Neocortical pyramidal neurons with axons emerging from dendrites are frequent in non-primates, but rare in monkey and human
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Evaluation Summary:
Wahle and colleagues investigate the pervasiveness of the non-canonical arrangement of axons emerging from dendrites rather than the soma of neocortical pyramidal cells of different mammalian species. Using a variety of anatomical techniques, the authors demonstrate that axons can originate directly from pyramidal cell dendrites in species as diverse as rodents, ferret, cats, pigs and primates. Cross-species comparisons indicate that non-primate brains have a higher proportion of axon-carrying-dendrites (AcD) than did brains of macaques or humans. This paper is of potential interest to a broad range of neuroscientists in reporting the distribution of this non-canonical structure and indicating that primate brains may potentially feature axons emanating from dendrites less commonly.
(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, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms ‘axon carrying dendrite’ (AcD) and ‘AcD neurons’ have been coined to describe this feature. In rodent hippocampus, AcD cells are shown to be functionally ‘privileged’, since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Here, we report on the diversity of axon origins in neocortical pyramidal cells of rodent, ungulate, carnivore, and primate. Detection methods were Thy-1-EGFP labeling in mouse, retrograde biocytin tracing in rat, cat, ferret, and macaque, SMI-32/βIV-spectrin immunofluorescence in pig, cat, and macaque, and Golgi staining in macaque and human. We found that in non-primate mammals, 10–21% of pyramidal cells of layers II–VI had an AcD. In marked contrast, in macaque and human, this proportion was lower and was particularly low for supragranular neurons. A comparison of six cortical areas (being sensory, association, and limbic in nature) in three macaques yielded percentages of AcD cells which varied by a factor of 2 between the areas and between the individuals. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In addition, interneurons assessed in developing cat and adult human cortex had AcDs at type-specific proportions and for some types at much higher percentages than pyramidal cells. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortex of non-primate taxa, which strikingly differ from primates where these cells are mainly found in deeper layers and white matter.
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Author Response:
Reviewer #2 (Public Review):
This is an interesting and scientifically rigorous report documenting atypical, dendritic locations for the emerging axon of pyramidal neurons. This is not an entirely new observation (the authors cite relevant publications, including Kole and Brette, 2018 and Mendizabal-Zubiaga et al., 2007), but still important, as a relatively overlooked fact with functional implications. A main feature of the present report is an exceptionally thorough cross-species survey, from which the authors conclude that, as compared with non-primates, the macaque and human brains have a lower proportion of neocortical pyramidal neurons with axon carrying dendrites. The results might be further supported by additional experiments, especially ultrastructural data, or by including more extensive developmental …
Author Response:
Reviewer #2 (Public Review):
This is an interesting and scientifically rigorous report documenting atypical, dendritic locations for the emerging axon of pyramidal neurons. This is not an entirely new observation (the authors cite relevant publications, including Kole and Brette, 2018 and Mendizabal-Zubiaga et al., 2007), but still important, as a relatively overlooked fact with functional implications. A main feature of the present report is an exceptionally thorough cross-species survey, from which the authors conclude that, as compared with non-primates, the macaque and human brains have a lower proportion of neocortical pyramidal neurons with axon carrying dendrites. The results might be further supported by additional experiments, especially ultrastructural data, or by including more extensive developmental data. There is a section on Development, but there is hardly any Discussion. However, these matters are raised and adequately treated by reference to the existing literature.
We cannot do EM with frozen material or DEPEX-cleared sections. The developmental aspects have been more extensivel discussed now, but we refrained from speculating too much, since we do not have physiological data.
Reviewer #3 (Public Review):
The authors used neuroanatomical techniques to study neocortical pyramidal neurons from several different mammalian species. Their message is that primate neocortex differs from that of other mammals in having substantially fewer cells with axons emanating from dendrites, rather than the canonical route from the soma. The authors employed a range of standard methods, ranging from tracer injection to Golgi impregnation to immunocytochemistry. The feature the authors report is undeniable; there clearly are axons that emanate from dendrites of neocortical pyramidal neurons. Prior studies have reported that these axons are more excitable, thus leading to the intriguing possibility of a fundamental architectural (and thus presumably functional) feature in how primate neocortex operates.
This is a provocative narrative, that leads to a number of interesting questions. However, I have reservations that the authors must address before I believe the claim that primates are really fundamentally different from other mammals in this respect. A strength but also a central limitation of this study is that different species were compared using different methods, and different areas were studied in different species. The authors make the implicit assumption that the prominence of this feature does not differ among cortical areas.
We initially considered it a strength of the study – looking into many area with many methods in many species. However, it seemed a bit like cherry-picking, and we now enlarged the data sets for a more systematic analysis. Please note, we assessed archived material. We are bound to what we have available. We now delivered areal comparisions, and I am afraid, the answer is NO, no remakable differences in the areas that we assessed in monkey and cat.
However, it is entirely plausible that the proportion of neurons with axon-carrying dendrites does differ among cortical areas. The authors also group neurons into 2 large populations: infra- and supragranular. But again, layers 2 and 3 differ from one another (as do layers 5 and 6) in the specific populations of pyramidal cells they contain (morphological and neurochemical types, inputs and outputs, etc.). Certainly many studies do group neurons into these broad populations, but for this kind of comparison relevant differences or similarities could have been lost. Comparisons among species ideally would have all been in the same layer and area.
As said, we are bound to what we have available. And this is more than what has ever been published on these question so far. The graph and the Tables to Figure 3B allow to compare species across the layers.
We are aware that pyramidal cells in the layers can differ. Looking into RNA seq papers, up to 19 types exist in mouse. How many could potentially then exist in human? There is no way of pulverizing our kind of analysis down to the level of 19 pyramidal cell types differing by some unexplained RNA signatures which so far exist only for mouse. The SMI-32 staining already “selects” for one subtype in that it stains preferentially so-called type 1 pyramidal cells (Molnar et al., 2006).
Another limitation is that the same method was not employed in different species. The reader needs to know that different methods reveal the same proportion of axon-carrying dendrites in a given area of a certain species. This should have been stated more clearly and earlier in the text; it took examination of the data tables to see this. The tables show that measurements were made in several different cortical areas. Can the authors provide any evidence that the proportion of neurons with axon-carrying dendrites does not differ in any one species among cortical areas?
We now provide areal comparisons for 5 fields in monkey (new Figure 4A) and visual fields in cat (new Fig. 4B), both with the same methods. We can even provide a within-individual comparison of brain areas and of methods. Another three areal values for the infant macaque have been plotted in Figure 3B.
Figure 3 description and/or legend needs to state clearly that different species' neocortex was studied in different areas (and if all Fig3 samples shown are from same layers).
Figure 3A is total cortex, Figure 3 B is by layers. Counting strategies are now described in detail in methods.
Supplementary Excel file suggests that for humans Golgi-Kopsch reveals fewer infragranular AcD-cells than Golgi-Cox (4.43 vs 1.39), while for adult macaques Golgi-Kopsch revealed fewer than biocytin injection or SMI-32/BetaIV-spectrin immunofluorescence (13.34 vs 7.98 vs 6.29). Since the human data relies on Golgi methods, the authors must reassure the readers that the comparison of species is validated by direct comparison of different methods.
The message that primates have fewer cells with axon-carrying dendrites than other mammals might therefore certainly be interesting but far less compelling. The message might be that primate neocortex is not qualitatively different from that of other species; instead they simply have somewhat fewer AcD-bearing neurons than other mammalian species. But even that more modest conclusion is suggested but not fully proven by the data here.
The referee was right at this point. Having doubled our data sets with more human data we now aggree: the Golgi method underestimates the AcD neurons simply because of optical limitations. We now extensively discuss the issue and we no longer do statistical analysis on human. The issue needs further investigation with more methods.
I was puzzled by Fig 4 not including primate tissue. If the message is that spine density does not differ in dendrites with and without axons, surely it would be important to include primate tissue in this comparison; the comparison between primates and on-primates is after all the core message of this study. I also do not think the values for each species for non-AcD and shared root should be connected by a line; I suggest instead there should simply be a scatter of values for each group with a large symbol indicating mean or median value of each group. This would facilitate comparison.
First to the graph on spines, now Figure 6. You have to connect the individual neurons by line, otherwise the major point can no longer be seen: the dendrites differ in spine counts, sometimes the AcD is higher than the other basals of the very same neuron, in the next cell the AcD had a lower count. Statistics did not even suggest a trend. We aggree that things may differ in immature neurons. Possibly, during early development the AcD gains advantages by means of its higher excitability.
Please read the methods part to this point, elegible neurons had to fullfil a number of criteria. We fully exploited the available material of rat and ferret; no more elegible neurons. We indeed tried the same in macaque. Section thickness 50 µm. We found exactly two neurons which fullfilled the criteria. We had no chance with this material given the enormous dimension of the pyramidal cell dendritic trees in monkey. They were simply cut. For this type of classical tracing studies, non-alternating section series were prepared and submitted to different types of staining. Section spacing was several hundred µm in each individual. No chance to “reconstruct” dendrites from adjacent sections, since there were no adjacent sections.
The core message of the study is still valid, also without the spine analysis in monkey.
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Evaluation Summary:
Wahle and colleagues investigate the pervasiveness of the non-canonical arrangement of axons emerging from dendrites rather than the soma of neocortical pyramidal cells of different mammalian species. Using a variety of anatomical techniques, the authors demonstrate that axons can originate directly from pyramidal cell dendrites in species as diverse as rodents, ferret, cats, pigs and primates. Cross-species comparisons indicate that non-primate brains have a higher proportion of axon-carrying-dendrites (AcD) than did brains of macaques or humans. This paper is of potential interest to a broad range of neuroscientists in reporting the distribution of this non-canonical structure and indicating that primate brains may potentially feature axons emanating from dendrites less commonly.
(This preprint has been reviewed by …
Evaluation Summary:
Wahle and colleagues investigate the pervasiveness of the non-canonical arrangement of axons emerging from dendrites rather than the soma of neocortical pyramidal cells of different mammalian species. Using a variety of anatomical techniques, the authors demonstrate that axons can originate directly from pyramidal cell dendrites in species as diverse as rodents, ferret, cats, pigs and primates. Cross-species comparisons indicate that non-primate brains have a higher proportion of axon-carrying-dendrites (AcD) than did brains of macaques or humans. This paper is of potential interest to a broad range of neuroscientists in reporting the distribution of this non-canonical structure and indicating that primate brains may potentially feature axons emanating from dendrites less commonly.
(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, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Wahle and colleagues surveyed the occurrence of the non-canonical of "axon carrying dendrites" of neocortical pyramidal cells across different mammalian species. Their results indicate that there are differences between rodents, pigs, cats and ferrets on the one hand and primates on the other. Pyramidal cells of primates (humans and monkeys) have overall a lower proportion of axon carrying dendrites in the gray matter, which seem to be more concentrated in infragranular layers and in white matter.
The major strength of the manuscript lie in the large scope of the systematic survey of pyramidal cells with axon carrying dendrites across a range of different mammalian species. The authors also include developmental data and results comparing different cortical layers as well as the results from white matter and …
Reviewer #1 (Public Review):
Wahle and colleagues surveyed the occurrence of the non-canonical of "axon carrying dendrites" of neocortical pyramidal cells across different mammalian species. Their results indicate that there are differences between rodents, pigs, cats and ferrets on the one hand and primates on the other. Pyramidal cells of primates (humans and monkeys) have overall a lower proportion of axon carrying dendrites in the gray matter, which seem to be more concentrated in infragranular layers and in white matter.
The major strength of the manuscript lie in the large scope of the systematic survey of pyramidal cells with axon carrying dendrites across a range of different mammalian species. The authors also include developmental data and results comparing different cortical layers as well as the results from white matter and gray matter. With the necessary diversity of histological source material necessary for this study, the authors should assess and discuss potential other sources of differences between the species and their strategy to overcome these more explicitly.
The presented data and scope of the study are impressive and shed light on the potential evolutionary differences in the cortical circuitry of primates in contrast to other mammals.
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Reviewer #2 (Public Review):
This is an interesting and scientifically rigorous report documenting atypical, dendritic locations for the emerging axon of pyramidal neurons. This is not an entirely new observation (the authors cite relevant publications, including Kole and Brette, 2018 and Mendizabal-Zubiaga et al., 2007), but still important, as a relatively overlooked fact with functional implications. A main feature of the present report is an exceptionally thorough cross-species survey, from which the authors conclude that, as compared with non-primates, the macaque and human brains have a lower proportion of neocortical pyramidal neurons with axon carrying dendrites. The results might be further supported by additional experiments, especially ultrastructural data, or by including more extensive developmental data. There is a section …
Reviewer #2 (Public Review):
This is an interesting and scientifically rigorous report documenting atypical, dendritic locations for the emerging axon of pyramidal neurons. This is not an entirely new observation (the authors cite relevant publications, including Kole and Brette, 2018 and Mendizabal-Zubiaga et al., 2007), but still important, as a relatively overlooked fact with functional implications. A main feature of the present report is an exceptionally thorough cross-species survey, from which the authors conclude that, as compared with non-primates, the macaque and human brains have a lower proportion of neocortical pyramidal neurons with axon carrying dendrites. The results might be further supported by additional experiments, especially ultrastructural data, or by including more extensive developmental data. There is a section on Development, but there is hardly any Discussion. However, these matters are raised and adequately treated by reference to the existing literature.
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Reviewer #3 (Public Review):
The authors used neuroanatomical techniques to study neocortical pyramidal neurons from several different mammalian species. Their message is that primate neocortex differs from that of other mammals in having substantially fewer cells with axons emanating from dendrites, rather than the canonical route from the soma. The authors employed a range of standard methods, ranging from tracer injection to Golgi impregnation to immunocytochemistry. The feature the authors report is undeniable; there clearly are axons that emanate from dendrites of neocortical pyramidal neurons. Prior studies have reported that these axons are more excitable, thus leading to the intriguing possibility of a fundamental architectural (and thus presumably functional) feature in how primate neocortex operates.
This is a provocative …
Reviewer #3 (Public Review):
The authors used neuroanatomical techniques to study neocortical pyramidal neurons from several different mammalian species. Their message is that primate neocortex differs from that of other mammals in having substantially fewer cells with axons emanating from dendrites, rather than the canonical route from the soma. The authors employed a range of standard methods, ranging from tracer injection to Golgi impregnation to immunocytochemistry. The feature the authors report is undeniable; there clearly are axons that emanate from dendrites of neocortical pyramidal neurons. Prior studies have reported that these axons are more excitable, thus leading to the intriguing possibility of a fundamental architectural (and thus presumably functional) feature in how primate neocortex operates.
This is a provocative narrative, that leads to a number of interesting questions. However, I have reservations that the authors must address before I believe the claim that primates are really fundamentally different from other mammals in this respect. A strength but also a central limitation of this study is that different species were compared using different methods, and different areas were studied in different species. The authors make the implicit assumption that the prominence of this feature does not differ among cortical areas. However, it is entirely plausible that the proportion of neurons with axon-carrying dendrites does differ among cortical areas. The authors also group neurons into 2 large populations: infra- and supragranular. But again, layers 2 and 3 differ from one another (as do layers 5 and 6) in the specific populations of pyramidal cells they contain (morphological and neurochemical types, inputs and outputs, etc.). Certainly many studies do group neurons into these broad populations, but for this kind of comparison relevant differences or similarities could have been lost. Comparisons among species ideally would have all been in the same layer and area.
Another limitation is that the same method was not employed in different species. The reader needs to know that different methods reveal the same proportion of axon-carrying dendrites in a given area of a certain species. This should have been stated more clearly and earlier in the text; it took examination of the data tables to see this. The tables show that measurements were made in several different cortical areas. Can the authors provide any evidence that the proportion of neurons with axon-carrying dendrites does not differ in any one species among cortical areas? Figure 3 description and/or legend needs to state clearly that different species' neocortex was studied in different areas (and if all Fig3 samples shown are from same layers). Supplementary Excel file suggests that for humans Golgi-Kopsch reveals fewer infragranular AcD-cells than Golgi-Cox (4.43 vs 1.39), while for adult macaques Golgi-Kopsch revealed fewer than biocytin injection or SMI-32/BetaIV-spectrin immunofluorescence (13.34 vs 7.98 vs 6.29). Since the human data relies on Golgi methods, the authors must reassure the readers that the comparison of species is validated by direct comparison of different methods.
The message that primates have fewer cells with axon-carrying dendrites than other mammals might therefore certainly be interesting but far less compelling. The message might be that primate neocortex is not qualitatively different from that of other species; instead they simply have somewhat fewer AcD-bearing neurons than other mammalian species. But even that more modest conclusion is suggested but not fully proven by the data here.
I was puzzled by Fig 4 not including primate tissue. If the message is that spine density does not differ in dendrites with and without axons, surely it would be important to include primate tissue in this comparison; the comparison between primates and on-primates is after all the core message of this study. I also do not think the values for each species for non-AcD and shared root should be connected by a line; I suggest instead there should simply be a scatter of values for each group with a large symbol indicating mean or median value of each group. This would facilitate comparison.
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