Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (Recommendations For The Authors):
This manuscript aims to understand the biological mechanisms underlying neuropsychiatric symptoms in Parkinson's disease by characterizing subtypes of neurons in the dorsal raphe nucleus and defining their susceptibility to the degeneration of dopaminergic and adrenergic systems in the brain. This study was well-designed, the results were presented beautifully, and the manuscript was well-written. Here are some comments that may help to improve the overall quality of this work.
We thank the reviewer for the kind comments.
Major concerns:
The current study utilized an intrastriatal 6-OHDA injection, which raises the possibility that the observed electrophysiological and morphological changes of DRN5-HT and DRNDA neurons (Figs 3-6) may be due to the direct effects of 6-OHDA to DRN5-HT and DRNDA neurons projecting to the dorsal striatum (at least for DRN5-HT neurons). This possibility requires further clarification and discussion.
6-OHDA is a catecholamine neurotoxin with low selectivity for serotonin neurons.
However, changes in the levels of serotonin have been observed with high doses of 6OHDA. In our study, we used lower concentrations of 6-OHDA, which did not affect the levels of serotonin (Suppl. Fig 4D), or the number of DRN5-HT neurons (Suppl. Fig. 5B). Concerning the possible effect of 6-OHDA on DRNDA neurons, we did not observe any modification in the number of these cells in response to the administration of 6-OHDA (Suppl. Fig. 5C), (lines 170-175).
How does the loss of nigrostriatal dopamine neurons affect the electrophysiology and morphology of DRNDA neurons (Figs. 5-6)? What are the potential circuit mechanisms?
The dopaminergic system in the midbrain and the DRN constitute two highly interconnected nuclei and hence there are multiple possible circuit mechanisms that could explain how loss of nigrostriatal dopaminergic neurons affects DRNDA neurons: First, DRNDA neurons are directly innervated by dopaminergic neurons in the SNc and VTA and hence loss of SNc inputs might evoke acute as well as homeostatic changes in DRNDA (Lin et al., 2020; Pinto et al., 2019). Second, midbrain dopaminergic neurons are in turn innervated by the DRN (Watabe-Uchida et al., 2012) and loss of postsynaptic dopaminergic neurons might affect all neuron types in the DRN that target the midbrain. Finally, GABAergic populations in the midbrain have been shown to target DRN5-HT neurons and might potentially also target other local cell types such as DRNDA (Li et al., 2019). Another possible pathway is the bidirectional connection between the striatum and the DRN (Pollak-Dorocic et al, 2014). DA depletion in the striatum may affect the GABAergic projection to the DRN and in turn modify the properties of postsynaptic DRN neurons.
The potential circuit mechanisms are now included in the introduction (lines 58-59).
Whether these intrastriatal 6-OHDA mice exhibited nonmotor deficits (e.g., anxiety) that may be related to the observed changes in the DRN? Such behavioral data would enhance the overall conclusions of this work.
The PD model utilized in this study displays non-motor deficits, including depression- and anxiety-like behavior (Masini et al. 2021, Ztaou et al., 2018). This is now highlighted in the manuscript (lines 167-169).
Minor issues:
The panels of Fig. 2 should be re-labelled to match the descriptions in the main text (L.
142-158).
Fig.2 now matches the descriptions in the main text.
Fig 4D was missing from the figure, which does not match the descriptions in the main text (L. 193-204:)
Fig. 4D includes the parameters describing the dendritic branching and starts with the last graph on the right in the second row of the panel.
Line 409: Extra "as" after "average"
Corrected in revised manuscript.
Fig 3G: Missed asterisks.
Corrected in revised manuscript (Fig. 3G)
Details of how action parameters were quantified should be stated and specified in the methods.
We have now added a section called ‘Quantification of electrophysiological parameters’ in the methods where we explain how the electrophysiological properties are defined and quantified (lines 407-439).
"Parkinson's disease" in the title should be revised to "parkinsonism"
Corrected in revised manuscript.
Reviewer #2 (Recommendations For The Authors):
(1) Throughout the paper, there are numerous inaccuracies and inconsistencies in the figures, which impede the clear understanding of this paper. For example, there are discrepancies between the labeling of the main figures (sub-panels) and the corresponding manuscript (Figure 2, Figure 4).
Corrected in the revised manuscript.
The statistical presentations are inaccurate in several figures (Figure 3E, 3G), making it difficult to distinguish which data is statistically meaningful. Furthermore, the number of cells presented in each figure is ambiguous in the figure legend. It would be better to avoid expressions such as 'n = 28 - 43 cells per group', as in line 456 (Figure 1I).
Please provide the exact number of cells for each graph.
We agree with the reviewer, and we have now added the precise n numbers for each panel in the corresponding legends in Fig 1, Fig 3, and Fig 5. Please note that some analysis was restricted to recordings where neurons fired close to their average spontaneous firing frequency (e.g. 1Hz for DRN5-HT) to allow for a fair comparison of the data across groups and that therefore the n numbers vary in different panels.
In some figures, the value of n in the graph seems different from the value of n in the figure legends (Figure 2G-I, Figure 4, Figure 6). Collectively, these inaccurate figures and the manuscript weaken the general credibility of the data presented.
We apologize for the misunderstanding, but in the type of chosen graph, equal values are overlapped. The numbers described in the figure legend are correct.
(2) Some of the authors' claims in this paper are not supported by quantitative analysis, but only by sample recording traces or simple descriptions. For example, in line 97, the authors mentioned, "no differences when comparing TH-positive to TH-negative neurons".
But there are no data actually analyzing these two groups in Supplementary Figure 2A.
In addition, in line 103, there is a claim that "DRN DA neurons showed that they share several properties characteristics of other DA populations located in the SNc and the ventral tegmental area". However, this claim is backed up only by a few sample traces in Figure 1E.
The statement (lines 110-111), "a relative constant action potential (AP) amplitude", is also not supported by appropriate quantitative analysis but only by sample recording traces.
In our study we found a small subset of DAT-tdTomato positive neurons which did not stain positive for TH after the slice recordings. In 5 of 6 of these neurons (recorded in sham), the electrophysiological properties did not differ from other TH-positive neurons. This is visualized in Suppl. Fig 2A. The absence of any statistical difference was also confirmed by a Mann Whiteny U test comparing the TH negative to the TH positive DRNDA neurons (no significant differences in all 6 of 6 properties shown in Suppl. Fig 2A). Additionally, all these cells were DAT-positive, further supporting their classification as dopaminergic neurons. Therefore, we suspect that the lack of TH staining is likely caused by the tissue processing itself. Please note that all our immunohistochemistry was run on slices after several hours of patch-clamping procedures. Finally, including or excluding this small subset of neurons in the present study does not change any of the results presented and data was therefore pooled. We have now clarified this in more detail in the results section and in Suppl. Fig 2A (lines 100-103).
We have moved the comparison of hallmark properties found in DRNDA neurons as well as in dopaminergic neurons in the midbrain from the results section to the discussion (lines 281-283).
The claim that DRN5HT neurons have a comparatively constant action potential amplitude compared to DRNDA neurons is supported by quantitative analysis shown in Fig 1I (left panel, “AP drop rate”), while the representative example traces are shown in Fig 1G.
(3) In the legend of Figure 2, the mouse used in this experiment is mentioned with two different names (wild-type mice in line 463 and sham-lesion mice in line 465). Is this a mistake? Or did the authors intentionally use the brain samples from sham-lesion mice for Figure 2?
Figure 2 shows data in control conditions (Sham-lesion in our case), both from wild-type and Dat-Tomato. The text has been changed to avoid misunderstandings.
(4) While the primary claim of this paper is the differential alterations of DRN 5-HT and DA neurons in a mouse PD model, the observed changes in the DRN neurons of the 'DA only lesion model' are comparatively minor to the 'DA and NA lesions model'.
Therefore, it looks like NA depletion has a more critical role in the DRN neurons of 6OHDA-lesion mice than DA depletion. To understand the results of this paper better, it would be great if the authors can provide additional data from the 'NA only lesion model'.
We agree with the reviewer, and we have now added a new set of experiments in which we selectively lesioned noradrenergic cells by injecting 6-OHDA unilaterally into the LC. The new data are presented in supplementary figure 6 in the revised manuscript. We find that selective lesioning of the NA system affects DRNDA and DRN5-HT neurons mildly, suggesting that the concomitant lesion of the DA and NA systems is particularly impactful (possibly because of interactions between these two systems).
(5) In Figure 3B and Figure 5B, only the 6-OHDA+DMI group shows significant differences from the sham group. This finding might be attributed to the effect of DMI itself, not to the nigrostriatal DA degeneration without NA degeneration. Thus, adding the 'DMI-only group' in all experiments will strengthen the conclusion of this paper.
The effect of one acute administration of desipramine was temporally limited to the stereotactic intervention (line 373-375), which was performed several weeks before the electrophysiological and morphological analyses. Given that the half-life of desipramine is approximately 24 hrs (Nagy and Johansson, 1975), we believe that its impact was limited to the neuroprotection of NA-neurons from 6-OHDA toxicity.
(6) DRN 5-HT neurons are known to exhibit cellular heterogeneity, and in particular their electrophysiological properties are quite heterogeneous (Bernat Kocsis. 2006; J.V. Schweimer. et al. 2011). Furthermore, 5-HT neurons in the distinct subregions of the DRN display different membrane properties (LaTasha K. Crawford, 2010). Therefore, not all DRN 5-HT neurons can be regarded as electrophysiologically identical. Given that the molecular identity of all recorded cells was confirmed with neurobiotin in this paper, it would be better to show that recorded cells are not biased toward certain subregions of DRN.
In addition, providing more comprehensive descriptions of the electrophysiological features used in PCA analysis would be beneficial in understanding the electrophysiological profiling of DRN neurons explained in this paper.
Although several studies have revealed electrophysiological and molecular heterogeneity within the DRN5-HT population, we did not observe any significant differences within the DRN5-HT neurons recorded in this study. We compared the properties of DRN5HT neurons recorded more anterior to those recorded in the posterior
DRN as well as neurons found in more ventral locations to those in more dorsal locations (data not shown). We would like to point out that the largest differences within serotonergic neuron populations described by previous studies were often found when comparing those located in the medial raphe nucleus (MRN) to those found in the DRN. Calizo et al., (2011) showed for example significant differences in the input resistance and AHP amplitude between MRN5HT and DRN5HT neurons. These two properties as well as the AP amplitude, AP threshold, AP duration, and tau did however not differ between DRN subregions in their study - and neither in ours. We extended our Suppl. Fig 1 and mapped the location of DRN5HT and DRNDA neurons recorded in sham (Suppl. Fig 1D).
Overall, we’ve sampled neurons along the anterior-posterior and dorsal-ventral axes of the DRN, while on the medial-lateral axis, recorded DRN neurons were located medially.
We agree with the reviewer that a comprehensive description of the electrophysiological features was missing in the manuscript, and we have therefore added a new section in the materials and methods where we explain in detail how each parameter was measured and analyzed (‘Quantification of electrophysiological parameters’, lines 407-439). This section also provides detailed information about the five properties underlying the PCA shown in figure 1 (i.e. delay to the first action potential, action potential drop rate, action potential rise time, duration of the afterhyperpolarization, and capacitance).
(7) Some sample images presented in this paper contain information that can conflict with the previous research. In Figures 4B and 6B, TH expression was significantly increased in the DMI pretreatment group compared to the control group. However, several studies have shown that the administration of DMI decreases TH expression levels (Komori et al.1992; Nestler et al.1990). Therefore, it would be great if the authors further explained how the pretreatment of DMI with 6-OHDA affects TH level within the DRN.
Figure 4B and 6B do not show any quantification of TH expression. The difference observed in the representative pictures is casual and due to the variable expression of TH across the slice. Moreover, as mentioned in the response to point 5, mice were subjected to a single injection of DMI immediately preceding the stereotactic intervention (line 373375). In contrast, the increase in TH expression reported by Komori et al. 1992 and Nestler et al. 1990 was observed in response to chronic (two weeks) administration of DMI.
(8) This paper lacks direct evidence to demonstrate whether DMI pretreatment could effectively protect against NA depletion. Therefore, in addition to TH expression levels, it is important to provide data to confirm the intact NA levels (or NA axons) after DMI treatment.
NA levels in the striatum were measured by Enzyme-linked immunosorbent assay and reported in Suppl.Fig.4 in the revised manuscript.
(9) It would be great if the authors specifically explained why 6-OHDA was injected into the striatum (neither MFB nor SNc) to make a mouse model of PD.
Mice were injected in the dorsal striatum to produce a partial bilateral lesion of the dopamine and noradrenaline systems. This model reproduces the initial stages of PD and also recapitulates several non-motor symptoms of PD, including affective disorders, which may be related to changes in serotonergic and dopaminergic transmission in the dorsal raphe. In contrast, injections in the MFB and SNc quickly produce a severe motor phenotype closer to a late stage of the disease and cannot be done bilaterally.
The striatal model has been successfully used in other publications (Kravitz et al., 2010, Masini et al., 2021, Ztaou et al., 2018, Chen et al., 2014, Branchi et al., 2008, Marques et al. 2019, Tadaiesky et al., 2008, Matheus et al., 2016, Silva et al., 2016).
(10) Supplementary Figures 2 and 3 were erroneously cut on the right side. These figure images should be replaced with the correct ones.
We thank the reviewer for noticing and we have now replaced the figures with the correct ones.
(11) There should be more explanations about tdTomato-positive but non-TH neurons in Supplementary Figure 2. It is strange to regard TH-negative neurons as DA neurons although these neurons have DA neuron-like electrophysiological properties. If these tdTomato-positive but non-TH neurons cannot release DA, can we say these are DA neurons?
In our study we found a small subset of DAT-tdTomato positive neurons which did not stain positive for TH afterwards. In 5 of 6 of these neurons (recorded in sham), the electrophysiological properties did not differ from other TH-positive neurons. This is visualized in Suppl. Fig 2A. The absence of any statistical difference was also confirmed by a Mann Whiteny U test comparing the TH-negative to the TH-positive DRNDA neurons (no significant differences in all 6 of 6 properties shown in SF2A). Additionally, all these cells were DAT-positive, further supporting their classification as dopaminergic neurons. Therefore, we suspect that the lack of TH staining is likely caused by the tissue processing itself. Please note that all our immunohistochemistry was run on slices after several hours of patch-clamping procedures. Finally, including or excluding this small subset of neurons in the present study does not change any of the results presented and data was therefore pooled. We have now clarified this in more detail in the results section and in Suppl. Fig 2A (lines 100-103).
Reviewer #3 (Recommendations For The Authors):
The authors report using a parametric statistical test, the t-test. The t-test makes the assumption that the data are normally distributed. Most biological data is not distributed normally, and with smaller datasets, it is difficult to say whether the underlying distribution would be normally distributed. I would recommend using the non-parametric versions of the same test (eg Mann-Whitney U test), which is likely to give a similar result while being more conservative given the potential for non-normal distribution.
All electrophysiological data were first tested for normality before running the corresponding statistical test (either t-test for normal distributed data or Mann-Whitney U test for non-normally distributed data). The morphological data are now analyzed by the Mann-Whitney U test (lines 484-494).
The authors state that mice were treated with 6-OHDA at 3 months, then brain slices were prepared 3 weeks later, making them about 4 months old. I could not find the age of sham/control mice and 6-OHDA/desipramine mice in the methods section. Were sham/controls and 6-OHDA slices prepared in an interleaved fashion?
Sham and 6-OHDA+DMI mice underwent surgery at 3 months and the brain slices were prepared 3 weeks later, as the 6-OHDA mice. We have now clarified this in the methods (line 381).
While desipramine is relatively selective as a norepinephrine reuptake inhibitor, it also can prevent serotonin reuptake. Could this mechanism also protect DRN neurons from the effects of 6-OHDA?
Even if desipramine has some affinity for the serotonin reuptake, this affinity is 100-fold less than the one described for the noradrenaline reuptake (Richelson and Pfenning, 1984, Gillman, 2007). Moreover, in our study the 6-OHDA injection in the dorsal striatum did not cause any direct damage to the DRN5-HT, as shown by the 5-HT measurement and DRN5-HT counting (Suppl. Fig. 4D, Suppl. Fig. 5A,B), so we can exclude that the effects observed in the DMI+6-OHDA group are related to a protection of the serotonergic system exerted by a single injection of desipramine.
On line 168, the authors use the abbreviation NA for noradrenergic. Was this abbreviation previously defined in the manuscript?
Yes, the abbreviation is defined in the introduction (line 73).
On line 45, the authors cite that the DRN-5HT subpopulation accounts for 30-50% of the DRN neurons. It would be helpful to know approximately what percentage of the DRN neurons belong to the DRNDA subpopulation as well.
To the best of our knowledge, there is unfortunately no detailed analysis of the prevalence of DRNDA neurons in mice available. Previous studies in rats have estimated that this population comprises around 1000 neurons (Descarries et al., 1986). According to Calizo et al. (2011), the number of any non-serotonergic neuron population (releasing dopamine or other neurotransmitters) in the DRN is one third to one tenth less than the number of DRN5-HT neurons. But please note that this study was also performed in rats (line 55).
While I appreciate that the authors did not over-interpret their findings, it would be useful to comment (in the Discussion) on how their findings could/should be used in interpreting other studies using 6-OHDA, as well as the relationship of their findings to loss of 5-HT and/or DRN neurons in Parkinson's Disease itself.
In the manuscript, we refer to the utility of the 6-OHDA model for the study of a wide range of non-motor symptoms. We have now described, in this model, how the loss of midbrain dopaminergic and noradrenergic neurons affects the electrophysiological and morphological properties of DRN5-HT and DRNDA neurons. This information will allow for a more precise assessment of the mechanisms involved in the affective and cognitive aspects of PD symptomatology (lines 354-356).
