急性大鼠癫痫模型海马神经元原发性单位后放电脉冲间隔特征

受到刺激后即刻出现的海马（hippocampus,HPC）原发性单位后放电是癫痫相关性细胞电活动的重要形式之一,其放电脉冲间隔（interspike interval,ISI）和串内平均频率（Hz）特征及其在网络癫痫形成中的作用值得探讨。实验用急性强直电刺激（60Hz,2S,0．4-0．6mA）大鼠右侧后背HPC（acute tetanization of the fight posterior dorsal hippocampus,以后简称ATPDH）或右侧尾壳核（acute tetanization of the fight caudate putamen nucleus,以后简称ATRC）诱导HPC或皮层网络癫痫,重点观察HPC神经元原发性单位后放电模式和上述的瞬时时间编码特征。结果表明：（1）HPC原发性单位后放电表现为两种不同的放电模式,即先易化后抑制或先抑制后易化,其ISI序列分别表现为先小后大的“头尾”式分布或先大后小的“尾头”式分布。（2）ATFDH主要引起“尾头”式（35／57串）、而ATRC主要引起“头尾”式（12／22串）ISI点分布的原发性单位后放电,串内“头”、“尾”平均持续时间均具有明显差异（P〈0．05）。（3）ATRC可以诱导双侧HPC单位后放电出现交互的“头尾”、‘呢头”式ISI点分布特征。（4）多串电刺激可以诱导HPC原发性单位后放电特征性ISI点分布重复显现。（5）特征性HPC原发性单位后放电伴随出现网络癫痫发作样高频电振荡。这提示：强直电刺激诱导的HPC神经元原发性单位后放电“头尾”或呢头”式ISI序列分布规律,可以较准确地反映所记录神经元的诱发性易化或抑制活动的程度,用于网络癫痫形成中单个成员细胞癫痫相关性电活动机制的分析。     

Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na⁺ ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λ_max of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pK_a values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.     

Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin

Background

Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents.

Methodology/Principal Findings

The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5–10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5–10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex.

Conclusions/Significance

The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state.     

Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q₁₀) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.     

In Vivo Optogenetic Control of Striatal and Thalamic Neurons in Non-Human Primates

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 10⁹ particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.     

Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans

By enabling a tight control of cell excitation, optogenetics is a powerful approach to study the function of neurons and neural circuits. With its transparent body, a fully mapped nervous system, easily quantifiable behaviors and many available genetic tools, Caenorhabditis elegans is an extremely well-suited model to decipher the functioning logic of the nervous system with optogenetics. Our goal was to establish an efficient dual color optogenetic system for the independent excitation of different neurons in C. elegans. We combined two recently discovered channelrhodopsins: the red-light sensitive Chrimson from Chlamydomonas noctigama and the blue-light sensitive CoChR from Chloromonas oogama. Codon-optimized versions of Chrimson and CoChR were designed for C. elegans and expressed in different mechanosensory neurons. Freely moving animals produced robust behavioral responses to light stimuli of specific wavelengths. Since CoChR was five times more sensitive to blue light than the commonly used ChR2, we were able to use low blue light intensities producing no cross-activation of Chrimson. Thanks to these optogenetics tools, we revealed asymmetric cross-habituation effects between the gentle and harsh touch sensory motor pathways. Collectively, our results establish the Chrimson/CoChR pair as a potent tool for bimodal neural excitation in C. elegans and equip this genetic model organism for the next generation of in vivo optogenetic analyses.     

Hippocampal afterdischarges and localization of the stimulating electrodes

Electrically evoked hippocampal afterdischarges are used as a model of partial epileptic seizures with a complex symptomatology and for testing anticonvulsants and toxic substances. Stimulating electrodes were implanted in the dorsal hippocampus of 16 laboratory rats and when the animals had recovered they were stimulated (15-s series, 8 Hz, pulse length 1 ms) with a voltage double the threshold value for a tissue response. The following features of the evoked afterdischarge were evaluated: the duration of the first phase of the afterdischarge, the duration of the non-active interphase, the duration of the second phase and the number of "wet dog shakes" (a constant accompaniment of hippocampal afterdischarges). Localization of the electrodes in the CA1 (n = 7) and CA3 (n = 7) region of the hippocampus made no difference to these parameters and in both cases the measured and evaluated data were the same. The afterdischarges were always accompanied by a marked orientation reaction. The study showed that when using macroelectrodes to stimulate the dorsal hippocampus, their localization in the CA1/CA3 is not of critical importance.     

Dizocilpine pretreatment suppresses the action of hypoxia on hippocampal epileptic afterdischarges in immature rats

Effect of dizocilpine (0.5 mg/kg i.p.) on epileptic afterdischarges elicited by low-frequency electrical stimulation of the dorsal hippocampus was studied in rat pups aged 12 and 18 days. Repeated elicitation of afterdischarges (ADs) in control animals resulted in a progressive increase of the duration of ADs in both age groups. Dizocilpine (MK-801) injected after the first afterdischarge suppressed this prolongation in 12-day-old rats only. Hypobaric hypoxia (simulated altitude of 9000 m for one hour) led to a marked prolongation of the first afterdischarge in both age groups with a tendency to shorter ADs after repeated stimulations. Dizocilpine potentiated this tendency in 12-day-old rat pups so that it became statistically significant. Administration of dizocilpine before hypoxia prevented the increase in duration of the first afterdischarge in both age groups.     

Optogenetic Delay of Status Epilepticus Onset in an In Vivo Rodent Epilepsy Model

Epilepsy is a devastating disease, currently treated with medications, surgery or electrical stimulation. None of these approaches is totally effective and our ability to control seizures remains limited and complicated by frequent side effects. The emerging revolutionary technique of optogenetics enables manipulation of the activity of specific neuronal populations in vivo with exquisite spatiotemporal resolution using light. We used optogenetic approaches to test the role of hippocampal excitatory neurons in the lithium-pilocarpine model of acute elicited seizures in awake behaving rats. Hippocampal pyramidal neurons were transduced in vivo with a virus carrying an enhanced halorhodopsin (eNpHR), a yellow light activated chloride pump, and acute seizure progression was then monitored behaviorally and electrophysiologically in the presence and absence of illumination delivered via an optical fiber. Inhibition of those neurons with illumination prior to seizure onset significantly delayed electrographic and behavioral initiation of status epilepticus, and altered the dynamics of ictal activity development. These results reveal an essential role of hippocampal excitatory neurons in this model of ictogenesis and illustrate the power of optogenetic approaches for elucidation of seizure mechanisms. This early success in controlling seizures also suggests future therapeutic avenues.     

Combining Microfluidics,Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.     

Comparative study of the epileptogenic effect of kainic acid injected into the cerebral cortex, hippocampus and amygdala in adult cats chronically implanted

Intracerebral injection of kainic acid in cerebral cortex, hippocampus or amygdala in cats chronically implanted showed that: 1) Hippocampus and amygdala presented a greater sensitivity than the cerebral cortex, while hippocampus presented a greater sensitivity than the amygdala to the generation of an epileptic focus. 2) Comparison of latency, mean duration of afterdischarges, and the mean time period to obtain the peak intensity of the afterdischarge in the three cited structures, showed that mean latency of the first afterdischarge was significantly shorter in hippocampus and amygdala compared with the cerebral cortex. Moreover the mean time period to reach the peak intensity of the afterdischarge was again shorter in the subcortical structures. 3) The epileptic foci both in hippocampus and amygdala were blocked by CNQX and muscimol. 4) The behavioral changes depended on the intensity of the epileptic process. Tonic-clonic convulsions appeared only when the motor cerebral cortex was involved. Finally, 5) kainic acid injections in hippocampus and amygdala elicited an intense neuronal destruction and gliosis of these structures. We conclude that intracerebral injection of low doses of kainic acid in cats represent a good model to study focal epileptic thresholds in the CNS.     

光敏感通道(Channelrhodopsin-2)——神经回路功能和神经系统疾病研究的新工具

光敏感通道(channelrhodopsin-2,ChR2)是一种受光脉冲控制的具有7次跨膜结构的非选择性阳离子通道蛋白,自1991年从莱茵衣藻中发现后被许多实验室所关注.依据ChR2可以快速形成光电流,使细胞发生去极化反应的电生理特性,ChR2已被广泛应用于神经系统的研究.与传统的神经系统研究方法如电生理技术、神经药理学方法相比,用光脉冲控制带有ChR2的神经元的活动,具有更高的空间选择性和特异性.ChR2作为光基因技术的核心组成部分,对神经科学是一个崭新的应用前景广泛的研究工具.近年来ChR2不仅应用于视觉、躯体感觉、听觉和嗅觉等多条感觉神经回路的形态和功能研究,还被应用于各种临床神经系统疾病的研究.本文总结了目前ChR2在神经系统中的研究进展,并对ChR2未来的应用前景作了进一步展望.     

PINP: A New Method of Tagging Neuronal Populations for Identification during In Vivo Electrophysiological Recording

Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons—mainly fast-spiking interneurons—by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Assessing cortico-hippocampal functional connectivity under anesthesia and kainic acid using generalized partial directed coherence

A significant challenge in modern neuroscience lies in determining the functional connectivity between discrete populations of neurones and brain regions. In this study, a variation of partial directed coherence, the generalized partial directed coherence (gPDC), along with a newly proposed critical value for gPDC, were applied on recorded local field potentials (LFPs) and single-unit activity, in order to assess information flow between medial prefrontal cortex (mPFC) and hippocampus and within the hippocampus of the rat brain, under isoflurane anesthesia and kainic acid-induced enhanced neuronal activity. Our findings suggest that, under anesthesia, there exists a continuous information flow from hippocampus towards mPFC, reversed mostly during activity bursts occurring in the mPFC. Moreover, there was a clear directional connection from the lateral towards medial dorsal hippocampus, most prominent in the beta frequency band (10–30 Hz). Kainic acid resulted in partially disrupting the reciprocal cortico-hippocampal connectivity and reversing the intra-hippocampal one. The biological implications of these findings on the effects of anesthesia and kainic acid in brain connectivity, along with implementation issues of gPDC analysis on field potentials and spike trains, are extensively discussed.     

Neuronal septal activity during hippocampal seizure discharges generation in acute model of epilepsy

The activity of the neurones of the medial septal region (MS) and the hippocampal EEG in control and during the appearance of seizure discharges provoked by electrical stimulation of the perforant path were investigated in the awake rabbit. During afterdischarge generation in the hippocampus the dense neuronal bursts separated by periods of inhibition were recorded in the MS. In one group of neurons the bursts of spikes coincided with the discharges in the hippocampus, in other group-occured during inhibitory periods. When the afterdischarge stopped, in the septal neurons with theta activity the disruption of theta pattern was recorded, which have been correlated with the occurrence of low amplitude high frequency (20-25 Hz) waves in the hippocampal EEG. As a rule, the neuronal activivity of the MS recovered much quickly than EEG of the hippocampus; in some cases the increasing of the theta regularity was observed. The definite accordance of the electrical activity of the hippocampus and MS during seizure discharges suggests that the septohippocampal system operate as integral nervous circuit in these conditions. Diverse in the temporal interrelations between the discharges of MS neurones and ictal discharges in the hippocampus in the different cells possible indicate that various groups of the septal nervous elements have different participation in the seizure development. Appearance of the high frequency bursts in the MS is a possible "precursor" of the seizure onsets.     

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects^1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea^7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of the cochlea has been used to evoke responses in the auditory nerve¹⁰. Nevertheless it requires higher energies than electrical stimulation^10,11 and uncertainty remains as to the underlying mechanism¹². Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)¹³ or virus-mediated expression of the ChR2-variant CatCh¹⁴. We used micro-light emitting diodes (µLEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.     

Structural model of channelrhodopsin

Channelrhodopsins (ChRs) are light-gated cation channels that mediate ion transport across membranes in microalgae (vectorial catalysis). ChRs are now widely used for the analysis of neural networks in tissues and living animals with light (optogenetics). For elucidation of functional mechanisms at the atomic level, as well as for further engineering and application, a detailed structure is urgently needed. In the absence of an experimental structure, here we develop a structural ChR model based on several molecular computational approaches, capitalizing on characteristic patterns in amino acid sequences of ChR1, ChR2, Volvox ChRs, Mesostigma ChR, and the recently identified ChR of the halophilic alga Dunaliella salina. In the present model, we identify remarkable structural motifs that may explain fundamental electrophysiological properties of ChR2, ChR1, and their mutants, and in a crucial validation of the model, we successfully reproduce the excitation energy predicted by absorption spectra.     

The Spatiotemporal Dynamics of Phase Synchronization during Epileptogenesis in Amygdala-Kindling Mice

The synchronization among the activities of neural populations in functional regions is one of the most important electrophysiological phenomena in epileptic brains. The spatiotemporal dynamics of phase synchronization was investigated to reveal the reciprocal interaction between different functional regions during epileptogenesis. Local field potentials (LFPs) were recorded simultaneously from the basolateral amygdala (BLA), the cornu ammonis 1 of hippocampus (CA1) and the mediodorsal nucleus of thalamus (MDT) in the mouse amygdala-kindling models during the development of epileptic seizures. The synchronization of LFPs was quantified between BLA, CA1 and MDT using phase-locking value (PLV). During amygdala kindling, behavioral changes (from stage 0 to stage 5) of mice were accompanied by after-discharges (ADs) of similar waveforms appearing almost simultaneously in CA1, MDT, as well as BLA. AD durations were positively related to the intensity of seizures. During seizures at stages 1~2, PLVs remained relatively low and increased dramatically shortly after the termination of the seizures; by contrast, for stages 3~5, PLVs remained a relatively low level during the initial period but increased dramatically before the seizure termination. And in the theta band, the degree of PLV enhancement was positively associated with seizure intensity. The results suggested that during epileptogenesis, the functional regions were kept desynchronized rather than hyper-synchronized during either the initial or the entire period of the seizures; so different dynamic patterns of phase synchronization may be involved in different periods of the epileptogenesis, and this might also reflect that during seizures at different stages, the mechanisms underlying the dynamics of phase synchronization were different.     

Ascorbate and Glutamate Release in the Rat Hippocampus After Perforant Path Stimulation: A "Dialysis Electrode" Study

Abstract: Excitatory amino acids have been proposed to play a critical role in the development and maintenance of epileptic seizures and in the development of neuronal damage. Previous animal studies of glutamate during seizures, however, have often failed to measure any rise in glutamate. We have overcome many of the problems of these studies by using an animal model in which epileptic afterdischarges are induced by stimulation of the perforant path, and glutamate and ascorbate are measured using a newly developed microdialysis electrode that combines the advantages of microdialysis and in vivo electrochemistry. We have successfully shown (1) a rise in glutamate after an epileptic afterdischarge, (2) a concomitant initial fall and then a later rise in ascorbate, and (3) progressive dwindling of this effect when afterdischarges are repeated within minutes, despite similar electroencephalographic responses. The possible mechanisms of these effects are discussed and include ascorbate/glutamate heteroexchange, reversal of the glutamate uptake mechanism, and augmentation of glutamate uptake after a seizure.