Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
The study addresses the organisation of synaptic connections from the medial to the lateral entorhinal cortex. Classic anatomical work has suggested these connections exist, but very little is known about their identity or functional impact. The manuscript argues that these projections are mediated by glutamatergic neurons, providing excitatory input from MEC to all layers of LEC, and by SST+ve interneurons sending inhibitory projections to L1 of LEC. This appears to be the most likely interpretation of the data, although in my opinion, more could be done to rule out the possible impact of the spread of the virus/tracer from the injection site.
While this concern might seem overly picky, the importance of this level of detail is nicely shown by the authors' previous work clarifying connectivity from postrhinal to entorhinal cortices through careful analysis of similar types of data (Doan et al. 2019). If additional analyses/data can address the concern here, then I think this will be an important set of fundamental results that will influence thinking about circuit mechanisms for spatial cognition and episodic memory. In particular, it will nicely add to an emerging view that MEC and LEC can interact directly, showing that the organisation of these interactions is asymmetric and identifying a potentially interesting long-range inhibitory pathway.
Reviewer #2 (Public review):
Summary:
The manuscript by Nilssen et al. presents a comprehensive study of the circuitry linking the medial and lateral entorhinal cortices (MEC and LEC). Using a combination of anatomical tracing, optogenetics, and in vitro electrophysiology, the authors convincingly demonstrate that the MEC sends both glutamatergic and long-range inhibitory SST+ GABAergic projections to the LEC, with distinct laminar and cell-type-specific targeting. Notably, they reveal that SST+ inhibitory projections selectively suppress the activity of layer IIa neurons, whereas excitatory inputs preferentially engage neurons in layers IIb and III, thereby differentially modulating hippocampal-projecting populations.
Strengths:
The experiments are carefully executed, the results are compelling, and the conclusions are well supported by the data. This work will be of broad interest to researchers studying memory circuits, cortical inhibition, and the organization of long-range connectivity.
Weaknesses:
Although the in vivo relevance of these connections remains to be determined, this is an important and timely contribution to our understanding of entorhinal-hippocampal interactions.
The request for validation of injection specificity and viral spread, as detailed in the comments and suggestions of the two reviewers has been provided in the revised version. We added supplementary figures 1,2 and 6 as well as an extra insert into the old supplementary figure 5, now supplementary figure 9.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Major Points:
(1) Interpretation of the retrograde labelling experiment in Figure 1A-C is challenging, as the spread of the tracer at the injection site is not shown. It's important to see the full dorsal-ventral extent of the injection site in order to establish that the labelling of neurons in MEC results from projections to LEC and not adjacent areas (including MEC).
As mentioned in our initial reply, we are fully aware of the risks associated with an incomplete assessment of injection sites and viral spread, so we provide a new Supplementary Fig. 1 showing 6 dorsoventral levels of the case shown in Fig. 1A. The injection site in case of FG often shows a core of damaged tissue with a halo of substantial unspecific fluorescence. Outside of the injection side, one only sees retrogradely labeled somata (often recognizable by FG signal clustered in lysosomes) and dendritic elements. As indicated in the legend, we report some tracer leakage along the needle track in temporal and perirhinal cortex, areas that receive only sparse MEC projections, but there is no apparent spread of the injection into MEC.
Numbers are also quite low. E.g., Figure 1A is N=1/2 for retrograde labelling experiments.
The reviewer would be correct if the experiments were meant to analyze MEC projections to LEC in full anatomical detail. This was not our intention (several papers addressed this pathway in detail), we merely aimed to distinguish between glutamatergic and potential GABAergic contributions to this pathway and to establish optimal coordinates in slices to prepare for the electrophysiological recording experiments. Two animals suffice for this purpose and using more would be against the aim of reducing the use of experimental animals as much as possible.
The rationale here for the use of AAV2-CAG-tdTomato as a retrograde tracer is unclear. My understanding is that this is more effective as an anterograde tracer. Some clarification and validation would be important.
The reviewer is correct that AAV2 is generally considered an effective anterograde tracer, but tracing the connectivity of entorhinal cortex with AAVs has been proven to be notoriously difficult, in particular retrograde tracing of inputs to layer II. In a neighboring lab in the centre, headed by Edvard and May-Britt Moser, it was established that AAV2 types show very efficient retrograde transport and that is why we decided to use the virus. Also, in our hands the virus showed excellent retrograde transport that served our purpose
(2) Interpretation of the anterograde experiments in Figure 1D-E would also benefit from showing evidence that the injection sites are restricted to MEC. It should be straightforward to make a supplemental figure showing labelling at all dorsoventral levels.
More careful analysis of the axon labelling in the dentate gyrus could also help make a case for the selectivity of the injection site for eGFP. In this case, only the intermediate portion of the molecular layer of the DG should be labelled. In the image shown, the labelled band is quite wide, but it's hard to tell if this reflects the plane of section or is because it also includes labelling in the outer molecular layer (which would be indicative of LEC expression).
We thank the reviewer for these two suggestions, and we have prepared a new Supplementary Fig. 2 in line with this.
Numbers are also on the low side for these experiments.
See our response above
(3) For optogenetic experiments in Figure 2, the selectivity of targeting of AAV to MEC is assessed through the specificity of labelling in the DG. This is great, but it's important to show that this specificity is maintained at all dorsoventral levels.
Higher resolution images of labelling in LEC could also be helpful. It's hard to tell from the images in 2A if labelling is axonal or is in the soma adjacent to the nuclear NeuN signal (which would indicate a lack of selectivity for MEC).
We thank the reviewer for these two suggestions and provide a new Supplementary Fig. 6, showing both the details of AAV1 being present only in neuropil in MEC not in somata as well as the specific labeling in the middle molecular layer of DG in detail. Including all dorsoventral levels would not provide additional information in view of the very well-established topographical organization of the entorhinal to dentate projection, reaching approximately 20 -25 % of the full long axis of DG (Van Groen et al., 2003)
In addition, we have again carefully screened all tissue from the electrophysiological experiments for possible leakage of virus from MEC to LEC. We decided to exclude recordings from one mouse, which had labelling in MEC that was close to the border with LEC. Neuron counts have therefore been adjusted (pages 7-9) and the example recording showing responses to TTX/4-AP exposure in Figure 2B has been exchanged.
(4) The analysis of excitatory and inhibitory opto-responses in Figure 2 is nice. It may be helpful to report quantification of the rise and decay kinetics of the synaptic currents. They appear much slower for the inhibitory input, which may be functionally important.
This would indeed be nice to add, but it would not significantly impact or change the main message of our study. Since the lab of the senior author (MPW) has been discontinued and the resources for conducting these analyses are not readily available anymore, we have found it difficult to comply with the reviewer’s request
(5) More direct evidence for SST axons projecting from MEC to LEC would strengthen the conclusions made. E.g., in experiments where the SST neurons are labelled, is it possible to follow the axons? Do they project as expected from the MEC to the LEC?
In our view the tracing data provide convincing evidence in support of a direct projection from MEC to LEC by SST neurons, as shown in horizontal brain sections where SST axons labelled in MEC of an SSTCre mouse projects within Layer I from the site of origin in MEC to Layer I of MEC (Supplementary Figure 3). Similar visualizations were not possible to obtain in our electrophysiological experiments where semicoronal slices were used. This cutting angle has been shown to be optimal to preserve most of the axon and the dendritic tree of LEC neurons (Tahvildari and Alonso, 2005; Canto and Witter 2012), but does not maintain the projection from MEC to LEC.
Minor Points:
(1) "These layers are heavily innervated by medial entorhinal axons (Figure 1F...". I don't see a 1F.
This has been corrected; should have been Figure 1E.
(2) Methods should report series resistance values for patch-clamp experiments (range and mean).
Fully agree and this information has now been added on page 22 of the manuscript:
Under Voltage clamp: ‘Recordings with series resistance ≤ 25 MΩ were accepted, with an average of 16.2 MΩ for voltage clamp recorded neurons (range, 4.9 – 24.9 MΩ).’
Under Current clamp: ‘All recordings (series resistance: 18.9 MΩ, 5.0 – 66.7 MΩ; mean, range) were included for analysis.’
Reviewer #2 (Recommendations for the authors):
(1) Please specify in the figure or, alternatively, in the figure legend which virus was used in each group shown in Figures 2H and 2I. This is somewhat confusing, since Figure 2E illustrates a specific combination of viruses and mouse lines that only corresponds to part of Figure 2H. While this information is provided in the text, including it directly in the figure would help the reader.
We thank the reviewer for this excellent suggestion, and we have implemented this in the new version of figure 2.
(2) In Figure 4, regarding the inputs from PIR, cLEC, and PER to LEC, the inhibitory components recruited by each input were not examined as thoroughly as for the MEC inputs. In fact, some inhibitory interneurons were double-labeled in the GAD67 mice (Figure 4B), which could also influence the responses of LEC neurons, especially for PER inputs. Recordings in Figure 4C appear to have been obtained near the reversal potential for inhibition, which may have prevented the observation of inhibitory effects. The authors could discuss this point in the Results.
The reviewer is correct and this issue is now addressed in the relevant section in the results (page 11):
‘It should be noted, however, that it is possible that inhibitory effects could have been masked in some recordings, due to the resting membrane potential in our recordings being close to the theoretical chloride equilibrium potential. This could be particularly relevant for the inputs from PER, an area where we found LEC-projecting GABAergic neurons (Fig. 4B) and which is known to provide long-distance inhibition to LEC (Pinto et al., 2006; Apergis- Schoute et al., 2007).’
(2) A diagram summarizing the known connections among MEC, LEC, and the hippocampal formation, highlighting the relevant cell types, layers, and the new connections identified in this study, would be a valuable addition, perhaps as a supplementary figure.
We appreciate the suggestion, though find a full summary of known connectivity a bit overdone. Instead, we included a new figure 6 that summarizes the main new findings of the paper in the context of LEC projections to the hippocampal formation.
(3) Although the main focus is on MEC-LEC connectivity, the experiments examining interactions with other cortical areas and converging inputs would benefit from a discussion of how MEC-driven inhibition of LEC might influence those inputs and shape the resulting output to the hippocampus. Including a short paragraph addressing this in the Discussion section would strengthen the manuscript.
Excellent suggestion although we did speculate briefly in the result section on the possible effect. We have added a short paragraph in the discussion (page 15), reiterating the part in the results (page 13, last paragraph of results). We also briefly discussed the potential functional relevance of the suppression of the pathway from layer IIa to DG-CA3/CA2 versus the facilitation of activity in the pathway from layers IIb/III to CA1 and subiculum (last section of the discussion).
(4) Lastly, it would be interesting to know what the main source of activation is for the SST long-range LEC projecting neurons. Are these neurons recruited in a feedback manner by the activity of MEC excitatory cells? I realize this question is beyond the scope of the present study, but if the authors have any data or insights related to this point, including a brief discussion would be valuable.
This is an interesting thought, and we have included a new supplementary figure (supplementary Figure 5) showing data from experiments mapping monosynaptic inputs to MEC SST neurons using rabies virus. Although it was not possible to target only MEC SST neurons that project to LEC, the data show which are the main extrinsic inputs to the population of MEC SST neurons, most likely including those that project to LEC.
