AgRP Neurons Can Increase Food Intake during Conditions of Appetite Suppression and Inhibit Anorexigenic Parabrachial Neurons.

To maintain energy homeostasis, orexigenic (appetite-inducing) and anorexigenic (appetite suppressing) brain systems functionally interact to regulate food intake. Within the hypothalamus, neurons that express agouti-related protein (AgRP) sense orexigenic factors and orchestrate an increase in food-seeking behavior. In contrast, calcitonin gene-related peptide (CGRP)-expressing neurons in the parabrachial nucleus (PBN) suppress feeding. PBN CGRP neurons become active in response to anorexigenic hormones released following a meal, including amylin, secreted by the pancreas, and cholecystokinin (CCK), secreted by the small intestine. Additionally, exogenous compounds, such as lithium chloride (LiCl), a salt that creates gastric discomfort, and lipopolysaccharide (LPS), a bacterial cell wall component that induces inflammation, exert appetite-suppressing effects and activate PBN CGRP neurons. The effects of increasing the homeostatic drive to eat on feeding behavior during appetite suppressing conditions are unknown. Here, we show in mice that food deprivation or optogenetic activation of AgRP neurons induces feeding to overcome the appetite suppressing effects of amylin, CCK, and LiCl, but not LPS. AgRP neuron photostimulation can also increase feeding during chemogenetic-mediated stimulation of PBN CGRP neurons. AgRP neuron stimulation reduces Fos expression in PBN CGRP neurons across all conditions. Finally, stimulation of projections from AgRP neurons to the PBN increases feeding following administration of amylin, CCK, and LiCl, but not LPS. These results demonstrate that AgRP neurons are sufficient to increase feeding during noninflammatory-based appetite suppression and to decrease activity in anorexigenic PBN CGRP neurons, thereby increasing food intake during homeostatic need.SIGNIFICANCE STATEMENT The motivation to eat depends on the relative balance of activity in distinct brain regions that induce or suppress appetite. An abnormal amount of activity in neurons that induce appetite can cause obesity, whereas an abnormal amount of activity in neurons that suppress appetite can cause malnutrition and a severe reduction in body weight. The purpose of this study was to determine whether a population of neurons known to induce appetite ("AgRP neurons") could induce food intake to overcome appetite-suppression following administration of various appetite-suppressing compounds. We found that stimulating AgRP neurons could overcome various forms of appetite suppression and decrease neural activity in a separate population of appetite-suppressing neurons, providing new insights into how the brain regulates food intake.

Estimation of Current and Future Physiological States in Insular Cortex.

Interoception, the sense of internal bodily signals, is essential for physiological homeostasis, cognition, and emotions. While human insular cortex (InsCtx) is implicated in interoception, the cellular and circuit mechanisms remain unclear. We imaged mouse InsCtx neurons during two physiological deficiency states: hunger and thirst. InsCtx ongoing activity patterns reliably tracked the gradual return to homeostasis but not changes in behavior. Accordingly, while artificial induction of hunger or thirst in sated mice via activation of specific hypothalamic neurons (AgRP or SFOGLUT) restored cue-evoked food- or water-seeking, InsCtx ongoing activity continued to reflect physiological satiety. During natural hunger or thirst, food or water cues rapidly and transiently shifted InsCtx population activity to the future satiety-related pattern. During artificial hunger or thirst, food or water cues further shifted activity beyond the current satiety-related pattern. Together with circuit-mapping experiments, these findings suggest that InsCtx integrates visceral-sensory signals of current physiological state with hypothalamus-gated amygdala inputs that signal upcoming ingestion of food or water to compute a prediction of future physiological state.

Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering.

Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.

Sustained NPY signaling enables AgRP neurons to drive feeding.

Artificial stimulation of Agouti-Related Peptide (AgRP) neurons promotes intense food consumption, yet paradoxically during natural behavior these cells are inhibited before feeding begins. Previously, to reconcile these observations, we showed that brief stimulation of AgRP neurons can generate hunger that persists for tens of minutes, but the mechanisms underlying this sustained hunger drive remain unknown (Chen et al., 2016). Here we show that Neuropeptide Y (NPY) is uniquely required for the long-lasting effects of AgRP neurons on feeding behavior. We blocked the ability of AgRP neurons to signal through AgRP, NPY, or GABA, and then stimulated these cells using a paradigm that mimics their natural regulation. Deletion of NPY, but not AgRP or GABA, abolished optically-stimulated feeding, and this was rescued by NPY re-expression selectively in AgRP neurons. These findings reveal a unique role for NPY in sustaining hunger in the interval between food discovery and consumption.

Identification of discrete, intermingled hypocretin neuronal populations.

Neurons in the lateral hypothalamic area that express hypocretin (Hcrt) neuropeptides help regulate many behaviors including wakefulness and reward seeking. These neurons project throughout the brain, including to neural populations that regulate wakefulness, such as the locus coeruleus (LC) and tuberomammilary nucleus (TMN), as well as to populations that regulate reward, such as the nucleus accumbens (NAc) and ventral tegmental area (VTA). To address the roles of Hcrt neurons in seemingly disparate behaviors, it has been proposed that Hcrt neurons can be anatomically subdivided into at least two distinct subpopulations: a "medial group" that projects to the LC and TMN, and a "lateral group" that projects to the NAc and VTA. Here, we use a dual retrograde tracer strategy to test the hypotheses that Hcrt neurons can be classified based on their downstream projections and medial/lateral location within the hypothalamus. We found that individual Hcrt neurons were significantly more likely to project to both the LC and TMN or to both the VTA and NAc than would be predicted by chance. In contrast, we found that Hcrt neurons that projected to the LC or TMN were mostly distinct from Hcrt neurons that projected to the VTA or NAc. Interestingly, these two populations of Hcrt neurons are intermingled within the hypothalamus and cannot be classified into medial or lateral groups. These results suggest that Hcrt neurons can be distinguished based on their downstream projections but are intermingled within the hypothalamus.

Dynamics of Gut-Brain Communication Underlying Hunger.

Communication between the gut and brain is critical for homeostasis, but how this communication is represented in the dynamics of feeding circuits is unknown. Here we describe nutritional regulation of key neurons that control hunger in vivo. We show that intragastric nutrient infusion rapidly and durably inhibits hunger-promoting AgRP neurons in awake, behaving mice. This inhibition is proportional to the number of calories infused but surprisingly independent of macronutrient identity or nutritional state. We show that three gastrointestinal signals-serotonin, CCK, and PYY-are necessary or sufficient for these effects. In contrast, the hormone leptin has no acute effect on dynamics of these circuits or their sensory regulation but instead induces a slow modulation that develops over hours and is required for inhibition of feeding. These findings reveal how layers of visceral signals operating on distinct timescales converge on hypothalamic feeding circuits to generate a central representation of energy balance.

Hunger neurons drive feeding through a sustained, positive reinforcement signal.

The neural mechanisms underlying hunger are poorly understood. AgRP neurons are activated by energy deficit and promote voracious food consumption, suggesting these cells may supply the fundamental hunger drive that motivates feeding. However recent in vivo recording experiments revealed that AgRP neurons are inhibited within seconds by the sensory detection of food, raising the question of how these cells can promote feeding at all. Here we resolve this paradox by showing that brief optogenetic stimulation of AgRP neurons before food availability promotes intense appetitive and consummatory behaviors that persist for tens of minutes in the absence of continued AgRP neuron activation. We show that these sustained behavioral responses are mediated by a long-lasting potentiation of the rewarding properties of food and that AgRP neuron activity is positively reinforcing. These findings reveal that hunger neurons drive feeding by transmitting a positive valence signal that triggers a stable transition between behavioral states.

Increasing frequency and fatality of poison control center reported exposures involving medication and multiple substances: data from reports of the American Association of Poison Control Centers 1984-2013.

CONTEXT: Medication use has become increasingly prevalent in the United States, with up trending use of both prescription and over the counter medication. The increasing use and availability of medication may be changing the nature of poisoning fatality. OBJECTIVE: To evaluate changes in fatal poisoning over time, with respect to fatalities involving one or more medications, using annual reports published by the American Association of Poison Control Centers (AAPCC). MATERIALS AND METHODS: AAPCC annual reports were reviewed from 1984 to 2013. Data from tables in each annual report titled Number of Substances Involved in Human Exposure Cases and Summary of Fatal Exposures were abstracted. Fatality rates and changes in these rates over time were calculated for exposures to 1, 2, or ≥ 3 substances. All substances detailed in Summary of Fatal Exposures tables were then coded as medication or non-medication. The percentage of fatalities involving 1, 2, or ≥ 3 medications was calculated and trended over time. Subset analysis was performed to compare the periods 1984-2005 and 2006-2013 in order to limit confounding from changes in reporting. Secondary analysis linking the number of substances cases were exposed to and the fatality rate was performed for data from 2006-2013. RESULTS: There were 59,866,357 human exposures and 29,659 fatalities reported from 1984 to 2013. There were 49.5 fatalities per 100,000 exposures. The majority of fatalities (52.2%) involved more than one substance, although multiple substances were involved in only 8.3% of exposures. Fatality rates increased over time and were higher for cases involving multiple substances. Medications were involved in 79.2% of fatalities, a percentage that increased from 70% in the 1980s to nearly 90% after 2010. In recent years, the majority of fatalities have involved multiple medications. For data from 2006-2013 there was a strong association between fatality rate and number of substances involved in an exposure (221 additional fatalities per 100,000 exposures for each additional substance involved in an exposure). DISCUSSION AND CONCLUSION: Multiple substance exposures have become a greater percentage of cases reported to the AAPCC and have higher fatality rates than single substance exposures. The majority of fatal poisonings reported to the AAPCC between 1984 and 2013 involved medication. The percentage of fatal poisonings involving medication increased over the interval, as did the percentage of fatalities involving more than one medication. Fatalities involving multiple medications are now the most common type of fatal poisoning reported to the AAPCC.