Dopamine and serotonin inversely modulate D2 medium spiny neurons to regulate cocaine reward

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
Classic theories propose opponent functions for striatal dopamine (DA) and serotonin (5-hydroxytryptamine; 5HT), with DA promoting approach and 5HT promoting patience or avoidance. How these neuromodulators regulate downstream circuits to achieve such antagonistic effects remains mysterious. Here, we mapped striatal 5HT receptor expression in mice to reveal preferential enrichment of putatively excitatory, Gq-coupled 5HT receptors on medium spiny neurons expressing inhibitory D2 DA receptors (D2-MSNs). Acute slice recordings from genetically identified striatal neurons showed that DA and 5HT inversely regulate D2-MSN firing and confirmed that 5HT’s excitatory effect on these cells is blocked by 5HT2a and 5HT2c receptor antagonists. Pharmacologically upregulating striatal 5HT release preferentially induced cfos expression in D2-MSNs, validating that 5HT’s excitatory effect on these cells also occurs in vivo. Finally, 5HT2c receptor loss-of-function in D2-MSNs, but not D1-MSNs, enhanced animals’ sensitivity to the behavioral effects of cocaine – a potent releaser of DA and 5HT – showing that 5HTergic excitation of D2-MSNs counteracts the reinforcing effects of striatal DA release. Altogether these results demonstrate that DA and 5HT inversely modulate D2-MSN activity to regulate cocaine reward , identifying a key circuit mechanism underlying the opponent behavioral effects of these important neuromodulators.

Introduction
Striatal DA release is crucial for animals to learn from and seek rewards1. Longstanding theories have proposed that 5HT works against DA by promoting behavioral suppression or driving aversive learning2–7. These influential notions of opponency are supported by pharmacology experiments showing that 5HT-releasing drugs blunt the DA-driven rewarding effects of drugs of abuse8–11, as well as by studies showing that 5HT activity can suppress reward-seeking behavior12–16. Consistent with an oppositional relationship between striatal DA and 5HT, we recently found that rewards trigger opposite striatal DA and 5HT responses and that both of these inverse signals are required for a stimulus to be maximally reinforcing17. However, it remains unclear how opponent DA and 5HT signals regulate striatal activity to exert opposing effects on behavior.

In the striatum, DA works at least in part by differentially modulating excitability and plasticity in two primary projection neuron classes. DA is thought to increase activity in medium spiny neurons expressing the Gs-coupled D1 DA receptor (D1-MSNs) and decrease activity in those expressing the Gi-coupled D2 receptor (D2-MSNs)18,19. In contrast, there is much more limited understanding of the effects of 5HT on striatal circuits. Owing to the limited spatial and cell-type resolution of transcriptomic20 and autoradiographic21 studies, respectively, how 5HT receptors are organized across striatal subregions and cell-types remains unclear. As a result, the mechanisms by which 5HT may modulate striatal physiology to counteract DA’s behavioral effects are poorly understood. Here, we map striatal 5HT receptor expression to reveal that putatively excitatory 5HT receptors are enriched on D2-MSNs and show that 5HTergic excitation counteracts striatal DA’s inhibitory effect on their activity in a manner that constrains the reinforcing properties of cocaine.

Results
First, we set out to identify which 5HT receptors are expressed on MSNs by mining a single-cell RNA-sequencing dataset of the mouse striatum22. We found that the vast majority (>90%) of MSNs express 5HT receptors, including members of the Gi-, Gs-, and Gq- coupled 5HT receptor families but not the ionotropic receptor 5HTR3 (Supplementary Fig. 1a-c). Of the 14 5HT receptor genes found in mice, 7 are widely expressed in MSNs (Htr1b, Htr1d, Htr1f, Htr2a, Htr2c, Htr4, Htr6; Supplementary Fig. 1d,e), and one more is known to be expressed in a unique minority MSN population in rodents22,23 and humans24 (Htr7). Some 5HT receptor genes showed differential expression between striatal patch and matrix compartments and many were correlated with expression of D1- Cor D2- MSN marker genes (Supplementary Fig. 1f,g). Together, these results suggest that 5HT receptor expression in striatal MSNs is nearly ubiquitous, restricted primarily to 8 receptor genes, and varied across cell-types and subregions.

To better understand striatal 5HT receptor organization, we next used fluorescence in situ hybridization to map expression of the subset of genes identified above. In each experiment, we labeled transcripts for the D1 receptor (Drd1a), the D2 receptor (Drd2), and one 5HT receptor gene, allowing us to identify putative D1- and D2- MSNs (pD1- and pD2-MSNs, respectively) and measure spatial location and receptor expression individually for each cell. Control samples labeled for the MSN marker gene Ppp1r1b validated that this approach accurately identifies MSNs which could be divided into pD1 and pD2 subpopulations based on their DA receptor expression profiles (see Methods and Supplementary Fig. 2a-e).

Expression of every 5HT receptor gene that we examined varied across cell-types and subregions (Fig. 1a-h). At the cell-type level, pD1- and pD2- MSNs both expressed Gi-, Gs-, and Gq- coupled 5HT receptors but showed quantitative differences in expression of individual receptor genes (Supplementary Fig. 3a-d). pD1-MSNs were preferentially enriched for Gi-coupled Htr1f and Gs-coupled Htr4 receptors which co-labeling experiments showed to be widely co-expressed in individual neurons, suggesting 5HT may have a relatively balanced net effect on the activity of these cells (Fig. 1 c,f and Supplementary Fig. 4a-c). By contrast, pD2-MSNs were most strongly enriched for the Gs-coupled Htr7 and Gq-coupled Htr2a and Htr2c receptors, suggesting that 5HT may have a net excitatory effect on the activity of these cells (Fig. 1d,e,h and Supplementary Fig. 4d-f). Spatially, individual 5HT receptor genes were preferentially expressed in dorsal (Htr7: DMS, DLS), ventral (Htr1f: NAcmedSh, NAccore, NAclatSh; Htr4: NAcmedSh, NAclatSh), medial (Htr2c: NAcmedSh, NAccore), or lateral (Htr2a: NAclatSh) striatal subregions, and some were enriched (Htr2a, Htr7) or depleted (Htr1b) in irregular patchy patterns consistent with striosome and matrix organization, respectively (Fig. 1i).

This spatial heterogeneity in 5HT receptor gene expression raised the question of which striatal subregions receive the most 5HT input. By analyzing labeled axons from dorsal raphe 5HT (DR5HT) neurons17 – the primary source of striatal 5HT input25 – we found that 5HT inputs were denser in the nucleus accumbens (NAc) compared to the dorsal striatum (consistent with reports in rats26–28 and primates29,30; Supplementary Fig. 5a,b), and densest in the posterior NAc medial shell (NAcmedSh; Fig. 1j and Supplementary Fig. 5c-g). Examining cell-type differences in 5HT receptor expression in this region further suggested that striatal pD1- and pD2-MSNs may be differentially regulated by 5HT, likely in a manner approximately opposite to their modulation by DA (Fig. 1k).

To test this hypothesis, we performed acute slice recordings from genetically identified D1- and D2- MSNs in the NAcmedSh and measured their responses to bath application of DA or 5HT (Fig. 2a). We did this in cell-attached mode to prevent dialyzing the cell – which interferes with the intracellular signal transduction cascades recruited by G-protein coupled receptors31,32– and in the presence of potassium channel blockers to induce spontaneous activity in normally quiescent MSNs, allowing us to detect both increases and decreases in MSN activity in response to DA or 5HT (Fig. 2b,c). In agreement with previous work18,19,32, we found that DA increased activity of D1-MSNs and decreased activity of D2-MSNs (Fig. 2d-g and Supplementary Fig. 6a,b). By contrast, bath application of 5HT did not significantly change D1-MSN activity, but it excited D2-MSNs (Fig. 2h-k and Supplementary Fig. 6c,d). This was consistent with these cell-types’ distinct 5HT receptor expression profiles and indicated that DA and 5HT differentially regulate the activity of the two primary striatal cell-types (Fig. 2l-m). In subsequent experiments, application of 5HT2a and 5HT2c receptor antagonists was sufficient to completely abolish 5HT’s excitatory effect on D2-MSNs, confirming that this effect is mediated by Gq-coupled 5HT2-family receptors (Fig. 2 n-o).

To examine whether the excitatory effect of 5HT on D2-MSNs observed in slice also occurs in vivo, we implanted Drd1a-TdTomato;Drd2-eGFP double reporter mice with drug infusion cannulas above the NAcmedSh, locally infused saline or the 5HT reuptake inhibitor escitalopram to enhance striatal 5HT signaling, and immunostained for the immediate early gene product cfos as a proxy for neural activity (Fig. 2p). While escitalopram-treated mice showed similar total numbers of cfos+ MSNs compared to saline-treated controls, cfos expression was preferentially localized to D1-MSNs in the saline-treated group and to D2-MSNs in the escitalopram-treated group, suggesting that 5HT also has an excitatory effect on D2-MSNs in vivo (Fig. 2q-t and Supplementary Fig. 7a). Importantly, this effect was unlikely to be explained by off-target effects of our pharmacological manipulations, as fiber photometry recordings with genetically encoded sensors for DA and 5HT confirmed that escitalopram robustly enhances striatal 5HT release with only modest effects on striatal DA (Supplementary Fig. 7b-c). Altogether, our electrophysiological recordings and cfos data suggest that 5HT generally counteracts DA’s effects on striatal physiology, and this effect is especially pronounced in D2-MSNs where excitatory, Gq-coupled 5HT receptors directly oppose DA’s inhibitory effect on these cells in slice and in vivo.

In a final set of experiments, we sought to test whether 5HT’s effects on striatal activity functionally oppose the behavioral effects of striatal DA signaling. We reasoned that if DA release drives reinforcement at least in part by inhibiting D2-MSNs, then 5HTergic excitation of the same neurons should constrain the rewarding properties of a stimulus that evokes release of both neuromodulators, such as cocaine. To test this hypothesis, we injected Drd1-Cre and Adora2a-Cre mice in the NAcmedSh with bicistronic viral vectors encoding the Cas9 endonuclease from Staphylococcus aureus (SaCas9)33 and single-guide RNAs for Htr2c or the control locus Rosa26 (Fig. 3a). In this way, we induced loss-of-function mutations of Htr2c – the most highly expressed Gq-coupled 5HT receptor in the NAcmedSh — in either D1- or D2- MSNs with cell-type specificity.

We then tested these mice on a subthreshold cocaine conditioned place preference (CPP) paradigm (Fig. 3b). We chose cocaine for this experiment because it evokes robust increases in striatal DA and 5HT34,35 and used a modest dose (5mg/kg) to enable us to detect increases in the CPP score without the confound of ceiling effects. Furthermore, cocaine reward related behaviors have been shown to require striatal DA release36,37, to be enhanced by D1-MSN activation38,39, and to be suppressed by D2-MSN activation38–40; making cocaine CPP a powerful assay to study how DA and 5HT regulate striatal circuits to control reward and reinforcement. We found that mice with Htr2c loss-of-function in D1-MSNs showed no difference in their locomotor or CPP responses to cocaine compared to Rosa26 controls (D1-sgHtr2c and D1-sgRosa, respectively; Fig. 3c-f). By contrast, mice with Htr2c loss-of-function in D2-MSNs showed a trend toward increased locomotor sensitization and exhibited an enhanced place preference for cocaine compared to Rosa26 controls (D2-sgHtr2c and D2-sgRosa, respectively; Fig. 3g-j). Importantly, these effects could not be explained by changes in general locomotion induced by our manipulations because neither the D1-sgHtr2c nor the D2-sgHtr2c group showed any detectable changes in locomotor behavior at baseline (Supplementary Fig. 8a). Additionally, the behavior of mice in the D1- and D2- sgRosa control groups was identical, further arguing against the possibility that off-target effects from our genetic manipulations could contribute to the behavioral changes observed in the D2-sgHtr2c group (Supplementary Fig. 8b-e). Instead, Htr2c loss-of-function in D2-MSNs, but not D1-MSNs, appeared to specifically enhance animals’ sensitivity to the behavioral effects of striatal DA release induced by cocaine (Fig. 3k-l). Consistent with this interpretation, behavioral measures of cocaine’s locomotor, sensitizing, and rewarding effects were significantly correlated for mice in the D2-sgHtr2c group but not for the D1-sgHtr2c or control groups (Fig. 3m-n). Altogether, these results suggest that 5HT2c receptor-mediated excitation of D2-MSNs constrains the DA-dependent reinforcing effects of cocaine, showing that 5HTergic modulation of striatal physiology functionally opposes the behavioral functions of striatal DA release.

Methods
Mice
C57BL6/J (Jackson Laboratory #000664), Drd1a-TdTomato (Jackson Laboratory #016204), Drd2-eGFP (Jackson Laboratory #030255), Drd1a-TdTomato;Drd2-eGFP (cross between Jackson Laboratory #016204 and #030255), Drd1-Cre (MMRRC #029178-UCD), and Adora2a-Cre (MMRRC #036158-UCD), and SERT-Cre (MMMRC #017260-UCD) mice of both sexes (>5 weeks old) were used in this study. Mice were housed at ~21°C with 30-70% humidity on a 12-hour light/dark cycle with ad-libitum access to food and water. All experimental procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care and the Administrative Panel on Biosafety.

Stereotactic surgeries
Stereotactic surgeries were performed as previously described17. Mice were anesthetized with isoflurane (4-5% induction, 1-2% maintenance) and a stereotaxic frame (Kopf instruments) was used to target injections and implantations to the following structures (coordinates are in mm, with AP and ML relative to bregma and DV relative to the skull surface): DR, AP -4.6, ML 0, DV -3.3; NAcmedSh, AP +1.0, ML +/-0.7, DV -4.3. Viral vectors (diluted to between 1012 and 1013 gc/ml, except for photometry viruses which were used at stock titer) were infused with a syringe pump (Harvard Apparatus) at a rate of 100-200 nL/min and allowed to diffuse for at least 5 min. Drug infusion guide cannulas and/or fiber optic cannulas were secured to the skull using screws (Antrin Miniature Specialties) and dental cement (Geristore). Viral incubation times were >3 weeks. Drug infusion guide cannulas placements were histologically verified post-hoc.

Fluorescence in situ hybridization mapping of 5HT receptor expression
Fluorescence in situ hybridization was performed as previously described86. Briefly, wild-type mice were deeply anesthetized with isofluorane and then decapitated. The brains were dissected and flash frozen in isopentane on dry ice. Coronal sections of the striatum (16 um thick, approximately from 0.74 to 1.70 mm anterior to bregma) were collected on a cryostat, and samples were prepared following the RNAscope Multiplex Fluorescent Reagent Kit v2 protocol from ACDBio, using 1 hr on ice cold PFA for fixation and 5 min treatment with Protease Plus for digestion. The following probes were used, all from ACDBio: Drd1a (#406491, #406491-C2), Drd2 (#406501-C3), Htr1b (#315861-C2), Htr1d (#315871-C2), Htr1f (#315881-C2), Htr2a (#401291-C2), Htr2c (#401001-C2), Htr4 (#408241), Htr6 (#411161), Htr7 (#401321-C2), and Ppp1r1b (#405901-C2). The Drd1a and Drd2 probes were used for all experiments, while the third labeled gene varied across different experiments, and slides were coverslipped with Fluoromount-G containing DAPI (Southern Biotech, 0100-20). Thus, all samples were labeled for DAPI, Drd1a, Drd2, and one more gene that varied across experiments (either a 5HT receptor gene or the MSN marker gene Ppp1r1b).

Stitched images of the entire striatum in each hemisphere were acquired on a Keyence BZ-X800 microscope running BZ-X800 Viewer software using a 10x, 0.45 NA objective with a resolution of ~0.755 um/pixel. Exposure times for the DAPI, Drd1a, and Drd2 channels was kept constant across all experiments. Owing to differences in the expression level of the various 5HT receptor genes and the MSN marker gene Ppp1r1b, exposure times for imaging the third channel varied across experiments, but within an experiment all samples were imaged with identical settings and quantifications presented show only relative, not absolute, differences to account for the different exposure times used. Subsequent image analysis was carried out in ImageJ to perform cell-detection based on the DAPI channel and then measure signal in the other fluorescent channels separately for each detected cell. Specifically, each fluorescent channel was preprocessed as follows. DAPI channel: background subtraction (rolling ball, radius 50 pixels), top-hat filter (radius 12 pixels), Mexican hat filter (radius 7 pixels). Drd1a and Drd2 channels: background subtraction (rolling ball, radius 50 pixels), top-hat filter (radius 12 pixels). 5HT receptor gene or Ppp1r1b channel: background subtraction (rolling ball, radius 50 pixels). All preprocessed channels were then duplicated and one copy of each channel was binarized as follows: DAPI, thresholding using Li’s algorithm87, opening, watershedding, and opening; Drd1a and Drd2 channels, thresholding using Li’s algorithm87, erosion; 5HT receptor gene and Ppp1r1b channel, thresholding (using the moments algorithm88 for 5HT receptor experiments and Otsu’s algorithm for Ppp1r1b experiments, followed by a dilation operation for Ppp1r1b-labeled samples only). Samples were then manually annotated for striatal subregions based on DAPI signal and the Paxinos brain atlas89 (excluding the Islands of Calleja), and cell-detection was done by applying the ImageJ Analyze Particles function (size = 64-324, circularity = 0.25-1.00) to the binarized DAPI channel. Cell-ROIs were then overlayed onto the preprocessed and binarized fluorescence channels to measure the mean brightness and percentage of area covered by fluorescence signal in each channel for each cell, and the coordinates and striatal subregion of each detected cell were also recorded.

Putative D1 and D2 MSNs were then identified on the basis of their Drd1a and Drd2 signals. First, cells with low DA receptor expression (defined as <75% area covered by Drd1a or Drd2 signal) were excluded. Then, a cell-type identity index was calculated as ([Drd1a area covered – Drd2 area covered] / [Drd1a area covered + Drd2 area covered]) for each cell. Cells with a score less than 0 were assigned the pD2-MSN label and cells with a score greater than 0 were assigned the pD1-MSN label. A very small minority of cells with a cell-type identity score equal to 0 (i.e. equal amounts of Drd1a and Drd2 signal) were excluded from further analysis (see Supplementary Fig. 2e). These image preprocessing steps and the chosen cutoff values were validated using a dataset where the MSN marker gene Ppp1r1b was labeled instead of a 5HT receptor gene, as shown in Supplementary Fig 2. This control experiment confirmed that the image analysis pipeline described above accurately identified MSNs and the resulting population of cells showed a clear bimodal distribution of Drd1a and Drd2 expression enabling us to assign pD1- and pD2- MSN identities accordingly.

After pD1- and pD2- MSNs were identified as described above, 5HT receptor expression was compared across striatal subregions and cell-types as follows. 5HT receptor expression reconstruction maps in Fig 1 were generated by selecting an example hemisphere for each experiment and plotting each pMSN in that hemisphere as a dot at its corresponding coordinates in the original image. Each dot was then colored by cell-type and scaled in size and opacity by the signal in the 5HT receptor channel for that cell (percentage of each cell’s area covered by 5HT receptor gene signal, scaled separately for each 5HT receptor gene using the scale function in R with center=FALSE and scale=TRUE). The relative expression bar graphs in Fig 1 show the percentage of each cell’s area covered by 5HT receptor gene signal averaged within each cell-type and subregion for each mouse and normalized to the highest expressing cell-type and subregion for that gene. Finally, the cell-type preference index graphs in Fig 1k and Supplementary Fig. 3 were generated by averaging the percentage of area covered by 5HT receptor signal across cells for each mouse within cell-types and subregions, and then calculating a cell-type preference index for each receptor with the formula ([average 5HTR area covered in pD1 cells - average 5HTR area covered in pD2 cells]/[ average 5HTR area covered in pD1 cells + average 5HTR area covered in pD2 cells]).

Fluorescence in situ hybridization mapping of 5HT receptor co-expression
Fluorescence in situ hybridization was performed as described above except that samples were co-labeled with probes from ACDBio for the Drd1a (#406491), Htr1f (#315881-C2), and Htr4 (#408241-C3) transcripts or the Drd2 (#406501), Htr2a (#401291-C2), Htr2c (#401001-C3) transcripts to evaluate co-expression of 5HT receptors enriched on D1- and D2-MSNs, respectively. Image acquisition and processing were done as described above, except that the preprocessing for the DAPI channel used a sharpening and top-hat filter (radius 7 pixels) operation in lieu of the Mexican hat filter. Cells that met DA receptor expression criteria were identified as pD1- and pD2- MSNs (>75% cell area covered by thresholded Drd1a and Drd2 signal, respectively) and the brightness and fraction of area covered by signal in the 5HT receptor gene channels were measured for each of those cells.

RNAseq analysis
RNAseq data shown in Supplementary Fig 1a-g originated from Stanley et al22. We performed original analyses on this data beginning with the count matrix, which listed the number of reads detected of each gene for every cell that met the original paper’s quality control standards, and the metadata matrix which contained the discrete and continuous cell-type identities assigned to each cell in the count matrix. Cells from the olfactory tubercle and islands of Calleja (continuous subtypes “OT.ruffle”, “OT.flat”, and “ICj”) were excluded from analysis and 5HT receptor genes that were not detected in any cell in the dataset were assigned a count value of 0 (Htr3a, Htr3b). For the remaining data, the expression level (as counts per million reads; cpm) and fraction of cells (as a percentage) expressing at least one read was calculated for each 5HT receptor gene and averaged within mice. For the patch vs matrix comparison, the same calculation was done separately for cells that were assigned patch or matrix sub-type identities in the original Stanley et al 22 paper on the basis of their gene expression profiles (patch: continuous subtypes “Htr7.vMedPat”, “Pat.Dorsolateral_CPu”, “Pat.Ventromedial_CPu”; matrix: continuous subtypes ““Mat.Dorsolateral_CPu”, “Mat.Ventromedial_CPu”). See Stanley et al.22 Methods for additional details on RNAseq data preprocessing and patch/matrix cell-type identity assignments. Finally, gene expression correlation analysis was done by calculating the Spearman correlation between scaled log expression levels for D1 and D2 marker genes and 5HT receptor genes for all cells pooled across mice.

Histology and Immunohistochemistry
Histology and immunohistochemistry were performed as previously described17. Mice were transcardially perfused with 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS), and the brains were postfixed in PFA overnight before being sectioned coronally to a thickness of 50 um on a vibratome. Sections were then washed three times for 10 min in PBS, blocked and permeabilized for 1 hr in a solution of 10% normal goat serum, 0.2% bovine serum albumin, and 0.5% triton-X, washed once for 5 min in PBS, incubated with primary antibodies in carrier solution (1% normal goat serum, 0.2% bovine serum albumin, and 0.5% triton-X) overnight on a shaker at room temperature, washed four times for 10 min, incubated for 2 hr with secondary antibodies in carrier solution, and finally washed four times for 10 min in PBS before being mounted on microscope slides and coverslipped with Fluoromount-G containing DAPI (Southern Biotech #0100-20). For axon tracing experiments, sections were immunostained for EYFP (primary: chicken anti-GFP, Aves #GFP-1020; secondary: goat anti-chicken 488, Invitrogen #A-11039) and/or mCherry (primary: rat anti-mCherry, Invitrogen #M11217; secondary: goat anti-rat 594, Invitrogen #11007). For immediate early gene experiments, sections were immunostained for cfos (primary: rabbit anti-cfos, Sysy #226008; secondary: goat anti-rabbit 647, Invitrogen # A-21244). Primary antibodies were used at a concentration of 1:1000 and secondary antibodies were used at a concentration of 1:750.

Axon tracing
The axon tracing data presented in Supplementary Fig. 5a-e are a new analysis of data that were collected and published previously17. For the DR5HT presynaptic bouton labeling experiments in Supplementary Fig. 5f-g, SERT-Cre mice were injected in the DR with 1 uL of AAV-DJ-hSyn-GFP-2A-synaptophysin-mRuby, immunostained for EYFP and mCherry, and example images were captured on a Nikon A1 confocal microscope.

Intra-NAc drug infusions and cfos screen
Intra-NAc drug infusion experiments were performed on Drd1a-TdTomato;Drd2-eGFP double transgenic mice. Guide cannulas (Protech International) were surgically implanted unilaterally 1 mm above the NAcmedSh (i.e. -3.3 mm DV) such that the tip of the infusion cannula (Protech International), which protruded 1 mm past the tip of the guide cannula, would reach the target coordinate on the day of the experiment. After recovery from surgery (>1 week), mice received 1-2 days of habituation to handling, to the open field behavioral arena, and to being tethered, and one sham intra-NAc infusion of saline. On the day of the experiment, the infusion cannula was attached via tubing to a 5 uL syringe (Hamilton) mounted on a syringe pump (Harvard apparatus) and threaded through the guide cannula to reach the target structure in each mouse. Mice then received either 500 nL saline or 2.5 ug of escitalopram oxalate (Tocris) dissolved in 500 nL of saline infused at a rate of 150 nL/min. Mice were allowed to recover for 5-10 min in the home cage after the infusion completed before being placed into the open field arena for 90 min and then were immediately perfused. The brains were processed for cfos immunohistochemistry as described above and imaged as follows. For each of 6-8 sections per mouse, we acquired one image of the NAcmedSh (AP +0.98-1.34 mm from bregma) on the implanted hemisphere (~300 um x ~300 um, 40x 1.3 NA objective, Nikon A1 confocal microscope running NIS Elements AR 5.02.00 software). The TdTomato, eGFP, and cfos channels of these images were then processed in ImageJ as follows: top hat filter (100 px radius), background subtraction (rolling ball radius 50 px), and thresholding with the moments algorithm 88. TdTomato+/cfos+ and eGFP+/cfos+ cells were then manually counted.

Slice electrophysiology
Coronal striatal slices were prepared as previously described86. Drd1a-tdTomato or Drd2-eGFP mice (P60-90) were decapitated following deep isoflurane anesthesia. The brain was quickly removed and placed in ice cold cutting solution (bubbled with 95%O2/5%CO2) consisting (in mM): 228 sucrose, 2.5 KCl, 1.0 NaH2PO4, 8 MgSO4, 26 NaHCO3, 20 glucose and 0.5 CaCl2. The forebrain was isolated, mounted on a cutting platform, and hemisected prior to slicing. Coronal slices (200 µm thick) containing the NAc were cut with a vibratome (Leica VT1000s) and transferred to a holding chamber containing warmed (30 C, bubbled with 95%O2/5%CO2) ASCF consisting (in mM): 119 NaCl. 2.5 KCl, 1 NaH2PO4, 1.3 MgCl2, 26 NaHCO3, 10 glucose and 2.5 CaCl2. Slices were incubated for 45 min and then kept at room temperature for 1-1.5 hrs before being transferred to the fixed recording chamber of an upright microscope (BX50WI, Olympus). Slices were perfused with ACSF (bubbled with 95%O2/5%CO2) and maintained at 30-32 C. D1- and D2- MSNs in the medial shell of posterior NAc slices were identified by epifluorescence. To prevent dialysis of intracellular contents, which can interfere with G-protein signaling cascades of interest in this study, cell-attached voltage-clamp recordings were made using patch electrodes containing (in mM): 146 NaCl, 10 HEPES (pH adjusted to 7.4 with NaOH)90. Electrode resistances ranged from 6-8 MΩ. Cell-attached recordings were maintained at a holding potential of -10 mV and resistances (> 1 GΩ) were continually monitored by delivering a 4 mV, 100ms voltage-pulse every 20 s. Cell-attached voltage-clamp recordings were acquired with a MultiClamp 700B (Molecular Devices) and recorded in gap free mode (PClamp 10, Molecular Devices). Signals were filtered at 2 KHz and digitized at 10 KHz (National Instruments BNC-2090 or Digidata 1320A, Axon Instruments). All drugs (Sigma Aldrich) were bath applied and diluted to their final concentrations from concentrated stocks (1000-2000x in dH20). Spontaneous action potentials (APs) were generated by addition of barium chloride (Ba2+, 0.5 mM) and 4-aminopyridine (4-AP, 0.15 mM) to the ACSF. Once steady AP firing had been achieved (~5 min) slices were exposed to either serotonin (5-HT, 25 µM) or dopamine (DA, 100 µM) for 4 min. Effects on AP frequency were quantified in a 3 min window before and the last 3 min of drug exposure. Excitability difference and excitability ratio measures were calculated by taking the difference and the ratio, respectively, of the AP frequencies measured during these time windows for each cell. In a subset of experiments evaluating the contribution of 5HT2a and 5HT2c receptors to the excitatory effects of 5HT on D2-MSNs, the 5HT2a antagonist MDL100907 (0.1 µM) and the 5HT2c antagonist SB242084 (0.1 µM) were included in the ACSF. APs were analyzed with MiniAnalysis software (Synaptosoft).

Photometry recordings with genetically encoded DA and 5HT sensors
Wildtype mice were injected with 800 nL of a 1:1 cocktail of AAV9-hSyn-GRABrDA2m and AAV9-hSyn-GRAB5HT3.0 viruses from WZBiosciences and implanted with fiber optic cannulas unilaterally above the NAcmedSh. After waiting >3 weeks for viral expression, mice were habituated to handling, to the behavioral arena, to being tethered, and to intraperitoneal (i.p.) injections for at least one day. On the day of the experiment, mice were placed into the behavioral arena, connected to optical patch cords and allowed to habituate for at least 3 minutes before we performed fiber photometry recordings. These consisted of a 10 minute baseline period followed by an i.p. injection of 50 mg/kg escitalopram oxalate (Tocris), after which the fiber photometry recording continued for at least 25 more minutes. Data shown correspond to a 30 minute window beginning 5 minutes before the i.p. injection.

Fiber photometry recordings were performed as previously described17,54. Briefly, GRAB sensors were excited by 465 nm and 560 nm frequency-modulated light and fluorescence signals were collected through low-autofluorescence patch cords, filtered through a fluorescence minicube, (Doric FMC6_IE(400-410)_E1(460-490)_F1(500-540)_E2(555-570)_F2(590-680)), measured with dedicated photodetectors for each channel (Newport, 2151), sampled at 1.1073 Khz, and demodulated with an Rz5P signal processor running Synapse software (Tucker-Davis Technologies)17. Data from each channel underwent preprocessing as follows: the data were de-bleached by fitting a mono-exponential decay function to the 10-minute pre-drug baseline period and subtracting it from the full recording trace, then the mean of the baseline period was used to calculate dF/F using the formula (F-Fmean) / Fmean, and the resulting data were filtered using the filtfilt function with an equally weighted 100 sample window54. In post processing, the data were smoothened using a rolling average over a window of 1000 samples, decimated by a factor of 20, and averaged across mice.

Conditioned place preference
To determine if 5-HT2c receptors in the NAcmedSh influence the positive reinforcing effects of cocaine, we bilaterally injected Drd1-Cre or Adora2a-Cre mice in the NAcmedSh with Cre-dependent CRISPR constructs targeting Htr2c or the control locus Rosa26 (500 nL per hemisphere of a 1:3 cocktail of AAV-FLEX-eGFP-KASH [addgene #154373] and AAV-FLEX-saCas9-U6-sgHtr2c [addgene # 249541] or AAV-FLEX-saCas9-U6-sgRosa26 [addgene #159914]; all produced by the Zweifel lab at University of Washington, Seattle). The guide sequences for these constructs are the following: sgHtr2c, GGTGTGACCTCGAAGTAACAT; sgRosa26, CTCGATGGAAAATACTCCGAG). After 6-8 weeks of expression, mice were allowed to explore a 2-sided CPP chamber with distinct tactile floors and wall patterns (Med Associates Inc.) in a 15 min pretest. The next morning, mice were confined to one side of the chamber for 15 min after receiving an injection of saline. Approximately 4 hrs later, mice were confined to the opposite side of the chamber immediately after receiving an injection of subthreshold cocaine HCl (5 mg/kg, i.p., Sigma-Aldrich), again for 15 min. This procedure was repeated over the next 2 days for a total of 3 conditioning sessions on each of the saline and cocaine paired sides. Locomotor activity (i.e., distance traveled) was recorded during each of the conditioning sessions. On the 5th day, mice were allowed to explore both sides of the CPP chamber in a 15 min posttest. Preference was calculated as the percentage of time spent in the cocaine-paired side of the chamber during the posttest. CPP was also measured by calculating time spent on the cocaine-paired side during the posttest (cocaine time - saline time) and as a difference score (posttest cocaine time - pretest cocaine time). Cocaine-paired sides were assigned in a counterbalanced and unbiased fashion such that the average preference for the cocaine-paired side during the pretest was ~50% for all groups.

Statistics and reproducibility
Statistical analyses were done in Graphpad Prism and R. Data that met assumptions of normality and equal variance (assessed by inspection of the fitted-values vs residuals and/or Q-Q plots) were analyzed using parametric tests, with two-way ANOVAs followed by Holm-Sidak or Bonferroni post-hoc tests for two-factor designs and t-tests or one-way ANOVAs followed by Holm-Sidak post-hoc tests for one factor designs. Paired or repeated measures and the Greenhouse-Geisser correction were used where appropriate. Data that did not meet assumptions of normality and equal variance underwent the Box-Cox transformation and parametric tests were then used on the transformed data, or the data were analyzed with nonparametric tests instead (Wilcoxon signed-rank test, Friedman test followed by Dunn’s multiple comparisons tests). All hypothesis tests were two-tailed and data are shown as mean +/- sem.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports new findings on the localization of 5-HT receptors in striatal cell populations. Eshel and colleagues\nfound that most striatal projection neurons (MSNs) express 5-HT receptors. they also found that there are stronger 5-HT\nprojections to the nucleus accumbens\ncompared to the dorsal striatum. Using slice physiology, they found that 5-HT excited D2+ MSNs. In vivo, they found that\nbupropion (DA reuptake inhibitor) preferentially induced cfos expression in D1 MSNs while escitalopram (5-HT reuptake\ninhibitor) preferentially induced cfos expression in D2 MSNs.DA and 5HT thus exert opposite effects on striatal neurons.\nOn the whole the results are promising and interesting. They support the common idea in the field that DA and 5-HT can\nhave opposite effects and provide an explanation for the opponent effects. However, the major limitation of this study is that it\nremains preliminary, and the novelty of the results is quite limited. My concerns are listed below.\n1. In the introduction it is stated that DA might promote approach while 5-HT might promote avoidance. But this study\ndoes not include any analysis of behavioral function. It is simply taken for granted that somehow increasing D2-MSN output would\ncause avoidance, but no evidence is provided. What's the in vivo behavioral consequences of the reuptake inhibitors?\n2. the evidence provided for the effect of 5-HT in vivo is very limited and preliminary. c-fos expression is used as the main\nmeasure. But can this effect be blocked pharmacologically using selective antagonists of various 5-HT receptors? How does\n5-HT excite D2+ MSNs? What's the mechanism?\n3. No data is provided on other striatal cell types, like fast-spiking interneurons or cholinergic interneurons. Can these cell\npopulations explain some of the in vivo observations?", "reviewer_2": "In this manuscript, Pinto and colleagues examine the serotonin receptor profile in dopaminoceptive neurons in the striatum\nand outline their functional consequences within this brain region. While much is known about this in terms of serotonin\npharmacology at dopamine-sensitive medium spiny neurons, less is known about cell-type-specific (i.e. D1 vs D2) 5-HT\nreceptor distribution and function. This is an important gap in knowledge of the serotonin-dopamine relationship. Using\npublicly available data single cell RNAseq data, the authors outline general patterns of 5-HT receptor expression in striatal\ncells, and follow this up with in situ hybridization for D1, D2, and individual 5-HT receptors observing some spatial\ndifferences in their distribution. They then used patch clamp electrophysiology to outline how 5-HT influences the activity of\nD1 and D2 neurons in the posteromedial shell of the NAc, observing a marked activation of D2 MSNs without much impact\non D1 MSNs. The authors then use local infusion of dopamine or serotonin reuptake inhibitors (bupriopion and\nescitalopram) in the NAc to examine impacts on neural activity of genetically tagged D1 or D2 neurons in vivo using Fos as\na marker. The findings were consistent with those from in vitro patch clamp studies. Overall, the studies appear well carried\nout, the findings are interesting and informative, and the statistics are appropriate. However, there is a general lack of\nrobustness to the findings, and while they will be useful to the field, do not fit into a larger story with behavioural significance.\nBecause of the limited scope of the work, I am not sure at this stage if it warrants publication in Nature Communications. I\nsummarize a few of my main issues below:\n• The characterization of 5-HT receptors throughout the striatum is useful and appears thorough but not particularly\ngroundbreaking.\n• It is very interesting to see the dichotomy of effect on D1 and D2 neurons between dopamine and serotonin, but the story\nseems to end there leaving this as an observation rather than a comprehensive study one might expect in this journal. Are\nthere additional studies that could be conducted to demonstrate how specific receptors in D2s might be mediating\nbehavioural processes known to be regulated by 5-HT?\n• The tracing studies revealing a unique distribution throughout the NAc are not particularly novel, and have been shown\npreviously using older approaches (e.g. Brown, Molliver, 2000). This paper also identifies that many caudal NAc medial\nshell 5-HT axons lack SERT – how does this relate to the present findings using SERT-cre mice to label DRN 5-HT\nprojections?\n• The patch clamp studies are strong and straightforward but I don’t quite follow the Fos staining experiment. Why are the\ncFos counts not compared against a control injection? I understand the goal is to show the shift in balance between D1 and\nD2, but it is difficult to frame this as a change from baseline. Further, how did the authors come to choose the doses of\nbupropion and escitalopram for these intra-NAc infusion studies?", "reviewer_3": "This manuscript by Cardozo Pinto et al. maps striatal 5HT receptor expression and assesses the effects of upregulated DA\nor 5HT activity on D1 and D2 MSNs in the nucleus accumbens medial shell region. Using previously published single cell\nRNAseq data, the authors find that 5HT receptor expression profiles differ by region and cell-type. Using cell attached\nrecordings from ex vivo slices, the authors find differing effects of DA vs. 5HT on D1 and D2 MSNs. Additionally, the authors\nfind that intra-NAc infusions of DA or 5HT reuptake inhibitors via cannula, preferentially induced cfos expression in D1 or\nD2 MSNs respectively (indicated by cfos expression). Overall, this paper is clear, and the data is interesting; however, a few\nconcerns are listed below.\nMinor concerns:\n- Quantification of patch vs. matrix areas was done in extended date figure 1f; however, the authors did not specify how they\ndetermined patch vs. matrix areas. This should be included in the methods.\n- In the text describing figure 1k and extended figure 3, authors describe regions as dorsal, ventral, medial, or lateral (lines\n50-52); however, figures use NAc Core, NAc LatSh, STR dm, NAc medSH, and STR dl. Wording should be consistent.\n- Figure 2: the authors should indicate the meaning of the grey arrows (and that the inset is the zoomed trace) in the figure\nlegend.\n- Statistics are not listed for extended data figure 5.\n- Authors need to elaborate on the summary data in figure 2I-M. How was excitability difference and ratio calculated? What\ndoes this mean?\n- I believe line 312, should reference Fig. 1K not 1J\nMajor concerns:\n- Clarification is needed on how the cell type preference index is generated. Authors state that this was “generated by\naveraging the percentage of area covered by 5HT receptor signal across all the cells in each subregion for each mouse, and\nthen calculating a cell-type preference index with the formula ([pD1-pD2]/[pD1+pD2]).” It is unclear how the use of the cell\nidentity index formula was combined with the percent coverage of each receptor subtype. If a cell is being defined as either\nD1 or D2 and this value is being used as a binary determinant, (as stated by “cells with a score less than 0 were assigned\nthe pD2-MSN label and cells with a score greater than 0 were assigned the pD1-MSN label”), the raw value should not be\nfactored in. This would also not be biologically accurate, as the percentage of cells that express both D1 and D2 receptors is\nlow, and cell identity is typically not defined on a sliding scale. Along these lines, if a lot of the cells had both D1 and D2\ncoverage this is problematic as this is generally a very low percentage of MSNs in the striatum and could indicate issues\nwith methodology.\n- Authors state that “pD1-MSNs preferentially expressed one Gi-coupled receptor (Htr1f) and one Gs-coupled receptor (Htr4),\nsuggesting 5HT could have a relatively balanced net effect on their activity (Fig. 1c,f).” The wording here (and describing the\npD2-MSNs expression) is misleading given what was measured.\no First, the wording suggests that D1s primarily express 2 subtypes; however, the data indicates that other receptor subtypes\nare also expressed, they are just in both D1 and D2 MSNs. The wording should be changed to indicate that this is not the\ncase, rather that authors are highlighting these two (Htr1f and Htr4) because they are the subtypes that are primarily\nexpressed in D1s but not D2s.\no Additionally, the wording suggests that a D1 MSN preferentially expresses both (one of each), but co-expression of\nreceptor subtypes was not measured. One population of MSNs could preferentially express Htr4 while a separate population\nexpresses Htr4. An MSN could express any combination of these subtypes. The suggestion that 5HT has a balanced effect\non an MSNs activity in this context requires that the same cells are expressing one Gi and one Gs. However, this is not\nmeasured. Along these lines, the receptor # can contribute to function, but it is not the only factor. For example, the\nlocation of expression along the MSN also contributes. Overall, language suggesting that results indicate 5HT has a balanced effect\non D1s should be softened as this is not directly measured."}, "author_response": "Nature Communications Manuscript NCOMMS-25-33991-T\nResponse to Referees\nWe are pleased to submit a revised version of our manuscript originally titled “Opponent regulation of\nstriatal output pathways by dopamine and serotonin”. We thank the referees for their thoughtful comments\nand overall enthusiasm for the work.\nReferee #1 writes “On the whole the results are promising and interesting. They support\nthe common idea in the field that DA and 5-HT can have opposite effects and provide an\nexplanation for the opponent effects.”\nReferee #2 recognizes that our study addresses “an important gap in knowledge of the\nserotonin-dopamine relationship”, calls our experiments “useful” and “thorough”, and\nfinds that “overall, the studies appear well carried out, the findings are interesting and\ninformative, and the statistics are appropriate”.\nReferee #3 remarks that “Overall, this paper is clear, and the data is interesting”.\nWe have read the reviewers’ feedback carefully and in response have modified the paper in a manner that\nwe hope they find addresses all their concerns. This has required undertaking an extensive set of additional\nexperiments and analyses over the course of half a year. Some of our most important revisions include:\n1. Quantification of co-expression between 5HTRs enriched in D1-MSNs or D2-MSNs. In new in\nsitu hybridization experiments, we find that Gi-coupled Htr1f and Gs-coupled Htr4 serotonin\n(5HT) receptors are widely co-expressed in D1-MSNs, consistent with the neutral effect of 5HT\non the excitability of these cells in our recording experiments. By contrast, expression of Gq-\ncoupled Htr2a and Htr2c was only weakly correlated in D2-MSNs, consistent with the distinct\nspatial distributions of these transcripts, suggesting that 5HT’s excitatory effect on D2-MSNs may\nbe mediated primarily by Htr2a in some subregions (e.g. NAclatSh) and by Htr2c in others (e.g.\nNAcmedSh, NAccore). We present these data in Extended Data Fig 4a-f.\n2. Identification of key receptors mediating 5HTergic excitation of D2-MSNs. Using\nelectrophysiological recordings in the presence of specific 5HT receptor antagonists, we find that\nblocking just two receptors – 5HT2a and 5HT2c – is sufficient to abolish the excitatory effect of\n5HT on D2-MSNs. These results indicate that the excitatory effect of 5HT on D2-MSNs is entirely\nmediated by the Gq-coupled 5HT2 family receptors that are specifically enriched in these neurons.\nWe present these data in Fig 2n-o.\n3. Addition of various new controls for the cfos immunostaining experiments. First, we performed\nphotometry recordings with genetically encoded sensors to characterize DA and 5HT release\nevoked by high dose bupropion or escitalopram. Consistent with our original cfos results, we found\nthat escitalopram evoked robust and persistent striatal 5HT release with comparatively minor\neffects on dopamine (DA) that decayed over time (Extended Data Fig 7b-c). By contrast, we were\nsurprised to find that high dose bupropion evoked robust and sustained release of both DA and\n5HT, including levels of evoked 5HT release comparable to those we observed with escitalopram.\nConsidering that bupropion may not have been an effective way to selectively enhance striatal DA\nsignaling as we originally intended, we no longer present cfos data from the bupropion group.\nInstead, we have added a saline control group to compare escitalopram evoked cfos expression to\nstriatal activity at baseline. We then re-analyzed data from saline and escitalopram groups using\nautomated background subtraction and thresholding procedures and redid the cell-counting\nblinded to experimental condition (Extended Data Fig 7a). With the benefit of this new control\ngroup and improved analysis pipeline, we find that escitalopram treated mice have more cfos+ D2-\nMSNs compared to saline controls, confirming that the excitatory effect of 5HT on D2-MSNs\nobserved in slice also occurs in vivo (Fig 2p-t). Together with the GRAB sensor validation of the\nspecificity of the escitalopram manipulation, we believe these revisions strengthen our claim that\n5HT preferentially excites D2-MSNs. The idea that DA preferentially excites D1-MSNs remains\nsupported by our electrophysiology recording data and is generally accepted in the field based on\nnumerous studies employing a wide variety of methodologies, a subset of which we cite here1,2\nand in the revised text. As a result, our conclusion from the aggregated electrophysiology and cfos\ndata – that DA excites D1-MSNs and inhibits D2-MSNs while 5HT excites D2-MSNs – remains\nunchanged.\n4. Establishing the behavioral function of DA and 5HT’s opponent effects on striatal neurons. Finally,\nwe examined the behavioral relevance of inverse regulation of D1- and D2-MSN activity by striatal\nDA and 5HT. We reasoned that if DA drives reinforcement at least in part by inhibiting D2-MSNs,\nthen 5HTergic excitation of the same neurons should constrain the rewarding properties of a\nstimulus that evokes release of both neuromodulators. To test this hypothesis, we used\nCRISPR/saCas9 targeted mutagenesis to induce loss-of-function mutations of Htr2c –the most\nhighly expressed Gq-coupled 5HT receptor in the NAcmedSh– in either D1- or D2- MSNs. We then\ntested these mice on a cocaine conditioned place preference (CPP) paradigm. We chose this assay\nbecause cocaine evokes robust increases in striatal DA and 5HT3,4 and because cocaine reward\nbehaviors have been shown to require striatal DA release5,6, to be enhanced by D1-MSN\nactivation7,8, and to be suppressed by D2-MSN activation7–9; making this a useful assay to study\nhow DA and 5HT regulate striatal circuits to control reward and reinforcement. We found that mice\nwith Htr2c loss-of-function in D1-MSNs showed no difference in their locomotor or CPP\nresponses to cocaine compared to controls. By contrast, mice with Htr2c loss-of-function in D2-\nMSNs showed a trend toward increased locomotor sensitization and exhibited an enhanced place preference for\ncocaine. Behavioral measures of cocaine’s locomotor, sensitizing, and rewarding effects were also\ncorrelated for mice in the D2 group but not for the D1 or control groups. \nAltogether, these results show that 5HT2c receptor-mediated excitation of D2-MSNs constrains\nthe DA-dependent behavioral effects of cocaine, showing that 5HTergic modulation of striatal\nphysiology opposes the reinforcing functions of striatal DA. Together with our earlier work on the\nopponent control of reinforcement by striatal DA and 5HT25, these results also suggest that inverse\nregulation of striatal output pathways by these modulators may be a key mechanism underlying\nreward learning more broadly. We present these data in Fig 3a-m and Extended Data Fig 8a-e.\nWe wish to highlight that although approaches for cell-type specific or receptor-specific circuit\nmanipulations are widely available, achieving both levels of specificity simultaneously remains\nextremely technically challenging. In our case they required establishing a new cross-institutional\ncollaboration with Dr. Larry Zweifel at the University of Washington to access conditional, single\nvector CRISPR/saCas9 targeted mutagenesis technology. We believe that the addition of these\ndifficult and technically sophisticated experiments substantially strengthens the conclusions of our\nstudy and meaningfully expands the scope of its implications. We hope the reviewer will agree.\nBelow, we reproduce the reviewers’ comments in blue italics and describe point-by-point the actions we\nhave taken to address their concerns.\nReviewer #1:\nThis manuscript reports new findings on the localization of 5-HT receptors in striatal cell populations.\nEshel and colleagues found that most striatal projection neurons (MSNs) express 5-HT receptors. They\nalso found that there are stronger 5-HT projections to the nucleus accumbens compared to the dorsal\nstriatum. Using slice physiology, they found that 5-HT excited D2+ MSNs. In vivo, they found that\nbupropion (DA reuptake inhibitor) preferentially induced cfos expression in D1 MSNs while escitalopram\n(5-HT reuptake inhibitor) preferentially induced cfos expression in D2 MSNs.DA and 5HT thus exert\nopposite effects on striatal neurons. On the whole the results are promising and interesting. They support\nthe common idea in the field that DA and 5-HT can have opposite effects and provide an\nexplanation for the opponent effects. However, the major limitation of this study is that it remains preliminary, and the\nnovelty of the results is quite limited. My concerns are listed below.\nWe thank the referee for their detailed evaluation of our work and support for the importance of our\nfindings. We address their specific concerns below.\n1. In the introduction it is stated that DA might promote approach while 5-HT might promote avoidance.\nBut this study does not include any analysis of behavioral function. It is simply taken for granted that\nsomehow increasing D2-MSN output would cause avoidance, but no evidence is provided. What's the in\nvivo behavioral consequences of the reuptake inhibitors?\nBecause the reuptake inhibitor infusions in our cfos experiments were only done unilaterally, we did not\nthink that this experiment was an adequate test for the behavioral consequences of 5HTergic excitation of\nD2-MSNs. However, we completely agreed with this reviewer that identifying a behavioral function for\nthe excitation of D2-MSNs by 5HT would substantially strengthen the paper, and as a result we have\nundertaken a substantial set of new experiments to address this important point.\nWe reasoned that if DA drives reinforcement at least in part by inhibiting D2-MSNs, then 5HTergic\nexcitation of the same neurons should constrain the rewarding properties of a stimulus that evokes release\nof both neuromodulators. To test this hypothesis, we used CRISPR/saCas9 targeted mutagenesis to induce\nloss-of-function mutations of Htr2c –the most highly expressed Gq-coupled 5HT receptor in the\nNAcmedSh– in either D1- or D2- MSNs. We then tested these mice on a cocaine conditioned place\npreference (CPP) paradigm. We chose this assay because cocaine evokes robust increases in striatal DA\nand 5HT3,4 and because cocaine reward behaviors have been shown to require striatal DA release5,6, to be\nenhanced by D1-MSN activation7,8, and to be suppressed by D2-MSN activation7–9; making this a useful\nassay to study how DA and 5HT regulate striatal circuits to control reward and reinforcement. We found\nthat mice with Htr2c loss-of-function in D1-MSNs showed no difference in their locomotor or CPP\nresponses to cocaine compared to controls. By contrast, mice with Htr2c loss-of-function in D2-MSNs\nshowed a trend toward increased locomotor sensitization and exhibited an enhanced place preference for\ncocaine. Behavioral measures of cocaine’s locomotor, sensitizing, and rewarding effects were also\ncorrelated for mice in the D2 group but not for the D1 or control groups. Altogether, these results show\nthat 5HT2c receptor-mediated excitation of D2-MSNs constrains the DA-dependent behavioral effects of\ncocaine, showing that 5HTergic modulation of striatal physiology opposes the reinforcing functions of\nstriatal DA. Together with our earlier work on the opponent control of reinforcement by striatal DA and\n5HT25, these results also suggest that inverse regulation of striatal output pathways by these neuro\nmodulators may be a key mechanism underlying reward learning more broadly. We present these data in\nFig 3a-m and Extended Data Fig 8a-e.\nmanipulations are widely available, achieving both levels of specificity simultaneously remains extremely\ntechnically challenging. In our case they required establishing a new cross-institutional collaboration with\nDr. Larry Zweifel at the University of Washington to access conditional, single vector CRISPR/saCas9\ntargeted mutagenesis technology. We believe that the addition of these difficult and technically\nsophisticated experiments substantially strengthens the conclusions of our study and meaningfully\nexpands the scope of its implications. We hope the reviewer will agree.\n2. the evidence provided for the effect of 5-HT in vivo is very limited and preliminary. c-fos expression is\nused as the main measure. But can this effect be blocked pharmacologically using selective antagonists of\nvarious 5-HT receptors? How does 5-HT excite D2+ MSNs? What's the mechanism?\nIn the revised manuscript, we now provide multiple independent lines of evidence to support our claim\nthat 5HT excites D2-MSNs in slice and in vivo. These include: 1) anatomical experiments showing\nenrichment of putatively excitatory 5HT2a and 5HT2c receptors on D2-MSNs (Fig 1d-e); 2) axon tracing\nstudies showing dense 5HT inputs to the NAc medial shell where 5HT2c receptors are most strongly\nenriched (Extended Data Fig 5a-g); 3) electrophysiology recordings showing 5HT excites D2-MSNs in\nacute brain slices (Fig 2 h-k); 4) slice recording experiments showing that the excitatory effects of 5HT\non D2-MSNs are abolished by 5HT2 receptor antagonists (Fig 2n-o); 5) pharmacological experiments\nshowing that infusion of a 5HT reuptake inhibitor increases cfos expression in D2-MSNs (Fig 2 p-t); and,\n6) new behavioral experiments demonstrating that loss-of-function of 5HT2c receptors in D2-MSNs\nenhances animals’ responsivity to the DA-dependent behavioral effects of cocaine (Fig 3a-m). We believe\nthat altogether these experiments provide converging and compelling evidence to show that 5HT excites\nD2-MSNs both in slice and in vivo and that this effect powerfully modulates reward related behaviors.\nMechanistically, we also show both in slice and in vivo that the underlying mechanism involves activation\nof 5HT2c receptors, which have previously been shown to increase neuronal excitability in the striatum\nby multiple intracellular mechanisms including decreasing inactivating potassium currents10,11. In future\nwork, we look forward to further elucidating the intracellular mechanisms for this excitation, but we think\nthese types of experiments would be beyond the scope of this paper because they would not substantially\naffect the primary conclusion of our study: that 5HT activates D2-MSNs to counteract the effects of DA\non striatal activity and behavioral reinforcement.\n3. No data is provided on other striatal cell types, like fast-spiking interneurons or cholinergic\ninterneurons. Can these cell populations explain some of the in vivo observations?\nWe chose to focus on striatal MSNs and not the interneurons for two reasons. First, our overarching goal\nwas to understand how 5HT modulates striatal activity to counteract DA’s reinforcing effects. Because\nDA is thought to drive reinforcement primarily by differentially modulating MSN populations that express\ndistinct DA receptors, we reasoned that understanding how 5HT receptors are organized to regulate the\nactivity of these same cells, as opposed to the interneurons, was the most relevant focus for our study.\nConsistent with this view, our new functional manipulation experiments show that exciting D2-MSNs via\n5HT2c receptors (i.e. counteracting the effects of inhibitory D2 receptors on these cells) is at least one\nway that 5HT opposes DA’s behavioral effects to regulate drug reinforcement. The second reason that we\ndid not pursue studying striatal interneurons is that, in contrast with the dearth of available data on 5HT’s\neffects on D1- and D2-MSNs, there is already a growing body of work investigating 5HT receptor\nexpression on both GABAergic12–15 and cholinergic16–20 striatal interneurons including the effects of 5HT\n4\nsignaling on the activity of these neurons and striatal circuits. While expanding on this work would be\ninteresting, we ultimately felt that investing the large amount of time and effort that would be required to\nextend this work to striatal interneurons would be out of scope. In response to feedback from this reviewer\nthough, we now cover the topic of DAergic and 5HTergic modulation of striatal interneurons in the\ndiscussion and highlight it as an important future direction:\n“In other behavioral contexts, the opponent regulation of striatal output pathways by DA and 5HT may\nbe complemented by additional actions reported for these neuromodulators in the striatum. These include\nmodulating the strength of corticostriatal62–64 and thalamostriatal65,66 synapses, differentially filtering\npresynaptic inputs onto MSNs66, and regulating lateral inhibition between D1 and D2 MSNs67–69. Notably,\na growing body of work suggests that DA and 5HT may inversely regulate some cholinergic interneurons\n–which express inhibitory D2 DA receptors and excitatory 5HT2-family receptors for 5HT– while exerting\nheterogeneous effects on various subtypes of GABAergic interneurons70–77. We anticipate that identifying\nhow each of these mechanisms contributes to behavioral functions ascribed to striatal DA and 5HT beyond\nreinforcement – including decision making78,79, sociability80–82, and aggression83–85 – will be an important\nfuture direction for the field.”\nReviewer #2:\nIn this manuscript, Pinto and colleagues examine the serotonin receptor profile in dopaminoceptive\nneurons in the striatum and outline their functional consequences within this brain region. While much is\nknown about this in terms of serotonin pharmacology at dopamine-sensitive medium spiny neurons, less\nis known about cell-type-specific (i.e. D1 vs D2) 5-HT receptor distribution and function. This is an\nimportant gap in knowledge of the serotonin-dopamine relationship. Using publicly available data single\ncell RNAseq data, the authors outline general patterns of 5-HT receptor expression in striatal\ncells, and follow this up with in situ hybridization for D1, D2, and individual 5-HT receptors observing some spatial\ndifferences in their distribution. They then used patch clamp electrophysiology to outline how 5-HT\ninfluences the activity of D1 and D2 neurons in the posteromedial shell of the NAc, observing a marked\nactivation of D2 MSNs without much impact on D1 MSNs. The authors then use local infusion of dopamine\nor serotonin reuptake inhibitors (bupriopion and escitalopram) in the NAc to examine impacts on neural\nactivity of genetically tagged D1 or D2 neurons in vivo using Fos as a marker. The findings were consistent\nwith those from in vitro patch clamp studies. Overall, the studies appear well carried out, the findings are\ninteresting and informative, and the statistics are appropriate. However, there is a general lack of\nrobustness to the findings, and while they will be useful to the field, do not fit into a larger story with\nbehavioural significance. Because of the limited scope of the work, I am not sure at this stage if it warrants\npublication in Nature Communications. I summarize a few of my main issues below:\nWe thank the referee for their consideration of our work and support for the importance of our findings.\nWe address their specific concerns below.\n• The characterization of 5-HT receptors throughout the striatum is useful and appears thorough but not\nparticularly groundbreaking.\nGiven that the differential expression of D1 vs D2 DA receptors between MSN classes is a cornerstone in\nour knowledge of basal ganglia function, we agree with the reviewer that the lack of a similar\nunderstanding for 5HT receptors represents “an important gap in knowledge of the serotonin-dopamine\nrelationship”. For this reason, we believe that our findings showing differential expression of 5HT\nreceptors between D1- and D2- MSNs– and particularly the enrichment of excitatory 5HT receptors on\nD2-MSNs that are inhibited by DA – represents an important advance for the field. It also sets up the\nremaining experiments in our manuscript, which include electrophysiology, in vivo activity mapping, and\n(new in the revision) a series of behavioral experiments.\n• It is very interesting to see the dichotomy of effect on D1 and D2 neurons between dopamine and\nserotonin, but the story seems to end there leaving this as an observation rather than a comprehensive\nstudy one might expect in this journal. Are there additional studies that could be conducted to\ndemonstrate how specific receptors in D2s might be mediating behavioural processes known to be\nregulated by 5-HT?\nWe completely agree with this reviewer that identifying the receptor(s) mediating 5HT’s excitatory effect\non D2-MSNs and elucidating their behavioral function would substantially strengthen the paper. As a\nresult, we have undertaken a substantial set of new experiments to address these important points.\nFirst, we sought to identify which receptors mediate 5HT’s excitatory effect on D2 MSNs. We\nhypothesized that this effect is mediated by the 5HT2a and 5HT2c receptors that we found to be\nconcentrated on D2-MSNs in our receptor expression mapping experiments. We tested this hypothesis by\nrepeating our cell-attached electrophysiology recordings experiments in the presence of antagonists\nspecific for these two receptors. We found that selectively blocking 5HT2a and 5HT2c receptors was\nsufficient to entirely abolish the excitatory effect of 5HT on D2-MSNs, identifying these receptors are\ncrucial mediators of 5HT’s excitatory effects on the D2-MSN population.\nWe then reasoned that if DA drives reinforcement at least in part by inhibiting D2-MSNs, then 5HTergic\nexcitation of the same neurons should constrain the rewarding properties of a stimulus that evokes release\nof both neuromodulators. To test this hypothesis, we used CRISPR/saCas9 targeted mutagenesis to induce\nloss-of-function mutations of Htr2c –the most highly expressed Gq-coupled 5HT receptor in the\nNAcmedSh– in either D1- or D2- MSNs. We then tested these mice on a cocaine conditioned place\npreference (CPP) paradigm. We chose this assay because cocaine evokes robust increases in striatal DA\nand 5HT3,4 and because cocaine reward behaviors have been shown to require striatal DA release5,6, to be\nenhanced by D1-MSN activation7,8, and to be suppressed by D2-MSN activation7–9; making this a useful\nassay to study how DA and 5HT regulate striatal circuits to control reward and reinforcement. We found\nthat mice with Htr2c loss-of-function in D1-MSNs showed no difference in their locomotor or CPP\nresponses to cocaine compared to controls. By contrast, mice with Htr2c loss-of-function in D2-MSNs\nshowed a trend toward increased locomotor sensitization and exhibited an enhanced place preference for\ncocaine. Behavioral measures of cocaine’s locomotor, sensitizing, and rewarding effects were also\ncorrelated for mice in the D2 group but not for the D1 or control groups. Altogether, these results show\nthat 5HT2c receptor-mediated excitation of D2-MSNs constrains the DA-dependent behavioral effects of\ncocaine, showing that 5HTergic modulation of striatal physiology opposes the reinforcing functions of\nstriatal DA. Together with our earlier work on the opponent control of reinforcement by striatal DA and\n5HT25, these results also suggest that inverse regulation of striatal output pathways by these neuro\nmodulators may be a key mechanism underlying reward learning more broadly. We present these data in\nFig 3a-m and Extended Data Fig 8a-e.\nmanipulations are widely available, achieving both levels of specificity simultaneously remains extremely\ntechnically challenging. In our case they required establishing a new cross-institutional collaboration with\nDr. Larry Zweifel at the University of Washington to access conditional, single vector CRISPR/saCas9\ntargeted mutagenesis technology. We believe that the addition of these difficult and technically\nsophisticated experiments substantially strengthens the conclusions of our study and meaningfully\nexpands the scope of its implications. We hope the reviewer will agree.\n• The tracing studies revealing a unique distribution throughout the NAc are not particularly novel, and\nhave been shown previously using older approaches (e.g. Brown, Molliver, 2000). This paper also\nidentifies that many caudal NAc medial shell 5-HT axons lack SERT – how does this relate to the present\nfindings using SERT-cre mice to label DRN 5-HT projections?\nWe appreciate the opportunity to contextualize our axon tracing results relative to previous work in the\nfield. First, we would like to point out that the main conclusion of our study is the novel finding that 5HT\nexcites D2-MSNs to counteract DA’s effects on striatal physiology and reward related behaviors. We\npresent the 5HT axon tracing data to justify our choice to focus on the NAc medial shell for our recording\nand manipulation experiments, but it is not the central finding of our work here. Even so, while we\nacknowledge that a qualitative concentration of 5HT axons in the NAc medial shell has been observed\nbefore, we believe that our axon tracing work offers two advantages over previous studies. First, to the\nbest of our knowledge this is the first systematic and quantitative analysis of the mesolimbic 5HT\nprojection. This analysis shows that the NAcmedSh is likely to be the primary locus of action for 5HT in the\nstriatum and we believe that this is an important contribution to the field because 5HT’s functions in this\nregion are understudied relative to the dorsal striatum which has been the focus of much previous work21–\n23. Second, our approach using viral tools for neuromodulatory axon tracing offers improved sensitivity\nover older methods, including the immunohistochemical techniques employed by Brown and Molliver24.\nWe believe that this difference in sensitivity explains the apparent discrepancy between our results and\ntheirs. Specifically, we think that what Brown and Molliver’ immunohistochemical approach failed to\ndetect NAcmedSh dorsal raphe inputs expressing lower levels of SERT than 5HT axons in adjacent striatal\nregions. Such low-SERT expressing axons could be below the minimal threshold of detection required to\nbe labeled using Brown and Molliver’s immunohistochemical techniques while still being brightly labeled\nin axon tracing studies like ours where even modest SERT expression still produces sufficient recombinase\nexpression in transgenic mice to drive GFP recombination and expression. Another, less likely, potential\nexplanation for the discrepancy between our results and those of Brown and Molliver could also be species\ndifferences given that their study used rats and ours uses mice. Most importantly though, we want to\nemphasize that our primary conclusions are not dependent on whether these axons express high or low\nlevels of SERT. What is central to our conclusions is that these axons release 5HT, which we have\npreviously demonstrated to be the case using genetically encoded sensors for 5HT in both the NAc core\nand NAc medial shell25, and that 5HT levels in this brain region are sensitive to escitalopram treatment,\nwhich we now validate with new experiments using genetically-encoded 5HT sensor recordings in this\nbrain region (Extended Data Fig 7b-c).\n• The patch clamp studies are strong and straightforward but I don’t quite follow the Fos staining\nexperiment. Why are the cFos counts not compared against a control injection? I understand the goal is\nto show the shift in balance between D1 and D2, but it is difficult to frame this as a change from\n7\nbaseline. Further, how did the authors come to choose the doses of bupropion and escitalopram for\nthese intra-NAc infusion studies?\nWe agree that the reviewer raises important points here and appreciate the chance to improve the rigor of\nour cfos immunolabeling experiments. We originally chose to use intra-NAc infusions of bupropion and\nescitalopram because this approach avoids potentially confounding effects of systemic injections which\nwould have elevated DA or 5HT levels throughout the entire brain. However, one drawback of this\napproach is that for most drugs there is no dose-response data available for the intra-cerebral route of\nadministration which makes it difficult to make an informed decision about drug dose. In this case, we are\nonly aware of a handful of studies using intra-cerebral infusions of escitalopram or bupropion, and because\ncfos is an indirect measure of neural activity with low sensitivity, we chose doses that were somewhat\nhigher in order to maximize our chance of detecting an effect. In response to this reviewer’s feedback\nthough, we sought to validate that high doses of escitalopram and bupropion selectively elevate striatal\nDA and 5HT, respectively, as we originally anticipated. To do this, we performed photometry recordings\nwith genetically encoded sensors for DA and 5HT following administration of high doses of escitalopram\nor bupropion. In these experiments, the drugs had to be delivered systemically owing to the technical\ndifficulty of combining photometry recordings with local drug infusions into the same brain region, but\nwe chose doses at the very high end of the typical range for i.p. delivery of both drugs (50 mg/kg) and\nreasoned that if very high dose bupropion/escitalopram delivered systemically was specific for DA/5HT\nthen the same drugs delivered at high doses intracerebrally were likely to be even more specific.\nConsistent with our original cfos results, we found that high dose escitalopram evoked robust and\npersistent striatal 5HT release with comparatively minor evoked DA activity which decayed over time\n(Extended Data Fig 7b-c). By contrast, we were surprised to find that high dose bupropion evoked robust\nand sustained release of both DA and 5HT, including levels of evoked 5HT release comparable to those\nwe observed with escitalopram. While this may have been due to off-target effects of systemically\nadministered bupropion, we could not rule out the possibility that bupropion may not have been an\neffective way to selectively enhance striatal DA signaling as we originally intended, and as a result we no\nlonger present cfos data from the bupropion group in the manuscript. Instead, we have taken this\nreviewer’s helpful suggestion and added a new saline control group to compare escitalopram evoked cfos\nexpression to striatal activity at baseline. We then re-analyzed data from saline and escitalopram groups\nusing automated background subtraction and thresholding procedures and redid the cell-counting\nblinded to experimental condition (Extended Data Fig 7a). With the benefit of this new control group and an\nimproved analysis pipeline, we found that escitalopram treated mice have more cfos+ D2-MSNs compared\nto saline controls, confirming that the excitatory effect of 5HT on D2-MSNs also occurs in vivo (Fig 2p-\nt). Together with the GRAB sensor validation of the specificity of the escitalopram manipulation, we\nbelieve these revisions strengthen our claim that 5HT preferentially excites D2-MSNs. The idea that DA\npreferentially excites D1-MSNs remains supported by our electrophysiology recording data and is\ngenerally accepted in the field based on numerous studies, a subset of which we cite here1,2 and in the\nrevised text. As a result, our conclusion from the aggregated electrophysiology and cfos data – that DA\nexcites D1-MSNs and inhibits D2-MSNs while 5HT excites D2- MSNs – remains unchanged. We are\ngrateful to this reviewer for their constructive feedback on these points.\nReviewer #3:\nThis manuscript by Cardozo Pinto et al. maps striatal 5HT receptor expression and assesses the effects\n8\nof upregulated DA or 5HT activity on D1 and D2 MSNs in the nucleus accumbens medial shell region.\nUsing previously published single cell RNAseq data, the authors find that 5HT receptor expression\nprofiles differ by region and cell-type. Using cell attached recordings from ex vivo slices, the authors\nfind differing effects of DA vs. 5HT on D1 and D2 MSNs. Additionally, the authors find that intra-NAc\ninfusions of DA or 5HT reuptake inhibitors via cannula, preferentially induced cfos expression in D1 or\nD2 MSNs respectively (indicated by cfos expression). Overall, this paper is clear, and the data is\ninteresting; however, a few concerns are listed below.\nWe thank the reviewer for their complimentary evaluation of our work and address their specific concerns\nbelow.\nMinor concerns:\n- Quantification of patch vs. matrix areas was done in extended date figure 1f; however, the authors did\nnot specify how they determined patch vs. matrix areas. This should be included in the methods.\nThank you for the chance to clarify this point. In the Methods we now specify “RNAseq data shown in\nExtended Data Fig 1a-g originated from Stanley et al22… [For this data] the expression level (as counts\nper million reads; cpm) and fraction of cells (as a percentage) expressing at least one read was calculated\nfor each 5HT receptor gene and averaged within mice. For the patch vs matrix comparison, the same\ncalculation was done separately for cells that were assigned patch or matrix sub-type identities in the\noriginal Stanley et al paper on the basis of their marker gene expression profiles (patch: continuous\nsubtypes “Htr7.vMedPat”, “Pat.Dorsolateral_CPu”, “Pat.Ventromedial_CPu”; matrix: continuous\nsubtypes ““Mat.Dorsolateral_CPu”, “Mat.Ventromedial_CPu”). See Stanley et al. Methods for additional\ndetails on RNAseq data preprocessing and patch/matrix cell-type identity assignments.”\n- In the text describing figure 1k and extended figure 3, authors describe regions as dorsal, ventral,\nmedial, or lateral (lines 50-52); however, figures use NAc Core, NAc LatSh, STR dm, NAc medSH, and\nSTR dl. Wording should be consistent.\nWe apologize for this oversight and in response we have revised all the figures and main text to make sure\nthe nomenclature for striatal subregions is consistent throughout (now NAcmedSh, NAccore, NAclatSh, DMS,\nDLS). The revised version of the specific portion of the text highlighted by the referee in this comment is\nreproduced here: “Spatially, individual 5HT receptor genes were preferentially expressed in dorsal (Htr7:\nDMS, DLS), ventral (Htr1f: NAcmedSh, NAccore, NAclatSh; Htr4: NAcmedSh, NAclatSh), medial (Htr2c:\nNAcmedSh, NAccore), or lateral (Htr2a: NAclatSh) striatal subregions….”\n- Figure 2: the authors should indicate the meaning of the grey arrows (and that the inset is the zoomed\ntrace) in the figure legend.\nAs requested, we have clarified this point in the legend for Fig 2 as follows: “In d,f,h,i,and n tick marks\nindicate where calibration pulse artifacts were manually removed, and in d,f,h, and i arrows indicate\ncorresponding locations on the example traces (left) and their magnified insets (right).”\n9\n- Statistics are not listed for extended data figure 5.\nWe apologize for this oversight. In the revised manuscript we include the results of these statistical tests\n(now corresponding to Extended Data Fig 6) in Supplementary Table 1.\n- Authors need to elaborate on the summary data in figure 2I-M. How was excitability difference and\nratio calculated? What does this mean?\nWe now include the requested clarification in the Methods, which reads as follows: “Effects on AP\nfrequency were quantified in a 3 min window before and the last 3 min of drug exposure. Excitability\ndifference and excitability ratio measures were calculated by taking the difference and the ratio,\nrespectively, of the AP frequencies measured during these time windows for each cell.”\n- I believe line 312, should reference Fig. 1K not 1J\nThat is correct and we have fixed it in the revised manuscript.\nMajor concerns:\n- Clarification is needed on how the cell type preference index is generated. Authors state that this was\n“generated by averaging the percentage of area covered by 5HT receptor signal across all the cells in\neach subregion for each mouse, and then calculating a cell-type preference index with the formula\n([pD1-pD2]/[pD1+pD2]).” It is unclear how the use of the cell identity index formula was combined\nwith the percent coverage of each receptor subtype. If a cell is being defined as either D1 or D2 and this\nvalue is being used as a binary determinant, (as stated by “cells with a score less than 0 were assigned\nthe pD2-MSN label and cells with a score greater than 0 were assigned the pD1-MSN label”), the raw\nvalue should not be factored in. This would also not be biologically accurate, as the percentage of cells\nthat express both D1 and D2 receptors is low, and cell identity is typically not defined on a sliding scale.\nAlong these lines, if a lot of the cells had both D1 and D2 coverage this is problematic as this is\ngenerally a very low percentage of MSNs in the striatum and could indicate issues with methodology.\nThank you for the opportunity to clarify this important point. Our analysis involves calculating two values:\na cell-type identity index which is calculated for each detected cell (used to classify putative MSNs as\npD1- or pD2- type MSN) and a cell-type preference index which is calculated for each 5HT receptor gene\n(used to evaluate cell-type differences in 5HT receptor gene expression). Below, we describe how each of\nthose measures are calculated, how we apply them in our analyses, and the specific changes we made to\nthe Methods to communicate this more clearly.\nAfter image preprocessing, cell-detection, and thresholding, our image analysis pipeline provides us with\nthe fraction of area covered by signal in each of the Drd1a, Drd2, and 5HT receptor gene channels for\nevery striatal cell detected in the image. We begin by using each cell’s DA receptor expression profile to\nseparate putative MSNs from other striatal cells such as glia. Cells with >75% area covered by Drd1a or\nDrd2 signal are classified as putative MSNs (pMSNs; Extended Data Fig 2b-c). For each pMSN, we then\ncalculate a cell-type identity index as (Drd1a area covered – Drd2 area covered) / (Drd1a area covered +\nDrd2 area covered) and use that to classify each cell as a pD1- or pD2- MSNs. This assignment is binary,\nnot graded, because as this reviewer notes cell identity is not defined on a sliding scale. pMSNs expressing\n10\nequal amounts of Drd1a and Drd2 are excluded from further analysis but these represent only a tiny\nfraction of the total number of pMSNs in our data set (the small grey bar at 0 on the x-axis in Extended\nData Fig 2e) and, as the reviewer points out, this agrees with previous work in the field that estimates the\nprevalence of these cells to be low. Only after pD1- and pD2- MSNs have been identified on the basis of\ntheir DA receptor expression in this fashion do we examine 5HT receptor expression in these cells.\nWe then analyze 5HT receptor expression as follows. First, we average the percentage of area covered by\n5HT receptor gene signal in pD1- and pD2- MSNs separately for each striatal subregion. After a\nnormalization step, this yields the data shown in the bar graphs in Fig 1a-h. To arrive at the cell-type\npreference index for each 5HT receptor in the NAcmedSh shown in Fig 1k, we simply take the NAcmedSh\ndata shown in the bar graphs of Fig 1a-h before the normalization step and take the difference over the\ntotal for each matching pair of values; i.e., for each cell-type and each subregion we calculate ([average\n5HTR area covered in pD1 cells - average 5HTR area covered in pD2 cells] / [average 5HTR area covered\nin pD1 cells + average 5HTR area covered in pD2 cells]).\nIn the original methods, the final calculation step for the cell-type preference index was described as\n([pD1-pD2]/[pD1+pD2]). As the reviewer points out, this may have been ambiguous because it could have\nbeen interpreted as comparing DA receptor expression, and not as comparing 5HT receptor expression\nbetween the pD1 and pD2 cell populations as we intended to convey. To clarify this point, we now clearly\ndifferentiate between formulas used to calculate the cell-type identity and cell-type preference indices in\nthe revised methods:\n“a cell-type identity index was calculated as ([Drd1a area covered – Drd2 area covered] / [Drd1a area\ncovered + Drd2 area covered]) for each cell…”\nAnd,\n“a cell-type preference index [was calculated] for each receptor with the formula ([average 5HTR area\ncovered in pD1 cells - average 5HTR area covered in pD2 cells]/[ average 5HTR area covered in pD1\ncells + average 5HTR area covered in pD2 cells]).\n- Authors state that “pD1-MSNs preferentially expressed one Gi-coupled receptor (Htr1f) and one Gs-\ncoupled receptor (Htr4), suggesting 5HT could have a relatively balanced net effect on their activity\n(Fig. 1c,f).” The wording here (and describing the pD2-MSNs expression) is misleading given what was\nmeasured. First, the wording suggests that D1s primarily express 2 subtypes; however, the data\nindicates that other receptor subtypes are also expressed, they are just in both D1 and D2 MSNs. The\nwording should be changed to indicate that this is not the case, rather that authors are highlighting\nthese two (Htr1f and Htr4) because they are the subtypes that are primarily expressed in D1s but not\nD2s.\nThank you for the opportunity to more clearly communicate the findings from our 5HT receptor mapping\nexperiments. As suggested by the reviewer, we have revised the text of the Results section to explicitly\nconvey that D1- and D2- MSNs express a variety of 5HT receptors with graded differences in expression\nfor specific receptor types that we focus on and describe in more detail. The revised text reads as follows:\n“At the cell-type level, pD1- and pD2- MSNs both expressed Gi-, Gs-, and Gq- coupled 5HT receptors\n11\nbut showed quantitative differences in expression of individual receptor genes (Extended Data Fig. 3a-\nd). pD1-MSNs were preferentially enriched for Gi-coupled Htr1f and Gs-coupled Htr4 receptors…”\no Additionally, the wording suggests that a D1 MSN preferentially expresses both (one of each), but co-\nexpression of receptor subtypes was not measured. One population of MSNs could preferentially express\nHtr4 while a separate population expresses Htr4. An MSN could express any combination of these\nsubtypes. The suggestion that 5HT has a balanced effect on an MSNs activity in this context requires\nthat the same cells are expressing one Gi and one Gs. However, this is not measured. Along these lines,\nthe receptor # can contribute to function, but it is not the only factor. For example, the location of\nexpression along the MSN also contributes. Overall, language suggesting that results indicate 5HT has\na balanced effect on D1s should be softened as this is not directly measured.\nThe reviewer makes a compelling argument here. Rather than tone down the language around these claims,\nwe were persuaded to go a step further and undertake additional experiments to directly address the\nquestion of co-expression of Htr1f and Htr4 receptors which are enriched in D1-MSNs. In new in situ\nhybridization experiments, we labeled transcripts for Htr1f, Htr4, and Drd1a in the same tissue and found\nthat Htr1f and Htr4 receptors are widely co-expressed in D1-MSNs. Furthermore, the level of expression\nof Htr1f was strongly correlated with the expression level of Htr4 on a cell-by cell basis (Extended Data\nFig 4a-c). Taken together with our earlier findings showing that D1 MSNs express a mix of Gs, Gi,\nand Gq coupled receptors, these new data help explain why 5HT had a balanced net effect on the activity of\nD1-MSNs which we observed in our electrophysiological recording experiments. We thank the reviewer\nfor their helpful suggestion for this additional experiment which we believe substantially improves the\npaper. As the reviewer astutely points out though, we cannot formally rule out the possibility that\nadditional factors, such as the subcellular localization of individual 5HT receptors, could still enable 5HT\nto increase membrane excitability in one neuronal compartment and decrease it in another (e.g. two\nbranches of a dendrite). Given that the focus of our study was to investigate how 5HT modulates the\nactivity of the principal populations of striatal neurons though, we argue that questions about the\nsubcellular functions of 5HT signaling in individual neurons are beyond the scope of the work we are\npresenting and we leave that for future studies to explore.\nFinally, we note that while preparing the source data file for the resubmission of this manuscript we\nidentified an analysis error related to Extended Data Fig 2c – the relevant panel is corrected in the revised\nmanuscript and our conclusions remain unchanged. We thank all three referees for the time they invested\nevaluating our work and thank them for their constructive feedback which we feel has substantially\nstrengthened the revised manuscript.\n1. Lahiri, A. K. & Bevan, M. D. Dopaminergic Transmission Rapidly and Persistently Enhances\nExcitability of D1 Receptor-Expressing Striatal Projection Neurons. Neuron106, 277-290.e6\n(2020).\n2. 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Cell Type–Specific Loss of BDNF Signaling Mimics Optogenetic Control of\nCocaine Reward. Science (1979)330, 385–390 (2010).\n8. Heinsbroek, J. A. et al. Loss of Plasticity in the D2-Accumbens Pallidal Pathway Promotes\nCocaine Seeking. The Journal of Neuroscience37, 757–767 (2017).\n9. Bock, R. et al. Strengthening the accumbal indirect pathway promotes resilience to\ncompulsive cocaine use. Nat Neurosci16, 632–638 (2013).\n10. North, R. A. & Uchimura, N. 5-Hydroxytryptamine acts at 5-HT2 receptors to decrease\npotassium conductance in rat nucleus accumbens neurones. J Physiol417, 1–12 (1989).\n11. Stefani, A., James Surmeier, D. & Kitai, S. T. Serotonin enhances excitability in neostriatal\nneurons by reducing voltage-dependent potassium currents. Brain Res529, 354–357\n(1990).\n12. Muñoz-Manchado, A. B. et al. Novel Striatal GABAergic Interneuron Populations Labeled in\nthe 5HT3a EGFP Mouse. Cerebral Cortex26, 96–105 (2016).\n13. Assous, M. et al. Identification and Characterization of a Novel Spontaneously Active Bursty\nGABAergic Interneuron in the Mouse Striatum. The Journal of Neuroscience38, 5688–5699\n(2018).\n14. Cains, S., Blomeley, C. P. & Bracci, E. Serotonin inhibits low threshold spike interneurons in\nthe striatum. J Physiol590, 2241–2252 (2012).\n-\n15. Blomeley, C. P. & Bracci, E. Serotonin excites fast spiking interneurons in the striatum.\nEuropean Journal of Neuroscience29, 1604–1614 (2009).\n-\n16. Vizi, E. S., Ha´rsing, L. G. & Zsilla, G. Evidence of the modulatory role of serotonin in\nacetylcholine release from striatal interneurons. Brain Res212, 89–99 (1981).\n17. Blomeley, C. & Bracci, E. Excitatory effects of serotonin on rat striatal cholinergic\ninterneurones. J Physiol569, 715–721 (2005).\n18. Bonsi, P. et al. Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the\nActivation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for\nExtrapyramidal Side Effects of Serotonin Reuptake Inhibitors. Neuropsychopharmacology\n32, 1840–1854 (2007).\n19. Virk, M. S. et al. Opposing roles for serotonin in cholinergic neurons of the ventral and dorsal\nstriatum. Proceedings of the National Academy of Sciences113, 734–739 (2016).\n20. Matityahu, L. et al. Synchronous activation of striatal cholinergic interneurons induces local\nserotonin release. BioRXiv vol. 2 537–8 Preprint at\nhttps://doi.org/10.1101/2024.11.03.621726 (2024).\n21. Li, Y. et al. Synaptic mechanism underlying serotonin modulation of transition to cocaine\naddiction. Science (1979)373, (2021).\n22. Spring, M. G. & Nautiyal, K. M. Striatal Serotonin Release Signals Reward Value. J Neurosci\n44, (2024).\n23. Cavaccini, A. et al. Serotonergic Signaling Controls Input-Specific Synaptic Plasticity at\nStriatal Circuits. Neuron98, 801-816.e7 (2018).\n24. Brown, P. & Molliver, M. E. Dual Serotonin (5-HT) Projections to the Nucleus Accumbens\nCore and Shell: Relation of the 5-HT Transporter to Amphetamine-Induced Neurotoxicity.\nThe Journal of Neuroscience20, 1952–1963 (2000).\n25. Cardozo Pinto, D. F. et al. Opponent control of reinforcement by striatal dopamine and\nserotonin. Nature639, 143–152 (2025).\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns and improved the manuscript.", "reviewer_2": "The authors have gone to great lengths to improve the quality, rigor, and impact of the paper. The new, careful additions to\nthe manuscript make it a much more complete story suitable for Nature Communications. I believe the manuscript is\nacceptable.", "reviewer_3": "In the revised manuscript, the authors provide additional evidence to support their conclusions that 5HT has an excitatory\neffect on D2 MSNs that modulates cocaine reward related behaviors. To address reviewer concerns, the authors perform\nadditional in situ hybridization experiments finding that Htr1f and Htr4 serotonin receptors are co-expressed in D1-MSNs,\nwhereas Htr2a and Htr2 are weakly correlated in D2-MSNs. Additional electrophysiological recordings suggest that blocking\n5HT2a and 5HT2c receptors is sufficient to abolish the excitatory effect of 5HT on D2-MSNs. Lastly, authors developed a\nCRISPR/saCas9 line to achieve receptor-specific loss of function of Htr2c in a D1 or D2 cell-type specific manor. CPP in these\nmice suggests that Htr2c loss of function in D2 but not D1 MSNs enhances place preference for cocaine. Together the\naddition of these new experiments clarify and strengthen the claims made by the authors and sufficiently address the\nreviewers concerns given the constraints."}, "author_response": "Nature Communications Manuscript NCOMMS-25-33991-A\nResponse to Referees\nWe are pleased to submit a revised version of our manuscript “Dopamine and serotonin inversely\nmodulate D2 medium spiny neurons to regulate cocaine reward”. We thank the referees for their\nthoughtful comments throughout the review process and were glad to see that we have now\nsatisfied all of their concerns. Below, we reproduce the reviewers’ feedback in blue italics and\nrespond point-by-point to their comments.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my concerns and improved the manuscript.\nWe thank this reviewer for their feedback throughout the review process and are glad to see that we have now\nsatisfied all of their concerns.\nReviewer #2 (Remarks to the Author):\nThe authors have gone to great lengths to improve the quality, rigor, and impact of the paper. The\nnew, careful additions to the manuscript make it a much more complete story suitable for Nature\nCommunications. I believe the manuscript is acceptable.\nWe thank this reviewer for their feedback throughout the review process and are pleased to see\nthat in their estimation the paper is now suitable for publication.\nReviewer #3 (Remarks to the Author):\nIn the revised manuscript, the authors provide additional evidence to support their conclusions\nthat 5HT has an excitatory effect on D2 MSNs that modulates cocaine reward related behaviors.\nTo address reviewer concerns, the authors perform additional in situ hybridization experiments\nfinding that Htr1f and Htr4 serotonin receptors are co-expressed in D1-MSNs, whereas Htr2a\nand Htr2 are weakly correlated in D2-MSNs. Additional electrophysiological recordings suggest\nthat blocking 5HT2a and 5HT2c receptors is sufficient to abolish the excitatory effect of 5HT on\nD2-MSNs. Lastly, authors developed a CRISPR/saCas9 line to achieve receptor-specific loss of\nfunction of Htr2c in a D1 or D2 cell-type specific manor. CPP in these mice suggests that Htr2c\nloss of function in D2 but not D1 MSNs enhances place preference for cocaine. Together the\naddition of these new experiments clarify and strengthen the claims made by the authors and\nsufficiently address the reviewers concerns given the constraints.\nWe thank this reviewer for their feedback throughout the review process and are pleased to see\nthat in their estimation the paper is now suitable for publication."}]

Decision letter