Activation of Pedunculopontine Tegmental Protein Kinase A: A Mechanism for Rapid Eye Movement Sleep Generation in the Freely Moving Rat

Subjects.

Experiments were performed on 56 adult male Wistar rats (Charles River Laboratories, Wilmington, MA) weighing between 200 and 300 g. The rats were housed individually at 24°C with ad libitum access to food and water. Lights were on from 7:00 A.M. to 7:00 P.M. (light cycle) and off from 7:00 P.M. to 7:00 A.M. (dark cycle). The principles for the care and use of laboratory animals in research, as outlined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, were strictly followed.

Surgical procedures for electrode and guide tube implantation.

All surgical procedures were performed stereotaxically under aseptic conditions and were in accordance with the guidelines approved by the institutional animal care and use committee (protocol AN-14084). Animals were anesthetized with pentobarbital (40 mg/kg, i.p.; Abbott Laboratories, Chicago, IL), placed in the stereotaxic apparatus, and secured using blunt rodent ear bars as described previously (Paxinos and Watson, 1997). The appropriate depth of anesthesia was judged by the absence of palpebral reflexes and the absence of a response to tail pinch. Core body temperature was maintained at 37 ± 1°C with a thermostatic heating pad and a rectal thermister probe. The scalp was cleaned and painted with povidone iodine. A scalp incision was made, and the skin was retracted. The skull surface was cleaned in preparation for electrode implantation. After completion of the surgical procedure, animals were administered saline (5 cc, s.c.) to prevent dehydration and ampicillin (50 mg per rat, i.m.; Bristol-Myers Squibb, Princeton, NJ) to control any potential postsurgical infection. Potential postoperative pain was controlled with buprenorphine (0.05 mg/kg, s.c.; Abbott Laboratories).

To record the behavioral states of vigilance, cortical electroencephalogram (EEG), dorsal neck muscle electromyogram (EMG), electrooculogram (EOG), hippocampal EEG (to record theta wave), and pontine EEG (to record pontine wave) recording electrodes were chronically implanted as described previously (Datta, 2000, 2002; Datta et al., 2002). In addition to these electrodes, 35 rats had a stainless steel guide tube (26 gauge), with an equal-length stylet inside, stereotaxically implanted 2 mm above the PPT (in relation to stereotaxic “0”: anterior, 1.0 mm; lateral, 1.8 mm; and horizontal, 3.0 mm) as described previously (Datta, 2002; Datta et al., 2002). All electrodes and guide tube were secured to the skull with dental acrylic. Electrodes were crimped to miniconnector pins and brought together in a plastic connector. Immediately after surgery, animals were placed in recovery cages and monitored for successful recovery from anesthesia and surgery. Successful recovery was gauged by the return of normal postures, voluntary movement, and grooming.

Adaptation recording session.

After the postsurgical recovery period of 3–7 d, rats were habituated to the experimenter, the sound-attenuated recording cage (electrically shielded; 76.2 × 45.72 × 45.72 cm), and free-moving polygraphic (Grass polygraph, model 79; Grass Instruments, Quincy, MA) recording conditions for 7–10 d. The last day of these adaptation recording sessions was determined when, for 3 consecutive days, the day-to-day variation of the percentage of REM sleep was <5% of the rats’ total amount of REM sleep. All adaptation–recording sessions were performed between 9:00 A.M. and 4:00 P.M., when rats are normally sleeping. During recovery, habituation, and free-moving recording conditions (adaptation recording sessions), all rats were housed under the same 12 h light/dark cycle with ad libitum access to food and water.

Polygraphic recordings and REM sleep deprivation setup.

To record cortical EEG, EMG, EOG, hippocampal EEG, and pontine EEG in a freely moving condition, each rat's head plug was mated to a male connector that was in turn connected to a commutator. Signals from this commutator were sent to a polygraph (Grass model 79; located in the next room) via the electrode board (located inside the recording chamber). To allow rats to move freely inside the recording cage while maintaining the head plug connection, a counterbalanced connecting cable and a mechanical pulley system (attached to the roof of the recording chamber) were used. In a separate room, polygraphic signs of the rat were continuously observed on a computer monitor to identify ongoing behavioral stages (Datta and Siwek, 2002; Ulloor et al., 2004).

For the purpose of REM sleep deprivation, the beginning of each REM sleep episode was identified by observation of ongoing polygraphic records. From the room adjacent to the rat, the experimenter pressed a mechanical lever within 2–3 s of REM sleep onset, the animal's head was gently lifted, and the animal was awakened. The detailed description and validation of this selective REM sleep deprivation method was described previously (Datta et al., 2004). One of the most important advantages of this REM sleep deprivation method is that this method successfully eliminates >75% of REM sleep without significantly reducing SWS.

Determination of behavioral states.

For the purpose of determining possible effects on sleep and wakefulness (W), polygraphic data were captured on-line with a computer using Gamma software (Grass product group; Astro-Med, West Warwick, RI). From this captured data, three behavioral states were distinguished and scored visually using Rodent Sleep Stager software (Grass product group; Astro-Med). These three states were W, SWS, and REM sleep. The physiological criteria for the identification of these wake–sleep states were described in detail previously (Datta et al., 2002, 2004). In the first study, which focused on PKA subunit analysis, the behavioral states of W, SWS, and REM sleep were scored in successive 5 s epochs. This epoch length allowed us to quantify the short periods of REM sleep (3–5 s) in the rats in which REM sleep was selectively terminated (Datta et al., 2004). In the second study, however, which focused on determining possible effects of Rp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (cAMPS) on sleep and wakefulness, the behavioral states of W, SWS, and REM sleep were scored in successive 10 s epochs.

Drug and vehicle for microinjections.

The drug used in the second study was a specific membrane-permeable inhibitor of cAMP-dependent PKA activation, Rp-cAMPS (molecular weight, 446.46), purchased from Sigma (St. Louis, MO). The Rp-cAMPS was dissolved in 0.9% saline to make four different doses (0.28, 0.55, 1.1, and 2.2 nmol/100 nl). 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. The selection of this drug was based on the selective inhibitory effect on intracellular cAMP–PKA activation (Van Haastert et al., 1984; Wang et al., 1991; Shi and Bunney, 1992). This commercially available Rp-cAMPS is suitable for our local microinjection studies because it is water soluble, cell permeable, has reversible effects, and has been used successfully for microinjection studies in behaving animals (Cook et al., 1995; Self and Nestler, 1995; Capece and Lydic, 1997; Punch et al., 1997; Self et al., 1998; Schafe and LeDoux, 2000).

Tissue collection and Western blotting for the quantification of PKA subunits.

After the rats were killed with CO₂, the brains were rapidly removed and quickly chilled with dry ice. These frozen brains were cut in the transverse plane in 300-μm-thick sections with the use of a Vibratome (series 3000; Technical Products International, St. Louis, MO). Then, under a dissecting microscope, the medial prefrontal cortex (mPFC), anterior hypothalamus (AHTh), PPT, and mPRF were dissected separately on an ice-chilled Petri dish. The dissected tissues from these four specific brain areas, containing tissues from both the left and right sides of the brain, of each individual rat were stored separately in prechilled microcentrifuge tubes at −80°C until assayed. As a result, levels of each PKA subunit were measured in each specific brain area using tissue collected from both hemispheres. Following are the descriptions for the spatial and anatomical areas of the brain (Paxinos and Watson, 1997) from which these tissues were collected. The mPFC tissue collection area: anteroposterior, between 2 and 4 mm anterior to the bregma; mediolateral, between midline and 2 mm lateral; dorsoventral, between dorsal surface and 3 mm deep from the surface. Anatomically, this area includes cingulate cortex (areas 1 and 2) and frontal cortex (area 2). The AHTh tissue collection area: anteroposterior, between 0.2 and 2.2 mm posterior to the bregma; mediolateral, between midline and 2 mm lateral; dorsoventral, between anterior commissure and optic chiasm. Anatomically, this area includes preoptic-anterior hypothalamus and a part of the lateral hypothalamus. The PPT tissue collection area: anteroposterior, between 7.7 and 8.7 mm posterior to the bregma; mediolateral, between 1.5 and 2.5 mm lateral to the midline; dorsoventral, between 5.5 and 6.5 mm from the dorsal surface of the brain. Anatomically, this area includes mainly the PPT. The mPRF tissue collection area: anteroposterior, between 7.7 mm and 8.7 mm posterior to the bregma; mediolateral, between 0.5 and 1.5 mm lateral to the midline; dorsoventral, between 7.0 and 8.0 mm from the dorsal surface of the brain. Anatomically, this area includes only the oral part of the pontine reticular nucleus.

The individual frozen tissue samples were rapidly sonicated (Sonic Dismembrator, model 100; Fisher Scientific, Pittsburgh, PA) in five volumes (w/v) of ice-cold mammalian CelLytic buffer (Sigma). The homogenates were then centrifuged at 10,000 rpm for 10 min at 4°C. The amount of protein in the supernatant (soluble fraction) of individual samples was then measured using Bradford's method (Bradford, 1976). Protein (∼25 μg) from each sample was heated (for 3 min at 95°C) in Laemmli's 1× protein loading buffer (Laemmli, 1970). These denatured samples were then loaded onto 12% SDS-polyacrylamide gels and resolved by standard electrophoresis in the Tris-glycine-SDS buffer, pH 8.3 (containing 25 mm Tris base, 192 mm glycine, and 0.1% SDS; at 80 constant volts for 2 h). Separated proteins were then electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) using Tris-glycine buffer, pH 8.4 (containing 25 mm Tris, 192 mm glycine, and 10% methanol). To probe any specific protein, membranes were pretreated with Tris-buffered saline, 5% nonfat dry milk, and 0.05% Tween 20 (TBST) solution. Binding of antibodies was also done under the same conditions. To detect the levels of different PKA subunits, anti-human PKA_Cα (clone 5B; 1:1500 dilution; BD Biosciences, San Jose, CA), anti-mouse PKA_RI (clone 18; 1:500 dilution; BD Biosciences), anti-human PKA_RIIα (clone 40; 1:1500 dilution; BD Biosciences), anti-human PKA_RIIβ (clone 45; 1:1500 dilution; BD Biosciences), and anti-actin (clone AC-40; 1:1500 dilution; Sigma) monoclonal primary antibodies were used. In this study, a ubiquitous protein, actin, was measured as a methodological control to ensure uniformity in our Western blot procedure. All of these primary antibody-binding reactions were done in a shaker at room temperature for 2 h. At the completion of primary antibody reactions, blots were washed in the TBST solution containing no dry milk (typically three washes, each for 5 min). After the final wash, blots were treated with secondary antibody [anti-mouse IgG (heavy plus light) HRP conjugate at 1:10,000 dilution; Promega, Madison, WI] diluted with the TBST buffer. After the secondary antibody reactions, blots were again washed in TBST solution containing no dry milk (three washes, each for 5 min). The resulting immunocomplexes were detected with a chemiluminescent substrate (SuperSignal West Femto; Pierce Biotechnology, Rockford, IL). The quantification of the immunoreactive band was performed using a Kodak imaging densitometer (PerkinElmer, Shelton, CT). Each sample was probed at least three times. The net intensities were equalized according to exposure time. For the quantification of PKA_Cα, PKA_RI, PKA_RIIα, PKA_RIIβ, and actin levels in the PPT, mPRF, mPFC, and AHTh, chemiluminescent values obtained from the control REM sleep group of rats were normalized. The averaged means of these normalized values were considered to be the 100% value. Finally, in the high and low REM sleep groups of animals, the chemiluminescent value in each band was calculated as a percentage level compared with the comparable normalized mean chemiluminescent value in the control REM sleep group. These percentage level values were then used for the statistical analysis.

Tissue collection and estimation of PKA activity.

The tissue collection procedure for the PKA activity in the PPT and mPRF is identical to the tissue collection procedure described above for the PKA subunit expression analysis. To estimate PKA activity, the frozen tissue (0.1–0.2 g) isolated from the brain was initially homogenized (Sonic Dismembrator; Fisher Scientific) in 500 μl of ice-cold cell lysis buffer (mammalian CelLytic reagent; Sigma). The homogenate was then centrifuged at 14,000 rpm for 15 min at 4°C to remove cell debris. The supernatant was saved as crude extract. For the activity assay, the crude extract was further purified by passing it through a mini spin column (Micro Bio-Spin 6 chromatographic column; Bio-Rad Laboratories); however, before loading the extract, the column was prepared and equilibrated. Excess of the suspension solution present with the matrix was removed, and 250 μl of cell lysis buffer was added to equilibrate it. Any remaining amount of liquid that remained in the column bed after equilibration was also removed by spinning the column at 5000 rpm for 30 s at 4°C. The 200 μl of crude extract was then loaded on the equilibrated column and spun at 5000 rpm for 2 min at 4°C. Afterward, the flow-through solution was collected and saved as cleared extract. To prevent any loss of enzymatic activity in the preparation, a glycerol solution was added to the cleared extract, creating a final concentration of 10%. An aliquot of this glycerol-mixed cleared extract was used to measure the protein concentration [by Bradford's method (Bradford, 1976)]. The rest was saved at −20°C until additional use.

In our study, PKA activity was measured using a nonradioactive assay method, the PepTag assay system that was developed by Promega (White and Shultz, 1992; Sahir et al., 2001; Goueli et al., 2004). The required assay kit was obtained from Promega. For the activity assay, empty 1.5 ml Eppendorf tubes (Eppendorf Scientific, Westbury, NY) were placed on ice and appropriately labeled (one for each experimental sample, one for the positive control, and another for the negative control). Once chilled, a 5 μl volume of PKA buffer (containing 100 mm Tris-HCl, pH 7.4, 50 mm MgCl₂, and 5 mm ATP) was poured into each tube, followed by a 5 μl volume of PKA substrate (containing 2 μg of colored PepTag A1 Kemptide protein). Next, 1 μl of company-supplied protector solution was added to each tube containing PKA buffer and substrate. At this point, a 10 μl volume of brain extract (containing 2 μg of total protein) was added only to tubes labeled with both experimental condition and anatomical site. No brain extract was added to either the positive or negative control tube. Instead, 10 μl of company-supplied standard enzyme [containing 20 ng of purified form of the PKA catalytic (C) subunit] was added to the positive control tube, and 10 μl of ice-cold deionized water was added to the negative control tube. After adding all of the required reagents and the appropriate enzyme, the final volume of each tube was adjusted to a total volume of 25 μl by adding 4 μl of ice-cold deionized water. The reaction mixes were then incubated at room temperature for 60 min. After incubation, the tubes were placed in a 95°C water bath for 10 min to stop the reaction. The samples were then quickly spun in a microfuge to pull down the condensate and ultimately loaded in a 1% agarose gel. The gel used for the study was prepared in 50 mm Tris-HCl buffer, pH 8.0, and was electrophoresed in the same solution. Electrophoresis continued for 20–30 min at 100 constant volts. After electrophoresis, the gel was photographed under UV light using a Kodak Image Station (440CF; Eastman Kodak, Rochester, NY). For quantitation of the bands, which represented both the phosphorylated and nonphosphorylated Kemptides, the image intensities were converted to OD by using the Kodak image analysis software.

Experimental design to measure the PKA subunit expression and activity at three different levels of REM sleep.

On the day after the last adaptation recording session, when day-to-day variation on the total amount of REM sleep stabilized, rats underwent a session of baseline sleep–wakefulness recordings (between 9:00 A.M. and 2:00 P.M.). The following day, experimental recording sessions began. During the experimental recording session, each animal was connected to the polygraphic recording system at 8:55 A.M. In the next room, polygraphic signs of the sleep–wake cycle were continuously observed on a computer monitor (between 9:00 A.M. and 2:00 P.M.). At this point, the 18 rats were randomly divided into three groups. The experimental design of these studies is summarized in Figure 1A. Group 1 (n = 6 rats) rats were recorded for 5 h (between 9:00 A.M. and 2:00 P.M.) of undisturbed sleep–wakefulness [hereafter, group 1 is labeled as “baseline/control REM sleep” (“BR”)]. The experimental protocol for group 2 (n = 6 rats) animals was almost identical to the protocol described above for the BR group, except that for group 2 animals, from 11:00 A.M. to 2:00 P.M., REM sleep episodes were selectively terminated at the beginning (within 3–5 s) of each episode while the animals were connected to the polygraphic recording system [hereafter, group 2 is labeled as “REM sleep deprived” (“RD”)]. In group 3 (n = 6 rats) animals, REM sleep episodes were selectively terminated between 9:00 A.M. and 12:00 P.M., after which they were allowed to have undisturbed sleep–wake [hereafter, group 3 is labeled as “REM sleep rebound” (“RR”)]. In summary, the main difference between these three groups of animals is the amount of REM sleep during the last 2 h (between 12:00 A.M. and 2:00 P.M.). The BR group had a normal amount of REM sleep. The RD group had ∼77% less REM sleep than the BR group. The RR group had 84% more REM sleep than the BR group (Fig. 1B). At 2:02 P.M., all rats were killed with CO₂ to collect brain tissues for the Western blotting as described above.

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Figure 1.

Experimental designs for low and high REM sleep conditions. A, Group 1 rats were allowed to have a 5 h period (between 9:00 A.M. and 2:00 P.M.) of undisturbed sleep–wake (S–W) cycle with REM sleep. Group 2 rats were allowed to have undisturbed S–W cycle with REM sleep between 9:00 and 11:00 A.M., after which REM sleep episodes were selectively terminated between 11:00 A.M. and 2:00 P.M. REM sleep episodes in group 3 rats were selectively terminated from the ongoing S–W cycle between 9:00 A.M. and 12:00 P.M., followed by undisturbed S–W with REM sleep between 12:00 P.M. and 2:00 P.M. B, Histograms showing the total percentages of time (mean ± SD) spent in W, SWS, and REM sleep during the last 2 h (between 12:00 and 2:00 P.M.) of S–W data. Note that compared with group 1, group 2 rats had 77% less and group 3 rats had 84% more REM sleep during the last 2 h of S–W recordings. Therefore, in this study, group 1 rats were categorized as having a normal amount of REM sleep, whereas group 2 and group 3 rats were categorized as having low REM sleep and high REM sleep, respectively. Post hoc Scheffé's F test (compared with group 1), ***p < 0.001.

In addition to these 18 rats, three other rats were subjected to similar experimental design to study PKA activity in the PPT and mPRF at three different REM sleep conditions. Of those three rats, one rat was subjected to the BR protocol, and this rat had 11.5% REM sleep. The second rat was subjected to RD protocol, and this rat had only 2% REM sleep. The third rat was subjected to RR protocol and had 20.1% REM sleep. At 2:02 P.M., these three rats were killed with CO₂ to collect brain tissues for estimating PKA activity in the PPT and mPRF as described above.

Experimental design to study the effects of Rp-cAMPS microinjection on the sleep–wake cycle.

After the adaptation recording sessions, microinjection sessions began. During experimental sessions, animals (n = 35 rats) were connected to the polygraphic recording system 15 min before a microinjection into the PPT. The microinjection system consisted of a 32 gauge stainless steel injector cannula with a 26 gauge collar that extended 2.0 mm beyond the implanted guide tube. The collar was connected to a 1.0 μl motor-driven Hamilton microsyringe with PE 20 tubing. While the animal was connected to the recording system, the stylet was removed, and an injector with either control vehicle (100 nl volume of 0.9% saline) or one of four doses of Rp-cAMPS (0.28, 0.55, 1.1, and 2.2 nmol/100 nl) was introduced through the guide tube for the injection. One minute after the insertion of the injector cannula, 100 nl of control saline or one of the four doses of Rp-cAMPS was microinjected over a 60 s period (Pump II Pico Plus; Harvard Apparatus, Holliston, MA). The injector cannula was gently withdrawn 2 min after the injection, and the stylet was reinserted into the guide tube. During the microinjection, animals were free to move around the cage with the cannula in place. Immediately after completion of the microinjection procedure, polygraphic variables were recorded continuously for 6 h (between 10:00 A.M. and 4:00 P.M.), when rats would normally be sleeping (Datta, 2000). Each of these rats received a single microinjection in either the right or the left PPT in a single experimental recording session. None of the rats was used for more than one microinjection recording session. At the end of all experimental sessions and before perfusion, 100 nl of black ink was microinjected 1 mm dorsal to the injection site for localizing the injection site as described previously (Datta, 2002; Datta et al., 2002).

Histological localization of injection site.

At the conclusion of the microinjection experiments, rats were deeply reanesthetized with pentobarbital (60 mg/kg, i.p.) 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–diaphorase staining and histological localization of injection sites as described previously (Datta et al., 2002; Datta and Prutzman, 2005). This NADPH–diaphorase staining was performed to identify the cholinergic cell compartment of the PPT. Histological identification showed that in all 35 rats, unilateral microinjections were placed within the cholinergic cell compartment of the PPT (Fig. 2).

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Figure 2.

Anatomical location of analyzed microinjection sites. Schematic coronal sections through the brainstem are illustrated at levels 1.70, 1.20, 1.00, and 0.70 mm anterior (labeled at the top right of each section). Open circles (n = 7) indicate the location of control saline, and filled circles (n = 28) indicate the location of Rp-cAMPS injector tips. 4, Trochlear nucleus; Aq, aqueduct; ATg, anterior tegmental nucleus; BIC, nucleus brachium inferior colliculus; CG, central gray; CnF, cuneiform nucleus; ctg, central tegmental tract; DR, dorsal raphe nucleus; dtg, dorsal tegmental bundle; IC, inferior colliculus; InCo, intercollicular nucleus; LL, lateral lemniscus; Me5, mesencephalic trigeminal tract/nucleus; MiTg, microcellular tegmental nucleus; mlf, medial longitudinal fasciculus; Pa4, paratrochlear nucleus; PMR, paramedian raphe; PnO, pontine reticular nucleus, oral; RR, retrorubral nucleus; rs, rubrospinal tract; scp, superior cerebellar peduncle; SPTg, subpeduncular tegmental nucleus; ts, tectospinal tract; VTg, ventral tegmental nucleus; xscp, decussation of superior cerebellar peduncle. Scale bar, 600 μm.

Data analysis of expression of PKA subunits and effect of Rp-cAMPS on variation in sleep–wake cycle.

To determine the effects of REM sleep manipulations on changes in the sleep–wake data and expression of PKA subunits, the polygraphic measures of the rats (n = 18) were analyzed to calculate the total percentages of time spent in W, SWS, and REM sleep in two different recording sessions: during the last adaptation recording session (between 9:00 A.M. and 2:00 P.M.) and during the last 2 h of the experimental recording session (between 12:00 P.M. and 2:00 P.M.), before they were killed to collect brain tissues. These sleep–wake data were subjected to one-factor ANOVAs and post hoc Scheffé's F tests to compare the total percentages of time spent in W, SWS, and REM sleep among the BR, RD, and RR groups (StatView statistical software; Abacus Concepts, Berkeley, CA). For the PKA subunit data, one-factor ANOVAs and post hoc Tukey's tests were used to compare the levels of PKA_Cα, PKA_RI, PKA_RIIα, PKA_RIIβ, and actin in the PPT, mPRF, mPFC, and AHTh among BR, RD, and RR groups using SigmaStat statistical software (Systat Software, Point Richmond, CA).

For the purpose of determining possible effects of Rp-cAMPS on sleep and wakefulness, the polygraphic measures provided the following dependent variables, which were quantified for each recording session: (1) percentage of recording time spent in W, SWS, and REM sleep; (2) latency to onset of the first episode of REM sleep after the onset of recordings; (3) total number of REM sleep episodes; and (4) mean duration of REM sleep episodes. The effects of the five different treatments [(1) control saline, (2) 0.28 nmol of Rp-cAMPS, (3) 0.55 nmol of Rp-cAMPS, (4) 1.1 nmol of Rp-cAMPS, and (5) 2.2 nmol of Rp-cAMPS] on the percentages of W, SWS, and REM sleep were statistically analyzed using a two-way ANOVA with time as a repeated-measure variable (six levels corresponding to six 1 h epochs after injections) and treatment as a between-subjects variable (five levels corresponding to the five different treatments). After a two-way ANOVA, post hoc Scheffé's F tests were done to determine the individual levels of significant difference between the control (saline) and the four different doses of Rp-cAMPS treatment protocols at six individual data points. The latency, number, and duration of REM sleep episodes were analyzed using a one-way ANOVA followed by post hoc Scheffé's F tests. Statistical analyses (two-way ANOVA, one-way ANOVA, and Scheffé's F test) were performed using StatView statistical software (Abacus Concepts).