Corwin R Butler1, Jeffery A Boychuk2, Francois Pomerleau3, Ramona Alcala4, Peter Huettl3, Yi Ai5, Johan Jakobsson6, Sidney W Whiteheart7, Greg A Gerhardt8, Bret N Smith9, John T Slevin10. 1. Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States. 2. Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Epilepsy Center, University of Kentucky, Lexington, KY, 40536, United States. 3. Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Brain Restoration Center, University of Kentucky, Lexington, KY, 40356, United States. 4. Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States. 5. Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States. 6. Wallenburg Neuroscience Center, Lund Stem Cell Center, Lund University, Lund, Sweden. 7. Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, United States; Veterans Affairs Medical Center, Lexington, KY, 40536, United States. 8. Epilepsy Center, University of Kentucky, Lexington, KY, 40536, United States; Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40536, United States; Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Brain Restoration Center, University of Kentucky, Lexington, KY, 40356, United States. 9. Department of Physiology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Epilepsy Center, University of Kentucky, Lexington, KY, 40536, United States; Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, 40536, United States; Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States. 10. Epilepsy Center, University of Kentucky, Lexington, KY, 40536, United States; Department of Neurology, College of Medicine, University of Kentucky, Lexington, KY, 40536, United States; Veterans Affairs Medical Center, Lexington, KY, 40536, United States; Brain Restoration Center, University of Kentucky, Lexington, KY, 40356, United States. Electronic address: jslevin@uky.edu.
Abstract
BACKGROUND: Genesis of acquired epilepsy includes transformations spanning genetic-to- network-level modifications, disrupting the regional excitatory/inhibitory balance. Methodology concurrently tracking changes at multiple levels is lacking. Here, viral vectors are used to differentially express two opsin proteins in neuronal populations within dentate gyrus (DG) of hippocampus. When activated, these opsins induced excitatory or inhibitory neural output that differentially affected neural networks and epileptogenesis. In vivo measures included behavioral observation coupled to real-time measures of regional glutamate flux using ceramic-based amperometric microelectrode arrays (MEAs). RESULTS: Using MEA technology, phasic increases of extracellular glutamate were recorded immediately upon application of blue light/488 nm to DG of rats previously transfected with an AAV 2/5 vector containing an (excitatory) channelrhodopsin-2 transcript. Rats receiving twice-daily 30-sec light stimulation to DG ipsilateral to viral transfection progressed through Racine seizure stages. AAV 2/5 (inhibitory) halorhodopsin-transfected rats receiving concomitant amygdalar kindling and DG light stimuli were kindled significantly more slowly than non-stimulated controls. In in vitro slice preparations, both excitatory and inhibitory responses were independently evoked in dentate granule cells during appropriate light stimulation. Latency to response and sensitivity of responses suggest a degree of neuron subtype-selective functional expression of the transcripts. CONCLUSIONS: This study demonstrates the potential for coupling MEA technology and optogenetics for real-time neurotransmitter release measures and modification of seizure susceptibility in animal models of epileptogenesis. This microelectrode/optogenetic technology could prove useful for characterization of network and system level dysfunction in diseases involving imbalanced excitatory/inhibitory control of neuron populations and guide development of future treatment strategies.
BACKGROUND: Genesis of acquired epilepsy includes transformations spanning genetic-to- network-level modifications, disrupting the regional excitatory/inhibitory balance. Methodology concurrently tracking changes at multiple levels is lacking. Here, viral vectors are used to differentially express two opsin proteins in neuronal populations within dentate gyrus (DG) of hippocampus. When activated, these opsins induced excitatory or inhibitory neural output that differentially affected neural networks and epileptogenesis. In vivo measures included behavioral observation coupled to real-time measures of regional glutamate flux using ceramic-based amperometric microelectrode arrays (MEAs). RESULTS: Using MEA technology, phasic increases of extracellular glutamate were recorded immediately upon application of blue light/488 nm to DG of rats previously transfected with an AAV 2/5 vector containing an (excitatory) channelrhodopsin-2 transcript. Rats receiving twice-daily 30-sec light stimulation to DG ipsilateral to viral transfection progressed through Racine seizure stages. AAV 2/5 (inhibitory) halorhodopsin-transfected rats receiving concomitant amygdalar kindling and DG light stimuli were kindled significantly more slowly than non-stimulated controls. In in vitro slice preparations, both excitatory and inhibitory responses were independently evoked in dentate granule cells during appropriate light stimulation. Latency to response and sensitivity of responses suggest a degree of neuron subtype-selective functional expression of the transcripts. CONCLUSIONS: This study demonstrates the potential for coupling MEA technology and optogenetics for real-time neurotransmitter release measures and modification of seizure susceptibility in animal models of epileptogenesis. This microelectrode/optogenetic technology could prove useful for characterization of network and system level dysfunction in diseases involving imbalanced excitatory/inhibitory control of neuron populations and guide development of future treatment strategies.
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