Abstracts

Rationale: Epileptic syndromes are frequently accompanied by changes in interneuron properties and numbers. Parvalbumin-positive (FS) interneurons exert strong perisomatic inhibition onto cortical pyramidal cells and can veto spike generation. To investigate their influence on cortical circuit properties we dynamically silenced FS interneurons via activation of the light-gated chloride pump halorhodopsin (eNpHR). We hypothesize that silencing of FS interneurons leads to a pronounced enhancement of cortical excitability. Methods: Mice (3-4 weeks old) expressing cre-recombinase under the control of the parvalbumin promoter (parv/cre-mice) were infected with an AAV vector ( AAV5-EF1a-DIO-eNpHR3.0-EYFP ) containing a doublefloxed eNpHR-EYFP construct controlled by the EF-1a promoter. Virus was stereotaxically injected into each hemisphere of the primary somatosensory cortex. Expression efficiency and localization were evaluated using EYFP fluorescence at time points from 10 days to 7.5 weeks post injection. Activation of eNpHR was achieved using a xenon arc lamp that illuminated the slice through the microscope objectives and a 593 /- 20 nm (yellow) bandpass filter. Inhibitory and excitatory postsynaptic responses were evoked by electrical stimulation and laser-scanning photostimulation/glutamate uncaging (LSPS), and cells were recorded in current and voltage clamp using standard techniques. Results: Strong EYFP expression was detected up to 1 mm from the injection site. Healthy fluorescent cells were observed 2-4 weeks post injection, but by 5 weeks post injection, many strongly fluorescent cells had formed inclusion bodies and were no longer suitable for electrophysiological recordings. Subsequent experiments were therefore conducted 2-3 weeks post injection. Fluorescent cells had light-activated currents of -195 /- 65 pA (n=14), corresponding to a hyperpolarization of 24 /- 10 mV. Stimulation in layer 2/3 elicited complex postsynaptic responses in layer 5 pyramidal cells, consisting of an early EPSC and a later IPSC, which presumably originated via mono- and disynaptic activation, respectively. In the presence of yellow light the IPSC component was reduced or absent in over 50% of recorded cells. In current clamp recordings, repetitive activation of eNpHR by trains of yellow light caused a 2-3-fold increase in spike output at all stimulation frequencies tested (10, 50 and 100 Hz). Conclusions: These experiments demonstrate the feasibility of a) directing eNpHR expression to FS cells in somatosensory cortex and b) eNpHR mediated disinhibition of pyramidal cells. These methods can now be used to investigate the specific role of FS interneurons in controlling epileptiform activity in slices, and seizures in vivo.