Modified viral-genetic mapping reveals local and global connectivity relationships of ventral tegmental area dopamine cells
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Evaluation Summary:
By addressing shortcomings in rabies viral-mediated labeling of monosynaptic inputs to ventral tegmental area dopamine neurons, this study provides a previously unattained precision local inputs to VTA dopamine neurons. Main findings include the preservation of a medial to lateral topography in the projection patterns within VTA microcircuitry, prominence of inhibition of DA neurons from the substantial nigra pars reticulata (SNr), DA-DA transmission, and inputs from raphe serotonin neurons. The precise local VTA connectivity described here is important for identifying how dopamine neurons compute reward, prediction, and movement-related signals during behavior, and thus is likely to be of interest to neuroscientists interest in those processes and the midbrain dopamine system.
(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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Abstract
Dopamine cells in the ventral tegmental area (VTA DA ) are critical for a variety of motivated behaviors. These cells receive synaptic inputs from over 100 anatomically defined brain regions, which enables control from a distributed set of inputs across the brain. Extensive efforts have been made to map inputs to VTA cells based on neurochemical phenotype and output site. However, all of these studies have the same fundamental limitation that inputs local to the VTA cannot be properly assessed due to non-Cre-dependent uptake of EnvA-pseudotyped virus. Therefore, the quantitative contribution of local inputs to the VTA, including GABAergic, DAergic, and serotonergic, is not known. Here, I used a modified viral-genetic strategy that enables examination of both local and long-range inputs to VTA DA cells in mice. I found that nearly half of the total inputs to VTA DA cells are located locally, revealing a substantial portion of inputs that have been missed by previous analyses. The majority of inhibition to VTA DA cells arises from the substantia nigra pars reticulata, with large contributions from the VTA and the substantia nigra pars compacta. In addition to receiving inputs from VTA GABA neurons, DA neurons are connected with other DA neurons within the VTA as well as the nearby retrorubal field. Lastly, I show that VTA DA neurons receive inputs from distributed serotonergic neurons throughout the midbrain and hindbrain, with the majority arising from the dorsal raphe. My study highlights the importance of using the appropriate combination of viral-genetic reagents to unmask the complexity of connectivity relationships to defined cells in the brain.
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Evaluation Summary:
By addressing shortcomings in rabies viral-mediated labeling of monosynaptic inputs to ventral tegmental area dopamine neurons, this study provides a previously unattained precision local inputs to VTA dopamine neurons. Main findings include the preservation of a medial to lateral topography in the projection patterns within VTA microcircuitry, prominence of inhibition of DA neurons from the substantial nigra pars reticulata (SNr), DA-DA transmission, and inputs from raphe serotonin neurons. The precise local VTA connectivity described here is important for identifying how dopamine neurons compute reward, prediction, and movement-related signals during behavior, and thus is likely to be of interest to neuroscientists interest in those processes and the midbrain dopamine system.
(This preprint has been reviewed by …
Evaluation Summary:
By addressing shortcomings in rabies viral-mediated labeling of monosynaptic inputs to ventral tegmental area dopamine neurons, this study provides a previously unattained precision local inputs to VTA dopamine neurons. Main findings include the preservation of a medial to lateral topography in the projection patterns within VTA microcircuitry, prominence of inhibition of DA neurons from the substantial nigra pars reticulata (SNr), DA-DA transmission, and inputs from raphe serotonin neurons. The precise local VTA connectivity described here is important for identifying how dopamine neurons compute reward, prediction, and movement-related signals during behavior, and thus is likely to be of interest to neuroscientists interest in those processes and the midbrain dopamine system.
(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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Reviewer #1 (Public Review):
Beier leveraged a more selective Rabies virus retrograde tracing method to identify, for the first time, local monosynaptic inputs to DAT+ dopamine neurons in the VTA. He identified a previously unappreciated preponderance of SNr inhibitory inputs, as well as local connectivity among neighboring DA neurons and inputs from serotonergic inputs of the raphe nucleus. Rigorous neuroanatomy is a prerequisite to identify how DA neurons compute reward, prediction, and movement signals during behavior.
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Reviewer #2 (Public Review):
This single-author study highlights an important caveat of currently prevalent monosynaptically-restricted rabies tracing methods in neuroscience, which complicate the interpretation of data on synaptic inputs surrounding injection sites. The author used a previously published, modified viral strategy (with weakened TVA expression) to overcome this shortcoming and revisit both global and local inputs to dopaminergic neurons of the ventral tegmental area. This study provides a useful tool for the field, along with significant new data, essentially correcting/updating a series of datasets from high profile papers originally mapping the inputs to DA neurons. This work can be strengthened in a few ways to enhance clarity and impact and can improve in addressing alternative, previous methods that may solve …
Reviewer #2 (Public Review):
This single-author study highlights an important caveat of currently prevalent monosynaptically-restricted rabies tracing methods in neuroscience, which complicate the interpretation of data on synaptic inputs surrounding injection sites. The author used a previously published, modified viral strategy (with weakened TVA expression) to overcome this shortcoming and revisit both global and local inputs to dopaminergic neurons of the ventral tegmental area. This study provides a useful tool for the field, along with significant new data, essentially correcting/updating a series of datasets from high profile papers originally mapping the inputs to DA neurons. This work can be strengthened in a few ways to enhance clarity and impact and can improve in addressing alternative, previous methods that may solve similar problems.
Related to Figure 1:
Previously published studies have attempted to address the problem of low background levels of TVA expression and non-Cre dependent labeling of inputs near injection sites. One solution to prevent this ectopic expression of TVA is to titrate concentration of helper virus for efficient labeling of starter cells while minimizing non-Cre mediated expression, e.g. Wickersham's group https://doi.org/10.3389/fnsyn.2020.00006. Overall, there is an omission of discussion of alternative, potentially far simpler solutions. For example, how do volume, titer, AAV serotype, promoter, and other batch effects could alter relative to AAV helper virus expression change the background level of non-Cre dependent TVA infection? Without varying these parameters extensively, especially given the diverse applications of this technology in hundreds of neuroscience labs, it is challenging to evaluate the depth of the problem with mapping local inputs.Related to Figure 2:
The author's use of dimensionality reduction to analyze the relationship across local and long-range inputs is compelling and offers new ways of dissecting large-scale monosynaptic rabies tracing data. The author used z-score of fractional counts for UMAP input data and this ultimately aligns to spatial location of input fibers within the VTA. Considering that the UMAP input data is fractional counts for the number of input cells, does the corresponding spatial location of fibers relate to the density of fibers or fibers that are contacting specific subsets of VTA neurons?Related to Figure 3:
The author utilizes his altered viral strategy to dissect local inhibition to VTA dopamine neurons. This method provides critical new quantitative information about sources of local inhibition within the VTA, but the information would be significantly strengthened in its impact if complemented by immunohistochemistry for specific cell type markers of GABAergic cell types. For example, the use of other probes beyond GAD with UMAP analyses might begin to answer whether subsets of DA neurons receive different quantity of inhibitory synaptic contacts or receive different types of inhibition from specific inhibitory cell types.Related to Figure 4:
Why is immunofluorescence used to study DA-DA connectivity as opposed to FISH as used for GABAergic input? They are essentially the same type of analyses but two different methodologies are used without justification. Why was this analysis restricted to only VTA (or neighboring) regions when other long-range Th+ DA neurons, such as those in the hypothalamus, might also make connections to VTA DA neurons? While the focus on local inputs makes sense in the context of the study and TVA spread, there is a missed opportunity to delve deeper and create a unique resource that contributes biological insight on TH+ neuronal network organization. -
Reviewer #3 (Public Review):
A number of prior studies have used variants of cell-type specific rabies viral tracing to examine the inputs to midbrain dopamine neurons. This method generally speaking requires cells to express both the TVA protein that allows transsynaptic viral jumps as well as infection with and expression of a rabies viral backbone expressing a reporter. Although a transsynaptic jump is relatively rare (estimated here and elsewhere to be ~10 presynaptic jumps per double-infected neuron), the very modest leak of in cell-type specific expression using the cre recombinase system can lead to small amounts of TVA expression in nominally non-cre expressing cells (in this case non-dopaminergic neurons) and some transsynaptic labeling as a result. In this study Beier takes advantage of a recently described point mutant of TVA …
Reviewer #3 (Public Review):
A number of prior studies have used variants of cell-type specific rabies viral tracing to examine the inputs to midbrain dopamine neurons. This method generally speaking requires cells to express both the TVA protein that allows transsynaptic viral jumps as well as infection with and expression of a rabies viral backbone expressing a reporter. Although a transsynaptic jump is relatively rare (estimated here and elsewhere to be ~10 presynaptic jumps per double-infected neuron), the very modest leak of in cell-type specific expression using the cre recombinase system can lead to small amounts of TVA expression in nominally non-cre expressing cells (in this case non-dopaminergic neurons) and some transsynaptic labeling as a result. In this study Beier takes advantage of a recently described point mutant of TVA that reduces efficiency of transsynaptic labeling and thus requires higher levels of TVA expression for effect labelling. As a result nominally non-cre expressing cells are even less likely to mediate transsynaptic labeling. Although the number of experiments is relatively small (3-4) there is quite a clear reduction in off-target infection using this strategy (very similar to background levels observed in mice with no cre expression). Beier identifies a particularly important use case for this approach - namely assessing inputs to dopaminergic neurons from non-dopaminergic neurons within the area of viral injection. Due to the relatively large fraction of non-dopaminergic neurons in the midbrain previous uses of rabies transsynaptic tracing methods can be overwhelmed by off-target expression. This is particularly relevant because it has long been known from detailed electron microscopy analysis and functional studies of synaptic input that dopamine neurons have substantial, predominant inhibitory inputs. However, inhibitory inputs are, in general, biased towards more anatomically local sources in general in the brain and also in the midbrain. Thus, prior work using similar tracing methods was biased towards long range excitatory inputs to midbrain dopamine neurons. Beier here provides an important, valuable, and complementary tracing dataset that likely more accurately captures the input to dopamine neurons locally and yet is also consistent with prior long range tracing data. The manuscript is relatively focused on comparing this work with prior anatomical work, especially that using rabies mediated transsynaptic tracing. This is understandable given its focus, however, a number of functional studies and other anatomical studies have made similar important points about the relevance of local connectivity and yet those studies were little discussed. The dataset is also derived from a small set of injections (4) and focused on a particular coordinate in lateral VTA with some labelling in medial SNc and more medial VTA. Thus some questions remain about how well these results are representative of the broader population of midbrain dopamine neurons and more subtle differences in local connectivity. The "convergence index" is similar here to studies with TVA (~7-8) presumably due to improved RABV efficiency that compensates for a partial LOF mutation in TVA. Nonetheless, the number of input cells labelled per postsynaptic dopamine neuron is still a very small fraction of total inputs raising some questions about the weather heterogeneity of input patterns is accurately assessed. Finally, potential tropism for specific input cell classes remains an unknown.
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