Synchronous activation of striatal cholinergic interneurons induces local serotonin release

Domain: biology
Published: 2026-03-16
Editorial outcome: major_revision

Abstract
Striatal cholinergic interneurons (CINs) can drive local dopamine release via nicotinic acetylcholine receptors (nAChRs) expressed on dopaminergic axons, but their role in modulating serotonin (5-HT) signaling is poorly understood. Here, we show that synchronous activation of CINs directly triggers local 5-HT release in the dorsal striatum via nAChRs expressed on serotonergic axons. This CIN–5-HT coupling is not detectable in the ventral striatum, despite its substantially denser serotonergic innervation. The nAChR-dependent release not only increases 5-HT levels in the dorsal striatum, but also expands the spatial footprint of serotonergic signaling. In Sapap3-/- mice, a model of obsessive-compulsive disorder (OCD)-like behavior, this mechanism is exaggerated due to a hypercholinergic state, selectively amplifying the nAChR-dependent component of monoamine release. These findings demonstrate a regionally confined form of acetylcholine–5-HT crosstalk in the striatum and identify CINs as regulators of 5-HT dynamics in both healthy and pathological states.

Introduction
As the main input nucleus of the basal ganglia, the dorsal striatum guides goal-directed and habit learning by plastically adjusting how it processes cortical inputs1,2. Most of the relevant processes are shaped, at least in part, by the monoamine serotonin (5-HT) – this includes long term depression at corticostriatal synapses3, lateral inhibition between spiny projection neurons4 and release of another monoamine (dopamine) from nigrostriatal projections5. As such, regulation of 5-HT signaling is crucial to striatal function6.

Dysregulated striatal 5-HT signaling has been implicated in numerous neurological, psychiatric and addictive disorders; strikingly, pathological changes in striatal 5-HT signaling may occur in tandem with changes in both dopamine and acetylcholine7–10. This may not be a coincidence. It has become apparent that monoamines and acetylcholine (ACh) can intimately shape each other’s release in the striatum: for example, dopamine regulates the excitability of striatal cholinergic interneurons11 (CINs), and ACh release from CINs directly facilitates dopamine release by engaging nicotinic ACh receptors (nAChRs) on nigrostriatal axons12–16. Whether such an interplay extends to 5-HT is unclear: it is well-established that 5-HT directly regulates the activity of CINs (and presumably ACh release) in a region-specific manner17,18, but evidence for direct functional control of 5-HT release by ACh is limited19–21. The main source of striatal 5-HT is a long range projection from the dorsal raphe nucleus22–25, and serotonergic axons within the striatum express nAChRs. We therefore sought to determine if CIN-derived ACh can directly facilitate local 5-HT release, in an nAChR-dependent way.

Here, we show that synchronous activation of CINs triggers local 5-HT release in the dorsal, but not ventral, striatum via nAChRs expressed on serotonergic axons. We further show that CIN–5-HT coupling expands the spatial extent of 5-HT release and is selectively amplified in the Sapap3-/- mouse model of obsessive–compulsive disorder (OCD)–like behavior.

Results
Local nAChR-dependent 5-HT release in the dorsal–but not ventral–striatum
We stereotaxically inoculated the dorsal striatum of mice with an adeno-associated virus (AAV) harboring the genetically-encoded green fluorescent 5-HT sensor, GRAB-5HT26, and when the sensor was expressed, we prepared acute brain slices through the dorsal striatum (Fig. 1a). Intrastriatal electrical stimulation induced a robust increase in GRAB-5HT fluorescence, which decayed over 10s of seconds, as measured using two-photon laser scanning microscopy (2PLSM) (Fig. 1b) (see Methods). Application of the non-selective nAChR antagonist mecamylamine (10 µM) significantly reduced the amplitude of the striatal GRAB-5HT response (averaged over the region-of-interest, see Methods; Fig. 1a–c), suggesting that, as is the case for dopamine (DA)12–16,27, nAChR activation also facilitates striatal 5-HT release [N=3 mice, n=9 slices, t 16=–5.57, P=4.25·10–5, linear-mixed effect model (LMEM)].

Bath application of DA (10 µM) had no effect on GRAB-5HT fluorescence in slices (Supplementary Fig. 1a–c), confirming that changes in sensor fluorescence were not the result of nonselective detection of DA26. We further confirmed the specificity of GRAB-5HT fluorescence in our experiments by demonstrating that 1) RS 23597-190 (35 µM; a selective antagonist of the 5-HT 2C receptor, on which the GRAB-5HT design is based) eliminated evoked fluorescence (N=3 mice, n=6 slices, t 10=–3.45, P=0.006, LMEM) (Supplementary Fig. 2a, b) and 2) citalopram (30 µM), a selective 5-HT reuptake inhibitor (SSRI), significantly reduced the amplitude of the 5-HT release (N=4 mice, n=12 slices, t 22=–4.64, P=1.3·10–4, LMEM), likely due to presynaptic inhibition of release by 5-HT 1B auto-receptors18,28–32, and significantly slowed the temporal decay of evoked fluorescence (N=4 mice, n=12 slices, t 22=–4.29, P=3·10–4, LMEM), neither of which could be attributed to the passage of time (Supplementary Fig. 2c–h).

Compared to the dorsal striatum, the ventral striatum receives substantially denser 5-HT innervation33,34. We therefore tested whether nAChRs also facilitate 5-HT release in ventral striatum. Surprisingly, we found that while electrical stimulation elicited robust increases in GRAB-5HT fluorescence in the ventral striatum, their amplitude was not reduced by mecamylamine (Fig. 1d–f N=4 mice, n=23 slices, t 40=0.399, P=0.692, LMEM), suggesting that nAChRs do not contribute to 5-HT release in the ventral striatum.

nAChRs extend the spatial range of striatal 5-HT release
We and others have shown that activation of nAChRs on nigrostriatal axon terminals not only increases the amount of DA release, but also nearly doubles the spatial range of its release12,13. We therefore measured the spatial extent of GRAB-5HT responses by quantifying evoked fluorescence at 100 µm-wide increments from the stimulating electrode and fitting a Lorentzian function to the maximal responses (N=3 mice, n=9 slices) as a function of distance (Fig. 1g, h). The Lorentzian fell off with a length scale of 183 µm under control conditions. Mecamylamine application not only reduced the overall amplitude of the response (t 158=–5.8, P=3.55·10–8, LMEM), but also significantly shortened the length scale of the Lorentzian to 126 µm (t 158=2.68, P=0.0081, LMEM), indicating that nAChRs increase the spatial extent of striatal 5-HT release by approximately 45% (Fig. 1h).

Optogenetic activation of CINs saturates striatal nAChR-dependent 5-HT release
We observed that intrastriatal electrical stimulation in the dorsal striatum promotes nAChR-sensitive release of 5-HT, but can endogenous ACh released from CINs drive nAChR-dependent release of striatal 5-HT on its own? To this end, we inoculated the dorsal striatum of ChAT-Cre mice (which express Cre recombinase under the choline acetyltransferase promoter in CINs) with a mixture of the AAVs harboring GRAB-5HT (broadly labeling the dorsal striatum) and DIO-channelrhodopsin-2 (ChR2)-mCherry (specifically labeling CINs) (see Methods) (Fig. 2a; Supplementary Fig. 3a). The two-photon laser scanning persedid not alter the electrophysiological behavior of CINs (N=5 mice, n=8 CINs, t 26=–1.35, P=0.18, LMEM) (Supplementary Fig. 3d–i). A brief (1 ms) 470 nm (32 mW) LED stimulation, which is sufficient to drive a time locked action potential in recorded CINs (Supplementary Fig. 3f), resulted in a significant increase in GRAB-5HT fluorescence, with a slow decay lasting tens of seconds (Fig. 2b). Evoked changes in GRAB-5HT fluorescence were not an optical artifact of blue light stimulation, as: 1) under our experimental conditions, 470 nm LED simulation on its own did not alter GRAB-5HT fluorescence (Supplementary Fig. 1d, e), and 2) CIN-dependent 5-HT release could be replicated by optogenetically activating CINs using the red-shifted opsin [using 1 ms, 590 nm (7.3 mW) LED pulses in dorsal striatal brainslices from ChAT-cre mice inoculated with a mixture of GRAB-5HT and cre-dependent ChrimsonR-tdTomato] (Supplementary Fig. 4). Thus, synchronous activation of CINs is sufficient to evoke 5-HT release in the dorsal striatum. The GRAB-5HT signal was completely abolished by 10 µM mecamylamine or dihydro-β-erythroidine (DHβE), suggesting that CIN-evoked 5-HT release is mediated by α4β2 type nAChRs [ChR2: N=3 mice, n=9 slices with mecamylamine, N=1 mouse, n=3 slices with DHβE (pooled) t 22=–7.9., P=7.3·10–8, LMEM; Chrimson: N=2 mice, n=11 slices (all with mecamylamine), t 20=–7.18, P=5.9·10–7, LMEM] (Fig. 2b, c; Supplementary Fig. 4b, c).

Strikingly, the increase in GRAB-5HT fluorescence induced by synchronous optogenetic activation CINs is similar to the nAChR-dependent component of the GRAB-5HT signal evoked by electrical stimulation (Fig. 2d) (N=9 mice, n=25 slices, t 23=0.01, P=0.99, LMEM), suggesting that CINs are likely the sole source of ACh that promotes release of 5-HT in the dorsal striatum. Consistent with the results presented above using electrical stimulation (Fig. 1d–f), optogenetic stimulation of CINs failed to elicit detectable 5-HT release in ventral striatum (Supplementary Fig. 5) (N=7 mice, n=25 slices, t 45=0.95 P=0.35, LMEM).

Is CIN control of 5-HT release direct, or are the key ACh receptors on striatal loci other than serotonergic axons? To rule out the involvement of postsynaptic nAChRs on GABAergic interneurons35, presynaptic nAChRs on glutamatergic inputs36 or muscarinic ACh receptors in general37,38 we repeated the above experiment in the presence of GABA, glutamate and muscarinic ACh receptor antagonists (10 µM SR95531, 2 µM CPG55845, 10 µM DNQX, 50 µM D-AP5 and 10 µM atropine). This antagonist cocktail had no significant effect on either the amplitude (N=3 mice, n=8 slices, t 21=–0.59, P=0.56, LMEM) or the temporal decay (t 21=–1.98, P=0.061, LMEM) of the CIN-evoked 5-HT release, which was eliminated by subsequent blockade of nAChRs (Fig. 2e–g). Together, these data suggest that ACh acts directly on nAChRs located on 5-HT fibers to mediate 5-HT release [t 21=–6.02, P=5.69·10-6 (ACSF vs. mec), t 21=–5.42, P=2.19·10-5 (cocktail vs. mec), LMEM]. Nevertheless, to determine whether nAChRs amplify a putative co-release of 5-HT from DA axons39, or any other mechanism by which excitation of striatal DA axons may indirectly cause the release of striatal 5-HT, we inoculated 1) the substantia nigra pars compacta (SNc) of TH-Cre mice (which express Cre-recombinase under the tyrosine hydroxylase promoter) with an AAV harboring DIO-ChR2-mCherry and 2) the dorsal striatum with an AAV harboring GRAB-5HT (Supplementary Fig. 6a–d). Direct optogenetic activation of DA fibers induced a small but significant increase in GRAB-5HT. The increase, however, was only about a fifth of the size of the GRAB-5HT signal generated by synchronous optogenetic activation of CINs (Fig. 2c, e, Supplementary Fig. 4) and was insensitive to mecamylamine, indicating that DA axons do not contribute to the nAChR-dependent release of striatal 5-HT (Supplementary Fig. 6e, f).

nAChR-dependent monoamine release is elevated in a mouse model of OCD
If CINs induce the release of 5-HT under physiological conditions, then in a pathophysiologically hypercholinergic striatum this mechanism should be amplified. We recently reported that Sapap3-/- mice, which display OCD-like motor behaviors such as compulsive grooming, exhibit a hypercholinergic phenotype in the striatum: the density of CINs is higher, their autonomous firing rate is higher, and they release more ACh in response to electrical stimulation in acute striatal slices40. We therefore asked whether cholinergic modulation of 5-HT release is amplified in Sapap3-/- mice. Indeed, intrastriatal electrical stimulation in the dorsal striatum induced significantly larger GRAB-5HT fluorescence signals in the dorsal striatum of Sapap3-/- mice, and this difference disappeared in the presence of mecamylamine (Fig. 3). Supporting the conclusion that elevated 5-HT release is the result of increased CIN-mediated ACh release, only the mecamylamine-sensitive component of the GRAB-5HT signal was elevated in Sapap3-/- mice (Fig. 3c), suggesting that basal striatal 5-HT release capacity is unaltered. Corroborating that ACh facilitates 5-HT release by a mechanism similar to the one by which it facilitates DA release, intrastriatal electrical stimulation also induced significantly higher nAChR-sensitive (but not nAChR-insensitive) striatal DA release in Sapap3-/- mice (Supplementary Fig. 7). Overall, this is consistent with the observation that 5-HT turnover (but not baseline levels) is elevated in Sapap3-/- mice41.

Methods
Animals
Experimental procedures adhered to and received prior written approval from the Institutional Animal Care and Use Committees (IACUC) of the Hebrew University of Jerusalem (HUJI, protocol number MD-20-16113-3) and Stony Brook University (SBU). Experiments were carried out in the following mouse strains: 1. C57BL/6J mice (Strain #: 000664; Jackson Laboratories, Bar Harbor, ME, USA); 2. ChAT-IRES-Cre (Δneo) transgenic mice (stock number 031661; Jackson Laboratories, Bar Harbor, ME, USA); 3. TH-Cre1 (Strain #: 008601; Jackson Laboratories, Bar Harbor, ME, USA, RRID:IMSR_JAX:008601); and 4. Sapap3 conditional knock-in mice40. All Sapap3-/- mice lacked both copies of the Sapap3 gene. Two-to-seven-month-old C57BL/6J and transgenic mice were used for experiments. Sapap3-/- mice were 3-7 months old, when they exhibited compulsive grooming. Sex was not considered as a factor in the design of the experiments, and mice of both sexes were used and their results pooled. All mice were housed under a 12 h light/dark cycle with food and water ad libitum. Ambient temperatures and humidity were 22°C ± 2°C and 50 ± 10%, respectively.

Stereotaxic surgeries
At HUJI. Mice were deeply anesthetized with isoflurane in a non-rebreathing system (2.5% induction, 1–1.5% maintenance) and placed in a stereotaxic frame (model 940, Kopf Instruments, Tujunga, CA, USA). Temperature was maintained at 35°C with a heating pad, antibiotic eye ointment was applied to prevent corneal drying, and animals were hydrated with a bolus of injectable saline (5 ml/kg) mixed with an analgesic (5 mg/kg meloxicam). To calibrate specific injection coordinates, the distance between bregma and lambda bones was measured and stereotaxic placement of the mice was readjusted to achieve maximum accuracy. A small hole was bored into the skull with a micro drill bit and a glass pipette with AAVs attached to Nanoject III system (Drummond) was slowly inserted at the relevant coordinates under aseptic conditions. The coordinates of dorsal striatum (DS) injection were: +0.5 mm anteroposterior (AP); +/- 2.3 mm mediolateral (ML); −2.8 mm dorsoventral (DV), and for the SNc: -3.1 mm anteroposterior (AP); +/- 1.2 mm mediolateral (ML); −4.2 mm dorsoventral (DV) relative to bregma using a flat skull position. The coordinates of the nucleus accumbens (NAc) core injection were: +1.5 mm anteroposterior (AP); +/- 0.9 mm mediolateral (ML); −4.1 mm dorsoventral (DV). To minimize backflow, solution was slowly injected (1 nl/second) and the pipette was left in place for 5–7 min before slowly retracting it. For 5-HT sensor experiments, a total amount of 500 nl of an adeno-associated virus harboring the green-fluorescent GRAB-5HT3.5 construct (AAV-9 hSyn-5HT3.5; 2.20 × 1013 vg/ml; WZ Biosciences Inc. Lot No. 20201210), recently renamed g5-HT2h21, was injected into DS or 300 nl to the NAc. For optogenetic stimulations of CINs a total amount of 650 nl adeno-associated virus cocktail of GRAB-5HT3.5 construct and ChR2 construct (AAV5/ Ef1a-DIO-hchR2(H134R)-mCherry; 4 × 1012 vg/ml; UNC vector core; Lot # AV4313-2A) or Chrimson construct (pAAV/ Syn-FLEX-rc[ChrimsonR-tdTomato]; 12 × 1012 vg/ml; Addgene #62723 – AAV5; Lot # v122159) was injected into DS of ChAT-IRES-Cre mice; for optogenetic stimulation of dopamine axons 500 nl of GRAB-5HT3.5 was injected into the DS and 250 nl of the ChR2 construct into the SNc of TH-Cre mice.

At SBU (experiments involving Sapap3-/- mice). Mice were induced and maintained under anesthesia via inhalation of vaporized isoflurane (3% induction, 1–2% for maintenance). Prior to any surgical procedures, sedation was confirmed via toe pinch, then animals were placed in a stereotaxic apparatus, the scalp was shaved and disinfected with 70% ethanol and Betadine solution, and eyes were covered with protective gel (Puralube Ophthalmic Ointment, Dechra Veterinary Products). A 1 µL Hamilton Neuros micro-volume syringe was used for unilateral DS injections (0.5-62 mm AP; −2.2-3 mm ML; −2.8 mm DV) and manipulated via a motorized stereotaxic injector (Quintessential Stereotaxic Injector QSI, Stoelting Co.) guided by Angle Two software (Leica Biosystems). 500 nl of AAV9-hSyn-5HT3.5 (≥1x1013 vg/ml) or AAV9-hSyn-dLight1.1 (≥1x1013 vg/ml) were injected over 5 min and a further 5 min was allowed for diffusion before slowly withdrawing the needle. All mice were administered analgesic (Meloxicam, 5 mg/kg, subcutaneous injection) immediately after surgery and monitored daily for 72 h.

Acute slice preparation
At HUJI. After prolonged incubation time (at least 3 weeks), inoculated mice were deeply anesthetized with ketamine (200 mg/kg)–xylazine (23.32 mg/kg) and perfused transcardially with ice-cold modified artificial cerebrospinal fluid (ACSF) bubbled with 95% O2–5% CO2, and containing (in mM): 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 0.5 CaCl2, 10 MgSO4, 0.4 ascorbic acid, 10 glucose, and 210 sucrose. The brain was removed and sagittal slices sectioned at a thickness of 275 µm were obtained in ice-cold modified ACSF. Slices were then submerged in ACSF, bubbled with 95% O2–5% CO2, and containing (in mM): 2.5 KCl, 126 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, and 10 glucose, and stored at room temperature for at least 1 h prior to recording.

At SBU (experiments involving Sapap3-/- mice). 2-5 weeks after surgery, acute coronal slices (275 µm) containing DS were obtained from mice following anesthetization with ketamine/xylazine (100 mg/kg / 7 mg/kg) and transcardial perfusion with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 3 KCl, 1 CaCl2, 1.5 MgCl2, 26 NaHCO3, 1 NaHPO4, and 14 glucose. Slices were cut using a VT-1000 S vibratome (Leica Microsystems, Buffalo Grove, IL) and transferred to a holding chamber where they were incubated at 32°C for 45 min in ASCF containing (in mM) 2 CaCl2 and 1 MgCl2, then kept at room temperature until recording. During recordings, slices were perfused with ACSF heated to 32°C. All solutions were continuously bubbled with carbogen (95% O2 and 5% CO2).

Slice visualization and 2PLSM imaging
At HUJI. Slices were transferred to the recording chamber of Femto2D-resonant scanner multiphoton system (Femtonics Ltd., Budapest, Hungary) and perfused with oxygenated ACSF at 32°C. A ×16, 0.8 NA water immersion objective was used to examine the slice using oblique illumination.

2PLSM monoamine imaging and electrical/optogenetic stimulation. The 2PLSM excitation source was a Chameleon Vision 2 tunable pulsed laser system (680–1080 nm; Coherent Laser Group, Santa Clara, CA). Optical imaging of GRAB-5HT3.5 signals was performed by using a 920-nm excitation beam (10 mW after the objective), cholinergic mCherry imaging was performed by using a 1060 nm excitation beam. The fluorescence emission was detected and collected with gated GaAsP photomultiplier tubes (PMTs) for high sensitivity detection of fluorescence photons as part of the Femto2D-resonant scanner. Optical and electrophysiological data were obtained using the software package MESc (Femtonics, Budapest, Hungary), which also integrates the control of all hardware units in the microscope, opotogenetics & electrical stimulator. The software automates and synchronizes the imaging signals and stimulations (electrical/optical). Data were extracted from the MESc package (Femtonics) to personal computers using custom-made code in MATLAB (MathWorks, Natick, MA, USA) code.

Fields-of-view (FoV) of approximately 307 µm × 307 µm were selected and imaged at 31 Hz. Scans were performed using 0.6 µm pixels. Regions-of-interest (ROI) where fluorescence was highly expressed in the slice were identified automatically via custom MATLAB code binarizing the image by pixel intensities. The system is also equipped with full-field 470 nm and 590 nm LED illumination, which was used to stimulate the CINs in the ChAT-IRES-Cre mice transfected with DIO-hchR2(H132R)-mCherry, or with FLEX-rc[ChrimsonR-tdTomato], respectively. We empirically chose a LED stimulus amplitude (32 mW and 7.3 mW, respectively, after the objective) that reliably gave measurable GRAB-5HT responses, and a pulse duration of 1 ms as this sufficed to reliably trigger a single action potential in CINs (Supplementary Fig. 3f). Five milliseconds before the LED pulse, the gated PMTs were turned off for a duration of 50 ms. Nevertheless, despite removal of the voltage from the dynode of the PMT the massive flux of blue photons creates a huge photocurrent on the GaAsP photocathode which subsequently saturate the sensor’s electronics leading to a light artifact in the signal lasting approximately 250 ms, evident even in non-expressing slices from wild type mice. Therefore, the first 258 ms (8 samples at 31 Hz) after any LED stimulation were removed from the data of all optogenetic experiments, which precludes observing the initial rise of the GRAB signals. When using ChrimsonR-tdTomato (Supplementary Fig. 4), for consistency, the same procedures were carried out. Electrical stimulation was carried out by a parallel bipolar Platinum-Iridium electrode with diameter of 125 µm and spacing of 500 µm (FHC, PBSA0575). The magnitude of the stimulus was controlled through a stimulus isolator (ISO-Flex, MicroProbes) while the frequency and duration were controlled by computer software (MESc, Femtonics). A total of 10 pulses (10 Hz, 2 ms duration, 3 mA) were delivered to elicit a maximal response (Supplementary Fig. 8)13. To obtain sufficient signal-to-noise-ratio each optogenetic experiment was repeated 9 times, and each electric stimulation experiment was repeated 4 times with a 60 second inter-stimulus-interval. Traces were averaged over all repetitions and smoothed with a Gaussian filter with a width of approximately 400 ms. Fluorescent changes over time (ΔF/F0) were calculated as ΔF/F0 = (F(t) − F0)/F0, where F is the maximal fluorescent value within 5 seconds of the stimulation recorded while evoking electrical / optogenetic stimulations; and where F0 denotes the averaged baseline fluorescence (1-3 s long, as appropriate for each experiment) before the stimulation.

Slopes for ACSF, Cocktail and Citalopram conditions were extracted by a linear fit from the peak amplitude to the end of the decay duration. In Fig. 2b, d and Supplementary Fig. 2a, amplitude and slopes were reported after subtraction of the slope of the mecamylamine condition to account for photobleaching using the DETREND command in MATLAB.

Time lapse imaging. One-second-long time lapse imaging (Supplementary Fig. 1) were taken with ~30 seconds interval. Each point was calculated as the mean fluorescence intensity in the ROI (method explained above) over entire recording session (parameter mentioned above). 10 µM DA or 10 µM 5-HT were diluted in ACSF (see above) and perfused onto recording chamber for ~15 minutes.

Estimation of GRAB-5HT spatial scale. In Fig. 1h, ΔF/F0 data points were fitted with a Lorentzian function a = [1 + (x/λ)2]−1 to extract the length scale λ of the fluorescence signals’ decay with distance. Individual mice were considered independent samples.

Whole cell recording and electrophysiology. For whole-cell current-clamp recordings from CINs, the pipette contained (in mM): 135.5 KCH3SO4, 5 KCl, 2.5 NaCl, 5 Na-phosphocreatine, 10 HEPES, 0.2 EGTA, 0.21 Na GTP, and 2 Mg ATP (pH = 7.3 with KOH, 280–290 mOsm/kg). CINs were identified by expression of red mCherry molecules along with their morphology, spontaneous firing and by the presence of a voltage-sag (due to their prominent HCN currents).

At SBU (Slice 2PLSM 5-HT and dopamine sensor imaging in experiments involving Sapap3-/- mice). Recordings were made using an Ultima Laser Scanning Microscope system (Bruker Nano, Inc, Middleton, WI) equipped with an Olympus 60X/1.0 water-dipping lens (Olympus, Center Valley, PA) and Chameleon Ultra II laser (Coherent, Inc., Santa Clara, CA). To evoke 5-HT or dopamine release, a bipolar stimulating microelectrode (FHC Inc., Bowdoin, ME) powered by an ISO-Flex Stimulus Isolator (Microprobes for Life Science) was embedded into the surface of the slice just outside the 60x objective field-of-view (200 µm2 FOV area). 2-photon laser line scans in the shape of a spiral (920 nm, 10 µs/pixel dwell, 21.2-83 ms/line) covering the entire FOV were acquired while delivering electrical stimuli (1 s at 10 Hz, 2 ms pulse width, 3 mA for 5-HT; single 1 ms pulse for dopamine). Line scans began 2 s before stimulation and continued for 57 s (5-HT) or 8 s (dopamine) after stimulation ended. 10 µM mec or DHβE were then perfused onto the slice for 10 min and the line scan protocol was repeated. For baseline and post-drug recordings, three 60 s recordings were averaged. ΔF/F0 was calculated as above.

Immunohistochemistry and confocal imaging
After viral injection (see stereotaxic surgeries), mice were deeply anesthetized with ketamine-xylazine followed by cold perfusion to the heart of PBS and 4% PFA. The brain was removed and kept overnight at 4°C in 4% PFA. The next day, the brain was washed in PBS (three times, 20 min each) before thin 50 µm coronal slices were made using a vibratome (Leica VT1000S). Slices were briefly washed with CAS-BLOCK (Life Technologies) before being incubated in CAS-BLOCK (300 µl) overnight with Goat anti Vesicular-Acetylcholine-transporter antibodies (ABN100MI, Thermo Fisher, LOT: 3974859, 1:200). The slices were then washed in PBS (three times, 20 min each) and incubated with CAS-BLOCK and secondary antibodies (Abcam ab6566, LOT: GRI159765-18, 1:200) for 3 hr. Antifade Mounting Medium (VECTASHIELD) was applied to prevent slice bleaching.

Confocal imaging was acquired by Spinning Disk confocal microscope (Nikon) using Yokogawa W1 Spinning Disk camera and a 50 µm pinhole and a 20x dry objective with 0.75 N.A (CFI PLAN APO VC). Excitation laser used were 488 nm, 561 nm, 638 nm to excite GRAB-5HT, mCherry, anti VAChT, respectively. Optical and data were obtained using the software package NIS (Nikon), which also integrates the control of all hardware units in the microscope. Data was extracted from the ND2 package to personal computers and visualized with Fiji software. Low-magnification images were acquired using Nikon SMZ-25 fluorescent stereoscope equipped with ×1 and ×2 objectives.

Statistical analysis
Due to the nested design of this experiment, wherein we sampled multiple slices from individual mice, we fit the data with a linear mixed-effects model (LMEM), where ΔF/F0 from each slice was modeled as a sum of a fixed effect product (e.g., drug vs. ACSF) plus uncorrelated random effects due to each individual mouse. When fitting the Lorentzian, the model was of a product of two fixed effect, which are the treatment and the distance. To fit the LMEMs to the data, we used the MATLAB (Mathworks) FITLME command. We report the number of mice, the number of slices, Student’s t dof statistic (dof = degrees of freedom) and two-sided P values. The null hypotheses were rejected if the P value was below 0.05. For the bootstrap analysis of spike latency, recordings were obtained from 9 cells across 5 animals. The latency of the first spike from the onset of each recording was measured and compared against a bootstrap distribution generated by sampling spike times from the first 5 seconds of the recording 100,000 times. LED-evoked spike latencies and spontaneous first-spike latencies were each tested against this bootstrap distribution (Supplementary Fig. 3g).

Reagents used
Mecamylamine hydrochloride (mec), Sigma-Aldrich, Lot # 019M4108V CAS: 826-39-1; Atropine sulfate salt monohydrate, Sigma-Aldrich, Lot # BCBH8339V CAS No.: 5908-99-6; SR95531 hydrobromide (Gabazine), Hello Bio, CAS: 104104-50-9; DNQX, Tocris, CAS: 2379-57-9; D-AP5, Hello Bio, CAS: 79055-68-8; CGP55845 hydrochloride, Hello Bio, CAS: 149184-22-5; Dihydro-β-erythroidine hydrobromide, Tocris, CAS: 29734-68-7; Citalopram hydrobromide (HB2142), Hello Bio, CAS [59729-32-7]; Dopamine hydrochloride (DA), Sigma-Aldrich, CAS No.: 62-31-7; RS 23597-190, Tocris, CAS No.: 149719-06-2. Serotonin (5HT), Sigma-Aldrich, CAS No.: 153-98-0.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Matityahu et al. investigates the synchronous activation of striatal cholinergic interneurons (CINs) and its impact on serotonin (5-HT) release. The authors present compelling evidence that synchronized CIN activation induces 5-HT release in the striatum via nicotinic acetylcholine receptors (nAChRs). This novel finding significantly advances our understanding of the interplay between acetylcholine (ACh) and serotonin, extending prior research focused primarily on ACh-dopamine interactions. The study’s relevance is further underscored by its use of a mouse model of OCD-like behavior, providing critical insights into monoamine dysregulation. The experimental design is robust and employs state-of-the-art techniques, including: Genetic tools (AAV-based expression of GRAB5-HT and ChR2), Imaging (two-photon laser scanning microscopy to visualize 5-HT release in acute striatal slices), Pharmacological validation (nAChR antagonists such as mecamylamine), Optogenetics (CIN stimulation in ChAT-Cre mice), and Sensor specificity (tests with selective 5-HT receptor antagonists and reuptake inhibitors). These well-controlled experiments and rigorous analyses strongly support the manuscript’s findings. This study provides a significant contribution to understanding striatal neurotransmitter interactions and highlights a novel role for CINs in regulating 5-HT release via nAChRs. The combination of innovative findings and rigorous methodology lays a strong foundation for future research. Addressing the major concerns, particularly excitation overlap and the lack of in vivo validation, would enhance the study’s impact and reliability. Below are my suggestions.\n\nMajor Concerns\n1.Frequency, Duration, and Amplitude of CIN Stimulation (Figure 2). The manuscript could benefit from additional data exploring the relationship between the frequency, duration, and amplitude of optogenetic CIN stimulation and the resulting serotonin release.\n2.Excitation Overlap Between Imaging and Stimulation. The use of 920 nm two-photon excitation for imaging the 5-HT sensor may activate ChR2 during baseline measurements, complicating result interpretation. Validation through electrophysiological recordings is recommended to confirm that 920 nm excitation does not depolarize CINs. Ideally, red-shifted channelrhodopsins like Chrimson or ChRmine should be used to avoid activation at 920 nm, ensuring cleaner data.\n3. In Vivo Validation. While the study convincingly demonstrates CIN-mediated 5-HT release in acute brain slices, it remains unclear whether this phenomenon translates to in vivo conditions. Given the ongoing debate about CIN-induced dopamine release in vivo (Taniguchi et al., eLife, 2024), robust in vivo evidence of serotonin release would significantly strengthen the conclusions and establish physiological relevance.\n\nMinor Concerns\n1.Figure 1b: The sample trace should display a longer baseline and post-stimulation duration to better visualize the full decay to baseline.\n2.Figure 1c: It would be informative to demonstrate whether mecamylamine (MEC) washout rescues the electrically evoked 5-HT signal.\n3.Figure 2: Are spontaneous GRAB5-HT events observed? If so, are these events dependent on nAChRs?\n4.Sensor Type Clarification: The authors should explicitly state whether they used a green or red GRAB5-HT sensor, as the excitation wavelength differs.", "reviewer_2": "Matiyahu et al. present a series of experiments combining pharmacology, optogenetics, and two-photon imaging to investigate cholinergic regulation of serotonin (5-HT) release in the striatum. They claim to show that synchronous activation of cholinergic interneurons (CIN) in the dorsal striatum induces local serotonin release via presynaptic nicotinic receptors, and that this effect is enhanced in a mouse model of OCD that has hyperactive CINs. The topic of the study is timely and of interest in light of recent work showing cholinergic regulation of dopamine release, which has garnered much attention in the field. Nevertheless, the evidence presented to support the claim of ACh-induced 5-HT release is not convincing, and I have serious concerns about the methodology in key experiments. I describe my concerns below.\n\nMajor concerns:\n1. In figure 1, the authors show that blocking nicotinic receptors in the striatum attenuates electrically-driven 5-HT release. This experiment is convincing, but as the authors acknowledge, similar results have already been reported (e.g. ref 16), and this experiment cannot disambiguate between direct and indirect mechanisms that could underlie the effect. The novelty of the study thus stems primarily from the claim that this effect is direct – i.e. that ACh activates nAChRs found directly on the 5-HT axons to drive 5-HT release – which is supported by the experiments in Fig 2. I have two serious concerns about those experiments.\na. First the experiment presented in Fig 2 suffer from serious methodological confounds. In Fig 2, the authors optogenetically activate CINs, see an increase in GRAB 5-HT sensor fluorescence, and show this increase is sensitive to nAChR antagonists but not Glu/GABA/mAChR blockers. However, in this crucial experiment supporting the paper’s main claim, the authors multiplex a blue-light activated opsin with a blue-light sensitive GRAB 5-HT sensor. Thus the pulse of blue light used to activate the ChR2 would be expected excite the GRAB sensor directly and elicit an increase in GRAB-5HT fluorescence that is independent of 5-HT release (i.e. artifactual). This is especially concerning given that blue light used for optogenetic stimulation is often ~1000x stronger than blue light powers used for recording from blue-light activated sensors such that artifacts driven this way may resemble real transients in their amplitude, that similar confounds have been shown to create artifactual fluorescent signals lasting several seconds such that they resemble real transients in their waveform and timecourse (Taniguchi et al. 2024 eLife), and that the authors note in the methods that they observed a stimulation artifact that persisted beyond the photostimulation window. In my view, this is a critical limitation of the manuscript that must be addressed somehow. Some examples of experiments that could address this include: repeating the experiments in Fig 2 with the GRAB 5-HTmut control sensor which is insensitive to serotonin, and/or repeating the experiments in Fig 2 with a red-light activated opsin. This is by no means an exhaustive list, the authors may think of other controls to address this point.\nb. My second concern with the experiment in Fig 2 is that it is the only piece of evidence in the entire study supporting the paper’s central claim of a direct activation of nAChRs on 5HT terminals. Converging lines of evidence would considerably strengthen the manuscript. This is especially important considering the technical limitations of experiments in Fig 2 as I describe above.\n2. The authors show that nAChR-dependent 5-HT release is enhanced in the SAPAP3 model of OCD which they have previously shown to have hyperactive CINs. While roughly consistent with the physiology data in the rest of the manuscript, the authors do not present any behavioral data. As such, there is no evidence to show that this physiological change has any relevance at all to the behavioral deficits of OCD, and the data feel somewhat disjointed from the rest of the study.\n3. The authors make statistical comparisons with pairs of t-tests where two-way anovas should be used (Fig. 3b, 3c, Supp Fig 5b, 5c)\n\nMinor concerns:\n1. All of the experiments are done in the dorsal striatum, but evidence suggests DR 5-HT inputs are somewhat sparse in the dorsal striatum and more concentrated in the nucleus accumbens (see Soghomonian et al. 1987 and some recent studies from Malenka’s group). Notably, CINs in the dorsal and ventral striatum may also have some important functional differences (ref 14). This literature should be discussed, and the authors may wish to examine to what extent their findings generalize to other striatal subregions, which would strengthen the paper.\n2. The laser power used in the optogenetic stimulation is not given. This is a crucial methodological detail that should be included in the paper.\n3. In the methods, a detailed description of how the “blanking” of stimulation artifacts was done is lacking.\n4. There are duplicates in the references.\n5. In the introduction, the authors write “The source of striatal 5-HT is a long range projection from the dorsal raphe nucleus(17)”, but Ref 17 does not show this – TpH staining cannot disambiguate between sources of 5-HT input to the striatum.\n6. The final sentence of the paper offers speculation that goes so far beyond the evidence presented that it is arguably not relevant to include", "reviewer_3": "The authors demonstrate that synchronous activation of striatal cholinergic interneurons can lead to serotonin release mediated by nicotinic receptors. These is accomplished using a specific serotonin sensor, optogenetic stimulation and pharmacological manipulations. This work is valuable demonstrating a new mechanism for reciprocal cholinergic-monoamine dynamical control. While this report is relevant to the field, there are a few methodological concerns:\n-There seems to be a lack of consistency in how data is acquired, processed and/or displayed. While for the method section for the experiments carried out in SBU the authors mention:\n“For baseline and post-drug recordings, three 60s recordings were averaged.”\nThere is no similar statement for recordings carried out at MUJI, example traces show what seems like a single trace Figures 1b, 1d, 2b and 2d. However, for figure 3a the trace is smooth consistent with averaging of multiple pulses. Data should be processed in a similar way to allow comparison between conditions. For the distributions of response amplitude shown in figures 1c,1e, 2c and 2e it is unclear if the dots correspond to a single pulse or an average of several pulses. The amplitude of the response can change due to many factors i.e. receptor desensitization. In the absence of any manipulation, how variable are the responses to optogenetic or electrical stimulation? This would perhaps address if several pulses can/should be averaged to quantify the response. Additionally, it is important to quantify the stability of it in the span of several minutes (a time course equivalent to pharmacological manipulations reported) to discard the possibility of responses decreasing on their own.\n-The authors do not offer any explanation or additional information on the parameters chosen to evoke 5HT release with electrical stimulation, is a train of stimulation necessary to evoke this response? Is this response saturating or can different parameters generate distinct responses? Additionally, there is no discussion on why only a single 2 ms light pulse is required to evoke a similar response.\n-While the authors center in the effect of the stimulation on the peak amplitude of the response, only in a few of the responses show the full timescale the fluorescent signal going down to baseline levels. For instance, figures 1b, 2b and 2d, the authors only show a short part of the response. This makes questions of the variability of the response’s time course. For instance, the sample trace shown in response to addition of the cocktail of antagonists fig 2d looks very similar to addition of citalopram supp fig 2d. The authors mention there is no difference in the peak amplitude, but the example traces show perhaps a change in release dynamics which is not calculated, displayed or discussed.\n-Additionally, there seems to be a different process of normalization of fluorescence across experiments that might generate inconsistencies:\nFor SBU: “ΔF/F0 was calculated using the average fluorescence of the first 1.5s of baseline for F0. The peak amplitude of the signal was defined as the maximal ΔF/F0 that occurred between 0-5s after stimulation.”\nFor MUJI: “Fluorescent changes over time (∆F/F0) datapoints were extracted such that ∆(cid:1)/(cid:1)= ((cid:1) − (cid:1))/(cid:1), where F is the maximal fluorescent value recorded while evoking electrical / optogenetic stimulations; F0 denotes the averaged baseline fluorescence.”\nBaseline should be selected consistently.\n-While the authors provide proof of concept that cholinergic interneurons can generate serotonin release in vitro, there is no discussion on when can this occur in vivo, instances of synchronous cholinergic activation in vivo are sparse and there is little exploration on the parameter space to evoke serotonin release.\n-For some figures the distributions do not include the full set of data points for example figure 1e, 3c and throughout supplementary figures.\n-For optogenetic stimulation it is unclear why the rise in response is absent, while a light artifact is to be expected, the stimulation is of very short duration to lose all the rise time for the response, reported tau for this sensor is 250 ms.\n-In Supplementary figure 2d calculating a decay tau would probably be more adequate than simply measuring the slope of the initial segment of the response.\n-Check and confirm viral serotype in the following statement:\nFor 5-HT sensor experiments, a total amount of 500 nl of an adeno-associated virus serotype 5 harboring the GRAB-5HT3.5 construct (AAV9hSyn-5HT3.5; 2.20 × 1013 vg/ml; WZ Biosciences Inc. Lot No. 20201210)\n-For the following statement:\nLine scans began 2s before stimulation and continued for 57s (5-HT) or 8 ms (dopamine) after stimulation ended.\nWas the time recorded for dopamine was 8 s instead of 8 ms?", "reviewer_4": "In the current manuscript, Matityahu et al demonstrated that CHAT interneurons of the striatum control local serotonin release.\nIn my view, that is new and critical for our field evidence that need to be published as soon as possible.\nI think that all the experiments and analyses were done properly.\nI think that the abstract introduction and discussion may be improved so that authors will explain the relevance of the work (that is huge) to a broad non-expert readership. Here are a couple of the suggestions that can be discussed.\nA) Both dopamine and serotonin set a \"behavioral state\"- here is a mechanism for how DA state and serotonin state interact and how the state is set.\nB) DA, serotonin, and CHAT are affected in many disorders like SUD, PTSD, depression ets - so this work has high relevance for the broad readership.\nC) Here is a more nuanced point, that is relevant to Dr Plotkin's work, and he is a leading expert on this. CHAT interneurons are on a border between striosomes and matrix, DA releases affect strisomes and matrix differently. Same probably with serotonin (there is evidence that 5HTc resepror enreached in striosomes). Maybe authors can speculate how striosomal matrix balances and d1 and d2 balances will be affected by DA-Serotonine-CHAT interaction, hope to see future experimental works in this direction.\nIn any case, the current manuscript is very novel and significant. Our field (striatum) and disorder field can benefit from this evidence greatly. Work supports the conclusions. The methodology is sound, and details are provided to understand and reproduce the results."}, "author_response": "Response: We thank the reviewer for this strong endorsement and we address the concerns below.\nMajor Concern 1:\nFrequency, Duration, and Amplitude of CIN Stimulation (Figure 2). The manuscript could benefit from additional data exploring the relationship between the frequency, duration, and amplitude of optogenetic CIN stimulation and the resulting serotonin release.\nResponse: This is a valid and interesting point, but one that we think is more efficiently addressed by clarifying the rationale that went into our experimental design in the Methods section. Based on our work examining CIN-dependent DA release (Matityahu et al. Nat Comms 2023), we found that only the first evoked CIN spike in a train contributes significantly to DA release, with subsequent spikes failing to contribute to further DA release. This implies that the intensity of optogenetic stimulation translates into how many CINs are recruited. As such, we empirically chose a LED stimulus amplitude that reliably gave measurable GRAB-5HT responses comparable to a single intrastriatal electrical stimulation. Avoiding trains of LED stimulation also had the practical advantage of minimizing photobleaching and ChR2 desensitization. We chose a duration of 1 ms stimulation as this sufficed to trigger a single action potential in CINs as we now show in the new Supplementary Fig. 3e. We have updated the Methods section to clarify this. That said, we conducted new experiments (Supplementary Fig. 7) to explore the relationship between stimulation frequency and 5-HT release using electrical stimulation, which has the advantage of avoiding confounds related to photobleaching and ChR2 desensitization.\nMajor Concern 2:\nExcitation Overlap Between Imaging and Stimulation. The use of 920 nm two-photon excitation for imaging the 5-HT sensor may activate ChR2 during baseline measurements, complicating result interpretation. Validation through electrophysiological recordings is recommended to confirm that 920 nm excitation does not depolarize CINs. Ideally, red-shifted channelrhodopsins like Chrimson or ChRmine should be used to avoid activation at 920 nm, ensuring cleaner data.\nResponse: To rule out this concern, we conducted new experiments (New Suppl. Fig. 3) to show that the two-photon resonance scanning procedure at the laser intensities we used to image GRAB-5HT neither induces firing nor affects the electrophysiological properties of CINs that express ChR2. Hence, there was no need to use red-shifted ChR2 here.\nMajor Concern 3:\nIn Vivo Validation. While the study convincingly demonstrates CIN-mediated 5-HT release in acute brain slices, it remains unclear whether this phenomenon translates to in vivo conditions. Given the ongoing debate about CIN-induced dopamine release in vivo (Taniguchi et al., eLife, 2024), robust in vivo evidence of serotonin release would significantly strengthen the conclusions and establish physiological relevance.\nResponse: We appreciate this comment, and are reproducing here our response to the Editors about this issue.\nLack of in vivo data:\nWe appreciate this comment, and are keenly interested in this point ourselves, but have come to the conclusion that the time and resources necessary to generate such data simply make it infeasible to include in this study. Let us justify our conclusion:\n• As we now highlight in the revised Discussion, even for dopamine — whose striatal innervation and evoked release are much more robust than those of 5-HT — the mechanism of CIN-driven, nAChR-dependent release has proven difficult to demonstrate conclusively in vivo. Despite over three decades of investigation, the field continues to rely primarily on in vitro approaches for functional validation of this phenomenon. This elusiveness is because CIN-mediated dopamine release is highly context-dependent. Attempts to observe CIN-evoked 5-HT release in vivo will need to overcome the fact that the field has not yet identified the proper context in which to test it (thus in vivo validation is a daunting task in itself, and one we feel is beyond the scope of our trailblazing mechanistic study).\n• On this backdrop, the current study provides the first demonstration that CINs can directly induce serotonin release through nicotinic receptors. This mechanism is localized, region-specific, scalable with CIN density, pathologically upregulated in a mouse model of compulsive behavior, and rigorously verified using a combination of optogenetics, pharmacology, and two-photon imaging. Given the known technical limitations of monitoring 5-HT release in vivo, we believe the absence of in vivo data does not diminish the significance or validity of our findings. Further, given the novelty and broad implications of this mechanistic finding, we believe it is better to constrain this study to its most impactful mechanistic insight so it can be disseminated to the community as quickly as possible – this is an opinion shared by Reviewer 4.\nWe’ll also note that reviewer 3 seemed to imply that a discussion of when this mechanism may occur in vivo could suffice.\n• We have summarized these issues in the revised Discussion:\n“We have provided ex vivo demonstration that activation of CINs leads to local nAChR-dependent 5-HT release. How may this mechanism be engaged in vivo? In the case of DA, where release is augmented by the same mechanism that we report for 5-HT, this has turned out to be a difficult question to answer – multiple labs over 3 decades have reported in vitro nAChR-dependent DA release37,45,46 driven by CINs12–16, but attempts to demonstrate how this mechanism is engaged in vivo15 have been met by skepticism or constrained success47–49. One thing that has become clear is that context is critical48,50,51. This is likely to be the case for modulation of 5-HT release in vivo as well – based on the work examining CIN-dependent DA release in vivo, it’s unlikely we would stumble upon a measurable CIN-dependent 5-HT release in the absence of an appropriately defined behavioral or circuit-state context. One clue comes from the observation that local transfusion of nicotine, albeit independent of CIN activity, has been shown to be necessary and sufficient to drive striatal 5-HT release, but only in the presence of a stressful stimulus (foot shock)21. The precise activity of CINs that would be required to evoke 5-HT in this context is unclear, and may not be on the same sub-second timescale of the coupling we describe. Thus, future work is needed to determine the behavioral context in which nAChR activation leads to time-locked local release of 5-HT, but we show here that the requisite mechanism is in place. Notably, CINs are recruited synchronously by salient events, usually by way of activation of their thalamic input52–54, so the CIN synchrony required to drive local 5-HT release is likely attained in vivo.”\nIn summary, three decades of work (and dozens of papers in high tiered journals) showing nAChR-dependent and more recently CIN-dependent dopamine release in vitro, failed to reach a consensus as to whether or when this phenomenon exists in vivo. In that light, we humbly feel that requiring the first paper showing CIN-5-HT coupling in vitro to also demonstrate it in vivo is a tall order. It is not something we could achieve in the framework of a revision of a timely manuscript. That said, we agree that this is an important issue, and now address it in depth in our revised Discussion (pasted above). We hope the reviewer will accept this more pragmatic strategy.\nMinor Concern 1:\nFigure 1b: The sample trace should display a longer baseline and post-stimulation duration to better visualize the full decay to baseline.\nResponse: Thank for this comment. We have replaced it with an example where you see the decay back to baseline.\nMinor Concern 2:\nFigure 1c: It would be informative to demonstrate whether mecamylamine (MEC) washout rescues the electrically evoked 5-HT signal.\nResponse: Unfortunately, in our hands, mecamylamine does not wash out in a reliable fashion.\nMinor Concern 3:\nFigure 2: Are spontaneous GRAB5-HT events observed? If so, are these events dependent on nAChRs?\nResponse: In a previous study (Matityahu et al. Nat Comms 2023) we estimated the expected frequency of spontaneous dopamine release events (using the GRAD-DA2m sensor) in a striatal area of similar size to what we used in the current study, also imaged with the same two-photon resonance scanning, and we found them to occur once a minute. Given that the density of 5-HT fibers in the same size region-of-interest is orders-of-magnitude sparser than DA fibers, we do not realistically expect to find spontaneous events of 5-HT release.\nMinor Concern 4:\nSensor Type Clarification: The authors should explicitly state whether they used a green or red GRAB-5HT sensor, as the excitation wavelength differs.\nResponse: Thank you for pointing out this omission. We now explicitly state in the Results and Methods that we used a green-fluorescent GRAB-5HT3.5 sensor.\nReviewer #2 (Remarks to the Author):\nMatiyahu et al. present a series of experiments combining pharmacology, optogenetics, and two-photon imaging to investigate cholinergic regulation of serotonin (5-HT) release in the striatum. They claim to show that synchronous activation of cholinergic interneurons (CIN) in the dorsal striatum induces local serotonin release via presynaptic nicotinic receptors, and that this effect is enhanced in a mouse model of OCD that has hyperactive CINs. The topic of the study is timely and of interest in light of recent work showing cholinergic regulation of dopamine release, which has garnered much attention in the field. Nevertheless, the evidence presented to support the claim of ACh-induced 5-HT release is not convincing, and I have serious concerns about the methodology in key experiments. I describe my concerns below.\nResponse: We thank the reviewer for their constructive comments and we address the concerns below.\nMajor Concern 1:\nIn figure 1, the authors show that blocking nicotinic receptors in the striatum attenuates electrically-driven 5-HT release. This experiment is convincing, but as the authors acknowledge, similar results have already been reported (e.g. ref 16), and this experiment cannot disambiguate between direct and indirect mechanisms that could underlie the effect. The novelty of the study thus stems primarily from the claim that this effect is direct – i.e. that ACh activates nAChRs found directly on the 5-HT axons to drive 5-HT release – which is supported by the experiments in Fig 2. I have two serious concerns about those experiments.\nResponse: Before we address the two technical concerns that will be raised, the reviewer brings up a critical point about the novelty of our study, which we would like to highlight here. In this study we provide multiple pieces of evidence that disambiguate between direct versus indirect mechanisms underlying 5-HT release, supporting the conclusion that 5-HT release is promoted by direct activation of nAChRs on 5-HT fibers by ACh release from CINs:\na. We used various synaptic blockers to rule out involvement of local circuitry. This was particularly important in order to rule out: 1) muscarinic ACh receptors (mAChRs) expressed broadly in the striatum; 2) nAChRs on GABAergic interneurons, which are the only other known striatal neuronal types to express mecamylamine/DHßE sensitive nAChRs post-synaptically; and 3) ionotropic glutamate receptor antagonists, to eliminate indirect confounds caused by potential nAChR-dependent alterations in glutamate release.\nb. We showed that the CIN-dependent component (evoked optogenetically) was equal to the nAChR-dependent (evoked with electrical stimulation) of 5-HT release. With additional data added from new experiments the equality is even clearer (P-value of 0.99 when comparing the two, see the new Supplementary Fig. 4).\nc. We found a much smaller component of 5-HT release when activating dopamine fibers optogenetically, but this negligible effect was NOT nAChR-dependent. Therefore, putative indirect release of 5-HT from DA fibers is not responsible for the phenomenon we describe.\nTaken together we feel that we have provided strong evidence that the 5-HT release is triggered through local recruitment of nACHRs on 5-HT fibers that are directly activated by ACh release from CINs.\nMajor Concern 1a:\nFirst the experiment presented in Fig 2 suffer from serious methodological confounds. In Fig 2, the authors optogenetically activate CINs, see an increase in GRAB 5-HT sensor fluorescence, and show this increase is sensitive to nAChR antagonists but not Glu/GABA/mAChR blockers. However, in this crucial experiment supporting the paper’s main claim, the authors multiplex a blue-light activated opsin with a blue-light sensitive GRAB 5-HT sensor. Thus the pulse of blue light used to activate the ChR2 would be expected excite the GRAB sensor directly and elicit an increase in GRAB-5HT fluorescence that is independent of 5-HT release (i.e. artifactual).\nResponse: We have now added a new control experiment showing that simply flashing a 470 nm blue LED on striatal slices that express GRAB-5HT does not cause any change in the fluorescence (Supplementary Fig. 1d). In the analysis, we show the maximal value of fluorescence attained in a 5 s window following the stimulation. This maximalist estimate of the effect (Supplementary Fig. 1e) revealed that fluorescent changes are negligible relative to those evoked in mice whose CINs express ChR2, and in fact do not differ from fluorescent responses following CIN activation in the presence of nAChR antagonists (see Fig. 2c).\nMajor Concern 1a (cont):\nThis is especially concerning given that blue light used for optogenetic stimulation is often ~1000x stronger than blue light powers used for recording from blue-light activated sensors such that artifacts driven this way may resemble real transients in their amplitude, that similar confounds have been shown to create artifactual fluorescent signals lasting several seconds such that they resemble real transients in their waveform and timecourse (Taniguchi et al. 2024 eLife), and that the authors note in the methods that they observed a stimulation artifact that persisted beyond the photostimulation window.In my view, this is a critical limitation of the manuscript that must be addressed somehow. Some examples of experiments that could address this include: repeating the experiments in Fig 2 with the GRAB 5-HTmut control sensor which is insensitive to serotonin,and/or repeating the experiments in Fig 2 with a red-light activated opsin. This is by no means an exhaustive list, the authors may think of other controls to address this point.\nResponse: Photoconversion as addressed in Taniguchi et al. is a concern with red-shifted fluorophores. The GRAB-5HT version we used is a green fluorescent sensor (a fact that we apologize for omitting in the original submission) based on circularly permuted EGFP, which is extremely stable, and has not been reported to undergo photo-switching or photoconverting in response to brief blue LED pulses. We apologize for our oversight in not stating the version of GRAB-5HT3.5 we used in the methods (now fixed), which we think led to this misunderstanding. For the sake of completeness, however, our previous experiments showing that the optogenetically-evoked GRAB-5HT signal is completely abolished by nAChR antagonists (mecamylamine, DHβE) also rule-out such an artifact. If the GRAB signal were due to a direct light artifact, it would not be selectively sensitive to nAChR blockade. Regardless, as stated in our response abovewe performed new control experiments in wild-type mice lacking ChR2. Blue light pulses sufficient for optogenetic stimulation did not elicit any GRAB-5HT fluorescence changes, confirming that the sensor was not artifactually activated (Supplementary Fig. 1d,e).\nMajor Concern 1b:\nMy second concern with the experiment in Fig 2 is that it is the only piece of evidence in the entire study supporting the paper’s central claim of a direct activation of nAChRs on 5HT terminals. Converging lines of evidence would considerably strengthen the manuscript. This is especially important considering the technical limitations of experiments in Fig 2 as I describe above.\nResponse: Fig. 2 is indeed the main piece of direct evidence, but as outlined above, it is not the only one. First, Fig. 2d,e rules out indirect contributions from 1) muscarinic ACh receptors (which within the striatum can modulate presynaptic release of various neurotransmitters, as well as impact striatal projection neuron excitability); 2) nAChRs on GABAergic interneurons; and 3) nAChRs on presynaptic glutamatergic afferents. We also ruled out the possibility that we were detecting 5-HT release from another possible source: dopamine fibers (Supplementary Fig. 5); Further, we’ve added new data to supplement a data set showing that both nAChR-dependent and CIN-dependent 5-HT release are exactly equal (Suppl. Fig. 4), suggesting that CINs saturate the nAChR-dependent 5-HT release evoked by electrical stimulation. We have also added new data (Fig. 2g,h) suggesting that the density of CINs is related to how much nAChR-dependent 5-HT is released: CIN-evoked 5-HT release is significantly smaller in nucleus accumbens (NAc) core, where CIN density is reduced, compared to the dorsal striatum (see below response to your Minor Concern 1). Finally, addressing the technical concerns outlined in comment 1a above should hopefully assuage some of the concern.\nMajor Concern 2:\nThe authors show that nAChR-dependent 5-HT release is enhanced in the SAPAP3 model of OCD which they have previously shown to have hyperactive CINs. While roughly consistent with the physiology data in the rest of the manuscript, the authors do not present any behavioral data. As such, there is no evidence to show that this physiological change has any relevance at all to the behavioral deficits of OCD, and the data feel somewhat disjointed from the rest of the study.\nResponse: We apologize if the Sapap3 data came across as disjointed. Confirming or disproving a causal connection between ACh-mediated 5-HT release and OCD is well beyond the scope of this study. Our impetus for including these data was simply to test the corroborating hypothesis that the mechanism of 5-HT release we describe would be enhanced in a striatum with elevated CIN-mediated ACh release, and hint at a pathophysiological relevance of this mechanism. In this scenario, we used the Sapap3 mice as a tool to test the generality of the mechanism, not as an attempt to define a causal mechanism of OCD. That would certainly be beyond the scope of this study. We have re-written the Discussion to hopefully better integrate these data. Given that Sapap3 deletion is a disease model, we do feel compelled to speculate upon the clinical meaning, but we kept our speculation reserved.\nMajor Concern 3:\nThe authors make statistical comparisons with pairs of t-tests where two-way anovas should be used (Fig. 3b, 3c, Supp Fig 5b, 5c)\nResponse: We appreciate the careful attention to this detail. The tests we used are not paired t-tests, but rather t statistics arising from the linear-mixed effect (LME) hierarchical model that is designed to conduct a broad range of hypothesis testing, including when an ANOVA test would be warranted (for truly independent samples) but corrects for repeated measures. We feel that using the LME for hypothesis testing is a strength of our experimental design, which respects the nested/hierarchical structure of our data.\nMinor Concern 1:\nAll of the experiments are done in the dorsal striatum, but evidence suggests DR 5-HT inputs are somewhat sparse in the dorsal striatum and more concentrated in the nucleus accumbens (see Soghomonian et al. 1987 and some recent studies from Malenka’s group). Notably, CINs in the dorsal and ventral striatum may also have some important functional differences (ref 14). This literature should be discussed, and the authors may wish to examine to what extent their findings generalize to other striatal subregions, which would strengthen the paper.\nResponse: Thank you for this important comment! We repeated this experiment, isolating the CIN-dependent 5-HT release using ChAT-Cre mice and optogenetics, to directly compare CIN-evoked 5-HT release between dorsal striatum and the nucleus accumbens (NAc). While we initially expected 5-HT release to be larger in the NAc (because the density of 5-HT fibers is higher), we observed the opposite (New Fig. 2g,h): the CIN-dependent 5-HT release was smaller in the NAc. This finding suggests that the density of CINs within a region may be the limiting factor determining the size of the effect, rather than the density of 5-HT fibers.\nMinor Concern 2:\nThe laser power used in the optogenetic stimulation is not given. This is a crucial methodological detail that should be included in the paper.\nResponse: We now report the power of the pulsed laser and the LED after the objective .\nMinor Concern 3:\nIn the methods, a detailed description of how the “blanking” of stimulation artifacts was done is lacking.\nResponse: We have added this information to the Methods: “Five millisecond before the LED pulse, the gated PMTs were turned off for a duration of 50 ms. Nevertheless, despite removal of the voltage from the dynode of the PMT the massive flux of blue photons create a huge photocurrent on the GaAsP photocathode which subsequently saturate the sensor’s electronics leading to a light artifact in the signal lasting approximately 250 ms, evident even in non-expressing slices from wild type mice. Therefore, the first 258 ms (8 samples at 31 Hz) after any LED stimulation were removed from the data of all optogenetic experiments, which precludes observing the initial rise of the GRAB signals.”\nSee also our response below to Reviewer 3’s Concern 8 below.\nMinor Concern 4:\nThere are duplicates in the references.\nResponse: Thank you for catching this. We have removed the duplicated references.\nMinor Concern 5:\n5. In the introduction, the authors write “The source of striatal 5-HT is a long range projection from the dorsal raphe nucleus(17)”, but Ref 17 does not show this – TpH staining cannot disambiguate between sources of 5-HT input to the striatum.\nResponse: We have rephrased the sentence in the Introduction to state that it is the main source, as well as added more appropriate references.\nMinor Concern 6:\n6. The final sentence of the paper offers speculation that goes so far beyond the evidence presented that it is arguably not relevant to include.\nResponse: With regard to the final sentence in the Discussion, we agree that we speculated on a pathological state that we did not present evidence on. We did feel that it was important to provide at least a hint of speculation/context regarding the potential pathophysiological relevance of the mechanism we described (something requested by Reviewer 4). We have edited the final paragraph to make the speculative nature clear. We hope that the final paragraph is now palatable.\nReviewer #3 (Remarks to the Author):\nThe authors demonstrate that synchronous activation of striatal cholinergic interneurons can lead to serotonin release mediated by nicotinic receptors. These is accomplished using a specific serotonin sensor, optogenetic stimulation and pharmacological manipulations. This work is valuable demonstrating a new mechanism for reciprocal cholinergic-monoamine dynamical control. While this report is relevant to the field, there are a few methodological concerns:\nResponse: We thank the reviewer for recognizing the value in this study and we address the concerns below.\nConcern 1:\nThere seems to be a lack of consistency in how data is acquired, processed and/or displayed. While for the method section for the experiments carried out in SBU the authors mention:\n“For baseline and post-drug recordings, three 60s recordings were averaged.”\nThere is no similar statement for recordings carried out at MUJI, example traces show what seems like a single trace Figures 1b, 1d, 2b and 2d. However, for figure 3a the trace is smooth consistent with averaging of multiple pulses. Data should be processed in a similar way to allow comparison between conditions. For the distributions of response amplitude shown in figures 1c,1e, 2c and 2e it is unclear if the dots correspond to a single pulse or an average of several pulses. The amplitude of the response can change due to many factors i.e. receptor desensitization. In the absence of any manipulation, how variable are the responses to optogenetic or electrical stimulation? This would perhaps address if several pulses can/should be averaged to quantify the response.\nResponse:We thank the reviewer for raising these points. In both laboratories, multiple measurements were performed for each condition, with the exact number depending on the signal-to-noise ratio, as the serotonin signals were small. To improve signal-to-noise, multiple traces were averaged (in no case did we or are we showing individual traces), and the maximal ΔF/F₀ value was what appears in the dots of boxplots. Notably, in response to your concern, ΔF/F₀ was now recalculated across datasets from both laboratories to ensure consistency, with F₀ now defined as the baseline fluorescence prior to stimulation for all measurement from both labs. Needless to say, that when we compare two conditions in the same experiment the processing of the data is identical. Because the GRAB-5HT responses were sampled at a different rate at HUJI (resonant laser scanning) and SBU (galvo-based laser scanning), in order to match the appearance of the traces we now filtered the data from both labs with a temporal filter of the same absolute of approximately 400 ms.\nWe have updated the Methods section to explicitly state the above and that individual measurements were performed with ≥60-second intervals between trials, minimizing the risk of receptor desensitization. Visual assessment of the individual traces did not reveal any glaring time dependent change in the trials we averaged, and because their number was judged to be both necessary and sufficient to get a robust averaged response (3-4 for electrical stimulation and 9 for optical stimulation) we could not find any meaningful way to break down this variability further in search of a time dependent component. It should be noted, that in the current version we have added a control showing that the averaged optogenetic response remains stable over 10 minutes (see next Concern) further strengthening our confidence in the lack of systematic change of the response over time.\nWe kindly direct the reviewer to the revised Methods section for further details. In particular, we now confirm that the analysis of the data from both labs were analyzed similarly.\nConcern 2:\nAdditionally, it is important to quantify the stability of it in the span of several minutes (a time course equivalent to pharmacological manipulations reported) to discard the possibility of responses decreasing on their own.\nResponse: We have added new data (Supplementary Fig. 2f-h) showing that the response does not change simply with the passage of time (10 minutes; the amount of time given for drugs to take effect).\nConcern 3:\nThe authors do not offer any explanation or additional information on the parameters chosen to evoke 5HT release with electrical stimulation, is a train of stimulation necessary to evoke this response? Is this response saturating or can different parameters generate distinct responses? Additionally, there is no discussion on why only a single 2 ms light pulse is required to evoke a similar response.\nResponse: For electrical stimulation with bi-polar electrodes we tried various configurations of pulses and frequencies and found that 10 pulses and 10 Hz gave us an optimal and robust GRAB-5HT response. As per the reviewer’s request we now show the outcome of this initial optimization (see new Supplementary Fig. 7). For optogenetic stimulation, the choice of a single 1 ms-long (2 ms was a typo in the original manuscript) optogenetic pulse was based on its ability to drive an action potential in ChR2-laden CINs, which we now show an example of in Supplementary Fig. 3e.\nPlease see our detailed response to Reviewer 1 who raised similar issues in their Concern 1.\nConcern 4:\nWhile the authors center in the effect of the stimulation on the peak amplitude of the response, only in a few of the responses show the full timescale the fluorescent signal going down to baseline levels. For instance, figures 1b, 2b and 2d, the authors only show a short part of the response. This makes questions of the variability of the response’s time course. For instance, the sample trace shown in response to addition of the cocktail of antagonists fig 2d looks very similar to addition of citalopram supp fig 2d. The authors mention there is no difference in the peak amplitude, but the example traces show perhaps a change in release dynamics which is not calculated, displayed or discussed.\nResponse: As explained in our response to the Editors above, we conducted additional experiments to obtain longer measurements of the decay, and have replaced several examples in Fig. 1b and Fig. 2b with traces that show the decay back to zero. These measurements also enabled us to demonstrate that the passage of time alone (e.g., 10 minutes) did not affect the amplitude or kinetics of decay. We did not conduct additional experiments (with longer traces) for the various drug applications shown in Fig. 2d,e and new Supplementary Fig. 2 c-e, but we have added new analyses demonstrating that the cocktail of antagonists has no effect on the decay kinetics (New Fig. 2f). We therefore also chose a better exemplar for Fig. 2d so as not to create the wrong impression.\nConcern 5:\nAdditionally, there seems to be a different process of normalization of fluorescence across experiments that might generate inconsistencies:\nFor SBU: “ΔF/F0 was calculated using the average fluorescence of the first 1.5s of baseline for F0. The peak amplitude of the signal was defined as the maximal ΔF/F0 that occurred between 0-5s after stimulation.”\nFor MUJI: “Fluorescent changes over time (∆F/F0) datapoints were extracted such that ∆ / = ( − )/ , where F is the maximal fluorescent value recorded while evoking electrical / optogenetic stimulations; F0 denotes the averaged baseline fluorescence.”\n𝐹Ba𝐹selin𝐹e sh𝐹oul𝐹d be selected consistently.\nResponse: We apologize for this oversight. Please refer to our response to Concern 1 above.\nConcern 6:\nWhile the authors provide proof of concept that cholinergic interneurons can generate serotonin release in vitro, there is no discussion on when can this occur in vivo, instances of synchronous cholinergic activation in vivo are sparse and there is little exploration on the parameter space to evoke serotonin release.\nResponse: We agree that a discussion of how/when CINs may evoke 5-HT release in vivo is warranted, and we have added this to the new Discussion section. Please see our response to Reviewer 1’s major concern 3. In the related text we also now point out how the requisite condition of CIN synchrony may and does arise in vivo.\nConcern 7:\nFor some figures the distributions do not include the full set of data points for example figure 1e, 3c and throughout supplementary figures.\nResponse: Thanks for catching this. All figures now include the full set of data points. These data points are included in the Source Data file included with the submission.\nConcern 8:\nFor optogenetic stimulation it is unclear why the rise in response is absent, while a light artifact is to be expected, the stimulation is of very short duration to lose all the rise time for the response, reported tau for this sensor is 250 ms.\nResponse: The apparent absence of rise time kinetics in our optogenetic responses is due to technical limitations in our detection system rather than the biological response itself. During the 470 nm optogenetic stimulation, our gated GaAsP PMTs experience saturation despite electrical gating of the dynode chain. This occurs because high-intensity blue photons continue to generate photoelectrons at the photocathode through the photoelectric effect, even when the PMT multiplication is disabled. The resulting direct photocurrent saturates the detection electronics.\nTo prevent corrupted data acquisition, we mask approximately 250 ms of data following each optogenetic pulse - the time required for our detection electronics to recover from saturation. This masking period unfortunately coincides with the expected rise time kinetics of GRAB-5HT responses. Given that GRAB-5HT has a reported tau of 250 ms, the initial rising phase of any optogenetically-evoked 5-HT response would occur precisely within this masked interval and is therefore not detectable with our current system.\nThe responses we report begin only after the detection system has fully recovered from the optogenetic flash artifact. While this prevents us from capturing the complete kinetic profile of the 5-HT response, it ensures that all analyzed data points represent genuine biological signal rather than optical artifacts. The absence of visible rise time kinetics should not be interpreted as evidence against optogenetic efficacy, but rather as a technical limitation imposed by the temporal overlap between the sensor's response kinetics and our system's recovery time. This is also explained in the revised Methods section.\nConcern 9:\nIn Supplementary figure 2d calculating a decay tau would probably be more adequate than simply measuring the slope of the initial segment of the response.\nResponse: We agree that this would have been the natural choice, but fitting exponentials didn’t work. We therefore decided to model it as a linear decay and use the slope of the decay as a measure of the rate of the kinetics.\nConcern 10:\nCheck and confirm viral serotype in the following statement:\nFor 5-HT sensor experiments, a total amount of 500 nl of an adeno-associated virus serotype 5 harboring the GRAB-5HT3.5 construct (AAV9hSyn-5HT3.5; 2.20 × 1013 vg/ml; WZ Biosciences Inc. Lot No. 20201210)\nResponse: Thanks for catching this, it was indeed serotype 9. We have corrected the Methods accordingly.\nConcern 11:\nFor the following statement:\nLine scans began 2s before stimulation and continued for 57s (5-HT) or 8 ms (dopamine) after stimulation ended.\nWas the time recorded for dopamine was 8 s instead of 8 ms?\nResponse: Good catch! 8 ms should indeed be 8s. We have corrected this in the text.\nReviewer #4 (Remarks to the Author):\nIn the current manuscript, Matityahu et al demonstrated that CHAT interneurons of the striatum control local serotonin release.\nIn my view, that is new and critical for our field evidence that need to be published as soon as possible.\nI think that all the experiments and analyses were done properly.\nI think that the abstract introduction and discussion may be improved so that authors will explain the relevance of the work (that is huge) to a broad non-expert readership. Here are a couple of the suggestions that can be discussed.\nA) Both dopamine and serotonin set a \"behavioral state\"- here is a mechanism for how DA state and serotonin state interact and how the state is set.\nB) DA, serotonin, and CHAT are affected in many disorders like SUD, PTSD, depression ets - so this work has high relevance for the broad readership.\nC) Here is a more nuanced point, that is relevant to Dr Plotkin's work, and he is a leading expert on this. CHAT interneurons are on a border between striosomes and matrix, DA releases affect strisomes and matrix differently. Same probably with serotonin (there is evidence that 5HTc resepror enreached in striosomes). Maybe authors can speculate how striosomal matrix balances and d1 and d2 balances will be affected by DA-Serotonine-CHAT interaction, hope to see future experimental works in this direction.\nIn any case, the current manuscript is very novel and significant. Our field (striatum) and disorder field can benefit from this evidence greatly. Work supports the conclusions. The methodology is sound, and details are provided to understand and reproduce the results.\nResponse: We thank the Reviewer for their enthusiasm about our work, and for recognizing its significance and timeliness. We have taken the reviewer’s advice and updated the Abstract, Introduction and Discussion to try to provide more context about the significance of our findings.\nExamples include:\n-Introduction – “Dysregulated striatal 5-HT signaling has been implicated in numerous neurological, psychiatric and addictive disorders; strikingly, pathological changes in striatal 5-HT signaling may occur in tandem with changes in both dopamine andacetylcholine7–10.”\n-Discussion – “On a final speculative note, evidence suggests that both dopamine and 5-HT may set “behavioral states” that shape learning and disease62. Our data suggest a mechanism by which CINs may facilitate interactions between such monoamine-dependent states.”\nPlease note two things. First, while we would love to expand further, text limits, and at the risk of belaboring fine points that interest primarily aficionados, prevented us from doing so. Second, we enthusiastically appreciate the reviewer’s point C. This is something that excites us very much. Our concern about broaching this point, however, was that it would require too much additional background text to reasonably fit within our limits."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In my earlier review of NCOMMS-24-80701A, I noted three areas where additional clarification or data could strengthen the work:\nStimulation parameters – Further characterization of the frequency, duration, and amplitude of CIN stimulation could help clarify the relationship to observed 5-HT release.\nPotential optical interaction – Additional validation to fully exclude possible ChR2 activation during 920 nm imaging would help confirm that the GRAB5-HT signals reflect physiological release rather than optical artifacts.\nPhysiological context – In vivo evidence would help establish the relevance of these findings beyond the slice preparation.\nFrom the revised materials and rebuttal, these points appear to remain only partially addressed. While the new data add information, my overall assessment of the strength of the central conclusions is unchanged.", "reviewer_2": "Matityahu et al. report evoked serotonin release following excitation of cholinergic interneurons in the striatum. In my original review, I expressed concern over a lack of optical crosstalk controls and supporting lines of evidence for key experiments in figure 2 which underpin the study’s most novel and interesting conclusion: that acetylcholine signaling evokes serotonin release directly by activating nicotinic receptors on serotonin axons.\nMajor:\nIn their rebuttal, the authors argue that there are in fact additional lines of evidence to support the claim that acetylcholine acts directly on nACHRs on serotonin axons – namely, that the evoked release is sensitive to mecamylamine, that it persists in the presence of glu/GABA antagonists, and that the fraction of CIN-dependent serotonin release is equal to the fraction of nACHR dependent serotonin release. Although the gold-standard experiment of knocking down/out the appropriate nACHR selectively from serotonin neurons was not done, this is understandable given that such experiments are laborious and require difficult to obtain transgenic tools. Thus - assuming that the optogenetic stimulation experiments are supported by adequate controls (see below for remaining concerns) - I found the authors’ argument here convincing and my original concern about a lack of converging evidence is satisfied. I would simply suggest that the authors add a discussion point proposing the knock down/out experiment to the field as a future direction.\nIn the revised manuscript, the authors have also added two control experiments to address the possibility of crosstalk between the optical stimulation used to stimulate ChR2 and the optical stimulation used to record GRAB 5HT responses. These controls are vitally important to the study’s main conclusions given that ChR2 has been shown to be excitable by 920 nm light which is the wavelength used for the GRAB 5HT recordings, and GRAB 5HT can be excited by 470 nm light which is the wavelength used to activate ChR2. In the first control, the authors show that applying a pulse of 470 nm light onto slices that do not express ChR2 fails to evoke a GRAB 5HT response (Sup Fig 1e). The raw data files indicate this was done for 16 slices. The number of mice used here is not reported and should be included in a future revision, but assuming this was done in more than one mouse overall the result seems convincing . In the second control experiment, the authors show that application of 920 nm laser scanning does not activate a ChR2 expressing cholinergic interneuron. However this appears to be limited to a single example trace from a single cell from a single mouse with no replication or quantification provided, and consequently the strength of the evidence provided here is weak. Additional controls that are standard and widely accepted in the field (e.g. replication with a red light activated opsin, replication with the serotonin-insensitive GRAB 5HTmut control sensor) were not done. Given the centrality of the optogenetic experiments in Fig 2 to the study’s main finding, I continue to find the controls that support the paper’s main conclusion somewhat underwhelming.\nThe authors also present new data comparing Ach-evoked serotonin release in the dorsal and ventral striatum. This is a valuable addition to the paper which will be of interest to many specialists in the field, but I have some questions about the data. First, why not include an example trace for a recording from the NAc experiments to allow visual comparison to the data from dorsal striatum? Second, the authors imply that CIN stimulation successfully evoked serotonin release in the NAc, just not as much as in dorsal striatum. However, the peak fluorescence responses in NAc appear on average < 0.5 % dF/F, comparable to peak response amplitude seen in the dorsal striatum after nACHR antagonist block (Fig 2e, pink) and even to peak response amplitude seen in slices without any opsin at all (Sup Fig 1e). This would seem to suggest that acetylcholine-evoked serotonin release does not occur to a detectable level at all in NAc – which is different than saying the evoked release was simply smaller than in dorsal striatum. The authors would do well to clarify this important point.\nFinally, in view of the reservations I describe above, I strongly believe that a more conservative interpretation in the manuscript would be appropriate. I will highlight two examples where the authors’ conclusions go beyond the evidence provided, but these are just examples - given how thin control experiments are in key parts of the paper I believe a more cautious tone is warranted throughout. As a first example, the authors argue that a quantitative difference in Ach-evoked serotonin release between NAc and dorsal striatum is caused by decreased CIN density in ventral striatum. However, the authors neither quantify CIN density nor rule out alternative explanations that could explain this effect; e.g. serotonin neurons projecting to these two areas could have different expression levels for nACHRs resulting in differential sensitivity to Ach. As a second example, the authors write in their rebuttal “Confirming or disproving a causal connection between ACh-mediated 5-HT release and OCD is well beyond the scope of this study”. Nevertheless the authors argue in the abstract that their data provide “mechanistic insight into monoaminergic dysregulation in compulsive behavioral disorders”. These statements appear somewhat at odds, and I argue that tamping down the claims in the manuscript is appropriate given that the paper does not measure behavior and as such does not provide any evidence to indicate that dysregulated Ach-5HT signaling has any impact whatsoever on compulsive behaviors.\nMinor:\nDescriptions of experimental procedures in the methods are much improved\nThe “false” dopamine release terminology in the discussion is confusing – this is real dopamine released from non-dopamine producing cells through a noncanonical mechanism", "reviewer_3": "The authors have thoroughly addressed all of my previous concerns, and the revisions have substantially improved the clarity and rigor of the manuscript. I have no further comments and am pleased to recommend it for publication. I’m excited to see these findings shared with the broader community, as they represent a valuable contribution to the field.", "reviewer_4": "All my initial concerns are resolved in the resubmitted version of the manuscript.\nI strongly support publication of this article"}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nComment 1:\nIn my earlier review of NCOMMS-24-80701A, I noted three areas where additional clarification or data could strengthen the work:\nStimulation parameters – Further characterization of the frequency, duration, and amplitude of CIN stimulation could help clarify the relationship to observed 5-HT release.\nResponse 1:\nWe appreciate the reviewer’s suggestion to further characterize the relationship between CIN stimulation parameters and 5-HT release. We note, however, that CIN-evoked GRAB-5HT signals in this preparation are intrinsically small (typically ~2% ΔF/F ), requiring averaging across multiple trials (9 repetitions) to achieve sufficient signal-to-noise for quantitative analysis. In preliminary experiments, lower stimulation inte₀nsities often failed to produce a detectable signal, necessitating the use of a stimulation amplitude that reliably evoked measurable responses.\nImportantly, in response to this concern in the previous revision, we already added new experiments directly comparing stimulation parameters (now Supplementary Fig. 8), showing that a brief train of 10 pulses at 10 Hz produced the most robust and reproducible 5-HT responses relative to other tested conditions. Systematic mapping across a wider range of intensities or frequencies would require many repeated long-duration recordings, which in our hands introduce substantial photobleaching, opsin desensitization, and cumulative optical artifacts. We therefore used a fixed stimulation protocol that was empirically optimized to maximize response amplitude while minimizing these confounds, consistent with the mechanistic goals of the study rather than exhaustive input–output characterization.\nComment 2:\nPotential optical interaction – Additional validation to fully exclude possible ChR2 activation during 920 nm imaging would help confirm that the GRAB5-HT signals reflect physiological release rather than optical artifacts.\nResponse 2:\nWe have performed new controls to exclude imaging-related confounds. We upgraded our system to include 590-nm stimulation and replicated nAChR-dependent 5-HT release in dorsal striatum using ChAT-Cre mice expressing the red-shifted opsin ChrimsonR, eliminating concerns related to blue-light–dependent artifacts (Supplementary Fig. 4). We also addressed additional technical concerns related to multiplexed stimulation by showing that 920-nm resonant scanning does not alter CIN firing rates and does not induce scan-locked phase-like firing (Supplementary Fig. 3).\nComment 3:\nPhysiological context – In vivo evidence would help establish the relevance of these findings beyond the slice preparation.\nFrom the revised materials and rebuttal, these points appear to remain only partially addressed. While the new data add information, my overall assessment of the strength of the central conclusions is unchanged.\nResponse 3:\nWe thank the reviewer for their careful reassessment of the revised manuscript. We note that in the original review, the reviewer concluded that “these well-controlled experiments and rigorous analyses strongly support the manuscript’s findings,” and that the central conclusions were viewed as robust. The present revision was therefore not intended to alter the core claims of the study, but rather to address specific technical and methodological concerns raised during review, particularly those related to optical and circuit-level controls. In doing so, we believe we have substantially strengthened the manuscript by 1) adding additional controls to address what appeared to be the biggest point of concern – artifacts related to multiplexed light stimulation and 2) bolstering our examination of 5-HT release in the ventral striatum, revealing a pronounced regional specificity of cholinergic control of serotonin release, and demonstrating that this mechanism operates selectively in defined striatal subregions. While we agree that in vivo validation would be valuable, identifying the precise behavioral and experimental contexts in which this mechanism is engaged in vivo is a major endeavor that we view as beyond the scope of the present mechanistic study (a view that aligns with the broader set of reviewer comments). We hope the Reviewer will agree that the revised manuscript clarifies and strengthens the mechanistic conclusions without changing their fundamental support.\nReviewer #2 (Remarks to the Author):\nComment 1:\nMatityahu et al. report evoked serotonin release following excitation of cholinergic interneurons in the striatum. In my original review, I expressed concern over a lack of optical crosstalk controls and supporting lines of evidence for key experiments in figure 2 which underpin the study’s most novel and interesting conclusion: that acetylcholine signaling evokes serotonin release directly by activating nicotinic receptors on serotonin axons.\nMajor:\nIn their rebuttal, the authors argue that there are in fact additional lines of evidence to support the claim that acetylcholine acts directly on nACHRs on serotonin axons – namely, that the evoked release is sensitive to mecamylamine, that it persists in the presence of glu/GABA antagonists, and that the fraction of CIN-dependent serotonin release is equal to the fraction of nACHR dependent serotonin release. Although the gold-standard experiment of knocking down/out the appropriate nACHR selectively from serotonin neurons was not done, this is understandable given that such experiments are laborious and require difficult to obtain transgenic tools. Thus - assuming that the optogenetic stimulation experiments are supported by adequate controls (see below for remaining concerns) - I found the authors’ argument here convincing and my original concern about a lack of converging evidence is satisfied. I would simply suggest that the authors add a discussion point proposing the knock down/out experiment to the field as a future direction.\nResponse 1:\nWe thank the Reviewer for their thoughtful and pragmatic summary. We have added the additional controls that were requested (see below). We have also added a sentence to the Discussion stating “The present study employed convergent pharmacological and circuit-based evidence to demonstrate CIN-dependent 5-HT release; future studies may utilize additional approaches, such as selectively knocking down nAChR subunit expression in serotonergic dorsal raphe neurons or testing 5-HT-insensitive GRAB variants, to further corroborate and expand upon these findings.”\nComment 2:\nIn the revised manuscript, the authors have also added two control experiments to address the possibility of crosstalk between the optical stimulation used to stimulate ChR2 and the optical stimulation used to record GRAB 5HT responses. These controls are vitally important to the study’s main conclusions given that ChR2 has been shown to be excitable by 920 nm light which is the wavelength used for the GRAB 5HT recordings, and GRAB 5HT can be excited by 470 nm light which is the wavelength used to activate ChR2. In the first control, the authors show that applying a pulse of 470 nm light onto slices that do not express ChR2 fails to evoke a GRAB 5HT response (Sup Fig 1e). The raw data files indicate this was done for 16 slices. The number of mice used here is not reported and should be included in a future revision, but assuming this was done in more than one mouse overall the result seems convincing.\nResponse 2:\nSorry about this oversight. This important control was indeed done in more than one mouse. We now explicitly report in Supplementary Fig.1e’s caption the number of slices and mice, which are n = 16 slices from N = 4 mice.\nComment 3:\nIn the second control experiment, the authors show that application of 920 nm laser scanning does not activate a ChR2 expressing cholinergic interneuron. However this appears to be limited to a single example trace from a single cell from a single mouse with no replication or quantification provided, and consequently the strength of the evidence provided here is weak. Additional controls that are standard and widely accepted in the field (e.g. replication with a red light activated opsin, replication with the serotonin-insensitive GRAB 5HTmut control sensor) were not done. Given the centrality of the optogenetic experiments in Fig 2 to the study’s main finding, I continue to find the controls that support the paper’s main conclusion somewhat underwhelming.\nResponse 3:\nAs explained above in our response to Reviewer 1, we have performed new controls to exclude imaging-related confounds. We show (now for multiple neurons) that 920-nm resonant scanning does not alter CIN firing rates and does not induce scan-locked phase-like firing (Supplementary Fig. 3). In addition, we upgraded our system to include 590-nm stimulation and added new data that replicated nAChR-dependent 5-HT release in the dorsal striatum using ChAT-Cre mice expressing the red-shifted opsin ChrimsonR, eliminating concerns related to blue-light–dependent artifacts (Supplementary Fig. 4). Taken together, we have added new experiments that control for the remaining concerns of potential optical confounds, which we believe strengthens the central conclusions of the manuscript.\nComment 4:\nThe authors also present new data comparing Ach-evoked serotonin release in the dorsal and ventral striatum. This is a valuable addition to the paper which will be of interest to many specialists in the field, but I have some questions about the data. First, why not include an example trace for a recording from the NAc experiments to allow visual comparison to the data from dorsal striatum? Second, the authors imply that CIN stimulation successfully evoked serotonin release in the NAc, just not as much as in dorsal striatum. However, the peak fluorescence responses in NAc appear on average < 0.5 % dF/F, comparable to peak response amplitude seen in the dorsal striatum after nACHR antagonist block (Fig 2e, pink) and even to peak response amplitude seen in slices without any opsin at all (Sup Fig 1e). This would seem to suggest that acetylcholine-evoked serotonin release does not occur to a detectable level at all in NAc – which is different than saying the evoked release was simply smaller than in dorsal striatum. The authors would do well to clarify this important point.\nResponse 4:\nWe are particularly grateful for your insistence that we clarify whether nAChR-dependent control of serotonin release is present in the ventral striatum. With additional experiments, we now show that although robust 5-HT release can be evoked by electrical stimulation in the ventral striatum, this response is not altered by mecamylamine, nor is there detectable 5-HT release upon synchronous optogenetic activation of cholinergic interneurons (CINs). These distinctions are now clearly incorporated into revised Figures 1 and 2 and strengthen the manuscript by revealing a pronounced regional specificity of cholinergic control of serotonin release. As requested, we have also added raw traces for the experiments in the ventral striatum (Fig. 1; Supplementary Fig. 5).\nComment 5:\nFinally, in view of the reservations I describe above, I strongly believe that a more conservative interpretation in the manuscript would be appropriate. I will highlight two examples where the authors’ conclusions go beyond the evidence provided, but these are just examples - given how thin control experiments are in key parts of the paper I believe a more cautious tone is warranted throughout. As a first example, the authors argue that a quantitative difference in Ach-evoked serotonin release between NAc and dorsal striatum is caused by decreased CIN density in ventral striatum. However, the authors neither quantify CIN density nor rule out alternative explanations that could explain this effect; e.g. serotonin neurons projecting to these two areas could have different expression levels for nACHRs resulting in differential sensitivity to Ach.\nResponse 5:\nAs explained above, we have now quantified the statistically significant difference between dorsal and ventral striatum, vis-à-vis CIN-drive 5-HT release. These findings show that such release is undetectable in the ventral striatum under our experimental conditions. That said, we agree and with the Reviewer and have toned down this argument. We reference a paper that we have since published in PNAS (https://doi.org/10.1073/pnas.2519939123) that supports the notion of a reduced density of CINs in ventral striatum, but we now acknowledge alternative possibilities. We have added the following sentence to the Discussion: “Alternatively, the 5-HT fibers that innervate dorsal and ventral striatum may have different nAChR expression profiles leading to differential sensitivity to ACh.”\nComment 6:\nAs a second example, the authors write in their rebuttal “Confirming or disproving a causal connection between ACh-mediated 5-HT release and OCD is well beyond the scope of this study”. Nevertheless the authors argue in the abstract that their data provide “mechanistic insight into monoaminergic dysregulation in compulsive behavioral disorders”. These statements appear somewhat at odds, and I argue that tamping down the claims in the manuscript is appropriate given that the paper does not measure behavior and as such does not provide any evidence to indicate that dysregulated Ach-5HT signaling has any impact whatsoever on compulsive behaviors.\nResponse 6:\nPoint taken, and we apologize for such inconstancy. We have toned down these claims throughout the manuscript, including removing the abstract text quoted above.\nComment 7:\nMinor:\nDescriptions of experimental procedures in the methods are much improved\nThe “false” dopamine release terminology in the discussion is confusing – this is real dopamine released from non-dopamine producing cells through a noncanonical mechanism\nResponse 7:\nWe have changed ‘”false”’ to ‘non-canonical’.\nReviewer #3 (Remarks to the Author):\nComment:\nThe authors have thoroughly addressed all of my previous concerns, and the revisions have substantially improved the clarity and rigor of the manuscript. I have no further comments and am pleased to recommend it for publication. I’m excited to see these findings shared with the broader community, as they represent a valuable contribution to the field.\nResponse:\nWe are grateful that the Reviewer shares our enthusiasm about the finding and also eagerly anticipates their publication in Nature Communications.\nReviewer #4 (Remarks to the Author):\nComment:\nAll my initial concerns are resolved in the resubmitted version of the manuscript.\nI strongly support publication of this article.\nResponse:\nWe thank the Reviewer for their strong support of our publication in Nature Communications."}, {"round": 3, "reviewer_comments": {"reviewer_2": "With the addition of new control experiments ruling out optical crosstalk and additional characterization of regional differences between Ach-evoked 5HT release in dorsal vs ventral striatum, the manuscript is much improved and I believe the study is adequate for publication without additional experiments. I believe the text requires some final revisions though\nmajor:\n1. The finding that ACh-evoked 5HT release does not occur in ventral striatum, where 5HT inputs are strongest, is an important contribution. This finding should be mentioned in the abstract.\nminor:\n2. In the discussion, the authors describe ongoing confusion in the field about what the behavioral role of Ach-evoked dopamine release in the striatum could be. There seems to have been progress on this from Malenka's group in recent months which the authors may wish to discuss, at their discretion: PMID 40667072"}, "author_response": "Response:\nThank you for supporting publication, and for pushing us to compare dorsal to ventral striatum. We now explicitly mention the difference in a dedicated sentence in the Abstract: “This CIN–5-HT coupling is not detectable in the ventral striatum, despite its substantially denser serotonergic innervation.”\nRegarding the Discussion, we have added a sentence and a citation to the beautiful suggested study from the Eshel and Malenka groups (now published in Nature), as well as another recent report by Arif Hamid examining CIN-DA coupling."}]

Decision letter
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ATTACHED REVIEWER REPORT — IMPORTANT
The text below is a complete reviewer report submitted as a PDF
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Assign it to whichever reviewer wrote 'see pdf' or had no inline text.
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