| Epilepsy, one common chronic brain disorder, is increasingly recognized as reflecting abnormality in terms of neuronal networks and clinical recurrent seizures, the prevalence rate is about 0.7-1%. Despite new antiepileptic drugs constantly being invented, targeting novel ion channel, GABAa receptor function and other sites, there are still about 30% of epilepsy patients do not respond to a variety of anti-epileptic drug and become refractory epilepsy, which may due to complex neural circuits/channel/ receptor mechanism underlying epilepsy. Meanwhile, the current available anti-epileptic drugs are only effective on seizure onset, but not on prevention of epileptogenesis. Temporal lobe epilepsy (TLE) is the most common type of refractory epilepsy, as high as 75% of the middle TLE with seizure focus at hippocampus and amygdala (hippocampus is the most common) eventually develop refractory epilepsy. If originated in the focal type of seizure activity spread to the whole brain, secondary generalized seizures (GS) would happen, up to 70% of patients with partial epilepsy occasionally experience secondary GS. Poor control of those secondary GS is the most important risk factor for sudden unexpected depth in epilepsy and seizure-related serious injuries. Surgery resecting seizure focus may allow some patients to have no seizure in a short term, however, limited patient are suitable for surgery and even 30% of patients after surgery still have seizure relapse in 3-5 years. Furthermore, the resected areas (hippocampus, amygdala and other limbic system structures) in surgery are closely related with brain function of learning memory and emotion, and a variety of patients are still suffering from cognitive dysfunction and other complications. Therefore, it is an urgent need to find other effective interventions for temporal lobe epilepsy treatment or novel drug targets.Electrical stimulation is a new selection for treatment of brain disorder, which convey a certain frequency, intensity, pulse duration of the electrical stimulation to a specific brain target, adjusting the neural tissue and its related neural projection, to achieve the therapeutic purpose. It is minimally invasive, adjustable and reversible (which can be removed after the device implantation) compared with surgery. Currently, vagus nerve stimulation (approved by the FDA), as one common adjuvant treatment of refractory epilepsy, has gradually been applied. However, the anti-epileptic effective rate of this approach is a low, and without no side effects, restricting its application. On the other hand, deep brain stimulation (DBS) in animal study and clinical trials have been shown to have good prospects, and a large multi-center clinical study were conducted in 2010 to investigated the effect of DBS at anterior thalamic nuclei on epilepsy, and the initial data showed some promising results. Recently, our previous studies have shown that low-frequency electrical stimulation (LFS) both targeting at seizure focus and other multiple regions outside seizure focus have significant anti-epileptic effect, and seem to be safer than high-frequency stimulation. However, due to the unclear mechanisms of DBS, the optimal stimulation target and parameters are still unknown, leading to many conflicting reports existing. Such as, the effect is not the same even in same targets target and some targets aggravate seizures. Therefore, it is necessary to clarify the mechanism of DBS and to study its antiepileptic characteristics. On the first part of this thesis, we have studied the LFS inhibits epileptogenesis by modulating the early network of the limbic system.The non-specific basis of electrical stimulation makes it difficult to destruct the neural circuit mechanism of epilepsy. Currently, optogenetics, the latest technology developed (top technology of 《Nature》 in 2010), that is microbial opsin genes can be introduced to achieve optical control of defined action potential patterns in specific targeted neuronal populations within freely moving mammals or other intact-system preparations to achieve gain-or loss-of-function of well-defined events. For example, ChR2 is a microbial cation channel, blue light can selectively open ChR2 channels, thereby depolarizing membrane of neurons and enhancing the firing rate, when specifically introduced into specific neurons; Arch is a microbial proton pump, yellow light can selectively open Arch pump, thereby hyperpolarizing membrane of neurons and lowing the firing rate of specific neurons. This technology has been gradually applied to destruct the neural circuit of pain, depression, anxiety, and other neuropsychiatric diseases. Thus, the emergence of this technology makes it possible to destruct the neural circuit of TLE and thus develop new specific approach. In the second part of the thesis, we focus on a disinhibitory microcircuit of subiculum mediates secondary GS in TLE and further develop specific intervention.Part 1 LFS inhibits epileptogenesis by modulating the early network of the limbic systemLFS is emerging as a new option for the treatment of epilepsy. The present study was designed to determine whether there is a crucial period for the treatment of epileptogenesis with LFS. LFS was delivered at different time-points to evaluate its anti-epileptogenic effect on amygdala-kindling rats. F18-fluoro-deoxyglucose small animal positron-emission tomography (microPET) and multi-channel EEG recording (MER) were used to investigate the dynamics of brain networks during epileptogenesis and LFS treatment. Interestingly, LFS delivered in the first 7 days significantly retarded the progression of behavioral seizure stages and shortened the afterdischarge duration (ADD), LFS delivered throughout the whole process resulted in similar effects. However, if LFS was delivered at the beginning of seizure stage 2 or 3 (5 ± 0.3 days during kindling acquisition), it had no anti-epileptogenic effect and even prolonged the ADD and enhanced synchronization of the EEGs. MicroPET study revealed a notable hypometabolism in the amygdala, piriform cortex, entorhinal cortex and other regions in the limbic system during the period from seizure stage 0 to stage 2 or 3. The glucose metabolism in those regions was specifically increased by LFS. MER further verified that an early network of afterdischarge spread was formed in those brain regions during kindling acquisition. Thus, we provided direct evidence that modulation of the early network in the limbic system is crucial for the anti-epileptogenic effect of LFS in amygdaloid-kindling rats.Part 2 A disinhibitory microcircuit of subiculum mediates secondary generalized seizure in temporal lobe epilepsySecondary GS is one major source of disability in TLE, however the underlying cellular or circuit mechanisms still remain unclear. Here we found the photostimulation of subicular GABA-ergic neurons (or PV interneurons) genetically targeted with channelrhodopsin-2 (ChR2) delayed GS acquisition from focal seizures by inhibiting the pyramidal neurons. While once GS were stably acquired, photostimulation of subicular GABA-ergic neurons (or PV subtype) entrained a large proportion of pyramidal neurons and thus aggravated the GS. The pro-epileptic effect was reversed by a selective NKCC1 inhibitor bumetanide or inactivation of GABA-ergic neurons, indicating a depolarized GABA-ergic signaling in subiculum. Further, selective inhibition of subicular pyramidal neurons to mimic activation of GABA-ergic neurons, with a proton pump archaerhodopsin-3 (Arch), but not a chloride pump Halorhodopsin-3.0 (NPHR3.0), protected against GS. These results demonstrated a disinhibitory microcircuit of subiculum mediates secondary GS in TLE, which may be of therapeutic interest in understanding the neural circuitry underlying pathological patterns in it and further controlling seizures with specific interventions. |
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