Dorsal raphe stimulation relays a reward signal to the ventral tegmental area via GluN2C NMDA receptors

  1. Giovanni Hernandez
  2. Willemieke M. Kouwenhoven
  3. Emmanuelle Poirier
  4. Karim Lebied
  5. Daniel Lévesque
  6. Pierre-Paul Rompré

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Abstract

Glutamate relays a reward signal from the dorsal raphe (DR) to the ventral tegmental area (VTA). However, the role of the different subtypes of N-methyl-D-aspartate (NMDA) receptors is complex and not clearly understood. Therefore, we measured NMDA receptors subunits expression in limbic brain areas. In addition, we studied the effects of VTA down-regulation of GluN2C NMDA receptor on the reward signal that arises from DR electrical stimulation.

Methods

Using qPCR, we identified the relative composition of the different Grin2a-d subunits of the NMDA receptors in several brain areas. Then, we used fluorescent in situ hybridization (FISH) to evaluate the colocalization of Grin2c and tyrosine hydroxylase (TH) mRNA in VTA neurons. To assess the role of GluN2C in brain stimulation reward, we downregulated this receptor using small interfering RNA (siRNA) in rats self-stimulating for electrical pulses delivered to the DR. To delineate further the specific role of GluN2C in relaying the reward signal, we pharmacologically altered the function of VTA NMDA receptors by bilaterally microinjecting the NMDA receptor antagonist PPPA.

Results

We identified GluN2C as the most abundant subunit of the NMDA receptor expressed in the VTA. FISH revealed that about 50% of TH-positive neurons colocalize with Grin2c transcript. siRNA manipulation produced a selective down-regulation of the GluN2C protein subunit and a significant reduction in brain stimulation reward. Interestingly, PPPA enhanced brain stimulation reward, but only in rats that received the nonactive RNA sequence.

Conclusion

The present results suggest that VTA glutamate neurotransmission relays a reward signal initiated by DR stimulation by acting on GluN2C NMDA receptors.

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  1. Version published to 10.1371/journal.pone.0293564
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    Reply to the reviewers

    Comment 1.

    The impact of this study would be greatly enhanced if the authors could provide electrophysiological results validating that intra-VTA infusion of Grin2c siRNA affects the excitability or discharge regularity of different populations of VTA neurons (at least dopaminergic and GABA neurons).

    *Reply. The reviewer is suggesting an additional experiment that constitutes a next logical step to further determining the role of NMDA receptors (NMDARs) containing the GluN2C subunit(s) in induction of EPSP in VTA TH+ and TH- neurons; such an experiment could be performed in vivo in behaviorally tested animals, but the recordings would have to be done in anesthetized animals 24h after VTA siRNAs microinjections; an alternative would be an in vitro experiment with slices that would maintain a functional link between dorsal raphe (DR) glutamatergic reward neurons and VTA neurons; we agree that this would constitute an important step forward, and that can be performed once our findings are published. *

    Comment 2.

    The strategy of targeting Grin2c transcripts with siRNAs is appropriate and the authors implemented a western blot validation to control the effectiveness of their approach. However, due to the large volume of siRNA injected into the VTA (500 nl), the authors should consider extending their western blot validation experiments to structures anatomically close to the VTA and rich in GluN2C subunit such as RMTg. The DRN would also be an interesting structure to show that GluN2C subunit expression is not affected by the siRNA approach.

    *Reply. Optogenetic studies have shown that the reward signal initiated by glutamatergic neurons in the dorsal raphe is transmitted to VTA neurons (Liu et al., 2014; McDevitt et al., 2014 Qi et al., 2014;), findings that are consistent with, and that were predicited by, our previous findings (Rompré and Miliaressis, 1985; Boye and Rompré, 2000; Ducrot et al 2013). Moreover, a large body of research carried out with local (VTA) drug injections generated data confirming the role of VTA neurons in reward (See for instance Wise and McDevittt, 2018). Thus, it is reasonable to hypothesizes that it is the reduction of GluN2c (NMDARs that contain this subunit) in VTA neurons that is responsible of the attenuation of reward. Such a hypothesis is further reinforced by our findings that GluN2c and its gene are expressed in VTA neurons. Why should it be within the RMTg? The reviewer likely knows many different studies that have shown that RMTg neurons play a key role in aversion; they provide a strong tonic inhibitory input to reward-relevant VTA neurons and more specifically to VTA DA neurons (see Zhou et al). RMTg neurons receives a strong excitatory input from the lateral habenula; activation of these neurons strongly inhibit reward. Consequently, a logical hypothesis is that a reduction of the glutamatergic excitation of RMTg caused by a reduction of NMDARs that contain GluN2Csubunits would have produce an enhancement of the reward signal, not an attenuation. *

    *The DR, site at which the electrical stimulation was delivered is located near 2 mm behind and 3 mm above the injection sites, it is very unlikely that the small volume injected hit the DR. *

    Comment 3.

    The authors should consider discussing or re-evaluating their findings from their 2015 study which suggested that the effect on reward induced by DRN stimulation was controlled by GluN2A-containing NMDARs most likely located on afferent terminals.

    *Reply. We cannot understand why we should re-evaluate or re-discuss these findings. It has been repeatedly shown that blockade of GluN2A containing NMDARs by local VTA PPPA and **R-CPP *microinjections enhances the reward signal initiated by DR electrical stimulation (Bergeron and Rompré, 2013; Ducrot et al., 2013; Hernandez et al., 2015; and the present study (control group); this enhancement effect was not attenuated by a decrease in NMDARs that contain GluN2A subunit(s) (Hernandez et al 2015). Our discussion is reasonable in view of these findings, and the hypothesis that these GluN2A containing NMDARs are located on afferent terminal explains the results.

    Comment 4.

    In Figure 2, the authors should quantify the results of the colocalization levels of Grin2c and TH in dopaminergic neurons of the substantia nigra pars compacta.

    Reply. As mentioned in our reply to comment 2, the quantification of VTA neurons was highly justified by a large body of the literature which is not the case for the substantia nigra pars compacta neurons.

    Comment 5.

    The results should be presented differently in figure 5 in order to be able to compare on the same graph and with the appropriate statistical analyses (two-way ANOVA), the impact of PPPA infusion or solvent in the SCRGluN2C or siRNA groups.

    Reply. Unfortunately, this is not possible because not all subjects were tested with PPPA.

    Comment 6. The authors should clarify the N=12 per condition in Figure 3, especially since 11 values for the control conditions are plotted on the histogram.

    *Reply. The reviewer is correct there are 11 Subjects in the control group and 12 in the active sirna group. We made the changes to the methods section. *

    Comment 7.

    Authors should standardize the way they cite literature throughout the manuscript (number or authors).

    Reply. Thanks for the suggestion changes have been made to standardize the citations.

    Comment 8.

    The authors should clarify sentences 102-105 of their introduction, which seem to conflict but ultimately describe similar results.

    Reply. Reviewer is correct the following sentences at the end the paragraph were deleted:

    *This hypothesis predicts that activation of a given subtype(s) potentiates DA burst firing and DA release, whereas activation of different subtype(s) increases the inhibitory drive to DA neurons. This idea is supported by data mentioned above and others showing that both activation and blockade of VTA NMDARs increase DA burst firing (French et al., 1993), accumbens DA release (Karreman et al., 1996; Westerink et al., 1996; Mathé et al., 1998; Kretschmer, 1999), and stimulate forward locomotion (Kretschmer, 1999; Cornish et al., 2001). Rodents also readily learn to directly self-administer the non-selective NMDAR antagonists, AP-7, into the VTA (David et al., 1998), showing that NMDAR blockade can have positive rewarding properties on its own. *

    Comment 9.

    The expression of different GluN2 subunits across different regions of the brain has been known since the early 90's as the authors acknowledged. In the abstract, the authors state that GluN2C is "the most abundant subunit of the NMDA receptor expressed in the VTA" (line 67). This idea that GluN2C is "the most abundantly expressed in DR and VTA compared to other Grin2 subunit transcripts" (line 425), is repeated throughout the paper. However, in the Results section they state that GluN2C is present at the same level as GluN2B; something that is also clearly visible in figure 1, where is also clear that GluN2A is also present almost at the same level. The emphasis that GluN2C has a larger representation over 2A and 2B in VTA is not necessary and misleading.

    *Reply. The point here was to bring attention to the reader to the expression of GLuN2C, the main target of the current study. As shown in Figure 1, GluN2c is indeed the most abundant in the VTA. *

    Comment 10

    1. Performing an immunoblot in tissue obtained with a tissue punch of the VTA, the authors confirmed that the GluN2C mRNA detected is translated into protein. Unfortunately, this important data is not showed, and it should be shown. Moreover, immunocytochemistry of GluN2C could help to identify the cellular type where the protein is expressed, something that could be key to better understand the role of NMDARs in the reward pathway. Are 2A/2B expressed in different cells that 2C? What type of cells express 2C? These are just a few of the question that a better and more detailed analysis of 2C expression could provide. Without this, the interpretation of results presented here, as well as previous results, regarding the role of NMDARs continuous being confusing.

    Reply. Because we measured GluN2C proteins within the VTA, we infer that some Grin2C detected in VTA TH+ and TH- neurons is translated into proteins, and reduction of the protein expression resulted into a selective attenuation of reward We added a supplementary fig showing the GluN2c protein in different brain regions.

    Comment 11.

    1. The largest number of 2C positive cells do not express TH complicating the interpretation that 2C is necessary to convey reward information in the DR-VTA circuit. Other effects due to downregulation of 2C could be responsible of the behavior changes observed. Although the authors offer an explanation for this, is not enough. They suggest that 2C maybe involved in a reduction of excitatory inputs into inhibitory interneurons that when downregulated should produce an opposite effect to what is observed. However, without knowing the identity of those GluN2C expressing cells this comment is only speculation and does not rule out a role for other GluN2C expressing cells that are not TH positive.

    *Reply. We do consider the hypothesis that the attenuation of reward is due to a reduction of GluN2C in TH- neurons, in fact we discuss both hypothesis, TH+ and TH-. Characterization of the reward-relevant neuronal pathway has been an important aim since Olds and Milner discovery. Our findings constitute, as mentioned in reply to comment 1, and important step forward, and indeed identification of the specific VTA cells that convey the reward signal is another important question that should be addressed. Our findings provide a strong ground to focus on GluN2C but not the other subunits. *

    Comment 12.

    1. In this line, there is no good explanation why treatment of animals with GluN2A blocker enhances the reward pathway only in animals treated with control siRNA. Two possibilities could explain this. 1) there is some sort of relationship between 2C and 2A that when 2C is absent, PPPA has no effect. Again, it could be important to know if 2C and 2A are expressed in the same cellular type; 2) the control siRNAs are not completely innocuous and may produce unknown effects that alter the functionality of VTA.

    *Reply. We believe that our data are strong and valid because the methods we used have been validated and the results with PPPA in control group are similar to those previously published. Previously we have shown that a reduction of VTA GluN2A proteins has no impact on reward per se nor on the enhancement of reward by PPPA (Hernandez et al., 2015). The hypothesis raised by the reviewer that 2C and 2A interact is incompatible with the findings that we obtained. Could it be a non-specific effects like tissue damage. In such a case however we would have observed a decrease in 2A subunits as well which is not the case. *

    Comment 13- 16

    1. Downregulating 2C suggests that this subunit is vital to relay a reward signal in VTA neurons. The following are comments regarding the analysis of the data of Fig 4 and 5.
    2. It is not clear how the maximum and minimum are estimated in order to fit a sigmoidal curve to the data. Are they average of the stable part? Where the error bars on each data point come from? What is the actual value and standard deviation of M50 values?

    *Reply: *As described in the self-stimulation training in the materials and method section

    “The data relating to the rate-frequency was fitted to a sigmoid described by the following equation y=Min+((Max-Min) )/(1+[10]^((x50-x)*p) ) where Min is the lower asymptote, Max is the upper asymptote, x50 is the position parameter denoting the frequency at which the slope of the curve is maximal, and p determines the steepness of the sigmoid curve. The resulting fit was used to derive an index of reward defined as the pulse-frequency sustaining a half-maximal rate of responding (M50). Self-stimulation behavior was considered stable when the M50 values varied less than 0.1 log unit for three consecutive days”

    The Max, Min and all the free parameters of the equation are the determine by the best-fit parameters by minimizing a chosen merit function. A merit function, also known as a figure-of-merit function, is a function that measures the agreement between data and the fitting model for a particular choice of the parameters. By convention, the merit function is small when the agreement is good. To optimize the merit function, it is necessary to select a set of initial parameter estimates and then iteratively refine the merit parameters until the merit function does not change significantly between iterations. The Levenberg-Marquardt algorithm has been used for nonlinear least squares calculations in the current implementation.

    As described in the self-stimulation training and material section.

    “.. Four stimulation sweeps were run daily, and the first sweep was considered a warm-up and discarded from the analysis”. The remaining 3 sweeps were fitted to a sigmoid and the parameters were obtained. The error bars correspond to the difference across each sweep”.

    • What is the actual value of the M50 SD? Don’t understand why this is relevant? We already provide the SEM.*
    1. This type of data is better analyzed by nonlinear regression analysis followed by ANOVA and some post hoc multiple comparison test.

    *Reply: We totally agree with the reviewer that is why the analysis was done fitting a sigmoid line. In fact, the fitting uses non-linear regression to compare the data points to the function, which in this case is a sigmoid function defined by the equation y=Min+((Max-Min) )/(1+[10]^((x50-x)*p) ) where Min is the lower asymptote, Max is the upper asymptote, x50 is the position parameter denoting the frequency at which the slope of the curve is maximal, and p determines the steepness of the sigmoid curve. In fact, intracranial self-stimulation data has been analysed using non-linear regression models since the seminal work by Coulombe and Miliaresis 1986 *[1]

    Indeed, after obtaining the results of the parameters, we follow it up with traditional statistics like t test and Anovas

    1. Given the large variance of individual data points in Figure 4 and 5, a stricter statistical analysis than a t-test is necessary.

    *Reply: Thanks for the suggestion, but we do not fully understand what the reviewer is suggesting. In general, the condition to apply or not a specific statistical test assumes about the underlying distribution the conditions required to conduct a t-test include: the measured values are in ratio scale or interval scale, simple random extraction, homogeneity of variance (i.e., the variability of the data in each group is similar), and normal distribution of data. The normality assumption means that the collected data follows a normal distribution, which is essential for parametric assumption. In all the data presented in figure 4-5 the assumptions are respected and checked (the data is measure in a continues scale, the group assignation was performed randomly, the data does not violate the normality assumption and the variance between the groups is similar. In the only case where the variance assumption was not held the Welsh correction was applied. *

    Comment 17

    1. Minor comments include the need to refer to figure panels in ascending order in the same sequence as they are described in the text.

    Reply: Thanks for the suggestion we made the required changes. Now the figure panels are in the same sequence as they are described.

    Comment 18

    The role of NMDARs in VTA are explained in a rather confusing manner in the introduction. Lines 104 to 106 need some rewording since it conveys that blockade of NMDARs stimulates reward and that an opposite effect is observed following the blockade of NMDARs.

    Reply. We simply report data from the literature. Each statement is supported by the relevant literature.

    [1] Coulombe and Miliaressis, “Fitting Intracranial Self-Stimulation Data with Growth Models.”

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Given the importance of glutamatergic synaptic transmission in the reward pathway from the dorsal raphe to VTA, the authors set to study the role of GluN2 subunits in a reward behavior. First, using qRT-PCR they quantify the amount of GluN2 subunits in different brain regions. They highlight the presence of GluN2C as the most abundant species of GluN2 subunits in VTA. They also identify that ~50% of TH positive neurons are also positive for GluN2C mRNA. Finally, they downregulate GluN2C in VTA using a commercially available siRNA. Knockdown of GluN2C reduces reward-seeking behavior as it increases the M50 and reduces the maximum response. The authors conclude that "VTA glutamate neurotransmission relays a reward signal initiated by DR stimulation by acting on GluN2C NMDA receptors".

    Major comments

    1. The expression of different GluN2 subunits across different regions of the brain has been known since the early 90's as the authors acknowledged. In the abstract, the authors state that GluN2C is "the most abundant subunit of the NMDA receptor expressed in the VTA" (line 67). This idea that GluN2C is "the most abundantly expressed in DR and VTA compared to other Grin2 subunit transcripts" (line 425), is repeated throughout the paper. However, in the Results section they state that GluN2C is present at the same level as GluN2B; something that is also clearly visible in figure 1, where is also clear that GluN2A is also present almost at the same level. The emphasis that GluN2C has a larger representation over 2A and 2B in VTA is not necessary and misleading.
    2. Previous reports showed that pharmacological blockade on GluN2B or downregulation of GluN2A, the most common GluN2 subunits in the brain, do not affect the nose-poke behavior the authors use here. The biophysical properties of GluN2C are very different from those of 2B and 2A, therefore the fact that 2C downregulation does affect the behavior observed makes an interesting case for the subunit.
    3. Performing an immunoblot in tissue obtained with a tissue punch of the VTA, the authors confirmed that the GluN2C mRNA detected is translated into protein. Unfortunately, this important data is not showed, and it should be shown. Moreover, immunocytochemistry of GluN2C could help to identify the cellular type where the protein is expressed, something that could be key to better understand the role of NMDARs in the reward pathway. Are 2A/2B expressed in different cells that 2C? What type of cells express 2C? These are just a few of the question that a better and more detailed analysis of 2C expression could provide. Without this, the interpretation of results presented here, as well as previous results, regarding the role of NMDARs continuous being confusing.
    4. The largest number of 2C positive cells do not express TH complicating the interpretation that 2C is necessary to convey reward information in the DR-VTA circuit. Other effects due to downregulation of 2C could be responsible of the behavior changes observed. Although the authors offer an explanation for this, is not enough. They suggest that 2C maybe involved in a reduction of excitatory inputs into inhibitory interneurons that when downregulated should produce an opposite effect to what is observed. However, without knowing the identity of those GluN2C expressing cells this comment is only speculation and does not rule out a role for other GluN2C expressing cells that are not TH positive.
    5. In this line, there is no good explanation why treatment of animals with GluN2A blocker enhances the reward pathway only in animals treated with control siRNA. Two possibilities could explain this. 1) there is some sort of relationship between 2C and 2A that when 2C is absent, PPPA has no effect. Again, it could be important to know if 2C and 2A are expressed in the same cellular type; 2) the control siRNAs are not completely innocuous and may produce unknown effects that alter the functionality of VTA.
    6. Downregulating 2C suggests that this subunit is vital to relay a reward signal in VTA neurons. The following are comments regarding the analysis of the data of Fig 4 and 5.
    7. It is not clear how the maximum and minimum are estimated in order to fit a sigmoidal curve to the data. Are they average of the stable part? Where the error bars on each data point come from? What is the actual value and standard deviation of M50 values?
    8. This type of data is better analyzed by nonlinear regression analysis followed by ANOVA and some post hoc multiple comparison test.
    9. Given the large variance of individual data points in Figure 4 and 5, a stricter statistical analysis than a t-test is necessary.
    10. Minor comments include the need to refer to figure panels in ascending order in the same sequence as they are described in the text.
    11. The role of NMDARs in VTA are explained in a rather confusing manner in the introduction. Lines 104 to 106 need some rewording since it conveys that blockade of NMDARs stimulates reward and that an opposite effect is observed following the blockade of NMDARs.

    Overall, the data and analysis still leave too many open questions and the role of GluN2C, vs the other subunits, is not clearly established.

    Significance

    The study is limited in its scope and possible interpretations. The role of GluN2 subunits in the relay of reward information is only incrementally advanced and still continuous to be confusing.

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    Referee #1

    Evidence, reproducibility and clarity

    This study by Hernandez et al provides a series of molecular, anatomical, and behavioral experiments exploring the distribution and the function of the NMDA Glun2C subunit in the ventral tegmental area (VTA). The authors demonstrate that reward signals originating from the dorsal raphe (DR) are carried to VTA neurons through the activation of GluN2C NMDA receptors. this study did not address the specific role of VTA dopaminergic neurons in mediating the reward signal. The major strengths of this paper are the quality of the FISH experiments and the well-established model of Brain Stimulation Reward targeting the Dorsal Raphe. This study is a follow-up of a previous study published by the same group (2015), which explored the expression of GluN2A/2D subunits in the VTA and their role in reward induced by dorsal raphe stimulation. This is an interesting and original manuscript. However, several issues, concerning the design of the experiment and the interpretation of the data, reduce my enthusiasm.

    The impact of this study would be greatly enhanced if the authors could provide electrophysiological results validating that intra-VTA infusion of Grin2c siRNA affects the excitability or discharge regularity of different populations of VTA neurons (at least dopaminergic and GABA neurons).

    The strategy of targeting Grin2c transcripts with siRNAs is appropriate and the authors implemented a western blot validation to control the effectiveness of their approach. However, due to the large volume of siRNA injected into the VTA (500 nl), the authors should consider extending their western blot validation experiments to structures anatomically close to the VTA and rich in GluN2C subunit such as RMTg. The DRN would also be an interesting structure to show that GluN2C subunit expression is not affected by the siRNA approach.

    The authors should consider discussing or re-evaluating their findings from their 2015 study which suggested that the effect on reward induced by DRN stimulation was controlled by GluN2A-containing NMDARs most likely located on afferent terminals.

    In Figure 2, the authors should quantify the results of the colocalization levels of Grin2c and TH in dopaminergic neurons of the substantia nigra pars compacta.

    The results should be presented differently in figure 5 in order to be able to compare on the same graph and with the appropriate statistical analyses (two-way ANOVA), the impact of PPPA infusion or solvent in the SCRGluN2C or siRNA groups.

    The authors should clarify the N=12 per condition in Figure 3, especially since 11 values for the control conditions are plotted on the histogram.

    Authors should standardize the way they cite literature throughout the manuscript (number or authors).

    The authors should clarify sentences 102-105 of their introduction, which seem to conflict but ultimately describe similar results.

    Significance

    This study is a follow-up of a previous study published by the same group (2015), which explored the expression of GluN2A/2D subunits in the VTA and their role in reward induced by dorsal raphe stimulation. This is an interesting and original manuscript.

  5. Version published to 10.1101/2020.07.26.222125v2 on bioRxiv
  6. Version published to 10.1101/2020.07.26.222125v1 on bioRxiv