The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia
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Evaluation Summary:
This multifaceted study focuses on neurons in the brain that produce a small peptide molecule known as GnRH, which is central to reproduction in its role as the releasing hormone for gonadotropins from the anterior pituitary. The findings will be of interest to scientists interested in the role of neuropeptides in determining normal brain function and the onset of neurodegenerative disorders. The authors provide a detailed anatomical and molecular characterization of a large, previously unnoticed population of GnRH neurons, located in basal ganglia (mainly putamen) in humans, which diverge from the population of GnRH neurons regulating the pituitary, in number (much larger), morphology and, possibly, origin (not from olfactory placode). Moreover, the study rekindles the idea that GnRH producing neurons in other regions of the brain outside the hypothalamus may be involved in neural processes unrelated to reproduction such as locomotion and decision making.
(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 #3 agreed to share their name with the authors.)
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Abstract
Human reproduction is controlled by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here, we report the discovery and characterization of additional ~150,000–200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Nearly all extrahypothalamic GnRH neurons expressed the cholinergic marker enzyme choline acetyltransferase. Similarly, hypothalamic GnRH neurons were also cholinergic both in embryonic and adult human brains. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen showed selective expression of GNRH1 and GNRHR1 autoreceptors in the cholinergic cell population and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher-order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. The role and changes of GnRH/GnRHR1 signaling in neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinson’s and Alzheimer’s diseases, need to be explored.
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Author Response:
Reviewer #1:
Skrapits et al., report on a population of GnRH neurons in the putamen that dwarfs the commonly studied hypothalamic population that regulates fertility. This laboratory performs very careful immunohistochemical studies and has included a number of controls to support this claim. These primarily include comparison of an overlapping staining pattern with multiple polyclonal antibodies, in situ hybridization and measurements of GnRH decapeptide with LC-MS/MS. While these are supportive, the question of the degradation product GnRH1-5, which has been brought up as a potential caveat in prior studies of extrahypothalamic populations as pointed out by the authors, does remain. This cleavage product was detected in their samples from the forebrain, albeit at lower levels. Even the identification of a large …
Author Response:
Reviewer #1:
Skrapits et al., report on a population of GnRH neurons in the putamen that dwarfs the commonly studied hypothalamic population that regulates fertility. This laboratory performs very careful immunohistochemical studies and has included a number of controls to support this claim. These primarily include comparison of an overlapping staining pattern with multiple polyclonal antibodies, in situ hybridization and measurements of GnRH decapeptide with LC-MS/MS. While these are supportive, the question of the degradation product GnRH1-5, which has been brought up as a potential caveat in prior studies of extrahypothalamic populations as pointed out by the authors, does remain. This cleavage product was detected in their samples from the forebrain, albeit at lower levels. Even the identification of a large population of cells producing the cleavage product would be of interest, but knowledge of the GnRH-related peptides in these cells is needed to point future studies in a fruitful direction.
We are grateful to the Reviewer for the careful revision of our manuscript and we appreciate the critical remarks and helpful suggestions. Our point-by-point responses follow the reviewer’s comments.
These immuno studies present a more complete and state-of-the art characterization of populations that have been hinted at in past work not only in primates, which is cited, but also in rodents (Skynner et al., J Neurosci 19:5955-5966), citation of which was overlooked. The authors should also comment on the extended exposure to primary antibodies in these studies, which has been reported to increase the number of GnRH neurons visualized during development in rodents (Wu et al., J Neurobiol 1997 Dec;33(7):983-98.) Also relevant to this point the statement on lines 379-380 is incorrect; the fluorescence of eGFP in these regions in the GnRH-GFP mice used has indeed been reported (Endocrinology, September 2008, 149(9):4596-4604) as has GnRH-GFP signal in another line of mice (Prog Neurobiol 63: 673- 686), and cells were also identified using GnRH promoter to drive beta galactosidease (J Neurosci 19:5955-5966).
Thank you very much for bringing these papers to our attention. We have read them carefully. Indeed, all of these transgenic models suggested extrahypothalamic GnRH expression in the developing mouse brain. However, none of them mentioned or showed GnRH (transgene) expression specifically in the CPU which would be more closely related to our work. As we have agreed to entirely omit the mouse data from the revised manuscript, citing these articles would not be relevant any more to our work.
The authors should also comment on the extended exposure to primary antibodies in these studies, which has been reported to increase the number of GnRH neurons visualized during development in rodents (Wu et al., J Neurobiol 1997 Dec;33(7):983-98.).
We have improved the Methods section by adding missing technical details. In the revised manuscript we indicate that the use of extended exposure times was only necessary when using the 100-μm-thick sections. We believe that this approach is still less sensitive than the standard protocol performed on much thinner floated sections ensuring better antibody penetration. Unfortunately, the use of thick sections was a necessary compromise to reduce excessive overcounting which would be difficult to correct using Abercrombies principle where optimal section thickness should exceed the Z dimension of the counted particles (Figure 1 – figure supplement 1). In our revised manuscript we have also made a statement in Discussion that the quantification results on thick sections likely represent an underestimate of real extrahypothalamic GnRH cell numbers, due to multiple technical factors that would be difficult to eliminate when studying postmortem tissues (Lines 308-311).
The authors also support their claims with RNAseq data. Performing these studies in human tissues is difficult because of the difficulty in controlling conditions and the data largely support their claims but some of the admitted quality limitations may warrant being more circumspect in their conclusions.
To extend their findings beyond enhanced anatomical characterization, the authors perform electrophysiologic studies of both putamen GnRH neurons and other putamen neurons identified in young mice. These data are not currently presented in a manner that allows a reader to determine if their conclusions from these studies are justified. Past work on GnRH action on hypothalamic GnRH neurons has indicated a dose dependence (Endocrinology 145(2):728-735), thus the current work should also examine dose effects before a putative direction of action for GnRH can be posited. Discussion of the central localization of GnRH receptors from other studies relative to their findings should also be discussed (Endocrinology 152: 1515-1526).
We appreciate these critical comments which we agree with. We have accepted to remove the neonatal mouse study from the revised manuscript in view of its poor relevance to the adult human putamen. We have also restricted the citation of rodent literature in the revised manuscript which now contains human studies only.
In the discussion, possible therapeutic actions of GnRH analogues are suggested. While exciting, this is not new and prior work examining patients on analogue therapy (for example Almeida et al., Psychoneuroendocrinology.2004;29(8):1071-1081 and Gandy et al JAMA.2001;285:2195-2196) should be cited.
We appreciate these suggestions, too. To avoid being too speculative, we now cite the suggested article by Almeida et al. in the context of the predictable reproductive side effects of GnRH analogues: “...reproductive side effects of GnRH analogues would limit the use of this strategy in clinical practice...” (Lines 424-427).
Reviewer #2:
The study beautifully illustrates the detection of a rather large population of GnRH neurons in the basal ganglia, by a convincing combination of neuroanatomical techniques in human brain specimens; techniques which are mastered by the authors and are well suited in terms of characterization of the GnRH neuronal system. The more conventional neuroanatomical techniques are further backed-up by modern molecular (RNA-seq) and biochemical (HPLC-MS) approaches. In addition, incorporation of a mouse model expressing GFP under the GnRH promoter adds some mechanistic dimension to the descriptive contents of the paper, which is a potential advantage, albeit it is not always clear that mouse and human data are fully convergent.
We are grateful to the Referee for the work devoted to the careful review of our manuscript.
Despite the strengths of the paper, this referee has identified several limitations, which need further elaboration, in order to avoid over-interpretation of the current dataset. Among these weaknesses, the authors should better clarify the number of individuals used for each analysis, and how representative the current findings are for both sexes and range of ages (and even pathological conditions) in humans.
The number of individuals is now clearly stated for all studies in the Results and the Methods sections. Tissue sources for each study are stated in Supplementary File 1. Several biological and methodological factors that may contribute to the heterogeneous labeling of human tissue samples are recognized (Lines 447-449). Unfortunately, it would not be possible to distinguish the effects of sex, age, health conditions, perimortal period and postmortem time on the detection of extrahypothalamic GnRH neurons in the different samples used in our study.
In addition, further discussion about the potential origin and relation (similarities and dissimilarities) with the hypophysiotropic population of GnRH neurons is deserved.
To address this comment, we have extended the discussion to better support our conclusion that extrahypothalamic GnRH neurons, as opposed to hypothalamic GnRH cells, are unlikely to originate from the olfactory placodes. We discuss that while Quanbeck et al. (1997) initially suggested that the equivalent neurons in the embryonic/fetal monkey brain might originate from the dorsal olfactory placode before olfactory pit formation, the later report of this laboratory by Terasawa and co-workers suggested that the increasing number of extrahypothalamic GnRH neurons might rather be derived from the ventricular wall of the telencephalic vesicle (Lines 305- 340).
Further, combination of human and mouse data is difficult at some places, since the mouse model do not express GFP in adulthood, and even no confirmation is provided that striatum neurons expressing GFP are actually producing GnRH at the neonatal period in the mouse.
In accordance with the above and other critical comments about the mouse study and also considering its limited relevance to the adult human brain, we have agreed to omit all mouse data from this manuscript.
Finally, although the implications of current findings are potentially large, the extended discussion of the present dataset in the context of neurological disease makes the paper over-speculative.
We appreciate the critical comment. We have eliminated the speculation from the Abstract and Discussion about the use of GnRH/GnRHR1 signaling to influence neurological disorders. Likely reproductive side effects of this approach have been brought up (Lines 424-427). An extended Discussion section points out that the “Receptor profile of human cholinergic interneurons may offer new therapeutics targets to treat neurodegenerative disorders” (Lines 397-427).
Reviewer #3:
The impetus for the study was the relatively recent demonstration by Casoni et al that, in man, a large number of GnRH neurons (approx. 8000) migrating from the olfactory placode during embryonic development follow a dorsal migratory route that takes them towards pallial and or subpallial structures, rather than along the more established ventral pathway that leads them to the hypothalamus where they subserve reproduction. The primary purpose of the experiments described were to determine the fate of the embryonic GnRH neurons that follow this ventral pathway and to begin to examine the biology of this interesting group of cells.
By and large, the varied array of contemporary imaging and molecular methods used are well described and the results are robust. Indeed, the application of such an armamentarium of approaches to study GnRH neurons in the human brain is a major strength of the paper.
Quantification of extrahypothalamic GnRH neuron number was performed using IHC with a guinea pig antibody, #1018. However, it appears that the standard procedure to establish specificity of an antibody, namely pre-absorption with authentic GnRH in the case of #1018, was not performed here nor presented in the original paper cited as describing this antibody (Hrabovszky et al 2011).
We are also very grateful to this Reviewer for the time and work invested in the critical review of our manuscript. We appreciate the suggestion to carry out a pre-absorption validation of antibody #1018, in addition to the positive control studies. We now report that pre-absorption of the primary antibody working solution with 0.1 μg/ml GnRH decapeptide eliminated all labeling from the human putamen. We have described (Lines 89-90; Lines 529-533; Lines 937-938) and illustrated (Figure 2A) these negative results.
The significance of the electrophysiological data derived from brain slices containing caudate-putamen (CPU) of a transgenic mouse (GnRH-GFP), in which GFP expressing cells were observed transiently in the CPU around postnatal day 4-7, is unclear. Regardless of what the outcome of the mouse experiments might have been, it seems highly unlikely that the discussion and implications of the data obtained from extrahypothalamic GnRH neurons in the human brain would have changed. Also the authors themselves "recognize that the neonatal mouse model has severe limitations."
In the light of criticism to the mouse study by the reviewers and the editor, we have agreed to remove the mouse electrophysiology and morphology blocks from the revised manuscript.
The aims of the authors have been more than realized: they have 1) provided novel and convincing characterization of extra-hypothalamic GnRH neurons in the human brain, 2) discovered that this population of neurons (>100,000) is far larger than previously considered, and 3) tentatively suggest that the additional extrahypothalamic GnRH neurons they have discovered may not originate from the olfactory placode,
The authors findings will almost certainly lead to further examination of the function of extrahypothalamic GnRH in normal brain function and neurodegenerative disorders associated with aging, which in turn may lead to new therapeutic applications of GnRH1 receptor ligands.
Returning to the authors suggestion that the additional extrahypothalamic GnRH neurons they have discovered may not originate from the olfactory placode, the Paragraph discussing this issue (beginning Line 319) confused me. Here, the authors state that it is unlikely that the large number of extrahypothalamic GnRH neurons in the putamen and related areas are identical to the 8000 observed by Casoni et al (2016) along the dorsal migratory route (the authors original aim was to follow the fate of these cells). Instead they suggest that they are homologus to the GnRH cells that, in the monkey leave, the olfactory placode before E30 (termed "early" GnRH neurons). If "early" GnRH neurons originate from the olfactory placode then why are the large numbers of GnRH neurons observed in the human Pu, and argued unlikely to be of placode origin, considered to be homologus to "early" GnRH neurons. In this regard, the relationship between the ChAT negative GnRH neurons in the nasal region of the GW11 human fetus and the "early" and "late" GnRH cells in the monkey fetus should be provided. In clarifying the above issue, the fact that Terasawa's studies utilized fetal rhesus monkeys should be explicitly stated in the Introduction and reinforced when they are discussed with the author's results. As written, the reader does not discover the developmental origin of Terasawa's monkeys until the Discussion.
We recognize the problem in our writing. We have improved the revised Results and Discussion sections in order to better articulate that extrahypothalamic GnRH neurons, either in monkeys or humans, are very unlikely to be of placodal origin. This conclusion is based on the high number of neurons both in monkeys (Terasawa et al.) and humans (our present study) (Lines 305-340).
In the Discussion the authors refer to GnRH deficient patients (Chan 2011). Homozygous mutations of GnRH1 are very rare and therefore it's perhaps not surprising that patients with such mutation have shed little light on function of extrahypothalamic GnRH. However, GnRHR1 loss of function mutations are much more common and have been known for nearly 25 years. Surely, a review of this literature would be worthwhile to see if any insight into dysfunction unrelated to reproduction emerges.
We greatly appreciate this comment. We have analyzed the clinical literature. We found that synkinesia, which would be a challenging candidate symptom in striatal dysfunctions, is quite common in Kallmann syndrome. However, synkinesia or other non-reproductive dysfunctions have not been characterized in GnRH deficient patients (Chan, 2011) or in the more common cases of GnRHR1 deficiency (Seminara, 1998 #511; Chevrier, 2011 #510), as we recognize (Lines 400-402).
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Evaluation Summary:
This multifaceted study focuses on neurons in the brain that produce a small peptide molecule known as GnRH, which is central to reproduction in its role as the releasing hormone for gonadotropins from the anterior pituitary. The findings will be of interest to scientists interested in the role of neuropeptides in determining normal brain function and the onset of neurodegenerative disorders. The authors provide a detailed anatomical and molecular characterization of a large, previously unnoticed population of GnRH neurons, located in basal ganglia (mainly putamen) in humans, which diverge from the population of GnRH neurons regulating the pituitary, in number (much larger), morphology and, possibly, origin (not from olfactory placode). Moreover, the study rekindles the idea that GnRH producing neurons in other …
Evaluation Summary:
This multifaceted study focuses on neurons in the brain that produce a small peptide molecule known as GnRH, which is central to reproduction in its role as the releasing hormone for gonadotropins from the anterior pituitary. The findings will be of interest to scientists interested in the role of neuropeptides in determining normal brain function and the onset of neurodegenerative disorders. The authors provide a detailed anatomical and molecular characterization of a large, previously unnoticed population of GnRH neurons, located in basal ganglia (mainly putamen) in humans, which diverge from the population of GnRH neurons regulating the pituitary, in number (much larger), morphology and, possibly, origin (not from olfactory placode). Moreover, the study rekindles the idea that GnRH producing neurons in other regions of the brain outside the hypothalamus may be involved in neural processes unrelated to reproduction such as locomotion and decision making.
(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 #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Skrapits et al., report on a population of GnRH neurons in the putamen that dwarfs the commonly studied hypothalamic population that regulates fertility. This laboratory performs very careful immunohistochemical studies and has included a number of controls to support this claim. These primarily include comparison of an overlapping staining pattern with multiple polyclonal antibodies, in situ hybridization and measurements of GnRH decapeptide with LC-MS/MS. While these are supportive, the question of the degradation product GnRH1-5, which has been brought up as a potential caveat in prior studies of extrahypothalamic populations as pointed out by the authors, does remain. This cleavage product was detected in their samples from the forebrain, albeit at lower levels. Even the identification of a large …
Reviewer #1 (Public Review):
Skrapits et al., report on a population of GnRH neurons in the putamen that dwarfs the commonly studied hypothalamic population that regulates fertility. This laboratory performs very careful immunohistochemical studies and has included a number of controls to support this claim. These primarily include comparison of an overlapping staining pattern with multiple polyclonal antibodies, in situ hybridization and measurements of GnRH decapeptide with LC-MS/MS. While these are supportive, the question of the degradation product GnRH1-5, which has been brought up as a potential caveat in prior studies of extrahypothalamic populations as pointed out by the authors, does remain. This cleavage product was detected in their samples from the forebrain, albeit at lower levels. Even the identification of a large population of cells producing the cleavage product would be of interest, but knowledge of the GnRH-related peptides in these cells is needed to point future studies in a fruitful direction.
These immuno studies present a more complete and state-of-the art characterization of populations that have been hinted at in past work not only in primates, which is cited, but also in rodents (Skynner et al., J Neurosci 19:5955-5966), citation of which was overlooked. The authors should also comment on the extended exposure to primary antibodies in these studies, which has been reported to increase the number of GnRH neurons visualized during development in rodents (Wu et al., J Neurobiol 1997 Dec;33(7):983-98.) Also relevant to this point the statement on lines 379-380 is incorrect; the fluorescence of eGFP in these regions in the GnRH-GFP mice used has indeed been reported (Endocrinology, September 2008, 149(9):4596-4604) as has GnRH-GFP signal in another line of mice (Prog Neurobiol 63: 673- 686), and cells were also identified using GnRH promoter to drive beta galactosidease (J Neurosci 19:5955-5966).
The authors also support their claims with RNAseq data. Performing these studies in human tissues is difficult because of the difficulty in controlling conditions and the data largely support their claims but some of the admitted quality limitations may warrant being more circumspect in their conclusions.
To extend their findings beyond enhanced anatomical characterization, the authors perform electrophysiologic studies of both putamen GnRH neurons and other putamen neurons identified in young mice. These data are not currently presented in a manner that allows a reader to determine if their conclusions from these studies are justified. Past work on GnRH action on hypothalamic GnRH neurons has indicated a dose dependence (Endocrinology 145(2):728-735), thus the current work should also examine dose effects before a putative direction of action for GnRH can be posited. Discussion of the central localization of GnRH receptors from other studies relative to their findings should also be discussed (Endocrinology 152: 1515-1526).
In the discussion, possible therapeutic actions of GnRH analogues are suggested. While exciting, this is not new and prior work examining patients on analogue therapy (for example Almeida et al., Psychoneuroendocrinology.2004;29(8):1071-1081 and Gandy et al JAMA.2001;285:2195-2196) should be cited.
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Reviewer #2 (Public Review):
The study beautifully illustrates the detection of a rather large population of GnRH neurons in the basal ganglia, by a convincing combination of neuroanatomical techniques in human brain specimens; techniques which are mastered by the authors and are well suited in terms of characterization of the GnRH neuronal system. The more conventional neuroanatomical techniques are further backed-up by modern molecular (RNA-seq) and biochemical (HPLC-MS) approaches. In addition, incorporation of a mouse model expressing GFP under the GnRH promoter adds some mechanistic dimension to the descriptive contents of the paper, which is a potential advantage, albeit it is not always clear that mouse and human data are fully convergent.Despite the strengths of the paper, this referee has identified several limitations, …
Reviewer #2 (Public Review):
The study beautifully illustrates the detection of a rather large population of GnRH neurons in the basal ganglia, by a convincing combination of neuroanatomical techniques in human brain specimens; techniques which are mastered by the authors and are well suited in terms of characterization of the GnRH neuronal system. The more conventional neuroanatomical techniques are further backed-up by modern molecular (RNA-seq) and biochemical (HPLC-MS) approaches. In addition, incorporation of a mouse model expressing GFP under the GnRH promoter adds some mechanistic dimension to the descriptive contents of the paper, which is a potential advantage, albeit it is not always clear that mouse and human data are fully convergent.Despite the strengths of the paper, this referee has identified several limitations, which need further elaboration, in order to avoid over-interpretation of the current dataset. Among these weaknesses, the authors should better clarify the number of individuals used for each analysis, and how representative the current findings are for both sexes and range of ages (and even pathological conditions) in humans. In addition, further discussion about the potential origin and relation (similarities and dissimilarities) with the hypophysiotropic population of GnRH neurons is deserved. Further, combination of human and mouse data is difficult at some places, since the mouse model do not express GFP in adulthood, and even no confirmation is provided that striatum neurons expressing GFP are actually producing GnRH at the neonatal period in the mouse. Finally, although the implications of current findings are potentially large, the extended discussion of the present dataset in the context of neurological disease makes the paper over-speculative.
-
Reviewer #3 (Public Review):
The impetus for the study was the relatively recent demonstration by Casoni et al that, in man, a large number of GnRH neurons (approx. 8000) migrating from the olfactory placode during embryonic development follow a dorsal migratory route that takes them towards pallial and or subpallial structures, rather than along the more established ventral pathway that leads them to the hypothalamus where they subserve reproduction. The primary purpose of the experiments described were to determine the fate of the embryonic GnRH neurons that follow this ventral pathway and to begin to examine the biology of this interesting group of cells.
By and large, the varied array of contemporary imaging and molecular methods used are well described and the results are robust. Indeed, the application of such an armamentarium of …
Reviewer #3 (Public Review):
The impetus for the study was the relatively recent demonstration by Casoni et al that, in man, a large number of GnRH neurons (approx. 8000) migrating from the olfactory placode during embryonic development follow a dorsal migratory route that takes them towards pallial and or subpallial structures, rather than along the more established ventral pathway that leads them to the hypothalamus where they subserve reproduction. The primary purpose of the experiments described were to determine the fate of the embryonic GnRH neurons that follow this ventral pathway and to begin to examine the biology of this interesting group of cells.
By and large, the varied array of contemporary imaging and molecular methods used are well described and the results are robust. Indeed, the application of such an armamentarium of approaches to study GnRH neurons in the human brain is a major strength of the paper.
Quantification of extrahypothalamic GnRH neuron number was performed using IHC with a guinea pig antibody, #1018. However, it appears that the standard procedure to establish specificity of an antibody, namely pre-absorption with authentic GnRH in the case of #1018, was not performed here nor presented in the original paper cited as describing this antibody (Hrabovszky et al 2011).
The significance of the electrophysiological data derived from brain slices containing caudate-putamen (CPU) of a transgenic mouse (GnRH-GFP), in which GFP expressing cells were observed transiently in the CPU around postnatal day 4-7, is unclear. Regardless of what the outcome of the mouse experiments might have been, it seems highly unlikely that the discussion and implications of the data obtained from extrahypothalamic GnRH neurons in the human brain would have changed. Also the authors themselves "recognize that the neonatal mouse model has severe limitations."
The aims of the authors have been more than realized: they have 1) provided novel and convincing characterization of extra-hypothalamic GnRH neurons in the human brain, 2) discovered that this population of neurons (>100,000) is far larger than previously considered, and 3) tentatively suggest that the additional extrahypothalamic GnRH neurons they have discovered may not originate from the olfactory placode,
The authors findings will almost certainly lead to further examination of the function of extrahypothalamic GnRH in normal brain function and neurodegenerative disorders associated with aging, which in turn may lead to new therapeutic applications of GnRH1 receptor ligands.
Returning to the authors suggestion that the additional extrahypothalamic GnRH neurons they have discovered may not originate from the olfactory placode, the Paragraph discussing this issue (beginning Line 319) confused me. Here, the authors state that it is unlikely that the large number of extrahypothalamic GnRH neurons in the putamen and related areas are identical to the 8000 observed by Casoni et al (2016) along the dorsal migratory route (the authors original aim was to follow the fate of these cells). Instead they suggest that they are homologus to the GnRH cells that, in the monkey leave, the olfactory placode before E30 (termed "early" GnRH neurons). If "early" GnRH neurons originate from the olfactory placode then why are the large numbers of GnRH neurons observed in the human Pu, and argued unlikely to be of placode origin, considered to be homologus to "early" GnRH neurons. In this regard, the relationship between the ChAT negative GnRH neurons in the nasal region of the GW11 human fetus and the "early" and "late" GnRH cells in the monkey fetus should be provided. In clarifying the above issue, the fact that Terasawa's studies utilized fetal rhesus monkeys should be explicitly stated in the Introduction and reinforced when they are discussed with the author's results. As written, the reader does not discover the developmental origin of Terasawa's monkeys until the Discussion.
In the Discussion the authors refer to GnRH deficient patients (Chan 2011). Homozygous mutations of GnRH1 are very rare and therefore it's perhaps not surprising that patients with such mutation have shed little light on function of extrahypothalamic GnRH. However, GnRHR1 loss of function mutations are much more common and have been known for nearly 25 years. Surely, a review of this literature would be worthwhile to see if any insight into dysfunction unrelated to reproduction emerges.
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