truly distinguishing characteristics of MMN [24,25] – true deviance detection. Adaptation in narrow frequency channels should not be considered as true deviance detection, since it is sensitive to the rarity of the deviant but not to the regularity of the standard. The current results are the first demonstration that oddball sequences might engage true deviance-detection mecha- nisms, rather than only adaptation in narrow frequency channels, already at the level of auditory cortex. Therefore, these findings strengthen the case for SSA in auditory cortex as an important contributor to the generation of MMN. Materials and Methods Preparation We used 26 adult female Sabra rats weighing 140–250 gm and 2 male Sabra rat juveniles, p27 and p34, for this study (Harlan Laboratories Ltd., Jerusalem, Israel). The joint ethics committee (IACUC) of the Hebrew University and Hadassah Medical Center approved the study protocol for animal welfare (protocols NS-06- 10041-3, NS-08-11349-3). The Hebrew University is an AAALAC International accredited institute. Animals were initially anesthetized with an intramuscular injection of ketamine (20–70 mg/kg, Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) and medetomidine (0.05–0.5 mg/kg, Domitor, Orion Pharma, Espoo, Finland). Additional smaller doses of ketamine were administered as needed to maintain anesthesia during surgery. Surgical level of anesthesia was verified by pedal-withdrawal reflex. The trachea was cannulated and the animal was fixed to a custom-made head holder [26], that left the scalp and ears free. The animal was ventilated through the tracheal cannula (10-15 mmH2O peak inlet pressure, 47/min, 15–30cc per stroke, 0.7–1.4 L/min) by a mixture of O2 and halothane (Rhodia Organique Fine Ltd., Bristol, UK) using a small-animal ventilator (model AWS, Hallowell EMC, MA), and a halothane vaporizer (VIP 3000, Matrx, NY). Once the animal was ventilated, ketamine anesthesia was discontinued, and halothane volume concentration was regulated around 0.5% to have a sufficient anesthesia depth. Throughout the experiment, respiration quality was monitored by continuously measuring the CO2 concentration in the tracheal cannula (Micro- cap, Oridion Medical Ltd., Jerusalem, Israel). The depth of anesthesia was judged by the lack of motion and resistance to the respirator, and levels of anesthetics and ventilation pressure were adjusted accordingly. Body temperature was monitored and maintained at 36–38uC using a rectal thermistor probe and a feedback-controlled heating pad (FHC Inc., ME). The left temporal portion of the skull was cleaned from skin, muscles, and connective tissue. A craniotomy was performed over the estimated location of left auditory cortex – 2.5mm–6.5mm posterior to and 2mm–6mm ventral to bregma [27]. A copper wire hook implanted in the neck muscles was used as the electrical reference. Electrophysiological recordings We recorded extracellularly from the auditory cortex using 1–4 glass-coated tungsten electrodes (Alpha-Omega Ltd., Nazareth- Illit, Israel), or a single micropipette. Metal electrodes were assembled together with separations of ,600 microns. The electrodes were lowered into the cortex using a microdrive (MP- 225, Sutter Instrument Company, Novato, CA). The electrical signals were pre-amplified (610), filtered between 3 Hz and 8 kHz to obtain both LFP and action potentials, and then amplified again, for a total gain of 65000 (MCP, Alpha- Omega, Nazareth Illit, Israel), to yield the raw signals. The raw Deviance Detection in Rat Auditory Cortex signals were sampled at 25 kHz and stored for off-line analysis. The analog signals were also sampled at 977 Hz after anti-aliasing filtering (RP2.1, TDT, Tucker-Davis Technologies, Alachua, FL), stored for LFP analysis, and used for online display. Sound stimulation system All experiments were conducted in a sound-proof chamber (IAC, Winchester, UK). Sounds were synthesized online using Matlab (The Mathworks, Inc., Natick, MA), transduced to voltage signals by a sound card (HDSP9632, RME, Germany), attenuated (PA5, TDT), and played through a sealed speaker (EC1, TDT) into the right ear canal of the rat. In several animals, an acoustic calibration was performed as follows. A post-auricular incision was made and the meatus was cut as close to the skull as possible. A custom-made brass cone with speaker and microphone inlets was fastened so as to cover and seal the meatus in front of the tympanic membrane. The microphone (model EK-3133-000, Knowles, England) was previously calibrated against a calibrated condenser microphone (Type 2633, Bru¨ el & Kjær, Denmark). Frequencies in the range of 1–64 kHz, with 20 frequencies per octave, were presented through the sealed speaker. When harmonic distortion in the microphone signal was larger than 0.5%, presentation level was reduced; the largest attenuation level at which harmonic distortion was lower than 0.5% was measured as well, and was found to be higher than 80 dB SPL for all tested frequencies. The intensity of stimuli during the experimental paradigm did not exceed this value. The calibration of the system was found to be stable across animals. The intensity deviations between the pairs of frequencies that are reported here did not exceed 610dB near the tympanic membrane. In those experiments where calibration was performed, the speaker, the microphone and the brass cone were left in their position throughout the experiment. For pure tones, attenuation level of 0 dB corresponded to about 100 dB SPL. Noise stimuli were synthesized at a spectrum level of 250 dB/sqrt(Hz) relative to pure tones at the same attenuation level. Experimental procedure Recording sites were selected by their response to a broad-band noise (BBN). We searched for sites while continuously presenting 200 ms BBN bursts (0–50 kHz) with inter-stimulus time interval (ISI, onset to onset) of 500 ms and a level of 30 dB attenuation. The LFP responses were averaged online, and the electrodes were positioned at the location and depth that showed the largest evoked LFP responses over all the electrodes. Once selected, we validated and recorded the BBN responses of the recording site using a sequence of 280 BBN bursts with duration of 200 ms, 10 ms linear onset and offset ramps, ISI of 500 ms, and seven different attenuation levels, between 0 and 60 dB with 10 dB steps, that were presented pseudo-randomly so that each level was presented 40 times. The main data were collected if noise threshold level was at least 30 dB attenuation and noise-evoked potentials changed regularly with level; otherwise, the electrodes were moved to a different location. Quasi-random frequency-level sequence of 777 tone bursts (50 ms duration, 5 ms onset/offset linear ramps, 500 ms ISI) at 37 frequencies (1–64 kHz, 6 tones/octave) and 7 attenuation levels (80–20 dB, 10dB steps, roughly corresponding to 20–80 dB SPL) were used to measure the frequency response area (FRA) of the recording site (3 presentations at each frequency-level combina- tion). When the FRA was narrow or not smoothly graded with level, 370 tone bursts (300 ms ISI) at 37 frequencies (1–64 kHz, or narrower ranges when the FRA was narrow) were presented at a fixed attenuation level, in order to better characterize the PLoS ONE | www.plosone.org 15 August 2011 | Volume 6 | Issue 8 | e23369