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 ²⁹), 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.