Skip to main content
PMC home page
eLife logoLink to eLife
. 2024 May 15;13:e95694. doi: 10.7554/eLife.95694

Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'

James Taniguchi 1,†, Riccardo Melani 1,†, Lynne Chantranupong 2, Michelle J Wen 2, Ali Mohebi 3, Joshua D Berke 3, Bernardo L Sabatini 2, Nicolas X Tritsch 1,✉
Editors: Joseph F Cheer4, Michael A Taffe5
PMCID: PMC11095934  PMID: 38748470

Abstract

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Research organism: Mouse

Introduction

Presynaptic modulation is a ubiquitous mechanism through which neural circuits control the amount of chemical transmitter that axons release per incoming action potential (Lovinger et al., 2022). Striatum-projecting midbrain dopamine (DA) neurons are no exception; many transmitters present in the striatum directly act on DA axons to facilitate or depress vesicular release of DA (Sulzer et al., 2016). However, the axons of DA neurons do stand out in their sensitivity to acetylcholine (ACh), whose release from striatal cholinergic interneurons in brain slices is potent enough to trigger axonal action potentials and locally evoke DA release (i.e., independently of somatic spiking activity) via the activation of β2-containing nicotinic ACh receptors on DA axons (Cachope et al., 2012; Threlfell et al., 2012; Wang et al., 2014; Mamaligas et al., 2016; Kramer et al., 2022; Liu et al., 2022; Matityahu et al., 2023). This mechanism was proposed to underlie observations that DA release in ventral striatum (nucleus accumbens, NAc) can increase even when the firing of DA neurons in the midbrain appears unchanged (Berke, 2018; Mohebi et al., 2019). However, direct evidence for ACh-evoked striatal DA release in vivo remains limited.

In their original report of ACh-evoked DA release, Cachope et al., 2012 presented an example recording whereby strong optogenetic activation of cholinergic interneurons for several seconds is accompanied by DA elevation in the NAc of a urethane-anesthetized mouse. More recently, we and others showed in mice and rats that the patterns of DA and ACh release in various striatal locations in vivo are strongly correlated on sub-second time-scales in a manner that is consistent with ACh-evoked DA release (Howe et al., 2019; Liu et al., 2022; Chantranupong et al., 2023; Krok et al., 2023; Mohebi et al., 2023). Yet, we were unable to observe strong changes in the dynamics of DA in the dorsal and lateral striatum of mice after molecular, pharmacological and optogenetic interference with ACh signaling (Chantranupong et al., 2023; Krok et al., 2023).

A recent paper (Mohebi et al., 2023) provided some of the most compelling evidence to date that optogenetic activation of channelrhodopsin-expressing striatal cholinergic interneurons drives DA release in the NAc of awake behaving rats, as measured with the red-shifted DA sensor RdLight1 (Patriarchi et al., 2020). However, one concern with these experiments is that mApple-based fluorescent sensors – including RdLight1 and the GRAB-rDA series (Zhuo et al., 2024) – may exhibit photoactivation (also known as ‘photoswitching’ or ‘photoconversion’), a process whereby mApple’s red fluorescence changes in the presence of blue light (Shaner et al., 2008). This phenomenon is one of the main downsides of the R-GECO family of red Ca2+ indicators, which also use mApple and grow brighter independently of Ca2+ for hundreds of milliseconds following brief flashes of blue light, limiting their use with optogenetics (Akerboom et al., 2013; Dana et al., 2016). In the case of RdLight1, it was previously shown that photoactivation effects are negligible when expressed in cultured kidney cells and imaged using light-scanning confocal microscopy (Patriarchi et al., 2020). Whether RdLight1 shows photoactivation in vivo under conditions routinely used in behavioral experiments (i.e., full-field fiber photometry and optogenetic stimulation) has not been investigated.

Results

Blue light evokes RdLight1 fluorescence transients in the absence of an opsin

To determine if blue light modifies the fluorescent properties of RdLight1 in the behaving brain, the laboratory of N.X.T. virally expressed RdLight1 in either the dorsolateral striatum (DLS) or NAc of wild-type mice (Figure 1A and B) and imaged RdLight1 fluorescence in vivo using fiber photometry while mice were head-fixed on a cylindrical treadmill (Figure 1C). Under standard continuous illumination conditions (565 nm excitation light, 30–50 μW at the tip of the fiber), we observed transient increases and decreases in red fluorescence consistent with established patterns of DA release (Howe and Dombeck, 2016; da Silva et al., 2018; Chantranupong et al., 2023; Krok et al., 2023; Markowitz et al., 2023; Mohebi et al., 2024), including spontaneous fluctuations during immobility and large-amplitude reward-evoked responses (Figure 1D–E).

Figure 1. Blue light evokes RdLight1 photoactivation transients resembling DA release.

(A) The red DA sensor RdLight1 was virally expressed in neurons of the nucleus accumbens (NAc) or dorsolateral striatum (DLS) of wild-type mice and imaged in vivo via chronically-implanted fiber optic cannulas. (B) Example fixed coronal sections from two mice stained for the nuclear marker DAPI (blue) and imaged by epifluorescence showing RdLight1 (red) expression in the NAc (left) and DLS (right). Scale bar: 1 mm. (C) Experimental setup for imaging RdLight1 by photometry while delivering blue light (470 nm) via the same fiber optic in awake behaving mice. RdLight1 was excited with continuous yellow-green light (565 nm) and red emitted fluorescence (580–680 nm) was collected via a dual color photometry minicube. (D) Representative RdLight1 transient aligned to water reward delivery in NAc (top) and DLS (bottom). Solid lines are the mean of 15 trials. Shaded area shows SEM. (E) Continuous RdLight1 photometric imaging during immobility in NAc (top) and DLS (bottom). Blue lines highlight 4 ms-long pulses of blue light (9 mW) delivered through the same fiber optic, each followed by a delayed RdLight1 transient. (F) Comparison of mean reward- and blue light-evoked RdLight1 transients recorded in N=4 mice in NAc and DLS. (G) RdLight1 fluorescence (emitted upon continuous excitation with 565 nm light) increases upon exposure to blue light pulses (470 nm; 9 mW at tip of patch cord) of various durations (1–6ms; color-coded) in the NAc (top) and DLS (bottom) of a representative mouse. Solid lines are the mean of 15 blue light presentations. Shaded area shows SEM. (H) The magnitude of blue light-evoked RdLight1 fluorescence transients (i.e., photoactivation) grows with the duration of blue light pulses in both NAc (top; N=8 mice) and DLS (bottom; N=8 mice).(I) Magnitude of RdLight1 fluorescence transients evoked by 6 ms-long blue light pulses of various intensities (0.3–9 mW, measured at tip of patch cord) in both NAc (top) and DLS (bottom). Data plotted on Log10-Log10 scales. (J) Representative RdLight1 photoactivation transients (red: mean of 10 individual traces shown in gray) imaged in NAc before and after systemic block of vesicular DA release with reserpine. (K) Magnitude of blue light-evoked (9 mW; 6ms pulse width) RdLight1 photoactivation before and after reserpine treatment in NAc (N=4 mice) and DLS (N=4 mice).

Figure 1.

Figure 1—figure supplement 1. Blue light evokes RdLight1 photoactivation in VLS.

Figure 1—figure supplement 1.

(A) Injection and fiber implantation scheme for mice expressing RdLight1 in the VLS (left). Images of sensor expression from a representative mouse (right; scale bar: 800 µm). (B) Mean (± SEM) RdLight1 photoactivation signal in the VLS in response to 1, 2, 4, or 6ms single pulses of blue light stimulation (10 mW) coupled with constant 565 nm illumination (30 µW). Colored vertical bars indicate laser stimulation artifacts which were removed. Mean maximal RdLight1 photoactivation signals recorded under the indicated stimulation widths are displayed. Each dot represents the average signal from a single mouse, and error bars denote the standard deviation (N=3 mice). (C) Mean (± SEM) RdLight1 photoactivation signal in the VLS in response to a pair of 4ms blue light pulses (10 mW) separated by the indicated inter-pulse intervals. Mean paired pulse ratios are displayed (magnitude of pulse #2/pulse #1), with each dot representing a single mouse, and the error bars denoting the standard deviation (N=3 mice).
Open in a new tab

Delivering blue light pulses (4 ms-long) through the same fiber at powers typically used for optogenetic manipulations (9 mW at the tip of the patch cord) evoked distinct transients in RdLight1 fluorescence resembling DA release (Figure 1E–F). In the NAc, these transients averaged 7.0 ± 0.1% ΔF/F in magnitude, peaked 115±4ms after light onset and decayed back to baseline within 1 s (τdecay: 422±21ms; N=8 mice). In the DLS, blue light-evoked transients were smaller (4.5 ± 0.2% ΔF/F) but showed similar kinetics (time from light onset to peak: 129±2ms; τdecay: 516±60ms; N=8 mice). In both regions, blue light-evoked transients scaled in magnitude with the duration (Figure 1G–H) and intensity of light pulses (Figure 1I). In parallel experiments taking place in the laboratory of B.L.S., comparable increases in RdLight1 fluorescence were observed in N=3 mice that expressed RdLight1 in the ventrolateral striatum (VLS; Figure 1—figure supplement 1), confirming their occurrence across a range of experimental conditions. These delayed fluorescent signals are specific to RdLight1, as they are not observed in mice expressing the red fluorescent protein tdTomato (not shown).

Blue light-evoked RdLight1 fluorescence transients do not reflect DA release

Do these blue light-evoked RdLight1 transients reflect a phasic elevation in extracellular DA? Several lines of evidence suggest that this is not the case. First, our wild-type mice do not express blue light-gated opsins to drive DA release and DA neurons are not thought to be intrinsically sensitive to blue light. Second, blue light-evoked transients show little trial-by-trial variability in their amplitudes and kinetics (Figure 1G and J). Third, blue light-evoked transients do not display short-term facilitation or depression under a variety of stimulation conditions (Figure 2 and Figure 1—figure supplement 1C), calling into question their synaptic origin. Fourth, we repeated the above experiments in a subset of mice treated with reserpine, an irreversible antagonist of the transporter required for loading DA into synaptic vesicles (Figure 1J). Under these conditions, spontaneous fluctuations in RdLight1 fluorescence vanished in both the DLS (N=4) and NAc (N=4), confirming the absence of activity-dependent DA release in vivo. By contrast, blue light-evoked RdLight1 transients did not disappear and, if anything, grew in amplitude and duration in both DLS and NAc (Figure 1J and K), demonstrating that they do not reflect synaptic release of DA.

Figure 2. RdLight1 photoactivation transients evoked by blue light pulse trains.

Figure 2.

Open in a new tab

(A) Experimental setup. (B) Mean (± SEM) RdLight1 photoactivation in response to 1, 4, 16, 32, or 64 blue light pulses (9 mW, 4ms width, 16 Hz frequency) in the NAc of a representative mouse. Green bar illustrates constant 565 nm illumination. Blue bars show when blue light is delivered. (C) Same as (B) for pairs of blue light pulses (9 mW, 4ms width) separated by 2.5, 5 and 10 s. (D) Mean paired-pulse ratio (magnitude of pulse #2/pulse #1) across three inter-pulse intervals in each of N=8 mice.

Discussion

Our results show that RdLight1 displays strong photoactivation following exposure to blue light under wide-field illumination conditions routinely used to monitor and manipulate neural activity in vivo. This photoactivation manifests as a prolonged, DA-independent increase in RdLight1 fluorescence that outlasts the blue light pulse and slowly decays back to baseline over hundreds of milliseconds, giving it the appearance of synaptically-released DA. Under our recording conditions, photoactivation remained detectable with as little as 0.3 mW blue light (Figure 1I), indicating that RdLight1 fluorescence should be interpreted with caution when combined with blue light in a variety of experimental conditions, including dual-color imaging of green and red fluorophores. In this case, the steady or rapidly-modulating (i.e., tens to hundreds of Hz) blue light might lead to constant activation of mApple but, due to its long photoswitching time, is unlikely to contribute to phasic and aperiodic transients in the red channel, which our experiments showed reflect DA release (Chantranupong et al., 2023; Krok et al., 2023). It is possible that the initial characterization of RdLight1 failed to reveal strong blue light-mediated photoactivation as it used laser-scanning microscopy (Patriarchi et al., 2020), which Roger Tsien and colleagues found in their original characterization of mApple produced significantly less photoswitching compared to continuous wide-field illumination (Shaner et al., 2008).

Our findings call into question the nature of the RdLight1 fluorescent transients reported in Figure 1 of the study by Mohebi et al., 2023. Given the similarity of our recordings in terms of response magnitude, timing and dynamics over a variety of stimulation parameters, it is likely that the light-evoked RdLight1 responses reported reflect this photoactivation effect. Although the study used 405 nm illumination to control for changes in fluorophore properties independent of ligand binding (i.e., the so-called isosbestic point), this deep blue wavelength has not been shown to be the isosbestic point for RdLight1. Indeed, the isosbestic point of commonly-used green dopamine sensors is more green-shifted (e.g. 440 nm; Sun et al., 2020), stressing the need for sensors to be published along with their full excitation/emission spectra, and for experimenters to tailor illumination wavelengths to their specific sensor. It also remains to be determined whether illumination at isosbestic wavelengths can contribute to or modify photoactivation.

Moving forward, additional experiments will be needed to determine conditions under which cholinergic interneurons locally evoke DA release in the striatum of behaving animals. If using RdLight1, experiments should include controls to account for the effects of blue light alone, such as expressing RdLight1 only (i.e., no opsin), RdLight1 and a fluorophore [e.g., green fluorescent protein (GFP)] or a mutated sensor that does not bind DA. Blue light illumination power should be kept as low as possible. Alternatively, optogenetic experiments may be designed using GFP-based DA sensors such as dLight1 (Patriarchi et al., 2018) or GRAB-DA3 (Zhuo et al., 2024) in combination with red-shifted optogenetic actuators (Klapoetke et al., 2014; Marshel et al., 2019), although this configuration is not without caveats either, as continuous blue light illumination may cause opsin activation.

Importantly, photoactivation is not unique to RdLight1. Other mApple-based sensors, including the GRAB-rDA and R-GenGAR-DA families of red DA sensors (Sun et al., 2020; Nakamoto et al., 2021; Zhuo et al., 2024), as well as the R-GECO family of Ca2 +indicators (Akerboom et al., 2013; Dana et al., 2016) show photoactivation when presented with blue light, with individual constructs differing in the polarity, magnitude and duration of photoactivation effects. Photoactivation also extends beyond mApple, as many fluorophores can modify their biophysical properties when presented with different wavelengths of light. It is, for instance, the biophysical principle on which some molecular tracking and super-resolution imaging methods are founded (Lippincott-Schwartz and Patterson, 2009; Chozinski et al., 2014). Experiments should therefore be designed carefully to control for these and other potential confounds, particularly when novel sensors are introduced.

Materials and methods

Animals

Procedures were performed in accordance with protocols approved by the NYU Grossman School of Medicine (NYUGSM; #IA16-02082) and Harvard Medical School (HMS; #IS0000571) Institutional Animal Care and Use Committees. Wild type mice (C57BL/6 J; Jackson Laboratory strain #000664; 12–18 weeks of age) were housed in group before surgery and singly after surgery under a reverse 12 hour light-dark cycle (dark from 6 a.m. to 6 p.m. at NYUGSM and 10 a.m. to 10 p.m. at HMS) with ad libitum access to food and water.

Stereotaxic surgery

Mice were prepared for intracranial infections of adeno-associated viruses (AAVs) as before (Chantranupong et al., 2023; Krok et al., 2023). Briefly, mice were anaesthetized with isoflurane, administered Ketoprofen (10 mg/kg, subcutaneous) or Carprofen (5 mg/kg, subcutaneous) and placed in a stereotaxic apparatus (Kopf Instruments), where a small craniotomy was drilled above the NAc (from Bregma: AP +1.0 mm, ML +0.75 mm), DLS (from Bregma: AP +0.5 mm, ML +2.5 mm), or VLS (from Bregma: AP +0.6 mm, ML +2.3 mm). 300 nL of AAV2/9.Syn.RdLight1 (CNP Viral Vector Core at the CERVO Research Center contribution) was injected (100 nL/min) at a depth of 3.7 mm below dura for NAc, 2.3 mm for DLS or 3.2 mm for VLS using a microsyringe pump (KD Scientific; Legato 111) connected to a pulled glass injection needle (100 μm tip; Drummond Wiretrol II). Fiber optics (NAc and DLS: 400 μm diameter core, 0.5 NA; RWD Life Science Inc; VLS: 200 μm diameter core, 0.48 NA; Doric) were implanted 100 μm above the injection site and cemented to the skull using C&B metabond (Parkell) along with a custom titanium headpost placed over lambda to allow for head fixation during recordings. Mice were allowed to recover in their home cage for 2–4 weeks before head-fixation and treadmill habituation, and recordings.

Fiber Photometry

RdLight1 photometry recordings were carried out by feeding constant, low-power yellow-green excitation light (565 nm LED, 30–50 μW at the tip of the patch cord; Thorlabs M565F3) to a fluorescence mini cube (FMC5_E1(460–490)_F1(500–540)_E2(555–570)_F2(580–680)_S; Doric) connected to the mouse’s fiber optic implant via a 0.48 NA patch cord (NAc and DLS: MFP_400/460/900–0.48_2 m_FCM-MF1.25; VLS: MFP_200/220/900_2 m_FCM-MF1.25; both from Doric). The red light emitted by RdLight1 was collected through the same patch cord and routed via the fluorescence mini cube and a second fiber optic (MFP_600/630/LWMJ-0.48_0.5 m_FCM-FCM; Doric) to a photoreceiver (Newport 2151) to produce a voltage that is proportional to the intensity of the emitted light. Voltages were digitized at 2 kHz with either a National Instruments acquisition board (NI USB-6343) or a Labjack (T7) and saved to disk as ‘trials/sweeps’ lasting 5–20 s in duration each using Wavesurfer software (Janelia). To characterize the photoactivation behavior of RdLight1, we delivered brief pulses (1–6ms in duration) of blue light (NAc, DLS: 470 nm LED, Thorlabs M470F3; VLS: 470 nm laser, Optoengine) to the brain via the same fluorescence mini cube and patch cord used for photometry. We tested a range of blue light powers (measured at the tip of the patch cord) frequently used for optogenetic manipulations in vivo. The timing, duration and intensity of blue light pulses were controlled digitally using Wavesurfer, with each stimulation parameter repeated at minimum 10 times per mouse/recording site. RdLight1 photoactivation responses are extremely stable over time and were reliably seen for the duration of 1 h-long recording sessions.

Fiber Photometry Analysis

Photometry signals were processed and analyzed offline using custom code (https://github.com/TritschLab/Taniguchi-Melani-2024; copy archived at TritschLab, 2024) in MATLAB (Mathworks) and Igor Pro 6.02 A (Wavemetrics). Raw voltages collected from the photoreceiver were down sampled to 1,000 Hz and converted to ‘percent changes in fluorescence’ using the equation ∆FF=F-F0F0 , where F0 is the mean baseline fluorescence, computed for each trial/sweep during a 1.5–2 s baseline window preceding the blue light stimulus (Taniguchi, 2024). Blue excitation light led to an instantaneous artifact in the red channel for the exact duration of the blue light pulse, which was blanked in display panels. For each experiment, 10–15 replicates were performed. In figures, gray traces represent single trials, while colored traces represent the mean of 10–15 trials, with standard error of the mean (SEM) shown as a shaded area. The properties of RdLight1 photoactivation transients [peak amplitude, latency to peak (i.e., from blue light onset to RdLight1 photoactivation peak) and decay time constant (i.e., time from peak to 37% of peak)] were measured for each mouse using averaged waveforms. Data are reported in the text and figures as mean ± SEM. N-values represent the number of mice.

Immunohistochemistry

Mice were deeply anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were post-fixed for 24 h and sectioned coronally (100 μm in thickness) using a vibratome (Leica; VT1000S). Brain sections were mounted on superfrost slides and coverslipped with ProLong antifade reagent with DAPI (Molecular Probes). RdLight1 fluorescence was not immuno-enhanced. Whole sections were imaged with an Olympus VS120 slide scanning microscope.

Reagents

To inhibit DA vesicular transport and prevent vesicular release of DA, mice were injected intraperitoneally with the irreversible vesicular monoamine transporter inhibitor reserpine (5 mg/kg) for 24 h prior to RdLight1 photometry.

Acknowledgements

This work was supported by the National Institutes of Health (R01MH130658 to NXT), a Irma T Hirschl Research Award (to NXT), a National Science Foundation Graduate Research Fellowship (to JT) and a Howard Hughes Medical Institute Hanna Gray Fellowship (to LC, BLS). We acknowledge the New York University Langone Health Department of Comparative Medicine for animal care and maintenance and the Neuroscience Institute’s imaging facilities for microscope availability.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Nicolas X Tritsch, Email: nicolas.tritsch@nyulangone.org.

Joseph F Cheer, University of Maryland School of Medicine, United States.

Michael A Taffe, University of California, San Diego, United States.

Funding Information

This paper was supported by the following grants:

  • National Institutes of Health R01MH130658 to Nicolas X Tritsch.

  • Irma T. Hirschl Trust to Nicolas X Tritsch.

  • National Science Foundation to James Taniguchi.

  • Howard Hughes Medical Institute to Lynne Chantranupong, Bernardo L Sabatini.

Additional information

Competing interests

No competing interests declared.

Author contributions

Formal analysis, Investigation, Methodology, Writing – review and editing.

Formal analysis, Investigation, Methodology, Writing – review and editing.

Formal analysis, Investigation, Methodology, Writing – review and editing.

Investigation.

Writing – review and editing.

Writing – review and editing.

Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing.

Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing.

Ethics

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were performed in accordance with protocols approved by the NYU Grossman School of Medicine (NYUGSM; #IA16-02082) and Harvard Medical School (HMS; #IS0000571) Institutional Animal Care and Use Committees.

Additional files

MDAR checklist
Source data 1. Source data for histograms presented in Figures 1 and 2.
elife-95694-data1.xlsx (13.2KB, xlsx)

Data availability

All data plotted in the manuscript's figures is provided in Source data 1.

References

  1. Akerboom J, Carreras Calderón N, Tian L, Wabnig S, Prigge M, Tolö J, Gordus A, Orger MB, Severi KE, Macklin JJ, Patel R, Pulver SR, Wardill TJ, Fischer E, Schüler C, Chen TW, Sarkisyan KS, Marvin JS, Bargmann CI, Kim DS, Kügler S, Lagnado L, Hegemann P, Gottschalk A, Schreiter ER, Looger LL. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Frontiers in Molecular Neuroscience. 2013;6:2. doi: 10.3389/fnmol.2013.00002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berke JD. What does dopamine mean? Nature Neuroscience. 2018;21:787–793. doi: 10.1038/s41593-018-0152-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cachope R, Mateo Y, Mathur BN, Irving J, Wang HL, Morales M, Lovinger DM, Cheer JF. Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing. Cell Reports. 2012;2:33–41. doi: 10.1016/j.celrep.2012.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chantranupong L, Beron CC, Zimmer JA, Wen MJ, Wang W, Sabatini BL. Dopamine and glutamate regulate striatal acetylcholine in decision-making. Nature. 2023;621:577–585. doi: 10.1038/s41586-023-06492-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chozinski TJ, Gagnon LA, Vaughan JC. Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging. FEBS Letters. 2014;588:3603–3612. doi: 10.1016/j.febslet.2014.06.043. [DOI] [PubMed] [Google Scholar]
  6. da Silva JA, Tecuapetla F, Paixão V, Costa RM. Dopamine neuron activity before action initiation gates and invigorates future movements. Nature. 2018;554:244–248. doi: 10.1038/nature25457. [DOI] [PubMed] [Google Scholar]
  7. Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, Hu A, Walpita D, Patel R, Macklin JJ, Bargmann CI, Ahrens MB, Schreiter ER, Jayaraman V, Looger LL, Svoboda K, Kim DS. Sensitive red protein calcium indicators for imaging neural activity. eLife. 2016;5:e12727. doi: 10.7554/eLife.12727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Howe MW, Dombeck DA. Rapid signalling in distinct dopaminergic axons during locomotion and reward. Nature. 2016;535:505–510. doi: 10.1038/nature18942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Howe M, Ridouh I, Allegra Mascaro AL, Larios A, Azcorra M, Dombeck DA. Coordination of rapid cholinergic and dopaminergic signaling in striatum during spontaneous movement. eLife. 2019;8:e44903. doi: 10.7554/eLife.44903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK-S, Boyden ES. Independent optical excitation of distinct neural populations. Nature Methods. 2014;11:338–346. doi: 10.1038/nmeth.2836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kramer PF, Brill-Weil SG, Cummins AC, Zhang R, Camacho-Hernandez GA, Newman AH, Eldridge MAG, Averbeck BB, Khaliq ZM. Synaptic-like axo-axonal transmission from striatal cholinergic interneurons onto dopaminergic fibers. Neuron. 2022;110:2949–2960. doi: 10.1016/j.neuron.2022.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krok AC, Maltese M, Mistry P, Miao X, Li Y, Tritsch NX. Intrinsic dopamine and acetylcholine dynamics in the striatum of mice. Nature. 2023;621:543–549. doi: 10.1038/s41586-023-05995-9. [DOI] [PubMed] [Google Scholar]
  13. Lippincott-Schwartz J, Patterson GH. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends in Cell Biology. 2009;19:555–565. doi: 10.1016/j.tcb.2009.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liu C, Cai X, Ritzau-Jost A, Kramer PF, Li Y, Khaliq ZM, Hallermann S, Kaeser PS. An action potential initiation mechanism in distal axons for the control of dopamine release. Science. 2022;375:1378–1385. doi: 10.1126/science.abn0532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lovinger DM, Mateo Y, Johnson KA, Engi SA, Antonazzo M, Cheer JF. Local modulation by presynaptic receptors controls neuronal communication and behaviour. Nature Reviews. Neuroscience. 2022;23:191–203. doi: 10.1038/s41583-022-00561-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mamaligas AA, Cai Y, Ford CP. Nicotinic and opioid receptor regulation of striatal dopamine D2-receptor mediated transmission. Scientific Reports. 2016;6:37834. doi: 10.1038/srep37834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Markowitz JE, Gillis WF, Jay M, Wood J, Harris RW, Cieszkowski R, Scott R, Brann D, Koveal D, Kula T, Weinreb C, Osman MAM, Pinto SR, Uchida N, Linderman SW, Sabatini BL, Datta SR. Spontaneous behaviour is structured by reinforcement without explicit reward. Nature. 2023;614:108–117. doi: 10.1038/s41586-022-05611-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marshel JH, Kim YS, Machado TA, Quirin S, Benson B, Kadmon J, Raja C, Chibukhchyan A, Ramakrishnan C, Inoue M, Shane JC, McKnight DJ, Yoshizawa S, Kato HE, Ganguli S, Deisseroth K. Cortical layer-specific critical dynamics triggering perception. Science. 2019;365:eaaw5202. doi: 10.1126/science.aaw5202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matityahu L, Gilin N, Sarpong GA, Atamna Y, Tiroshi L, Tritsch NX, Wickens JR, Goldberg JA. Acetylcholine waves and dopamine release in the striatum. Nature Communications. 2023;14:6852. doi: 10.1038/s41467-023-42311-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mohebi Ali, Pettibone JR, Hamid AA, Wong J-MT, Vinson LT, Patriarchi T, Tian L, Kennedy RT, Berke JD. Dissociable dopamine dynamics for learning and motivation. Nature. 2019;570:65–70. doi: 10.1038/s41586-019-1235-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mohebi A, Collins VL, Berke JD. Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation. eLife. 2023;12:e85011. doi: 10.7554/eLife.85011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mohebi A, Wei W, Pelattini L, Kim K, Berke JD. Dopamine transients follow a striatal gradient of reward time horizons. Nature Neuroscience. 2024;27:737–746. doi: 10.1038/s41593-023-01566-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakamoto C, Goto Y, Tomizawa Y, Fukata Y, Fukata M, Harpsøe K, Gloriam DE, Aoki K, Takeuchi T. A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro. Molecular Brain. 2021;14:173. doi: 10.1186/s13041-021-00882-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patriarchi T, Cho JR, Merten K, Howe MW, Marley A, Xiong W-H, Folk RW, Broussard GJ, Liang R, Jang MJ, Zhong H, Dombeck D, von Zastrow M, Nimmerjahn A, Gradinaru V, Williams JT, Tian L. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science. 2018;360:eaat4422. doi: 10.1126/science.aat4422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Patriarchi T, Mohebi A, Sun J, Marley A, Liang R, Dong C, Puhger K, Mizuno GO, Davis CM, Wiltgen B, von Zastrow M, Berke JD, Tian L. An expanded palette of dopamine sensors for multiplex imaging in vivo. Nature Methods. 2020;17:1147–1155. doi: 10.1038/s41592-020-0936-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nature Methods. 2008;5:545–551. doi: 10.1038/nmeth.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sulzer D, Cragg SJ, Rice ME. Striatal dopamine neurotransmission: regulation of release and uptake. Basal Ganglia. 2016;6:123–148. doi: 10.1016/j.baga.2016.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sun F, Zhou J, Dai B, Qian T, Zeng J, Li X, Zhuo Y, Zhang Y, Wang Y, Qian C, Tan K, Feng J, Dong H, Lin D, Cui G, Li Y. Next-generation GRAB sensors for monitoring dopaminergic activity in vivo. Nature Methods. 2020;17:1156–1166. doi: 10.1038/s41592-020-00981-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taniguchi J. Tritschlab fiber Photometry analysis custom code. a3d3572GitHub. 2024 https://github.com/TritschLab/Taniguchi-Melani-2024
  30. Threlfell S, Lalic T, Platt NJ, Jennings KA, Deisseroth K, Cragg SJ. Striatal dopamine release is triggered by synchronized activity in cholinergic interneurons. Neuron. 2012;75:58–64. doi: 10.1016/j.neuron.2012.04.038. [DOI] [PubMed] [Google Scholar]
  31. TritschLab Taniguchi-Melani-2024. swh:1:rev:bf4ebf3e1b8ec9d5331382fe1c93541275ce696cSoftware Heritage. 2024 https://archive.softwareheritage.org/swh:1:dir:103b6ac24b964a6ee132f1b208ef02011ade880f;origin=https://github.com/TritschLab/Taniguchi-Melani-2024;visit=swh:1:snp:cf00242abd48099ef9f754f17164176d704aceab;anchor=swh:1:rev:bf4ebf3e1b8ec9d5331382fe1c93541275ce696c
  32. Wang L, Shang S, Kang X, Teng S, Zhu F, Liu B, Wu Q, Li M, Liu W, Xu H, Zhou L, Jiao R, Dou H, Zuo P, Zhang X, Zheng L, Wang S, Wang C, Zhou Z. Modulation of dopamine release in the striatum by physiologically relevant levels of nicotine. Nature Communications. 2014;5:3925. doi: 10.1038/ncomms4925. [DOI] [PubMed] [Google Scholar]
  33. Zhuo Y, Luo B, Yi X, Dong H, Miao X, Wan J, Williams JT, Campbell MG, Cai R, Qian T, Li F, Weber SJ, Wang L, Li B, Wei Y, Li G, Wang H, Zheng Y, Zhao Y, Wolf ME, Zhu Y, Watabe-Uchida M, Li Y. Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo. Nature Methods. 2024;21:680–691. doi: 10.1038/s41592-023-02100-w. [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision letter

Editor: Joseph F Cheer1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'" for consideration by eLife. Your article has been evaluated by Michael Taffe as Senior Editor, Joseph Cheer as Reviewing Editor, and two reviewers (who have opted to remain anonymous).

Please consider the comments and suggestions made by the two reviewers (please see below) and respond accordingly. Please also address the editorial issues.

Reviewer #1:

The comment by Taniguchi et al. systematically and convincingly demonstrates that the red dopamine sensor RdLight1 is photoactivated in response to blue light in the absence of blue light-activated opsins. Moreover, the dynamics of RdLight1 responses to blue light closely mimic the classic dynamics of dopamine transients and would be difficult to distinguish without proper controls. The magnitude of the artifact scales with blue light duration and intensity, is reproducible across several regions of striatum (and in separate laboratories), is robust to dopamine depletion with reserpine, and shows no evidence of depression with repeated stimulation. Taken together, these data highlight an important caveat for the use of RdLight1 with blue-light-activated opsins. This issue has implications for both the interpretation of the paper by Mohebi, Collins and Berke and for the wider audience of scientists utilizing fluorescent sensors in their work. While the findings of this comment show definitively that Figure 1 of Mohebi et al. is contaminated by an artifact, the remaining figures of the paper add valuable knowledge to our limited understanding of the role of cholinergic interneurons in modulating dopamine activity in the striatum in vivo. It is important to note that this comment is an example of responsible and transparent work that reflects how science should be conducted. Both the authors of the original paper and of this comment showed commendable scientific integrity in producing this comment. The fact that these studies were done across laboratories shows not only open collaboration, which should be applauded but also adds an extra layer of rigor.

1. It is still unclear if dual-color fiber photometry (as is used in some of the remaining Mohebi et al. figures) would be subject to the same artifact, as the light intensities generally used for fiber photometry are much lower than for optogenetics and lower than were tested in this comment. In the future, all authors making use of fluorescent sensors can use the approaches demonstrated in this comment to conduct proper controls for their own experiments.

2. The authors correctly point out that many mApple-based fluorescent sensors have been shown to have similar photoswitching artifacts (Zhuo et al., 2023), but should be careful about singling out mApple as the sole issue. Doing so could lead researchers to falsely assume non-mApple-based sensors are safe from such artifacts. Researchers should be reminded of the need to run appropriate controls for all new sensors.

3. The authors mention that the 405 nm wavelength has not been shown to be an appropriate isosbestic wavelength for RdLight1. This provides an opportunity to highlight the need for those developing new sensors to carefully test and publish the full excitation and emission spectra of their sensors in the future.

Reviewer #2:

In the present Comment Taniguchi et al. address an important methodological issue concerning the primary data collection in Mohebi et al. (2023) that substantially changes the interpretation and impact of the original article. In Mohebi et al., the authors used a red-shifted fluorescent dopamine sensor, RdLight1, to measure dopamine release in behaving rodents. They then simultaneously activated a population of acetylcholine-releasing interneurons (ChIs) using a blue-light gated channel rhodopsin (ChR2). They found that evoking action potentials in the ChIs with blue light led to transient increases in the RdLight1 signal that were interpreted as dopamine release. This result was the key observation for the principal conclusions of their study.

However, the present Comment clearly demonstrates through new data that blue light stimulation also directly 'activates' the RdLight1 sensor to produce a signal that appears as dopamine but is in fact not dopamine. Taniguchi et al. thoroughly tested and verified these findings. These results directly challenge the principal findings from Mohebi et al., including the observation that dopamine release is linearly scaled with intensity and duration of activation of cholinergic interneurons (i.e. no short-term depression).

It should be noted that Mohebi and Berke collaborated on this Comment and acknowledge the methodological error in their original publication. This Comment should be published with no need for additional data. I have no substantive concerns.

There are two changes to the text I would ask the authors to make.

1. The language used in a sentence in the Introduction implies that previous work examining the function of nicotinic receptors on dopaminergic fibers implicated these receptors as the sole drivers of all dopamine release in the dorsal and lateral striatum. This is because it is written that "DA dynamics in the dorsal and lateral striatum were found to persist even after …. interference with ACh signaling". Likely everyone would agree that the majority of "DA dynamics" should persist in these conditions. Please amend this section to use more precise language giving appropriate context to these experiments and results. For example, "previous results were unable to detect a change in DA dynamics…"

2. The authors of this Comment are experts in RdLight1, and similar sensors based on mApple. It would be a tremendous benefit to the field if the authors could leverage that expertise here to identify other sensors that could be impacted by the present results.

a. Are there other red fluorescent sensors that are based on mApple?

b. If the authors had used mApple instead of tdTomato in the experiment discussed in the Results, do they think they would have seen a signal like the one shown by RdLight1? Or is the problem with RdLight1 due to the molecular alterations necessary to make it respond to dopamine?

Please also make the following editorial revisions:

a) Abstract

Reading the article without the cover letter, it is not clear that the author list includes two authors from the paper that is being criticized (Mohebi and Berke): it would be good if this could be made clear by revising the abstract as follows:

It is widely believed that acetylcholine modulates the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors, but there is less evidence for this mechanism in vivo. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release, as measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Measurements of dopamine using RdLight1 should, therefore, be interpreted with caution when combined with optogenetics. In light of these results (which were obtained by a multi-laboratory collaboration that included Mohebi and Berke), and the results of other studies that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

b) Results section

The statement "In a separate laboratory..." will confuse readers: please revise the Results section to make clear where the different experiments were performed.

eLife. 2024 May 15;13:e95694. doi: 10.7554/eLife.95694.sa2

Author response

Reviewer #1:

The comment by Taniguchi et al. systematically and convincingly demonstrates that the red dopamine sensor RdLight1 is photoactivated in response to blue light in the absence of blue light-activated opsins. Moreover, the dynamics of RdLight1 responses to blue light closely mimic the classic dynamics of dopamine transients and would be difficult to distinguish without proper controls. The magnitude of the artifact scales with blue light duration and intensity, is reproducible across several regions of striatum (and in separate laboratories), is robust to dopamine depletion with reserpine, and shows no evidence of depression with repeated stimulation. Taken together, these data highlight an important caveat for the use of RdLight1 with blue-light-activated opsins. This issue has implications for both the interpretation of the paper by Mohebi, Collins and Berke and for the wider audience of scientists utilizing fluorescent sensors in their work. While the findings of this comment show definitively that Figure 1 of Mohebi et al. is contaminated by an artifact, the remaining figures of the paper add valuable knowledge to our limited understanding of the role of cholinergic interneurons in modulating dopamine activity in the striatum in vivo. It is important to note that this comment is an example of responsible and transparent work that reflects how science should be conducted. Both the authors of the original paper and of this comment showed commendable scientific integrity in producing this comment. The fact that these studies were done across laboratories shows not only open collaboration, which should be applauded but also adds an extra layer of rigor.

1. It is still unclear if dual-color fiber photometry (as is used in some of the remaining Mohebi et al. figures) would be subject to the same artifact, as the light intensities generally used for fiber photometry are much lower than for optogenetics and lower than were tested in this comment. In the future, all authors making use of fluorescent sensors can use the approaches demonstrated in this comment to conduct proper controls for their own experiments.

Although we cannot know for sure whether the flashes of blue light used to image GCaMP6 (expressed in cholinergic interneurons) can evoke photoactivation of RdLight1 under the original experimental conditions, we do not believe they account for the large RdLight1 fluorescence transients depicted in Figures 2 and 3 of the study by Mohebi et al. for several reasons. First, as the Reviewer correctly points out, photometry typically uses much less power than optogenetics (<100 uW vs. >1 mW). Our data suggest that the magnitude of the photoactivation artifact scales with blue light power (see our Figure 1I). Thus, while some photoactivation may take place any time blue light is applied, it is likely exceedingly small compared to the fluorescence transients evoked by dopamine. Second, the flashes of blue light used to image GCaMP6 were delivered at a constant rate and intensity. As such, they could not account for the large and aperiodic RdLight1 fluorescence transients observed. Lastly, close inspection of the example traces reveals that many RdLight1 and GCaMP6 transients occur independently of one another, providing additional evidence that one process does not drive the other. We added a paragraph at in the discussion to that effect:

“RdLight1 fluorescence should be interpreted with caution when combined with blue light in a variety of experimental conditions, including dual-color imaging with alternating stimulation of green and red fluorophores. In this case, the steady or rapidly-modulating (i.e., tens to hundreds of Hz) blue light might lead to constant activation of mApple but, due to its long photoswitching time, is unlikely to contribute to phasic and aperiodic transients in the red channel.”

2. The authors correctly point out that many mApple-based fluorescent sensors have been shown to have similar photoswitching artifacts (Zhuo et al., 2023), but should be careful about singling out mApple as the sole issue. Doing so could lead researchers to falsely assume non-mApple-based sensors are safe from such artifacts. Researchers should be reminded of the need to run appropriate controls for all new sensors.

We thank the Reviewer for this comment. We added a paragraph at the end of the discussion to emphasize this point:

“Importantly, photoactivation is not unique to RdLight1: other mApple-based sensors, including the GRAB-rDA and R-GenGAR-DA families of red DA sensors, as well as the R-GECO family of Ca2+ indicators also show photoactivation when presented with blue light, with individual constructs differing in the polarity, magnitude and duration of photoactivation effects. Photoactivation also extends beyond mApple, as many fluorophores can modify their biophysical properties when presented with different wavelengths of light. It is, for instance, the biophysical principle on which some molecular tracking and super-resolution imaging methods are founded. Experiments should therefore be designed carefully to control for these and other potential confounds, particularly when novel sensors are introduced.”

3. The authors mention that the 405 nm wavelength has not been shown to be an appropriate isosbestic wavelength for RdLight1. This provides an opportunity to highlight the need for those developing new sensors to carefully test and publish the full excitation and emission spectra of their sensors in the future.

We agree with the Reviewer. We added the following sentence in the discussion:

“Indeed, even the isosbestic point of commonly used green dopamine sensors is more green-shifted (e.g. 440 nm), stressing the need for sensors to be published along with their full excitation/emission spectra, and for experimenters to tailor illumination wavelengths to their specific sensor.”

Reviewer #2:

In the present Comment Taniguchi et al. address an important methodological issue concerning the primary data collection in Mohebi et al. (2023) that substantially changes the interpretation and impact of the original article. In Mohebi et al., the authors used a red-shifted fluorescent dopamine sensor, RdLight1, to measure dopamine release in behaving rodents. They then simultaneously activated a population of acetylcholine-releasing interneurons (ChIs) using a blue-light gated channel rhodopsin (ChR2). They found that evoking action potentials in the ChIs with blue light led to transient increases in the RdLight1 signal that were interpreted as dopamine release. This result was the key observation for the principal conclusions of their study.

However, the present Comment clearly demonstrates through new data that blue light stimulation also directly 'activates' the RdLight1 sensor to produce a signal that appears as dopamine but is in fact not dopamine. Taniguchi et al. thoroughly tested and verified these findings. These results directly challenge the principal findings from Mohebi et al., including the observation that dopamine release is linearly scaled with intensity and duration of activation of cholinergic interneurons (i.e. no short-term depression).

It should be noted that Mohebi and Berke collaborated on this Comment and acknowledge the methodological error in their original publication. This Comment should be published with no need for additional data. I have no substantive concerns.

There are two changes to the text I would ask the authors to make.

1. The language used in a sentence in the Introduction implies that previous work examining the function of nicotinic receptors on dopaminergic fibers implicated these receptors as the sole drivers of all dopamine release in the dorsal and lateral striatum. This is because it is written that "DA dynamics in the dorsal and lateral striatum were found to persist even after …. interference with ACh signaling". Likely everyone would agree that the majority of "DA dynamics" should persist in these conditions. Please amend this section to use more precise language giving appropriate context to these experiments and results. For example, "previous results were unable to detect a change in DA dynamics…"

We thank the Reviewer for their suggestion. We modified the highlighted sentence to read:

“Yet, we were unable to observe strong changes in the dynamics of DA in the dorsal and lateral striatum of mice after molecular, pharmacological and optogenetic interference with ACh signaling.”

2. The authors of this Comment are experts in RdLight1, and similar sensors based on mApple. It would be a tremendous benefit to the field if the authors could leverage that expertise here to identify other sensors that could be impacted by the present results.

a. Are there other red fluorescent sensors that are based on mApple?

Yes. All of the red fluorescent neuromodulator sensors developed to date use circular permutated (cp) mApple, including the RdLight (Patriarchi et al., 2020), GRAB-rDA (Sun et al., 2020; Zhuo et al., 2023) and R-GenGAR-DA (Nakamoto et al., 2021) families of red DA sensors. The GRAB family of red serotonin sensors also uses cpmApple (Deng et al., 2024). We now write a sentence in the discussion to that effect:

“Importantly, photoactivation is not unique to RdLight1: other mApple-based sensors, including the GRAB-rDA and R-GenGAR-DA families of red DA sensors, as well as the R-GECO family of Ca2+ indicators show photoactivation when presented with blue light, with individual constructs differing in the polarity, magnitude and duration of photoactivation effects.”

b. If the authors had used mApple instead of tdTomato in the experiment discussed in the Results, do they think they would have seen a signal like the one shown by RdLight1? Or is the problem with RdLight1 due to the molecular alterations necessary to make it respond to dopamine?

Having not tried the suggested experiment, it is difficult to answer the question in the affirmative, but our hypothesis is that we would. In the original paper describing the development of mApple, Shaner, Tsien and colleagues noted that mApple displays ‘complex reversible photoswitching behavior’ that is more pronounced with continuous wide-field illumination than with laser-scanning microscopy (Shaner et al., 2008). Interestingly, their attempts at fixing this problem using mutagenesis failed. We also know that all red DA sensors developed to date (by several groups, independently) show some form of photoactivation, although individual variants differ in the polarity, magnitude and duration of such effects (see Nakamoto et al., 2021; Zhuo et al., 2023). Lastly, photoactivation also extends to mApple-based Ca2+ indicators (Akerboom et al., 2013; Dana et al., 2016) and to fluorophores not based on mApple (Patterson and Lippincott-Schwartz 2002; Lippincott-Schwartz and Patterson 2009). Photoactivation is therefore neither specific to RdLight1, nor to the molecular alterations necessary to turn mApple into a fluorescent reporter for dopamine (i.e. circular permutation of mApple and insertion into one of the intracellular loops of a G protein-coupled DA receptor).

References cited in this response

Akerboom J, Carreras Calderon N, Tian L, Wabnig S, Prigge M, Tolo J, Gordus A, Orger MB, et al. (2013). Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6: 2.

Dana H, Mohar B, Sun Y, Narayan S, Gordus A, Hasseman JP, Tsegaye G, Holt GT, et al. (2016). Sensitive red protein calcium indicators for imaging neural activity. ELife 5: e12727.

Deng F, Wan J, Li G, Dong H, Xia X, Wang Y, Li X, Zhuang C, et al. (2024). Improved green and red GRAB sensors for monitoring spatiotemporal serotonin release in vivo. Nat Methods DOI: 10.1038/s41592-024-02188-8.

Lippincott-Schwartz J and Patterson GH (2009). Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11): 555-565.

Nakamoto C, Goto Y, Tomizawa Y, Fukata Y, Fukata M, Harpsoe K, Gloriam DE, Aoki K and Takeuchi T (2021). A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro. Mol Brain 14(1): 173.

Patriarchi T, Mohebi A, Sun J, Marley A, Liang R, Dong C, Puhger K, Mizuno GO, et al. (2020). An expanded palette of dopamine sensors for multiplex imaging in vivo. Nat Methods 17(11): 1147-1155.

Patterson GH and Lippincott-Schwartz J (2002). A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297(5588): 1873-1877.

Shaner NC, Lin MZ, Mckeown MR, Steinbach PA, Hazelwood KL, Davidson MW and Tsien RY (2008). Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5(6): 545-551.

Sun F, Zhou J, Dai B, Qian T, Zeng J, Li X, Zhuo Y, Zhang Y, et al. (2020). Next-generation GRAB sensors for monitoring dopaminergic activity in vivo. Nat Methods 17(11): 1156-1166.

Zhuo Y, Luo B, Yi X, Dong H, Miao X, Wan J, Williams JT, Campbell MG, et al. (2023). Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo. Nat Methods DOI: 10.1038/s41592-023-02100-w.

Please also make the following editorial revisions:

a) Abstract

Reading the article without the cover letter, it is not clear that the author list includes two authors from the paper that is being criticized (Mohebi and Berke): it would be good if this could be made clear by revising the abstract as follows:

It is widely believed that acetylcholine modulates the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors, but there is less evidence for this mechanism in vivo. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release, as measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Measurements of dopamine using RdLight1 should, therefore, be interpreted with caution when combined with optogenetics. In light of these results (which were obtained by a multi-laboratory collaboration that included Mohebi and Berke), and the results of other studies that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Thank you for this suggestion. In consultation with the authors, we agreed to implemented the first suggestion (“Mohebi, Collins and Berke recently reported that…”) but not the second (“In light of these results (which were obtained by a multi-laboratory collaboration that included Mohebi and Berke)…”) for the reason that Drs. Mohebi and Berke did not contribute to data collection. We did, however, include a new paragraph in the discussion describing how the various laboratories came together to produce this manuscript.

b) Results section

The statement "In a separate laboratory..." will confuse readers: please revise the Results section to make clear where the different experiments were performed.

To clarify where the different experiments were performed, we now mention in the result section whether results were obtained in the laboratory of N.X.T. or B.L.S.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    MDAR checklist
    elife-95694-mdarchecklist1.docx (99.5KB, docx)
    Source data 1. Source data for histograms presented in Figures 1 and 2.
    elife-95694-data1.xlsx (13.2KB, xlsx)

    Data Availability Statement

    All data plotted in the manuscript's figures is provided in Source data 1.

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

    RESOURCES