Main Text

DA neurons projecting from the VTA to the striatum [1–3] are involved in various cognitive functions, and DA dysregulation has been associated with several neuropsychiatric and neurodegenerative disorders [4–7]. Tryptophan-hydroxylase-positive (TpH+) neurons synthesize 5-HT in the dorsal raphe nucleus (DRN) and modulates synaptic transmission in the VTA, NAc and DS. Behavioral and pharmacological studies suggest that 5-HT, acting in concertation with DA, may affect reward-seeking, decision making, associative learning, and motor planning [8, 9].

Two conflicting hypotheses regarding the interaction between the DA and 5-HT systems exist. The opponency hypothesis [10–12] suggests that DA and 5-HT have opposite functions: while DA promotes behavioral activation, 5-HT, by contrast, suppresses behavior. Pharmacological studies indicate DA and 5-HT agonists or antagonists have opposite effects on reward processing [13, 14], feeding behavior [14], and self-stimulation [15]. Recent in vivo fiber photometry recordings further support this oppositional dynamic, suggesting that DA and 5-HT exhibit inverse activity patterns in the striatum in response to reward [16] and following choice actions [17]. Moreover, 5-HT can inhibit DA activity in the VTA and substantia nigra, leading to reduced DA release in the striatum[10, 18].

Alternatively, 5-HT could directly enhance DA activity or share properties that facilitate reinforcement processing similar to DA. Local 5-HT infusion into the striatum can increase extracellular DA levels [19]. Additionally, 5-HT can facilitate DA’s behavioral effects. For example, selective serotonin reuptake inhibitors (SSRIs) increase 5-HT, and lead to a modulative effect in DA self-stimulation thresholds [20] and enhancement of cocaine’s reinforcing effects [21]. In contrast, depletion of 5-HT or its precursor blocks cocaine’s rewarding effects [22], and impair d-amphetamine’s ability to decrease impulsivity [23]. Furthermore, serotonergic activity in the dorsal raphe interacts with DA, encoding complementary aspects of reward processing [24].

The above-listed discrepancies suggest complex mechanisms underlying DA-5-HT crosstalk that remain largely elusive, underscoring a complex interplay driven by receptor-specific interactions and overlapping projections. For example, 5-HT modulation of DA activity occurs through 5-HT1A, 5-HT1B, and 5-HT2A receptor activation, which enhances DA release in the striatum and prefrontal cortex. In contrast, 5-HT2C receptor activity inhibits DA release in mesolimbic regions [25].Additionally, lesion of DA neurons decreases 5HT spontaneous firing in DRN, whereas lesion of 5-HT neurons enhanced the firing activity of DA neurons in VTA [26].

Both DA and 5-HT are involved in reinforcement and decision-making in opposing and complementary ways [27]. An imbalance between these systems is implicated in various psychiatric disorders, including depression, schizophrenia, compulsion, and addiction [28–30]. However, these interactions are complex and not simply oppositional or coherent, requiring further investigation into their synaptic crosstalk and plasticity mechanisms.

There is increasing recognition of the functional and anatomical overlap between VTA DA neurons and DRN 5-HT neurons, particularly in the striatum, where both systems have dense projections and colocalizations [16, 31]. However, few studies have addressed the bidirectional interactions between the DA and 5-HT systems in response to specific neuronal stimulation, and the interaction between dorsal raphe (DR) serotonergic neurons and striatal DA transmission remains controversial. To address this gap, we investigated the effects of selective activation of DA and serotonin neurons on both DA and 5-HT release in the striatum. Recent advancements in GPCR sensors and refined optogenetic tools allow for real- time observation of neurotransmitter dynamics, enabling targeted interventions and establishing causal links. By stimulating DA neurons in the VTA and 5-HT neurons in the DRN, we aimed to investigate the reciprocal interactions between these systems and elucidate their crosstalk mechanisms.

Our findings demonstrate that stimulation of DA neurons in the VTA alters DA but not 5-HT release in the striatum, suggesting that the 5-HT system in the striatum operates relatively independently of VTA DA activity. In contrast, stimulation of 5-HT neurons in the DRN impacts mainly 5-HT with a minor impact on DA levels in the striatum, highlighting the reciprocal regulatory effects of serotonergic activity on DA circuits. These results provide new insights into the striatum’s bidirectional interplay between DA and 5-HT systems.

Understanding the mechanisms underlying DA-5-HT crosstalk is critical for advancing our knowledge of neuromodulatory systems and developing therapeutic interventions for disorders characterized by imbalances in these networks. By simultaneously assessing DA and 5-HT release in response to targeted neuronal activation, this study contributes to a more comprehensive understanding of how these systems interact to regulate striatal function.

Results

Activating VTA DA neurons increases DA but not 5HT in the striatum

We tested whether optogenetic stimulation of VTA DA neurons results in DA and/or 5-HT in the striatum. We injected AAV-FLEX-ChrimsonR-tdTomato into the VTA of DAT-Cre mice and AAV-dLight X.XX or AAV-GRAB5HT in the NAc or DS to monitor DA or 5-HT levels in the striatum (Figure 1B, C). We found that optogenetic stimulation of VTA DA neurons (Figure 1A) evoked a rapid and robust release of DA in NAc and DS, at a level within the same range as DA increase evoked by passive cocaine injection at 15mg/kg (Figure 1D).

Fig 1.

Conversely, we did not observe any change in 5-HT levels in either NAc or DS in response to VTA DA stimulation. In contrast, cocaine readily increased 5-HT levels in the two striatal regions (Figure 1G). These findings contradict the finding that optogenetic stimulation of serotonergic neurons in the DRN led to increased DA concentration in the striatum, as measured by microdialysis [31]. These conflicting results may arise from differences in stimulation protocols, measurement modalities, neuronal subpopulations recruited, or variations in experimental conditions, highlighting the complexity of 5-HT-DA interactions in the striatum. Together, these results suggest that VTA DA neurons modulate DA release but not 5-HT release in the striatum.

Activating DRN SERT neurons increases 5HT and transiently DA in the striatum

Then, we tested whether stimulation of DRN SERT neurons produces a release of DA or 5- HT in the striatum (Figure 2B). We tested the effects of DRN SERT optogenetic stimulation. We found optogenetic stimulation of DRN SERT neurons evoked rapid and robust release of 5-HT in DS at a level that is comparable to the DA increase evoked by passive cocaine injection at 15mg/kg (Figure 2C). On the contrary, we did not observe any significant change in DA levels in either NAc or DS in response to DRN SERT stimulation. In contrast, cocaine could readily increase 5-HT levels in the two regions (Figure 2E).

Fig 2.

Some studies have reported that electrical stimulation of the DRN inhibits DA neuron activity, suggesting a suppressive influence on DA release[32–34], whereas other findings report that electrical stimulation of the DRN induces DA release in the NAc [35]. We observed a small and transient increase in DA levels in NAc after DR SERT stimulation (Figure 2C, insert), significantly different from the baseline 5 seconds after the stimulation (Figure 2D).

Glutamate-5HT co-release of DR afferents in the VTA

To further investigate this transient crosstalk, we examined the synaptic afferents onto midbrain and striatal neurons in the acute slice preparation, as DR projections have been claimed to release glutamate in addition to serotonin (REF). Combining optogenetic projection targeting and whole-cell recordings (Figure 3A) allowed parsing the synaptic currents in identified regions. Glutamatergic innervation was absent in DS (Figure 3B) and anterior NAc Shell (Figure 3C) but observed in OFC (Figure 3D) and posterior NAc Shell (Figure 3E), as well as the VTA (Figure 3F). These results suggest that DRN SERT neurons modulate 5-HT releases in the striatum and indirectly affect DA release. This might be explained by the 5-HT neurons projecting from the DRN also sending glutamatergic projections to VTA, stimulating DA neurons and causing a brief release of DA in the striatum.

Fig 3.

Discussion

Our findings demonstrate that optogenetic stimulation of DRN SERT and VTA DA neurons results in minimal crosstalk between striatal DA and 5-HT, indicating that these neurotransmitter systems operate largely independently. This challenges earlier studies that reported DRN stimulation increases striatal DA [31] — a discrepancy likely due to differences in measurement techniques. Microdialysis combined with HPLC may fail to fully distinguish 5-HT from DA because 5-HT shares similar physicochemical properties and overlapping electrochemical signals with 3-methoxytyramine (3-MT, a metabolite of DA)[36].

We employed advanced GPCR-based sensors with high selectivity and sensitivity to overcome these limitations, ensuring a more accurate assessment of neurotransmitter dynamics [37, 38]. Stimulation of DRN SERT neurons elicited a robust, rapid increase in striatal 5-HT levels—comparable to the DA surge induced by cocaine—yet did not significantly alter DA levels in the NAc or DS, except for a transient increase in the NAc. This modest DA change is consistent with previous findings[35] and may be explained by DRN 5-HT neurons projecting to the VTA and co-release glutamate, which can also excite VTA DA neurons and evoke DA release in NAc .

Moreover, our analysis revealed that the 5-HT-glutamate co-release mechanism was present in the OFC and posterior NAc shell but absent in the DS and anterior NAc shell, underscoring the region-specific effects of DRN SERT activation. Our results refine the understanding of DArgic and serotonergic interactions in the striatum, highlighting their functionally distinct roles. These insights provide a foundation for future research into the interplay between these systems in cognitive and behavioral functions, with implications for neuropsychiatric disorders.

Methods

Animals

DAT-IRES-Cre (B6.SJL-Slc6a3^{tm1.1(cre)Bkmn}/J) and SERT-Cre mice (B6.129(Cg)- Slc6a4tm1(cre)Xz/J) were from the Jackson Laboratory. Upon the arrival of the animals a period of 7 days of habituation was made. Both male and female mice aged from 8-12 weeks were used and group housed in a temperature and humidity-controlled environment, under a 12 h light/dark cycle, and provided with food and water ad libitum.Weights and genders were distributed homogeneously among the groups if possible. All behavioral procedures were performed during the light cycle. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Geneva and by the animal welfare committee of the Cantonal of Geneva, in accordance with Swiss law (GE131A_33975).

Virus injection and implantation

Mice (age 8-12 weeks) were deeply anesthetized with a mixture of Isoflurane (w/v, induction 5%, maintenance 2%, Attane^tm) and O₂ (Compact anesthesia station from Minerve) during surgery and then secured in a stereotaxic frame (Angle One). Before craniotomy, body temperature was maintained at 37 °C with a temperature controller system and Lacryvisc (Alcon, Switzerland) was applied to prevent the eyes from drying. Lidocaine was applied on the surface of the epicranium. An incision was made to expose the bregma and lambda point of the skull. The skull above the target area was thinned with a dental drill and carefully removed. Viruses were injected with glass pipette at a rate of 50 nl/min. The amount of virus per injection site was 350-500 nl. After injection, the pipette was left in the place for 10 min to allow diffusion of the virus. The skin was sutured and disinfected after the injection. 500 µl saline was i.p. injected. Paracetamol (2 mg/ml in the water bottle) was given orally for the next 2-4 days.

(anterior posterior (AP): −3.28; medio–lateral (ML): −0.9; dorso-ventral (DV): −4.3; with a 10° angle)

To express opsins, AAV8-syn-Flex-Chrimson-tdTomato (UNC) was injected in VTA (anterior posterior (AP): −3.28; medio–lateral (ML): −0.9; dorso-ventral (DV): −4.3; with a 10° angle) of DAT-Cre mice or DRN (AP: −4.5; ML: 0; DV: −3) of SERT-Cre mice. To record DA release, an AAV5-CAG-dLight1.2 (400 nl, from Addgene) was injected in NAc (AP: +1.6; ML: ±0.75; DV: −4.3) and DS (AP: +1.3; ML: ±0.1; DV: −3.3). To record 5-HT release, AAV9-hSyn-5-HT3.0 (400 nl, from BrainVTA) was injected in NAc (AP: +1.6; ML: ±0.75; DV: −4.3) and DS (AP: +1.3; ML: ±0.1; DV: −3.3).

During the same surgical procedure, three screws were fixed into the skull to secure the optical implant. An optic fiber (0.4 mm diameter, MFC_400/430_0.48_4mm_ZF2.5(G)FLT, Doric Lenses) was implanted 150 µm above the virus injection site for in vivo fiber photometry recording. Another optic fiber (0,2 mm diameter, FOC-W-1.25-200-0.37-5.0, Inper) was implanted 200 µm above the virus injection site for optogenetic stimulation. Then the optic fibers were fixed to the skull with dental cement. After surgery mice were given 7 days of recovery and were habituated to handling.

For electrophysiological studies examining DRN SERT connectivity, AAV8-syn-Flex- Chrimson-tdTomato (UNC) was injected in DRN (AP: −4.5; ML: 0; DV: −3) of SERT-Cre mice.

Behavioral Paradigm

After 4-5 week of viral expression, mice were habituated to handling and to the connection cable for three day before testing. On the testing day mice were connected to the optogenetics cable and fiber photometry cable and placed in a transparent custom build open-field (30 X 30 X 20 cm) for 3 min of habituation before the start of recording. Then, 5 min of baseline recording were made. Animal received 5 pulses of laser stimulations, each composed of 30 bursts separated by 250 ms (each burst consisted of 5 laser pulses of 4-ms pulse width at 20 Hz). After another 5 minutes of baseline recording, animals received i.p. injection of cocaine (15mg/kg), and the neuronal activity was recorded for another 40 minutes.

Optogenetic stimulation

Optic fiber for optogenetic stimulation of mice were connected via patch cords (MFO-F- W1.25-200-0,37-100, Inper) to a rotary joint (FRJ_1 × 2_FC-2FC; Doric Lenses, Quebec, Canada), suspended above the operant chamber. A second patch cord was connected from the rotary joint to an orange DPSS laser (SDL-593–100 mW; Shanghai Dream Lasers; Shanghai, China) positioned outside of the context. Laser power was typically 15–20 mW measured at the end of each patch cord. A mechanical shutter was used to control laser output (SR474 driver with SR476 shutter head; Stanford Research Systems, aligned using a connectorized mechanical shutter adaptor; Doric Lenses).

Fiber Photometry recordings

Fiber photometry was performed similar to before (33). During recordings, excitation (470 nm, M470F3, Thorlabs) and control LED light (405 nm, M405FP1, Thorlabs) was passed through excitation filters and focused onto a patch cord. Both LED lights were sinusoidally modulated at 211 and 531 Hz (470 nm and 405 nm light, respectively) and light was passed through a mini cube (FMC4_AE(405)_E(450-490)_F(500-550)_S) onto the optic fiber patch cable (MFP_400/430/1100–0.48_4 m_FC-ZF2.5, Doric Lenses) that was connected to the chronically implanted fiber (MFC_400/430- 0.48_6mm_ZF2.5(G)_FLT, Doric Lenses). Light intensity at the tip of the patch cable was around 0.25 mW. Emission light travelled back through the same fibers onto the mini cube and a photo-receiver (Newport 2151, Doric Lenses), after which it was digitized, demodulated and stored using a signal processor (RZ5P, Tucker Davis Technologies).

The sample rate was 101.725 Hz, signals were low-pass filtered online at 3 Hz.

The data were analyzed using MATLABR2024 (MathWorks). First the signal during baseline acquisition originating from the 405 nm excitation source was linearly regressed to the signal originating from the 470 nm excitation source, and scale to the 470 nm originating signal. ΔF/F was then computed as (470⃞nm signal – fitted 405⃞nm signal)/fitted 405⃞nm signal. The average ΔF/F signal in the baseline periods before experimental intervention was then subtracted to normalize the signal to baseline. Finally, the normalized signal was binned into appropriate time bins in the graphs and analyses.

Electrophysiology recording ex vivo

SERT-Cre mice were injected with AAV-FLEX-Chirmsom-tdT in the DRN to detect potential glutamate corelease from 5-HT neurons. Three weeks after injection, mice were deeply anesthetized with 5% isoflurane and subsequently decapitated after confirmation of absent of toe and tail reflexes.

92⃞mM NMDG, 20⃞mM HEPES, 25⃞mM glucose, 30⃞mM NaHCO3, 2.5⃞mM KCl, 1.2⃞mM NaH2PO4 (pH 7.35, 303–306⃞mOsm) and saturated with 95% O2/5% CO2. Coronal slices (300 µm) containing the OFC, DS, NAc anterior, NAc posterior and VTA were prepared at a speed of 0.07 mm/s using a vibratome (Leica, VT1200). After slicing, sections were allowed to recover at 34°C in bubbled NMDG-based for 5-10 min, before being transferred to a holding chamber filled with modified holding aCSF saturated with 95% O2/5% CO2 containing 92⃞mM NaCl, 20⃞mM HEPES, 25⃞mM glucose, 30⃞mM NaHCO₃, 2.5⃞mM KCl, 1.2⃞mM NaPO₄ (pH 7.35, 303–306⃞mOsm) at room temperature for at least 1⃞h.

For recordings, slices were kept at 30 °C in a recording chamber perfused with 1.5 – 2.0 ml/min ACSF containing 126⃞mM NaCl, 2.5⃞mM KCl, 1.4⃞mM NaH₂PO₄, 1.2⃞mM MgCl₂, 2.4⃞mM CaCl₂, 25⃞mM NaHCO₃ and 11⃞mM glucose (303– 305⃞mOsm). Neurons were visualized with a ×40 water-immersion objective on an Olympus BX5iWI inverted microscope equipped with infrared-differential interference contrast (IR- DIC) optics and epifluorescence (Olympus Corp). Td-tomato-positive cells were classified based on strong fluorescence, while td-tomato-negative cells were those adjacent to td- tomato-positive cells, but lacking fluorescence. Whole cell recordings were amplified (Multiclamp 700B, Axon Instruments), filtered at 5 kHz and digitized at 20 kHz (National Instruments Board PCI-MIO-16E4, Igor, Wave Metrics). Data were rejected if the access resistance changed more than 20%. Optogenetic EPSCs were evoked with single or paired pulses of 0.1 Hz, with an interstimulus interval of 50 ms.), and recorded in the presence of PTX (100 µM, Tocris).

Histological analysis

Brains were fixed with 4% paraformaldehyde (PFA) for at least 24 h and sliced with vibratome (Leica, VT1200). 50 µm thick slices were washed with 3 times PBS for 5 min and blocked with 10% bovine serum albumin (BSA) dissolved in 0.5% TritonX-100 for 1 hour at room temperature. Slices were incubated with primary antibodies dissolved in blocking buffer overnight at 4 ⃞, followed by 4 times 15 min wash with PBS at room temperature.

After that, slices were incubated with fluorescent-conjugated secondary antibodies dissolved in blocking buffer for 2 h at room temperature followed by 4 times 15 min wash with PBS. Last, slices were mounted using mounting medium containing DAPI (Fluoroshiel, Abcam). Primary and secondary antibodies are listed below: Rabbit anti GFP (1:500, Invitrogen, A11122, RRID: AB_221569); Goat anti Rabbit 488 (1:500, Invitrogen, A11008, RRID: AB_143165).

Statistical analysis

Statistical analysis was performed in GraphPad Prism 9. For all tests, the significance threshold was placed at α = 0.05. Gaussian distribution was evaluated using D’Agostino & Pearson normality test. Multiple comparisons were first subject to mixed-factor ANOVA defining both between- and/or within-group factors. Where significant main effects or interaction between factors were found (P < 0.05), further comparisons were made by a two-tailed Student’s t-test with Bonferonni corrections applied when appropriate (that is, the level of significance equaled 0.05 divided by the number of comparisons). Mann- Whitney or Friedman test were used for non-Gaussian distributions when appropriate. Single comparisons of between- or within-group measures were made by two-tailed non-paired or paired Student’s t-test, respectively.

Acknowledgements

We thank all the Lüscher lab members for their comments on the manuscript. This work was supported by the Swiss National Science Foundation (grant no. 310030_219470) and European Union’s Horizon 2020 Research and Innovation Program DEEPER project (grant no. 101016787) to C.L.

Additional information

Author information

Conceptualization: YL, CL

Investigation: YL, JET

Formal Analysis: YL, JET

Visualization: YL, CL, JET

Funding acquisition: CL

Supervision: CL

Writing – original draft: YL, CL

Writing – review & editing: YL, CL, JET

Materials & correspondence

Corresponding author email: christian.luscher@unige.ch

Funding

Swiss National Science Foundation (310030_219470)

European Union (101016787)