On the first day, the hole used for the virus injection was enlarged and the dura removed but on subsequent days the hole was simply cleaned with saline. The optrode assembly was fixed to a manipulator and lowered into the CA1 pyramidal layer. The hole was then sealed with liquid agar (1.5%) applied at near body Protein Tyrosine Kinase inhibitor temperature. Aluminum
foil was folded around the entire optrode assembly, which both served as a Faraday cage and prevented the mice from seeing the light emitted by the optical fibers. After the CA1 pyramidal layer had been reached, the mice were allowed to recover completely from the anesthesia. Recording sessions typically lasted for 1 h, during which the animal’s behavior alternated between periods of running and immobility. After each recording session, the probe was removed and the hole was filled with a mixture of bone wax and paraffin oil, and covered with silicon sealant (Kwik-sil; WPI). Each mouse was subjected to a maximum
of four recording sessions (one session per day). A diode-pumped solid-state laser (561 nm, 100 mW; Crystalaser) controlled selleck products by transistor–transistor logic (TTL) pulses was used for NpHR activation. To adjust the intensity of the laser, a neutral density filter wheel was placed in front of the beam. An optrode with four optical fibers was used (Fig. 2B), so the laser beam was first split with beam splitters (ThorLabs no. CM1-BS1) and diverted by reflecting mirrors (Thorlabs no. CM1-P01) into four separate fiber ports (ThorLabs no. PAF-X-7-A). Long single-mode optical fibers connected the fiber ports to the optrode fibers as described for the rat experiments. The behavior MG132 hardware (valves, motorized doors and light-beam sensing switches) and the laser power supply were connected to a computer board (no. NI PCI-6221; National Instruments) and controlled by custom-made LabView (National Instruments) and Python programs. Neurophysiological signals were acquired continuously at 32 552 kHz on a 128-channel DigiLynx system (Neuralynx, Inc). The wideband signals were digitally high-pass filtered (0.8–5 kHz) offline for spike
detection or low-pass filtered (0–500 Hz) and down-sampled to 1252 kHz for local field potentials. Spike sorting was performed semi-automatically, using KlustaKwik (available at http://osiris.rutgers.du), followed by manual adjustment of the clusters (Harris et al., 2000). Additional data analysis was done using custom Matlab routines. A well-known problem with short electric pulses, typically used for stimulation, is that they activate the neurons in a highly synchronous manner. As a result, spike waveforms of nearby neurons get superimposed and blended into population spikes (complex waveforms), and isolation of single neurons by clustering methods using spike waveform features becomes compromised. The same problem is expected when using short light pulses to activate ChR2-expressing neurons.