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Feeding is critical for survival, and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs.Obesity and anorexia nervosa are two health conditions related to food intake. Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is, why animals seek and consume food. Measuring an animal’s food intake is usually done by weighing food containers. However, this can be inaccurate due to the small amount of food that rodents eat. As for studying feeding motivation, this can involve calculating the number of times an animal presses a lever to receive a food pellet. These tests are typically conducted in hour-long sessions in temporary testing cages, called operant boxes. Yet, these tests only measure a brief period of a rodent's life. In addition, it takes rodents time to adjust to these foreign environments, which can introduce stress and may alter their feeding behaviour. To address this, Matikainen-Ankney, Earnest, Ali et al. developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention. This ‘Feeding Experimentation Device’ (FED3) features a pellet dispenser and two ‘nose-poke’ sensors to measure total food intake, as well as motivation for and learning about food rewards. The battery-powered, wire-free device fits in standard home cages, enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals. This means researchers can relate data to circadian rhythms and meal patterns, as Matikainen-Ankney did here. Moreover, the device software is open-source so researchers can customise it to suit their experimental needs. It can also be programmed to synchronise with other instruments used in animal experiments, or across labs running the same behavioural tasks for multi-site studies. Used in this way, it could help improve reproducibility and reliability of results from such studies. In summary, Matikainen-Ankney et al. have presented a new practical solution for studying food-related behaviours in mice and rats. Not only could the device be useful to researchers, it may also be suitable to use in educational settings such as teaching labs and classrooms.

SummaryFeeding is critical for survival and disruption in the mechanisms that govern food intake underlie disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation: The Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs. In this paper we demonstrate the utility of FED3 in a range of experimental paradigms.In BriefUsing a novel, high-throughput home cage feeding platform, FED3, Matikainen-Ankney et al. quantify food intake and operant learning in groups of mice conducted at multiple institutions across the globe. Results include rates of operant efficiency, circadian feeding patterns, and operant optogenetic self-stimulation.HighlightsThe Feeding Experimentation Device version 3(FED3) records food intake and operant behavior in rodent home cages.Analysis of food intake includes total intake, meal pattern analysis, and circadian analysis of feeding patterns.FED3 also allows for operant behavioral assays to examine food learning and motivation.

Background Genetic factors influence the physical and neurobehavioral manifestations of prenatal alcohol exposure (PAE). Animal models allow the investigation of specific genes that confer vulnerability to, or protection from, birth defects associated with fetal alcohol spectrum disorders (FASDs). The objective of the present experiments was to determine if genetic alterations in the Sonic Hedgehog (Shh) signaling pathways affect the vulnerability to PAE-induced skeletal defects involving the forelimbs and/or hindlimbs. Method Wild-type C57BL/6J female mice were bred with males in which one copy of the Shh or Gli2 genes had been knocked out, to produce litters with both wild-type (+/+) and heterozygous (+/−) embryos. Alcohol doses (two injections of 2.9 g/kg, 4 hours apart) or vehicles were administered starting at gestational day (GD) 9.25, 9.5, or 9.75, a critical exposure time for inducing limb defects. Limb defects were examined at GD 17 using a dysmorphology scale based on abnormalities ranging from increased interdigital spacing to the deletion of multiple fingers and the ulna. Results Alcohol treatment caused a high incidence of forelimb defects, particularly on the right side, that was higher in Shh+/− and Gli2+/− fetuses compared to wild-type fetuses. Dysmorphology scores were also significantly higher in the Shh+/− and Gli2+/− mice. Conclusions These results extend previous findings demonstrating enhanced sensitivity to PAE-induced craniofacial dysmorphology and support the hypothesis that genetic alterations in the Shh signaling pathway influences the vulnerability to alcohol-induced birth defects. Moreover, these results emphasize the importance of understanding the interactions between genes and prenatal exposure to alcohol or other teratogens. Birth Defects Research, 2017.© 2017 Wiley Periodicals, Inc.

Background While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb. Methods Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression. Results The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated. Conclusion Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research (Part A), 2016.© 2016 Wiley Periodicals, Inc.

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

Smoking tobacco products is the leading cause of preventable death worldwide. Coordinated efforts have successfully reduced tobacco cigarette smoking in the United States; however, electronic cigarettes (e-cigarette) and other electronic nicotine delivery systems (ENDS) recently have replaced traditional cigarettes for many users. While the clinical risks associated with long-term ENDS use remain unclear, advancements in preclinical rodent models will enhance our understanding of their overall health effects. This review examines the peripheral and central effects of ENDS-mediated exposure to nicotine and other drugs of abuse in rodents and evaluates current techniques for implementing ENDS in preclinical research.

Background MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure. Methods On GD7, pregnant Mns1+/- dams were administered 2 doses of ethanol (5.8 g/kg total) or vehicle 4 hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1 = normal, 2 to 7 = defects escalating in severity). Craniofacial and brain abnormalities were also assessed. Results Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1+/- (64.29%) and Mns1-/- mice (92.31%). PAE Mns1-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum. Conclusions These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

In many experiments using fetal mice, it is necessary to determine the sex of the individual fetus. However, other than genotyping for sex-specific genes, there is no convenient, reliable method of sexing mice between gestational day (GD) 16.5 and GD 18.0. We designed a rapid, relatively simple visual method to determine the sex of mouse fetuses in the GD 16.5-GD 18.0 range that can be performed as part of a routine morphological assessment. By examining the genitalia for the presence or absence of key features, raters with minimal experience with the method were able to correctly identify the sex of embryos with 99% accuracy, while raters with no experience were 95% accurate. The critical genital features include: the presence or absence of urethral seam or proximal urethral meatus; the shape of the genitalia, and the presence or absence of an area related to the urethral plate. By comparing these morphological features of the external genitalia, we show a simple, accurate, and fast way to determine the sex of late stage mouse fetuses. Integrating this method into regular morphological assessments will facilitate the determination of sex differences in fetuses between GD 16.5 and GD 18.0.

While pharmacological activation of the Hedgehog (HH) signaling pathway may have therapeutic benefits for developmental and adult diseases, its teratogenic potential is of concern. The membrane molecule Smoothened (SMO) transduces HH signaling and can be acutely modulated by antagonists and agonists. The objective of the current experiments was to determine how maternal treatment with the Smo agonist, SAG, affects the developing limb.Pregnant C57BL/6J mice received a single injection of SAG (15, 17, or 20 mg/kg, i.p.) or its vehicle on gestational day (GD) 9.25, the time of limb bud induction. Embryos were examined on GD 15 for gross dysmorphology and skeletal staining was performed to visualize the number and type of digits on the fore- and hindlimbs. Additionally, in situ hybridization was performed 4 hr after GD 9.25 SAG administration to determine SAG's effects on Gli1 and Gli2 mRNA expression.The most prevalent effect of SAG was the dose-dependent induction of pre-axial polydactyly; defects ranged from a broad thumb to the duplication of two finger-like digits on the preaxial side of the thumb. The highest SAG dose was effective in ca. 80% of the embryos and increased Gli1 and Gli2 mRNA expression in the limb bud, with Gli1 mRNA being the most upregulated.Preaxial polydactyly can be caused in the developing embryo by acute maternal administration of a Smo agonist that activates HH signaling. These results are consistent with the preaxial polydactyly induced in developmental disorders associated with mutations in HH signaling genes.Birth Defects Research 109:49-54, 2017. © 2016 Wiley Periodicals, Inc.