Non-uniform distribution of dendritic nonlinearities differentially engages thalamostriatal and corticostriatal inputs onto cholinergic interneurons
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Evaluation Summary:
This manuscript addresses the cellular and dendritic physiology of cholinergic interneurons in the striatum. The authors use a creative integration of electrophysiology and optical methods to investigate this distinctive cell type, which is critically important at the intersection of motivated behavior and disease. They uncover a mechanism through which two separate active conductances - the hyperpolarization-activated h-current (HCN) and the persistent sodium current (NaP) - act in concert to selectively boost synaptic input from the thalamus onto proximal dendrites of cholinergic interneurons.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The tonic activity of striatal cholinergic interneurons (CINs) is modified differentially by their afferent inputs. Although their unitary synaptic currents are identical, in most CINs cortical inputs onto distal dendrites only weakly entrain them, whereas proximal thalamic inputs trigger abrupt pauses in discharge in response to salient external stimuli. To test whether the dendritic expression of the active conductances that drive autonomous discharge contribute to the CINs’ capacity to dissociate cortical from thalamic inputs, we used an optogenetics-based method to quantify dendritic excitability in mouse CINs. We found that the persistent sodium (NaP) current gave rise to dendritic boosting, and that the hyperpolarization-activated cyclic nucleotide-gated (HCN) current gave rise to a subhertz membrane resonance. This resonance may underlie our novel finding of an association between CIN pauses and internally-generated slow wave events in sleeping non-human primates. Moreover, our method indicated that dendritic NaP and HCN currents were preferentially expressed in proximal dendrites. We validated the non-uniform distribution of NaP currents: pharmacologically; with two-photon imaging of dendritic back-propagating action potentials; and by demonstrating boosting of thalamic, but not cortical, inputs by NaP currents. Thus, the localization of active dendritic conductances in CIN dendrites mirrors the spatial distribution of afferent terminals and may promote their differential responses to thalamic vs . cortical inputs.
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Author Response
Reviewer #1 (Public Review):
This study addresses the important question of understanding the cellular physiology of cholinergic interneurons in the striatum. These interneurons play a key role in learning and performance of motivated behaviors, and are central to movement disorders, psychiatric disease, and addiction. Their unique physiology, which includes tonic pacemaking activity and active conductances that shape integration of dendritic inputs, is critical to their function but is still incompletely understood. The authors cleverly integrate a series of innovative electrophysiological and optical approaches to gain insight into dendritic physiology of these neurons. Their creative approach yields some interesting and novel findings. However, there are technical and conceptual concerns that need to be addressed …
Author Response
Reviewer #1 (Public Review):
This study addresses the important question of understanding the cellular physiology of cholinergic interneurons in the striatum. These interneurons play a key role in learning and performance of motivated behaviors, and are central to movement disorders, psychiatric disease, and addiction. Their unique physiology, which includes tonic pacemaking activity and active conductances that shape integration of dendritic inputs, is critical to their function but is still incompletely understood. The authors cleverly integrate a series of innovative electrophysiological and optical approaches to gain insight into dendritic physiology of these neurons. Their creative approach yields some interesting and novel findings. However, there are technical and conceptual concerns that need to be addressed before these results can be readily interpreted. Some refinement of analysis and presentation, and potentially some additional experiments, will therefore be required to strengthen the conclusions and facilitate interpretation of the results.
We believe that with several new sets of experiments and simulations, we have successfully refined the analysis and addressed the technical and conceptual problems. Indeed, we strengthened the conclusion with a novel pharmacological experiment that provided model-independent evidence of proximal-only boosting.
Major concerns:
- This manuscript focuses on differential physiology of proximal and distal dendrites contribute to physiological activity and integration of inputs in cholinergic interneurons, suggesting that NaP and HCN currents act in concert to selectively boost inputs onto proximal dendrites (from thalamus), relative to inputs onto distal dendrites (from cortex). The results presented in Figures 1-4 are consistent with a distinct physiology of proximal-vs-distal dendrites based on purely electrical properties. Indeed, Figure 5 initially appears consistent with this model as well, since thalamic inputs (onto proximal dendrites) are boosted by an NaP conductance, while cortical inputs (onto distal dendrites) are not. This raises a key conceptual question: why are cortical inputs onto distal dendrites not boosted? Any depolarization of distal dendrites must pass through proximal dendrites before reaching the recording electrode at the soma. Shouldn't this signal be subject to the same active and passive conductances, and consequently the same boosting that shapes thalamic inputs onto proximal dendrites?
You are absolutely right in the case of a linear model (passive or quasi-linear). However, for a nonlinear system, there can be preferential boosting of proximal inputs. The new Appendix 2, addresses this point with computer simulations.
- The quasi-linear approach to characterizing active and passive membrane properties is promising, and the choice of a cable-based model is well supported. However, the model itself is rather opaque, which limits confidence in the interpretation of the results. Additional analysis and description should be presented to alleviate concerns about whether the experimental data, which has a limited number of measurable values, may be over-fit by a model with too many free parameters. For example, why is the radius of the dendrite a free parameter that is allowed to vary in the full field vs proximal experiment (Lines 253-256) - and isn't it a serious red flag that the value returned for proximal dendrites is smaller than for the full field? Additional tables (e.g. fixed and free parameters and how they were determined), and figures (plots of how those parameters influence the fits, and how the parameters interact with one another) would considerably strengthen confidence in the conclusions drawn by the authors.
Thank you very much for this comment. We have added in the new ms a table with all the parameters fit in the various figures, and have discussed the possible pitfalls of overfitting. Most importantly, we have provided a new appendix (#1) to the manuscript that explains the effects of the various model parameters in a systematic fashion, beginning with a passive dendrites, followed by the effects of boosting and then the effect of restorative currents that give rise to resonances. This appendix addresses the questions raised by the reviewer regarding how the various parameters influence the fits.
We apologize, if we created a confusion, with respect to the meaning of the parameter r. It does not represent the radius of the dendrites (which is not explicitly represented at all, only implicitly through the space constant) but rather the electrotonic range of illumination. We indeed find that the fits consistently estimate a value of r for the proximal illumination which is smaller than that estimated for the full-field illumination, as it should.
Finally, our new pharmacological demonstration of differential boosting in the case of proximal vs. fullfield illumination (see above) is entirely independent of the quasi-linear model fit. So for the main thrust of the ms, which is to demonstrate a proximal localization of nonlinearities and its correspondence to the spatial localization of excitatory afferent inputs, this is now achieved, at least vis-à-vis the NaP current, independently of the qausilinear model. However, we still find the model useful as it is used to estimate the distribution of HCN currents and provides a framework to think about how to manipulate dendritic nonlinearities experimentally.
- Technically, the use of ChR2 to modulate dendritic currents is creative. While the authors rightly acknowledge that activation/deactivation kinetics of the ChR2 channel will contribute to filtering, this important point should be expanded with additional analysis and potentially with new experiments. Of particular concern is the transition of ChR2 channels to an inactivated state over the comparatively long oscillating light pulse in Figure 3 Inactivation of ChR2 is prominent over this timescale and would precisely co-vary with the shift in oscillation frequency. To address this, the authors should present a direct measurement of this inactivation and account for it in their analysis of the chirp data. Alternatively, the chirp stimulus could be presented backwards (starting at high frequency), so that comparison of forwards-vs-backwards chirp recordings could disentangle this artefact. Either one or both of these additional experiments would be critical for interpreting the roll-off in photocurrent responses at high frequencies reported in Figure 3.
Touché! You were spot on with this critique and we were wrong. We have now conducted several new experiments (that appear in the main text and in Figure 3 and all its supplements) that show that including ChR2 kinetics explicitly in the model fits actually makes the fits more self-consistent and removes some of the glaring differences between the results from the somatic voltage perturbations (Figures 1–2) and the optogenetic illumination (Figure 3). So as per your request, we have now presented a direct measurement of the deactivation (Figure 3–figure supplement 1) and we have played the “chirp” backwards (Appendix 1–figure 2) to address the issue of inactivation.
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Evaluation Summary:
This manuscript addresses the cellular and dendritic physiology of cholinergic interneurons in the striatum. The authors use a creative integration of electrophysiology and optical methods to investigate this distinctive cell type, which is critically important at the intersection of motivated behavior and disease. They uncover a mechanism through which two separate active conductances - the hyperpolarization-activated h-current (HCN) and the persistent sodium current (NaP) - act in concert to selectively boost synaptic input from the thalamus onto proximal dendrites of cholinergic interneurons.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with …
Evaluation Summary:
This manuscript addresses the cellular and dendritic physiology of cholinergic interneurons in the striatum. The authors use a creative integration of electrophysiology and optical methods to investigate this distinctive cell type, which is critically important at the intersection of motivated behavior and disease. They uncover a mechanism through which two separate active conductances - the hyperpolarization-activated h-current (HCN) and the persistent sodium current (NaP) - act in concert to selectively boost synaptic input from the thalamus onto proximal dendrites of cholinergic interneurons.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study addresses the important question of understanding the cellular physiology of cholinergic interneurons in the striatum. These interneurons play a key role in learning and performance of motivated behaviors, and are central to movement disorders, psychiatric disease, and addiction. Their unique physiology, which includes tonic pacemaking activity and active conductances that shape integration of dendritic inputs, is critical to their function but is still incompletely understood. The authors cleverly integrate a series of innovative electrophysiological and optical approaches to gain insight into dendritic physiology of these neurons. Their creative approach yields some interesting and novel findings. However, there are technical and conceptual concerns that need to be addressed before these results …
Reviewer #1 (Public Review):
This study addresses the important question of understanding the cellular physiology of cholinergic interneurons in the striatum. These interneurons play a key role in learning and performance of motivated behaviors, and are central to movement disorders, psychiatric disease, and addiction. Their unique physiology, which includes tonic pacemaking activity and active conductances that shape integration of dendritic inputs, is critical to their function but is still incompletely understood. The authors cleverly integrate a series of innovative electrophysiological and optical approaches to gain insight into dendritic physiology of these neurons. Their creative approach yields some interesting and novel findings. However, there are technical and conceptual concerns that need to be addressed before these results can be readily interpreted. Some refinement of analysis and presentation, and potentially some additional experiments, will therefore be required to strengthen the conclusions and facilitate interpretation of the results.
Major concerns:
This manuscript focuses on differential physiology of proximal and distal dendrites contribute to physiological activity and integration of inputs in cholinergic interneurons, suggesting that NaP and HCN currents act in concert to selectively boost inputs onto proximal dendrites (from thalamus), relative to inputs onto distal dendrites (from cortex). The results presented in Figures 1-4 are consistent with a distinct physiology of proximal-vs-distal dendrites based on purely electrical properties. Indeed, Figure 5 initially appears consistent with this model as well, since thalamic inputs (onto proximal dendrites) are boosted by an NaP conductance, while cortical inputs (onto distal dendrites) are not. This raises a *key conceptual question*: why are cortical inputs onto distal dendrites not boosted? Any depolarization of distal dendrites must pass through proximal dendrites before reaching the recording electrode at the soma. Shouldn't this signal be subject to the same active and passive conductances, and consequently the same boosting that shapes thalamic inputs onto proximal dendrites?
The quasi-linear approach to characterizing active and passive membrane properties is promising, and the choice of a cable-based model is well supported. However, the model itself is rather opaque, which limits confidence in the interpretation of the results. Additional analysis and description should be presented to alleviate concerns about whether the experimental data, which has a limited number of measurable values, may be over-fit by a model with too many free parameters. For example, why is the radius of the dendrite a free parameter that is allowed to vary in the full field vs proximal experiment (Lines 253-256) - and isn't it a serious red flag that the value returned for proximal dendrites is smaller than for the full field? Additional tables (e.g. fixed and free parameters and how they were determined), and figures (plots of how those parameters influence the fits, and how the parameters interact with one another) would considerably strengthen confidence in the conclusions drawn by the authors.
Technically, the use of ChR2 to modulate dendritic currents is creative. While the authors rightly acknowledge that activation/deactivation kinetics of the ChR2 channel will contribute to filtering, this important point should be expanded with additional analysis and potentially with new experiments. Of particular concern is the transition of ChR2 channels to an inactivated state over the comparatively long oscillating light pulse in Figure 3 Inactivation of ChR2 is prominent over this timescale and would precisely co-vary with the shift in oscillation frequency. To address this, the authors should present a direct measurement of this inactivation and account for it in their analysis of the chirp data. Alternatively, the chirp stimulus could be presented backwards (starting at high frequency), so that comparison of forwards-vs-backwards chirp recordings could disentangle this artefact. Either one or both of these additional experiments would be critical for interpreting the roll-off in photocurrent responses at high frequencies reported in Figure 3.
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Reviewer #2 (Public Review):
The paper by Oz and colleagues uses optogenetics and whole-cell patch clamp recordings from striatal cholinergic interneurons (CINs) to investigate their dendritic nonlinearities, in particular the hyperpolarization-activated h-current (HCN) and the persistent sodium current (NaP). The experiments are motivated by an elegant model for phase-shift and dendritic nonlinearity analysis and also support the firing patterns of putative CINs in sleeping monkeys. Using perisomatic and wide-field photostimulation, 2-photon imaging, and optogenetic circuit interrogation, the authors show the role of persistent sodium current in supporting action-potential backpropagation in CIN dendrites and synaptic amplification. The functional implications of the resonant properties of CINs are demonstrated in extracellular …
Reviewer #2 (Public Review):
The paper by Oz and colleagues uses optogenetics and whole-cell patch clamp recordings from striatal cholinergic interneurons (CINs) to investigate their dendritic nonlinearities, in particular the hyperpolarization-activated h-current (HCN) and the persistent sodium current (NaP). The experiments are motivated by an elegant model for phase-shift and dendritic nonlinearity analysis and also support the firing patterns of putative CINs in sleeping monkeys. Using perisomatic and wide-field photostimulation, 2-photon imaging, and optogenetic circuit interrogation, the authors show the role of persistent sodium current in supporting action-potential backpropagation in CIN dendrites and synaptic amplification. The functional implications of the resonant properties of CINs are demonstrated in extracellular recordings from sleeping monkeys, showing modulation of the firing patterns in CINs but not striatal projection neurons. The results are interesting and the data presented is of high quality, combining several different methods and species.
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