The hypothesis that histaminergic neurons are involved in brain arousal is supported by many studies. However, the effects of the selective long-term abolition of histaminergic neurons on the sleep-wake cycle, indispensable in determining their functions, remain unknown. We have compared brain histamine(HA)-immunoreactivity and the cortical-EEG and sleep-wake cycle under baseline conditions or after behavioral or pharmacological stimuli in wild-type (WT) and knock-out mice lacking the histidine decarboxylase gene (HDC-/-). HDC-/-mice showed an increase in paradoxical sleep, a decrease in cortical EEG power in theta-rhythm during waking (W), and a decreased EEG slow wave sleep/W power ratio. Although no major difference was noted in the daily amount of spontaneous W, HDC-/-mice showed a deficit of W at lights-off and signs of somnolence, as demonstrated by a decreased sleep latencies after various behavioral stimuli, e.g., WT-mice placed in a new environment remained highly awake for 2-3 hr, whereas HDC-/-mice fell asleep after a few minutes. These effects are likely to be attributable to lack of HDC and thus of HA. In WT mice, indeed, intraperitoneal injection of alpha-fluoromethylhistidine (HDC-inhibitor) caused a decrease in W, whereas injection of ciproxifan (HA-H3 receptor antagonist) elicited W. Both injections had no effect in HDC-/-mice. Moreover, PCR and immunohistochemistry confirmed the absence of the HDC gene and brain HA-immunoreactive neurons in the HDC-/-mice. These data indicate that disruption of HA-synthesis causes permanent changes in the cortical-EEG and sleep-wake cycle and that, at moments when high vigilance is required (lights off, environmental change em leader ), mice lacking brain HA are unable to remain awake, a prerequisite condition for responding to behavioral and cognitive challenges. We suggest that histaminergic neurons also play a key role in maintaining the brain in an awake state faced with behavioral challenges.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histaminesynthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDCdeficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes. ß

Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock-gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR-based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferroni's corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59-38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H 1 , H 2 , and H 3 ) mediate these actions. To define the contribution of the histamine H 1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. Biological activity of histamine has been characterized mainly from pharmacological methods using its antagonists and agonists. These studies have demonstrated that histamine plays an important role in various physiological reactions such as inflammation and gastric acid secretion. In the mammalian central nervous system (CNS), histamine is thought to modulate many functions of the hypothalamus that exhibit circadian and other rhythms, such as arousal state, locomotor activity, feeding, and drinking (1-4). The histaminergic neuron system arises from the tuberomammillary nucleus of the posterior hypothalamus receiving inputs mainly from the limbic system and projects efferent nerve fibers to almost all parts of the brain (5-9). Autoradiographic binding studies show that three types of receptors are widely expressed in brain, not only on neurons but also on astrocytes and blood vessels (10, 11). However, much remains to be cleared about its roles in the CNS. To study the function of histamine neuron system, histamine H 1 receptor (H1R)-deficient mice were generated by homologous recombination. Considering the unique distribution pattern of the histamine neuron system, circadian rhythm, and exploratory behavior in a new environment were studied here in mice lacking H1R. MATERIALS AND METHODSConstruction of Targeting Vector. The H1R gene was isolated from a mouse 129͞ola-embryonic stem (ES)͞ EMBLE3 genomic library. A BglII-EcoRI 6.4-kb fragment containing DNA from mostly 3Ј of the H1R gene was subcloned into PUC19 at the sites of BamHI and EcoRI. A 0.8-kb ApaI-EcoRI fragment containing 5Ј upstream sequences was subcloned into the BamHI site of pMC1 poly(A) (Stratagene) in blunted end ligation. The 1.9-kb fragment with 5Ј upstream sequence and Neo-poly(A) cassette including promoter (excised with SalI and XhoI from the vector) was integrated at the site of SalI into the PUC19 with 6.4-kb fragment.Gene Targeting and Generation of Mutant Mice. The vector was linearized at a unique SalI site of the plasmid. Linearized targeting vector was electroporated in E14 ES cells, and neo r colonies were selected as previously described (12). Homologous recombinants were screened at first by PCR with a primer located upstream of the short arm of the homology (5Ј-GA-AGTATCTGGCTCTGAGTG-3Ј) and a primer complementary to the neo r gene sequence (5Ј-GCGTTGGCTACCCGT-GATAT-3Ј) and verified by Southern blotting. Genomic DNA from PCR positive clones was digested with EcoRI and hybridized with probe A, a neo probe (probe B), and probe D. ES cells carrying the H1R mutation were injecte...