- Oct 2019
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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 underlie these effects.
Read more in SciTechDaily.
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- Sep 2019
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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, whereas 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.
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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, whereas activation of POMC-expressing neurons decreases food intake.
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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, i.e. the vGlut2 PSTh neurons, was also found to increase food intake.
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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 and reduce food intake whereas 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.
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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 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.
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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 requirements are met.
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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 in a way similar to food deprivation.
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Anterograde AAV-ChIEF-tdTomato labeling
Infection of the neurons with tdTomato-tagged AAV allows the projection of the ZI GABA axons to be visualized.
The authors used this method to determine where in the brain these neurons project to.
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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, i.e. are more active in food deprived mice. Further, inputs from other neurons that excite/activate ZI GABA neurons are increased.
Therefore, increased ZI GABA neuron activity correlates with hunger.
See this HHMI BioInteractive video that explains how information is transmitted between neurons: Molecular mechanism of synapse function.
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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.
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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, e.g. 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 (e.g. those that express the GABA transporter VGAT) and 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 how mice optogenetics are used this video.
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.
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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 involvement in fear, learning, arousal, and feeding behaviors.
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axonal projections
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.
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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.
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- May 2019
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K. M. Kendrick, M. R. Hinton, B. A. Baldwin, Brain Res. 550, 165–168 (1991).
Kendrick and colleagues measured GABA in the ZI in live 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.
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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 authors hypothesize 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.
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excited by
Activated by.
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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.
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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.
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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 (i.e. high fidelity). Hz (hertz) refers to the number of times the light flashes per second, i.e. 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
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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.
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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.
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lateral hypothalamic neurons
A region of the brain in close proximity to ZI known to promote food intake.
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ChIEF-tdTomato was selectively expressed in ZI GABA neurons
The optogenetic tool ChIEF-tdTomato was found to be expressed only the ZI GABA neurons.
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can exhibit characteristics of binge eating (1–3)
Zahodne, Amami, and Novakova studied Parkinson's patients and 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.
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glutamate
An excitatory neurotransmitter, which causes neurons to become activated.
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parasubthalamic nucleus
A part of the brain located below the thalamus, which functions in motor control.
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ghrelin
A hormone produced in the gut in response to food deprivation.
Also known as the "hunger hormone," ghrelin increases appetite and food intake and energy storage.
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excitatory
Neurons that express excitatory neurotransmitters cause downstream neurons to become activated.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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 downstream of ZI GABA neurons and have an opposing effect on food intake and body weight.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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- Apr 2019
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energy homeostasis
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.
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ablation
A technique in which a neuron population of interest is killed. It allows the researchers to understand the function of a cell population.
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- Mar 2019
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antagonize
Serve an opposite function to.
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In vivo
In the living animal.
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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.
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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.
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orexigenic
Promotes appetite and food intake.
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neuronal substrate
Indicates the part of the brain that underlies a specific behavior or cognitive or physiological process.
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