Kacmaz, Halil

AbstractBackgroundIn preclinical studies whole gut transit (WGT) in mice is a gold‐standard “leading‐edge” approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known.MethodsWe used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild‐type Bl6/C57 mice under the standard 12 h light–dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light–dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed.Key ResultWhen complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit.Conclusion & InferencesDuring the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

BACKGROUND Enteroendocrine cells (EECs) in the gastrointestinal (GI) epithelium are specialized sensors of luminal chemicals and forces. Upon stimulation, EECs release a variety of neurotransmitters and hormones, but cellular mechanisms of excitation‐secretion coupling are poorly understood. We recently discovered that a population of EECs expresses the mechanosensitive ion channel Piezo2, which is necessary for force‐dependent intracellular Ca2+ increase and serotonin release. However, the molecular mechanism that links Piezo2 and serotonin release is unknown. In other sensory systems, the Cacna1h ‐encoded t‐type voltage‐gated calcium channel Cav3.2 is important in mechanotransduction for linking mechanical activation with neurotransmitter release. AIM to determine if Cav3.2 is expressed and functionally relevant in EECs. METHODS Quantitative RT‐PCR for Cacna1h mRNA expression was evaluated in colon epithelium of control and NeuroD1 Cre x RiboTag mice, in which actively transcribed mRNA specifically from EECs is tagged with hemagglutinin (HA). NeuroD1 Cre x tdTomato colon cross‐sections were fixed and immunolabeled with anti‐Cav3.2 antibody. Single primary NeuroD1 cre x GCaMP5 EECs were mechanically stimulated and Cav3.2 role in mechanotransduction was tested by pharmacology (Ca2+ free, nickel, mibefradil, ω‐agatoxin IVA) and Cacna1h siRNA knockdown. RESULTS Murine colon epithelium expressed Cav3.2 mRNA. In NeuroD1 Cre x RiboTag mouse, HA co‐localized with EEC marker Chromogranin A (Chga). NeuroD1+ EEC ribosomal trapped mRNA was enriched for markers of epithelium (Vil1), EEC (Chga), and enriched 7.0‐fold for Cav3.2 (n=3, p<0.05 compared to whole epithelium). Immunohistochemistry in NeuroD1 Cre x tdTomato showed that Cav3.2 co‐localized specifically with tdTomato (NeuroD1+) EECs. In primary NeuroD1+ EECs, Cav3.2 inhibition by all pharmacologic agents and siRNA knockdown inhibited mechanically induced Ca2+ response (96±1% for Ca2+ free, 71±9% nickel, 61±6% mibefradil, 80±4% ω‐agatoxin IVA and 96±1% Cav3.2 siRNA, n=3–18, p<0.05 compared to control untreated cells or cells treated with non‐targeted siRNA). CONCLUSIONS Cav3.2 is densely and specifically expressed in mouse colon EECs and it is important for regulating Ca2+ in EEC mechanotransduction. Future studies will determine how Cav3.2 channels contribute to EEC electrical excitability, excitation‐secretion coupling, and ultimately EEC Cav3.2 roles in GI physiology. Support or Funding Information Supported by NIH DK106456, NIH DK084567, and AGA RSA This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

AbstractBackgroundGastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading‐edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies.MethodWe have developed an easily achievable, labor‐saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time‐lapse video for offline analysis.Key ResultWe describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10‐15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach.Conclusion & InferencesA novel automated, inexpensive, and easily assembled whole gut transit setup is labor‐saving and allows minimal disruption to animal behavior compared with the conventional approach.