Slow Wave Oscillation-induced And Oscillation-triggered By Veratridine In Rat Dorsal Root Ganglion Neurons

Dorsal root ganglion (DRG) is considered as the first stage of the sensory pathway. The frequency and temporal patterns of spontaneous activity originated from DRG neurons play important role in determining the quality and intensity of sense. The recent study also found that the patterns of firing series were nearly related to the distribution and activity of sodium channels. DRG neurons are primary sensory neurons. Under the effect of injury or inflammation, the excitability of DRG neurons will often enhance abnormally and it becomes the pacemaker of chronic pain signal. The firing responses are as follows: decrease in threshold, which is response sensitively; complex firing patterns; the responses are catastrophic. Although there is evidence that the sodium current and sodium channel expression of the pacemaker increases significantly, how the block of inactivation gate of sodium channel-the circle structure modulating the activity of sodium channels will affect the excitability of primary sensory neurons and what the relationship between the block of inactivation gate and the occurrence of chronic pain especially triggered pain is still not very clear.Veratridine is an inhibitor of inactivation gate of sodium channels, which binds to D2-S6 site in sodium channel a -subunit. It blocks the inactivation and shifts the voltage dependence of activation to hypolarization. Consequently sodium channels keep open at the resting membrane potential. Recently it was found that veratridine can cause burst firing with different patterns in hippocampal neurons, which not only was considered as a cellular model of epilepsy seizure but also suggest that the inactivation gate of sodium channels play important role in the formation of firing patterns. Aconitine is also an inhibitor of inactivation gate of sodium channels, but it binds to D3-S6 site. Previous study have found that veratridine which binds to D2-S6 can induce oscillatory firing with high frequency in part of injured DRG neurons. However, what the effect of aconitine on firing patterns in injured DRG neurons is not clear. Whether or not the block of different sites of sodium channel inactivation gate will produce different effects on firing patterns is also under study.The present study was performed on a model of chronically compressed rat DRG through the single fiber and intracellular recording in vivo. We observed and analyzed the transformative characteristics and the membrane potential mechanism of firing series induced by these two inhibitors of inactivation gate of sodium channels in type A DRG neurons. Furthermore, on the basis of above results, the phenomenon, stimulation condition and the mechanism of potassium channels were also studied. The present study established a base of understanding the relationship between the inactivation gate of sodium channels and firingpatterns and exploring the relationship between firing patterns and chronic pain.Results:The first part: Effects of veratridine and aconitine on firing patterns and the membrane potential mechanism of slow wave oscillation (SWO) induced by veratridine in injured DRG neurons were studied using single fiber recording in vivo and intracellular recording in vivo respectively.1. In normal group, after 30 s-10 min bath of veratridine (3-10 umol/L) in L5 DRG, the irregular firing from part of DRG neurons can develop into slow wave oscillation firing. The majority interspike interval (ISI) changed from large to small, then from small to large continuously. The whole track of ISI change formed oscillation waves with different shapes, i.e. the firing density is comparatively high at the trough of oscillation but comparatively low at the wave crest of oscillation. The oscillation had the following characteristics:(1) The oscillatory period is comparatively long and the majority period is 30-120 s, so it is called SWO. Meanwhile, the oscillatory duration is about 10-40 s and the oscillatory interval is about 30-80 s.(2) The range of ISI changes, i.e. the oscillatory magnitude is comparatively large. The minimum of ISI oscillation is 2 ms while the maximum is 40 ms, which means that the firing frequency change from tens of to hundreds of Hz.2. In normal DRG neurons with spontaneous activity, aconitine (10-200 umol/L) can transform the irregular firing into period firing. When the background firing was periodic pattern, the firing patternkept constant while the firing frequency increased.3. In normal group, veratridine can induce SWO in part of resting neurons while aconitine (200 umol/L) cannot induce firing in part of resting neurons.4. In injured group, spontaneous activity with different patterns, i.e. bursting, irregular, period also can be transformed into SWO under the effect of veratridine. In addition, neurons with SWO in injured DRG neurons (61%) had higher proportion than in normal DRG neurons (45%). So we chose the injured DRG to mainly study.5. In injured group, under the effect of aconitine, the bursting and irregular firing from the majority of injured DRG neurons can be developed into periodic firing. When the background firing was periodic pattern, the firing pattern kept constant while the firing frequency increased gradually.6. In injured group, veratridine can also induce SWO in part of resting neurons.7. In injured group, aconitine (200 u mol/L) did not induce firing in resting units. However, Ca2+-free or TEA can induce firing.8. In the same neuron, veratridine caused slow wave oscillations of ISIs, while aconitine caused periodic firing.9. If only the firing patterns have changed under the effect of veratridine, then increasing the concentrations of the reagent, the transformative firing patterns would not change basically. Neither did aconitine.10. After the application of veratridine (5-10 n mol/L), membrane potential had an alternate change with long-term depolarization and repolarization. On the top of depolarization, high frequency ofaction potentials occurred in a bursting oscillatory pattern. It is suggested that the occurrence of SWO is related to the alternate fluctuation of depolarization and repolarization of membrane potential.The second part: The phenomenon, produce condition and mechanismof triggered oscillation under the effect of veratridine.1. After the bath of veratridine (1.5-5 y mol/L) in L5 injured DRG, the subthreshold concentration which can induce SWO, DRG neurons can keep silent. Here, touch or pressure on the cutaneous receptive fields or electrical stimulus of sciatic nerve can trigger part of DRG neurons to produce high-frequency SWO firing, termed triggered oscillation. The oscillation had the following characteristics: (1) The oscillatory duration is comparatively long (from a few seconds to 1-2 min). The triggered condition is peripheral afferent impulse. (2) Once the triggered oscillation has happened, the oscillatory frequency, duration and pattern would not change with the increase in stimulation intensity, which is different from afterdischarge significantly. It is suggested that the triggered stimulation and oscillation are two nearly correlative and mutually independent courses.2. Peripheral afferent impulses of the same neuron can induce triggered oscillation in DRG neurons, termed as triggered oscillation with the same nerve.3. The stimulus of sciatic nerve can induce multiple oscillations in DRG neurons, but each oscillatory frequency, duration and patternwere mutually different.4. The retrorse stimulus of dorsal root can also trigger oscillations in DRG neurons, termed as cross-triggered oscillation.5. Once the stimulation intensity has come to the threshold that can trigger oscillation, the oscillatory frequency, duration and pattern would not change with the increase in the stimulation intensity.6. The threshold of cross-triggered oscillation would increase if we decreased the concentration of extracellular potassium ions ([K+]o). On the contrary, the threshold of cross-triggered oscillation would decrease if we increased the [K^o- It is suggested that the x occurrence of cross-triggered oscillation is related to the increase in [K+]o and the according depolarization of membrane potential.Conclusions:1. Veratridine can induce part of DRG neurons to produce slow wave oscillation firing while aconitine induce period firing. It is suggested that different sites of sodium channel inactivation gate can induce different firing patterns.2. Slow wave oscillation induced by veratridine is related to the alternate fluctuation of depolarization and repolarization of membrane potential.3. Under the effect of veratridine with the subthreshold concentration, peripheral afferent impulses can trigger slow wave oscillation, including two basic modes: triggered oscillation with the same nerve and cross-triggered oscillation.4. The triggered stimulation and triggered oscillation are two nearly correlative and mutually independent courses. It is suggested that afferent impulses arouse the intrinsic oscillatory firing course in DRG neurons.