Department of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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.
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RESULTS |
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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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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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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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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.
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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
).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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