Extrasynaptic GABA_A Receptors in Rat Pontine Reticular Formation Increase Wakefulness

Abstract

Study Objectives:

Gamma-aminobutyric acid (GABA) causes phasic inhibition via synaptic GABA_A receptors and tonic inhibition via extrasynaptic GABA_A receptors. GABA levels in the extracellular space regulate arousal state and cognition by volume transmission via extrasynaptic GABA_A receptors. GABAergic transmission in the pontine reticular formation promotes wakefulness. No previous studies have determined whether an agonist at extrasynaptic GABA_A receptors administered into the pontine reticular formation alters sleep and wakefulness. Therefore, this study used gaboxadol (THIP; agonist at extrasynaptic GABA_A receptors that contain a δ subunit) to test the hypothesis that extrasynaptic GABA_A receptors within the pontine reticular formation modulate sleep and wakefulness.

Design:

Within/between subjects.

Setting:

University of Michigan.

Patients or Participants:

Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 10).

Interventions:

Microinjection of gaboxadol, the nonsubtype selective GABA_A receptor agonist muscimol (positive control), and saline (negative control) into the rostral pontine reticular formation.

Measurements and Results:

Gaboxadol significantly increased wakefulness and decreased both nonrapid eye movement sleep and rapid eye movement sleep in a concentration-dependent manner. Relative to saline, gaboxadol did not alter electroencephalogram power. Microinjection of muscimol into the pontine reticular formation of the same rats that received gaboxadol increased wakefulness and decreased sleep.

Conclusion:

Tonic inhibition via extrasynaptic GABA_A receptors that contain a δ subunit may be one mechanism by which the extracellular pool of endogenous GABA in the rostral pontine reticular formation promotes wakefulness.

Anesthesia, gaboxadol, microinjection, sedative-hypnotics, THIP

INTRODUCTION

GABAergic transmission regulates states of sleep and wakefulness.^1–3 Drugs prescribed to treat sleep disorders⁴ and most general anesthetic agents^5,6 act at GABA_A receptors. GABA causes fast phasic inhibition via synaptic GABA_A receptors and tonic inhibition via extrasynaptic GABA_A receptors.^7–9 Extrasynaptic receptors are expressed outside synapses and mediate volume transmission, which effectively modulates the excitability of widely distributed neuronal networks. Extrasynaptic GABA_A receptors have higher affinity and slower desensitization rates than synaptic GABA_A receptors, and are activated by low ambient GABA levels in the extracellular space.^8,10 Sedative-hypnotic agents, anesthetic drugs, alcohol, and neurosteroids act at extrasynaptic GABA_A receptors.^6,9,11–14 Emerging evidence suggests that extrasynaptic GABA_A receptors may be potential therapeutic targets for enhancing cognition, as well as for treatment of pain, neuropsychiatric illnesses, and sleep disorders.^9,15,16

Data from microinjection studies^17–20 and measures of endogenous GABA^21,22 indicate that the pontine reticular nucleus, oral part (PnO)²³ in the rodent is the functional homologue of the rostral gigantocellular tegmental field in the cat.²⁴ For clarity, the term rostral pontine reticular formation is used in this article when referring to these brainstem regions in cat or rat. The rostral pontine reticular formation is part of the ascending reticular activating system²⁵ and contains networks that regulate rapid eye movement (REM) sleep, wakefulness,^3,26 and neurophysiologic traits of anesthesia.^27,28 In the rostral pontine reticular formation, GABA acts to promote wakefulness and suppress sleep in the cat,^20,22,29 rat,^{17,19,21,29–31} and mouse.³² GABA_A receptors that contain a δ subunit are localized extrasynaptically^7–9,11 and are present within the rostral pontine reticular formation.³³ The effects of GABAergic transmission on sleep and wakefulness vary on a brain region-by-region basis,^1,34,35 and no previous studies have investigated whether tonic inhibition by extrasynaptic GABA_A receptors in the rostral pontine reticular formation regulates sleep and wakefulness.

Gaboxadol (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol; THIP) is an agonist at GABA_A receptors that contain a δ subunit.^7,11,36 Systemic administration of gaboxadol to rats^37–39 and humans^40–44 decreases sleep latency and increases sleep duration with no suppression of REM sleep. This study tested the hypothesis that microinjection of gaboxadol into the rostral pontine reticular formation increases wakefulness and decreases sleep.

MATERIALS AND METHODS

Animals and Drug Solutions

All procedures using animals were approved by the University Committee on Use and Care of Animals and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (The National Academies Press, Eighth Edition, Washington DC, 2011). Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 10) weighing 235 to 250 g were purchased from Charles River Laboratories (Wilmington, MA), housed in the Unit for Laboratory Animal Medicine facilities with ad libitum access to food and water, and kept on a 12-h light/dark cycle (lights on at 06:00). Rats were allowed a minimum of 7 days after arrival for acclimation to the new housing and laboratory environment before surgery.

Gaboxadol hydrochloride (THIP hydrochloride; an agonist at GABA_A receptors that contain a δ subunit) and muscimol (an agonist at all subtypes of GABA_A receptors) were purchased from Sigma-Aldrich (St. Louis, MO). Gaboxadol and muscimol were dissolved in sterile saline (0.9%). A stock solution (10 mM) of gaboxadol was made, divided in aliquots, and stored at -80°C for subsequent use. For each experiment, one aliquot of the stock solution was allowed to thaw at room temperature and diluted to achieve the desired final concentration for intra-cerebral microinjections. Solutions of muscimol were prepared immediately before use.

Surgical Procedures and Conditioning

Surgical preparation of rats with electrodes for recording states of sleep and wakefulness, and a guide cannula for intracerebral microinjections was performed according to protocols and procedures described previously.^30,31,45 Rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) and placed in a Kopf Model 962 small animal stereotaxic apparatus fitted with a Model 906 rat anesthesia mask (David Kopf Instruments, Tujunga, CA). Core body temperature was maintained at 37-38°C using a warm water-filled pad connected to a recirculating heat pump (Gaymar Industries, Orchard Park, NY). Inhaled isoflurane concentration and core body temperature were monitored throughout the surgery using a Cardiocap™/5 (Datex-Ohmeda, Louisville, CO). After the skull was exposed, three screw electrodes (8IE36320SPCE, Plastics One, Roanoke, VA) for recording the cortical electroencephalogram (EEG) were implanted above the dura mater at the following stereotaxic coordinates (relative to bregma): 1.00 mm anterior, 1.50 mm lateral; 2.00 mm posterior, 1.50 mm lateral; and 2.00 mm posterior, 1.27 mm lateral.²³ Two electrodes were implanted bilaterally in the dorsal neck muscles, and one electrode was implanted subcutaneously (indifferent) above the neck. These electrodes were used for recording the electromyogram (EMG). Electrodes for recording the EMG were made of AS632 biomed wire (Cooner Wire Company, Chatsworth, CA) soldered to an electrical gold socket contact (8IE3630XXXXE, Plastics One). A guide cannula (8IC315GSPCXC, Plastics One) was aimed 3 mm above the pontine reticular nucleus, oral part (PnO; rostral pontine reticular formation) at 8.40 mm posterior to bregma, 1.0 mm lateral to the midline, and 6.2 mm below the skull surface.²³ All electrodes were inserted into a six-pin electrode pedestal (MS363, Plastics One). The pedestal, recording electrodes, and microinjection guide cannula were fixed to the skull with three anchor screws (MPX-0080-02PC-C, Small Parts, Inc., Miami Lakes, FL) and dental acrylic (Lang Dental Manufacturing Company, Inc., Wheeling, IL).

After recovery from the surgical procedure, rats were conditioned for the microinjection procedure for at least 1 week. Conditioning sessions consisted of daily tethering from the electrode pedestal to a six-channel cable (363-441/6, Plastics One), handling, removing, and reinserting the stylet (8IC315DCSSSC, Plastics One) from the guide cannula simulating a microinjection procedure, and acclimation to the recording chamber (Raturn®, Bioanalytical Systems, West Lafayette, IN). In the recording chamber, rats had ad libitum access to food and water. Prior to the first microinjection, a 2-h baseline recording was performed to assess the quality of the signals. Rats were considered to be ready to enter the microinjection protocol when the latency to onset of the first nonrapid eye movement (NREM) sleep episode was less than 30 min.

Microinjections, Sleep Recordings, and Quantification of Arousal State

Rats were tethered and placed in their respective recording chambers the night prior to each experiment. All microinjections were performed during wakefulness between 09:00 and 10:00. Unilateral microinjections (100 nL over 60 sec) of saline (vehicle control) and drug solutions were made using a Model 7001 microliter syringe (Hamilton Company, Reno, NV). The syringe was mounted in a manual microdrive and connected to a microinjector (Internal Cannula 8IC315IXXXXC; 33 gauge, outer diameter = 0.20 mm, inner diameter = 0.10 mm; Plastics One) by a piece of polyethylene PE-20 tubing (Thermo Fisher Scientific, Waltham, MA). Microinjections of gaboxadol (0, 0.1, 1, 10, 100 pmol/100 nL; 0, 0.01766, 0.1766, 1.766, 17.66 ng, respectively) were made in random order, and only one microinjection was made into the same rat per week. After all concentrations of gaboxadol had been tested, a microinjection of muscimol (100 pmol/100 nL; 11.4 ng) was made as a positive control, because microinjection of muscimol into rat rostral pontine reticular formation has been shown to increase wakefulness and decrease sleep.^17,19

EEG and EMG were recorded continuously for 4 h after the microinjection. Electrophysiologic signals were amplified, filtered (EEG, 0.3 - 25 Hz; EMG, 10 - 100 Hz), digitized at 128 Hz, and analyzed using Icelus Data Acquisition and Analysis Software.⁴⁶ Electrode pedestals from each rat were connected via the six-channel cable to a minielectrode board (F-15EB/B1, Grass Instruments, Warwick, RI), a Grass model 15LT amplifier system, and a 12-bit data acquisition card (6024E, National Instruments, Austin, TX). States of wakefulness, NREM sleep, and REM sleep were scored manually in 10-sec epochs by two investigators, one of whom was blinded to the treatment condition. Agreement in sleep scoring was greater than 93%.

As described previously,^30,32,45 fast Fourier transform (FFT) data were obtained by analyzing EEG signals in 0.5-Hz increments in 2-sec bins. Five consecutive 2-sec bins were then averaged to generate one FFT for each 10-sec epoch. Averages of 10-sec epochs over 5 min for states of wakefulness, NREM sleep, and REM sleep that occurred between 30 to 120 min after a microinjection were used to quantitatively assess whether gaboxadol (1 pmol/100 nL) altered EEG power.

Statistical Analyses

Statistical analyses were performed in consultation with the University of Michigan Center for Statistical Consultation and Research using Statistical Analysis System v9.2, (SAS Institute, Cary, NC) and Prism v5.0a for Mac OS X (GraphPad Software Inc., La Jolla, CA). The effects of gaboxadol on the percentage of time spent in sleep and wakefulness and the duration of NREM sleep episodes were evaluated using a linear mixed model for a randomized incomplete block design, followed by a Dunnett test adjusted for multiple comparisons. This model was also used to test whether there was a significant drug effect on EEG power during wakefulness, NREM sleep, and REM sleep. Concentration response data were fit to the equation Y = B+(T-B)/(1+10^((LogEC50-X)*HillSlope)), where B represents the lower limit for the dependent variable (e.g., percentage of wakefulness), T is the upper limit for the dependent variable, X is the logarithm of the concentration of gaboxadol, and Y is the dependent variable. Regression analyses were used to obtain the coefficient of determination (r²) and calculate the percentage of the response accounted for by the concentration of gaboxadol. Because there were so few or no episodes of REM sleep after injection of gaboxadol, a negative binomial regression was used to evaluate drug effects on the number of REM episodes. Because most rats did not have REM sleep after administration of gaboxadol (e.g., REM latency > 4 h), the effect of gaboxadol on the latency to the first REM sleep episode was assessed by survival analysis. Effects of muscimol were evaluated using paired t-test. P < 0.05 was considered statistically significant. Data are reported as mean ± standard error of the mean.

Histologic Confirmation of Microinjection Sites

After completion of the final experiment, rats were deeply anesthetized and decapitated. Brains were removed from the skull, blocked, frozen, and 40-μm coronal sections were cut in a cryostat (Leica Microsystems, Nussloch, Germany). Sections were slide mounted, dried, fixed with hot paraformaldehyde vapor, and stained with cresyl violet. All sections containing microinjection sites were digitized and the stereotaxic coordinates were defined by comparison with a rat brain atlas.²³

RESULTS

The goal of the current study was to determine whether δ-subunit-containing extrasynaptic GABA_A receptors in the pontine reticular formation regulate sleep and wakefulness. Thus, four concentrations of gaboxadol were microinjected into rat rostral pontine reticular formation. Figure 1 shows representative patterns of sleep and wakefulness after micro-injections in one rat. Figure 2A-C show group data for the effect of gaboxadol on the amount of wakefulness, NREM sleep, and REM sleep in 10 rats. Gaboxadol significantly increased the time spent in wakefulness (F = 4.67; df = 4,31; P = 0.0046), and decreased NREM sleep (F = 4.82; df = 4,31; P = 0.0039) and REM sleep (F = 3.24; df = 4,31; P = 0.0247) in a concentration-dependent manner. The concentration of gaboxadol accounted for a significant percentage of the variance in the amount of wakefulness (96%), NREM sleep (94%), and REM sleep (98%). Gaboxadol significantly (F = 4.15; df = 4,30; P = 0.0086) decreased the average duration of NREM sleep episodes (Figure 2D). The number of REM episodes was significantly (F = 6.28; df = 4,31; P = 0.0008) decreased by all concentrations of gaboxadol (Figure 2E). There was no effect of gaboxadol on the number of NREM sleep episodes. Gaboxadol also increased the latency to the onset of the first REM sleep episode (Figure 2F). Survival analysis revealed that after microinjection of gaboxadol, the probability of not having REM sleep remained elevated until the end of the 4-h recording period (i.e., the latency to the first REM sleep episode increased or REM sleep did not occur). Relative to saline, the probability of REM sleep not occurring after administration of gaboxadol was significantly greater (P = 0.0003) for all concentrations. NREM sleep latency was not significantly altered by gaboxadol. Analysis using a linear mixed model revealed that, relative to control, there was no significant effect of gaboxadol on EEG power during wakefulness, NREM sleep, and REM sleep.

Figure 1

Gaboxadol altered the temporal distribution of sleep and wakefulness. A, The schematic sagittal view of the brain, modified from a rat brain atlas,²³ illustrates a cannula inserted into the pontine reticular nucleus, oral part (PnO) for microinjection of saline or drug. B-D, Hypnograms show the time course of sleep and wakefulness recorded from the same rat for 4 hours after microinjection of saline (B), and two concentrations of gaboxadol (1 pmol/100 nL, C; 100 pmol/100 nL, D). Time 0 on the abscissa indicates the end of the 1-min period during which saline or drug was microinjected into the PnO. The lowest, intermediate, and highest black bars represent the occurrence and duration of wakefulness, nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep, respectively.

Figure 2

Gaboxadol caused a concentration-dependent increase in wakefulness and decrease in sleep. Concentration-response curves show percent state for wakefulness (A), NREM sleep (B), and REM sleep (C), mean duration of NREM sleep episodes (D), and the number of REM sleep episodes (E) during 4 hours after microinjection of saline and gaboxadol. F, The graph plots the probability of no REM sleep occurring after a microinjection of saline or gaboxadol. Survival analysis revealed that the probability in the control group (black line) decreased to zero before reaching 100 min after microinjection (i.e., all rats had REM sleep). Increasing concentrations of gaboxadol (colored lines) increased the latency to REM sleep. Asterisks indicate significant differences from control (0 pmol). NREM, nonrapid eye movement sleep; REM, rapid eye movement sleep; SEM, standard error of the mean.

As a positive control, recordings of sleep and wakefulness were obtained after microinjection of muscimol (100 pmol) into the rostral pontine reticular formation. Muscimol increased the amount of wakefulness by 43% (t = 4.49; df = 8; P = 0.002), decreased NREM sleep by 60% (t = 4.59; df = 8; P = 0.0018), and decreased REM sleep by 78% (t = 2.79; df = 8; P = 0.023). These results are based on data from nine rats.

All microinjection sites used for the concentration-response study of gaboxadol and subsequent microinjections of muscimol were localized to the pontine reticular nucleus, oral part (Figure 3). Histologic measurements indicated that the average stereotaxic coordinates²³ for these microinjection sites in mm were 8.4 ± 0.07 posterior to bregma, 1.2 ± 0.06 lateral to the midline, and 8.4 ± 0.06 below the skull surface.

Figure 3

All microinjection sites (n = 10) were localized to the pontine reticular nucleus, oral part (PnO) and are represented by black dots on coronal brainstem plates.²³ The numbers on the right side of each plate indicate the distance (mm) from bregma. The vertical lines drawn on the sagittal schematic of the rat brain²³ (bottom left) indicate the anterior-to-posterior range of the microinjection sites. The digitized coronal section (top right) shows a representative microinjection site (arrow) in the PnO.²³

DISCUSSION

This study showed for the first time that microinjection of gaboxadol into the rostral pontine reticular formation of the rat caused a concentration-dependent increase in wakefulness and decrease in sleep. Gaboxadol is an agonist at extrasynaptic GABA_A receptors that contain a δ subunit.^7,9,11,36,47 The results are consistent with the interpretation that GABAergic transmission in the rostral pontine reticular formation promotes wakefulness, and demonstrate that extrasynaptic GABA_A receptors in the pontine reticular formation can regulate sleep and wakefulness. The role of extrasynaptic GABA_A receptors in the pontine reticular formation relative to sleep-wake regulation and the limitations of this study are discussed in the following paragraphs.

GABAergic Transmission in the Rostral Pontine Reticular Formation Promotes Wakefulness

The effects of GABAergic transmission on sleep and wakefulness vary as a function of brain region.^1,3,34,35 For example, microinjection of the GABA_A receptor agonist muscimol into the anterior hypothalamus,⁴⁸ basal forebrain,⁴⁹ or periaqueductal gray⁵⁰ increases wakefulness, NREM sleep, or REM sleep, respectively. Several lines of work support the conclusion that GABAergic transmission in the rostral pontine reticular formation promotes wakefulness and suppresses sleep. Microinjection of a GABA receptor agonist^{17,19,20,32,51} or drugs that increase levels of endogenous GABA^29,31 into the rostral pontine reticular formation increases wakefulness and decreases sleep. Conversely, administration of a GABA_A receptor antagonist^{17–20,32,52} or drugs that decrease endogenous GABA levels^20,29,31 into the rostral pontine reticular formation decreases wakefulness and increases sleep. Consistent with the wakefulness-promoting role of GABAergic transmission in the rostral pontine reticular formation, endogenous GABA levels are greatest during wakefulness and lowest during REM sleep.^21,22 Furthermore, systemic administration of drugs that eliminate wakefulness decrease GABA levels in the rostral pontine reticular formation.^29,45

GABAergic neurons in the rostral pontine reticular formation express hypocretinergic receptors⁵³ and are excited by hypocre-tins.⁵⁴ Administration of hypocretin-1 to the rostral pontine reticular formation increases GABA levels in the rostral pontine reticular formation and increases the amount of wakefulness.³¹ The increase in wakefulness caused by hypocretin-1 is blocked by coadministration of the GABA_A receptor antagonist bicuculline to the rostral pontine reticular formation.³⁰ This finding suggests that hypocretinergic and GABAergic transmission can interact within the rostral pontine reticular formation to increase wakefulness.

Neuronal sources of GABAergic input to the rostral pontine reticular formation that likely regulate arousal state include the lateral hypothalamic area,^55,56 central nucleus of the amygdala,⁵⁶ periaqueductal gray,^56,57 and a column of GABAergic neurons localized to the midbrain and pons that project to the contra-lateral rostral pontine reticular formation.⁵⁸ GABAergic transmission inhibits both acetylcholine release⁵² and GABAergic interneurons⁵⁴ via GABA_A receptors within the rostral pontine reticular formation. These data are compatible with the hypothesis that during wakefulness GABAergic transmission in the rostral pontine reticular formation exerts tonic inhibitory control on cholinergic mechanisms that promote REM sleep, as well as on GABAergic interneurons that inhibit the ascending reticular activating system. Future studies are needed to clarify the neural networks by which GABA in the rostral pontine reticular formation promotes wakefulness.

Extrasynaptic GABA_A Receptors in the Rostral Pontine Reticular Formation Regulate Sleep and Wakefulness

Extracellular GABA levels in the rostral pontine reticular formation of the cat^22,29 and the rat²¹ vary in a state-specific manner. GABA collected by microdialysis originates mainly from nonsynaptic release into the extracellular space by the neuron-glia unit, and modulates neural excitability by volume transmission.^59–62 Furthermore, extrasynaptic GABA_A receptors that contain a δ subunit are expressed in the pontine reticular formation.³³ Thus, these lines of evidence suggest that the extrasynaptic pool of GABA in the rostral pontine reticular formation regulates arousal state via extrasynaptic GABA_A receptors. Consistent with this interpretation, microinjection of gaboxadol into rat rostral pontine reticular formation increased wakefulness and decreased both NREM and REM sleep (Figures 1 and 2). The concentration-dependent nature of the response to gaboxadol indicates that the effects on arousal state were receptor-mediated.

In the current study, microinjection of muscimol into the rostral pontine reticular formation of the same rats that received gaboxadol served as a positive control. Muscimol significantly increased wakefulness and decreased NREM and REM sleep. The effects of muscimol were consistent with data from previous studies in the rat,^17,19 and confirmed that gaboxadol was delivered to the same region of the pontine reticular formation into which the nonsubtype selective GABA_A receptor agonist muscimol increases wakefulness and suppresses REM sleep. Potential cellular sources of ambient GABA that mediates volume transmission by extrasynaptic GABA_A receptors include diffusion of synaptically released GABA away from the synaptic cleft,^14,63–67 as well as nonvesicular (i.e., nonsynaptic) release from neurons and glia.^{60,63,65,68–70} Ambient GABA levels in the rostral pontine reticular formation are greatest during wakefulness.^21,22,29 Thus, one likely mechanism by which GABA in the rostral pontine reticular formation increases wakefulness is by tonic inhibition of sleep-promoting structures via extrasynaptic GABA_A receptors.

Limitations and Conclusions

The current study was limited by the fact that there are no selective antagonists for the δ-subunit-containing subtype of extrasynaptic GABA_A receptors. Decreasing wakefulness with a selective antagonist would demonstrate that endogenous GABA in the pontine reticular formation promotes wakefulness, at least in part, by activating these δ-subunit-containing receptors. A second limitation is that the neuronal networks by which GABA in the rostral pontine reticular formation alters sleep and wakefulness remain incompletely understood. These details can be addressed by future studies designed to identify the downstream pathways and synaptic mechanisms that are modulated by GABA in the rostral pontine reticular formation.

The current results suggest that tonic inhibition via δ-subunit-containing extrasynaptic GABA_A receptors is one mechanism by which GABA promotes wakefulness and decreases sleep. The data reported here support the conclusion that the extracellular pool of endogenous GABA in rat rostral pontine reticular formation regulates arousal state by volume transmission via extrasynaptic GABA_A receptors.

ACKNOWLEDGMENTS

Supported by a J. Christian Gillin, MD Research Grant (to Dr. Vanini) from the Sleep Research Society, National Institutes of Health grant MH45361 (to Dr. Baghdoyan), and the Department of Anesthesiology. The authors thank Dr. R. Lydic for critical comments, S. Jiang, B.S. and M.A. Norat, B.S. for expert assistance, and K. Welch, MS, MPH of the University of Michigan Center for Statistical Consultation and Research for help with statistical analyses.