A role for glial fibrillary acidic protein (GFAP)-expressing cells in the regulation of gonadotropin-releasing hormone (GnRH) but not arcuate kisspeptin neuron output in male mice

  1. Charlotte Vanacker
  2. R Anthony Defazio
  3. Charlene M Sykes
  4. Suzanne M Moenter

  • Curated by eLife

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    Evaluation Summary:

    The study by Vanacker et al examines the effect of activating GFAP expressing cells within the POA and ARC on the activity of GnRH and TAC2 neurons as well as LH levels. The authors found that activation of GFAP POA cells activated GnRH neurons and increased circulating LH levels, limited to intact males. Activation of ARC GFAP cells failed to alter TAC2 activity or LH levels. Inhibition of POA GFAP cells or activation of POA GFAP cells in females failed to alter the firing rate of GnRH neurons. The study is largely well done and clearly presented.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

GnRH neurons are the final central neural output regulating fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (KNDy neurons) are considered the main regulator of GnRH output. GnRH and KNDy neurons are surrounded by astrocytes, which can modulate neuronal activity and communicate over distances. Prostaglandin E2 (PGE2), synthesized primarily by astrocytes, increases GnRH neuron activity and downstream pituitary release of luteinizing hormone (LH). We hypothesized that glial fibrillary acidic protein (GFAP)-expressing astrocytes play a role in regulating GnRH and/or KNDy neuron activity and LH release. We used adeno-associated viruses to target designer receptors exclusively activated by designer drugs (DREADDs) to GFAP-expressing cells to activate Gq- or Gi-mediated signaling. Activating Gq signaling in the preoptic area, near GnRH neurons, but not in the arcuate, increases LH release in vivo and GnRH firing in vitro via a mechanism in part dependent upon PGE2. These data suggest that astrocytes can activate GnRH/LH release in a manner independent of KNDy neurons.

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  1. Version published to 10.7554/elife.68205 on eLife
  2. eLife

    Author Response:

    Reviewer #1:

    The use of DREADDs to modulate astrocyte signaling and evaluate the contribution of these glial cells to the control of the GnRH system is relevant, timely and innovative. The authors provide a combination of compelling neuroanatomical data, electrophysiological recordings and LH measures that support their key findings in males. The calcium imaging experiments are rigorously performed but the data need to be validated on a larger number of animals. The authors also explore possible sex differences in the process but several caveats need to be overcome before reaching a conclusion on this aspect. Several additional points should be addressed in order to improve the manuscript, as elaborated below.

    1. It would be relevant to provide an estimation of the fraction of GnRH or KNDy neuron populations surrounded by infected astrocytes. This data would be interesting to discuss in relation with previous work showing that activation of only a fraction of GnRH neurons can induce LH release.

    This information has been added to the results and discussion parts (line 64-69 and 245-247 and 255-256).

    1. In the characterization of cell targeting, the authors should specify whether GFAP+ alpha tanycytes lining the dorsal part of the arcuate nucleus were also infected by viral constructs injected into the arcuate nucleus.

    This point has been added to the results, in the characterization of cell targeting part (line 76-77).

    1. Calcium imaging analyses were performed on 1 to 2 animals per group, which is below the minimum number of animals required for statistical analyses. In all experiments, a minimum of 3 animals per group is required.

    We agree philosophically that the statistical analysis of small n is not ideal. Our goal with the calcium imaging was to test if treatment with CNO altered intracellular calcium as a technical control, not to make any statement about the dynamics of this response. We were frankly surprised that significant P values emerged given the low numbers. We included these for transparency but could delete if that is the wish of the editorial group.

    1. To evaluate whether PGE2 mediates the effect of astrocyte activation on GnRH neuron firing, the authors pretreated slices with a mix of EP1 and EP2 antagonists. The rationale for choosing this combination should be explained considering that EP1 was previously shown not to be involved in the stimulatory effect of PGE2 on GnRH neuronal activity (Clasadonte et al., 2011, PMID: 21896757).

    As the reviewer points out, the main effect of PGE2 on GnRH neurons is likely through EP2. We chose a mix of EP1 and EP2 because EP1 is also expressed in GnRH neurons and we wanted to block all potential PGE2 receptors present on GnRH neurons.

    1. It is not clear whether the characterization data shown in figure 1 are also applicable for the experiments performed on females. If it is not the case, the data obtained in female should be added.

    The characterization done in Figure 1 was in males. A similar colocalization pattern emerged in Figure 6 with the co-injection of AAV5-GCaMP6 and AAV5-Gq, with the exception of higher infection of cells with neuronal morphology likely attributable to the longer post injection period.

    1. As fairly pointed out by the authors, there are major caveats in the experiments performed in females. They indicate that recordings were not made at the same moment of the day between males and females but also that the time post-surgery significantly differed between the 2 sexes (less than 2 months in males vs 5 months in females). Therefore, any conclusion about a possible sex difference can unfortunately not be drawn from these data. These experiments need to be reproduced in a rigorously controlled manner in order to reach a definitive conclusion.

    We agree the female data are limited by caveats, which we attempted to be transparent about. As mentioned in our response above, we still feel providing these data while acknowledging their limitations may be of use to others working in this area. It is likely to be quite some time before we have a replacement set of female data and if the present data can inform the experimental design of others, we’d like to share it.

    1. No electrophysiological recordings are shown. Representative recordings of GnRH and KNDy neuronal activity should be added to the figures.

    We have added representative recordings of GnRH and KNDy neurons to figure 4.

  3. eLife

    Evaluation Summary:

    The study by Vanacker et al examines the effect of activating GFAP expressing cells within the POA and ARC on the activity of GnRH and TAC2 neurons as well as LH levels. The authors found that activation of GFAP POA cells activated GnRH neurons and increased circulating LH levels, limited to intact males. Activation of ARC GFAP cells failed to alter TAC2 activity or LH levels. Inhibition of POA GFAP cells or activation of POA GFAP cells in females failed to alter the firing rate of GnRH neurons. The study is largely well done and clearly presented.

    (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. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The use of DREADDs to modulate astrocyte signaling and evaluate the contribution of these glial cells to the control of the GnRH system is relevant, timely and innovative. The authors provide a combination of compelling neuroanatomical data, electrophysiological recordings and LH measures that support their key findings in males. The calcium imaging experiments are rigorously performed but the data need to be validated on a larger number of animals. The authors also explore possible sex differences in the process but several caveats need to be overcome before reaching a conclusion on this aspect. Several additional points should be addressed in order to improve the manuscript, as elaborated below.

    1. It would be relevant to provide an estimation of the fraction of GnRH or KNDy neuron populations surrounded by infected astrocytes. This data would be interesting to discuss in relation with previous work showing that activation of only a fraction of GnRH neurons can induce LH release.

    2. In the characterization of cell targeting, the authors should specify whether GFAP+ alpha tanycytes lining the dorsal part of the arcuate nucleus were also infected by viral constructs injected into the arcuate nucleus.

    3. Calcium imaging analyses were performed on 1 to 2 animals per group, which is below the minimum number of animals required for statistical analyses. In all experiments, a minimum of 3 animals per group is required.

    4. To evaluate whether PGE2 mediates the effect of astrocyte activation on GnRH neuron firing, the authors pretreated slices with a mix of EP1 and EP2 antagonists. The rationale for choosing this combination should be explained considering that EP1 was previously shown not to be involved in the stimulatory effect of PGE2 on GnRH neuronal activity (Clasadonte et al., 2011, PMID: 21896757).

    5. It is not clear whether the characterization data shown in figure 1 are also applicable for the experiments performed on females. If it is not the case, the data obtained in female should be added.

    6. As fairly pointed out by the authors, there are major caveats in the experiments performed in females. They indicate that recordings were not made at the same moment of the day between males and females but also that the time post-surgery significantly differed between the 2 sexes (less than 2 months in males vs 5 months in females). Therefore, any conclusion about a possible sex difference can unfortunately not be drawn from these data. These experiments need to be reproduced in a rigorously controlled manner in order to reach a definitive conclusion.

    7. No electrophysiological recordings are shown. Representative recordings of GnRH and KNDy neuronal activity should be added to the figures.

  5. eLife

    Reviewer #2 (Public Review):

    The study by Vanacker et al builds upon previous literature demonstrating the PGE2 from astrocytes activates GnRH neurons. The authors demonstrate that chemogenetic activation of GFAP cells in the POA activates neighboring GnRH neurons via a PGE2 dependent mechanism. This may have implications on circulating LH levels as well as thermogenic and tachycardic conditions. The study is largely well done and clearly presented. There is some confusion/concerns about inclusion/exclusion of data within graphs, number of animals used for study, validation of animal models, and interpretation of physiological drivers of the activity or phenotype observed.

  6. Version published to 10.1101/2021.03.10.434805v1 on bioRxiv