Quantitative evaluation of orofacial motor function in mice: The pasta gnawing test, a voluntary and stress-free behavior test.

BACKGROUND: Evaluation of motor deficits in rodents is mostly restricted to limb motor tests that are often high stressors for the animals. NEW METHOD: To test rodents for orofacial motor impairments in a stress-free environment, we established the pasta gnawing test by measuring the biting noise of mice that eat a piece of spaghetti. Two parameters were evaluated, the biting speed and the biting peaks per biting episode. To evaluate the power of this test compared to commonly used limb motor and muscle strength tests, three mouse models of Parkinson's disease, amyotrophic lateral sclerosis and Niemann-Pick disease were tested in the pasta gnawing test, RotaRod and wire suspension test. RESULTS: Our results show that the pasta gnawing test reliably displays orofacial motor deficits. COMPARISON WITH EXISTING METHODS: The test is especially useful as additional motor test in early onset disease models, since it shows first deficits later than the RotaRod or wire suspension test. The test depends on a voluntary eating behavior of the animal with only a short-time food deprivation and should thus be stress-free. CONCLUSIONS: The pasta gnawing test represents a valuable tool to analyze orofacial motor deficits in different early onset disease models.

Comparisons of chewing rhythm, craniomandibular morphology, body mass and height between mothers and their biological daughters.

OBJECTIVE: To study and compare the relationships between mean chewing cycle duration, selected cephalometric variables representing mandibular length, face height, etc., measured in women and in their teenage or young-adult biological daughters. DESIGN: Daughters were recruited from local high schools and the University of Michigan School of Dentistry. Selection criteria included healthy females with full dentition, 1st molar occlusion, no active orthodontics, no medical conditions nor medication use that could interfere with normal masticatory motor function. Mothers had to be biologically related to their daughters. All data were obtained in the School of Dentistry. Measurements obtained from lateral cephalograms included: two "jaw length" measures, condylion-gnathion and gonion-gnathion, and four measures of facial profile including lower anterior face height, and angles sella-nasion-A point (SNA), sella-nasion-B point (SNB) and A point-nasion-B point (ANB). Mean cycle duration was calculated from 60 continuous chewing cycles, where a cycle was defined as the time between two successive maximum jaw openings in the vertical dimension. Other variables included subject height and weight. Linear and logistic regression analyses were used to evaluate the mother-daughter relationships and to study the relationships between cephalometric variables and chewing cycle duration. RESULTS: Height, weight, Co-Gn and Go-Gn were significantly correlated between mother-daughter pairs; however, mean cycle duration was not (r(2)=0.015). Mean cycle duration was positively correlated with ANB and height in mothers, but negatively correlated with Co-Gn in daughters. CONCLUSIONS: Chewing rate is not correlated between mothers and daughters in humans.

Gene networks, occlusal clocks, and functional patches: new understanding of pattern and process in the evolution of the dentition.

Our understanding of the evolution of the dentition has been transformed by advances in the developmental biology, genetics, and functional morphology of teeth, as well as the methods available for studying tooth form and function. The hierarchical complexity of dental developmental genetics combined with dynamic effects of cells and tissues during development allow for substantial, rapid, and potentially non-linear evolutionary changes. Studies of selection on tooth function in the wild and evolutionary functional comparisons both suggest that tooth function and adaptation to diets are the most important factors guiding the evolution of teeth, yet selection against random changes that produce malocclusions (selectional drift) may be an equally important factor in groups with tribosphenic dentitions. These advances are critically reviewed here.

Mastication markedly affects mandibular condylar cartilage growth, gene expression, and morphology.

INTRODUCTION: Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing affects condylar cartilage growth of the mandible. METHODS: Mice were fed diets with varying hardness. Genes specific to neural crest-derived cells were measured by real-time polymerase chain reaction to compare the expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen. Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase chain reaction was used to assess the expression levels of osteopontin and type X collagen. RESULTS: Markers including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and Musashi1 expression were lower in mice fed a hard diet. After 4 weeks, numerous osteopontin and type X collagen-positive cells were observed in mice fed a mixed diet. CONCLUSIONS: Mastication affects the balance between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attributed to the presence of neural crest-derived cells.

Tbx1 is required autonomously for cell survival and fate in the pharyngeal core mesoderm to form the muscles of mastication.

Velo-cardio-facial/DiGeorge syndrome, also known as 22q11.2 deletion syndrome, is a congenital anomaly disorder characterized by craniofacial anomalies including velo-pharyngeal insufficiency, facial muscle hypotonia and feeding difficulties, in part due to hypoplasia of the branchiomeric muscles. Inactivation of both alleles of mouse Tbx1, encoding a T-box transcription factor, deleted on chromosome 22q11.2, results in reduction or loss of branchiomeric muscles. To identify downstream pathways, we performed gene profiling of microdissected pharyngeal arch one (PA1) from Tbx1(+/+) and Tbx1(-/-) embryos at stages E9.5 (somites 20-25) and E10.5 (somites 30-35). Basic helix-loop-helix (bHLH) transcription factors were reduced, while secondary heart field genes were increased in expression early and were replaced by an increase in expression of cellular stress response genes later, suggesting a change in gene expression patterns or cell populations. Lineage tracing studies using Mesp1(Cre) and T-Cre drivers showed that core mesoderm cells within PA1 were present at E9.5 but were greatly reduced by E10.5 in Tbx1(-/-) embryos. Using Tbx1(Cre) knock-in mice, we found that cells are lost due to apoptosis, consistent with increase in expression of cellular stress response genes at E10.5. To determine whether Tbx1 is required autonomously in the core mesoderm, we used Mesp1(Cre) and T-Cre mesodermal drivers in combination with inactivate Tbx1 and found reduction or loss of branchiomeric muscles from PA1. These mechanistic studies inform us that Tbx1 is required upstream of key myogenic genes needed for core mesoderm cell survival and fate, between E9.5 and E10.5, resulting in formation of the branchiomeric muscles.

From molecules to mastication: the development and evolution of teeth.

Teeth are unique to vertebrates and have played a central role in their evolution. The molecular pathways and morphogenetic processes involved in tooth development have been the focus of intense investigation over the past few decades, and the tooth is an important model system for many areas of research. Developmental biologists have exploited the clear distinction between the epithelium and the underlying mesenchyme during tooth development to elucidate reciprocal epithelial/mesenchymal interactions during organogenesis. The preservation of teeth in the fossil record makes these organs invaluable for the work of paleontologists, anthropologists, and evolutionary biologists. In addition, with the recent identification and characterization of dental stem cells, teeth have become of interest to the field of regenerative medicine. Here, we review the major research areas and studies in the development and evolution of teeth, including morphogenesis, genetics and signaling, evolution of tooth development, and dental stem cells.

Genomic alterations in breast cancer patients in betel quid and non betel quid chewers.

Betel Quid (BQ) chewing independently contributes to oral, hepatic and esophageal carcinomas. Strong association of breast cancer risk with BQ chewing in Northeast Indian population has been reported where this habit is prodigal. We investigated genomic alterations in breast cancer patients with and without BQ chewing exposure. Twenty six BQ chewers (BQC) and 17 non BQ chewer (NBQC) breast cancer patients from Northeast India were analyzed for genomic alterations and pathway networks using SNP array and IPA. BQC tumors showed significantly (P<0.01) higher total number of alterations, as compared with NBQC tumors, 48 ± 17% versus 32 ± 25 respectively. Incidence of gain in fragile sites in BQC tumors were significantly (P<0.001) higher as compared with NBQC tumors, 34 versus 23% respectively. Two chromosomal regions (7q33 and 21q22.13) were significantly (p<0.05) associated with BQC tumors while two regions (19p13.3-19p12 and 20q11.22) were significantly associated with NBQC tumors. GO terms oxidoreductase and aldo-keto reductase activity in BQC tumors in contrast to G-protein coupled receptor protein signaling pathway and cell surface receptor linked signal transduction in NBQC tumors were enriched in DAVID. One network "Drug Metabolism, Molecular Transport, Nucleic Acid Metabolism" including genes AKR1B1, AKR1B10, ETS2 etc in BQC and two networks "Molecular Transport, Nucleic Acid Metabolism, Small Molecule Biochemistry" and "Cellular Development, Embryonic Development, Organismal Development" including genes RPN2, EMR3, VAV1, NNAT and MUC16 etc were seen in NBQC. Common alterations (>30%) were seen in 27 regions. Three networks were significant in common regions with key roles of PTK2, RPN2, EMR3, VAV1, NNAT, MUC16, MYC and YWHAZ genes. These data show that breast cancer arising by environmental carcinogens exemplifies genetic alterations differing from those observed in the non exposed ones. A number of genetic changes are shared in both tumor groups considered as crucial in breast cancer progression.

Interleukin-6 maintains glucose homeostasis to support strenuous masseter muscle activity in mice.

The cytokine interleukin-6 (IL-6) is released from working skeletal muscles and reportedly plays key roles in their glucose homeostasis. However, it is unclear whether IL-6 plays such roles in the masseter muscle (MM), which is important in normal and pathological chewing behaviors, such as bruxism and/or prolonged clenching. When restrained (R+) in a narrow cylinder blocked at the front end with a thin plastic strip, a mouse gnaws away (G+) the strip to escape. The absolute weight of plastic gnawed away serves as an index of MM activity. Using this model, we examined the roles of IL-6 in MM with the following results. R+G+ increased the expression levels of IL-6 and glucose transporter 4 (Glut4) mRNAs in MM and the serum level of IL-6 protein. IL-6-deficient mice exhibited about 60% less gnawing activity than wild-type mice at 3-4 h after the start of R+G+, slower recovery of glycogen levels (indicating poorer glucose supply) in MM after R+G+, and no significant change in Glut4 mRNA in MM upon R+G+. During an R+G+ test conducted after "training" (repeated R+G+ sessions), wild-type mice exhibited greater gnawing activity than untrained controls, but no increase in IL-6 mRNA in MM. IL-6 mRNA increased in MM when hard food was eaten by mice raised on soft food for 3 weeks from weaning, but not in those raised on (accustomed to) hard food. Thus, IL-6 may maintain glucose homeostasis in MM in support of unusually strenuous activity, but not of accustomed activity levels.

Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice.

Pharmacogenomics is yet to fulfill its promise of manifestly altering clinical medicine. As one example, a predictive test for tardive dyskinesia (TD) (an adverse drug reaction consequent to antipsychotic exposure) could greatly improve the clinical treatment of schizophrenia but human studies are equivocal. A complementary approach is the mouse-then-human design in which a valid mouse model is used to identify susceptibility loci, which are subsequently tested in human samples. We used inbred mouse strains from the Mouse Phenome Project to estimate the heritability of haloperidol-induced activity and orofacial phenotypes. In all, 159 mice from 27 inbred strains were chronically treated with haloperidol (3 mg kg(-1) per day via subdermal slow-release pellets) and monitored for the development of vacuous chewing movements (VCMs; the mouse analog of TD) and other movement phenotypes derived from open-field activity and the inclined screen test. The test battery was assessed at 0, 30, 60, 90 and 120 days in relation to haloperidol exposure. As expected, haloperidol caused marked changes in VCMs, activity in the open field and extrapyramidal symptoms (EPS). Unexpectedly, factor analysis demonstrated that these measures were imprecise assessments of a latent construct rather than discrete constructs. The heritability of a composite phenotype was ∼0.9 after incorporation of the longitudinal nature of the design. Murine VCMs are a face valid animal model of antipsychotic-induced TD, and heritability estimates from this study support the feasibility of mapping of susceptibility loci for VCMs.

Myofiber properties of mouse mylohyoid muscle in the growth period.

The mouse mylohyoid muscle belongs to the mastication-related suprahyoid muscle group. It shows a plate-like morphology and forms the mouth floor. There have been no reports on the characteristics of the mouse mylohyoid muscle fibers, and especially on their functional role during ingestion action, and many points remain unclear. We examined the mouse mylohyoid muscle at both the transcriptional and protein levels by RT-PCR, immunohistochemistry, and Western Blotting. MyHC-2b, which is expressed in almost all head and neck muscles and is thought to play a role in rapid mastication movement, was not detected in the mouse mylohyoid muscle. This result suggests that the mouse mylohyoid muscle has a special function and does not directly function during ingestion.

Expression profiling reveals heightened apoptosis and supports fiber size economy in the murine muscles of mastication.

Distinctions between craniofacial and axial muscles exist from the onset of development and throughout adulthood. The masticatory muscles are a specialized group of craniofacial muscles that retain embryonic fiber properties in the adult, suggesting that the developmental origin of these muscles may govern a pattern of expression that differs from limb muscles. To determine the extent of these differences, expression profiling of total RNA isolated from the masseter and tibialis anterior (TA) muscles of adult female mice was performed, which identified transcriptional changes in unanticipated functional classes of genes in addition to those attributable to fiber type. In particular, the masseters displayed a reduction of transcripts associated with contractile and cytoskeletal load-sensing and anabolic processes, and heightened expression of genes associated with stress. Associated with these observations was a significantly smaller fiber cross-sectional area in masseters, significantly elevated load-sensing signaling (phosphorylated focal adhesion kinase), and increased apoptotic index in masseters compared with TA muscles. Based on these results, we hypothesize that masticatory muscles may have a fundamentally different strategy for muscle design, compared with axial muscles. Specifically there are small diameter fibers that have an attenuated ability to hypertrophy, but an increased propensity to undergo apoptosis. These results may provide insight into the molecular basis for specific muscle-related pathologies associated with masticatory muscles.

Compulsive behavior in the 5-HT2C receptor knockout mouse.

The efficacy of serotonergic pharmacotherapy indicates that serotonin (5-HT) plays a role in the treatment, if not the etiology, of obsessive-compulsive disorder (OCD). While some clinical evidence implicates 5-HT(2C) receptors in this disorder, a definitive function has yet to be validated. We hypothesized that 5-HT(2C) receptor knockout (KO) mice may display compulsive-like behavior. This paper describes characterization of several distinct, highly organized behaviors in mice lacking functional 5-HT(2C) receptors, which supports a compulsive-like syndrome.Compulsive-like behavior was assessed in male 5-HT(2C) receptor KO and wildtype (WT) mice. Chewing of non-nutritive clay, chewing patterns on plastic-mesh screens, and the frequency of head dipping were measured. 5-HT(2C) receptor KO mice chewed more clay, produced a distinct pattern of "neat" chewing of plastic screens and exhibited reduced habituation of head dipping activity compared to WT mice. We conclude that the 5-HT(2C) receptor null mutant mouse provides a promising model of compulsive behavior and a means to further explore the role of 5-HT in OCD.

The role of genetic and early environmental factors in determining apomorphine susceptibility.

RATIONALE: There is ample evidence that rats show large individual differences in their response to dopaminergic drugs, such as apomorphine. OBJECTIVE: The aim of the present study was to investigate the role of genetic and (early) environmental factors in determining the adult susceptibility to apomorphine. Four experiments were performed: In experiment 1, the original selective breeding of rats for apomorphine susceptibility (leading to APO-SUS and APO-UNSUS rats) was extended and replicated in an independent group of Wistar rats. In experiment 2, APO-SUS males were cross-bred with APO-UNSUS females and vice versa. In experiment 3 APO-SUS litters were cross-fostered to APO-UNSUS mothers or infostered to unknown APO-SUS mothers and vice versa. In experiment 4 APO-SUS and APO-UNSUS rats were maternally deprived on postnatal day 9, for a single 24-h period. METHODS: Adult rats were subcutaneously injected with 1.5 mg/kg apomorphine and their gnawing response was automatically recorded in a gnawing box for 45 min. RESULTS: In experiment 1, the original breeding was extended up to generation 24, leading to a strong and consistent difference in gnawing scores. The replication experiment also succeeded in differentiating APO-SUS and APO-UNSUS. The cross breeding experiments showed that the APO-SUS/UNSUS offspring showed gnawing scores in between the original selection lines. Cross-fostering APO-SUS with APO-UNSUS significantly reduced the gnawing response in the offspring, whereas it did not affect the gnawing score in the APO-UNSUS animals. Maternal deprivation had the opposite effect: increase in gnawing response in APO-UNSUS, with no effect in APO-SUS. CONCLUSION: The results show a clear-cut contribution of both genetic and early environmental factors to the susceptibility of apomorphine.

Quantitative trait loci affecting methamphetamine responses in BXD recombinant inbred mouse strains.

Individual differences in most behavioral and pharmacological responses to abused drugs are dependent on both genetic and environmental factors. The genetic influences on the complex phenotypes related to drug abuse have been difficult to study using classical genetic analyses. Quantitative trait locus (QTL) mapping is a method that has been used successfully to examine genetic contributions to some of these traits by correlating allelic variation in polymorphic genetic markers of known chromosomal location with variation in drug-response phenotypes. We evaluated several behavioral responses to multiple doses of methamphetamine (METH) in C57BL/6J (B6), DBA/2J (D2), and 25 of their recombinant inbred (BXD RI) strains. Stereotyped chewing, horizontal home cage activity, and changes in body temperature after 0, 4, 8, or 16 mg/kg METH, as well as stereotyped climbing behavior after 16 mg/kg METH, were examined. Associations (p < 0.01) between METH sensitivity and allelic status at multiple microsatellite genetic markers were subsequently determined for each response. QTLs were provisionally identified for each phenotype, some unique to a particular behavior and others that appeared to influence multiple phenotypes. Candidate genes suggested by these analyses included several that mapped near genes relevant for the neurotransmitters acetylcholine and glutamate. The locations of QTLs provisionally identified by this analysis were compared with QTLs hypothesized in other studies to influence methamphetamine- and cocaine-related phenotypes. In several instances, QTLs appeared to overlap, which is consistent with idea that common neural substrates underlie some responses to psychostimulants.

[Interbreed differences in cattle by "phenes"]. / Mezhporodnye razlichia krupnogo rogatogo skota po "fenam".

Significant interbreed differences (between Simmentals and Black-and-White cattle) for 16 phenotypes of molar surface structure were detected in milk-cows. In Black-and-White cattle, 15 phenotypic variants of the head colour marks correlating with the body colour were found.

Genetic and epigenetic regulation of craniofacial development.

This paper reviews a series of odontometric, anthropometric and cephalometric investigations of genetic and epigenetic regulation of dental, occlusal and craniofacial development. The results show that genes encoded with the X and Y chromosomes regulate the development of tooth crowns and roots. The Y chromosome seems to affect general somatic growth by regulating both cell function and mitotic activity. The effects of the X chromosome seem to be more restricted and include influences on the growth of cartilaginous structures and dental enamel. The findings also indicate that high masticatory stress promotes jaw growth and decreases occlusal variation, supporting the hypothesis that masticatory activity regulates occlusal and craniofacial development. In addition, the findings suggest that nerve growth may affect bone growth in the craniofacial skeleton.