) due to that the glial cell is an inactive one. Now we know that this is not true.Researches have proved that the glial cell have multiple functions in CNS. Firstly, the glial cells are located surrounding the synapses of neurons. Secondly, the glial cells can synthesise and secrete sort of neurotransmitters, express receptors, construct the carriers, uptake and transport the neurotransmitters in the synaptic cleft. Many functions and receptors expressing are both manifested in glial cells and neurons. The glial cells take a key role in modulation of CNS. The role of them in the generating and modulating in central respiration is still little known.Applying the fluoroacetate or methionine sulfoximine (MS), blockage of the tricarboxylic acid cycle (TAC), to mammals or the brainstem slices, the rhythmical respiration will be depressed. When the substrate of TAC isocitrate or glutamine was administrated, the RRDA would be fully recovery. It showed that the normal metabolism of the glial cells is important in the generating and modulating of rhythmical respiration. In the modulating precedure of glial cells, it is still unknown that:1)Changes of electrophysiological characteristics of respiratory neurons.2) The mRNA expressing of glial cell receptors and transmitters in mNRF.The aims of this study are:1)Make the brainstem slice preparations containing the mNRF from neonate rats, investigating the effects of doxapram and propofol on RRDA by using suction electrode from the hypoglossal nerve (Ⅻ) root, and the role of normal metabolism of glial cells in the modulating procedure.2) Investigating the changes of RRDA when the normal metabolism of glial cells were disturbed, further studies including the changes of electrophysiology characteristics of respiratory neurons by using extracellular recording technique.3) Using Real-time reverse transcription quantitative polymerase chain reaction (Real-time RT-PCR) to investigate the expressing of glial glycine transporter 1 (Gly T1) and glial cell-line derived neurotrophic factor (GDNF).Experiment 1:Effects of doxapram on the respiratory rhythmical discharge activity in the brainstem slice of neonatal ratsTo investigate the effects of doxapram on the respiratory rhythmical discharge activity (RRDA) in brainstem slices of neonatal rats.Experiments were performed on in vitro neonatal rat brainstem slices which contain the medial region of the nucleus retrofacialis (mNRF). The slices were perfused with modified Kreb's solution (MKS), and the RRDA in the hypoglossal nerve was recorded by suction electrode. Thirty Sprague-Dawle neonatal rats (of either sex,0-3 d old) were randomly divided into 6 equal groups:group I (control group):in which preparations were perfused with MKS only. GroupⅡtoⅣ(doxapram groups):preparations were perfused continuously with different concentrations of doxapram(2,5,10μmol/L) in the MKS respectively. Group V(propofol group):were perfused with propofol (20μmol/L). Group VI(propofol-doxapram group):were perfused with propofol (20μmol/L) plus doxapram (5μmol/L) continuously. The discharge time course of inspiratory (TI), expiratory (TE), respiratory cycle (RC) and intergrity amplitude of inspiratory discharge (IA) were recorded at 1,3,5,10,15,30 min after the drugs were applied. In control group, groupⅡand groupⅥ, there was no significant RRDA change in the whole course of the experiment (P>0.05). In groupsⅢandⅣ, the TI, IA were increased significantly (P<0.05), the TE reduced significantly after doxapram were applied at 5 min (P<0.05), the RC was shortened at 10 min in groupⅣonly. In group V,the TI and IA were reduced, and the RC, TE were increased significantly (P<0.05).The results suggest that doxapram(>5μmol/L) can directly stimulate the RRDA and can prevent propofol-induced (20μmol/L) depression effects in brainstem slice of neonatal rats. Actions upon the inspiratory neurons in mNRF mediate the major effects of doxapram.In order to investigate the changes of electrophysiological characteristics of respiratory neurons, whether glial cells are involved in the exciting procedure, we performed the following experiments.Experiment 2:The role of glial cells in the exciting effects of doxapram on brainstem slice in vitroPart 1:Effects of normal metabolism of glial cells on RRDA of brainstem slices in vitro This study tested whether the glial cells are involved in the exciting effects of doxapram on brainstem slice in vitro. Experiments were performed in brainstem slice preparations from neonatal rats. The medial area of nucleus retrofacialis (mNRF) and the hypoglossal nerve (Ⅻnerve) were contained in the preparations. The slices were perfused with modified Kreb's solution (MKS), and the rhythmical respiratory discharge activity (RRDA) was simultaneously recorded from theⅫnerve by using suction electrodes, including the discharge time course of inspiratory (Ti), expiratory (Te), respiratory cycle (RC) and intergrity amplitude of inspiratory discharge (IA). After applying of doxapram (5μM) to the MKS for 10 min, Ti and IA increased significantly (85.0±25.0%,13.2±2.5% respectively, P<0.05), the Te and the RC decreased significantly(19.0±1.4%,12.8±1.4% respectively, P<0.05) when compared with control group. When the methionine sulfoximine (MS,10μM),a blockage of glutamine synthetase was applied, all the exciting effects of doxapram on RRDA were reversed. After the glutamine (20μM) was applied to the MKS for 10 min, the exciting effects were revealed again. Our results suggest that the normal metabolism of glial cells takes a key role in the modification of the RRDA in the slices. In conclusion, glial cells are involved in the exciting effects of doxapram on brainstem slice in vitro.In order to investigate the changes of electrophysiological characteristics of respiratory neurons in the exciting procedure, we continue the following experiment.Part 2:Effects of normal metabolism of glial cells on RRDA of I-neurons in mNRFBrainstem slice preparations were made from 5 neonatal rats (SD), after the RRDA of theⅫrootlets were recorded stabilized, simultaneous extracellular recording technique was performed by using the glass microelectrode (filled with 0.5 mol/L CH3COONa) fixed by the micro-manipulator. Group 1 was perfused with 50μmol/L MS for 20 min. After the MS has been washout and the RRDA recovery, the slices were perfused with 30μmol/L glutamine for 20 min, the RRDA of I-neurons in mNRF was recorded simultaneously.After applying 50μmol/L MS to MKS for 10 min, the RC and TE of the I-neurons increased by 48.13% and 53.69% respectively (P<0.01),the TI, IA and PFn (the peak discharge frequency) decreased by 25.88%,27.14%,41.89% respectively (P<0.01).After washout and perfused with 30μmol/L Glutamine for 10 min, the RC, TE decreased 37.70%,65.76% respectively (P<0.01),no significant change of TI, IA and PFn (P>0.05), which induce the depressing effects on the RRDA of the brainstem slices.The results suggest that:1)The metabolism of glial cells are involved in the modulation of basic rhythmical respiration in the slices.2) Disturbing the glial cells metabolism may be induced the input of inhibitory synapse.The glial glycine transporter 1(Gly T1)and the glial cell-line derived neurotrophic factor (GDNF) were proposed to be involved in the modulating peocedures. Whether are they expressed in the mNRF? The following study is aim to answer this question.Experiment 3 The expression of mRNA of Gly T1 and GDNF gene in mNRF "island".To investigate the expression of mRNA of Gly T1 and GDNF gene in mNRF "island", we used Real-time RT-PCR (reverse transcriptase PCR) technique.1 The expressing of Gly T1 in mNRF: Ten SD neonatal rats (SPF class) were randomly divided into two groups (n=5), the mNRF "Island" were isolated from the brainstem slices. Brain tissue surrounding the nucleus of trigeminal nerve were used as control because the Gly T1 and GDNF were richly expressed in this area. The upstream primer was 5'- AGA TGA TGC TGG GGT TCC CAC-3',downstream primer was:5'-CGC CGA AGA CCA CTC CCTC-3'.The length of the amplification fragment was 229 bp; The control gene wasβ-actin of rats, The upstream primer was 5'-AGC CAT GTA CGT AGC CAT CC-3', downstream primer was:5'-CTC TCA GCT GTG GTG GTG AA-3',The length of the amplification fragment was 228 bp.2 The expressing of GDNF in mNRF:The making prearations and groups were similar to Gly T1 experiment. The upstream primer was 5'-AGA TGA TGC TGG GGT TCC CAC-3',downstream primer was:5'-CGC CGA AGA CCA CTC CCT C-3'.The results suggest that mRNA of Gly T1 and GDNF were both highly expressed in mNRF "island" and the quantity of expression were 4.08±3.08 (t=5.112, P=0.000),2.25±2.47 (t=3.532,P=0.003) times of that of the brain tissues surrounding to the nuclei of trigeminal nerve, where the Gly T1 and GDNF expressed plentifully according to the literatures reported. The results would support the hypothesis that the Gly T1,GDNF are involved in the generating and modulating of the rhythmical respiration.Conclusions1)Doxapram (>5μmol/L) can directly stimulats the RRDA and can prevent propofol-induced (20μ/L) depression effects in brainstem slice of neonatal rats. Actions upon the inspiratory neurons in mNRF mediates the major effects of doxapram.2) The metabolism of glial cells takes a key role in the modulation of basic rhythmical respiration in the slices, and involved in the exciting effects of doxapram on brainstem slice in vitro.3) Disturbing the glial cells metabolism may induced the input of inhibitory synapse, which induce the depressing effects on the RRDA of the brainstem slices.4) The mRNA of Gly T1 and GDNF were both highly expressed in mNRF, these may be the important neurotransmitters of glial cells in the generation and modulation of central respiration.