Abstract
Study Objective:
Caffeine, an adenosine A1 and A2a receptor antagonist, is a widely consumed stimulant and also used for the treatment of hypersomnia; however, the wake-promoting potency of caffeine is often not strong enough, and high doses may induce side effects. Caffeine is metabolized to paraxanthine, theobromine, and theophylline. Paraxanthine is a central nervous stimulant and exhibits higher potency at A1 and A2 receptors, but has lower toxicity and lesser anxiogenic effects than caffeine.
Design:
We evaluated the wake-promoting efficacy of paraxanthine, caffeine, and a reference wake-promoting compound, modafinil, in a mice model of narcolepsy, a prototypical disease model of hypersomnia. Orexin/ataxin-3 transgenic (TG) and wild-type (WT) mice were subjected to oral administration (at ZT 2 and ZT14) of 3 doses of paraxanthine, caffeine, modafinil, or vehicle.
Results:
Paraxanthine, caffeine, and modafinil significantly promoted wakefulness in both WT and narcoleptic TG mice and proportionally reduced NREM and REM sleep in both genotypes. The wake-promoting potency of 100 mg/kg p.o. of paraxanthine during the light period administration roughly corresponds to that of 200 mg/kg p.o. of modafinil. The wake-promoting potency of paraxanthine is greater and longer lasting than that of the equimolar concentration of caffeine, when the drugs were administered during the light period. The wake-promotion by paraxanthine, caffeine, and modafinil are associated with an increase in locomotor activity and body temperature. However, the higher doses of caffeine and modafinil, but not paraxanthine, induced hypothermia and reduced locomotor activity, thereby confirming the lower toxicity of paraxanthine. Behavioral evaluations of anxiety levels in WT mice revealed that paraxanthine induced less anxiety than caffeine did.
Conclusions:
Because it is also reported to provide neuroprotection, paraxanthine may be a better wake-promoting agent for hypersomnia associated with neurodegenerative diseases.
Citation:
Okuro M; Fujiki N; Kotorii N; Ishimaru Y; Sokoloff P; Nishino S. Effects of paraxanthine and caffeine on sleep, locomotor activity, and body temperature in orexin/ataxin-3 transgenic narcoleptic mice. SLEEP 2010;33(7):930-942.
Keywords: Narcolepsy, caffeine, paraxanthine, cataplexy, modafinil, mice
CAFFEINE, IN THE FORM OF CAFFEINE-CONTAINING BEVERAGES OR OVER-THE-COUNTER CAFFEINE TABLETS (100 MG OR 200 MG), IS A WIDELY USED WAKE-promoting substance, used to reduce sleepiness in normal people.1,2 Caffeine is also widely used for patients with primary (such as narcolepsy) and secondary hypersomnia (hypersomnia associated with neurological diseases); these patients self-medicate with caffeine before they visit the sleep clinic to receive more potent medication.2
Caffeine is a non-selective antagonist for adenosine receptors (A1 and A2a receptors), and blockade of these receptors is believed to mediate its stimulant effects.1 One of the limitations of caffeine for treating hypersomnia is its low wake-promoting potency, while high doses may induce side effects such as anxiety, tremors, headache, and gastrointestinal irritation.
Caffeine is demethylated to 3 active metabolites, paraxanthine, theobromine, and theophylline, and C-8 hydroxylated to 1,3,7-trimethyluric acid (TMU).3–5 The interspecies variations in caffeine metabolism, related to cytochrome P4501A enzyme, exist; and the percentages of the production of these 4 metabolites are reported to be 59.9%, 8.7%, 5.0%, and 26.4% in humans, and 19.1%, 10.8%, 21.7%, and 47.8% in mice.5
Like caffeine, paraxanthine is a central nervous stimulant. In addition, caffeine and paraxanthine have similar anti-adenosine actions, though the literature has consistently reported that paraxanthine exhibits slightly higher binding potencies for adenosine A1 and A2a receptors6 and both lower toxicity and lesser anxiogenic effects than caffeine.7 This may be explained by the pharmacological properties of other caffeine metabolites, such as theophylline, which often induce nausea, diarrhea, tachycardia, and arrhythmias.8 A recent study demonstrated that paraxanthine provides protection against dopaminergic cell death via ryanodine receptor stimulation.9 Therefore, paraxanthine may be more effective than caffeine for wake-promotion in normal people and treatment of hypersomnia in those with cardiovascular and neurological diseases.
Therefore, we evaluated the wake-promoting efficacy of paraxanthine in wild-type (WT) and narcoleptic mice models and compared it with those of caffeine and modafinil. Effects of these compounds on locomotor activity, core body temperature and anxiety levels were also compared. Narcoleptic mice were included since narcolepsy is the prototypical primary hypersomnia, and orexin/ataxin-3 narcoleptic mice share the major pathophysiology of human narcolepsy (hypocretin cell ablation).10,11 As such, the observed efficacy in narcoleptic mice is an accurate predictor of efficacy for treatment of pathological sleepiness.
MATERIAL AND METHODS
Animals
Eight orexin/ataxin-3 transgenic (TG) narcoleptic mice from the congenic line (N9, backcrossed to C57BL/6) and 8 respective WT littermates were used. Each group consisted of 4 males and 4 females, 168-323 days old (mean age ± SEM, 243 ± 13 days) (n = 8; mean body weight, 28.9 ± 0.7 g), littermate WT mice (n = 8; mean body weight, 27.3 ± 0.5 g). In a separate session, effects of pharmacological compounds on anxiety levels were evaluated in 10 C57BL/6 WT mice (n = 10; 5 males and 5 females; mean age, 223 ± 18 days; mean body weight, 29.8 ± 0.6 g) with the marble burying test and elevated plus maze.12–15 The entire study was approved by and conducted in accordance with the guidelines of Stanford's Administrative Panel on Laboratory Animal Care.
Headstage and Telemetry Implant Surgery
The mice were surgically prepared for electroencephalogram (EEG) and electromyogram (EMG) recordings with a headstage attached to a cable recorder.11 Under 3% isoflurane anesthesia, 2 of 4 electrodes for the EEG (stainless steel screws) were screwed into the skull 1.5 mm lateral and 1.5 mm anterior to the bregma (over the motor cortex); the other 2 were screwed 3 mm lateral and 1 mm anterior to the lambda (over the visual cortex), and 2 EMG electrodes (multistranded stainless steel wires) were inserted into the neck extensor muscle. The 6 leads for these electrodes were attached to one 2 × 3 pin header that was secured to the skull using dental acrylic.
In order to evaluate the effects of drug administrations on the locomotor activity and core body temperature, a telemetry-implanting device (G2 E-Mitter, Mini Mitter, OR) was implanted in the abdominal cavity of each mouse.
After surgery, surgical wounds, animal behaviors, and body weight were monitored, with analgesic (carprofen) and antibiotic (enrofloxacin) supplied as needed.
Data Collection
After 2 weeks of recovery period from the surgery, the mice were moved to specially modified Nalgene microisolator cages equipped with a low-torque slip-ring commutator (Biella Engineering, Irvine CA), and the cages were placed in the recording chamber. The headstage of the animal was connected to a slip-ring commutator through 15 to 20 cm of light-weight 6-strand shielded signal cable (NMUF6/30-4046SJ; Cooner Wire, Chatsworth, CA). The commutator output was connected to the amplifier. The animals were allowed full freedom of movement in the recording cages. The EEG–EMG signals were acquired using Grass Instruments (West Warwick, RI) model 12 amplifiers. The EEG and EMG signals, digitally filtered (30 Hz Low Pass Filter for EEG; 10–100 Hz Band Pass Filter for EMG), were captured at 128 Hz using a sleep recording system (Vital Recorder; Kissei Comtec, Matsumoto, Japan). EEG signals collected with ipsilateral bipolar EEG electrodes placed over motor and visual cortices together with the bipolar EMG signals were used for sleep scoring. Each mouse was housed in its own individual recording cage. Room temperature was maintained at 24 ± 1°C throughout experimentation. The cages were housed in custom-designed stainless steel cabinets with individual ventilated compartments. Food and water were available ad libitum. A 24-h light-dark cycle (12 h lights on, 12 h off) was maintained throughout the study (lights on at zeitgeber time [ZT] = 0 at 07:00 am).
Drug Administrations
In order to evaluate the pharmacological effects of paraxanthine, caffeine, and modafinil, we administered the drugs during both light and dark periods; effects on sleep, locomotor activity, and temperature were monitored. A stomach sonde (KN-348, Natsume Seisakusho, Japan) was attached to a 1 mL disposable syringe, and all drug solutions were adjusted to 0.3 mL/30g of body weight. Before initiating drug sessions, each mouse was acclimated to oral drug administration with the stomach sonde, and the vehicle session was repeated at least 3 times in each animal.
Paraxanthine was obtained from Pierre Fabre Research Institute, (Castres, France), and caffeine was purchased from Sigma-Aldrich (MO, USA). Modafinil was obtained from Laboratore L. Lafon (Maisons-Alfort, France). Three drug doses (plus vehicle) of each compound (6.25 mg/kg. 25 mg/kg, and 100 mg/kg p.o. for paraxanthine; 5.8 mg/kg, 23.2 mg/kg, and 92.8 mg/kg p.o. for caffeine [equimolar for paraxanthine doses]; 50 mg/kg, 100 mg/kg, and 200 mg/kg p.o. for modafinil) were administered to each animal at ZT 2 and ZT 14. Sleep data for the 6 h following administrations were captured and analyzed. In addition, cumulative wake amounts after the highest dose and the respective vehicle administrations of each compound were calculated for 24 h after the drug administrations to evaluate the cumulative wake surplus (over the vehicle sessions). The vehicle for paraxanthine and caffeine administrations was saline, while 10% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, Natick, MA) in saline was used for the modafinil administrations. We used the cross-over design for caffeine and paraxanthine administrations, and each session included one independent vehicle session. The caffeine session was initiated in half of the mice (both TG and WT mice), while the other half received paraxanthine; the dark period administrations were carried out after finishing light period experiments. In each session, the order of the drug doses was randomized. The modafinil session was initiated after the caffeine and paraxanthine sessions, and the order of modafinil doses was randomized in each light and dark period experiment.
The administrations at ZT 2 were done with lights on, while those at ZT 14 were done under a dim red light. The same mice used for the sleep studies were used in all drug studies, and a minimum 7 days of drug washout was employed between 2 consecutive drug administrations.
Sleep Scoring
The sleep stage of each 10-sec epoch was visually scored using our standard criteria, with 50% or more of a particular state in each epoch required to score the epoch.11 Briefly, wakefulness (Wake) is characterized by a desynchronized, low-amplitude, mixed-frequency (> 4 Hz) EEG and high EMG activity. Rhythmic theta/alpha (7-9 Hz) waves with high EMG activities may also appear. NREM is characterized by a synchronized, high-amplitude, low-frequency (0.25-4 Hz) EEG and reduced EMG activity compared to wakefulness. EEG activity in REM sleep is similar to that in wakefulness with desynchronized, mixed-frequency, low-amplitude waves. EMG activity during REM sleep is reduced even further than during NREM sleep and is completely absent in many cases. Some muscle twitching may be apparent in the EMG trace during REM sleep. During the REM sleep, rhythmic theta/alpha (7-9 Hz) waves with reduced EMG activity may be dominant, but not always, as this has been shown in other species.16,17 In addition, the number of direct transitions from wakefulness to REM sleep (DREM) was determined during the dark period. In our scoring criteria for DREM, four preceding 10-sec epochs must be wake; this criterion gave best specificity and sensitivity for detecting abnormal REM sleep transitions specific for narcoleptic mice.11 Sleep state changes were recorded when at least one 10-sec epoch was scored as having a different sleep stage; state episode length was defined as length of continuous single state episode. If the polygraph signals of some mice were not sufficient to score sleep stage with accuracy (especially poor EMG), data from these animals were excluded and a minimum of 6 animals were included for data analysis.
Polygraphic Data Analysis
Cumulative amount of each vigilance state (Wake, NREM, and REM [and DREM for the dark period]) was calculated and plotted for 6 h with a 10-min interval after drug administrations during the light and dark periods. The number of episodes of each vigilance state during 6 h after the drug administrations during the light period and dark period was also assessed. The mean episode duration in each vigilance state during 6 h after the drug administrations was calculated by dividing the total time spent in each vigilance state by the number of episodes.
To evaluate the recovery from drug induced wakefulness, we also calculated the cumulative wake surplus with a 1-h interval, defined as cumulative wake time (sec) minus the corresponding mean vehicle cumulative wake time (sec) value at a given time point for 24 h after the drug administrations.
Locomotor and Core Temperature Data Analysis
Locomotor and temperature data were acquired with the telemetry receiver (Series 4000, Mini Mitter, OR) and the Vital View software (Mini Mitter, OR). Each telemetry receiver was calibrated for temperature measure before implantation. These telemetric implants were not operated with a battery, and no battery replacement or additional calibration was required during the study. Locomotor activity counts and temperature of each mouse were calculated in each 10-min time bin, respectively, and the mean (± SEM) values of locomotor counts (counts/10 min) and temperature (°C) after each drug administration were plotted from 1 h before to 6 h after the drug administrations.
Anxiety Levels
The effects of paraxanthine (100 mg/kg, p.o.), caffeine (92.8 mg/kg, p.o.), and saline on anxiety levels were evaluated during light period. The drug administration was done at ZT 2; the marble burring test was carried out 90 min after the drug administration, followed by the elevated plus maze. The order of the drug administration was randomized.
(a) Marble burying test: Marble burying was measured by placing a single mouse in a polypropylene cage (22 cm × 16 cm × 12 cm) containing 12 glass marbles (1.5 cm diameter) evenly spaced on 3 cm deep sawdust. No food or water was present during the observation period. Mice have a tendency to bury objects, such as glass marbles, present in their environment. The cage was covered with a metal grid, and the number of marbles at least two-thirds covered was counted after 30 min. The number of marbles covered during the test has been shown to be an index of increased anxiety behavior.12,13
(b) Elevated plus maze: mice were placed in the center of an apparatus consisting of 2 opposing runways. One of the runways had closed arms (6 cm × 30 cm) of the maze, with walls 15 cm high. The other runway consisted of open arms (7 cm × 30 cm) of the maze, with no walls. The maze was elevated to 70 cm above the floor. Behavior of the mice in the maze was monitored for 5 min. The number of entries and time spent in the open arms was recorded. Increased time spent in closed arms has been shown to be an index of increased anxiety behavior.13–15
Statistics
Statistical significances of the effects on sleep parameters (cumulative total time and mean duration of each sleep stage), locomotor activity, and core body temperature were evaluated using multi-way analysis of variance (ANOVA) using SYSTAT 11 (Systat Software, Inc, Chicago, IL), with genotype, period (dark and light), and treatment (dose) as the independent variables. Cumulative total counts for the locomotor activity and mean values for the core body temperature during 6 h post drug administrations were considered for the statistical assessment.
Statistics were carried out on the data for 0-360 minutes, and P < 0.05 (2-tailed) was considered as the level of statistical significance.
The effects of the compounds on anxiety levels were analyzed using one-way ANOVA and a Tukey post hoc test.
RESULTS
Effects at Light (Resting) Period
Sleep stage analysis
We did not observe any difference in drug effects between male and female mice, and thus the pooled data of both sexes are presented. We observed potent wake-promoting effects of paraxanthine (6.25-100 mg/kg, p.o.) in both WT and TG narcoleptic mice during the light period (Figure 1). The low (6.25 mg/kg) dose had a small wake-promoting effect (P < 0.05 for WT mice, P < 0.05 for narcoleptic mice at 6-h time points), whereas the middle and high doses potently increased the cumulative amount of wakefulness (P < 0.001 for both WT and narcoleptic mice) (Figure 1, Table 1). Paraxanthine significantly reduced NREM sleep (dose effect; P < 0.001) and REM sleep (dose effect; P < 0.001), and the highest does of paraxanthine almost completely prevented sleep over 4 h in WT mice. Paraxanthine significantly reduced NREM sleep (dose effect; P < 0.001) in TG mice, but the reduction of REM sleep did not reach a significant level in TG mice. Both WT and TG narcoleptic mice responded well to paraxanthine, though slightly smaller effects (as seen in smaller percent changes from the baseline) were observed for all doses in narcoleptic mice compared to those in WT mice (Table 1).
Figure 1.
Effects of paraxanthine, caffeine and modafinil on wake/sleep amounts during the light-on period (0-6 h post-dosing) in wild-type (WT) and narcoleptic (TG) mice. Three drug doses and respective vehicles were orally administered at ZT 2, and sleep parameters were monitored for 6 h after the drug administration. Cumulative amounts (second) of wake, NREM, and REM sleep are displayed. Paraxanthine; dose effect (P < 0.001), genotype effect (P = 0.121), dose × genotype interaction (P = 0.108). Caffeine; dose effect (P < 0.001), genotype effect (P = 0.157), dose × genotype interaction (P = 0.288). Modafinil; dose effect (P < 0.001), genotype effect (P = 0.552), dose × genotype interaction (P = 0.634).
Table 1.
Effects of paraxanthine, caffeine, and modafinil on total wake, NREM, and REM sleep amounts and during the light-on period in wild-type (WT) and narcoleptic (TG) mice
| PARAXANTHINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 9040 ± 1142 | 8701 ± 390 |
| 6.25 | 10108 ± 632 (+11.8%)* | 9383 ± 361 (+7.8%) | |
| 25 | 12989 ± 377 (+43.7%)*** | 11423 ± 377 (+31.3%)* | |
| 100 | 18693 ± 1037 (+106.8%)*** | 16044 ± 377 (+84.4%)*** | |
| NREM | Vehicle | 11873 ± 1029 | 12136 ± 447 |
| 6.25 | 10500 ± 597 (−11.6%) | 11210 ± 415 (−7.6%) | |
| 25 | 8111 ± 391 (−31.7%) | 9390 ± 391 (−22.6%)* | |
| 100 | 2685 ± 874 (−77.4%)*** | 4934 ± 391 (-59.3%)*** | |
| REM | Vehicle | 1043 ± 220 | 684 ± 112 |
| 6.25 | 993 ± 141 (−4.9%) | 859 ± 106 (+25.5%) | |
| 25 | 744 ± 126 (−28.7%) | 701 ± 126 (+2.5%) | |
| 100 | 208 ± 118 (−80.0%)** | 421 ± 126 (−38.4%) | |
| CAFFEINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 11518 ± 601 | 10834 ± 346 |
| 5.8 | 15468 ± 227 (+34.3%)*** | 13961 ± 521 (+28.9%)** | |
| 23.2 | 15950 ± 209 (+38.5%)*** | 14531 ± 209 (+34.1%)*** | |
| 92.8 | 18924 ± 553 (+64.3%)*** | 17722 ± 553 (+63.6%)*** | |
| NREM | Vehicle | 9697 ± 553 | 10383 ± 329 # |
| 5.8 | 5867 ± 251 (−39.5%)** | 7324 ± 566 (−29.5%)** | |
| 23.2 | 5428 ± 192 (−44.0%)** | 6748 ± 523 (−35.0%)*** | |
| 92.8 | 2616 ± 540 (−73.0%)*** | 3776 ± 470 (−63.6%)*** | |
| REM | Vehicle | 385 ± 126 | 384 ± 94 |
| 5.8 | 265 ± 84 (−31.2%) | 314 ± 77 (−18.2%) | |
| 23.2 | 222 ± 56 (−42.4%)* | 321 ± 85 (−16.3%) | |
| 92.8 | 60 ± 25 (−84.4%)* | 102 ± 42 (−73.4%)* | |
| MODAFINIL | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 9110 ± 601 | 8553 ± 356 |
| 50 | 12948 ± 467 (+42.1%)*** | 11960 ± 191 (+39.0%)** | |
| 100 | 14928 ± 1197 (+63.9%)*** | 14523 ± 1197 (+70.0%)*** | |
| 200 | 17982 ± 1260 (+97.4%)*** | 16923 ± 1197 (+98.0%)*** | |
| NREM | Vehicle | 11736 ± 481 | 12473 ± 374 |
| 50 | 8180 ± 473 (−12.3%) | 9016 ± 169 (−27.7%)** | |
| 100 | 6345 ± 1040 (−33.2%)* | 6763 ± 1040 (−45.8%)*** | |
| 200 | 3513 ± 1232 (−62.3%)*** | 4550 ± 1040 (−63.5%)*** | |
| REM | Vehicle | 688 ± 98 | 567 ± 120 |
| 50 | 472 ± 44 (−27.9%) | 624 ± 81 (+10.1%) | |
| 100 | 418 ± 167 (−33.0%) | 314 ± 167 (−44.6%) | |
| 200 | 105 ± 43 (−83.9%)** | 127 ± 167 (−77.6%)** | |
Three drug doses and respective vehicles were orally administered at ZT 2, and sleep parameters were monitored for 6 h after the drug administration. Statistical significance for each parameter between doses (*P < 0.05, **P < 0.01, ***P < 0.001) and between genotypes (#P < 0.05) using multi-way ANOVA.
Equimolar doses of caffeine (5.8-92.8 mg/kg p.o.) also showed dose-dependent wake promotion in both WT and TG narcoleptic mice (dose effect; P < 0.001) (Figure 1). Caffeine significantly reduced NREM sleep (dose effect; P < 0.001) and REM sleep (dose effect; P < 0.001), and the highest doses almost completely prevented sleep over 3 h in WT mice and 2 h in TG narcoleptic mice. Both WT and narcoleptic mice responded well to caffeine, though slightly smaller effects were observed for all doses in narcoleptic mice compared to those in WT mice (Table 1). However, these differences were of lower magnitude than those seen after paraxanthine administration.
As a reference, we also evaluated the wake-promoting effects of modafinil in the same mice during the light period (Figure 1, Table 1). Modafinil (50, 100, and 200 mg/kg, p.o.) showed dose-dependent wake promotion in both WT and TG narcoleptic mice (dose effect: P < 0.001). Modafinil also significantly reduced NREM sleep (dose effect: P < 0.001) and REM sleep in both genotypes (dose effect: P < 0.001). Responses to modafinil were almost identical for WT and TG narcoleptic mice. The wake-promoting potency of modafinil at 200 mg/kg p.o. roughly corresponded to that of paraxanthine at 100 mg/kg p.o. and was slightly more potent than that of caffeine at 92.8 mg/kg p.o.
Wake-promoting effects of all 3 compounds were associated with an increase in mean duration of wake and a decrease in mean duration of NREM and REM sleep, typically after the highest dose drug administration; but statistical significance was observed only for some drug doses (Table 2)
Table 2.
Effects of paraxanthine, caffeine, and modafinil on mean epoch duration of wake, NREM, and REM sleep amounts and during the light-on periods in wild-type (WT) and narcoleptic (TG) mice
| PARAXANTHINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 75.7 ± 8.7 | 90.9 ± 10.5 |
| 6.25 | 80.3 ± 15.3 | 107.1 ± 8.9 | |
| 25 | 87.6 ± 13.5 | 111.4 ± 21.0 | |
| 100 | 124.6 ± 42.2 | 205.0 ± 81.6* | |
| NREM | Vehicle | 145.9 ± 17.0 | 136.3 ± 14.2 |
| 6.25 | 135.9 ± 9.9 | 122.7 ± 9.8 | |
| 25 | 103.5 ± 9.1 | 115.4 ± 8.6 | |
| 100 | 95.0 ± 8.51** | 99.2 ± 15.4* | |
| REM | Vehicle | 75.4 ± 10.4 | 68.3 ± 9.5 |
| 6.25 | 80.6 ± 4.7 | 67.9 ± 14.6 | |
| 25 | 65.9 ± 12.0 | 51.1 ± 4.1 | |
| 100 | 54.6 ± 28.0 | 54.7 ± 8.6 | |
| CAFFEINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 78.0 ± 6.5 | 73.9 ± 5.5 |
| 5.8 | 128.5 ± 10.8 | 105.7 ± 12.7 | |
| 23.2 | 111.3 ± 8.1 | 112.5 ± 7.9 | |
| 92.8 | 212.8 ± 59.7*** | 134.0 ± 14.2* ## | |
| NREM | Vehicle | 73.5 ± 7.2 | 81.5 ± 7.8 # |
| 5.8 | 64.3 ± 4.4 | 62.7 ± 5.2 | |
| 23.2 | 66.3 ± 2.6 | 73.8 ± 5.5 | |
| 92.8 | 51.8 ± 9.9 | 45.2 ± 6.2** | |
| REM | Vehicle | 56.5 ± 4.0 | 31.5 ± 9.7 |
| 5.8 | 61.3 ± 15.0 | 51.6 ± 7.0 | |
| 23.2 | 66.3 ± 19.5 | 64.6 ± 11.3 | |
| 92.8 | 31.0 ± 12.3 | 40.8 ± 14.5 | |
| MODAFINIL | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 81.2 ± 8.5 | 83.7 ± 4.9 |
| 50 | 107.0 ± 20.2 | 84.6 ± 6.2 | |
| 100 | 86.2 ± 9.0 | 89.7 ± 6.5 | |
| 200 | 241.7 ± 80.5** | 309.8 ± 61.4*** | |
| NREM | Vehicle | 131.2 ± 8.3 | 141.7 ± 10.7 |
| 50 | 108.8 ± 16.7 | 97.4 ± 6.6 | |
| 100 | 92.8 ± 7.7 | 94.3 ± 9.6 | |
| 200 | 142.3 ± 45.2 | 92.0 ± 19.6 | |
| REM | Vehicle | 68.7 ± 8.1 | 63.1 ± 7.5 |
| 50 | 76.8 ± 7.5 | 71.0 ± 6.4 | |
| 100 | 65.7 ± 3.1 | 52.9 ± 3.9 | |
| 200 | 41.3 ± 16.6* | 27.0 ± 14.5** | |
Three drug doses and respective vehicles were orally administered at ZT 2, and sleep parameters were monitored for 6 h after the drug administration. Statistical significance for each parameter between doses (*P < 0.05, **P < 0.01, ***P < 0.001) and between genotypes (#P < 0.05, ##P < 0.01) using multi-way ANOVA.
Effects on locomotor activity and core temperature
The wake-promoting effects of paraxanthine, especially at high dose, were associated with increases in locomotor activity in both WT and TG narcoleptic mice (dose effect; P < 0.002). Paraxanthine significantly elevated body temperature in both WT and narcoleptic mice (dose effect; P < 0.001). Locomotor and temperature effects observed after paraxanthine administrations were smaller in TG mice, and the P-values of genotype effects were P = 0.109 (nonsignificant) for locomotor activity and P = 0.026 (significant) for temperature.
Effects of caffeine on locomotor activity and core body temperature were different from those observed after paraxanthine administration, especially for the highest dose administration. Although the middle dose of caffeine increased locomotor activity and temperature, the highest dose of caffeine did not increase locomotor activity (for initial 3 h for WT and over 6 h for TG). More strikingly, the highest dose of caffeine did not immediately elevate temperature in WT mice and reduced temperature in TG narcoleptic mice for 3 h. The narcoleptic mice were more sensitive to caffeine in exhibiting this paradoxical temperature change.
Modafinil also significantly enhanced the locomotor activity in both WT and TG narcoleptic mice (P = 0.017) (Figure 2). It was noted that much larger locomotor enhancement was seen in WT mice, and the genotype effect was significant (P < 0.001). The effect on locomotor activity after the middle dose was greater than that of the highest dose in TG mice.
Figure 2.
Effects of paraxanthine, caffeine and modafinil on locomotor activity and body temperature during the light-on period (0-6 h post-dosing) in wild-type (WT) and narcoleptic (TG) mice. Three drug doses and respective vehicles were orally administered at ZT 2, and locomotor counts and body temperature were monitored for 6 h after the drug administration. Locomotor activity: Paraxanthine; dose effect (P < 0.01), genotype effect (P = 0.109), dose × genotype interaction (P = 0.474). Caffeine; dose effect (P < 0.01), genotype effect (P = 0.403), dose × genotype interaction (P = 0.310). Modafinil; dose effect (P < 0.05), genotype effect (P < 0.001), dose × genotype interaction (P < 0.05). Body temperature: Paraxanthine; dose effect (P < 0.001), genotype effect (P < 0.05), dose x genotype interaction (P = 0.793). Caffeine; dose effect (P < 0.05), genotype effect (P = 0.174), dose × genotype interaction (P = 0.083). Modafinil; dose effect (P = 0.064), genotype effect (P = 0.757), dose × genotype interaction (P = 0.853).
Modafinil also elevated body temperature in both WT and narcoleptic mice. The magnitude of temperature elevation after the highest dose administration was smaller than that of the middle dose. This suggested that a higher dose of modafinil may produce hypothermic effects and that narcoleptic mice may be more susceptible to exhibiting this response, thereby masking locomotor activation.
Effects at Dark (Active) Period
Sleep stage analysis
The same experiment was repeated by administering compounds during the active period (Figures 3 and 4, Table 3). Similar to the effects observed during the light period, paraxanthine showed dose-dependent increased wakefulness (dose effect; P < 0.001) and decreased both NREM (dose effect; P < 0.001) and REM sleep (dose effect; P < 0.001) in both WT and TG narcoleptic mice during the dark period. During active periods, narcoleptic mice spent less time in wakefulness and more time in NREM and REM sleep. However, both genotypic classes demonstrated similar levels of response to paraxanthine. It should also be noted that low drug doses of paraxanthine (6.25 mg/kg) given to narcoleptic animals brought the wake and NREM amounts to the levels of WT mice (Figure 3).
Figure 3.
Effects of paraxanthine, caffeine and modafinil on wake/sleep amounts during the dark period (0-6 h post-dosing) in wild-type (WT) and narcoleptic (TG) mice. Three drug doses and respective vehicles were orally administered at ZT 14, and sleep parameters were monitored for 6 h after the drug administration. Cumulative amounts (sec) of wake, NREM, and REM sleep are displayed. Paraxanthine; dose effect (P < 0.001), genotype effect (P = 0.094), dose x genotype interaction (P = 0.463). Caffeine; dose effect (P < 0.001), genotype effect (P < 0.001), dose × genotype interaction (P = 0.902). Modafinil; dose effect (P < 0.001), genotype effect (P < 0.001), dose × genotype interaction (P = 0.279).
Figure 4.
Effects of paraxanthine, caffeine and modafinil on locomotor activity and body temperature during the dark period (0-6 h post-dosing) in wild-type (WT) and narcoleptic (TG) mice. Three drug doses and respective vehicles were orally administered at ZT 14, and locomotor counts and body temperature were monitored for 6 h after the drug administration. Locomotor activity: Paraxanthine; dose effect (P < 0.01), genotype effect (P = 0.520), dose × genotype interaction (P = 0.562). Caffeine; dose effect (P < 0.05), genotype effect (P < 0.05), dose × genotype interaction (P = 0.951). Modafinil; dose effect (P < 0.001), genotype effect (P < 0.01), dose × genotype interaction (P = 0.183). Body temperature: Paraxanthine; dose effect (P = 0.067), genotype effect (P < 0.05), dose × genotype interaction (P = 0.595). Caffeine; dose effect (P < 0.001), genotype effect (P = 0.829), dose × genotype interaction (P = 0.963), Modafinil; dose effect (P = 0.630), genotype effect (P = 0.311), dose × genotype interaction (P = 0.907).
Table 3.
Effects of paraxanthine, caffeine, and modafinil on total wake, NREM, and REM sleep amounts during the dark period in wild-type (WT) and narcoleptic (TG) mice
| PARAXANTHINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 13787 ± 463 | 12148 ± 418 |
| 6.25 | 15149 ± 550 (+28.2%)* | 12830 ± 452 (+5.6%) | |
| 25 | 16914 ± 606 (+43.1%)** | 16173 ± 606 (+33.1%)** | |
| 100 | 19230 ± 778 (+62.7%)*** | 16930 ± 606 (+39.4%)** | |
| NREM | Vehicle | 7588 ± 422 | 8590 ± 363 |
| 6.25 | 6396 ± 545 (−1.7%) | 8238 ± 427 (−4.1%) | |
| 25 | 4589 ± 586 (−29.5%) | 4947 ± 586 (−42.4%)** | |
| 100 | 2321 ± 776 (−64.3%)*** | 4382 ± 586 (−49.0%)** | |
| REM | Vehicle | 225 ± 93 | 738 ± 68 |
| 6.25 | 56 ± 18 (−71.1%) | 517 ± 52 (−30.0%)* ### | |
| 25 | 97 ± 22 (−49.6%) | 435 ± 22 (−41.1%)** ### | |
| 100 | 49 ± 29 (−74.8%) | 240 ± 22 (−67.5%)*** # | |
| DREM | Vehicle | n.d. | 124 ± 55 |
| 6.25 | n.d. | 15 ± 15 (−87.9%) | |
| 25 | n.d. | 45 ± 0 (−63.7%) # | |
| 100 | n.d. | 48 ± 0 (−61%) # | |
| CAFFEINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 12303 ± 747 | 11266 ± 420 |
| 5.8 | 15235 ± 447 (+23.8%)*** | 14163 ± 436 (+25.7%)*** | |
| 23.2 | 16852 ± 391 (+37.0%)*** | 15414 ± 391 (+36.8%)*** # | |
| 92.8 | 19410 ± 267 (+57.8%)*** | 17764 ± 391 (+57.7%)*** # | |
| NREM | Vehicle | 8955 ± 772 | 9714 ± 423 |
| 5.8 | 6125 ± 389 (−31.6%)*** | 7114 ± 421 (−26.8%)*** | |
| 23.2 | 4532 ± 397 (−49.4%)*** | 5679 ± 397 (−41.5%)*** | |
| 92.8 | 2184 ± 266 (−75.6%)*** | 3766 ± 397 (−61.2%)*** # | |
| REM | Vehicle | 342 ± 33 | 508 ± 107 |
| 5.8 | 240 ± 110 (−29.8%) | 311 ± 66 (−38.6%) | |
| 23.2 | 216 ± 98 (−36.8%) | 489 ± 98 (−3.7%) # | |
| 92.8 | 6 ± 6 (−98.2%)* | 52 ± 98 (−89.8%)** | |
| DREM | Vehicle | n.d. | 113 ± 55 |
| 5.8 | n.d. | 11 ± 11 (−89.8%)** | |
| 23.2 | n.d. | 19 ± 0 (−83.3%)** | |
| 92.8 | n.d. | 18 ± 0 (−84.0%)* | |
| MODAFINIL | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 14108 ± 258 | 11356 ± 479 |
| 50 | 17271 ± 746 (+42.8%)** | 13023 ± 163 (+14.7%)** ## | |
| 100 | 18139 ± 966 (+50.0%)*** | 15292 ± 966 (+34.7%)*** | |
| 200 | 18319 ± 913 (+51.5%)*** | 14132 ± 966 (+24.4%)*** ## | |
| NREM | Vehicle | 7437 ± 266 | 9557 ± 475 ## |
| 50 | 4310 ± 744 (−32.4%) | 7926 ± 132 (−17.1%) # | |
| 100 | 3361 ± 896 (−47.3%)** | 5903 ± 896 (−38.2%)** ## | |
| 200 | 3279 ± 913 (−48.6%)** | 6991 ± 896 (−26.8%)* | |
| REM | Vehicle | 35 ± 108 | 566 ± 108 |
| 50 | 10 ± 143 (−60.6%) | 501 ± 143 (−11.4%) ### | |
| 100 | 86 ± 86 (+112.1%) | 297 ± 86 (−47.6%)* | |
| 200 | 2 ± 86 (−93.9%) | 391 ± 86 (−30.8%) ## | |
| DREM | Vehicle | n.d. | 121 ± 24 |
| 50 | n.d. | 150 ± 30 (+24%) # | |
| 100 | n.d. | 108 ± 22 (−10.7%) | |
| 200 | n.d. | 85 ± 17 (−29.8%) | |
Three drug doses and respective vehicles were orally administered at ZT 14, and sleep parameters were monitored for 6 h after the drug administration. Statistical significance for each parameter between doses (*P < 0.05, **P < 0.01, ***P < 0.001) and between genotypes (#P < 0.05, ##P < 0.01, ###P < 0.001) using multi-way ANOVA.
Effects of caffeine were similar to paraxanthine, increasing wakefulness in dose-dependent fashion (dose effect; P < 0.001) and decreasing both NREM (dose effect; P < 0.001) and REM sleep (dose effect; P < 0.002) in both WT and TG narcoleptic mice during the dark period.
Modafinil also significantly enhanced wakefulness (dose effect; P < 0.001), and reduced NREM (dose effect; P < 0.002), but induced nonsignificant reduction in REM sleep (dose effect; P = 0.50) in both WT and TG narcoleptic mice. However, the effects on wake and NREM were saturated at lower doses, and the higher dose of modafinil did not produce high efficacy.
We have recently shown that our DREM evaluation, especially during active periods, is an objective measure of abnormal transition of REM sleep, specific for the murine model of narcolepsy.11 We found that paraxanthine and caffeine significantly decreased DREM episodes during the active period (P < 0.05 for paraxanthine and P < 0.01 for caffeine), while modafinil did not have any noticeable effects on DREM (Figure 3).
Wake-promoting effects of all 3 compounds, especially for higher dose administration, were associated with an increase in mean duration of wake and a decrease in mean duration of NREM and REM sleep, but statistical significance was observed only for some drug doses (Table 4).
Table 4.
Effects of paraxanthine, caffeine, and modafinil on mean epoch duration of wake, NREM, and REM sleep amounts and during the dark periods in wild-type (WT) and narcoleptic (TG) mice
| PARAXANTHINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 164.7 ± 15.1 | 110.4 ± 8.8 # |
| 6.25 | 431.3 ± 191.2 | 136.4 ± 9.6 | |
| 25 | 130.1 ± 64.3 | 1844.6 ± 1495.9 | |
| 100 | 7725.1 ± 1984.8*** | 1201.3 ± 627.2 ### | |
| NREM | Vehicle | 112.0 ± 10.1 | 95.4 ± 8.5 |
| 6.25 | 94.4 ± 14.7 | 81.0 ± 5.8 | |
| 25 | 80.4 ± 12.9* | 55.0 ± 10.5** | |
| 100 | 3.1 ± 3.1*** | 33.0 ± 3.5*** # | |
| REM | Vehicle | 46.4 ± 13.7 | 91.1 ± 9.1 ## |
| 6.25 | 12.9 ± 12.6* | 74.0 ± 12.2 ## | |
| 25 | 0.0 ± 0** | 58.6 ± 16.7 ## | |
| 100 | 1.4 ± 1.4** | 33.3 ± 14.6** | |
| DREM | Vehicle | n.d. | 7.1 ± 7.1 |
| 6.25 | n.d. | 5.0 ± 5.0 | |
| 25 | n.d. | 0.0 ± 0.0 | |
| 100 | n.d. | 50.0 ± 19.3** ### | |
| CAFFEINE | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 158.2 ± 12.8 | 103.6 ± 4.8 |
| 5.8 | 114.3 ± 14.2 | 120.6 ± 4.1 | |
| 23.2 | 114.3 ± 14.2 | 120.6 ± 4.1 | |
| 92.8 | 204.6 ± 31.9*** | 139.0 ± 16.3 ## | |
| NREM | Vehicle | 89.0 ± 8.9 | 91.3 ± 7.9 |
| 5.8 | 71.3 ± 10.3 | 71.9 ± 6.8 | |
| 23.2 | 71.3 ± 10.3 | 71.9 ± 6.8 | |
| 92.8 | 40.4 ± 5.4*** | 36.4 ± 2.4*** | |
| REM | Vehicle | 63.5 ± 12.4 | 78.9 ± 8.4 |
| 5.8 | 90.7 ± 20.9 | 74.0 ± 14.3 | |
| 23.2 | 90.7 ± 20.9 | 74.0 ± 14.3 | |
| 92.8 | 6.0 ± 6.0* | 41.3 ± 15.1* | |
| DREM | Vehicle | n.d. | 27.5 ± 18.9 |
| 5.8 | n.d. | 11.4 ± 11.4 | |
| 23.2 | n.d. | 11.4 ± 11.4 | |
| 92.8 | n.d. | 9.0 ± 9.0 | |
| MODAFINIL | |||
|---|---|---|---|
| Dose (mg/kg) | WT | TG | |
| Wake | Vehicle | 191.1 ± 15.4 | 102.1 ± 8.5 # |
| 50 | 374.2 ± 126.0* | 120.4 ± 5.3 ## | |
| 100 | 363.7 ± 126.0 | 120.4 ± 5.3 | |
| 200 | 274.8 ± 60.7 | 166.0 ± 35.1 | |
| NREM | Vehicle | 126.3 ± 6.8 | 90.1 ± 5.1 # |
| 50 | 135.7 ± 19.8 | 86.9 ± 4.4 # | |
| 100 | 135.7 ± 19.8 | 86.9 ± 4.4 ## | |
| 200 | 129.2 ± 21.8 | 74.6 ± 12.3 # | |
| REM | Vehicle | 32.3 ± 14.0 | 65.1 ± 6.9 |
| 50 | 11.4 ± 5.8 | 57.3 ± 8.2 ### | |
| 100 | 11.4 ± 5.8 | 57.3 ± 8.2 # | |
| 200 | 3.3 ± 2.1 | 63.9 ± 7.1* ### | |
| DREM | Vehicle | n.d. | 24.3 ± 17.4 |
| 50 | n.d. | 30.0 ± 14.6 # | |
| 100 | n.d. | 30.0 ± 14.6 | |
| 200 | n.d. | 17.1 ± 9.2 | |
Three drug doses and respective vehicles were orally administered at ZT 14, and sleep parameters were monitored for 6 h after the drug administration. Statistical significance for each parameter between doses (*P < 0.05, **P < 0.01, ***P < 0.001) and between genotypes (#P < 0.05, ##P < 0.01, ###P < 0.001) using multi-way ANOVA.
Effects on locomotor activity and core temperature
We found that paraxanthine increased locomotor activity and temperature in dose-dependent fashion in WT mice. In TG narcoleptic mice, similar effects on locomotion and temperature were observed after low and middle doses of paraxanthine. Initial rises of locomotion and temperature were dampened after the high dose administration of paraxanthine, and dose dependency of locomotor activity was observed only 3 h after the drug administrations.
Caffeine also increased locomotor activity in both WT and TG narcoleptic mice. However, at the highest dose administration, the initial rise in locomotor activity was dampened in both WT and TG mice, the results consistent with the previously reported effects in WT mice.18 For caffeine, doses up to 23.5 mg/kg p.o. had little effect on body temperature, but the highest dose (92.8 mg/kg) significantly reduced body temperature both in WT and TG mice, and the maximum effect (more than −5°C) occurred about 2 h after the drug administration, with hypothermia lasting over 6 h.
Effects on anxiety levels
We found that caffeine (P = 0.035), but not paraxanthine (P = 0.98), significantly increased anxiety levels compared to the saline session, evaluated by marble burying test (Table 5). Similarly, caffeine significantly increased anxiety levels (P = 0.0013) evaluated by the plus maze test, while increase in anxiety levels by paraxanthine was only marginally significant (P = 0.06).
Table 5.
Effects of paraxanthine and caffeine on anxiety levels
| Marble burying test (number of objects hide) | Elevated plus maze (sec spent in closed arm) | |
|---|---|---|
| Saline | 1.3 ± 0.45 | 65.7 ± 5.0 |
| Paraxanthine | 1.5 ± 0.63 | 113.4 ± 5.9 |
| Caffeine | 4.2 ± 1.1* | 145.2 ± 23.3** |
| ANOVA | 0.023 ± 12.0 | 0.002 ± 4.1 |
P < 0.05,
P < 0.01.
Post hoc Tukey test vs. saline. P value for the difference between paraxanthine and caffeine sessions in marble burying test was 0.062, and that between saline and paraxanthine sessions was 0.052.
Sleep Rebound after Paraxanthine, Caffeine, and Modafinil Administration
In order to evaluate the long-term effects and rebound sleep after the wake-promoting effects of paraxanthine, caffeine, and modafinil, cumulative wake surplus (over the respective vehicle sessions) for the highest dose of all 3 compounds after light and dark period administration were calculated and displayed (Figure 5). The wake-promotion of the highest dose of the 3 compounds lasted for 6-8 h (except the dark period caffeine administration), but the recovery patterns differed among the compounds. During light period drug administration, sleep rebound was nonexistent after wake promotion by paraxanthine and minor for modafinil and caffeine. During dark period drug administration, cumulative wake surplus was balanced after 12 h for WT and after 20-24 h for narcoleptic mice, suggesting they slept more when wake-promoting effects subsided. Wake-promoting effects of caffeine during the dark period drug administration lasted over 12 h and were much more robust than those of paraxanthine and modafinil. It should be noted that significant hypothermia was associated with the prolonged wakefulness after administration of the highest dose of caffeine.
Figure 5.
Cumulative wake surplus for 24 h after administrations of paraxanthine (100 mg/kg, p.o.), caffeine (92.8 mg/kg p.o.), and modafinil (200 mg/kg) at ZT2 (light period) and ZT 14 (dark period) in wild-type (WT) and narcoleptic (TG) mice. “Cumulative wake surplus” (sec) was calculated by the cumulative wake amount after the drug administrations minus that after the vehicle administrations.
DISCUSSION
Paraxanthine, one of the major metabolites of caffeine in animals, significantly promoted wakefulness in both WT and narcoleptic mice. Paraxanthine proportionally reduced NREM and REM sleep in both genotypes. The wake-promoting potency of 100 mg/kg p.o. of paraxanthine during the light period administration roughly corresponds to that of 200 mg/kg p.o. of modafinil, a non-amphetamine wake-promoting compound currently used for the treatment of human narcolepsy. The wake-promoting potency of paraxanthine is greater than that of the equimolar concentration of caffeine when the drugs were administered during the light period. The difference was well contrasted by analysis of wake surplus for 24 h after the highest dose drug administration during light period (Figure 5). Although all three compounds enhanced wakefulness similarly for 6-8 h, paraxanthine did not produce any sleep rebound. As previously reported by several authors,19,20 modafinil produced only mirror sleep rebound, and caffeine exhibited a similar pattern. Both WT and narcoleptic mice responded to paraxanthine, caffeine, and modafinil in the same way, and similar enhancements of wake amount were observed in both WT and TG mice. These results suggest that the wake-promoting effects of these compounds do not require intact hypocretin neurotransmission. Considering the fact that wake-promoting effects of paraxanthine are comparable to modafinil, there may be a clinical application of paraxanthine in human narcolepsy.
Wake promotion by paraxanthine, caffeine, and modafinil during the light period was associated with an increase in locomotor activity and body temperature. However, the highest doses of caffeine and modafinil, but not paraxanthine, induced significant hypothermia (for the highest dose of caffeine in TG mice) or dampened these effects (smaller temperature elevation than that of middle dose, observed for the highest dose of modafinil in WT and TG mice and the highest dose of caffeine in WT mice), suggesting that the wake-promoting effect of paraxanthine is untainted in the dose range tested.
Significant wake-promoting effects were also seen for all three drugs administered during the active phase. Due to the high baseline wake amount during the active phase, percent increases in wake amount (as well as percent reductions in NREM and REM sleep) were smaller than those observed during the light period. In contrast to the results of the light period, smaller wake enhancements were seen in narcoleptic mice after paraxanthine and modafinil administration.
As we and other authors previously demonstrated, wake and sleep fragmentation during the active period is one of the major characteristics of sleep abnormalities seen in narcoleptic mice.10,11 This finding was reported mostly from undisturbed baseline data for the entire (12 h) dark period. Our current study is a pharmacological study, and the drug effects were monitored for 6 h, with the mice receiving drug administration using a stomach sonde. The vehicle administration itself subsequently induced behavioral activation and enhanced wake and locomotor activity. We also noted that baseline REM sleep amount (after vehicle administration) was very low for some drug sessions (e.g., modafinil dark period session in WT mice); this may be caused by a technical issue in oral drug administration with the stomach sonde, since REM sleep is easily disrupted by behavioral manipulations.
Nevertheless, the tendency for wake fragmentation during vehicle sessions were still evident in TG mice (Table 4). Tendency for larger REM sleep amount in TG mice during dark period are also observed. All three wake-promoting compounds prolonged the mean duration of wakefulness in both WT and TG mice (and shortened the mean duration of NREM and REM sleep). It should be noted that the low dose of these compounds, especially paraxanthine and modafinil, were able to adjust the level of wake fragmentation in TG to those of baseline WT levels, suggesting that low doses may be enough to manage the sleep problems (i.e., sleepiness) in narcoleptic subjects.
As has been reported by several authors,21,22 we observed a significant drop in body temperature after the high dose of caffeine administration, and this likely interfered with the locomotor enhancement by caffeine. However, larger and longer lasting wake promotion was still observed in response to the highest dose of caffeine during dark phase experiments (Figures 4 and 5), suggesting that the significant hypothermia induced by caffeine administration did induce sedation, but did not induce sleep.
In contrast to caffeine, paraxanthine, administered during the dark period, increased temperature and locomotor activity in dose-dependent fashion in WT mice. However, the highest dose of paraxanthine inhibited the initial rise in temperature in TG mice (Figure 4). Locomotor activation after the highest dose of paraxanthine was also dampened at the initial phase in these mice, while locomotor activity was increased during the second half of the 6-h recording period (Figure 4). This initial effect, however, did not interfere with the wake-promoting effects of paraxanthine. Modafinil did not significantly increase body temperature, but locomotor activity was increased significantly. The highest dose of modafinil also induced hypothermia in narcoleptic mice and interfered with locomotor activation. In contrast to effects seen in response to paraxanthine and caffeine, the highest dose of modafinil reduced wakefulness in TG mice, suggesting that hypothermia by modafinil enhanced sleep. Due to these adverse effects, the wake-promoting potencies of the three compounds during the active period were difficult to rank.
Willie et al.20 previously reported that hypocretin-deficient narcoleptic mice (i.e., preproorexin knockout mice) are more sensitive to modafinil (10-100 mg/kg i.p.) and enhanced wake-promoting effects, compared to their littermate WT mice, are seen in these mice during the dark period drug administration. However, we observed an attenuated wake-promoting effect by modafinil in (hypocretin neuron-ablated) TG narcoleptic mice.
The discrepancy of the results may be due to the difference in the hypocretin status in two different mice models; a small portion of hypocretin neurons are left in ataxin/orexin TG mice, in contrast to the complete loss of hypocretin ligand in gene knockout mice. The difference in the route of administration and more prominent hypothermia in TG mice that interfered with the wake-promoting effects of modafinil also need to be considered.
Both paraxanthine and caffeine, but not modafinil, reduced DREM during active periods. This result may suggest that paraxanthine and caffeine may have anticataplectic effects, since we recently demonstrated that effects on DREM during active periods in the murine narcoleptic model may be predictive for those on human cataplexy.11 However, it should be also noted that REM sleep and abnormal REM sleep (i.e., DREM) can be easily reduced by many nonspecific manipulations, and we need to account for the possibility that the temperature drops in response to paraxanthine and caffeine may nonspecifically reduce DREM in TG mice.
Large amount of caffeine intake can induce anxiety in humans. It is also reported that acute administrations of caffeine in mice induce anxiety levels with plus maze or open field evaluations.15,18 We also found that acute caffeine administration enhances anxiety levels in mice evaluated by marble burying test and elevated plus maze. In contrast, paraxanthine had no effects in marble burying test (compared to the vehicle session) and only moderately enhanced anxiety levels evaluated with the open maze.
Our results demonstrated that pharmacological properties of paraxanthine were different from those of caffeine. Compared with caffeine, paraxanthine: (1) produced more potent wake-promoting effects; (2) did not produce any sleep rebound when administered during the light period; (3) did not induce significant hypothermia, thereby indicating that influences on locomotion by temperature changes were minimal; and (4) did not enhance anxiety levels.
The higher potency of paraxanthine vs caffeine in promoting wake may be attributed to its higher potency at adenosine receptors: Ki values are 19 and 37 μM at A1 receptor and 1.1 and 2.5 μM at A2 receptor for paraxanthine and caffeine, respectively (Sokoloff, unpublished results). The absence of sleep rebound after paraxanthine treatment cannot be attributed to particular pharmacokinetic features of paraxanthine; plasma clearances of caffeine and paraxanthine do not differ in man and rats,23 and it is unlikely that major differences occur in mice. It should be also noted that paraxanthine failed to generalize to the caffeine cue in rats trained to discriminate caffeine,24 which indicate that the pharmacological actions of the two methylxanthines differ.
An identified pharmacological difference between caffeine and paraxanthine is the ability of the latter compound to activate ryanodine receptors and to produce a mild elevation of intracellular calcium in dopamine neurons.9 Elevation of intracellular calcium facilitates opening of calcium-sensitive potassium channels, which can increase neuronal firing regularity.25 Although the relationship between such a possible alteration of dopamine neuron function and wake maintenance is not understood, all wake-promoting compounds currently used for the treatment of hypersomnia, including amphetamines and modafinil, enhance dopaminergic neurotransmissions.26,27 Thus, it is interesting to evaluate if an additional action of paraxanthine on dopamine neurons accounts for its advantageous in vivo wake-promoting effects with respect to caffeine.
Since caffeine, but not paraxanthine, exhibited marked hypothermic effects, this effect is likely mediated by caffeine itself, or its active metabolite, theobromine, or theophylline. The receptor binding data of caffeine and paraxanthine also suggest that hypothermia is not mediated by antagonism of adenosine receptors, since both compounds have similar, yet slightly distinct affinities for adenosine receptors. Durcan et al.21 studied mechanisms of caffeine- and other alkylxanthine-induced hypothermia and suggested that inhibition of calcium-independent phosphodiesterase action, rather than blockade of adenosine receptors, is involved in the hypothermic effects. Our results further demonstrated that this effect can be differentiated from the wake-promoting effects of alkylxanthines.
Caffeine is the most widely used wake-promoting substance, in the form of over-the-counter (OTC) or caffeine-containing beverages or snacks. Side effects of caffeine include agitation, anxiety, tremors, rapid breathing, and insomnia. Non-negligible portions of caffeine metabolize to theobromine, and theophylline in humans and experimental animals. Although a large variation of interspecies difference in caffeine metabolism is known,5 influences of these compounds on caffeine's adverse effects need to be considered.
Theophylline (used mostly in therapy for respiratory disease such as asthma or chronic obstructive pulmonary disease) has side effects such as nausea, diarrhea, increase in heart rate, and arrhythmias, and has a narrow therapeutic window.8 Similarly, theobromine has a lower potency as a central nervous system stimulant than caffeine, but more potently stimulates the heart.8
Interestingly, it has been recently shown that paraxanthine also provides protection against dopaminergic cell death.9 Epidemiological evidence suggests that caffeine or its metabolites reduce the risk of developing Parkinson disease, possibly by protecting dopaminergic neurons.28 Guerreiro et al. showed that paraxanthine, but not caffeine, was strongly protective against neurodegeneration and loss of synaptic function in a culture system of selective dopaminergic cell death.9 The protective effect of paraxanthine was not mediated by blockade of adenosine receptors or by elevation of intracellular cAMP levels, but rather by an increase in free cytosolic calcium via the activation of ryanodine receptors.9 Consistent with these observations, both paraxanthine and ryanodine, the preferential agonist of ryanodine receptors, were protective in an unrelated paradigm of mitochondrial toxin-induced dopaminergic cell death.
Considering the fact that sleepiness is one of the disabling symptoms of Parkinson disease, and sleepiness is often associated with other neurological conditions (i.e., hypersomnia due to medical condition),29,30 paraxanthine may be a better wake-promoting formula for normal individuals and those suffering from neurological conditions, and the possibility of using paraxanthine as a treatment of these conditions in humans should be further evaluated.
DISCLOSURE
This research was supported by a research grant from Pierre Fabre Research Institute. Dr. Sokoloff is an employee of Pierre Fabre Research Institute and is co-inventor of a Patent Application claiming therapeutic utilizations of paraxanthine. The other authors have indicated no financial conflicts of interest.
ACKNOWLEDGMENTS
The authors thank Nicolle Chan for scoring the sleep EEG and Cayde Ritchie and Stephanie Newland for editing the manuscript.
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