p.) anesthesia. For EMG recordings, fine tungsten wires (A-M Systems) were threaded into the whisker pad. After 4–7 days of habituation to head fixation, craniotomies over vM1 and S1 (<0.5 mm in diameter) were established SNS-032 nmr under isoflurane anesthesia using stereotactic coordinates (from bregma, vM1: 1 mm rostral, 1 mm lateral; S1: 1.5 mm caudal, 3.5 mm lateral). Recordings from waking mice commenced at least 1–2 hr after surgery, allowing recovery from anesthesia such that the animals appeared to be behaving normally in their own cages prior to head fixation. For

recordings under anesthesia, 2- to 3-month-old mice were sedated with chlorprothixene (5 mg/kg, i.p.) and anesthetized with urethane (0.7 g/kg, i.p.). The head-holder was adhered to the skull, and two or three craniotomies were established over vM1, S1, and V1 (from bregma, V1: 3.5 mm caudal, 2.25 mm lateral). For focal muscimol injections, a glass pipette containing 2 mM muscimol (Tocris) was lowered into vM1 or VPM (from bregma, VPM: 1.8 mm caudal, 1.5 mm lateral, 3 mm ventral) and slowly volume injected (0.5–0.7 μl over 10 min). Recordings were conducted 1–2 hr after muscimol injection. LFP/MUA signals were obtained with tungsten microelectrodes (0.3–1 MΩ resistance, FHC) or 16 channel multielectrode arrays (A16, 177 μm2 site area, NeuroNexus). Single microelectrodes were targeted to layer V at depths ranging from 750

to 850 μm, Fulvestrant whereas multielectrode arrays spanned the full cortical depth. Signals were processed through a preamplifier (Multichannel Systems) and amplifier (A-M Systems 3500), band-pass filtered between 0.3 and 5 kHz, and digitized at 10 kHz (Power 1401, CED). Blind” whole-cell recordings in vivo (Margrie et al., 2002) and IR-DIC guided whole-cell

recordings in vitro were targeted to layer V neurons. Standard patch pipettes (4–6 MΩ) were used containing 130 mM K-gluconate, 7 mM KCl, 4 mM Mg-ATP, 10 mM to Na-phosphocreatine, 0.3 mM Na-GTP, 10 mM HEPES, 0.2%–0.4% biocytin (pH 7.3 with KOH). Signals were processed using an AxoClamp-2B or Multiclamp 700B (Axon Instruments), filtered at 10 kHz, and digitized at 20–40 kHz. ChR2 was activated by an LED-based light source (460 nm, Prizmatix) and multimode optical fiber (0.37 NA, 300 μm diameter, 30 mW/mm2 maximum intensity at fiber terminus for stimulation in vM1; 0.48 NA, 1 mm diameter, 120 mW/mm2 maximum intensity for stimulation in S1 both in vivo and in vitro). The optical fiber was positioned at the meningeal surface above vM1 or S1 (in vivo) or approximately 1 mm above the brain slice (in vitro). Whereas continuous ramp illumination was used for vM1 stimulation, continuous or high-frequency repetitive illumination was used for axonal stimulation in vivo. Ramps were used instead of square pulse stimuli to minimize onset transient responses. Prolonged vM1 stimulation under anesthesia neither evoked whisker movements nor disrupted the spontaneous slow rhythmic whisker twitching in lightly anesthetized mice.