Endogenous GABA Levels in the Pontine Reticular Formation Are Greater during Wakefulness than during Rapid Eye Movement Sleep

Animals, chemicals, and drugs.

All procedures using animals were approved by the University of Michigan Committee on Use and Care of Animals and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies, 1996) and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Academies, 2003). Adult, male cats (n = 6) that were bred for research were purchased from Harlan Laboratories. The advantages of using cat for in vivo studies that aim to quantify changes in endogenous neurotransmitters as a function of sleep and wakefulness have been discussed in detail previously (Vazquez and Baghdoyan, 2003; Vanini et al., 2008). Briefly, accurate and reliable behavioral-state-specific measures of GABA require long-lasting and consolidated episodes of sleep and wakefulness. Compared with rodent, cat has long duration episodes of NREM sleep and REM sleep. Thus, the cat provides an optimal species for overcoming the temporal limitations of microdialysis coupled to HPLC for testing the hypothesis that GABA levels in the pontine reticular formation change as a function of sleep state.

Chemicals for Ringer's solution, o-phosphoric acid, sodium phosphate dibasic, sucrose, and formalin were purchased from Thermo Fisher Scientific. HPLC-grade methanol, acetonitrile, sodium tetraborate decahydrate, β-mercaptoethanol, GABA, and neostigmine bromide were obtained from Sigma-Aldrich. o-phthaldialdehyde was purchased from Mallinckrodt. Solutions for microinjections were made immediately before use.

In vivo microdialysis and microinjection studies.

Cats were instrumented in a sterile operating suite as described previously (Vazquez and Baghdoyan, 2003; Vanini et al., 2008). Briefly, cats were anesthetized with isoflurane and placed in a stereotaxic frame (David Kopf Instruments). Standard recording electrodes for objective assessment of sleep and wakefulness (Ursin and Sterman, 1981) were implanted, and a craniotomy was created over the cerebellum to provide access for stereotaxic placement of a microdialysis probe in the pontine reticular formation. The recording electrodes and a pair of parallel stainless-steel tubes custom designed to accommodate the Kopf 880 head holder were cemented to the skull with dental acrylic. These tubes and the Kopf 880 holder were used during subsequent experiments to place the head in stereotaxic position without the need for anesthesia. This procedure permitted collection of microdialysis samples during states of sleep and wakefulness. Cats were allowed to recover from surgery and were conditioned to sleep in the laboratory for 3–4 weeks before experiments began.

All experiments were conducted at the same time of day. Microdialysis sample collection began between 10:30 A.M. and 12:00 P.M., and experiments lasted 4–6 h. For each experiment, a custom-made microdialysis probe (CMA/10 or 12; CMA/Microdialysis) with a shaft length of 70 mm and polycarbonate or PAES membrane (20 kDa cutoff, 2 mm length, 0.5 mm diameter) was inserted into a stainless-steel guide tube that was aimed stereotaxically for the pontine reticular formation. Stereotaxic coordinates for dialysis aim sites ranged from 1.0 to 3.0 mm posterior to zero, 1.0 to 2.5 mm lateral to the midline, and 4.0 to 6.0 mm below zero (Berman, 1968). Each dialysis site was used once. Dialysis aim sites were separated from each other by at least 1 mm in the anterior–posterior and mediolateral planes. Microdialysis probes were perfused continuously with Ringer's solution (147.0 mm NaCl, 2.4 mm CaCl₂, 4.0 mm KCl, 1.0 mm MgSO₄, pH 6.0) at a flow rate of 3.0 μl/min using a CMA/400 syringe pump.

Figure 1 illustrates the time course for data collection and the within-subjects study design used for measuring GABA during electrographically and behaviorally identified states of sleep and wakefulness. Samples collected during the first 120 min of dialysis (Fig. 1A, phase 1) were used to ensure that detectable and stable levels of GABA were obtained from the pontine reticular formation (Vanini et al., 2008). Samples collected during minutes 120–170 of dialysis (Fig. 1A, phase 2) provided control levels of GABA. During phases 1 and 2, cats were kept awake most of the time (Fig. 1B,C) by auditory stimulation and gentle handling. Thereafter (Fig. 1A, phase 3), cats were allowed to sleep ad libitum, and additional dialysis samples were collected during objectively confirmed states of wakefulness, NREM sleep, and REM sleep (Fig. 1B,C).

Microinjection of cholinomimetics into the pontine reticular formation causes the rapid onset of a REM sleep-like state. This approach has been used productively to study REM sleep physiology (Baghdoyan et al., 1984; Morales et al., 1987; Datta et al., 1993; Garzón et al., 1997; Kubin, 2001; Lydic and Baghdoyan, 2005; Márquez-Ruiz and Escudero, 2009) and confers the advantage of producing long-duration episodes of REM sleep (Fig. 1C). Thus, GABA levels were also measured during the REM sleep-like state (REM^Neo) caused by microinjecting the acetylcholinesterase inhibitor neostigmine (6 μg/0.25 μl; 20 nmol) into the pontine reticular formation. The purpose of these experiments was to determine whether causing REM sleep to occur would cause a significant decrease in GABA levels. Stereotaxic coordinates used to deliver neostigmine to the pontine reticular formation were 3.0–3.5 mm posterior to zero, 2.0 mm lateral, and 4.0 mm below zero (Berman, 1968). Figure 1C illustrates that unilateral microinjections were made contralateral to the dialysis site during NREM sleep and that dialysis samples were collected during REM^Neo after the collection of dialysis samples during spontaneously occurring sleep and wakefulness.

At the end of each experiment, the dialysis probe was removed from the brain and a microinjector was inserted into the guide cannula to deliver nontransportable, colored fluorescent microspheres (0.1 μl; FluoSpheres; Invitrogen). The object of this procedure was to uniquely label each dialysis site to determine whether GABA levels vary along the anterior-to-posterior, medial-to-lateral, and dorsal-to-ventral extent of the rostral pontine reticular formation. After the microspheres were delivered, the microinjector and guide cannula were removed from the brain, the craniotomy was sealed with bone wax, and the animal was returned to its home cage in the Unit for Laboratory Animal Medicine.

The percentage of GABA recovered by the dialysis membrane in vitro was determined before and after each experiment. Mean ± SEM recovery of GABA by all dialysis membranes was 7.7 ± 0.4%. Experiments were excluded from the group data if in vitro recovery of GABA changed significantly in the same direction as the change observed in the levels of brain GABA. This exclusion criterion insured that changes in GABA levels were not an artifact of dialysis membrane dynamics.

Quantification of GABA.

Detection and quantification of GABA in microdialysis samples obtained from the pontine reticular formation have been described in detail (Watson et al., 2007, 2008; Vanini et al., 2008) and are overviewed here. An aliquot (12.5 μl) of each dialysis sample was loaded into an autosampler and mixed with 6.0 μl of a derivatization solution [5.0 mm o-phthaldialdehyde, 1.8 mm β-mercaptoethanol, 97.1 mm borate buffer, 2.5% (v/v) methanol, pH 9.3]. A portion (10 μl) of the derivatized solution was injected into a Shiseido CAPCELL PAK C-18 separation column (JM Science), and the derivatized samples were carried through the HPLC system (ESA) in a mobile phase [100 mm sodium phosphate buffer, 25% (v/v) methanol, and 3% (v/v) acetonitrile, pH 6.75] at a flow rate of 0.6 μl/min. Automation of the HPLC system and analysis of GABA peaks (see Fig. 2C) were performed with EZChrom Elite chromatography data system (Scientific Software). The amount of GABA in each dialysis sample was calculated by comparison with a standard curve, which was generated by nine known concentrations of GABA ranging from 0.0055 (detection limit) to 0.913 pmol/10 μl.

Electrophysiological recordings of sleep and wakefulness and power spectral analysis.

The electroencephalogram (EEG) (Fig. 1B, inset), electrooculogram, and dorsal neck electromyogram were recorded during microdialysis sample collection. Behavioral observations also were obtained. These measures made it possible to objectively assess states of sleep and wakefulness in real time. The physiological signals were recorded using a Grass model 7D polygraph (Grass Instruments). Signals were digitized and analyzed using a Power CED1401 data acquisition unit and Spike2 software (Cambridge Electronic Design). EEG power spectral analyses were performed to determine whether the cortical EEG recorded during REM^Neo differed from the EEG recorded during spontaneous REM sleep. Frontal EEG power spectra were generated by averaging artifact-free, 10 s epochs from spontaneous REM sleep and REM^Neo episodes in 1 Hz increments ranging from 0.5 to 60 Hz. The mean EEG power during REM sleep and REM^Neo was normalized and statistically compared in the frequency bands of 0.5–4 (delta), 5–8 (theta), 9–16 (sigma), 17–29 (beta), and 30–60 (gamma) Hz.

Histological localization of dialysis sites.

After the last dialysis experiment, cats were deeply anesthetized with isoflurane, injected with pentobarbital (35–40 mg/kg, i.v.), and perfused transcardially with cold saline (0.9%) followed by formalin (10%). Brains were removed and fixed in formalin (10%) for 3–4 weeks. Thereafter, the brainstem block was transferred for cryoprotection to sucrose (30%) in formalin (10%) for 7 d. Serial, sagittal sections (40 μm thick) of the brainstem were cut on a sliding microtome and float-mounted onto chrom-alum-coated glass slides. Alternate sections were stained with cresyl violet and coverslipped using Permount (Thermo Fisher Scientific). All sections containing microdialysis sites were digitized, and the stereotaxic coordinates of the dialysis sites were assigned by comparison with an atlas of the cat brainstem (Berman, 1968). The midpoint of the 2-mm-long dialysis membrane was used to assign stereotaxic coordinates in the anterior–posterior and dorsoventral planes. Alternate, nonstained sections were coverslipped with Gel/Mount (Biomeda) and used to localize fluorescent beads that marked the microdialysis sites. These microspheres contained blue, red, or yellow-green dyes with excitation and emission wavelengths of 365/415, 580/605, and 505/515 nm, respectively. Digital photographs of brain sections containing fluorescent beads were obtained with a DP70 camera attached to an Olympus BH-2 microscope (Olympus America).

Data analysis.

Statistical tests were performed in consultation with the University of Michigan Center for Statistical Consultation and Research using programs developed by Statistical Analysis System, version 9.1.3 (SAS Institute); GBStat, version 6.5.6 (Dynamic Microsystems); and Prism, version 5.0a for Mac OS X (GraphPad Software). The data distribution for measures of GABA levels did not meet the assumptions of the general linear model. Thus, to test whether there was an effect of arousal state or dialysis site on GABA levels, the data were analyzed by fitting a linear mixed model. This model included variance components for animal and for experiments nested within animal, to capture the extra variance introduced by these two factors. Within-groups degrees of freedom were conservatively constrained to 10 based on a between-within design, with cat being the subject in the analysis. The post hoc Tukey–Kramer procedure, adjusted for multiple comparisons, was used to evaluate differences in GABA levels between arousal states. EEG power-by-frequency during REM sleep and REM^Neo was compared by t test with Bonferroni's correction. Data are reported as mean ± SEM, and a value of p < 0.05 was considered statistically significant.