Ered to 0.8?.9 in the same gas mixture as before. This anaesthetic level was characterized by an EEG dominated by 3? Hz theta waves (mean level, see fig. 1), with no signs of desynchronization during noxious stimulation. The blood pressure was stable also during noxious stimulation. Experiments were terminated after any signs of deterioration, i.e. precipitous drops in blood pressure or expiratory pCO2 levels.SDBefore comp/after comp = experiments with EEG dominant frequency compensation. doi:10.1371/journal.pone.0053966.tStimulation and recordings; Nobiletin web protocol and drug administrationRecordings of LCEPs and EEG were made from the contralateral cortical surface hindpaw representation area with fine silver ball-tipped electrodes (,0.3 mm diameter). See the Data analysis section for filtering parameters. Tactile input was used to locate the cortical representation of the glabrous skin of the digits, arch and heel of the left hind paw [12,14,19]. A hand-held mechanical stimulator with a blunt metal probe 18325633 (0.8 mm diameter) attached to a coil, was used for tactile stimulation. The probe was displaced by a current pulse generated by a Grass stimulator. The stimulation was adjusted to cause a light touch of the skin, without any visible joint movement. Radiant heat pulses emitted by a CO2laser (Irradia, Sweden; wavelength 10.6 mm, output power 15 W, beam diameter 3.0 mm, pulse duration 20 26001275 to 32 ms) were used to elicit LCEP. These stimulation energies have previously been shown to reliably evoke late cortical field potentials (onset latency exceeding 180 ms) in rat SI through the activation of cutaneous nociceptive C fibres [14,19]. No visible 478-01-3 site damage to the skin was observed using this stimulation. The pulse duration was adjusted to the local paw temperature (27?4uC) [27]. This corresponds to approximately 2? times the threshold for evoking LCEP. CO2 laser stimulation, consisting of trains of 16 pulses at a frequency of 1.0 Hz, of the glabrous skin of the hind paw was made to obtain averaged LCEPs. The stimulation sites were randomized in order to avoid repeated stimulation of the same sites (to avoid desensitization of C-nociceptors). In the beginning of each experiment, a baseline was obtained from at least 4 averaged LCEPs. The time interval between averages was set to 10 minutes. The first LCEP recording was not used in the analysis, as a stable baseline was obtained after the first train. EEG was sampled at regular intervals (for 45 s approximately every 5 minutes), always with at least two minutes pause after noxious stimulation. See figure 2 for an overview of the stimulation protocol. After control recordings, Midazolam 10 mmol/kg or Morphine 1 or 3 mg/kg was administered through the right jugular vein.The drug doses used were within the range of effective doses found previously in various models of nociceptive transmission [28?0]. After drug, averaged LCEPs were collected as above. Due to pharmacodynamics the first averaged LCEP obtained 5 minutes after drug was not used. Instead, 3 separate averaged LCEPs starting at fifteen minutes after drug application was used for analysis. In some experiments the level of Isoflurane was lowered to 0.6?.7 from 0.8?.9 (the level of oxygen/nitrous oxide was kept constant throughout the experiments) after drug administration to reverse the dominant EEG frequency to control level (see data analysis section).Data analysisThe signals (10 kHz sampling frequency) were amplified and filtered using Digitimer.Ered to 0.8?.9 in the same gas mixture as before. This anaesthetic level was characterized by an EEG dominated by 3? Hz theta waves (mean level, see fig. 1), with no signs of desynchronization during noxious stimulation. The blood pressure was stable also during noxious stimulation. Experiments were terminated after any signs of deterioration, i.e. precipitous drops in blood pressure or expiratory pCO2 levels.SDBefore comp/after comp = experiments with EEG dominant frequency compensation. doi:10.1371/journal.pone.0053966.tStimulation and recordings; protocol and drug administrationRecordings of LCEPs and EEG were made from the contralateral cortical surface hindpaw representation area with fine silver ball-tipped electrodes (,0.3 mm diameter). See the Data analysis section for filtering parameters. Tactile input was used to locate the cortical representation of the glabrous skin of the digits, arch and heel of the left hind paw [12,14,19]. A hand-held mechanical stimulator with a blunt metal probe 18325633 (0.8 mm diameter) attached to a coil, was used for tactile stimulation. The probe was displaced by a current pulse generated by a Grass stimulator. The stimulation was adjusted to cause a light touch of the skin, without any visible joint movement. Radiant heat pulses emitted by a CO2laser (Irradia, Sweden; wavelength 10.6 mm, output power 15 W, beam diameter 3.0 mm, pulse duration 20 26001275 to 32 ms) were used to elicit LCEP. These stimulation energies have previously been shown to reliably evoke late cortical field potentials (onset latency exceeding 180 ms) in rat SI through the activation of cutaneous nociceptive C fibres [14,19]. No visible damage to the skin was observed using this stimulation. The pulse duration was adjusted to the local paw temperature (27?4uC) [27]. This corresponds to approximately 2? times the threshold for evoking LCEP. CO2 laser stimulation, consisting of trains of 16 pulses at a frequency of 1.0 Hz, of the glabrous skin of the hind paw was made to obtain averaged LCEPs. The stimulation sites were randomized in order to avoid repeated stimulation of the same sites (to avoid desensitization of C-nociceptors). In the beginning of each experiment, a baseline was obtained from at least 4 averaged LCEPs. The time interval between averages was set to 10 minutes. The first LCEP recording was not used in the analysis, as a stable baseline was obtained after the first train. EEG was sampled at regular intervals (for 45 s approximately every 5 minutes), always with at least two minutes pause after noxious stimulation. See figure 2 for an overview of the stimulation protocol. After control recordings, Midazolam 10 mmol/kg or Morphine 1 or 3 mg/kg was administered through the right jugular vein.The drug doses used were within the range of effective doses found previously in various models of nociceptive transmission [28?0]. After drug, averaged LCEPs were collected as above. Due to pharmacodynamics the first averaged LCEP obtained 5 minutes after drug was not used. Instead, 3 separate averaged LCEPs starting at fifteen minutes after drug application was used for analysis. In some experiments the level of Isoflurane was lowered to 0.6?.7 from 0.8?.9 (the level of oxygen/nitrous oxide was kept constant throughout the experiments) after drug administration to reverse the dominant EEG frequency to control level (see data analysis section).Data analysisThe signals (10 kHz sampling frequency) were amplified and filtered using Digitimer.
