Protocol for Mice Behavioral Analysis in Response to Predator Cues

Mice exhibit defensive behaviors in response to various predator cues. When a mouse “senses”a predator at a close distance, it exhibits freezing behavior. Alternatively, when it senses bodilyexcretions from a predator, it escapes from the area. These behaviors are evolutionary responses topredators that help their increase survival. How animals sense the different types of predator-derivedcues and induce appropriate behaviors in response to the specific predator cues have largely remainedelusive.In this study, we aimed to establish a method to analyze mouse behavioral responses toward variousforms of predator-derived biological samples, such as cat saliva, which contain chemical cues. Wecategorized mouse responses to predator cue exposure as freezing, fear assessment, or exploratorybehavior, each of which is triggered by different levels of fear that the animal is experiencing. Thebehaviors were quantified manually and compared between the animals exposed to control andpredator-cue stimuli. We show that this protocol is effective in analyzing levels of fear in mice asthere is a significant increase in the occurrence of fear-based behaviors in mice exposed to cat saliva.Developing a strong protocol for quantifying fear-related behaviors is essential to understand brainmechanisms underlying behavioral responses induced by different types of predator cues in mice.Moreover, the present protocol can be further utilized to understand how different levels of fear areprocessed in an animal’s brain circuitry

Protocol for Mice Behavioral Analysis in Response to Predator Cues

Mice exhibit defensive behaviors in response to various predator cues. When a mouse “senses”a predator at a close distance, it exhibits freezing behavior. Alternatively, when it senses bodilyexcretions from a predator, it escapes from the area. These behaviors are evolutionary responses topredators that help their increase survival. How animals sense the different types of predator-derivedcues and induce appropriate behaviors in response to the specific predator cues have largely remainedelusive.In this study, we aimed to establish a method to analyze mouse behavioral responses toward variousforms of predator-derived biological samples, such as cat saliva, which contain chemical cues. Wecategorized mouse responses to predator cue exposure as freezing, fear assessment, or exploratorybehavior, each of which is triggered by different levels of fear that the animal is experiencing. Thebehaviors were quantified manually and compared between the animals exposed to control andpredator-cue stimuli. We show that this protocol is effective in analyzing levels of fear in mice asthere is a significant increase in the occurrence of fear-based behaviors in mice exposed to cat saliva.Developing a strong protocol for quantifying fear-related behaviors is essential to understand brainmechanisms underlying behavioral responses induced by different types of predator cues in mice.Moreover, the present protocol can be further utilized to understand how different levels of fear areprocessed in an animal’s brain circuitry

Rapid Assessment of Insect Steroid Hormone Entry Into Cultured Cells

Steroid hormones control development and homeostasis in a wide variety of animals by interacting with intracellular nuclear receptors. Recent discoveries in the fruit fly Drosophila melanogaster revealed that insect steroid hormones or ecdysteroids are incorporated into cells through a membrane transporter named Ecdysone Importer (EcI), which may become a novel target for manipulating steroid hormone signaling in insects. In this study, we established an assay system that can rapidly assess EcI-mediated ecdysteroid entry into cultured cells. Using NanoLuc Binary Technology (NanoBiT), we first developed an assay to detect ligand-dependent heterodimerization of the ecdysone receptor (EcR) and retinoid X receptor (RXR) in human embryonic kidney (HEK) 293T cells. We also developed HEK293 cells that stably express EcI. By combining these tools, we can monitor ecdysteroid entry into the cells in real time, making it a reliable system to assess EcI-mediated steroid hormone incorporation into animal cells

A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila

Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies

Coadaptation of the chemosensory system with voluntary exercise behavior in mice.

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process. Although the role of the vomeronasal chemosensory receptors in VWR activity remains to be determined, the current results suggest that these vomeronasal chemosensory receptors are important quantitative trait loci for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals

Increased body weight in mice with fragile X messenger ribonucleoprotein 1 (Fmr1) gene mutation is associated with hypothalamic dysfunction.

Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene are linked to Fragile X Syndrome, the most common monogenic cause of intellectual disability and autism. People affected with mutations in FMR1 have higher incidence of obesity, but the mechanisms are largely unknown. In the current study, we determined that male Fmr1 knockout mice (KO, Fmr1-/y), but not female Fmr1-/-, exhibit increased weight when compared to wild-type controls, similarly to humans with FMR1 mutations. No differences in food or water intake were found between groups; however, male Fmr1-/y display lower locomotor activity, especially during their active phase. Moreover, Fmr1-/y have olfactory dysfunction determined by buried food test, although they exhibit increased compulsive behavior, determined by marble burying test. Since olfactory brain regions communicate with hypothalamic regions that regulate food intake, including POMC neurons that also regulate locomotion, we examined POMC neuron innervation and numbers in Fmr1-/y mice. POMC neurons express Fmrp, and POMC neurons in Fmr1-/y have higher inhibitory GABAergic synaptic inputs. Consistent with increased inhibitory innervation, POMC neurons in the Fmr1-/y mice exhibit lower activity, based on cFOS expression. Notably, Fmr1-/y mice have fewer POMC neurons than controls, specifically in the rostral arcuate nucleus, which could contribute to decreased locomotion and increased body weight. These results suggest a role for Fmr1 in the regulation of POMC neuron function and the etiology of Fmr1-linked obesity

Increased body weight in mice with fragile X messenger ribonucleoprotein 1 (Fmr1) gene mutation is associated with hypothalamic dysfunction

Abstract Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene are linked to Fragile X Syndrome, the most common monogenic cause of intellectual disability and autism. People affected with mutations in FMR1 have higher incidence of obesity, but the mechanisms are largely unknown. In the current study, we determined that male Fmr1 knockout mice (KO, Fmr1 −/y ), but not female Fmr1 −/−, exhibit increased weight when compared to wild-type controls, similarly to humans with FMR1 mutations. No differences in food or water intake were found between groups; however, male Fmr1 −/y display lower locomotor activity, especially during their active phase. Moreover, Fmr1 −/y have olfactory dysfunction determined by buried food test, although they exhibit increased compulsive behavior, determined by marble burying test. Since olfactory brain regions communicate with hypothalamic regions that regulate food intake, including POMC neurons that also regulate locomotion, we examined POMC neuron innervation and numbers in Fmr1 −/y mice. POMC neurons express Fmrp, and POMC neurons in Fmr1 −/y have higher inhibitory GABAergic synaptic inputs. Consistent with increased inhibitory innervation, POMC neurons in the Fmr1 −/y mice exhibit lower activity, based on cFOS expression. Notably, Fmr1 −/y mice have fewer POMC neurons than controls, specifically in the rostral arcuate nucleus, which could contribute to decreased locomotion and increased body weight. These results suggest a role for Fmr1 in the regulation of POMC neuron function and the etiology of Fmr1-linked obesity