Summary data are reported as mean ± SD, and all statistical tests were Student’s t test unless noted otherwise. After recordings, slices were fixed overnight in 4% paraformaldehyde solution in PBS, at 4°C. To confirm the injection site,

samples were imaged with a confocal or a tiling wide-field imaging microscope (LSM 510 or Axio Imager Z2, Zeiss). To identify the recorded cells, biocytin was reacted to Selleckchem CX-5461 streptavidin conjugated with Alexa 594 (Invitrogen) in 0.1% PBS-Tx overnight and samples were imaged with a Zeiss LSM 510 and 710 confocal microscope. The fluorescence intensity of confocal images was analyzed by image processing in ImageJ. Two to five weeks postinjection rats were anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg). A head-fixing plate was glued on the skull a small craniotomy was performed over the right bulb, ipsilateral to the injected AON, and the dura was removed. Extracellular signals from MCs were recorded with sharp tungsten electrodes (1–10 MΩ; FHC). Breathing

signals were monitored with a piezoelectric stress sensor (Kent Scientific) that was wrapped around the learn more mouse thorax. MCs were identified based on depth, respiration related firing pattern, and by monitoring the activity levels in more superficial layers. ChR2 was activated with a blue laser (450 nm, ∼60 mW/mm2 on the brain surface). Stimuli consisted of a pair of 40 ms pulses of light delivered 50 ms apart. Light intensity for in vivo experiments was greater than that used for in vitro experiments to ensure adequate penetration of the light through tissue. In both sets of experiments, light intensity and duration was kept within limits that typically do not cause heating effects in tissue (Cardin et al., 2010; Han, 2012).

second Odors were delivered from a custom-built olfactometer containing the following odors: methyl tiglate, ethyl valerate, isopropyl tiglate, ethyl butyrate, hexanal, heptanal, and isoamyl acetate. All odors were dissolved in diethyl-phthalate to a concentration of 10%. Odors were delivered by a stream of clean air (0.6 l/m) that was passed through vials containing the diluted odors. The airflow at the nose port was constant to ensure that that the responses obtained are not caused by a sudden change in air flow near the nose. Odors were delivered for 5 s every 45 s. Signals were amplified and filtered: 300 Hz to 5 kHz (A-M systems). Both breathing and MC activity signals were acquired at 20 kHz sampling and digitized with 16 bit precision (National Instruments). Data were analyzed using MATLAB (MathWorks). Spikes were sorted manually based on their projections in the principal component space and a refractory period was used for validation. Only single unit data are presented here. For analysis of the breathing signals, we defined peak inhalation as phase zero.