Uneven balance of power between hypothalamic peptidergic neurons in the control of feeding

Characterization of Tg POMC-ChR2 mice. (A) Genetic design for ChR2-eYFP expression in the Arc. (B) Expression of ChR2-eYFP in the Arc. (C) Representative images showing colocalization of POMC-positive neurons with ChR2-eYFP expressing cells in the Arc. Inset shows ChR2-eYFP expression (green) in POMC neurons (red). (Scale bar, 50 µm.) (Scale bar in Inset, 20 µm.) (D) Representative images showing colocalization of AgRP-positive neurons with ChR2-eYFP expressing cells in the Arc. Inset shows ChR2-eYFP expression (green) in a subgroup of AgRP neurons (red). Yellow arrows point to ChR2-eYFP-positive AgRP neurons; white arrows point to ChR2-eYFP-negative AgRP neurons. (Scale bar, 50 µm.) (Scale bar in Inset, 20 µm.) (E and F) Representative whole-cell current-clamp recordings of ChR2-eYFP-positive neurons in the Arc from acute slices. To replicate the stimulation pattern used in the hot-plate test, the neuron was stimulated with 15-ms light pulses at 10 Hz for 6 min (indicated by blue bar). After the 6-min period of action potential firing, the light was turned off to show the specificity of the response to light stimulation. Action potential firing resumed at 10 Hz when the light in turned back on. Insets show individual action potential firing at 10 Hz at the times indicated by the numbers. To replicate the stimulation pattern used in the food intake study, the neuron was stimulated with 15-ms light pulses at 10 Hz for 30 s (indicated by blue bar) every other 30 s for 30 min. For simplicity, only the first stimulation period (during minute 1; Left trace) and the last stimulation period (during minute 30; Right trace) are shown. Insets show individual action potential firing at 10 Hz at the times indicated by the numbers. n = 25 cells, 15 slices from 8 mice. (G) Statistics of cFos-positive neurons in ChR2-eYFP expressing cells in the region below the implanted optic fiber in the Arc with or without optic stimulation. Five percent of cFos-positive neurons expressed in ChR2-eYFP neurons in the no-optic stimulation group (n = 3 mice, 30 cFos-positive neurons, 522 ChR2-eYFP neurons) versus 61% in the light-stimulation group (n = 3 mice, 543 cFos-positive neurons, 901 ChR2-eYFP neurons). Unpaired two-tailed t test: t(4) = 13.63, ***P < 0.001. Mean ± SEM. (H) Representative images showing the induction of cFos (white) in POMC-positive (red) ChR2-eYFP expressing neurons (green) (yellow arrows) following blue light stimulation in the Arc. POMC-positive neurons were immunostained with anti–β-endorphin antibody. (Scale bar, 20 µm.) (I) Representative images showing the induction of cFos (white) in AgRP-positive (red) ChR2-expressing neurons (green) (yellow arrows) following light stimulation in the Arc. (Scale bar, 20 µm.)

Selective activation of POMC neurons in the Arc suppresses food intake in fasted viral POMC-ChR2 mice. (A) Blue light stimulation of POMC neurons induced a reduction of food intake in 4-h fasted viral POMC-ChR2 mice, F(2, 40) = 18.99, P < 0.0001; ****P < 0.0001; n = 21 mice. (B) Light stimulation of POMC neurons led to an increased latency for the mice to lick their paws during the hot-plate test, F(2, 30) = 8.82, P < 0.01; **P < 0.01; n = 16 mice. (C) Increased latency induced by light stimulation was blunted by pretreatment with opioid antagonist naloxone (10 mg/kg) in the hot-plate test, F(2, 28) = 6.797, P < 0.01; *P < 0.05; **P < 0.01; n = 15 mice. Repeated-measures one-way ANOVA followed by Turkey’s test were used for all above statistics. Box plots show median, mean (+), lower and upper quartiles (boxes), and minima and maxima (whiskers).

Activation of Arc neurons derived from POMC-expressing lineage increases food intake in Tg POMC-ChR2 mice. (A) Repeated-measures two-way ANOVA revealed a significant genotype × day interaction, F(2, 82) = 9.44, P < 0.001, for the hot-plate test. Blue light stimulation of Arc neurons in Tg POMC-ChR2 mice led to an increased latency for the animals to lick their paws, F(2, 52) = 21.91, P < 0.0001; ****P < 0.0001; n = 27 mice. Latency for single transgenic littermate control mice without ChR2-eYFP expression to lick their paws was unchanged, F(2, 30) = 1.06, P = 0.36; n = 16 mice, 8 mice per single transgenic mouse line. (B) Increased latency induced by light stimulation in Tg POMC-ChR2 mice was reduced by pretreatment with opioid antagonist naloxone, F(2, 24) = 15.2, P < 0.0001; ***P < 0.001, ****P < 0.0001; n = 13 mice. (C) Repeated-measures two-way ANOVA revealed a significant genotype × day interaction, F(2, 54) = 76.59, P < 0.0001, for the food intake study. Light stimulation of Arc neurons evoked a robust increase in food intake in Tg POMC-ChR2 mice, F(2, 24) = 68.28, P < 0.0001; ****P < 0.0001; n = 13 mice. Food intake for control mice was unchanged following light stimulation of Arc, F(2, 30) = 1.19, P = 0.32; n = 16 mice. Box plots show median, mean (+), lower and upper quartiles (boxes), and minima and maxima (whiskers).

Anatomical analysis of Arc POMC neuronal projections and the extent of activation in viral POMC-ChR2 mice. (A) eYFP-expressing Arc POMC neurons and their connectivity shown as a max intensity z-projection image. Representative CLARITY processed image shows prominent arcuate connectivity with the paraventricular, dorsomedial, and lateral hypothalamus, as well as the zona incerta, periaqueductal gray, and lateral septum in the viral POMC-ChR2 mouse brain slice (4-mm thickness). (Scale bar, 1500 µm.) (B) Magnified view shows a more detailed eYFP expression pattern in the Arc in the viral-injected hemisphere. (Scale bar, 600 µm.) (C) Neuronal activation in the brain following light stimulation in the Arc of viral POMC-ChR2 mice. ISH revealed cFos mRNA expression was increased in limited brain regions in response to selective activation of Arc POMC neurons. Images were quantitatively analyzed with results shown in SI Appendix, Table S2. Representative images are shown ranging from Bregma 1.10 mm (indicated by the no. 1) through Bregma −2.46 mm (indicated by the no. 6).

Anatomical analysis of the projections of Arc neurons derived from POMC-expressing progenitors and the extent of activation in Tg POMC-ChR2 mice. (A) Labeled Arc neurons and their connectivity shown as a max intensity z-projection image. Representative image shows prominent arcuate connectivity with the dorsomedial, paraventricular, and lateral hypothalamus in the Tg POMC-ChR2 mouse brain slice (2-mm thickness) processed with the iDISCO method. (Scale bar, 1000 µm.) (B) Magnified view shows detailed eYFP expression pattern in the Arc. (Scale bar, 500 µm.) (C) ISH revealed cFos mRNA expression was intensively increased in broader brain areas in response to activation of Arc neurons of Tg POMC-ChR2 mice. Images were quantified with results shown in SI Appendix, Table S3. Representative images are shown ranging from Bregma 1.10 mm (indicated by the no. 1) through Bregma −2.12 mm (indicated by the no. 8).