Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state

Abstract

In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues.

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Figure 1: AgRP stimulation recapitulates fasting-related foraging and reduced territoriality.
Figure 2: MeA-projecting AgRP neurons evoke hunger and inhibit territorial aggression.
Figure 3: Npy1R neurons in the MeA can evoke aggression and inhibit feeding.
Figure 4: The posterior BNST is a secondary target of AgRP and receives direct input from Npy1RMeA neurons.
Figure 5: Identification of Vgat-expressing NPY-responsive cells in the MeA that received direct input from AgRP neurons and project to the pBNST.
Figure 6: Npy1RMeA neurons that project to the pBNST evoke territoriality.

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Acknowledgements

We thank K. Kafer and M. Chiang for technical assistance generating the new line of mice and maintaining the mouse colonies. We thank M.A. Patterson and B.C. Jarvie for careful reading of this paper and the entire Palmiter laboratory for discussions and critiques. We thank D.J. Anderson and L. Lo (Caltech) for generously providing H129Δ-fs-TK-TT. We thank E. Strakbein at U.W. Scientific Instruments for the development of tools and apparatus used in this manuscript. This work was supported by funds from the Hilda Preston Davis Foundation (S.L.P.), the US National Institutes of Health (R01DK068098, R01NS038809, M.J.K. and O.K.R.; R01MH094536, L.S.Z.; R01DA024908, R.D.P.), a Marie Sklodowska-Curie award (H2020-MSCA-IF-2014-658352, E.S.), a Ramón y Cajal fellowship (RyC-2012-11873, A.Q.), European Research Council Starting Grant NEUROMITO (ERC-2014-StG-638106, A.Q.) and MINECO Proyectos I+D de Excelencia (SAF2014-57981P, A.Q.).

Author information

S.L.P. designed the study under the guidance of R.D.P.; J.Q. gathered and analyzed electrophysiological data in the laboratories of M.J.K. and O.K.R.; M.E.S. gathered and analyzed electrophysiology data in the laboratory of L.S.Z.; E.S. performed the RiboTag pulldown and quantitative PCR in the laboratory of A.Q.; C.C.N. performed single-cell PCR on harvested cells after whole-cell recordings; R.D.P. generated the Agrpcre and Npy1rcre lines of mice; F.D.B. assisted with behavior experiments and blind scoring; R.D.P., M.J.K., O.K.R. and L.S.Z. provided laboratory space and resources to conduct the experiments; S.L.P. and R.D.P. wrote the manuscript with revisions and input from all contributing authors.

Correspondence to Stephanie L Padilla.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 AgRP-expressing cells are active in the state of starvation and act to prioritize behavior and physiology to promote food seeking and conserve energy.

(a) Model of AgRP-directed behaviors and physiology. (b) AgRP neurons that project to the Npy1r-expressing cells in the MeA are likely involved in decreasing territorial behaviors to facilitate food seeking in the state of starvation. (c) AgRP neurons that project to the PVH influence feeding but do not influence territorial behavior.

Supplementary Figure 2 Profile of fasted residents.

(a) Fasted animals learn to avoid the shock-associated chamber when the food-challenge assay is performed in the absence of food or food-associated cues (ad lib n = 6; 48-hr Fast n = 6). (b) Territorialized resident animals are significantly heavier than intruders. (c) 24-hr fasted residents display decreased territorial aggression (n = 6). (d) Time distribution of nose-nose interactions and escape behaviors (including rearing and jumping), comparing the ad libitum and fasted state of an individual mouse. (e,f) Average scores: nose-to-nose (e) and escape behavior (f) (n = 9). Error bars represent the mean ± SEM.

Supplementary Figure 3 In the presence of food, AgRP-stimulated animals display little aggression toward an intruding conspecific.

(a) Home-cage aggression of AgRP stimulated animals in the presence of food (n = 7) was significantly less than non-stimulated controls (sal, n = 10; Fig. 1e). (b) Food was presented following 30 sec of exposure to an intruder. The blue bars represent the time that each animal spent engaged with the food during the trial. Stimulated animals spend 47.2 ± 3.4% of the trail engaged with the food and consumed 0.34 ± 0.3 g of food during the 10-min trial.

Supplementary Figure 4 Fluorescent bead targeting and fiber placement. Validation of stereotaxic MeA targeting.

(a) Fluorescent image (left) compared to a matching coronal atlas (right). Scale bar, 200 μm. Fiber placement characterization in the MeA (b) and PVH (c). (b,c) Dapi stain (left), blue circles represent the diameter and ventral position of the fiber relative to the targeted bregma position (middle). Scale bar, 200 μm. Insert (right) is a composite of all fiber tracks identified in the targeted region. Colored circle sizes represent the thickness of the fiber track at the targeted bregma positions: MeA (−1.5 mm), PVH (−0.8 mm)

Supplementary Figure 5 Npy1R is expressed throughout the MeA.

Percent distribution of cells in the rostral, mid and caudal MeA

Supplementary Figure 6 Silencing of Npy1RMeA neurons does not alter the innate response to a threatening environment.

Npy1rCre-expressing cells in the MeA were bilaterally injected with either AAV1-DIO-GFP:TetTox or AAV1-DIO-YFP. Npy1RMeA silencing did not significantly change anxiety assessed by time spent in the exposed arms of an elevated maze. Comparing TetTox, n = 4 to YFP controls, n = 4. Error bars represent the mean ± SEM.

Supplementary Figure 7 Few if any cells in the ARH project to the pBNST; also targeting for experiment Figure 4d.

(a) Retrobead targeting of the pBNST. An image of the fluorescent injection site (left) is paired with a matching coronal atlas image (right). Scale bar, 200 μm. (b) RetroBeads retained in the ARH, scale bar, 100 μm. (c) tdT expression following injection of H129D-fs-TK-TT into the ARH of an AgrpCre animal; scale bar 200 μm. (d) Green RetroBeads injected into the pBNST; scale bar 200 μm. (e) Distribution of aggressive behaviors in: ad libitum mice (left), Npy1RMEA::hM3Dq + CNO (center), and Npy1RMEA::ChR2 with light stimulation of fibers in the pBNST (right).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 2376 kb)

Supplementary Methods Checklist (PDF 388 kb)

Npy1RMeA activation enhances home-cage aggressive behaviour

Attack behavior demonstrated by CNO-induced activation of Npy1RMeA neurons that express hM3Dq. CNO was delivered by intraperitoneal injection 30 min prior to the test. The intruder test was performed during the dark cycle and recorded with an infrared light. (AVI 14125 kb)

Npy1RMeA activation can evoke violent aggression

Violent attack of an anesthetized intruder demonstrated by CNO-induced activation of Npy1RMeA neurons that express hM3Dq. CNO was delivered by intraperitoneal injection 30 min prior to the test. The intruder test was performed during the dark cycle and recorded with an infrared light. (AVI 17099 kb)

Npy1RMeA → pBNST circuit activation enhances territorial aggression

Nudging behavior demonstrated by light-induced activation of Npy1RMeA neurons that express ChR2 and project to the pBNST. Light was delivered at 10 Hz with 5-ms pulses for 5 s followed by 2 s light-off recovery. The intruder test was performed during the dark cycle and recorded with an infrared light. (AVI 16943 kb)

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Padilla, S., Qiu, J., Soden, M. et al. Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state. Nat Neurosci 19, 734–741 (2016) doi:10.1038/nn.4274

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