Aversion encoded in the subthalamic nucleus

  1. Gian Pietro Serra
  2. Adriane Guillaumin
  3. Jérome Baufreton
  4. François Georges
  5. Åsa Wallén-Mackenzie

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Abstract

Activation of the subthalamic nucleus (STN) is associated with the stopping of ongoing behavior via the basal ganglia. However, we recently observed that optogenetic STN excitation induced a strong jumping/escaping behavior. We hypothesized that STN activation is aversive. To test this, place preference was assessed. Optogenetic excitation of the STN caused potent place aversion. Causality between STN activation and aversion has not been demonstrated previously. The lateral habenula (LHb) is a critical hub for aversion. Optogenetic stimulation of the STN indeed caused firing of LHb neurons, but with delay, suggesting the involvement of a polysynaptic circuit. To unravel a putative pathway, the ventral pallidum (VP) was investigated. VP receives projections from the STN and in turn projects to the LHb. Optogenetic excitation of STN-VP terminals caused firing of VP neurons and induced aversive behavior. This study identifies the STN as critical hub for aversion, potentially mediated via an STN-VP-LHb pathway.

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  1. eLife

    ###Reviewer #3:

    In this paper the authors show for the first time that optogenetic activation of the subthalamic nucleus (STN) is aversive and can drive avoidance behavior. This effect may be mediated by polysynaptic activation of the Lateral habenula, which they show is activated following optogenetic activation of the STN. They propose that the STN may excite glutamatergic neurons in the ventral pallidum that in turn project to and excite the lateral habenula. The authors do mention that other pathways may mediate the aversive effects but no other pathways are tested.

    Overall this paper presents a simple and clear demonstration that optogenetic activation of the subthalamic is aversive. It may be that this effect involves activation of the ventral pallidum and the lateral habenula but the evidence provided to support this possibility is weak and currently uncompelling.

    Major issues,

    -While it has not to my knowledge been reported that activation of the STN can drive aversive responses there are a number of lines of evidence that suggested it should be the case. None of these are mentioned in the paper and should be discussed. First the STN is part of the indirect pathway in the basal ganglia. Previous work has shown through optogenetic and other methods that the indirect pathway striatal neurons in the dorsal and ventral striatum can drive aversive responses and are involved in aversive learning (for a critical review that discusses this literature see Soares-Cunha et al., 2016). In line with this, recordings of the indirect pathway have also shown that this pathway is preferentially involved in processing aversive information, for example STN neurons are activated by nociceptive information and are needed for appropriate behavioural responses to nociceptive stimuli (Pautrat et al., 2018), STN neurons are also activated by aversive stimuli and by negative reward prediction error (Breysse et al., 2015). The paper needs to discuss their findings in the context of this and other previous work (these references are just examples and not an exhaustive list) that supports the role of the indirect pathway in processing aversive information.

    -Another topic that should be discussed is the heterogeneity of the STN. The authors themselves mention that the STN is composed of distinct spatio-molecular domains. This may well be relevant as rabies tracing from the EP neurons that project to the habenula and from the glutamatergic neurons in the ventral pallidum has revealed that they receive the majority of their input from the parasubthalamic nucleus and not from the core of the STN (Stephenson-Jones et al., 2016, Stephenson-Jones et al., 2020, Tooley et al., 2018). This raises the possibility that the aversive responses from the STN are primarily driven by neurons in the pSTN. The authors could test this point by restricting their ChR2 expression to one or the other region of the STN. At the moment all example images show that expression is in both the STN and pSTN. This possibility should be discussed.

    -The authors mention that they perform selective activation for the STN-VP pathway by stimulating the STN terminals in the VP. It is not clear that this will selectively activate this pathway. If the STN neurons that project to the VP also project to other areas then these will likely also be activated due to back propagating action potentials driven by the ChR2 stimulation. More work needs to be done to determine if the VP is really the pathway that mediates the aversive effect. Additional work including multi-colour retrograde tracing, selective inactivation of the VP projection while stimulating the STN or stimulating the STN fibers in the VP while inactivating the STN cell bodies would be needed to really determine if the VP is important for mediating the aversive effect. This may be beyond the scope of what the authors want to do but would be needed to support a claim that their evidence "provide strong support for a STN-VP-LHb is a pathway for aversion".

    -The title should not include the word encoded as there were no experiments performed in this paper that looked at any aspect of coding in the STN.

  2. eLife

    ###Reviewer #2:

    In this manuscript Serra et al. demonstrate that stimulation of subthalamic nucleus (STN) neurons can drive place avoidance and delayed (presumably bisynaptic) excitation of lateral habenula (LHb) neurons. They also show that STN inputs to the ventral pallidum (VP) can drive place avoidance and excitation of VP neurons. While the potential role of a STN-VP-LHb of driving aversion and avoidance is intriguing, the manuscript leaves many open questions regarding the nature of STN's role in mediating aversion, as well as the circuit mechanisms governing STN-induced avoidance.

    Major Comments:

    1. STN in aversion: The manuscript addresses the role of the STN in mediating "aversion" in a very limited manner, despite the framing of the title ( "Aversion encoded in the subthalamic nucleus"). Based on the title I expected data showing that STN activity is correlated with the aversiveness of stimuli, or data showing that STN activity is required for aversion processing. Instead the authors show that STN stimulation can drive avoidance, which does not necessarily mean that this activity drives "aversion" per se. Data showing that STN represents the aversiveness of stimuli or that activity here is necessary for avoidance or other responses to aversive stimuli would strengthen the point. Currently the evidence for the statement made by the title is weak.

    2. Claims about the role of the STN->VP->LHb pathway in the abstract and elsewhere in the text: The authors demonstrate that activation of STN terminals in VP recapitulates their RTPP avoidance effects, but they do not directly demonstrate that these effects are mediated by downstream VP->LHb connectivity. They show that activation of STN terminals in VP results in excitation of VP units, but it remains unknown whether STN neurons specifically target/activate VP neurons that project to LHb, and/or whether they target VP glutamate neurons specifically (the primary cell type in the VP->LHb pathway that mediates aversion). The current data set does not demonstrate either that that a) STN-induced activity changes are LHb are predominantly mediated by VP (as opposed to EP or GP or other connections), or that b) avoidance elicited by STN->VP activation is mediated by LHb activity. Therefore, statements throughout the manuscript about the STN-VP-LHb circuit are not supported.

    3. Statistical analysis: The authors provide comprehensive statistical information for their behavioral experiments, but not for the electrophysiology. It appears that individual neurons were treated as independent measurements even when they were recorded from the same subject, though in some cases it is not clear how many mice were recorded from (e.g. 1G, 5D, 5E). If multiple measurements were taken from the same subjects, then this should be taken into account in the statistical analysis (such as by including subject as a random effect in an ANOVA or linear mixed model).

  3. eLife

    ###Reviewer #1:

    In this study, Serra et al. attempted to study the circuit responsible for aversive behavior in mice. They had previously observed that subthalamic nucleus (STN) excitation induced aversive jumping behavior. The authors proposed that the indirect projection to the lateral habenula (LHb) via the ventral pallidum (VP) could be involved. They used Pitx2-Cre mice for STN-specific gene expression and performed real-time place preference paradigm (RT-PP) and elevated plus maze (EPM) as a means to study aversive behavior. Overall, the findings in this study are potentially important as they describe a previously unknown role of the STN, and its downstream targets, in aversive behavior. However, the authors have not convincingly demonstrated the pathway involved. The evidence so far is rather circumstantial and the arguments made were based entirely on gain-of-function experiments using ChR2. As outlined below here are a number of significant concerns that need to be addressed.

    Major:

    1. The authors should demonstrate the effectiveness and specificity of Pitx2-Cre in driving ChR2 expression. What was the cellular expression pattern within the STN? Did the authors observe ChR2 expression in 100% of STN neurons? Did it label any non-neuronal cells? Did the neighboring regions also express ChR2? According to Papathanou et al., that is likely to be the case. The authors should provide a more rigorous histological examination. Otherwise, a more in-depth discussion is needed to address how these concerns would confound the interpretation of results.

    2. It is interesting that Pitx2/ChR2-eYFP mice avoided STN-photostimulation by spending less time in the light-paired compartment. It should be discussed why not the compartment where STN is stimulated is not completely avoided.

    3. It is unclear if the mice jumped in this study, as the authors had previously observed. Was there any other movement-related behavioral changes?

    4. In Figure 1B, it seems like the entry into Compartments A and B of Pitx2/ChR2-eYFP on Day 5 and 6 is not very different. However, in Figure 1C, the representative heatmap shows a difference. In contrast, in Figure 1B, it seems like the entry into Compartments A and B of Pitx2/ChR2-eYFP on Day 9 is equal. Whereas in Figure 1C, the representative heatmap shows substantial entries. It would be helpful to have an explanation for the discrepancies.

    5. Assuming that the STN excitation duration is 10 seconds upon entry to the "Light" compartment, do the mice remain in the "Light" compartment? If the mice are only stimulated at the entry point of the "Light" compartment, do they just remain there and avoid exiting (as a means to avoid reentry)? As 10 seconds is a long time period for the mice to move around, is the stimulation continued if they then switch to the neutral compartment before the end of the 10-second stimulation period?

    6. It is not exactly clear the point of the first EPM experiment with 10 minutes of stimulation of STN neurons or their terminals. That is a very long time period; it is very likely plasticities were induced with such a paradigm and would confound the study.

    7. The STN-VP slice experiment does not really address any of the circuit questions they proposed to answer. The STN-VP connection is already known. It would be more interesting if the authors show the specific connection between STN and the glutamatergic VP neurons, as they speculate as the downstream target of the STN. This is an important point because of the complex cellular composition within the VP.

    8. It would be important to show that direct optogenetic stimulation of glutamatergic neurons within the VP produced the same phenotype. At the very least, the authors should locally infuse glutamatergic blockers into the VP to examine if the effects with STN stimulation can in fact be blocked.

    9. Both Figures 1 and 5 show a rather low density of STN fiber in the VP and they are restricted to about one-third of the VP. The involvement of the STN-VP circuit in mediating the observed behavior is less than convincing. On the other hand, there are no investigations of whether direct connections to other known targets are involved in the aversive response.

    10. All optogenetic interrogations were based on ChR2 stimulation. As antidromic spikes can propagate to other collateral branches in other synaptic targets of STN neurons (i.e., the GP, EP, and/or SNr), orthogonal approaches are needed to decisively show STN-VP circuit is involved.

    11. What is the latency of STN-driven spiking in LHb? The latency in the peri-stimulus time histogram Figure 4 looks too short to be a polysynaptic event. It also does not match up with that stated in the text (i.e., 10 ms, line 212). This is not a trivial matter as synaptic delays can provide important clues for whether mono- vs polysynaptic events are involved.

    12. In Figure 5E, DNQX and APV did not completely block the evoked currents. A more rigorous examination is needed if multiple neurotransmitters were released.

    13. As anxiety and aversive behaviors are often dichotomous between males and females, the authors should comment on whether there were any sex differences observed.

    14. Some of the sample sizes are very small (only 3-5).

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    While the demonstration that stimulation of subthalamic nucleus (STN) neurons produces avoidance is potentially interesting, the circuit basis of this effect was not well established. Specifically, the proposed functional connection of STN with lateral habenula through ventral pallidum was not clearly demonstrated and the STN stimulation findings on their own represent a more minor advance.

  5. Version published to 10.1101/2020.07.09.195610 on bioRxiv