Theme : Dreams

A GABAergic Pontine Reticular System Is Involved in the Control of Wakefulness and Sleep

Ming-Chu Xi, Francisco R. Morales and Michael H. Chase
Department of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA
Abstract
The present work is the first in a series of studies designed to examine the role of a brainstem GABAergic system in the control of the behavioral states of sleep and wakefulness. GABA, muscimol (a GABAA receptor agonist) and bicuculline methiodide (a GABAA receptor antagonist) were microinjected, separately, into the nucleus pontis oralis (NPO) in three chronic, unanesthetized cats. The effects of these microinjections on the behavioral states of sleep and wakefulness were then examined. The injection of either GABA or muscimol induced wakefulness; quiet sleep and active sleep were suppressed. In contrast, the injection of bicucculline induced a prolonged state that was similar to naturally-occurring active sleep. These findings indicate the existence of GABAergic processes capable of controlling the activity of neurons within the NPO that are involved in the control of sleep and waking states. Specifically, these data suggest that cells within the NPO must be tonically inhibited by a GABAergic brainstem system in order for the state of wakefulness to be generated and maintained.

Current Claim: A GABAergic brainstem system plays a critical role in the control of the activity of neurons within the NPO that are involved in the control of sleep and waking states.

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Convergent lines of evidence indicate that the core of the brainstem reticular formation contains neuronal populations, with different neurotransmitter phenotypes, which are involved in the generation and maintenance of wakefulness, quiet sleep and active sleep (Batini et al., 1959a, 1959b; Steriade and McCarley, 1990; Jones, 1991). A crucial structure is the nucleus pontis oralis (NPO), which is located in the pontine tegmentum (Taber, 1961; Chase and Morales, 1990; Steriade and McCarley, 1990; Siegel, 1994). Neurons within this nucleus receive an abundant supply of cholinergic, glutamatergic, catecholaminergic and histaminergic afferents which are thought to be involved in the executive control of sleep and wakefulness (Kosaka et al., 1987; Mitani et al., 1988; Jones, 1991; Lai and Siegel, 1991; Lin et al., 1996). In addition, NPO neurons are innervated by a rich plexus of GABAergic fibers (Kosaka et al., 1987; Jones, 1991; Ford et al., 1995). The function that is subserved by this innervation is unknown, which is enigmatic when one considers the fact that GABA is the most abundant inhibitory neurotransmitter in the central nervous system (CNS), and that GABAergic systems figure prominently in critically important circuits and gating mechanisms throughout the CNS (Sivilotti and Nistri, 1991; Mody et al., 1994; Thompson, 1994).
The present report presents the results of the first in a series of systematic studies designed to elucidate the functions of GABAergic innervation of cells in the NPO. Accordingly, we examined the behavioral states of cats following the microinjection of GABA and GABAA receptor agonists and antagonists into the NPO. Both GABA and the GABAA receptor agonist, muscimol, were found to induce wakefulness. In contrast, the GABAA receptor antagonist, bicuculine, evoked a prolonged state similar to naturally-occurring active sleep. This is the first evidence that a GABAergic inhibitory system is involved in the maintenance of wakefulness, which suggests that the occurrence of this state is not solely dependent on excitatory transmission within the reticular activating system. In addition, the data indicate that when this inhibitory control is not present, the state of active sleep arises. Accordingly, we suggest that these data establish a new foundation and perspective for understanding the neural mechanisms involved in the control of wakefulness as well as sleep.

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Three 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). Briefly, under halothane anesthesia, using sterile surgical procedures, cats were implanted with electrodes for recording the electroencephalogram (EEG), electrooculogram (EOG) and electromyogram (EMG). A Winchester plug, connected to these electrodes, and a chronic head-restraining device were bonded to the calvarium with acrylic cement. A hole 4-5 mm in diameter, which was drilled in the calvarium overlying the cerebellar cortex, was covered with bonewax. This hole provided subsequent access for a cannula that was used for drug microinjections.
After recovery from surgery, all cats were head-restrained for 5-6 hours a day, for two weeks, for adaptation to the recording conditions. Following the adaptation period, carbachol (0.25 µl, 22 mM) was injected into the rostral pontine reticular formation (L:2, P:3, and H:-4 [Berman, 1968]) using a 2-µl Hamilton syringe in order to determine the optimal stereotaxic coordinates for the NPO in each animal. 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 active sleep with a latency shorter than 4 min. In experimental sessions, all of which were conducted between 10:00 and 16:00, GABA (0.25 µl, 200 mM in saline), muscimol (0.25 µl, 10 mM in saline), or bicuculline (0.25 µl, 10 mM in saline) were 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 GABA, bicuculline or muscimol. The injections were delivered, unilaterally, over a period of 1 minute. All injections were carried out while the animals were in quiet sleep, except for 3 injections of GABA and 2 injections of muscimol which were delivered during active sleep, and 2 injections of muscimol which were delivered during the carbachol-induced active sleep-like state. No injections were carried out during control sessions.

The EEG, EOG and EMG were recorded on a video cassette recorder by means of a PCM recording adapter (Vetter Co., Model 4000) for subsequent off-line analysis. Polygraphic recordings, which were divided into 30-sec epochs, were used to construct hypnograms. States of wakefulness (W), quiet sleep (QS) and active sleep (AS) were scored according to standard polygraphic and behavioral criteria (Ursin and Sterman, 1981). Experimental data are expressed as means ± SEM. The statistical significance of the difference between sample means was evaluated using the two-tailed unpaired Student's t-test and an analyses of variance (ANOVA). The criterion chosen to discard the null hypothesis was p< 0.05.

At the conclusion of the microinjection 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 Nembutal and perfused with saline followed by a solution of 10% formaldehyde. Coronal serial sections of brainstem tissue were examined to verify the injection sites.

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Figure 1

Figure 2

Figure 3

Forty microinjections were made into the pontine reticular formation to examine the effects of GABA as well as a GABAA receptor agonist and antagonist on behavioral states. Of these 40 injections, 35 were placed in the NPO (Fig. 1D) and 5 were directed to a region of the brainstem that was 1 to 3 mm posterior to the NPO.
Effects of GABA
Microinjections of GABA into the NPO during either the state of active sleep (n=3) or quiet sleep (n=2) induced wakefulness. A polygraphic recording of this waking effect of GABA is presented in Fig. 1A. The latency to the onset of wakefulness, as measured from the time of the beginning of the injection of GABA, was 4 minutes. This episode of GABA-induced wakefulness lasted for 25 minutes.

Because of the accepted notion that GABAA receptors mediate most of the postsynaptic inhibitory actions of GABA in the CNS (Thompson 1994; Smith and Olsen, 1995), the effect of microinjections of the GABAA receptor agonist, muscimol, and the GABAA receptor antagonist, bicuculline were examined.

Effects of muscimol
A series of hypnograms showing the effects of microinjections of saline and muscimol in a representative cat are shown in Figures 2A, B and C. Injections of saline (vertical arrows in Fig. 2B) did not change the duration and temporal distribution of sleep and wakefulness compared with that observed during the control recording session (Fig. 2A). In contrast to saline, the injection of muscimol (arrow in Fig. 2C) during quiet sleep induced a prolonged episode of wakefulness which lasted 124 minutes, except for one brief episode of quiet sleep. Note that this injection of muscimol blocked the subsequent occurrence of both quiet and active sleep for a period of 128 and 150 minutes, respectively.

The effects of muscimol on the percentage of time that the cats spent in different behavioral states during the first hour following the injection are presented in Fig. 3. Compared to the percentage observed following saline injections (n=8), injections of muscimol (n=10) significantly increased the time spent in wakefulness (263%; p<0.01) and significantly reduced the time spent in active sleep (75%; p<0.05) and quiet sleep (69%; p<0.01).

In order to examine the effect of muscimol for a longer period, a determination was made of the percentages of time spent in different behavioral states for the entire 4-hour recording session following the injections. These results are summarized in Table 1. The quantitative changes in the percentage of time spent in wakefulness, quiet and active sleep during the 4-hr recording period were similar to those observed during the first hour, which demonstrates a long duration of effect of muscimol.

The effect of microinjection of muscimol on the latency of active sleep was studied by measuring the latency to the onset of the first active sleep episode following the injection. Injections of muscimol significantly increased the mean latency of active sleep (Table 1).

Effects of bicuculline
Microinjections of bicuculline induced, with a short latency, a behavioral state that was similar to naturally-occurring active sleep. The polygraphic recordings from an episode of the active sleep-like state that occurred following the injection of bicuculline are shown in Fig. 1B. The bicuculline-induced state and naturally occurring active sleep appear to be indistinguishable on the basis of the polygraphic recordings (Fig. 1C). Fig. 2D is a hypnogram showing the effect of the injection of bicuculline. When injected during quiet sleep (vertical arrow in Fig. 2D), it induced a short-latency (3 minutes), long-duration (53 minutes) episode of an active sleep-like state.

The effects of bicuculline injections on the percentage of time spent in different behavioral states were also examined during the first hour following the injection as well as throughout the 4-hour recording period (Fig. 3 and Table 1). During the first hour following the injection of bicuculline, the cats spent most of their time in active sleep. The increase in the percentage of time spent in active sleep following the injection of bicuculline (n=12) was statistically significant compared to that following the injection of saline (n=8; 575%, p<0.01; Fig. 3). This increase was accompanied by significant decreases of 80% and 64% in the time spent in either quiet sleep or wakefulness (Fig. 3; p<0.01 and p<0.05, respectively). There was also a statistically significant increase of 175% in the time spent in active sleep during the 4-hour recording period following the injection (Table 1; p<0.01). The changes in the total time spent in either quiet sleep or wakefulness during the 4-hour recording period, however, were not statistically significant (Table 1). The above data indicate that the effect of bicuculline was mainly due to changes that occurred during the first hour following the injection. The bicuculline injection induced an active sleep-like state with a short latency (bicuculline: 2.5 ± 0.5 min., n=12 vs. saline: 40.7 ± 4.1 min., n=8; p<0.01; Table 1).

Five injections, which were placed in a region outside and posterior to the NPO (P: 4.3 ± 0.2 mm [Berman 1968]), did not induce active sleep nor did it produce statistically significant changes in either the percentage of time spent in sleep or wakefulness or in the latency of active sleep. These results suggest that the bicuculline effects were site specific and localized to the NPO.

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This study is the first demonstration that the microinjection of GABA or muscimol into the NPO induces wakefulness and suppresses both active sleep and quiet sleep. On the other hand, the injection of bicuculline induces, with a short latency, a behavioral state that is similar to naturally-occurring active sleep.

The preceding data provide evidence indicating that a GABAergic system is involved in the control of the generation of the behavioral states of wakefulness and active sleep. When this particular GABAergic system is activated, it functions to abolish sleep and to produce wakefulness. When the activity of this GABAergic system is decreased or is suppressed, the state of active sleep occurs. Therefore, it is possible that an active sleep gating mechanism exists within the NPO that is closed during wakefulness (and possibly during quiet sleep as well) and is opened during active sleep. The operation of this gating mechanism apparently functions in such a way that wakefulness is maintained when the activity of GABAergic synaptic transmission in the NPO is predominant, so that the activity of AS-ON neurons in the NPO, whose discharge is selectively related to active sleep, are tonically inhibited; active sleep occurs when this GABAergic synaptic transmission is suppressed. The recent unit-recording data by Sakai and Koyama (1996) also suggest that some of the AS-ON cells in the pons are under tonic GABAergic inhibitory control, which supports the preceding hypothesis. The interactions of the presently proposed GABAergic system with cholinergic input to the NPO from the laterodorsal tegmental nuclei (LDT) and pedunculopontine tegmental nuclei (PPT) (Baghdoyan et al., 1984, 1987; George et al., 1964; Morales et al., 1987) remains to be determined.
With regard to the location of the somas of GABAergic neurons of this system, there are two likely possibilities: (1) the GABAergic somas belong to local interneurons which are located within the NPO, or (2) they are located outside the NPO and send axons to this area. In support of the first possibility, anatomical data have demonstrated that GABAergic neurons are distributed in and in the vicinity of the NPO (Kosaka et al., 1987; Jones, 1991; Ford et al., 1995). On the other hand, anatomical data also indicate that there exist a large number of GABAergic neurons whose axons project over long distances in the CNS (Mugnaini and Oertel, 1985; Ford et al., 1995). It is thus possible that long axonal GABAergic projecting neurons innervate NPO neurons which are responsible for the control of wakefulness and sleep. Further studies are required to determine the site of origin of the GABAergic system described in the present report.

Three types of GABA receptors have been distinguished on the basis of their pharmacological properties and the physiological consequences of their activation: these are the GABAA, GABAB and GABAC receptor subtypes (Thompson, 1994). Among these subtypes, GABAA receptors are known to mediate most postsynaptic inhibitory transmission in the CNS (MacDonald and Olsen, 1994; Thompson, 1994; Smith and Olsen, 1995). Therefore, in the present study we focused on the effects of microinjections of a GABAA receptor agonist or antagonist into the NPO. However, it is possible that inhibitory actions mediated by GABAB and GABAC receptor subtypes in the NPO are also involved in the control of sleep and wakefulness. In this regard, we have found in preliminary studies that the injection of a GABAB receptor antagonist, 2-OH saclofen, while less effective than bicuculline, also appears to be capable of inducing active sleep.

In summary, the present results indicate that wakefulness is maintained by GABAergic synaptic transmission within the NPO, while the suppression of GABAergic synaptic transmission in this same area produces an active sleep-like state. We therefore conclude that there is a GABAergic system which plays a critical role in the control of the activity of NPO neurons that are involved in generating and maintaining the behavioral states of sleep and wakefulness.

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This work was supported by the following grants from the U.S. Public Health Service: NS 23426, NS 09999 and MH 43362. We thank Dr. J.K. Engelhardt for his critical comments regarding this manuscript.

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