Chemicals, Solutions, and Materials
Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride, potassium chloride, and calcium chloride were purchased from Fisher Scientific (Pittsburgh, PA, USA). All solutions were made with 18.2 MΩ MilliQ water (Millipore Corporation, Billerica, MA, USA). Ringer’s solution consisted of 147 mM NaCl, 1.22 mM CaCl2, 2.68 mM KCl, and 1.01 mM MgSO4 at pH 6.0. Electrophoresis buffer contained 10 mM sodium tetraborate and 0.9 mM hydroxypropyl-β-cyclodextrin (0.8 molar substitution) at pH 9.5. Derivatization solution was comprised of 42.6 mM o-phthaldialdehyde, 148.6 mM β-mercaptoethanol, 9 mM sodium tetraborate, 0.81 mM hydroxypropyl-β-cyclodextrin and 10% (vol/vol) methanol at pH 9.5. Fused silica capillary was purchased from Polymicro Technologies (Phoenix, AZ, USA).
Animals, Surgery, and Conditioning
All experiments were approved by the University of Michigan Committee on Use and Care of Animals and adhered to guidelines established by the Guide for the Care and Use of Laboratory Animals, Eighth Edition (National Academies Press, Washington DC, 2011). Male, adult, Crl:CD*(SD) (Sprague Dawley) rats weighing 235 – 250 g were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed in a temperature and humidity controlled environment. Rats were kept on a 12 h light/dark cycle (lights on at 0600), had ad libitum access to food and water, and were allowed a minimum of one week for adjustment to the housing facilities before undergoing any procedures.
Detailed descriptions of surgical methods for implanting a guide tube for microdialysis and electrodes for recording the cortical EEG and electromyogram (EMG) have been published (Watson et al. 2007
, Watson et al. 2008
, Brevig et al. 2010
). Briefly, isoflurane (2.5% for induction; 1.5 – 2.0% for maintenance; delivered in 100% O2
) was used to anesthetize rats during surgery. Three electrodes for recording the cortical EEG were positioned above the primary and secondary motor cortices. Two electrodes for recording the EMG were inserted into dorsal neck muscles, and one indifferent electrode was placed between the skin and muscle layers above the spine. Three anchor screws were implanted into the skull to hold the head cap in place. A guide tube (8IC312GP0XXC; Plastics One Inc., Roanoke, VA, USA) containing a stylet (8IC312DCXXXC, Plastics One, Inc.) was aimed (Paxinos & Watson 2007
) 1 mm above the PnO (from bregma: 8.4 mm posterior, 1.2 mm lateral, 8.4 mm ventral). The EEG and EMG electrode leads, electrode pedestal, and guide tube were cemented in place with dental acrylic. Rats were given three days to recover from surgery and an additional two weeks for conditioning before being used for experiments. During the recovery period, rats were conditioned to handling, being housed in a Raturn experiment cage (Bioanalytical Systems Inc, West Lafayette, IN, USA), and being tethered to the six-channel cable (used for recording EEG and EMG signals) for at least 8 h per day. This conditioning paradigm has been successfully used previously (Brevig et al. 2010
, Watson et al. 2007
, Watson et al. 2008
). For each experiment, rats were placed in the Raturn bowl the afternoon before the start of the experiment.
In Vivo Microdialysis and CE-LIF
Microdialysis probes were constructed according to a previously described method (Pettit & Justice 1989
). Briefly, two lengths of fused silica capillary were offset by 1 mm, bound together with a small drop of cyanoacrylate, and fed through a guide tube connector (8IC313ICPNIT, Plastics One Inc.). The tips of the capillaries were then encased in a semi-permeable membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) that was plugged on one end with polyimide resin (Alltech Associates, Deerfield, IL, USA). The regenerated cellulose membrane had a molecular weight cut-off of 18 kDaltons. The membrane was bonded to the capillaries and all non-active areas of the microdialysis probe (from tip to connector) were coated with polyimide resin. The final maximum dimensions of each dialysis probe were 1 mm active length with an outer diameter of 240 μm. Once screwed into position on the guide tube, the microdialysis membrane extended 1 mm below the bottom of the guide tube. The microdialysis probe was perfused at a flow rate of 0.4 μL/min for all experiments.
Coupling in vivo
microdialysis on-line to CE-LIF has been described previously (Bowser & Kennedy 2001
, Lada et al. 1997
, Smith et al. 2004
) and is schematized in . Briefly, the outlet of the microdialysis probe was connected on-line to a CE-LIF system. Derivatization solution and dialysate were mixed in a 1:2.5 ratio. Derivatized dialysate was then injected electrokinetically onto the separation capillary via the flow-gate interface. As the analytes migrated along the separation capillary, they passed through a transparent window of the capillary. A laser was focused through this window to induce fluorescence. Emitted fluorescence was collected by a photo-multiplier tube. The current from the photo-multiplier tube was amplified and then collected by software written using LabVIEW™ 8.0 (National Instruments, Austin, TX, USA). Concentrations of amino acids were quantified using the Cutter analysis program (Shackman et al. 2004
Figure 1 Schematic of the capillary electrophoresis (CE) instrument. Drawing on the lower left indicates placement of a microdialysis probe in the brain of an awake rat. Dialysate from the probe flows through the derivatization capillary into a flow-gate interface (more ...)
Recording and Analysis of Sleep and Wakefulness
EEG and EMG electrodes were connected to a Model 1700 differential AC amplifier (A-M Systems Inc., Sequim, WA, USA) via a six-channel cable. The amplified signal was then sent to a Micro 1401 mkII data acquisition unit (Cambridge Electronic Design Ltd. (CED), Cambridge, UK) for signal processing and storage (on a personal computer) by Spike2 version 5.14 software (CED). EEG and EMG signals were analyzed in 10-s bins for classification as wakefulness, NREM sleep, or REM sleep. Wakefulness displayed low amplitude, mixed frequencies in the EEG and moderate to high muscle tone in the EMG. NREM sleep was characterized by the presence of delta waves (0.5 – 4.0 Hz) or spindles (8.5 – 14.0 Hz) in the EEG and a decrease (compared to wakefulness) in the amplitude of the EMG. REM sleep was characterized by a predominant peak in the theta range (4.5 – 8.0 Hz) of the EEG and a further reduction of EMG amplitude. Two investigators, one of whom was blinded to the experimental conditions, scored every 10-s bin of each recording as wakefulness, NREM sleep, or REM sleep. A minimum of 98% agreement between the two investigators was achieved for each sleep record.
At the beginning of each experiment, the computer collecting data from the CE-LIF system and the computer collecting data from the EEG and EMG recording system were synchronized to a National Institute of Standards and Technology Internet time server to ensure correct time alignment between the data files. The time for the dialysate to travel from the microdialysis membrane to the detection window was calculated prior to each experiment. The mean ± SEM travel time was 8 min 40 s ± 56 s. For each experiment, a microdialysis probe was inserted into the implanted guide tube 2 h before the start of sample collection. This allowed adequate time for concentrations of amino acids to stabilize. This pre-experiment insertion time was determined by preliminary experiments (data not shown) and conformed to or exceeded pre-experiment insertion times previously published for measures of glutamate and GABA (Smith et al. 2004
, Ferrario et al. 2008
, Watson et al. 2007
, Watson et al. 2008
). Sample collection began between 1400 and 1600. At the end of each experiment, the rat was placed in its home cage and returned to its homeroom. Rats (n = 10) were allowed to sleep ad libitum
prior to and during the experiment.
Histological Verification of Microdialysis Sites
Each rat was used for one experiment. At least three days after the experiment, rats were deeply anesthetized and decapitated. Brains were quickly removed, frozen, and placed in a Leica model CM3050S cryostat (Leica Microsystems, Nussloch, Germany). Serial, coronal sections (40 μm) were mounted on chrom-alum coated slides, fixed with hot paraformaldehyde vapor, and stained with cresyl violet. All sections containing the microdialysis site were digitized and compared to a stereotaxic atlas of the rat brain (Paxinos & Watson 2007
) for determining the three-dimensional coordinates (in mm) of the dialysis site. Only experiments in which the microdialysis membrane was localized to the PnO were included in the data analysis.
Statistical analyses were performed in consultation with the University of Michigan Center for Statistical Consultation and Research. Descriptive and inferential statistics were performed using GB-Stat™ PPC 6.5.6 (Dynamic Microsystems Inc., Silver Spring, MD, USA) and SAS version 9.2 (SAS Institute, Cary, NC, USA). Neurochemical data were analyzed using a linear mixed-model, repeated-measures ANOVA followed by Tukey-Kramer multiple comparisons tests. Data comparing episode duration for wakefulness, NREM sleep, and REM sleep to the duration of the decrease in amino acid concentration were analyzed using simple regression ANOVA.