A Study Of The Effects Of Nikethamide On Basic Rhythmic Respiration And Respiratory Neurons Of The MNRF

Keeping normal rhythmic respiration is essential for life. It is important to make it clear that which site is the precise site for respiratory rhythm generation and the mechanisms underlying respiratory rhythmogenesis. There are many diseases such as sudden infant death syndrome (SIDS), congenital central hypoventialation syndrome (CCHS), central respiratory failture, central respiratory rhythm disturbance, which are due to abnormality of respiratory center. So making the mechanisms underlying respiratory rhythmogenesis clear is not only meanful in physiology but also for prevention and cure respiratory central diseases.Our researches have demonstrated that the medial area of nucleus retrofacialis (mNRF) is the site of respiratory rhythmogenesis. Smith stated that the pre-Botzinger complex (PBC) was the site of respiratory rhythmogenesis in 1991.mNRF and PBC are located in rostrol ventrolateral medulla. The anatomical position of them is of difference, but they are overlapped partly. The site of basic respiratory rhythmogenesis has been ascertained in the rostrol ventrolateral medulla. There are two hypothesis for the mechanism of respiratory rhythmogenesis: pacemaker neuron hypothesis and neurons network hypothesis. Pacemaker neuron hypothesis has been supported by a majority of the present findings. We had found Expiratory-Inspiratory phase spanning (E-I PS) neuron maybe is pacemaker on mNRF in vivo and in vitro in neonate rats. The 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 pacemaker neurons. Foreign scholars haveidentified pacemaker neuron as cadmium-insensitive and cadmium-sensitive. Cadmium-insensitive pacemakers are characterized by bursting that persist in the presence of Cadmium, dependent on persistent and transient sodium current (I_NaP and I_NaT) and blocked by TTX. Cadmium-sensitive pacemakers are characterized by bursting that is blocked in the presence of Cadmium, dependent on Ca²⁺-activated nonspecific cationic current (I_CAN) and blocked by flufenamic acid (FFA).Nikethamide (N, N-diethylnicotinamide) has been usedwidely in clinic. It is generally used in three directions as follow. First, it is used as a respiratory central stimulator, nikethamide can excite respiratory center selectively. Second nikethamide can decrease the aminopherase and jaundice levels of infant Thirdly, niketnamide comprise polarity part and nonpolarity part, it can be used as chaotropic agent to dissolve drugs which is difficult to dissolve in water. Although nikethamide has been used widerly but its mechanisms is still unknown.5-HT_2A receptors have been found in many positions of the CNS, including the cerebral cortex, basal ganglia, hippocampus, thalamus, cerebellum, and hypothalamus. 5-HT_2A receptror agonists have excitory effects on respiratory activity. Activation of 5-HT_2A receptors with l-(2,5-dimethoxy-4-iodophenyl)-2-am inopropane (DOI) increases the frequency of respiratory activity. Blockade of endogenously activated 5-HT_2A receptors decreases the frequency, amplitude, and regularity of respiratory population activity. An ERK activation pathway has been confirmed in rat renal mesangial cells as follows: 5-HT _2A receptor-> G(q) protein-> phospholipase C-> diacylglycerol-> protein kinase C (PKC)->NAD(P)H oxidase-> reactive oxygen species (ROS, i.e., H2O2 and superoxide) -> mitogen-activated extracellular signal-regulated kinase-> extracellular signal-regulated kinase (ERK).To explore the effects of nikethamide on respiratory-related rhythmic discharge activity and respiratory neurons in the mNRF in vitro, and explore whether exist interaction between nikethamide and 5-HT_2A receptors pathway, we designed this study as following: 1. To explore the effects of nikethamide on activity (RRDA) and discharge activity of respiratory neurons, simultaneous recording of the hypoglossal nerve (Xlln) respiratory-related rhythmic discharge (RRDA) with suction electrode and the respiratory neuronal discharge with microelectrodes in the mNRF on the brainstem slices in vitro. 2. Simultaneous recording of RRDA and the respiratory neuronal discharge. To observe the roles of 5-HT_2A receptors in respiratory neurons. 3. To explore whether the interaction exist between nikethamide and 5-HT_2A receptors pathway on respiratory center, RRDA was recorded with suction electrode on brainstem slices. 4. To explore weather exist the interaction between nikethamide and GABAa receptors pathway on respiratory center, RRDA was recorded with suction on brainstem slices. 5. Simultaneous recording of RRDA and the respiratory neuronal discharge was performed with whole cell patch in the mNRF on the brainstem slice in vitro. To observe transient and the persistent sodium current changes of respiratory pacemaker neurons and respiratory neurons respectively before and after nikethamide perfusion.1. Effects of nikethamide on respiratory-related rhythmic discharge activity recorded from hypoglossal nerve rootlets and discharge activity of respiratory neurons in mNRF.The possible role of nikethamide in RRDA was investigated by perfused different concentration of nikethamide, 0, 0.5,1, 3, 5, 7, 10μg/mL in modified Kreb's solution (ACSF). Nikethamide increased RRDA at the range of concentration from 0.5 to 7μg/mL, 5μg/mL being the most effective concentration of nikethamide on RRDA of transverse medullary slices, in inspiratory time (TI)( F=7.263, P=0.000), integral amplitude (IA)( F=31.80, P=0.000), and respiratory cycle (RC)( F=6.430, P=0.001)(Repeat measures, n=6). While the concentration of nikethamide reachedlOμg/mL RRDA changed into the deep, very slow inspiratory mode. It suggested nikethamide has a depressive effect on RRDA in this concentration.According to the response to nikethamide, the respiratory neurons in mNRF were classified into two groups. One group was the changed in discharge amplitude (t=12.346, P=0.000) it was significant, but change in discharge frequency was nonsignificant (t=0.502, P=0.637) (paired-samples t test, n=6); and another group was changed with significance in discharge frequency (t=6.576, P=0.003) but not in discharge amplitude (t=1.832, P=0.126) (paired-samples t test, n=5).On the basis of those experimental results, nikethamide may stimulate respiration by exciteing respiratory neuron and neurons in respiratory related nucleus.2. Effects of DOI and ketanserine on inspiratory neuronsDOI prolonged TI( P=0.004 vs control group), ketanserine shortened TI(P=0.002 vs control group) (F=50.593, P=0.000); DOI increased IA (P=0.001 vs control group), ketanserine decreased IA (P=0.004 vs control group) (F=50.694, P=0.000); DOI shortend RC (P=0.008 vs control group), ketanserine prolonged RC (P=0.003 vs control group) (F=68.778, P=0.000); DOI increased peak discharge frequency (PF) (P=0.008 vs control group), ketanserine decreased PF (P=0.003 vs control group) (Repeat measures, n=7).5-TH_2A receptors are involved in the modulation of discharge activity of inspiratory neurons. Actived 5-TH_2A receptors play a role of stimulation on inspiratory neurons and RRDA, block 5-TH_2A receptors have an opposite effect.3. Effects of 5-HT_2A receptor in the increased of RRDA by nikethamide in neonatal rats transverse medullary slice.DOI increased RRDA in TI (t=10.225, P=0.000), IA (t=14.143, P=0.000), and RC (t=7.817, P=0.001). Ketanserine decreased RRDA in TI (t=11.62, P=0.000), IA (t=12.147, P=0.000) and RC (t=7.21, P=0.001). Ketanserine plus DOI had no significant effects on RRDA (TI t=0.342 P=0.746; IA t=1. 179 P=0.291; RC t=0.168 P=0.873). (Those groups were texted by paired-samples t test, n=6.) The effects of nikethamide on RC (P=0.002 vs nikethamide group, F=44.132 P=0.000) and IA (P=0.001 vs nikethamide group, F=81.025 P=0.000) were totally and partially reversed respectively by additional application of ketanserine, but the effect of nikethamide on TI (P=0.83 vs nikethamide group, F=47.762 P=0.000) was not influenced by ketanserine (Repeat measures, n=6).Experimental results show ketanserine can partially block the stimulating effects of nikethamide on RRDA, it suggests nikethamide can increase the RRDA partly via 5-TH_2A receptor.4. Roles of GABAa receptor in respiratory enhancement induced by nikethamide in neonatal rats.Within the concentration range of 10 to 40μmol/L, GABA decreased RRDA in TI (F=43.07, P=0.000), IA (F=93.97, P=0.000), RC (F=8.968, P=0.001) (Repeat measures, n=5). 40μmol/L GABA was the most effective concentration decreasing RRDA in TI, IA, RC. 10μmol/L bicuculline could increase RRDA in TI (t=8.257, P=0.001), IA (t=10.786, P=0.000) and RC (t=8.968, P=0.001). 10μmol/L bicuculline plus 40μmol/L GABA had no significant effects on RRDA [TI (t=0.384, P=0.721), IA (t=1.596, P=0.186), RC (t=1.295, P=0.265)] (Those two groups were texted by paired-samples t test, n=5). The effects of nikethamide plus bicuculline were significantly increased in TI (P=0.006 vs nikethamide group, F=63.837 P=0.000) and IA (P=0.003 vs nikethamide group, F=126.395 P=0.000) compared with nikethamide alone, but RC (P=0.642 vs nikethamide group, F=44.23 P=0.000) had no significant influence by nikethamide plus bicuculline compared with nikethamide alone (Repeated measures, n=6).Experimental results show GABAa receptors were involved in the modulation of RRDA, 40μmol/L GABA was the most effective concentration to depress RRDA. Blocking GABAa receptors can stimulate RRDA based on the role of nikethamide.It suggests nikethamide can increase the RRDA partly via GABAa receptor.5. Effects of nikethamide on sodium current and sodium channel of respiratory pacemaker neurons and inspiratory neurons.5.1 Effects of nikethamide on burst of respiratory pacemaker neuronsNikethamide significantly increased the bursts duration (t=4.885 P=0.016), burst amplitude (t=11.676 P=0.001) and spike frequency (t=4.886 P=0.016) of respiratory pacemaker neurons and inspiratory neuron (paired-samples t test, n=4).5.2 Effects of nikethamike on transient sodium current and persistent sodium current of respiratory pacemaker neurons and inspiratory neurons.Experiment was performed in voltage-clamp configuration. Voltage steps from -80 to +20 mV and slow voltage ramps from -80 to +20 mV (90 mV/ sec) were applied to elicit transient and persistent sodium currents, respectively. The persistent sodium current elicited by Voltage ramp were increased by nikethamide too. Both the persistent and transient inward currents recorded in the Cd-insensitive pacemaker neurons were sensitive to TTX. The transient sodium currents were increased by nikethamide in pacemaker neurons (t=15.135 P=0.001) (paired-samples t test, n=4) and inspiratory neuron (t=6.798 P=0.001) (paired-samples t test, n=6). The transient sodium currents were increased by nikethamide in pacemaker neurons (t=9.886 P=0.002) (paired-samples t test, n=4) and inspiratory neuron (t=4.077 P=0.01) (paired-samples t test, n=4) too. There were no differences between pacemaker neurons and inspiratory neurons in transient (t=1.177 P=0.273) and persistent (t=1.291 P=0.233) sodium currents (independent-samples t test). The experimental results suggest that nikethamide stimulates respiratory neurons due to increasing sodium conductance.The ampulite of persistent sodium currents and the change ratio of persistent sodium currents before and after nikethamide of pacemaker neurons are larger than those of inspiratory neuron. So we think that persistent sodium current are involved with "pacemaker" properties, but its role is not decisive, there are other factors contribute to "pacemaker" properties of pacemaker neuron.5.3 Effects of nikethamide on steady activation curves and steady inactivation curves of sodium channel in respiratory pacemaker neurons.Nikethamide makes sodium channel steady activation curves shift left vs control. This means the threshold of sodium channel is lower than control, channel begin open at a lower potential. Nikethamide makes sodium channel steady inactivation curves shift right vs control. This means the inactivation threshold of sodium channel is higher than control, channel begin to close at a higher potentialFrom those experimental results we thought nikethamide increase sodium current due to it change the kinetics of sodium channel, insreases its open probability, decreases its close probability.Conclusion:â‘ Nikethamide can increase discharge activity of respiratory-relate rhythm discharge activity and inspiratory neurons.â‘¡Endogenous 5-HT and 5-HT_2A receptors are involved in the modulation of discharge activity of inspiratory neurons and respiratory rhythmogenesis.â‘¢Activation of 5-HT _2a receptors have excitory effects on rhythmic respiration, nikethamide increases the RRDA partly via 5-TH_2A receptors.â‘£Activation of GABA_a receptors in mNRF have depressive effects on rhythmic respiration, nikethamide increases the RRDA partly via GABA receptors.⑤Nikethamide increases transient and persistent sodium currents, perhaps it is the reason why nikethamide can increase RRDA level.Nikethamide changes kinetics to increase the excitability of neurons.â‘¥Persistent sodium currents is are involved with "pacemaker" property, but its role is not decisive, there are other factor contribute to "pacemaker" property of pacemaker neuron.