- Apr 2019
binge eating, which can at times lead to obesity
Binge eating disorder in humans is characterized by overeating and can lead to weight gain.
For interviews with sufferers of the condition and scientists working towards understanding the disorder, read more in BBC News: (https://www.bbc.com/news/health-45680281)
To determine the role of the ZI in feeding and body weight regulation
The authors set out to understand how the ZI influences food intake and to map the circuits in the brain that underlies these effects.
Read more in SciTechDaily: (https://scitechdaily.com/yale-researchers-shed-light-on-binge-eating/)
Read more in Scientific American: (https://www.scientificamerican.com/article/electric-brain-stimulation-offers-binge-eating-clue/)
much-studied lateral hypothalamus
The lateral hypothalamus is a region neighboring the ZI. Stimulation of this site has been shown to substantially increase food intake and is also hedonic.
hat 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).
Anorexia induced by dietary deficiency in a single indispensable amino acid was found to induce expression of c-fos, a protein known to be present in active neurons, in the PSTh.
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) (27, 28).
Analysis of the inputs to PVH VGlut2 neurons showed that these neurons receive input from the parasubthalamic nucleus, a brain region with previously described roles in appetite.
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).
To confirm that the caspase virus was killing cells, a tdTomato reporter, which makes the cells red under a fluorescent microscope, was injected at the same time as the caspase virus.
The authors found that few tdTomato cells were present in mice that also received the caspase, compared to control mice that were injected with the tdTomato only. Thus, the caspase virus efficiently killed the PVT neurons.
Intraperitoneal CNO produced an increase in food intake during a 3-hour trial (fig. S9D).
The CNO was injected into the peritoneal/abdominal cavity. It travels through the circulation and crosses the blood brain barrier and affects its target cells.
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 (25, 26) (fig. S9, A and B).
Silencing of neurons in the PVT that receive input from ZI GABAergic neurons should increase food intake given that these neurons are inhibited by ZI GABA neurons, which increase food intake.
The authors used a chemogenetic approach in which a modified (DREADD) receptor is expressed in the neurons using AAVs. The receptor is activated specifically by a synthetic drug (CNO) that has no other biological effect.
The authors used this approach over an optogenetic method to silence the neurons as currently available optogenetic tools for inhibition are not very efficient.
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).
The authors selectively killed ZI GABA neurons by using an AAV to express a caspase in these neurons. Caspase-3 is an enzyme that induces cell death.
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).
Mice that received stimulation of ZI GABA terminals in the PVT activated spent more time in the light compartment in the presence of high-fat food even though mice usually avoid places that are brightly lit. This means that the stimulation of the ZI to PVT projection was able to overcome the aversive nature of the light.
o 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 (19, 20). As soon as the laser was turned off, the mice stopped eating.
The authors wanted to compare the latency of eating onset upon stimulation of ZI projections to the PVT to that of AgRP neurons which are known to promote food intake in response to hunger. They found that mice start to eat much faster than mice did in previous reports where AgRP neurons were stimulated or when AgRP projections to the PVT were stimulated.
They did this by intermittently turning on the stimulation light for 10 seconds followed by 30 seconds of no stimulation. With each 10 second of light on, they measured how long it took for the mice to begin eating.
These results are consistent with an early report that lesions in the area of the ZI can alter food intake (18).
The ZI has been shown to be important for food intake that occurs in response to low levels of blood glucose. Rats with ablated ZI eat less food under these conditions.
photostimulation of ZI VGAT-ChIEF-tdTomato terminals in the PVT evoked GABA-mediated inhibitory currents in PVT vGlut2-GFP neurons (Fig. 2D).
Stimulation of ZI GABA neuron projections (terminals) in the PVT with blue light led to inhibition of PVT excitatory neurons. This finding confirms the rabies tracing result and shows that the connection between ZI neurons and PVT neurons is inhibitory.
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 (16, 17).
The authors bred two different mouse lines together: one parent expressed Cre in VGAT-positive neurons and the other parent expressed a protein that emits green fluorescence (GFP) in VGlut2-positive neurons.
The researchers than used the offspring of this cross to record from GFP-positive cells in a slice and ask whether VGAT cells in the ZI provide input to these neurons.
We asked whether the PVT may be a critical target for ZI regulation of food intake.
The authors showed that ZI GABA neurons send input to the PVT. They then assessed through further experimentation whether these PVT neurons are involved in food intake. Thus, they have used scientific questioning and evidence to build on their model of how the ZI controls feeding behavior.
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).
The authors recorded the activity of the ChIEF-expressing neurons in brain slices using electrodes. Stimulating the slice with blue light activated the ChIEF-expressing neurons, causing them to fire in the same pattern with which they were stimulated (ie. high fidelity). Hz (hertz) refers to the number of times the light flashes per second. ie. 20Hz corresponds to 20 flashes of light per second which caused the neurons to fire 20 times per second.
This virtual lab demonstrates electrophysiological recordings of neurons: Neurophysiology Virtual Lab
Food deprivation lasting 24 hours increased ZI GABA neuron activity and excitatory neurotransmission to these neurons
Recordings made in brain slices show that ZI GABA neurons fire more ie. are more active in food deprived mice. Further, inputs from other neurons that excite/activate ZI GABA neurons are increased.
See this video that explains how information is transmitted between neurons: Molecular mechanism of synapse function
Therefore, increased ZI GABA neuron activity correlates with hunger.
Refers to energy balance, in which an organism's energy intake (food) and outflow (energy expenditure) is coordinated to achieve no overall energy surplus or deficit.
previous observations that some ZI cells project to the PVT (13, 14),
This shows that science builds on the replication of studies to increase confidence in findings. Further, science advances by current studies building on those from the past.
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).
Ghrelin is produced in the stomach. Its synthesis is increased by food deprivation.
Application of Ghrelin to the brain slices containing ZI GABA neurons increases their activity similar to food deprivation.
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) (10, 11)] bilaterally into the rostral ZI of vesicular GABA transporter (VGAT)–Cre mice that express Cre recombinase in GABA neurons
To target a neuron population of interest, eg. those that express GABA, scientists use genetically modified viruses (AAVs) to deliver proteins into the brain (such as optogenetic tools).
This is achieved by using two tools: 1) a mouse line that expressed the enzyme Cre recombinase in a specific population of neurons (eg. those that express the GABA transporter VGAT). 2) an AAV that expresses an optogenetic protein only in the presence of Cre. The AAV is injected into the brain region of interest in the Cre mice. This AAV has a tdTomato tag which allows the injection site to be visualized under a fluorescent microscope.
For further information on these tools see: Optogenetics- YouTube
The ZI in both hemispheres of the brain was injected with the AAV (bilaterally), with the region lying towards the front of the brain (rostral) being targeted. The optogenetic tool used (ChIEF) activates neurons when blue light is shone on the cells.
K. M. Kendrick, M. R. Hinton, B. A. Baldwin, Brain Res. 550, 165–168 (1991).
Kendrick and colleagues measured GABA in the ZI in awake sheep. They found that in food-deprived animals, GABA was increased in the ZI upon the sight and ingestion of food. This response did not occur when a non-food object was presented.
sheep may release γ-aminobutyric acid (GABA) from the ZI in response to the sight or ingestion of food (6, 7).
Kendrick and colleagues found that the neurotransmitter GABA was increased in the ZI in sheep when the animals were presented with and ingested food.
can exhibit characteristics of binge eating (1–3)
Zahodne, Amami and Novakova studied Parkison's patients are found that many subjects exhibited binge-eating behavior. Those that had received deep-brain stimulation of the subthalamus were more likely to display this behavior.
A technique in which a neuron population of interest is killed. It allows the researchers to understand the function of a cell population.
- Author's experiments
- Previous work
- News and policy links
- Connect to learning standards
- Results and conclusions
- References and notes
- Mar 2019
19. Y. Aponte, D. Atasoy, S. M. Sternson, Nat. Neurosci. 14, 351–355 (2011)
This paper was one of the first to show that two populations of neurons in the arcuate nucleus of the hypothalamus have opposing effects on food intake. They demonstrated that opotogenetic activation of AgRP-expressing neurons increases food intake while activation of POMC-expressing neurons decreases food intake.
18. B. A. Stamoutsos, R. G. Carpenter, L. Grossman, S. P. Grossman, Physiol. Behav. 23, 771–776 (1979).
The authors show that rats administered with 2-deoxy-D-glucose (2-DG) increase their food intake while this is abolished in animals with ZI lesions. 2-DG is a modified glucose molecule that inhibits the breakdown of glucose, leading to low levels of blood glucose. This increases food intake in order to restore blood glucose levels. The reduced food intake in 2-DG treated rats with ZI lesions suggests that the ZI is necessary for food intake in response to low blood glucose.
13. J. S. Lee, E. Y. Lee, H. S. Lee, Brain Res. 1598, 97–113 (2015).
The authors performed retrograde mapping from the PVT and showed that neurons in the ZI directly project to the PVT. These cells were shown to express the protein cocaine- and amphetamine-related transcript (CART).
Stimulation of PSTh glutamatergic neuron terminals in the PVT inhibited food intake (Fig. 4L).
PVT VGlut2 neurons decrease food intake. Therefore stimulation of excitatory neurons upstream of them ie. the vGlut2 PSTh neurons was also found to increase food intake.
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).
The authors confirmed that stimulation of the PSTh terminals in the PVT was able to activate PVT neurons. ie. induce excitatory activity in the PVT cells.
This confirms the rabies tracing result and shows that the connection between the PSTh and the PVT is functional.
Serve an opposite function to.
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).
The increase in food intake and body weight gain with PVT neuron ablation shows that these cells are important for body weight maintenance.
This finding is opposite to what was found when ZI GABA neurons were ablated, suggesting that PVT neurons are downsteam of ZI GABA neurons and have an opposing effect on food intake and body weight.
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).
The authors assessed the role of the neurons downstream (postsynaptically) of the ZI neurons that project to the PVT. They examined how food intake was affected when PVT excitatory neurons were optogentically stimulated.
Given that GABA is an inhibitory neurotransmitter, the PVT neurons would normally be inhibited when the ZI to PVT projection is active. Thus, when the PVT neurons are stimulated, food intake should increase. Indeed, this is what the authors found.
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).
The authors gave the mice a small amount of food to eat overnight, which meant that they were hungry during the experiment. Therefore, control mice commenced eating with short latency at at the onset of the stimulation protocol.
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).
When the ZI GABA neurons were killed, the mice could no long maintain their normal body weight and ate less food than control mice that had their ZI GABA neurons intact. This suggests that these cells are required for normal food intake and body weight maintenance.
After the days of photostimulation were completed, mice showed a significantly reduced food intake compared with that of controls (Fig. 3H).
The authors measured daily food intake for 15 days during the stimulation protocol. They then continued measuring daily food intake in the absence of stimulation.
The mice likely reduced their food consumption when the stimulation protocol ceased due to satiety signals that normally prevent overeating in the absence of the manipulation of the ZI to PVT pathway.
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.
The author hypothesise that because stimulation of the ZI to PVT pathway evokes a large increase in food intake in a short amount of time, long-term stimulation should lead to weight gain.
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).
Motivational valence is the degree to which something is perceived as pleasurable (positive valence) or aversive (negative valence). The authors hypothesised that because the ZI to PVT projection promotes intake of foods that are pleasurable to eat and also makes mice overcome their aversion to light in order to eat that food, that stimulation of this pathway is pleasurable or rewarding for the mice.
They tested this by placing the animals in a box with two identical compartments. The mice were able to freely move around the box. On one side of the chamber the mice received stimulation of their ZI-PVT neurons while the stimulation was turned off when the mice were on the other side.
That the mice preferred to spend more time in the compartment where they received stimulation of the ZI-PVT pathway suggest that stimulation of the neurons is pleasurable for the animal.
food intake was measured when food was put in a brightly illuminated chamber in a two-chamber light-or-dark conflict test
Mice were placed in a chamber with two compartments- one with no lights and one brightly illuminated. Mice are innately averse to light and so will usually spend more time in the unlit compartment.
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).
The arcuate nucleus, a subregion of the hypothalamus has been shown to be a very important brain region that controls food intake
Neurons within this region have been shown to control feeding in a reciprocal manner. POMC-expressing neurons are activated by satiety signals are reduce food intake while AgRP-expressing neurons are activated by starvation and increase food intake.
These neurons have been proposed to be the neural substrate for satiety and hunger respectively.
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
The authors found that blocking the function of the GABA receptor (GABA-A receptor) in the PVT could blunt the increase in food intake mediated by stimulation of ZI terminals in the PVT. This suggests that GABA is an important neurotransmitter underlying this effect.
The limitations of this approach include that the authors cannot control the spread of the GABA-A receptor blocker or that other neurotransmitters might be involved.
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).
This suggests that even though ZI GABA neurons project to other brain regions in addition to the PVT, the PVT projection appears to be the most important mediator of increased food intake. This is because ZI GABA cell body stimulation and stimulation of the projections to the PVT evoked similar degrees of food intake.
Although mice prefer sweet and high-fat foods when stimulation is off,
Mice will avidly consume high-fat and sweet foods even when not hungry. This is because these foods are palatable, meaning that they are pleasurable or rewarding to eat and so will be consumed even when nutrition requibments are met.
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).
The authors conclude that the food intake seen with ZI stimulation is not because the manipulation increases gnawing behavior directed at any object, but directs behavior towards edible sources of food.
In control mice with tdTomato expression, consumption was only 4% of their 24-hour intake during the same period (Fig. 1E).
Lack of food intake in a control animal that was injected with an AAV expressing only a fluorescent protein shows that the result seen in ChIEF expressing mice was not due to non-specific effects of AAV injection or protein expression in the neurons.
In the living animal.
Satiety feedback signals can thus attenuate ZI-induced feeding.
Signals from the body inform the brain that sufficient food has been eaten (satiety). These include release of hormones from the gut as well as stomach distention.
The finding that ZI-stimulated mice will not eat indefinitely suggests that satiety feedback mechanisms are still intact.
Food deprivation lasting 24 hours increased inhibitory synaptic neurotransmission to PVT glutamate neurons (fig. S4)
In food deprived mice, PVT glutamate neurons receive more inhibitory inputs compared to fed mice. These may come from ZI inhibitory GABA neurons which the authors showed have increased activity upon food deprivation.
confirmed that PVT glutamate neurons receive strong and direct innervation from ZI neurons (Fig. 2, B and C, and fig. S3).
The authors confirm their anterograde tracing findings using retrograde tracing to show that ZI GABA neurons send projections to excitatory neurons in the PVT.
Cre recombinase–dependent rabies virus–mediated monosynaptic retrograde pathway tracing in vGluT2–Cre recombinase mice
The authors identified the neurons that lie upstream and provide input to PVT neurons.
They targeted excitatory PVT neurons using vGluT2-Cre mice and used a modified rabies virus that traffics into neurons that provide input to the starting population of cells.
Anterograde AAV-ChIEF-tdTomato labeling
Infection of the neurons with tdTomato-tagged AAV allows the projection of the ZI GABA axons to be visualised.
The authors used this method to determine where in the brain these neurons project to.
VGAT-Cre mice with ChIEF expression, bilateral laser stimulation (20 Hz) in the ZI increased food intake
When ZI GABA neurons are activated by delivering blue light into the brains of the mice, the mice eat a large amount of food.
ad libitum high-fat food intake
The amount of food eaten when the mice are allowed to eat as much or as often as they like.
lateral hypothalamic neurons
A region of the brain in close proximity to zona incerta known to promote food intake.
ChIEF-tdTomato was selectively expressed in ZI GABA neurons
The optogenetic tool ChIEF-tdTomato was found to be expressed only the ZI GABA neurons.
deep brain stimulation
A procedure in which electrodes are implanted into specific parts of the brain, allowing electrical stimulation of a target region.
It is most commonly used as a treatment for Parkinson's disease and other movement disorders as well as obsessive-compulsive disorder and depression.
Promotes appetite and food intake.
An excitatory neurotransmitter which causes neurons to become activated
A part of the brain located below the thalamus which functions in motor control.
A hormone produced in the gut in response to food deprivation. It increases appetite and food intake and energy storage.
Neurons that express excitatory neurotransmitters and cause downstream neurons to become activated.
paraventricular thalamus (PVT)
A subregion of a part of the brain called the thalamus.
The PVT has been shown to have a broad range of function including in fear, learning, arousal and feeding behavior.
The axon is a long-thin part of the neuron that facilitates communication between neurons. Axons extend from the cell body of a neuron to other parts of the brain, allowing communication between different brain regions.
γ-aminobutyric acid (GABA)
An inhibitory neurotransmitter. Neurons communicate with each other by releasing neurotransmitters. Neurons respond to inhibitory neurotransmitters by reducing their activity.
zona incerta (ZI)
A part of the brain.
The function of this area is poorly described but is thought to regulate behavior of an animal in response to internal (such as hunger) and external (such as pain) sensory cues.
A technique that uses light to control the activity of cells, most commonly neurons, in living animals. The cells are genetically modified to express ion channels that are sensitive to light. Shining light on the neurons changes their activity allowing scientists to understand the role of the neuron in a given behavior or physiological process.
Indicates the part of the brain that underlies a specific behavior or cognitive or physiological process.