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

Bernstein et al. investigate the role of neuropeptide signaling in ventral tegmental dopamine neurons (VTA_DA), 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 VTA_DA 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 VTA_DA 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 VTA_DA 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.