A neuropeptide-specific signaling pathway for state-dependent regulation of the mesolimbic dopamine system

  1. Mollie X. Bernstein
  2. Omar Koita
  3. Marta Trzeciak
  4. Andrew Fan
  5. Daniel T. McAuley
  6. Seung-Woo Jin
  7. Larry S. Zweifel

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Abstract

Dopamine (DA)-producing neurons of the ventral tegmental area (VTA) regulate consummatory behavior in a state-dependent manner (e.g. when hungry or thirsty). The mechanisms by which and extent to which DA neurons are regulated by these interoceptive signals are poorly understood. Here, we identify transient receptor potential canonical type 6 (TRPC6) channels as selective mediators of neuropeptide receptor-induced calcium signaling in VTA-DA neurons. These channels regulate DA neuron activity and consummatory behavior in a state-dependent manner. We find that TRPC6 channels regulate distinct aspects of neuropeptide-induced calcium signals in DA neurons but make little contribution to calcium dynamics associated with metabotropic neurotransmitter receptor signaling. We further show that TRPC6 channels regulate scalable reward valuation and consummatory behavior in hungry but not thirsty mice. These findings demonstrate that neuropeptide-and neurotransmitter-activated G-protein coupled receptors (GPCRs) regulate cellular calcium dynamics through distinct mechanisms, and that TRPC6 channels are important determinants of how animals respond to different homeostatic demands.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/18006667.

    Bernstein et al. investigate the role of neuropeptide signaling in ventral tegmental dopamine neurons (VTADA), a population implicated in both natural and drug rewards. Using CRISPR-Cas9 mutagenesis in mice, they knock down transient receptor potential canonical type 6 (TRPC6) channels, which are highly expressed in these neurons and are downstream of several neuropeptides. While TRPC6-knockdown had no effect on VTADA neurons' intrinsic properties, it reduced the likelihood and magnitude of neural responses to neuropeptide receptor activation. Behaviorally, TRPC6-mutant mice exhibited altered licking for sucrose after food restriction. This effect was mirrored by in-vivo calcium imaging, where food restricted (FR) TRPC6 mutants' neural activity did not scale with sucrose concentration like in control mice or water restricted (WR) TRPC6 mutants.

    These results provide important mechanistic insight into VTADA neuron function and address the fundamental question of how neuropeptides and neurotransmitters can affect neural activity in parallel. The data presented supports the authors' conclusion that TRPC6 channels are functionally downstream of neuropeptides and that they shape VTADA calcium dynamics. However, the behavioral phenotype is subtle and state-specificity for hunger versus thirst is only somewhat supported by data from the head-fixed multi-sucrose task.

    Strengths:

    The authors used a wide range of techniques -CRISPR, slice calcium imaging, whole cell patch clamp electrophysiology, and in-vivo calcium imaging- to interrogate the function of TRPC6 channels, a largely unexplored target involved in motivated behaviors.

    Weaknesses:

    The authors' distinction between hunger and thirst is complicated by the use of sucrose solutions in behavioral assays, which makes it hard to disentangle the roles of internal state, caloric content, palatability, hydration, and expectation of future sucrose rewards. The argument for state-specificity would be strengthened by single-cell imaging and a more comprehensive characterization of TRPC6-knockdown mice in various consummatory behaviors.

    Major points:

    1.      In-vivo single-cell imaging would help find a more interpretable change in neural activity in the mutant, especially because figure 2D suggests a reduction of oscillating cells, which would be masked by bulk fiber photometry.

    2.      While using the same sucrose task is valuable because it allows comparison of lick rates across FR and WR, additional behavior experiments would greatly aid in clarifying the results in figures 4 and 5:             

    2a.      Figure 4D shows that TRPC6-KD mice lick equally for 20% and 30% sucrose - is this because they cannot distinguish them? Or because they don't have a preference? Are they less motivated to consume overall? The authors might do a choice assay to clarify these questions, such as a 30-minute two-bottle (20% vs 30% sucrose) preference test in TRPC6-KD vs wild-type hungry mice.

    2b.      In figures 4 and 5, the authors generally see smaller effects under WR compared to FR. This may be due to the expectation of future sucrose in this task or a documented food seeking bias in thirsty mice (Eiselt et al., 2021, PMID: 33972802). To confirm that TRPC6 effects are specific for hunger, it would be helpful to include an experiment with only water in thirsty mice. Another factor is the degree of deprivation – it would be helpful to assess TRPC6-mutants after more severe (e.g. 48h) water deprivation.

    2c.      Figures 4 and 5: To demonstrate that their findings are related to homeostatic state and not just palatability, the authors could use a less palatable caloric food like gelled food, diluted ensure, or intralipid in their multi-spout assays. The reference above also includes a recipe for gelled food which is compatible with head fixed feeding (PMID: 33972802, see Methods: Gelled hydrated food).

    Minor points:

    -          For all figures, please consider including the label "n.s." when there is no significant difference for clarity.

    -          Figure 2D (and corresponding main text): including the % of each cell type would make the pie charts easier to interpret.

    -          One optional experiment to strengthen the neuropeptide hypothesis would be repeating the slice experiments in tissue from hungry and thirsty mice to see how deprivation affects intrinsic and evoked activity ex-vivo.

    -          Figure 3A, 4A: On the schematic, consider clarifying that the viruses used were cre-dependent.

    -          Figure 4: It is hard to see increased licking for water in thirsty mice, especially for the TRPC6 group. To show this, the authors could move supplemental figures 2A and B to figure 4 or include a statistical comparison of water licks in WR vs FR.

    -          Figure 5G: In the main text, the authors mislabel increased responses to water and low sucrose under WR as decreases.

    -          For the RPE-like signal (Figure 5G), including a brief discussion of what is known about this type of inhibitory dynamics in the VTA would provide helpful context.

    -          Figures 4C,F and 5A,D: Since data are presented as averages across all trials, it might be interesting to include a comparison of the first 10 trials and the last 10 trials of each session in the supplementary data. This could address whether reduced homeostatic need across trials affects the VTA and behavior.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The authors declare that they did not use generative AI to come up with new ideas for their review.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/17992300.

    Review for "A neuropeptide-specific signaling pathway for state-dependent regulation of the mesolimbic dopamine system"

    Yue Yu, Theresa Haunold

    Preprint doi: https://doi.org/10.1101/2025.07.17.665396

    Summary:

    In this preprint, the authors use mouse models to investigate the mechanisms and extent to which dopamine (DA) neurons in the ventral tegmental area (VTA) are regulated by interoceptive signals associated with internal states such as hunger and thirst, and how these signals shape consummatory behavior. Using CRISPR/SaCas9-mediated mutagenesis and ex vivo calcium imaging, the authors demonstrate that transient receptor potential canonical type 6 (TRPC6) channels selectively regulate neuropeptide receptor-induced calcium signaling in VTA-DA neurons, while exerting minimal influence on calcium responses driven by neurotransmitter receptors. Whole-cell patch-clamp recordings indicate that Trpc6 mutagenesis does not alter the intrinsic excitability of VTA-DA neurons. Finally, in vivo calcium imaging during a multi-sucrose consumption task in head-restrained mice shows that TRPC6 channels modulate scalable reward evaluation and consummatory behavior in hungry, but not thirsty, mice.

    Strengths:

    By combining CRISPR/SaCas9-mediated mutagenesis with ex vivo calcium imaging and whole-cell patch-clamp recordings, this preprint provides a robust experimental framework to investigate how hunger- and thirst-related neuropeptides modulate VTA-DA neuron activity and to assess their intrinsic excitability. In vivo calcium imaging during a multi-sucrose consumption task further links neuronal activity to real-time consummatory behavior.

    Weakness:

    The reliance on bulk fiber photometry limits insight into how individual VTA-DA neurons encode hunger- versus thirst-related behaviors. In addition, the behavioral design using sucrose at varying concentrations may complicate interpretation of internal-state specificity. Finally, the in vivo calcium signals associated with altered reward scaling in sgTrpc6 mice appear modest, raising the question of whether TRPC6 loss produces a sufficiently robust change in DA neuron activity to account for the corresponding behavioral phenotype.

    Major comments:

    1. In Figure 4 and 5, the conclusions regarding TRPC6-dependent modulation of state-specific reward processing would be substantially strengthened by single-cell calcium imaging during water and food consumption under thirst and hunger. Because the current in vivo data rely on bulk fiber photometry, it remains unclear how individual VTA-DA neurons encode these behaviors and whether TRPC6 loss affects specific neuronal subpopulations that drive the observed phenotype.

    2. In Figure 4C-E and Figure 5A-C, both groups exhibited comparable licking behavior and calcium responses across all five solutions, leading the authors to conclude that TRPC channels are dispensable for reward valuation and consummatory behavior in thirsty mice. However, varying sucrose concentrations may confound the evaluation of water's reward value under thirst. To strengthen this conclusion, the authors could consider assessing licking behavior and performing fiber photometry using water alone in both groups.

    3. In Figure 4 and Figure 5, the in vivo calcium signals associated with altered reward scaling in sgTrpc6 mice appear modest, with significant differences emerging only between the 20% and 30% sucrose conditions. This raises questions about whether TRPC6 loss produces a functionally meaningful change in DA neuron activity sufficient to account for the behavioral phenotype. To strengthen the interpretation, it would be helpful to assess neural responses to additional, compositionally distinct food stimuli, such as lab chow, comparing Ensure versus diluted Ensure, or Ensure versus quinine, as shown in prior work (doi.org/10.1016/j.cell.2020.07.031).

    4. In Figure 4F-H and Figure 5D-F, the sgTrpc6 group exhibited impaired assessment of reward value and scalable liking behavior for 20% and 30% sucrose in hungry mice. To further verify this deficit, a two-bottle preference assay directly comparing 20% and 30% sucrose could determine whether hungry mice in both groups can properly discriminate between these concentrations.

    Minor comments:

    1.        In Figure 2A, the authors note no significant difference in the proportion of responsive versus nonresponsive cells. A brief discussion or potential explanation would help contextualize this finding.

    2.        In Figure 2D and corresponding text, reporting the percentage of each neuron type observed in control and Trpc6 knockdown conditions would aid interpretability.

    3.        On page 7, in the text accompanying Figure 2F, please consider noting that VTA-DA neurons in the sgTrpc6 group showed a significant reduction in the proportion of responsive versus nonresponsive cells at 1uM NTS, and update the text as needed to improve reader understanding.

    4.        The authors might consider moving Figure 3A to the first figure, as it provides a helpful schematic of the viral injection strategy used throughout the paper.

    5.        To aid interpretation, please consider indicating "NS" for all comparisons that do not reach statistical significance.

    6.        On page 6, confirm the reference to Fig. 1A should be corrected to Fig. 2A and update as needed to improve reader understanding.

    Competing interests

    The authors declare that they have no competing interests.

    Use of Artificial Intelligence (AI)

    The authors declare that they did not use generative AI to come up with new ideas for their review.

  3. Version published to 10.1101/2025.07.17.665396 on bioRxiv