014 Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex

Introduction Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance within cortical circuits. This balance is impaired in several disorders that are also associated with altered diurnal rhythms, yet few studies examined diurnal rhythms of PNNs or PV cells. Methods We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0, 6, 12, and 18. To examine changes in inhibitory and excitatory inputs to PV cells, we measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs was conducted to determine the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA:NMDA) at ZT18 vs. ZT6. Finally, the number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, was also assessed. Results Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark compared with the light phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. There was an increased AMPA:NMDA ratio at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing OTX2 showed a strong trend toward an increase from ZT6 to ZT18, with no differences in non-PV-containing cells. Conclusion Diurnal fluctuations in PNNs and PV cells alter cortical excitatory:inhibitory balance. Detailed understanding of how these fluctuations are regulated should provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms. Ongoing studies are examining diurnal fluctuations in downstream signaling after PNN removal. Support (if any) Washington State University Alcohol and Drug Abuse Research Program, NIH GM134789 (JHH); NIH DA033404 (BAS), DA040965 (BAS, TEB, SAA); NIH NS078498 (JPW); NIH P30 NS061800 (SAA); and Agence Nationale de la Recherche ANR-18-CE16-0013-01 (AP and AAD).

Influence of industry standard feeding frequencies on behavioral patterns and rumen and fecal bacterial communities in Holstein and Jersey cows

This study aimed to evaluate the effects of feeding frequency on behavioral patterns and on diurnal fermentation and bacteriome profiles of the rumen and feces in Holstein and Jersey cows. Ten Holstein and 10 Jersey cows were offered a TMR (53:47 forage-to-concentrate ratio dry matter basis) for ad libitum consumption and were randomly allocated within breed to one of the following feeding frequencies: (1) TMR delivered 1×/d (at 0600 h) or (2) TMR delivered 2×/d (at 0600 and 1800 h). The experiment lasted for 28 d with the first 14 d for cow adaptation to the Calan gates and the next 14 d for data collection. On d 23 and 24, an observer manually recorded the time budget (time spent lying, eating, drinking, standing, and milking), rumination activity, and number of visits to the feeding gate from each animal. On d 28, 5 concomitant collections of rumen and fecal samples were performed at intervals of 6 h via esophageal tubing and fecal grab, respectively. The bacteriome composition from these samples was determined through sequencing of the V4 region of the 16S rRNA gene. Feeding frequency did not affect behavioral patterns; however, Holstein cows spend more time lying (15.4 vs. 13.5 ± 0.8 h) and ruminating (401 vs. 331 ± 17.5 min) than Jersey cows. Fermentation profiles were similar by feeding frequency in both breeds. While no major diurnal fluctuations were observed in the fecal bacterial community from both breeds, diurnal fluctuations were identified in the rumen bacterial community from Holstein cows which appeared to follow pH responses. Overall, the bacterial community composition was not differentiated by industry standard feeding frequencies but was differentiated by breed and sample type.

Acute Beetroot Juice Supplementation Attenuates Morning-Associated Decrements in Supramaximal Exercise Performance in Trained Sprinters

Diurnal fluctuations in power output have been well established with power loss typically occurring in morning (AM) times. Beetroot juice (BRJ) is a source of dietary nitrate that possess ergogenic properties, but it is unknown if ingestion can mitigate performance decrements in the morning. The purpose of this study was to examine the effects of acute BRJ supplementation on diurnal fluctuations in anaerobic performance in trained sprinters. Male Division 1 National Collegiate Athletic Association (NCAA) sprinters (n = 10) participated. In a double-blinded crossover study design, participants completed three counterbalanced exercise trials under different conditions: Morning–placebo (8:00 HR, AM-PL), Morning–BRJ (8:00 HR, AM-BRJ), and Afternoon–no supplement (15:00 HR, PM). For each trial, participants completed 3 × 15 s Wingate anaerobic tests separated by 2 min of rest. Each trial was separated by a 72 h washout period. Mean power output (p = 0.043), anaerobic capacity (p = 0.023), and total work (p = 0.026) were significantly lower with the AM-PL condition compared to PM. However, BRJ supplementation prevented AM losses of mean power output (p = 0.994), anaerobic capacity (p = 0.941), and total work (p = 0.933) in the AM-BRJ compared to the PM condition. Rate of perceived exertion was not significantly different between any conditions (p = 0.516). Heart rate was significantly lower during the AM-BRJ condition compared to AM-PL (p = 0.030) and PM (p < 0.001). Findings suggest anaerobic capacity suffers during AM versus PM times in trained sprinters, but BRJ ingestion abolishes AM-associated decrements in performance.

Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques

Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17β-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine.