Striatal Crosstalk Between Dopamine and Serotonin Systems

Reviewer #1 (Public review):

Summary:

In this study, Liu et al use optogenetics and genetically encoded neuromodulator sensors to test the extent to which dopamine neuron stimulation produces striatal serotonin release, and vice versa. The study is timely given growing interest in dopamine/serotonin interactions and in the context of recent work showing bidirectional and dynamic regulation of striatal dopamine by another neuromodulator, acetylcholine. The authors find that striatal dopamine and serotonin afferents function largely independently, with dopamine neuron stimulation producing no striatal serotonin release and serotonin neuron stimulation producing minimal striatal dopamine release. This work will inform future work seeking to dissect the contributions of striatal dopamine, serotonin, and their interactions to various motivated behaviors. While the paper's main conclusions are adequately supported (see Strengths), additional controls and experiments would significantly broaden the paper's impact (see Weaknesses). Finally, this draft of the work is poorly presented with numerous errors, omissions, and inconsistencies evident throughout the text and the figures that should be addressed.

Strengths:

The study employs optogenetic stimulation simultaneously with fiber photometry recording of dopamine or serotonin release measured with genetically encoded sensors. These methods are state-of-the-art, offering tighter temporal control compared to pharmacological methods for manipulating dopamine and serotonin and improved selectivity over techniques like electrochemistry and microdialysis used to record neuromodulator release in previous studies on the subject. As a result, the paper's main conclusions are well supported.

Weaknesses:

(1) The electrophysiology experiments in Figure 3 are only tangentially related to the focus of the study, and their findings are almost entirely irrelevant to the paper's main conclusions. The results of these experiments are also not novel. Glutamate corelease from 5HT neurons has been previously shown, including in the OFC and VTA (Ren et al, 2018, Cell, McDevitt et al, 2014, Cell Rep, Liu et al 2014, Neuron; and others). The authors should explain more clearly what they think these data add to the manuscript and/or consider removing them altogether.

(2) Related to the point above, as far as I can tell, the only value the electrophysiology data add is to suggest that perhaps activation of serotonin neurons may drive minimal striatal dopamine release via glutamate corelease in the VTA. The evidence provided in this version of the manuscript is insufficient to support that claim, but the manuscript would be significantly strengthened if the authors tested this hypothesis more directly. One way to do that could be to stimulate serotonin axons in the striatum (as opposed to the serotonin cell bodies) and record striatal dopamine release. A complementary anatomical approach would be to use retrograde tracing to test whether the DR 5HT neurons projecting to the striatum are the same or different from the VTA projecting population.

(3) The findings would be strengthened by the addition of a fluorophore-only control group lacking opsin expression in all experiments in Figures 1 and 2.

(4) The experiment of stimulating serotonin neurons and recording serotonin release in the NAc was not performed. It would be useful to be able to compare the magnitudes of evoked serotonin release in these two striatal regions, though it is not central to the main claims of the paper.

(5) The interpretation of the results from Figure 2 is described inconsistently throughout the manuscript. The title implies there is significant crosstalk between the dopamine and serotonin systems. The abstract calls the crosstalk "transient", which is a description of its temporal dynamics, not its magnitude. Then the introduction figures and discussion all suggest the crosstalk is minimal. I suggest the authors describe the main findings - minimal crosstalk between the dopamine and serotonin systems - clearly and consistently in the title, abstract, and main text.

Reviewer #2 (Public review):

Summary:

This brief communication aims to clarify interactions between the dopamine (DA) and serotonin (5HT) systems of mice. The authors use optogenetic stimulation of DA neurons in the VTA or of 5HT neurons in the DRN, while monitoring the fluorescence of DA and 5HT sensors in the nucleus accumbens (NAc) and dorsal striatum (DS) using fiber photometry. The authors report on a small release of DA in the NAc following DRN stimulation, which they attribute to glutamate co-release onto VTA DA neurons using slice electrophysiology. The authors also report on cocaine-induced 5-HT release in the striatum.

Strengths:

This is a topic well worth studying.

Weaknesses:

In its current form, this is an incomplete and underpowered study that does little to clarify the complicated relationship that exists between DA and 5HT in the mammalian brain under physiological conditions or during cocaine use.

Reviewer #3 (Public review):

The authors suggest that the small release of DA may be due to a release of glutamate from DRN 5-HT neurons to the VTA that stimulates weakly and in a transient fashion the VTA DA neurons, which in the end, produce a transient and small release of DA in the NAc.

Their findings give more information on the previously reported complex and partial known crosstalk between 5-HT and DA in the NAc.

I only have some minor concerns about the manuscript:

(1) In Figure 2F, there is a missing curve for 5-HT in NAc. Besides, the legend shows n=2, making it difficult to perform statistical analysis with that data.

(2) In Figure 3, the use of NBQX/AP5 is shown, but it is not mentioned either in the methodology or in the discussion. What is the meaning of those results?

(3) Line 98 compares results from two different places of stimulation. The results are related to stimulation in the VTA, but the comparison indicates that the stimulation was made in the DRN.

(4) If the release of 5-HT in Nac does not occur, it needs to be precise in the abstract that 5-HT is released in the dorsal striatum (DS) but not in the NAc (line 19).

(5) Be consistent with the way you mention the 5-HT neurons. For example, in lines from 106 to 119, SERT neurons are used. Previously, 5-HT neurons were used.

(6) There are several points of confusion when referring to the figures, making the text difficult to follow because the text explains something that is not shown in the figure cited.

Author response:

We appreciate the reviewers’ insightful feedback and propose to undertake an extensive revision of the manuscript to strengthen our findings and underscore the significance of this work. We remain convinced that our study offers critical insights into the largely independent dopamine and serotonin neural circuits. Nevertheless, we concur that substantial revisions are warranted, as the current organization may not be ideal to showcase the central findings. In particular, we will increase the number of animals to address data variability and enhance the reproducibility of the observed effects. We also recognize the need to perform additional control experiments and to include complementary anatomical tracing studies. Moreover, we will reformat the manuscript and conduct additional analyses to emphasize that evoked dopamine and serotonin release originate from distinct loci with minimal crosstalk. To address all of these points thoroughly, we estimate that a 12-month revision period will be required.