Evidence That Wakefulness and REM Sleep Are Controlled by a GABAergic Pontine Mechanism

Ming-Chu Xi, Francisco R. Morales, and Michael H. Chase

Department of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Xi, Ming-Chu, Francisco R. Morales, and Michael H. Chase. Evidence That Wakefulness and REM Sleep Are Controlled by a GABAergic Pontine Mechanism. J. Neurophysiol. 82: 2015-2019, 1999. The pontine microinjection of the inhibitory neurotransmitter GABA and its agonist induced prolonged periods of wakefulness in unanesthetized, chronic cats. Conversely, the application of bicuculline, a GABAA antagonist, resulted in the occurrence of episodes of rapid eye movement (REM) sleep of long duration. Furthermore, administration of antisense oligonucleotides against glutamic acid decarboxylase (GAD) mRNA into the same area produced a significant decrease in wakefulness and an increase in REM sleep. Microinjections of glycine, another major inhibitory neurotransmitter in the CNS, and its antagonist, strychnine, did not have any effect on the behavioral states of sleep and wakefulness. These data argue forcibly that 1) GABAergic neurons play a pivotal role in determining the occurrence of both wakefulness and REM sleep and 2) the functional sequelea of inhibitory GABA actions within the pontine reticular formation are excitatory directives and/or behaviors.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present report brings new interdisciplinary evidence to bear on a fundamentally important question in a variety of areas ranging from behavior to neuropharmacology, i.e., whether GABAergic neurons play a key role in determining whether wakefulness or rapid eye movement (REM) sleep occurs at any given point in time. Because wakefulness and REM sleep are mutually exclusive states, they must be some mechanisms to initiate and/or suppress either state for the other to take place on the basis that excitatory processes promote both wakefulness [by activation of the reticular activation system (Moruzzi and Magoun 1949; Steriade et al. 1990)] and REM sleep [by activation of cholinergic brain stem mechanisms (Jones 1991; Rye 1997; Siegel 1994; Steriade and McCarley 1990)]. The question addressed in this report is whether there is, and, if so, the nature of, a neuronal switch whose position determines the occurrence of either REM sleep or wakefulness.

To pursue this objective, we developed the concept of a neuronal switch based on the hypothesis that GABAergic neurons suppress the activity of REM sleep neurons of the nucleus pontis oralis (NPO) in the pontine reticular formation. The fact that GABA might participate in this putative pontine mechanism is supported by anatomic data (Ford et al. 1995; Kosaka et al. 1988). Accordingly, in the present study we examined the behavioral responses of chronic, unanesthetized cats following microinjections of 1) GABA, 2) a GABAA receptor agonist and antagonist, 3) antisense to glutamic acid decarboxylase (GAD), 4) glycine, and 5) a glycine receptor antagonist into the NPO.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Six adult cats were used in the present study. The animals were prepared for monitoring behavioral states and for drug administration, as previously described (Yamuy et al. 1993). After recovery from surgery, all cats were head-restrained for 5-6 h a day, for 2 wk, for adaptation to the recording conditions.

After the adaptation period, carbachol (0.25 µl, 4 mg/ml) was injected into the rostral pontine reticular formation to determine the optimal stereotaxic coordinates for the NPO [L: 2.0, P: 2.5 to 3.0, H: -3.5 to -4.0 (Berman 1968)] in each animal using a 2-µl Hamilton syringe. The syringe was connected to a remote-controlled hydraulic micropositioner. For the purpose of this study, the effective NPO region was defined by the stereotaxic coordinates at which an injection of carbachol induced REM sleep with a latency shorter than 4 min. In experimental sessions, all of which were conducted between 10:00 and 16:00 h local time, GABA (0.25 µl, 200 mM in saline), muscimol (0.25 µl, 10 mM in saline), bicuculline (0.25 µl, 10 mM in saline), glycine (0.25 µl, 100 mM in saline), or strychnine (0.25 µl, 15 mM in saline) was microinjected into the NPO. In all cats control solutions of saline (0.25 µl) were injected into the same site that received the injections of drugs. The injections were delivered, unilaterally, over a period of 1 min. All injections were carried out while the animals were in wakefulness, non-REM (NREM) sleep or REM sleep. No injections were carried out during control sessions.

Phosphorothioate oligos were obtained from Bio Synthesis (Lewisville, TX). A 15-mer S-oligo complementary to the region spanning the translation start codon for cat GAD (GAD67) was constructed. The sequence of the antisense oligos to GAD was 5'-CGC-CAT-CAG-CAG-CTC-GGT-3'. The scrambled sequence for control S-oligos was 5'-CAG-GTG-CAT-ATC-CCG-3'. The italic letters indicate the sequence to the translation start codon. In injection sessions, which were conducted between 10:00 and 16:00 h, three cats that were head-restrained received a bilateral microinjection of either antisense oligos to GAD (1 µl, 1 µg/µl in saline) or control oligos (1 µl, 1 µg/µl in saline) into the NPO using a 2-µl Hamilton syringe. The effective NPO region was defined by the method described above. The injections were delivered over a period of 20 min during the first hour of each recording session. Pre- and postinjection recordings were carried out 1 day before and on the 5 days subsequent to the day of the injection under the same recording conditions and recording hours that were utilized on the day of the injection. No abnormal behavior was observed after the injection of antisense in these animals. The animals sat quietly in the recording apparatus throughout the recording sessions and approached the investigators readily both before and after these experimental sessions.

Polygraphic records were obtained in each recording session. States of wakefulness (W), NREM sleep (NREM), and REM sleep (REM) were scored according to standard polygraphic and behavioral criteria. Experimental data are expressed as means ± SE. The statistical significance of the difference between sample means was evaluated using the two-tailed unpaired Student's t-test and analyses of variance (ANOVA). At the conclusion of the experiments, the site of drug injection was marked with 0.5 µl of a 2% solution of Chicago sky blue dye in 0.5 M Na-acetate. The animal was then anesthetized with pentobarbital sodium (Nembutal) and perfused with 10% formaldehyde. Coronal serial sections of brain stem tissue were examined to verify the injection sites.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Microinjections of the endogenous inhibitory transmitter, GABA, into the NPO in three cats during either REM sleep (n = 3) or NREM sleep (n = 2) induced wakefulness with a short latency. The mean latency to the onset of wakefulness, as measured from the time of the beginning of the injection of GABA, was 3.8 ± 0.9 min. A polygraphic recording of this waking effect of GABA is presented in Fig. 1C. This episode of GABA-induced wakefulness lasted for 25 min.



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Fig. 1. Anatomic location of effective injections site (n = 11) in the rostral pons of six cats (A). Schematic frontal planes of cat brain stem are illustrated at level P 2.5 and P 3.0. Sites where injections were delivered to the left and right side are indicated by circles and squares, respectively. Representative polygraphic recordings of an episode of spontaneous rapid eye movement (REM) sleep (B), an episode of wakefulness that occurred following the injection of GABA (C), and a REM sleep episode that was induced following an injection of bicuculline, a GABAA receptor antagonist (D). The injection of GABA was performed during a spontaneous REM sleep episode. Note that bicuculline-induced REM sleep appears indistinguishable from a spontaneous episode of REM sleep; however, the former lasted 52 min, almost 8 times longer than the mean time of spontaneous active sleep episodes. EEG, electroencephalogram; EOG, electrooculogram; EMG, electromyogram. All vertical bars: 100 µV. BC, brachium conjunctivum; LC, locus coeruleus.

The effect of a microinjection of the GABAA receptor agonist, muscimol, during NREM and REM sleep was examined in four cats and also found to induce prolonged wakefulness. A series of hypnograms showing the effects of microinjections of muscimol and saline in a representative cat are shown in Fig. 2. The injection of muscimol (arrow in Fig. 2C) during NREM sleep induced, with a short latency, a prolonged episode of wakefulness that was about four times longer than those that occurred during control and saline sessions. The effects of muscimol on the percentages of time that cats spent in different behavioral states during 4-h recording sessions following its injections are presented in Table 1. Compared with the percentages observed following saline injections, injections of muscimol significantly increased the time spent in wakefulness (118%, P < 0.01). This increase was accompanied by significant decreases of the time spent in REM sleep (63%, P < 0.01) and NREM sleep (59%, P < 0.01). Injections of muscimol also significantly increased the mean latency of active sleep (muscimol: 105.9 ± 27.2 min, n = 12 vs. saline: 40.1 ± 4.0 min, n = 10; P < 0.05). These data suggest that cells within the NPO must be tonically inhibited by a GABAergic brain stem system in order for the state of wakefulness to be generated and maintained.



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Fig. 2. Hypnograms of control (A), saline (B), muscimol (C), and bicuculline (D) recording sessions from a representative cat. The duration and temporal distribution of behavioral states during a session with 2 injections of saline (vertical arrows in B) were similar to those observed during control sessions. Note that the injection of muscimol (vertical arrow in C), which was made during a non-REM (NREM) sleep episode, immediately induced wakefulness and blocked the subsequent occurrence of both NREM sleep and REM sleep for 105 and 165 min, respectively. In contrast, an injection of bicuculline into the nucleus pontis oralis (NPO; down-arrow  in D) induced a short-latency (3 min), long-duration (53 min) episode of REM sleep. Effects of GABA (F), muscimol (G), and bicuculline (H) during each of the 4 h after the injections on the percentage of time spent in 3 behavioral states. Note the long-lasting effect of muscimol, which was usually present more than 2 h after the injection. The effect of bicuculline, however, occurred mainly during the 1st hour. The vertical bar represents SE.


                              
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Table 1. Effects of GABA, muscimol, and bicuculline injections on wakefulness and sleep

On the other hand, microinjections of bicuculline, a GABAA receptor antagonist, in four cats induced REM sleep with a short latency. The bicuculline-induced state and naturally occurring active sleep appeared to be indistinguishable on the basis of the polygraphic recordings (Fig. 1, B and D). When injected during NREM sleep (vertical arrow in Fig. 2D) or wakefulness, a long-duration episode of REM sleep arose. The effects of bicuculline injections on the percentages of time spent in different behavioral states were examined during 4-h recording periods after the injection (Table 1). The increase in the percentages of time spent in REM sleep following the injection of bicuculline (203%, P < 0.01) was statistically significant compared with that following the injection of saline. The mean latency of bicuculline-induced REM sleep was significantly shorter than that following saline injections (bicuculline: 2.5 ± 0.5 min., n = 10 vs. saline: 40.7 ± 4.1 min., n = 10; P < 0.01). On the other hand, there was no statistically significant difference in the time spent in NREM sleep. Bicuculline was also injected at different concentrations (1, 5, 8, and 10 mM). A positive correlation was found between the dose of bicuculline and the mean percentage of time spent in REM sleep during the first hour after the injection of the drug (1 mM: 24.8 ± 4.1%, n = 4; 5 mM: 58.0 ± 4.5%, n = 3; 8 mM: 69.3 ± 6.9%, n = 3; 10 mM: 74.2 ± 5.6%, n = 10; Y = 5.0X + 28.1, r = 0.95, P < 0.05, Spearman rank correlation coefficient). Eight injections (muscimol, n = 3; bicuculline, n = 5), which were placed in a region posterior to the NPO, did not produce statistically significant changes in either the percentage of time spent in sleep or wakefulness or in the latency of REM sleep. These results suggest that the effects of GABAA agonist and antagonist were site specific and localized to the NPO.

The effect of bilateral injections, into the NPO, of phosphorothioated antisense oligonucleotides (S-oligos) against the GABA synthetic enzyme, GAD, was then examined in three cats. Figure 3 presents the mean percentages of time spent in wakefulness, NREM, and REM sleep on the day before the injection, the day of the injection, and the following 5 days. Note that on the day of the injection and the first postinjection day, antisense produced a significant decrease in wakefulness and an increase in REM sleep, which occurred due to the generation of longer duration episodes of REM sleep. These changes were maximal on the first postinjection day. On day 2 following injections, there was a rebound increase in the percentage of the time spent in wakefulness and a decrease in REM sleep. The percentages of time spent in NREM sleep following injections of antisense, however, were not significantly different from those following control sense injections. Intracerebral administration of antisense oligonucleotides targeted at GAD mRNA has been shown to specifically decrease the expression of GAD protein and subsequently the levels of GABA availability (Bannai et al. 1998; McCarthy et al. 1994). Thus we believe that the preceding data demonstrate that GABA actions are critical for wakefulness to occur.



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Fig. 3. Effect of antisense on the mean percentages of time spent in different behavioral states during 5-h recording sessions on 7 consecutive days in 3 cats. Each circle and vertical bar represent the mean and SE, respectively. Filled and open circles represent the data of antisense and control sense injections, respectively. Data of day 0 (the injection day) was collected 1 h after the 1st injection of antisense. Wakefulness on day 0: antisense 34.7 ± 2.4%, n = 4 vs. control sense 44.0 ± 2.6%, n = 3; REM sleep on day 0: antisense 18.6 ± 1.3%, n = 4 vs. control sense 12.1 ± 0.8%, n = 3; wakefulness on day 1 (the 1st postinjection day): antisense 30.4 ± 0.9%, n = 4 vs. control sense 43.7 ± 3.4%, n = 3; REM sleep on day 1: antisense 21.0 ± 0.9%, n = 4 vs. control sense 12.6 ± 0.5%, n = 3; ** P < 0.01; * P < 0.05.

As a control for the unique importance of GABA and to determine whether a glycinergic inhibitory system might also be involved in the control of wakefulness and REM sleep, behavioral-states responses to microinjections of glycine and its antagonist into the NPO were examined. Microinjections of neither glycine (n = 5) nor its antagonist, strychnine (n = 7) had any effect on the duration and temporal distribution of the states of wakefulness or sleep (Table 1). These data suggest that, unlike the NPO GABAergic system, glycinergic pontine reticular mechanisms do not play a direct, critical role in the control of the behavioral states of wakefulness and sleep.


    DISCUSSION
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The present data provide the first evidence that a GABAergic inhibitory system is involved in the control of wakefulness and REM sleep. We suggest that REM sleep neurons within the NPO are tonically inhibited by GABAergic neurons and that when this inhibition takes place, wakefulness, but not REM sleep occurs. This conclusion is supported by the finding that when GABAergic synaptic transmission in the NPO is suppressed, for example, by the injection of either a GABAA antagonist or antisense to GAD (which interferes with the synthesis of GABA), REM sleep arises. The concept that REM-sleep neurons in the pons are under tonic GABAergic inhibitory control is also supported by recent unit recording data (Sakai and Koyama 1996). However, the present data were obtained following the pharmacological and antisense manipulation of GABAergic synaptic transmission in the NPO. Additional studies are needed to confirm the physiological significance of these GABA-related effects and the precise fashion in which GABAergic synaptic transmission is involved in the processes that regulate behavioral states.

The present results highlight the critical importance of taking into consideration the site within the brain when evaluating the response to a pharmacological agent. For example, Sastre et al. (1996) reported that muscimol, when injected into the periaqueductal gray, promotes REM sleep, whereas we found that it induced wakefulness when applied within the NPO. Furthermore, potent hypnotics, such as benzodiazepines, are known to act on the GABA ionophore and enhance the postsynaptic inhibitory actions of GABA (Thompson 1994). Therefore benzodiazepines can not be producing their hyponotic effects by acting on the GABA system in the NPO. These sleep inducing effects of GABA contrast with the GABAergic pontine-dependent waking drive described in the present report. Thus it appears to be only in the NPO that GABA functions to promote wakefulness and its antagonists to selectively produce REM sleep.

It is interesting to note that the effects of the GABA agonist and antagonist were localized to the NPO where REM sleep is also induced by the injection of cholinergic agonists (Baghdoyan et al. 1993; George et al. 1964; Yamamoto et al. 1990; Yamuy et al. 1993). Why are both cholinergic and GABAergic input present in the NPO? Although a number of studies have demonstrated that a pontine cholinergic mechanism is involved in the generation of REM sleep (Jones 1991; Rye 1997; Siegel 1994; Steriade and McCarley 1990), we suggest that the pontine GABAergic processes described in the present study are responsible for the generation and maintenance of wakefulness as well as functioning as part of the mechanisms that control REM sleep.

The generation of REM sleep directly following wakefulness, without the usual preceding period of NREM sleep when an antagonist to the GABA receptor is administrated, suggests that there is a distinct separation between the structures responsible for REM sleep and NREM sleep. This is consistent with the current belief that the structures responsible for NREM generation are located in forebrain regions (Sherin et al. 1996; Szymusiak and McGinty 1990).


    ACKNOWLEDGMENTS

We thank Dr. J. K. Engelhardt for critical comments regarding the manuscript.

This work was supported by National Institutes of Health Grants NS-23426, NS-09999, and MH-43362.


    FOOTNOTES

Address for reprint requests: M. H. Chase, Dept. of Physiology, 53-231 CHS, UCLA School of Medicine, Los Angeles, CA 90095-1746.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 March 1999; accepted in final form 17 June 1999.


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