Article Figures & Data
Figures
- Figure 1.
Genetic ablation of LHA glutamatergic neurons increases caloric intake and enhances body weight gain. A, Schematic for viral injection of AAV2-FLEX-taCasp3-TEVp into the LHA of VGlut2-ires-Cre mice. B, C, Confocal images (20×) demonstrating decreased VGlut2 expression in VGlut2LHA::taCasp3 (C) compared with VGlut2LHA::Control (B) mice. Fx, Fornix; DMH, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle; D, dorsal; L, lateral; M, medial; V, ventral. Scale bars, 200 μm. D, Ablation of LHA glutamatergic neurons significantly potentiated weight gain induced from a calorie-dense diet. E, Ablation of LHA glutamatergic neurons significantly increased caloric intake as measured by the average daily calories consumed over the last 7 d of exposure to the calorie-dense diet. F, Ablation of LHA glutamatergic neurons significantly increased fat intake as measured by the average grams of fat consumed over the last 7 d of exposure to the calorie-dense diet. G, H, Open-field velocity was not significantly different between VGlut2LHA::taCasp3 and VGlut2LHA::Control mice. I, J, Open-field center time was not significantly different between VGlut2LHA::taCasp3 and VGlut2LHA::Control mice.
- Figure 2.
The anterior LHA sends a glutamatergic projection to the LHb. A, Schematic for injection of red retrobeads into the LHb of a C57BL/6J mouse. B, Confocal image of the LHA showing LHb-projecting LHA neurons. Scale bar, 20 μm. C, Quantification of LHb-projecting LHA neurons along the anterior–posterior axis of the LHA. LHb-projecting neurons are significantly denser in the anterior LHA compared with the posterior LHA. Ant, Anterior; Post, posterior. D, Confocal images of coronal sections showing expression of ChR2-eYFP in the LHA and LHb (inset) of a VGlut2LHA::ChR2 mouse. Scale bar, 500 μm. E, Confocal images of coronal sections showing expression of ChR2-eYFP in the LHA and LHb (inset) of a VgatLHA::ChR2 mouse. Scale bar, 500 μm. F, VGlut2LHA-LHb::ChR2-eYFP fluorescence intensity compared with VGatLHA-LHb::ChR2-eYFP fluorescence intensity in and around the LHb. MHb, Medial habenula; DG, dentate gyrus; LDT, lateral dorsal thalamus; MDT, medial dorsal thalamus.
- Figure 3.
Characterization of neurotransmitter release from VGlut2LHA-LHb::ChR2 terminals. A, B, Postsynaptic light-evoked EPSCs recorded from LHb neurons at −70 mV are significantly attenuated following bath-application of 10 μm DNQX. C, D, Postsynaptic light-evoked IPSCs recorded from LHb neurons at +10 mV are significantly attenuated by bath-application of 10 μm gabazine. E–G, Cell-attached recordings demonstrating a significant increase in the spontaneous firing rate of a subset LHb neurons in response to a 1 s 20 Hz optical stimulation of VGlut2LHA-LHb::ChR2 terminals in the LHb. H, I, Cell-attached recordings demonstrating a decrease in the spontaneous firing rate from a LHb neurons in response to a 1 s 20 Hz optical stimulation of VGlut2LHA-LHb::ChR2 terminals in the LHb (n = 1 cell). J, One second 20 Hz optical stimulation of VGlut2LHA-LHb::ChR2 terminals in the LHb resulted in 73.7% of LHb neurons showing a net excitation (n = 14 cells), 21.1% of LHb neurons showing <20% change in firing (n = 4 cells), and 5.3% of LHb neurons showing a net inhibition (n = 1 cell, n = 19 neurons total). K, Asynchronous release (gray trace) in LHb neuron following optical stimulation of VGlut2LHA-LHb::ChR2 terminals. Asynchronous release is reduced (purple trace) following bath-application of EGTA-AM. L, mEPSC frequency (Hz) is significantly increased during the 1 s following optical stimulation compared with the 1 s before optical stimulation. M, mEPSC frequency (percentage baseline) is significantly reduced following bath-application of EGTA-AM.
- Figure 4.
Optogenetic modulation of the VGlut2LHA-LHb circuit acutely modulates feeding and reward-related behaviors. A, Schematic diagram showing optical fiber placements for VGlut2LHA-LHb::ChR2 (n = 5), VGlut2LHA-LHb::Control (n = 7), and VGlut2LHA-LHb::NpHR3.0 (n = 7) in the LHb on corresponding coronal brain atlas plates. B, Confocal images of coronal sections showing expression of NpHR3.0-eYFP in the LHA (Scale bars: left, 100 μm; bottom right, 20 μm) and LHb (Scale bar, 20 μm) of a VGlut2-ires-cre mouse. C, D, Representative heat maps showing time spent in particular locations from VGlut2LHA-LHb::ChR2 (B) and VGlut2LHA-LHb::NpHR3.0 (C) during the real-time place preference task. E, VGlut2LHA-LHb::ChR2 spent less time on the side of the chamber paired with stimulation than VGlut2LHA-LHb::Control mice. VGlut2LHA-LHb::NpHR3.0 spent more time on the side of the chamber paired with stimulation than VGlut2LHA-LHb::Control mice. F, VGlut2LHA-LHb::NpHR3.0 licked more for a palatable liquid during a free-licking task than VGlut2LHA-LHb::Control mice. G, VGlut2LHA-LHb::NpHR3.0 had more licking bouts than VGlut2LHA-LHb::Control mice. H, VGlut2LHA-LHb::NpHR3.0 had a significantly lower interlick-bout interval than VGlut2LHA-LHb::Control mice. I, VGlut2LHA-LHb::NpHR3.0 licked more for a palatable liquid adulterated with 0 mm quinine during a free-licking task compared with VGlut2LHA-LHb::Control mice. VGlut2LHA-LHb::NpHR3.0 did not lick more for a palatable liquid adulterated with 2.5 mm quinine during the free-licking task compared with VGlut2LHA-LHb::Control mice.
