Protocol for Rat Sleep EEG

Transcription

1 Protocol for Rat Sleep EEG Subjects Male Spraue Dawley rats weihin rams at the time of surery are used. Food and water are available ad libitum throuhout the experiment. Rats are roup housed prior to surery, and followin surery they are housed individually in polypropylene shoebox caes (47 x 26 x 20 cm). A 12h:12h liht:dark cycle and constant room temperature are maintained. Surery Sleep-wake states are identified electrophysioloically usin chronically implanted EEG (N=4) and EMG (N=2) electrodes. The electrodes are implanted usin stereotaxic coordinates. Anesthesia and analesia are maintained usin the followin dru cocktails: AGENT DRUG (concentration) DOSE Induction Ketamine 90m/ (100m/ml) k Induction Xylazine (20m/ml) 9m/k Induction Acepromazine 2m/k (10m/ml) Maintenan Isoflurane/O ce 2% Analesia Metacam (5m/ml) 1m/k Hydration Lactated Riner's 10m/ solution k Reversal Antisedan (5m/ml) 1m/k ROUTE Inhalan t The EEG electrodes used are stainless-steel jeweller's screws threaded into holes drilled throuh locations on the skull as described (Mistlberer et al, 1983, 1987; Pollock & Mistlberer, 2003). Slow wave and spindle neocortical EEG activity are obtained by differential recordins from two electrodes positioned in the lateral parietal (P) and lateral occipital (O) cortices. Hippocampal theta rhythms are

2 obtained by differential recordins from two electrodes positioned in the dorsal hippocampus (H) and the frontal cortex (F) as a reference. EMG is recorded from two subcutaneous wire electrodes implanted between the occipital bone and the neck muscle (M). An extra screw (X) is placed on the skull above the cerebellum to provide additional anchor support. The followin stereotaxic coordinates relative to landmarks on the skull, based upon the rat brain stereotaxic atlas by Paxinos & Watson (1997), are used to determine electrode placements: LOCATION A-P (mm) M-L (mm) D-V (mm) Parietal -1.0 from cortex Brema Occipital +1.0 from cortex Lambda Hippocampus -4.3 from Brema Frontal cortex +3.0 from Brema The pins from all 6 electrodes are connected to a protective plastic headcap. The entire electrode assembly is insulated and bonded to the skull with dental acrylic.

3 Dru test procedure & sleep recordin Followin one week recovery from surery, an additional week is spent handlin the rats (for 15 min/day) and allowin them to habituate to sleep recordin conditions. Durin this habituation period, sample sleep recordins are taken to ensure proper functionin of the electrodes and to determine polyraphic sinal amplitude cutoffs for each individual rat (see below). Recordin chambers consist of plexilass chambers (37 x 34 x 53 cm) within electrically-shielded and sound-attenuatin enclosures (Model E3125AA-3 Animal Chest, Grason-Stadler Co., West Concord, MA). Each recordin chamber is equipped with commutators (Plastics One Co.) to allow free movement of the animals while bein connected to EEG recordin cables, overhead passive infrared motion detectors (modified versions of Model , RadioShack Co.) to detect locomotor activity, and observational windows throuh which video recordins can be taken. Fresh air is continuously pumped into the recordin chambers, which are housed in a temperature- and liht-controlled isolation room with a white noise enerator. Durin experimental recordins, no person is present in the isolation room to prevent possible disturbances of sleep. Followin this adaptation period, the rats are housed in the recordin chambers for periods of two consecutive days: one day for a baseline recordin followed by a second day for a dru/vehicle test recordin session. Prior to each baseline recordin day, the animals are placed in the recordin chambers at the beinnin of the dark period (their usual wake phase of the circadian cycle). On each recordin day, the rats are removed from their recordin chambers for a brief period (30 min.) immediately after liht onset (the beinnin of their usual sleep phase) in order to chane their food, water, and beddin. On dru test days, each animal receives an injection immediately before bein returned to the recordin chamber. As soon as the animal is reconnected to the EEG recordin cable, the baseline or dru/vehicle test recordin commences, and continues for 23.5 hours. This procedure permits assessment of the wake promotin effects of the dru when sleep propensity is hihest; by recordin continuously for a full day, extended wakin effects and/or subsequent rebound increases in sleep (if any) are detectable. At the end of each 2-day recordin period, the rats are permitted 2 days of recovery before the next 2-day recordin session, thus providin a four-day interval between injections. Each rat is tested with this procedure for each condition in the study desin (e.., 5 times to assess vehicle control, 3 dru doses, and a positive control, the latter bein caffeine, modafinil or amphetamine). The order of treatments is balanced across rats.

4 Baseline day1 Experimental Schedule One week recovery followin surery One week habituation to sleep recordins Test Baseline Test day1 day2 day2 Baseline Test Baseline day3 day3 day4 Baseline Test day5 day5 Test day4 Polyraphic sinals are amplified and bandpass filtered ( and Hz for EEG and EMG, respectively) by a polysomnoraph (Grass Model 9, Grass Instruments Co.). Alon with sinals from the motion detectors, these polyraphic sinals are diitized (samplin rate of 250 Hz) and stored on a computer usin data collection software (SleepSin, Kissei Comptec Co.) for subsequent off-line analysis. Data analysis Waveform reconition analysis of EEG sinals is performed usin the followin parameters: WAVEFORMS Slow waves Theta rhythms Spindles EEG DERIVATION Parietal- Occipital Hippocampal- Frontal Parietal- Occipital AMP MIN AMP MAX FRQ MIN FRQ MAX HALF- WAVE COUNTS +67% 1 Hz 3 Hz 1-50% +50% 5 Hz 9 Hz % 10 Hz 15 Hz 4 Slow wave and spindle amplitude cutoffs are set relative to typical desychronized EEG levels seen in active wakefulness and REM sleep. Theta rhythm amplitude cutoffs are set relative to the typical theta rhythm amplitude exhibited in REM sleep. Behavioral states are scored in 10-second epochs accordin to standard criteria (Pollock & Mistlberer, 2003). Each epoch is

5 classified as to the predominant state. The followin seven sleep/wake states are reconized: BEHAVIORAL STATE MOTIO N EMG SLOW WAVE S SPINDLE S Locomotor + Wakin Active - ++ Quiet THETA RHYTHMS Slow wave - ± + NREM Transitional - ± + sleep Lowamplitude REM sleep The minimum EMG interal cutoff for active wakefulness is set to within 25% of maximal EMG interal levels, which can be determined when the rat is first placed in the recordin chamber and is actively locomotin. To accommodate phasic twitches which can occur within REM sleep, the maximum EMG interal cutoff for low-amplitude sleep and REM sleep is set to within 300% of minimal EMG interal levels, which can be observed durin epochs of REM sleep with complete muscle atonia. Sleep-wake states (averaed into hourly time intervals throuhout the recordin day) are raphed and evaluated for dru and time effects usin repeated measures ANOVA and Bonferroni post-hoc tests (GraphPad Prism,Version 4.00, GraphPad Software, Inc.). Behavioral state episode durations and frequencies, and fast Fourier transform analysis of delta power (1-4 Hz) in all SWS epochs, are similarly analyzed.