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NEUROPHARMACOLOGY
Departments of Anesthesiology (R.B., R.L., H.A.B.) and Pharmacology (R.B., H.A.B.), University of Michigan, Ann Arbor, Michigan
Received October 13, 2005; accepted December 7, 2005.
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Abstract |
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-thio)triphosphate autoradiography. ACh release was measured using in vivo microdialysis and high-performance liquid chromatography. Hypocretin-1-stimulated G protein activation was significantly decreased by pertussis toxin, demonstrating that some hypocretin receptors in rat PnO activate inhibitory G proteins. Hypocretin-1-stimulated ACh release was not blocked by pertussis toxin, supporting the conclusion that the hypocretin receptors modulating ACh release in rat PnO activate stimulatory G proteins. Hypocretin-1 and -2 each caused a concentration-dependent increase in ACh release with similar potencies, indicating that hcrt-r2 modulates ACh release in PnO. Hypocretin-1 caused a significantly greater increase in ACh release than hypocretin-2, suggesting a role for hcrt-r1 in the modulation of PnO ACh release. Taken together, these data provide the first evidence that hypocretin receptors in rat PnO signal via inhibitory and stimulatory G proteins and that ACh release in rat PnO is modulated by hcrt-r2 and hcrt-r1. One mechanism by which hypocretin promotes arousal may be to increase ACh release in the pontine reticular formation.
; Peyron et al., 1998; Sakurai et al., 1998). Physiological roles of the hypocretin peptides include promoting behavioral arousal and locomotor activity, as well as modulating food intake and energy homeostasis (Kukkonen et al., 2002; Smart and Jerman, 2002). Impaired hypocretinergic signaling underlies the human sleep disorder narcolepsy (Nishino et al., 2000; Thannickal et al., 2000), and defects in hypocretin peptides or receptors produce animal models of narcolepsy (Chemelli et al., 1999; Lin et al., 1999). The mechanisms by which hypocretins modulate arousal are not well understood and may include excitation (de Lecea et al., 1998) of brainstem neurons that promote behavioral arousal and cortical activation (Kilduff and Peyron, 2000). Hypocretinergic neurons discharge at their fastest rates during active wakefulness (Lee et al., 2005; Mileykovskiy et al., 2005), and hypocretins have excitatory effects on noradrenergic neurons of the locus coeruleus, serotonergic neurons of the dorsal raphe, and cholinergic neurons of the laterodorsal and pedunculopontine tegmental nuclei (for review, see Kukkonen et al., 2002).
Hypocretin-1 and -2 alter cell excitability via two receptor subtypes (hcrt-r1 and hcrt-r2) (Sakurai et al., 1998). Hypocretin-1 has relatively high affinity for both receptor subtypes, whereas hypocretin-2 has higher affinity for hcrt-r2 than for hcrt-r1 (Sakurai et al., 1998; Smart et al., 2001). Both hypocretin receptor subtypes couple to guanine nucleotide binding (G) proteins (Sakurai et al., 1998). Pertussis toxin is a standard pharmacological tool for blocking signal transduction mediated by inhibitory G proteins (Carty, 1994), and work with in vitro expression systems has shown that hcrt-r2 couples to both pertussis toxin-insensitive and -sensitive G proteins (Zhu et al., 2003). The G protein subtypes coupled to each hypocretin receptor subtype in vivo remain to be determined and may vary across brain regions.
The present study focused on functional effects of hypocretin in one arousal promoting brainstem region, the pontine reticular nucleus oral part (PnO). The PnO is the rostral portion of rat pontine reticular formation (Paxinos and Watson, 1998). In vivo and in vitro approaches were combined with the goal of identifying mechanisms by which the arousal-promoting peptide hypocretin acts within the PnO, which comprises an important part of the ascending reticular activating system. In vitro autoradiography was used to test the hypothesis that treatment of the PnO with pertussis toxin inhibits hypocretin-1-stimulated [35S]guanylyl-5'-O-(-thio)triphosphate ([35S]GTPS) binding in the PnO. A second series of experiments used in vivo microdialysis to test the hypothesis that treatment of the PnO with pertussis toxin inhibits hypocretin-1-stimulated ACh release in the PnO. Finally, in vivo microdialysis was used to test the hypothesis that hypocretin-1 and -2 each cause a concentration-dependent increase in PnO ACh release. The concentration-response curves for hypocretin-1- and -2-stimulated ACh release were compared with the goal of identifying the hypocretin receptor subtype modulating ACh release in the PnO.
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Materials and Methods |
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). Stereotaxic coordinates for the PnO were 8.6 mm posterior to bregma, 1.2 mm from the midline, and 9.2 mm ventral to the skull surface. Animals were kept warm and observed continuously during recovery from anesthesia.
Chemicals, Materials, and Microdialysis Probes. Hypocretin-1 (human, rat, and mouse) was purchased from California Peptide Research (Napa, CA). Hypocretin-2 (rat, mouse) was obtained from AnaSpec, Inc. (San Jose, CA). Pertussis toxin was obtained from Sigma-Aldrich (St. Louis, MO) and List Biological Laboratories (Campbell, CA). [D-Ala2, N-Me-Phe4, Gly5] enkephalin (DAMGO), GDP, guanosine 5'-O-(-thio)triphosphate, N6-p-sulfophenyladenosine (SPA), and all other chemicals were purchased from Sigma-Aldrich. Reflection autoradiography film (Kodak X-OMAT Blue XB-1) and [35S]GTPS were obtained from Perkin Elmer Life Science Products (Boston, MA).
CMA/11 microdialysis probes (Cuprophane membrane: l mm long, 0.24 mm in diameter, 6-kDa cut-off; CMA Microdialysis, North Chelmsford, MA) were used for drug delivery to the PnO and for simultaneous collection of endogenous ACh. Probes were connected to a CMA/100 pump that delivered a constant 2.0 µl/min flow rate. Dialysis probes were perfused continuously with Ringer's solution (147 mM NaCl, 4.0 mM KCl, 2.4 mM CaCl2, and 10 µM neostigmine bromide, pH 5.8–6.2). All drugs delivered by microdialysis were dissolved in Ringer's solution.
Quantification of ACh Using High-Performance Liquid Chromatography with Electrochemical Detection. Dialysis samples were injected manually into an high-performance liquid chromatography-electrochemical detection system (Bioanalytical Systems, West Lafayette, IN) that separated ACh and choline and converted ACh into hydrogen peroxide. Electrochemically active hydrogen peroxide was detected by a platinum electrode and generated a chromatogram that was stored digitally and analyzed using ChromGraph software (Bioanalytical Systems). ACh chromatograms generated by dialysis samples were compared with a standard curve produced from known amounts of ACh (0.05–1 pmol). A standard curve was generated before every experiment. ACh was quantified as picomoles per 15 min.
Before each experiment, a dialysis probe was placed into a solution of known ACh concentration. Five 30-µl samples were collected and analyzed for ACh to calculate percent of ACh recovered by the probe. At the end of each dialysis experiment, the dialysis probe was removed from the brain and placed again in a solution of known ACh concentration to collect an additional five 30-µl samples for calculating percent recovery of ACh. Pre- and postexperimental probe recoveries were compared by Student's t test. The present study includes data from experiments in which probe recoveries did not change significantly in the direction of the hypothesized drug effect. This procedure ensured that measured changes in ACh resulted from drug effects and were not an artifact of intraexperimental changes in dialysis membrane properties.
Histological Localization of Dialysis Sites. Two to 3 days following each dialysis experiment, animals were deeply anesthetized and decapitated. The brainstem was cut serially to obtain 40-µm-thick coronal sections through the pontine reticular formation. Tissue sections were mounted on chrome alum-coated slides, fixed in paraformaldehyde vapor (80°C), and stained with cresyl violet. Stained sections were digitized using a Cohu CCD camera (Cohu, Inc., San Diego, CA) with a Micro Nikon 60-mm objective (Nikon, Tokyo, Japan), the Scion Image 1.62c version of NIH Image (Scion Corporation, Frederick, MD), and a G3 Apple Macintosh computer (Apple Computer, Cupertino, CA). Stereotaxic coordinates were assigned to each dialysis probe site by comparing the digitized images and actual tissue sections with the plates in a rat brain atlas (Paxinos and Watson, 1998).
Methods for Testing the Hypothesis That Pertussis Toxin Blocks Hypocretin-1-Stimulated G Protein Activation: In Vivo Pertussis Toxin Microinjection Followed by in Vitro [35S]GTPS Autoradiography. Pertussis toxin was administered to intact animals by microinjection into the PnO before brain removal and sectioning. To perform an in vivo microinjection, rats were anesthetized as described above, and a stainless steel guide tube was aimed stereotaxically for the PnO. A stainless steel microinjector was inserted through the guide tube, and pertussis toxin (0.25 µg/0.25 µl) was delivered to the PnO. Control rats received a unilateral PnO microinjection (0.25 µl) of vehicle solution consisting of 0.1 M sodium phosphate buffer and 0.5 M sodium chloride. Rats were decapitated 48 h postinjection when ADP-ribosylation of G proteins was nearly complete (van der Ploeg et al., 1991). Brains were frozen in a bilayer composed of bromobutane and isopentane (–30°C), and the pons was sectioned coronally from bregma –8.80 to –7.30 mm (Paxinos and Watson, 1998) using a Leica CM3050 S cryostat (Leica Microsystems, Nussloch, Germany). Serial sections were thaw-mounted pair-wise onto chrome alum-coated slides. Slide-mounted tissue sections were dried in a desiccator (4°C) for 2 h, then stored at –80°C until used for [35S]GTPS autoradiography.
The binding assay for in vitro [35S]GTPS autoradiography has been described previously (Bernard et al., 2003). Assay buffer contained 50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, and 100 mM NaCl, pH 7.4. Slide-mounted tissue sections were brought to room temperature and incubated for 2 h in assay buffer containing 0.04 nM [35S]GTPS and 2 mM GDP and one of the following four treatment conditions: hypocretin-1 (200 nM), DAMGO (3 µM) as a positive control, unlabeled guanosine 5'-O-(-thio)triphosphate (10 µM) for nonspecific binding, or basal [35S]GTPS binding in the absence of exogenous ligands. The incubation period was terminated by rinsing the tissue sections in ice-cold 50 mM Tris-HCl buffer, pH 7.0, followed by ice-cold deionized water. Sections were dried overnight and placed in light-proof stainless steel cassettes (Fisher Scientific, Pittsburgh, PA) along with 14C microscale standards (31–883 nCi/g, Amersham Biosciences, Arlington Heights, IL) and X-OMAT Blue XB-1 film (Eastman Kodak Company, Rochester, NY). After a 72-h exposure period, films were developed with a Kodak X-OMAT model 2002A film processor. Tissue sections were fixed and stained with cresyl violet, and autoradiograms and stained tissue sections were digitized as described above.
The PnO is a bilateral structure, and [35S]GTPS binding was measured on both the injected and noninjected side of the PnO for rats microinjected with pertussis toxin and rats microinjected with vehicle. The PnO was identified on the cresyl violet-stained tissue sections according to a rat brain atlas (Paxinos and Watson, 1998). A digital outline of the PnO was transferred to the matching position in each autoradiogram, and optical density was quantified. Optical density measures were converted to total [35S]GTPS binding (nanocuries per gram) using a 14C standard curve and a 14C correction factor for 35S. Specific PnO [35S]GTPS binding was obtained by subtracting the mean nonspecific binding value for the PnO from every individual total [35S]GTPS binding measurement. Individual specific [35S]GTPS binding measures were averaged by treatment condition for each animal. Data were analyzed using descriptive statistics, one-way analysis of variance (ANOVA) for repeated measures, and Tukey/Kramer multiple comparisons test (GB-Stat version 6.5.6; Dynamic Microsystems, Inc., Silver Spring, MD). The 
level was set at p < 0.05, and power calculations were performed to ensure using the smallest number of animals required to achieve a 
level of 80%.
Methods for Testing the Hypothesis That Pretreatment of the PnO with Pertussis Toxin Inhibits Hypocretin-1-Stimulated ACh Release in the PnO: In Vivo Microinjection Followed by in Vivo Microdialysis. Animals were surgically prepared with guide tubes aimed for the PnO and microinjected with pertussis toxin or pertussis toxin vehicle as described above. Forty-eight hours after pertussis toxin administration, rats were anesthetized with isoflurane, and a dialysis probe was placed through the guide tube into the PnO. Dialysis samples (30 µl) were collected every 15 min. Five samples were collected during dialysis with Ringer's to establish baseline levels of ACh release. A CMA/110 liquid switch then was turned to deliver Ringer's containing hypocretin-1 (100 µM) to the probe. Five sequential dialysis samples were collected during continuous PnO administration of hypocretin-1. The probe then was removed from the brain. Brainstems were processed for cresyl violet histology, as described above.
Positive control experiments were performed using dialysis administration of the adenosine A1 receptor agonist SPA (300 µM). Adenosine A1 receptors couple to pertussis toxin-sensitive G proteins (Ribeiro et al., 2003), and SPA decreases ACh release in cat pontine reticular formation (Tanase et al., 2003). Thus, pretreatment with pertussis toxin was predicted to inhibit the SPA-induced decrease in ACh release, and pretreatment with pertussis toxin vehicle was predicted to have no effect on the SPA-induced decrease in ACh release.
ACh release during dialysis administration of hypocretin-1 or SPA was expressed as a percentage of baseline ACh measured during dialysis with Ringer's for each experiment. Data were analyzed by descriptive statistics and two-way ANOVA (SAS software, release 9.1.2; SAS Institute, Inc., Cary, NC). A significant ANOVA was followed by a Tukey/Kramer multiple comparisons test. The level for statistical significance was set at p < 0.05. Power calculations ascertained that all analyses used the minimum number of animals needed to achieve a level of 80%.
Methods for Testing the Hypothesis That Hypocretin-1 and -2 Each Cause a Concentration-Dependent Enhancement of ACh Release in the PnO: In Vivo Microdialysis. Animals were anesthetized as described above and a dialysis probe was aimed unilaterally for the PnO. No microinjections were made before dialysis for these studies. Five control samples (75 min) were collected during dialysis with Ringer's before hypocretin-1 or -2 delivery was initiated by turning a CMA/110 liquid switch. Four concentrations of hypocretin-1 and -2 (0.1, 1, 10, and 100 µM) were used. Each rat was used for only one experiment, and only one concentration of hypocretin was tested per experiment. Five dialysis samples (75 min) were collected during continuous hypocretin delivery. When the last dialysis sample was collected, the probe was removed from the brain, the scalp was closed, and the animal recovered from anesthesia. Brains were processed for cresyl violet histology 2 to 3 days later, as described above.
Data were analyzed using descriptive and inferential statistics. The level for statistical significance was set at p < 0.05. The smallest number of animals required to achieve a level of 80% was established using power calculations. One-way ANOVA and Dunnett's multiple comparisons test (GB-Stat) were used to determine whether each peptide concentration significantly increased ACh release. Nonlinear regression analysis (GraphPad Prism software version 4.0a for Macintosh; GraphPad Software Inc., San Diego, CA) was used to fit sigmoid curves to the concentration-response data according to the following equation: percentage of Ringer's ACh release = basal percent Ringer's release + (maximum percent Ringer's release–basal percent Ringer's release)/(1 + 10(log EC50 – X)), where X is the logarithm of the concentration of hypocretin-1 or -2. Regression analyses provided the coefficient of determination (r2) for each of the peptides, as well as the concentration of hypocretin-1 and -2 that produced a half-maximal increase in ACh release (EC50). Two-way ANOVA for repeated measures (SAS) was used to compare the concentration-response curves for ACh release stimulated by hypocretin-1 and -2. Bonferroni post hoc tests identified statistically significant differences in ACh release caused by equimolar concentrations of hypocretin-1 and -2.
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Results |
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S Binding Was Inhibited by Pertussis Toxin. Figure 1 summarizes the results of experiments testing the hypothesis that pertussis toxin inhibits hypocretin-1-stimulated G protein activation. Pertussis toxin (Fig. 1A) or vehicle solution (Fig. 1D) were microinjected into one side of the PnO 48 h before brain removal for autoradiography. The contralateral side of the PnO served as the noninjected control. Figure 1, B, C, E, and F, summarizes the results of the quantitative autoradiography. The total number of [35S]GTPS binding measurements contributing to each figure is reported below. To avoid inflated degrees of freedom for statistical analyses, measurements were averaged by rat. This resulted in a sample size of n = 4 as shown in Fig. 1, B and C, and n = 3 in Fig. 1, E and F.
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S binding on the noninjected and pertussis toxin-injected sides of the PnO. Results are based on 386 measurements from four rats. ANOVA revealed a significant (F = 19.5; df = 3,12; p < 0.001) treatment main effect on [35S]GTPS binding. On the noninjected side of the PnO, hypocretin-1 significantly (p < 0.01) increased [35S]GTPS binding over basal levels (28.9%). In contrast, on the pertussis toxin-injected side of the PnO, hypocretin-1 did not significantly increase [35S]GTPS binding over basal levels. On the pertussis toxin-injected side of the PnO, hypocretin-1-stimulated [35S]GTPS binding was significantly (p < 0.01) lower (–25.1%) than on the noninjected side of the PnO.
Figure 1C illustrates the results of positive control experiments that used the µ opioid receptor agonist DAMGO to stimulate [35S]GTPS binding. Results are based on 384 measurements from four rats. Repeated measures ANOVA revealed a significant (F = 42.3; df = 3,12; p < 0.0001) treatment main effect on [35S]GTPS binding. On the noninjected side of the PnO, DAMGO significantly (p < 0.01) increased [35S]GTPS binding (95.6%) over basal levels. On the pertussis toxin-injected side of the PnO, the DAMGO-stimulated increase (49.6%) in [35S]GTPS binding was significantly (p < 0.01) decreased (–37.3%) below the DAMGO-stimulated increase on the contralateral, noninjected PnO.
Additional control experiments involved unilateral microinjection of the pertussis toxin vehicle into the PnO 48 h before brain removal for autoradiography (Fig. 1D). Figure 1E summarizes the effects of vehicle microinjection on basal and hypocretin-1-stimulated [35S]GTPS binding. Results are based on 339 measurements from three rats. Repeated measures ANOVA showed a significant (F = 22.7; df = 2,9; p < 0.001) treatment main effect on [35S]GTPS binding. On the noninjected side of the PnO, [35S]GTPS binding was significantly (p < 0.01) increased over basal levels by hypocretin-1 (19.7%). On the side of the PnO that received a vehicle microinjection, hypocretin-1-stimulated [35S]GTPS binding also was significantly (p < 0.01) increased (18.7%) over basal binding. Specific [35S]GTPS binding levels for basal and hypocretin-1-treated sections on the noninjected side of the PnO were not different from binding levels of the corresponding treatment conditions on the vehicle-microinjected side of the PnO.
Figure 1F shows the effects of vehicle microinjection on basal and DAMGO-stimulated [35S]GTPS binding in the PnO. Results are based on 340 measurements from three rats. Repeated measures ANOVA revealed a significant (F = 182.9; df = 2,9; p < 0.0001) treatment main effect on [35S]GTPS binding. On the noninjected side of the PnO, [35S]GTPS binding was significantly (p < 0.01) increased over basal levels by DAMGO (82.6%). On the vehicle-injected side of the PnO, DAMGO-stimulated [35S]GTPS binding also was significantly (p < 0.01) increased (81.0%) over basal binding. Specific [35S]GTPS binding levels for basal and DAMGO-treated sections on the noninjected side of the PnO were not different from binding levels of the corresponding treatment conditions on the vehicle-microinjected side of the PnO.
The stereotaxic coordinates of histologically localized microinjection sites (Fig. 1, A and D) were compared using the Bonferroni t test. Mean ± S.E.M. stereotaxic coordinates for pertussis toxin injection sites (8.54 ± 0.18 mm posterior to bregma; 1.61 ± 0.07 mm lateral to bregma; 8.50 ± 0.04 mm ventral to bregma) were not significantly different from the stereotaxic coordinates for vehicle microinjection sites (8.34 ± 0.21 mm posterior to bregma; 1.48 ± 0.16 mm lateral to bregma; 8.47 ± 0.09 mm ventral to bregma).
Pertussis Toxin Did Not Inhibit Hypocretin-1-Stimulated ACh Release. Figure 2 localizes the microdialysis sites used to test the hypothesis that pretreatment with pertussis toxin blocks hypocretin-1-stimulated ACh release. All probe sites were located in the PnO. Bonferroni t test revealed that the average ± S.E.M. stereotaxic coordinates for pertussis toxin injection sites (8.04 ± 0.11 mm posterior to bregma; 1.38 ± 0.07 mm lateral to bregma; 9.18 ± 0.12 mm ventral to bregma) were not significantly different from the stereotaxic coordinates for vehicle injection sites (8.13 ± 0.06 mm posterior to bregma; 1.18 ± 0.04 mm lateral to bregma; 9.13 ± 0.10 mm ventral to bregma).
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The effects of pertussis toxin or vehicle pretreatment on hypocretin-1-stimulated ACh release were tested in six experiments summarized by Fig. 3A. Two-way ANOVA was based on 60 dialysis samples, accounted for as follows: five samples per dialysis condition x two dialysis conditions (Ringer's versus hypocretin-1) x two pretreatment conditions (vehicle injection versus pertussis toxin injection) x three rats per pretreatment condition. There was a significant effect of dialysis condition (Ringer's versus hypocretin-1) on ACh release (F = 176.6; df = 1,52; p < 0.0001), no significant effect of pretreatment (vehicle versus pertussis toxin), and no significant interaction. Hypocretin-1 significantly (p < 0.0001) increased ACh release in the PnO after microinjection of vehicle (67.1%) and after microinjection of pertussis toxin (54.6%). ACh levels were not different between pertussis toxin and vehicle-injected rats. Mean ± S.E.M. basal (Ringer's) ACh levels (picomoles per 15 min) were 0.077 ± 0.006 for vehicle injection experiments and 0.086 ± 0.005 for pertussis toxin injection experiments (Fig. 3A).
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Hypocretin-1 and -2 Each Caused a Concentration-Dependent Increase in ACh Release. Figure 4 summarizes the location of all dialysis sites used for the concentration-response studies. Figure 4A shows representative histological sections from a rat that received hypocretin-1 (left) and a rat that received hypocretin-2 (right). Histological analysis of all hypocretin-1 (Fig. 4B) and -2 (Fig. 4C) dialysis sites confirmed that measures of ACh were obtained from the PnO. Analysis of the stereotaxic coordinates for the dialysis sites determined that mean ± S.E.M. probe sites for hypocretin-1 delivery (8.00 ± 0.07 mm posterior to bregma; 1.30 ± 0.06 mm lateral to bregma; 8.98 ± 0.05 mm ventral to bregma; n = 12 experiments) were not significantly different from probe sites for hypocretin-2 delivery (8.30 ± 0.11 mm posterior to bregma; 1.33 ± 0.07 mm lateral to bregma; 8.98 ± 0.07 mm ventral to bregma; n = 12 experiments). Mean ± S.E.M. in vitro ACh recovery of probes used to deliver hypocretin-1 (6.0 ± 0.2%) and -2 (6.4 ± 0.3%) also was not significantly different. These measures provided important controls, indicating that differences reported below (Figs. 5 and 6) in the magnitude of the ACh response to equimolar concentrations of hypocretin-1 and -2 were not artifacts caused by differences in intrapontine site of hypocretin delivery or differences in amounts of ACh recovered by the dialysis probes.
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Figure 5 summarizes ACh release during Ringer's (control) dialysis and during dialysis with four concentrations of hypocretin-1 (Fig. 5, A–D) and -2 (Fig. 5, E–H). Each bar represents mean ± S.E.M. of ACh release obtained from three experiments. Variability among the five Ringer's control ACh samples was similar for all experiments. Dialysis with 0.1 µM hypocretin-1 (Fig. 5A) or 0.1 µM hypocretin-2 (Fig. 5E) did not alter ACh release. Figure 5B shows that 1 µM hypocretin-1 significantly increased ACh release during the second 15-min sampling period. In contrast, 1 µM hypocretin-2 did not increase ACh release (Fig. 5F). Dialysis with 10 and 100 µM hypocretin-1 (Fig. 5, C and D) caused significant increases in ACh release. In contrast to Fig. 5, E to G, only the 100 µM concentration of hypocretin-2 significantly increased ACh release (Fig. 5H). There was no significant difference between mean ± S.E.M. basal (Ringer's) ACh levels for experiments delivering hypocretin-1 (0.141 ± 0.006 pmol/15 min, Fig. 5, A–D) and experiments delivering hypocretin-2 (0.142 ± 0.005 pmol/15 min, Fig. 5, E–H). This finding supports the interpretation that differences in the magnitude of the ACh response to equimolar concentrations of hypocretin-1 and -2 were not artifacts resulting from differences in basal levels of ACh release.
Figure 6 shows the concentration-response curves for hypocretin-1- and -2-stimulated ACh release. Two-way ANOVA was based on 119 dialysis samples, accounted for as follows: (five samples per peptide concentration x four peptide concentrations x two peptides x three rats per peptide concentration) – one missing value. Two-way ANOVA revealed a significant drug main effect (F = 10.1; df = 1,111; p = 0.0019), a significant concentration main effect (F = 28.9; df = 3,111; p < 0.0001), and no significant interaction. A post hoc Bonferroni test revealed that 10 and 100 µM hypocretin-1 caused a significantly (p < 0.05) greater increase in ACh release than respective, equimolar concentrations of hypocretin-2.
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Hypocretin Receptors Activate Pertussis Toxin-Sensitive G Proteins in Rat PnO. In vitro [35S]GTPS autoradiography has been used successfully to quantify the functional activity of many different G protein coupled receptors (Sóvágó et al., 2001). Agonist-activated [35S]GTPS binding in the brain is thought to preferentially label inhibitory (Gi/o, Gz), rather than stimulatory (Gs, Gq), G proteins (Sóvágó et al., 2001; Harrison and Traynor, 2003; Laitinen, 2004). Pertussis toxin selectively inactivates Gi/o proteins, and pertussis toxin has been shown to be effective following microinjection into rat striatum (van der Ploeg et al., 1991), ventral tegmental area (Gronier and Rasmussen, 1999), substantia nigra compacta (Gronier and Rasmussen, 1999), and hippocampus (Tzavara et al., 2003). The present finding that PnO microinjection of pertussis toxin decreased hypocretin-1-stimulated [35S]GTPS binding in the PnO (Fig. 1B) is the first direct evidence that hypocretin activates inhibitory G proteins in rat pontine reticular formation. This result is consistent with the interpretation that some hypocretin receptors in rat PnO couple to Gi/o proteins. An alternative explanation is that PnO hypocretin receptors indirectly activate Gi/o proteins via G protein cross talk (Vazquez-Prado et al., 2003) or heterodimerization of hypocretin receptors (Karteris and Randeva, 2003) with other Gi/o protein-coupled receptors (Devi, 2001).
To further test the interpretation that hypocretin-1 activates Gi/o proteins in rat PnO, the effects of pertussis toxin on DAMGO-stimulated [35S]GTPS binding also were quantified. The µ opioid agonist DAMGO was used as a positive control because µ opioid receptors couple exclusively to Gi/o proteins (Birnbaumer et al., 1990). DAMGO-stimulated [35S]GTPS binding was significantly reduced by pretreatment with pertussis toxin (Fig. 1C, compare filled bars), confirming that microinjection of pertussis toxin effectively inactivated Gi/o proteins in the PnO. DAMGO-stimulated [35S]GTPS binding was not completely eliminated by pretreatment with pertussis toxin (Fig. 1C, pertussis toxin injection, hatched versus filled bar, 
). A higher concentration of pertussis toxin than was used in the present study may be required to eliminate the remaining DAMGO-stimulated G protein activation. Alternatively, the remaining DAMGO-stimulated G protein activation may have resulted from activating µ opioid receptors that couple to pertussis toxin-insensitive inhibitory G proteins (Gz) (Ho and Wong, 2001).
Additional control experiments were performed by microinjecting the vehicle solution for pertussis toxin into the PnO before [35S]GTPS autoradiography (Fig. 1D). Compared with no injection, vehicle injection did not alter specific [35S]GTPS binding levels for basal (Fig. 1E, compare hatched bars), hypocretin-1 (Fig. 1E, compare solid bars), and DAMGO (Fig. 1F, compare solid bars) treatment conditions. These data demonstrate that vehicle injection did not alter [35S]GTPS binding and confirm that the block of DAMGO- and hypocretin-1-stimulated [35S]GTPS binding was due to the pharmacological actions of pertussis toxin. Hypocretin-stimulated [35S]GTPS binding in rat pons is known to be a receptor-mediated response because it is concentration-dependent and blocked by specific hypocretin receptor antagonists (Shiba et al., 2002; Bernard et al., 2003, 2005). Therefore, Fig. 1 results support the conclusion that some hypocretin receptors in rat PnO couple to pertussis toxin-sensitive inhibitory G proteins. This conclusion is consistent with in vitro electrophysiological data showing that hypocretin-induced alterations in cell excitability are inhibited by pertussis toxin (Hoang et al., 2003; van den Top et al., 2003; Zhu et al., 2003; Holmqvist et al., 2005).
Hypocretin-1-Stimulated ACh Release in the PnO Is Mediated by Pertussis Toxin-Insensitive G Proteins. The present study aimed to determine whether one mechanism by which hypocretin modulates ACh release includes activation of pertussis toxin-sensitive G proteins in rat PnO. Using the same protocol shown to be effective for blocking hypocretin-1-stimulated G protein activation (Fig. 1), pertussis toxin was microinjected into the PnO 48 h before dialysis administration of hypocretin-1 or SPA (Fig. 2). Positive control experiments used the adenosine A1 receptor agonist SPA, which is known to activate pertussis toxin-sensitive G proteins (Ribeiro et al., 2003) and inhibit ACh release (Tanase et al., 2003). Pertussis toxin blocked the SPA-induced decrease in ACh release (Fig. 3B), confirming that Gi/o proteins were inactivated. In contrast, the hypocretin-1-induced increase in ACh release was not blocked by pertussis toxin (Fig. 3A), suggesting that hypocretin-1-stimulated ACh release is mediated by noninhibitory G proteins. Additional control experiments established that ACh release was not altered due to vehicle microinjection (Fig. 3).
The conclusion that hypocretin receptors modulating ACh release in PnO couple to stimulatory G proteins is consistent with the neuroexcitatory activity of hypocretin (de Lecea et al., 1998). Hypocretin receptors also have been suggested to signal through Gq proteins (van den Pol et al., 1998; Zhu et al., 2003), which are known to stimulate phosphoinositol turnover and increase intracellular Ca2+. Hypocretin-1-stimulated calcium release in Chinese hamster ovary cell lines (Smart et al., 1999) and in hypothalamic neurons (van den Pol et al., 1998) involves phospholipase C and/or protein kinase C. Alternatively, hypocretin-1 could stimulate ACh release via pertussis toxin-insensitive inhibitory G proteins (Knott et al., 1993). However, the present observation that pertussis toxin inhibits hypocretin-1-stimulated G protein activation (Fig. 1) suggests that in rat PnO, some hypocretin receptors couple to inhibitory G proteins that are indeed pertussis toxin-sensitive. In addition, the Fig. 3 results show for the first time that hypocretin-1-stimulated ACh release in the PnO involves activation of pertussis toxin-insensitive G proteins.
Both Hypocretin Receptor Subtypes Modulate ACh Release in Rat PnO. The discovery that hypocretin-1 stimulates ACh release via noninhibitory G proteins encouraged further studies aiming to reveal the hypocretin receptor subtype modulating ACh release in the PnO (Fig. 4). Hypocretin-1 and -2 each caused a concentration-dependent increase in ACh release (Figs. 5 and 6). The two peptides showed only about a 2-fold difference in EC50 values (Fig. 6), consistent with the interpretation that hcrt-r2 modulates ACh release in the PnO (Sakurai et al., 1998; Smart et al., 1999, 2001). At concentrations of 10 and 100 µM, hypocretin-1 caused a significantly greater increase in ACh release than hypocretin-2 (Fig. 6), suggesting that hcrt-r1 also may be involved in modulating PnO ACh release. Both hypocretin receptor subtypes are expressed in rat PnO (Greco and Shiromani, 2001). Thus, these data provide novel evidence that ACh release in rat PnO is modulated by hcrt-r2 and hcrt-r1.
Functional Implications. One new finding presented here is that rat PnO contains a population of hypocretin receptors that activate pertussis toxin-sensitive G proteins (Fig. 1). The function served by hypocretinergic activation of putatively inhibitory G proteins is unknown. The present study also showed, for the first time, that hypocretin-1 causes a pertussis toxin-insensitive increase in ACh release within the PnO (Fig. 3), and that both peptides evoke a concentration-dependent increase in PnO ACh release (Fig. 6). These data provide the first functional evidence that hypocretin receptors in rat PnO activate stimulatory G proteins. Increasing ACh release in PnO may be one mechanism by which hypocretin promotes brain activation. Cholinergic transmission in the pontine reticular formation participates in generating the cortical activation characteristic of wakefulness and rapid eye movement sleep (Lydic and Baghdoyan, 2005), and microinjection of hypocretin-1 into cat pontine reticular formation can trigger rapid eye movement sleep (Xi et al., 2002). Interactions between cholinergic and GABAergic transmission in the pontine reticular formation promote wakefulness, particularly when GABAergic transmission is enhanced (Xi et al., 2004). Most recently, hypocretin-1 has been shown to cause a concentration-dependent increase in PnO GABA levels (Soto-Calderon et al., 2005). Taken together, these findings are consistent with the interpretation that hypocretin promotes arousal, in part, by increasing ACh release in the PnO.
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ABBREVIATIONS: hcrt-r1, hypocretin receptor subtype 1; hcrt-r2, hypocretin receptor subtype 2; PnO, pontine reticular nucleus oral part; [35S]GTPS, [35S]guanylyl-5'-O-(-thio)triphosphate; ACh, acetylcholine; DAMGO, [D-Ala2, N-Me-Phe4, Gly5] enkephalin; SPA, N6-p-sulfophenyladenosine; ANOVA, analysis of variance.
Address correspondence to: Dr. Helen A. Baghdoyan, University of Michigan, Department of Anesthesiology, 7433 Medical Sciences Building I, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0615. E-mail: helenb{at}umich.edu
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