Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation

  1. Ali Mohebi
  2. Val L Collins
  3. Joshua D Berke

  • Curated by eLife

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    eLife assessment

    This study by Mohebi, Collins, and Berke presents valuable findings on the control of the neurotransmitter dopamine by cholinergic interneurons, a sparse but important subclass of neurons with the ventral striatum, a key brain region involved in motivational behaviors. The evidence supporting the claims of the authors is solid, although the inclusion of a key experiment presenting causality between cholinergic neuron activity and dopamine release during behavior is needed. The work will be of broad interest to neuroscientists in the fields of motivation and decision-making.

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Abstract

Motivation to work for potential rewards is critically dependent on dopamine (DA) in the nucleus accumbens (NAc). DA release from NAc axons can be controlled by at least two distinct mechanisms: (1) action potentials propagating from DA cell bodies in the ventral tegmental area (VTA), and (2) activation of β2* nicotinic receptors by local cholinergic interneurons (CINs). How CIN activity contributes to NAc DA dynamics in behaving animals is not well understood. We monitored DA release in the NAc Core of awake, unrestrained rats using the DA sensor RdLight1, while simultaneously monitoring or manipulating CIN activity at the same location. CIN stimulation rapidly evoked DA release, and in contrast to slice preparations, this DA release showed no indication of short-term depression or receptor desensitization. The sound of unexpected food delivery evoked a brief joint increase in CIN population activity and DA release, with a second joint increase as rats approached the food. In an operant task, we observed fast ramps in CIN activity during approach behaviors, either to start the trial or to collect rewards. These CIN ramps co-occurred with DA release ramps, without corresponding changes in the firing of lateral VTA DA neurons. Finally, we examined the effects of blocking CIN influence over DA release through local NAc infusion of DHβE, a selective antagonist of β2* nicotinic receptors. DHβE dose-dependently interfered with motivated approach decisions, mimicking the effects of a DA antagonist. Our results support a key influence of CINs over motivated behavior via the local regulation of DA release.

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  1. Version published to 10.7554/elife.85011 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    The manuscript by Mohebi et al. examines a critical open question regarding the interaction of cholinergic interneurons of the striatum and transmitter release from dopaminergic axons in behaving animals. Activation of cholinergic interneurons in the striatum can evoke dopamine release in brain slices and in vivo as measured with voltammetry. However, it remains an open question in what context and to what extent this acetylcholine-mediated dopamine occurs in behaving animals. Here, the authors argue that CIN activity triggers dopamine release in the nucleus accumbens which encodes the motivation to obtain a reward through increasing "ramps" of dopamine release. Their data suggest that the ramps are not reflected in the firing of dopaminergic neurons. Rather, they provide compelling evidence that the ramps of dopamine release correlate with ramps in cholinergic interneuron activity as measured with GCaMP6. What's more, the authors show that ACh-mediated dopamine release has no paired-pulse depression, a striking result that differs from all prior ex vivo brain slice data. The manuscript is extremely well written and the data are of very high quality. Overall, this study represents an important step forward in our understanding of how ACh-mediated dopamine release regulates behavior, and more broadly how axons can generate behaviors independently from somatic activity.

    Major comments

    1. The complete absence of any short-term plasticity in CIN-mediated dopamine release is a striking result that is important for the field. The authors should strengthen this result with additional quantitative analysis demonstrating the lack of STP. They have analyzed paired-pulse ratios, but they should analyze this for stimuli at the higher frequencies (4 Hz, etc) that are more physiologically relevant. For example, Fig 1e shows a CIN-evoked DA release at many optically-stimulated frequencies. The authors should quantify short-term plasticity by generating fits of the single stimulus signal and comparing the mathematical sum predicted from 4 stim DA signals at different frequencies to the recorded data. A similar analysis has been done with Ca signals (Koester and Sakmann, 2000).

    Thank you for this very helpful suggestion. We have performed this analysis as recommended, and now confirm the lack of STP even at the higher frequencies (see new Supplementary Figure 1).

    1. The authors show that optical activation of CINs results in DA release as measured by dLight. To clearly establish that these signals are generated by DA release driven by nicotinic receptors (and not a partial effect of some unknown artifact), it would be useful to show that the optical CIN-evoked dLight signals shown in Fig. 1 are inhibited by nicotinic receptor antagonists such as DHbE. This control experiment would significantly strengthen the result shown here.

    We agree that combining drug manipulations with photometry would be useful, but as noted above this is not a methodology in our current technical repertoire.

    1. Similarly, the authors show clear correlations between CIN activity and DA release during behavior. The authors should consider determining whether CINs play a causal role in triggering DA release during behavior. For example, does infusion of DHbE in the NAc prevent the light-mediated DA release during behavior? As an alternative hypothesis, some groups have been suggesting that CIN activity has almost no direct influence over DA. Therefore, testing whether a causal relationship exists between CINs and DA release would be an important experiment in addressing these two opposing viewpoints.

    As noted above we are not currently able to combine drug manipulations with photometry in behaving animals.

    1. The ramps that are described in this manuscript are an order of magnitude faster (increasing over 100s of milliseconds) than ramps described in other studies that occur over seconds. In fact, the two signals may be completely different functionally. Discussion of this topic would be helpful.

    Dopamine ramps have indeed been reported over multiple different time scales, and as discussed in Berke 2018, this seems to reflect the duration of the approach behavior. We think further discussion of this topic is better saved for another paper, especially as we are now actively studying ramping over longer time scales (Krausz et al. 2023).

    Reviewer #3 (Public Review):

    This report by Mohebi et al. provides new answers to old questions by showing that the activity of striatal cholinergic interneurons (CINs) escalates progressively during specific reward-related behaviors and that this correlates with previously observed ramps in dopamine (DA) release in the nucleus accumbens core. The report is strong and provides evidence for the authors' hypothesis that DA ramps are independent of DA neuron activity, but are instead the result of CIN activity and corresponding acetylcholine (ACh) release. The authors further demonstrate that the fidelity of CIN activation and consequent driving of DA release is even more robust in vivo than observed ex vivo slice preparations, which is fundamental for understanding the role of ACh-DA interactions in behavior. The findings complement the authors' previous evidence ventral tegmental area (VTA) DA neuron firing patterns do not show a ramping pattern; the previously reported VTA data are appropriately included here (in Fig. 3) to illustrate the absence of VTA firing during the time-locked increases in CIN activity and DA release. The present studies stop short of showing a direct link between CIN activity and DA release, however, which would require examining DA release during behavior in the presence of an antagonist of nicotinic ACh receptors. The authors also extend the understanding of the regulation of DA release by acetylcholine (ACh) by showing that optical activation of CINs in vivo promotes DA release responses that do not attenuate with repetitive stimulation. This contrasts with previous results in ex vivo striatal slices in which ACh-evoked DA release has been found to decline progressively from rundown and/or receptor desensitization. The authors propose that in vivo, AChE may be more effective in curtailing local ACh levels than in slices because of the slightly lower temperature typically used for slice studies, as well as the use of superfusion that might facilitate some AChE washout (AChE inhibitors are still effective in slices, of course). Overall, the report not only provides evidence for the cellular substrate for DA ramps but also shows the robustness of ACh-driven DA release in vivo. A few points to strengthen the report are listed below.

    1. The authors give a few details about how CINs were activated at the beginning of the results, but say only that DA dynamics were monitored using fiber photometry. Given that the methods are at the end, a brief summary should be given here to indicate whether this means direct monitoring of DA or indirect via GCaMP, for example. It would be helpful to note the sensor used in the abstract, as well. In this light, as it were, RdLight1 should be described upon the first mention.

    We have now clarified in both abstract and text that we are using the direct DA sensor RdLight1.

    1. The authors show that infusion of DHbE in the NAc likelihood of decisions to approach the center port, as did antagonism of DA receptors. This supports the authors' argument that ramping of CIN activity and consequent ACh release underlies observed ramps in DA release. However, to show a causal interaction requires testing whether the observed DA ramps are absent after DHbE infusion in the NAc, under the same conditions that attenuated behavior.

    As noted above we are not currently able to combine drug manipulations with photometry in behaving animals.

    1. In Fig. 3, the y-axis title for the upper panels should specify VTA, not simply "rate". This is stated in the legend, but should also be specified in the figure panel.

    We have updated the y-axis titles in this figure.

    1. A recent preprint in BioRxiv by AC Krok, NX Tritsch et al. shows a related correlation between ACh and DA release in vivo in a reward task, as well as differences in other conditions. This report shows also that cortical input to CINs indeed plays a role, as suggested in the concluding sections of the present report. Consideration of the data in the preprint in the context of the present results could be valuable for the field.

    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.

  3. eLife

    eLife assessment

    This study by Mohebi, Collins, and Berke presents valuable findings on the control of the neurotransmitter dopamine by cholinergic interneurons, a sparse but important subclass of neurons with the ventral striatum, a key brain region involved in motivational behaviors. The evidence supporting the claims of the authors is solid, although the inclusion of a key experiment presenting causality between cholinergic neuron activity and dopamine release during behavior is needed. The work will be of broad interest to neuroscientists in the fields of motivation and decision-making.

  4. eLife

    Reviewer #1 (Public Review):

    The paper by Mohebi, Collins, and Berke describes the interactions between cholinergic interneurons and dopamine (DA) release in the core of the nucleus accumbens (NAc) in rats. The cholinergic triggering of DA release has been a debated issue in recent years, and this study provides data supporting cholinergic-dependent DA release.

    The authors first show that optogenetic activation of cholinergic interneurons (CINs) induces DA release in the NAc, increasing with pulse width, frequency, and train pulse duration. They next show using simultaneous imaging of CIN calcium activity and DA release using RdLight that both are correlated in their response to sensory stimuli and entry to reward port in freely moving rats. They show that while CIN activity and DA release show ramping activity before entry to the center and food ports, such ramping is not seen in the spiking activity of DA cells. lastly, the authors show that blocking nicotinic receptors in the NAc by injection of DHBE impairs task performance, with similar (albeit weaker) effects as the DA antagonist flupenthixol. The uncoupling between DA release and DA cell firing, under certain conditions, has been shown by the authors in a previous paper (Mohebi et al, 2019). Here, the authors add the CINs calcium activity during the same task, showing that the dynamics of CIN activity resemble that of DA release. The results presented show correlations between CIN activity and DA release during behavior, however, the role of CINs in controlling DA release is not tested directly. The data presented in the paper are clear and it is well written. However, there are a few issues that need to be addressed, including some key experiments that could directly test the functional role of CIN-induced DA release.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript by Mohebi et al. examines a critical open question regarding the interaction of cholinergic interneurons of the striatum and transmitter release from dopaminergic axons in behaving animals. Activation of cholinergic interneurons in the striatum can evoke dopamine release in brain slices and in vivo as measured with voltammetry. However, it remains an open question in what context and to what extent this acetylcholine-mediated dopamine occurs in behaving animals. Here, the authors argue that CIN activity triggers dopamine release in the nucleus accumbens which encodes the motivation to obtain a reward through increasing "ramps" of dopamine release. Their data suggest that the ramps are not reflected in the firing of dopaminergic neurons. Rather, they provide compelling evidence that the ramps of dopamine release correlate with ramps in cholinergic interneuron activity as measured with GCaMP6. What's more, the authors show that ACh-mediated dopamine release has no paired-pulse depression, a striking result that differs from all prior ex vivo brain slice data. The manuscript is extremely well written and the data are of very high quality. Overall, this study represents an important step forward in our understanding of how ACh-mediated dopamine release regulates behavior, and more broadly how axons can generate behaviors independently from somatic activity.

    Major comments
    1. The complete absence of any short-term plasticity in CIN-mediated dopamine release is a striking result that is important for the field. The authors should strengthen this result with additional quantitative analysis demonstrating the lack of STP. They have analyzed paired-pulse ratios, but they should analyze this for stimuli at the higher frequencies (4 Hz, etc) that are more physiologically relevant. For example, Fig 1e shows a CIN-evoked DA release at many optically-stimulated frequencies. The authors should quantify short-term plasticity by generating fits of the single stimulus signal and comparing the mathematical sum predicted from 4 stim DA signals at different frequencies to the recorded data. A similar analysis has been done with Ca signals (Koester and Sakmann, 2000).

    2. The authors show that optical activation of CINs results in DA release as measured by dLight. To clearly establish that these signals are generated by DA release driven by nicotinic receptors (and not a partial effect of some unknown artifact), it would be useful to show that the optical CIN-evoked dLight signals shown in Fig. 1 are inhibited by nicotinic receptor antagonists such as DHbE. This control experiment would significantly strengthen the result shown here.

    3. Similarly, the authors show clear correlations between CIN activity and DA release during behavior. The authors should consider determining whether CINs play a causal role in triggering DA release during behavior. For example, does infusion of DHbE in the NAc prevent the light-mediated DA release during behavior? As an alternative hypothesis, some groups have been suggesting that CIN activity has almost no direct influence over DA. Therefore, testing whether a causal relationship exists between CINs and DA release would be an important experiment in addressing these two opposing viewpoints.

    4. The ramps that are described in this manuscript are an order of magnitude faster (increasing over 100s of milliseconds) than ramps described in other studies that occur over seconds. In fact, the two signals may be completely different functionally. Discussion of this topic would be helpful.

  6. eLife

    Reviewer #3 (Public Review):

    This report by Mohebi et al. provides new answers to old questions by showing that the activity of striatal cholinergic interneurons (CINs) escalates progressively during specific reward-related behaviors and that this correlates with previously observed ramps in dopamine (DA) release in the nucleus accumbens core. The report is strong and provides evidence for the authors' hypothesis that DA ramps are independent of DA neuron activity, but are instead the result of CIN activity and corresponding acetylcholine (ACh) release. The authors further demonstrate that the fidelity of CIN activation and consequent driving of DA release is even more robust in vivo than observed ex vivo slice preparations, which is fundamental for understanding the role of ACh-DA interactions in behavior. The findings complement the authors' previous evidence ventral tegmental area (VTA) DA neuron firing patterns do not show a ramping pattern; the previously reported VTA data are appropriately included here (in Fig. 3) to illustrate the absence of VTA firing during the time-locked increases in CIN activity and DA release. The present studies stop short of showing a direct link between CIN activity and DA release, however, which would require examining DA release during behavior in the presence of an antagonist of nicotinic ACh receptors. The authors also extend the understanding of the regulation of DA release by acetylcholine (ACh) by showing that optical activation of CINs in vivo promotes DA release responses that do not attenuate with repetitive stimulation. This contrasts with previous results in ex vivo striatal slices in which ACh-evoked DA release has been found to decline progressively from rundown and/or receptor desensitization. The authors propose that in vivo, AChE may be more effective in curtailing local ACh levels than in slices because of the slightly lower temperature typically used for slice studies, as well as the use of superfusion that might facilitate some AChE washout (AChE inhibitors are still effective in slices, of course). Overall, the report not only provides evidence for the cellular substrate for DA ramps but also shows the robustness of ACh-driven DA release in vivo. A few points to strengthen the report are listed below.

    1. The authors give a few details about how CINs were activated at the beginning of the results, but say only that DA dynamics were monitored using fiber photometry. Given that the methods are at the end, a brief summary should be given here to indicate whether this means direct monitoring of DA or indirect via GCaMP, for example. It would be helpful to note the sensor used in the abstract, as well. In this light, as it were, RdLight1 should be described upon the first mention.

    2. The authors show that infusion of DHbE in the NAc likelihood of decisions to approach the center port, as did antagonism of DA receptors. This supports the authors' argument that ramping of CIN activity and consequent ACh release underlies observed ramps in DA release. However, to show a causal interaction requires testing whether the observed DA ramps are absent after DHbE infusion in the NAc, under the same conditions that attenuated behavior.

    3. In Fig. 3, the y-axis title for the upper panels should specify VTA, not simply "rate". This is stated in the legend, but should also be specified in the figure panel.

    4. A recent preprint in BioRxiv by AC Krok, NX Tritsch et al. shows a related correlation between ACh and DA release in vivo in a reward task, as well as differences in other conditions. This report shows also that cortical input to CINs indeed plays a role, as suggested in the concluding sections of the present report. Consideration of the data in the preprint in the context of the present results could be valuable for the field.

  7. Version published to 10.1101/2022.11.06.515335v1 on bioRxiv