Septins assemble on the cortex and restore normal cell shape by retracting aberrantly protruding membranes and promoting cortical contraction during amoeboid motility.

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Pregnancy and childbirth are both natural occurring events, but still little is known about the signaling mechanisms that induce contractions. Throughout the world, premature labor occurs in 12% of all pregnancies with 36% of infant deaths resulting from preterm related causes [1]. Even though the cause of preterm labor can vary, understanding alternative signaling pathways which affect muscle contraction could provide additional treatment options in stopping premature labor. The uterus is composed of smooth muscle which is innervated with a plexus of nerves that cover the muscle fibers. Smooth muscle can be stimulated or modulated by many sources such as neurotransmitters [dopamine], hormones [estrogen], peptides [oxytocin] and amines. In this study, we are focusing on the biogenic monoamine tyramine which is produced in the tyrosine catecholamine biosynthesis pathway. Tyramine is known to be associated with peripheral vasoconstriction, increased cardiac output, increased respiration, elevated blood glucose and the release of norepinephrine. Our research has found tyramine, and its specific receptor TAAR1 [2], to be localized within mouse uterus and that this monoamine can induce uterine contractions at levels similar to oxytocin.Research that focuses on reproduction commonly use techniques involving immunohistochemistry or histological staining to identify localization of key modulators and receptors or they observe general morphology in fixed whole or sectioned tissue [ Fig. 1]. In studies involving muscle tissue, force transduction measurements allow the researcher to quantify differences in contractile force. These methods can be used with the addition of agonists and antagonists which help decipher the mechanisms that may modulate the contraction. A drawback of these methods is that muscle contraction is a complex system that relies on feedback mechanisms and without observing the tissue in real time, the researcher would be making interpretations based on random snapshots in time. To overcome this limitation, we have developed an imaging technique that can observe muscle signaling dynamics in real time.This protocol was approved through the IACUC protocol 15-1388T at Arizona State University and the mouse uterine tissues were derived from GFP LifeAct transgenic mice [3]. These mice contain a transgene encoding a 17-amino acid peptide called LifeAct, which binds F-actin. This peptide is coexpressed with GFP which enables the actin cytoskeleton to be imaged both in vivo and in vitro. Actin is a key component of muscle tissue and this transgenic model allows researchers the ability to visualize muscle contractions. Combining this tissue with the TAAR1 receptor antibody conjugated to a fluorescent probe [antibody labeling kit, Thermo Fisher, #A20186] is a tool that can be used to visualize the actual response and timing of a receptor mediated event. Imaging was conducted using a dipping lens on a Leica SP5 confocal microscope which allowed the tissue to continue normal peristaltic contractile activity. Time-...

Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.

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