Long-range inhibitory axons from medial entorhinal cortex target lateral entorhinal neurons projecting to the hippocampal formation

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.

Author response:

Reviewer #1:

The issue on validation of injection sites and viral spread is an important one, and we are fully aware of the risks associated with an incomplete assessment. Note that in the supplementary material, section on ‘Brain area identification’ we write the following: ‘In all neuroanatomical tracing experiments, correct placement of tracer injections into the four different areas (MEC, PER, PIR and LEC) was carefully evaluated based on known cytoarchitectonic features (see below). Electrophysiological experiments were initiated after our neuroanatomical experiments had verified the correct surgery coordinates for interrogating pathways to LEC from MEC, PIR, PER and cLEC. In patch-clamp experiments, viral injections were considered to hit the intended target area whenever the axonal innervation patterns in LEC were consistent with the patterns obtained in our neuroanatomical tracing experiments. To ensure that our injections were placed in MEC, without unintended spread to LEC, we examined the innervation patterns in DG.

In agreement with the current understanding of entorhinal innervation of DG in rodents (Steward, 1976; van Groen et al., 2003), injections targeting MEC or LEC resulted in axonal labelling in the middle one-third or outer one-third of the molecular layer of DG, respectively. Cases where the injection had clearly spread to LEC, evident from the laminar distribution of labelling in DG and labelled cell bodies in LEC, were excluded from analysis.’

In our view this provides sufficient security that we did not by mistake included intrinsic LEC projections into our dataset. In the result section, we addressed this issue as well by stating that: ‘We carefully checked all sections at and close to the levels we used for our experiments and did not observe any virally labelled neurons in LEC.’ In case of electrophysiological experiments, one normally does not secure whole brain material to exclude viral spread, but since for each animal we did record from multiple adjacent thick slices and in none did we find indications of including LEC. Finally, we included an analysis of SST projections originating from LEC (suppl Figure 1). As can be seen from panel C the local SST axonal pattern in LEC is markedly different form that seen following an injection in MEC. We aim to provide additional supplementary detail of this and include that in the text of the revised version.

Reviewer #2:

The remark that the in vivo relevance of these connections remains to be determined is absolutely correct and in the discussion we only speculated on this, since we currently do not have functional data of sufficient quality to address this. However, in an earlier version of the paper, still accessible on bioRxiv (https://biorxiv.org/cgi/content/short/2022.11.29.518323v1), we did include data on changes in expression of the immediate early gene cFos in LEC layer IIa cells upon manipulation of the SST projections from MEC within the context of conspecific memory. These data resulted in a non-significant trend, but we do not have the time, nor the financial means to extent that dataset. Therefore we cannot revise the paper in this respect.