Abstracts

Rationale: Targeted control of neural systems to establish causality between neuronal activity and behavior has been difficult to achieve until the introduction of optogenetics, a technique to place light sensitive proteins (opsins) into neurons to regulate transmembrane ion conductance. Membrane potentials and neurotransmitter (NT) release, can be modulated by using blue light-activated channel rhodopsin (ChR2, depolarization) or yellow light-activated halorhodopsin (NpHR, hyperpolarization). The goal of these studies was to use optical modulation of hippocampal neurons to better understand entorhinal cortical-hippocampal networks that promote epileptogenesis by measuring variations in membrane potentials and real-time glutamate (Glu) NT release, and to induce/delay seizure development. Methods: Hippocampal Virus Injection: AAV5-hSyn-hChR2(H134R)-EYFP (~10e14 genome/ml), AAV5-hSyn-eNpHR 3.0-EYFP (~10e14 genome/ml); 1 l per single injection into DG (AP: -4.4 mm; ML: -2.2 mm; DV: -3.8) Laser Source/Optical Fiber Configuration: Glass Fiber optic (ز00 m Core, 0.39 NA); Laser source - blue (OBIS LS 488 nm, 100mW), yellow (LaserGlow 594 nm, 100mW) Electrochemistry: Glu-selective measurements, S2 microelectrode array (MEA) configuration, FAST16mkIII potentiostat (Quanteon LLC), second-by-second recordings (10 Hz data display), mPD size exclusion layer. Guide cannula for optical fiber 200 m from surface of MEA Electrophysiology: Whole cell patch-clamp, isolated and visualized DG Granule Cells; Optical activation: Sutter Lambda XL shutter system coupled to an Olympus BX51WI microscope with fluorescent optics. Behavioral Studies: ChR2 rats - Optical fiber implanted in DG, stimulated with blue light 2x day @ 50 mW; NpHR rats - kindling with bipolar electrode implanted in amygdala (100-800 A, 1 sec 2x day); optical fiber implanted in DG for yellow light stimulation (~40 mW) during kindling. Results: Opsin distribution: Enhanced Yellow Fluorescent protein expression (YFP) showed bilateral distribution of AAV5-CHR2-Syn-EYFP 6 weeks after a single injection into right DG. Merge of ChR2-YFP and GAD67-RFP showed localization of markers with GAD67- RFP in GABA cell bodies and ChR2-YFP in bouton-like clumps in granule cell neuritic field. Similar results were seen with NpHR-YFP. Light-induced Glu release: Blue laser-induced release of Glu in rat DG using MEA + waveguide array. Pulse trains (100 pulses) with variable pulse durations and constant blue laser activations using a range of 5-50 mW laser intensities were used to produce reproducible, but light dose-dependent, increases in extracellular Glu. Behavior: Blue light stimulation of DG kindled 2 rats to Racine stage 1 (7 stimuli, lost head gear), 1 to stage 2 (27 stimuli), 1 to stage 3 (15 stimuli) and 1 to stage 5 (18 stimuli). Following yellow light activation 5 secs prior to electrical stimulation of amygdala and throughout the seizures, NpHR animals required more stimulations (P < 0.03) to achieve stage 4/5 seizures (21.4 5.1 [SD]; n=5) than shams (11.0 5.4; n=4) or historical controls (8.2 3.1). Electrophysiology: AAV5-Syn-ChR2-EYFP resulted in predominantly direct activation responses whereas AAV5-Syn-NpHR-EYFP resulted in synaptic activation responses of dentate granule cells. Conclusions: Results demonstrate the feasibility of in vivo real-time measure by MEAs and exquisite control with optogenetics of neuronal/network Glu release. AAV5-Syn-ChR2-EYFP is expressed in Glu neurons; AAV5-Syn-NpHR-EYFP is expressed in GABA neurons, allowing selective activation to modify behavior. Blue light stimulation induced kindling in ChR2 rats; yellow light delayed amygdala kindling in NpHR rats. Funding: DARPA N66001-09-C-2080 The Department of Veterans Affairs