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Carefully monitoring the depth of anesthesia throughout the surgical procedures is crucial during in vivo neurophysiological studies. Although the variability in delivering inhalational anesthetics among the anesthetic vaporizers is very likely, this issue has been commonly ignored. Such variability could produce incongruities in results obtained from in vivo studies that can be erroneously interpreted largely due to inherent biological error. The purpose of this study, therefore, was to investigate the depth of anesthesia using the cortical EEG recording technique with carefully increased concentrations of isoflurane (ISO), a common inhalational anesthetic agent. The present study demonstrates the existence of concentration-dependent distinct changes in EEG waveform activity during the shift from light to deep ISO anesthesia in rats. The study was performed across three cohorts of male Sprague-Dawley rats (cohort I - control, cohort II - sleep-deprived continuously for 9 h and cohort III - recovery for 48 h). Initially, a relatively low isoflurane concentration (ISO%) produced continuous large-amplitude slow-wave baseline EEG activity within the delta and theta bandwidths (0-8 Hz). As the ISO% was increased the slow-wave activities fragmented into a burst-suppression pattern. As the concentration of ISO increased further the number of burst-suppression events decreased further and disappeared leaving an isoelectric EEG pattern. Further increases in ISO% (3% relative ISO) resulted in the emergence of FLAS-waves during very deep ISO%. Increasing the ISO% exerted a dose-response curve for burst events and FLAS-waves. This suggests that the cortical EEG can serve as a reliable biomarker of stable and consistent distinct planes of anesthesia by monitoring the desired number of burst events per minute. Since sleep and anesthesia share similar characteristics, it was appropriate to test whether sleep deprivation exerted any effect on the EEG activities during ISO anesthesia. The present study demonstrated that the dose-response curve for ISO on burst events was shifted to the right following sleep deprivation. Additionally, the finding of FLAS-waves in rats during very deep ISO anesthesia following burst-suppression events and the subsequent isoelectric period suggest a new arena of the brain physiology that may have multiple implications for the study of pain, coma, aging, cognition, etc.