Binge-eating on the brain

brain food

Editor's Introduction

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation

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Have you ever wondered why some foods are so difficult to stop eating? Well, you're not alone. Over 2% of Americans will be clinically diagnosed with binge eating disorder in their lifetime. The authors of this paper have pinpointed a specific part of the brain that might be responsible for this kind of overeating behavior. Using a mouse model, the scientists examined how a region of brain cells, known as zona incerta GABA neurons, affects feeding on high-fat and sweet foods. This study offers a better understanding of binge eating behaviors in humans and sheds light on why some food are just so hard to stop eating.

Paper Details

Original title
Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation
Original publication date
Reference
Vol. 356 no. 6340 pp. 853-859
Issue name
Science
DOI
10.1126/science.aam7100

Abstract

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons.

Report

Patients receiving deep brain stimulation of the subthalamus, including the zona incerta (ZI), for the treatment of movement disorders can exhibit characteristics of binge eating (13), a common eating disorder characterized by recurrent episodes of consuming large quantities of food, particularly highly palatable food (45). It is not clear why stimulation in the subthalamus would evoke eating, although sheep may release γ-aminobutyric acid (GABA) from the ZI in response to the sight or ingestion of food (67).

The ZI is one of the least-studied regions of the brain, despite its robust projections throughout the brain (89). To determine the role of the ZI in feeding and body weight regulation, we injected Cre recombinase–inducible adeno-associated viruses (AAV) expressing the optogenetic channelrhodopsin-like ChIEF fused with a tdTomato reporter [AAVdj-CAG-DIO-ChIEF-tdTomato (driven by the CAG promoter) (1011)] bilaterally into the rostral ZI of vesicular GABA transporter (VGAT)–Cre mice that express Cre recombinase in GABA neurons (Fig. 1A). ChIEF-tdTomato was selectively expressed in ZI GABA neurons but not in lateral hypothalamic neurons (fig. S1). Laser stimulation (1 to 20 Hz) evoked depolarizing currents in ZI ChIEF-tdTomato–expressing VGAT neurons tested with whole-cell recording in brain slices, displaying a high-fidelity correspondence with stimulation frequency (Fig. 1B). In VGAT-Cre mice with ChIEF expression, bilateral laser stimulation (20 Hz) in the ZI increased food intake, with mice rapidly consuming 35.4% of their 24-hour ad libitum high-fat food intake in just 10 min (Fig. 1, C to E, and movie S1). In control mice with tdTomato expression, consumption was only 4% of their 24-hour intake during the same period (Fig. 1E). When stimulation of 10 min ON followed by 30 min OFF was repeated four times, ZI-VGAT-ChIEF mice consumed 74% of their normal 24-hour food intake, whereas control mice consumed only 22% (Fig. 1E). Food deprivation lasting 24 hours increased ZI GABA neuron activity and excitatory neurotransmission to these neurons (Fig. 1, F to J). Ghrelin, a hormone that signals a reduced gut energy state (12), excited ZI GABA neurons and increased excitatory synaptic input onto these neurons (Fig. 1, K to M, and fig. S2).

 

BingeEating_1
Fig. 1 Optogenetic activation of ZI GABA neurons rapidly evokes binge-like eating. (A) Red fluorescent image shows restricted expression of ChIEF-tdTomato in the ZI after AAV-ChIEF-tdTomato was bilaterally injected into the ZI of VGAT-Cre mice. Scale bar, 500 μm. (B) Optogenetic activation with varying frequency of a ZI GABA neuron in a brain slice. (C) Schematic illustration showing the location of optical fiber tips implanted above the ZI on both sides of the brain. (D) High-fat food intake during 10 min and four times 10 min from control mice with tdTomato and mice with ChIEF-tdTomato, both with ZI expression. For the 10-min trial, continuous light stimulation (10 ms, 20 Hz) was supplied to the ZI. For the 4 ×10-min trial, 10-min light stimulation (10 ms, 20 Hz) was followed by 30 min without stimulation, repeated four times. (E) High-fat food intake over 10 min and four times 10-min trial as a percentage of unstimulated 24-hour intake (100%). (F) Action potentials evoked by 100-pA current injection in ZI GABA neurons in brain slices of mice fed or fasted for 24 hours. (G) Firing rate at different levels of current injection from ZI GABA neurons in brain slices of mice fed or fasted for 24 hours. (H) Excitatory postsynaptic currents (EPSCs) in ZI GABA neurons of mice fed or fasted for 24 hours. (I) EPSC frequency from ZI GABA neurons of mice fed (n = 12 cells from each of four mice) or mice fasted for 24 hours (n = 13 cells from four mice). (J) EPSC amplitude from ZI GABA neurons in mice fed (n = 12 cells from four mice) or mice fasted for 24 hours (n = 13 cells from four mice). (K) Ghrelin (100 nM) excites a ZI GABA neuron. (L) Ghrelin depolarizes ZI GABA neurons. (M) Ghrelin increases the firing rate of ZI GABA neurons. Statistical analysis for comparison between two groups: Two-way analysis of variance (ANOVA) with Bonferroni post hoc comparison for (D) and (E); unpaired t test for (G), (I), (L) and (M). *P < 0.05; **P < 0.01; ***P < 0.001.
Panels A, B, and C

Panel A: An image of the mouse ZI showing the location of the ChIEF-tdTomato expression in the ZI GABA neurons acquired with a fluorescent microscope.

Panel B: Electrophysiology recordings from ChIEF-tdTomato expressing ZI GABA neurons. The cells fired in response to different frequencies of blue light stimulation.

Panel C: A scheme depicting the placement of optic fibers above the mouse ZI for delivery of blue light for optogenetics experiments.

Panels D, E, F, and G

High-fat food intake (measured in kCal) of ChIEF-expressing and control mice during two different paradigms of ZI GABA neuron optogenetic stimulation. Left: 10 minutes stimulation. Right: 10 minutes simulations repeated four times. Each circle depicts behavior of an individual animal.

Panel E represents the same data as seen in Panel D with food intake expressed as a percentage of the animal's food intake over a 24-hour period in the absence of optogenetic stimulation of ZI GABA neurons.

Panel F: An action potential is a measure of the flow of electricity along the axon of a neuron when it fires. In order to make a neuron fire, the authors injected current (100-pA) into the cell using an electrode.

The recordings show example action potentials evoked from ZI GABA cells in fed and fasted mice. ZI GABA neurons fired more action potentials in fasted mice. For more information on action potentials, check out this activity from HHMI BioInteractive.

Panel G: The graph depicts the firing rate of ZI GABA neurons in response to different amounts of injection current. The neurons fired more in the fasted state and in response to larger injections of current.

Panels H, I, and J

Panel H: Excitatory postsynaptic currents (EPSCs) are a measure of how much excitatory input a cell receives. They are quantified by recordings from a cell in a brain slice using an electrode. The recordings are example EPSCs from ZI GABA neurons in fed and fasted mice.

Panel I: The frequency (number per second, represented in Hertz(Hz)) of EPSCs recorded from ZI GABA neurons in fed and fasted mice. ZI GABA neurons received more excitatory inputs in the fasted state.

Panel J: Similar to Panel I, showing the amplitude (size) of EPSCs. Notice that ZI GABA neurons received larger excitatory inputs in the fasted state.

Panels K, L, and M

Panel K: A recording from a ZI GABA neuron in a brain slice in the presence of ghrelin. The horizontal bar depicts when ghrelin was applied to the slice. Ghrelin caused the ZI GABA neuron to fire.

Panel L: Depolarization is a change in the electrical properties of the cell. A cell is more likely to fire action potentials when the charge (voltage) inside a neuron is less negative. The voltage inside ZI GABA neurons was measured in the absence and presence of ghrelin using an electrode. Ghrelin increased the charge inside ZI GABA cells making them more likely to fire action potentials.

Learn more about the electrical activity of neurons with this activity from HHMI BioInteractive: https://www.hhmi.org/biointeractive/electrical-activity-neurons

Panel M: The firing rate of ZI GABA neurons was measured using an electrode in the presence and absence of ghrelin. Ghrelin increased the firing rate of ZI GABA neurons.

 

Anterograde AAV-ChIEF-tdTomato labeling of ZI GABA cells in VGAT-Cre mice showed strong axonal projections into the paraventricular thalamus (PVT) (Fig. 2A), consistent with previous observations that some ZI cells project to the PVT (1314), a brain area that may contribute to energy homeostasis (15). Cre recombinase–dependent rabies virus–mediated monosynaptic retrograde pathway tracing in vGluT2–Cre recombinase mice confirmed that PVT glutamate neurons receive strong and direct innervation from ZI neurons (Fig. 2, B and C, and fig. S3). Food deprivation lasting 24 hours increased inhibitory synaptic neurotransmission to PVT glutamate neurons (fig. S4). We asked whether the PVT may be a critical target for ZI regulation of food intake. We crossed VGAT-Cre mice with vGlut2-GFP mice in which neurons expressing vesicular glutamate transporter (vGlut2) were labeled with green fluorescent protein (GFP) to study whether ZI GABA neurons release synaptic GABA to inhibit PVT glutamate neurons (1617). One month after AAV-ChIEF-tdTomato was injected into the ZI of these mice (Fig. 2A), photostimulation of ZI VGAT-ChIEF-tdTomato terminals in the PVT evoked GABA-mediated inhibitory currents in PVT vGlut2-GFP neurons (Fig. 2D). In vivo stimulation (20 Hz) of axon terminals from ZI GABA neurons to PVT glutamate neurons (VGATZI-PVT) evoked food-foraging behavior (movie S2). Continuous stimulation (20 Hz) for 10 min increased the intake of high-fat, sweet, and regular foods (Fig.2E) in mice with ZI-VGAT-ChIEF-tdTomato expression. No effect of laser stimulation on high-fat food intake was detected in control mice with AAV-tdTomato in the ZI (Fig. 2F). The total feeding time for ZI VGAT-ChIEF-tdTomato mice was 7.1 ± 0.5 min compared with 0.3 ± 0.1 min for controls (Fig. 2G). Photostimulation of ZI-PVT inhibitory axons evoked gnawing, but not eating, of nonnutritional wood sticks (fig. S5, A and B); photostimulation leading to food intake eliminated subsequent evoked stick gnawing. A priori wood gnawing had no effect on photostimulation-evoked food intake (fig. S5, C and D). We then measured food intake of the same mice during three successive trials of 10-min laser stimulation with a 5-min interval without photostimulation between the trials. The food intake for the first trial was 4.95 ± 0.80 kcal. The amount for the second trial was reduced substantially to 0.72 ± 0.29 kcal and 0.49 ± 0.25 kcal, respectively (Fig. 2H). Satiety feedback signals can thus attenuate ZI-induced feeding.

 

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Fig. 2 Optogenetic activation of ZI GABA axon terminals in PVT rapidly evokes eating with preference for high-fat food. (A) Anterograde mapping of ZI GABA neuron projections to PVT. (Top left) Schematic shows bilateral injection of AAV-ChIEF-tdTomato into ZI and placement of fiber optic tip above PVT. (Bottom left) Red fluorescent image shows strong projection to PVT from ZI-VGAT-ChIEF-tdTomato neurons. D3V, dorsal third ventricle. Scale bar, 100 μm. (Right) ZI axons in PVT. Scale bar, 5 μm. (B) Retrograde mapping of presynaptic neurons to PVT glutamate neurons. (Top left) Schematic shows the strategy for tracing presynaptic ZI projections to PVT glutamate neurons. TVA, the avian tumor virus receptor A; RVdg, glycoprotein–deleted rabies virus (RV). (Top right) Selective expression of RV-GFP (green) and TVA-mCherry (red) in PVT. Scale bar, 300 μm. (Bottom) PVT neurons detected with TVA-mCherry (left), RV-GFP (middle) and merged image (right) shows originating cells (yellow, expressing both GFP and mCherry). Scale bar, 20 μm. (C) RV-labeled presynaptic neurons in ZI. LH, lateral hypothalamus. Scale bars: top, 300 μm; bottom, 20 μm. (D) Optogenetically evoked inhibitory postsynaptic currents (IPSCs) of PVT vGlut2 neurons at 1, 5, 10, and 20 Hz (membrane potential clamped at –40 mV). In bicuculline (Bic, 30 μM), 1-Hz pulses evoked no obvious current. (E) Photostimulation (10 ms, 20 Hz) of ZI-VGAT-ChIEF neuron terminals in PVT increases food intake during 10-min trial. (F) Photostimulation of PVT has no effect on food intake of VGAT-Cre mice after control AAV-tdTomato injection into ZI. (G) Cumulative time during eating by VGAT-ChIEF mice during 10-min photostimulation (10 ms, 20 Hz). (H) Food intake induced by photostimulation is greater in first 10-min trial, and reduced in second and third trial, with a 5-min interval between photostimulations. (I) VGATZI-PVT photostimulation increased preference for high-fat food. (J) Food intake for 24 hours with photostimulation of ZI VGAT neurons or VGATZI-PVT terminals. For 4 ×10-min trial, 10-min light stimulation (10 ms, 20 Hz) followed by 30-min no stimulation, repeated four times. (K) Bic attenuates optogenetic stimulation of food intake in PVT. Statistical analysis for comparison between two groups: two-way ANOVA with Bonferroni post hoc comparison for (E) and (J); unpaired t test for (G); one-way ANOVA repeated measure with Bonferroni post hoc comparison for (H); two-way ANOVA repeated measure with Bonferroni post hoc comparison for (I) and (K). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Panel A

Top left: A scheme depicting injection of an AAV-delivering ChIEF-tdTomato into ZI GABA neurons and placement of an optic fiber above the PVT for stimulation of ZI terminals into this region.

Bottom left: A fluorescent of tdTomato terminals of ZI GABA neurons in the PVT.

Right: A higher magnification view of the image in the bottom left.

Panels B and C

Panel B top left: A scheme of the monosynaptic rabies tracing strategy used to identify neurons that provide input to the PVT VGlut2 neurons.

The TVA + G proteins (red in the fluorescent images) are expressed in the PVT VGlut2 neurons. These are necessary for the rabies virus to enter the neurons and replicate. The EnvA + RVdG (green) proteins are necessary fro the rabies virus to traffic into upstream neurons, marking them with a green fluorescent protein and therefore marking both the PVT VGlut2 neurons (yellow) and the cells upstream of them (green).

Panel C top: Rabies virus-expressing cells in the ZI upstream of the PVT VGlut2 neurons.

Panel C bottom: A higher magnification image of the ZI in the above image.

Panels, D, E, F, and G

Panel D: Inhibitory postsynaptic currents (IPSCs) are a measure of how much inhibitory input a neuron receives. The authors examined whether ZI GABA neurons inhibit PVT VGlut2 neurons by recording from them in brain slices while optogenetically activating ZI GABA neurons. The PVT VGlut2 neurons responded with IPSCs upon optogenetic stimulation of different frequencies (measured in Hz). This response was blocked by blocking receptors of the inhibitory neurotransmitter GABA using the drug bicuculline.

Panel E: Food intake upon optogenetic stimulation of ZI GABA projections to the PVT. Food intake of different types of food was increased during the stimulation period compared to when the blue light was off (no stimulation).

Panel F: Food intake of control mice expressing tdTomato virus in their ZI GABA neurons. Optogenetic stimulation of mice expressing this control viurs had no effect on food intake.

Panel G: Total time spent eating by mice expressing ChIEF in their ZI GABA neurons during the optogenetic stimulation.

Panels H, I, J, and K

Panel H: Food intake during optogenetic stimulation of ZI GABA neurons during three consecutive 10-minute stimulation periods.

Panel I: Food intake, expressed as a percentage of normal food intake, of sweet and high-fat foods of mice receiving ZI GABA neuron stimulation. Stimulation increased preference for high-fat foods.

Panel J: A comparison of the effect of ZI GABA cell body (also known as the soma) optogenetic stimulation and stimulation of ZI terminals in the PVT on food intake. Since both manipulations had the same effect, all of the effect of the ZI GABA neurons on food intake can be accounted for by the ZI GABA neurons that project to the PVT.

Panel K: The GABA receptor antagonist bicuculline was infused into the PVT prior to stimulation of the projections into the PVT. This strongly reduced the ZI-PVT mediated food intake, showing that this effect is mediated by GABA release in the PVT.

ZI-stimulated mice showed a preference for high-fat and sweet foods over normal food (Fig. 2I). Although mice prefer sweet and high-fat foods when stimulation is off, laser stimulation increased the relative preference for high-fat food (Fig. 2I). When normal, sweet, and high-fat foods were all available, mice consistently chose high-fat food during laser stimulation of ZI axons in the PVT (movie S3). ZI GABA neurons project to multiple brain regions, including the hypothalamus and midline thalamus (fig. S6). We therefore measured the relative contribution of stimulation of ZI somata with selective stimulation of ZI axons targeting the PVT. Stimulation of ZI VGAT cell bodies or VGATZI-PVT terminals in the PVT evoked similar levels of feeding (Fig. 2J). To further confirm the importance of the VGATZI-PVT projection in mediating ZI GABA neuron control of food intake, the type A GABA (GABAA) receptor antagonist bicuculline (Bic) was microinjected into the PVT 10 min before photostimulation of VGATZI-PVT axon terminals. Bic attenuated photostimulation-evoked feeding (Fig. 2K). That Bic did not completely block photostimulation-evoked food intake could be a diffusion limitation of Bic after application, or ZI VGAT-Cre neurons may coexpress other neurotransmitters responsible for the remaining action. These results are consistent with an early report that lesions in the area of the ZI can alter food intake (18).

Stimulation of anorexigenic proopiomelanocortin (POMC) cells in the hypothalamic arcuate nucleus leads to a reduction in feeding slowly over the succeeding 24 hours, whereas stimulation of orexigenic hypothalamic neurons expressing agouti-related peptide (AgRP) leads to what has previously been considered to be a rapid increase in feeding with mean latency to eat of 6.1 min (range: 1.9 to 13.8 min) (19). To test the time course and efficiency of optogenetic activation of VGATZI-PVT inhibitory inputs to evoke feeding, we used a laser stimulation protocol of 10 s ON (20 Hz) followed by 30 s OFF for more than 20 min to study ZI axon stimulation in PVT brain slices and feeding behavior. Stimulation of ZI axons with this protocol hyperpolarized and inhibited PVT glutamatergic neurons each time the light was activated (Fig. 3A). Mice immediately started feeding for each of the 30 successive trials of ZI axon laser stimulation (Fig. 3B and movie S4). The mean latency to initiate feeding was 2.4 ± 0.6 s when we used laser stimulation of 20 Hz (Fig. 3C). This is almost 100 times faster than that reported for optogenetic stimulation of the AgRP neuron soma and 500 times faster than stimulation of AgRP-PVT axon terminals (1920). As soon as the laser was turned off, the mice stopped eating. To test further whether photostimulation of VGATZI-PVT terminals evokes compulsive eating, food intake was measured when food was put in a brightly illuminated chamber in a two-chamber light-or-dark conflict test. Mice spent only 20% of their time in the brightly lit chamber with high-fat food when the laser was off, suggesting an aversion to the light (Fig. 3D). In spite of the light aversion, photostimulation of VGATZI-PVTterminals significantly increased the time mice spent on the illuminated side to 61% when high-fat food was available (Fig. 3D). Photostimulation increased high-fat food intake in bright light (Fig. 3E).

 

BingeEating_3
Fig. 3 Binge-like eating evoked by optogenetic activation of ZI GABA axon terminals produces rapid increase in body weight and shows positive hedonic association. (A) Photostimulation of ChIEF-expressing ZI axons reduces activity of PVT glutamate neuron in brain slice during repeated 10-s photostimulations (10 ms, 20 Hz) with 30-s rest interval. (B) Latency for representative mouse to rapidly initiate feeding in response to photostimulation over 30 consecutive trials. Photostimulation protocol same as in (A). (C) Latency of feeding initiation using 10-s photostimulation (10-ms pulses) at 10, 20, and 40 Hz. (D) Photostimulation increases time in lit chamber during light-dark conflict test. (E) Light-dark conflict test shows photostimulation increases high-fat food intake in brightly lit chamber. (F) Real-time place-preference data show tracks of control (left) and ZI VGAT-ChIEF mouse (right) in photostimulation-paired (photostim.) and nonpaired chambers. (G) Time (%) that control and ChIEF-tdTomato mice stay in photostimulation-paired chamber during a 20-min trial. (H) Daily food intake of control tdTomato and ChIEF-tdTomato mice with photostimulation of VGATZI-PVTterminals for 5 min (20 Hz) every 3 hours repeated over 14 days (shaded box) then continued without photostimulation (unshaded). F1,230 = 343.9, P < 0.0001, two-way ANOVA. (I) Body weight of tdTomato control and ChIEF-tdTomato mice with a 5-min (20-Hz) photostimulation of VGATZI-PVT terminals every 3 hours repeated over 14 days (shaded box), then continued without photostimulation (unshaded). Data were from the same mice tested in (D). F1,230 = 73.45, P < 0.0001, two-way ANOVA. (J) Weekly food intake from control and ZI VGAT neuron ablation. Ablation versus control: F1,99 = 55.84, P < 0.0001. (K) Body weight gain of mice from control group and ZI VGAT neuron ablation. Ablation versus control: F1,99 = 60.12, P < 0.0001. Statistical analysis for comparison between two groups: Paired t test for (D) and (E); unpaired t test for (G); two-way ANOVA with Bonferroni post hoc comparison for (J) and (K). *P < 0.05; **P < 0.01; ***P < 0.001.
Panels A, B, C, D, and E

Panel A: Example electrophysiology recording from a PVT VGlut2 neuron during stimulation of ZI GABA neurons. Each time the blue light was on (denoted by elevated 'On' state), the PVT VGlut2 neuron was inhibited, i.e. stopped firing. This effect shows that PVT VGlut2 neurons are inhibited by ZI GABA neurons.

Panel B: Latency to eating onset of an example mouse receiving blue light stimulation of its ZI GABA-PVT neurons in the same pattern as in Panel A.

Panel C: Latency of mice to feed with photostimulation onset with different frequencies of blue light stimulation. Mice ate much more quickly when they received 10Hz stimulation than for the other frequencies tested.

Panel D: The light-dark conflict test assesses whether mice will overcome their innate aversion to light when their PVT-projecting ZI GABA neurons are stimulated. Indeed, the mice spent more time on the lighted side when the stimulation light was on. This behavior suggest that activation of these neurons has positive hedonic associations, meaning that it makes the mice feel good.

Panel E: Although mice prefer to eat high-fat foods, presentation of this food in the lighted side of the light-dark conflict test decreases consumption. This was overcome by stimulation of PVT-projecting ZI GABA neurons, suggesting that this positive hedonic association overcomes the aversion of the animals to light.

Panels F, G, H, and I

Panel F: Tracks that show the location of a representative control mouse and a mouse receiving ZI GABA-PVT neuron stimulation in the real-time place-preference task. When the mice were in the side of the chamber marked "Photostim.," the blue light was turned on.

Panel G: Control (Ctrl) mice spent approximately 50% of their time on either side of the chamber while ChIEF mice spent more time in the side of the chamber in which they received ZI-PVT photostimulation.

Panel H: Daily food intake of mice receiving ZI-PVT photostimulation vs. controls. The mice received photostimulation for 14 days (shaded box) during which time their food intake was increased. On subsequent days after photostimulation had ceased, the mice ate less than controls. This effect is likely to compensate for the increase in food intake during the photostimulation period.

Panel I: Body weights of mice in the same experiment in Panel H. The reduction in body weight once the photostimulation had ended reflects the reduction in food intake in H.

Panels J and K

Weekly food intake of control mice and mice after surgery—during which the ZI GABA neurons were ablated (removed) using a caspase virus which leads to cell death. Food intake was increased when the mice were given normal food and when they were given high-fat food (shaded bar). This effect demonstrates that ZI GABA neurons are required for normal food intake.

Panel K: Weekly body weight gain after surgery of the same mice as in Panel J. Control mice gained weight after surgery, especially on a high-fat diet whereas mice who had their ZI GABA neurons removed gained much less weight.

Binge eating has been linked to a reward-system disorder (2122). To test the hypothesis that the VGATZI-PVT pathway is involved in a reward state, we explored the motivational valence of VGATZI-PVT in mice by using a two-chamber place preference test. In the absence of available food, optogenetic activation of the VGATZI-PVT pathway evoked a significant preference for the chamber associated with laser stimulation compared with the control chamber (Fig. 3, F and G).

To test whether activation of the VGATZI-PVT inhibitory pathway leads to body weight gain, we selectively photostimulated this pathway for only 5 min every 3 hours over a period of 2 weeks. Photostimulation increased food intake and body weight of mice with ChIEF-tdTomato expression in ZI GABA neurons (Fig. 3, H and I). After the days of photostimulation were completed, mice showed a significantly reduced food intake compared with that of controls (Fig. 3H). The body weight of mice that showed an increase with ZI GABA neuron photostimulation gradually returned to the prestimulation body weight level of controls (Fig. 3I), consistent with the perspective that the mice return to a normal body weight set point (23) in the absence of continuing ZI activation. To test whether ZI GABA neurons exert long-term effects on energy homeostasis, we microinjected AAV-flex-taCasp3-TEVp, which expresses caspase-3 (24), into the ZI of VGAT-Cre mice to selectively ablate ZI GABA neurons (fig. S7). Ablation of ZI GABA neurons decreased long-term food intake and reduced body weight gain by 53% over 8 weeks (Fig. 3, J and K).

To explore the neuronal pathway postsynaptic to the VGATZI-PVT axon terminals, we injected Cre-inducible AAV-ChIEF–tdTomato selectively into the PVT of vGlut2-Cre mice (Fig. 4A and fig. S8A). In brain slices, laser stimulation excited PVT ChIEF-tdTomato–expressing glutamatergic neurons (Fig. 4C). Laser stimulation (20 Hz) above the PVT of ChIEF-tdTomato mice significantly inhibited normal, sweet, and high-fat food intake during 1-hour tests (Fig. 4D and fig. S8B). The mean latency for mice to stop eating was 6.1 ± 2.0 s after the laser (20 Hz) was turned on (Fig. 4E). After mice were partially fasted with only 60% of the normal food available during the preceding night, laser stimulation (20 Hz, 10 min ON followed by 10 min OFF, two times) of ChIEF-expressing PVT vGluT2 neurons reduced food intake (Fig. 4, F to H).

 

BingeEating_4
Fig. 4 Direct and indirect activation of glutamatergic PVT neurons postsynaptic to ZI axons inhibits food intake. (A) Fluorescent image shows restricted expression of ChIEF-tdTomato in PVT after AAV Cre-dependent ChIEF-tdTomato injected into PVT of vGlut2-Cre mice. Scale bar, 100 μm. (B) Implanted optical fiber tip above PVT. (C) Photostimulation (10-ms pulses) at 5, 10, and 20 Hz excites vGlut2-ChIEF neurons in PVT. (D) Photostimulation of PVT vGlut2-ChIEF neurons decreases intake of normal, sweet, and high-fat (HF) foods over 1 hour. (E) Latency in eating cessation in control and PVT vGlut2-ChIEF mice. (F) (Top) Four consecutive 10-min trial periods with first and third paired with stimulation (10 ms, 20 Hz). Food intake of control and vGlut2-ChIEF mice with partial food restriction during each period. (G) Total food intake is reduced by photostimulation in PVT of vGlut2-ChIEF mice with food restriction over 40 min compared with controls. (H) Food intake ratio of photostimulated to unstimulated periods in food-restricted control and vGlut2-ChIEF mice. (I) (Left) Schematic shows strategy for tracing presynaptic PSTh projections to PVT glutamate neurons. (Middle and right) Green images show fluorescent RV-labeled presynaptic PSTh neurons. cp, Cerebral peduncle. (J) Schematic shows bilateral AAV-ChIEF-tdTomato injections in parasubthalamic nucleus (PSTh) and placement of fiber optic tip above PVT. (Left bottom) Restricted expression of ChIEF-tdTomato in PSTh after AAV-ChIEF-tdTomato injection in vGlut2-Cre mice. Scale bar, 500μm. (Right bottom) PSTh vGlut2 neurons project to PVT. Scale bar, 100 μm. (Top) Higher magnification shows PSTh vGlut2 neuron terminals in PVT. Scale bar, 5 μm. (K) EPSCs in PVT glutamate neurons evoked by photostimulation (10-ms pulses) at 1, 5, 10, and 20 Hz (membrane potential clamped at –70 mV). In 2-amino-5-phosphonopentanoic acid (AP5) (50 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 μM), photostimulation of 1 Hz evoked no obvious current. (L) Photostimulation (10 ms, 20 Hz) of excitatory vGlut2 neuron terminals in PVT projecting from the PSTh decreased food intake. Statistical analysis for comparison between two groups: paired t test for (D) and (L); one-way ANOVA with Bonferroni post hoc comparison for (E) and (F); unpaired t test for (G) and (H). *P < 0.05; **P < 0.01; ***P < 0.001.
Panels A, B, and C

Panel A: A fluorescent image of the mouse PVT showing the location of ChIEF-tdTomato expression in PVT VGlut2 neurons acquired with a fluorescent microscope.

Panel B: A scheme depicting the placement of an optic fiber above the PVT for optogenetic stimulation experiments.

Panel C: Electrophysiology recordings from ChIEF-tdTomato expressing PVT VGlut2 neurons. The cells fired in response to different frequencies of blue light stimulation.

Panels D and E

Panel D: Food intake of mice expressing ChIEF in their PVT VGlut2 neurons in the presence and absence of optogenetic stimulation. The mice ate normal, sweet, and high-fat foods during the stimulation. This behavior shows that PVT VGlut2 neurons are necessary for food intake.

Panel E: Latency to stop eating after optogenetic stimulation of control and ChIEF-expressing mice. The latency to cease eating was strongly reduced in ChIEF-expressing mice upon stimulation onset.

Panels F, G, and H

Panel F top: Photostimulation protocol in which the mice were subjected to alternating periods of photostimulation followed by no photostimulation. The mice were partially fasted prior to the experiment and so they were hungry. Bottom: Food intake of control and ChIEF-expressing mice during each time block above. Despite being hungry, the ChIEF-expressing mice ate less during the ON periods. Therefore, stimulation of the PVT VGlut2 neurons was sufficient to suppress hunger-induced food intake.

Panel G: Total food intake during the stimulation ON periods in Panel F.

Panel H: Food intake of the mice in Panel F expressed as a ratio of the total amount during the ON stimulation over the total amount during the OFF stimmultion.

Panels I and J

Panel I left: A scheme of the monosynaptic rabies tracing strategy used to identify neurons that provide input to the PVT VGlut2 neurons (see figure 2B for more information). Neurons that express rabies tagged with green fluorescent protein are depicted in the PSTh. Center: Rabies-expressing neurons in the PSTh. Right: A higher magnification view of the labelled neurons on the left.

Panel J top left: Scheme depicting AAV injection of ChIEF into the PSTh VGlut2 neurons and optic fiber implantation above the PVT. Bottom left: An image of the mouse PSTh showing the location of ChIEF-tdTomato expression in the PSTh VGlut2 neurons acquired with a fluorescent microscope. Bottom right: A fluorescent image of tdTomato terminals of PSTh VGlut2 neurons in the PVT. Top right: A higher magnification view of the image at the bottom right.

Panels K and L

Panel K: The authors examined whether PVT VGlut2 neurons received excitatory input from PSTh neurons by recording EPSCs in PVT VGlut2 neurons in brain slices while optogenetically stimulating PSTh VGlut2 neurons. The VGlut2 PVT neurons responded with IPSCs upon stimulation of various frequencies. The effect was blocked by addition of drugs to the brain slices that inhibit receptors for excitatory neurotransmissions (AP5 and CNQX). This effect shows that PSTh VGlut2 neurons excite VGlut2 PVT neurons by releasing excitatory neurotransmitters.

Panel L: Intake of sweet food by mice receiving stimulation of PSTh VGlut2 terminals in the PVT. Each connected circle is the intake of an individual mouse during the OFF and ON periods. Food intake of each mouse was reduced during the stimulation compared to its intake of food in the absence of stimulation.

A chemo-genetic designer receptor exclusively activated by designer drugs (DREADD) was used to test the hypothesis that silencing the cells postsynaptic to ZI GABA axons, the PVT glutamate neurons, would enhance food intake. We injected Cre-inducible AAV5-hSyn-HA-hM4D(Gi)-IRES-mCherry coding for the clozapine-N-oxide (CNO) receptor into the PVT of vGlut2-Cre mice (2526) (fig. S9, A and B). CNO inhibited PVT neurons with hM3D(Gi)-mCherry receptor expression (fig. S9C). Intraperitoneal CNO produced an increase in food intake during a 3-hour trial (fig. S9D). To test whether PVT neuronal activity affects body weight gain, we microinjected AAV-flex-taCasp3-TEVp into the PVT of vGlut2-Cre mice to induce Cre-dependent caspase expression and selectively ablate PVT glutamatergic neurons. To confirm that PVT vGlut2 neurons were killed by the virus-generated caspase-3, we injected the Cre-dependent reporter construct AAV-tdTomato simultaneously with AAV-flex-taCasp3-TEVp to corroborate that reporter-expressing neurons were absent after selective caspase expression. With coinjection, little tdTomato expression was detected, whereas many cells were detected with injections of AAV-tdTomato by itself, consistent with the elimination of vGluT2 neurons in the PVT (fig. S10, A to D). Ablation of PVT vGluT2 neurons substantially increased both food intake and body weight gain for an extended period (16-week study) (fig. S10, G and H).

In our monosynaptic retrograde tracing with Cre-dependent rabies virus, although less robust than the projection from the ZI, we found a substantial projection to PVT glutamate neurons from the parasubthalamic nucleus (PSTh) (Fig. 4I and fig. S11) (2728). That the PSTh may be involved in feeding is suggested by increased c-fos expression in the PSTh during anorexia induced by amino acid deficiency (29). To test whether the PSTh maintains an excitatory input to PVT glutamatergic neurons that could serve to antagonize binge-like eating evoked by VGATZI-PVT inhibitory pathway activation, we injected Cre-inducible AAV-ChIEF–tdTomato bilaterally into the PSTh of vGlut2-Cre mice (Fig. 4J). Restricted expression of ChIEF–tdTomato was observed in the PSTh cell bodies (Fig. 4J) and in large numbers of PSTh axon terminals in the PVT (Fig. 4J). Brain slice electrophysiology confirmed that optogenetic activation of PSTh glutamatergic neuron terminals in the PVT evoked strong glutamate-mediated postsynaptic excitatory currents in PVT vGlut2-GFP neurons, suggesting a functional role for PSTh glutamate neurons in the synaptic excitation of PVT glutamate neurons (Fig. 4K). Stimulation of PSTh glutamatergic neuron terminals in the PVT inhibited food intake (Fig. 4L). Furthermore, optogenetic activation of the vGlut2PSTh-PVT excitatory pathway in a two-chamber place-preference test generated a significant aversion associated with the laser stimulation–paired chamber (fig. S12).

Together, our data demonstrate a powerful inhibitory projection from the ZI to the PVT that can reliably generate rapid and substantial eating. That the ZI GABA cells may participate in energy homeostasis is suggested by electrophysiological data showing increased activity of these cells after food deprivation and in the presence of the empty gut–signaling peptide ghrelin. Based on retrograde rabies virus and anterograde AAV tracing, ZI axonal projections to the excitatory neurons of the PVT appear more robust than those from other known regions of the brain involved in food intake, suggesting the ZI is not a minor component; furthermore, optogenetic stimulation of the ZI generated a more robust feeding response than stimulation of the much-studied lateral hypothalamus, further suggesting that the ZI can play a substantive role in enhancing food consumption. Our study provides a potential explanation for why clinical deep brain stimulation in the ventral thalamus near the ZI can increase binge eating.

 

Supplementary Materials

www.sciencemag.org/content/356/6340/853/suppl/DC1

Materials and Methods

Figs. S1 to S12

References

Movies S1 to S4

 

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