agrp neurons  (Addgene inc)

Journal: Acta physiologica (Oxford, England)

Article Title: Fam163a knockdown and mitochondrial stress in the arcuate nucleus of hypothalamus reduce AgRP neuron activity and differentially regulate mitochondrial dynamics in mice.

doi: 10.1111/apha.70020

Figure Lengend Snippet: FIGURE 3 Fam163a knockdown and rotenone reduce the firing rate of ARCAgRP neurons. (A and D) Representative immunostaining against FAM163A. White arrows depict the co-expression of GFP and FAM163A (Scale bars: [A and C] 50 μm and [B and D] 20 μm). (E) GFP expression in AgRP neurons containing brain slice of AgRP-Cre mouse. (Representative image depicting Cell-attached loose-seal recording from an AgRP neuron; recording pipette is highlighted with red-dashed lines, Scale bar: 15 μm). (F and H) Representative loose- seal recording traces for each group. (G) Effect of Fam163a KD on firing rate of AgRP neurons (n = 3–4 mice for each group; Neg. Ctr. n = 70 neurons, Fam163a KD n = 63 neurons) (I) Effect of rotenone on firing rate of AgRP neurons (n = 3–4 mice for each group; DMSO n = 46 neurons, rotenoe n = 52 neurons) (Data are mean ± standard deviation. Statistical analysis: Mann–Whitney U test; *p = 0.017; **p = 0.0062).

Article Snippet: Each Agrp- ires- cre mouse also received an injection of AAV pCAG- FLEX- EGFP- WPRE (AddGene, 51502- AAV9) to label AgRP neurons for electrophysiological recordings.113,114

Techniques: Knockdown, Immunostaining, Expressing, Slice Preparation, Transferring, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Model: AgRP axon stimulation could decrease cAMP production by releasing neuropeptide NPY that binds to NPY receptors and/or by releasing AgRP that prevents MC4R receptor agonism. (B) Setup for optogenetic Chrimson stimulation of AgRP axons in the PVH in awake, head-fixed mice while measuring cAMP activity in PVH MC4R neurons using the sensor cADDis via a GRIN lens. (C) Example cAMP traces during 10 trials of photostimulation (8 s, 30 Hz, 60 s inter-trial interval; red shade) from a fed session show cAMP decrements (blue traces) that occurred on different trials for different cells. Traces show –ΔF/F 0 . (D) Summary plot of single-trial cADDis traces across cells shows an all-or-none distribution. Traces are sorted by peak fluorescence decrements in the 20-40 s window. Inset shows distribution of peak intensities, color-coded blue for hits and gray for misses, with the x-axis on a log scale. n = 2283 trials from 4 mice. (E) Odds of obtaining 2-4 hits (decrements) in a row are well modeled by the dice model (P n = [P 1 ] n ). n = 1190 fasted and 1093 fed trials from 4 mice. (F-G) In all trials (F) and in hit trials only (G), cAMP decrements are greater in amplitude and more persistent in the fasted state (n = 1190 fasted and 1093 fed trials from 4 mice). No-stimulation control trials show almost no hits. (H-I) cAMP decrements show greater amplitude (H) and persistence (I) in fasted than in fed mice (n = 13 FOVs from 4 mice, paired t-test). (J-K) I.c.v. pre-infusion of 10 nmol BIBP3226 (NPY1R antagonist) and 10 nmol CGP71683 (NPY5R antagonist) reduced both the hit rate (J) and amplitude (K) of cAMP decrements in fed mice. n = 680 ACSF and 909 antagonist trials from 2 mice. (L) Model: NPY release from AgRP axons and αMSH release from POMC axons respectively trigger stochastic and state-dependent cAMP increments and decrements in PVH MC4R neurons.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Activity Assay, Fluorescence, Control

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Mean hit rates of AgRP axon stimulation–induced cAMP decrements in PVH MC4R neurons are not different between fasted and fed states. No-stimulation condition in both fasted and fed states shows a low hit rate. n = 1093-1928 trials from 4 mice. Bootstrap comparison of hit rates. Mean ± 95% C.I. (B) cAMP decrements last more than 5 min in fasted mice and 1-2 min in fed mice. n = 262-355 hits from 4 mice. (C) Complete surgical setup for combining i.c.v. infusion, optogenetic axon stimulation, and cAMP recording through the GRIN lens. Chrimson is virally expressed in AgRP neurons in the arcuate, and cADDis is virally expressed in PVH MC4R neurons. A GRIN lens is placed above PVH, and a cannula is placed in the posterior lateral ventricle. (D) Histology of cADDis expression (green) in PVH MC4R neurons and Chrimson-tdTomato expression (red) in AgRP axons. In the left panel, dotted lines delineate the GRIN lens track. In the right panel, white dotted line delineates the anterior side of the infusion cannula inserted in the lateral ventricle (LV; cyan dotted line) which contains choroid plexus (ChP). (E) Single-mouse fluorescence lifetime traces of cADDis in PVH MC4R neurons in response to infusion of 0.5 nmol NPY. n = 3 mice. (F) Single field-of-view summary of decrease in cAMP in PVH MC4R neurons in response to infusion of 0.5 nmol NPY (n = 11 FOVs from 3 mice). (G) Pre-infusing NPY1R antagonist BIBP3226 (10 nmol) together with NPY5R antagonist CGP71683 (10 nmol) reduced the hit rate of AgRP stimulation–induced cAMP decrements in PVH MC4R neurons (n = 680-909 trials from 2 mice). (H) Cumulative distribution of magnitudes of single-trial cAMP decrements (measured with cADDis) indicates a decrease in magnitudes when BIBP3226 and CGP71683 were pre-infused before optogenetic stimulations (n = 55-144 hits from 2 mice). (I-J) Pre-infusing BIBP3226 and CGP71683 reduced the magnitude of AgRP stimulation–induced cAMP decrements without affecting the persistence of these cAMP decrements (n = 6 FOVs from 2 mice, t-test).

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Comparison, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) To measure NPY release in slice, Chrimson is expressed in AgRP neurons to allow axon stimulation in PVH, and npyLight is expressed in the PVH to capture NPY release. These experiments used fed mice. (B) The fluorescence of npyLight increases in response to NPY (200 nM; n = 10 slices from 4 mice). (C-F) When we increased the duration of 15.5 Hz photostimulation eight-fold, from 2 s to 16 s, the hit rate of npyLight transients increases from 14% to 21% (C-D, n = 16439-19969 trials from 7 mice), and hit amplitudes remain unchanged (E-F) (n = 2690, 2293, 2871, 2982 hits). (G) If neuropeptide signaling is spatially discrete (‘Discrete’), hits and misses should be uncorrelated between pairs of regions-of-interest (ROIs) from a soma and distant neurite belonging to the same cell. If hits involve neuropeptide release that diffuses across a large volume (“Large volume”), or if cAMP could diffuse intracellularly (‘cAMP diffusion’), hits and misses should be correlated for pairs of ROIs from the same cell. (H-I) During AgRP axon stimulation, neurite cAMP decrements are also stochastic (H) and strengthened in the fasted state (I). n = 6048-9371 trials from 4 mice. (J) Neurite cAMP signals averaged across trials containing soma hits or soma misses are similar and smaller in magnitude, indicating decoupling of soma and neurite signals. n = 2379 fasted and 3138 fed trials from 4 mice. (K-M) During POMC axon stimulation, neurite cAMP increments are also stochastic (K) and are larger and more sustained in the fed state (L). Neurite traces averaged across trials containing soma hits or soma misses were similar and smaller in magnitude, again indicating decoupling of soma and neurite signals (M). K-L: n = 4356-5122 trials from 4 mice. M: n = 1350 fasted and 1363 fed trials. (N) By calculating the probability of concurrent hits or concurrent misses in two somas (left model), we found that correlation decreases as the distance between the two increases. The distance beyond which the probability of concurrent hits reaches chance levels is consistently ∼100 μm for cADDis recordings during AgRP (left) and POMC stimulation (middle). The same ∼100 μm distance threshold is also seen with inter-ROI correlations in npyLight recordings during AgRP stimulation (right). See also . (O) Stochastic release leads to temporally and spatially discrete neuropeptide signaling.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Fluorescence, Diffusion-based Assay

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Hypothesis for state-dependent cAMP signaling: In the fed state, elevated basal αMSH levels curb experimentally induced cAMP decrements. Likewise, in the fasted state, increased basal NPY levels curb cAMP increments. This hypothesis is tested by mimicking the presence of competing peptides. (B-C) In fasted mice, MTII pre-administration (3 mg/kg, i.p.) attenuated the amplitude and persistence of AgRP stimulation–induced cAMP decrements. n = 95-114 hits and 6 FOVs from 3 mice, paired t-test. (D-E) In fasted mice, αMSH pre-infusion (1 nmol, i.c.v.) reduced hit amplitude and persistence of AgRP stimulation–induced cAMP decrements. n = 391-417 trials and 6 FOVs from 2 mice, paired t-test. (F-G) In fed mice, NPY pre-infusion (0.5 nmol, i.c.v.) reduced hit amplitude and persistence of POMC stimulation–induced cAMP increments. n = 1311-1667 trials and 6 FOVs from 2 mice, paired t-test.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques:

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Feeding rapidly decreases AgRP neuron activity while increasing POMC neuron activity, which should result in persistently elevated cAMP. (B) Periodic milkshake delivery to head-fixed mice results in transient and time-locked spikes in lick rate that are reliably observed in fasted and fed mice. Lick rates are greater in fasted than in fed mice. n = 6 mice. (C) cAMP in PVH MC4R neurons climbs up to a steady state level within ∼4 one-minute trials in both conditions, albeit from a higher initial level in the fed state. Gray traces: mean soma cADDis lifetime changes per FOV. Blue and red traces: mean across FOVs. n = 7-11 FOVs from 6 mice. (D-E) In the climbing epoch where cAMP is increasing (Trials 1-4), cAMP increments are stochastic. The hit rate is lower in the steady-state epoch (Trials 5-15) (E). D: n = 561-646 trials from 6 mice, E: 1542-1759 trials. (F-G) Persistent cAMP increments in the fasted (F) and fed states (G). F: n = 2103 trials from 6 mice, G: 2405 trials. (H) Either chemogenetic inhibition of AgRP neurons (hM4Di and i.p. injection of CNO) or optogenetic stimulation of POMC neurons (ChR2) suppresses dark-phase feeding in freely-moving mice, but the suppression is strongest when the two manipulations are combined (n = 11-17 mice, one-way ANOVA), consistent with decreased peptide competition. (I) Continuous low-level (∼15 μW average) stimulation of biPAC in PVH MC4R neurons reduces cumulative lick rate during an ad libitum binge assay in head-fixed, fasted mice (n = 8 mice for each condition). (J) Experimental design: in control sessions, fasted mice lick during tone to obtain reward (milkshake) with no biPAC stimulation. In experimental sessions, brief biPAC stimulation was delivered 5 s before 50% of the trials, while the other 50% trials did not involve stimulation. In control sessions, no trials contained stimulation. (K) Lick rate during tone declines faster in the experimental session and does not recover after 10 min wait (n = 8 mice, t-test). (L) Lick rates are lower in experimental sessions, but there is no difference between stimulation and no-stimulation trials within an experimental session (n = 8 mice, one-way ANOVA). (M) The decrease in lick rate during tone presentation over 10 trials scales with the average number of biPAC stimulations delivered in the intervening time (n = 8 mice).

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Activity Assay, Inhibition, Injection, Control

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Example field view of npyLight expression in PVH (green) and Chrimson-tdTomato expression in AgRP axons (red). (B-E) Summary heatmaps of single-trial npyLight signals in response to 2 s (B), 4 s (C), 8 s (D), or 16 s (E) of Chrimson photostimulation (n = 16439-19969, trials from 7 mice). Trials are sorted by peak intensity. Inset: distribution of peak intensities, color-coded red for hits and gray for misses, with x-axis on a log scale. (F) The hit rates of AgRP stimulation–induced NPY signals (measured with npyLight) are mostly well described using the dice model, with a modest, sublinear increase in probability of release with increasing stimulation duration (n = 16439-19969 trials from 7 mice). (G) In an example neuron with a clearly visible soma (S) and two associated neurites (A and B), POMC stimulation–induced cAMP increments are spatially localized and occur on different trials for the soma and for each neurite. (H) Left: example neurite segmentation by Cellpose 2.0 nuclear model that is retrained by manual neurite segmentation (see Methods). Right: During both AgRP axon stimulation and POMC axon stimulation, the hit rate per area is non-zero in neurites (N) but is lower in neurites than soma (S; n = 10-13 FOVs from 8 mice, one-way ANOVA). (I) Model: during spatially restricted peptide release, ROIs that are closer to each other than the impact diameter are more likely to receive the same peptide signal than ROIs that are further apart. (J) The truth table of exclusive not-or (XNOR), a metric of concurrence between pairs of binary events, A and B. When cAMP responses in two regions-of-interest concur on a given trial (e.g. both exhibit a hit or both exhibit a miss), the XNOR value equals one. (K) Probability of concurrent hits or misses between soma-neurite pairs drops as the distance between the two ROIs increases. The distance beyond which concurrence of cAMP responses drops to chance levels is ∼100 μm during both AgRP stimulation (left) and POMC stimulation (right). (L) During AgRP axon stimulation in both fasted (blue) and fed (red) states, the soma-neurite signals are consistently decorrelated from each other. This is evident using F1-score analysis of binary hit/miss data, XNOR analysis of binary hit/miss data, or Pearson correlation of continuous ΔF/F 0 data. Vertical lines are experimental data, and shaded area shows the bootstrapped distribution (100,000 iterations, with replacement). P-value indicates two-tailed probability that actual concurrence estimate (F1, XNOR or Pearson correlation) falls outside the mean of shuffled values. (M) Same as L, but for POMC axon stimulation.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) To measure potential axon excitability differences across states, which could lead to differences in peptide release, we first co-expressed Chrimson and Axon-GCaMP6s in AgRP neurons and, in separate experiments, in POMC neurons. In both cases, we implanted an optic fiber in PVH to photostimulate Chrimson-expressing axons and to record the resulting calcium transients in these same axons. Axon calcium transients evoked by 25-Hz, 8-s Chrimson stimulation in AgRP (left) and POMC axons (right) were similar in fasted and fed states (n = 9 mice). (B) Brief 10 Hz, 1 s Chrimson stimulation of AgRP axons triggers stronger calcium transients in the fed state (when NPY signaling is weaker). The same photostimulation of POMC axons triggers stronger calcium transients in the fasted state (when αMSH signaling is weaker). These differences are presumably due to lower baseline activity of AgRP axons and POMC axons in the fed and fasted state, respectively. n = 9 mice. Note that these differences argue against a major contribution of presynaptic excitability to the state-dependent differences in cAMP responses: AgRP stimulation drives weaker PVH MC4R cAMP responses in the fed state, despite the slightly stronger 1-s stimulation-evoked AgRP axon calcium signals in the fed state. Similarly, POMC stimulation drives weaker PVH MC4R cAMP responses in the fasted state, despite the slightly stronger 1-s stimulation-evoked POMC axon calcium signals in the fasted state. (C-D) We used blue-light activation of the optogenetic adenylyl cyclase, biPAC, to bypass endogenous peptide receptor signaling and directly produce cAMP in PVH MC4R neurons in slices, while monitoring cAMP dynamics with the sensor cADDis. cAMP produced by biPAC photostimulation (1x 100 ms or 2x 2 s) in PVH MC4R neurons is cleared by endogenous PDEs at similar rates in fasted and fed states, arguing against state-dependent cAMP degradation (n = 8 slices from 4 mice). (D) MTII injection (3 mg/kg, i.p.) elevates cAMP in PVH MC4R neurons using two-photon fluorescence lifetime imaging in vivo (n = 6 FOVs from 3 mice, one-way ANOVA). (E) MTII pre-injection does not change the hit rate of AgRP stimulation–induced cAMP decrements (n = 473-517 trials from 3 mice). (F-G) αMSH pre-injection does not change the hit rate of AgRP stimulation–induced cAMP decrements (F) but reduces hit magnitudes (G). n = 391-473 trials from 2 mice, bootstrap comparison of hit rates. (H-I) NPY pre-infusion does not change the hit rate of POMC stimulation–induced cAMP increments (H) but reduces hit magnitudes (I). n = 1311-1667 trials from 2 mice, bootstrap comparison of hit rates. (J-L) When analyzing two consecutive hits of AgRP stimulation–induced cAMP decrements, the second decrement is larger than the first (n = 13 FOVs from 4 mice). Amplitudes are calculated as –ΔF/F 0 means in the 20-40 s window following stimulation onset. Baselines are calculated separately for first and second hits to prevent lingering elevation from the first hit from contributing to the calculations of the second. (M-O) The same as J-L but for POMC stimulation–induced cAMP increments (n = 10 FOVs from 4 mice). (P-Q) To more directly manipulate the total amount of peptide released in a local region of PVH, we presented groups of ten 8-s photostimulations of AgRP and POMC axons with a much shorter inter-stimulation interval (2 s instead of 52 s; other experimental parameters were not modified). The shorter inter-stimulation intervals within each 98-s stimulation sequence should decrease the degree to which peptides released during each 8-s stimulation diffuse away or are broken down by peptidases (Xiong et al ., 2022; Turner et al ., 1985) between trials, resulting in greater accumulation of extracellular neuropeptide levels that could overcome endogenous competition from opposing neuropeptides (illustrated in P). Consistent with this prediction, these 98-s groups of AgRP or POMC axon stimulations drove cAMP decrements (P; 42-44% hit rate per group) and increments (Q; 45-48% hit rate) that were long-lasting (>8 min after the last pulse) and insensitive to hunger state. P: n = 112-167 trials from 4 mice, Q: n = 97-139 trials from 4 mice.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Expressing, Activity Assay, Activation Assay, Produced, Injection, Fluorescence, Imaging, In Vivo, Comparison, Modification, Sequencing

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A-B) Injection of ghrelin (2.5 mg/kg, i.p.), which stimulates AgRP neurons and inhibits POMC neurons, induces a robust decrease in cAMP in PVH MC4R neurons in fed mice. (C) cAMP levels are stable in PVH MC4R neurons in the absence of feeding (n = 8 FOVs from 3 mice), arguing against any non-stationarities due to elapsed time within a session. (D-E) Trial-average cAMP increments in the fasted (D) and fed states (E) in the first four trials. D: n = 561 trials from 6 mice, E: 646 trials. (F) In an assay where fasted mice lick during a tone (blue) to obtain reward (orange, milkshake), well-trained food-restricted mice start licking during the tone but the lick rate gradually decreases over 50 trials. (G-H) In experimental sessions (purple), in which biPAC stimulation was delivered in 50% of the trials, lick rates (G) and success rates (H) drop off faster than in control sessions (gray), and do not recover after 10 min without additional cues or reward deliveries. n = 8 mice, t-test. (I) Within the experimental session, no difference in success rate (rate at which the mouse exhibited licking following the tone but prior to the reward delivery) was observed between stimulation trials (purple, ‘Y’) and no-stimulation trials (purple, ‘N’). n = 8 mice, one-way ANOVA. (J) Over a window of 10 trials, the decrease in lick rate during reward (left), and success rate of correct licking following the cue (right) scale with the total number of biPAC stimulations delivered across the last ten trials (n = 8 mice). Together with panel I, this suggests that the effects of cAMP increments are gradual (i.e., they do not affect same-trial performance) and cumulative across minutes.

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Injection, Control

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: Spatially localized plumes of neuropeptides NPY and αMSH are released stochastically in the PVH (A), where they compete to control cAMP in PVH MC4R neurons (B). Feeding reduces AgRP neuron activity while elevating POMC neuron activity, a combination that results in sustained cAMP elevation in PVH MC4R neurons for tens of minutes (C). In turn, elevated cAMP gradually potentiates feeding-related excitatory inputs to satiety-promoting PVH MC4R neurons (D), thereby promoting satiation (E).

Article Snippet: For experiments that measure the impact of simultaneous POMC neuron activation and AgRP neuron inhibition on feeding, AAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362-AAV8) and AAV8-FREX-ChR2-EYFP (gift from Henning Fenselau) were injected bilaterally in the arcuate nucleus of AgRP-IRES-Cre;POMC-Dre mice (1:1 mixture, 150 nl total; Bregma: AP -1.5 mm, ML ±0.3 mm, DV -5.9 to -5.75 mm).

Techniques: Control, Activity Assay

Journal: Journal of neurochemistry

Article Title: Chemogenetic approaches to identify metabolically important GPCR signaling pathways: therapeutic implications

doi: 10.1111/jnc.15314

Figure Lengend Snippet: Summary of in vivo ‘DREADD studies’ investigating signaling pathways regulating glucose and energy homeostasis. This cartoon summarizes data obtained with mutant mice expressing DREADDs in pancreatic β- and α-cells (a), adipocytes (b), hepatocytes (c), skeletal muscle cells (d), and AgRP (e) and POMC neurons (f) of the arcuate nucleus of the hypothalamus. For simplicity, only some key signaling factors/pathways and metabolic phenotypes are listed. See text for details.

Article Snippet: A recent study ( Bunner et al., 2020 ) showed that a single bout of acute exercise increases the activity of ARC AgRP neurons, resulting in increased food intake.

Techniques: In Vivo, Protein-Protein interactions, Mutagenesis, Expressing

Journal: Journal of neurochemistry

Article Title: Chemogenetic approaches to identify metabolically important GPCR signaling pathways: therapeutic implications

doi: 10.1111/jnc.15314

Figure Lengend Snippet: Potential therapeutic strategies to improve glucose hemostasis and/or reduce obesity, as suggested by the use of DREADD technology in mice

Article Snippet: A recent study ( Bunner et al., 2020 ) showed that a single bout of acute exercise increases the activity of ARC AgRP neurons, resulting in increased food intake.

Techniques: Activation Assay, Activity Assay, Inhibition, Blocking Assay, Fat

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: AgRP is a candidate for the evening feeding rhythm-induced insulin resistance. Real time quantitative PCR in 24-h ( n = 3–4). ( A ) AgRP ( B ) NPY ( C ) POMC. Red, blue, and black lines represent the Evening, Morning, and Ad-lib groups, respectively. Bottom panel shows the area under the curve of each gene mRNA circadian expression. * p < 0.05 vs. Ad-lib group.

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: AgRP is responsible for the evening feeding rhythm-induced insulin resistance. ( A ) AgRP mRNA expression in the hypothalamus after intracerebroventricular antisense oligonucleotide (ASO) administration (twice a week) in the Evening group. ( B ) insulin tolerance test (ITT). ( C – E ) Akt phosphorylation in soleus ( n = 3) ( C ), TG content in gastrocnemius ( n = 6) ( D ), FAS expression in soleus ( E ) following ASO treatment. ( F , G ) VO 2 in the dark and light period ( F ) and circadian RQ over 24 h ( n = 6) following ASO treatment in the each Evening group. Data are presented as mean ± standard error. * p < 0.05 vs. Ad-lib group. $ p < 0.05 vs. Akt phosphorylation of each group without insulin.

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: Hypo thalamic glucocorticoid sensitivity is critical for evening feeding-induced AgRP expression. ( A ) Circadian rhythm of plasma corticosterone levels (left) and areas under the curve (right) ( n = 4). ( B ) Two enzymes expression required for glucocorticoid synthesis, Cyp11a1 and StAR, in the adrenal glands ( n = 4). ( C ) Effect of oral administration of the glucocorticoid receptor antagonist RU-486 (20 mg/kg/day) for 2 weeks on AgRP expression of the hypothalamus ( n = 5). ( D ) Effect of adrenalectomy and 2 weeks of corticosterone supplementation (Adx) on AgRP expression of the hypothalamus ( n = 5). Data are presented as mean ± standard error. * p < 0.05 vs. Ad-lib group. ** p < 0.01 vs. Ad-lib group. ## p < 0.01 vs. Morning group.

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: AgRP neuron-specific GR deficient mice has no insulin tolerance even in the Evening feeding schedule. ( A ) Immunohistochemistry of the hypothalamic arcuate nucleus in WT and AgRP-GR-KO mice. The scale bars indicate 100 μm. ( B ) Food intake in WT and AgRP-GR-KO mice. ( C ) Alteration of body weight in WT and AgRP-GR-KO mice. ( D ) Circadian change of oxygen consumption in WT and AgRP-GR-KO mice. White column shows light period, black column shows dark period of VO 2 in bottom panels. ( E ) Insulin tolerance in Evening group WT and AgRP-GR-KO mice ( n = 6). * p < 0.05 vs. Ad-lib group.

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: AgRP neuron-specific GR deficient mice does not show insulin resistance in skeletal muscle even in the Evening feeding schedule with decrease in AgRP expression. ( A ) Hypothalamic AgRP expression in WT and AgRP-GR-KO mice ( n = 6). ( B ) Plasma glucocorticoid level in WT and AgRP-GR-KO mice ( n = 6). ( C ) Insulin-induced Akt phosphorylation in the soleus in WT and AgRP-GR-KO mice ( n = 3–5). ( D ) FAS expression in the soleus in WT and AgRP-GR-KO mice in the Ad-lib and Evening groups ( n = 3–5). ( E ) JNK phosphorylation in soleus of WT or AgRP-GR-KO mice ( n = 3–4). Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01 vs. Ad-lib group. $ p < 0.05 vs. Akt phosphorylation of each group without insulin.

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Clinical Proteomics, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle

doi: 10.3390/ijms221910831

Figure Lengend Snippet: Effect of hepatic vagotomy on AgRP expression in ( A ) WT and ( B ) AgRP-GR-KO mice. ( A ) Hypothalamic AgRP, NPY, POMC and GR expression in WT mice with or without hepatic vagotomy ( n = 5). ( B ) Hypothalamic AgRP, NPY, POMC, and GR expression in AgRP-GR-KO mice in the Ad-lib group with or without hepatic vagotomy ( n = 5). Data are presented as mean ± SE, n = 4. * p < 0.05, ** p < 0.01 vs. Ad-lib (Sham).

Article Snippet: Floxed GR mice (B6.Cg-Nr3c1tm1.1Jda/J) and AgRP neuron-specific Cre-recombinase mice (Agrptm1[cre]Lowl/J) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing