A synaptic amplifier of hunger for regaining body weight in the hypothalamus

Abstract

Restricting caloric intake effectively reduces body weight, but most dieters fail long-term adherence to caloric deficit and eventually regain lost weight. Hypothalamic circuits that control hunger drive critically determine body weight; yet, how weight loss sculpts these circuits to motivate food consumption until lost weight is regained remains unclear. Here, we probe the contribution of synaptic plasticity in discrete excitatory afferents on hunger-promoting AgRP neurons. We reveal a crucial role for activity-dependent, remarkably long-lasting amplification of synaptic activity originating from paraventricular hypothalamus thyrotropin-releasing (PVH^TRH) neurons in long-term body weight control. Silencing PVH^TRH neurons inhibits the potentiation of excitatory input to AgRP neurons and diminishes concomitant regain of lost weight. Brief stimulation of the pathway is sufficient to enduringly potentiate this glutamatergic hunger synapse and triggers an NMDAR-dependent gaining of body weight that enduringly persists. Identification of this activity-dependent synaptic amplifier provides a previously unrecognized target to combat regain of lost weight.

Keywords: AgRP neurons; TRH-expressing neurons; chemogenetics; electrophysiology; hunger drive; hypothalamus; intersectional genetics; optogenetics; synaptic plasticity; weight regain.

Figure 1:. Weight loss evokes input-specific forms of potentiation in discrete excitatory afferents onto AgRP neurons

A) Schematic of the approach used to characterize plastic changes in distinct glutamatergic afferents onto AgRP neurons. ChR2-mCherry was virally expressed in PVH^TRH or DMH^Vglut2 neurons. B) Representative traces of light-evoked postsynaptic currents in AgRP neurons under control conditions (black) and after replacing external calcium by strontium (grey), which desynchronizes the timing of vesicle fusion. Blue tics indicate light pulses (473 nm, 5 ms), and arrows indicate single quantal excitatory postsynaptic currents (qEPSCs). C, D) Representative traces of postsynaptic currents recorded from fed or fasted mice following light stimulation of PVH^TRH (C) or DMH^Vglut2 (D) inputs in presence of strontium (left). Quantification of light-evoked qEPSCs (le-qEPSCs) was performed on a 300 ms time window 25 ms after the light pulse. The summary of le-qEPSCs obtained from AgRP neurons show that fasting increases the quantal frequency of PVH^TRH input (C; N = 4/5 mice, fed/fasted), whereas DMH^Vglut2 input increases in quantal amplitude (D; N = 5/5 mice). E, F) Representative traces and summary of paired-pulse ratio (PPR) and coefficient of variation (CV) in PVH^TRH (E; N = 7/7 mice) and DMH^Vglut2 (F; N = 4/3 mice) inputs onto AgRP neurons in fed and fasted mice. G, H) Representative traces of light-evoked AMPAR- and NMDAR-mediated currents recorded at −70mV and +40mV, respectively (left). PVH^TRH (G; N = 7/7 mice) or DMH^Vglut2 (H; N = 4/3 mice) input was optically stimulated in fed or fasted mice. Summary of AMPAR-mediated le-EPSCs amplitude show that DMH^Vglut2 input to AgRP neurons is increased by fasting. All data are presented as mean ± s.e.m.; * p < 0.05, ** p < 0.01; two-tailed unpaired Student’s t-test. Scale bars: B, 25 pA, 50 ms; C, D, E, and F, 30 pA, 50 ms; G and H, 30 pA, 30 ms.

Figure 2:. Activity of PVH^TRH neurons is required for regaining body weight after fasting.

A) Experimental schematic: A Cre-dependent viral approach was used to express the Ivermectin (IVM)-responsive glycine receptor hGlyR for selective inhibition of PVH^TRH or DMH^Vglut2 neurons. Mice were injected with vehicle (VEH) or IVM before food removal at the onset of the dark cycle, followed by 16-hours of fasting. B) Representative trace of current clamp recordings from an hGlyR-expressing neuron. IVM wash-in blocks action-potential firing. Scale bars: 30 mV, 2 min. C, D) Left: Immunostainings showing site-specific hGlyR-mCherry expression in the PVH of Trh-ires-Cre (C) and in the DMH of Slc17a6-ires-Cre (D) mice. Scale bars: 100 µm. IVM/hGlyR-mediated inhibition of PVH^TRH neurons reduces food intake after fasting, and this effect strengthens over time (hours 8 and 24; C). In the same mice, regain of lost body weight is diminished over the week following fasting (C, right). Selective inhibition of DMH^Vglut2 neurons does not significantly affect food intake or body weight regain after fasting (D). All data are presented as mean ± s.e.m.; * p < 0.05, *** p < 0.001, **** p < 0.0001; ordinary two-way ANOVA followed by Sidak’s multiple comparisons test.

Figure 3:. Potentiation of PVH^TRH input to AgRP neurons is driven by PVH^TRH neuron activity

A) Representative histological images and analysis of Fos expression in PVH^TRH neurons that express Adcyap1 (PACAP) from fed or fasted mice assessed by FISH. Scale bars represent 10 μm. (N = 4/4 mice). B) Schematic of recordings from AgRP neurons following in vivo chemogenetic manipulations of PVH^TRH neurons. Mice were injected with AAVs to express hGlyR or hM3Dq selectively in PVH^TRH neurons. C) Representative traces (top) of spontaneous excitatory postsynaptic currents (sEPSCs) in mice expressing hGlyR in PVH^TRH neurons injected with VEH or IVM before fasting (scale bars: 25 pA, 2 s). Inhibition of PVH^TRH neuron activity reduces the frequency, but not the amplitude, of sEPSCs in AgRP neurons in fasted mice (N = 2/2 mice). D) Fed mice expressing hM3Dq in PVH^TRH neurons were administered with vehicle (VEH) or CNO at the onset of the light cycle. Frequency, but not amplitude, of sEPSCs in AgRP neurons is significantly increased in mice treated with CNO 4 hours before brain slices are prepared (N = 2/2 mice; scale bar: 25 pA, 2 s). E) Because chemogenetic stimulation of PVH^TRH neurons acutely increases food intake (Figure S2B and ), we tested the influence of circuit-specific activation on upregulation of sEPSC frequency. For this, hM3Dq was unilaterally expressed in PVH^TRH neurons. In AgRP neurons recorded from the ipsilateral (Ipsi), but not from the contralateral (Contra), site of the stimulated PVH^TRH neurons, sEPSC frequency is significantly upregulated, consistent with increased activity of the PVH^TRH➔AgRP circuit as the functionally relevant mechanism for the amplification of excitatory drive onto AgRP neurons (N = 2/2 mice). F) Schematic of the optogenetic approach to assess circuit-specific plasticity upon PVH^TRH neuron activation (top left). Representative fluorescence images showing expression of ChR2-EYFP (green) and hM3Dq-mCherry (magenta) in the PVH (top right; scale bars, 20 µm). Note the co-expression of both viruses in PVH^TRH neurons. Representative traces (bottom left) of currents obtained from optical stimulation of PVH^TRH afferents in presence of strontium from mice treated with VEH or CNO 4 hours before slice preparation (scale bars, 30 pA, 50 ms). Summary of le-qEPSC recorded from AgRP neurons show that PVH^TRH neuron activation significantly increases quantal frequency, but not amplitude, demonstrating that circuit activation amplifies activity of PVH^TRH➔AgRP synapses (N = 5/4 mice). All data are presented as mean ± s.e.m.; * p < 0.05, ** p < 0.01, ***p < 0.001; two-tailed unpaired Student’s t-test.

Figure 4:. Elevated activity of the excitatory PVH^TRH➔AGRP circuit persists in circumstances in which weight gain is promoted

A) Timeline showing the experimental scheme for determination of plasticity at PVH^TRH➔ AgRP synapses after an overnight fast. Ad libitum food intake and body weight changes were determined at indicated time points. B) Summary of food intake (left) show that ad libitum intake was significantly increased for 4 days after an overnight fast. This effect diminished over time (days 2–4) and dissipated at day 7. Right: Body weight development in the same mice. C) Left: Schematic of recordings from AgRP neurons following optical stimulation of PVH^TRH afferents in presence of strontium from mice refed for 2 or 7 days after fasting, or kept on the caloric maintenance paradigm (CMP; D) before slice preparation. Right: Summary of le-qEPSC recorded from AgRP neurons show that quantal frequency, but not amplitude, is increased on day 2 after refeeding and on day 7 after CMP, demonstrating amplification of PVH^TRH➔AgRP synaptic activity in conditions in which weight gain is promoted (N = 4/3/4/3 mice). D) Timeline showing the experimental scheme for the CMP. After an overnight fast, mice were kept on the CMP for 6 days, during which they were provided the amount of food they consumed on average in ad libitum control conditions (3.8 g per day; bottom). E) Summary of food intake (left) and body weight (right) show that ad libitum intake and body weight were increased at day 7 when mice were kept on the CMP for 6 days after fasting. F) Effects of the CMP as well as 7 days of a caloric restriction paradigm (CR 7; Fig. S4) on Fos expression in PVH^TRH that express Adcyap1 (PACAP) assessed by FISH. Scale bars represent 10 μm. (N = 4/4 mice). G) Summary of Fos expression in AgRP neurons in fed mice, in mice that were kept on the CMP after fasting, and in mice that were subjected to the CR for 7 days. (N = 4/4/3 mice) All data are presented as mean ± s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; repeated-measures one-way ANOVA (B and E) and ordinary one-way ANOVA (C, F, and G).

Figure 5:. Brief, high-frequency stimulation potentiates PVH^TRH➔AgRP synapses and promotes feeding

A) Schematic of the ex vivo approach used for high frequency photostimulation (HFpS; 10 minutes, 50 Hz) of PVH^TRH➔AgRP synapses. The brief HFpS was applied to PVH^TRH neuron terminals in the ARC. Two hours later, slices were transferred to strontium-containing ACSF and le-qEPSCs were recorded from AgRP neurons. Representative fluorescence image shows expression of ChR2-mCherry (magenta) in originating from PVH^TRH neuron terminals and NPY-hrGFP (green) from AgRP neurons (scale bar, 20 µm). B) Representative traces of light-evoked currents recorded in strontium-containing solution from unstimulated slices (control), and from slices that were subjected to HFpS in absence or presence of D-AP5 (scale bars, 30 pA, 50 ms). Summary of le-qEPSCs show that HFpS for 10 minutes increases the quantal frequency, but not the quantal amplitude, of PVH^TRH input onto AgRP neurons. When the NMDAR antagonist D-AP5 is present during light illumination, HFpS fails to increase the quantal frequency (N = 4/8/3 mice). C) Schematic of the in vivo approach. The quantal frequency of PVH^TRH input is significantly increased in AgRP neurons when brain slices are prepared 4 hours after the brief HFpS delivered in vivo (N = 2/2 mice). D) 24-hour food intake was significantly increased in mice that were subjected to HFpS, compared to control condition (no light illumination). This increase was prevented when mice were pretreated with the NMDAR antagonist MK-801 before HFpS, and food intake in these mice did not significantly differ from control condition. Right: Feeding significantly increased at hour 1 and hours 1–4 following HFpS and tended to increase at hours 8–24. Data are presented as mean ± s.e.m.; * p < 0.05; ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (B, and D, left), two-tailed unpaired Student’s t-test (C), and two-tailed paired Student’s t-test (D, right).

Figure 6:. NMDAR signaling is required for long-lasting hyperphagia upon PVH^TRH➔AgRP circuit activation

A) Schematic of the chemogenetic approach used for selective stimulation of PVH^TRH neurons (left), and representative fluorescence image showing hM3Dq-mCherry (magenta) in the PVH (right; scale bar, 100 µm). B) Co-administration of the NMDAR antagonist MK-801 significantly reduces the increase in quantal frequency of PVH^TRH input onto AgRP neurons upon hM3Dq-induced activation of PVH^TRH neurons (N = 4/3/4 mice). C) 24-hour food intake was increased in hM3Dq-expressing mice that received a single injection of CNO, compared to control conditions (vehicle, VEH injected). Co-administration of MK-801 significantly diminishes the increase in 24-hour food intake (left). Summary of food intake measured at different time points show that the acute increase in feeding (1–4- and 4–8-hours’ time windows) is reduced by MK-801 co-administration, whereas the effect 8–24-hour after CNO administration is completely blocked, consistent with an activity-dependent, NMDAR-dependent signaling that leads to amplification of synaptic excitation in the PVH^TRH➔AgRP circuit to promote a long-lasting increase in feeding. D) Schematic of the genetic approach for simultaneous PVH^TRH neuron activation and AgRP neuron inhibition. Mice expressing Dre-recombinase in PVH^TRH neurons and Cre-recombinase in AgRP neurons (Trh-p2a-Dre; Agrp-ires-Cre mice) were injected with an AAV to express a Dre-dependent (roxed) hM3Dq-mCherry construct in the PVH, and a Cre-dependent (floxed) hGlyR-mCherry construct in the ARC (left; see methods). Representative fluorescence images showing hM3Dq-mCherry and hGlyR-mCherry in the PVH and ARC, respectively (magenta; scale bars, 100 µm). E) Increased food intake upon hM3Dq-induced activation of PVH^TRH neurons is blocked in mice whose AgRP neuron activity is simultaneously inhibited through IVM-mediated activation of hGlyR. F) Schematic of the chemogenetic approach to determine the necessity of glutamatergic transmission in driving the food consumption upon PVH^TRH neuron activation. For selective deletion of Vglut2 from PVH^TRH neurons, Trh-p2a-Dre mice with loxp-flanked Slc17a6 alleles (Vglut2, Trh-p2a-Dre; Slc17a6^fl/fl mice) were injected with a Dre-dependent AAV to express Cre-recombinase. An AAV expressing Cre-dependent-hM3Dq was co-injected. Representative fluorescence images showing co-expression of Trh mRNA (green) and hM3Dq-mCherry in PVH^TRH neurons (magenta; scale bars, 50 µm). G) Food intake is significantly increased in mice expressing hM3Dq in PVH^TRH neurons (Dre+) using the genetic approach shown in F, compared to control mice (Dre-recombinase negative). This increase was absent in Trh-p2a-Dre; Slc17a6^fl/fl mice Cre-/Dre-dependently lacking Vglut2 in PVH^TRH neurons (Slc17a6^fl/fl), demonstrating that Vglut2-dependent glutamatergic transmission is necessary for the acute and long-term increases in food consumption upon circuit stimulation. All mice were co-injected with AAV-FREX-Cre and AAV-FLEX-hM3Dq into the PVH, and received an administration of CNO at the onset of the light cycle. Data are presented as mean ± s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (B, C, E, and G, left), and ordinary two-way ANOVA followed by Tukey’s multiple comparisons test (C, E, and G, right).

Figure 7:. Activation of the PVH^TRH➔AgRP circuit evokes persistent gaining of body weight

A) Mice expressing hM3Dq in PVH^TRH neurons significantly increase body weight following a single CNO injection. When the NMDAR antagonist MK-801 is co-administered with CNO, body weight is not affected. B) Summary of food intake show that food consumption is increased for 2 days following a single injection of CNO. This effect diminishes over time (day 2), and is not followed by reduced food intake over the next 7 days. C) Body weight change measured 7 days after a single injection. Persistent body weight gain is present after a single CNO injection in mice expressing hM3Dq in PVH^TRH neurons, but not in control mice (mCherry-expressing). CNO fails to increase body weight when NMDAR are blocked by co-administration of MK-801 with CNO. D) Body weight increases further in mice expressing hM3Dq in PVH^TRH neurons following single CNO injections with intervals of two weeks. This body weight gain is blocked by co-administration of MK-801. All data are presented as mean ± s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (A, C), repeated-measures one-way ANOVA followed by Tukey’s multiple comparisons test (B), and ordinary two-way ANOVA followed by Tukey’s multiple comparisons test (D).