Dalbywashington2309

The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. selleckchem Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.