While many risk factors are linked to atherosclerosis, including blood cholesterol, physical inactivity, age, hypertension, obesity and smoking, these factors don't apply in roughly 15 percent of first-time cardiovascular disease CVD events. What's more, heart attacks due to atherosclerosis are rare even in some of our closest genetic relatives, including chimps ; that applies to chimps with human-like risk factors, too - such as hypertension and physical inactivity. The team also looked at the effects of eating red meat, finding that when the modified mice were fed a Neu5Gc-rich diet — mimicking red meat intake — the risk of atherosclerosis increased again.
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Hrh1 expression was generally lower in astrocytes than in neurons, suggesting that neuronal H 1 receptors comprise majority of H 1 receptors in the brain. Histamine is produced in histaminergic neurons originating in the hypothalamic TMN; it is possible that this may be a compensatory mechanism for impaired H 1 receptor signaling.
However, under baseline conditions, histamine release and the concentration of its metabolite 1-methylhistamine are strongly correlated. Therefore, the stable concentration of 1-methylhistamine suggests that histamine release remained unchanged. We assessed behaviour that is reportedly linked to H 1 receptor signalling, including exploratory and anxiety-like behaviour, memory, learning, aggression, and circadian rhythms.
Exploratory behaviour of cKO mice was not affected in a novel environment. Although several researchers have reported the involvement of H 1 receptors in exploratory behaviour and found it to be decreased in conventional Hrh1 KO mice 7 , 8 , our data did not support these observations. The memory tasks conducted indicated that memory function is complex and involves different cell signaling pathways in the brain. Unlike in a previous study that used conventional Hrh1 mice 33 , the spatial memory function was not affected by conditional Hrh1 deletion.
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Mice with astrocytic Hrh1 deficiency did not display any changes. H 1 receptors are known to be important for memory and long-term potentiation in the hippocampus 34 , 35 , but the role of H 1 receptors on astrocytes in memory has not been established. To the best of our knowledge, these results show for the first time that neuronal, but not astrocytic, H 1 receptors are important for recognition memory. Further, we observed that anxiety-like behaviour was significantly decreased in both cKO mouse models.
In agreement with our findings, an Hrh1 knockout study and a pharmacological study using the H 1 receptor antagonist diphenhydramine reported that H 1 receptor signalling mediated anxiety-like behaviour 8 , Additionally, our results indicated that both neuronal and astrocytic H 1 receptors played a crucial role in anxiety. Our data suggest that both cell types can independently regulate anxiety-like behaviour, but further studies are required to validate the roles of neuronal and astrocytic H 1 receptors in this process.
The debate on how histamine modulates aggression continues to this day 8 , 36 , 37 , The present study reports, for the first time, that H 1 receptors on astrocytes regulate aggressive behaviour. In an earlier study with conventional Hrh1 KO mice, resident mice were significantly less aggressive compared to controls 8.
However, a noticeable difference in the period of individual housing among the previous and the present study, six months versus one weeks, has to be considered when comparing the findings. Other publications by Ray et al. The authors concluded that H 1 and H 2 receptors possess interlinked signalling that can alter aggressive behaviour. The hypothesis regarding the interlinked nature of H 1 and H 2 receptor signalling was further explored by Alonso et al.
As astrocytes express both H 1 and H 2 receptors, the question arises whether these receptors have opposing roles and counteract each other in the context of aggression. Our results emphasise the role of H 1 receptors on astrocytes in aggressive behaviour.
Nevertheless, further research is required to fully understand the role of astrocytic H 1 receptor signalling and its potential link to H 2 receptor signalling in the context of aggressive behaviour. The importance of H 1 receptor activation in arousal and wakefulness is well-established 12 , 32 , 41 , and our data support the concept of astrocytic H 1 receptors as a key player in this process.
Conventional Hrh1 KO mice showed altered sleep behaviour, with decreased time spent in the wake stage during the early dark phase 32 , but none of the cKO mouse models exhibited this phenotype. There are different scenarios that could explain why the observed phenotype is different from the one previously observed. First, to date, the role of H 1 receptors has been studied with conventional KO mice, which may have had additional impact on the phenotype in previous studies.
In contrast, Cre-dependent cKO mice still express the gene of interest until Cre expression ensures cell-specific recombination at the loxP sites and subsequent gene deletion.
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Therefore, we cannot rule out the possibility that previous observations were associated with these differences between the two mouse models. Second, it is possible that H 1 receptors on astrocytes and neurons jointly regulate sleep behaviour, and conditional gene deletion is insufficient to cause significant changes. It might be possible that further reduction of Hrh1 expression in astrocytes is required to induce a stronger phenotype, possibly comparable to that of the conventional KO mouse.
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To further explore the reasons for reduced vigilance, we investigated the quality of wakefulness and sleep in cKO mice by analysing EEG power spectra of all groups. An increase in delta waves during wakefulness has been described by Parmentier et al. Delta waves are a major indication for insufficient cortical activation and are associated with a drowsy state and low vigilance during wakefulness.
Moreover, histamine perfusion significantly decreases the amount of delta waves during wakefulness We hypothesise that deletion of H 1 receptors from astrocytes prevented activation of signalling cascades, which usually ensure sufficient cortical activity after awakening to maintain high vigilance. H 1 receptor-mediated signalling has the ability to adequately stimulate cortical activity through the activation of cholinergic and serotonergic neurons 2. It is possible that Hrh1 -expressing astrocytes play a critical role in exciting these neurons through the release of bioactive molecules.
Concluding from the obtained results, we propose that the histaminergic system is crucial to maintain wakefulness with high vigilance after awakening, and that this signalling is mediated through the activation of H 1 receptors on astrocytes.
However, deletion of Hrh1 in astrocytes did not affect the normal sleep-wake cycle, quantity, or quality of sleep, because neither sleep latencies nor the duration of sleep episodes were affected. Wakefulness has typically been associated with H 1 receptors, but previous studies did not confirm distinct roles of neurons and astrocytes in this process.
This is the first report indicating that H 1 receptors on astrocytes are important for the maintenance of high vigilance after awakening. Given the incomplete deletion of Hrh1 in astrocytes, it is possible that we are currently observing a phenotype that can somewhat cope with the partial gene deletion resulting in impaired wakefulness, but not disturbed sleep behaviour. It will be of great interest to study spontaneous locomotor activity, sleep, and wakefulness in a mouse model that can achieve complete Hrh1 deletion in astrocytes.
This study provided new functional insights regarding the role of H 1 receptors on neurons and astrocytes, and novel Hrh1 cKO mice were highlighted as a powerful tool to study cell-specific functionality. Nevertheless, several limitations of this study need to be addressed by future research. First, we did not provide data showing the distribution of neuronal and astrocytic H 1 receptors in different brain regions.
Currently, there are no good antibodies against the H 1 receptor, and we were unable to design a probe with high sensitivity and specificity for in situ hybridization purposes. Second, this study did not investigate the roles of brain regions implicated in the observed phenotypic changes. Adeno-associated virus AAV -mediated delivery of Cre-recombinase can selectively achieve genetic engineering in targeted cell populations and represents an advanced strategy to examine brain circuits involved in H 1 receptor-mediated behaviour in the future.
For instance, AAV-mediated regulation of neuronal-specific Cre recombinase expression in the hippocampus will enable investigation of the roles of hippocampal H 1 receptors in memory function. Third, future research should examine the relationship between observed behavioural changes and their underlying molecular mechanisms, as we did not investigate the underpinning cellular changes in cKO mice. It is well established that astrocytes respond to H 1 receptor activation in various ways, including glutamate homeostasis, energy metabolism and inflammatory processes. Thus, it will be important to study molecular changes and their consequences in the cKO mouse, because an interpretation of the observed phenotype can only be speculative at this moment.
Finally, astrocyte-specific KO mouse models that will achieve better Hrh1 deletion have to confirm our observations as current negative results, especially the results that are opposing to those in studies with conventional KO mice, might be a result of incomplete Cre recombination in astrocytes.
In conclusion, our study stresses the importance of H 1 receptors on neurons and astrocytes in distinct physiological processes. Our findings contribute to our understanding of how H 1 receptors on different cell types are involved in the development of pathological states and highlight astrocytes as a novel therapeutic target. Unless otherwise described, mice were housed in groups with up to five animals per cage with ad libitum access to a commercial standard diet Labo MR stock; Nosan Corporation, Yokohama, Japan and water.
All animals and gene-recombination experiments were given ethical approval from the Tohoku University Center for Laboratory Research and Tohoku University Center for Gene Research, respectively. Ltd Chiba, Japan. Briefly, a targeting vector was used to introduce two loxP sites into exon 3 of the allele of interest, flanking the coding sequence of Hrh1 Fig.
Cg-Tg Gfap-cre Cell-specific deletion of Hrh1 was confirmed with real-time PCR. Primary astrocytes and neurons were isolated at P0—P1 as previously described with some modifications The tissue was homogenized and incubated with 0. Cells for neuronal cell culture were resuspended in Neurobasal medium Life Technologies containing B supplement Life Technologies and 0. The medium was changed every 3 days until cells were used for further studies. Genomic DNA from mouse tail-cut samples was obtained as described by Truett et al.
The supernatant was subjected to PCR analysis. Cells were lysed and homogenised before chloroform was added. After phase separation, the aqueous phase was used to perform ethanol precipitation and purify the genomic DNA. Male mice aged 8—12 weeks positively genotyped for one of the three experimental groups were assessed in a series of behavioural tests for exploratory behaviour, anxiety, learning, memory, circadian activity, and aggression.
All behavioural experiments were performed during the dark period. During the open field, light-dark box, elevated plus-maze, Y-maze, and locomotor activity tests, the movement tracking software automatically assessed the behaviour of the mice. We avoided to keep mice in isolated environment except for resident-intruder test and home cage locomotor activity test. There were eight photocells on each side that tracked movements.
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The two outer photo cells on each side Spatial working memory of cKO mice was assessed using the Y-maze test Mice were placed at the end of one of the arms and were allowed to move freely in the maze. An alternation was defined as entries into all three arms on consecutive choices. The novel object recognition test was used to assess working memory of cKO mice The test was performed for 3 days with one session per day.
The objects were fixed at two positions with the same distance to the walls. The preference ratio indicates the time spent at the new object relative to the total exploration time. Changes in the preference ratio among the experimental groups during the test session were assessed using two-way ANOVA and Bonferroni post-hoc test. The elevated plus-maze test was conducted as previously described with minor modifications The light intensity at the central platform was lux. Home cage locomotor activity was assessed as previously described The resident-intruder test was performed as previously described Mice residents were housed individually for 1 week prior to the experimental day.
Testing was performed in the home cage of the resident. The mice were allowed to move freely and interact during the test. The test was recorded and subsequently, the attack latency, number of attacks, and cumulative attack time were scored.