The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward

Yasmine Sherafat; Malia Bautista; J. P. Fowler; Edison Chen; Amina Ahmed; Christie D. Fowler

doi:10.1523/ENEURO.0191-20.2020

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RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
Christie Fowler

Submitted on: 16 February 2021
RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
Eric Turner

Submitted on: 14 December 2020

Submitted on: (16 February 2021)
Page navigation anchor for RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
Christie Fowler, Associate Professor, University of California, Irvine
In the ELetter from Eric Turner dated 14 December 2020, the author provides several arguments regarding the description of the pathway from the IPN to the vHipp with was published in Sherafat et al. However, the authors mainly provide semantic arguments of debate, which doesn’t provide a significant context to change interpretation of the findings or conclusions of the manuscript. Moreover, several issues are noted with their arguments.

In the first argument, the authors indicate an issue with the characterization of the IPN-vHIPP being “previously undefined”. The Sherafat et al paper implies that the function of the pathway has been previously undefined, which is a valid statement. While prior literature describes the presence of the pathway from the IPN to the vHipp and identified it as consisting of serotonergic projections, as noted in the citations within the manuscript and further citations provided by the commentary authors, the function of the circuit had been previously undefined until these studies, as was indicated by Sherafat et al. Since it is not an extensive review paper, an exhaustive list of all the prior papers demonstrating the presence of this circuit was not provided, since the foundation of the studies was to define function and the cited literature in the manuscript highlights the discovery of the circuit. Thus, the purpose and findings of the current study are not replicated in the citations provided, as this paper is the first to provide a...
Show More

In the ELetter from Eric Turner dated 14 December 2020, the author provides several arguments regarding the description of the pathway from the IPN to the vHipp with was published in Sherafat et al. However, the authors mainly provide semantic arguments of debate, which doesn’t provide a significant context to change interpretation of the findings or conclusions of the manuscript. Moreover, several issues are noted with their arguments.

In the first argument, the authors indicate an issue with the characterization of the IPN-vHIPP being “previously undefined”. The Sherafat et al paper implies that the function of the pathway has been previously undefined, which is a valid statement. While prior literature describes the presence of the pathway from the IPN to the vHipp and identified it as consisting of serotonergic projections, as noted in the citations within the manuscript and further citations provided by the commentary authors, the function of the circuit had been previously undefined until these studies, as was indicated by Sherafat et al. Since it is not an extensive review paper, an exhaustive list of all the prior papers demonstrating the presence of this circuit was not provided, since the foundation of the studies was to define function and the cited literature in the manuscript highlights the discovery of the circuit. Thus, the purpose and findings of the current study are not replicated in the citations provided, as this paper is the first to provide a mechanistic understanding of the function of the pathway.

With regard to the population of serotonergic cells being included within the IPN or MnR, this is a largely a semantic argument. As noted in the first paragraph by the ELetter author, the region of investigation does lie within the IPN. Thus, while the MnR is in close proximity, it is a subjective evaluation as to whether the population of cells should be associated with the raphe or the IPN. Given the location within the IPN, the characterization of the IPN as containing serotonergic cells is valid, as is contended in the Sherafat et al. manuscript and other papers in the literature.

While the focus on the specific IPA subregion of notation that the commentary authors suggest could have been suggested, the Sherafat et al. paper focused on the projection cells from the IPN to the vHIPP. Since a systematic analysis of all the possible IPN-vHipp projections were not characterized and not all of the IPN-vHIPP cells co-labeled as serotonergic, it would have been misleading to define the subregion in such a manner, as these projection cells were not always confined to the discrete IPA subregion along the rostrocaudal axis of the IPN, and thus, the more general IPN terminology is most appropriate. Indeed, prior reports have found cells projecting to the dorsal HIPP from the IPN, which are also serotonergic. The focus on the serotonergic cells was functional in these studies to determine whether such neurotransmitter signaling may underlie the behavioral effects found through receptor activation in the vHIPP, as performed with pharmacological approaches combined with circuit manipulation.

Finally, with regard to the volume of the virus injection and size of the brain region, Sherafat et al. employed an intersectional strategy. A retrovirus was injected into the vHIPP that expressed cre, and a floxed DREADD virus was injected into the IPN. Thus, only cells in the IPN that project to the vHIPP contain both viruses. This advanced technique thereby provides selective DREADD-mediated inhibition of the IPN projections into the vHIPP. Given this selectivity and the directed injections into the IPN (not a specific subregion), any potential floxed DREADD virus that would have spread to other cells in the IPN, or outside of the IPN boundaries, would not have resulted in expression of the DREADD receptor, since both viruses are necessary to permit receptor expression. The virus localization as being restricted in the IPN region was also confirmed as noted by the authors.

Finally, the resulting inhibition of the circuit with DREADDs is in direct contrast to that previously demonstrated with inhibition of serotonergic cells in the raphe for the forced swim test, as noted in the Sherafat manuscript. Thus, this behavioral data provides further evidence that the serotonergic raphe cells were not involved in the behavior exhibited, as a directly opposing behavioral effect was found.

Therefore, Turner’s conclusion in the ELetter that “no clear conclusions can be drawn from the data as presented.” is unfounded based on the data presented within the manuscript.
Show Less

Competing Interests: Author on the published paper. No other competing interests.
Submitted on: (14 December 2020)
Page navigation anchor for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
Eric Turner, Professor of Psychiatry, Seattle Childrens' Research Institute; University of Washington
The goal of this recent report by Sherafat et al. was to determine the behavioral effect of manipulating the activity of neurons in the interpeduncular nucleus (IP) that project to the ventral hippocampus (vHipp) using an intersectional retrograde viral strategy to express an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Specifically, they injected a retrograde adeno-associated virus expressing cre recombinase (rAAV-cre) into the vHipp, together with the injection of a cre-dependent AAV expressing hM4Di (inhibitory DREADD), or a control AAV, into the IP. The effects of the DREADD ligand clozapine-N-oxide (CNO) administration in DREADD-expressing and control mice were then examined in several behavioral models. In preliminary experiments, the IP neurons retrogradely labeled from the vHipp in this manner were shown to be serotonergic using co-localization of a genetic reporter for retrogradely transported cre-recombinase (ROSA-CAG-tdTomato), together with serotonin immunolabeling.

Although this IP-vHipp pathway is described in this work as “previously undefined”, several prior reports not cited by the authors have examined serotonergic neurons in the rodent IP (Singhaniyom et al., 1982; Hamill et al., 1984; Hemmendinger and Moore, 1984), traced the projection of the IP to the hippocampus (Baisden et al., 1979; Shibata and Suzuki, 1984; Groenewegen et al., 1986; Lima et al., 2017), and have combined retrograde tract-tracing plus se...
Show More

The goal of this recent report by Sherafat et al. was to determine the behavioral effect of manipulating the activity of neurons in the interpeduncular nucleus (IP) that project to the ventral hippocampus (vHipp) using an intersectional retrograde viral strategy to express an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Specifically, they injected a retrograde adeno-associated virus expressing cre recombinase (rAAV-cre) into the vHipp, together with the injection of a cre-dependent AAV expressing hM4Di (inhibitory DREADD), or a control AAV, into the IP. The effects of the DREADD ligand clozapine-N-oxide (CNO) administration in DREADD-expressing and control mice were then examined in several behavioral models. In preliminary experiments, the IP neurons retrogradely labeled from the vHipp in this manner were shown to be serotonergic using co-localization of a genetic reporter for retrogradely transported cre-recombinase (ROSA-CAG-tdTomato), together with serotonin immunolabeling.

Although this IP-vHipp pathway is described in this work as “previously undefined”, several prior reports not cited by the authors have examined serotonergic neurons in the rodent IP (Singhaniyom et al., 1982; Hamill et al., 1984; Hemmendinger and Moore, 1984), traced the projection of the IP to the hippocampus (Baisden et al., 1979; Shibata and Suzuki, 1984; Groenewegen et al., 1986; Lima et al., 2017), and have combined retrograde tract-tracing plus serotonergic markers to identify the neurotransmitter used in this pathway (Montone et al., 1988; Quina et al., 2017). In these studies, the IP neurons that express serotonergic markers and/or project to the hippocampus have been largely confined to a small area in the most caudal/dorsal part of the IP, consistent with the area shown in Figure 1 of Sherafat et al. In publications from 1984 onward, following the standardization of the IP nomenclature (Lenn and Hamill, 1984), this area has been referred to as the apical subnucleus of the IP (IPA), a designation that has been adopted in standard atlases for the rat and mouse (Paxinos and Watson, 1998; Paxinos and Franklin, 2001). If Sherafat et al. had identified the area of interest as the IPA, their work could have been more easily placed in the context of the prior research.

The prior work on the projections from the IP to the hippocampus may affect how readers interpret the results presented. As discussed in a recent paper (Quina et al., 2017), the molecular characteristics and anatomical connections of the IPA argue that it should be considered as the most rostral part of the median raphe (MnR) group of serotonergic neurons, rather than as an IP subnucleus. Three major points support this view: 1) Rather than being “a discrete population of neurons outside of the raphe nuclei”, the serotonergic IPA is anatomically continuous with the serotonergic neuronal population in the median raphe (MnR, B8 serotonin neurons). The identification of the IPA as a separate population of serotonergic neurons is essentially an artifact of sectioning in the coronal plane. This was noted in the earliest descriptions of these neurons (Singhaniyom et al., 1982). The continuity of the IPA with the MnR can also be seen in Allen Brain Atlas in situ hybridization data for serotonergic markers (Ng et al., 2009), where it is most easily visualized in the sagittal plane. One example is the ISH data for the serotonin transporter (Slc6a4, SERT, Allen Brain Atlas experiment 2624). In in the parasagital view shown in image 18/20 of this series, the IPA can be seen as a small cell group continuous with the rostral MnR, which is designated CS in the Allen nomenclature:
https://mouse.brain-map.org/experiment/siv?id=2624
2) The IPA does not resemble the largest and most-studied IP subnuclei, the rostral IP (IPR) and caudal IP (IPC), which do not contain serotonergic neurons, but instead are principally GABAergic (Hsu et al., 2013). 3) The hallmark of the IP is its highly conserved innervation by the medial habenula (MHb), to form the “habenulopeduncular pathway”, and the function of this pathway has been the focus of numerous recent studies (McLaughlin et al., 2017). The IPR and IPC receive inputs from the ventral MHb which expresses choline acetyltransferase, while the lateral IP (IPL) receives inputs from the dorsal MHb, which expresses the neuropeptide substance P. However, the IPA does not appear to receive inputs from either of these MHb systems (Quina et al., 2017). Thus both the gene expression characteristics and connectivity of the IPA link it to the serotonergic raphe, not the IP.

This continuity between the IPA and MnR has implications for interpreting the behavioral experiments presented by Sherafat et al. Extensive projections from the MnR to the hippocampus, including the vHipp, have been demonstrated using both classical tract-tracing methods (Vertes et al., 1999) and tract tracing with Cre-dependent adeno-associated viruses (AAV) in mice expressing Cre-recombinase from the Slc6a4 (serotonin transporter, SERT) gene locus (Muzerelle et al., 2016). The IPA is a small nucleus, contained within an area of approximately 100μm x 100μm x 200μm in the rostrocaudal, mediolateral, and dorsoventral dimensions, respectively. The volume of AAV injected in these hM4Di labeling experiments, 0.5 microliters, is thus ~250 times the volume of the targeted structure. Such an injection could not be confined to the IPA, and would be very likely to spread extensively into the immediately adjacent and much larger population of vHipp-projecting neurons in the MnR, unless somehow the retrograde transport of cre recombinase is selective for the IPA. However, no data are presented that address the extent of labeling with hM4Di in these injections, whether it is restricted to the IPA, extends into the MnR, or into other nearby populations of non-serotonergic neurons that may also have hippocampal projections and thus might express cre recombinase.

In conclusion, neurotransmitter identity, anatomy, and connectivity suggest that the intended target area in these studies, the IPA, should be considered as part of the MnR (B8) serotonergic cell group. In any case, it seems likely that vHipp-projecting neurons from the MnR proper would be labeled with silencing DREADDs using the methods described in this study. It is possible that the behavioral experiments presented reveal new information about the role of the serotonergic raphe system in the regulation of the behaviors examined, but no clear conclusions can be drawn from the data as presented.

References

Baisden RH, Hoover DB, Cowie RJ (1979) Retrograde demonstration of hippocampal afferents from the interpeduncular and reuniens nuclei. Neurosci Lett 13:105-109.

Groenewegen HJ, Ahlenius S, Haber SN, Kowall NW, Nauta WJ (1986) Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat. J Comp Neurol 249:65-102.

Hamill GS, Olschowka JA, Lenn NJ, Jacobowitz DM (1984) The subnuclear distribution of substance P, cholecystokinin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine-beta-hydroxylase, and serotonin in the rat interpeduncular nucleus. J Comp Neurol 226:580-596.

Hemmendinger LM, Moore RY (1984) Interpeduncular nucleus organization in the rat: cytoarchitecture and histochemical analysis. Brain Res Bull 13:163-179.

Hsu YW, Tempest L, Quina LA, Wei AD, Zeng H, Turner EE (2013) Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus. J Neurosci 33:18022-18035.

Lenn NJ, Hamill GS (1984) Subdivisions of the interpeduncular nucleus: a proposed nomenclature. Brain Res Bull 13:203-204.

Lima LB, Bueno D, Leite F, Souza S, Goncalves L, Furigo IC, Donato J, Jr., Metzger M (2017) Afferent and efferent connections of the interpeduncular nucleus with special reference to circuits involving the habenula and raphe nuclei. J Comp Neurol 525:2411-2442.

McLaughlin I, Dani JA, De Biasi M (2017) The medial habenula and interpeduncular nucleus circuitry is critical in addiction, anxiety, and mood regulation. J Neurochem 142 Suppl 2:130-143.

Montone KT, Fass B, Hamill GS (1988) Serotonergic and nonserotonergic projections from the rat interpeduncular nucleus to the septum, hippocampal formation and raphe: a combined immunocytochemical and fluorescent retrograde labelling study of neurons in the apical subnucleus. Brain Res Bull 20:233-240.

Muzerelle A, Scotto-Lomassese S, Bernard JF, Soiza-Reilly M, Gaspar P (2016) Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem. Brain Struct Funct 221:535-561.

Ng L, Bernard A, Lau C, Overly CC, Dong HW, Kuan C, Pathak S, Sunkin SM, Dang C, Bohland JW, Bokil H, Mitra PP, Puelles L, Hohmann J, Anderson DJ, Lein ES, Jones AR, Hawrylycz M (2009) An anatomic gene expression atlas of the adult mouse brain. Nat Neurosci 12:356-362.

Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th Edition. San Diego: Academic Press.

Paxinos G, Franklin KBJ (2001) The mouse brain in stereotaxic coordinates, 2nd Edition. San Diego, Calif. London: Academic.

Quina LA, Harris J, Zeng H, Turner EE (2017) Specific connections of the interpeduncular subnuclei reveal distinct components of the habenulopeduncular pathway. J Comp Neurol 525:2632-2656.

Shibata H, Suzuki T (1984) Efferent projections of the interpeduncular complex in the rat, with special reference to its subnuclei: a retrograde horseradish peroxidase study. Brain Res 296:345-349.

Singhaniyom W, Wreford NG, Guldner FH (1982) Distribution of 5-hydroxytryptamine-containing neuronal perikarya in the rat interpeduncular nucleus. Neurosci Lett 30:51-55.

Vertes RP, Fortin WJ, Crane AM (1999) Projections of the median raphe nucleus in the rat. J Comp Neurol 407:555-582.
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Competing Interests: None declared.

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RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
Christie Fowler

Published on: 16 February 2021
RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
Eric Turner

Published on: 14 December 2020

Published on: (16 February 2021)
Page navigation anchor for RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
RE: Turner E-Letter for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward”
Christie Fowler, Associate Professor, University of California, Irvine
In the ELetter from Eric Turner dated 14 December 2020, the author provides several arguments regarding the description of the pathway from the IPN to the vHipp with was published in Sherafat et al. However, the authors mainly provide semantic arguments of debate, which doesn’t provide a significant context to change interpretation of the findings or conclusions of the manuscript. Moreover, several issues are noted with their arguments.

In the first argument, the authors indicate an issue with the characterization of the IPN-vHIPP being “previously undefined”. The Sherafat et al paper implies that the function of the pathway has been previously undefined, which is a valid statement. While prior literature describes the presence of the pathway from the IPN to the vHipp and identified it as consisting of serotonergic projections, as noted in the citations within the manuscript and further citations provided by the commentary authors, the function of the circuit had been previously undefined until these studies, as was indicated by Sherafat et al. Since it is not an extensive review paper, an exhaustive list of all the prior papers demonstrating the presence of this circuit was not provided, since the foundation of the studies was to define function and the cited literature in the manuscript highlights the discovery of the circuit. Thus, the purpose and findings of the current study are not replicated in the citations provided, as this paper is the first to provide a...
Show More

In the ELetter from Eric Turner dated 14 December 2020, the author provides several arguments regarding the description of the pathway from the IPN to the vHipp with was published in Sherafat et al. However, the authors mainly provide semantic arguments of debate, which doesn’t provide a significant context to change interpretation of the findings or conclusions of the manuscript. Moreover, several issues are noted with their arguments.

In the first argument, the authors indicate an issue with the characterization of the IPN-vHIPP being “previously undefined”. The Sherafat et al paper implies that the function of the pathway has been previously undefined, which is a valid statement. While prior literature describes the presence of the pathway from the IPN to the vHipp and identified it as consisting of serotonergic projections, as noted in the citations within the manuscript and further citations provided by the commentary authors, the function of the circuit had been previously undefined until these studies, as was indicated by Sherafat et al. Since it is not an extensive review paper, an exhaustive list of all the prior papers demonstrating the presence of this circuit was not provided, since the foundation of the studies was to define function and the cited literature in the manuscript highlights the discovery of the circuit. Thus, the purpose and findings of the current study are not replicated in the citations provided, as this paper is the first to provide a mechanistic understanding of the function of the pathway.

With regard to the population of serotonergic cells being included within the IPN or MnR, this is a largely a semantic argument. As noted in the first paragraph by the ELetter author, the region of investigation does lie within the IPN. Thus, while the MnR is in close proximity, it is a subjective evaluation as to whether the population of cells should be associated with the raphe or the IPN. Given the location within the IPN, the characterization of the IPN as containing serotonergic cells is valid, as is contended in the Sherafat et al. manuscript and other papers in the literature.

While the focus on the specific IPA subregion of notation that the commentary authors suggest could have been suggested, the Sherafat et al. paper focused on the projection cells from the IPN to the vHIPP. Since a systematic analysis of all the possible IPN-vHipp projections were not characterized and not all of the IPN-vHIPP cells co-labeled as serotonergic, it would have been misleading to define the subregion in such a manner, as these projection cells were not always confined to the discrete IPA subregion along the rostrocaudal axis of the IPN, and thus, the more general IPN terminology is most appropriate. Indeed, prior reports have found cells projecting to the dorsal HIPP from the IPN, which are also serotonergic. The focus on the serotonergic cells was functional in these studies to determine whether such neurotransmitter signaling may underlie the behavioral effects found through receptor activation in the vHIPP, as performed with pharmacological approaches combined with circuit manipulation.

Finally, with regard to the volume of the virus injection and size of the brain region, Sherafat et al. employed an intersectional strategy. A retrovirus was injected into the vHIPP that expressed cre, and a floxed DREADD virus was injected into the IPN. Thus, only cells in the IPN that project to the vHIPP contain both viruses. This advanced technique thereby provides selective DREADD-mediated inhibition of the IPN projections into the vHIPP. Given this selectivity and the directed injections into the IPN (not a specific subregion), any potential floxed DREADD virus that would have spread to other cells in the IPN, or outside of the IPN boundaries, would not have resulted in expression of the DREADD receptor, since both viruses are necessary to permit receptor expression. The virus localization as being restricted in the IPN region was also confirmed as noted by the authors.

Finally, the resulting inhibition of the circuit with DREADDs is in direct contrast to that previously demonstrated with inhibition of serotonergic cells in the raphe for the forced swim test, as noted in the Sherafat manuscript. Thus, this behavioral data provides further evidence that the serotonergic raphe cells were not involved in the behavior exhibited, as a directly opposing behavioral effect was found.

Therefore, Turner’s conclusion in the ELetter that “no clear conclusions can be drawn from the data as presented.” is unfounded based on the data presented within the manuscript.
Show Less

Competing Interests: Author on the published paper. No other competing interests.
Published on: (14 December 2020)
Page navigation anchor for RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
RE: “The Interpeduncular-Ventral Hippocampus Pathway Mediates Active Stress Coping and Natural Reward” by Sherafat et al., eNeuro, 7, 2020
Eric Turner, Professor of Psychiatry, Seattle Childrens' Research Institute; University of Washington
The goal of this recent report by Sherafat et al. was to determine the behavioral effect of manipulating the activity of neurons in the interpeduncular nucleus (IP) that project to the ventral hippocampus (vHipp) using an intersectional retrograde viral strategy to express an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Specifically, they injected a retrograde adeno-associated virus expressing cre recombinase (rAAV-cre) into the vHipp, together with the injection of a cre-dependent AAV expressing hM4Di (inhibitory DREADD), or a control AAV, into the IP. The effects of the DREADD ligand clozapine-N-oxide (CNO) administration in DREADD-expressing and control mice were then examined in several behavioral models. In preliminary experiments, the IP neurons retrogradely labeled from the vHipp in this manner were shown to be serotonergic using co-localization of a genetic reporter for retrogradely transported cre-recombinase (ROSA-CAG-tdTomato), together with serotonin immunolabeling.

Although this IP-vHipp pathway is described in this work as “previously undefined”, several prior reports not cited by the authors have examined serotonergic neurons in the rodent IP (Singhaniyom et al., 1982; Hamill et al., 1984; Hemmendinger and Moore, 1984), traced the projection of the IP to the hippocampus (Baisden et al., 1979; Shibata and Suzuki, 1984; Groenewegen et al., 1986; Lima et al., 2017), and have combined retrograde tract-tracing plus se...
Show More

The goal of this recent report by Sherafat et al. was to determine the behavioral effect of manipulating the activity of neurons in the interpeduncular nucleus (IP) that project to the ventral hippocampus (vHipp) using an intersectional retrograde viral strategy to express an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Specifically, they injected a retrograde adeno-associated virus expressing cre recombinase (rAAV-cre) into the vHipp, together with the injection of a cre-dependent AAV expressing hM4Di (inhibitory DREADD), or a control AAV, into the IP. The effects of the DREADD ligand clozapine-N-oxide (CNO) administration in DREADD-expressing and control mice were then examined in several behavioral models. In preliminary experiments, the IP neurons retrogradely labeled from the vHipp in this manner were shown to be serotonergic using co-localization of a genetic reporter for retrogradely transported cre-recombinase (ROSA-CAG-tdTomato), together with serotonin immunolabeling.

Although this IP-vHipp pathway is described in this work as “previously undefined”, several prior reports not cited by the authors have examined serotonergic neurons in the rodent IP (Singhaniyom et al., 1982; Hamill et al., 1984; Hemmendinger and Moore, 1984), traced the projection of the IP to the hippocampus (Baisden et al., 1979; Shibata and Suzuki, 1984; Groenewegen et al., 1986; Lima et al., 2017), and have combined retrograde tract-tracing plus serotonergic markers to identify the neurotransmitter used in this pathway (Montone et al., 1988; Quina et al., 2017). In these studies, the IP neurons that express serotonergic markers and/or project to the hippocampus have been largely confined to a small area in the most caudal/dorsal part of the IP, consistent with the area shown in Figure 1 of Sherafat et al. In publications from 1984 onward, following the standardization of the IP nomenclature (Lenn and Hamill, 1984), this area has been referred to as the apical subnucleus of the IP (IPA), a designation that has been adopted in standard atlases for the rat and mouse (Paxinos and Watson, 1998; Paxinos and Franklin, 2001). If Sherafat et al. had identified the area of interest as the IPA, their work could have been more easily placed in the context of the prior research.

The prior work on the projections from the IP to the hippocampus may affect how readers interpret the results presented. As discussed in a recent paper (Quina et al., 2017), the molecular characteristics and anatomical connections of the IPA argue that it should be considered as the most rostral part of the median raphe (MnR) group of serotonergic neurons, rather than as an IP subnucleus. Three major points support this view: 1) Rather than being “a discrete population of neurons outside of the raphe nuclei”, the serotonergic IPA is anatomically continuous with the serotonergic neuronal population in the median raphe (MnR, B8 serotonin neurons). The identification of the IPA as a separate population of serotonergic neurons is essentially an artifact of sectioning in the coronal plane. This was noted in the earliest descriptions of these neurons (Singhaniyom et al., 1982). The continuity of the IPA with the MnR can also be seen in Allen Brain Atlas in situ hybridization data for serotonergic markers (Ng et al., 2009), where it is most easily visualized in the sagittal plane. One example is the ISH data for the serotonin transporter (Slc6a4, SERT, Allen Brain Atlas experiment 2624). In in the parasagital view shown in image 18/20 of this series, the IPA can be seen as a small cell group continuous with the rostral MnR, which is designated CS in the Allen nomenclature:
https://mouse.brain-map.org/experiment/siv?id=2624
2) The IPA does not resemble the largest and most-studied IP subnuclei, the rostral IP (IPR) and caudal IP (IPC), which do not contain serotonergic neurons, but instead are principally GABAergic (Hsu et al., 2013). 3) The hallmark of the IP is its highly conserved innervation by the medial habenula (MHb), to form the “habenulopeduncular pathway”, and the function of this pathway has been the focus of numerous recent studies (McLaughlin et al., 2017). The IPR and IPC receive inputs from the ventral MHb which expresses choline acetyltransferase, while the lateral IP (IPL) receives inputs from the dorsal MHb, which expresses the neuropeptide substance P. However, the IPA does not appear to receive inputs from either of these MHb systems (Quina et al., 2017). Thus both the gene expression characteristics and connectivity of the IPA link it to the serotonergic raphe, not the IP.

This continuity between the IPA and MnR has implications for interpreting the behavioral experiments presented by Sherafat et al. Extensive projections from the MnR to the hippocampus, including the vHipp, have been demonstrated using both classical tract-tracing methods (Vertes et al., 1999) and tract tracing with Cre-dependent adeno-associated viruses (AAV) in mice expressing Cre-recombinase from the Slc6a4 (serotonin transporter, SERT) gene locus (Muzerelle et al., 2016). The IPA is a small nucleus, contained within an area of approximately 100μm x 100μm x 200μm in the rostrocaudal, mediolateral, and dorsoventral dimensions, respectively. The volume of AAV injected in these hM4Di labeling experiments, 0.5 microliters, is thus ~250 times the volume of the targeted structure. Such an injection could not be confined to the IPA, and would be very likely to spread extensively into the immediately adjacent and much larger population of vHipp-projecting neurons in the MnR, unless somehow the retrograde transport of cre recombinase is selective for the IPA. However, no data are presented that address the extent of labeling with hM4Di in these injections, whether it is restricted to the IPA, extends into the MnR, or into other nearby populations of non-serotonergic neurons that may also have hippocampal projections and thus might express cre recombinase.

In conclusion, neurotransmitter identity, anatomy, and connectivity suggest that the intended target area in these studies, the IPA, should be considered as part of the MnR (B8) serotonergic cell group. In any case, it seems likely that vHipp-projecting neurons from the MnR proper would be labeled with silencing DREADDs using the methods described in this study. It is possible that the behavioral experiments presented reveal new information about the role of the serotonergic raphe system in the regulation of the behaviors examined, but no clear conclusions can be drawn from the data as presented.

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Competing Interests: None declared.

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