Obtaining rewards typically takes work: time-consuming sequences of unrewarded actions before the reward is reached. Deciding when such work is worthwhile is an essential aspect of adaptive behavior. Dopamine (DA) in the nucleus accumbens (NAc) is a critical modulator of such motivated decision-making (Berke, 2018; Salamone and Correa, 2012), especially choices to approach potential rewards (Ikemoto and Panksepp, 1999; Nicola, 2010). Yet how NAc DA release is itself dynamically regulated to achieve appropriate motivation is not well understood.

Some features of DA release mirror the spiking of DA cell bodies. Burst firing (or strong artificial stimulation) of ventral tegmental area (VTA) DA cell bodies is accompanied by a corresponding pulse of NAc DA release (Mohebi et al., 2019; Phillips et al., 2003; Suaud-Chagny et al., 1992). This pulse can encode reward prediction error (RPE), an important learning signal (Schultz et al., 1997). Other aspects of DA release appear to be dissociated from spiking. Enhancing motivation by increasing reward availability does not change VTA DA cell firing rates (Cohen et al., 2012; Mohebi et al., 2019), but does boost NAc Core DA levels, as measured on a time scale of minutes by microdialysis (Hamid et al., 2016; McCullough and Salamone, 1992). On faster time scales (seconds), many groups have reported ramps in NAc DA as animals approach rewards (Hamid et al., 2016; Howe et al., 2013; Roitman et al., 2004; Wassum et al., 2012). These ramps may reflect discounted estimates of future reward (value), a useful motivational signal. Yet we found no evidence for corresponding ramps in spiking rate among identified DA cells in the lateral VTA (Mohebi et al., 2019), which provide the major DA input to NAc Core (Breton et al., 2019).

An alternative means of controlling DA release involves local circuit components within the NAc (Sulzer et al., 2016). Prominent among these are cholinergic interneurons (CINs) which, despite constituting only 1–2% of striatal neurons, provide a very dense network of fibers releasing acetylcholine (ACh) intermeshed with the DA axon network (Descarries et al., 1996). DA axons possess nicotinic ACh receptors (nAChRs) that specifically contain the β2 subunit (β2*; Jones et al., 2002). Activation of nAChRs is sufficient to locally evoke action potentials in DA axons, and consequent DA release, even in the absence of DA cell bodies (Liu et al., 2022; Threlfell et al., 2012; Cachope and Cheer, 2014; Zhou et al., 2001).

Yet how CINs contribute to NAc DA dynamics in behaving animals is largely unknown. Slice studies suggest that DA release is evoked only by highly synchronous activation of multiple CINs (Threlfell et al., 2012), as might occur in response to a salient cue (Kimura et al., 1984). Furthermore, DA release in slices shows a lack of summation to repetitive CIN stimulation (Threlfell et al., 2012) and strong paired-pulse depression (Cachope et al., 2012; Wang et al., 2014). This is apparently due to rapid desensitization of nAChRs (Giniatullin et al., 2005; Quick and Lester, 2002) and feedback inhibition of CINs via DA D2 receptors (Shin et al., 2017). These features might limit the ability of CINs to sculpt motivation-related DA release, including ramping. Furthermore, results on CIN contributions to motivation are mixed. Suppression of CINs has been reported to produce a depression-like state in some behavioral tests (Warner-Schmidt et al., 2012) but to boost motivation in others (Collins et al., 2019).

We examined the relationships between CIN activity, DA release, and motivation. We took advantage of recent advances in optical DA sensors (Patriarchi et al., 2020) and a specific operant task in which we previously found a dissociation between DA spiking and release (Mohebi et al., 2019). We report that CINs can indeed control DA release in awake behaving animals, but with quite distinct temporal characteristics compared to slices. We find that CIN population activity ramps up during approach behaviors in parallel with DA ramps. Furthermore, selective pharmacological blockade of NAc β2* nAChRs interferes with motivated approach, as does blockade of DA receptors. Our results provide convergent evidence supporting the possibility that CINs are a key mechanism regulating motivation via local control of DA release.

Local regulation of striatal DA by ACh has long been established in brain slices (Giorguieff et al., 1976). However, how this influence shapes DA signals in vivo has remained obscure. As a result, cholinergic modulation of DA release has not typically been incorporated into functional accounts of DA dynamics (Berke, 2018), which have instead emphasized the faithful transmission of RPEs encoded in DA cell spiking. An accurate characterization of local modulation of NAc DA in behaving animals is essential not simply for understanding the neural control of motivation, but also altered physiological states evoked by disorders or abused drugs (notably, nicotine).

Seminal slice studies reported that while CINs can profoundly influence DA release via β2* nicotinic receptors, this effect shows strong short-term depression that can take minutes to recover (Cachope et al., 2012; Threlfell et al., 2012). If these desensitizing mechanisms apply in the awake behaving case, we might expect little functional contribution of CINs to DA signaling, and that stimulating CINs would not evoke appreciable extra DA release. Our results clearly demonstrate otherwise: stimulating CINs evoked strong DA release in a frequency-dependent manner, with no evidence of depression. This result both confirms the presence of powerful ACh modulation of DA release under physiological conditions and demonstrates that this modulation has the dynamic range to be a major, continuing influence over behaviorally relevant DA signals.

What can explain the discrepancy between ex vivo and in vivo experiments? One possibility involves the very different conditions for acetylcholinesterase (AChE), an enzyme that provides critical constraints over ACh signaling dynamics. AChE functions very rapidly, almost approaching the rate of ACh diffusion (Dvir et al., 2010; Quinn, 1987), but this rate will be slower at the lower temperature of slice experiments compared to whole animals. Furthermore, AChE is soluble in the extracellular matrix (Sirviö et al., 1989), and so can be partly washed away in the slice preparation. Taken together, released ACh in the slice may escape degradation in the synaptic space for longer than normal, and thereby provoke desensitization of a larger proportion of nAChRs.

One of the most prominent reported features of striatal CINs is a reliable pause in response to reward-predicting cues, in contrast to DA release (Morris et al., 2004). However, at least in NAc we found that both DA release and CIN activity increase rapidly in response to such cues (see also Atallah et al., 2014; Howe et al., 2019). Of course, bulk CIN GCaMP signals are at best an incomplete readout of either CIN firing or the spatiotemporal dynamics of ACh release. In particular, imaging of CINs GCaMP in the dorsal striatum found a mismatch between signals at cell bodies and the rest of the field of view (considered to be neuropil; Rehani et al., 2019). Understanding whether control of DA release via nAChRs directly reflects CIN firing will require more experiments adding either optogenetic tagging of CINs (Duhne, 2022) or high-resolution voltage imaging (Piatkevich et al., 2019). One related important direction for future studies is to understand why some transient fluctuations in CIN activity evoke DA release (Liu et al., 2022) but others do not (e.g., Figure 2b).

In animals performing an operant task, we demonstrated a correspondence between increasing ramps in NAc DA release during motivated approach and increasing NAc CIN activity (measured with GCaMP), at times when VTA DA cell firing is not increasing. This observation supports the idea that CINs can drive behaviorally relevant DA release even in the absence of DA firing changes. We further showed that, in the same task, interfering with NAc β2* nAChRs reduces the likelihood that rats perform the motivated approach behavior. However, one limitation of this study is that we did not causally establish that CINs are directly driving DA release, via β2* nAChRs, at the specific moments of DA ramping during approach. In subsequent studies, we hope to do this through combined local pharmacology, optogenetics, and photometry in behaving animals, as technical advances permit. Designing a conclusive manipulation experiment is not trivial; for example, if CINs tonically promote DA release, simple suppression of CINs could interfere with DA ramps during approach, even if CINs do not normally drive ramping.

Given that ACh can promote NAc DA release and thereby motivation, a continuing conundrum is to explain long-standing evidence for opposing roles for striatal DA and ACh in behavioral control (Mcgeer et al., 1961; Hikida et al., 2001; Collins et al., 2019). One potentially relevant behavioral dimension is whether actions are prompted by external events versus being ‘self-initiated.’ Increases in both (presumed) CIN activity and striatal DA release have been reported in conjunction with self-timed actions (e.g., Lee et al., 2006; Hamilos et al., 2021). This seems consistent with our observation of prominent joint DA and CIN activity ramps in longer-latency trials in our operant task, where rats are not simply reacting to the trial start light, but rather approaching at a variable time after its onset. However, if CIN control of NAc DA release is always important for self-initiated, motivated behavior, DHβE should suppress the hazard rate of approach across a wide range of delays after the light onset. In fact, both DHβE and FLU particularly depressed approach behavior in the 1–3 s range after light onset, with the hazard rate returning to control levels by ~4 s (Figure 4d). This suggests that the NAc joint ACh-DA mechanisms investigated here may be particularly involved in deciding whether it is worthwhile reorganizing ongoing behavior to respond to an external cue (Blazquez et al., 2002).

Finally, if CINs do indeed help locally sculpt motivational aspects of DA release, a natural question becomes how relevant CIN signals are themselves generated. CINs receive inputs from a wide range of areas that can supply motivational information and influence DA release, including the basolateral amygdala (Floresco et al., 1998), parafascicular thalamus (Bradfield et al., 2013), frontal cortex (Kosillo et al., 2016), and long-range GABAergic subpopulations in the VTA (Al-Hasani et al., 2021; Brown et al., 2012). They also sample the state of surrounding networks of striatal projection cells, allowing them to potentially extract useful decision variables (Franklin and Frank, 2015). Resolving how motivational signals are computed remains an ongoing challenge, but monitoring and manipulating each of these various circuit components within the same behavioral context is a promising approach.

All data generated or analyzed during this study will be made publicly available on Dryad (http://doi.org/10.7272/dryad.Q68P5XST).

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Decision letter after peer review:

Thank you for submitting your article "Cholinergic Interneurons Drive Motivation by Promoting Dopamine Release in the Nucleus Accumbens" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Michael Taffe as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Although there is significance to the paper, all reviewers are of the mindset that the authors need to show that, at physiologically relevant patterns of activity, CINs drive DA release in a behaviorally-relevant manner (manipulating CIN activity while at the same time measuring behaviorally evoked DA fluorescence, for example). This experiment is crucial in light of recent papers showing a disconnect between the two processes in behaving animals (the recent work of the Sabatini and Tritsch labs).

2) It is also very important for the authors to show that optically evoked DA fluorescence is indeed dependent on a4b2 receptors (see Mateo et al., 2017).

3) Please thoroughly explain the observation of a lack of frequency-dependent depression, which greatly contrasts with previously published reports (Cachope et al., 2012; Threlfell et al., 2012).

4) Please clarify whether independent single-unit VTA DA neuron data was collected for this study.

Reviewer #1 (Recommendations for the authors):

Detailed comments follow below.

– Figure 1 shows that synchronous optogenetic activation of CINs induces DA release in vivo. These results confirm those obtained in previous slice experiments, although the lack of frequency-dependent depression is intriguing. The authors discuss potential reasons such as the different ACH-E dynamics in slices vs in vivo, temperature effects, and more. Could this difference also be due to the methods used to measure DA release (voltammetry vs optical)?

– The dual wavelength imaging presented in figures 2-3 shows a high similarity between the DA signal and CIN activity in response to auditory stimuli and approach to the reward port. Are the simultaneous measurements completely independent? No chance of "cross talk" between the channels? Would the results hold if the imaging of CIN calcium activity and DA release were measured separately in different animals, or simultaneously but in different hemispheres?

– It is also not completely clear if the spiking data was obtained simultaneously or if it is actually the data from a previous paper by the lab. It says in the figure legend that the spiking activity of DA neurons during this task was previously reported, but does this mean that only the imaging data was acquired simultaneously while the spiking data are from different animals?

– Figure 3 indeed shows that the ramping in DA release is not accompanied by ramping in VTA cell spiking but with a ramping of CIN activity. While this does not contradict the potential involvement of CINs in mediating the DA ramp, it does not prove it.

– One open question regarding the local DA release vs spiking activity is whether CIN-induced release generates spikes that backpropagate to the cell bodies. Using the experimental setup presented in figure 3, the authors are positioned to answer this question by optogenetically activating CINs and recording backpropagating spikes in VTA cells.

– The last figure (4) shows a decrease in performance in the operant task following the application of DHBE in the NAc. The impact of the DA agonist FLU was clearly larger, and the DHBE application was effective only in higher concentrations. The changes in behavioral performance are clear in both FLU and DHBE-30ug applications. This does not, however, prove that the impact of DHBE is mediated via DA release. Such a strong manipulation of nicotinic receptor-mediated transmission would affect other cells in the NAc, including several types of GABAergic interneurons (See papers by Assous 2021, 2022). It is therefore not clear that the data presented in Fig, 4 adds to our understanding of CIN-mediated DA release.

– This leads me to the main concern, that the role of CINs in mediating DA release is not directly shown. It is shown that synchronous optogenetic activation of CINs indeed causes DA release (Figure 1). It is also shown that CIN and DA release are often correlated, such as in approach-related ramping and sensory responses. However, whether CIN-induced DA release underlies these correlated activities is not proven. This could be done by suppressing CIN activity and showing the impact on DA release. Will the ramping be affected? Will the sensory responses decrease? Manipulation of CIN activity can be done by optogenetic or chemogenetic inhibition while imaging DA release.

– Two recent studies by the Sabatini and Tritsch labs address similar questions regarding the interactions between CINs and DA release. Although the striatal regions tested are not the same as in this study, a common claim is that there is little impact of CINs on DA release in behaving mice and that to a large degree their fluctuations are negatively correlated. It would be important to discuss the discrepancies between these recent studies, in addition to the comparison with older slice studies.

Reviewer #2 (Recommendations for the authors):

– The data presented here suggests that "ramp" increases in spiking do not occur. However, this stands in contrast to data from the Witten lab showing that ramping does occur in somatic spiking. Could a difference in recording region or experimental approach account for this difference?

– The authors mention in the discussion that "stimulating CINs evoked strong DA release…". Strong relative to what? The signals in this study are reported as normalized values or presented as z-scores in nearly all instances. The authors should provide clearer justification based on absolute data values for why they think the CIN-evoked DA signals are strong.

– In the final paragraph of the results, flupenthixol is probably more accurately described as a "non-selective dopamine receptor antagonist."

– It would be helpful to give readers an idea of where electrical VTA DA neurons were made (lateral, medial, etc).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Cholinergic Interneurons Drive Motivation by Promoting Dopamine Release in the Nucleus Accumbens" for further consideration by eLife. Your revised article has been evaluated by Michael Taffe (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The reviewers find that your rebuttal is fine on all points, except for the key experiment showing that cholinergic interneurons causally drive motivation by promoting dopamine release. In light of the fact that you could not complete the required experiments, this needs to be thoroughly addressed as a limitation in the discussion. All reviewers suggest toning down the message in the title and in the introduction (lines 86-88), to claim that the results support the possibility of CINs sculpting DA release, as this is not directly and unambiguously demonstrated.

Essential revisions:

Reviewer #1 (Recommendations for the authors):

Detailed comments follow below.

– Figure 1 shows that synchronous optogenetic activation of CINs induces DA release in vivo. These results confirm those obtained in previous slice experiments, although the lack of frequency-dependent depression is intriguing. The authors discuss potential reasons such as the different ACH-E dynamics in slices vs in vivo, temperature effects, and more. Could this difference also be due to the methods used to measure DA release (voltammetry vs optical)?

We did not perform a direct comparison of voltammetry vs optical methods in this manuscript, but based on prior work by others and ourselves we think this is unlikely to be a critical factor. When used appropriately both methods are capable of following sub-second DA dynamics, and have sufficient dynamic range to examine both facilitating and depressing effects. As just one example, Rice and Cragg (2004) used voltammetry in slices and observed short-term depression of DA release to multiple electrical pulses. However this depression was not an artifact of the recording method, as it was alleviated in the presence of nicotine. In our in vivo photometry experiments we carefully selected stimulation parameters to evoke transients that are comparable to naturally occurring DA transients, and we are not aware of any reason to think that we would have not seen depression of this response if it were present.

– The dual wavelength imaging presented in figures 2-3 shows a high similarity between the DA signal and CIN activity in response to auditory stimuli and approach to the reward port. Are the simultaneous measurements completely independent? No chance of "cross talk" between the channels? Would the results hold if the imaging of CIN calcium activity and DA release were measured separately in different animals, or simultaneously but in different hemispheres?

It is indeed important to consider cross-talk, but we can rule it out because:

1) In a previous study (Patriarchi, Mohebi et al., Nature Methods 2020), we demonstrated that cross-talk between RdLight1 and GCaMP6f is negligible.

2) In simultaneous measurements we observe clear examples of transients in CIN activity that do not correspond to DA transients, and vice versa (examples in Figure 2 of the current manuscript)

3) The current manuscript does include both simultaneous measurements (Figure 2), and recordings in separate animals (Figure 3). In each case we observe concurrent ramps in CIN GCaMP6f and RdLight1 signals during motivated approach behaviors. In contrast, at other times (reward delivery at Side-In, Figure 3b) we see a large increase in DA release without a corresponding large change in CIN GCaMP signal. DA release at that time instead mirrors a large increase in DA cell firing, consistent with the idea that DA release can be controlled in multiple ways in behaving animals.

– It is also not completely clear if the spiking data was obtained simultaneously or if it is actually the data from a previous paper by the lab. It says in the figure legend that the spiking activity of DA neurons during this task was previously reported, but does this mean that only the imaging data was acquired simultaneously while the spiking data are from different animals?

We have now clarified in the text that for the operant task (Figure 3), we used three separate cohorts of rats to record (1) spiking of DA cells, (2) photometry of dLight, and (3) photometry of CIN activity, and clarified that data from the first two cohorts was previously reported in Mohebi et al. 2019.

– Figure 3 indeed shows that the ramping in DA release is not accompanied by ramping in VTA cell spiking but with a ramping of CIN activity. While this does not contradict the potential involvement of CINs in mediating the DA ramp, it does not prove it.

We acknowledge that our current study has significant limitations in definitively establishing that CIN activity drives behavior-linked DA ramps. In principle, a helpful experiment would be to simultaneously monitor DA release while antagonizing the nAChRs on DA terminals, during behaviors when DA ramps normally occur. Such an experiment is not within our current technical capabilities. Furthermore, even this experiment might not be conclusive: for example, if there is “baseline” DA release evoked by CINs, then suppressing CINs could interfere with observed ramping even if CINs do not normally drive ramping. For these reasons we believe it is appropriate to proceed with the current data set, while acknowledging its limitations (which we do in the revised Discussion).

– One open question regarding the local DA release vs spiking activity is whether CIN-induced release generates spikes that backpropagate to the cell bodies. Using the experimental setup presented in figure 3, the authors are positioned to answer this question by optogenetically activating CINs and recording backpropagating spikes in VTA cells.

Whether CIN-induced DA release causes DA spikes that backpropagate all the way back to cell bodies is a very interesting question, but it is beyond the scope of the current manuscript. To date we have not performed experiments in which we record DA cell spiking while optogenetically activating CINs.

– The last figure (4) shows a decrease in performance in the operant task following the application of DHBE in the NAc. The impact of the DA agonist FLU was clearly larger, and the DHBE application was effective only in higher concentrations. The changes in behavioral performance are clear in both FLU and DHBE-30ug applications. This does not, however, prove that the impact of DHBE is mediated via DA release. Such a strong manipulation of nicotinic receptor-mediated transmission would affect other cells in the NAc, including several types of GABAergic interneurons (See papers by Assous 2021, 2022). It is therefore not clear that the data presented in Fig, 4 adds to our understanding of CIN-mediated DA release.

If we had observed that DHBE infusion into NAc does not affect initiation of trials in our instrumental task, this would have contradicted our hypothesis that CIN control over DA release via nAChRs contributes to motivation to work. Observing that we do see such an effect is therefore useful support for our hypothesis. However we agree that it is certainly possible that more indirect effects are involved instead, and we have updated the Discussion section to reflect this point.

– This leads me to the main concern, that the role of CINs in mediating DA release is not directly shown. It is shown that synchronous optogenetic activation of CINs indeed causes DA release (Figure 1). It is also shown that CIN and DA release are often correlated, such as in approach-related ramping and sensory responses. However, whether CIN-induced DA release underlies these correlated activities is not proven. This could be done by suppressing CIN activity and showing the impact on DA release. Will the ramping be affected? Will the sensory responses decrease? Manipulation of CIN activity can be done by optogenetic or chemogenetic inhibition while imaging DA release.

As noted above, our study has limitations that we are now careful to acknowledge in the Discussion.

– Two recent studies by the Sabatini and Tritsch labs address similar questions regarding the interactions between CINs and DA release. Although the striatal regions tested are not the same as in this study, a common claim is that there is little impact of CINs on DA release in behaving mice and that to a large degree their fluctuations are negatively correlated. It would be important to discuss the discrepancies between these recent studies, in addition to the comparison with older slice studies.

We have also noted those pre-prints with interest, even though they investigated different brain regions using different approaches. There are established differences between CIN-DA interactions in dorsal vs. ventral striatum that we suspect are relevant here. But given the rapid pace of developments in this subfield, we prefer not to speculate too much at this point and instead review the overall body of work once it is published.

Reviewer #2 (Recommendations for the authors):

– The data presented here suggests that "ramp" increases in spiking do not occur. However, this stands in contrast to data from the Witten lab showing that ramping does occur in somatic spiking. Could a difference in recording region or experimental approach account for this difference?

These are both possibilities. In particular we note that the ramps reported by the Witten lab are actually ramps in the calcium activity of DA cells, monitored using GCaMP6, which has different dynamics and may signal distinct processes compared to spiking.

– The authors mention in the discussion that "stimulating CINs evoked strong DA release…". Strong relative to what? The signals in this study are reported as normalized values or presented as z-scores in nearly all instances. The authors should provide clearer justification based on absolute data values for why they think the CIN-evoked DA signals are strong.

By "strong," we simply mean that the CIN-evoked DA signals are larger than most spontaneous fluctuations (Figure 1b), and comparable in signal:noise ratio to the large transients evoked by natural reward cues.

– In the final paragraph of the results, flupenthixol is probably more accurately described as a "non-selective dopamine receptor antagonist."

Agreed. Given this comment and that of Reviewer 3 we have now modified the text to say "a non-selective D1 and D2 DA receptor antagonist".

– It would be helpful to give readers an idea of where electrical VTA DA neurons were made (lateral, medial, etc).

We have now modified the text to specify lateral VTA.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The reviewers find that your rebuttal is fine on all points, except for the key experiment showing that cholinergic interneurons causally drive motivation by promoting dopamine release. In light of the fact that you could not complete the required experiments, this needs to be thoroughly addressed as a limitation in the discussion. All reviewers suggest toning down the message in the title and in the introduction (lines 86-88), to claim that the results support the possibility of CINs sculpting DA release, as this is not directly and unambiguously demonstrated.

In response to the issue you mentioned we have:

– Amended the Title, to make the claim about the relationship between CIN-evoked DA release and the control of motivation via nicotinic receptors less strong.

– Amended the Introduction. In the specific place you flagged we now state that our results merely support the possibility that CINs regulate motivation via local control of DA.

– Amended the Discussion. We now have both an explicit statement describing the limitation of this experiment in not combining causal manipulations together with DA sensing, together with a line noting that designing an interpretable experiment along these lines is not as simple as it might seem.

Author details

1. Ali Mohebi

   Department of Neurology, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7291-3448

2. Val L Collins

   Department of Neurology, University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

3. Joshua D Berke

   1. Department of Neurology, University of California, San Francisco, San Francisco, United States
   2. Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, San Francisco, United States
   3. Neuroscience Graduate Program, University of California, San Francisco, San Francisco, United States
   4. Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Investigation, Visualization, Project administration, Writing - review and editing

   For correspondence

   joshua.berke@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1436-6823

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