Expressions of glial glycine transporter 1 and glial cell line-derived neurotrophic factor in the medial area of nucleus retrofacialis / 中华神经医学杂志

Objective To investigate the mRNA expressions of glial glycine transporter 1 (GlyT1) and glial cell line-derived neurotrophic factor (GDNF) genes in the medial area of nucleus retrofacialis (mNRF).Methods Ten neonatal SD rats (aged 0-3 d) were randomly divided into control and experimental groups (n=5); the mNRF "islands" were isolated from the brainstem slices in rats of the experimental group,while trigeminal nucleus surrounding brain tissues were chosen in rats of the control group.GlyT1 and GDNF gene expressions were detected using real time quantitative PCR (RT-PCR).Results The expressions of GlyT1 and GDNF genes in the mNRF "island" were both significantly higher than those in the controls (t=5.112,P=0.000; t=3.532,P=0.003).Conclusion ThemRNA expressions of GlyT1 and GDNF genes are high in the mNRF; however,the roles of them in generation and modulation in rhythmical respiration and the mechanisms between neurons and both GlyT1 and GDNF should be further studied.

Involvement of cAMP-PKA pathway in group Ⅱ metabotropic glutamate receptors-mediated regulation of respiratory rhythm from neonatal rat brainstem slice / 生理学报

The study aims to identify the role of cAMP-PKA pathway in the group Ⅱ metabotropic glutamate receptors (mGluRs)-mediated regulation of respiratory rhythm from the brainstem slice. Neonatal (aged 0-3 d) Sprague-Dawley rats of either sex were used. The brainstem slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared, and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O2 and 5% CO2) bubbling, and ended in 3 min. Respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve rootlets was recorded by suction electrode. Eighteen brainstem slice preparations were divided into 3 groups. In group 1, group Ⅱ mGluRs specific antagonist (2S)-α-ethylglutamic acid (EGLU) was added into the perfusion solution for 10 min. In group 2, after application of Forskolin for 10 min, washout with MKS, the slice was perfused with Rp-cyclic 3', 5'-hydrogen phosphorothioate adenosine triethylammonium salt (Rp-cAMPS) alone for another 10 min. In group 3, after application of Rp-cAMPS for 10 min, additional EGLU was added into the perfusion for another 10 min. The results showed EGLU shortened respiratory cycle (RC), but the changes of integral amplitude (IA) and inspiratory time (TI) were not statistically significant. Forskolin induced significant decreases in RC, and increased TI, IA. Rp-cAMPS could make the opposite effect compared with the changes of RRDA with Forskolin. The effect of EGLU on the RRDA was inhibited after blocking the cAMP-PKA pathway. Taken together, cAMP-PKA pathway may play an important role in the group Ⅱ mGluRs-mediated regulation of RRDA in the brainstem slice of neonatal rats.

Effect of doxapram on the respiratory rhythmical discharge activity in the brainstem slice of neonatal rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of doxapram on the respiratory rhythmical discharge activity (RRDA) in the brainstem slices of neonatal rats.</p><p><b>METHODS</b>Thirty neonatal SD rats (of either sex, 0-3 days old) were randomly divided into 6 equal groups (groups I-VI), and the brainstem slices which contained the medial region of the nucleus retrofacialis (mNRF) were prepared. All the slices were perfused with modified Kreb's solution (MKS), and in group I (control group), the slices were perfused with MKS only; in groups II to IV, the slices were perfused with doxapram in MKS continuously at the concentrations of 2, 5, and 10 micromol/L, respectively; in groups V and VI, the slices were perfused with 20 micromol/L propofol and 20 micromol/L propofol plus 5 micromol/L doxapram, respectively. The RRDA in the hypoglossal nerve was recorded by suction electrode. The discharge time course of the inspiratory (TI), expiratory (TE), respiratory cycle (RC) and integral amplitude of the inspiratory discharge (IA) were recorded at 1, 3, 5, 10, 15, and 30 min after the application of the drugs.</p><p><b>RESULTS</b>The hypoglossal nerve in groups I, II and VI showed no significant changes of RRDA in the entire course of the experiment (P>0.05). In groups III and IV, the TI, IA increased and TE decreased significantly 5 min after doxapram application (P<0.05), and the RC was shortened only at 10 min. In group V, the TI and IA decreased and the RC and TE increased significantly after the drug application (P<0.05).</p><p><b>CONCLUSION</b>Doxapram (>5 micromol/L ) can directly stimulate the RRDA and prevent propofol-induced inhibitory effects in the brainstem slice of neonatal rats, and the effects are mediated by its actions upon the inspiratory neurons in the mNRF.</p>

Histamine H1 receptors modulate the discharge activities of inspiratory neurons in the medial region of neonatal rat nucleus retrofacialis ex vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of histamine H1 receptors in modulating the discharge activities of the inspiratory neurons in the nucleus retrofacialis of neonatal rats.</p><p><b>METHODS</b>Brainstem slices were obtained from neonatal rats containing the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve (XII nerve) rootlets retained. The rhythmic discharges of the inspiratory neurons (I neurons) and activities of the XII nerve rootlets were simultaneously recorded using microelectrodes and suction electrodes, respectively. The role of H1 receptors in modulation of the discharge activities of the inspiratory neurons was investigated using the H1 receptor agonist histamine and its specific antagonist pyrilamine dissolved in modified Kreb's solution for slice perfusion.</p><p><b>RESULTS</b>Histamine shortened the respiratory cycle (RC) and expiratory time (TE) of the neurons in the brain slices, and pyrilamine produced the opposite effects. Neither histamine nor pyrilamine affected the inspiratory time (TI), integral amplitude (IA) or the peak discharge frequency (PF) of the I neurons.</p><p><b>CONCLUSION</b>H1 receptors play an excitatory role in the modulation of the discharge activities of the inspiratory neurons in neonatal rat brainstem slices.</p>

Effects of glycine on the discharge activities of the inspiratory neurons in neonatal rat medulla oblongata slices / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of glycine on the discharge activities of inspiratory neurons in the medulla oblongata slices of neonatal rats.</p><p><b>METHODS</b>Neonatal rat medulla oblongata slices including the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets retained were prepared. The activity of the inspiratory neurons in the mNRF and the respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve rootlets were simultaneously recorded using microelectrodes and suction electrodes, respectively. The effect of glycine on the rhythmical respiration was investigated by administration of glycine receptor agonist and its specific antagonist strychnine (STR) into a modified Kreb's perfusion solution (MKS).</p><p><b>RESULTS</b>Glycine significantly shortened the inspiratory time (TI) and integral amplitude (IA) of the inspiratory neurons and decreased the peak frequency (PFn). By contrast, STR induced a decrease in the expiratory time (TE) and respiratory cycle (RC) of the inspiratory neurons, but the peak frequency was not significantly affected.</p><p><b>CONCLUSION</b>Glycine and its receptor play a role in the modulation of the rhythmical respiration, and glycine is probably involved in the phase-switching between expiration and inspiration by affecting the inspiratory neurons.</p>

Group II metabotropic glutamate receptors is involved in the modulation of respiratory rhythmical discharge activity in neonatal rat medullary brain slices / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of group II metabotropic glutamate receptors in the modulation of basic respiratory rhythm.</p><p><b>METHODS</b>Neonatal (0-3 days) SD rats of either sex were used. The medulla oblongata brain slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared, and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O2 and 5% CO2) within 3 min. The brain slices were quickly transferred to a recording chamber and continuously perfused with oxygen-saturated MKS at a rate of 4-6 ml/min at 27-29 degrees celsius. Eighteen medulla oblongata slices were divided into 3 groups and treated for 10 min with group II metabotropic glutamate receptor-specific agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC) (at concentrations of 10, 20, 50 micromol/L), group II metabotropic glutamate receptor antagonist (2S)-alpha-ethylglutamic acid (EGLU) (300 micromol/L), or APDC (50 micromol/L)+EGLU (300 micromol/L) after a 10 min APDC (50 micromol/L) application. Respiratory rhythmical discharge activity (RRDA) of the rootlets of the hypoglossal nerve was recorded by suction electrodes.</p><p><b>RESULTS</b>APDC produced a dose-dependent inhibitory effect on the RRDA, prolonging the respiratory cycle and expiratory time and decreasing the integral amplitude and inspiratory time. EGLU induced a significant decrease in the respiratory cycle and expiratory time. The effect of APDC on the respiratory rhythm was partially reversed by the application of APDC+EGLU.</p><p><b>CONCLUSION</b>Group II metabotropic glutamate receptors are probably involved in the modulation of the RRDA in isolated neonatal rat brainstem slice.</p>

Effects of A68930 on rhythmical respiratory discharge in isolated neonatal rat brainstem slice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of dopamine-1 receptor in the modulation of basic respiration rhythm.</p><p><b>METHODS</b>Newborn SD rat (0-3 days, n=20) brain stem slices containing the medial region of the nucleus retrofacialis (mNRF) were prepared with the hypoglossal nerve roots retained. The respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve was recorded using suction electrodes on these preparations, and the effects of dopamine-1 receptor on RRDA were investigated by application of the specific agonist of dopamine-1 receptor A68930 at different concentrations (0, 1, 2, and 5 micromol/L) in the perfusion solution.</p><p><b>RESULTS</b>The respiratory cycles (RC) and the expiratory time (TE) decreased progressively with gradual increment of the integrated amplitude (IA) after A68930 administration, and their changes were the most conspicuous at 5 min after the administration. A68930 at the concentrations of 2 and 5 micromol/L resulted in the most obvious changes in RC, TE, and IA (P<0.05), but IA exhibited no significant variation at 1 min after perfusion with 2 micromol/L A68930 (P>0.05). RC and TE were gradually shortened after treatment with increasing concentrations of A68930, which also caused gradual increment of IA, and at the concentration of 5 micromol/L, RC, TE, and IA all showed the most obvious changes (P<0.01).</p><p><b>CONCLUSIONS</b>Dopamine-1 receptor plays a role in the modulation of RRDA in isolated neonatal rat brainstem slice. A68930 may increase the frequency of respiration by shortening TE and enhance the respiratory activity by increasing the amplitude of inspiratory discharge of the respiratory neurons.</p>

Effect of glial cell metabolism on respiratory rhythmical discharge activity in neonatal rat medulla oblongata slices / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of glial cell metabolism in the generation and regulation of central respiratory rhythm.</p><p><b>METHODS</b>The medulla oblongata slices (600-700 microm) containing the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets retained from 12 neonatal (0-3 days) Sprague-Dawley rats were prepared and perfused with modified Kreb's solution (MKS). Upon recording of respiratory rhythmical discharge activity (RRDA) of the rootlets of the hypoglossal nerve, the brain slices were treated with glial cell metabolism antagonist L-methionine sulfoximine (L-MSO, 50 micromol/L) for 20 min followed by application of glial cell metabolism agonist L-glutamine (L-GLN, 30 micromol/L) for 20 min, or with L-MSO for 20 min with additional L-GLN for 20 min. The changes in the RRDA of the rootlets of the hypoglossal nerve in response to the treatments were recorded.</p><p><b>RESULTS</b>L-MSO prolonged the respiratory cycle (RC) and expiratory time (TE), and reduced the integral amplitude (IA) and the inspiratory time (TI) in the brain slices. L-GLN induced a significant decrease in RC and TE, but IA and TI showed no obvious variations. The effect of L-MSO on the respiratory rhythm was reversed by the application of L-GLN.</p><p><b>CONCLUSION</b>Glial cell metabolism may play an important role in the modulation of RRDA in neonatal rat brainstem.</p>

Identification and classification of respiratory pacemaker neurons in the medial region of nucleus retrofacialis in neonatal rats / 生理学报

The aim of the present study is to supply direct experimental proof that the medial region of nucleus retrofacialis (mNRF) is the site generating basic rhythm of respiration. Medullary slices of neonatal Sprague-Dawley rats, including hypoglossal nerve root and mNRF, were made according to Suzue's method. Simultaneous recordings of the respiratory rhythmic discharge activity (RRDA) in hypoglossal nerve root with suction electrode and the respiratory neuronal discharge in the mNRF with whole cell patch clamp were performed on the brainstem slice in vitro. Not only the electrophysiological characteristics of pacemaker and non-pacemaker neurons, but the cadmium-sensitivity of pacemaker neurons was observed. The nature of the discharge in the respiratory pacemaker neurons in mNRF was spontaneous, rhythmical and voltage-dependent burst behavior, generating an ectopic burst in response to a depolarizing or hyperpolarizing current. There were no significant differences in capacitance of membrane (C(m)), input resistance of membrane (R(m)) and leak inward current (I(leak)) between the pacemaker neurons and the non-pacemaker ones. Moreover, most pacemaker neurons in the mNRF were Cd(2+)-insensitive. In conclusion, these results support the idea that the mNRF is the site generating basic respiratory rhythm.

GABA A receptor participates in respiratory enhancement induced by nikethamide in neonatal rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of GABA A receptor in nikethamide-induced respiratory enhancement in the medullary slices of neonatal rats.</p><p><b>METHODS</b>Ex vivo medullary slices of neonatal rats (1 to 3 days old) containing the medial region of the nucleus retrofacialis with the hypoglossal nerve rootlets were prepared and perfused with modified Kreb's solution to record respiration-related rhythmic discharge activity (RRDA) from the hypoglossal nerve rootlets using suction electrodes. Thirty RRDA-positive slices were randomized into 5 equal groups and perfused with nikethamide (at concentrations of 0.5, 1, 3, 5, 7, and 10 microg/ml with the optimal nikethamide concentration determined), GABA (at 10, 20, 40, and 60 micromol/ to determine the optimal concentration), 10 micromol/ bicuculline, 10 micromol/ bicuculline plus 40 micromol/L GABA, and 5 microg/ml nikethamide followed by 5 microg/ml nikethamide plus 10 micromol/ bicuculline after wash out, respectively.</p><p><b>RESULTS</b>Nikethamide increased RRDA at the concentrations of 0.5-7 microg/ml, and 5 microg/ml nikethamide showed the most distinct effect on the inspiratory time (TI), integral amplitude (IA), and respiratory cycle (RC). GABA at 40 micromol/ showed the most effective inhibition of RRDA in terms of TI, IA, and RC. Bicuculline at 10 micromol/ could increase the IA, TI and RC, but the combination of 10 micromol/ bicuculline and 40 micromol/ GABA had no significant effects on RRDA. Compared with nikethamide used alone, nikethamide plus bicuculline significantly increased TI and IA without affecting RC.</p><p><b>CONCLUSION</b>Nikethamide can enhance RRDA of the hypoglossal nerve rootlets in the medullary slices of neonatal rats, and the effect can be partially mediated by the GABA A receptor.</p>

Role of histamine H(1) and H(2) receptors in the modulation of respiratory rhythmical discharge in medulla oblongata slice preparation of neonatal rats / 生理学报

The present study was carried out to determine the role of histamine H(1) and H(2) receptors in the generation of basic respiratory rhythm. Neonatal (aged 0-3 d) Sprague-Dawley rats of either sex were used. The medulla oblongata slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O(2) and 5% CO(2)), and ended in 3 min. Respiratory rhythmical discharge activity (RRDA) of the rootlets of hypoglossal nerve was recorded by suction electrode. Thirty medulla oblongata slice preparations were divided into 5 groups. In groups I, II and III, histamine (5 μmol/L), H(1) receptor specific antagonist pyrilamine (10 μmol/L) and H(2) receptor specific antagonist cimetidine (5 μmol/L) was added into the perfusion solution for 15 min separately. In group IV, after application of histamine for 15 min, additional pyrilamine was added into the perfusion for another 15 min. In group V, after application of histamine for 15 min, additional cimetidine was added into the perfusion for another 15 min. The discharges of the roots of hypoglossal nerve were recorded. Signals were amplified and band-pass filtered (100-3.3 kHz). Data were sampled (1-10 kHz) and stored in the computer via BL-420 biological signal processing system. Our results showed that histamine significantly decreased the respiratory cycle (RC) and expiratory time (TE), but changes of integral amplitude (IA) and inspiratory time (TI) were not statistically significant. Pyrilamine induced significant increases in RC and TE, but changes of TI and IA were not statistically significant. Cimetidine had no effects on RC, TE, TI and IA of RRDA. The effect of histamine on the respiratory rhythm was reversed by additional application of pyrilamine but not cimetidine. Taken together, with the results mentioned above, histamine H(1) receptors but not H(2) receptors may play an important role in the modulation of RRDA in the medulla oblongata slice preparation of neonatal rats.

Role of 5-HT(2A) receptor in increase in respiratory-related rhythmic discharge activity by nikethamide in neonatal rat transverse medullary slices / 生理学报

To investigate the effects of nikethamide on the generation and modulation of rhythmic respiration of neonatal rats and the role of 5-HT(2A) receptor in this course, experiments were performed on the transverse medullary slices of neonatal rats (both sexes, 1-3 d) in vitro. The slices containing the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets were prepared in which the respiratory-related rhythmic discharge activity (RRDA) was recorded from the hypoglossal nerve rootlets by suction electrode. The possible role of nikethamide on RRDA was investigated by administration of an agonist of 5-HT(2A) receptor, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and an antagonist of 5-HT(2A) receptor, ketanserine, dissolved in modified Krebos solution (MKS). Thirty slices were randomly divided into five groups: Group 1: the slices were perfused with different concentrations of nikethamide (0.5, 1, 3, 5, 7, 10 μg/mL), and the most effective concentration was selected; Group 2: the slices were perfused with DOI (40 μmol/L); Group 3: the slices were perfused with ketanserine (40 μmol/L); Group 4: the slices were perfused with ketanserine + DOI; Group 5: the slices were perfused with nikethamide, then perfused with nikethamide + ketanserine after washout of nikethamide. Nikethamide increased RRDA in transverse medullary slices at 0.5-7 μg/mL, and 5 μg/mL was the most effective concentration. DOI increased RRDA with prolonged inspiratory time (TI), increased integral amplitude (IA), and shortened respiratory cycle (RC). Ketanserine decreased RRDA with shortened TI, decreased IA and prolonged RC. Ketanserine + DOI had no significant effects on RRDA. The effects of nikethamide on RC and IA were totally and partially reversed by additional application of ketanserine, but the effect of nikethamide on TI was not influenced by ketanserine. It is proposed that nikethamide increases RRDA partly via 5-HT(2A) receptors.

Glycine is involved in the modulation of respiratory rhythmical discharge activity in neonatal rat medullary brain slices / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the role of glycine (Gly) in the generation and modulation of basic respiratory rhythm.</p><p><b>METHODS</b>Neonatal (0-3 days) SD rats of either sex were used in this study. The medulla oblongata brain slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared, and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O(2) and 5% CO(2)) within 3 min. The rhythmical respiratory discharge activity (RRDA) of the hypoglossal nerve rootlets was recorded using suction electrode. Eighteen medulla oblongata slice preparations were divided into 3 groups and treated for 20 min with Gly receptor specific agonist Gly (10 micromol/L), Gly receptor antagonist strychnine (STR, 1 micromol/L), or Gly+STR after a 20 min Gly application. The changes in RRDA of the hypoglossal nerve rootlets were observed.</p><p><b>RESULTS</b>Gly significantly decreased the inspiratory time and integral amplitude (IA), but the changes of respiratory cycle (RC) and expiratory time (TE) were not statistically significant. STR induced a decrease in expiratory time and respiratory cycle without significantly affecting the inspiratory time or integral amplitud. The effect of Gly on the respiratory rhythm was partially reversed by additional application of STR.</p><p><b>CONCLUSION</b>Gly may play an important role in the modulation of RRDA in the medulla oblongata slice of neonatal rats.</p>

D(1)-dopamine receptor is involved in the modulation of the respiratory rhythmical discharge activity in the medulla oblongata slice preparation of neonatal rats in vitro / 生理学报

To explore the role of D(1)-dopamine receptor in the modulation of basic respiratory rhythm, neonatal (0-3 d) Sprague-Dawley rats of either sex were used. The medulla oblongata slice was prepared and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous ventilating 95% O2 and 5% CO2 and ended in 3 min. A 600-700 mum single transverse slice containing the hypoglossal nerve roots and some parts of the ventral respiratory group was cut. The preparation was quickly transferred to a recording chamber and continuously perfused with oxygen-saturated MKS at a rate of 4-6 mL/min at 27-29 degrees C. Ten medulla oblongata slice preparations were randomly divided into two groups. In group I, the preparations were perfused with perfusion solution containing D(1)-dopamine receptor specific agonist cis-(+/-)-1-(Aminomethyl)-3,4-dihydro-3-phenyl-1H-2-Benzopyran-5,6-Diolhy-drochlo-ride (A68930, 5 mumol/L) for 10 min first; after washing out, the preparations were then perfused with perfusion solution containing D(1)-dopamine receptor specific antagonist R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390, 2 mumol/L) for 10 min. In group II, after perfusion with A68930 for 10 min, the preparations were perfused with additional A68930 + SCH-23390 for 10 min. Respiratory rhythmical discharge activity (RRDA) of the rootlets of hypoglossal nerve was recorded by suction electrodes. The results showed that A68930 shortened the respiratory cycle (RC) and expiratory time (TE) with an increase in the integral amplitude (IA). However, SCH-23390 significantly prolonged RC and TE, and decreased IA with a decrease in inspiratory time (TI). Moreover, the effect of A68930 on the respiratory rhythm was partially reversed by additional application of A68930 + SCH-23390. These results indicate that D(1)-dopamine receptor is possibly involved in the modulation of the RRDA in the isolated neonatal rat brainstem slice.

Modulatory Effect of 5-Hydroxytryptamine 2A Receptor on Discharge Activities of Inspiratory Neurons in the Medial Region of Nucleus Retrofacialis of Neonatal Rats in Vitro / 实用儿科临床杂志

Objective To explore the modulatory effect of 5-HT2A receptors on the discharge activities of inspiratory neurons in medial region of nucleus retrofacialis of neonatal rats. Methods Experiments were performed in vitro brainstem slice preparations from neonatal rats. These preparations included the medial region of nucleus retrofacialis with the hypoglossal nerve rootlets retained. The rhythmic discharges of the inspiratory neurons and activities of the hypoglossal nerve rootlets were simultaneously recorded by using microelectrodes and suction electrodes, respectively. Roles of 5-HT2A receptors in modulation of the discharge activities of inspiratory neurons were investigated by administration of the 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodopbenyl)-2-aminopropane (DOI), and its specific antagonist ketanserine dissolved in modified Kreb's solution for perfused slices. Results In DOI group, the inspiratory time (TI) was (0.864±0.07)s, expiratory time (TE) was (10.78±1.06)s, respiratory cycle (RC) was (11.79±1.64)s, integral amplitude (IA) was (357.98±37.21)(μV·s) and the peak discharge frequency (PF) was (37.83±3.66)Hz. In control group, they were (0.68±0.06)s, (13.89±2.14)s, (14.77±1.92)s, (273.57±24.39)(μV·s), and (29.92±4.50)Hz, there were significant differences between the 2 groups (Pa<0.01). In ketanserine group, TI was (0.55±0.07)s, TE and RC were (18.43±3.28)s and (20.17+2.91)s respectively, IA and PF were (214.37±33.52)(μV·s) and (22.17±3.92)Hz, there were significant differences between ketanserine group and DOI, control group (Pa<0.01). Conclusion 5-HT2A receptors take part in modulate the discharge activities of inspiratory neurons in neonatal rat brainstem slices.

5-HT(1A) receptors are involved in the modulation of respiratory rhythmical discharge activity in the medulla oblongata slice preparation of neonatal rats / 生理学报

The present study was carried out to determine the role of 5-HT(1A) receptors in the generation and modulation of basic respiratory rhythm. Neonatal (aged 0-3 d) Sprague-Dawley rats of either sex were used. The medulla oblongata slice was prepared and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O2 and 5% CO2), and ended in 3 min. In cold MKS, a 600-700 microm single transverse slice was cut, which was rostral to the edge of area postrema and retained the hypoglossal nerve roots and some parts of the ventral respiratory group. The preparation was quickly transferred to a recording chamber and continuously perfused with carbogen-saturated MKS at a rate of 4-6 mL/min at 27-29 degrees C. Glass adsorb-electrodes containing Ag-AgCl needle were attached to the ventral roots of the hypoglossal nerve. Respiratory rhythmical discharge activity (RRDA) of the rootlets of hypoglossal nerve was recorded. Ten medulla oblongata slice preparations were divided into two groups. In group I, 5-HT(1A) receptor specific agonist (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide (8-OHDPAT, 20 micromol/L) was added into the perfusion solution for 10 min first, after washing out, the 5-HT(1A) antagonist [4-iodo-N-[2-[4-methoxyphenyl]-1-piperazinyl]ethyl]-N-2-pyridynyl-benzamide hydrochloride] (PMPPI, 10 micromol/L) was applied to the perfusion solution for 10 min. In group II, after application of 8-OHDPAT for 10 min, additional PMPPI was added into the perfusion solution for 10 min. The discharges of the rootlets of hypoglossal nerve were recorded. Signals were amplified and band-pass filtered (100-3.3 kHz). Data were sampled (1-10 kHz) and stored in the computer via BL-420 biological signal processing system. Our results showed that 8-OHDPAT increased the respiratory cycle (RC) and expiratory time (TE) as well as reduced the integral amplitude (IA), but the changes of the inspiratory time (TI) were not statistically significant. PMPPI induced a significant decrease in RC, TE and TI, but the changes of IA were not statistically significant. The effect of 8-OHDPAT on the respiratory rhythm was partially reversed by additional application of PMPPI. Taken together with previous results, 5-HT(1A) receptors may play an important role in the modulation of RRDA in the medulla oblongata slice preparation of neonatal rats.

Effects of 8-OH-DPAT on respiratory rhythmical discharge activity in isolated neonatal rat brainstem slice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of 5-HT1A receptors in the generation and modulation of basic respiration rhythm.</p><p><b>METHODS</b>Brainstem slices of 20 newborn SD rats (0-3 days) were prepared and respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve was recorded by suction electrode on these preparations including the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets retained, and the effects of 5-HT1A receptors on RRDA were investigated by application of specific agonist of 5-HT1A receptors 8-OH-DPAT in the perfusion solution. The 20 neonatal rats were divided into 4 groups and the brainstem slices were perfused continuously for 10 min with different concentrations of 8-OH-DPAT (1, 5, 10, 20 micromol/L, respectively). RRDA was recorded before and 1, 3, 5 min after 8-OH-DPAT perfusion.</p><p><b>RESULTS</b>The respiratory cycles (RC) varied significantly between the different time points of 8-OH-DPAT administration (F=181.219, P<0.001), which was the shortest before 8-OH-DPAT administration and increased progressively after administration till reaching the maximum 5 min after the administration. The RC also varied significantly between different 8-OH-DPAT concentrations (F=61.675, P<0.001). At each time point after 8-OH-DPAT administration, RC was the shortest with 1 micromol/L and maximum with 20 micromol/L 8-OH-DPAT. A crossover effect was observed between the time and administered 8-OH-DPAT concentration (F=22.940, P<0.001). The integrated amplitude (IA) was significantly different between different time points of 8-OH-DPAT administration (F=20.949, P<0.001), and the application of 10 and 20 micromol/L 8-OH-DPAT resulted in significant IA decrement (F=5.050, P=0.017; F=51.389, P=0.001, respectively). Different concentrations of 8-OH-DPAT also significantly affected IA (F=41.027, P<0.001), and at each time point after administration, IA was the maximum with 1 micromol/L and minimum with 20 micromol/L 8-OH-DPAT, also showing a crossover effect between time and 8-OH-DPAT concentration (F=3.483, P=0.002).</p><p><b>CONCLUSIONS</b>8-OH-DPAT induces a dose-dependent increase in RC and a dose-dependent inhibition of the IA and burst frequency, with long-lasting inhibitory effect on the inspiration. 5-HT1A receptors play an important role in the modulation of the RRDA in isolated neonatal rat brainstem slice.</p>

Role of 5-HT2A-receptors coupled with superoxide anion in the medial area of nucleus retrofacialis / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of 5-HT2A-receptors coupled with superoxide anion (O2-) on respiratory regulation signal transductionin passageway in the medial area of nucleus retrofacialis (mNRF).</p><p><b>METHODS</b>mNRF island was prepared from medullary slices of neonatal SD rats according to Johnson's method and transferred separately into 24-well culture plates with reagents according to protocol, followed by incubation for 60 min at 37 degrees C in a humidified incubator with 5% CO2. Absorbance of 100 microl supernatant was measured by spectrophotometry at 550 nm and the effect of 5-HT and 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, agonist of 5-HT2A-receptors) on O2- generation in the mNRF was observed, along with the inhibition of this effect by ketanserin (antagonist of 5-HT2A-receptors) and alpha-lipoic acid (alpha-LA, a antioxidant).</p><p><b>RESULTS</b>5-HT concentration-response curve demonstrated that absorbance peak occurred at 1 micromol/L without further increment with higher concentration. DOI concentration-response curve showed the absorbance peak at 20 micromol/L without further increment. 5-HT and DOI significantly increased the absorbance with comparable effects. Ketanserin and alpha-LA significantly decreased the absorbance generated by 5-HT and DOI.</p><p><b>CONCLUSION</b>Activation of 5-HT2A receptors results in obvious O2- production in mNRF, suggesting that 5-HT2A receptors regulate respiratory function in association with O2-.</p>

5-HT1A receptors are involved in the modulation of discharge activities of biphasic expiratory neurons and inspiratory neurons / 中华神经医学杂志

Objective To determine whether 5-HT1A receptors could modulate the discharge activities of biphasic expiratory neurons(BE-neurons)and inspiratory neurons (I-neurons).Methods Brainstem slices from newborn SD rats(0～3d)were made according to the method of Suzue,et al.These preparations included the medial region of the nucleus retrofacialis (mNRF)with the hypoglossal nerve rootlets retained.Respiratory rhythmical discharge activities (RRDA)of BE-neurons/I-neurons in mNRF and activities of the hypoglossal nerve(Ⅻ nerve)were simultaneously recorded by using extracellular microelectrodes and suction electrode,respectively.The effects of 5-HT1A receptors on the respiratory rhythm were investigated by application of 5-HT1A receptor specific agonist 8-OH-DPAT and its specific antagonist PMPPI in the perfusion solution.Results 8-OHDPAT induced a significant increase in the respiratory cycles(RC)and the expiratory time (TE) as well as a decrease in the integral amplitude(IA) and the peak discharge frequency (PFn) of RRDA of BE-neurons and I-neurons. By contrast, PMPPI produced opposite effects on the BE-neurons and I-neurons except that PMPPI changed IA and PFn of BE-neurons/I-neurons nonsignificantly. Conclusion 5-HT1A receptors are involved in the phase-switching between expiration and inspiration by affecting BE-neurons;5-HT1A receptors are also possibly involved in the modulation of rhythmical respiration through the inhibitory synaptic input from I-neurons.

Isolation and physiological characterization of mesenteric arterial smooth muscle cells / 南方医科大学学报

Vascular smooth muscle cells (VSMCs) are crucial for studying the mechanism and regulation of vasogenic response, but so far no ideal method has been reported for their isolation. We established a VMSC isolation method based on two-step digestion, with which a large population of single smooth muscle cells could be obtained. After cell isolation, the potassium current, cytosolic calcium concentration, reactivity to caffeine were measured, and the results demonstrated that the cells were highly viable and exhibited reactivity to the vascular constrictor to meet various needs in experimental research.