Characterization of the wake-promoting agents with reference to histamine-mediated transmission using knockout mouse models

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As HA neurons are thought to play a crucial role in maintaining cortical activation and waking, one may ask whether modafinil induces sustained wakefulness via activation of histaminergic neurons. The same question may be addressed regarding psychostimulants even though a predominant dopaminergic mechanism exists. The question has become more intriguing since reports in the rat of a c-fos expression in histaminergic tuberomammillary nucleus [41] and an increase in hypothalamic HA outflow [42], both seen with large doses of modafinil. In regard to H₃R-antagonists, one may also question the importance of histamine transmission in their arousal effects and if there is an involvement of other neurotransmitters also involved in waking [8; 9] and controlled by H₃-receptors. To test the histaminergic hypothesis regarding the mechanisms of action of amphetamine, modafinil and ciproxifan in waking, their effects in KO mouse models in which histamine-mediated transmission is altered, e.g., HDC and H₁-, H₂- and H₃- receptor KO mice was studied. As previously reported, these KO mice are able to maintain, under the basal non-challenged conditions and despite qualitative change, a daily amount of waking near to that of wild type (WT) mice [16; 48-50], probably due to the compensatory mechanisms elaborated by brain plasticity.

A group (n=9) of 129Sv genetic-background inbred WT and HDC KO mice were recorded simultaneously, as previously described, to compare the sleep-wake effects of the W-promoting agents given i.p. during the light phase [16]. These mice were generated according to procedures previously described [51] and their genotypes confirmed using PCR. The doses used for amphetamine, modafinil and ciproxifan were 1, 32 and 1 mg/kg respectively as these approximated equal potency in terms of wake induction. As presented above, all agents at the indicated doses caused, in all WT models (129Sv as well as C57/Black6/J genetic background), increased waking at the expense of slow wave sleep and paradoxical sleep as compared with placebo (Figures 6, 7; Table 3).

In the HDC-KO mice, the same amphetamine and modafinil doses increased waking and decreased slow wave sleep and paradoxical sleep during the sleeping phase, the effect being identical or slightly superior to that seen in WT animals. In contrast, the same ciproxifan dosing (or higher doses up to 10 mg/kg, data not shown) had no effect on either the cortical EEG or the sleep-wake states (Figures 6, 7; Table 3).

HDC-KO mice lack endogenous HA synthesis and HA-containing neurons in the brain [16; 51]. The present data thus indicate that the sleep-wake effects of ciproxifan, but not those of amphetamine or modafinil, depend on histamine-mediated transmission. However, these data do not appear to support a direct excitation of histamine neurons in the mechanism of action of modafinil or amphetamine-like psychostimulants. C-fos expression in the histaminergic tuberomammillary nucleus and diffuse brain areas [41] or the HA release [42] seen in the rat after large doses of modafinil is thus likely to be the consequence of sustained waking rather than a modafinil-mediated excitation, as both c-fos expression and histamine release are positively correlated to waking [43-45;52]. Using c-fos as a marker in the cat supports this hypothesis. Indeed, examination of c-fos labeling after modafinil dosing but before an established long duration wake state revealed sparse c-fos expression in the histaminergic tuberomammillary nucleus and other brain regions [53]. Additionally in either normal [15] or brainstem-transectioned [4] cats, expression of c-fos in histamine neurons has only been seen with ciproxifan and not with other waking substances including modafinil, as already mentioned, and psychostimulants like amphetamine and methylphenidate [53], indicating that only stimuli specific to the histaminergic system may induce c-fos expression within histaminergic cell bodies. However, it cannot be excluded that histaminergic neurons may be indirectly involved in modafinil-induced waking, as a significant decrease in GABA outflow in the posterior hypothalamus is seen in vivo after modafinil dosing [46; 47] and so HA or orexin neurons located in this region could be disinhibited and so enhance waking.

Since only ciproxifan-mediated arousal depends on HA, this compound was further characterized using C57/Black6/J background KO mice devoid of H₁- (n = 8), or H₂- (n = 8) or H₃- (n = 12) receptors. These mouse genotypes were generated respectively according to the previously described procedures and both WT and KO littermates were identified using PCR [54-56].

Ciproxifan (1 mg/kg, i.p.) increased waking (+50-86% over a 4h recording) and cortical fast rhythms in all WT mouse groups during the sleeping period, whereas it had no effect in either H₁- or H₃-receptor KO littermates. Interestingly, the effects of ciproxifan on the cortical EEG and waking were intact in H₂-receptor KO-mice, the increase in waking being similar in KO (+ 93% over 4h) than WT littermates (+85%) (Table 3). These data confirm the pharmacological selectivity of ciproxifan for H₃ -receptors already demonstrated with imetit and the essential role of the H₃-receptor in its arousal effect. Although recent studies indicate that both ciproxifan and imetit may process weak activity at H₄-receptors [34;57;58] and although H₃-receptors also regulate the availability of neurotransmitters other than HA, e.g., norepinephrine, acetylcholine and 5HT, that are also involved in sleep-wake control, the results generated from KO mice indicated that the effect of ciproxifan on EEG and sleep-wake parameters selectively depend on H₃-receptor and histamine-mediated transmission. The fact that the waking effect of ciproxifan was observed in the H₂-receptor KO mouse but absent in the H₁-receptor KO genotype [48] confirms the dominant, if not exclusive, importance of H₁-receptors in the postsynaptic mechanisms of histaminergic arousal. Together, these data validate the earlier hypothesis [4; 20] that H₃R-antagonists, via dis-autoinhibition of presynaptic H₃-receptors, enhance the turnover and activity of histaminergic neurons, increasing synaptic HA that in turn activates postsynaptic H₁-receptors, promoting wakefulness and improving vigilance.