The Electrophysiological Mechanism Of Epileptogenesis In Veratridine-treated Rat Hippocampal CA1 Pyramidals

The mechanism of epileptogenesis is always being the focus of researchers of brain neuroscience. By far its cellular mechanism has not been fully understood. Corresponding to the epilepsy mark "spike" in EEG, "paroxymal depolarizing shift(PDS)" was recorded in single neuron by intracellular recording. In this topic, small dose of veratridine, a kind of neurotoxin, induced such PDS epileptiform activities on rat hippocampal CAl pyramidal neurons. It is similar to the electrophysiologic fits of human temporal lobe epilepsy. And related cellular electrophysiologic mechanism was investigated on this slice model.In our study, whole-cell recording by infrared visual patch clamp on brain slice was adopted. Through stimulating on neuron bodies or presynaptic collaterals, blocking varied ion channels, we observed the physiological feature of epileptogenesis induced by veratridine, investigated the function of persistent sodium current(I_NaP) and Ih current, and observed the excitability of CAl pyramidals and their responsibility to stimuli under thebackground of veratridine perfusion.Main results 1. The morphological feature and electrophysilogicalcharacteristics of rat hippocampal CA1 neurons under theinfrared visual patch clamp recordingA. Under the infrared video, each typical CA1 pyramidal neuron had one slender basal dendrite stretching out from its body. Yet only bodies could been seen in interneurons.B. Under normal conditions, pyramidal neurons had got no or low rate of spontaneous discharges, their average frequencies are not above 3/s. Interneurons had high rate of spontaneous discharges, their frequencies were about 5~30/s. On the action potentials(AP), pyramidal neyrons had DAPs (depolarization after-potentials), no HAPs (hyperpolarization after-potentials) or smaller HAPs. While interneurons had no DAPs but bigger HAPs. Under the current clamp configuration, when stimulated with depolarizing step, pyramidais showed "voltage frenquency adaption" phenomena, some of which even with after discharges. Interneurons did not show voltage frenquency adaption at all.C. Under normal conditions, of 160 CA1 neurons, 144 pyramidalsshared -62. 8 + 6. 42mV of average membrane potential, 18.2 ±4. 16M Q of average membrane input resistence, 23.2 + 4.15ms of average membrane time constant. 16 interneurons had -50. 2 + 3. 12mV of average membrane potential, 41.4 + 6.85M Q of average membrane input resistence, 60.45 +10.92ms of average membrane time constant. There existed difference statistically between these two kinds of neurons. 2. The electrophysiological feature of epileptiform activities ofrat hippocampal CA1 pyramidals induced by small dose ofveratridineA. When small dose of veratridine was added to the ACSF, 85 out of 90 (90%) pyramidals showed persistent oscillation of"paroxymal depolarizing shift (PDS) " , similar to epileptic seizures reported. With perfusion of veratridine, CA1 pyramidal neuron membrane tended to hyperpolarizing direction. The number of single AP increased and membrane slightly depolarized before the occurrence of first PDS . Each PDS were followed by a 5~10mV hyperpolarization. And then , the neuron membrane slowly depolarized again until the next PDS broke out. The bursting pattern was rather consistent in a given neuron at a given time. In some nuerons, the runsof PDS lasted as long as 90min.B. According to veratridine concentration, neurons were dividedinto three groups: 0. 3u M group(n= 10), 0. 5uM(n=15) and 0. 8 uM (n=10). Their latent periods of the first PDSs were: 30 ±12s, 20±8s, 10±5s. Therefore higher the concentration of veratridine, shorter the latent period of PDS. In 45 CA1 pyramidals, there was no correlation between veratridine concentration /perfusion duration and PDS numbers /persistent duration.C. Holding the membrane potential at different levels, In some range(-60mV-—80mV, for example, varied with individuals), with the hyperpolarization of membrane potential, the amplitude of PDS (bigger slow wave) became bigger, single PDS prolonged, AP numbers riding on the PDS increased. It suggested the ability of veratridine inducing PDS was restricted by membrane potential.D. In hippocampal slice, single stimulation of the Schaffer collaterals input induce a EPSP or AP on the postsynaptic CA1 pyramidal. When such synaptic transmission was completely blocked by the mixture of CNQX (5uM) + AP-5 (12. 5 u M) +Bic (10 u M), the runs of PDS induced by small dose (0. 3uM~0. 8uM ) of veratridine still occurred.3. The current mechanism of epileptiform discharges induced by veratridine on CA1 pyramidal neurons (1) The relation between INap and PDS generation A. under the voltage clamp configuration, before and after PDS epileptiform discharges induced by 0. 5u M veratridine, a series of depolarizing step (2. 5mV, 800ms) were given to construct an I-V curve. With the increase of holding potential, I-V relationship tended to non-linearity. This deviation is called the "depolarizing rectification" . Under the condition of veratridine exposure, neurons exhibiting PDS burstings had enhanced depolarizing rectification. When low concentration of TTX (80nM) was applied in the presence of veratridine, runs of PDS wereblocked, and depolarizing rectification relieved.B. under the voltage clamp configuration, holding membrane potential at different levels (-75mV~-55mV ), a series of depolarizing step (5mV, 800ms) were given. The steady-state current was plotted at 770ms against depolaring voltage before and after TTX. Subthreshold TTX-sensitive INap was calculated by subtracting the step current in the absence of TTX from that in the presence of TTX. When neurons were treated with 0.5 y M veratridine, TTX-sensitive INai> became bigger. Especially when holding potential was depolarized more than - 65mv, it increased statistically.C. 15uM~20uM phenytoin (PHT) terminated PDS epileptiform activities in the presence of veratridine. When various concentration of PHT were added into ACSF in a increasing manner, we found the inter-bursting interval prolonged with the inreasing of drug concentration, and the duration of per PDS did not shorted. It suggested PHT may get rid of epileptiform activities by inhibiting the generation of PDS.(2)The relation between Ih and PDS generationA. Under the voltage clamp configuration, a series of hyperpolarizing step (lOraV, 1500ms) were given to measure Ih at different membrane potential levels (~73~-163mV ) before and after the PDS. We found in the course of PDS generation, Ih decreased gradually.B. Under the background of PDS activities induced by small dose of veratridine, 3mM CsCl, a kind of Ih blocker, was addedinto ACSF. It did not blocked the epileptiform discharges.C. Under the background of PDS activities induced by small dose of veratridine, 6uM gabapentin was added into ACSF. It did not blocked the epileptiform discharges, either. 4. Changes of CA1 pyramidals' excitibility and PDS trigger factorsunder the background of PDS activities induced by small doseof veratridineA. During 0. 5 u M veratridine perfusion, spontaneous discharges were observed per 2 minutes, and depolarizing ramp stimulations(0 — 300pA, 1000ms) were given under current clamp configuration to measure the AP number during the stimulation, the latent period and threshold of the first AP during the stimulation. A hyperpolarizing step (400pA, 200ms) was given to measure the membrane input resistence. We found in the process of epileptogenesis induced by veratridine , the distance between membrane potential and threshold potential became smaller and smaller, membrane input resistence became increasing bigger, triggered APs bacame more and more.B. 64 out of 90 PDSs in 20 pyramidals induced by small dose of veratridine showed membrane potential oscillation before PDS occurred. The oscillation' s amplitude, duration varied. PDS finally broke out riding on the last oscillation.C. Under normal conditions, given depolarizing current step stimulation, whether increasing stimulating intensity or prolonging stimulating duration, both could make CA1pyramidals discharged burstingly. Given increasing intensity of Schaffer collateral stimulation, the postsynaptic CA1 pyramidals showed APs from no APs, burstings from APs.D. Under the background of small dose of veratridine perfusion, before spontaneous PDSs occurred, PDS epileptiform activities were triggered if given small depolarizing step stimulation on neuron body or excitatory input from presynapse (under normal condition, these stimulations were not sufficient to trigger a bursting). Afer spontaneous PDSs occurred, above stimulations made PDSs appeared ahead if in suitable periods.Main conclusion1. Applying with infrared visual patch clamp technique, we succeeded in recording the physiological activities of rat CA1 pyramidal neurons. The pyramidals were distinguished from interneurons by their imagings and electrophysiological characteristics.2.Small dose of veratridine induced PDS epileptiform activities on rat hippocampal CM pyramidals. Such activities were dose dependent, membrane voltage dependent and intrinsic. 3. During the epileptogenesis by veratridine, INap was enlarged. Blocking INal. terminated PDS discharges. In the course of epileptogenesis , Ih current decreased gradually. But whether blocking or enhancing it did not get rid of the PDSs. So INaP play a key role in the activities induced by veratridine.