Metabolic sensing in AgRP neurons integrates homeostatic state with dopamine signalling in the striatum

Article activity feed

1. 

   Version published to 10.7554/elife.72668 on eLife

   Jan 12, 2022
2. 

   eLife

   Dec 20, 2021

   Author Response:

   Reviewer #1 (Public Review):

   The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release. The authors use of contemporary methods to monitor the kinetics of of AgRP neuron activity (fiber photometry) and dopamine release (GRAB-DA) in response to feeding fed or …

   Author Response:

   Reviewer #1 (Public Review):

   The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release. The authors use of contemporary methods to monitor the kinetics of of AgRP neuron activity (fiber photometry) and dopamine release (GRAB-DA) in response to feeding fed or fasted mice with PB chips is commendable. The authors acknowledge that the neural circuits linking changes in AgRP neurons activity to release of dopamine is indirect because AgRP neurons do not directly synapse onto dopamine neurons; thus, their findings provide intriguing correlations without a clear understanding of the circuit(s) involved. The authors clearly demonstrate that the Crat knockout (KO) mice do not respond to PB chips the same was as WT mice; the KO mice respond to the first chip normally but responses to additional chips are blunted. The mechanisms underlying the blunted response are assumed to be due to a failure of metabolic sensing, but the mechanisms involved are not explored.

   1.1 We would like to thank the reviewer for reviewing our manuscript. Our studies use AgRP crat deletion as a model of impaired metabolic sensing to examine how metabolic sensing in AgRP neurons controls dopamine signaling, as this question has not been previously addressed. Our previous proteomic analysis of AgRP neurons in WT and KO mice highlighted numerous differences in metabolic pathways, mitochondrial function and synaptic control mechanisms (1). Although defining the mechanisms responsible for impaired metabolic sensing will be important for future studies, this does not affect the conclusions in this current manuscript – that metabolic sensing in AgRP neurons is required for normal dopamine signaling in the nucleus accumbens and dorsal striatum in response to caloric foods. Similar studies have employed genetic approaches to modify insulin signaling in AgRP neurons by deleting phosphatases regulating insulin signaling, not to define the mechanism of phosphatase function, but to assess the impact of altered insulin signaling in AgRP neurons (2).

   Reviewer #2 (Public Review):

   In their manuscript, Reichenbach et al perform experiments to demonstrate that knocking the metabolic enzyme carnitine acetyltransferase (Crat) out of hunger-promoting AgRP neurons impairs an animal's ability to accurately sense its nutritional state and thus decreases motivation to work for food and attenuates neural response to palatable food rewards. They accomplish this using in vitro and in vivo neural recording techniques, and thoughtful behavioral approaches. Specifically they show 1) impaired responses of AgRP neurons to glucose (in vitro) and sensory cues predicting food (in vivo), 2) impaired striatal dopamine release in response to palatable food presentation, and 3) decreased motivation to work for palatable rewards.

   Their work largely substantiates their conclusions. Their experiments are well described, and the phenotypes observed are generally clear. The data partially explain prior behavioral studies performed on these conditional knockout mice. Moreover, their data are consistent with and a valuable addition to previously published data showing how dorsal and ventral striatum differentially respond to nutrient intake with ventral striatum dopamine release increasing in response to sweet taste and dorsal striatum dopamine increasing in response to rewarding post-ingestive effects of nutrients. This study will be of interest to a fairly broad community of feeding, hypothalamus, and dopamine researchers.

   A limitation of this study is that it does not adequately address the possibility that decreased AgRP neuron responses to food presentation may be related to altered in vivo baseline activity or attenuated fasting-induced hyperactivity of these neurons.

   2.1 In these current studies, we have shown under fasted conditions that WT AgRP and KO AgRP neurons have similar electrophysiological properties ex vivo (Fig 1). For example, resting membrane potential, input resistance and spontaneous firing frequency are not different between WT and KO AgRP neurons. We also see that ghrelin-induced food intake and AgRP activity, as measured by population calcium activity, does not differ between genotypes, further supporting the idea that AgRP neurons respond normally to pharmacologic challenges. Finally, we have now added new data that shows impaired in vivo AgRP activity in response ip glucose (Figure 1-figure supplement 1J-M), which supports altered glucose responsiveness with ex vivo slice recordings (Fig 1). Based on this in vivo and ex vivo evidence we assume baseline activity is not different in vivo but rather responses to glucose are impaired.

   While their slice studies show mostly normal ex vivo electrophysiologic properties of these neurons, in other models ex vivo and in vivo measurements of AgRP neuron activity are not directly correlated. Specifically Kristen O'Connell's group has shown increased baseline AgRP neuron activity in diet-induced obese (DIO) mice that is not further increased by fasting in slice (Baver et al, 2014).

   2.2 We appreciate the reviewers comment and knowledge of the literature but respectfully point out that our studies have been conducted on mice fed a chow diet.

   By contrast, Michael Krashes's group recently shown that DIO mice have reduced baseline AgRP neuron activity using a fiber photometry approach in vivo (Mazzone et al, 2020). Of note, decreased baseline or fasting-induced AgRP neuron activity would not necessarily diminish the impact of the rest of the results presented. Moreover, it is not necessarily a question that must be answered by this study, but it should be acknowledged as a possibility that is important to test.

   2.3 We thank the reviewer for these comments but we would also like to point out that the paper by Mazzone (2020) (7) does not show a difference in baseline AgRP neural activity with photometry in HFD vs chow fed animals but rather that HFD feeding affects the change in activity to food presentation (either chow or HFD), a similar effect was recently reported by Beutler and colleagues (8).

   This is also a good opportunity to emphasise that Mazzone and colleagues use the same approach to quantify changes in population calcium changes as in our study. In their study, they used df/f (%) in which the change df/f after food presentation is compared to a baseline df/f period, hence the designation df/f (%). This normalised approach is also regularly used by Knight and colleagues, the pioneers in the use of GCaMP to measure population activity of AgRP neurons (8-10).

   This is the same approach as we have used, except that we call this a z-score, which is the statistical convention for this normalisation approach. Moreover, this normalisation approach is commonly used for statistical analysis with fibre photometry and miniscope approaches to measure calcium dynamics as an index of neural activity (4,5,7-9,11-19). Normalisation is essential since df/f depends on numerous factors including 1) GCaMP expression, 2) illumination wavelength, 3) light intensity, 4) cell type, 5) quality of surgery (ie gliosis), 6) position of fibre optic implant and because normalisation is required for in vivo calcium imaging, all studies are likely subjected to a similar experimental limitation of potential differences baseline cell firing frequency. For these reasons highlighted above, calcium imaging is not typically used to estimate baseline differences in activity, rather it is most useful to examine neural responses to different stimuli with the magnitude of change in neural activity to a given stimulus encoding meaningful information. This is why df/f (%) or z-score normalisation is important and standard across most studies (4,5,7-9,11-19).

   Additional minor concerns do not significantly dampen my generally positive opinion of the study. These include: 1) the lack of feeding data associated with AgRP neuron fiber photometry responses,

   2.4 We designed these experiments so that mice were given single PB pellets, weighing approximately 70 mg, and all was consumed during exposure – therefore all mice ate the approximate amount. We have now described this in the methods.

   “Peanut butter chips were measured to ~70mg per pellet and one pellet was given per trial. Mice consumed all of this peanut butter chip during each trial such that no differences in consumption were observed.”

   We have also added the additional data. During each trial the time to peanut butter consumption was not different between genotypes (new data - Figure 1-figure supplement 1G,H,I)

   1. analysis of operant GRAB-DA data by pellet retrieval event rather than by mouse, and

   2.5 We also recognised this as a weakness and have now repeated these experiments using multiple additional animals. All operant GRAB-DA data in the accumbens and dorsal striatum is now presented as the averaged nose poke or pellet responses recorded for each mouse. Data points in Fig 4 and Fig 6 now reflect analysis by mouse.

   We have also updated this in the methods section “For DA photometry during PR session, data are presented as the averaged dopamine response for each animal. On average mice collected ~3.5 pellets during the PR in ad libitum-fed conditions and ~6 pellets when fasted (Fig 4O).”

   1. incompletely described inclusion criteria for mice in photometry studies.

   2.6 – For AgRP photometry studies, only mice with a maximal z-score response to IP ghrelin greater than 4 were included for analysis, ensuring differences in AgRP neural activity to food cues were not related to differences in GCaMP expression or illumination rates.

   We have added the following text to the manuscript. “For AgRP GCaMP6 photometry studies, an increase in activity to ghrelin response was used as an index of correct viral expression and fibre optic placement. Only mice with a maximal peak z-score of >4 were included for analysis in experimental group, using this criterion 5/20 mice, across both WT and KO mice, were excluded for experimentation (Figure 1-figure supplement 1N-O)”.

   In addition, increases in dopamine release before contact with PB in the NAc or dorsal striatum (Fig 2L-P; Fig 5L,M) and before pellet retrieval (in Fig 4K-N; Fig 6 J, K) is similar between genotypes, suggesting equivalent capacity to increase dopamine release under stimuli not affected by AgRP input. Thus, genotype difference in response to palatable food or sucrose pellets could not be due to differences in GRAB-DA expression in the NAc. Moreover, a postmortem analysis was conducted to identify the localization of GFP expression (Figure 7).

   We have added the following text. “GRAB-DA responses in WT and KO mice were similar on approach to PB and prior to pellet retrieval in both the NAc and dorsal striatum showing that genotype differences in response to palatable food or sucrose pellets could not be due to differences in GRAB-DA expression in the NAc. Moreover, a post mortem analysis was conducted to identify the localization of GFP expression (Figure 7)”.

   References

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   2. Dodd GT, Kim SJ, Mequinion M, Xirouchaki CE, Bruning JC, Andrews ZB, Tiganis T. Insulin signaling in AgRP neurons regulates meal size to limit glucose excursions and insulin resistance. Science advances. 2021;7(9).
   3. Goldstein N, McKnight AD, Carty JRE, Arnold M, Betley JN, Alhadeff AL. Hypothalamic detection of macronutrients via multiple gut-brain pathways. Cell Metabolism. 2021;33:1-12.
   4. Garfield AS, Shah BP, Burgess CR, Li MM, Li C, Steger JS, Madara JC, Campbell JN, Kroeger D, Scammell TE, Tannous BA, Myers MG, Andermann ML, Krashes MJ, Lowell BB. Dynamic GABAergic afferent modulation of AgRP neurons. Nature Neuroscience. 2016;19(12):1628-1635.
   5. Berrios J, Li C, Madara JC, Garfield AS, Steger JS, Krashes MJ, Lowell BB. Food cue regulation of AGRP hunger neurons guides learning. Nature. 2021;595(7869):695-700.
   6. Cavalcanti-de-Albuquerque JP, de-Souza-Ferreira E, de Carvalho DP, Galina A. Coupling of GABA Metabolism to Mitochondrial Glucose Phosphorylation. Neurochem Res. 2021.
   7. Mazzone CM, Liang-Guallpa J, Li C, Wolcott NS, Boone MH, Southern M, Kobzar NP, Salgado ID, Reddy DM, Sun FM, Zhang YJ, Li YL, Cui GH, Krashes MJ. High-fat food biases hypothalamic and mesolimbic expression of consummatory drives. Nature Neuroscience. 2020;23(10):1253-+.
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   13. Daviu N, Fuzesi T, Rosenegger DG, Rasiah NP, Sterley TL, Peringod G, Bains JS. Paraventricular nucleus CRH neurons encode stress controllability and regulate defensive behavior selection. Nat Neurosci. 2020;23(3):398-410.
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   16. Livneh Y, Ramesh RN, Burgess CR, Levandowski KM, Madara JC, Fenselau H, Goldey GJ, Diaz VE, Jikomes N, Resch JM, Lowell BB, Andermann ML. Homeostatic circuits selectively gate food cue responses in insular cortex. Nature. 2017;546(7660):611-+.
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   Read the original source
3. 

   eLife

   Sep 9, 2021

   Evaluation Summary:

   The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release.

   Read the original source
4. 

   eLife

   Sep 9, 2021

   Reviewer #1 (Public Review):

   The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release. The authors use of contemporary methods to monitor the kinetics of of AgRP neuron activity (fiber photometry) and dopamine release (GRAB-DA) in response to feeding fed or fasted mice with PB chips …

   Reviewer #1 (Public Review):

   The authors demonstrate that selective inactivation of carnitine acetyltransferase (Crat) – a key metabolic enzyme – in AgRP neurons attenuates the response of AgRP neurons to peanut butter (PB) chips, the release of dopamine in the nucleus accumbens, and the motivation to work for food when mice are fasted. The strength of this study is the demonstration that metabolic sensing by AgRP neurons is somehow linked to dopamine release in the nucleus accumbens, but a weakness is that it is unclear how the lack of Crat in AgRP neurons affects their responsiveness to PB chips or how AgRP neurons regulate dopamine release. The authors use of contemporary methods to monitor the kinetics of of AgRP neuron activity (fiber photometry) and dopamine release (GRAB-DA) in response to feeding fed or fasted mice with PB chips is commendable. The authors acknowledge that the neural circuits linking changes in AgRP neurons activity to release of dopamine is indirect because AgRP neurons do not directly synapse onto dopamine neurons; thus, their findings provide intriguing correlations without a clear understanding of the circuit(s) involved. The authors clearly demonstrate that the Crat knockout (KO) mice do not respond to PB chips the same was as WT mice; the KO mice respond to the first chip normally but responses to additional chips are blunted. The mechanisms underlying the blunted response are assumed to be due to a failure of metabolic sensing, but the mechanisms involved are not explored.

   Read the original source
5. 

   eLife

   Sep 9, 2021

   Reviewer #2 (Public Review):

   In their manuscript, Reichenbach et al perform experiments to demonstrate that knocking the metabolic enzyme carnitine acetyltransferase (Crat) out of hunger-promoting AgRP neurons impairs an animal's ability to accurately sense its nutritional state and thus decreases motivation to work for food and attenuates neural response to palatable food rewards. They accomplish this using in vitro and in vivo neural recording techniques, and thoughtful behavioral approaches. Specifically they show 1) impaired responses of AgRP neurons to glucose (in vitro) and sensory cues predicting food (in vivo), 2) impaired striatal dopamine release in response to palatable food presentation, and 3) decreased motivation to work for palatable rewards.

   Their work largely substantiates their conclusions. Their experiments are well …

   Reviewer #2 (Public Review):

   In their manuscript, Reichenbach et al perform experiments to demonstrate that knocking the metabolic enzyme carnitine acetyltransferase (Crat) out of hunger-promoting AgRP neurons impairs an animal's ability to accurately sense its nutritional state and thus decreases motivation to work for food and attenuates neural response to palatable food rewards. They accomplish this using in vitro and in vivo neural recording techniques, and thoughtful behavioral approaches. Specifically they show 1) impaired responses of AgRP neurons to glucose (in vitro) and sensory cues predicting food (in vivo), 2) impaired striatal dopamine release in response to palatable food presentation, and 3) decreased motivation to work for palatable rewards.

   Their work largely substantiates their conclusions. Their experiments are well described, and the phenotypes observed are generally clear. The data partially explain prior behavioral studies performed on these conditional knockout mice. Moreover, their data are consistent with and a valuable addition to previously published data showing how dorsal and ventral striatum differentially respond to nutrient intake with ventral striatum dopamine release increasing in response to sweet taste and dorsal striatum dopamine increasing in response to rewarding post-ingestive effects of nutrients. This study will be of interest to a fairly broad community of feeding, hypothalamus, and dopamine researchers.

   A limitation of this study is that it does not adequately address the possibility that decreased AgRP neuron responses to food presentation may be related to altered in vivo baseline activity or attenuated fasting-induced hyperactivity of these neurons. While their slice studies show mostly normal ex vivo electrophysiologic properties of these neurons, in other models ex vivo and in vivo measurements of AgRP neuron activity are not directly correlated. Specifically Kristen O'Connell's group has shown increased baseline AgRP neuron activity in diet-induced obese (DIO) mice that is not further increased by fasting in slice (Baver et al, 2014). By contrast, Michael Krashes's group recently shown that DIO mice have reduced baseline AgRP neuron activity using a fiber photometry approach in vivo (Mazzone et al, 2020). Of note, decreased baseline or fasting-induced AgRP neuron activity would not necessarily diminish the impact of the rest of the results presented. Moreover, it is not necessarily a question that must be answered by this study, but it should be acknowledged as a possibility that is important to test.

   Additional minor concerns do not significantly dampen my generally positive opinion of the study. These include: 1) the lack of feeding data associated with AgRP neuron fiber photometry responses, 2) analysis of operant GRAB-DA data by pellet retrieval event rather than by mouse, and 3) incompletely described inclusion criteria for mice in photometry studies.

   Read the original source
6. 

   eLife

   Sep 9, 2021

   Reviewer #3 (Public Review):

   Reichenbach et al tested the hypothesis whether metabolic sensing in AgRP neurons is required to increase food reward motivation by influencing dopamine release in the striatum. As a model for disrupted metabolic sensing they employed their previous described mouse model that specifically lacks carnitine acetyltransferase (Crat) in AgRP neurons. They confirm using electrophysiology the appropriateness of the model and then conduct a series of elegant experiments measuring short-term dopamine release by fibre optics in response to different feeding manipulations in the nucleus accumbens and striatum. In a final experiment they then also test for longer term responses to Dopamine release utilising 18F-fDOPA in combination with positron electron tomography. Their data reveals that reduced metabolic sensing in …

   Reviewer #3 (Public Review):

   Reichenbach et al tested the hypothesis whether metabolic sensing in AgRP neurons is required to increase food reward motivation by influencing dopamine release in the striatum. As a model for disrupted metabolic sensing they employed their previous described mouse model that specifically lacks carnitine acetyltransferase (Crat) in AgRP neurons. They confirm using electrophysiology the appropriateness of the model and then conduct a series of elegant experiments measuring short-term dopamine release by fibre optics in response to different feeding manipulations in the nucleus accumbens and striatum. In a final experiment they then also test for longer term responses to Dopamine release utilising 18F-fDOPA in combination with positron electron tomography. Their data reveals that reduced metabolic sensing in the knockout mouse model reduces acute dopamine release in the NAc but not in the dorsal striatum, while in the longer time frame (30 min) the dorsal striatum is also affected. In summary the authors conclude that metabolic sensing in AgRP neurons is necessary to integrate homeostatic regulatory processes in the AgRP neurons with hedonic aspects of dopamine signalling in reward pathways. Taken together, the experiments are well-executed and the results justify the main conclusions of the study.

   Read the original source
7. 

   Version published to 10.1101/2021.03.22.436393v2 on bioRxiv

   Jul 22, 2021
8. 

   Version published to 10.1101/2021.03.22.436393v1 on bioRxiv

   Mar 23, 2021