Introduction

1. Effects of DC Electric Field on Synaptic Plasticity and Epileptiform Activities in Thalamocingulate circuitryWei-Pang Chang 1, Jing-Yun Chen 2 ,and Bai-Chuang Shyu 31,3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, R.O.C. 2Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, R.O.C Spontaneous PTZ induced seizure 500 3 s Anodic DC field mediated potentiation Introduction Seizure affects 1 % of population, and 30 % among them suffered from drug-resistant epilepsy. Clinical application of transcranial magnetic stimulation, transcranial current stimulation (tDCS) and direct current (DC) field stimulation provide non-invasive approaches for the treatment of drug-resistant seizures. Previous studies showed that field stimulation could modulate synaptic plasticity as well as influence epileptiform activities in various brain regions, such as in motor cortex and hippocampus. However, seldom research focus on the field effect on synaptic transmission within thalamocortical system, the important circuitry in sensory processing and in generating epileptiform activities. The medial dorsal (MD) thalamus are heavily connected to anterior cingulate cortex (ACC) and medial prefrontal cortex (mPFC) and could regulate seizure activities in cortical regions. Seizure generated in mPFC and ACC are often drug-resistant and alternative treatment such as field stimulation needs to be evaluated in this brain region. Therefore, the current study is aimed to investigate the effect of DC field stimulation on the changes of thalamo-cingulate synaptic plasticity and seizure-like activities generated within this circuitry. Results Field modulation on thalamic evoked epileptiform activities A B C A C D E F D B Figure 2. A. Cartoon outline indicated the relative position of brain slice and electric field. Electrical stimulation is in the thalamus and channels in cortex (green square) were selected for subsequent analysis. B. Comparison among thalamic evoked baseline activities and during 20 µA anodic DC stimulation. C. Negative slope of thalamic evoked activities were plotted against time. Noted that after anodic DC stimulation, the potentiation could last. Some channel are not subjected to potentiation. D Potential profile was plotted as isopotential profile. E. Cartoon outlined indicated the electric field was changed to cathodic stimulation. F. None of the evoked responses recorded by each channe was potentiated. Figure 6.A. Cartoon outline indicated the relative position of brain slice and electric filed. B Typical examples of thalamic evoked seizure-like activities in 100 µA anodic DC, control and 100 µA cathodic DC group. Noted that anodic DC application augmented thalamic evoked seizure-like activities while cathodic DC suppress them. C. Isopotential changes of first 100ms after thalamus stimulation. Noted that anodic DC application enhance the propagation of seizure-like activities. D. Statistical results showed that anodic DC application significantly augment thalamic evoked seizure-like activities, while cathodic DC significantly suppress the duration of thalamus evoked seizure-like activities. Anodic DC mediated potentiation is NMDA-R dependent Aim 1. To investigate the mechanism of DC field on synaptic transmission in thalamocingulate circuitry Furosemide block the augmentation effect of anodic DC A B C 2.To investigate the mechanism of DC field on modulation of seizure like activities in thalamo-cingulate circuitry. Materials and Methods A C MEA recording MEA 500/30ir (6x10MEA: 30 µm in electrode diameter; the electrode spacing is 500 µm) were used. Slices were continuously perfused with warmed and oxygenated aCSF in rate of 10 ml per minute. The local field potential (LFP) at each electrode was recorded against the bath electrode. Data were acquired by the PC based data software MC_Rack (Multi Channel Systems). Data were analyzed with MEA-Tools (U. Egert 1998) and lab-made program written in MATLAB 6.0 (The MathWorks, Inc, MA, USA). D E B Brain slice preparation Slices were prepared from 2~4 weeks anaesthetized C57/B6 mice. After decapitation the brain was transferred into oxygenated ACSF for 3 min. To keep MT-ACC pathway, the brains were trimmed 2 sagittal cuts from lateral and 2 cuts at ventral side by hand. The brain block was glued on a flexible plastic sheet and was made a slight cut just above the turning point of MT-ACC pathway. Then the sheet was unfolded and glued on the stage of Vibratome and 500 µm slices were made on it. Figure 3. A. Cartoon outline indicates the relative position of brain slice and electric field. B. The slope of thalamic evoked activities were plotted against time. Noted that application of APV 50 µM could block anodic DC mediated potentiation. C. Comparison of thalamic evoked activities among baseline, during application of 20 µA anodic DC and post control under the effect of NMDA-receptor antagonist APV 50 µM. D. The potentiation effect of anodic DC in control and in APV groups. E. Statistical results showed that APV could block anodic DC mediated potentiation. Furosemide block anodic DC mediated potentiation Figure 7.A. Ensemble seizure-like activities in 4-AP+BIM and furosemide application group. Noted that furosemide application suppress seizure-like activities. B. Statistical results showed that furosemide application could significantly lower the amplitude and duration of seizure-like activities. C. Electrical stimulation in thalamus could evoke seizure-like activities in the cortex under the application of furosemide 1.5mM. However, the augmentation effect of anodic DC on seizure-like activities were blocked by the application of furosemide. A B C Cathodic DC attenuate PTZ-induced seizure-like activities in vivo Generation of electric fields Uniform electric fields were generated by passing current between two parallel AgCl-coated silver wires place inside the MEA chamber. Currents were generated by a stimulator (A-M Systems, Inc. Carlsborg, WA, USA) under the control of a pulse generator (STG 1002, Multi Channel Systems) A C Figure 4. A. Cartoon outline indicated the relative position of brain slice and electric field. B. Comparison of thalamus evoked cortical activities among baseline, during DC application and post control under furosemide 1.5mM application C. The slope of thalamic evoked activities were plotted against the time under the effect of furosemide. Noted that application of furosemide 1.5mM could block DC mediated potentiation. Effect of DC field on spontaneous seizure-like activities Experimental setup B A A B D C D B Figure 8.A. Cartoon outline indicated the relative position of recording and stimulating electrodes within the brain. Red and Green dot indicated the location of field stimulation electrodes. B. During anodic DC application, electrical stimulation in contralateral corpus callosum could induce seizure like activities. C. Results showed that anodic DC could promote seizure, while cathodic DC application could suppress seizure. D. Cathodic DC could suppress spiking activities elicited by corpus callosum stimulation. Summary Synaptic transmission : 1. nDC-mediated potentiation might be caused by increasing concentration of neurotransmitters. 2. Potentiation is NMDA receptor dependent Seizure-like activities : 1. pDC could suppress spontaneous and thalamic-evoked cingulate seizure-like activities, while nDC has opposite effects 2. Field effect on seizure-like activities might caused by the alteration of extracellular potassium concentration which is mediated by NKCC cotransporter. Figure 5. A. Typical seizure-like activities were selected from different treatments. During continuous 20 µA anodic DC treatment, the seizure-like activities are enhanced, while during continuous 20 µA cathodic DC treatment, the seizure-like activities were suppressed. While two other direction of field did not modulate seizure-like activities. B. The statistical summary showed that anodic DC can significantly enhance seizures, while cathodic DC has opposite effect. C. Upper panel: different strength and direction of field was applied, lower panel: seizure duration were recorded in a time lapse experiment. D. Typical traces selected from different treatments were depicted. Figure 1. A. Cartoon outline depicts the experimental setup. Slices were continuously perfused with oxygenated ACSF. Electrical signal was mointered by axon 2A, and field was generated by sliver wire connect to a AM pulse generator. B. Left panel: Golgi stain showed the axon-dendritic orientation in ACC. Right panel: The applied electrical field is in parallel with the axon-dendritic orientation.