Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

Chemokine ligand 28 (CCL28) negatively regulates trabecular bone mass by suppressing osteoblast and osteoclast activities.

INTRODUCTION: Bone metabolism imbalances cause bone metabolism diseases, like osteoporosis, through aging. Although some chemokines are known to be involved in bone mass regulation, many have not been investigated. Thus, the present study aimed to investigate the role of chemokine ligand 28 (CCL28) on bone metabolism. MATERIALS AND METHODS: To investigate the role of CCL28 on bone metabolism, 10-week-old male wild-type and Ccl28 knockout (Ccl28 KO) mice were analyzed. Microcomputed tomography analysis and bone tissue morphometry were used to investigate the effect of Ccl28 deficiency on the bone. CCL28 localization in bone tissue was assumed by immunohistochemistry. Osteoblast and osteoclast markers were evaluated by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. Finally, in vitro experiments using MC3T3-E1 and bone marrow macrophages revealed the direct effect of CCL28 on osteoblast and osteoclast. RESULTS: This study showed that Ccl28 deficiency significantly increased bone mass and the number of mature osteoblasts. Immunoreactivity for CCL28 was observed in osteoblasts and osteoclasts on bone tissue. Additionally, Ccl28 deficiency promoted osteoblast and osteoclast maturation. Moreover, CCL28 treatment decreased osteoblast and osteoclast activities but did not affect differentiation. CONCLUSION: In summary, this study indicated that CCL28 is one of the negative regulators of bone mass by suppressing osteoblast and osteoclast activities. These results provide important insights into bone immunology and the selection of new osteoporosis treatments.

Deletion of the PDZ-binding kinase (Pbk) gene does not affect male fertility in mice.

The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.

Glutamate input in the dorsal raphe nucleus as a determinant of escalated aggression in male mice.

Although the dorsal raphe nucleus (DRN) has long been linked to neural control of aggression, little is known about the regulatory influences of the DRN when an animal engages in either adaptive species-typical aggressive behavior or escalated aggression. Therefore it is important to explore which neurotransmitter inputs into the DRN determine the escalation of aggression in male mice. Previously, we observed that microinjection of the GABAB receptor agonist baclofen into the DRN escalates aggressive behavior in male mice. Here, we used a serotonin (5-HT) neuron-specific GABAB receptor knock-out mouse to demonstrate that baclofen acts on nonserotonergic neurons to escalate aggression. Intra-DRN baclofen administration increased glutamate release, but did not alter GABA release, within the DRN. Microinjection of l-glutamate into the DRN escalated dose-dependently attack bites toward an intruder. In vivo microdialysis showed that glutamate release increased in the DRN during an aggressive encounter, and the level of glutamate was further increased when the animal was engaged in escalated aggressive behavior after social instigation. Finally, 5-HT release was increased within the DRN and also in the medial prefrontal cortex when animals were provoked by social instigation, and during escalated aggression after social instigation, but this increase in 5-HT release was not observed when animals were engaged in species-typical aggression. In summary, glutamate input into the DRN is enhanced during escalated aggression, which causes a phasic increase of 5-HT release from the DRN 5-HT neurons.

Cell adhesion molecule contactin-associated protein 3 is expressed in the mouse basal ganglia during early postnatal stages.

Cell adhesion molecules play important roles in the development of the nervous system. Among the contactin-associated protein (Caspr; also known as Cntnap) family, which belongs to the neurexin superfamily of proteins, Caspr and Caspr2 are indispensable for the formation and maintenance of myelinated nerves. In contrast, a physiological role for Caspr3 remains to be elucidated. This study examines the expression and localization of Caspr3 in the mouse brain using newly generated Caspr3 antibodies. Caspr3 was expressed abundantly between the first and the second postnatal weeks. During this period, Caspr3 was localized especially to the basal ganglia, including the striatum, external segment of the globus pallidus, and substantia nigra, and no gross abnormalities were apparent in the basal ganglia of Caspr3 knockout mice. In the striatum, Caspr3 was expressed by a subpopulation of medium spiny neurons that constitute the direct and indirect pathways. Caspr3 immunostaining was observed as punctate around the cell bodies as well as in the soma. These Caspr3 signals did not, however, overlap with those of synaptic markers. Our findings suggest that Caspr3 may play an important role in basal ganglia development during early postnatal stages.

Enhanced prepulse inhibition and low sensitivity to a dopamine agonist in HESR1 knockout mice.

Transcription factor Hesr family genes are important in neuronal development. We demonstrated previously that HESR1 and HESR2 modified expression of the dopamine transporter (DAT) reporter gene. HESR-family genes have been investigated in development, but their functions, especially in relation to behaviors regulated by dopamine, in adult animals remain unclear. In the present study, we investigated the effects of Hesr1 and Hesr2 on behavior. A behavioral test battery to examine spontaneous activity, anxiety-like behavior, aggressive behavior, pain sensitivity, and sensorimotor gating was conducted in Hesr1 and Hesr2 knockout (KO) mice. Enhanced prepulse inhibition (PPI), which is a form of sensorimotor gating, was observed in only Hesr1 KO mice; other behavioral traits were mostly comparable to wild-type animals in both the Hesr1 and the Hesr2 KO lines. Next, we used a dopamine agonist, apomorphine, to confirm the involvement of the dopaminergic system. Injection of apomorphine reduced the enhanced PPI in Hesr1 KO mice. Additionally, dose-dependent sensitivity to the agonist was lower in the Hesr1 KO mice than in wild-type mice, suggesting that the enhanced PPI resulted from this alteration in dopamine sensitivity. Furthermore, DAT mRNA was downregulated in Hesr1 KO mice, whereas the dopamine D1 and D2 receptors were comparable. These findings suggest Hesr1 to be a novel factor that affects dopamine sensitivity and the sensorimotor gating system.

Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.

Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.

Association of tameness and sociability but no sign of domestication syndrome in mice selectively bred for active tameness.

Domesticated animals have been developed by selecting desirable traits following the initial unconscious selection stage, and now exhibit phenotypes desired by humans. Tameness is a common behavioural trait found in all domesticated animals. At the same time, these domesticated animals exhibit a variety of morphological, behavioural, and physiological traits that differ from their wild counterparts of their ancestral species. These traits are collectively referred to as domestication syndrome. However, whether this phenomenon exists is debatable. Previously, selective breeding has been used to enhance active tameness, a motivation to interact with humans, in wild heterogeneous stock mice derived from eight wild inbred strains. In the current study, we used tame mice to study how selective breeding for active tameness affects behavioural and morphological traits. A series of behavioural and morphological analyses on mice showed an increased preference for social stimuli and a longer duration of engagement in non-aggressive behaviour. However, no differences were observed in exploratory or anxiety-related behaviours. Similarly, selection for tameness did not affect ultrasonic vocalisations in mice, and no changes were observed in known morphological traits associated with domestication syndrome. These results suggest that there may be a link between active tameness and sociability and provide insights into the relationship between tameness and other behaviours in the context of domestication.

Identification of both copy number variation-type and constant-type core elements in a large segmental duplication region of the mouse genome.

BACKGROUND: Copy number variation (CNV), an important source of diversity in genomic structure, is frequently found in clusters called CNV regions (CNVRs). CNVRs are strongly associated with segmental duplications (SDs), but the composition of these complex repetitive structures remains unclear. RESULTS: We conducted self-comparative-plot analysis of all mouse chromosomes using the high-speed and large-scale-homology search algorithm SHEAP. For eight chromosomes, we identified various types of large SD as tartan-checked patterns within the self-comparative plots. A complex arrangement of diagonal split lines in the self-comparative-plots indicated the presence of large homologous repetitive sequences. We focused on one SD on chromosome 13 (SD13M), and developed SHEPHERD, a stepwise ab initio method, to extract longer repetitive elements and to characterize repetitive structures in this region. Analysis using SHEPHERD showed the existence of 60 core elements, which were expected to be the basic units that form SDs within the repetitive structure of SD13M. The demonstration that sequences homologous to the core elements (>70% homology) covered approximately 90% of the SD13M region indicated that our method can characterize the repetitive structure of SD13M effectively. Core elements were composed largely of fragmented repeats of a previously identified type, such as long interspersed nuclear elements (LINEs), together with partial genic regions. Comparative genome hybridization array analysis showed that whereas 42 core elements were components of CNVR that varied among mouse strains, 8 did not vary among strains (constant type), and the status of the others could not be determined. The CNV-type core elements contained significantly larger proportions of long terminal repeat (LTR) types of retrotransposon than the constant-type core elements, which had no CNV. The higher divergence rates observed in the CNV-type core elements than in the constant type indicate that the CNV-type core elements have a longer evolutionary history than constant-type core elements in SD13M. CONCLUSIONS: Our methodology for the identification of repetitive core sequences simplifies characterization of the structures of large SDs and detailed analysis of CNV. The results of detailed structural and quantitative analyses in this study might help to elucidate the biological role of one of the SDs on chromosome 13.

Function of gastrin-releasing peptide receptors in ocular itch transmission in the mouse trigeminal sensory system.

The prevalence of allergic conjunctivitis in itchy eyes has increased constantly worldwide owing to environmental pollution. Currently, anti-allergic and antihistaminic eye drops are used; however, there are many unknown aspects about the neural circuits that transmit itchy eyes. We focused on the gastrin-releasing peptide (GRP) and GRP receptor (GRPR), which are reportedly involved in itch transmission in the spinal somatosensory system, to determine whether the GRP system is involved in itch neurotransmission of the eyes in the trigeminal sensory system. First, the instillation of itch mediators, such as histamine (His) and non-histaminergic itch mediator chloroquine (CQ), exhibited concentration-dependent high levels of eye scratching behavior, with a significant sex differences observed in the case of His. Histological analysis revealed that His and CQ significantly increased the neural activity of GRPR-expressing neurons in the caudal part of the spinal trigeminal nucleus of the medulla oblongata in GRPR transgenic mice. We administered a GRPR antagonist or bombesin-saporin to ablate GRPR-expressing neurons, followed by His or CQ instillation, and observed a decrease in CQ-induced eye-scratching behavior in the toxin experiments. Intracisternal administration of neuromedin C (NMC), a GRPR agonist, resulted in dose-dependent excessive facial scratching behavior, despite the absence of an itch stimulus on the face. To our knowledge, this is the first study to demonstrate that non-histaminergic itchy eyes were transmitted centrally via GRPR-expressing neurons in the trigeminal sensory system, and that NMC in the medulla oblongata evoked facial itching.

Downregulation of chemokine receptor 9 facilitates CD4+CD8αα+ intraepithelial lymphocyte development.

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4+ T cells can develop into CD4+CD8αα+ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4+CD8αα+ IELs, but CCR9 deficiency results in CD4+CD8αα+ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4+CD8αα+ IELs in Ccr9-/- mice. CD4+ T cells isolated from the epithelium of Ccr9-/- mice also display increased expression of Cbfß2, and the genomic occupancy modification of Cbfß2 expression reveals its important function in CD4+CD8αα+ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4+CD8αα+ IEL differentiation.

Epigenetics in autism and other neurodevelopmental diseases.

Autism was previously thought to be caused by environmental factors. However, genetic factors are now considered to be more contributory to the pathogenesis of autism, based on the recent findings of mutations in the genes which encode synaptic molecules associated with the communication between neurons. Epigenetic is a mechanism that controls gene expression without changing DNA sequence but by changing chromosomal histone modifications and its abnormality is associated with several neurodevelopmental diseases. Since epigenetic modifications are known to be affected by environmental factors such as nutrition, drugs and mental stress, autistic diseases are not only caused by congenital genetic defects, but may also be caused by environmental factors via epigenetic mechanism. In this chapter, we introduce autistic diseases caused by epigenetic failures and discuss epigenetic changes by environmental factors and discuss new treatments for neurodevelopmental diseases based on the recent epigenetic findings.

Efficient genome editing in wild strains of mice using the i-GONAD method.

Wild mouse strains have been used for many research studies, because of the high level of inter-strain genetic and phenotypic variations in them, in addition to the characteristic phenotype maintained from wild mice. However, since application of the current genetic engineering method on wild strains is not easy, there are limited studies that have attempted to apply gene modification techniques in wild strains. Recently, i-GONAD, a new method for genome editing that does not involve any ex vivo manipulation of unfertilized or fertilized eggs has been reported. We applied i-GONAD method for genome editing on a series of wild strains and showed that genome editing is efficiently possible using this method. We successfully made genetically engineered mice in seven out of the nine wild strains. Moreover, we believe that it is still possible to apply milder conditions and improve the efficiencies for the remaining two strains. These results will open avenues for studying the genetic basis of various phenotypes that are characteristic to wild strains. Furthermore, applying i-GONAD will be also useful for other mouse resources in which genetic manipulation is difficult using the method of microinjection into fertilized eggs.

The role of cell-autonomous circadian oscillation of Cry transcription in circadian rhythm generation.

The current model of the mammalian circadian clock describes cell-autonomous and negative feedback-driven circadian oscillation of Cry and Per transcription as the core circadian rhythm generator. However, the actual contribution of this oscillation to circadian rhythm generation remains undefined. Here we perform targeted disruption of cis elements indispensable for cell-autonomous Cry oscillation. Mice lacking overt cell-autonomous Cry oscillation show robust circadian rhythms in locomotor activity. In addition, tissue-autonomous circadian rhythms are robust in the absence of overt Cry oscillation. Unexpectedly, although the absence of overt Cry oscillation leads to severe attenuation of Per oscillation at the cell-autonomous level, circadian rhythms in Per2 accumulation remain robust. As a mechanism to explain this counterintuitive result, Per2 half-life shows cell-autonomous circadian rhythms independent of Cry and Per oscillation. The cell-autonomous circadian clock may therefore remain partially functional even in the absence of overt Cry and Per oscillation because of circadian oscillation in Per2 degradation.

SDOP-DB: a comparative standardized-protocol database for mouse phenotypic analyses.

UNLABELLED: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information. AVAILABILITY: SDOP-DB (http://www.brc.riken.jp/lab/bpmp/SDOP/).

B6-MSM consomic mouse strains reveal multiple loci for genetic variation in sucrose octaacetate aversion.

Based on crosses among inbred strains derived principally from M. m. domesticus, sucrose octaacetate (SOA) aversion in laboratory mice has been thought for many years to be controlled by a single genetic locus (Soa) located on distal chromosome (Chr) 6. To expand knowledge of the genetic basis underlying SOA aversion, we have studied the M. m. molossinus derived strain (MSM) and MSM consomic strains on a M. m. domesticus (C57BL/6J: B6) host background. Using two-bottle preference procedures, MSM mice avoided 0.1 mM and 1 mM SOA while B6 mice were indifferent to 0.1 mM and exhibited slight aversion to 1 mM SOA. Preference tests of 16 available consomic strains implicated Chr 2, 4 and 15 in SOA aversion in addition to the prominent effect of the known Soa locus on Chr 6 (implicated by study of two congenic strains). The originally defined Soa locus is presumably associated with one or more members of the cluster of Tas2r genes on distal Chr 6 that code for bitter taste receptors. Our results point to the possible role of established Tas2r genes on Chr 2 and 15, as well as to genes not coding for bitter receptors (Chr 4), in SOA aversion. SOA aversion emerges from this consomic screen as being definitively under polygenic control. The genetic diversity captured by the MSM model system is shown to be a valuable tool to complement the limited genetic variation in the commonly used stocks derived from M m. domesticus.

Combined change of behavioral traits for domestication and gene-networks in mice selectively bred for active tameness.

Tameness is a major element of animal domestication and involves two components: motivation to approach humans (active tameness) and reluctance to avoid humans (passive tameness). To understand the behavioral and genetic mechanisms of active tameness in mice, we had previously conducted selective breeding for long durations of contact and heading toward human hands in an active tameness test using a wild-derived heterogeneous stock. Although the study showed a significant increase in contacting and heading with the 12th generation of breeding, the effect on other behavioral indices related to tameness and change of gene expression levels underlying selective breeding was unclear. Here, we analyzed nine tameness-related traits at a later stage of selective breeding and analyzed how gene expression levels were changed by the selective breeding. We found that five traits, including contacting and heading, showed behavioral change in the selective groups comparing to the control through the generations. Furthermore, we conducted cluster analyses to evaluate the relationships among the nine traits and found that contacting and heading combined in an independent cluster in the selected groups, but not in the control groups. RNA-Seq of hippocampal tissue revealed differential expression of 136 genes between the selection and control groups, while the pathway analysis identified the networks associated with these genes. These results suggest that active tameness was hidden in the control groups but became apparent in the selected populations by selective breeding, potentially driven by changes in gene expression networks.

QTL analysis of measures of mouse home-cage activity using B6/MSM consomic strains.

The activity of mice in their home cage is influenced greatly by the cycle of light and dark. In addition, home-cage activity shows remarkable time-dependent changes that result in a prominent temporal pattern. The wild-derived mouse strain MSM/Ms (MSM) exhibits higher total activity in the home cage than does C57BL/6 (B6), a commonly used laboratory strain. In addition, there is a clear strain difference in the temporal pattern of home-cage activity. This study aimed to clarify the genetic basis of strain differences in the temporal pattern of home-cage activity between MSM and B6. Through the comparison of temporal patterns of home-cage activity between B6 and MSM, the pattern can be classified into five temporal components: (1) resting phase, (2) anticipation phase, (3) 1st phase, (4) 2nd phase, and (5) 3rd phase. To identify quantitative trait loci (QTLs) involved in these temporal components, we used consomic strains established from crosses between B6 and MSM. Five consomic strains, for Chrs 2T (telomere), 3, 4, 13, and 14, showed significantly higher total activity than B6. In contrast, the consomic strains of Chrs 6C (centromere), 7T, 9, 11, and 15 were less active than B6. This indicates that multigenic factors regulate the total activity. Further analysis showed an impact of QTLs on the temporal components of home-cage activity. The present data showed that each temporal component was regulated by different combinations of multigenic factors, with some overlap. These temporal component-related QTLs are important to understand fully the genetic mechanisms that underlie home-cage activity.

Genetic mapping of social interaction behavior in B6/MSM consomic mouse strains.

Genetic studies are indispensable for understanding the mechanisms by which individuals develop differences in social behavior. We report genetic mapping of social interaction behavior using inter-subspecific consomic strains established from MSM/Ms (MSM) and C57BL/6J (B6) mice. Two animals of the same strain and sex, aged 10 weeks, were introduced into a novel open-field for 10 min. Social contact was detected by an automated system when the distance between the centers of the two animals became less than approximately 12 cm. In addition, detailed behavioral observations were made of the males. The wild-derived mouse strain MSM showed significantly longer social contact as compared to B6. Analysis of the consomic panel identified two chromosomes (Chr 6 and Chr 17) with quantitative trait loci (QTL) responsible for lengthened social contact in MSM mice and two chromosomes (Chr 9 and Chr X) with QTL that inhibited social contact. Detailed behavioral analysis of males identified four additional chromosomes associated with social interaction behavior. B6 mice that contained Chr 13 from MSM showed more genital grooming and following than the parental B6 strain, whereas the presence of Chr 8 and Chr 12 from MSM resulted in a reduction of those behaviors. Longer social sniffing was observed in Chr 4 consomic strain than in B6 mice. Although the frequency was low, aggressive behavior was observed in a few pairs from consomic strains for Chrs 4, 13, 15 and 17, as well as from MSM. The social interaction test has been used as a model to measure anxiety, but genetic correlation analysis suggested that social interaction involves different aspects of anxiety than are measured by open-field test.

Ultradian components in the locomotor activity rhythms of the genetically normal mouse, Mus musculus.

Ultradian periodicities in physiological processes have been reported for a wide variety of organisms and may appear as bouts in locomotor activity. In some instances, this temporal organization can be related to some ethological strategy. In mice, however, ultradian rhythms have been reported largely in animals with circadian pacemakers disrupted either by genetic or surgical manipulation. Using analysis techniques capable of resolving periodicities in the ultradian range in the presence of strong diel periodicity, we found unequivocal evidence of ultradian rhythms in mice entrained to an light:dark cycle. We collected locomotor activity data of individuals from 11 genetically disparate strains of mice whose activity was recorded in 12 h:12 h L:D photoperiods for 3 days. Data were subjected to maximum entropy spectral analysis and autocorrelation, both before and after filtering to remove the 24-h periodicity. We found that every strain had a majority of individuals with strong ultradian rhythms ranging from ~3 to ~5 h. These periodicities were commonly visible in individual animals both in high-pass-filtered and in unfiltered data. Furthermore, when all raw data from a given strain were pooled to get a 24-h ensemble average across all animals and days, the rhythms continued to be discernable. We fitted Fourier series to these form estimates to model the frequency structure of each strain and found significant effects of strain and an interaction between period and strain indicating significant genetic variation for rhythmicity in the ultradian range. The techniques employed in this study should have wider use in a range of organisms and fields.