State-dependent central synaptic regulation by GLP-1 is essential for energy homeostasis

Le Wang; Rohan H Savani; Yi Lu; Matteo Bernabucci; Jorge Luis-Islas; Erin Park; Ishnoor Singh; Wei Xu; Abdelfattah El Ouaamari; Michael B Wheeler; Harvey J Grill; Mark A Rossi; Zhiping P Pang

doi:10.1038/s42255-025-01305-x

. Author manuscript; available in PMC: 2026 Mar 9.

Published in final edited form as: Nat Metab. 2025 Jun 4;7(7):1443–1458. doi: 10.1038/s42255-025-01305-x

State-dependent central synaptic regulation by GLP-1 is essential for energy homeostasis

Le Wang ^1,², Rohan H Savani ^1,², Yi Lu ^1,², Matteo Bernabucci ¹, Jorge Luis-Islas ^1,², Erin Park ^1,², Ishnoor Singh ^1,³, Wei Xu ⁴, Abdelfattah El Ouaamari ⁵, Michael B Wheeler ³, Harvey J Grill ⁶, Mark A Rossi ^1,^2,⁷, Zhiping P Pang ^1,^2,^7,^8,^*

PMCID: PMC12967307 NIHMSID: NIHMS2146516 PMID: 40467923

Abstract

Central glucagon-like peptide-1 (GLP-1), secreted by a distinct population of the nucleus tractus solitarius neurons, suppresses feeding but the exact mechanisms of action in the brain remain unclear. Here, we investigate a descending circuit formed by GLP-1 receptor (GLP-1R) neurons in the paraventricular hypothalamic nucleus (PVN^GLP-1R) projecting to dorsal vagal complex (DVC) of the brainstem in mice. PVN^GLP-1R→DVC synapses release glutamate and are augmented by GLP-1. Chemogenetic activation of PVN^GLP-1R→DVC suppresses feeding. Under energy deficit (i.e. hunger) state, synaptic strength is weaker but is more profoundly augmented by GLP-1R activation than under energy-replete state. In obese condition, the dynamic synaptic changes in this circuit are disrupted. Optogenetic activation of PVN^GLP-1R→DVC projections suppresses food intake energy state-dependently, and blocking its synaptic release or ablating GLP-1Rs in the presynaptic neurons impairs metabolic health. These findings suggest that the state-dependent synaptic regulation by GLP-1 in PVN^GLP-1R→DVC descending circuit is important for energy homeostasis.

Glucagon-like peptide-1 (GLP-1) is secreted by L-cells in the small intestine and a discrete population of neurons located in the brain stem (i.e. nucleus tractus solitarius, NTS¹) and plays a significant role in controlling food intake and maintaining glucose homeostasis². GLP-1 analogs are clinically effective in treating type 2 diabetes and obesity², but they have been reported to be associated with adverse events, including nausea, vomiting, diarrhea, and gallbladder issues^2-4. The lack of a complete mechanistic understanding of GLP-1 receptor (GLP-1R) signaling in both the peripheral nervous system and the central nervous system (CNS) impedes the development of more effective clinical interventions. The peripheral potentiation of insulin release does not explain the weight loss associated with GLP-1 analogs in humans². Because central and peripheral GLP-1R signaling suppress feeding via independent gut-brain pathways⁵, it is appealing to develop specific strategies to activate only the central GLP-1R signaling pathway to treat obesity via suppression of feeding^1,2,6,7. In the brain, it has been demonstrated that GLP-1R signaling can enhance excitatory synaptic transmission in the lateral hypothalamic area⁸ and the paraventricular hypothalamic nucleus (PVN)⁹, two brain regions that are essential in regulating food intake. Moreover, ablation of GLP-1R in the PVN leads to obesity and hyperphagia⁹. It has also been shown that GLP-1R signaling in the brain stem, including the NTS and the area postrema (AP), plays a key role in regulating food intake^6,10. However, the role of GLP-1R signaling in regulating descending pathways to the brainstem remains uninvestigated. Dissecting the CNS synaptic and neurocircuit mediation of endogenous central GLP-1 release and subsequent function in the regulation of metabolism will contribute to understanding the regulation by GLP-1 in the brain and will also provide an opportunity to further improve GLP-1 analog therapeutics².

GLP-1R signaling enhances PVN^GLP-1R→DVC presynaptic release of glutamate

To identify the downstream neural targets of PVN^GLP-1R expressing neurons, we conducted ‘SynaptoTag’ assisted circuit tracing. We injected AAV-DIO-SynaptoTag2 (a Cre-dependent vector expressing mCherry and Syb2-EGFP to label presynaptic boutons) into the PVN of GLP-1R-ires-Cre mice, enabling quantitative assessment of the number of synapses formed by SynaptoTag-AAV-infected neurons in a target region¹¹ (Extended Data Fig. 1a). Among many brain structures, we observed dense expression of mCherry and Syb2-EGFP in the dorsal vagal complex (DVC), composed of the AP, NTS, and dorsal motor nucleus of the vagus nerve (DMV). Additionally, we also found PVN^GLP-1R neurons projecting to the lateral parabrachial nucleus (LPBN), locus coeruleus (LC), and median eminence (ME), consistent with previous studies^12,13 (Extended Data Fig. 1b-c). The DVC is a key central region in regulating metabolism^14,15 and mediating GLP-1R signaling-induced food intake suppression^6,16. To further verify this PVN^GLP-1R→DVC descending projection, we performed retrograde tracing by injecting AAVrg-hSyn-DIO-EGFP in the DVC of GLP-1R-ires-Cre mice and systematically quantifying retrogradely labeled PVN^GLP-1R→DVC neurons (Extended Data Fig. 1d&e). PVN^GLP-1R→DVC neurons are mainly located in the posterior PVN (Extended Data Fig. 1f). We next investigated whether these PVN^GLP-1R→DVC neurons also send collateral projections to other brain regions. We injected AAVrg-DIO-Flpo in DVC and AAV-fDIO-EYFP in the PVN of GLP-1R-ires-Cre mice, which expresses cytosolic EYFP and labels any axon collaterals that exist in these PVNDVC neurons and systematically examined their projections (Extended Data Fig. 2a). Among all downstream targets (i.e. DVC, LC, ME, LPBN) identified using SynaptoTag2 tracing (Extended Data Figure 1b&c), no strong EYFP-labeled terminals were observed, suggesting these neurons have little to no collateralization (Extended Data Fig. 2b). To further confirm this, we utilized a dual-color retrograde tracing strategy by injecting AAVrg-DIO-EYFP in the DVC and AAVrg-DIO-mCherry into the LPBN or LC. Consistently, we did not observe a high density of overlapping cells between PVN^GLP-1R→DVC (EYFP-positive) neurons with PVN^GLP-1R neurons projecting to LPBN or LC (mCherry-positive) (Extended Data Fig. 2c-f).

We then asked if PVN^GLP-1R neurons form functional synapses with DVC neurons using Channelrhodopsin-2 (ChR2) assisted circuit mapping. We injected AAV-DIO-ChR2-EYFP into the PVN of GLP-1R-ires-Cre mice and conducted whole-cell patch clamp electrophysiology in DVC neurons (Fig. 1a). Consistent with results from ‘SynaptoTag’ tracing (Extended Data Fig. 1a-c), we observed dense axon terminals in the DVC, especially the DMV, as visualized with choline acetyltransferase (ChAT) immunostaining (Fig. 1b-d). We recorded robust optically evoked excitatory postsynaptic currents (oEPSCs) that were blocked by the AMPA receptor blocker cyanquixaline (CNQX) (Fig. 1e), indicating that PVN^GLP-1R neurons form glutamatergic synapses with DVC neurons. Comparing oEPSC amplitude and the connectivity ratio among the DVC neurons, our data indicate that the DMV receives stronger PVN^GLP-1R synaptic input compared to the NTS and AP (Fig. 1f).

Figure 1: — a. Experimental paradigm for tracking PVN GLP-1R neuronal projections.

b. Representative image of the PVN with ChR2-EYFP labeled GLP-1R neurons (n>3 mice).

c. Representative image of the DVC with ChR2-EYFP labeled projections (n>3 mice).

d. Representative image of ChR2-EYFP labeled projections in DMV (immunostained for ChAT) and NTS (n>3 mice).

e. Optogenetically evoked- CNQX-sensitive EPSCs in the DVC neurons (two-tailed paired t-test, t (20) =6.473, p< 0.0001, n=20 cells/12 mice).

f. Amplitudes of oEPSCs in DVC neurons with numbers of responsive cells/total recorded cells indicated in different regions of the DVC. (one-way Brown-Forsythe ANOVA, F (2,53.03) = 34.1, p < 0.0001, Dunnett’s T3 multiple comparisons test, p < 0.0001 vs. DMV).

g. Representative traces of AMPAR-oEPSCs before and after application of Exn-4.

h. Pooled data (two-tailed Wilcoxon matched-pairs signed rank test, p= 0.0008, n=29 cells/12 mice).

i. Representative traces of paired-pulse oEPSCs before and after application of Exn-4.

j. Quantifications of paired pulse ratios (PPRs) (two-tailed paired t-test, t (24) =4.519, p=0.0002, n=24 cells/12 mice).

k. AMPAR- and NMDAR-mediated oEPSCs were recorded at holding potentials of −70 mV and +60 mV, respectively. NMDAR- oEPSCs were measured at 50 ms after stimulation (dashed lines).

l. Pooled data of AMPAR/NMDAR-EPSCs ratio (two-tailed Wilcoxon matched-pairs signed rank test, p=0.1688, n=15 cells/12 mice).

m. Representative traces of oEPSCs in DVC neurons after replacing 2.5 mM Ca²⁺ with 5 mM strontium (Sr²⁺).

n. Representative traces of AMPAR-mediated oEPSCs before and after application of Exn-4 in Sr²⁺.

o. Quantification of AMPAR-mediated asynchronous oEPSC amplitudes before and after Exn-4 application in Sr²⁺ (two-tailed paired t-test, t (10) = 1.559, p = 0.1535, n = 10 cells/3 mice)

p. Quantification of delayed asynchronous quantal frequencies before and after Exn-4 application (two-tailed paired t-test, t (10) = 5.275, p = 0.0005, n = 10 cells/3 mice).

q. Representative traces of oEPSCs in the absence or presence of H89.

r. Pooled data and quantification. (two-tailed paired t-test, t (10) =0.8904, p=0.3964, n=10 cells/3 mice).

Data are presented as mean ± SEM. **p< 0.01; ***p< 0.001; ****p< 0.0001.

Since PVN^GLP-1R neurons project to the DVC and release glutamate, we hypothesized that GLP-1 signaling regulates synaptic release at this synapse via presynaptically expressed GLP-1Rs. To test this hypothesis, we recorded PVN^GLP-1R→DVC oEPSCs in DMV neurons before and after applying the GLP-1R agonist Exendin-4 (Exn-4)¹⁷. As expected, PVN^GLP-1R→DVC oEPSCs are significantly augmented by Exn-4 (Fig. 1g&h). Consistent with our hypothesis of a presynaptic mechanism, paired-pulse ratios (PPR) of oEPSCs in the presence of Exn-4 were reduced (Fig. 1i&J), suggesting an increase in presynaptic release probability. To further support this, we also recorded AMPAR and NMDAR oEPSCs, at holding potentials of −70 mV and +60 mV respectively, and calculated the ratio of AMPAR/NMDAR oEPSCs. No statistical differences were found in AMPAR/NMDAR-oEPSCs ratio before and after the application of Exn-4 (Fig. 1k&l), once again indicating regulation of presynaptic release probability. Moreover, we also observed an increase in spontaneous synaptic release frequency but not amplitude (Extended Data Fig. 3a-c). Additionally, we injected a Cre-dependent ChR2-EYFP construct into the DVC of Glp-1r-ires-Cre mice and immunostained for ChAT. We find there to be little overlap between the two neuronal populations (Extended Data Fig. 3 d&e).

To further substantiate the conclusion that GLP-1R signaling enhances PVN^GLP-1R→DVC synaptic release, we recorded oEPSCs in the presence of strontium (Sr²⁺), which leads to asynchronous quantal release of neurotransmitter vesicles following stimulation^18,19. As expected, replacing extracellular Ca²⁺ with Sr²⁺ reduced the peak amplitude of evoked release. Delayed synaptic vesicle release became apparent and persisted for about 300 ms post-stimulation (Fig. 1m). In the presence of Exn-4, the frequency of desynchronized synaptic vesicle release significantly increased, without changes in amplitude (Fig. 1n-p). In addition, the initial synchronous peak of oEPSCs in the presence of Sr²⁺ showed augmentation following Exn-4 application (Extended Data Fig. 3 f), consistent with observations in standard Ca²⁺-containing extracellular solution (Fig. 1g). Collectively, these data provide strong evidence that GLP-1R signaling enhances PVN^GLP-1R→DVC synaptic release via a presynaptic mechanism.

Furthermore, since we previously showed that GLP-1R activates protein kinase A (PKA) to regulate synaptic transmission in the PVN²⁰, we hypothesized that a similar signaling cascade is involved in regulating presynaptic release probability. Indeed, we found that blocking PKA signaling with H-89 abolished the augmentation of PVN^GLP-1R→DVC synaptic release by Exn-4 (Fig. 1q&r).

Activation of PVN^GLP-1R→DVC suppresses feeding

We previously showed that activation of GLP-1 neuronal projections to the PVN suppresses food intake²⁰. However, it is unclear whether the activation of PVN^GLP-1R→DVC neurons, a subset of the whole PVN^GLP-1R population, is sufficient to inhibit food intake. To this end, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) chemogenetics²¹. To express hM₃Dq DREADDs in PVN^GLP-1R→DVC neurons, we injected AAVrg-EF1a-DIO-Flpo in the DVC and AAV-hSyn-fDIO-hM₃D(Gq)-mCherry in the PVN of GLP-1R-ires-Cre mice (Fig. 2a&b). The application of clozapine-N-oxide (CNO, 10 μM) resulted in depolarization of membrane potential and increased action potential firing in hM₃Dq-expressing PVN^GLP-1R neurons in brain slices (Fig. 2c&d). Chemogenetic activation of PVN^GLP-1R→DVC neurons in behaving mice with 1mg/kg CNO profoundly suppressed food intake regardless of light/dark cycle and energy state (Fig. 2e and Extended Data Fig. 4b&c). Mice injected with the vehicle control (saline) showed no significant difference in food intake (Extended Data Fig. 4a). The suppression of food intake after chemogenetic activation of PVN^GLP-1R→DVC neurons did not result from increased anxiety or impaired locomotion (Extended Data Fig. 4e-i). Moreover, we also investigated whether activation of the PVN^GLP-1R→DVC pathway directly regulates blood glucose metabolism. We found no significant effects during the glucose tolerance test after CNO administration (Extended Data Fig. 4d). These results indicate that the activity of PVN^GLP-1R→DVC neurons is sufficient to selectively regulate food intake behavior.

Figure 2: — a. Experimental paradigm for virus delivery for chemogenetic manipulating of PVN^GLP-1R→DVC neurons.

b. Representative image of AAV-fDIO-hM₃Dq-mCherry expression in the PVN^GLP-1R→DVC neurons (n=7 mice)

c. Representative traces of action potential firing after CNO application.

d. Quantification of resting membrane potentials (RMP) after CNO application in PVN hM₃Dq- or control-virus expressing cells (two-tailed paired t-test, t (6) =4.339, p=0.0074, n=6 cells/2 mice).

e. Food consumption upon activation of PVN^GLP-1R→DVC neurons in the dark cycle (two-way ANOVA, the main effect of Group: F (1, 14)=16.48, p=0.0012; main effect of Time: F (1.317, 18.43)=100.7, p<0.0001; interaction between Group and Time: F (3, 42)=9.659, p<0.0001; Sidak's multiple comparisons test vs. control: 30 min p=0.0187; 60 min p=0.0037; 120 min p=0.0032; 180 min p=0.0133; control n=9 mice, hM₃Dq n=7 mice).

f. Experimental paradigm for virus delivery for fiber photometry analysis.

g. Representative image of GCaMP6f expression in the PVN^GLP-1R→DVC neurons (n=5 mice).

h. Representative traces of calcium activity in the fasted and fed state in PVN^GLP-1R→DVC neurons. “x” indicates detected peaks in calcium dynamics.

i. Frequency and amplitude quantification of PVN^GLP-1R→DVC neurons’ calcium activity in fasted and fed states (frequency two-tailed Wilcoxon matched-pairs signed rank test, p=0.0049; amplitude two-tailed Wilcoxon matched-pairs signed rank test, p>0.9999, n=100, 20 trial per mice (pooled from 5-minute baseline of food/object presentation experiments) from 5 mice).

j. Heatmap plots showing calcium responses of PVN^GLP-1R→DVC neurons during food or object presentations and under different energy states.

k. Quantification of area under the curve (AUC). (one-way ANOVA, F (3,16) = 6.251, p =0.0052, n=5 mice).

Data are presented as mean ± SEM. *p< 0.05; **p< 0.01.

PVN^GLP-1R→DVC neuronal responses to food presentation are energy state dependent

Overall, the neuronal activity of PVN^GLP-1R neurons increases during food access²². To test whether the PVN^GLP-1R→DVC neuronal subset responds to food, we utilized fiber photometry to record their real-time activity dynamics during feeding behaviors ^22-24. To express the genetically encoded Ca²⁺ sensor GCaMP6f in PVN^GLP-1R→DVC neurons, we injected AAVrg-EF1a-DIO-Flpo in the DVC and AAV-hSyn-fDIO-GCaMP6f in the PVN of GLP-1R-ires-Cre mice (Fig. 2f&g). Using fiber photometry in behaving mice, we found that these neurons showed significant increases in activity during grooming and in response to tail pick stress (Extended Data Fig. 5a-d). Interestingly, we also observed that the relative basal activity of these neurons was reduced with overnight fasting (i.e. hunger or energy deficiency state) compared to ad libitum feeding (i.e. energy sufficient state), suggesting that PVN^GLP-1R→DVC neuronal activity is modified by energy state (energy sufficient vs. deficient) (Fig. 2h-i). We next asked whether PVN^GLP-1R→DVC neuronal activity is affected by food presentation in an energy state-dependent manner. We presented chow pellets or non-edible objects to mice in two different metabolic states: energy deficient (fasted) or energy replete (ad libitum fed). Interestingly, food presentation to fasted mice significantly increased PVN^GLP-1R→DVC neuronal activity, but not when mice were fed. In contrast, the introduction of a non-food object had no effect on PVN^GLP-1R→DVC activity (Fig. 2j&k). Furthermore, we also investigated whether food accessibility is necessary for the induction of PVN^GLP-1R→DVC activity. Mice were presented with a chow pellet confined within an enclosed tea ball, allowing them to see and smell the food directly without consuming it. Consistent with the responses to food presentation, PVN^GLP-1R→DVC neuronal activity in fasted mice, but not fed mice, showed a prominent increase in neuronal activity in response to inaccessible food. Meanwhile, inaccessible non-edible objects did not induce changes in neuronal activity (Extended Data Fig. 5e&f). Overall, these data strongly suggest that PVN^GLP-1R→DVC neuronal responses to food cues are energy state-dependent.

State-dependent synaptic plasticity in the PVN^GLP-1R→DVC neuronal circuit

Intake of large volumes of highly palatable diet activates GLP-1 releasing neurons²⁵, increases immediate early gene cFos expression in the PVN, and increases PVN^GLP-1R neurons’ spontaneous action potential firing²². Given that food intake-induced neuronal activity in the PVN^GLP-1R→DVC pathway is dependent on energy state (Fig. 2j-k), we hypothesized that GLP-1 regulation of synaptic transmission within this neural circuit (Fig. 1g-r) is also energy state-dependent. To test this hypothesis, we again conducted whole-cell patch clamp recordings to interrogate the changes in electrophysiological activity between fasted (energy deficient) and well-fed (energy replete) states.

We first characterized the intrinsic membrane properties of PVN^GLP-1R→DVC neurons after fasting. We injected AAVrg-hSyn-DIO-EGFP in the DVC of GLP-1R-ires-Cre mice and conducted patch clamp recordings from PVN^GLP-1R→DVC neurons (Extended Data Fig. 6a). Despite overnight fasting, no detectable differences in membrane capacitance and membrane resistance, and a slight change in resting membrane potential (RMP), were found (Extended Data Fig. 6b-d). We also measured the spontaneous activity and input-output relationship (current injection and action potential numbers) of PVN^GLP-1R→DVC neurons but found no differences between fasted and well-fed conditions (Extended Data Fig. 6e-j). These data indicate that the membrane excitability of PVN^GLP-1R→DVC neurons was unaffected by energy state.

Next, we investigated synaptic release from PVN^GLP-1R→DVC neurons after overnight fasting (Fig. 3a&b). We found a remarkable decrease in oEPSCs mediated by both AMPAR (recorded at a holding potential of −70 mV) and NMDAR (recorded at a holding potential of +60 mV, measured at 50ms after optical stimulation) with no change in the AMPAR/NMDAR oEPSC ratio in fasted mice (Fig. 3c-f). These data suggested that the presynaptic release probability of glutamate at the PVN^GLP-1R→DVC synapse is reduced when body energy is low. Because circulating GLP-1 levels are circadian and postprandially related^26-29, it is conceivable that after overnight fasting, circulating GLP-1 levels are lower than under ad libitum feeding (energy replete state). Since GLP-1R signaling augments synaptic strength at this synapse (Fig. 1), we therefore hypothesized that synaptic transmission at this synapse will be more sensitive under the state of hunger because of low GLP-1 levels. Consistent with this hypothesis, we found a larger relative increase in synaptic transmission under the state of hunger (Fig. 3g-k). This result highlights the regulatory effects of GLP-1 on synaptic transmission and the dynamic synaptic strength changes contributing to energy replenishment to maintain metabolic homeostasis.

Figure 3: — a. Experimental paradigm for AAV-DIO-ChR2-EYFP injection, electrophysiology

b. Food intake manipulations in mice.

c. Representative traces and quantification of AMPAR-mediated oEPSCs

d. Pooled data of AMPAR EPSCs (two-tailed Mann-Whitney test, p= 0.0005, Fed n=21 cells/3 mice, Fasted n=30 cells/4 mice),

e. Pooled data of NMDAR EPSCs (two-tailed Mann-Whitney test, p<0.0001, Fed n=21 cells/3 mice, Fasted n=29 cells/4 mice),

f. AMPAR/NMDAR EPSC ratio under different conditions (two-tailed Mann-Whitney test, p=0.52, Fed n=21 cells /3 mice, Fasted n=29 cells/4 mice).

g. Representative traces of AMPAR oEPSCs with or without Exn-4 under different energy states

h. Pooled data of AMPAR oEPSCs under Fed condition (two-tailed paired t-test, t (11) =2.802, p=0.0187, Fed n=11 cells/4 mice).

i. Pooled data of oEPSCs under fasting (Wilcoxon matched-pairs signed rank test, p=0.002; Fasted n=10 cells/3 mice).

j. Representative traces of AMPAR-mediated oEPSCs (normalized to control levels) with or without Exn-4 under fed or fasted states.

k. Pooled data (two-tailed t-test, t (21) =3.036, p=0.0068, Fed n=11 cells/4 mice, Fasted n=10 cells/3 mice).

Data are presented as mean ± SEM. *p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001.

High-fat diet-induced obesity blunts the activity of PVN^GLP-1R→DVC neurons

Energy homeostasis is perturbed in obesity, and we hypothesized that energy state-dependent synaptic plasticity at the PVN^GLP-1R→DVC synapse would be disrupted in the obese state. To test this hypothesis, we investigated synaptic transmission in a high-fat diet (HFD)-induced obesity (DIO) mouse model.

To label PVN^GLP-1R→DVC neurons, we again injected AAVrg-hSyn-DIO-EGFP in the DVC of GLP-1R-ires-Cre mice. After 12 weeks of HFD/control diet exposure (Extended Data Fig. 7a&b), we first examined the intrinsic properties of PVN^GLP-1R→DVC neurons. We found minor changes in the membrane properties including RMP but no change in cell capacitance (Extended Data Fig. 7c&d). We also observed a trend toward slightly increased spontaneous action potential firing frequency but no changes in the input-output relationship of current injection with induced action potential firing (Extended Data Fig. 7e-j). Thus, excitability is only modestly affected in these cells in DIO mice.

We then investigated whether excitatory synaptic strength of PVN^GLP-1R→DVC neurons is altered under DIO (Fig. 4a&b). We found a significant decrease in both AMPAR and NMDAR-mediated oEPSCs with no change in the AMPAR/NMDAR oEPSC ratio in HFD-fed mice compared with those fed normal chow (Fig. 4c-f). These data indicate that the presynaptic release of glutamate at the PVN^GLP-1R→DVC synapse is reduced in DIO mice. Since fasting had a significant impact on PVN^GLP-1R→DVC synaptic release in normal chow-fed mice (Fig. 3a-f), we tested if we could observe a similar impact of energy state on synaptic strength at this synapse in DIO subjects. However, we found no change in oEPSCs mediated by AMPAR and NMDAR between different energy states (Fig. 4g-j), with PVN^GLP-1R→DVC synapse release in DIO mice, either when fed or fasted, resembling that of fasted state synapse release in normal chow-fed mice. These data suggest that the dynamic changes in synaptic strength (presynaptic release probabilities) at the PVN^GLP-1R→DVC synapse are disrupted in DIO mice.

Figure 4: — a. Experimental paradigm for AAV-DIO-ChR2-EYFP injection and electrophysiology experiments.

b. Different animal groups fed with chow or HFD.

c. Representative traces of AMPAR-mediated oEPSCs under different conditions.

d. Pooled data of AMPAR-oEPSCs. (two-tailed Mann-Whitney test, p<0.0001, control n=25 cells/3 mice, HFD n=23 cells/3 mice).

e. NMDAR-oEPSCs (two-tailed Welch's t test, p<0.0001, control n=25 cells/3 mice, HFD n=21 cells/3 mice).

f. AMPAR/NMDAR oEPSC ratio (two-tailed t-test, t (46) =1.716, p=0.093, control n=25 cells/3 mice, HFD n=21 cells/3 mice) in control or HFD animal.

g. Representative traces of oEPSCs under conditions as indicated.

h. Pooled data of AMPAR oEPSCs (two-tailed t-test, t (38) =0.3835, p=0.7036, Fed n=19 cells/3 mice, Fasted n=19 cells/3 mice),

i. Pooled data of NMDAR oEPSCs (two-tailed t-test, t (36) =1.071, p=0.2919, Fed n=18 cells/3 mice, Fasted n=18 cells/3 mice),

j. Pooled data of AMPAR/NMDAR oEPSCs ratio (two-tailed Mann-Whitney test, p = 0.2387, Fed n=18 cells/3 mice, Fasted n=18 cells/3 mice) under different energy states in HFD-induced obese animals.

k. Representative traces of AMPAR-mediated oEPSCs before and after application of Exn-4 in HFD-induced obese animals

l. Pooled data under conditions shown in k. (two-tailed paired t-test, t (8) =3.014, p=0.0195, n=8 cells/7 mice).

m. Representative traces of AMPAR-mediated oEPSCs after control or Liraglutide i.p. 2h injection.

n. Pooled data. Two-tailed Welch's t-test, p =0.0012, control n=19 cells/3 mice (same data showed in h, fed HFD), Liraglutide n=25 cells/3 mice) in HFD obese mice.

Data are presented as mean ± SEM. *p< 0.05; **p< 0.01; ****p< 0.0001.

Since GLP-1R agonists are effective in obese humans in reducing appetite and body weight², we hypothesized that GLP-1R signaling is still intact in DIO mice to regulate synaptic transmission. Indeed, the application of Exn-4 significantly increased PVN^GLP-1R→DVC synaptic release in ex vivo slice recordings (Fig. 4k&l). We further hypothesized that systemically administered GLP-1R agonists, such as liraglutide³⁰, may exert actions on the PVN^GLP-1R→DVC pathway. To this end, we injected 400 μg/kg liraglutide intraperitoneally in DIO mice and conducted brain slice electrophysiological recordings 2 hours after injection. We found that liraglutide-injected DIO mice showed enhanced PVN^GLP-1R→DVC oEPSCs mediated by AMPAR and NMDAR (Fig. 4m&n and Extended Data Fig. 8). These results indicate that GLP-1R mediated signaling is intact in DIO mice and systemic administration of GLP-1 analogs may suppress feeding by augmenting PVN^GLP-1R→DVC synaptic strength.

Activation of PVN^GLP-1R→DVC projections suppresses food intake in an energy-dependent manner

Next, we evaluated the effect of optogenetic activation of the PVN^GLP-1R→DVC projections on food intake. To accomplish this, we expressed ChR2 or EYFP (as a control) in PVN^GLP-1R neurons and implanted optic fibers in the DVC (Fig 5a&b). As predicted, photoactivation of PVN^GLP-1R neuronal axons in DVC strongly suppressed feeding (stimulation for 30 minutes vs. subsequent 30 minutes), while control mice showed no differences (Fig. 5c-e, Extended Data Fig. 9a).

Figure 5: — a. Experimental paradigm for ChR2/EYFP injection and optical fiber implantation.

b. Representative image of the DVC showing the expression of ChR2-EYFP (n=7 mice).

c. Experimental paradigm for chow intake in mice.

d. Quantification of fasting-induced refeeding chow intake for ChR2/EYFP mice (470 nm 20 Hz, 1s on, 0.5 s off). (two-tailed paired t-test, t (6) =0.4582, p=0.666, EYFP n=6 mice).

e. Quantitation of ChR2 chow intake measurement (two-tailed paired t-test, t (7) =6.386, p=0.0007, ChR2 n=7 mice).

f. Experimental paradigm of EYFP/ChR2 mice during consumption of sucrose.

g. Raster plot of licks of fasted EYFP/ChR2 before and after 12 weeks of HFD during consumption of 10% sucrose during control (black; no stimulation) or test periods (blue; photostimulation, 470 nm 20 Hz, 1s on, 0.5 s off).

h. Normalized sucrose licking rate of fasted ChR2 and ChR2 after 12 weeks of HFD mice during control (black; no stimulation) or test intervals (blue; photostimulation, 470 nm 20 Hz, 1s on, 0.5 s off). (two-way ANOVA, the main effect of Group: F (3, 22) = 176.7, p<0.0001; main effect of Time: F (1.274, 28.03) = 6.310, p=0.0127; interaction between Group and Time: F (6, 44) = 0.4783, p=0.8208; Sidak's multiple comparisons test vs. EYFP: Light On ChR2 p<0.0001; EYFP n= 6 mice, ChR2 n=7 mice).

Data are presented as mean ± SEM. ***p< 0.001; ****p< 0.0001.

Parts of Figure 5c&f were sourced from https://scidraw.io/ under the Creative Commons license (CC-BY).

Since the dynamic changes in synaptic transmission were disrupted in the PVN^GLP-1R→DVC circuit in DIO mice, we hypothesized that prolonged HFD feeding would impair the ability of PVN^GLP-1R→DVC stimulation to suppress feeding. To test this, we utilized a head-fixed sucrose (palatable food) consumption paradigm in mice (Fig. 5f) under different energy states (fasting, fed, and DIO). Mice were well-trained to consume sucrose solution by licking at a spout. Consistent with chow feeding (Fig.5a-e), we found that photostimulation of PVN^GLP-1R projections in the DVC significantly reduced sucrose licking rates (i.e., food intake) (Fig. 5g&h). In fact, we observed a significant reduction in sucrose licking rates in ChR2 mice during photostimulation in both fasted and fed conditions (Fig. 5g&h, Extended Data Fig. 9d&e), suggesting that even under the hunger (energy deficient) state, activation of the PVN^GLP-1R→DVC circuit is sufficient to suppress feeding. We then fed these mice with 60% HFD for 12 weeks (i.e., DIO) and tested the impact of HFD on food intake suppression mediated by PVN^GLP-1R→DVC projections (Extended Data Fig. 9b&c). Consistent with our hypothesis, DIO impaired the food intake suppression induced by photoactivation of PVN^GLP-1R axons in the DVC (Fig. 5g&h, Extended Data Fig. 9d&e).

One explanation of the reduction of food intake during photostimulation of the PVN^GLP-1R→DVC projection was aversion induced by activation of this pathway. To test this, we conducted a test for real-time place preference. Ad libitum fed mice were placed in a rectangular arena in which half of the area was paired to optogenetic stimulation. In this task, mice expressing ChR2 or EYFP in PVN^GLP-1R→DVC neurons spent similar time in the light-paired and unpaired sides, indicating that activation of this pathway is likely not aversive (Extended Data Fig. 9f&g).

Overall, these data suggest that within the PVN^GLP-1R→DVC circuit, synaptic strength is dynamically changing with energy state (i.e. stronger synaptic transmission when energy replete, weaker when energy depleted (Fig. 3). This dynamic synaptic plasticity is disrupted in DIO mice (Fig. 4c-f) but nevertheless can be rescued by GLP-1R agonists (Fig. 4k-n). Photostimulation of the PVN^GLP-1R→DVC circuit strongly suppresses food intake, and DIO impairs the function of this circuit on feeding suppression (Fig 5).

Knockout (KO) of presynaptic GLP-1Rs and inactivation of PVN^GLP-1R→DVC neuronal synaptic release causes body weight gain

Having demonstrated that GLP-1R signaling regulates synaptic strength in the PVN^GLP-1R→DVC descending circuit and the involvement of this in controlling feeding, we asked whether GLP-1Rs in this circuit are necessary for maintaining energy homeostasis. To achieve specific knockout in PVN^GLP-1R→DVC neurons, we injected AAVrg-FLPo-WPRE-hGHpA in the DVC and AAV-EF1a-fDIO-Cre in the PVN of GLP-1R^flox/flox mice³¹. This widely accepted gene manipulation strategy^32-34 allows Cre recombinase expression and thus ablates GLP-1R specifically in DVC-projecting PVN^GLP-1R neurons (Fig. 6a). Compared to control mice, PVN^GLP-1R→DVC GLP-1R KO mice exhibited significant body weight gain and increased daily normal chow intake (Fig. 6b&c). These results suggest that GLP-1R signaling in this pathway is vital to controlling energy homeostasis, likely via regulating synaptic release.

Figure 6: — a. Experimental paradigm for KO of GLP-1R in mice.

b. Quantification of body weight after KO of GLP-1R in PVN^GLP-1R→DVC neurons (two-way ANOVA, Group effect: F(1, 19)=5.775, p=0.0266; Time effect: F (1.474, 28)=17.29, p<0.0001; interactions: F(6, 114)=4.183, p=0.0008; n=9 and 12 mice, control and KO groups, respectively).

c. Quantification of daily food intake (two-tailed t-test, t (12)=2.848, p=0.0173, control 4 mice, KO 8 mice).

d. Experimental paradigm for inactivation of synaptic release by TeNT.

e. Representative image of the PVN showing the expression of TeNT-GFP (n=9 mice).

f. Quantification of body weight gain (two-way ANOVA, Group effect: F(1, 15)=4.841, p=0.0439; Time effect: F(1.282, 18.64)=20.84, p<0.0001; interaction: F(11, 160)=3.258, p=0.0005; control 8 mice, TeNT 9 mice).

g. Quantification of daily food intake with or without TeNT expression (two-tailed t-test, t(17) =1.123, p=0.0165; control 8 mice, TeNT 9 mice).

h. Fasting glucose levels (two-tailed t-test, t(16) =2.381, p=0.0371; control 7 mice, TeNT 9 mice).

i. Insulin tolerance test (two-way ANOVA, Group effect: F(1, 14)=5.679, p=0.0319; Time effect: F (2.062, 28.87)=23.9, p<0.0001; interaction: F(3, 42)=3.508, p=0.0233; Sidak's multiple comparisons: 15 min p=0.3564; 30 min p=0.0097; 60 min p=0.295; control 7 mice, TeNT 9 mice).

j. Liver weights in TeNT-injected and control mice (two-tailed Welch's t-test, t(15)=2.246, p=0.0471; control 6 mice, TeNT 9 mice, same as in k-m).

k. Subcutaneous WAT weights (two-tailed Welch's t-test, t(15)=2.586, p=0.023).

l. Perigonadal WAT weights (two-tailed Welch's t-test, t(15)=1.162, p=0.2726)

m. Brown fat (BAT) weights (two-tailed t-test, t(15)=1.515, p=0.1547).

n. Experimental paradigm for TeNT and Liraglutide application in HFD-mice.

o. Body weight change (two-way ANOVA, Group effect: F(1, 12) =0.009758, p=0.9229; Time effect: F(1.539, 18.47) = 60.56; interaction: F (5, 60) =0.5699, p=0.7227; n=7 in both groups);

p. Cumulative food intake. (two-way ANOVA, Group effect: F(1, 12) =0.958, p=0.347; Time effect: F(1.212, 14.55) =84.55, p<0.0001; interaction: F(5, 114) =1.092, p=0.3743; n=7 in both groups).

Data are presented as mean ± SEM. *p< 0.05; **p< 0.01.

Parts of Figure 6n were drawn by adapting pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 4.0 Unported License (https://creativecommons.org/licenses/by/4.0/).

To directly test this possibility, we expressed tetanus toxin (TeNT) light chain in PVN^GLP-1R→DVC neurons to irreversibly silence synaptic release by cleaving the synaptic vesicle protein synaptobrevin³⁵. In GLP-1R-ires-Cre mice, we injected AAVrg-DIO-FLPo in the DVC and AAV-CMV-fDIO-TeNT or a control virus in the PVN (Fig. 6d&e). TeNT-expressing mice gained significantly more body weight and exhibited increased daily food intake compared to controls (Fig. 6f&g). We further examined glucose metabolism at 13 weeks after inactivating PVN^GLP-1R→DVC synaptic transmission and found that the inactivation group exhibited elevated fasting blood glucose levels, and decreased insulin tolerance (ITT) (Fig. 6h&i). Since chemogenetic activation of PVN^GLP-1R→DVC neurons did not affect GTT (Extended Data Fig. 4d), the blood glucose impairment was likely a consequence of obesity induced by inactivation. Moreover, an altered respiratory exchange ratio (RER) was also observed (Extended Data Fig. 10 a-e). The TeNT group also displayed an increase in liver and subcutaneous white adipose tissue (WAT) weight, while the perigonadal WAT and brown adipose tissue weight remained unchanged (Fig. 6j-m).

It has been suggested that the pharmacological effects of systemically applied GLP-1 analogs induce food suppression via peripheral organs and the brain independently⁵. It is of interest to investigate if the PVN^GLP-1R→DVC circuit mediates the anorexigenic effects of liraglutide. In DIO mice with or without PVN^GLP-1R → DVC inactivation (by expressing TeNT or EYFP in these neurons), we administered 200 μg/kg liraglutide intraperitoneally for 5 consecutive days. We found that liraglutide significantly reduced food intake and body weight in both TeNT and EYFP DIO mice (Extended Data Fig. 10 f-h). However, there were no significant differences in cumulative food intake or body weight reduction between the two groups (Fig. 6n-p).

Collectively, these data strongly indicate that GLP-1R signaling and synaptic transmission in the hypothalamic-brain stem descending circuit, via PVN^GLP-1R→DVC neurons, contribute to physiological energy balance regulation but are not required for the anorexigenic effects of systematic application of GLP-1 analog.

Discussion

GLP-1 analogs are effective in treating type 2 diabetes and obesity, but the physiological functions, pharmacological targets, and brain circuits that mediate these important effects are not fully understood². Therefore, unraveling the CNS function of GLP-1R signaling is crucial. This study reveals a hypothalamus-brain stem descending pathway as an important mediator of GLP-1R signaling, feeding behavior, and body weight homeostasis. We find that GLP-1R signaling in the PVN^GLP-1R→DVC descending pathway regulates synaptic plasticity to maintain energy homeostasis. Disruption of synaptic transmission or ablation of GLP-1R in this circuit causes obesity. In DIO mice, the state-dependent synaptic plasticity in the circuit is disrupted but still responsive to GLP-1. Our study highlights that this dynamic synaptic plasticity in the PVN^GLP-1R→DVC descending circuit regulated by GLP-1 is crucial for maintaining energy homeostasis.

While previous studies have demonstrated the role of descending oxytocinergic PVN to caudal brainstem pathway in regulating feeding³⁶, we show a role for presynaptic GLP-1R signaling in this descending pathway in this study. Experiments involving administration of GLP-1R antagonists or genetic manipulation of GLP-1R expression lend support for the role of both hypothalamic and hindbrain GLP-1Rs in the physiological control of food intake^30,37. However, central and peripheral GLP-1 systems suppress eating via independent gut-brain circuits⁵. Clinical studies suggest that GLP-1 reduces postprandial glycemic excursions but no longer inhibits food intake or the rate of gastric emptying in non-diabetic normal-weight men after truncal vagotomy and pyloroplasty³⁸. Therefore, hindbrain vagal circuits must be critical for endogenous GLP-1 action and the pharmacological responses to GLP-1R agonists, as recently reported⁶. Nevertheless, it is still unclear how GLP-1R signaling translates to feeding regulation via descending circuits. In this study, we address this question and find that GLP-1R-mediated signaling in the PVN^GLP-1R→DVC circuit is required for maintaining energy homeostasis. We showed that 1) GLP-1 regulates PVN^GLP-1R→DVC glutamate release via the PKA pathway in the presynaptic compartment (Fig. 1); 2) both chemogenetic and optogenetic activation of the PVN^GLP-1R→DVC circuit strongly suppresses food intake, and the suppression can be modulated by different energy states and DIO (Fig 2&5); 3) knockout of GLP-1Rs in presynaptic neurons in the PVN^GLP-1R→DVC circuit causes body weight gain (Fig. 6a-c); 4) blockade of synaptic transmission in the PVN^GLP-1R→DVC circuit increases body weight and induces deficits in glucose metabolism (Fig. 6d-m); and 5) the PVN^GLP-1R→DVC circuit is not required for the anorexigenic effects of liraglutide (Fig. 6n-p, Extended Data Fig. 10 f-h). Nevertheless, due to the long debate on the involvement of central GLP-1R-mediated signaling in regulating metabolism^5,20,39, more defined pathway and cell-type specific manipulation of GLP-1R expression levels in the PVN^GLP-1R neurons including those projecting to other brain regions beyond DVC will be needed in the future.

Our loss-of-function (inactivation of synaptic release) data and the state-dependent synaptic plasticity we describe demonstrate that the descending PVN^GLP-1R→DVC circuit is a crucial target for endogenous GLP-1. NTS GLP-1-producing neurons are within the caudal brain stem, and it is conceivable that the major source of brain stem GLP-1 is from the local production of the NTS GLP-1 neurons, given that somatodendritic release of neuropeptides can occur^40,41. Moreover, the identity of downstream neurons in the DVC remains to be determined. It is well-documented that multiple brainstem neuron populations⁴², including GLP-1 neurons, GABAergic neurons⁴³, neurons expressing ChAT⁴⁴, leptin receptor-expressing neurons, calcitonin receptors neurons, cholecystokinin-expressing neurons⁴⁵, as well as proopiomelanocortin-expressing neurons⁴⁶, collectively suppress food intake. Whether or not PVN^GLP-1R neurons project to NTS GLP-1-producing neurons to form a positive feedback loop remains to be elucidated in future studies. It has also been shown that descending motor vagal circuits regulate not only regulating food intake but also fat absorption and stress-induced immune response^47-49. Future work is required to investigate brain-gut actions involving PVN^GLP-1R→DVC neurons.

Circulating GLP-1 levels, mainly derived from intestinal L-cells, increase postprandially^27,28,50. In the brain, GLP-1 released from NTS neurons is likely the main source of GLP-1, and food intake activates these neurons; therefore, brain GLP-1 availability may also increase postprandially^51,52. NTS GLP-1 neurons were shown to be activated by mechanical feedback from the gut, tracked food intake, and promoted long-lasting satiety⁵³. In this study, we showed that synaptic transmission in the PVN^GLP-1R→DVC circuit is crucial for energy metabolism and found that synaptic plasticity in this circuit is dynamically regulated by energy states (Fig. 3): when energy is replete, PVN^GLP-1R→DVC synaptic release is potentiated, suppressing further food intake. When energy is deficient (i.e. in hunger), PVN^GLP-1R→DVC synaptic release is weak, therefore allowing food intake. We further demonstrated that synaptic plasticity in this circuit is regulated by GLP-1, which has a more profound impact on synaptic release in energy-deficient states (e.g., after overnight fasting). In ad libitum-fed mice, there were higher GLP-1 levels compared to the fasted state, which precluded further increases in synaptic strength when an exogenous GLP-1 analog was applied. We speculate that the dynamic changes of brain GLP-1 with mealtime are the main regulator of synaptic plasticity in the PVN^GLP-1R→DVC circuit, and changes in PVN^GLP-1R→DVC synaptic strength regulate food intake behavior. Future work will focus on linking the contributions of these changes in synaptic strength to the in vivo neuronal responses to food intake. It has been shown that metabolic states such as fasting and high-fat feeding could rewire feeding-related circuits and form new synapses^54,55; whether synapse formation changes and rewires in the PVN^GLP-1R→DVC circuit remains to be studied. Nevertheless, our fiber photometry data clearly demonstrated that these PVN^GLP-1R→DVC neurons are energy state-dependently responsive to food cues.

It has been reported that DIO remodels neurocircuits in the hypothalamic arcuate nucleus⁵⁵ and the lateral hypothalamus⁵⁶. In our study, we observed a marked reduction in PVN^GLP-1R→DVC synaptic strength in DIO mice (Fig. 4c-f). This is somewhat surprising—since it has been reported that fasting GLP-1 levels are increased in obesity⁵⁷, we would expect that if GLP-1 levels are higher in the brains of DIO mice, synaptic transmission would be stronger. Another alternative explanation is that the GLP-1R is desensitized due to the higher GLP-1 levels in the obese subjects. However, this appears not to be the case since both in vitro applied GLP-1 analog Exn-4 and systematically applied liraglutide facilitated synaptic release in the PVN^GLP-1R→DVC pathway (Fig. 4k-n), suggesting that GLP-1R mediated signaling appeared to be intact in obese mice. Moreover, there was no significant change in these synaptic responses between ad libitum fed and overnight fasting in DIO mice (Fig. 4g-i). In mice fed normal chow, synaptic strength is likely regulated by changing brain GLP-1 levels in different energy states. Therefore, we speculate endogenous GLP-1 production or release in DIO is defective, consistent with a previous report demonstrating reductions in oral glucose-induced GLP-1 release in obese patients⁵⁸. This also explains why the administration of exogenous GLP-1 analogs is effective in obese patients. Although our data do not provide further molecular insights into how obesity causes synaptic strengths modifications, our observations are in line with the hypothesis of defective GLP-1 production and release in DIO, as failure to appropriately regulate synaptic plasticity according to energy state is suspected to underlie overeating. We thus argue that the dynamic changes in brain GLP-1 levels regulate synaptic strength depending on energy state to regulate feeding. We thus propose that therapeutics mimicking the endogenous dynamic changes of GLP-1 levels may be more effective for patient health.

GLP-1 analogs such as liraglutide have robustly anorexigenic effects but also cause side effects, including nausea, vomiting, diarrhea, and gallbladder issues^2-4. The anorectic and nausea-inducing effects of these agents are shown to be mediated by the activation of GLP-1 receptors within the brain^3,6. Recent studies have demonstrated that AP, NTS⁶, dorsomedial hypothalamus⁷, and dorsolateral septum⁵⁹ GLP-1R neurons are required for the GLP-1 receptor agonists’ anorexigenic effects. It has also been shown that GLP-1R-expressing neurons in the AP are the main culprits of the aversive effects of systematically applied GLP-1R agonists⁶. Our data suggest that chemogenetic and optogenetic activation of the PVN^GLP-1R→DVC pathway robustly suppressed feeding without apparent aversive avoidance (Fig 2,5 and Extended Data Fig. 9). However, TeNT ablation in DIO mice with liraglutide administration suggests that the PVN^GLP-1R→DVC pathway is not required for anorexigenic effects (Fig. 6n-p, Extended Data Fig. 10 f-h). The interpretation of these results could be complex. Nevertheless, this data is also consistent with the CRISPR-mediated knockdown of GLP-1Rs in the PVN not altering the anorectic properties of liraglutide⁵⁹. Moreover, the therapeutic effects of GLP-1R agonists on reducing feeding and body weight are not entirely dependent on central GLP-1 release, as NTS GLP-1 neuron activation augments semaglutide-induced eating suppression⁵. Future studies are needed to determine whether GLP-1 signaling in the PVN enhances the effects of GLP-1 receptor agonists on food intake and other aspects of metabolic regulation such as the regulation of gastrointestinal functions via the autonomic parasympathetic nervous system.

Methods

Animals.

All studies and procedures involving mice were approved by the Rutgers University Institutional Animal Care and Use Committee (IACUC) and by the National Institute of Health (NIH) guidelines. The mice used in this study were 5-20 weeks old, housed, and bred in the Child Health Institute of New Jersey animal facility. Mice were housed in a 12-hour light–dark cycle (6:00 on, −18:00, off), room temperature 21-23 °C, humidity 30-70%. Homozygous GLP-1R-ires-Cre⁶⁰ (C57bl/6 hybrid background), and GLP-1R-flox³⁰ (C57bl/6 hybrid background) mice were used in the present study. Mice had ad libitum access to chow (TestDiet 5058-PicoLab) and water unless otherwise indicated (fasting studies and high fat diet (HFD, Cat#D12492, Research Diets, Inc)). In all cases, when possible, mice were randomized according to body weight in each experimental group. The investigators were blinded to the treatment when possible.

AAV vectors and stereotaxic injections.

The AAV used in this study include: AAV-DIO-SynaptoTag2 (obtained from Dr. Wei Xu at UT Southwestern Medical Center-AAV_DJ), AAV-EF1a-double-floxed-hChR2 (H134R)-EYFP-WPRE-HGHpA (Addgene, Cat# 20298-AAV9), AAV-hSyn-DIO-EGFP (Addgene, Cat# 50457-AAVrg), AAV-hSyn-DIO-mCherry (Addgene, Cat# 50459-AAVrg), AAV-Ef1a-DIO EYFP (Addgene, Cat# 27056-AAV9), AAV-EF1a-DIO-FLPo-WPRE-hGHpA (Addgene, Cat# 87306-AAVrg), AAV-EF1a-Flpo (Addgene, Cat# 55637-AAVrg), AAV-EF1a-fDIO-Cre (Addgene, Cat# 121675-AAV9), AAV-Ef1a-fDIO-GCaMP6f (Addgene, Cat# 128315-AAV1), AAV-hSyn-fDIO-hM3D (Gq)-WPREpA (Addgene, Cat# 154868-AAV8), AAV-Ef1a-fDIO-mCherry (Addgene, Cat# 55641-AAV1), and AAV-CMV-fDIO-TeNT-EYFP (obtained from Dr. Wei Xu at UT Southwestern Medical Center-AAV_DJ). AAVs were injected using a Nanoject III^® with 1nL/s speed stereotactically in 5-7 weeks old male mice under isoflurane anesthesia. The injection coordinates used for the DVC were: Antero-Posterior (AP): −7.5 mm from bregma; mediolateral (ML): ± 0.5 mm; dorsoventral (DV): −4.9 mm. The injection coordinates used for the PVN were: AP −0.92 mm from bregma; ML: ± 0.18 mm; DV: −4.75 mm. The injection coordinates used for the LPBN: AP −5.05 mm from bregma, ML ±1.25 mm, DV: −2.88 to −2.83 mm. The injection coordinates used for the LC: AP: −5.45 mm from bregma, ML ±1.1 mm, DV −4.2 to −3.7 mm. Viral-mediated gene expression was allowed for 3-4 weeks before experimental manipulation. Injection sites were confirmed post hoc by cutting brain sections and inspecting them under a stereoscope or microscope in all mice reported in this study.

Histology and Immunohistochemistry assay.

Mice were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4. The brains were postfixed in 4% PFA overnight and then moved to 30% sucrose for at least 24 hours. Coronal brain cryosections (50 μm) were cut thereafter. For immunohistochemistry experiments, sections were incubated in blocking buffer (4% bovine serum albumin, BSA, 1% goat serum, 0.2% Triton X-100 in PBS) for 1 hour and then incubated overnight with the primary antibody at 4°C. The next day, brain sections were washed with blocking buffer at least 3 times, at least 5 min each, incubated with secondary antibody at room temperature for 2 hours, and washed with PBS. Brain sections were then mounted onto glass slides with Fluoroshield (with or without DAPI). Images were acquired with a Zeiss LSM700 confocal microscope. Z-stack images captured the entire thickness of the section at 10 μm steps for images taken with a 20 × objective (N.A. 0.8).

Antibodies.

The primary antibodies used in this study were goat anti-ChAT (Sigma; AB144P; 1:500) and chicken anti-GFP (Invitrogen, A10262, 1:500). The 2^nd antibodies used were Goat anti-Chicken IgY (H+L), Alexa Fluor^™ 488, (Invitrogen, Catalog # A11039, 1:500); Donkey anti-Goat IgG (H+L), Alexa Fluor 633, (Invitrogen, Catalog # A-21082, 1:500).

Electrophysiology.

Brain slice electrophysiology and data analysis ^20,61 were conducted as the following: mice were deeply anesthetized with isoflurane and decapitated, and brains were removed and quickly immersed in the cold (4°C) oxygenated cutting solution containing (in mM): 50 sucrose, 2.5 KCl, 0.625 CaCl₂, 1.2 MgCl₂, 1.25 NaH2PO₄, 25 NaHCO₃, and 2.5 glucose. Coronal cerebral cortex slices, 300 μm in thickness, were cut using a vibratome (VT 1200S; Leica). Brain slices were collected in artificial cerebrospinal fluid (ACSF) and bubbled with 5%CO₂ and 95%O₂. The ACSF contained (in mM): 125 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 2.5 glucose. After 1 hour of recovery, slices were transferred to a recording chamber and constantly perfused with bath solution (33°C) at a flow rate of 2 ml/min. To record EPSCs, picrotoxin (50 mM, Sigma) was added to block inhibitory postsynaptic currents mediated by GABA_A receptors. For some experiments, GLP-1 analog Exn-4 (100 nM, Tocris) was added to the bath solution to activate GLP-1Rs. For a subset of experiments (Fig. 1q-r), H-89 (10μM) was applied to block the PKA pathway. Patch pipettes with a resistance of 5-8 MΩ were made from borosilicate glass (World Precision Instruments) with a pipette puller (PC-10, Narishige) and filled with the pipette solution containing (in mM): 126 K-Gluconate, 4 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na₂-GTP, 10 phosphocreatine (pH to 7.2 with KOH) for current and voltage-clamp recordings. After the whole-cell patch clamp was achieved, spontaneous EPSCs were recorded at a holding potential of −70 mV. Optogenetically evoked EPSCs, i.e. oEPSCs, were recorded at a holding potential of −70 mV for the AMAPAR-mediated component. To record NMDAR-mediated EPSCs, the holding potential was changed to +60mV, where both AMPAR-EPSC and NMDAR-EPSCs could be recorded, but AMPAR EPSCs would decay to baseline within 50ms. AMPAR/NMDAR-EPSCs ratios were calculated as the peak amplitude of oEPSC at a holding potential of −70 mV over the amplitude of oEPSCs at 50 ms after photostimulation. For analyzing quantal synaptic release from the PVN^GLP-1R terminals in the DVC, strontium (Sr²⁺, 5mM) was used to substitute the 2.5 mM of Ca²⁺ in the extracellular solution¹⁸. Delayed asynchronous synaptic release within a 300 ms window 25 ms after optogenetic stimulation was used for the analysis of the evoked quantal events¹⁹. MultiClamp 700B, Digitizer 1440, and pClamp 10.5.0.9 (Molecular Devices, USA) were used for data acquisition. All data were analyzed offline using ClampFit 10.5.0.9 (Molecular Devices, USA) software. Experiments with Exn-4 recording were performed only on DMV neurons to prevent post-synaptic GLP-1Rs confounding data interpretation.

Fiber Photometry.

We injected AAVrg- EF1a-DIO-Flpo in the DVC and AAV-hSyn-fDIO-GCaMP6f in the PVN of GLP-1R-ires-Cre mice. GCaMP6f fluorescence was collected through an optical fiber unilaterally implanted above the PVN (diameter: 400 μm; length: 5.0 mm; NA: 0.5). Recordings took place at least 3 weeks after virus delivery. A continuous blue LED at 465 nm and UV at 405 nm served as an excitation light source (Thorlabs), modulated at 211hz and 333hz respectively, and delivered to a filtered minicube (FMC6, Doric Lenses) before connecting through optic fibers to a rotary joint to allow for movement. GCaMP fluorescence was detected by a visible femtowatt photoreceiver (Tucker- Davis Technologies, Model 2151) with the gain set to DC low. The light was then converted to electrical signals and demodulated by a real-time processor (Tucker- Davis Technologies, RZ5P).

Data was collected through the software Synapse (TDT) and exported via Browser (TDT). Fluorescence emission was captured by Tucker-Davis Technologies hardware at about 1 kHz. Collected data were then processed in Python. Fluorescence data was downsampled to 10 Hz after smoothing with a 100 ms rolling average filter. The isosbestic 405 nm data channel was linearly scaled to the 465 nm channel and used as f0 to calculate ΔF/F = (f – f0)/f0. The change in fluorescence was normalized to the mean change of the whole recording. This data stream was then aligned around each manually annotated behavioral event, with the baseline mean of 10-30 seconds before the event being subtracted out. After this correction, the average and standard error across all events was obtained and visualized. The area under the curve (AUC) was calculated using event-aligned and background-subtracted ΔF/F (tail pick: 10s pre vs. post; grooming: 60s pre vs. post; food-related events: AUC of 3 minutes post subtracted by AUC of 1-minute pre-event). To calculate the frequency and amplitude of baseline calcium transients, we applied Scipy’s find_peaks function (1s distance, prominence of 1.4) to the 5-minute baseline (zero-phase filtered, no downsampling) of food and object accessible/inaccessible presentation experiments (4 experiments per mouse, yielding a total of 20 experiments). Data, as presented in figures (not for statistics), were then smoothed and downsampled further.

Sensory detection experiments were conducted as described²². Experiments were conducted in the afternoon. Mice were fasted for 12 to 16 hours by food deprivation. They were habituated to the open arena for at least 30 minutes before beginning of experimentation. For presentation experiments, photometry data was obtained as mice were presented with either a chow pellet or non-edible object (falcon tube). For tea ball presentation experiments, mice were first presented with either chow or an object in an inaccessible tea ball for 20 minutes. Then, the tea ball was opened, and the contents were accessible for 20 minutes. For all these experiments, 10 minutes of baseline photometry data was first obtained.

Chemogenetics.

For the activation of PVN^GLP-1R→DVC neurons, we injected 150 nL of AAV-EF1a-DIO-FLPo-WPRE-hGHpA (Cat# 87306-AAVrg) in the DVC (Antero-Posterior (AP) −7.5 mm from bregma; mediolateral (ML) ±0.5 mm, dorsal-ventral (DV): −4.9-5.0 mm.) and AAV-hSyn-fDIO-hM3D(Gq)-mCherry-WPREpA (Cat# 154868-AAV8) or AAV-Ef1a-fDIO mCherry (Cat# 114471-AAV8) in PVN (Antero-Posterior (AP) −0.92 mm from bregma; mediolateral (ML) ±0.18 mm, dorsal-ventral (DV): −4.7-4.8 mm).

Glucose Tolerance Test (GTT).

GTT was conducted in overnight fasted mice as described elsewhere ⁶². At time 0, blood glucose was measured to set a baseline. 1 mg/kg body weight CNO was injected intraperitoneally (i.p.). After 30 minutes, 1 g/kg body weight of 20% dextrose solution was injected i.p., and blood samples were collected from the tail vein at 15, 30, 60, and 120 minutes. For GTT, injection volume was calculated as follows: Injection volume (μL) = mouse weight (Kg) x $\frac{\frac{2 g}{k g} g l u c o s e}{0.2 g ∕ m l}$ = mouse weight (kg) x 10 ml/kg glucose= mouse weight (g) x 10

Insulin Tolerance Test (ITT).

ITT was conducted in mice after fasting of 6 hours as described elsewhere ⁶². At time 0, blood glucose was measured to set a baseline. Insulin (1 unit/kg ITT) was injected i.p., blood samples were collected from the tail vein at 15, 30, and 60 minutes. The plasma glucose was measured by a glucometer (FreeStyle Lite system, Abbott Diabetes Care CA, USA).

Injection volume (μL) = mouse weight (kg) x $\frac{\frac{1 u n i t}{k g} i n s u l i n}{0.1 u n i t ∕ m l}$ = mouse weight (kg) x 10 ml/kg insulin= mouse weight (g) x 10.

Comprehensive Lab Animal Monitoring System (CLAMS) assay.

Mice were placed into metabolic phenotyping cages (Columbus Instruments, OH, USA). Data was recorded every 15 minutes. Data was collected for 3 days (both light and dark cycles) after 2 days of habituation to single housing within the cages.

Food Intake.

Mice were single housed before the experiment on a 12 h light/dark cycle with ad libitum access to water and were fasted overnight for 12 hours (9 pm-9 am), 0.5 g of food was given to reduce animal anxiety. The following day, each animal's weight was measured, CNO was prepared, and 1mg/kg BW CNO was injected i.p. into each mouse. After 30 minutes, food was added to their cage. Standard chow intake was measured at t=0,0.5,1,2,3 and 24 hours. After the experiment, mice were grouped into their respective cages.

Open Field Locomotor Activity.

The open field test was performed as previously described⁶³. Briefly, mice were placed in the middle of a custom-made 45 x 45 cm square open field arena for 10 minutes. The time spent in the center and total distance traveled were quantified using DeepLabCut version 2.2 and region of interest selection⁶⁴. The pose estimation model (ResNet-50-based with default parameters) was trained for 600,000 iterations on 200 frames of open field arena data extracted from 10 videos. 95% of these frames were used for training and 5% for testing. Estimation data was processed using custom software adapted from DLC2Kinematics and region of interest tools. The inner three-fifths of the arena was defined as the center zone. Time in the center zone was calculated using the center of the mouse’s body. During analysis, investigators were also blinded to the experimental group.

Light-dark box.

The light/dark box is viewed as an ethological model of anxiety, placing into competition the drives to remain safe and to explore novel environments. Mice were placed in the light side of a commercial light-dark box apparatus (AlfaSci) consisting of one dark and one illuminated compartment connected by a door. Time spent in each zone, latency to enter the dark zone, and number of entries were measured for 10 minutes.

Optogenetics.

For the photoactivation of PVN^GLP-1R→DVC neurons, we injected 150 nL pAAV-EF1a-double-floxed-hChR2 (H134R)-EYFP-WPRE-HGHpA or pAAV-hSyn-DIO-EGFP in PVN (Antero-Posterior (AP) −0.92 mm from bregma; mediolateral (ML) ±0.18 mm, dorsal-ventral (DV) −4.7-4.8 mm). An optical fiber was implanted above the DVC (Antero-Posterior (AP) −7.56mm from bregma; mediolateral (ML): 0 mm, dorsal-ventral (DV): −3.6 mm; diameter: 200 μm; length: 5.0 mm; NA: 0.37).

Free chow feeding behavior.

Mice were habituated to the behavioral context in daily 10 minute sessions for 3 days before experiments. For the fasted refeeding test, mice were food-restricted the day before the test. Mice were presented with a regular chow pellet and allowed to feed. The weight of the food pellet, including the food debris left on the cage floor after the test, was measured to calculate the food intake. Optogenetic stimulation was started just after the mice were put into the testing cage for 30 min, then the light was off for 30 min. The food intake was measured for 1 h. All of the feeding tests were performed between 1 p.m. and 6 p.m.

Head-fixed sucrose licking behavior.

Optogenetic stimulation was performed as previously described^56,65. Following recovery 3-4 weeks from surgery and prior to optogenetic stimulation experiments, mice were habituated to head fixation for 3 days, during which lick-induced drops of sucrose (10% sucrose in water; 2–2.5μL) were delivered intermittently for 15 minutes through a gravity-driven, solenoid-controlled lick spout. On optogenetic test days, mice were head-fixed and connected to a 473-nm DPSS laser.

Test sessions consisted of 5 min laser off (pre), 5 min laser on, and 5 min off (post, 15 min total) in which ~2μL of 10% sucrose solution was delivered via a tube placed directly in front of the mouse’s mouth when mice licked at the spout. Photostimulation was performed using laser stimulation (20 Hz, 5 ms pulse width 1s on, 0.5 s off, 10 mW). Licks were recorded during the entire session (baseline, stimulation, and post-stimulation periods). Mice were kept food-restricted at 85–90% baseline body weight during initial conditioning. Once the mice displayed sufficient licking behavior, they were either kept ad libitum fed or fasted overnight for each of the optogenetic stimulation experiments. After experiments were performed in the fed and fasted states, mice were put on HFD (60% animal fat calories; Research Diet Cat# D12492) for 12 weeks. Optogenetic experiments were resumed, with mice kept either ad libitum fed or fasted for 24 hours through each of the experiments. Licking data was normalized to the average of the first 5 minutes off (baseline) of sessions to calculate the normalized lick rate.

Real-time place preference paradigm (RTPP).

As described before⁶⁶, mice (chow fed) were connected to the laser via optic fiber patch cables and habituated to a rectangular acrylic arena (20 × 20 × 42 cm). The position of the mouse within the chamber was tracked by a camera above the chamber. The test session lasted 15 min, and the laser-paired side of the chamber was counterbalanced. Every time the mouse entered the stimulation side, 10 ms, 470 nm light pulses at 20 Hz and ~7.2 mW (measured at the tip of optic fibers) were delivered intracranially for the activation experiment. Customized MATLAB code was used to determine the mouse’s position based on the contrast of the mouse’s fur coat and coordinate the delivery of light pulses.

Ablation of GLP-1R in PVN^GLP-1R→DVC neurons.

To achieve specific ablation of PVN^GLP-1R→DVC neurons, we injected AAVrg-FLPo-WPRE-hGHpA in the DVC and AAV-EF1a-fDIO-Cre in the PVN in GLP-1R^flox/flox mice. Body weight was monitored every week post-surgery (week 0). On week 6, mice were separated into individual cages to adapt to their surroundings for one day. Food intake was measured from day 3 to day 8.

Tetanus toxin (TeNT) light chain inactivation of synaptic transmission.

In GLP-1R-ires-Cre mice, we injected AAVrg-DIO-FLPo in the DVC and AAV-CMV-fDIO-TeNT-EYFP or control virus in the PVN. Body weight was monitored every week post-surgery (week 0). On week 7, mice were separated into individual cages to adapt to their surroundings for one day. Food intake was measured from day 3 to day 8. During week 12, all mice were placed into metabolic cages (Columbus Instruments, Comprehensive Lab Animal Monitoring System (CLAMS), OH, USA) to adapt to their surroundings 12 hr before measurement. All metabolic data were collected in both day and night phases. In the DIO liraglutide experiments, GLP-1R-ires-Cre mice were fed a HFD for 12 weeks before receiving AAVrg-DIO-FLPo injections in DVC and AAV-CMV-fDIO-TeNT-EYFP injections in PVN. After 4 weeks of TeNT or EYFP AAV expression, food intake and body weight were measured for 2 days prior to liraglutide administration. Both TeNT and EYFP mice were then injected with 200 μg/kg liraglutide daily for 5 consecutive days. Average daily food intake during baseline (2 days) and liraglutide treatment (5 days), along with cumulative body weight change and food intake, was recorded.

Statistical information.

No statistical methods were used to pre-determine sample sizes, but our sample sizes are similar to those reported in previous publications ^20,22,65. Statistical analysis was performed using GraphPad Prism 10.0. All the data is presented as mean ± standard error of the mean (SEM). Data were tested for normality using the Shapiro-Wilk test. Non-significant results (p>0.05) are not displayed. A p-value of less than 0.05 was considered statistically significant. All details of the statistical information can be found in the figure legends.

Data exclusions.

Viral expression was confirmed by post hoc histological examination. Off-target injected mice were excluded in the final data summary. In the optogenetic head-fixed experiments, fasted mice with fewer than 100 licks in 15 minutes after several days of training were considered unable to learn the paradigm and were also excluded.

Extended Data

Extended Data Fig. 3: — a. Representative image of a recorded DVC neuron labeled with neurobiotin. DMV neurons were visualized with ChAT immunostaining (n>3 mice). b. Representative traces of sEPSCs with or without Exn-4 (100 nM). c. Pooled data of frequency and amplitude of sEPSCs recorded in DVC neurons (frequency: two-tailed Wilcoxon matched-pairs signed rank test, p <0.0001; amplitude: two-tailed Wilcoxon matched-pairs signed rank test, p = 0.5876; n=35 cells/12 mice). d. The vast majority of DMV preganglionic motor neurons (ChAT-positive) do not express GLP-1R. GLP-1R-expressing neurons are visualized via ChR2-EYFP expression. e. Enlarged view of the area shown in panel d. n=3 mice. f. Quantification of AMPAR-mediated synchronous oEPSC (i.e., first peak) amplitudes before and after Exn-4 application with 5 mM Sr²⁺in external ACSF (two-tailed paired t-test, t(10)=2.935, p=0.0166, n=10 cells/3 mice). Note that these traces are the same from Figure 1 n, but to emphasize the synchronous release phase for quantifications. Data are presented as mean ± SEM. *p< 0.05; ****p< 0.0001.

Extended Data Fig. 4: — a. Food intake consumption after i.p. saline injection (control) in GLP1R-ires-Cre mice expressing hM₃Dq- or control-virus in PVN^GLP-1R→DVC neurons during the dark cycle (related to Figure 2e) (two-way ANOVA, Group effect: F(1, 14)=2.492, p=0.1368; Time effect: F(3, 42)=89.98, p<0.0001; interaction: F(3, 42)=0.6374, p=0.5952, control n=9 mice, hM₃Dq n=7 mice). b. Food intake upon activation of PVN^GLP-1R→DVC neurons in a fasted-refeeding paradigm during the light cycle (two-way ANOVA, main effect of Group: F(1, 14)=10.83, p=0.0054; main effect of Time: F(1.570, 21.98)=60.54, p<0.0001; interaction between Group and Time: F(3, 42)=1.921, p=0.1408; Sidak's multiple comparisons test vs. control: 30 min p=0.0056; 60 min p=0.0188; 120 min p=0.0296; 180 min p=0.0346; control n=9 mice, hM₃Dq n=7 mice). c. Food intake consumption upon activation of PVN^GLP-1R→DVC neurons in the fed light cycle (two-way ANOVA, Group effect: F(1, 14)=6.781, p=0.0208; Time effect: F(1.903, 26.64)=21, p<0.0001; interaction: F(3, 42)=3.899, p=0.0152; Sidak's multiple comparisons test vs. control: 30 min p=0.063; 60 min p=0.2495; 120 min p=0.0623; 180 min p=0.0256; control n=9 mice, hM₃Dq n=7 mice). d. Glucose tolerance test with or without chemogenetic activation (two-way ANOVA, Group effect: F(1, 14)=1.663, p=0.2181; Time effect: F(2.740, 38.36)=355.1, p<0.0001; interaction: F(4, 56)=1.342, p=0.2659; control n=9 mice, hM₃Dq n=7 mice). e. Representative traces of animal exploration in the open field with or without chemogenetic activation of PVN^GLP-1R→DVC neurons. f. Time spent in the center of the open field (two-tailed t-test, t (16) =1.617, p=0.1282; control n=9 mice, hM₃Dq n=7 mice). g. Total traveled distance in the open field (two-tailed t-test, t(16)=1.283, p=0.2205; control n=9 mice, hM₃Dq n=7 mice). h. Quantification of light-dark box assay for anxiety-like behaviors: time spent in the dark zone (two-tailed t-test, t(16)=0.1509, p=0.8822; control n=9 mice, hM₃Dq n=7 mice). i. Dark zone entries (two-tailed t-test, t(16)=0.0516, p=0.9596; control n=9 mice, hM₃Dq n=7 mice). Data are presented as mean ± standard error of the mean (SEM). *p< 0.05; **p< 0.01.

Extended Data Fig. 5: — a. Average response of fiber photometry data showing calcium dynamics of PVN^GLP-1R→DVC neurons during self-grooming behavior. b. Pooled data (two-tailed paired t-test, t(5) = 2.822, p = 0.0477; n=5 mice). c. Average response of fiber photometry calcium dynamics of PVN^GLP-1R→DVC neurons during tail picking-induced stress d. Pooled data (two-tailed paired t-test, t(5)=4.109, p = 0.0147; n=5 mice). e. Heatmap showing calcium dynamics of PVN^GLP-1R→DVC neurons during different food/object tea ball drop stimuli across different energy states. f. Quantitation of the areas under the curve (AUC) of calcium response (one-way ANOVA, F(3,16)=4.383, p=0.0197, n=5 mice). Data are presented as mean ± SEM. *p< 0.05.

Extended Data Fig. 6: — a. Experimental paradigm. b. Intrinsic electrophysiological characterization of PVN^GLP-1R→DVC neurons. Summary of capacitance (two-tailed t-test, t(52)=0.6357, p=0.5279; Fed n=24 cells/3 mice, Fasted n=28 cells/3 mice). c. Input resistance (two-tailed Mann-Whitney test, p= 0.7134; Fed n=24 cells/3 mice, Fasted n=28 cells/3 mice). d. Resting membrane potential (two-tailed Mann-Whitney test, p=0.0049; Fed n=24 cells/3 mice, Fasted n=28 cells/3 mice). e. Representative traces of spontaneous action potentials (sAPs). f. Quantification of sAP frequency (two-tailed Mann-Whitney test, p=0.3148; Fed n=24 cells/3 mice, Fasted n=28 cells/3 mice). g. Representative traces of neurons responding to ramping current injection. Insert show the current injection protocol. h. Quantification of ramping current injection action potential firing number (two-tailed Mann-Whitney test, p=0.3461; Fed n=25 cells/3 mice, Fasted n=27 cells/3 mice). i, Representative traces of neurons in response to stepped current injection. Insert shows the current injection protocol. j. Plot of the number of APs as a function of injected current (two-way ANOVA, Group effect: F(1, 48)=0.7961, p=0.3767; Time effect: F(1.340, 64.33)=98.47, p<0.0001; interaction: F(5, 240)=0.3641, p= 0.8728; Fed n=24 cells/3 mice, Fasted n=26 cells/3 mice). Data are presented as mean ± SEM. **p< 0.01.

Extended Data Fig. 7: — a. Experimental paradigm. b. Body weight of control and HFD-induced obese animals (two-tailed t-test, t (11) = 4.671, p=0.0012; control n=5 mice, HFD n=6 mice). c. Intrinsic electrophysiological characterization of PVN^GLP-1R→DVC neurons. Summary of capacitance (c) (two-tailed Mann-Whitney test, p=0.2638; control n=20 cells/3 mice, HFD n=32 cells/3 mice). d. Resting membrane potential (RMP) (two-tailed t-test, t (51) = 3.212, p=0.0023; control n=20 cells/3 mice, HFD n=31 cells/3 mice). e. Representative traces of spontaneous action potentials (sAPs). f. Pooled data of sAP frequency (two-tailed t-test, t (20) = 2.042, p= 0.0561; control n=6 cells/3 mice, HFD n=14 cells/3 mice). g. Representative traces of neurons in response to ramp current injection. Inset shows the current injection protocol. h. Quantification plot of ramping current injection-induced AP firing number (two-tailed Mann-Whitney test, p=0.5784; control n=20 cells/3 mice, HFD n=30 cells/3 mice). i. Representative traces of neurons in response to stepped current injection. Inset shows the current injection protocol. j. Plot of the number of APs as a function of injected current (two-way ANOVA, Group effect: F(1, 48)=0.1073, p=0.7447; Time effect: F(1.362, 64.97)=157.7, p<0.0001; interaction: F(10, 477) = 0.7476, p=0.6795; control n=20 cells/3 mice, HFD n=30 cells/3 mice). Data are presented as mean ± SEM. **p< 0.01.

Extended Data Fig. 8: — a. Representative traces of AMPAR-oEPSCs and NMDAR oEPSCs in HFD-fed mice with or without i.p. injection of 400 μg/kg liraglutide. b. Pooled data of NMDAR-oEPSCs (two-tailed t-test, t (43) = 2.561, p=0.0153; HFD n=18 cells/3 mice, Liraglutide n=25 cells/3 mice). c. AMPAR/NMDAR oEPSCs ratio (two-tailed t-test, t(43)=1.391, p=0.1718; HFD n=18 cells/3 mice, Liraglutide n=18 cells/3 mice). Data are presented as mean ± SEM. *p< 0.05.

Extended Data Fig. 9: — a. Quantification of fasted-refeeding normalized chow intake for ChR2/EYFP mice (normalized to EYFP/ChR2 off average chow intake, 470 nm 20 Hz, 1s on, 0.5 s off; two-tailed Mann-Whitney test, p=0.0146, EYFP n=6 mice, ChR2 n=7 mice). b. Experimental paradigm for testing sucrose licking in mouse in a head-fixed format. Animals were injected with AAV-ChR2 or EYFP into the PVN, and fiber optics were implanted in the DVC. c. Quantification of body weight changes after 12 weeks of HFD-DIO (two-tailed paired t-test, t(13)=4.257, p=0.0011; EYFP n=6 mice, ChR2 n=7 mice). d. Raster plots of licks (consumption of 10% sucrose) of energy-replete (fed) ChR2 and EYFP mice (post 12 weeks HFD) after pre (5 min), during blue light photostimulation (470 nm 20 Hz, 1s on, 0.5 s off, 5min), and 5 min post optogenetic stimulation. e. Normalized sucrose licking rates before, during and after optogenetic activation (two-way ANOVA, Group effect: F (3, 22) = 1.335e+017, p<0.0001; Time effect: F (1.294, 28.47) = 13.27, p=0.0005; interaction between Group and Time: F (6, 44) = 0.5878, p=0.7381; Sidak's multiple comparisons test vs. EYFP Light On ChR2 p=0.0003; EYFP n= 6 mice, ChR2 n=7 mice). f. Representative heatmaps depicting the time spent in the real-time place preference task. The colorbar denotes time spent in an area normalized to the maximum time spent. g. Preference of EYFP and ChR2 mice for the photostimulation chamber (470 nm, 20 Hz) (two-tailed Mann-Whitney test, p=0.9452; EYFP n=6 mice, ChR2 n=7 mice). Data are presented as mean ± SEM. *p< 0.05; **p< 0.01; ***p< 0.001. Parts of Extended Data Fig. 9b&f were sourced from https://scidraw.io/ under the Creative Commons license (CC-BY).

Extended Data Fig. 10: — a. Energy expenditure (EE) (two-way ANOVA, the main effect of Group: F(1, 14)=0.01004, p= 0.9216; main effect of Time: F(6.449, 90.29) = 4.545, p=0.0003; interaction between Group and Time: F(159, 2226)=1.054, p=0.3132; control n=7 mice, TeNT n=9 mice). b. The volume of carbon dioxide produced (VCO²) (two-way ANOVA, the main effect of Group: F(1, 14)=0.1053, p=0.7503; main effect of Time: F (6.291, 88.08)=4.765, p=0.0002; interaction between Group and Time: F(159, 2226) = 0.9634, p=0.6126; control n=7 mice, TeNT n=9 mice). c. Respiratory exchange ratio (RER) (two-way ANOVA, Group effect: F (1, 14)=4.844, p= 0.0450; Time effect: F(3.463, 48.49)=2.22, p=0.0893; interaction: F (159, 2226) = 0.442, p>0.9999; control n=7 mice, TeNT n=9 mice). d. Oxygen consumed (VO²) (two-way ANOVA, Group effect: F(1, 14)=0.003172, p=0.9559; Time effect: F(6.303, 88.25)=3.659, p=0.0023; interaction: F(119, 1666)=1.157, p=0.1256; control n=7 mice, TeNT n=9 mice). e. Average locomotor activity of TeNT and control (GFP) animals (two-way ANOVA, Group effect: F(1, 12)=1.862, p= 0.1974; Time effect: F(8.698, 104.4)=2.654, p=0.0089; interaction between Group and Time: F(159, 1908)=1.101, p=0.1919; control n=6 mice, TeNT n=8 mice). f. Experimental paradigm for inactivating PVN^GLP-1R→DVC synaptic release. Cre-dependent expression of Flpo and Flpo-dependent expression of TeNT in PVN^GLP-1R →DVC neurons. g. Average daily food intake of EYPF HFD-fed obese animals’ baseline for 2 days and after i.p. injection of 200 μg/kg liraglutide for 5 days (two-tailed paired t-test, t(7)=4.658, p = 0.0035; EYFP n=7 mice). h. Average daily food intake consumption of TeNT HFD-fed obese animals’ baseline for 2 days and after i.p. injection of 200 μg/kg liraglutide for 5 days (two-tailed paired t-test, t(7)=5.595, p = 0.0014; TeNT n=7 mice). Data are presented as mean ± SEM. **p< 0.01.

Acknowledgments

We thank Dr. Hyokjoon Kwon for help on the comprehensive Lab Animal Monitoring System (CLAMS) assay; Azeddine Tahiri and Naureen Hammed for help on the ITT and GTT experiments; Michael Yang for help with part of the histology and Dr. Junbing Wu for the helpful discussions. This study was supported by grants from the Robert Wood Johnson Foundation to the Child Health Institute of New Jersey (RWJF grant #74260, Z.P.P), NIH NIMH RF1MH120144 (Z.P.P.), NIH NIDDK R01DK131452 (Z.P.P.), NIH NIDDK R01DK122167 (A.E.), startup funding from New York Medical College (A.E.), CIHR PJT 180576 (M.B.W.), NSERC 72067156 (M.B.W.) NIH NIDDK R01DK136641 (M.A.R.), and the Whitehall Foundation Grant (#2022-12-051, M.A.R.). L.W. was supported by the New Jersey Governor’s Council for Medical Research and Treatment of Autism Postdoctoral Fellowship (CAUT24DFP) and the NExT-Metabolism Pilot Award (500301). I. S. was supported by an NSERC/CREATE fellowship. Some artwork used in Figures 5 and 6 was adapted from Servier Medical Art (http://https://smart.servier.com/).

Footnotes

Conflict of Interests

The authors declare no conflict of interest.

Data Accessibility

The data and datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Original histological images can be found at figshare⁶⁷.

Code Accessibility

Code used to analyze fiber photometry and open-field data can be found at https://github.com/RohanSavani/PVN_GLP1R.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data and datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Original histological images can be found at figshare⁶⁷.

Code used to analyze fiber photometry and open-field data can be found at https://github.com/RohanSavani/PVN_GLP1R.

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[R38] 38.Plamboeck A. et al. The effect of exogenous GLP-1 on food intake is lost in male truncally vagotomized subjects with pyloroplasty. Am J Physiol Gastrointest Liver Physiol 304, G1117–1127 (2013). 10.1152/ajpgi.00035.2013 [DOI] [PubMed] [Google Scholar]

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PERMALINK

State-dependent central synaptic regulation by GLP-1 is essential for energy homeostasis

Le Wang

Rohan H Savani

Yi Lu

Matteo Bernabucci

Jorge Luis-Islas

Erin Park

Ishnoor Singh

Wei Xu

Abdelfattah El Ouaamari

Michael B Wheeler

Harvey J Grill

Mark A Rossi

Zhiping P Pang

Abstract

GLP-1R signaling enhances PVNGLP-1R→DVC presynaptic release of glutamate

Figure 1: PVNGLP-1R→DVC descending circuit is regulated by GLP-1R-mediated signaling.

Activation of PVNGLP-1R→DVC suppresses feeding

Figure 2: State-dependent PVNGLP-1R→DVC neuronal activity suppresses feeding.

PVNGLP-1R→DVC neuronal responses to food presentation are energy state dependent

State-dependent synaptic plasticity in the PVNGLP-1R→DVC neuronal circuit

Figure 3: State-dependent synaptic plasticity of PVNGLP-1R→DVC neurons.

High-fat diet-induced obesity blunts the activity of PVNGLP-1R→DVC neurons

Figure 4: High-fat diet (HFD) induced obesity blunts the state-dependent synaptic plasticity of PVNGLP-1R→DVC neurons.

Activation of PVNGLP-1R→DVC projections suppresses food intake in an energy-dependent manner

Figure 5: HFD blunts optogenetic activation of the PVNGLP-1R→DVC projection induced feeding suppression.

Knockout (KO) of presynaptic GLP-1Rs and inactivation of PVNGLP-1R→DVC neuronal synaptic release causes body weight gain

Figure 6: Chronic perturbation of PVNGLP-1R→DVC neurons.

Discussion

Methods

Animals.

AAV vectors and stereotaxic injections.

Histology and Immunohistochemistry assay.

Antibodies.

Electrophysiology.

Fiber Photometry.

Chemogenetics.

Glucose Tolerance Test (GTT).

Insulin Tolerance Test (ITT).

Comprehensive Lab Animal Monitoring System (CLAMS) assay.

Food Intake.

Open Field Locomotor Activity.

Light-dark box.

Optogenetics.

Free chow feeding behavior.

Head-fixed sucrose licking behavior.

Real-time place preference paradigm (RTPP).

Ablation of GLP-1R in PVNGLP-1R→DVC neurons.

Tetanus toxin (TeNT) light chain inactivation of synaptic transmission.

Statistical information.

Data exclusions.

Extended Data

Extended Data Fig. 1: PVNGLP-1R neurons project to different downstream targets.

Extended Data Fig. 2: PVNGLP-1R→DVC neurons do not have apparent collateral projections.

Extended Data Fig. 3: GLP-1R mediated signaling enhances DVC neuron synaptic inputs.

Extended Data Fig. 4: Chemogenetic activation of PVNGLP-1R→DVC neurons via hM3Dq suppresses food intake.

Extended Data Fig. 5: Fiber Photometry measurement of PVNGLP-1R→DVC neuronal calcium activity in responding to various sensory inputs.

Extended Data Fig. 6: Electrophysiological characterization of PVNGLP-1R→DVC neurons under different energy states.

Extended Data Fig. 7: Electrophysiological characterization of PVNGLP-1R→DVC neurons in HFD-induced obesity animals.

Extended Data Fig. 8: Liraglutide augments PVNGLP-1R→DVC synaptic release in HFD-fed mice.

Extended Data Fig. 9: Impact of optogenetic activation of PVNGLP-1R projections in the DVC on food intake and aversion.

Extended Data Fig. 10: Comprehensive metabolic analyses of animals after inactivation of PVNGLP-1R→DVC synaptic release and daily food consumption.

Acknowledgments

Footnotes

Data Accessibility

Code Accessibility

REFERENCES

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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GLP-1R signaling enhances PVN^GLP-1R→DVC presynaptic release of glutamate

Figure 1: PVN^GLP-1R→DVC descending circuit is regulated by GLP-1R-mediated signaling.

Activation of PVN^GLP-1R→DVC suppresses feeding

Figure 2: State-dependent PVN^GLP-1R→DVC neuronal activity suppresses feeding.

PVN^GLP-1R→DVC neuronal responses to food presentation are energy state dependent

State-dependent synaptic plasticity in the PVN^GLP-1R→DVC neuronal circuit

Figure 3: State-dependent synaptic plasticity of PVN^GLP-1R→DVC neurons.

High-fat diet-induced obesity blunts the activity of PVN^GLP-1R→DVC neurons

Figure 4: High-fat diet (HFD) induced obesity blunts the state-dependent synaptic plasticity of PVN^GLP-1R→DVC neurons.

Activation of PVN^GLP-1R→DVC projections suppresses food intake in an energy-dependent manner

Figure 5: HFD blunts optogenetic activation of the PVN^GLP-1R→DVC projection induced feeding suppression.

Knockout (KO) of presynaptic GLP-1Rs and inactivation of PVN^GLP-1R→DVC neuronal synaptic release causes body weight gain

Figure 6: Chronic perturbation of PVN^GLP-1R→DVC neurons.

Ablation of GLP-1R in PVN^GLP-1R→DVC neurons.

Extended Data Fig. 1: PVN^GLP-1R neurons project to different downstream targets.

Extended Data Fig. 2: PVN^GLP-1R→DVC neurons do not have apparent collateral projections.

Extended Data Fig. 4: Chemogenetic activation of PVN^GLP-1R→DVC neurons via hM₃Dq suppresses food intake.

Extended Data Fig. 5: Fiber Photometry measurement of PVN^GLP-1R→DVC neuronal calcium activity in responding to various sensory inputs.

Extended Data Fig. 6: Electrophysiological characterization of PVN^GLP-1R→DVC neurons under different energy states.

Extended Data Fig. 7: Electrophysiological characterization of PVN^GLP-1R→DVC neurons in HFD-induced obesity animals.

Extended Data Fig. 8: Liraglutide augments PVN^GLP-1R→DVC synaptic release in HFD-fed mice.

Extended Data Fig. 9: Impact of optogenetic activation of PVN^GLP-1R projections in the DVC on food intake and aversion.

Extended Data Fig. 10: Comprehensive metabolic analyses of animals after inactivation of PVN^GLP-1R→DVC synaptic release and daily food consumption.