rAAVs (AAV2/1; synapsin-1 promoter) were injected into the primary somatosensory cortex (S1) of 2–3 week old C57Bl/6Crl wild-type mice. Two weeks after injection, mice were anaesthetized with 2% isoflurane, and a 1.5mm circular craniotomy was performed over the injection site as previously described 43 (link). Cells were recorded with a patch pipette containing (in mM): 10.0 KCl, 140 K-gluconate, 10.0 HEPES, 2.0 MgCl2, 2.0 CaCl2, 0.05 Alexa 594, pH 7.25, 290 mOsm. For recording and stimulation a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, California) was used. In whole cell mode, action potentials were evoked by 2–5 ms long current injections; in cell attached mode currents up to 100 nA were necessary. The Ti:Sapphire laser (Mai Tai, Spectro-Physics, CA) was tuned to 910 nm for GCaMP3 imaging. Fluorescence images were simultaneously acquired using a custom-built, two-photon laser-scanning microscope equipped with a 40X, 0.8 NA objective (Olympus, Tokyo, Japan). Frame scans were acquired at 15 Hz (256×32 pixels) for a period of 3 seconds.
For imaging awake, head-fixed running mice, virus injection and surgery were identical to the anesthetized condition, except that the injection and craniotomy were performed over the primary whisker and forelimb motor area (M1). In addition, local (Marcaine) and general (Buprenorphine, 0.1mg/kg IP and Ketoprofen, 5mg/kg SC) anesthetics were administered. After full recovery on a heating pad the animals were head restrained, but allowed to run freely on a linear treadmill. Action potentials were recorded using a loose-seal cell attached configuration with patch pipettes filled with buffer (in mM: 125 NaCl, 2.5 KCl, 25.0 glucose, 10.0 HEPES, 2.0 CaCl2, 2.0 MgSO4, 0.05 Alexa 594; pH 7.4, 285 mOsm), and signals were amplified using a MultiClamp 700B (Molecular Devices, Sunnyvale, California). To confirm the identity of recorded neurons, each recording was terminated by breaking into the cell and filling with red pipette solution. During the imaging sessions the animals were kept alert by sporadic acoustic stimuli (clapping) or by presenting a pole or mild air puffs to the whisker field. Images were acquired at frame rates of 4–8 Hz at a resolution of 256×512 pixels using a 16X, 0.8 NA water immersion objective (Nikon USA, Lewisville, TX). All images acquired while awake were corrected for movement artifacts using the ImageJ plug-in TurboReg (http://bigwww.epfl.ch/thevenaz/turboreg/ ). ΔF/F was calculated by subtracting the baseline fluorescence level (F0, 35th percentile of total fluorescence) from the actual fluorescence level and normalized to F0.
For imaging awake, head-fixed running mice, virus injection and surgery were identical to the anesthetized condition, except that the injection and craniotomy were performed over the primary whisker and forelimb motor area (M1). In addition, local (Marcaine) and general (Buprenorphine, 0.1mg/kg IP and Ketoprofen, 5mg/kg SC) anesthetics were administered. After full recovery on a heating pad the animals were head restrained, but allowed to run freely on a linear treadmill. Action potentials were recorded using a loose-seal cell attached configuration with patch pipettes filled with buffer (in mM: 125 NaCl, 2.5 KCl, 25.0 glucose, 10.0 HEPES, 2.0 CaCl2, 2.0 MgSO4, 0.05 Alexa 594; pH 7.4, 285 mOsm), and signals were amplified using a MultiClamp 700B (Molecular Devices, Sunnyvale, California). To confirm the identity of recorded neurons, each recording was terminated by breaking into the cell and filling with red pipette solution. During the imaging sessions the animals were kept alert by sporadic acoustic stimuli (clapping) or by presenting a pole or mild air puffs to the whisker field. Images were acquired at frame rates of 4–8 Hz at a resolution of 256×512 pixels using a 16X, 0.8 NA water immersion objective (Nikon USA, Lewisville, TX). All images acquired while awake were corrected for movement artifacts using the ImageJ plug-in TurboReg (
