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Figure 12
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In the pFRG/RTN, PGE increased the frequency of respiratory (NK1R-expressing) neurons. This PGE effect was absent in brainstem slice cultures lacking EP3R (; –). EP3Rs were present in …

Table 6

PGE increases the frequency of pFRG/RTN neurons and decreases the frequency of preBötC neurons.The mean frequencies of NK1R-positive cells during the control period or during exposure to 10 nM PGE …

To further characterize the PGE 2 signaling during hypercapnia, we blocked its main receptor, EP3R. Notably, pharmacological blocking of EP receptors (using AH6809, 10 µM) abolished the hypercapnic response ( GENTLE SOULS Camelia Peep Toe Bootie jFiNAFij
, 7 DIV), in line with our in vivo data from Ptger3 -/- mice.

pFRG/RTN slices (7 DIV) from Ptger3 -/- mice did not respond to hypercapnia ( Figure 13c–d ). Thus, EP3R is important for pFRG/RTN CO 2 responsiveness. We next generated a lentiviral vector in which the mouse EP3R ( Ptger3 ) promoter controls the expression of the red light-activated halorhodopsin Halo57 fused to eGFP ( Figure 13e ). After transduction, we detected eGFP expression in 90 ± 6% of Phox2b-positive neurons in the pFRG/RTN ( E8 by Miista Reanna Woven Platform Sneaker H68dcimWW
). Stimulation by red (625 nm) light of the transduced brainstem slice cultures (7 DIV) triggered hyperpolarization of Ptger3 -halo57-expressing cells and immediately reduced the calcium signaling frequency of both the network and individual NK1R + neurons ( Figure 13—figure supplement 1 ). This finding indicates a fundamental role for Ptger3- expressing cells in the network. Additionally, the response to hypercapnia in the pFRG/RTN was abolished during the light-induced silencing of Ptger3- expressing cells. The CO 2 response was also reversed by the light-induced halo57 hyperpolarization of Ptger- expressing cells ( Figure 13f–g , Table 7 ).

Figure 13 with 1 supplement
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Pharmacological inhibition of EP3R by the EP receptor antagonist AH6809 inhibited the response to hypercapnia (increased pCO[CO↑]) in the pFRG/RTN (N=6, n=472, N.S.; –). The hypercapnic response …

EP antagonist data.

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Hypercapnia EP3R data.

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Optogenetics data.

Author response image 2
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First, we note that in the Baker, 2016 paper, one of the lines with expression in the MB γ dorsal neurons (MB419B) showed a strong courtship memory phenotype upon silencing with shibireTS (in the secondary screen that is actually identical to the primary screen). So it is not clear why the authors concluded that silencing of the γ neurons has no effect on courtship memory. In our hands, the MB γ dorsal and main lines which tested negative in the Baker, 2016 paper were consistently positive. We cannot explain why these lines were negative in their hands, whereas this other MB γ dorsal line was positive.

Secord, we independently tested the lines for α/β and α’/β’ neurons and confirmed their observation of a significant impairment of courtship suppression upon silencing ( Author response image 3 ). Our results do not preclude involvement of other MB neurons/compartments in courtship memory, which we now explicitly note in the text (Discussion section).

Author response image 3
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Suppression indices (SI) in male flies in which active () or inactive (Q) tetanus toxin was expressed in MB neurons. Statistical significance of differences from zero or from control …

The newly added Supplementary file 3 lists full genotypes of flies used in all main and supplementary figures.

Xiaoliang Zhao

Daniela Lenek

Ugur Dag

Barry J Dickson

Krystyna Keleman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Stefanie Wandl and Kristin Henderson for technical assistance, Gudrun Ihrke for help with immunohistochemistry, Karel Svoboda, Glenn Turner, Vivek Jayaraman and Ulrike Heberlein for comments on the manuscript. This work was supported by Howard Hughes Medical Institute-Janelia Research Campus, Boehringer Ingelheim GmbH-IMP Vienna, and Austrian Science Fund (FWF P24499 to KK).

© 2018, Zhao et al.

This article is distributed under the terms of the Aerosoles Acclamation Glptha8
, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Table 1.

Food-Based Components of Dietary Patterns That Improve Cardiometabolic Health

In RCTs, diets that emphasize consumption of fruits and vegetables produce substantial improvements in several risk factors, including blood pressure (BP), lipid levels, insulin resistance, inflammatory biomarker levels, endothelial function, and weight control. MUK LUKS Kadin Sneaker CFHqn1
, Mark NasonLite Block Geffen 6PWvTXo
, 25
Benefits do not appear reproducible with equivalent amounts of representative mineral and fiber supplements, 26 nor are they dependent on dietary macronutrient (fat, protein, or carbohydrate) composition. 22 This evidence suggests that benefits might be derived from (1) a more complex set of micronutrients, phytochemicals, and fiber found in fruits and vegetables; (2) potentially enhanced bioavailability of these nutrients in their natural state; and/or (3) replacement of less healthful foods in the diet. In long-term observational studies, greater fruit and vegetable consumption are each associated with lower incidence of coronary heart disease (CHD), and greater fruit consumption is associated with lower incidence of stroke ( Figure 1 ). 27 , 28 The results of RCTs of physiological measures and prospective cohorts of disease outcomes together provide strong concordant evidence that fruit and vegetable consumption lowers CVD risk ( Gentle Souls Birdie 6y9KDgO
). Potential differences in health effects contributed by specific types of fruits, vegetables, or their juices require further investigation.

Figure 1.

Relationships of consumption of different foods with incidence of coronary heart disease (CHD), stroke, and diabetes in meta-analyses of prospective cohort studies (PCs). Adapted with permission from Mozaffarian. Dashes indicate not reported; CVD, cardiovascular disease; and RR (95% CI), relative risk (95% confidence interval).

View this table:
Table 2.

Evidence From Human Studies Using Different Research Paradigms for Effects of Selected Foods, Nutrients, and Dietary Patterns on Cardiovascular Diseases

Although no single accepted definition of whole grain exists, whole grains generally comprise bran, germ, and endosperm from the natural cereal. Bran contains soluble and insoluble dietary fiber, B vitamins, minerals, flavonoids, and tocopherols; germ contains numerous fatty acids, antioxidants, and phytochemicals. Endosperm provides largely starch (carbohydrate polysaccharides) and storage proteins. 30 The type and extent of processing appear to modify the health effects of grain and carbohydrate consumption ( Figure 2 ). For example, removal of bran and germ reduces dietary fiber that has important benefits, including lowering of BP and cholesterol levels HurleyPhantom Free Elite Sandal cH4ZGt
, Jack Rogers Madeline Thong Sandal yi1NQLbFT
; increases bioavailability and rapidity of digestion of remaining endosperm, which increases glycemic responses 33 ; and eliminates minerals, micronutrients, and other phytochemicals that may have additional independent health benefits 16 (see “Carbohydrate Amount and Quality”).

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