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  • 1. Subimal Datta J Neurophysiol 97:3841-3850, 2007. First published Apr 4, 2007; doi:10.1152/jn.00263.2007 You might find this additional information useful... This article cites 98 articles, 20 of which you can access free at: http://jn.physiology.org/cgi/content/full/97/6/3841#BIBL This article has been cited by 1 other HighWire hosted article: Protein Kinase A in the Pedunculopontine Tegmental Nucleus of Rat Contributes to Regulation of Rapid Eye Movement Sleep S. Datta and F. Desarnaud J. Neurosci., September 15, 2010; 30 (37): 12263-12273. [Abstract] [Full Text] [PDF] Updated information and services including high-resolution figures, can be found at: http://jn.physiology.org/cgi/content/full/97/6/3841 Additional material and information about Journal of Neurophysiology can be found at: http://www.the-aps.org/publications/jn Downloaded from jn.physiology.org on October 28, 2010 This information is current as of October 28, 2010 . Journal of Neurophysiology publishes original articles on the function of the nervous system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2007 by the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at http://www.the-aps.org/.
  • 2. J Neurophysiol 97: 3841–3850, 2007. First published April 4, 2007; doi:10.1152/jn.00263.2007. Activation of Pedunculopontine Tegmental PKA Prevents GABAB Receptor Activation–Mediated Rapid Eye Movement Sleep Suppression in the Freely Moving Rat Subimal Datta Sleep and Cognitive Neuroscience Laboratory, Department of Psychiatry, Boston University School of Medicine, Boston, Massachusetts Submitted 8 March 2007; accepted in final form 3 April 2007 Datta S. Activation of pedunculopontine tegmental PKA prevents rats have identified several different types of cells whose firing GABAB receptor activation–mediated rapid eye movement sleep rates correlate with both wakefulness and REM sleep (Datta suppression in the freely moving rat. J Neurophysiol 97: 3841–3850, 1995; Datta and Siwek 2002; El-Mansari et al. 1989; Steriade 2007. First published April 4, 2007; doi:10.1152/jn.00263.2007. The pedunculopontine tegmental (PPT) GABAergic system plays a crucial et al. 1990; Thakkar et al. 1998). Some of the recent pharma- role in the regulation of rapid eye movement (REM) sleep. I recently cological studies in cats and rats have shown that the activation reported that the activation of PPT GABAB receptors suppressed of PPT kainate type glutamate receptors induces REM sleep Downloaded from jn.physiology.org on October 28, 2010 REM sleep by inhibiting REM-ON cells. One of the important mech- (Datta 2002; Datta and Siwek 2002; Datta et al. 2002). Other anisms for GABAB receptor activation–mediated physiological action studies have shown that the activation of PPT GABAB recep- is the inhibition of the intracellular cAMP-dependent protein kinase A tors inhibit PPT REM-ON cells activity and suppresses REM (cAMP-PKA) signaling pathway. Accordingly, I hypothesized that sleep (Datta et al. 2003; Ulloor et al. 2004). Despite tremen- the PPT GABAB receptor activation–mediated REM sleep suppres- dous progress in the identification of specific neurotransmitters sion effect could be mediated through inhibition of cAMP-PKA and receptors involved in the regulation of the PPT cells activation. To test this hypothesis, a GABAB receptor selective neuronal activity and REM sleep, very few attempts have been agonist, baclofen hydrochloride (baclofen), cAMP-PKA activator, Sp- adenosine 3 ,5 -cyclic monophosphothioate triethylamine (SpCAMPS), made to study the possible intracellular signal transduction and vehicle control were microinjected into the PPT in selected mechanisms of the PPT that may be involved in the regulation combinations to determine effects on sleep-waking states of chroni- of REM sleep. cally instrumented, freely moving rats. Microinjection of SpCAMPS It is known that the GABAB receptors couple to Gi/Go G (1.5 nmol) induced REM sleep within a short latency (12.1 3.6 min) proteins (Couve et al. 2000; Kerr and Ong 1995; Mody et al. compared with vehicle control microinjection (60.0 6.5 min). On 1994; Robbins et al. 2001; Sivilotti and Nistri 1991; Takahashi the contrary, microinjection of baclofen (1.5 nmol) suppressed REM et al. 1998; Thompson 1994). It is also known that Gi/Go G sleep by delaying its appearance for 183 min; however, the sup- proteins inhibit adenylyl cyclase (AC) and inhibition of AC pression of REM sleep by baclofen was prevented by a subsequent prevents activation of the cAMP-protein kinase A (PKA) microinjection of SpCAMPS. These results provide evidence that the activation of cAMP-PKA within the PPT can successfully block the signal transduction pathway (Gilman 1987; Marinissen and GABAB receptor activation–mediated REM sleep suppression effect. Gutkind 2001). Because it has been shown that the activation These findings suggest that the PPT GABAB receptor activation– of GABAB receptors within the PPT suppresses activity of mediated REM sleep regulating mechanism involves inactivation of REM-ON cells and REM sleep in the freely moving rat (Ulloor cAMP-PKA signaling in the freely moving rat. et al. 2004), it is reasonable to suggest that the PPT GABAB receptor activation–induced suppression of REM sleep may have been caused by the inhibition of AC that ultimately INTRODUCTION prevented activation of cAMP-PKA. In support of this sugges- Research in the past 10 yr, ranging from developmental tion, two recent studies have shown that the local application of maturation to genetic model of Alzheimer’s, has provided drugs that inhibit AC activity and cAMP-PKA activation in the evidence that the cholinergic cells in the pedunculopontine PPT suppress REM sleep (Bandyopadhya et al. 2006; Datta tegmentum (PPT) and laterodorsal tegmentum (LDT) are crit- and Prutzman 2005). Although these two studies have shown ically involved in the physiological regulation of rapid eye that the inhibitions of both AC and cAMP-PKA activation in movement (REM) sleep (Datta 1995; Deurveilher and Hen- the in the PPT suppresses REM sleep (Bandyopadhya et al. nevin 2001; Garcia-Rill 1997; Kobayashi et al. 2004a,b; Zhang 2006; Datta and Prutzman 2005), no studies have shown that et al. 2005). The PPT, situated in the dorsolateral mesopontine the activation of either AC or cAMP-PKA in the PPT could tegmentum, contains a prominent group of cholinergic and increase REM sleep. It has also not been shown that the some noncholinergic neurons, which project widely throughout GABAB receptor activation–mediated REM sleep suppression the brain stem and forebrain (for reviews, see Datta 1995; effect could be rescued and/or bypassed by the activation of Garcia-Rill 1991; Jia et al. 2003; Jones 2004; Lai et al. 1993). intracellular cAMP-PKA. Single cell recordings from the PPT/LDT of behaving cats and In this study, I examined the changes in sleep-wake behavior of freely moving rats after activation of cAMP-PKA, GABAB Address for reprint requests and other correspondence: S. Datta, Sleep and Cognitive Neuroscience Lab., Dept. of Psychiatry, Boston Univ. School of The costs of publication of this article were defrayed in part by the payment Medicine, M-902, 715 Albany St., Boston, MA 02118 (E-mail: of page charges. The article must therefore be hereby marked “advertisement” SUBIMAL@BU.EDU). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. www.jn.org 0022-3077/07 $8.00 Copyright © 2007 The American Physiological Society 3841
  • 3. 3842 S. DATTA receptor, or both cAMP-PKA and GABAB receptor within the relation to sterotaxic “0”: anterior, 1.0 mm; lateral, 1.8 mm; and PPT. Our results show that the activation of PPT cAMP-PKA horizontal, 3.0 mm) as described previously (Datta 2002; Datta et al. induces REM sleep. Although activation of PPT GABAB 2002). receptors suppresses REM sleep, these results also show that the activation of cAMP-PKA could rescue REM sleep from the Intracerebral microinjections and experimental design GABAB receptor activation–induced suppression. The results After the adaptation recording sessions, microinjection sessions of these experiments indicate that, in the PPT, GABAB recep- began. During experimental sessions, animals were connected to the tor activation–mediated REM sleep regulation involves inacti- polygraphic recording system 15 min before the first microinjection vation of the cAMP-PKA system. into the PPT. The microinjection system consisted of a 32-gauge stainless steel injector cannula with a 26-gauge collar that extended METHODS 2.0 mm beyond the implanted guide tube. The collar was connected to a 1.0- l motor-driven Hamilton microsyringe with PE 20 tubing. In Subjects and housing this study, a double-microinjection protocol was adopted as described earlier (Datta 2002; Datta et al. 1993). Briefly, each microinjection Experiments were performed on 28 adult male Sprague-Dawley session consisted of two injections in the same site with a 15-min rats (Charles River, Wilmington, MA) weighing between 250 and interval between the first and second injections. While the animal was 350 g. The rats were housed individually at 24°C with food and water connected to the recording system, the stylett was removed, and a provided ad libitum with lights on from 7:00 AM to 7:00 PM (light control vehicle-filled (100 nl volume of 0.9% saline) or any one of the cycle) and off from 7:00 PM to 7:00 AM (dark cycle). The principles for two drug-filled (1.5 nmol in 100 nl, SpCAMPS or Baclofen) injectors the care and use of laboratory animals in research, as outlined by the were introduced through the guide tube for the first injection. One National Institutes of Health Publication no. 85–23 (1985), were Downloaded from jn.physiology.org on October 28, 2010 minute after the insertion of the injector cannulae, 100 nl of control strictly followed. saline or any one of the two drugs was microinjected over a 60-s period (Pump II Pico Plus, Harvard Apparatus, Holliston, MA). The Drug and vehicle for microinjections cannula was gently withdrawn 2 min after the injection, and the stylett was reintroduced inside the guide tube. For the second injection, In this study, GABAB receptors in the PPT were activated by a control saline or SpCAMPS was injected into the same site using the GABAB receptor–specific agonist, baclofen hydrochloride (baclofen; same injector system as described for the first injection. During the molecular weight: 250.13), purchased from Sigma Chemicals (St. microinjections, the animals were free to move around the cage with Louis, MO). The selection of baclofen was based on the selective the cannulae in place. Because of the extended tubing, the injections agonistic effects on GABAB receptors (Bowery 1993; Enna and could be made without restraining mobility of the animal. Immedi- Maggi 1979; Falch et al. 1986; Fromm et al. 1990; Hill and Bowery ately after completion of the microinjection procedure, polygraphic 1981; Hong and Henry 1991; Kerr and Ong 1995; Misgeld et al. 1995; variables were recorded continuously for 6 h (between 10:00 AM and Paredes and Agmo 1989; Ulloor et al. 2004). In addition to specificity, 4:00 PM), when rats would normally be sleeping (Datta 2000). Each of the above studies have shown that baclofen is readily soluble in water these rats received a total of two microinjections (100 nl each) into the and accessible to extracellular receptors. Baclofen was dissolved in PPT (left or right side) in a single experimental recording session. 0.9% saline to make 1.5 nmol/100 nl dose. The optimum dose of None of the rats were used for more than one microinjection recording baclofen (1.5 nmol/100 nl) was predetermined from an earlier dose- session. response study (Ulloor et al. 2004). Another drug used in this study The injection protocol was designed so that 28 rats were divided was an activator of cAMP-PKA activity, Sp-cAMPS triethylamine equally into four groups (n 7 rats/group). The first group received (SpCAMPS; molecular weight 446.46), also purchased from Sigma saline saline injections (control group) into the PPT. The second Chemicals. The selection of this drug was based on the selective group received baclofen saline injections (GABAB receptor acti- activation effect on intracellular cAMP-PKA activity (Capece and vation group) into the PPT. The third group received baclofen Lydic 1997; Cook et al. 1995; Punch et al. 1997; Wang et al. 1991). SpCAMPS injections (GABAB receptor PKA activation group) In addition to the selectivity, SpCAMPS was suitable for this study into the PPT. The fourth group received SpCAMPS saline injec- because these studies have shown that it is water soluble, cell tions (PKA activation group) into the PPT. At the end of experimental membrane permeable, has reversible effects, and has been used session and before perfusion, with the use of same injector used for successfully in microinjection studies in behaving animals (Capece saline or drugs, 100 nl of black ink was microinjected 1 mm dorsal to and Lydic 1997; Cook et al. 1995; Punch et al. 1997; Wang et al. each injection site for localizing the injection sites as described earlier 1991,1993). SpCAMPS was dissolved in 0.9% saline to make 1.5 (Datta 2002; Datta et al. 2001a). nmol/100 nl dose. This 0.9% saline (100 nl) was also used for the vehicle control microinjection. Control saline and drug solutions were freshly prepared under sterile conditions before each use. Determination of behavioral states and data analysis For the purpose of determining possible effects on sleep and Surgical procedures and implantation of electrodes wakefulness, polygraphic data were captured on-line in a computer using “Gamma” software (Grass Product Group, Astro-Med, West Treatment of the animals and surgical procedures were in accor- Warwick, RI). From this captured data, three behavioral states were dance with an approved institutional animal welfare protocol (AN- distinguished and scored visually using “Rodent Sleep Stager” soft- 14084). Efforts were made to minimize the number of animals used ware (Grass Product Group, Astro-Med) as described earlier (Datta and their suffering. Rats were anesthetized with pentobarbital sodium 2002; Datta et al. 2004). The behavioral states of wakefulness (W), (40 mg/kg, ip), placed in the stereotaxic apparatus, and secured using slow-wave sleep (SWS), and REM sleep were scored in successive blunt rodent ear bars (Paxinos and Watson 1997). With the use of 10-s epochs. The polygraphic measures provided the following de- sterile procedures, cortical EEG, dorsal neck muscle EMG, and pendent variables, which were quantified for each recording session: hippocampal EEG (to record theta waves) recording electrodes were 1) percentage of recording time spent in W, SWS, and REM sleep; 2) chronically implanted, as described elsewhere (Datta 2000). In addi- latency to onset of the first episode of REM sleep after the onset of tion, a stainless steel guide tube (26 gauge), with an equal-length recordings; 3) total number of REM sleep episodes; and 4) mean stylett inside, was stereotaxically implanted 2 mm above the PPT (in duration of REM sleep episodes. The effects of the four different J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 4. PPT GABAB RECEPTOR AND PKA IN REM SLEEP REGULATION 3843 treatments (saline saline; baclofen saline; baclofen SpCAMPS; SpCAMPS saline) on the percentages of W, SWS, and REM sleep were statistically analyzed using a two-way ANOVA with time as a repeated-measure variable (6 levels corresponding to 6 1-h epochs after injections) and treatment as a between-group variable (4 levels corresponding to the 4 different treatment groups). After a two-way ANOVA, post hoc Scheffe F tests were done to determine the individual levels of significant difference between the control (sa- line saline) and the three different drug treatment protocols at six individual data points. Individual post hoc tests were also done to compare between three different drug treatment protocols. The la- tency, number, and duration of REM sleep episodes were analyzed using a one-factor ANOVA followed by post hoc Scheffe F tests. Statistical analyses (2-way ANOVA, 1-factor ANOVA, and Scheffe F test) were performed with the use of StatView statistical software (Abacus Concepts, Berkeley, CA). Histological localization of injection site At the conclusion of the microinjection experiments, rats were Downloaded from jn.physiology.org on October 28, 2010 deeply reanesthetized with pentobarbital sodium (60 mg/kg, ip) and perfused transcardially with heparinized cold phosphate buffer (0.1 M, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and processed for NADPH-diapho- rase staining and histological localization of injection sites as de- scribed earlier (Datta 2002; Datta et al. 2001a). This NADPH- FIG. 1. Anatomical location of analyzed microinjection sites. Schematic diaphorase staining was done to identify the cholinergic cell compart- coronal sections through the brain stem are shown at levels A: 1.70, A: 1.20, ment of the PPT (Bredt and Snyder 1992; Bredt et al. 1991; Datta A: 1.00, and A: 0.70 (labeled at top right). E (n 7), location of control saline saline; F (n 7), location of baclofen saline injection injector tips; 2002; Datta et al. 1997; Hope et al. 1991; Vincent et al. 1983). ‚ (n 7), baclofen Sp-adenosine 3 ,5 -cyclic monophosphothioate trieth- ylamine (SpCAMPS); f (n 7), location of SpCAMPS saline injection injector tips. 4, trochlear nucleus; Aq, aqueduct; ATg, anterior tegmental RESULTS nucleus; BIC, nucleus brachium inferior colliculus; CG, central gray; CnF, cuneiform nucleus; ctg, central tegmental tract; DR, dorsal raphe nucleus; dtg, Figure 1 shows the anatomical location of microinjection dorsal tegmental bundle; IC, inferior colliculus; InCo, intercollicular nucleus; sites. Histological identification showed that all four combina- LL, lateral lemniscus; Me5, mesencephalic trigeminal tract/nucleus; MiTg, tions of microinjections (control saline saline, baclofen microcellular tegmental nucleus; Mlf, medial longitudinal fasciculus; Pa4, paratrochlear nucleus; PMR, paramedian raphe; PnO, pontine reticular nu- saline, baclofen SpCAMPS, and SpCAMPS saline) were cleus, oral; PPT, pedunculopontine tegmental nucleus; RR, retrorubral nucleus; within the PPT (Fig. 1). The microinjection sites were anatom- rs, tectospinal tract; scp, superior cerebellar peduncle; SPTg, subpeduncular ically comparable between four different treatment groups; tegmental nucleus; ts, tectospinal tract; VTg, ventral tegmental nucleus. Scale therefore the analysis below quantifies the effects of ba- bar, 600 m. clofen saline (n 7 rats), baclofen SpCAMPS (n 7 Effects on wakefulness rats), and SpCAMPS saline (n 7 rats) microinjected into the PPT on the W, SWS, and REM sleep states. The changes in the percentage of time spent in W after Figure 2 shows representative sleep-wake architectures for microinjections are summarized in Fig. 3. Two-way ANOVA the 6-h postinjection recording sessions (10:00 AM to 4:00 PM) indicated a significant main effect of treatment (df 3, F starting immediately after each of four different sets of micro- 9.32, P 0.001), time (df 5, F 19.24, P 0.001), and a injections. The figure shows that the latency between the end of significant treatment time interaction (df 15, F 5.62, microinjection and the first episode of REM sleep was much P 0.001) on the total percentage of time spent in W. The shorter after microinjections of SpCAMPS saline compared results of post hoc analysis (Scheffe F tests) on total percentage with after saline saline. In contrast, with microinjections of of time spent in W are presented in Fig. 3. Post hoc analysis baclofen saline, REM sleep latency was about 2 h longer indicated that the microinjections of baclofen saline and than after saline saline. However, after baclofen Sp- baclofen SpCAMPS reduced total percentages of W signif- CAMPS microinjections, REM sleep latency was shorter than icantly (F 44.40 and 17.89; P 0.001) compared with the after saline saline and after baclofen saline microinjec- microinjections of saline saline (control group), only during tions. Microinjections of baclofen saline decreased latency the first hour. During the second hour, baclofen saline and of the first episode and increased duration of each SWS baclofen SpCAMPS microinjected groups total percentages episodes compared with after saline saline, SpCAMPS of W were less compared with saline control microinjections, saline, or baclofen SpCAMPS. For about 2 h after micro- but this reduction did not reach significance. Next, the total injections of SpCAMPS saline, baclofen saline, and percentages of W in the baclofen SpCAMPS and SpCAMPS baclofen SpCAMPS, the duration of W episodes were saline microinjection groups were compared (Scheffe F test) shorter compared with after saline microinjections. These re- with the baclofen saline microinjection group. This test sults showed that these microinjections into the PPT did, in revealed that the total percentage of W in the baclofen fact, change the sleep-wake architecture of the rat. SpCAMPS group was significantly higher only in the first hour J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 5. 3844 S. DATTA second hour (F 4.16; P 0.05). These results show that the application of baclofen into the PPT causes reduction in the total percentage of W. These results also showed that the application of SpCAMPS into the baclofen microinjection site attenuates baclofen-induced W-reducing effect. Effects on SWS The changes in the percentage of time spent in SWS after microinjections into the PPT are summarized in Fig. 4. A two-way ANOVA indicated a significant main effect of treat- ment (df 3, F 32.57, P 0.001), time (df 5, F 14.19, P 0.001), and a significant treatment time interaction (df 15, F 3.61, P 0.001) for total percentage of time spent in SWS. The results of post hoc analysis (Scheffe F test) for the total percentage of time spent in SWS are presented in Fig. 4. Post hoc analysis indicated that, after microinjections of baclofen saline, the total percentages of SWS were signifi- Downloaded from jn.physiology.org on October 28, 2010 cantly increased during the first (F 41.81; P 0.001), second (F 5.0; P 0.01), and third (F 11.12; P 0.001) FIG. 2. Examples of sleep-wake architecture after microinjections into hours of the postinjection period compared with after control pedunculopontine tegmental (PPT). These microinjections were saline saline saline microinjections. Similar comparisons revealed saline (Sal Sal), baclofen saline (baclofen Sal), SpCAMPS baclofen that, after baclofen SpCAMPS microinjections, total per- (baclofen SpCAMPS), and SpCAMPS saline (SpCAMPS saline). These four 6-h continuous step histograms plot occurrence and duration of centage of SWS increased significantly (F 9.25; P 0.001) polygraphically and behaviorally defined wakefulness (W), slow-wave sleep only during the first hour compared with after saline saline (S), and rapid eye movement (REM) sleep (R) after microinjections. Note that microinjections. The next set of post hoc tests (Scheffe F test) after microinjections of baclofen saline, 1st episode of REM sleep was compared total percentages of SWS in the baclofen SpCAMPS delayed compared with after saline saline microinjections. Also note appearance of 1st episode of REM sleep was much earlier after microinjections and SpCAMPS saline microinjection groups with the ba- of baclofen SpCAMPS and SpCAMPS saline than after microinjections clofen saline microinjection group. This test revealed that of saline saline. the total percentages of SWS in the baclofen SpCAMPS and SpCAMPS saline groups were significantly less during the (F 5.99; P 0.01), but in the SpCAMPS saline group, first (F 11.78; P 0.001 and F 53.15; P 0.001), second total percentages of wakefulness were significantly higher in (F 3.26; P 0.05 and F 6.84; P 0.01), and third (F the first (F 35.04; P 0.001) and second hour (F 3.12; 18.89; P 0.001 and F 6.11; P 0.001, respectively) hours P 0.05). Similar post hoc tests between SpCAMPS of the postinjection period. A similar post hoc test between the baclofen and SpCAMPS saline groups revealed that the total baclofen SpCAMPS and SpCAMPS saline groups re- percentages of W in the SpCAMPS saline group were vealed that the total percentages of SWS in the SpCAMPS significantly higher in the first (F 12.15; P 0.001) and saline group were significantly less in the first (F 14.89; P FIG. 3. Total percentages of wakefulness after microinjections of baclofen FIG. 4. Total percentages of slow-wave sleep after microinjections of and SpCAMPS into PPT. Bars represent percentages (means SE) of baclofen and SpCAMPS into PPT. Bars represent percentages (means SE) wakefulness during each of the 6-h periods after injections of saline saline, of slow-wave sleep during each of 6-h periods after injections of saline baclofen saline, baclofen SpCAMPS, and SpCAMPS saline. Post hoc saline, baclofen saline, baclofen SpCAMPS, and SpCAMPS saline. tests (Scheffe F-test): *comparison with control saline saline, comparison Post hoc tests (Scheffe test): *comparison with control saline saline, with baclofen saline, and #comparison with baclofen SpCAMPS. or comparison with baclofen saline, and #comparison with baclofen # P 0.05; P 0.01; ***P 0.001. SpCAMPS. P 0.05; ** or P 0.01; *** or or ###P 0.001. J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 6. PPT GABAB RECEPTOR AND PKA IN REM SLEEP REGULATION 3845 0.001) hour. These results show that the application of baclofen SpCAMPS saline into the PPT, Fig. 6 shows the effects on into the PPT increases SWS. These results also show that the latency, number, and duration of REM sleep episodes after application of SpCAMPS into the baclofen microinjection site these microinjections. One-factor ANOVA indicated a signif- attenuates baclofen-induced SWS increase. icant change in the latency of the first episode of REM sleep between different treatments (df 3, F 1795.17, P 0.001). Post hoc (Scheffe F test) analyses indicated that the Effects on REM sleep latency to the first episode of REM sleep was significantly The changes in the percentage of time spent in REM sleep higher (F 734.34, P 0.001) after microinjections of after microinjections into the PPT are summarized in Fig. 5. A baclofen saline compared with after microinjections of two-way ANOVA indicated a significant main effect of treat- control saline saline. In contrast, latency of the first episode ment (df 3, F 75.94, P 0.001), time (df 5, F 44.92, of REM sleep was significantly reduced after microinjections P 0.001), and treatment time interaction (df 15, F of baclofen SpCAMPS (F 69.69, P 0.001) and 11.81, P 0.001) on total percentage of time spent in REM SpCAMPS saline (F 110.53, P 0.001) compared with sleep. The results of post hoc analysis (Scheffe F test) on total after control saline saline microinjection. The second set of percentage of time spent in REM sleep are presented in Fig. 5. post hoc analyses indicated that the REM sleep latencies after Microinjections of baclofen saline into the PPT suppressed microinjections of baclofen SpCAMPS and SpCAMPS REM sleep completely for the first 3 h of the postinjection saline were significantly less compared with REM sleep la- period. During the fourth hour after injection, REM sleep tency after baclofen saline microinjection. In addition to began to reappear, but the total percentages of REM sleep were changes in REM sleep latency, one-factor ANOVA indicated a Downloaded from jn.physiology.org on October 28, 2010 significantly less both during the fourth (F 21.05, P significant change in the total number of REM sleep episodes 0.001) and fifth (F 3.82, P 0.05) hours compared with between different treatments (df 3, F 59.42, P 0.001). their saline saline control values. On the contrary, in the first Post hoc analyses indicated that the total number of REM sleep postinjection hour, the total percentages of REM sleep were episodes was significantly (F 31.07, P 0.001) decreased significantly higher in the baclofen SpCAMPS (F 17.72, after baclofen saline microinjection compared with the P 0.001) and SpCAMPS saline (F 26.5, P 0.001) control saline saline microinjection. Post hoc analyses also groups compared with after microinjections of the control indicated that the total number of REM sleep episodes after saline saline group. Similar comparisons after baclofen microinjections of baclofen SpCAMPS and SpCAMPS saline microinjection revealed that the total percentages of saline were not significantly different compared with after REM sleep after microinjections of both baclofen SpCAMPS microinjection of saline saline. Compared with after micro- and SpCAMPS saline were significantly higher during postin- injection of baclofen saline, the total number of REM sleep jection hours 1–5. The total percentages of REM sleep were not episodes were significantly higher after baclofen SpCAMPS significantly different between the baclofen SpCAMPS and (F 33.91, P 0.001) and after SpCAMPS saline (F SpCAMPS saline microinjection groups. These results in- 50.0, P 0.001) microinjections. Similar one-factor ANOVA dicate that the microinjection of baclofen into the PPT suppress and post hoc analyses did not reveal any significant difference REM sleep, and this baclofen-induced REM sleep–suppressing in the mean duration of REM sleep episodes between different effect could be eliminated by the subsequent application of treatment groups. These results showed that the reduction in SpCAMPS. the total percentage of time spent in REM sleep after micro- Having documented the changes in REM sleep after micro- injection of baclofen saline was caused by an increase in the injections of baclofen saline, baclofen SpCAMPS, and latency and decrease in the number of REM sleep episodes. These results also showed that the increased REM sleep after baclofen SpCAMPS and SpCAMPS saline are only caused by a decrease in the REM sleep latency. DISCUSSION In this study, I examined the role of cAMP-PKA signal transduction pathway in the PPT for the regulation of GABAB receptor activation–induced REM sleep suppression. The prin- cipal findings of this study are that 1) microinjection of the GABAB receptor selective agonist, baclofen, into the PPT suppressed REM sleep, 2) microinjection of the cAMP-PKA activator, SpCAMPS, into the PPT increased REM sleep, and 3) microinjection of SpCAMPS into the PPT blocked the baclofen-induced REM sleep suppression effect. These results showed that the activation of PPT cAMP-PKA increases REM FIG. 5. Total percentages of REM sleep after microinjections of baclofen sleep and also blocks PPT GABAB receptor activation–induced and SpCAMPS into PPT. Bars represent percentages (means SE) of REM REM sleep suppression. The findings are discussed in relation sleep during each of 6-h periods after injections of saline saline, baclofen saline, baclofen SpCAMPS, and SpCAMPS saline. Post hoc tests to ongoing efforts to understand the PPT neurotransmitter (Scheffe test): *comparison with control saline saline and comparison with receptor activation– dependent intracellular signaling mecha- baclofen saline. *or P 0.05; *** or P 0.001. nisms for the regulation of REM sleep. J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 7. 3846 S. DATTA al. 2003; Moon-Edley and Graybiel 1983; Reese et al. 1995; Scarnati and Florio 1997; Scarnati et al. 1988; Steininger et al. 1992). The possibility that endogenous GABAergic neuro- transmission in the PPT may modulate PPT cell activity is also supported by the presence of different types of GABA recep- tors and GABAergic fibers in the PPT (Bowery et al. 1987; Chu et al. 1990; Kosaka et al. 1987; Mugnaini and Oertel 1985). Collectively, the results of this study and results of earlier studies discussed above provide conclusive evidence to support that the endogenous GABA released in the PPT regu- lates physiological REM sleep and wakefulness by activating GABAB receptors. This study showed that the activation of PPT cAMP-PKA by local application of SpCAMPS, an activator of cAMP-PKA activation, increases REM sleep. This result suggests that the activation of PPT cAMP-PKA is involved in the generation of REM sleep. Because earlier studies have shown that the inhi- bition of AC (Datta and Prutzman 2005) and cAMP-PKA (Bandyopadhya et al. 2006) in the PPT suppresses REM sleep, Downloaded from jn.physiology.org on October 28, 2010 it is reasonable to suggest that the cAMP-PKA intracellular FIG. 6. Effects on latency, total number, and mean duration of REM sleep signaling system in the PPT is critically involved in the episodes observed after microinjection of baclofen and SpCAMPS into PPT. Data presented as mean SE. Note that compared with control saline regulation of PPT cell activity and REM sleep. Besides these saline, microinjections of baclofen saline increased latency for 1st episode studies, a number of other studies in behaving cats and rats of REM sleep and decreased number of REM sleep episodes. On the contrary, have looked at the involvement of signal transduction path- microinjections of baclofen SpCAMPS and SpCAMPS saline decreased ways in the pontine reticular formation (PRF), another site latency for 1st episode of REM sleep. Post hoc tests (Scheffe test): *compar- ison with control saline saline and comparison with Baclofen saline. known to be involved in the regulation of REM sleep (Capece *** or P 0.001. and Lydic 1997; Marks and Birabil 2000; Shuman et al. 1995). These studies have indicated that the signal transduction path- Neurotransmitter receptor activation–mediated excitation way activated by muscarinic cholinergic receptors involves a and inhibition of PPT cells are important processes for the pertussis toxin–sensitive G protein, AC, cAMP, and PKA. In regulation of REM sleep (Datta 1995). For the excitation the rat, this signal transduction pathway in the PRF is also process, pharmacological and single-cell recording studies shown to be involved in spontaneous REM sleep (Marks and have shown that the activation of PPT kainate type glutamate Birabil 2000). Thus it is reasonable to conclude that, depending receptors induces REM sleep (Datta 2002; Datta and Siwek on the anatomical site and involved neurotransmitter receptors, 2002; Datta et al. 2002). For the inhibition process, studies activation of the intracellular cAMP-PKA signaling system is have shown inhibitory neurotransmitter GABA activates excited and/or inhibited for the regulation of REM sleep. GABAB receptors and suppresses REM sleep through inhibi- The results of this study also showed that the local activation tion of PPT REM-ON cells (Datta et al. 2003; Ulloor et al. of intracellular cAMP-PKA blocked PPT GABAB receptor 2004). In this study, I showed that the local application of activation–induced REM sleep suppression. It is known that GABAB receptor selective agonist suppresses REM sleep. the activation of GABAB receptors is capable of inhibiting AC These results are in agreement with the idea that the GABAer- (Couve et al. 2000; Kerr and Ong 1995; Mody et al. 1994; gic system in the PPT is involved in the regulation of REM Robbins et al. 2001; Sivilotti and Nistri 1991; Takahashi et al. sleep by inhibiting PPT cells. Other studies have consistently 1998; Thompson 1994), and inhibition of AC prevents activa- shown that the activation of PPT cells increases wakefulness tion of the cAMP-PKA signal transduction pathway (Gilman and/or REM sleep (Datta 2002; Datta and Siwek 1997; Datta et 1987; Marinissen and Gutkind 2001). Thus it is logical to al. 2001a,b, 2002; Garcia-Rill 1991; Garcia-Rill et al. 1990, suggest that the PPT GABAB receptor activation–mediated 2001; Radulovacki et al. 2004). Thus the activation or inhibi- REM sleep suppression effect may have been caused by the tion of PPT cells correspondingly increases or decreases REM inhibition of cAMP-PKA signal transduction pathway. Earlier sleep. In addition to the suppression of REM sleep, our results studies have shown that the inhibition of both AC and cAMP- showed that the activation of GABAB receptors in the PPT PKA in the PPT suppresses REM sleep (Bandyopadhya et al. reduces the total percentages of wakefulness. This observation 2006; Datta and Prutzman 2005). is also consistent with the observation of another study that has The results of this study are particularly valuable given shown the intracerebroventricular application of GABAB re- recent evidence from studies in both academic and industrial ceptor antagonist increases wakefulness and REM sleep in the settings indicating that novel compounds designed to modify rat (Gauthier et al. 1997). Anatomical studies demonstrating intracellular signaling molecules have promising therapeutic that the PPT receives GABAergic inputs from many parts of potential for the treatment of neurodegenerative disorders, the brain, including the substantia nigra and local neurons, endogenous depression, and other psychiatric disorders (Coyle further support the suggestion that the GABAergic system and Duman 2003; Gould and Manji 2002; Gunawardena and could modulate the physiological acitvity of PPT cells as well Goldstein 2004; Paez-Pereda 2005; Tortora and Ciardiello as behavioral states of wakefulness and REM sleep. (Beckstead 2002). Because the phenomenology and neurotransmitters in- 1983; Carpenter et al. 1981; Jackson and Crossman 1981; Jia et volved in narcolepsy, cataplexy, hypnogogic hallucinations, J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 8. PPT GABAB RECEPTOR AND PKA IN REM SLEEP REGULATION 3847 depression, and other major psychiatric and neurological dis- away from the PPT microinjection sites. Thus it is highly orders are closely related to those of normal REM sleep improbable that microinjected drugs used in this study (100 nl (Garcia-Rill 1997; Garcia-Rill et al. 2003; Rye 1997), there volume and 1.5 nmol dose) diffused into the LDT and subco- may be a convergence of mechanisms at the level of the brain eruleus nucleus. stem. Therefore the results of these experiments will be im- The results of this study also confirm a well-established fact portant in developing rational schematics of pharmacological that the activation of PPT is positively involved in the regula- therapies for these persistent and debilitating disorders. tion of REM sleep (for reviews, see Datta 1995; McCarley I chose to study chronically implanted freely moving rats 2004). Contrasting this view, a recent publication claimed that because this preparation provides the most physiological ap- the total amount of REM sleep increases in the rat after PPT proach to a longitudinal analysis of the behavioral and elec- lesion (Lu et al. 2006). This study also claimed that lesioning trographic events related to the wake-sleep cycle (Datta and the subcoeruleus alpha [labeled as sublaterodorsal nucleus Siwek 2002; Datta et al. 2002; Ulloor et al. 2004). After the (SLD)] suppressed REM sleep (Lu et al. 2006). However, adaptation period of each experimental condition, the rats numerous previous PPT lesion studies in both cats (Shouse and showed regular values of wake and sleep stages. Thus the Siegel 1992; Webster and Jones 1988) and rats (Deurveilher microinjections of a pharmacologically active selective signal and Hennevin 2001) have shown that the elimination PPT transduction-altering activator that affected electrographic cholinergic cells suppressed physiological REM sleep. More- signs of wake-sleep patterns could be evaluated accurately over, one study has shown that rats with inbred cholinergic (Capece and Lydic 1997; Marks and Birabil 2000). Moreover, hyperfunction have excess REM sleep (Shiromani et al. 1988). understanding the role of the intracellular signal transduction More recently, it has also been shown that, in the animal model Downloaded from jn.physiology.org on October 28, 2010 pathway in a behaviorally active living organism is a vital step of Alzheimer’s, impaired REM sleep is caused by the reduction toward creating ways to converge functional biochemistry and of cholinergic cells in the PPT (Zhang et al. 2005). Collec- integrative physiology. However, a major limitation of the tively, these lesion studies provided much stronger and defin- microinjection method relates to the diversity in the neuro- itive evidence to indicate that the intact PPT cholinergic cells chemical and functional nature of the neuronal population are critical for the generation of REM sleep. affected by the drug application. In this study, a GABAB Assuming PPT cholinergic cells are necessary for the gen- receptor selective antagonist, baclofen, and a selective activa- eration of REM sleep, a careful analysis of previous studies tor of cAMP-PKA, SpCAMPS, were microinjected into the reveals a potential oversight in the study of Lu et al. (2006). PPT. I injected these drugs into the part of the PPT (pars Stimulation studies have shown that the electrical and chemical compacta) where most cells are known to be cholinergic stimulation of the PPT increases active wakefulness in rats and (Mesulam et al. 1983; Rye et al. 1987) and express REM-ON cats (Datta and Siwek 1997; Datta et al. 2001b; Garcia-Rill type of activity (Datta and Siwek 2002). Nonetheless, I ac- 1991; Garcia-Rill et al. 1987). On the other hand, using an knowledge that if there are noncholinergic and REM-OFF cells excitotoxin lesion technique, studies have shown that the located within the targeted site, they will also be affected by elimination of PPT cells suppresses motivational behaviors the application of these drugs. Another limitation of the mi- (Bechara and van der Kooy 1989, 1992; Olmstead and Franklin croinjection method is the extent of diffusion of injected 1994); and consequently PPT lesioned rats spend more time in materials. In recent years, several laboratories, including our quiet wakefulness by reducing active wakefulness. Cortical own, have improved this technique tremendously in many EEG waves amplitudes and frequencies are very similar during different ways. Recent microinjection studies have shown that quiet wakefulness and REM sleep (Datta and Hobson 2000). a major advantage of the microinjection technique is its ability During quiet wakefulness, EMG sign of muscle tone in the rat to stimulate a relatively circumscribed neuronal pool (Bandyo- is also very low, especially in PPT-lesioned rats. Thus it is padhya et al. 2006; Capece and Lydic 1997; Datta et al. possible that while identifying sleep-wake states, Lu et al. 2001a,b, 2003; Marks and Birabil 2000). This view is sup- (2006) might have misidentified some quiet wake episodes as ported by biophysical studies of drug diffusion (Nicholson REM sleep episodes. It is also known that the lesion of nucleus 1985), and distribution kinetics (Herz and Teschemacher 1971; subcoeruleus alpha suppresses REM sleep muscle atonia (Hen- Martin 1991). Diffusion data obtained from radiolabeled drug dricks et al. 1982; Sakai 1980; Sanford et al. 2001; Shouse and studies have shown that 30 min after brain microinjections of Siegel 1992). Despite a reduction in REM sleep muscle atonia 500-nl volumes, 72% of the drug remained within a radius of in these lesioned animals, the total amount of REM sleep is 0.75 mm (Yaksh and Rudy 1978). In this study, the target area relatively unchanged. Similar to REM sleep muscle atonia, it of the 100-nl drug solutions injected into the PPT (approximate has also been shown that the lesion of P-wave (one of the size: length: 1.0; height: 1.0; and width: 0.8 mm), were well cardinal features of REM sleep) generator suppresses P-wave within the diffusion limit of this small volume. Moreover, in activity in the rat and cat during REM sleep (Datta and Hobson our recent microinjection mapping study in the PPT, it was 1995; Mavanji et al. 2004). In these P-wave generator–lesioned shown that 100 nl of glutamate with drug concentrations rats and cats, the total amount of wakefulness, SWS, and REM similar to those used in this proposal were ineffective when sleep remains relatively unchanged. Therefore in these REM injections were 0.5 mm away from the center of the PPT site sleep sign generator–lesioned animals, identification of REM (Datta 2002; Datta et al. 2001a,b). Additionally, using a similar sleep is relatively more challenging than the identification of microinjection technique, I showed that ibotenic microinjec- sleep-wake states in normal animals. It is likely that the tion-induced cell loss in the brain stem of rats was limited to a SLD-lesioned rats of Lu et al. (2006) also exhibited some REM maximum radius of 0.35 mm. Other sleep-wake–related struc- sleep episodes without muscle atonia. Therefore they might tures that are close to the PPT microinjection sites are the LDT have failed to identify some of those dissociated REM sleep and subcoeruleus nucleus. These two structures are 1 mm episodes. This could explain why the study of Lu et al. (2006) J Neurophysiol • VOL 97 • JUNE 2007 • www.jn.org
  • 9. 3848 S. DATTA presents paradoxical results, such as reduced REM sleep ac- Datta S, Hobson JA. Suppression of ponto-geniculo-occipital waves by companying an SLD lesion. Indeed, lesion of subcoeruleus neurotoxic lesions of pontine caudo-lateral peribrachial cells. Neuroscience 67: 703–712, 1995. nucleus (part of SLD) does not change total amount of REM Datta S, Hobson JA. The rat as an experimental model for sleep neurophys- sleep or wakefulness (Mavanji et al. 2004; Sanford et al. 1994). iology. Behav Neurosci 114: 1239 –1244, 2000. In conclusion, this study showed that the inactivation of Datta S, Mavanji V, Patterson EH, Ulloor J. Regulation of rapid eye cAMP-PKA in the PPT is a critical step for the GABAB movement sleep in the freely moving rat: local microinjection of serotonin, receptor activation–mediated regulation of REM sleep. The norepinephrine, and adenosine into the brainstem. Sleep 26: 513–520, 2003. results also suggest that the activation of cAMP-PKA in the Datta S, Mavanji V, Ulloor J, Patterson EH. Activation of phasic pontine- wave generator prevents rapid eye movement sleep deprivation-induced PPT is an important step to induce REM sleep. These data learning impairment in the rat: a mechanism for sleep-dependent plasticity. provide a novel perspective on the regulatory aspect of PPT J Neurosci 24: 1416 –1427, 2004. cell activity in the regulation of REM sleep. These novel Datta S, Patterson EH, Siwek DF. Endogenous and exogenous nitric oxide in findings are critical for our complete understanding of the basic the pedunculopontine tegmentum induces sleep. Synapse 27: 69 –78, 1997. mechanisms in REM sleep regulation. Datta S, Patterson EH, Spoley EE. Excitation of the pedunculopontine tegmental NMDA receptors induces wakefulness and cortical activation in ACKNOWLEDGMENTS the rat. J Neurosci Res 66: 109 –116, 2001a. Datta S, Prutzman SL. 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