New insights into anatomical connectivity along the anterior–posterior axis of the human hippocampus using in vivo quantitative fibre tracking
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Evaluation Summary:
The work presented herein presents a novel method to characterize hippocampal-cortical anatomical network connectivity. These important results have the potential to generate new hypotheses and influence future queries into the hippocampal-cortical 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 #2 agreed to share their name with the authors.)
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Abstract
The hippocampus supports multiple cognitive functions including episodic memory. Recent work has highlighted functional differences along the anterior–posterior axis of the human hippocampus, but the neuroanatomical underpinnings of these differences remain unclear. We leveraged track-density imaging to systematically examine anatomical connectivity between the cortical mantle and the anterior–posterior axis of the in vivo human hippocampus. We first identified the most highly connected cortical areas and detailed the degree to which they preferentially connect along the anterior–posterior axis of the hippocampus. Then, using a tractography pipeline specifically tailored to measure the location and density of streamline endpoints within the hippocampus, we characterised where these cortical areas preferentially connect within the hippocampus. Our results provide new and detailed insights into how specific regions along the anterior–posterior axis of the hippocampus are associated with different cortical inputs/outputs and provide evidence that both gradients and circumscribed areas of dense extrinsic anatomical connectivity exist within the human hippocampus. These findings inform conceptual debates in the field and emphasise the importance of considering the hippocampus as a heterogeneous structure. Overall, our results represent a major advance in our ability to map the anatomical connectivity of the human hippocampus in vivo and inform our understanding of the neural architecture of hippocampal-dependent memory systems in the human brain.
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Author Response
Reviewer #1 (Public Review):
- The connectivity patterns along the anterior-posterior hippocampal axis broadly follow an anterior-posterior cortical bias, such that posterior regions, e.g. the visual cortex, are preferentially connected to the hippocampal tail, and anterior regions, e.g. the temporal pole, are preferentially connected to the hippocampal head. The authors focus on the twenty regions with the highest connectivity profiles, which appears to capture the majority of all connections. However, some of the present structural connectivity patterns differ in interesting ways from previously described cortical networks reported in resting-state fMRI studies. Most notably, the medial PFC and orbitofrontal regions combined account for less than 1% of all connections in the present investigation (Table S1 & S2). This …
Author Response
Reviewer #1 (Public Review):
- The connectivity patterns along the anterior-posterior hippocampal axis broadly follow an anterior-posterior cortical bias, such that posterior regions, e.g. the visual cortex, are preferentially connected to the hippocampal tail, and anterior regions, e.g. the temporal pole, are preferentially connected to the hippocampal head. The authors focus on the twenty regions with the highest connectivity profiles, which appears to capture the majority of all connections. However, some of the present structural connectivity patterns differ in interesting ways from previously described cortical networks reported in resting-state fMRI studies. Most notably, the medial PFC and orbitofrontal regions combined account for less than 1% of all connections in the present investigation (Table S1 & S2). This is an interesting contrast to functional investigations which tend to find that these regions cluster with the aHPC (e.g., Adnan et al. 2016 Brain Struct Func; Barnett et al. 2021 PLoS Biol; Robinson et al. 2016 NeuroImage). In contrast, the present DWI results suggesting preferential pHPC-medial parietal connectivity dovetail with those observed in fMRI studies. It seems important to discuss why these differences may arise: whether this is a differentiation between structural and functional networks, or whether this is due to a difference in methods.
We thank Reviewer 1 for making this important point and agree that these observations are deserving of further expansion. We have now included additional text where we place the surprising observation of sparse connectivity between PFC regions and the hippocampus more firmly in the context of recent evidence and argue that these observations suggest a potential differentiation between structural and functional networks.
We have included the following text in the discussion (pp. 16-17, lines 439-457);
“While many of our observed anatomical connections dovetail nicely with known functional associations, patterns of anatomical connectivity strength did not always mirror well characterised functional associations between the hippocampus and cortical areas. For example, a surprising observation from our study was that only weak patterns of anatomical connectivity were observed between the hippocampus and the ventromedial prefrontal cortex (vmPFC) and other frontal cortical areas. This lies in contrast to well documented functional associations between these regions (46-48). Our observation, however, supports a growing body of evidence that direct anatomical connectivity between the hippocampus and areas of the PFC may be surprisingly sparse in the human brain. For example, Rosen and Halgren (49) recently reported that long range connections between the hippocampus and functionally related frontal cortical areas may constitute fewer than 10 axons/mm2 and more broadly observed that axon density between spatially distant but functionally associated brain areas may be much lower than previously thought. Our observation of sparse anatomical connectivity between the hippocampus and PFC mirrors this recent work and suggests a potential differentiation between structural and functional networks as they relate to the hippocampus. It remains possible, however, that methodological factors may contribute to these differences. We return to this point later in the discussion. A future dedicated study aimed at assessing whether the well characterised functional associations between the hippocampus and vmPFC are driven by sparse direct connections or primarily by intermediary structures is necessary to address this issue in an appropriate level of detail.”
- While the analytic pipeline is described in sufficient detail in the Methods, it is somewhat unclear to a non-DWI expert what the major methodological advance is over prior approaches. The authors refer to a tailored processing pipeline and 'an advance in the ability to map the anatomical connectivity (p. 5), but it's not immediately clear what these entail. It would be useful to highlight the key methodological differences or advances in the Introduction to help with the interpretation of the similarities and differences with previous connectivity findings.
We have now included a brief description in the Introduction highlighting the key methodological advances used in the current study.
We have included the following text in the Introduction (pp. 4-5, lines 130-144);
“In typical fibre-tracking studies, we cannot reliably ascertain where streamlines would naturally terminate, as they have been found to also display unrealistic terminations, such as in the middle of white matter or in cerebrospinal fluid (39). While methods have been proposed to ensure more meaningful terminations (40), for example, with terminations forced at the grey matter-white matter interface (gmwmi), this approach is still not appropriate for characterising terminations within complex structures like the hippocampus. A key methodological advance of our approach was to remove portions of the gmwmi inferior to the hippocampus (where white matter fibres are known to enter/leave the hippocampus). This allowed streamlines to permeate the hippocampus in a biologically plausible manner. Importantly, we combined this with a tailored processing pipeline that allowed us to follow the course of streamlines within the hippocampus and identify their ‘natural’ termination points. These simple but effective methodological advances allowed us to map the spatial distribution of streamline ‘endpoints’ within the hippocampus. We further combined this approach with state-of-the-art tractography methods that incorporate anatomical information (40) and assign weights to each streamline (41) to achieve quantitative connectivity results that more faithfully reflect the biological accuracy of the connection’s strength (39).”
- Related to the point above, it was a bit unclear to me how the present connections map onto canonical white matter tracts. In Fig., 4A, the tracts are shown for a single participant, but it would be helpful to map or quantify know how many of the connections for a given hippocampal subregion are associated with a given tract to provide a link to prior work or clarify the approach. A fairly large body of prior research on hippocampal white matter connectivity has focused on the fornix, but it's a little difficult to align these prior findings with the connectivity density results in the current paper.
We thank Reviewer 1 for this comment and agree this would be an interesting avenue to pursue. However, the reliable segmentation of white matter fibre bundles is currently an area of contention in the DWI community. This pervasive and problematic issue was highlighted in a recently published large multi-site study that revealed a high degree of variability in how white matter bundles are defined, even from the same set of whole-brain streamlines (Schilling et al., 2021, Neuroimage. Nov; 243:118502. https://pubmed.ncbi.nlm.nih.gov/34433094/). This means that, even if we were to choose a particular method to segment white matter bundles, our results would not be readily translatable to those reported in previous DWI studies. This significantly limits meaningful comparison and/or interpretation. Indeed, such an approach may paradoxically take away from the detailed characterisations we have achieved in the current study. As highlighted in that study, it is now paramount that consensus is reached in this field to define criteria to reliably and reproducibly define white matter fibre bundles. Once that is achieved, we plan to conduct a follow-up study to characterise this in more detail, with bundles that will be able to be reliably reproduced by others.
- Finally, on a more speculative note: based on the endpoint density maps, there seems to be a lot of overlap between the EDMs associated with different cortical regions (which makes sense given the subregion results). Does this effectively mean that the same endpoints may be equally connected with multiple different cortical regions? Part of the answer can be found in Fig. 3D showing the combined EDM for three different regions, but how spatially unique is each endpoint? This is likely not a feasible question to address analytically but it might be helpful to provide some more context for what these maps represent and how they might relate to differences across individuals.
The primary aim of the current analysis was to characterise broad patterns of endpoint density captured by our averaged group level analysis. However, Reviewer 1 is astute in assuming that, although there is overlap in the group averaged endpoint density maps (EDMs) associated with different cortical areas, at the single participant level, there are both overlaps and spatial uniqueness in the location of individual endpoints. For example, while group level analysis revealed that area V1 and area V2 showed preferential connectivity with overlapping regions of the posterior medial hippocampus, when visualising individual endpoints associated with each of these areas at the single participant level, we can see that some endpoints overlap while others display spatially unique patterns (see image below). Although a more in-depth analysis of individual variability in these patterns was beyond the scope of this investigation (as noted on Page14; Lines 379-381), we agree with Reviewer 1 that this is an important point to note in the manuscript. We have, therefore, included additional text touching on this and have included a new Supplementary Figure (Page 42; also see below) to emphasise that, at the single participant level, different cortical areas display both overlapping and spatially unique endpoints within specific regions of the hippocampus (using areas V1 and V2 as an example).
We have included the following text in the Results section (pp. 14, lines 370-379);
“Finally, while we observed clear overlaps in the group averaged EDMs associated with specific cortical areas, a closer inspection of individual endpoints at the single participant level revealed that endpoints associated with different cortical areas displayed both overlapping and spatially unique characteristics within these areas of overlap. For example, at the group level, areas V1 and V2 showed preferential connectivity with overlapping regions of the posterior medial hippocampus (see Supplementary Figure S5) while, at the single participant level, individual endpoints associated with each of these areas display both overlapping and spatially unique patterns (see Supplementary Figure S6). This suggests that, while specific cortical areas display overlapping patterns of connectivity within specific regions of the hippocampus, subtle differences in how these cortical regions connect within these areas of overlap likely exist.”
Reviewer #2 (Public Review):
Dalton and colleagues present an interesting and timely manuscript on diffusion weighted imaging analysis of human hippocampal connectivity. The focus is on connectivity differences along the hippocampal long axis, which in principle would provide important insights into the neuroanatomical underpinnings of functional long axis differences in the human brain. In keeping with current models of long-axis organisation, connectivity profiles show both discrete areas of higher connectivity in long axis portions, as well as an anterior-to-posterior gradient of increasing connectivity. Endpoint density mapping provided a finer grained analysis, by allowing visualisation of the spatial distribution of hippocampal endpoint density associated with each cortical area. This is particularly interesting in terms of the medial-lateral distribution with hippocampal head, body and tail. Specific areas map to precise hippocampal loci, and some hippocampal loci receive inputs from multiple cortical areas.
This work is well-motivated, well-written and interesting. The authors have capitalised on existing data from the Human Connectome Project. I particularly like the way the authors try to link their findings to human histological data, and to previous NHP tracing results.
Many thanks.
- There are some important surprises in the results, particularly the relatively strong connectivity between hippocampus and early visual areas (including V1) and low connectivity with areas highly relevant from functional perspectives, such as the medial prefrontal cortex (rank order by strength of connectivity 7th and 78th of all cortical structures, respectively). This raises a concern that the fibre tracking method may be joining hippocampal connections with other tracts. In particular, given the anatomical proximity of the lateral geniculate nucleus to the body and tail of the hippocampus, the reported V1 connectivity potentially reflects a fusion of tracked fibres with the optic radiation. In visualizing the putative posterior hippocampus-to-V1 projection (Figure 4B, turquoise), the tract does indeed resemble the optic radiation topography. Although care was taken to minimise the hippocampus mask 'spilling' into adjacent white matter, this was done with focus on the hippocampal inferior margin, whereas the different components of the optic radiation lie lateral and superior to the hippocampus.
We agree with Reviewer 2 that our observations relating to area V1 could be the result of limitations inherent to current tracking methodology. Indeed, probabilistic tracking can result in tracks mistakenly ‘jumping’ between fibre bundles. Unfortunately, primarily due to limitations in image resolution, we do not believe that we can categorically rule this possibility out in the current dataset beyond the measures we have already taken in our analysis pipeline. We have now included additional text in the Discussion acknowledging and emphasising this possible limitation of our study.
We have included the following text in the Discussion section (Page 25; Lines 694-699);
“Also, we cannot rule out that some connections observed in the current study may result from limitations inherent to current probabilistic fibre-tracking methods whereby tracks can mistakenly ‘jump’ between fibre bundles (e.g. for connections between the posterior medial hippocampus and area V1 due to the proximity to the optic radiation), especially in “bottleneck” areas. Again, future work using higher resolution data may allow more targeted investigations necessary to confirm or refute the patterns we observed here.”
Beyond the possibility of tracks jumping between fibre bundles, we feel it is important to emphasise that an integral part of our analysis was the detailed attention we took to minimise mask ‘spillage’ of the entire hippocampus mask. It is not the case that we primarily focussed on inferior portions of the hippocampus as stated by Reviewer 2. Equal focus was paid to medial, lateral and superior portions of the mask which lie adjacent to visual thalamic nuclei, the optic radiation posteriorly and a number of other structures. We can see that our description relating to this lacked the necessary detail to convey this important point clearly and we apologise for the confusion. We have, therefore, included additional text in the Methods section clarifying this further.
We have included the following text in the Methods section (Page 26; Lines 751-755);
“We took particular care to ensure that all boundaries of the hippocampus mask (including inferior, superior, medial and lateral aspects) did not encroach into adjacent white or grey matter structures (e.g., amygdala, thalamic nuclei). This minimised the potential fusion of white matter tracts associated with other areas with our hippocampus mask.”
These points notwithstanding, our results support recently observed structural and functional associations between the posterior hippocampus and early visual processing areas. We agree that these findings are potentially of great conceptual importance for how we think about the hippocampus and its connectivity with primary sensory cortices in the human brain and we have now included a brief comment relating to this in the Discussion.
We have included the following text in the Discussion (Page 23-24; Lines 638-644);
“However, this observation supports recent reports of similar patterns of anatomical connectivity as measured by DWI in the human brain (38) and functional associations between these areas (43, 60). Collectively, these findings are potentially of great conceptual importance for how we think about the hippocampus and its connectivity with early sensory cortices in the human brain and open new avenues to probe the degree to which these regions may interact to support visuospatial cognitive functions such as episodic memory, mental imagery and imagination.”
- A second concern pertains to the location of endpoint densities within the hippocampus from the cortical mantle. These are almost entirely in CA1/subiculum/presubiculum. It is, however, puzzling why, in Supp Figure 2, the hippocampal endpoints for entorhinal projections is really quite similar to what is observed for other cortical projections (e.g., those from area TF). One would expect more endpoint density in the superior portions of the hippocampal cross section in head and body, in keeping with DG/CA3 termination. I note that streamlines were permitted to move within the hippocampus, but the highest density of endpoints is still around the margins.
We agree with Reviewer 2 that, in relation to the entorhinal cortex, we would expect to see more endpoint density in areas aligning with the dentate gyrus (DG) and CA3 regions of the hippocampus. We noted in the discussion that “Despite the high-quality HCP data used in this study, limitations in spatial resolution likely restrict our ability to track particularly convoluted white-matter pathways within the hippocampus and our results should be interpreted with this in mind”. We believe that this limitation applies to pathways between the entorhinal cortex and DG/CA3. We have now included additional text specifically noting that this limitation likely affects our ability to track streamlines as they relate to DG/CA3. A targeted investigation of this effect using higher resolution diffusion MRI data may help address this issue, and this will be the subject of future work.
We have included the following text in the Discussion (Page 25; Lines 690-693);
“Indeed, this may explain the surprising lack of endpoint density observed in the DG/CA4-CA3 regions of the hippocampus where we would expect to see high endpoint density associated with, for example, the entorhinal cortex which is known to project to these regions. Future dedicated studies using higher resolution data are needed to assess these pathways in greater detail.”
- On a related point, the use of "medial" and "lateral" hippocampus can be confusing. In the head, CA2/3 is medial to CA1, but so are subicular subareas, just that the latter are inferior.”
We agree that applying the terms ‘medial’ and ‘lateral’ to our three-dimensional representations can lead to some ambiguities and confusion. We have included a new description defining our use of these terms in the Results section.
We have included the following text in the Results section (Page 10; Lines 268-273).
“In relation to nomenclature, our use of the term ‘medial’ hippocampus refers to inferior portions of the hippocampus aligning with the distal subiculum, presubiculum and parasubiculum. Our use of the term ‘lateral’ hippocampus refers to inferior portions of the hippocampus aligning with the proximal subiculum and CA1. In instances that we refer to portions of the hippocampus that align with the DG or CA3/2 we state these regions explicitly by name”.
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Evaluation Summary:
The work presented herein presents a novel method to characterize hippocampal-cortical anatomical network connectivity. These important results have the potential to generate new hypotheses and influence future queries into the hippocampal-cortical 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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript presents novel results which suggest that networks of cortical regions show different patterns of structural connectivity with hippocampal subregions. The results build on prior work, but also provide a spatially precise characterization of whole-brain structural connectivity patterns along the anterior-posterior hippocampal gradient. The paper is well-motivated and well-written. The authors discuss their findings in the context of previous investigations in non-human primates, and draw a number of parallels between these bodies of evidence. However, there were also some interesting differences between the connectivity patterns uncovered in resting-state fMRI and those identified using the present approach. It would also be helpful to highlight the key methodological advances or differences …
Reviewer #1 (Public Review):
This manuscript presents novel results which suggest that networks of cortical regions show different patterns of structural connectivity with hippocampal subregions. The results build on prior work, but also provide a spatially precise characterization of whole-brain structural connectivity patterns along the anterior-posterior hippocampal gradient. The paper is well-motivated and well-written. The authors discuss their findings in the context of previous investigations in non-human primates, and draw a number of parallels between these bodies of evidence. However, there were also some interesting differences between the connectivity patterns uncovered in resting-state fMRI and those identified using the present approach. It would also be helpful to highlight the key methodological advances or differences compared to prior work to contextualize the present findings.
1. The connectivity patterns along the anterior-posterior hippocampal axis broadly follow an anterior-posterior cortical bias, such that posterior regions, e.g. the visual cortex, are preferentially connected to the hippocampal tail, and anterior regions, e.g. the temporal pole, are preferentially connected to the hippocampal head. The authors focus on the twenty regions with the highest connectivity profiles, which appears to capture the majority of all connections. However, some of the present structural connectivity patterns differ in interesting ways from previously described cortical networks reported in resting-state fMRI studies. Most notably, the medial PFC and orbitofrontal regions combined account for less than 1% of all connections in the present investigation (Table S1 & S2). This is an interesting contrast to functional investigations which tend to find that these regions cluster with the aHPC (e.g., Adnan et al. 2016 Brain Struct Func; Barnett et al. 2021 PLoS Biol; Robinson et al. 2016 NeuroImage). In contrast, the present DWI results suggesting preferential pHPC-medial parietal connectivity dovetail with those observed in fMRI studies. It seems important to discuss why these differences may arise: whether this is a differentiation between structural and functional networks, or whether this is due to a difference in methods.
2. While the analytic pipeline is described in sufficient detail in the Methods, it is somewhat unclear to a non-DWI expert what the major methodological advance is over prior approaches. The authors refer to a tailored processing pipeline and 'an advance in the ability to map the anatomical connectivity (p. 5), but it's not immediately clear what these entail. It would be useful to highlight the key methodological differences or advances in the Introduction to help with the interpretation of the similarities and differences with previous connectivity findings.
3. Related to the point above, it was a bit unclear to me how the present connections map onto canonical white matter tracts. In Fig., 4A, the tracts are shown for a single participant, but it would be helpful to map or quantify know how many of the connections for a given hippocampal subregion are associated with a given tract to provide a link to prior work or clarify the approach. A fairly large body of prior research on hippocampal white matter connectivity has focused on the fornix, but it's a little difficult to align these prior findings with the connectivity density results in the current paper.
4. Finally, on a more speculative note: based on the endpoint density maps, there seems to be a lot of overlap between the EDMs associated with different cortical regions (which makes sense given the subregion results). Does this effectively mean that the same endpoints may be equally connected with multiple different cortical regions? Part of the answer can be found in Fig. 3D showing the combined EDM for three different regions, but how spatially unique is each endpoint? This is likely not a feasible question to address analytically but it might be helpful to provide some more context for what these maps represent and how they might relate to differences across individuals.
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Reviewer #2 (Public Review):
Dalton and colleagues present an interesting and timely manuscript on diffusion weighted imaging analysis of human hippocampal connectivity. The focus is on connectivity differences along the hippocampal long axis, which in principle would provide important insights into the neuroanatomical underpinnings of functional long axis differences in the human brain. In keeping with current models of long-axis organisation, connectivity profiles show both discrete areas of higher connectivity in long axis portions, as well as an anterior-to-posterior gradient of increasing connectivity. Endpoint density mapping provided a finer grained analysis, by allowing visualisation of the spatial distribution of hippocampal endpoint density associated with each cortical area. This is particularly interesting in terms of the …
Reviewer #2 (Public Review):
Dalton and colleagues present an interesting and timely manuscript on diffusion weighted imaging analysis of human hippocampal connectivity. The focus is on connectivity differences along the hippocampal long axis, which in principle would provide important insights into the neuroanatomical underpinnings of functional long axis differences in the human brain. In keeping with current models of long-axis organisation, connectivity profiles show both discrete areas of higher connectivity in long axis portions, as well as an anterior-to-posterior gradient of increasing connectivity. Endpoint density mapping provided a finer grained analysis, by allowing visualisation of the spatial distribution of hippocampal endpoint density associated with each cortical area. This is particularly interesting in terms of the medial-lateral distribution with hippocampal head, body and tail. Specific areas map to precise hippocampal loci, and some hippocampal loci receive inputs from multiple cortical areas.
This work is well-motivated, well-written and interesting. The authors have capitalised on existing data from the Human Connectome Project. I particularly like the way the authors try to link their findings to human histological data, and to previous NHP tracing results.
I do, however, have some concerns about the interpretation of the results.
There are some important surprises in the results, particularly the relatively strong connectivity between hippocampus and early visual areas (including V1) and low connectivity with areas highly relevant from functional perspectives, such as the medial prefrontal cortex (rank order by strength of connectivity 7th and 78th of all cortical structures, respectively). This raises a concern that the fibre tracking method may be joining hippocampal connections with other tracts. In particular, given the anatomical proximity of the lateral geniculate nucleus to the body and tail of the hippocampus, the reported V1 connectivity potentially reflects a fusion of tracked fibres with the optic radiation. In visualizing the putative posterior hippocampus-to-V1 projection (Figure 4B, turquoise), the tract does indeed resemble the optic radiation topography. Although care was taken to minimise the hippocampus mask 'spilling' into adjacent white matter, this was done with focus on the hippocampal inferior margin, whereas the different components of the optic radiation lie lateral and superior to the hippocampus.
A second concern pertains to the location of endpoint densities within the hippocampus from the cortical mantle. These are almost entirely in CA1/subiculum/presubiculum. It is, however, puzzling why, in Supp Figure 2, the hippocampal endpoints for entorhinal projections is really quite similar to what is observed for other cortical projections (e.g., those from area TF). One would expect more endpoint density in the superior portions of the hippocampal cross section in head and body, in keeping with DG/CA3 termination. I note that streamlines were permitted to move within the hippocampus, but the highest density of endpoints is still around the margins.
On a related point, the use of "medial" and "lateral" hippocampus can be confusing. In the head, CA2/3 is medial to CA1, but so are subicular subareas, just that the latter are inferior.
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