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Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48109
Submitted 2 February 2004; accepted in final form 10 June 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Hobson and Pace-Schott 2002; Lydic and Baghdoyan 2003; McCarley 1999; Pace-Schott and Hobson 2002; Reinoso-Suarez et al. 2001; Saper et al. 2001). Cholinergic neurotransmission in the pontine reticular formation is a principle component of the neuronal network generating the rapid eye movement (REM) stage of sleep (Steriade and McCarley 1990). Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in brain, and many drugs used clinically to produce sleep or anesthesia enhance GABAergic transmission at GABAA receptors (Campagna et al. 2003; Lancel 1999). The present study focused on the REM sleep-related interaction between pontine cholinergic and GABAergic neurotransmission based on the following rationale.
The pontine reticular formation does not contain cholinergic neurons and receives cholinergic projections from the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei (reviewed in Steriade and McCarley 1990). LDT/PPT neurons release acetylcholine (ACh) in the pontine reticular formation (Lydic and Baghdoyan 1993), and during REM sleep, ACh release is significantly increased in the pontine reticular formation over wakefulness and non-REM (NREM) sleep levels (Kodama et al. 1990; Leonard and Lydic 1995). Direct administration of cholinomimetics into the pontine reticular formation causes a concentration dependent enhancement of REM sleep that is blocked by atropine, suggesting that cholinergic transmission at pontine reticular formation muscarinic receptors contributes to REM sleep generation (reviewed in Baghdoyan 1997). Further support for the role of pontine cholinergic neurotransmission in REM sleep generation comes from studies showing that the discharge rate of putatively cholinergic LDT/PPT neurons is significantly increased prior to and during REM sleep (El Mansari et al. 1989; Kayama et al. 1992; Steriade et al. 1990), electrical stimulation of LDT/PPT neurons increases REM sleep (Thakkar et al. 1996), and neurotoxic lesions of the LDT/PPT decrease REM sleep in amounts proportional to the loss of cholinergic neurons (Webster and Jones 1988). Taken together, these data provide strong support for the role of ACh, the LDT/PPT, and the pontine reticular formation in generating REM sleep.
GABAergic neurons are distributed throughout cholinergic (LDT/PPT) and noncholinergic cholinoceptive regions of the pons, including the pontine reticular formation (Ford et al. 1995; Mugnaini and Oertel 1985). GABAergic neurons in several nuclei of the pons, including the LDT/PPT, have been shown to be active during REM sleep based on expression of the protein c-Fos (Maloney et al. 1999; Torterolo et al. 2000–2002). The GABAA receptor ligands muscimol (agonist) and bicuculline (antagonist) have opposite effects on sleep and wakefulness when microinjected into the same pontine reticular formation sites where cholinomimetics enhance REM sleep. Bicuculline triggers a REM sleep-like state with a short latency, whereas muscimol increases wakefulness and suppresses sleep (Xi et al. 1999).
The mechanisms by which blocking transmission at GABAA receptors in the pontine reticular formation enhances REM sleep are unknown but may involve removing GABAergic inhibition of ACh release from LDT/PPT terminals. GABA inhibits the release of ACh in other brain regions (Giorgetti et al. 2000; Materi and Semba 2001; Moor et al. 1998; Vazquez and Baghdoyan 2003b), and enhancing cholinergic transmission in the pontine reticular formation increases REM sleep (reviewed in Baghdoyan 1997). Therefore this study tested the hypothesis that blocking GABAergic transmission in the pontine reticular formation by dialysis administration of bicuculline increases ACh release in the pontine reticular formation. Preliminary reports of these data have been presented (Baghdoyan et al. 2002; Vazquez and Baghdoyan 2003a).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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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 U. S. Department of Agriculture Animal Welfare Act and the American Physiological Society's Guiding Principles in the Care and Use of Animals (http://www.the-aps.org/publications/journals/guide.htm#note2). The rationale for performing these studies in cat has been reviewed recently (Vazquez and Baghdoyan 2003b). One advantage of cat over rat or mouse is the comparatively long duration of cat NREM sleep and REM sleep epochs, allowing collection of dialysis samples during periods comprised entirely of each sleep stage (Kodama et al. 1990; Leonard and Lydic 1995, 1997; Marrosu et al. 1995; Vazquez and Baghdoyan 2001; Vazquez et al. 2002). Second, the large size of the cat brain stem (Berman 1968) provides a high level of anatomic resolution and facilitates accurate placement of microdialysis probes. Third, bicuculline-induced REM sleep enhancement was discovered in cat (Xi et al. 1999).
Sterile surgical procedures that have been described previously (Vazquez and Baghdoyan 2003b) were used to implant cats (n = 12) with standard recording electrodes for objectively identifying states of sleep and wakefulness (Ursin and Sterman 1981). A permanent craniotomy was created over the cerebellum by surgically removing a small portion of the skull and replacing the bone with sterile Bone Wax (Ethicon, Sommerville, NJ) (Baghdoyan et al. 1998; Leonard and Lydic 1997; Lydic and Baghdoyan 2002). The craniotomy provided access to the pontine reticular formation during subsequent dialysis experiments. Dental acrylic was used to fix the electrodes to the skull and to secure two stainless steel sleeves that attach to a Kopf 880 semi-chronic head holder (David Kopf Instruments, Tujunga, CA). The Kopf 880 device permits the placement of an animal in stereotaxic position without tissue contact. The head holder and the absence of pain receptors in the brain makes it possible to place dialysis probes in specific brain regions of an unanesthetized animal (Leonard and Lydic 1997; Lydic and Baghdoyan 1993, 2002; Vazquez and Baghdoyan 2001; Vazquez et al. 2002).
Chemicals and microdialysis probes
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The GABAA receptor antagonist bicuculline methiodide and the GABAA receptor agonist muscimol were dissolved in Ringer solution [which contained (in mM)147 NaCl, 2.4 CaCl2, and 4.0 KCl and 10 µM neostigmine bromide; pH 5.8–6.2] and were made fresh prior to each experiment. Concentrations of bicuculline used to perfuse the dialysis probe were 0.03, 0.1, 0.3, 1, 3, and 10 mM. Muscimol (0.1 mM) was delivered to the probe either alone or with bicuculline (0.3 mM).
Microdialysis probes (CMA Microdialysis, North Chelmsford, MA) had a shaft length of 70 mm and a polycarbonate dialysis membrane (20-kDa cutoff, 2-mm length, 0.5 mm diam). A CMA/100 dialysis pump was used for continuous perfusion (3 µl/min) of dialysis probes with Ringer solution. Immediately prior to placing a dialysis probe in the brain and immediately after removal of a dialysis probe from the brain the percent of ACh recovered by the probe was determined in vitro (Baghdoyan et al. 1998; Lydic and Baghdoyan 2002; Tanase et al. 2003; Vazquez and Baghdoyan 2003b). Pre- and postexperimental probe recovery values were compared by t-test. Results from an experiment were included in the final data set only if there was no significant change in probe recovery during the experiment. Average ± SD of ACh recovered was 7.8 ± 2.0%, which is typical for this type of dialysis probe (Tanase et al. 2003; Vazquez and Baghdoyan 2003b).
Dialysis during general anesthesia
ACh release in the dorsal tegmental field (Kodama et al. 1990) and the medial pontine reticular formation (Leonard and Lydic 1995, 1997) increases significantly during REM sleep compared with NREM sleep and wakefulness. Therefore the first part of this study used general anesthesia to eliminate REM sleep and hold arousal state constant. This procedure has been shown to be particularly useful for studies involving microdialysis delivery of drugs to the pontine reticular formation during simultaneous collection of endogenous ACh (Baghdoyan et al. 1998; Bernard et al. 2003). Inhalation anesthesia is ideal for this purpose because end tidal anesthetic concentration can be monitored continuously using spectrophotometry. As described previously in detail (Baghdoyan et al. 1998; Tanase et al. 2003; Vazquez and Baghdoyan 2003b), every experiment began by mask induction with halothane (3–4% in oxygen). The trachea was intubated, the cat was mechanically ventilated and fixed in stereotaxic position, and monitors were placed to measure delivered and end tidal concentrations of halothane, CO2, core body temperature, blood-oxygen saturation, and heart rate. Continuous monitoring of autonomic parameters and end tidal halothane concentration helped ensure adequate depth of anesthesia. End tidal halothane was maintained at 1.4–1.5% throughout the dialysis experiments.
A microdialysis probe was aimed stereotaxically for the pontine reticular formation, located within the gigantocellular tegmental field (Berman 1968). Dialysis samples (30 µl) were collected every 10 min and ACh content was quantified as described in the following text. Stable levels of ACh release were observed within 30 min after probe placement in the brain. Five sequential dialysis samples were collected during perfusion of the dialysis probe with Ringer solution to determine control levels of ACh release (Baghdoyan et al. 1998; Vazquez and Baghdoyan 2003b). A liquid switch (CMA/110) then was turned to deliver Ringer solution containing bicuculline, muscimol, or a mixture of bicuculline and muscimol. Five sequential dialysis samples were obtained during drug administration. After collection of the last sample, the dialysis probe was removed from the brain, and the craniotomy was closed with sterile bone wax. Halothane administration was discontinued, the ventilator was turned off, and the animal was extubated when it demonstrated regular breathing. Animals were kept under continuous observation in the laboratory until fully recovered from anesthesia and were returned to their home cages in the Unit for Laboratory Animal Medicine.
Only one concentration of bicuculline was administered per experiment. A minimum of 7 days was allowed between experiments in the same animal. Dialysis probe aim sites in the same animal were separated by 
1 mm in the rostrocaudal and mediolateral planes, and the same dialysis site was used only once to avoid possible alterations in ACh release caused by probe-induced glial scarring (Moore et al. 1995).
Dialysis and polygraphic recordings during sleep and wakefulness
The effects of bicuculline on ACh release also were studied in unanesthetized cats that displayed spontaneous epochs of wakefulness, NREM sleep, and REM sleep. For every experiment, a Grass Model 7D polygraph (Grass Instruments, West Warwick, RI) was used to record the cortical electroencephalogram (EEG), electrooculogram (EOG), electromyogram (EMG), ponto-geniculo-occipital (PGO) waves, and respiratory rate. These physiological measures were used for objectively scoring states of sleep and wakefulness according to standard, previously described criteria (Lydic and Baghdoyan 2002; Ursin and Sterman 1981; Vazquez and Baghdoyan 2001; Vazquez et al. 2002).
An experiment began by placing an animal in stereotaxic position using the Kopf 880 semi-chronic head holder. A stainless steel guide tube was then stereotaxically aimed for the pontine reticular formation. A dialysis probe was inserted into the guide tube while the animal was awake. Dialysis samples (30 µl) were collected during polygraphically defined states of wakefulness, NREM sleep, and REM sleep while the dialysis probe was perfused with Ringer solution to obtain control levels of ACh release for each of the three states (Leonard and Lydic 1997; Vazquez and Baghdoyan 2001; Vazquez et al. 2002). A CMA/110 liquid switch then was activated to deliver Ringer containing bicuculline, and dialysis samples continued to be collected during objectively defined states of sleep and wakefulness. After collection of the last dialysis sample, the dialysis probe and guide tube were removed from the brain, the craniotomy was closed, and the animal was returned to its home cage.
Quantification of ACh
ACh content of the dialysis samples was quantified using a Bioanalytical Systems (BAS, West Lafayette, IN) high-performance liquid chromatography and electrochemical detection (HPLC/EC) system and a standard curve generated prior to every experiment. As previously described (Baghdoyan et al. 1998; Vazquez and Baghdoyan 2003b; Vazquez et al. 2002), dialysis samples were injected into the HPLC/EC system immediately after collection. Samples were transported through the system (1 ml/min) by a Na2HPO4 mobile phase (50 mM, pH 8.5). A column containing acetylcholinesterase produced H2O2 in amounts proportional to the ACh in the sample, and H2O2 was detected by a platinum electrode (500 mV applied potential) referenced to a Ag+/AgCl electrode. A continuous record of the chromatograms was obtained using a flatbed recorder. Chromatograms were simultaneously digitized and stored to disk with ChromGraph software (BAS). ChromGraph also was used to calculate the amount of ACh in each dialysis sample, expressed as pmol/10 min.
Histological confirmation of dialysis probe placement in the pontine reticular formation
Stereotaxic coordinates used to aim dialysis probes for the pontine reticular formation ranged from posterior (P) 1.5 to P3.0, lateral (L) 1.0 to L2.5, and horizontal (H) –4.0 to H-6.0 (Berman 1968) on either side of the brain stem. Probe placement was confirmed histologically, as described previously (Baghdoyan et al. 1998; Leonard and Lydic 1997; Tanase et al. 2003). Animals were anesthetized and brains were perfused in situ with 10% formalin, removed, and soak-fixed in formalin. The brain stem then was cryoprotected in formalin containing 30% sucrose. Serial, sagittal, brain stem sections (40 µm thick) were cut on a freezing microtome, float mounted onto chrome alum coated glass slides, and stained with cresyl violet. All tissue sections containing a dialysis probe-induced lesion were digitized and the stereotaxic coordinates of the dialysis sites were defined according to a cat brain stem atlas (Berman 1968).
Statistical analyses
Data analysis included descriptive and inferential statistics. Significant drug effects on pontine ACh release were determined by one-way ANOVA, post hoc multiple comparisons test (Dunnett's or Tukey-Kramer), and t-test. The ANOVA model was adjusted for unequal variance when needed as determined by Levene's test of equality of error variances. The degrees of freedom for the F test were adjusted to compensate for unequal variance, resulting in a noninteger value for the denominator degrees of freedom. Dunnett's T3, which takes into account unequal variances between groups, was used for post hoc multiple comparisons with the adjusted ANOVA model. Concentration response data were fit to a sigmoid curve using nonlinear regression analysis (GraphPad Prism version 4.0a for Macintosh, GraphPad Software, San Diego, CA) with the following equation: ACh release = basal release + (maximal release-basal release)/[1 + 10^(log EC50 – X)], where X is the logarithm of the bicuculline concentration. The coefficient of determination (r2) and concentration of bicuculline that produced a 50% increase in ACh release (EC50) were calculated using this equation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Figure 1A schematizes simultaneous collection of ACh and drug delivery using the same dialysis probe. Figure 1B illustrates a typical histological section used to localize microdialysis sites. Probe placement ranged from approximately P1 to P3, L1 to L2.5, and H-5 to H-7 (the H coordinate indicates the deepest part of the 2-mm-long dialysis membrane). These stereotaxic coordinates (based on the sagittal plates in Berman 1968) provide confirmation that measures of ACh were obtained from the pontine reticular formation. Figure 1C shows an example of chromatograms used to quantify ACh during control (Ringer solution) dialysis (Fig. 1C, left) and during dialysis with Ringer solution containing bicuculline (Fig. 1C, right).
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) were stable across the 50-min period of dialysis with Ringer solution. The lowest concentration of bicuculline (0.03 mM, Fig. 2A, 
) did not alter ACh release. A 10-fold greater concentration of bicuculline (0.3 mM, Fig. 2B, ) caused a clear enhancement of ACh release after 10 min of delivery. Delivering 3 mM bicuculline to the dialysis probe increased ACh release within the first 10 min (Fig. 2C, ).
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The foregoing data obtained during general anesthesia established that pharmacological antagonism of GABAA receptors in the pontine reticular formation increased ACh release in the pontine reticular formation. Because volatile anesthetics have direct actions on the GABAA receptor (Campagna et al. 2003), the effects of bicuculline on ACh release next were studied without the use of halothane to rule out possible confounding effects of the anesthetic. Polygraph recordings were used to distinguish among wakefulness (Fig. 5A), NREM sleep (B), and REM sleep (C). Figure 5D shows that REM sleep during dialysis delivery of bicuculline (REMBIC) appears to be indistinguishable from spontaneously occurring REM sleep (Fig. 5C), consistent with previous reports (Xi et al. 1999). ACh chromatograms to the right of the polygraph records illustrate typical ACh measures obtained during each arousal state.
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, 1997). ANOVA demonstrated a significant effect of arousal state on ACh release (F = 28.0; df = 2, 77; P < 0.001). ACh release was significantly (P < 0.01) greater during REM sleep than during NREM sleep or wakefulness (Tukey-Kramer multiple comparisons test). The effects of four bicuculline concentrations on ACh release during sleep and wakefulness are summarized by Fig. 6. ANOVA revealed a significant concentration main effect of bicuculline on ACh release during wakefulness (Fig. 6A; F = 7.5; df = 4, 16.3; P = 0.001) and during REM sleep (Fig. 6C; F = 9.7; df = 4, 5.8; P = 0.01). Dunnett's T3 post hoc multiple comparisons test showed that perfusion of the dialysis probe with 1 mM bicuculline significantly (P < 0.01) increased ACh release during wakefulness (Fig. 6A). Although 3 mM bicuculline increased ACh release during wakefulness by 261% over control levels, this increase was not statistically significant because of the small sample size (n = 4) and large variability (coefficient of variation = 39%). The amount of time spent in NREM sleep was reduced by lower concentrations of bicuculline (0.1 and 0.3 mM), and NREM sleep was completely suppressed by higher bicuculline concentrations (1 and 3 mM). Thus ANOVA was not applied to the ACh measures obtained during NREM sleep (Fig. 6B) due to the small sample size. During REM sleep (Fig. 6C), Dunnett's T3 indicated that dialysis with 1 and 3 mM bicuculline significantly (P < 0.01) increased ACh release.
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Behavioral effects of bicuculline
Dialysis administration of higher bicuculline concentrations to the unanesthetized cat caused a number of behavioral effects, including REM sleep enhancement, motor activation with circling behavior, and motor atonia during wakefulness. Animals that received higher concentrations of bicuculline during general anesthesia also exhibited circling behavior on recovery from anesthesia. Systematic examination of these effects was outside the scope of this study.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). The present findings support the interpretation that one mechanism by which pontine administration of bicuculline enhances REM sleep includes increasing ACh release in the pontine reticular formation. The results are discussed in the following text within the context of existing knowledge about the role of pontine GABA and ACh in REM sleep generation. GABAergic regulation of REM sleep
Sedative hypnotics that enhance the actions of GABA, such as benzodiazepines and barbiturates, generally increase NREM sleep and decrease REM sleep when administered systemically to humans or animals (Lancel 1999). However, when delivered intracranially to experimental animals, the effects of GABA on sleep and wakefulness vary significantly as a function of brain region. GABA has been shown to promote wakefulness, NREM sleep, or REM sleep, depending on the brain region of action (reviewed in Baghdoyan and Lydic 2002). In the locus coeruleus, GABAergic inhibition of waking-active neurons has been proposed to promote REM sleep generation (Kaur et al. 1997; Mallick et al. 2001). This proposal is supported by the findings that GABA tonically inhibits locus coeruleus neurons during sleep (Gervasoni et al. 1998) and that endogenous GABA levels in the locus coeruleus are greater during REM sleep than during wakefulness or NREM sleep (Nitz and Siegel 1997). GABAergic neurons in the locus coeruleus and dorsal raphe nucleus have been shown to be active during recovery REM sleep after sleep deprivation (Maloney et al. 1999). These findings are consistent with the concept that GABAergic inhibition of wakefulness-promoting monoaminergic neurons contributes to the generation of REM sleep (Jones 1990b).
In contrast to the locus coeruleus, pontine reticular formation GABA is thought to promote wakefulness and inhibit REM sleep. Early studies in rat noted that pontine reticular formation microinjection of muscimol increased wakefulness and suppressed NREM sleep, whereas microinjection of bicuculline into the same pontine reticular formation sites produced a nonsignificant decrease in the latency to onset of both NREM sleep and REM sleep (Camacho-Arroyo et al. 1991). Compelling evidence that pontine reticular formation GABA promotes wakefulness and inhibits REM sleep has come from microinjection studies in cat. These data show that pontine reticular formation administration of bicuculline causes a REM sleep-like state. Bicuculline significantly increases the amount of time spent in the REM sleep-like state and decreases the latency to onset of the REM sleep-like state (Xi et al. 1999). Microinjection of muscimol increases wakefulness and suppresses sleep (Xi et al. 1999).
The discovery that bicuculline injected into the rostral portion of the pontine reticular formation of cat significantly increased REM sleep time and decreased REM sleep latency (Xi et al. 1999) has prompted similar studies in rat and mouse focused on homologous regions of the pontine reticular formation. The first study to report bicuculline-induced REM sleep enhancement in rat used iontophoresis and localized an effective region to the subdorsolateral nucleus (Boissard et al. 2002), also named the dorsal subcoeruleus nucleus (Paxinos and Watson 1998). Unilateral microinjection of bicuculline into the dorsal subcoeruleus nucleus also has been reported to produce a REM sleep-like state (Pollock and Mistlberger 2003). Microinjection studies in rat demonstrated REM sleep enhancement caused by bicuculline bilaterally administered into the oral part of the rat pontine reticular formation (reticularis pontis oralis) (Sanford et al. 2003). Bicuculline-induced REM sleep enhancement did not occur after microinjections into the caudal part of the rat pontine reticular formation (reticularis pontis caudalis) (Sanford et al. 2003). Preliminary data indicate that microinjection of bicuculline into the rostral pontine reticular formation of C57BL/6J mouse evokes a REM sleep-like state (Falgout et al. 2003). Dialysis sites used in the present study were in the rostral and medial portion of the cat pontine reticular formation, homologous to the pontine reticular formation, oral part, in rat (Paxinos and Watson 1998) and mouse (Franklin and Paxinos 2001).
The GABAB antagonist phaclofen has been shown to significantly increase REM sleep when microinjected into cat pontine reticular formation, although the enhancement was less robust than with the GABAA antagonist bicuculline (Xi et al. 2001). Similar to muscimol, the GABAB agonist baclofen increased wakefulness and decreased NREM sleep after microinjection into the rostral pontine reticular formation (Xi et al. 2001). The effects of phaclofen and baclofen on pontine reticular formation ACh release remain to be investigated. Most recently, baclofen has been shown to inhibit REM sleep when microinjected into rat PPT (Ulloor et al. 2004). Baclofen also blocked the discharge of PPT neurons that selectively increase their firing rates during REM sleep (putatively cholinergic REM-on cells) (Ulloor et al. 2004). Cholinergic PPT neurons project to the pontine reticular formation (Jones 1990a; Mitani et al. 1988; Semba 1993; Shiromani et al. 1988) where they release ACh (Lydic and Baghdoyan 1993). Thus it is likely that PPT administration of baclofen inhibits ACh release in the pontine reticular formation.
Evocation of a REM sleep-like state or REM sleep traits during dialysis administration of bicuculline
Microinjection of bicuculline into the rostral pontine reticular formation of cat (Xi et al. 1999), rat (Sanford et al. 2003), and mouse (Falgout et al. 2003) significantly increases REM sleep. Dialysis administration of bicuculline in the present study enhanced REM sleep but not as consistently or with the same magnitude as has been reported after microinjection delivery of bicuculline in cat (Xi et al. 1999, 2001). Potential reasons for these differences include different routes of bicuculline delivery (microinjection verses microdialysis) and different sites of bicuculline administration within the pontine reticular formation. Different routes of administration would cause differences in the amount of bicuculline delivered to the tissue, and bicuculline-induced REM sleep enhancement has been shown to be concentration dependent (Sanford et al. 2003; Xi et al. 2001). Furthermore, bicuculline-induced REM sleep enhancement is site dependent within the pontine reticular formation. Effective microinjection sites in cat pontine reticular formation (Xi et al. 1999, 2001) are located more dorsally than most of the dialysis sites used for the present study. Iontophoretic studies mapping dorsal portions of the rat pontine reticular formation showed that the effective region for enhancing REM sleep with bicuculline was restricted to the subdorsolateral nucleus (Boissard et al. 2002). Microinjection studies in rat have shown that sites in the rostral pontine reticular formation (Sanford et al. 2003) or dorsal subcoeruleus nucleus (Pollock and Mistlberger 2003) were more effective for enhancing REM sleep with bicuculline than sites in the caudal pontine reticular formation (Pollock and Mistlberger 2003; Sanford et al. 2003).
Bicuculline caused motor atonia during wakefulness. Microinjection of cholinomimetics into cat pontine reticular formation can evoke individual REM sleep traits, such as motor atonia or PGO waves, dissociated from the state of REM sleep. These state-trait dissociations have been shown to be dependent on the intrapontine site of cholinomimetic administration (reviewed in Baghdoyan 1997). The dissociated REM sleep trait of motor atonia observed during wakefulness in the present study may be accounted for, in part, by the site of bicuculline administration. A systematic mapping study is required to investigate this possibility.
The evocation of motor activation and circling by dialysis administration of bicuculline is consistent with previous reports of REM sleep disruption due to circling following microinjection of bicuculline into the caudal part of the rat pontine reticular formation (Pollock and Mistlberger 2003; Sanford et al. 2003) and by microinjecting higher concentrations of bicuculline to the rostral part of the rat pontine reticular formation (Sanford et al. 2003). Motor activation also has been reported in rat after iontophoretic application of bicuculline to the dorsal subcoeruleus nucleus (Boissard et al. 2002). The neural circuits mediating bicuculline-induced circling have been discussed in detail (Sanford et al. 2003).
Limitations and conclusions
The data presented in this report suggest that GABAA receptors in cat pontine reticular formation modulate ACh release. This conclusion is limited, however, because the only drug tested was bicuculline methiodide. In thalamic slices from young rats, bicuculline methiodide has been shown to alter cell excitability by actions that are independent of GABAA receptor antagonism (Debarbieux et al. 1998). Future studies can test additional more selective GABAA antagonists, such as picrotoxin, for their ability to increase ACh release in vivo in cat pontine reticular formation. Testing the effects of GABAB antagonists and GABA uptake inhibitors on ACh release can also address the present limitations.
The synaptic location of the pontine reticular formation GABAA receptors modulating ACh release remains to be identified. Pontine reticular formation neurons are not cholinergic, and anatomical and functional data demonstrate that cholinergic input to the pontine reticular formation arises from the LDT/PPT (Jones 1990a; Lydic and Baghdoyan 1993; Mitani et al. 1988; Semba 1993; Shiromani et al. 1988; Steriade and McCarley 1990). The GABAA receptors that modulate ACh release could be localized presynaptically on LDT/PPT terminals in the pontine reticular formation or postsynaptically on pontine reticular formation neurons that project back to the LDT/PPT.
One implication of the present findings is that GABA levels in the pontine reticular formation are significantly greater during wakefulness than during REM sleep. A preliminary report of studies in rat supports this prediction (Marks et al. 2003). A second implication of the current findings is that increasing ACh release in the pontine reticular formation is one mechanism by which blockade of pontine GABAA receptors enhances REM sleep. Cholinergic neurotransmission at pontine reticular formation muscarinic receptors has been proposed to be the final common pathway through which a variety of neuroactive molecules contribute to REM sleep generation (reviewed in Baghdoyan and Lydic 2002; Lydic and Baghdoyan 2003).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. A. Baghdoyan, Dept. of Anesthesiology, The University of Michigan, 7433 Medical Sciences Bldg. I, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0615 (E-mail: helenb{at}umich.edu).
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