1. Evaluation Summary:

    This work clearly demonstrates an important role for two specific sodium-permeable ion channels for maintaining the pacemaker-like firing of midbrain dopamine neurons. These neurons have a key role in motivation, reinforcement and locomotion, and have been implicated in Parkinson's disease and multiple neuropsychiatric disorders. The authors also find that the regular firing of these cells is robustly maintained even when one of the two channels is knocked out, through upregulation of the level of the other channel.

    (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 #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    This paper examines the ionic currents underlying the pacemaker firing in midbrain dopamine cells, neurons that have a key role in motivation, reinforcement and locomotion, and have been implicated in multiple neuropsychiatric disorders. The authors first demonstrate that blocking TRPC3 receptors or all TRPC channels prevents repetitive firing; however, surprisingly, the TRPC3 KO mouse has normal pacemaking in the DA cells. The authors then provide good evidence supporting the idea that in the TRPC3 KO mouse there is upregulation of NALCN leak channels, and that these substitute for the normal pacemaking contribution by TRPC3. Although it was previously demonstrated that TRPC channel family blockers such as 2-APB inhibit pacemaker firing, these compounds have many off-target effects as well. The present paper adds strongly to the argument that TRPC3 channels make an important contribution to pacemaking in these cells, and the suggestion that TRPC3 channels and NALCN channels together are the workhorses of this feature in dopamine cells is well-supported. In my opinion, the authors' conclusions are justified, and this paper makes a solid contribution to our understanding of pacemaker currents in these cells; the results may also have relevance to other neurons that exhibit similar firing properties and may utilize the same underlying conductances.

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  3. Reviewer #2 (Public Review):

    Ki Bum Um and collaborators show that application of a TRPC3-channel inhibitor (Pyr10) ablates spontaneous firing and decreases membrane potential in midbrain dopaminergic neurons recorded from brain slices and acutely dissociated neurons from young mice, whereas HCN or voltage-activated calcium channels do not contribute under these conditions to pacemaking because firing is not affected by inhibitors of these two channel types. Injection of current in the presence of Pyr10 can restore spontaneous firing, with a firing frequency proportional to the magnitude of the injected current, indicating that Pyr10 did not affect neuronal properties other than the leak current that contributes to subthreshold depolarization. Neurons from TRPC3-deficient mice exhibit spontaneous firing as in wild-type, and their membrane potential becomes insensitive to Pyr10 and another wide-spectrum TRPC channel inhibitor, indicating that another type of channel must compensate for the absence of TRPC3. Application of a NALCN channel inhibitor (L-703,606) in neurons from TRPC3 KO animals, as well from wild type, has a comparable effect to that of Pyr10 on firing and membrane potential, suggesting that NALCN can compensate for the absence of TRPC3, and that both NALCN and TRPC3 contribute to the current that drives periodic firing in these neurons. Consistently, the authors find that neurotensin-sensitive NALCN currents are enhanced in TRPC3 KO mice, as well as mRNA and protein levels for this channel detected by RTPCR of tissue and single cells or immunofluorescence, respectively. Finally, the authors estimate the contribution of each channel to the subthreshold membrane depolarization by measuring membrane potential in the presence of pharmacological inhibitors and estimate that TRPC3 and NALCN each contribute >30%, with a remaining third of the current remaining unaccounted for.

    The data are clearly presented and of high quality, and the observations are robust, proving appropriate support for the authors' conclusions, which represent a substantive advancement regarding a long-standing and relevant open question in the field of neuroscience. No direct measurements of the NALCN and TRPC3-mediated currents are performed. No evidence for dose-dependence of Pyr10 or L-703,606 is provided, which would lend further support that the observed effects on membrane potential are caused by a reduction in channel-mediated currents that occurs when they bind the inhibitors. In addition, the specificity of the TRPC3 inhibitor Pyr10 is not discussed; this would be relevant because the cited references do not establish its specificity and in turn show some inhibitory effect on TRPC6. It remains a possibility that TRPC6 and TRPC7 could form heteromers with TRPC3 channels and contribute to pacemaking, but this consideration certainly does no affect the authors' conclusions. Importantly, the number of mice used for recordings is not indicated in all figures.

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  4. Reviewer #3 (Public Review):

    The authors set out to identify the main ion channels that underlie the regular pacemaking of the dopamine neurons of the substantia nigra compacta. Previous work has pointed to sodium permeable ion channels, but the identity of these channels has been elusive because of the nonspecificity of some pharmacological tools and the difficulties in interpreting gene knockouts.

    This work provides convincing evidence for a key role for two particular Na-permeable channels, TRPC3 and NALCN. It shows the puzzling discrepancy that TRPC3 blockers abolish pacemaking but that in TRPC3 knockout animals, pacemaking is normal--and it nicely resolves this discrepancy, first by showing that TRPC3 blockers no longer abolish pacemaking (arguing against a nonspecific effect of the blockers) and then by showing that NALCN channels are upregulated in the knockout animals. A newer drug that targets NALCN channels is used, in combination with TRPC3 blockers, to dissect the relative contributions of these two channels (and of other channels) to the pacemaking.

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