Effects of modafinil, psychostimulants and H3-receptor antagonists on the mouse cortical EEG and sleep-wake cycle.

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To compare the wake promoting effects of H₃R-antagonists in comparison to modafinil and classical psychostimulants, C57/Black6/J genetic background mice (n = 22, Charles River, France) were implanted with electrodes to monitor the cortical EEG and sleep-wake cycle according to previously described methods [16]. Briefly, All mouse strains used in this study were housed individually in transparent barrels (f 20 cm, height 30 cm) in an insulated sound-proofed recording room maintained at an ambient temperature of 22 ± 1°C and on a 12h light/dark cycle (lights-on at 7h00), food and water being available ad libitum. Polygraphic recordings were performed after administration of placebo or the drugs and scored as described [16] by 30s epochs for wakefulness (W), slow wave sleep and paradoxical (PS or REM) sleep. Cortical EEG power spectra were analyzed for consecutive 30-sec epochs within the frequency range of 0.4-60 Hz using a fast Fourier transformation routine by the CED-spike 2 analysis system. Statistical evaluation was performed using ANOVA followed by Dunnett’s t test. Each animal served as its own control.

D-amphetamine (1, 4 and 8 mg/kg, Sigma, St. Louis, MO USA), caffeine (10, 30 and 100 mg/kg, Sigma), modafinil (10, 30 and 100 mg/kg, Cephalon, France), thioperamide (10, 30 and 100 mg/kg, Sigma) and ciproxifan (1, 3 and 10 mg/kg, Sigma) were administered i.p. at the light phase (11h30) when the animals slept most of the time at baseline (defined as sleeping period). As shown in Table 1 and Figures 1 and 2, all compounds at the doses used increased the time spent awake. The wake effect, occurring as early as the first hour after dosing, was accompanied by delayed sleep latencies (Table 1; Figures 1 and 2), the duration of the effect on waking being dose-dependant. The increase in wakefulness was at the expense of both slow wave sleep (SWS) and paradoxical sleep (PS). Compared with modafinil and psychostimulants, the effects of the two H₃R-antagonists had several characteristics:

Prompt awakening effect

The waking effect of H₃R-antagonists occurred quickly in the mouse and was promptly terminated, similar to that seen in the cat [4; 20; 30]. At a dose 10-fold higher than the minimally effective one, the waking effect was prolonged to two more hours whereas at an 8-10 fold higher dose, the waking effect of modafinil, amphetamine and caffeine produced additional periods of 3, 4 and more than 7h respectively (Figure 2). In addition to the bioavailability of H₃R-antagonists, their short-lasting effect probably reflects the fact that the HA released by H₃R-antagonists is rapidly eliminated and that there are no known transporter mechanisms involved in HA catabolism that could be affected by H₃R-antagonists. Indeed, released HA is instantly inactivated by the enzyme, histamine-N-methyltransferase. HA is also known to have a faster turnover rate than other neurotransmitters with the exception of acetylcholine [1;29]. H₃R-antagonists (e.g. ciproxifan) also have no interactions with monoamine transporters [data not shown]. In contrast, the psychostimulant effects of amphetamine depend on an inhibition of the dopamine transporter (DAT) in addition to an enhancement of monoamine release and blockade of monoamine oxidase activity. Modafinil binds with moderate affinity to DAT (~ 4-7 µM) [31-33]. However, the dopaminergic mechanisms involved in the waking effect of modafinil may be relatively pronounced in the mouse [32] compared with other experimental species. The relative short lasting effect of H₃R-antagonists demonstrated here, if extrapolatable to humans, may be an advantage for their potential therapeutic use, i.e. maintaining daytime wakefulness, followed by normal nocturnal sleep.

2.2 Quiet and alert waking

In mice as in other species, psychostimulants such as amphetamine and caffeine markedly increased behavioral activity and locomotion in addition to EEG arousal, whereas no overt behavioral excitation occurs during wakefulness following modafinil, thioperamide and ciproxifan. Animals were quiescent for the majority of time presenting a level of activity similar to waking seen during baseline recording. The most notable difference seen in this study between H₃R-antagonists and other wake-promoting agents involved the qualitative aspect of waking, i.e., cortical EEG. Whereas all the compounds used caused a clear suppression of cortical slow waves (d and slow q bands, mainly 0.8-5 Hz), H₃R-antagonists like ciproxifan were distinct from other compounds due to their effect on cortical fast rhythms (b and g bands, 20-60 Hz). Thus, amphetamine and modafinil enhanced waking behavior without increasing cortical fast activity. Conversely, the “quiet” waking induced by ciproxifan was accompanied by a marked enhancement in cortical fast rhythms (Figure 1). Thus, the mean total power of cortical fast rhythms (20-60 Hz) during waking after ciproxifan dosing is increased by 9±2% (p < 0.05, ANOVA) compared with that seen after placebo in the same mice (n=7, data obtained from 120 consecutive samples of 30s wake episodes after placebo or ciproxifan dosing in each mouse). Similar results have been obtained in the cat [4; 30 and data not shown].

The marked wake-improving effect of ciproxifan demonstrated in several species is consistent with the concept of a predominant role of histaminergic neurons in cortical activation during waking. Because the occurrence of cortical fast rhythms is closely associated with the so-called higher mental activities, e.g., attention, alertness, and leaning, these results thus indicate that waking elicited by H₃R-antagonists is of a high level of vigilance and that the histaminergic system plays a role not only in waking, the basis for all other high brain functions, but also in some cognitive processes. These data also suggest that clinically suitable H₃R-antagonists might be designated as a therapeutic approach for vigilance disorders associated with cognitive deficiency [4;20;30,34]. Finally, ciproxifan, through activation of histamine neurons as demonstrated by their dense c-fos expression after dosing, restored a sustained cortical activation in comatose or hypersomniac cats after acute or chronic brainstem transection, respectively [4]. This clear arousing effect suggests that drug-like H₃R-antagonists may have the ability to restore cortical activation in comatose or brain-traumatized patients. 

Absence of sleep rebound

The long lasting waking elicited by amphetamine and caffeine, but not that induced by modafinil, was followed by a significant sleep rebound, mainly consisting of slow wave sleep (Tables 2 and 3) and by an increase in power spectral density of cortical slow activity (data not shown). These data are in agreement with those previously obtained in the cat [35; 36] and rat [37; 38]. Like modafinil, but unlike amphetamine or caffeine, the waking effects of thioperamide and ciproxifan were followed by a sleep-wake cycle with an amount of both slow wave sleep and paradoxical sleep similar to that seen during baseline recording, indicating no significant sleep rebound. The significance of the different effects of the studied agents on subsequent sleep remains unclear as the mechanisms and functions of sleep rebound are far from well understood. It has previously speculated that an overuse or exhaustion of catecholamines, such as the enhanced prolonged release associated with the use of amphetamine (but presumably not with that of modafinil), may be one cause of sleep rebound following the amphetamine-induced arousal and behavioral excitation [36]. One function of sleep rebound would, therefore, be to restore the physiological and functional levels of catecholaminergic neurons after over activity and also to allow the brain to recover from the deleterious effects of catecholamine systems during sustained waking. In support of this assumption, one of the few genes in the rat brain which is significantly induced and proportionally expressed after sleep deprivation is that of arylsulfotransferase, a final enzyme responsible for the catabolism of catecholamines in rodents [39; 40]. Thus the absence of sleep rebound associated with modafinil could also be interpreted as absence of catecholamine exhaustion as the waking effect of modafinil does not seem to depend on endogenous catecholamines [35]. Moreover, no signs of direct neuronal depolarization/excitation on target cells have been reported for modafinil, even though diffuse expression of immediate early gene c-fos [41], or enhanced histamine release [42] occurred with high doses of modafinil. These effects can be attributed to a direct consequence of the sustained waking induced by modafinil rather than a direct pharmacological targeted action per se. Indeed, c-fos expression is a state-dependent phenomenon, occurring most densely in large brain areas after spontaneous or induced wakefulness [43-45]. Alternatively, one of the major effects of modafinil is the induction of a marked decrease in GABA outflow in the critical brain regions involved in sleep-wake control including the posterior hypothalamus and preoptic area [46, 47]. Wakefulness seen with modafinil could then result from a disinhibition of brain arousal systems, e.g., the HA and orexin containing cells in the posterior hypothalamus known for their crucial role in the maintenance of waking [4-6; 9]. A quiet waking state resulting from this disinhibitory mechanism would therefore have a different effect on the subsequent sleep rebound to that seen with amphetamine.

The reasons why H₃R-antagonist-induced waking is not associated with a sleep rebound remain to be determined. In any proposed hypothesis, the above-mentioned associated characteristics, including quiet waking, prompt and short-lasting effect, rapid HA turnover, the lack of an overuse of catecholamines and the lack of potent interactions with monoamine transporters may be contributory. Whatever these underlying mechanisms might be, the absence of sleep rebound observed with modafinil and H₃R-antagonists, is critical in the clinical setting in terms of quality of life outcomes.