No evidence from complementary data sources of a direct glutamatergic projection from the mouse anterior cingulate area to the hippocampal formation
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Evaluation Summary:
In this study, the authors attempted to further examine the existence of a potential direct projection from the anterior cingulate cortex to the hippocampus which has important functional implication but is currently supported by only one major publication in the literature. They used distinct anterograde et retrograde viral tracing strategies and analyzed the data available in the Allen atlas but found no evidence in support of the existence of this connection.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
The connectivity and interplay between the prefrontal cortex and hippocampus underpin various key cognitive processes, with changes in these interactions being implicated in both neurodevelopmental and neurodegenerative conditions. Understanding the precise cellular connections through which this circuit is organised is, therefore, vital for understanding these same processes. Overturning earlier findings, a recent study described a novel excitatory projection from anterior cingulate area to dorsal hippocampus. We sought to validate this unexpected finding using multiple, complementary methods: anterograde and retrograde anatomical tracing, using anterograde and retrograde adeno-associated viral vectors, monosynaptic rabies tracing, and the Fast Blue classical tracer. Additionally, an extensive data search of the Allen Projection Brain Atlas database was conducted to find the stated projection within any of the deposited anatomical studies as an independent verification of our own results. However, we failed to find any evidence of a direct, monosynaptic glutamatergic projection from mouse anterior cingulate cortex to the hippocampus proper.
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Author Response:
We thank the reviewers for their positive, constructive feedback. However, we would like to take the opportunity now to briefly address one comment from Reviewer #1:
In addition, only pyramidal cells were considered here but long-range projecting GABA neurons have been recently reported in the prelimbic cortex (preprint from Malik et al.,
- which suggests that this could be a possibility in the anterior cingulate cortex as well. So even if the starter cells identified in the present study were sufficient to detect other inputs to the hippocampus, I am not sure it is sufficient to completely rule out the existence of a scarce and potentially inhibitory projection to the hippocampus.
We are quantifying the number of starter cells in our rabies experiment and will report these in the revised manuscript. However, the …
Author Response:
We thank the reviewers for their positive, constructive feedback. However, we would like to take the opportunity now to briefly address one comment from Reviewer #1:
In addition, only pyramidal cells were considered here but long-range projecting GABA neurons have been recently reported in the prelimbic cortex (preprint from Malik et al.,
- which suggests that this could be a possibility in the anterior cingulate cortex as well. So even if the starter cells identified in the present study were sufficient to detect other inputs to the hippocampus, I am not sure it is sufficient to completely rule out the existence of a scarce and potentially inhibitory projection to the hippocampus.
We are quantifying the number of starter cells in our rabies experiment and will report these in the revised manuscript. However, the preprint from Malik et al. reported that optogenetic stimulation of the inhibitory projection from PFC did not evoke IPSCs in CA1 pyramidal cells (0/38 neurons tested). As such, if a similar inhibitory projection from ACC does indeed exist (it appears that it does, based on informal conversations with other researchers on social media) then we would not detect it in our monosynaptic rabies experiment, as we used the Emx1-cre mouse line to specifically map presynaptic inputs onto hippocampal glutamatergic neurons. Indeed, the preprint from Malik and colleagues provides evidence of a direct route by which prefrontal cortex can drive feedforward inhibition in CA1 while avoiding pyramidal cells. How the function of this projection differs from the indirect route via nucleus reuniens which, as we recently reported in another preprint (Andrianova et al., 2021, bioRxiv 2021.09.30.462517), also appears selective for interneurons over pyramidal cells, provides an exciting avenue for future research.
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Evaluation Summary:
In this study, the authors attempted to further examine the existence of a potential direct projection from the anterior cingulate cortex to the hippocampus which has important functional implication but is currently supported by only one major publication in the literature. They used distinct anterograde et retrograde viral tracing strategies and analyzed the data available in the Allen atlas but found no evidence in support of the existence of this connection.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The prefrontal cortex and the hippocampus are key brain regions for a number of major cognitive processes. While the direct neural routes connecting these brain regions have been well documented in the hippocampus-to-prefrontal direction, top-down messages from the prefrontal cortex to the hippocampus were thought to be essentially indirectly conveyed. The discovery of a direct prefrontal cortex-to-hippocampus pathways was thus a major and surprising discovery (Rajasethupath et al., Nature 2015). This has not really been confirmed so far by any subsequent work. The present study attempted to examine this issue by relying on multiple viral tracing strategies, both retrograde and anterograde, and also by analyzing data currently available in the Allen brain atlas. No evidence in support of the existence of …
Reviewer #1 (Public Review):
The prefrontal cortex and the hippocampus are key brain regions for a number of major cognitive processes. While the direct neural routes connecting these brain regions have been well documented in the hippocampus-to-prefrontal direction, top-down messages from the prefrontal cortex to the hippocampus were thought to be essentially indirectly conveyed. The discovery of a direct prefrontal cortex-to-hippocampus pathways was thus a major and surprising discovery (Rajasethupath et al., Nature 2015). This has not really been confirmed so far by any subsequent work. The present study attempted to examine this issue by relying on multiple viral tracing strategies, both retrograde and anterograde, and also by analyzing data currently available in the Allen brain atlas. No evidence in support of the existence of this projection was found which raises important methodological issues. The work conducted here is rigorous and the authors clearly invested a fair amount of efforts to find out about this projection. Off course, a formal demonstration that something does not exist is logically impossible but the present attempt has a clear value.
As strengths, they authors relied on multiple and complementary viral approaches to examine the extent to which the anterior cingulate cortex may directly contact the hippocampus, using both a rabies-based approach (as in the original paper) and retroAAVs for the retrograde strategy and more standard AAVs for the anterograde work. The fact that all these approaches were effective and consistent in reporting known direct hippocampal projections from multiple brain regions (other than the ACC) demonstrate that the present work has value. The confirmation from data mining in the Allen brain atlas adds further to this.
Regarding potential weaknesses, I was a bit concerned by the low number of starter cells apparent in figure C (rabies-based approach). If very few are actually encountered, it somehow undermines the approach. A more complete description of the number of starter cells considered could clarify this issue. In addition, only pyramidal cells were considered here but long-range projecting GABA neurons have been recently reported in the prelimbic cortex (preprint from Malik et al., 2021) which suggests that this could be a possibility in the anterior cingulate cortex as well. So even if the starter cells identified in the present study were sufficient to detect other inputs to the hippocampus, I am not sure it is sufficient to completely rule out the existence of a scarce and potentially inhibitory projection to the hippocampus. I was also wondering whether the use of more classic but well established retrograde tracers (e.g. Fluorogold, Ctb) may have been useful here as viral vectors often come with their own limitations and specific tropisms and the particular retrograde viral vector used here was found to only bring quite disappointing findings (basically "off-target" labeling). Taken together, these two issues thus leave the entire retrograde approach with some possible flaws. I think it would be important that the authors clarify this by further discussion and/or new data/analyses.
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Reviewer #2 (Public Review):
The study by Andrianova et al. on the existence or non-existence of projections from the anterior cingulate area to the hippocampus provides a confirmation of the non-existence of this direct projection. Earlier data had not shown the existence of this connection; this was confirmed by the current experiments. The provided data are supporting the claim of the manuscript. The important difference between their data and the earlier "suggested" connection is a more careful analysis of possible labeling in neighboring brain areas which can easily lead to incorrect conclusions. Even so, it should be pointed out that proving something does not exist is harder then showing something could exist. Furthermore, they do point out that anterograde/retrograde tracers do not work exactly the same. Multiple tracers should …
Reviewer #2 (Public Review):
The study by Andrianova et al. on the existence or non-existence of projections from the anterior cingulate area to the hippocampus provides a confirmation of the non-existence of this direct projection. Earlier data had not shown the existence of this connection; this was confirmed by the current experiments. The provided data are supporting the claim of the manuscript. The important difference between their data and the earlier "suggested" connection is a more careful analysis of possible labeling in neighboring brain areas which can easily lead to incorrect conclusions. Even so, it should be pointed out that proving something does not exist is harder then showing something could exist. Furthermore, they do point out that anterograde/retrograde tracers do not work exactly the same. Multiple tracers should always be used to confirm data. Finally, the putative direct connection was shown not to exist; this is important for modeling the flow of data between the hippocampus and anterior cingulate area.
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