Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
The anatomical connectivity of the claustrum and the role of its output projections has, thus far, not been studied in detail. The aim of this study was to map the outputs of the endopiriform (EN) region of the claustrum complex, and understand their functional role. Here the authors have combined sophisticated intersectional viral tracing techniques, and ex vivo electrophysiology to map the neural circuitry of EN outputs to vCA1, and shown that optogenetic inhibition of the EN→vCA1 projection impairs both social and object recognition memory. Interestingly the authors find that the EN neurons target inhibitory interneurons providing a mechanism for feedforward inhibition of vCA1.
Strengths:
The strength of this study was the application of a multilevel analysis approach combining a number of state-of-the-art techniques to dissect the contribution of the EN→vCA1 to memory function.
Weaknesses:
Some authors would disagree that the vCA1 represents a 'node for recognition of familiarity' especially for object recognition although that is not to say that it might play some role in discrimination, as shown by the authors. I note however that the references provided in the Introduction, concerning the role of vCA1 in memory refer to anxiety, social memory, temporal order memory, and not novel object recognition memory. Given the additional projections to the piriform cortex shown in the results, I wonder to what extent the observations may be explained by odour recognition effects.
We have added references demonstrating that the ventral hippocampus contributes to object recognition memory in rodents (Broadbent NJ et al., Learn Mem 2010; Titulaer J et al., Front Behav Neurosci 2021).
The odor recognition effect is an interesting perspective that we have also considered. However, in our object recognition test, the same odor (70% EtOH) was used for both objects, yet the mice were able to discriminate between the familiar and novel objects. This suggests that the likelihood of the odor cue contributing to their performance in object discrimination test is low.
In addition, I wondered whether the impairments in discrimination following Chemogenetic inhibition of the EN→vCA1 were due to the subject treating the novel and familiar stimuli as either both novel- which might be observed as an increase in exploration, or both stimuli as familiar, with a decrease in overall exploration.
We thank the reviewer for rising this interesting point. We analyzed the total exploration time (i.e., time in interaction zones in familiar and novel) during social discrimination test. The data is added to Fig. S9. Total exploration time was not affected by CNO treatment. This indicates inhibition of ENvCA1-proj. neurons reduced interaction time with the novel conspecific and increased interaction time with the familiar conspecific. The subject mice seem to give even weight on familiar and novel stimuli.
Reviewer #2 (Public Review):
Summary:
Yamawaki et al., conducted a series of neuroanatomical tracing and whole-cell recording experiments to elucidate and characterise a relatively unknown pathway between the endopiriform (EN) and CA1 of the ventral hippocampus (vCA1) and to assess its functional role in social and object recognition using fibre photometry and dual vector chemogenetics. The main findings were that the EN sends robust projections to the vCA1 that colateralise to the prefrontal cortex, lateral entorhinal cortex, and piriform cortex, and these EN projection neurons terminate in the stratum lacunosum-moleculare (SLM) layer of distal vCA1, synapsing onto GABAergic neurons that span across the Pyramidal-Stratum Radiatum (SR) and SR-SML borders. It was also demonstrated that EN input disynaptically inhibits vCA1 pyramidal neurons. vCA1 projecting EN neurons receive afferent input from the piriform cortex, and from within EN. Finally, fibre photometry experiments revealed that vCA1 projecting EN neurons are most active when mice explore novel objects or conspecifics, and pathway-specific chemogenetic inhibition led to an impairment in the ability to discriminate between novel vs. familiar objects and conspecifics.
This is an interesting mechanistic study that provides valuable insights into the function and connectivity patterns of afferent input from the endopiriform to the CA1 subfield of the ventral hippocampus. The authors propose that the EN input to the vCA1 interneurons provides a feedforward inhibition mechanism by which novelty detection could be promoted. The experiments appear to be carefully conducted, and the methodological approaches used are sound. The conclusions of the paper are supported by the data presented on the whole.
We thank the reviewer for their positive comments on our work.
The authors used dual retrograde tracing and observed that the highest percentage (~30%) of vCA1 projecting EN cells also projected to the PFC. They then employed an intersectional approach to show the presence of collaterals in other cortical areas such as the entorhinal cortex and piriform cortex in addition to the PFC. However, they state that 'Projection to prefrontal cortex was sparse relative to other areas, as expected based on the retrograde labeling data' (referring to Figure 2K) and subsequently appear to dismiss the initial data set indicating strong axonal projections to the PFC.
Our interpretation is that 70% of the ENCA1-proj. population does not send collaterals to the PFC, suggesting that the PFC is not a major target for this population (unlike vCA1 where 100% of its population projects). This hypothesis is supported by our axon branching study, which showed lower axon density in the PFC compared to vCA1 (and other regions). We revised the text to 'much sparser relative to that of vCA1' (line 101) to facilitate a direct comparison with the retrograde and anterograde labeling study.
Since this is a relatively unknown connection, it would be helpful if some evidence/discussion is provided for whether the EN projects to other subfields (CA3, DG) of the ventral hippocampus. This is important, as the retrograde tracer injections depicted in Figure 1B clearly show a spread of the tracer to vCA3 and potentially vDG and it is not possible to ascertain the regional specificity of the pathway.
We addressed the potential caveat associated with the retrograde tracer injection, as mentioned by the reviewer, by performing intersectional axon branching analysis. This analysis demonstrated that EN axons are primarily located in the SLM of the distal CA1 subfield (Figs. 2, 3, S2). However, we occasionally observed very weak labeling in the CA3 or dentate gyrus. We modified our text (lines 106-108) and figure (Fig. S2D) to account for this.
The vCA1 projecting EN cells appear to originate from an extensive range along the AP axis. Is there a topographical organization of these neurons within the vCA1? A detailed mapping of this kind would be valuable.
This is an interesting question for future research. Our data show a non-uniform distribution of this cell type, suggesting the potential for topographic organization.
Given this extensive range in the location of vCA1 EN originating cells, how were the targets (along the AP axis) in EP selected for the calcium imaging?
Using our injection coordinates, ENvCA1-proj. neurons were consistently labeled at high density just posterior to the bregma (Fig. 1J). Therefore, we targeted this region for our imaging.
The vCA1 has extensive reciprocal connections with the piriform cortex as well, which is in close proximity to the EN. How certain are the authors that the chemogenetic targeting was specific to the EN-vCA1 connection?
We performed histology on every animal used in the behavioral study to examine the specificity of hM4D expression, and only included those with specific labeling in the EN.
Raw data for the sociability and discrimination indices should be provided so that the readers can gain further insight into the nature of the impairment.
The raw data for total interaction time during the social discrimination test has been added (Fig. S9F).
Line 222: It is unclear how locomotor activity informs anxiety in the behavioral tests.
The degree of exploratory behavior in a novel context is generally considered to infer anxiety levels in rodents. We have added a review paper (Ref 44, Prut, 2003) that discusses this point.
Figure 7 title; It is stated that activity of EN neurons 'predict' social/object discrimination performance. However, caution must be exercised with this interpretation as the correlational data are underpowered (n=5-8). Furthermore, the results show a significant correlation between calcium event ratios and the discrimination index in the social discrimination test but not the object discrimination test.
We added the sample size for EN calcium imaging during the object recognition memory test (Fig. 7G). The updated data indicate a significant correlation between EN activity and the object recognition index (N = 9, Pearson R = 0.8, p = 0.01).
We have changed the title of Figure 7 to 'Activity of ENvCA1-proj. neurons correlates with social/object discrimination performance’.
While both male and female mice were included in the anatomical tracing and recording experiments, only male mice were used for behavioral tests.
The female behavior was highly inconsistent in the control condition of our social recognition memory paradigm; therefore, we decided to conduct the study with males. We will design a new behavioral paradigm for future studies to address this challenge.
Reviewer #1 (Recommendations For The Authors):
(1) It is not clear how the relative number of vCA1 projecting neurons in Figure 1H was acquired, not enough detail is presented in the methods section. To what extent could these data have been affected by differences in the size or anatomical position of the injection site in vCA1, which judging from the example fluorescent image in Figure 1B also appears to include CA3.
We used AMaSiNe (Song et al. 2020) to semi-automatically quantify fluorescently labeled presynaptic neurons. This open-source software identifies the number and location of these cells across different regions based on the Allen Mouse Brain Common Framework. To control for transfection variability (e.g., due to slight differences in injection volume or site), we normalized the presynaptic cell count in each region by the total number of cells in regions of interest. We performed for N = 5 brain and found consistent trend as seen in Fig. 1H (grey lines).
We have added the detailed method of quantification in the Materials and Methods section (line 393).
(2) For a number of the results, the full statistical values are not presented in the Results section or figure legend.
We have included the full statistical values in the figure legends of the revised manuscript.
(3) It is not clear how much virus was injected in the different experiments (tract racing, electrophysiology, behaviour, etc.). The methods state 50-100ul, but there is no further detail in the results or figure legends.
We have included the injected volumes of the virus in the revised manuscript.
(4) Figure 2 mentions the CLA complex (line 702) but this is not defined in the text. Although the introduction does refer to the claustrum complex, there is no acronym.
We have corrected the manuscript accordingly.
(5) Line 131- 'we recorded from 3-4 GABAergic neurons' - presumably this is in each animal?
We recorded 3 to 4 GABAergic neurons sequentially from the same slice to compare input strength. We have edited the text to clarify this (line 134).
Reviewer #2 (Recommendations For The Authors):
Figure 3C: It is not clear what the dashed lines labelled proximal and distal represent.
It is the proximal and distal vCA1 regions where GFP signals were measured for Fig. 3D. We have modified the figure legend to clarify this (line 736).
Figure 5D: what do the different colors represent? Different colors for one brain?
I assume that the reviewer meant to refer to Fig. 4D instead of Fig. 5D. In Fig. 4D, one color indicates starter cells in one brain. To clarify this, we have edited the figure legend (line 748).
Figure S6E: The images are low resolution and it is hard to decipher the exact locations of labeled neurons. Please provide more guidance (e/g/. labeling areas of interest).
We have added reference lines and labels in Figure S6E.
Some details are missing: what was the volume of AAV injected for each site/experiment; how was CNO made, and where was it purchased from?
We have added this information (lines 330-331; 431-434).
