Central amygdala circuits modulate food consumption through a positive-valence mechanism

TLDR: This work found that GABAergic serotonin receptor 2a-expressing CeA neurons modulate food consumption, promote positive reinforcement and are active in vivo during eating, and showed that CeAHtr2a neurons receive inputs from feeding-relevant brain regions.

Abstract: The complex behaviors underlying reward seeking and consumption are integral to organism survival The hypothalamus and mesolimbic dopamine system are key mediators of these behaviors, yet regulation of appetitive and consummatory behaviors outside of these regions is poorly understood The central nucleus of the amygdala (CeA) has been implicated in feeding and reward, but the neurons and circuit mechanisms that positively regulate these behaviors remain unclear Here, we defined the neuronal mechanisms by which CeA neurons promote food consumption Using in vivo activity manipulations and Ca2+ imaging in mice, we found that GABAergic serotonin receptor 2a (Htr2a)-expressing CeA neurons modulate food consumption, promote positive reinforcement and are active in vivo during eating We demonstrated electrophysiologically, anatomically and behaviorally that intra-CeA and long-range circuit mechanisms underlie these behaviors Finally, we showed that CeAHtr2a neurons receive inputs from feeding-relevant brain regions Our results illustrate how defined CeA neural circuits positively regulate food consumption

Figures

Figure 3. CeA Htr2a neurons increase activity during food consumption 1085 a, Position of the GRIN lens above GCaMP6s-expressing CeAHtr2a-Cre neurons. b, Scheme of free-1086

Figure 5. CeA neuronal modulators of feeding behavior form reciprocal inhibitory 1135 connections. 1136 a, Scheme of whole-cell recording from CeAHtr2a- neurons in Htr2a-Cre;tdTomato slices 1137

Figure 4. CeAHtr2a neuron activity is positively reinforcing and modulates reward 1110 consumption. 1111

Figure 2. CeAHtr2a neurons are required for normal food consumption. 1063 a, Viral delivery of AAV-flex-dtA into the CeA of Htr2a-Cre mice. b, Representative images of 1064

Figure 6. CeAHtr2a neuron inhibition of the PBN is rewarding and modulates 1156 consummatory behaviour. 1157

Figure 1. CeAHtr2a neurons increase food consumption. 1024 a, Representative image of the CeA from Htr2a-Cre;floxed-lacZ mouse immunostained for-β-Gal 1025

Full text

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Central amygdala circuits modulate food consumption through a positive valence
1
mechanism
2
3
Amelia M. Douglass
1
*, Hakan Kucukdereli
1
*, Marion Ponserre
1
*, Milica Markovic
2
, Jan
4
Gründemann
2
, Cornelia Strobel
1
, Pilar L. Alcala Morales
1
, Karl-Klaus Conzelmann
3
, Andreas
5
Lüthi
2
, & Rüdiger Klein
1,4,5
6
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1 Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany
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2 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel,
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Switzerland
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3 Max von Pettenkofer-Institute & Gene Center, Ludwig-Maximilians-University Munich,
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Feodor-Lynen-Strasse 25, 81377 Munich, Germany
12
4 Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany
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* Equal contribution
14
5 Corresponding author
15
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 2, 2017. ; https://doi.org/10.1101/145375doi: bioRxiv preprint

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Summary
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The complex behaviors underlying the pursuit and consumption of rewards are integral to an
17
organism’s survival. The hypothalamus and mesolimbic dopamine system are key mediators of
18
these behaviors, yet regulation of appetitive and consummatory behaviors outside of these regions
19
is not well understood. The central nucleus of the amygdala (CeA) is implicated in feeding and
20
reward behavior, but the specific neural players and circuit mechanisms that positively regulate
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these behaviors remain unclear. Here, we define the neuronal mechanisms by which the CeA
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promotes consumption of food. We show, using in vivo activity manipulations and Ca
2+
imaging,
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that CeA GABAergic neurons expressing the serotonin receptor 2a (Htr2a) modulate food
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consumption in multiple contexts, promote positive reinforcement and are active in vivo during
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eating. We demonstrate using electrophysiology, anatomical tracing methods and in vivo
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optogenetics that both intra-CeA and long-range circuit mechanisms underlie these functional
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effects. Finally, we show that CeA
Htr2a
neurons are poised to regulate food consumption through
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inputs from feeding-relevant brain regions. Our study highlights a mechanism by which defined
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CeA neural circuits positively regulate food consumption.
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Introduction
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Survival of an organism relies on the ability to seek out and consume natural rewards in an ever-
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changing environment. To do so, animals must attend to salient environmental stimuli and execute
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complex motivated behavioral sequences to successfully seek and consume the reward. A network
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of brain regions, involving most notably the lateral hypothalamus (LH), ventral tegmental area
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(VTA) and nucleus accumbens (NAc), mediate goal-directed reward seeking and consumption
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behavior by increasing the saliency of environment cues and promoting positive reinforcement
1-
37
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 2, 2017. ; https://doi.org/10.1101/145375doi: bioRxiv preprint

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. The CeA, a forebrain structure of striatal origin, is important for processing salient stimuli and
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orchestrating the appropriate behavioral response. This region is comprised of a highly
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interconnected network of inhibitory GABAergic neurons that are functionally classified based on
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expression of molecular markers, several of which have described roles in fear
5-8
, anxiety
9,10
and
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appetite suppression
11
. Although the CeA has a reported role in magnifying reward saliency
12-14
,
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modulating food consumption
11,14
, and promoting appetitive behaviors
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, the cellular
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heterogeneity and high degree of neural interconnectivity in this region has precluded an insight
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into the specific neural players and underlying circuits that positively regulate food consumption.
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Here we report that a molecularly defined population of CeA neurons positively modulates food
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consumption. Using optogenetic and pharmacogenetic tools, we demonstrate that CeA neurons
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expressing the serotonin receptor Htr2a promote food consumption and positive reinforcement.
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Deep brain calcium imaging revealed that CeA
Htr2a
neurons consistently increase activity during
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eating. We show, using optogenetic and rabies tracing techniques, a local intra-CeA circuit
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mechanism by which CeA
Htr2a
neurons exert their functions. Further, we reveal that CeA
Htr2a
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neurons promote feeding and positive reinforcement through long-range inhibition of cells within
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the parabrachial nucleus (PBN), a brain region known to process aversive gustatory and sensory
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signals
16-18
. Finally, we demonstrate that PBN-projecting CeA
Htr2a
neurons receive distinct
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monosynaptic inputs from brain regions with known roles in feeding behaviors. Together these
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findings reveal the specific neural players within the CeA that positively regulate eating behavior.
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certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 2, 2017. ; https://doi.org/10.1101/145375doi: bioRxiv preprint

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Results
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CeA
Htr2a
neurons modulate food consumption
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To identify CeA neural subpopulations that positively regulate food consumption, we searched the
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GENSAT transgenic mouse collection for a mouse line that permitted genetic access to CeA
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neurons mutually exclusive of the anorexigenic population marked by expression of protein kinase
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C-δ (PKCδ)
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. Of the top candidates, the BAC transgenic line Htr2a-Cre (KM208) was found to
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be highly expressed within the CeA. Our characterization of the line by breeding Htr2a-Cre mice
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to a LacZ reporter line confimed the faithful representation of Htr2a+ neurons in the CeA
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(Supplementary Fig. 1a, b) and revealed no overlap with CeA
PKCδ
neurons (Fig. 1a, b), but partial
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overlap with other CeA genetic markers (Supplementary Fig. 1c-f). Physiologically, CeA
Htr2a
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neurons were found to be homogeneous and exhibited late firing properties (Fig. 1c, d). Given that
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CeA
Htr2a
and CeA
PKCδ
neurons were mutually exclusive populations, we determined whether
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CeA
Htr2a
neurons promoted food intake. First, we virally targeted CeA
Htr2a
neurons in Htr2a-Cre
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mice with Cre-dependent stimulatory hM3Dq designer receptors exclusively activated by designer
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drugs (DREADDs)
19,20
and performed a free-feeding assay (Fig. 1e, f and Supplementary Fig. 2a-
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d). Acute activation of CeA
Htr2a
neurons by intraperitoneal injection of the DREADD ligand
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clozapine-N-oxide (CNO) in satiated mice increased food intake compared to controls by
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increasing the total time that the animals spent feeding (Fig. 1g and Supplementary Fig. 2e-h). The
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animals preferred food over clay pellets of similar size and hardness to the food (Fig. 2h and
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Supplementary Fig. 2i-k), indicating that activation of CeA
Htr2a
neurons specifically led to food
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intake rather than ill-directed consummatory behavior.
77
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 2, 2017. ; https://doi.org/10.1101/145375doi: bioRxiv preprint

5
We next asked whether activation of CeA
Htr2a
neurons could increase consumption under
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anorexigenic conditions where motivation to find and consume food is low
21-24
. Indeed,
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chemogenetic activation of CeA
Htr2a
neurons in fasted mice decreased the appetite suppressant
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effects of lithium chloride (LiCl) and lipopolysaccharide (LPS) that mimic toxic foods and
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bacterial infections respectively
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(Fig. 1i). Activation of CeA
Htr2a
neurons also rescued the effect
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of quinine-spiked bitter food which normally reduces food intake
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, without affecting the
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sensitivity of the mice to bitter tastants (Supplementary Fig. 2l-m). Together, these data
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demonstrate that CeA
Htr2a
neurons promote food consumption even in the absence of physiological
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need and under conditions when the motivation to consume food is low.
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We further examined whether activation of CeA
Htr2a
neurons would also increase the effort made
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to obtain food by assessing the behavior of CeA
Htr2a
::hM3Dq mice in a food-seeking progressive-
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ratio task. Here, active nose-pokes were rewarded with a food pellet on a progressive-ratio 2 (PR2)
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schedule. We compared the performance of CeA
Htr2a
::hM3Dq mice in two consective sessions
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where either CNO or saline was injected prior to the experiment. CNO-treated CeA
Htr2a
::hM3Dq
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did not show a difference in the number of active nose-pokes or the number of consecutive nose-
92
pokes made to obtain a single pellet (breakpoint) compared to their performance after saline
93
treatment (Fig. 2j and Supplementary Fig. 2o). Thus, activation of CeA
Htr2a
neurons evokes
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increased consumption without affecting the motivation to work for food.
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We additionally found that anxiety-like and locomotor behaviors of CeA
Htr2a
::hM3Dq mice were
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not significantly different from controls (Supplementary Fig. 2p-s), suggesting the altered
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consummatory behavior is unlikely to result from altered locomotion.
98
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 2, 2017. ; https://doi.org/10.1101/145375doi: bioRxiv preprint