Neural basis for regulation of vasopressin secretion by anticipated disturbances in osmolality

  1. Angela Kim
  2. Joseph C Madara
  3. Chen Wu
  4. Mark L Andermann
  5. Bradford B Lowell

  • Curated by eLife

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    Evaluation Summary:

    This study investigates the important question of how vasopressin neurons, which are critical for fluid balance, are rapidly activated or inhibited when mice begin to eat or drink. The study presents useful anatomic data on connectivity between these neurons and other structures and tests a broad range of possible inputs that could mediate these effects. The conclusions are largely supported by the data.

    (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

Water balance, tracked by extracellular osmolality, is regulated by feedback and feedforward mechanisms. Feedback regulation is reactive, occurring as deviations in osmolality are detected . Feedforward or presystemic regulation is proactive, occurring when disturbances in osmolality are anticipated . Vasopressin (AVP) is a key hormone regulating water balance and is released during hyperosmolality to limit renal water excretion. AVP neurons are under feedback and feedforward regulation. Not only do they respond to disturbances in blood osmolality, but they are also rapidly suppressed and stimulated, respectively, by drinking and eating, which will ultimately decrease and increase osmolality. Here, we demonstrate that AVP neuron activity is regulated by multiple anatomically and functionally distinct neural circuits. Notably, presystemic regulation during drinking and eating are mediated by non-overlapping circuits that involve the lamina terminalis and hypothalamic arcuate nucleus, respectively. These findings reveal neural mechanisms that support differential regulation of AVP release by diverse behavioral and physiological stimuli.

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  1. Version published to 10.7554/elife.66609 on eLife
  2. eLife

    Author Response:

    Reviewer #2:

    This study investigates the important question of which afferent circuits are responsible for the rapid, presystemic regulation of AVP neurons by food and water. Using a combination of rabies tracing, CRACM, and chemogenetic silencing in conjunction with photometry, the authors conclude that the MnPO transmits presystemic fluid signals whereas food signals are relayed by unidentified neurons in the ARC. The paper presents a collection of useful data investigating the anatomic connectivity between cell types in the LT and elsewhere and tests a broad range of possible inputs that could mediate these presystemic effects. However there are interpretational problems with the experiments that investigate functional connectivity (which pathways transmit which signals).

    Major points:

    1. The conclusion that the ARC is the source of the presystemic signal that activates AVP neurons in response to food is based on an experiment in which the effect of silencing the VMH and DMH individually on a photometry trace is subtracted from the effect of silencing DMH, VMH and ARC simultaneously. This is problematic given the broad and variable spread of such injections, the difficulty of completely and selectively hitting a single nucleus, and the lack of characterization. It also seems likely that there could be synergistic effects of inhibiting multiple adjacent nuclei. From the data in Figure 7F, it appears that most of the effect is driven by a few animals, which also raises the question of sources of variability.

    We completely agree with the reviewer. However, we want to emphasize that given its anatomical size and location and molecular and functional complexity, ARC is an extremely difficult region to study especially without knowing an identity of the neuronal population we are targeting. As mentioned in the manuscript (“This approach allowed for effective silencing of the ARC with hM4Di, which is difficult to achieve with restricted injection of cre-independent AAVs into the ARC” lines 418-420), subtractive approach was the most reasonable and efficient strategy we could take to address the question. However, as mentioned in our response to Essential revisions #4, we have attempted, though without much success, multiple approaches to more specifically identify the circuit.

    1. The effect of silencing DMH/VMH/ARC on food intake is not reported. If these mice eat less or more slowly, this would explain the partial reduction in the presystemic activation of AVP neurons.

    We now provide food intake and latency data for DMH/VMH/ARC silencing experiments (Figure 7-figure supplement 4). Feeding behavior was not affected during the experiment. Therefore, reduced feeding-related presystemic response in AVP neurons is not indirectly caused by slower or less food consumption.

    1. It is not clearly reported to what extent the chemogenetic silencing of the MnPO/OVLT in the mice used in Fig. 2 and 5 reduces the amount of water consumed and how this relates to the dynamics in each animal/trial. This is confounding in two ways. Given that the silencing does not fully block drinking, this implies that the MnPO/OVLT silencing is incomplete (based on Augustine 2018), and thus the negative photometry result in Fig. 5 is hard to interpret. Conversely, if silencing reduces drinking partially, which seems likely, then this behavioral change could account for the reduced presystemic inhibition of AVP neurons. It is hard to see how the direct effect of MnPO on AVP neural dynamics could be separated from its effects on behavior in this experiment. While there is a pre-ingestive response in the AVP neurons (which does not have this confound), in several experiments in Fig. 2 this is approx. 1% dF/F.

    Please refer to our response to Essential revisions #1.

    "It is a valid concern and we now discuss this point in our manuscript (lines 507-512). However, for the following reasons, we do not believe it is likely that reduced presystemic suppression of AVP neurons is mainly driven by behavioral changes. First, pre-ingestive, cue-induced suppression observed prior to any drinking behavior is completely blocked by silencing of MnPO/OVLTVgat neurons (Figure 5). Second, MnPO/OVLT neurons provide direct excitatory and inhibitory synaptic inputs to AVP neurons with high probability connections (~100% and ~50%, respectively, Figure 1). Finally, SON-projecting, putative AVP-regulating MnPO/OVLT neurons show water- related presystemic responses that resemble those seen in AVP neurons (Figure 3). Altogether, these factors strongly support the notion that the reduced presystemic suppression of AVP neurons upon silencing of MnPO/OVLT input is primarily caused by direct reduction of the influence of MnPO/OVLT input onto AVP neurons."

    1. MnPO neurons are heterogeneous in their dynamics, especially the MnPO-GABA neurons, and for this reason ruling out a possible mechanism based on a photometry trace is challenging. For example, compare the interpretation of the photometry recordings of MnPO-Glp1r neurons in (Augustine, 2018) with the results of single cell imaging of the same neurons in (Zimmerman, 2019)).

    We completely agree with the reviewer that MnPO neurons are extremely heterogeneous. To address this issue, we specifically recorded the activity of SON- projecting MnPO/OVLT neurons that will certainly include the population that is directly involved in AVP neuron regulation. Please note that the SON is almost exclusively comprised of neuroendocrine AVP and Oxytocin neurons.

    Reviewer #3:

    This manuscript by Kim and colleagues explores the circuit that communicates food- and water-intake-related presystemic regulations to vasopressinergic endocrine output neurons. Although previous work from several labs had observed food and water intake related anticipatory signaling in cell types in several lamina terminalis (LT) nuclei, the functional significance of these remained unexplored. Here, the authors demonstrate that the neural circuits underlying foo- and water-related presystemic signals are anatomically dissociable at the level of the vasopressin secreting endocrine output neurons. The authors use viral retrograde tracing to identify candidate anatomical regions that could communicate food and water intake related anticipatory signals to VP neurons in the SON and PVN. They show that excitatory neurons in LT nuclei SFO and MnPO/OVLT and inhibitory neurons in the latter make direct synaptic connections onto VP neurons in the SON and PVN. They also perform chemogenetic silencing experiments to elucidate the functional importance of LT and other brain structures for presystemic VP regulation. They show that MnPO/OVLT is important for water drinking but not food intake related presystemic regulation. Furthermore, the authors survey several brain regions that could provide the food intake-related input to VP neurons identifying the arcuate nucleus as the likely source. The experiments in this paper are generally rigorous. Addressing the following points should improve the manuscript:

    • In Figure 2, DREADD was used to suppress the activity of the LT. However, the virus construct is a general promoter, and no data is provided to demonstrate that CNO/DREADD works in this system or cells. In particular, there is no behavioral effect by CNO inhibition of SFO or MnPO/OVLT. To confirm these negative data, slice ephys or similar method should be used to confirm the efficiency of chemogenetic manipulation.

    We now provide slice electrophysiology data showing effective silencing of MnPO/OVLT and SFO neurons by CNO/hM4Di (Figure 2-figure supplement 3).

    • Although the authors revealed the anatomic sites relevant for different kinds of presystemic regulation of VP neurons, the causal role of specific cell types in these structures that provide this input remains untested/unclear. The manuscript would be significantly more impactful if this was addressed. Specifically, whether excitatory and inhibitory populations in the MnPO/OVLT indeed mediate the pre- and post-ingestive effects on presystemic VP neuronal activity as suggested by GCaMP imaging should be tested.

    We thank the reviewer for suggesting this experiment. The data is now presented in Figure 5.

    • The authors rule out two cell populations in ARC as a potential source of food-related presystemic effects on VP neurons. They do suspect a specific cell type in ARC (OXTR+ neurons), which should be tested.

    Please refer to our response to Essential revisions #4.

    "We agree that this is an extremely interesting question, and we have ambitiously attempted multiple approaches to identify an ARC neuronal population that provide feeding-related presystemic signal to AVP neurons. A main obstacle in targeting glutamatergic ARC population is that we currently do not have a specific genetic marker for these neurons. We could not use Oxtr-cre mice that were used in our original study because we saw cre expression around the SON that prevented us from using Oxtr- cre;AVP-cre mice to selectively target ARC Oxtr and AVP neurons in the same animal. We also attempted using Vglut2-flp;AVP-cre mice but achieving restricted hM4Di expression in the ARC with viral injection was extremely challenging and we decided that the experiment is too inefficient to be completed “in a timely manner”.

    As we now present in Figure 7-figure supplement 4, DMH/VMH/ARC silencing did not alter feeding behavior. Therefore, reduced feeding-related presystemic response in AVP neurons are not indirectly caused by slower or less food consumption."

  3. eLife

    Evaluation Summary:

    This study investigates the important question of how vasopressin neurons, which are critical for fluid balance, are rapidly activated or inhibited when mice begin to eat or drink. The study presents useful anatomic data on connectivity between these neurons and other structures and tests a broad range of possible inputs that could mediate these effects. The conclusions are largely supported by the data.

    (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.)

  4. eLife

    Reviewer #1 (Public Review):

    The authors of this paper use contemporary circuit-mapping techniques to identify the excitatory and inhibitory inputs to AVP neurons in the hypothalamus. They then use chemogenetic methods to inhibit potential inputs to AVP neurons while imaging calcium activity of the AVP neurons by fiber photometry to identify the relevant inputs for pre-systemic inhibition of AVP neurons. They conclude that the presystemic regulation AVP neurons by drinking and eating are mediated by non-overlapping neural circuits - drinking inhibits AVP neurons via decreased activity of glutamatergic MnPo/OVLT and SFO neurons and an increased activity of GABAergic MnPO/OVLT neurons. Whereas AVP neuron activation in response to eating comes from unknown neurons in the arcuate nucleus. Overall, this is a comprehensive analysis of the neural circuitry controlling presystemic AVP neuron activity.

    A major disappointment in the study is the failure to identify the neurons in the ARC thT provide excitatory input to AVP neurons in response to eating. The authors suggest that the rapid activation of AVP neurons is likely to glutamatergic (lines 492-497) and go on to suggest the Oxtr-expressing neurons are a good candidate. Thus, it is surprising that they tested Pomc- and Agrp-expressing neurons and not the glutamatergic Oxtr-expressing neurons.

  5. eLife

    Reviewer #2 (Public Review):

    This study investigates the important question of which afferent circuits are responsible for the rapid, presystemic regulation of AVP neurons by food and water. Using a combination of rabies tracing, CRACM, and chemogenetic silencing in conjunction with photometry, the authors conclude that the MnPO transmits presystemic fluid signals whereas food signals are relayed by unidentified neurons in the ARC. The paper presents a collection of useful data investigating the anatomic connectivity between cell types in the LT and elsewhere and tests a broad range of possible inputs that could mediate these presystemic effects. However there are interpretational problems with the experiments that investigate functional connectivity (which pathways transmit which signals).

    Major points:

    1. The conclusion that the ARC is the source of the presystemic signal that activates AVP neurons in response to food is based on an experiment in which the effect of silencing the VMH and DMH individually on a photometry trace is subtracted from the effect of silencing DMH, VMH and ARC simultaneously. This is problematic given the broad and variable spread of such injections, the difficulty of completely and selectively hitting a single nucleus, and the lack of characterization. It also seems likely that there could be synergistic effects of inhibiting multiple adjacent nuclei. From the data in Figure 7F, it appears that most of the effect is driven by a few animals, which also raises the question of sources of variability.

    2. The effect of silencing DMH/VMH/ARC on food intake is not reported. If these mice eat less or more slowly, this would explain the partial reduction in the presystemic activation of AVP neurons.

    3. It is not clearly reported to what extent the chemogenetic silencing of the MnPO/OVLT in the mice used in Fig. 2 and 5 reduces the amount of water consumed and how this relates to the dynamics in each animal/trial. This is confounding in two ways. Given that the silencing does not fully block drinking, this implies that the MnPO/OVLT silencing is incomplete (based on Augustine 2018), and thus the negative photometry result in Fig. 5 is hard to interpret. Conversely, if silencing reduces drinking partially, which seems likely, then this behavioral change could account for the reduced presystemic inhibition of AVP neurons. It is hard to see how the direct effect of MnPO on AVP neural dynamics could be separated from its effects on behavior in this experiment. While there is a pre-ingestive response in the AVP neurons (which does not have this confound), in several experiments in Fig. 2 this is approx. 1% dF/F.

    4. MnPO neurons are heterogeneous in their dynamics, especially the MnPO-GABA neurons, and for this reason ruling out a possible mechanism based on a photometry trace is challenging. For example, compare the interpretation of the photometry recordings of MnPO-Glp1r neurons in (Augustine, 2018) with the results of single cell imaging of the same neurons in (Zimmerman, 2019)).

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript by Kim and colleagues explores the circuit that communicates food- and water-intake-related presystemic regulations to vasopressinergic endocrine output neurons. Although previous work from several labs had observed food and water intake related anticipatory signaling in cell types in several lamina terminalis (LT) nuclei, the functional significance of these remained unexplored. Here, the authors demonstrate that the neural circuits underlying foo- and water-related presystemic signals are anatomically dissociable at the level of the vasopressin secreting endocrine output neurons. The authors use viral retrograde tracing to identify candidate anatomical regions that could communicate food and water intake related anticipatory signals to VP neurons in the SON and PVN. They show that excitatory neurons in LT nuclei SFO and MnPO/OVLT and inhibitory neurons in the latter make direct synaptic connections onto VP neurons in the SON and PVN. They also perform chemogenetic silencing experiments to elucidate the functional importance of LT and other brain structures for presystemic VP regulation. They show that MnPO/OVLT is important for water drinking but not food intake related presystemic regulation. Furthermore, the authors survey several brain regions that could provide the food intake-related input to VP neurons identifying the arcuate nucleus as the likely source. The experiments in this paper are generally rigorous. Addressing the following points should improve the manuscript:

    • In Figure 2, DREADD was used to suppress the activity of the LT. However, the virus construct is a general promoter, and no data is provided to demonstrate that CNO/DREADD works in this system or cells. In particular, there is no behavioral effect by CNO inhibition of SFO or MnPO/OVLT. To confirm these negative data, slice ephys or similar method should be used to confirm the efficiency of chemogenetic manipulation.

    • Although the authors revealed the anatomic sites relevant for different kinds of presystemic regulation of VP neurons, the causal role of specific cell types in these structures that provide this input remains untested/unclear. The manuscript would be significantly more impactful if this was addressed.
    Specifically, whether excitatory and inhibitory populations in the MnPO/OVLT indeed mediate the pre- and post-ingestive effects on presystemic VP neuronal activity as suggested by GCaMP imaging should be tested.

    • The authors rule out two cell populations in ARC as a potential source of food-related presystemic effects on VP neurons. They do suspect a specific cell type in ARC (OXTR+ neurons), which should be tested.

  7. Version published to 10.1101/2021.01.27.428388v1 on bioRxiv